U.S. patent application number 17/229013 was filed with the patent office on 2021-11-25 for novel gold-based nanocrystals for medical treatments and electrochemical manufacturing processes therefor.
This patent application is currently assigned to Clene Nanomedicine, Inc.. The applicant listed for this patent is David A. Bryce, Anthony Lockett, Mikhail Merzliakov, Mark Gordon Mortenson, D. Kyle Pierce, Reed N. Wilcox. Invention is credited to David A. Bryce, Anthony Lockett, Mikhail Merzliakov, Mark Gordon Mortenson, D. Kyle Pierce, Reed N. Wilcox.
Application Number | 20210361699 17/229013 |
Document ID | / |
Family ID | 1000005740433 |
Filed Date | 2021-11-25 |
United States Patent
Application |
20210361699 |
Kind Code |
A1 |
Mortenson; Mark Gordon ; et
al. |
November 25, 2021 |
Novel Gold-Based Nanocrystals for Medical Treatments and
Electrochemical Manufacturing Processes Therefor
Abstract
The present invention relates to novel gold nanocrystals and
nanocrystal shape distributions that have surfaces that are
substantially free from organic impurities or films. Specifically,
the surfaces are "clean" relative to the surfaces of gold
nanoparticles made using chemical reduction processes that require
organic reductants and/or surfactants to grow gold nanoparticles
from gold ions in solution. The invention includes novel
electrochemical manufacturing apparatuses and techniques for making
the gold-based nanocrystals. The invention further includes
pharmaceutical compositions thereof and the use of the gold
nanocrystals or suspensions or colloids thereof for the treatment
or prevention of diseases or conditions for which gold therapy is
already known and more generally for conditions resulting from
pathological cellular activation, such as inflammatory (including
chronic inflammatory) conditions, autoimmune conditions,
hypersensitivity reactions and/or cancerous diseases or conditions.
In one embodiment, the condition is mediated by MT (macrophage
migration inhibiting factor).
Inventors: |
Mortenson; Mark Gordon;
(North East, MD) ; Pierce; D. Kyle; (Elkton,
MD) ; Bryce; David A.; (Elkton, MD) ; Wilcox;
Reed N.; (Littleton, CO) ; Lockett; Anthony;
(Leeds, GB) ; Merzliakov; Mikhail; (Parkville,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mortenson; Mark Gordon
Pierce; D. Kyle
Bryce; David A.
Wilcox; Reed N.
Lockett; Anthony
Merzliakov; Mikhail |
North East
Elkton
Elkton
Littleton
Leeds
Parkville |
MD
MD
MD
CO
MD |
US
US
US
US
GB
US |
|
|
Assignee: |
Clene Nanomedicine, Inc.
North East
MD
|
Family ID: |
1000005740433 |
Appl. No.: |
17/229013 |
Filed: |
April 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17223336 |
Apr 6, 2021 |
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17229013 |
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16535672 |
Aug 8, 2019 |
10980832 |
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17223336 |
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15465092 |
Mar 21, 2017 |
10449217 |
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16535672 |
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13382781 |
Dec 28, 2012 |
9603870 |
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PCT/US10/41427 |
Jul 8, 2010 |
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15465092 |
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61294690 |
Jan 13, 2010 |
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61263648 |
Nov 23, 2009 |
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61249804 |
Oct 8, 2009 |
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61235574 |
Aug 20, 2009 |
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61228250 |
Jul 24, 2009 |
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61226153 |
Jul 16, 2009 |
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61223944 |
Jul 8, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C30B 29/60 20130101;
A61K 9/14 20130101; A61K 33/24 20130101; C30B 29/02 20130101; C30B
7/12 20130101; A61K 9/10 20130101; A61K 9/0095 20130101; Y02A 50/30
20180101; B22F 1/0022 20130101; B22F 9/00 20130101; A61K 33/242
20190101; A61K 9/08 20130101; A61K 47/02 20130101; B22F 2001/0037
20130101; B82Y 30/00 20130101 |
International
Class: |
A61K 33/242 20060101
A61K033/242; B22F 1/00 20060101 B22F001/00; B22F 9/00 20060101
B22F009/00; B82Y 30/00 20060101 B82Y030/00; C30B 7/12 20060101
C30B007/12; C30B 29/02 20060101 C30B029/02; C30B 29/60 20060101
C30B029/60; A61K 9/10 20060101 A61K009/10; A61K 47/02 20060101
A61K047/02; A61K 9/00 20060101 A61K009/00; A61K 9/14 20060101
A61K009/14; A61K 33/24 20060101 A61K033/24; A61K 9/08 20060101
A61K009/08 |
Claims
1. A method for treating a patient with amyotrophic lateral
sclerosis disease, comprising administering to a patient in need
thereof an effective amount of a pharmaceutically acceptable
suspension comprising: a.) pharmaceutical grade water; b.) at least
one processing enhancer comprising sodium bicarbonate; c.) gold
nanocrystals suspended in said water forming a suspension, wherein
said gold nanocrystals: i.) have surfaces that do not have organic
chemical constituents adhered or attached to said surfaces: ii.)
have a mode particle size of less than about 50 nm; iii.) are
present in said suspension at a concentration of at least 2 ppm;
and d.) said suspension having a pH of between about 5 to about
9.5, said gold nanocrystals having a zeta potential of about -20 my
or lower at a temperature of about 25'C, said zeta potential being
determined by measuring the electrophoretic mobility of the gold
nanocrystals in the suspension, and the suspension does not contain
chloride ions.
2. The method of claim 1, wherein said suspension is administered
orally.
3. A method for treating a patient with amyotrophic lateral
sclerosis disease, comprising administering to a patient in need
thereof an effective amount of a pharmaceutically acceptable
suspension comprising: a.) water; b.) at least one processing
enhancer; c.) gold nanocrystals suspended in said water forming a
suspension, wherein said gold nanocrystals: i) have surfaces that
do not have organic chemical constituents adhered or attached to
said surfaces; ii.) have a mode particle size of less than about 50
nm; iii.) are present in said suspension at a concentration of at
least 2 ppm; and d.) said suspension having a pH of between about 5
to about 9.5, said gold nanocrystals having a zeta potential of
about -20 my or lower at a temperature of about 25.degree. C., said
zeta potential being determined by measuring the electrophoretic
mobility of the gold nanocrystals in the suspension.
4. The method of claim 3, wherein said suspension is administered
orally.
5. A method for treating a patient with amyotrophic lateral
sclerosis disease, comprising administering to a patient in need
thereof an effective amount of a pharmaceutical suspension
comprising: a.) water and sodium bicarbonate dissolved therein,
said suspension medium having a pH of between about 5 to about 9.5;
b.) shaped gold nanocrystals in said suspension medium forming a
suspension, said shaped gold nanocrystals having a zeta potential
of about -30 mV or lower at a temperature of about 25.degree. C.,
said zeta potential being determined by measuring the
electrophoretic mobility of the shaped gold nanocrystals in the
pharmaceutical suspension; and wherein said shaped gold
nanocrystals: i.) have surfaces that do not have organic chemical
constituents adhered or attached to said surfaces: ii.) have a mode
particle size of less than about 30 nm; iii.) are present in said
suspension at a concentration of at least about 2 ppm; and iv.)
comprise triangle and pentagon shapes.
6. The method of claim 5, wherein said suspension is administered
orally.
7. The method of claim 1, wherein said gold nanocrystals have a
zeta potential of about -30 mV or lower.
8. The method of claim 1, wherein said gold nanocrystals have a
zeta potential of about -40 mV or lower.
9. The method of claim 1, wherein said gold nanocrystals have a
zeta potential of about -50 mV or lower.
10. The method of claim 1, wherein said gold nanocrystals have
shapes comprising faces with spatially extended low index crystal
planes, said shapes appearing as triangles and pentagons when dried
from suspension on a surface.
11. The method of claim 10, wherein said shaped gold nanocrystals
further comprise shapes which appear as hexagons and diamond shapes
when dried from suspension on a surface.
12. The method of claim 1, wherein said gold nanocrystals are
present at a concentration of about 2-200 ppm.
13. The method of claim 1, wherein said mode particle size is
within a range of about 8-18 nm and said pH is between about 8 to
about 9.5.
14. The method of claim 13, wherein said gold nanocrystals are
shaped and have low Miller index crystal planes arranged into
shapes comprising triangles and pentagons.
15. The method of claim 14, wherein said shaped gold nanocrystals
having said low Miller index crystal planes further comprise shapes
of hexagons and diamonds.
16. The method of claim 1, wherein said gold nanocrystals are
shaped and include at least one low Miller index crystal plane
selected from the group of crystal planes consisting of {111},
{110} and {100}.
17. The method of claim 1, wherein said gold nanocrystals are
shaped and comprise at least one low Miller index {111} crystal
plane.
18. The method of claim 1, wherein said gold nanocrystals have a
mode particle size of less than about 30 nm.
19. The method of claim 1, wherein said gold nanocrystals have a
mode particle size within a range of about 8-18 nm.
20. The method of claim 3, wherein said gold nanocrystals are
present at a concentration of about 2-200 ppm.
21. The method of claim 5, wherein said gold nanocrystals are
present at a concentration of about 2-200 ppm.
22. The method of claim 3, wherein said at least one processing
enhancer comprises at least one material selected from the group of
materials consisting of sodium bicarbonate, sodium carbonate,
potassium bicarbonate, potassium carbonate, trisodium phosphate,
disodium phosphate, monosodium phosphate, potassium phosphates or
other salts of carbonic acid.
23. The method of claim 3, wherein said suspension does not contain
chloride ions.
24. A method for treating a patient with amyotrophic lateral
sclerosis disease comprising administering orally to a patient in
need thereof an effective amount of a pharmaceutically acceptable
suspension comprising: a.) water and NaHCO.sub.3 dissolved therein;
b.) gold nanocrystals suspended in said water forming a suspension,
wherein said gold nanocrystals: i.) have surfaces that do not have
organic chemical constituents adhered or attached to said surfaces;
ii.) have a mode particle size of less than about 50 nm; iii.) are
present in said pharmaceutical suspension at a concentration of at
least 2 ppm iv.) are shaped and comprise at least one low Miller
index crystal plane selected from the group of crystal planes
consisting of {111}, {110} and {100}; and c.) said suspension
having a pH of between about 8 to about 9.5, said gold nanocrystals
have a zeta potential of about -30 mV or lower at a temperature of
about 25.degree. C., said zeta potential being determined by
measuring the electrophoretic mobility of the gold nanocrystals in
the pharmaceutical suspension, and the pharmaceutical suspension
does not contain chloride ions.
25. The method of claim 24, wherein said gold nanocrystals have
shapes comprising faces with spatially extended low index crystal
planes, said shapes appearing as triangles and pentagons when dried
from suspension on a surface.
26. The method of claim 25, wherein said shaped gold nanocrystals
further comprise hexagon and diamond shapes.
27. The method of claim 24, wherein said at least one low Miller
index crystal plane comprises a crystal plane {111}.
Description
[0001] The present application is a division of U.S. application
Ser. No. 17/223,336 (filed Apr. 6, 2021). U.S. application Ser. No.
17/223,336 is a division of Ser. No. 16/535,672 (filed Aug. 8,
2019), which was a division of U.S. application Ser. No. 15/465,092
(filed Mar. 21, 2017), now U.S. Pat. No. 10,449,217 (issued Oct.
22, 2019). U.S. application Ser. No. 15/465,092 is a division of
U.S. application Ser. No. 13/382,781 (filed Dec. 28, 2012, now U.S.
Pat. No. 9,603,870 (issued Mar. 28, 2017). U.S. application Ser.
No. 13/382,781 is a U.S. national stage entry of International
Application No. PCT/US2010/41427, filed Jul. 8, 2010. Said
international application claims priority to seven other US patent
applications: 1) U.S. Ser. No. 61/223,944 filed on Jul. 8, 2009; 2)
U.S. Ser. No. 61/226,153 filed on Jul. 16, 2009; 3) U.S. Ser. No.
61/228,250 filed on Jul. 24, 2009; 4) U.S. Ser. No. 61/235,574
filed on Aug. 20, 2009; 5) U.S. Ser. No. 61/249,804 filed on Oct.
8, 2009; 6) U.S. Ser. No. 61/263,648 filed on Nov. 23, 2009; and 7)
U.S. Ser. No. 61/294,690 filed on Jan. 13, 2010.
FIELD OF THE INVENTION
[0002] The present invention relates to novel gold nanocrystals and
nanocrystal shape distributions that have surfaces that are
substantially free from organic or other impurities or films.
Specifically, the surfaces are "clean" relative to the surfaces of
gold nanoparticles made using chemical reduction processes that
require organic reductants and/or surfactants to grow gold
nanoparticles from gold ions in solution.
[0003] The invention includes novel electrochemical manufacturing
apparatuses and techniques for making the gold-based nanocrystals.
The invention further includes pharmaceutical compositions thereof
and the use of the gold nanocrystals or suspensions or colloids
thereof for the treatment or prevention of diseases or conditions
for which gold therapy is already known and more generally for
conditions resulting from pathological cellular activation, such as
inflammatory (including chronic inflammatory) conditions,
autoimmune conditions, hypersensitivity reactions and/or cancerous
diseases or conditions. In one embodiment, the condition is
mediated by MIF (macrophage migration inhibiting factor).
BACKGROUND OF THE INVENTION
Gold Salts
[0004] Robert Koch is credited with discovering the bacteriostatic
effect of gold cyanide on Mycobacterium tuberculosis. It was
subsequently observed that patients with tuberculosis often
benefited from a reduction in certain inflammatory conditions when
given gold salt injections for the disease. This observed reduction
in inflammation led to aurothiolates being used by Forestier in
1927 as a treatment for rheumatoid arthritis (Panyala, 2009)
(Abraham, 1997). The early gold-based products were typically
injected in an intramuscular, or subcutaneous manner (and later in
an intraarterial manner) and some are still available today and/or
still being used to treat rheumatoid arthritis.
[0005] Specifically, it has been known for many years that certain
gold compounds possess anti-inflammatory activity. For example, (i)
sodium gold thiomalate (also referred to as "gold sodium
thiomalate"), marketed as Myocrisin and related chemical versions,
marketed as Myochrisine and Myochrisis; (ii) sodium gold
thioglucose (also referred to as "gold sodium thioglucose"),
marketed as Solganol; (iii) sodium gold thiosulfate, marketed as
Sanocrysin and related chemical versions, marketed as Crisalbine,
Aurothion and Sanocrysis; and (iv) sodium gold
thiopropanolsulfonate, marketed as Allocrysine, have been used in
the treatment of rheumatoid arthritis (Sadler, 1976; Shaw, 1999;
Eisler, p. 133, 2004). Only monovalent gold salts were believed to
exhibit therapeutic effects for the treatment of rheumatoid
arthritis. In 1961 the Empire Rheumatism Council affirmed that
injectable gold salts showed efficacy and gold salts remain a
widely used method of treatment of progressive rheumatoid arthritis
(Ueda, 1998).
[0006] Treatment with various gold salts has also been suggested,
or anecdotally observed, to be effective in a range of other
diseases, including asthma, HIV, malaria and cancer. A considerable
body of evidence exists in these diseases, in both human and animal
models, suggesting that gold may be a viable treatment option for
these areas of unmet medical need (Dabrowiak, 2009).
Oral Gold
[0007] More recently, an oral gold product, 2,3,4,6-Tetra-o-acetyl
I-thio B-D-glucopyranosato-S-(triethyl-phosphine), marketed as
Auranofin.RTM. or Ridaura.RTM. in several parts of the world, has
become available (Ho & Tiekink, 2005, Dabrowiak, 2009).
Auranofin.RTM. was approved by the FDA for human use in the
mid-1980's; and Auranofin.RTM. had the advantage of being orally
absorbed, but was considered to be less effective than the
injectable gold thiolates (Sadler, 1976; Shaw 1999).
Toxicology of Gold Salts and Oral Gold
[0008] Historically, toxicity has limited the use of all injectable
and oral gold-based therapies, with anywhere from 30-50% of
patients terminating various gold-based treatments due to
undesirable or intolerable side effects. The side effects of many
conventional gold therapies include rashes or mucocutaneous effects
(e.g., pruritus, dermatitis and stomatitis); hematologic changes
(e.g., thrombocytopenia); protein in the urine (proteinuria);
inflammation of the mouth; reduction in the number of circulating
leukocytes; decreased number of blood platelets; aplastic anemia
due to organ damage; lung abnormalities; adverse immune reactions,
such as eosinophilia, lymphadenopathy, hypergamma globulinemia;
severe hypotension, angina, myocardial infarction, nephrotoxicity
and nephrotic syndrome; hepatitis; colitis; and chrysiasis
(pigmentation) of the cornea, lens, and skin (Eisler, p. 133-134,
2004). The most common side effect of chrysotherapy was skin
toxicity, accounting for up to 60% of all adverse reactions,
especially lichenoid eruptions and non-specific dermatitis (Eisler,
p. 133-134, 2004). These side effects are believed to be related to
the formulations used (e.g., carrier molecule, oxidation state of
the gold in the compound, etc.), rather than the gold itself (Ho
& Tiekink, 2005).
[0009] Payne and Arena in 1978 reported the subacute and chronic
toxicity of several oral gold compounds, including Auranofin.RTM.,
in rats, compared to an injected gold control. Sprague Dawley rats
were dosed for periods of 6 weeks, 6 months and one year. In a
follow-up study, the 1-year investigation was repeated with
sequential kills and a modified dosing regimen.
[0010] The target organs identified by this study were the stomach
and kidney. Gastric changes consisted of superficial erosions of
the mucosa extending up to 1/3 of the thickness of the mucosa and
covering up to 5% of its surface area. This change was dose-related
and was associated with loss of body weight. Healing lesions were
also evident. In the kidney of rats given SK&F 36914 for six
months there was enlargement of cortical tubular epithelial cells
(cytomegaly). In addition, there was a dose-related enlargement of
the nucleus (karyomegaly), with evidence of pleomorphic and
multinucleate cells. In the 1-year study similar changes were seen,
but in addition renal cortical cell adenomas were seen in a
dose-related incidence (0/38, 3/39, 6/37 and 8/37 for control, low,
intermediate and high dose respectively). In a repeated 1-year
study an unexpectedly high incidence of mortality occurred. This
was attributed to ileocaecal lesions that progressed to ulceration
that appeared to perforate the gut wall in a number of cases.
Presumably death resulted from acute infectious peritonitis. In the
injected controls, gold sodium thiomalate was administered by
intra-muscular injection once weekly for a year and, in a second
study, once weekly for 46 weeks and then daily for 330 days. In the
1-year study, renal tubular cell karyomegaly was observed and renal
cell adenoma was seen in 1/16 females but not in males. In the
21-month study all surviving rats showed karyomegaly of the renal
cortical tubular epithelium and cystic tubules were frequently
observed. Renal adenomas, occasionally multiple, were seen in 8/8
females and 3/7 males surviving to 21 months (Payne & Arena,
1978). Similar results were seen in dogs (Payne & Arena, The
subacute and chronic toxicity of SK&F 36914 and SK&F
D-39162 in dogs, 1978).
[0011] Szabo et al 1978 a reported the effects of gold-containing
compounds, including Auranofin.RTM. on pregnant rats and fetuses.
The effects of gold sodium thiomalate and the oral gold compound
Auranofin.RTM. on maternal and fetal toxicity and teratogenicity
were investigated. Oral gold was administered by intubation on days
6-15 of pregnancy, while gold sodium thiomalate was administered on
days 6-15 by subcutaneous injection. This was a standard exposure
period in such studies and this exposure is considered to be
equivalent to the first trimester of a human pregnancy. Standard
procedures were used to examine fetuses and group sizes were
adequate for the purpose of the study. Maternal and fetal toxicity
was evident, and fetuses of gold sodium thiomalate-dosed animals
showed a pattern of dose-related malformations. The doses used led
to death of a proportion of the dams and showed marked effects on
body weight (including actual weight loss at the start of dosing)
and reduced food consumption. The malformations included skeletal
anomalies, external malformations and degrees of hydrocephalus and
ocular defects. SK&F D-39162 did not affect food intake or
weight gain, but was also associated with reductions in fetal
weight compared to controls. The only major defect found with
SK&F D-39162 treatment was edema. There was no evidence of an
effect of gold sodium thiomalate on implantation, resorption, fetal
number or fetal weight in the gold sodium thiomalate-treated
animals. These authors concluded that the effects on the fetus were
indirect and were attributable to accumulation of gold in the
lysosomes of the visceral yolk sac epithelium, with consequent
inhibition of vital enzymes involved in fetal nutrition. This
hypothesis was advanced to explain the teratogenicity of other
chemicals and could be plausible (Szabo, Guerriero, & Kang, The
effects of gold containing compounds on pregnant rats and their
fetuses, 1978).
[0012] Szabo et al 1978b reported the effects of gold-containing
compounds on pregnant rabbits and fetuses. In this study pregnant
rabbits were dosed from days 6-18 of pregnancy. Gold sodium
thiomalate was administered by sub-cutaneous injection and oral
compounds were given by intubation. Both routes of administration
led to maternal deaths and abortions were also observed in
surviving animals. Dose-related decreases in maternal food
consumption, leading to actual body weight losses, were observed at
the higher doses of both injected and oral gold. Effects were also
evident on litter sizes, numbers of resorptions and mean fetal
weights. Fetal anomalies and malformations were also observed,
primarily in the abdomen (gastroschisis and umbilical hernia), with
a lower incidence of anomalies affecting the brain, heart lungs and
skeleton. The authors concluded that the incidence of abdominal
anomalies, exceeding all of their historical control data,
indicated a specific sensitivity in the rabbit to such an effect of
gold (Szabo, DiFebbo, & Phelan, 1978).
[0013] Based on these studies, oral administration of relatively
high doses of gold-containing compounds was associated with a
dose-related incidence of erosions of the gastric mucosa and, in a
longer duration study, of significant ileocaecal lesions (including
ulceration) that caused the deaths of a number of animals.
Examination of the data presented suggested that the gastric
lesions were typical of a marked direct local effect on the mucosa.
The renal cortical tubular epithelium was another target tissue,
perhaps through the development of high local concentrations during
the concentration of the urine. The cortical tubular epithelium
lesions progressed from karyomegaly to adenoma formation in a
significant number of animals. Although this is a benign tumor it
cannot be ignored in terms of risk assessment. However, it is also
notable that lesions of the rodent kidney are relatively common,
particularly in males, but these appeared to affect females
relatively more than males in these studies.
[0014] The gastric lesions occurred after administration of
relatively large amounts of gold solutions. There was also a
suggestion in these studies that the important toxic agent is ionic
gold (e.g., Au (III) or Au.sup.3+). Lesions of this type are also
produced by many NSAID agents used in the treatment of various
forms of arthritis and are generally considered to be a manageable,
albeit undesirable, side effect. Accordingly, the absence of such
negative effects would constitute an advantage over existing
gold-based therapies.
[0015] Cheriathundam and Alvares in 1996 evaluated the effects of
sodium gold thiolate and Auranofin.RTM. on liver and kidney markers
and metallothionein levels in the Sprague Dawley rat and three
strains of mouse (Swiss-Webster, C3H/Hej and DBA/2J). In the rat,
gold sodium thiolate led to a 7-fold increase in liver
metallothionein levels, whereas in the mouse strains
metallothionein levels increased 2-fold in the Swiss-Webster and
about 5-fold in the inbred strains. Gold sodium thiolate led to
only minimal changes in renal metallothionein levels in the mouse
strains. The liver marker serum ALAT was not altered by gold sodium
thiolate in any of the species or strains tested. BUN, an indicator
of kidney function, was elevated 3-fold in rats but not in any of
the mouse strains. These data are consistent with the observation
that gold sodium thiolate is nephrotoxic in rats and humans, but it
is interesting to note the lack of evidence of nephrotoxicity in
the mouse (Cheriathundam & Alvares, 1996).
[0016] The observation of embryonic toxicity and fetal defects
after treatment of pregnant animals of two species suggests the
possibility that gold in many, if not all previously used forms,
represents a developmental risk. This has parallels with many other
current RA therapies, in which methotrexate, for example, is
subject to label warnings regarding potential harmful effects on
the fetus.
[0017] Several possible pharmacological actions contributing to
both clinical efficacy and adverse reactions have been identified
for oral gold. For example, Walz and his colleagues showed that
Auranofin.RTM. inhibited carrageenan-induced edema in rats in a
dose-related fashion in concentrations of 40, 20 and 10 mg/kg with
maximum inhibition of 86% at the highest dose, and a serum gold
level of approximately 10 .mu.g/mL. The two basic ligands of
Auranofin.RTM., namely triethylphosphine oxide and
2,3,4,6-tetra-o-acetyl-1-thio- -D glucopyranose did not show any
significant biological activity, and gold sodium thiomalate, gold
thioglucose and thiomalic acid did not significantly affect rat paw
edema. Auranofin.RTM. was shown to significantly suppress adjuvant
arthritis, whereas the ligands were without any effect.
Auranofin.RTM. inhibited antibody dependent complement lysis.
Auranofin.RTM. has been shown to inhibit the release of lysosomal
enzymes such as -glucuronidase and lysozyme from stimulated
polymorphs. Auranofin.RTM. is a potent inhibitor of antibody
dependent cellular cytotoxicity exhibited by polymorphs from
adjuvant arthritic rats. Auranofin.RTM. is a much more potent
inhibitor of superoxide production than gold sodium thiomalate. In
an immune phagocytosis assay, gold sodium thiomalate showed no
inhibitory activity at a concentration of 40 times that of
Auranofin.RTM., causing marked inhibition (Walz, DiMartino,
Intocca, & Flanagan, 1983).
[0018] Walz and his colleagues also stated that Auranofin.RTM. was
more potent than gold sodium thiomalate as an inhibitor of
cutaneous migration, chemotaxis and phagocystosis by peripheral
blood monocytes. Lipsky and his colleagues showed that
Auranofin.RTM., like gold sodium thiomalate, inhibited
lymphoblastogenesis in vitro by directly inhibiting of mononuclear
phagocytes. However, Auranofin.RTM. also had an inhibitory effect
on lymphocyte function, not observed with gold sodium thiomalate.
Inhibition of monocytes was achieved with concentrations of
Auranofin.RTM. which were 10 to 20-fold lower than those of the
gold sodium thiomalate (Walz, DiMartino, Intocca, & Flanagan,
1983).
[0019] In general, patients with active rheumatoid disease have a
decreased capacity for either mitogen-stimulated
lymphoblastogenesis or for lymphoblastogenesis induced by the mixed
lymphocyte reaction. Although patients initially treated with gold
sodium thiomalate first showed some suppression of
mitogen-stimulated lymphoblastogensis, those who eventually
responded to the drug showed normal lymphocyte responsiveness in
vitro. In contrast, within a few weeks of patients receiving
Auranofin.RTM., lymphocyte responsiveness was markedly inhibited.
Thus, Auranofin.RTM. exhibits a powerful immunosuppressant effect
in vitro at an order of magnitude less than the injectable gold
compounds, most likely due to the major differences in the
pharmacological properties of the oral compounds versus the
injectable gold-thiol compounds (Dabrowiak, 2009).
[0020] Adverse reactions were the major limiting factor to the use
of oral gold compounds such as Auranofin.RTM., in that
approximately 30-50% of treated patients developed some form of
toxicity (Dabrowiak, 2009) (Kean & Anastassiades, 1979) (Kean
& Kean, The clinical Pharmacology of Gold, 2008).
[0021] Skin rash was the most common negative side effect and some
form of rash occurred in approximately 30% of patients. Most
lesions occurred on the hands, forearm, trunk and shins, but
occasionally occurred on the face and were slightly erythematous
with scaly patches, 1-10 cm in size, resembling a seborrheic rash.
Severe problems of skin rash in the form of nummular eczema, total
exfoliation and intense pruritis have been recorded as rare.
[0022] Oral ulcers (painful and pain free) resembling the aphthous
ulcer, occurred in approximately 20% of patients who received
injectable gold therapy. The development of a mouth ulcer was a
definite contraindication to continuation of gold therapy since it
was known that oral ulceration could herald pemphigold-like bullous
skin lesions.
[0023] The frequency of protein urea varied widely (0-40%) in the
studies reported by Kean and Anastassiades, most likely reflecting
different definitions as to what constitutes protein urea. In these
studies there are no well documented cases of any long term serious
or permanent renal damage due to gold therapy; however microscopic
haematuria was a cause for discontinuing oral gold treatment (Kean
& Anastassiades, 1979).
[0024] Thrombocytopenia due to gold compounds occurred as two
distinct types: the more usual was associated with platelet surface
IgG antibody and the other less common was secondary to bone marrow
suppression. The genetic marker HLA DR3 may indicate an increased
risk of a patient developing thrombocytopenia associated with
platelet surface antibodies.
[0025] Idiopathic toxicities in the form of cholestatic jaundice or
acute enterocolitis have also been associated with the injectable
gold compounds, particularly gold sodium thiomalate, but have not
been reported with oral gold.
[0026] The deposition of elemental gold in the lens of the eye and
the cornea has been reported, but this did not seem to result in
any specific damage to visual acuity.
[0027] Specific to oral gold therapy was the development of loose
soft stools, usually in the first month of therapy. The lower
incidence of altered stools in later treatment months may be
related to an earlier drop-out of those patients susceptible to the
diarrhea. The development of frank watery diarrhea occurred in 2-5%
of patients and appeared to be dose-related.
[0028] In general the adverse event incidence is lower with oral
gold than injectable gold, but can still be substantial.
[0029] A second major drawback to the use of available gold-based
treatments is the very slow onset of efficacy. Patients often must
continue treatment with, for example, gold salts for three to six
months before experiencing any significant benefit. This long wait
for any observed benefit is a major impediment to patient
compliance and therefore adversely affects efficacy in use.
[0030] The knowledge of the pharmacokinetic profiles of gold is
largely centered on the measurement of the element Au, but not much
is known of the gold structure (e.g., its chemical or physical or
crystalline structure) when the gold is present in various tissues
or organs.
[0031] After oral ingestion, oral gold complexes are rapidly, but
incompletely, absorbed. The gold moiety of the injectable gold
complex seems to be rapidly absorbed into the circulation after
intramuscular injection. In blood circulation, Auranofin.RTM. (or
ligands thereof) seem to be bound predominately to albumin.
Specifically, after oral administration of radiolabeled
Auranofin.RTM. to human volunteers, approximately 25% of the
administered dose was detected in the blood plasma, with peak
concentrations of 6-9 .mu.g/100 mL being reached within 1-2 hours.
The plasma half-life was on the order of 15-25 days with almost
total body elimination after 55-80 days. Only about 1% of
radiolabeled Au was detectable after 180 days, whereas up to 30% of
gold from gold sodium thiomalate was detected at this time. The
gold was widely distributed throughout the reticulo-endothelial
system, particularly in the phagocytic cells of the liver, bone
marrow, lymph nodes, spleen, and also in the synovium. Deposition
in the skin occurred and it has been observed that there may be a
quantitative correlation between the amount of gold in the dermis
and the total dose of gold given. Electron dense deposits of gold
were also observed in the tubular cells of the kidney, another site
rich in sulphydryl-containing enzymes, but the presence of gold
associated with the glomerulus does not appear to be common (Walz,
DiMartino, Intocca, & Flanagan, 1983) (Dabrowiak, 2009).
Gold Nanoparticles
[0032] Other formulations of gold have been and continue to be
developed, most of which utilize gold nanoparticles made by a
variety of chemical reduction techniques; and some of which utilize
an underwater plasma arcing technique; and most of which result in
various stable or partially stable gold colloids or gold
nanoparticle suspensions.
Colloidal Gold Nanoparticles by Chemical Reduction
[0033] Michael Faraday made the first colloidal gold suspension by
chemical reduction methods around the 1850's (Faraday, 1857).
Faraday used reduction chemistry techniques to reduce chemically an
aqueous gold salt, chloroaurate (i.e., a gold (III) salt),
utilizing either phosphorous dispersed into ether (e.g.,
CH.sub.3--CH.sub.2--O--CH.sub.2--CH.sub.3), or carbon disulfide
(i.e, CS.sub.2), as the reductant.
[0034] Today, most colloidal gold preparations are made by a
reduction of chloric acid (hydrogen tetrachloroaurate) with a
reductant like sodium citrate to result in "Tyndall's purple."
There are now a variety of "typical" reduction chemistry methods
used to form colloidal gold. Specifically, several classes of
synthesis routes exist, each of which displays different
characteristics in the final products (e.g., colloidal gold
nanoparticles) produced thereby. It has been noted that in addition
to the strength, amount and type of the reductant utilized, the
action of a stabilizer (i.e., the chemical utilized in the solution
phase synthesis process) is critical (Kimling, 2006).
[0035] While Faraday introduced colloidal gold solutions, the
homogenous crystallization methods of Turkevich and Frens (and
variations thereof) are most commonly used today and typically
result in mostly spherical-shaped particles over a range of
particle sizes (Kimling, 2006). Specifically, most current methods
start with a gold (III) complex such as hydrogen tetrachloroaurate
(or chloric acid) and reduce the gold in the gold complex to gold
metal (i.e., gold (0) or metallic gold) by using added chemical
species reductants, such as Na thiocyanate, White P, Na.sub.3
citrate & tannic acid, NaBH.sub.4, Citric Acid, Ethanol, Na
ascorbate, Na.sub.3 citrate, Hexadecylaniline and others (Brown,
2008). However, another chemical reduction technique uses sodium
borohydride as a chemical species reductant for AuP (Ph.sub.3)
(Brown, 2008). Depending on the particular processing conditions
utilized in these chemical reduction processes, the sizes of these
mostly spherical nanoparticles formed range from about lnm to about
64 nm in diameter (Brown, 2008). Additionally, specific thermal
citrate reduction methods utilized by Kimling resulted in a small
fraction of triangular-shaped particles, in addition to
spherical-shaped particles, with the triangular-shaped species at
most being about 5% (Kimling 2006).
[0036] Additional work has focused on controlling shapes of
colloidal metal nanoparticles. Biologists and biochemists have long
understood that "structure dictates function" with regard to
protein functioning. Gold nanoparticles of different shapes also
possess different properties (e.g., optical, catalytic, biologic,
etc.). Controlling nanoparticle shape provides an elegant approach
to, for example, tune nanoparticles optically. While all gold
nanoparticles contain a lattice that is face-centered cubic, if
permitted or caused by certain processing conditions, gold
nanoparticles can adopt a variety of crystalline shapes ranging
from irregular ellipsoids with defect loaded surfaces (e.g., steps)
to polyhedra with comparatively limited surface defects. Different
crystalline morphologies are associated with different crystal
planes (or sets of crystal planes). However, some of the most
common gold nanoparticle morphologies are not composed of single
domains, but rather are made of twinned planes (Tao, 2008).
[0037] Yuan, et al. recognized that non-spherical-shaped gold
nanoparticles could be most readily achieved by providing seed
crystals from a borohydride reduction of a gold salt (i.e.,
HAuCl.sub.4 or auric acid). The seed crystals were then placed into
contact with the same gold salt in solution with the chemical
species NH.sub.2OH, CTAB and sodium citrate being added as
reductants and/or surfactants (e.g., capping agents). Several
different crystalline shapes were formed by this approach including
triangular, truncated triangular, hexagonal layers and
pseudo-pentagonal. Yuan concluded that variations in processing by
using different chemical reduction techniques can influence the
physical and chemical properties of the resulting particles. The
researchers noted that the choice of a capping agent was a key
factor in controlling the growth (and shape) of the nanoparticles
(Yuan, 2003).
[0038] The process described and used by Yuan is known as
"heterogeneous nucleation" where seed particles are produced in a
separate synthetic step. Thus, this type of shape control can be
considered an overgrowth process (Tao, 2008). Many chemical
reduction techniques utilize this more complex two-step
heterogeneous nucleation and growth process. However, others use a
single step homogenous nucleation whereby seed crystals are first
nucleated, and nanoparticles are then formed from the nucleated
seed crystals. Typically, a series of chemical reactions occur
simultaneously in homogeneous nucleation. A main goal in homogenous
nucleation is to balance the rate of nucleation against the rate of
crystal growth and to control particle size because both nucleation
and growth proceed by the same chemical process(es) (Tao,
2008).
[0039] Metal nanoparticle synthesis in solution(s) commonly
requires the use of surface-active agents (surfactants) and/or
amphiphilic polymers as stabilizing agents and/or capping agents.
It is well known that surfactants and/or amphiphilic polymers serve
critical roles for controlling the size, shape and stability of
dispersed particles (Sakai, 2008).
[0040] Some of the most common crystal morphologies observed in
crystalline gold nanoparticles (for example in heterogeneous
nucleation processes) do not consist of single crystals or single
domains, but rather particles containing multiple crystal domains,
often bounded by twin planes. A regular decahedron (also referred
to as a pentagonal bi-pyramid) is an equilibrium shape bound
completely by triangular (III) facets and can be thought of as five
tetrahedral sharing a common edge along a fivefold axis. These
structures are commonly observed for nanocrystalline particles
synthesized by metal evaporation onto solid substrates and seeded
heterogeneous nucleation reduction chemistry approaches (Tao,
2008). However, for nanoparticles synthesized by the methods of
Turkevich and Frens, decahedra are difficult to observe because
they function as favorable seeds for the growth of nanowires and
nanorods (Tao, 2008). Thus, a variety of shapes can be achieved by
controlling processing conditions, along with the amounts and types
of surfactants and capping agents added and used during the
reduction chemistry approaches attributed to Turkevich and
Frens.
[0041] In each of the colloidal gold compositions produced by
reduction chemistry approaches, it is apparent that a surface
coating comprising one or more elements of the reductant and/or the
surfactant or capping agent will be present on (or in) at least a
portion of the suspended gold nanoparticles. The use of a reductant
(i.e., a reducing agent) typically assists in suspending the
nanoparticles in the liquid (e.g., water). However, the reducing
agent coating or surface impurity is sometimes added to or even
replaced by surfactant coatings or capping agents. Such
reductant/surfactant coatings or films can be viewed as impurities
located on and/or in the metal-based nanoparticles and may result
in such colloids or sols actually possessing more of the properties
of the protective coating or film than the gold nanoparticle per se
(Weiser, p. 42, 1933).
[0042] For example, surfactants and amphiphilic polymers become
heavily involved not only in the formation of nanoparticles (thus
affecting size and shape), but also in the nanoparticles per se.
Surface properties of the nanoparticles are modified by reductant
coatings and/or surfactant molecule coatings (Sperling, 2008).
[0043] Absorption of a hydrophobic tail, a hydrophilic head group
and certain counter ions (at least in the case of the use of ionic
surfactants) on the surface of nucleated particles, as well as
complexation of metal ions with surfactants and/or amphiphilic
polymers with the formed particles, all can influence the shape of
the nanoparticles, the surface of the nanoparticles and/or alter
the functioning of the nanoparticles (Sakai, 2008).
[0044] Different surface chemistries or surface films (e.g., the
presence of reductant by-product compositions and/or thicknesses
(e.g., films) of reductant by-products) can result in different
interactions of the gold nanoparticles with, for example, a variety
of proteins in an organism. Biophysical binding forces (e.g.,
electrostatic, hydrophobic, hydrogen binding, van der Waals) of
nanoparticles to proteins are a function not only of the size,
shape and composition of the nanoparticles, but also the type of
and/or thickness of the surface impurities or coating(s) on the
nanoparticles. The Turkevich and Frens methods (and variations
thereof) for making gold nanoparticles are the most widely
understood and utilized chemical reduction processes. The use of a
citric acid or sodium citrate reductant results in citrate-based
chemistries (e.g., a citrate-based coating) on the surface of the
gold nanoparticle (i.e., also referred to as citrate-stabilized)
(Lacerda, 2010).
[0045] Further, Daniel et al. reviewed the major gold nanoparticle
formation techniques, including the chemical synthesis and assembly
processes including: (1) citrate reduction, which results in "a
rather loose shell of [citrate-based] ligands" attached to the gold
nanoparticles; (2) a variation of the citrate reduction method
which uses a citrate salt and an amphiphile surfactant (for size
control); (3) the "Brust-Schiffrin" methods which result in thiol
or thiolate ligands "that strongly bind gold"; (4) methods that
result in sulfur-containing ligands including xanthates,
disulfides, dithiols, trithiols and resorcinarene tetrathiols; and
(5) other ligands that relate to phosphine, phosphine oxide,
amines, carboxylates, aryl isocyanides, and iodides (which can
replace citrate coatings). The authors reiterated statements
attributed to Brust regarding formed gold nanoparticles: "The
resulting physical properties are neither those of the bulk metal
nor those of the molecular compounds, but they strongly depend on
the particle size, interparticle distance, nature of the protecting
organic shell, and shape of the nanoparticles." (Daniel, 2004)
[0046] While the organic ligands present on the gold nanoparticles
(e.g., citrate-based ligands or coatings or films) helps to
stabilize the gold nanoparticles in the liquid to prevent the
nanoparticle from, for example, being attached to other
nanoparticles and agglomerating and/or settling out of suspension
due to, for example, gravity, these organic-based ligands (e.g.,
organic shells) are impurities (i.e, relative to the underlying
gold nanoparticle) and contribute to the gold nanoparticle's
interaction with proteins in a living system. Such coating(s) or
film(s) can have strong biological influences (Lacerda, 2010).
[0047] Further, Wang et al concluded that the commonly used
citrate-reduced gold nanoparticles interfere with the uptake of
gold nanoparticles relative to reductant and stabilizer-free
colloidal solutions (Wang, 2007).
[0048] Likewise, Lacerda, et al. stated that a better understanding
of the biological effects of nanoparticles requires an
understanding of the binding properties of the in-vivo proteins
that associate themselves with the nanoparticles. Protein
absorption (or a protein corona) on nanoparticles can change as a
function of nanoparticle size and surface layer composition and
thickness. Lacerda concluded that the protein layers that "dress"
the nanoparticle control the propensity of the nanoparticles to
aggregate and strongly influence their interaction with biological
materials (Lacerda, 2010).
Cleaning Colloidal Gold Nanoparticles Made by Chemical Reduction
Techniques
[0049] In some cases, the reductant surface coating or film is
permitted to remain as an impurity on the surface of the
nanoparticles, but in other cases, it is attempted to be removed by
a variety of somewhat complex and costly techniques. When removed,
the coating typically is replaced by an alternative composition or
coating to permit the nanoparticles to stay in suspension when
hydrated. The influence of purity on the chemistry and properties
of nanoparticles is often overlooked; however, results now indicate
that the extent of purification can have a significant impact
(Sweeney, 2006). These researchers noted that sufficient
purification of nanoparticles can be more challenging that the
preparation itself, usually involving tedious, time-consuming and
wasteful procedures such as extensive solvent washes and fractional
crystallization. Absent such purification, the variables of surface
chemistry-related contaminants on the surface of chemically reduced
nanoparticles affects the ability to understand/control basic
structure-function relationships (Sweeney, 2006).
[0050] Subsequent processing techniques may also require a set of
washing steps, certain concentrating or centrifuging steps, and/or
subsequent chemical reaction coating steps, all of which are
required to achieve desirable results and certain performance
characteristics (e.g., stabilization due to ligand exchange,
efficacy, etc.) for the nanoparticles and nanoparticle suspensions
(Sperling, 2008). In other cases, harsh stripping methods are used
to ensure very clean nanoparticle surfaces (Panyala, 2009).
[0051] Thus, others have concluded that the development of gold
nanoparticles in the management, treatment and/or prevention of
diseases is hampered by the fact that current manufacturing methods
for gold nanoparticles are by-and-large based on chemical reduction
processes. Specifically, Robyn Whyman, in 1996, recognized that one
of the main hindrances in the progress of colloidal golds
manufactured by a variety of reduction chemistry techniques was the
lack of any "relatively simple, reproducible and generally
applicable synthetic procedures" (Whyman 1996). There are many
variations of the original reduction chemistry techniques taught by
Faraday each of which can produce colloidal gold having a variety
of different physical properties (e.g., alone or in suspension) and
reductant coatings, all of which can result in different
efficacy/toxicity profiles when used in or with living cells. None
of these techniques meet Whyman's criteria. Accordingly, a
relatively simple, reproducible and generally applicable
manufacturing approach for making gold nanocrystals would be
welcomed. Further, the ability of such a manufacturing approach to
be compliant with FDA cGMP requirements would be even more
valuable.
[0052] Others have begun to recognize the inability to extricate
completely adverse physical/biological performance of the formed
nanoparticles from the chemical formation (i.e., chemical
reduction) processes used to make them. In this regard, even though
somewhat complex, expensive and non-environmentally friendly,
washing or cleaning processes can be utilized to alter or clean the
surface of nanoparticles produced by reduction chemistry, elements
of the chemical process may remain and affect the surface of
nanoparticles (and thus their functioning). Moreover, the presence
of certain chemicals during the nanoparticle formation process
affects the morphology (i.e, size and/or shape) of the forming
nanoparticles. Certain possible desirable morphologies (shapes)
known to exist in gold-based crystalline systems are not readily
observed in many products produced by these reduction chemistry
techniques.
Other Techniques for Making Colloidal Gold
[0053] Obtaining a surfactant and reducer-free (e.g., no
stabilizing, capping or reducing agents added to achieve reduction
of gold ionic species) has become a goal of certain researchers who
apparently understand some adverse consequences of
reductant/surfactant coatings being present from reduction
chemistry approaches. For example, ultrasound techniques have been
used whereby a 950 kHz frequency is applied to an aqueous hydrogen
tetrachloraurate solution. Spherical gold nanoparticles in the
range of 20-60 nm were prepared at temperatures above 50.degree.
C., while relatively larger triangular plates and some hexagonal
spheres coexisted when the mixture was processed below 50.degree.
C. (Sakai, 2008).
[0054] X-ray irradiation of HAuCl.sub.4 has been developed to
obtain reductant and stabilizer-free gold nanoparticles so as not
to "jeopardize" biocompatibility issues in biomedical applications.
The authors speculated that they generated the required electrons
for chemical reduction of Au.sup.+ by using "intense" X-ray beams
to create a hydrogen-free radical electron donor (Wang, 2007).
[0055] Another older and more complex technique for minimizing or
eliminating the need for reducing agents and/or minimizing
undesirable oxidation products of the reductant utilizes
.gamma.-irradiation from a .sup.60Co source at a dose rate of
1.8.times.10.sup.4 rad/h. In this instance, Au (CN).sub.2 was
reduced by first creating hydrated electrons from the radiolysis of
water and utilizing the hydrated electrons to reduce the gold ions,
namely:
e.sub.aq.sup.-+Au (CN).sub.2.fwdarw.Au.sup.0+2CN.sup.- (Henglein,
1998).
[0056] It is known that the surface of the gold nanoparticle may be
further processed by adding chemical species, such as polyethylene
glycol (PEG), or other specific ligands. In this regard, extensive
work has occurred in therapies for cancer where PEG-coated gold
nanoparticles are induced by a variety of techniques to migrate to
a cancer or tumor site and are thereafter irradiated with, for
example, infrared or radio waves to heat and destroy cancer cells
(Panyala, 2009). Surface PEGylation is also known to increase the
blood half-life of nanoparticles; and polysorbate-80 can improve
the blood-brain-barrier transport of nanoparticles (Teixido &
Giralt, 2008).
Colloidal Gold by Underwater Arcing
[0057] Also known in the art are methods for making gold
nanoparticles by an underwater arcing method. This method was first
pioneered by Bredig in the late 1800's. Bredig used a direct
current to create an underwater arc between two wires. Bredig used
a current of 5-10 amps and a voltage of 30-110 volts. In some
cases, Bredig also used 0.001N sodium hydroxide instead of pure
water. Bredig thought of his process as pulverizing the metallic
electrodes. Bredig obtained hydrosols of gold in this manner
(Weiser, pp. 9-17, 45-46, 1933).
[0058] Svedberg later improved on Bredig's process by utilizing a
high frequency arc instead of the direct current arc of Bredig.
Svedberg pointed out that the arc permits the formation of a metal
gas which subsequently condenses into particles of colloidal
dimensions. Much debate surrounded the exact mechanisms of the
process; however vaporization of the metal was viewed as being
important (Weiser, pp. 9-17, 45-46, 1933).
[0059] The parameters of greatest interest to Svedberg in
controlling the electric pulverization process to form colloid
solutions were, a) the rate of pulverization, b) the ratio of
sediment to total metal dispersed, c) the extent of decomposition
of the medium, and d) the dependence of (a)-(c) on the current
characteristics. The amount of sediment achieved by the Bredig and
Svedberg processes ranged from about 30% to about 50%, under a
variety of processing conditions (Kraemer, 1924).
[0060] More recent work with the Bredig process on palladium was
performed by Mucalo, et al. These investigators tested the theory
of whether the metallic particles in Bredig sols were "impure" due
to impurities from the concurrent electrolyte decomposition of the
electrolyte and oxidized material thought to form during arcing
(Mucalo, 2001). These investigators utilized modem surface
analytical techniques (i.e., XPS, or "x-ray photoelectron
spectroscopy") to determine differences in surface speciation as a
function of pH. At lower pH's a grey-black unstable material was
produced. At higher pH's, the sol was more stable, but still
completely aggregated within 1-2 weeks. Nanoparticles produced
consisted of irregularly-shaped spheres. While materials produced
at both higher and lower pH's were mostly metallic in character,
the surface characteristics of these unstable colloids were
different. The higher pH Bredig sols resulted in a thicker outer
oxide layer on the unstable nanoparticles (Mucalo, 2001).
[0061] The methods of Bredig and Svedberg were subsequently
improved on by others to result in a variety of underwater
arc-based methods. However, common to each of these underwater
arcing methods is the result of somewhat irregularly-shaped
metallic-based spheres. In this regard, the nanoparticles produced
by the Bredig or Svedberg processes are non-specific,
spherical-like shapes, indicative of a metal-based vaporization
followed by rapid quench methods, the nanoparticles being coated
with (and/or containing) varying amounts of different oxide-based
materials.
Toxicology of Colloidal Gold Nanoparticles
[0062] A review on the toxicology of gold nanoparticles was
performed by Johnston, et al. and reported in 2010. There were four
intravenous exposure routes summarized for both mice and rats and
an intratracheal approach for rats. Regarding the four intravenous
studies summarized, Johnston, et al. reported that tissue sites of
accumulation, in order of quantity were liver-spleen in 3 of 4
tests and liver-lung in 1 of 4 tests (i.e., highest gold
nanoparticle accumulation was in the liver). Specifically, the four
intravenous tests reported by Johnston et al. are summarized below
(Johnston, 2010).
[0063] The tissue distribution of metal particles, following
exposure via a variety of routes (Johnston, et al., 2010).
TABLE-US-00001 Tissue sites of Size Exposure accumulation (in Paper
NP (nm) Route order of quantity) Conclusion Cho, et al., Gold 13
Intravenous Liver, spleen, The primary sites of 2009 (PEG (mice)
kidney, lung, accumulation are the coated) brain liver and spleen,
NP's accumulate within macrophages De Jong, et al., Gold 10, 50,
Intravenous Liver, spleen, Wider organ distribution 2008 100, 250
(rats) lungs, kidneys, of smaller particles, heart, brain, whereas
larger particles thymus, testis were restricted to the liver and
spleen Semmler- Gold 4 and Intravenous Liver, spleen, Small
particles Behnke, et al., 18 (rats) kidneys, skin, demonstrate a
more 2008 GIT, heart widespread accumulation/distribution Sonavane,
et al., Gold 15, 50, Intravenous Liver, lung, Wider tissue
distribution 2008b 100, 200 (mice) kidneys, spleen, for smaller
particles- brain 15- and 50-nm NP's accumulated within the
[0064] Johnston, et al. were critical of a variety of uncertainties
introduced into a number of the reviewed toxicology studies
including that certain conclusions (made by others) regarding
toxicity as a function of only particle size were not accurate.
Specifically, Johnston, et al. reported that Pan et al (in 2007)
concluded that 1.4 nm gold nanoparticles were the most toxic gold
nanoparticles tested out of a range of nanoparticle sizes,
including 1.2 nm diameter gold nanoparticles. While Pan, et al.
believed there to be a difference in toxicity profile as a function
of size, Johnston, et al. noted that the 1.4 nm particles were made
by the investigators themselves and the 1.2 nm particles were
obtained from an outside company (thus suggesting that there were
different surface characteristics of both nanoparticles). Johnston,
et al. concluded that "agglomerations states or surface chemistry"
were the reason(s) for differential performance with both being
"known to alter particle behavior and toxicity" (Johnston,
2010).
[0065] Johnston, et al. also concluded that experimental setup
influences toxicity results; and that the tissue distribution of
gold nanoparticles in an organism is a function of the exposure
route, as well as size, shape and surface chemistry of
nanoparticles. Additionally, they observed that the liver appears
to be the primary site of accumulation and speculated that result
is due to the presence of macrophages in the liver. They also noted
that nanoparticle uptake is probably a result of the type and
extent of protein binding occurring on the surface of the
nanoparticles (e.g., a protein corona) which is a function of the
size, shape and surface coating on the nanoparticles. In
particular, they noted that the ability of a variety of cell types
to internalize nanoparticles by, for example, endocytosis. This
endocytosis mechanism which appeared to be a function of particle
shape, as well as particle surface characteristics, such as protein
absorption on the surface thereof. In other words, biological
uptake is a function of shape, size and charge; and is also very
serum-dependent (Johnston, 2010).
Efficacy of Colloidal Gold
[0066] Work by Abraham and Himmel (reported in 1997) disclosed the
use of colloidal gold in the treatment of 10 patients who
previously did not respond to a variety of other gold-based
treatments. The colloidal gold used in the study was made by a
variation of the standard "citrate method" of Maclagan and Frens
with "several proprietary modifications." Maltodextrins (Food
Grade) were used at a concentration of 2.5% to prevent auto
aggregation of the gold particles (Abraham, 2008). The sizes of the
colloid particles produced were reported as being less than 20 nm,
as confirmed by a process of passing the colloidal suspension
through a 20 nm filter (i.e, produced by Whatman Anotop).
Subsequent TEM work caused Abraham to conclude that 99% of the
particles produced were less than 10 nm. Sodium benzoate was also
added (Abraham, 2008).
[0067] The colloidal gold suspension resulted in a 1,000 mg/L
(i.e., 1,000 pm) concentration. The dosage level provided to each
patient varied between 30 mg/day and 60 mg/day, with most dosages
being 30 mg/day, for a 24-week period. These dosages were taken
orally. Table 1 therein lists the patient's sex, age and previous
conditions and/or treatments. The article concludes that 9 of the
10 patients "improved markedly by 24 weeks of intervention"
(Abraham & Himmel, 1997). Abraham also reported a lowering of
certain cytokine concentrations including IL-6 and TNF (Abraham,
2008).
[0068] Work on collagen-induced arthritis in rats by Tsai concluded
that nanogold particles bound to the protein VEGF and that such
binding was the reason for an improved clinical performance of rats
that were intra-articularly injected with colloidal gold. In this
case, the injected colloidal gold was prepared by the standard
chemical reduction method of utilizing a gold chloroaurate reduced
with sodium citrate. Tsai, et al. reported that the gold
nanoparticles were spherical having an approximate diameter of 13
nm, as measured by transmission electron microscopy. The
concentration of the intra-articular solution was 180 .mu.g/ml
(i.e., 180 ppm). The intra-articular injection was made one time,
either on day 7 or day 10 after induction of CIA (Tsai, 2007).
[0069] Brown, et al. disclosed in 2007 that a standard colloidal
gold preparation (referred to as Tyndall's purple) was prepared by
standard chemical reduction methods, namely, the reduction of
chloroauric acid with sodium citrate. The average particle size of
the gold nanoparticles produced was 27+/-3 nm. This colloidal gold
was dispersed in isotonic sorbitol and injected by a parenteral and
subcutaneous approach into rats that experienced experimentally
induced arthritis. The dose injected was at a concentration of 3.3
.mu.g/kg. Brown, et al. also disclosed that colloidal gold, when
administered subcutaneously, was approximately 1,000 times more
effective than the comparative sodium aurothiomalate. Brown, et al.
also disclosed that the colloidal gold was ineffective when given
orally and concluded that the ineffectiveness was due to
coagulation of the gold nanoparticles in the presence of gastric
juice and sodium chloride (Brown, 2007).
[0070] Brown, et al. reviewed alternative preparation methods for
colloidal gold having a variety of sizes and shapes (Brown, 2008).
Brown, et al. disclosed in Table 2 a variety of properties
associated with "nano-gold hydrosol." The authors concluded that
the studies conducted by them (and reviewed by them) "suggest that
gold nanoparticle (Au0)-based drugs may play a role in future
clinical therapies targeted to regulating macrophages" (Brown,
2008).
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SUMMARY OF THE INVENTION
[0112] New gold nanocrystals are provided that have nanocrystalline
surfaces that are substantially free (as defined herein) from
organic or other impurities or films. Specifically, the surfaces
are "clean" relative to those made using chemical reduction
processes that require chemical reductants and/or surfactants to
grow gold nanoparticles from gold ions in solution. The majority of
the grown gold nanocrystals have unique and identifiable surface
characteristics such as spatially extended low index, crystal
planes {111}, {110} and/or {100} and groups of such planes (and
their equivalents). Resulting gold nanocrystalline suspensions or
colloids have desirable pH ranges such as 4.0-9.5, but more
typically 5.0-9.5 and zeta potential values of at least -20 mV, and
more typically at least -40 mV and even more typically at least -50
mV for the pH ranges of interest.
[0113] The shapes and shape distributions of these gold
nanocrystals prepared according to the manufacturing process
described below include, but are not limited to, triangles (e.g.,
tetrahedrons), pentagons (e.g., pentagonal bipyramids or
decahedrons), hexagons (e.g., hexagonal bipyramids, icosahedrons,
octahedrons), diamond (e.g., octahedrons, various elongated
bipyramids, fused tetrahedrons, side views of bipyramids) and
"others". The shape distribution(s) of nanocrystals (i.e., grown by
various embodiments set forth herein) containing the aforementioned
spatially extended low index crystal planes (which form the
aforementioned shapes) and having "clean" surfaces is unique.
Furthermore, the percent of tetrahedrons and/or pentagonal
bipyramids formed in the nanocrystalline suspensions is/are also
unique.
[0114] Any desired average size of gold nanocrystals below 100 nm
can be provided. The most desirable crystalline size ranges include
those having an average crystal size or "mode" (as measured and
determined by specific techniques disclosed in detail herein and
reported as "TEM average diameter") that is predominantly less than
100 nm, and more typically less than 50 nm, even more typically
less than 30 nm, and in many of the preferred embodiments disclosed
herein, the mode for the nanocrystal size distribution is less than
21 nm and within an even more preferable range of 8-18 nm.
[0115] Any concentration of gold nanoparticle can be provided
according to the invention. For example, concentrations of these
gold nanocrystals can be a few parts per million (i.e., .mu.g/ml or
mg/l) up to a few hundred ppm, but are typically in the range of
2-200 ppm (i.e., 2 .mu.g/ml-200 g/ml) and more often in the range
of 2-50 ppm (i.e., 2 .mu.g/ml-50 .mu.g/ml) and even more typically
5-20 ppm (i.e., 5 .mu.g/ml-20 .mu.g/ml).
[0116] A novel process is provided to produce these unique gold
nanocrystals. The process involves the creation of the gold
nanocrystals in water. In a preferred embodiment, the water
contains an added "process enhancer" which does not significantly
bind to the formed nanocrystals, but rather facilitates
nucleation/crystal growth during the electrochemical-stimulated
growth process. The process enhancer serves important roles in the
process including providing charged ions in the electrochemical
solution to permit the crystals to be grown. These novel
electrochemical processes can occur in either a batch,
semi-continuous or continuous process. These processes result in
controlled gold nanocrystalline concentrations, controlled
nanocrystal sizes and controlled nanocrystal size ranges; as well
as controlled nanocrystal shapes and controlled nanocrystal shape
distributions. Novel manufacturing assemblies are provided to
produce these gold nanocrystals.
[0117] Pharmaceutical compositions which include an effective
amount of these gold nanocrystals to treat medical conditions are
also provided. The pharmaceutical composition can provide any
desired systemic dosage, as a non-limiting example, 0.1 mg/kg/day
or less, or 0.05 mg/kg/day or less, or even more typically 0.025
mg/kg/day or less, or most typically 0.001 mg/kg/day or less.
[0118] Since these gold nanocrystals have substantially cleaner
surfaces than the prior available gold nanoparticles, and can
desirably contain spatially extended low index crystallographic
planes forming novel crystal shapes and/or crystal shape
distributions, the nanocrystals appear to be more biologically
active (and may be less toxic) than spherical-shaped nanoparticles,
as well as nanoparticles (or nanocrystals) containing surface
contaminants such as chemical reductants and/or surfactants that
result from traditional chemical reduction processes. Therefore,
medical treatments may be affected at lower dosages of gold.
[0119] Pharmaceutical compositions are provided that are
appropriate for systemic or topical use, including oral,
intravenous, subcutaneous, intraarterial, buccal, inhalation,
aerosol, propellant or other appropriate liquid, etc, as described
further in the Detailed Description of the Invention.
[0120] These substantially surface-clean or surface-pure gold
crystals can be used to treat any disorder for which gold therapy
is known, which includes a broad range of inflammatory and
autoimmune disorders as well as certain infectious diseases (e.g.,
HIV, aids malaria, and Chagas disease) and cancer. Descriptions of
many of these uses are provided in the Background of the Invention,
above.
[0121] It has now been surprisingly discovered as part of this
invention that the gold nanocrystals inhibit macrophage migration
inhibitory factor ("MIF"). It is believed that this is the first
disclosure of such activity of gold nanocrystals (or
nanoparticles), and may provide a scientific basis to understand
the range of medical uses for gold nanocrystals to date. It also
provides a scientific basis to conclude that the gold nanocrystals
will be effective against other diseases which are mediated by
macrophage migration inhibitory factor. In addition, it has been
identified that these gold nanocrystals inhibit IL-6 but not IL-10.
For example, because MIF and/or IL-6 is/are indicated in a large
variety of conditions and/or biological signaling pathways, such
finding confirms that the novel gold nanocrystals will be effective
for the treatment or prevention of diseases or conditions resulting
from pathological cellular activation, such as inflammatory
(including chronic inflammatory) conditions, autoimmune conditions,
hypersensitivity reactions and/or cancerous diseases or
conditions.
[0122] Further, by following the inventive electrochemical
manufacturing processes of the invention, these gold-based metallic
nanocrystals can be alloyed or combined with other metals in
liquids such that gold "coatings" may occur on other metals (or
other non-metal species such as SiO.sub.2, for example) or
alternatively, gold-based nanocrystals may be coated by other
metals. In such cases, gold-based composites or alloys may result
within a colloid or suspension. Further, certain composites which
include both gold and other metals can also be formed.
[0123] Still further, gold-based metallic nanocrystals suspensions
or colloids of the present invention can be mixed or combined with
other metallic-based solutions or colloids to form novel solution
or colloid mixtures (e.g., in this instance, distinct metal species
can still be discerned).
BRIEF DESCRIPTION OF THE FIGURES
[0124] FIGS. 1a, 1b and 1c show schematic cross-sectional views of
a manual electrode assembly according to the present invention.
[0125] FIGS. 2a and 2b show schematic cross-sectional views of an
automatic electrode control assembly according to the present
invention.
[0126] FIGS. 3a-3d show four alternative electrode control
configurations for the electrodes 1 and 5 controlled by an
automatic device 20.
[0127] FIGS. 4a-4d show four alternative electrode configurations
for the electrodes 1 and 5 which are manually controlled.
[0128] FIGS. 5a-5e show five different representative embodiments
of configurations for the electrode 1.
[0129] FIG. 6 shows a cross-sectional schematic view of plasmas
produced utilizing one specific configuration of the electrode 1
corresponding to FIG. 5e.
[0130] FIGS. 7a and 7b show a cross-sectional perspective view of
two electrode assemblies that can be utilized.
[0131] FIGS. 8a-8d show schematic perspective views of four
different electrode assemblies arranged with planes parallel to
flow direction F.
[0132] FIGS. 9a-9d show schematic perspective views of four
different electrode assemblies arranged with planes perpendicular
to flow direction F.
[0133] FIGS. 10a-10e show a variety of cross-sectional views of
various trough members 30.
[0134] FIGS. 11a-11h show perspective views of various trough
members 30, with FIGS. 11c and 11d showing an atmosphere control
device 35' and FIG. 11d showing a support device 34.
[0135] FIGS. 12a and 12b show various atmosphere control devices 35
for locally controlling the atmosphere around electrode set(s) 1
and/or 5.
[0136] FIG. 13 shows an atmosphere control device 38 for
controlling atmosphere around substantially the entire trough
member 30.
[0137] FIG. 14 shows a schematic cross-sectional view of a set of
control devices 20 located on a trough member 30 with a liquid 3
flowing therethrough and into a storage container 41.
[0138] FIGS. 15a and 15b show schematic cross-sectional views of
various angles .theta.1 and 02 for the trough members 30.
[0139] FIGS. 16a, 16b and 16c show perspective views of various
control devices 20 containing electrode assemblies 1 and/or 5
thereon located on top of a trough member 30.
[0140] FIGS. 16d, 16e and 16f show AC transformer electrical wiring
diagrams for use with different embodiments of the invention.
[0141] FIG. 16g shows a schematic view of a transformer 60 and
FIGS. 16h and 16i show schematic representations of two sine waves
in phase and out of phase, respectively.
[0142] FIGS. 16j, 16k and 16l each show schematic views of eight
electrical wiring diagrams for use with 8 sets of electrodes.
[0143] FIG. 17a shows a view of gold wires 5a and 5b used in the
trough section 30b of FIG. 22a in connection with Examples 8, 9 and
10.
[0144] FIG. 17b shows a view of the gold wires 5a and 5b used in
the trough section 30b of FIG. 21a in connection with Examples 5, 6
and 7.
[0145] FIG. 17c shows the electrode configuration used to make
sample GB-118 in Example 16.
[0146] FIGS. 17d-17f show the devices 20 used in Examples 1-4 for
suspensions GT032, GT031, GT019 and GT033 and to make Samples
GB-139, GB-141 and GB-144 in Example 16.
[0147] FIGS. 17g, 17h, 17i and 7k show wiring diagrams used to
control the devices 20 used in Examples 1-4 and 16.
[0148] FIGS. 17j and 17l show wiring diagrams used to power devices
20.
[0149] FIGS. 17m-17n show alternative designs for the devices 20.
The device 20 in FIG. 17n was used in Example 18.
[0150] FIGS. 18a and 18b show a first trough member 30a wherein one
or more plasma(s) 4 is/are created. The output of this first trough
member 30a flows into a second trough member 30b, as shown in FIGS.
19a and 19b.
[0151] FIGS. 19a and 19b are schematics of two trough members 30a
and 30b having two different electrode 5 wiring arrangements
utilizing one transformer (Examples 8-10) and utilizing two
transformers (Examples 5-7).
[0152] FIGS. 20a-20h are alternatives of the apparatus shown in
FIGS. 19a and 19b (again having different electrode 5 wiring
arrangements and/or different numbers of electrodes), wherein the
trough members 30a' and 30b' are contiguous.
[0153] FIGS. 21a-21g show various trough members 30b in connection
with FIGS. 20a-h and various Examples herein.
[0154] FIGS. 22a and 22b show trough members 30b in connection with
FIGS. 19a, 19b and 20 and various Examples herein.
[0155] FIGS. 23a-23d show various schematic and perspective views
of an alternative trough embodiment utilized in Example 19.
[0156] FIG. 24a shows a schematic of an apparatus used in a batch
method whereby in a first step, a plasma 4 is created to condition
a fluid 3.
[0157] FIGS. 24b and 24c show a schematic of an apparatus used in a
batch method utilizing wires 5a and 5b to make nanocrystals in
suspension (e.g., a colloid) in association with the apparatus
shown in FIG. 24a and as discussed in Examples herein.
[0158] FIG. 25a is a representative TEM photomicrograph of gold
nanocrystals from dried suspension GD-007 made according to Example
5.
[0159] FIG. 25b shows the particle size distribution histogram from
TEM measurements for the nanocrystals of suspension GD-007 made
according to Example 5.
[0160] FIG. 25c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
5.
[0161] FIG. 25d is a representative TEM photomicrograph of gold
nanocrystals from dried suspension GD-007 made according to Example
5.
[0162] FIG. 25e shows the energy dispersive x-ray pattern of the
interrogation beam point of the nanocrystal from suspension
GD-007.
[0163] FIG. 25f shows the experimental setup for collecting plasma
emission data (e.g., irradiance).
[0164] FIG. 25g shows the Au electrode plasma irradiance from
200-300 nm generated by the apparatus shown in FIG. 25f.
[0165] FIG. 25h shows the Au1 electrode plasma irradiance from
200-300 nm generated by the apparatus shown in FIG. 25f.
[0166] FIG. 25i shows the Au electrode plasma irradiance from
300-400 nm generated by the apparatus shown in FIG. 25f.
[0167] FIG. 25j shows the Au electrode plasma irradiance from
400-500 nm generated by the apparatus shown in FIG. 25f.
[0168] FIG. 26a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GD-016 made according to Example
6.
[0169] FIG. 26b shows the particle size distribution from TEM
measurements for the nanocrystals made according to Example 6.
[0170] FIG. 26c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
6.
[0171] FIG. 27a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GD-015 made according to Example
7.
[0172] FIG. 27b shows the particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
7.
[0173] FIG. 27c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
7.
[0174] FIG. 28a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-018 made according to Example
8.
[0175] FIG. 28b shows the particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
8.
[0176] FIG. 28c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
8.
[0177] FIG. 29a is a representative TEM photomicrograph of gold
nanoparticles from dried solution GB-019 made according to Example
9.
[0178] FIG. 29b shows the particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
9.
[0179] FIG. 29c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
9.
[0180] FIG. 30a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-020 made according to Example
10.
[0181] FIG. 30b shows particle size distribution histogram from TEM
measurements for the nanocrystals made according to Example 10.
[0182] FIG. 30c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
10.
[0183] FIG. 31a is a representative TEM photomicrograph of gold
nanocrystals from dried solution 1AC-202-7 made according to
Example 11.
[0184] FIG. 31b shows the particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
11.
[0185] FIG. 31c shows the dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
11.
[0186] FIG. 32a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GT-033 made according to Example
4.
[0187] FIG. 32b shows the particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
14.
[0188] FIG. 32c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
4.
[0189] FIG. 33a is a representative TEM photomicrograph of gold
nanocrystals from dried solution 1AC-261 made according to Example
12.
[0190] FIG. 33b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
12.
[0191] FIG. 34a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-154 (20 Hz sine wave) made
according to Example 13.
[0192] FIG. 34b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
13.
[0193] FIG. 35a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-157 (40 hz sinewave) made
according to Example 13.
[0194] FIG. 35b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
GB-157.
[0195] FIG. 36a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-159 (60 Hz sine wave) made
according to Example 13.
[0196] FIG. 36b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-159.
[0197] FIG. 37a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-161 (80 Hz sine wave) made
according to Example 13.
[0198] FIG. 37b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-161.
[0199] FIG. 38a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-173 (100 Hz sine wave) made
according to Example 13.
[0200] FIG. 38b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-173.
[0201] FIG. 39a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-156 (300 Hz sine wave) made
according to Example 13.
[0202] FIG. 39b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-156.
[0203] FIG. 40 is a schematic diagram of the electrical setup used
to generate the nanocrystals in solutions GB-166, GB-165, GB-162,
GB-163 and GB-164.
[0204] FIG. 41 shows a schematic of the electrical wave forms
utilized in solutions GB-166, GB-165 and GB-162.
[0205] FIG. 42a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-166 (60 Hz sine wave) made
according to Example 14 FIG. 42b shows a particle size distribution
histogram from TEM measurements for the nanocrystals made according
to GB-166.
[0206] FIG. 43a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-165 (60 Hz square wave) made
according to Example 14.
[0207] FIG. 43b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-165.
[0208] FIG. 44a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-162 (60 Hz triangle wave) made
according to Example 14.
[0209] FIG. 44b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-162.
[0210] FIG. 45 is a schematic of the triangular-shaped electrical
wave forms utilized to generate samples in accordance with GB-163
and GB-164.
[0211] FIG. 46a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-163 (max duty cycle triangle
wave) made according to Example 15.
[0212] FIG. 46b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-163.
[0213] FIG. 47a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-164 (min duty cycle triangle
wave) made according to Example 15.
[0214] FIG. 47b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-164.
[0215] FIG. 48a1 is a representative TEM photomicrograph of gold
nanocrystals from dried suspension GB-134 made according to Example
16.
[0216] FIG. 48a2 is a representative TEM photomicrograph of gold
nanocrystals from dried suspension GB-134 made according to Example
16.
[0217] FIG. 48b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to Example
16.
[0218] FIG. 48c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
16.
[0219] FIGS. 49a1, a2-FIGS. 61a1, a2 show two representative TEM
photomicrographs for dried samples GB-098, GB-113, GB-118, GB-120,
GB-123, GB-139, GB-141, GB-144, GB-079, GB-089, GB-062, GB-076 and
GB-077, respectively, made according to Example 16.
[0220] FIGS. 49b-61b show the particle size distribution histogram
from TEM measurements for the nanocrystals corresponding to dried
samples GB-098, GB-113, GB-118, GB-120, GB-123, GB-139, GB-141,
GB-144, GB-079, GB-089, GB-062, GB-076 and GB-077, respectively,
made according to Example 16.
[0221] FIGS. 49c-61c show dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals corresponding to samples
GB-098, GB-113, GB-118, GB-120, GB-123, GB-139, GB-141, GB-144,
GB-079, GB-089, GB-062, GB-076 and GB-077, respectively, made
according to Example 16; and FIG. 54d shows current as a function
of time for GB-139 made in accordance with Example 16.
[0222] FIGS. 54d, 55d and 56d show measured current (in amps) as a
function of process time for the samples GB-139, GB-141 and GB-144
made according to Example 16.
[0223] FIG. 61d shows the UV-Vis spectral patterns of each of the
14 suspensions/colloids made according to Example 16 (i.e., GB-098,
GB-113 and GB-118); (GB-120 and GB-123); (GB-139); (GB-141 and
GB-144); (GB-079, GB-089 and GB-062); and (GB-076 and GB-077) over
an interrogating wavelength range of about 250 nm-750 nm.
[0224] FIG. 61e shows the UV-Vis spectral patterns for each of the
14 suspensions over an interrogating wavelength range of about 435
nm-635 nm.
[0225] FIG. 62a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-151 made according to Example
18.
[0226] FIG. 62b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-151.
[0227] FIG. 63a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-188 made according to Example
18.
[0228] FIG. 63b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-188.
[0229] FIG. 64a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-175 made according to Example
18.
[0230] FIG. 64b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-175.
[0231] FIG. 65a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-177 made according to Example
18.
[0232] FIG. 65b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-177.
[0233] FIG. 66a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-176 made according to Example
18.
[0234] FIG. 66b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-176.
[0235] FIG. 67a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-189 made according to Example
18.
[0236] FIG. 67b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-189.
[0237] FIG. 68a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-194 made according to Example
18.
[0238] FIG. 68b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-194.
[0239] FIG. 69a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-195 made according to Example
18.
[0240] FIG. 69b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-195.
[0241] FIG. 70a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-196 made according to Example
18.
[0242] FIG. 70b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-196.
[0243] FIG. 71a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-198 made according to Example
18.
[0244] FIG. 71b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-198.
[0245] FIG. 72a is a representative TEM photomicrograph of gold
nanocrystals from dried solution GB-199 made according to Example
18.
[0246] FIG. 72b shows a particle size distribution histogram from
TEM measurements for the nanocrystals made according to GB-199.
[0247] FIG. 72c shows the UV-Vis spectral patterns of each of the
11 suspensions/colloids made according to Example 18 (i.e., GB-151,
GB-188, GB-175, GB-177, GB-176, GB-189, GB-194, GB-195, GB-196,
GB-198 and GB-199) over an interrogating wavelength range of about
250 nm-750 nm.
[0248] FIG. 72d shows the UV-Vis spectral patterns for each of the
11 suspensions over an interrogating wavelength range of about 435
nm-635 nm.
[0249] FIGS. 73a1 and FIG. 73a2 show two representative TEM
photomicrographs for sample Aurora-020.
[0250] FIG. 73b shows the particle size distribution histogram from
TEM measurements for the nanoparticles corresponding to dried
sample Aurora-020.
[0251] FIG. 73c shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanoparticles corresponding to sample
Aurora-020.
[0252] FIGS. 74a1, a2-FIGS. 80a1, a2 show two representative TEM
photomicrographs for dried samples GA-002, GA-003, GA-004, GA-005,
GA-009, GA-011 and GA-013, respectively.
[0253] FIGS. 74b-80b show the particle size distribution histogram
from TEM measurements for the nanocrystals corresponding to dried
samples GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013,
respectively.
[0254] FIGS. 74c-80c show dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals corresponding to samples
GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013,
respectively.
[0255] FIG. 81a is a perspective view of a comparative Bredig-arc
apparatus utilized to make representative/comparative gold
nanoparticles.
[0256] FIG. 81b is a cross-sectional view of a comparative
Bredig-arc apparatus utilized to make representative/comparative
gold nanoparticles.
[0257] FIG. 82a is a representative TEM photomicrograph of gold
nanoparticles from dried solution ARCG-05 made according to Example
21.
[0258] FIG. 82b is a particle size distribution histogram from TEM
measurements for the nanoparticles made according to ARCG-05.
[0259] FIGS. 83a-90a show representative TEM photomicrographs for
eight comparative commercially available colloidal gold products
discussed in Example 22.
[0260] FIGS. 83b-90b shows the particle size distribution
histograms from TEM measurements for the nanoparticles
corresponding to the eight comparative commercially available
colloidal gold products discussed in Example 22.
[0261] FIG. 90c shows the UV-Vis spectral patterns of each of the 7
of the 8 commercially available gold nanoparticle suspensions
discussed in FIG. 22a (Utopia Gold, SNG911219, Nanopartz,
Nanocomposix 15 nm, Nanocomposix 10 nm, Harmonic Gold and MesoGold)
over an interrogating wavelength range of about 250 nm-750 nm.
[0262] FIG. 90d shows the UV-Vis spectral patterns for 7 of the 8
commercially available gold nanoparticle suspensions discussed in
FIG. 22a (Utopia Gold, SNG911219, Nanopartz, Nanocomposix 15 nm,
Nanocomposix 10 nm, Harmonic Gold and MesoGold) over an
interrogating wavelength range of about 435 nm-635 nm.
[0263] FIG. 91 is a graph showing Zeta Potentials.
[0264] FIG. 92 is a graph showing conductivity.
[0265] FIG. 93 shows dynamic light scattering data (i.e.,
hydrodynamic radii) for the nanocrystal suspension GD-006 made
according to Example 23a.
[0266] FIGS. 94a-94d show graphically amounts of four different
cytokines produced by human PBMCs when antagonized by LPS in the
presence of different amounts of GB-079.
[0267] FIG. 95 is a graph showing the results from a
collagen-induced arthritis ("CIA") model in mice showing control
water, two experimental mixtures (i.e., GT-033 and GD-007) and
contrasting the measured experimental results with results from a
typical steroid model (i.e., not measured in this model).
[0268] FIGS. 96a-96d show representative photomicrographs of cross
sections of mouse paw joints at various stages of arthritis.
[0269] FIGS. 97a-97e show representative photomicrographs of cross
sections of mouse paw joints at various stages of arthritis.
[0270] FIG. 98 is a graph showing results from an Experimental
Auto-Immune Encephalitis ("EAE") model in Biozzi mice showing the
percent of animals developing symptoms in the water Control Group 1
versus the GB-056 Treatment Group 2.
[0271] FIG. 99 is a graph showing results from an Experimental
Auto-Immune Encephalitis ("EAE") model in Biozzi mice showing the
average clinical disease score for the water Control Group 1 versus
the GB-056 Treatment Group 2.
[0272] FIGS. 100a, 100b, 100c, 100d and 100e are representative TEM
photomicrographs of gold nanocrystals from dried solution GB-056
made in accordance with Example 17.
[0273] FIG. 101a shows the particle size distribution histogram
from TEM measurements for the gold nanocrystals made according to
Example 17.
[0274] FIG. 101b shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
17.
[0275] FIGS. 102a, 10[0]2b, 10[0]2c and 10[0]2d are representative
TEM photomicrographs of the same gold nanocrystals from dried
solution GB-056 made in accordance with Example 17 after serving as
the test compound for 24 hours in the EAE test of Example 26.
[0276] FIG. 103a shows the particle size distribution histogram
from TEM measurements for the gold nanocrystals made according to
Example 17 after serving as the test compound for 24 hours in the
EAE test of Example 26.
[0277] FIG. 103b shows dynamic light scattering data (i.e.,
hydrodynamic radii) for gold nanocrystals made according to Example
17 after serving as the test compound for 24 hours in the EAE test
of Example 26.
[0278] FIGS. 104a, 104b and 104c are representative TEM
photomicrographs of the same gold nanoparticles from dried solution
GB-056 made in accordance with Example 17 after serving as the test
compound for 24 hours in the EAE test of Example 26.
[0279] FIG. 105a shows the particle size distribution histogram
from TEM measurements for the nanocrystals made according to
Example 17 after serving as the test compound for 24 hours in the
EAE test of Example 26.
[0280] FIG. 105b shows dynamic light scattering data for the
nanocrystals made according to Example 17 from Day 4-Day 5.
[0281] FIG. 106 shows the average weight gain of all mice over a
long-term study according to Example 27.
[0282] FIG. 107 shows the average amount of treatment and control
liquids consumed for all mice over a long-term study according to
Example 27.
[0283] FIG. 108 shows the average weight gain of all mice over a
35-day study according to Example 28.
[0284] FIG. 109 shows the average amount of treatment and control
liquids consumed for all mice over a 35-day study according to
Example 28.
[0285] FIG. 110 shows the amount of gold found in the feces of mice
according to Example 28.
[0286] FIG. 111 shows the amount of gold found in the urine of mice
according to Example 28.
[0287] FIG. 112 shows the amount of gold found in the organs and
blood of mice according to Example 28.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0288] I. Novel Gold Nanocrystals
[0289] New gold nanocrystals are provided that have nanocrystalline
surfaces that are substantially free from organic or other
impurities or films. Specifically, the surfaces are "clean"
relative to those made using chemical reduction processes that
require chemical reductants and/or surfactants to form gold
nanoparticles from gold ions in solution. The new gold nanocrystals
are produced via novel manufacturing procedures, described in
detail herein. The new manufacturing procedures avoid the prior use
of added chemical reductants and/or surfactants (e.g., organic
compounds) or other agents which are typically carried along in, or
on, the particles or are coated on the surface of the chemically
reduced particles; or the reductants are subsequently stripped or
removed using undesirable processes which themselves affect the
particle.
[0290] In a preferred embodiment, the process involves the
nucleation and growth of the gold nanocrystals in water which
contains a "process enhancer" or "processing enhancer" (typically
an inorganic material or carbonate or such) which does not
significantly bind to the formed nanocrystals, but rather
facilitates nucleation/growth during electrochemical-stimulated
growth process. The process enhancer serves important roles in the
process including providing charged ions in the electrochemical
solution to permit the crystals to be grown. The process enhancer
is critically a compound(s) which remains in solution, and/or does
not form a coating (e.g., an organic coating), and/or does not
adversely affect the formed nanocrystals or the formed
suspension(s), and/or is destroyed, evaporated, or is otherwise
lost during the electrochemical process. A preferred process
enhancer is sodium bicarbonate. Examples of other process enhancers
are sodium carbonate, potassium bicarbonate, potassium carbonate,
trisodium phosphate, disodium phosphate, monosodium phosphate,
potassium phosphates or other salts of carbonic acid or the like.
Further process enhancers may be salts, including sodium or
potassium, of bisulfite or sulfite. Still other process enhancers
to make gold nanocrystals for medical applications under certain
conditions may be other salts, including sodium or potassium, or
any material that assists in the electrochemical growth processes
described herein; which is not substantially incorporated into or
onto the surface of the gold nanocrystals; and does not impart
toxicity to the nanocrystals or to the suspension containing the
nanocrystals.
[0291] Desirable concentration ranges for the processing enhancer
include typically 0.01-20 grams/gallon (0.0026-2.1730 mg/ml), more
typically, 0.1-7.5 grams/gallon (0.0264-1.9813 mg/ml) and most
typically, 0.5-2.0 grams/gallon (0.13210-0.5283 mg/ml).
[0292] Because the grown gold nanocrystals have "bare" or "clean"
surfaces of gold metal (e.g., in the zero-oxidation state) the
surfaces are highly reactive or are highly biocatalytic (as well as
highly bioavailable). The nanocrystals are essentially surrounded
by a water jacket. These features provide increased efficacy in
vivo relative to nanoparticle surfaces that contain, for example,
organic material present from reduction chemistry processes. The
"clean" surfaces may also reduce the toxicity of the nanocrystals,
over those nanoparticles that contain coated or "dressed" surfaces.
The increased efficacy of these "clean" gold nanocrystals may
provide an increased therapeutic index via a lower dose needed to
achieve a therapeutic effect. A comparative mouse model example
herein (Example 25) compares an inventive gold nanocrystal
suspension to Auranofin, a commercially available and FDA-approved
gold drug, This Example shows that these novel gold nanocrystals,
in mice, are at least 5 times more active than Auranofin in the
well-accepted collagen induced arthritis model of inflammation in
rheumatoid arthritis.
[0293] Specifically, the comparative mouse model (Example 25)
compares the dose levels demonstrating efficacy using an inventive
crystal suspension to the dose levels demonstrating efficacy using
Auranofin, a commercially available and FDA-approved gold-based
drug, Example 25 shows that these novel gold nanocrystals, in mice,
achieve efficacy at a dose level at least 17 times lower than the
effective dose level of Auranofin in the well accepted collagen
induced arthritis model of inflammation in the mouse, and 5 times
lower than the gold content contained in the effective dose level
of Auranofin. Thus, comparing relative efficacy levels of the novel
gold nanocrystal to those of the gold-based drug Auranofin, and to
only the gold content of those of the Auranofin, the relative
potency of the novel gold nanocrystals is 17 times greater than
Auranofin and 5 times greater than the gold contained in the
Auranofin.
[0294] This potency advantage means that treatment efficacy can be
achieved at a much lower dose level (17.times. lower dose than
Auranofin, 5.times. lower dose than the gold contained in
Auranofin), or alternatively, that potentially much greater
efficacy can be achieved at equivalent dose levels.
[0295] There are other important advantages of the novel
nanocrystals in two other dimensions: relative toxicity, and
relative speed of onset of benefits. With respect to both observed
relative toxicity, and observed relative speed of onset of
benefits, in an animal model, the novel gold nanocrystals are
significantly different and significantly outperform Auranofin, the
only orally administrated, FDA-approved gold-based pharmaceutical
product in the prior art.
[0296] In a preferred embodiment, the nanocrystals are not dried
before use but instead used in the liquid they were formed in
(i.e., forming a suspension) or a concentrate or a reconstituted
concentrate thereof. It appears that completely removing these
crystals from their suspension (e.g., completely drying) may, in
certain cases, affect the surface properties of the crystals,
(e.g., partial oxidation may occur) and/or may affect the ability
to rehydrate the crystals by, for example, altering the initially
formed water jacket. This suggests that it may be optimal to use
sterile pharmaceutical grade water (i.e., USP) and the
aforementioned process enhancers in the manufacturing
processes.
[0297] The gold nanocrystals made according to this invention can
also be used for industrial applications where gold reactivity is
important (e.g., catalytic and/or electrochemical processes) but
pharmaceutical grade products are not required. When prepared for
non-pharmaceutical uses, the gold nanocrystals can be made in a
wider variety of solvents and with a wider variety of process
enhancers, depending on the application.
[0298] According to the processes herein, the gold nanocrystals can
be grown in a manner that provides unique and identifiable surface
characteristics such as spatially extended low index, crystal
planes {111}, {110} and/or {100} and groups of such planes (and
their equivalents). The shapes of the gold nanocrystals prepared
according to the processes described herein include, but are not
limited to, triangles (e.g., tetrahedrons), pentagons (e.g.,
pentagonal bipyramids or decahedrons), hexagons (e.g., hexagonal
bipyramids, icosahedrons, octahedrons), diamond (e.g., octahedrons,
various elongated bipyramids, fused tetrahedrons, side views of
bipyramids) and "others". The percent of nanocrystals (i.e., grown
by various embodiments set forth herein) containing the
aforementioned spatially extended low index crystal planes and
having "clean" surfaces is another novel feature of the invention.
Furthermore, the percent of tetrahedrons and/or pentagonal
bipyramids formed or present in the nanocrystalline suspensions
is/are also unique.
[0299] In a preferred embodiment the percent of pentagonal
bipyramids is at least about 5%, or is in a range of about 5%-35%,
and more typically at least about 10%, or is in a range of about
10%-35%, and even more typically, at least about 15%, or is in a
range of about 15%-35%, and still more typically, at least about
25%, and in some cases at least about 30%.
[0300] In another preferred embodiment the percent of tetrahedrons
is at least 5%, or is in a range of about 5%-35%, and more
typically at least about 10%, or is in a range of about 10%-35%,
and even more typically, at least about 15%, or is in a range of
about 15%-35%, and still more typically, at least about 25%, and in
some cases at least about 30%.
[0301] Still further, the combination of pentagonal bipyramids and
tetrahedrons is at least about 15%, or is in a range of about
15%-50%, and more typically at least about 20%, or is in a range of
about 20%-50%, and even more typically, at least about 30%, or is
in a range of about 30%-50%, and still more typically, at least
about 35%, and in some cases at least about 45%.
[0302] Still further, the combination of pentagonal bipyramids,
tetrahedrons, octahedrons and hexagonal is at least about 50%, or
is in a range of about 50%-85%, and more typically at least about
60%, or is in a range of about 60%-85%, and even more typically, at
least about 70%, or is in a range of about 70%-85%, and still more
typically, at least about 70%, and in some cases at least about
80%.
[0303] Any desired average size of gold nanocrystals below 100 nm
can be provided. The most desirable crystalline size ranges include
those having an average crystal size or "mode" (as measured and
determined by specific techniques disclosed in detail herein and
reported as "TEM average diameter") that is predominantly less than
100 nm, and more typically less than 50 nm, even more typically
less than 30 nm, and in many of the preferred embodiments disclosed
herein, the mode for the nanocrystal size distribution is less than
21 nm and within an even more preferable range of 8-18 nm.
[0304] Resulting gold nanocrystalline suspensions or colloids can
be provided that have or are adjusted to have target pH ranges.
When prepared with, for example, a sodium bicarbonate process
enhancer, in the amounts disclosed in detail herein, the pH range
is typically 8-9, which can be adjusted as desired.
[0305] The nature and/or amount of the surface change (i.e.,
positive or negative) on formed nanoparticles or nanocrystals can
have a large influence on the behavior and/or effects of the
nanoparticle/suspension or colloid. For example, protein coronas
such as albumin coronas formed in vivo can be influenced by surface
charge or surface characteristics of a nanoparticle. Such surface
charges are commonly referred to as "zeta potential". It is known
that the larger the zeta potential (either positive or negative),
the greater the stability of the nanoparticles in the solution
(i.e., the suspension is more stable). By controlling the nature
and/or amount of the surface charges of formed nanoparticles or
nanocrystals, the performance of such nanoparticle suspensions can
be controlled.
[0306] Zeta potential is known as a measure of the electro-kinetic
potential in colloidal systems and is also referred to as surface
charge on particles. Zeta potential is the potential difference
that exists between the stationary layer of fluid and the fluid
within which the particle is dispersed. A zeta potential is often
measured in millivolts (i.e., mV). The zeta potential value of
approximately 20-25 mV is an arbitrary value that has been chosen
to determine whether or not a dispersed particle is stable in a
dispersion medium. Thus, when reference is made herein to "zeta
potential", it should be understood that the zeta potential
referred to is a description or quantification of the magnitude of
the electrical charge present at the double layer.
[0307] The zeta potential is calculated from the electrophoretic
mobility by the Henry equation:
U E = 2 .times. .times. .times. zf .function. ( ka ) 3 .times.
.eta. ##EQU00001##
where z is the zeta potential, U.sub.E is the electrophoretic
mobility, .epsilon. is a dielectric constant, .eta. is a viscosity,
f(ka) is Henry's function. For Smoluchowski approximation
f(ka)=1.5.
[0308] Zeta potentials ("ZP") for the gold nanocrystals prepared
according the methods herein typically have a ZP of at least -20
mV, more typically at least about -30 mV, even more typically, at
least about -40 mV and even more typically at least about -50
mV.
[0309] II. Use of Novel Gold Nanocrystals
[0310] The gold nanocrystals of the present invention can be used
to treat any disorder for which gold therapy is known to be
effective, which includes a broad range of inflammatory and
autoimmune disorders as well as certain infectious diseases and
cancer. Descriptions of many of these uses are provided in the
Background of the Invention, above, or otherwise, in more detail
below.
[0311] The subject to be treated may be human or another animal
such as a mammal. Non-human subjects include, but are not limited
to primates, livestock animals (e.g., sheep, cows, horses, pigs,
goats), domestic animals (e.g., dogs, cats), birds and other
animals (e.g., mice, rats, guinea pigs, rabbits).
[0312] Importantly, it has now been surprisingly discovered as part
of this invention that the gold nanoparticles (and in particular
the gold nanocrystals described in detail herein) inhibit
macrophage Migration Inhibitory Factor ("MIF"). It is believed that
this is the first disclosure of such activity of gold
nanoparticles, and may provide a scientific basis to understand the
range of medical uses for gold compositions to date. It also
provides a scientific basis to conclude that the gold nanoparticles
will be effective against other diseases which are mediated by
macrophage migration inhibitory factor. In addition, it has been
identified that these gold nanocrystals inhibit IL-6 but not IL-10.
Because MIF and/or IL-6 is/are indicated in a large variety of
conditions and/or biological signaling pathways, such finding
confirms that the novel gold nanocrystals will be effective for the
treatment or prevention of diseases or conditions resulting from
pathological cellular activation, such as inflammatory (including
chronic inflammatory) conditions, autoimmune conditions, certain
infections, hypersensitivity reactions and/or cancerous diseases or
conditions.
[0313] MIF is a macrophage derived multifunctional cytokine
important in a number of pro-inflammatory events. MIF was
originally described as a product of activated T-lymphocytes that
inhibits the random migration of macrophages. While MIF was
initially found to activate macrophages at inflammatory sites, MIF
has now been shown to mediate a range of signaling agents in the
immune system. MIF has been shown to be expressed in human and
animal diseases or conditions which include infection,
inflammation, injury, ischemia and/or malignancy. MIF appears to
have a key role in cell proliferation, cell differentiation,
angiogenesis and wound healing. MIF also seems to mediate
glucocorticoid (steroids) activity by counteracting at least some
of their anti-inflammatory effects.
[0314] As shown in Examples 25 and 26, the nanocrystalline
compositions of the present invention are very effective in the
animal models for CIA and EAE. A connection between these two
animal models (as well as human disease state) is the presence of
MIF.
[0315] Recent studies have indicated that monoclonal antibody
antagonism of MIF may be useful in the treatment of sepsis, certain
types of cancers and delayed type hypersensitivity. It appears that
sepsis is triggered by an over-reaction of the inflammation and
immune systems. In certain infections, upon attack by
microorganisms, the innate immune system reacts first, whereby
neutrophils, macrophages and natural killer cells ("NK cells") are
mobilized. Cytokines (and MIF) thus play an important role as
mediators, which regulate activation and differentiation of these
cells. Finally, the innate immune system interacts with the
adaptive immune system via these and other stimulating molecules,
upon which the adaptive immune system has the ability of
constructing an immunological memory in addition to providing
pathogen specific protection.
[0316] MIF is seen as a major mediator in sepsis, as MIF incites
the production of TNF, other pro-inflammatory cytokines and
eicosanoids, induces the expression of TLR-4, which recognizes LPS,
and appears to resist in activating the innate immune response. MIF
and glucocorticoids act as antagonists and are at least partially
responsible for regulating the inflammatory reaction. MIF has an
inhibiting effect on glucocorticoids, which typically inhibit
inflammation.
[0317] Therapeutic antagonism of MIF can provide "steroid-sparing"
effects or can even be therapeutic in "steroid-resistant" diseases.
Unlike other pro-inflammatory molecules, such as certain cytokines,
the expression and/or release of MIF is coupled to (e.g., can be
induced by) glucocorticoids. MIF seems to be able to antagonize the
effects of glucocorticoids. MIF has a major role in regulating
pro-inflammatory cytokines. This has been shown to be the case for
macrophages secreting TNF, IL-1.beta., IL-6 and IL-8. MIF also
regulates IL-2 release. MIF also has a role in regulating T cell
proliferation. In vivo, MIF exerts a powerful
glucocorticoid-antagonist effect in models including endotoxic
shock and experimental arthritis (e.g., collagen-induced arthritis
or "CIA" models, such as the one utilized in a later example herein
and models of other inflammatory conditions and immune diseases
including colitis, multiple sclerosis (i.e., the EAE model
discussed in greater detail in Example 26), atherosclerosis,
glomerulonephritis, uveitis and certain cancers).
[0318] Further, MIF has recently been shown to be important in the
control of leukocyte-endothelial interactions. Leukocytes interact
with vascular endothelial cells in order to gain egress from the
vasculature into tissues. The role of MIF in these processes has
been demonstrated to affect leukocyte-endothelial adhesion and
migration. These processes seem to be an essential part of nearly
all inflammatory diseases, and also for diseases less
well-identified as inflammatory including, for example,
atherosclerosis.
[0319] MIF is also expressed in plants (thus "MIF" may also refer
to plant MIF) and where appropriate, the inventive gold nanocrystal
suspensions (e.g., comprising aqueous gold-based metal nanocrystals
and/or mixtures of gold nanocrystals and other metal(s) and/or
alloys of gold nanocrystals with other metal(s) and/or a
combination therapy approach) may be used in botanical/agricultural
applications such as crop control.
[0320] MIF is a key cytokine in switching the nature of the immune
response. The immune response has two effector mechanisms. The Th1
immune response generates cytotoxic T cells that kill pathogens and
damaged/defunct cells. The Th2 response generates antibodies that
facilitate phagocytosis and activate complement. The role of MIF in
determining the polarization of the immune system is dependent on
other cytokines such as IL-10. IL-10 is a potent anti-inflammatory
cytokine that blocks the action of MIF on Th1 cells and leads to
the generation of a Th2 response. In the absence of IL-10 MIF will
stimulate Th1 cells to produce a cytotoxic response. IL10 is
produced by Monocytes and B cells in response to stimulation,
whereas MIF is, for example, independently produced and stored in
the pituitary and T cells. MIF therefore plays an important role in
both T Cytotoxic cell mediated diseases--such as rheumatoid
arthritis and Crohns, and antibody mediated diseases such as
idiopathic thrombocytopenia.
[0321] Without wishing to be bound by any particular theory or
explanation, when reference is made herein to "one or more
signaling pathway(s)" it should be understood as meaning that MIF,
or at least one protein associated with MIF (e.g., including
receptor sites such as CD74 receptor sites) is/are implicated in
the innate immune system (e.g., NK and phagocyte cells, complement
proteins (e.g., C5a) and/or inflammatory pathways) and the adaptive
immune systems (e.g., the T cell dependent cytotoxicity (Th1) and
antibody (Th2) pathways). For example, when MIF is involved in the
Th1 signaling pathway generating T Cytotoxic cells other proteins
such as, for example, IL6, TNF, and other cytokines are also
involved.
[0322] When the Th1 signaling pathway is overactive, a variety of
diseases can result, such as rheumatic diseases, connective tissue
diseases, vasculitides, inflammatory conditions, vascular diseases,
ocular diseases, pulmonary diseases, cancers, renal diseases,
nervous system disorders, complications of infective disorders,
allergic diseases, bone diseases, skin diseases, Type 1 Diabetes,
Crohn's Disease, MS and gastrointestinal diseases, etc.
Accordingly, by reducing the amount of MIF function associated with
this particular Th1 signaling pathway, chronic disease conditions
can be mitigated.
[0323] In contrast, again without wishing to be bound by any
particular theory or explanation, when the Th2 signaling pathway is
over-active, the production of various antibodies occurs leading to
diseases such as, for example and including, hemolytic anemia, ITP
(Idiopathic Thrombocytopenic Purpura), Hemolytic Disease of the
newborn, etc. Furthermore, over-activity of this Th2 signaling
pathway can result in an under-activity of the Th1 pathway, thus
permitting various parasites or cancers to thrive. For example, in
the case of malaria where over production of one or more homologues
of MIF leads to the generation of an ineffective antibody response
that is ineffective against the parasite (e.g., it is plausible
that a variety of crystal forms or homologues of MIF (or
equivalents thereto) are made or presented by a variety of
bacteria, parasites, virus, fungi, etc., each of which may have
different reactivity relative to, for example, "ordinary" human
MIF, and which may alter host immune response so as to create at
least local environments of "immune privilege"). Accordingly, by
reducing the amount of MIF function associated with this particular
Th2 signaling pathway, other disease conditions can be
mitigated.
[0324] Still further, without wishing to be bound by any particular
theory or explanation, MIF also has a role in driving the signaling
pathway associated with innate immunity. This pathway involves the
activation of natural killer ("NK") cells, phagocytes and other
non-specific pathogen cell types and certain proteins such as
complement proteins (e.g., C5a). Excess MIF (and/or MIF
homologues), or similar effects of the same, can result in
undesirable over-expression or over-reaction in this particular
signaling pathway as seen in multiple organ failure as a result of
sepsis. Examples include the Systemic Inflammatory Response
Syndrome (SIRS). Accordingly, by reducing the amount of MIF
activity associated with this particular signaling pathway many
inflammatory diseases can be mitigated.
[0325] Accordingly when endogenous MIF is present (e.g., in excess
under local environmental conditions), as measured by, for example,
known body fluid measuring techniques such as ELISA, spectroscopy,
etc., it is possible that one or more innate or adaptive immune
system signaling pathways may over-express, over activate or
over-produce inflammatory/immunological components. If for example,
one or more forms of MIF present causes the production of an
excessive T Cytotoxic response, or an excessive antibody response
or an exaggerated NK/phagocyte cell response, human disease can
result. When, for example, too many T Cytotoxic cells are
expressed, a variety of chronic inflammatory conditions can result.
Similarly when excessive Th2 or innate responses are facilitated by
MIF, other diseases are produced.
[0326] Still further, it is also known that malaria parasites, and
other parasites such as nematodes and filarial worms, and some
cancers produce certain types of exogenous or non-regulated MIF or
MIF homologues. Again, without wishing to be bound by any
particular theory or explanation, it appears that exogenous
expression of MIF, or its homologues, leads to stimulation of the
Th2 signaling pathway, and may be an attempt by the parasite, or
the tumor, (i.e., "the invader") to create a state where the immune
response is activated by MIF or its homologues such that that the
activated particular signaling pathway is not detrimental to the
tumor or the parasite, etc.
[0327] With regard to, for example, a malaria parasite, the
parasite may stimulate the Th2 signaling pathway by providing
excess exogenous MIF resulting in the production of antibodies
rather than T Cytotoxic cells. However, such antibodies do not
typically harm the parasite. Therefore, the parasite appears to
create at least a local area of immune privilege. In this regard if
an alternative pathway such as, for example, the Th1 pathway, or
the natural killer (NK) cell pathway, can be re-activated, damage
could then occur to the parasite (e.g., the immune system could
remove the parasite). However, if excess antibodies or other
immune/inflammatory products are created, for example, as a result
of the preferential activation of the Th2 pathway, it is possible
that the excess antibodies will end up cross-linking to various
cell sites or activating other immunological molecules. When such
cross-linking or activation occurs, a very large inflammatory
response could result. Without wishing to be bound by any
particular theory or explanation, it is possible that this
inflammatory response is precisely the response that occurs in
women who are pregnant and are infected with malaria making them
vulnerable to severe malaria, and the anemia of malaria. It is
believed that pregnant women are particularly susceptible to this
effect, due to the immunological effects of the placenta in
promoting a Th2 response and sequestering parasites in this
immune-privileged zone.
[0328] Again, without wishing to be bound by any particular theory
or explanation, cancer cells also express MIF apparently in an
attempt to at least partially control immune response thereto
and/or promote their own growth. In this regard, it appears that
cancer cells are also attempting to manipulate the immune system to
follow the Th2 signaling pathway, in contrast to the Th1 signaling
pathway which could damage or kill the cancer cells. For example,
by causing local immune privilege to be created, there is no (or
little) particular risk to cancer cells. In contrast, if MIF was to
stimulate the Th1 signaling pathway, then a cytokine
cell/inflammatory response may result, causing damage or death to
the cancer cells (e.g., the tumor could be naturally eliminated by
the immune system).
[0329] Again, without wishing to be bound by any particular theory
or explanation, children possess an immature immune system,
particularly the innate and Th1 pathways. This immaturity in some
children results in altered MIF metabolism. It thus appears that
the modulation of MIF in children could result in the prevention or
improvement of infectious or inflammatory diseases.
[0330] Accordingly, without wishing to be bound by any particular
theory or explanation, the inventive gold nanocrystal suspensions
of the present invention can be used to modify one or more
signaling pathways (e.g., Th1 signaling pathway, Th2 signaling
pathway and/or innate immunity pathway) either alone or in
conjunction with other therapies that modulate signaling pathways.
Thus, by interacting with or controlling the MIF (or MIF homologue)
associated with one or more signaling pathway(s), various
immunological responses can be turned on and/or can be turned off.
Accordingly, the response along the Th1 and Th2 signaling pathways
for the creation of T Cytotoxic cells or antibodies can be turned
on, or can be turned off (e.g., the Th1-Th2 switch can be
controlled to direct more or less of either immune pathway being
invoked). Similarly, the innate immune system and resultant
inflammation can be turned on or can be turned off.
[0331] With the knowledge that one or more signaling pathways can
be turned on/off, very important therapeutic treatments can thus
occur. For example, a variety of surrogate endpoints can be
monitored or examined for a variety of different diseases,
including, for example, many cancers. For example, the antigen,
"carcino-embryonic antigen" or "CEA" is a known surrogate endpoint
marker for the amount of tumor or the amount of tumor burden
present in a variety of different cancers. For example, it is known
that the higher the CEA amount, the more tumors there are
associated with ovarian cancer, breast cancer, colon cancer, rectal
cancer, pancreatic cancer, lung cancer, etc. In this regard, the
amount of carcino-embryonic antigen can be measured by, for
example, drawing blood and testing for the presence of CEA by known
techniques including, for example, ELISA and certain spectroscopy
techniques. In this regard, once blood is drawn and a measurement
is made to determine the amount of CEA, the extent of treatment
required (e.g., the dose, duration and/or the amount) can be driven
by monitoring the change in the amount of CEA measured. For
example, if 15-45 ml of 10 ppm product is taken 2-3 times per day,
monitoring of the amount of CEA could cause an increase in dosage,
or a decrease in dosage, depending on the desired outcome.
[0332] Likewise, prostate cancer has a known surrogate endpoint of
"prostate-specific antigen" or "PSA". This surrogate endpoint can
also be monitored by drawing blood and searching for the same by
ELISA techniques.
[0333] Still further, various cancers, like melanoma (e.g., ocular,
etc.) also express antigens for example "GP100" and/or "Melan-A".
These surrogate endpoints can also be determined by drawing blood
from a patient and then measuring by similar ELISA or
spectrographic techniques for the amount of antigen present. In all
such cases, the presence of antigen can cause an increase/decrease
in the amount of therapeutic treatment provided.
[0334] The following "Table A" sets forth a number of known "Tumor
Markers" and associated cancers, as well as where biological
samples are drawn to measure such markers.
TABLE-US-00002 TABLE A Common Tumor Markers Currently in Use Tumor
Markers Cancers Usual sample AFP Liver, germ cell cancer Blood
(Alpha-feto protein) of ovaries or testes B2M Multiple myeloma and
Blood (Beta-2 microglobulin) lymphomas CA 15-3 Breast cancer and
others, Blood (Cancer antigen 15-3) including lung, ovarian CA 19-9
Pancreatic, sometimes Blood (Cancer antigen 19-9) colorectal and
bile ducts CA-125 Ovarian Blood (Cancer antigen 125) Calcitonin
Thyroid medullary Blood carcinoma CEA Colorectal, lung, breast,
Blood (Carcino-embryonic thyroid, pancreatic, liver, antigen)
cervix, and bladder Chromogranin A Neuroendocrine tumors Blood
(CgA) (carcinoid tumors, neuro- blastoma) Estrogen receptors Breast
Tissue hCG (Human chorionic Testicular and tropho- Blood, urine
gonadotropin) blastic disease Her-2/neu Breast Tissue Monoclonal
Multiple myeloma and Blood, urine immunoglobulins Waldenstrom's
macro- globulinemia Progesterone receptors Breast Tissue PSA
(Prostate specific Prostate Blood antigen), total and free
Thyroglobulin Thyroid Blood Other Tumor Markers Less Widely Used
BTA Bladder Urine (Bladder tumor antigen) CA 72-4 Ovarian Blood
(Cancer antigen 72-4) Des-gamma-carboxy Hepatocellular Blood
prothrombin (DCP) carcinoma (HCC) EGFR (Her-1) Solid tumors, such
as of Tissue the lung (non small cell), head and neck, colon,
pancreas, or breast NSE Neuroblastoma, small cell Blood
(Neuron-specific enolase) lung cancer NMP22 Bladder Urine Prostatic
acid Metastatic prostate cancer, Blood phosphatase (PAP) myeloma,
lung cancer Soluble Mesothelin- Mesothelioma Blood Related Peptides
(SMRP)
[0335] Still further, various diseases of immune and inflammation
dysfunction, like rheumatoid arthritis and Crohn's can be assessed
using inflammatory markers such as C Reactive Protein (CRP) or
erythrocyte sedimentation rate (ESR). These surrogate endpoints can
also be determined by drawing blood from a patient and then
measuring by visual ELISA or spectrographic techniques for the
amount of marker present. In all such cases, the change in an
inflammatory/immune marker can cause an increase/decrease in the
amount of therapeutic treatment provided.
[0336] Still further, various antibody-based diseases, like
hemolytic anemia or Rhesus disease, can be monitored by the
concentration of specific antibodies present. These surrogate
endpoints can also be determined by drawing blood from a patient
and then measuring, by similar ELISA or spectrographic techniques,
for the amount of antibody present. In all such cases, the presence
of antibody can cause an increase/decrease in the amount of
therapeutic treatment provided.
[0337] Inhibitors or modifiers of MIF and/or one or more of MIF's
signaling pathway(s) may also be used in implantable devices such
as stents. Accordingly, in a further aspect the present invention
provides an implantable device, preferably a stent, comprising:
[0338] (i) a reservoir containing at least one compound of
metallic-based compound comprising gold solutions or colloids and
mixtures and alloys thereof; and
[0339] (ii) means to release or elute the inhibitor or modifier
from the reservoir.
[0340] According to the invention therefore, there are a variety of
indications that the nanocrystalline gold-based therapies of the
present invention will have desirable efficacy against including
various autoimmune diseases, tumors, or chronic or acute
inflammatory conditions or diseases, disorders, syndromes, states,
tendencies or predispositions, etc., selected from the group
comprising:
[0341] rheumatic diseases (including but not limited to rheumatoid
arthritis, osteoarthritis, psoriatic arthritis, Still's disease)
spondyloarthropathies (including but not limited to ankylosing
spondylitis, reactive arthritis, Reiter's syndrome), crystal
arthropathies (including but not limited to gout, pseudogout,
calcium pyrophosphate deposition disease), Lyme disease,
polymyalgia rheumatica;
[0342] connective tissue diseases (including but not limited to
systemic lupus erythematosus, systemic sclerosis, scleroderma,
polymyositis, dermatomyositis, Sjogren's syndrome);
[0343] vasculitides (including but not limited to polyarteritis
nodosa, Wegener's granulomatosis, Churg-Strauss syndrome);
[0344] inflammatory conditions or tendencies including consequences
of trauma or ischemia; sarcoidosis;
[0345] vascular diseases including atherosclerotic vascular disease
and infarction, atherosclerosis, and vascular occlusive disease
(including but not limited to atherosclerosis, ischemic heart
disease, myocardial infarction, stroke, peripheral vascular
disease), and vascular stent restenosis;
[0346] ocular diseases including uveitis, corneal disease, iritis,
iridocyclitis, and cataracts;
[0347] autoimmune diseases (including but not limited to diabetes
mellitus, thyroiditis, myasthenia gravis, sclerosing cholangitis,
primary biliary cirrhosis);
[0348] pulmonary diseases (including but not limited to diffuse
interstitial lung diseases, pneumoconiosis, fibrosing alveolitis,
asthma, bronchitis, bronchiectasis, chronic obstructive pulmonary
disease, adult respiratory distress syndrome);
[0349] cancers whether primary or metastatic (including but not
limited to prostate cancer, colon cancer, bladder cancer, kidney
cancer, lymphoma, lung cancer, melanoma, multiple myeloma, breast
cancer, stomach cancer, leukemia, cervical cancer and metastatic
cancer);
[0350] renal diseases including glomerulonephritis, interstitial
nephritis;
[0351] disorders of the hypothalamic-pituitary-adrenal axis;
[0352] nervous system disorders including multiple sclerosis,
Alzheimer's disease, Parkinson's Disease, Huntington's disease;
[0353] diseases characterized by modified angiogenesis (e.g.,
diabetic retinopathy, rheumatoid arthritis, cancer) and
endometriosis;
[0354] infectious diseases, including but not limited to bacterial,
parasites or viral, including HIV, HBV, HCV, tuberculosis, malaria,
and worms (including the current FDA designated neglected diseases
of the developing world).
[0355] complications of infective disorders including endotoxic
(septic) shock, exotoxic (septic) shock, infective (true septic)
shock, complications of malaria (e.g., cerebral malaria and
anemia), other complications of infection, and pelvic inflammatory
disease;
[0356] transplant rejection, graft-versus-host disease;
[0357] allergic diseases including allergies, atopic diseases,
allergic rhinitis;
[0358] bone diseases (e.g., osteoporosis, Paget's disease);
[0359] skin diseases including psoriasis, eczema, atopic
dermatitis, UV(B)-induced dermal cell activation (e.g., sunburn,
skin cancer);
[0360] diabetes mellitus and its complications;
[0361] pain, testicular dysfunctions and wound healing;
[0362] gastrointestinal diseases including inflammatory bowel
disease (including but not limited to ulcerative colitis, Crohn's
disease), peptic ulceration, gastritis, esophagitis, liver disease
(including but not limited to cirrhosis and hepatitis).
[0363] In one embodiment, the disease or condition is selected from
the group consisting of rheumatoid arthritis, osteo arthritis,
systemic lupus erythematosus, ulcerative colitis, Crohn's disease,
multiple sclerosis, psoriasis, eczema, uveitis, diabetes mellitus,
glomerulonephritis, atherosclerotic vascular disease and
infarction, asthma, chronic obstructive pulmonary disease, HIV,
HBV, HCV, tuberculosis, malaria, worms, and cancer(s).
[0364] III. Pharmaceutical Compositions
[0365] Pharmaceutical compositions which include an effective
amount of the gold nanocrystals to treat any of the medical
conditions described in this application are also provided. In a
preferred embodiment, the gold nanocrystals are administered in an
orally delivered liquid, wherein the gold nanocrystals remain in
the water of manufacture which may be concentrated or
reconstituted, but preferable not dried to the point that the
surfaces of the gold nanocrystals become completely dry or have
their surfaces otherwise altered from their pristine state of
manufacture.
[0366] Based on experiments, it appears that the present gold
nanocrystals are a more potent form of gold than prior art
gold-based materials, including both FDA-approved gold-based
pharmaceutical products, and non-FDA-approved gold colloids, due to
the substantially clean very active crystalline surfaces. Because
of this, it is expected that significantly lower doses of the
present nanocrystals can be used, than dose levels required by
prior art compositions, including the oral gold product
Auranofin.
[0367] For example, in the widely accepted collagen induced
arthritis mouse model, a standard dose is 40 mg/kg/day of
Auranofin, which is approximately 1 mg/mouse/day of Auranofin, and
0.30 mg gold/day of gold contained in Auranofin. This standard
Auranofin dose level appears to give an equivalent response to that
resulting from a dose of about 0.06 mg/day of the gold nanocrystals
of the present invention (Example 25). Thus, in such experiment,
the present nanocrystals were calculated to be 17 times more potent
than was the Auranofin, and 5 times more potent than the gold
species contained in the Auranofin.
[0368] The standard FDA-approved dose level for Auranofin in humans
is 6 mg/day, or 0.9 mg/kg/day. The gold contained in that human
dose levels of Auranofin is 1.74 mg, or 0.025 mg/kg. Given the
relative potency of the novel gold nanocrystals compared to that of
Auranofin, as demonstrated in the live animal model, an approximate
human dose level for the novel gold nanocrystal can be calculated
by dividing the human dose level for Auranofin by the relative
potency factor of 17.times., or by dividing the human dose level of
the gold contained in the Auranofin by the relative potency factor
of 5.times.. This results in an approximate human dose level for
the novel gold nanocrystals of 0.35 mg/day, versus the 6 mg/day
required for Auranofin, and 1.74 mg/day required for gold contained
in Auranofin. 0.35 mg/day, for a 70 kg human being, is a dose of
0.005 mg/kg/day.
[0369] It is normal in developing dosing levels to establish a
range of one order of magnitude or more surrounding an estimated
mg/kg dose. In this case, if the approximate suggested base dose is
1/17 that of the base dose of Auranofin, or 0.348 mg/day, which is
0.005 mg/kg/day, this suggests that an effective dosing range for
Auranofin-like efficacy with the novel nanocrystals can be achieved
at dosing levels of 0.005 mg/kg/day, and even greater efficacy at
levels in the range of 0.01 mg/kg/day or 0.25 mg/kg/day.
[0370] It is important to recognize that in pharmaceutical products
the objective is to establish the minimum dose necessary to achieve
efficacy, thus minimizing potential for toxicity or complications.
A new orally administered product with significantly greater
potency can achieve efficacy at dose levels below those of prior
art products, and/or can achieve substantially greater efficacy at
equivalent dose levels.
[0371] Moreover, it is observed in animal trials that toxicity
levels of the novel nanocrystals are low, even at maximum dose
levels, which means that even at higher dose levels there is less
toxicity than with current products such as Auranofin.
[0372] It has also been observed in mice that a therapeutic effect
is seen faster than with Auranofin, which has a typical onset of
action of weeks, compared to days for the present nanocrystals (See
Example 25). This is a major advantage in use, since it means
patients enjoy relief sooner, and are much more likely to continue
to comply with the regimen and thus continue to benefit from the
product.
[0373] It has further been observed that the present gold
nanocrystals have a better therapeutic index than Auranofin due to
the lower dose required to achieve efficacy and the associated
lower toxicity.
[0374] It is also important to recognize that to have real value as
a pharmaceutical treatment, a product must be manufacturable under
high pharmaceutical-grade manufacturing, sourcing, and quality
control standards, as defined by the FDA as Good Manufacturing
Practice (GMP). Conventional gold nanoparticles are made by a
variety of methods, most of which involve chemical reduction
processes. There appear to be no current chemical reduction or
other conventional processes for production of gold nanoparticles
which comply with GMP, and given the nature of these processes, it
appears that GMP compliance, if possible, will be extremely
challenging and will require substantial time, money, and inventive
engineering to achieve. The process by which the present novel gold
nanocrystals are produced is designed to be GMP compliant,
establishing another major difference and advantage of the present
gold nanocrystals.
[0375] While clinical trials are required to confirm the
therapeutically efficacious dose, it is reasonable to conclude that
doses ranging from 0.05 mgs or more (or 0.1, 0.5, 1.0, 2.0 mg or
more) to 10 mg or more per dosage (once, twice or multiple times
per day) are effective in a human to treat any of the conditions
described herein. Given the low toxicity of these gold
nanocrystals, for more problematic disorders it is appropriate to
use at higher dose levels, including but not limited dosages of 10
mgs or more, such as 20 mg or more per dosage.
[0376] Any concentration of gold nanocrystals can be provided
according to the invention. For example, concentrations of these
gold nanocrystals can be a few parts per million (i.e., .mu.g/ml or
mg/l) up to a few hundred ppm, but are typically in the range of
2-200 ppm (i.e., 2 .mu.g/ml-200 .mu.g/ml) and more often in the
range of 2-50 ppm (i.e., 2 .mu.g/ml-50 .mu.g/ml). A typical
convenient concentration may be around 5-20 .mu.g/ml, and more
typically about 8-15 .mu.g/ml.
[0377] Pharmaceutical compositions are provided that are
appropriate for systemic or topical use, including oral,
intravenous, subcutaneous, intra-arterial, buccal, inhalation,
aerosol, propellant or other appropriate liquid, etc, as described
further herein, including specific gels or creams discussed in
Example 23.
[0378] Alternatively, suitable dosages of active ingredient may lie
within the range of about 0.1 ng per kg of body weight to about 1 g
per kg of body weight per dosage. The dosage is typically in the
range of 1 .mu.g to 1 g per kg of body weight per dosage, such as
is in the range of 1 mg to 1 g per kg of body weight per dosage. In
one embodiment, the dosage is in the range of 1 mg to 500 mg per kg
of body weight per dosage. In another embodiment, the dosage is in
the range of 1 mg to 250 mg per kg of body weight per dosage. In
yet another preferred embodiment, the dosage is in the range of 1
mg to 100 mg per kg of body weight per dosage, such as up to 50 mg
per kg of body weight per dosage. In yet another embodiment, the
dosage is in the range of 1 .mu.g to 1 mg per kg of body weight per
dosage.
[0379] Suitable dosage amounts and dosing regimens can be
determined by the attending physician or veterinarian and may
depend on the desired level of inhibiting and/or modifying
activity, the particular condition being treated, the severity of
the condition, whether the dosage is preventative or therapeutic,
as well as the general age, health and weight of the subject.
[0380] The gold nanocrystals contained in, for example, an aqueous
medium, colloid, suspension, foam, gel, paste, liquid, cream or the
like, may be administered in a single dose or a series of doses.
While it is possible for the aqueous medium containing the
metallic-based nanocrystals to be administered alone in, for
example, colloid form, it may be acceptable to include the active
ingredient mixture with other compositions and or therapies.
Further, various pharmaceutical compositions can be added to the
active ingredient(s)/suspension(s)/colloid(s).
[0381] Accordingly, typically, the inventive gold nanocrystal
suspensions or colloids (e.g., comprising aqueous gold-based metal
and/or mixtures of gold and other metal(s) and/or alloys of gold
with other metal(s) and/or a combination therapy approach) are
administered in conjunction with a second therapeutic agent. More
typically, the second therapeutic agent comprises a
glucocorticoid.
[0382] In a further aspect of the invention, there is provided a
pharmaceutical composition comprising the inventive gold
nanocrystal suspensions or colloids (e.g., comprising aqueous
gold-based metal and/or mixtures of gold and other metal(s) and/or
alloys of gold with other metal(s) and/or a combination therapy
approach) together with a pharmaceutically acceptable carrier,
diluent or excipient. The formulation of such compositions is well
known to those skilled in the art. The composition may contain
pharmaceutically acceptable additives such as carriers, diluents or
excipients. These include, where appropriate, all conventional
solvents, dispersion agents, fillers, solid earners, coating
agents, antifungal and/or antibacterial agents, dermal penetration
agents, ibuprofen, ketoprofen, surfactants, isotonic and absorption
agents and the like. It will be understood that the compositions of
the invention may also include other supplementary physiologically
active agents. Still further, a large variety of dietary
supplements and homeopathic carriers can also be utilized.
Specifically, choices of such ingredients can be based in part on
known functionality or use of these ingredients such that when
combined with active ingredients of the invention, additive or
synergistic affects can be achieved.
[0383] The carrier should be pharmaceutically acceptable in the
sense of being compatible with the other ingredients in the
inventive gold nanocrystal suspensions and not injurious (e.g.,
toxic at therapeutically active amounts) to the subject.
Compositions include those suitable for oral, rectal, inhalational,
nasal, transdermal, topical (including buccal and sublingual),
vaginal or parenteral (including subcutaneous, intramuscular,
intraspinal, intravenous and intradermal) administration. The
compositions may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy,
homeopathy and/or dietary supplements. Such methods include the
step of bringing into association the inventive metallic-based
nanocrystals or suspensions with the carrier which constitutes one
or more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing into association one
or more active ingredients in the solution/colloid under
appropriate non-reactive conditions which minimize or eliminate, to
the extent possible, negative or adverse reactions.
[0384] Depending on the disease or condition to be treated, it may
or may not be desirable for the inventive gold nanocrystal
suspensions or colloids to cross the blood/brain barrier.
[0385] Thus, the gold nanocrystal suspensions or colloids of the
present invention may be manufactured to be of desirable size,
desirable crystal plane(s) and/or desirable shapes or shape
distributions, etc (as discussed elsewhere herein) to assist in
crossing the blood/brain barrier.
[0386] Gold nanocrystal suspensions according to the present
invention suitable for oral administration are presented typically
as a stable solution, colloid or a partially stable suspension in
water. However, such gold nanocrystals may also be included in a
non-aqueous liquid, as discrete units such as liquid capsules,
sachets or even tablets (e.g., drying-out suspensions or colloids
to result in active ingredient gold-based nanocrystals so long as
such processing does not adversely affect the functionality of the
pristine gold nanocrystal surfaces) each containing a predetermined
amount, of, for example, the gold nanocrystal active ingredient; as
a powder or granules; as a solution, colloid or a suspension in an
aqueous or as non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The gold nanocrystal
active ingredient may also be combined into a bolus, electuary or
paste.
[0387] A tablet made from the inventive gold nanocrystal
suspensions or colloids (e.g., comprising aqueous gold-based
nanocrystals and/or alloys of gold with other metal(s) and/or a
combination therapy approach) and other materials or compounds may
be made by, for example, first drying the suspension or colloid,
collecting residual dried material and by compression or molding,
forcing the powder into a suitable tablet or the like. For example,
compressed tablets may be prepared by compressing in, a suitable
machine, the active ingredient nanocrystals, for example, the
metallic-based nanocrystals, in a free-flowing form such as a
powder or granules, optionally mixed with a binder (e.g., inert
diluent, preservative, disintegrant (e.g., sodium starch glycolate,
cross-linked polyvinyl pyrrolidone, cross-linked sodium
carboxymethyl cellulose)) surface-active or dispersing agent.
Molded tablets may be made by, for example, molding or pressing in
a suitable machine a mixture of the powdered compound moistened
with an inert liquid diluent. The tablets may optionally be coated
or scored and may be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile. Tablets may optionally be provided with an
enteric coating, to provide for release in parts of the gut other
than the stomach.
[0388] Compositions suitable for topical administration in the
mouth include lozenges comprising suspensions or colloids
containing one or more active ingredient(s) gold nanocrystal in a
flavored base, such as sucrose and acacia or tragacanth gum;
pastilles comprising the gold nanocrystal active ingredient in an
inert base such as a gelatin and a glycerin, or sucrose and acacia
gum; and mouthwashes comprising the gold nanocrystal active
ingredient in a suitable liquid carrier.
[0389] The inventive gold nanocrystal suspensions or colloids
(e.g., comprising aqueous gold-based metal and/or mixtures of gold
and other metal(s) and/or alloys of gold with other metal(s) and/or
a combination therapy approach) may also be administered
intranasally or via inhalation, for example by atomizer, aerosol or
nebulizer means for causing one or more constituents in the
solution or colloid (e.g., the gold nanocrystals) to be, for
example, contained within a mist or spray.
[0390] Compositions suitable for topical administration to the skin
may comprise the gold nanocrystals of the invention suspended in
any suitable carrier or base and may be in the form of lotions,
gel, creams, pastes, ointments and the like. Suitable carriers
include mineral oil, propylene glycol, polyoxyethylene,
polyoxypropylene, emulsifying wax, sorbitan monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol, carbopol and water.
[0391] Transdermal devices, such as patches, may also be used to
administer the compounds of the invention.
[0392] Compositions for rectal administration may be presented as a
suppository with a suitable carrier base comprising, for example,
cocoa butter, gelatin, glycerin or polyethylene glycol.
[0393] Compositions suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0394] Compositions suitable for parenteral administration include
aqueous and non-aqueous isotonic sterile injection suspensions or
colloids which may contain anti-oxidants, buffers, bactericides and
solutes which render the composition isotonic with the blood of the
intended recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents and thickening agents. The
compositions may be presented in unit-dose or multi-dose sealed
containers, for example, ampoules and vials, and may be stored in a
freeze-dried (lyophilised) condition requiring only the addition of
the sterile liquid carrier, for example water for injections,
immediately prior to use. Extemporaneous injection solutions,
colloids and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0395] Preferred unit dosage compositions are those containing a
daily dose or unit, daily sub-dose, as herein above described, or
an appropriate fraction thereof, of the active ingredient.
[0396] It should be understood that in addition to the gold
nanocrystal active ingredients particularly mentioned above, the
compositions of this invention may include other agents
conventional in the art having regard to the type of composition in
question, for example, those suitable for oral administration may
include such further agents as binders, sweeteners, thickeners,
flavoring agents, disintegrating agents, coating agents,
preservatives, lubricants, time delay agents and/or position
release agents. Suitable sweeteners include sucrose, lactose,
glucose, aspartame or saccharine. Suitable disintegrating agents
include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan
gum, bentonite, alginic acid or agar. Suitable flavoring agents
include peppermint oil, oil of wintergreen, cherry, orange or
raspberry flavoring. Suitable coating agents include polymers or
copolymers of acrylic acid and/or methacrylic acid and/or their
esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable
preservatives include sodium benzoate, vitamin E, alpha-tocopherol,
ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
Suitable lubricants include magnesium stearate, stearic acid,
sodium oleate, sodium chloride or talc. Suitable time delay agents
include glyceryl mono stearate or glyceryl distearate.
[0397] Further, by following the inventive electrochemical
manufacturing processes of the invention, these gold-based metallic
nanocrystals can be alloyed or combined with other metals in
liquids such that gold "coatings" may occur on other metals (or
other non-metal species such as SiO.sub.2, for example) or
alternatively, gold-based nanocrystals may be coated by other
metals. In such cases, gold-based composites or alloys may result
within a colloid or suspension. Further, certain composites which
include both gold and other metals can also be formed.
[0398] Still further, gold-based metallic nanocrystals suspensions
or colloids of the present invention can be mixed or combined with
other metallic-based solutions or colloids to form novel solution
or colloid mixtures (e.g., in this instance, distinct metal species
can still be discerned).
[0399] IV. Method of Manufacturing Gold Nanocrystals
[0400] A novel process is provided to produce these unique gold
nanocrystals. The process involves the creation of the gold
nanocrystals in water. In a preferred embodiment, the water
contains an added "process enhancer" which does not significantly
bind to the formed nanocrystals, but rather facilitates
nucleation/crystal growth during the electrochemical-stimulated
growth process. The process enhancer serves important roles in the
process including providing charged ions in the electrochemical
solution to permit the crystals to be grown. These novel
electrochemical processes can occur in either a batch,
semi-continuous or continuous process. These processes result in
controlled gold nanocrystalline concentrations, controlled
nanocrystal sizes and controlled nanocrystal size ranges; as well
as controlled nanocrystal shapes and controlled nanocrystal shape
distributions. Novel manufacturing assemblies are provided to
produce these gold nanocrystals.
[0401] In one preferred embodiment, the gold-based nanocrystal
suspensions or colloids are made or grown by electrochemical
techniques in either a batch, semi-continuous or continuous
process, wherein the amount, average particle size, crystal
plane(s) and/or particle shape(s) and/or particle shape
distributions are controlled and/or optimized to achieve high
biological activity and low cellular/biologic toxicity (e.g., a
high therapeutic index). Desirable average crystal sizes include a
variety of different ranges, but the most desirable ranges include
average crystal sizes that are predominantly less than 100 nm and
more typically, for many uses, less than 50 nm and even more
typically for a variety of, for example, oral uses, less than 30
nm, and in many of the preferred embodiments disclosed herein, the
mode for the nanocrystal size distribution is less than 21 nm and
within an even more preferable range of 8-18 nm, as measured by
drying such solutions and constructing particle size histograms
from TEM measurements (as described in more detail herein).
Further, the particles desirably contain crystal planes, such
desirable crystal planes including crystals having {111}, {110}
and/or {100} facets, which can result in desirable crystal shapes
and desirable crystal shape distributions and better performance
than gold spherical or randomly-shaped particles.
[0402] Further, by following the inventive electrochemical
manufacturing processes of the invention, these gold-based metallic
nanocrystals can be alloyed or combined with other metals in
liquids such that gold "coatings" may occur on other metals (or
other non-metal species such as SiO.sub.2, for example) or
alternatively, gold-based nanocrystals may be coated by other
metals. In such cases, gold-based composites or alloys may result
within a colloid or suspension. Further, certain composites which
include both gold and other metals can also be formed.
[0403] Still further, gold-based metallic nanocrystals suspensions
or colloids of the present invention can be mixed or combined with
other metallic-based solutions or colloids to form novel solution
or colloid mixtures (e.g., in this instance, distinct metal species
can still be discerned).
[0404] Methods for making novel metallic-based nanocrystal
suspensions or colloids according to the invention relate generally
to novel methods and novel devices for the continuous,
semi-continuous and batch manufacture of a variety of constituents
in a liquid including micron-sized particles, nanocrystals, ionic
species and aqueous-based compositions of the same, including,
nanocrystal/liquid(s), solution(s), colloid(s) or suspension(s).
The constituents and nanocrystals produced can comprise a variety
of possible compositions, concentrations, sizes, crystal planes
(e.g., spatially extended low index crystal planes) and/or shapes,
which together can cause the inventive compositions to exhibit a
variety of novel and interesting physical, catalytic, biocatalytic
and/or biophysical properties. The liquid(s) used and
created/modified during the process can play an important role in
the manufacturing of, and/or the functioning of the constituents
(e.g., nanocrystals) independently or synergistically with the
liquids which contain them. The particles (e.g., nanocrystals) are
caused to be present (e.g., created and/or the liquid is
predisposed to their presence (e.g., conditioned)) in at least one
liquid (e.g., water) by, for example, typically utilizing at least
one adjustable plasma (e.g., created by at least one AC and/or DC
power source), which adjustable plasma communicates with at least a
portion of a surface of the liquid. However, effective constituent
(e.g., nanocrystals) suspensions or colloids can be achieved
without the use of such plasmas as well.
[0405] Metal-based electrodes of various composition(s) and/or
unique configurations or arrangements are preferred for use in the
formation of the adjustable plasma(s), but non-metallic-based
electrodes can also be utilized for at least a portion of the
process. Utilization of at least one subsequent and/or
substantially simultaneous adjustable electrochemical processing
technique is also preferred. Metal-based electrodes of various
composition(s) and/or unique configurations are preferred for use
in the electrochemical processing technique(s). Electric fields,
magnetic fields, electromagnetic fields, electrochemistry, pH, zeta
potential, chemical/crystal constituents present, etc., are just
some of the variables that can be positively affected by the
adjustable plasma(s) and/or adjustable electrochemical processing
technique(s) of the invention. Multiple adjustable plasmas and/or
adjustable electrochemical techniques are preferred in many
embodiments of the invention to achieve many of the processing
advantages of the present invention, as well as many of the novel
nanocrystals and nanocrystal compositions which result from
practicing the teachings of the preferred embodiments to make an
almost limitless set of inventive aqueous solutions, suspensions
and/or colloids.
[0406] In the continuous process embodiments of the invention, at
least one liquid, for example water, flows into, through and out of
at least one trough member and such liquid is processed,
conditioned, modified and/or effected by said at least one
adjustable plasma and/or said at least one adjustable
electrochemical technique. The results of the continuous processing
include new constituents in the liquid, micron-sized particles,
ionic constituents, nanocrystals (e.g., metallic-based
nanocrystals) of novel and/or controllable size, hydrodynamic
radius, concentration, crystal sizes and crystal size ranges,
crystal planes, spatially extended low index crystal planes,
crystal shapes and distributions of crystal shapes and,
composition, zeta potential, pH and/or properties, such
nanocrystal/liquid mixture being produced in an efficient and
economical manner.
[0407] In a preferred embodiment, the process involves the
nucleation and growth of the gold nanocrystals in water which
contains a "process enhancer" or "processing enhancer" (typically
an inorganic material) which does not significantly bind to the
formed nanocrystals, but rather facilitates nucleation/growth
during electrochemical-stimulated growth process. The process
enhancer serves important roles in the process including providing
charged ions in the electrochemical solution to permit the crystals
to be grown. The process enhancer is critically a compound(s) which
remains in solution, and/or does not form a coating (e.g., an
organic coating), and/or does not adversely affect the formed
nanocrystals or the formed suspension(s), and/or is destroyed,
evaporated, or is otherwise lost during the electrochemical
process. A preferred process enhancer is sodium bicarbonate.
Examples of other process enhancers are sodium carbonate, potassium
bicarbonate, potassium carbonate, trisodium phosphate, disodium
phosphate, monosodium phosphate, potassium phosphates or other
salts of carbonic acid or the like. Further process enhancers may
be salts, including sodium or potassium, of bisulfite or sulfite.
Still other process enhancers to make gold nanocrystals for medical
applications under certain conditions may be other salts, including
sodium or potassium, or any material that assists in the
electrochemical growth processes described herein; and any material
is not substantially incorporated into or onto the surface of the
gold nanocrystals; and does not impart toxicity to the nanocrystals
or to the suspension containing the nanocrystals.
[0408] Desirable concentration ranges for the processing enhancer
include typically 0.01-20 grams/gallon (0.0026-2.1730 mg/ml), more
typically, 0.1-7.5 grams/gallon (0.0264-1.9813 mg/ml) and most
typically, 0.5-2.0 grams/gallon (0.13210-0.5283 mg/ml).
[0409] For example, certain processing enhancers may dissociate
into positive ions (cations) and negative ions (anions). The anions
and/or cations, depending on a variety of factors including liquid
composition, concentration of ions, applied fields, frequency of
applied fields, waveform of the applied filed, temperature, pH,
zeta potential, etc., will navigate or move toward oppositely
charged electrodes. When said ions are located at or near such
electrodes, the ions may take part in one or more reactions with
the electrode(s) and/or other constituent(s) located at or near
such electrode(s). Sometimes ions may react with one or more
materials in the electrode (e.g., when NaCl is used as a processing
enhancer, various metal chloride (MCl, MCl.sub.2, etc.) may form).
Such reactions may be desirable in some cases or undesirable in
others. Further, sometimes ions present in a solution between
electrodes may not react to form a product such as MCl, MCl.sub.2,
etc., but rather may influence material in the electrode (or near
the electrode) to form metallic nano-crystals that are "grown" from
material provided by the electrode. For example, certain metal ions
may enter the liquid 3 from the electrode 5 and be caused to come
together (e.g., nucleate) to form constituents (e.g., ions,
nanocrystals, etc.) within the liquid 3.
[0410] Further, it is important to select a process enhancer that
will not impart toxicity to the gold nanocrystal or the liquid that
the crystal is in to maximize pharmaceutical acceptability. For
example, for certain applications, chloride ion may be undesired if
it creates gold chloride salts which may have toxicity.
[0411] Further, depending upon the specific formed products,
drying, concentrating and/or freeze drying can also be utilized to
remove at least a portion of, or substantially all of, the
suspending liquid, resulting in, for example, partially or
substantially completely dehydrated nanocrystals. If solutions,
suspensions or colloids are completely dehydrated, the metal-based
species should be capable of being rehydrated by the addition of
liquid (e.g., of similar or different composition than that which
was removed). However, not all compositions/colloids of the present
invention can be completely dehydrated without adversely affecting
performance of the composition/colloid. For example, many
nanocrystals formed in a liquid tend to clump or stick together (or
adhere to surfaces) when dried. If such clumping is not reversible
during a subsequent rehydration step, dehydration should be
avoided.
[0412] In general, it is possible to concentrate, several folds,
certain solutions, suspensions or colloids of gold made according
to the invention, without destabilizing the composition. However,
complete evaporation is difficult to achieve due to, for example,
agglomeration effects. In many of the embodiments disclosed herein,
such agglomeration effects seem to begin at an approximate volume
of 30% of the initial or starting reference volume being removed
from the suspension or colloid. Additionally, one can evaporate off
a certain volume of liquid and subsequently reconstitute or
add-back the amount of liquid evaporated to achieve a very similar
product, as characterized by, for example, FAAS, DLS, and UV-Vis
techniques. For Example, two 500 ml suspensions of nanocrystalline
colloidal gold, made by techniques similar to those to manufacture
GB-139 (discussed in detail in the Examples section herein) were
each placed into a glass beaker and heated on a hot plate until
boiling. The suspensions were evaporated to 300 mL and 200 mL,
respectively, and later reconstituted with that amount of liquid
which was removed (i.e., with water purified by deionization and
reverse osmosis ("DI/RO") water in 200 mL and 300 mL quantities,
respectively) and subsequently characterized. Additionally, in
another instance, two GB-139 suspension were again evaporated to
300 mL and 200 mL and then characterized without rehydration. It
was found that these dehydration processes had little to no
detrimental effects on the nanocrystal sizes or nanocrystal shapes
(i.e., the nanocrystal size range and nanocrystal shape
distributions did not change dramatically when the GB-139 colloid
was dehydrated; or dehydrated and rehydrated to its initial gold
concentration or ppm level).
[0413] One important aspect of the invention involves the creation
of at least one adjustable plasma, which adjustable plasma is
located between at least one electrode positioned adjacent to
(e.g., above) at least a portion of the surface of a liquid (e.g.,
water) and at least a portion of the surface of the liquid itself.
The liquid is placed into electrical communication with at least
one second electrode (or a plurality of second electrodes) causing
the surface of the liquid to function as an electrode, thus taking
part in the formation of the adjustable plasma. This configuration
has certain characteristics similar to a dielectric barrier
discharge configuration, except that the surface of the liquid is
an active electrode participant in this configuration.
[0414] Each adjustable plasma utilized can be located between the
at least one electrode located above a surface of the liquid and a
surface of the liquid due to at least one electrically conductive
electrode being located somewhere within (e.g., at least partially
within) the liquid. At least one power source (in a preferred
embodiment, at least one source of volts and amps such as a
transformer or power source) is connected electrically between the
at least one electrode located above the surface of the liquid and
the at least one electrode contacting the surface of the liquid
(e.g., located at least partially, or substantially completely,
within the liquid). The electrode(s) may be of any suitable
composition and suitable physical configuration (e.g., size and
shape) which results in the creation of a desirable plasma between
the electrode(s) located above the surface of the liquid and at
least a portion of the surface of the liquid itself.
[0415] The applied power (e.g., voltage and amperage) between the
electrode(s) (e.g., including the surface of the liquid functioning
as at least one electrode for forming the plasma) can be generated
by any suitable source (e.g., voltage from a transformer) including
both AC and DC sources and variants and combinations thereof.
Generally, the electrode or electrode combination located within
(e.g., at least partially below the surface of the liquid) takes
part in the creation of a plasma by providing voltage and current
to the liquid or solution. However, the adjustable plasma is
actually located between at least a portion of the electrode(s)
located above the surface of the liquid (e.g., at a tip or point
thereof) and one or more portions or areas of the liquid surface
itself. In this regard, the adjustable plasma can be created
between the aforementioned electrodes (i.e., those located above at
least a portion of the surface of the liquid and a portion of the
liquid surface itself) when a breakdown voltage of the gas or vapor
around and/or between the electrode(s) and the surface of the
liquid is achieved or maintained.
[0416] In one embodiment of the invention, the liquid comprises
water (or water containing certain processing enhancer(s)), and the
gas between the surface of the water and the electrode(s) above the
surface of the water (i.e., that gas or atmosphere that takes part
in the formation of the adjustable plasma) comprises air. The air
can be controlled to contain various different water content(s) or
a desired humidity which can result in different compositions,
concentrations, crystal size distributions and/or crystal shape
distributions of constituents (e.g., nanocrystals) being produced
according to the present invention (e.g., different amounts of
certain constituents in the adjustable plasma and/or in the
solution or suspension can be a function of the water content in
the air located above the surface of the liquid) as well as
different processing times required to obtain certain
concentrations of various constituents in the liquid, etc. Specific
aspects of the adjustable plasma 4 are discussed in greater detail
in Examples 5-7.
[0417] The breakdown electric field at standard pressures and
temperatures for dry air is about 3MV/m or about 30 kV/cm. Thus,
when the local electric field around, for example, a metallic point
exceeds about 30 kV/cm, a plasma can be generated in dry air.
Equation (1) gives the empirical relationship between the breakdown
electric field "E.sub.c" and the distance "d" (in meters) between
two electrodes:
E c = 3 .times. 0 .times. 0 .times. 0 + 1 . 3 .times. 5 d .times.
kV / m Equation .times. .times. 1 ##EQU00002##
Of course, the breakdown electric field "E.sub.c" will vary as a
function of the properties and composition of the gas or vapor
located between electrodes. In this regard, in one preferred
embodiment where water (or water containing a processing enhancer)
is the liquid, significant amounts of water vapor can be inherently
present in the air between the "electrodes" (i.e., between the at
least one electrode located above the surface of the water and the
water surface itself which is functioning as one electrode for
plasma formation) and such water vapor should have an effect on at
least the breakdown electric field required to create a plasma
therebetween. Further, a higher concentration of water vapor can be
caused to be present locally in and around the created plasma due
to the interaction of the adjustable plasma with the surface of the
water. The amount of "humidity" present in and around the created
plasma can be controlled or adjusted by a variety of techniques
discussed in greater detail later herein. Likewise, certain
components present in any liquid can form at least a portion of the
constituents forming the adjustable plasma located between the
surface of the liquid and the electrode(s) located adjacent (e.g.,
along) the surface of the liquid. The constituents in the
adjustable plasma, as well as the physical properties of the plasma
per se, can have a dramatic influence on the liquid, as well as on
certain of the processing techniques (discussed in greater detail
later herein).
[0418] The electric field strengths created at and near the
electrodes are typically at a maximum at a surface of an electrode
and typically decrease with increasing distance therefrom. In cases
involving the creation of an adjustable plasma between a surface of
the liquid and the at least one electrode(s) located adjacent to
(e.g., above) the liquid, a portion of the volume of gas between
the electrode(s) located above a surface of a liquid and at least a
portion of the liquid surface itself can contain a sufficient
breakdown electric field to create the adjustable plasma. These
created electric fields can influence, for example, behavior of the
adjustable plasma, behavior of the liquid (e.g., influence the
crystal state of the liquid) behavior of constituents in the
liquid, etc.
[0419] In this regard, FIG. 1a shows one embodiment of a point
source electrode 1 having a triangular cross-sectional shape
located a distance "x" above the surface 2 of a liquid 3 flowing,
for example, in the direction "F". An adjustable plasma 4 can be
generated between the tip or point 9 of the electrode 1 and the
surface 2 of the liquid 3 when an appropriate power source 10 is
connected between the point source electrode 1 and the electrode 5,
which electrode 5 communicates with the liquid 3 (e.g., is at least
partially below the surface 2 of the liquid 3).
[0420] The adjustable plasma region 4, created in the embodiment
shown in FIG. 1a can typically have a shape corresponding to a
cone-like structure or an ellipsoid-like structure, for at least a
portion of the process, and in some embodiments of the invention,
can maintain such shape (e.g., cone-like shape) for substantially
all of the process. The volume, intensity, constituents (e.g.,
composition), activity, precise locations, etc., of the adjustable
plasma(s) 4 will vary depending on a number of factors including,
but not limited to, the distance "x", the physical and/or chemical
composition of the electrode 1, the shape of the electrode 1, the
power source 10 (e.g., DC, AC, rectified AC, the applied polarity
of DC and/or rectified AC, AC or DC waveform, RF, etc.), the power
applied by the power source (e.g., the volts applied, which is
typically 1000-5000 Volts, and more typically 1000-1500 Volts, the
amps applied, electron velocity, etc.) the frequency and/or
magnitude of the electric and/or magnetic fields created by the
power source applied or ambient, electric, magnetic or
electromagnetic fields, acoustic fields, the composition of the
naturally occurring or supplied gas or atmosphere (e.g., air,
nitrogen, helium, oxygen, ozone, reducing atmospheres, etc.)
between and/or around the electrode 1 and the surface 2 of the
liquid 3, temperature, pressure, volume, flow rate of the liquid 3
in the direction "F", spectral characteristics, composition of the
liquid 3, conductivity of the liquid 3, cross-sectional area (e.g.,
volume) of the liquid near and around the electrodes 1 and 5,
(e.g., the amount of time (i.e., dwell time) the liquid 3 is
permitted to interact with the adjustable plasma 4 and the
intensity of such interactions), the presence of atmosphere flow
(e.g., air flow) at or near the surface 2 of the liquid 3 (e.g.,
fan(s) or atmospheric movement means provided) etc., (discussed in
more detail later herein).
[0421] The composition of the electrode(s) 1 involved in the
creation of the adjustable plasma(s) 4 of FIG. 1a, in one preferred
embodiment of the invention, are metal-based compositions (e.g.,
metals such as gold and/or alloys or mixtures thereof, etc.), but
the electrodes 1 and 5 may be made out of any suitable material
compatible with the various aspects (e.g., processing parameters)
of the inventions disclosed herein. In this regard, while the
creation of a plasma 4 in, for example, air above the surface 2 of
a liquid 3 (e.g., water) will, typically, produce at least some
ozone, as well as amounts of nitrogen oxide and other components
(discussed in greater detail elsewhere herein). These produced
components can be controlled and may be helpful or harmful to the
formation and/or performance of the resultant constituents in the
liquid (e.g., nanocrystals) and/or, nanocrystal suspensions or
colloids produced and may need to be controlled by a variety of
different techniques, discussed in more detail later herein.
Further, the emission spectrum of each plasma 4, as shown for
example in Examples 5-7, is also a function of similar factors
(discussed in greater detail later herein). As shown in FIG. 1a,
the adjustable plasma 4 actually contacts the surface 2 of the
liquid 3. In this embodiment of the invention, material (e.g.,
metal) from the electrode 1 may comprise a portion of the
adjustable plasma 4 (e.g., and thus be part of the emission
spectrum of the plasma) and may be caused, for example, to be
"sputtered" onto and/or into the liquid 3 (e.g., water).
Accordingly, when metal(s) are used as the electrode(s) 1, a
variety of constituents (such as those shown in Examples 5-7) can
be formed in the electrical plasma, resulting in certain
constituents becoming part of the processing liquid 3 (e.g.,
water), including, but not limited to, elementary metal(s), metal
ions, Lewis acids, Bronsted-Lowry acids, metal oxides, metal
nitrides, metal hydrides, metal hydrates and/or metal carbides,
etc., can be found in the liquid 3 (e.g., for at least a portion of
the process and may be capable of being involved in
simultaneous/subsequent reactions), depending upon the particular
set of operating conditions associated with the adjustable plasma 4
and/or subsequent electrochemical processing operations. Such
constituents may be transiently present in the processing liquid 3
or may be semi-permanent or permanent. If such constituents are
transient or semi-permanent, then the timing of subsequent
reactions (e.g., electrochemical reactions) with such formed
constituents can influence final products produced. If such
constituents are permanent, they should not adversely affect the
desired performance of the active ingredient nanocrystals.
[0422] Further, depending on, for example, electric, magnetic
and/or electromagnetic field strength in and around the liquid 3
and the volume of liquid 3 exposed to such fields (discussed in
greater detail elsewhere herein), the physical and chemical
construction of the electrode(s) 1 and 5, atmosphere (naturally
occurring or supplied), liquid composition, greater or lesser
amounts of electrode(s) materials(s) (e.g., metal(s) or derivatives
of metals) may be found in the liquid 3. In certain situations, the
material(s) (e.g., metal(s) or metal(s) composite(s)) or
constituents (e.g., Lewis acids, Bronsted-Lowry acids, etc.) found
in the liquid 3 (permanently or transiently), or in the plasma 4,
may have very desirable effects, in which case relatively large
amounts of such materials will be desirable; whereas in other
cases, certain materials found in the liquid 3 (e.g., by-products)
may have undesirable effects, and thus minimal amounts of such
materials may be desired in the liquid-based final product.
Accordingly, electrode composition can play an important role in
the materials that are formed according to the embodiments
disclosed herein. The interplay between these components of the
invention are discussed in greater detail later herein.
[0423] Still further, the electrode(s) 1 and 5 may be of similar
chemical composition (e.g., have the same chemical element as their
primary constituent) and/or mechanical configuration or completely
different compositions (e.g., have different chemical elements as
their primary constituent) in order to achieve various compositions
and/or structures of liquids and/or specific effects discussed
later herein.
[0424] The distance "y" between the electrode(s) 1 and 5; or 1 and
1 (shown later herein) or 5 and 5 (shown later herein) is one
important aspect of the invention. In general, when working with
power sources capable of generating a plasma under the operating
condition, the location of the smallest distance "y" between the
closest portions of the electrode(s) used in the present invention
should be greater than the distance "x" in order to prevent an
undesirable arc or formation of an unwanted corona or plasma
occurring between the electrode (e.g., the electrode(s) 1 and the
electrode(s) 5) (unless some type of electrical insulation is
provided therebetween). Features of the invention relating to
electrode design, electrode location and electrode interactions
between a variety of electrodes are discussed in greater detail
later herein.
[0425] The power applied through the power source 10 may be any
suitable power which creates a desirable adjustable plasma 4 under
all of the process conditions of the present invention. In one
preferred mode of the invention, an alternating current from a
step-up transformer is utilized. Preferred transformer(s) 60 (see
e.g., FIGS. 16d-16l) for use in various embodiments disclosed
herein, have deliberately poor output voltage regulation made
possible by the use of magnetic shunts in the transformer 60. These
transformers 60 are known as neon sign transformers. This
configuration limits current flow into the electrode(s) 1/5. With a
large change in output load voltage, the transformer 60 maintains
output load current within a relatively narrow range.
[0426] The transformer 60 is rated for its secondary open circuit
voltage and secondary short circuit current. Open circuit voltage
(OCV) appears at the output terminals of the transformer 60 only
when no electrical connection is present. Likewise, short circuit
current is only drawn from the output terminals if a short is
placed across those terminals (in which case the output voltage
equals zero). However, when a load is connected across these same
terminals, the output voltage of the transformer 60 should fall
somewhere between zero and the rated OCV. In fact, if the
transformer 60 is loaded properly, that voltage will be about half
the rated OCV.
[0427] The transformer 60 is known as a Balanced Mid-Point
Referenced Design (e.g., also formerly known as balanced midpoint
grounded). This is most commonly found in mid to higher voltage
rated transformers and most 60 mA transformers. This is the only
type transformer acceptable in a "mid-point return wired" system.
The "balanced" transformer 60 has one primary coil 601 with two
secondary coils 603, one on each side of the primary coil 601 (as
shown generally in the schematic view in FIG. 16g). This
transformer 60 can in many ways perform like two transformers. Just
as the unbalanced midpoint referenced core and coil, one end of
each secondary coil 603 is attached to the core 602 and
subsequently to the transformer enclosure and the other end of each
secondary coil 603 is attached to an output lead or terminal. Thus,
with no connector present, an unloaded 15,000-volt transformer of
this type, will measure about 7,500 volts from each secondary
terminal to the transformer enclosure but will measure about 15,000
volts between the two output terminals. These exemplary
transformers 60 were utilized to form the plasmas 4 disclosed in
the Examples herein. However, other suitable transformers (or power
sources) should also be understood as falling within the metes and
bounds of the invention. Further, these transformers 60 are
utilized exclusively in Examples 1-4 herein. However, different AC
transformers 50 and 50a (discussed elsewhere herein) are utilized
for the electrodes 5/5' in most of the other examples disclosed
herein.
[0428] In another preferred embodiment, a rectified AC source
creates a positively charged electrode 1 and a negatively charged
surface 2 of the liquid 3. In another preferred embodiment, a
rectified AC source creates a negatively charged electrode 1 and a
positively charged surface 2 of the liquid 3. Further, other power
sources such as RF power sources and/or microwave power sources can
also be used with the present invention. In general, the
combination of electrode(s) components 1 and 5, physical size and
shape of the electrode(s) 1 and 5, electrode manufacturing process,
mass of electrodes 1 and/or 5, the distance "x" between the tip 9
of electrode 1 above the surface 2 of the liquid 3, the composition
of the gas between the electrode tip 9 and the surface 2, the flow
rate (if any) and/or flow direction "F" of the liquid 3, the amount
of liquid 3 provided, type of power source 10, frequency and/or
waveform of the power output of the power source 10, all contribute
to the design, and thus power requirements (e.g., breakdown
electric field) required to obtain a controlled or adjustable
plasma 4 between the surface 2 of the liquid 3 and the electrode
tip 9.
[0429] In further reference to the configurations shown in FIG. 1a,
electrode holders 6a and 6b are capable of being lowered and raised
by any suitable means (and thus the electrodes are capable of being
lowered and raised). For example, the electrode holders 6a and 6b
are capable of being lowered and raised in and through an
insulating member 8 (shown in cross-section). The mechanical
embodiment shown here includes male/female screw threads. The
portions 6a and 6b can be covered by, for example, additional
electrical insulating portions 7a and 7b. The electrical insulating
portions 7a and 7b can be any suitable material (e.g., plastic,
polycarbonate, poly (methyl methacrylate), polystyrene, acrylics,
polyvinylchloride (PVC), nylon, rubber, fibrous materials, etc.)
which prevent undesirable currents, voltage, arcing, etc., that
could occur when an individual interfaces with the electrode
holders 6a and 6b (e.g., attempts to adjust the height of the
electrodes). Likewise, the insulating member 8 can be made of any
suitable material which prevents undesirable electrical events
(e.g., arcing, melting, etc.) from occurring, as well as any
material which is structurally and environmentally suitable for
practicing the present invention. Typical materials include
structural plastics such as polycarbonates, plexiglass (poly
(methyl methacrylate), polystyrene, acrylics, and the like.
Additional suitable materials for use with the present invention
are discussed in greater detail elsewhere herein.
[0430] FIG. 1c shows another embodiment for raising and lowering
the electrodes 1, 5. In this embodiment, electrical insulating
portions 7a and 7b of each electrode are held in place by a
pressure fit existing between the friction mechanism 13a, 13b and
13c, and the portions 7a and 7b. The friction mechanism 13a, 13b
and 13c could be made of, for example, spring steel, flexible
rubber, etc., so long as sufficient contact or friction is
maintained therebetween.
[0431] Preferred techniques for automatically raising and/or
lowering the electrodes 1, 5 are discussed later herein. The power
source 10 can be connected in any convenient electrical manner to
the electrodes 1 and 5. For example, wires 11a and 11b can be
located within at least a portion of the electrode holders 6a, 6b
(and/or electrical insulating portions 7a, 7b) with a primary goal
being achieving electrical connections between the portions 11a,
11b and thus the electrodes 1, 5.
[0432] FIG. 2a shows another schematic of a preferred embodiment of
the invention, wherein an inventive control device 20 is connected
to the electrodes 1 and 5, such that the control device 20 remotely
(e.g., upon command from another device or component) raises and/or
lowers the electrodes 1, 5 relative to the surface 2 of the liquid
3. The inventive control device 20 is discussed in more detail
later herein. In this one preferred aspect of the invention, the
electrodes 1 and 5 can be, for example, remotely lowered and
controlled, and can also be monitored and controlled by a suitable
controller or computer (not shown in FIG. 2a) containing an
appropriate software program (discussed in detail later herein). In
this regard, FIG. 2b shows an electrode configuration similar to
that shown in FIG. 2a, except that a Taylor Cone "T" is utilized
for electrical connection between the electrode 5 and the surface 2
(or effective surface 2') of the liquid 3. Accordingly, the
embodiments shown in FIGS. 1a, 1b and 1c should be considered to be
a manually controlled apparatus for use with the techniques of the
present invention, whereas the embodiments shown in FIGS. 2a and 2b
should be considered to include an automatic apparatus or assembly
20 which can remotely raise and lower the electrodes 1 and 5 in
response to appropriate commands. Further, the FIG. 2a and FIG. 2b
preferred embodiments of the invention can also employ computer
monitoring and computer control of the distance "x" of the tips 9
of the electrodes 1 (and tips 9' of the electrodes 5) away from the
surface 2; or computer monitoring and/or controlling the rate(s)
which the electrode 5 is advanced into/through the liquid 3
(discussed in greater detail later herein). Thus, the appropriate
commands for raising and/or lowering the electrodes 1 and 5 can
come from an individual operator and/or a suitable control device
such as a controller or a computer (not shown in FIG. 2a).
[0433] FIG. 3a corresponds in large part to FIGS. 2a and 2b,
however, FIGS. 3b, 3c and 3d show various alternative electrode
configurations that can be utilized in connection with certain
preferred embodiments of the invention. FIG. 3b shows essentially a
mirror image electrode assembly from that electrode assembly shown
in FIG. 3a. In particular, as shown in FIG. 3b, with regard to the
direction "F" corresponding to the flow direction of the liquid 3,
the electrode 5 is the first electrode which communicates with the
fluid 3 when flowing in the longitudinal direction "F" and contact
with the plasma 4 created at the electrode 1 follows. FIG. 3c shows
two electrodes 5a and 5b located within the fluid 3. This
particular electrode configuration corresponds to another preferred
embodiment of the invention. In particular, as discussed in greater
detail herein, the electrode configuration shown in FIG. 3c can be
used alone, or in combination with, for example, the electrode
configurations shown in FIGS. 3a and 3b. Similarly, a fourth
possible electrode configuration is shown in FIG. 3d. In this FIG.
3d, no electrode(s) 5 are shown, but rather only electrodes 1a and
1b are shown. In this case, two adjustable plasmas 4a and 4b are
present between the electrode tips 9a and 9b and the surface 2 of
the liquid 3. The distances "xa" and "xb" can be about the same or
can be substantially different, as long as each distance "xa" and
"xb" does not exceed the maximum distance for which a plasma 4 can
be formed between the electrode tips 9a/9b and the surface 2 of the
liquid 3. As discussed above, the electrode configuration shown in
FIG. 3d can be used alone, or in combination with one or more of
the electrode configurations shown in FIGS. 3a, 3b and 3c. The
desirability of utilizing particular electrode configurations in
combination with each other with regard to the fluid flow direction
"F" is discussed in greater detail later herein.
[0434] Likewise, a set of manually controllable electrode
configurations, corresponding generally to FIG. 1a, are shown in
FIGS. 4a, 4b, 4c and 4d, all of which are shown in a partial
cross-sectional view. Specifically, FIG. 4a corresponds to FIG. 1a.
Moreover, FIG. 4b corresponds in electrode configuration to the
electrode configuration shown in FIG. 3b; FIG. 4c corresponds to
FIG. 3c and FIG. 4d corresponds to FIG. 3d. In essence, the manual
electrode configurations shown in FIGS. 4a-4d can functionally
result in similar materials produced according to certain inventive
aspects of the invention as those materials produced corresponding
to remotely adjustable (e.g., remote-controlled by computer or
controller means) electrode configurations shown in FIGS. 3a-3d.
The desirability of utilizing various electrode configuration
combinations is discussed in greater detail later herein.
[0435] FIGS. 5a-5e show perspective views of various desirable
electrode configurations for the electrode 1 shown in FIGS. 1-4 (as
well as in other Figures and embodiments discussed later herein).
The electrode configurations shown in FIGS. 5a-5e are
representative of a number of different configurations that are
useful in various embodiments of the present invention. Criteria
for appropriate electrode selection for the electrode 1 include,
but are not limited to the following conditions: the need for a
very well defined tip or point 9, composition, mechanical
limitations, the ability to make shapes from the material
comprising the electrode 1, conditioning (e.g., heat treating or
annealing) of the material comprising the electrode 1, convenience,
the constituents introduced into the plasma 4, the influence upon
the liquid 3, etc. In this regard, a small mass of material
comprising the electrodes 1 shown in, for example, FIGS. 1-4 may,
upon creation of the adjustable plasmas 4 according to the present
invention (discussed in greater detail later herein), rise to
operating temperatures where the size and or shape of the
electrode(s) 1 can be adversely affected. In this regard, for
example, if the electrode 1 was of relatively small mass (e.g., if
the electrode(s) 1 was made of gold and weighed about 0.5 gram or
less) and included a very fine point as the tip 9, then it is
possible that under certain sets of conditions used in various
embodiments herein that a fine point (e.g., a thin wire having a
diameter of only a few millimeters and exposed to a few hundred to
a few thousand volts; or a triangular-shaped piece of metal) would
be incapable of functioning as the electrode 1 (e.g., the electrode
1 could deform undesirably or melt), absent some type of additional
interactions (e.g., internal cooling means or external cooling
means such as a fan, etc.). Accordingly, the composition of (e.g.,
the material comprising) the electrode(s) 1 may affect possible
suitable electrode physical shape due to, for example, melting
points, pressure sensitivities, environmental reactions (e.g., the
local environment of the adjustable plasma 4 could cause
undesirable chemical, mechanical and/or electrochemical erosion of
the electrode(s)), etc.
[0436] Moreover, it should be understood that in alternative
preferred embodiments of the invention, well defined sharp points
are not always required for the tip 9. In this regard, the
electrode 1 shown in FIG. 5e comprises a rounded tip 9. It should
be noted that partially rounded or arc-shaped electrodes can also
function as the electrode 1 because the adjustable plasma 4, which
is created in the inventive embodiments shown herein (see, for
example, FIGS. 1-4), can be created from rounded electrodes or
electrodes with sharper or more pointed features. During the
practice of the inventive techniques of the present invention, such
adjustable plasmas can be positioned or can be located along
various points of the electrode 1 shown in FIG. 5e. In this regard,
FIG. 6 shows a variety of points "a-g" which correspond to
initiating points 9 for the plasmas 4a-4g which occur between the
electrode 1 and the surface 2 of the liquid 3. Accordingly, it
should be understood that a variety of sizes and shapes
corresponding to electrode 1 can be utilized in accordance with the
teachings of the present invention. Still further, it should be
noted that the tips 9, 9' of the electrodes 1 and 5, respectively,
shown in various Figures herein, may be shown as a relatively sharp
point or a relatively blunt end. Unless specific aspects of these
electrode tips 9, 9' are discussed in greater contextual detail,
the actual shape of the electrode tip(s) 9, 9' shown in the Figures
should not be given great significance.
[0437] FIG. 7a shows a cross-sectional perspective view of the
electrode configuration corresponding to that shown in FIG. 2a (and
FIG. 3a) contained within a trough member 30. This trough member 30
has a liquid 3 supplied into it from the back side identified as 31
of FIG. 7a and the flow direction "F" is out of the page toward the
reader and toward the cross-sectioned area identified as 32. The
trough member 30 is shown here as a unitary piece of one material,
but could be made from a plurality of materials fitted together
and, for example, fixed (e.g., glued, mechanically attached, etc.)
by any acceptable means for attaching materials to each other.
Further, the trough member 30 shown here is of a rectangular or
square cross-sectional shape, but may comprise a variety of
different and more desirable cross-sectional shapes (discussed in
greater detail later herein). Accordingly, the flow direction of
the fluid 3 is out of the page toward the reader and the liquid 3
flows past each of the electrodes 1 and 5, which are, in this
embodiment, located substantially in line with each other relative
to the longitudinal flow direction "F" of the fluid 3 within the
trough member 30. This causes the liquid 3 to first experience an
adjustable plasma interaction with the adjustable plasma 4 (e.g., a
conditioning reaction) and subsequently then the conditioned fluid
3 is permitted to interact with the electrode(s) 5. Specific
desirable aspects of these electrode/liquid interactions and
electrode placement(s) or electrode locations within the trough
member 30 are discussed in greater detail elsewhere herein.
[0438] FIG. 7b shows a cross-sectional perspective view of the
electrode configuration shown in FIG. 2a (as well as in FIG. 3a),
however, these electrodes 1 and 5 are rotated on the page 90
degrees relative to the electrodes 1 and 5 shown in FIGS. 2a and
3a. In this embodiment of the invention, the liquid 3 contacts the
adjustable plasma 4 generated between the electrode 1 and the
surface 2 of the liquid 3, and the electrode 5 at substantially the
same point along the longitudinal flow direction "F" (i.e., out of
the page) of the trough member 30. The direction of liquid 3 flow
is longitudinally along the trough member 30 and is out of the
paper toward the reader, as in FIG. 7a. Various desirable aspects
of this electrode configuration are discussed in greater detail
later herein.
[0439] FIG. 8a shows a cross-sectional perspective view of the same
embodiment shown in FIG. 7a. In this embodiment, as in FIG. 7a, the
fluid 3 first interacts with the adjustable plasma 4 created
between the electrode 1 and the surface 2 of the liquid 3.
Thereafter the plasma influenced or conditioned fluid 3, having
been changed (e.g., conditioned, modified, or prepared) by the
adjustable plasma 4, thereafter communicates with the electrode(s)
5 thus permitting various electrochemical reactions to occur, such
reactions being influenced by the state (e.g., chemical
composition, pH, physical or crystal structure, excited state(s),
etc., of the fluid 3 (and constituents, semi-permanent or
permanent, within the fluid 3)) discussed in greater detail
elsewhere herein. An alternative embodiment is shown in FIG. 8b.
This embodiment essentially corresponds in general arrangement to
those embodiments shown in FIGS. 3b and 4b. In this embodiment, the
fluid 3 first communicates with the electrode 5, and thereafter the
fluid 3 communicates with the adjustable plasma 4 created between
the electrode 1 and the surface 2 of the liquid 3. In this
embodiment, the fluid 3 may have been previously modified prior to
interacting with the electrode 5.
[0440] FIG. 8c shows a cross-sectional perspective view of two
electrodes 5a and 5b (corresponding to the embodiments shown in
FIGS. 3c and 4c) wherein the longitudinal flow direction "F" of the
fluid 3 contacts the first electrode 5a and thereafter contacts the
second electrode 5b in the direction "F" of fluid flow.
[0441] Likewise, FIG. 8d is a cross-sectional perspective view and
corresponds to the embodiments shown in FIGS. 3d and 4d. In this
embodiment, the fluid 3 communicates with a first adjustable plasma
4a created by a first electrode 1a and thereafter communicates with
a second adjustable plasma 4b created between a second electrode 1b
and the surface 2 of the fluid 3.
[0442] FIG. 9a shows a cross-sectional perspective view and
corresponds to the electrode configuration shown in FIG. 7b (and
generally to the electrode configuration shown in FIGS. 3a and 4a
but is rotated 90 degrees relative thereto). All of the electrode
configurations shown in FIGS. 9a-9d are situated such that the
electrode pairs shown are located substantially at the same
longitudinal point along the trough member 30, as in FIG. 7b.
[0443] Likewise, FIG. 9b corresponds generally to the electrode
configuration shown in FIGS. 3b and 4b, and is rotated 90 degrees
relative to the configuration shown in FIG. 8b.
[0444] FIG. 9c shows an electrode configuration corresponding
generally to FIGS. 3c and 4c, and is rotated 90 degrees relative to
the electrode configuration shown in FIG. 8c.
[0445] FIG. 9d shows an electrode configuration corresponding
generally to FIGS. 3d and 4d and is rotated 90 degrees relative to
the electrode configuration shown in FIG. 8d.
[0446] The electrode configurations shown generally in FIGS. 7, 8
and 9, all can create different results (e.g., different
conditioning effects for the fluid 3, different pH's in the fluid
3, different nanocrystals sizes and size distribution, different
nanocrystal shapes and nanocrystal shape distributions, and/or
amounts of constituents (e.g., nanocrystal matter) found in the
fluid 3, different functioning of the fluid/nanocrystal
combinations (e.g., different biologic/biocatalytic effects),
different zeta potentials, etc.) as a function of a variety of
features including the electrode orientation and position relative
to the fluid flow direction "F", cross-sectional shape and size of
the trough member 30, and/or amount of the liquid 3 within the
trough member 30 and/or rate of flow of the liquid 3 within the
trough member 30 and in/around the electrodes 5a/5b, the thickness
of the electrodes, the number of electrode pairs provided and their
positioning in the trough member 30 relative to each other as well
as their depth into the liquid 3 (i.e., amount of contact with the
liquid 3), the rate of movement of the electrodes into/through the
liquid 3 (which maintains or adjusts the surface profile or shape
if the electrodes), the power applied to the electrode pairs, etc.
Further, the electrode compositions, size, specific shape(s),
number of different types of electrodes provided, voltage applied,
amperage applied and/or achieved within the liquid 3, AC source
(and AC source frequency and AC waveform shape, duty cycle, etc.),
DC source, RF source (and RF source frequency, duty cycle, etc.),
electrode polarity, etc., can all influence the properties of the
liquid 3 (and/or the nanocrystals formed or contained in the liquid
3) as the liquid 3 contacts, interacts with and/or flows past these
electrodes 1, 5 and hence resultant properties of the materials
(e.g., the nanocrystals produced and/or the suspension or colloid)
produced therefrom. Additionally, the liquid-containing trough
member 30, in some preferred embodiments, contains a plurality of
the electrode combinations shown in FIGS. 7, 8 and 9. These
electrode assemblies may be all the same configuration or may be a
combination of various different electrode configurations
(discussed in greater detail elsewhere herein). Moreover, the
electrode configurations may sequentially communicate with the
fluid "F" or may simultaneously, or in parallel communicate with
the fluid "F". Different exemplary and preferred electrode
configurations are shown in additional figures later herein and are
discussed in greater detail later herein in conjunction with
different constituents formed (e.g., nanocrystals and solutions or
nanocrystal suspensions or colloids produced therefrom).
[0447] FIG. 10a shows a cross-sectional view of the liquid
containing trough member 30 shown in FIGS. 7, 8 and 9. This trough
member 30 has a cross-section corresponding to that of a rectangle
or a square and the electrodes (not shown in FIG. 10a) can be
suitably positioned therein.
[0448] Likewise, several additional alternative cross-sectional
embodiments for the liquid-containing trough member 30 are shown in
FIGS. 10b, 10c, 10d and 10e. The distance "S" and "S'" for the
preferred embodiment shown in each of FIGS. 10a-10e measures, for
example, between about 0.25'' and about 6'' (about 0.6 cm-15 cm).
The distance "M" ranges from about 0.25'' to about 6'' (about 0.6
cm-15 cm). The distance "R" ranges from about 1/2'' to about 7''
(about 1.2 cm to about 17.8 cm). All of these embodiments (as well
as additional configurations that represent alternative embodiments
are within the metes and bounds of this inventive disclosure) can
be utilized in combination with the other inventive aspects of the
invention. It should be noted that the amount of liquid 3 contained
within each of the liquid containing trough members 30 is a
function not only of the depth "d", but also a function of the
actual cross-section. Briefly, the amount of liquid 3 present in
and around the electrode(s) 1 and 5 can influence one or more
effects of the adjustable plasma 4 upon the liquid 3 as well as the
electrochemical interaction(s) of the electrode 5 with the liquid
3. Further, the flow rate of the liquid 3 in and around the
electrode(s) 1 and 5 can also influence many of properties of the
nanocrystals formed in the resulting colloids or suspensions. These
effects include not only adjustable plasma 4 conditioning effects
(e.g., interactions of the plasma electric and magnetic fields,
interactions of the electromagnetic radiation of the plasma,
creation of various chemical species (e.g., Lewis acids,
Bronsted-Lowry acids) within the liquid, pH changes, temperature
variations of the liquid (e.g., slower liquid flow can result in
higher liquid temperatures and/or longer contact or dwell time with
or around the electrodes 1/5 which can also desirably influence
final products produced, such as size/shape of the formed
nanocrystals, etc.) upon the liquid 3, but also the concentration
or interaction of the adjustable plasma 4 with the liquid 3.
Similarly, the influence of many aspects of the electrode 5 on the
liquid 3 (e.g., electrochemical interactions, temperature, etc.) is
also, at least partially, a function of the amount of liquid
juxtaposed to the electrode(s) 5. All of these factors can
influence a balance which exists between nucleation and growth of
the nanocrystals grown in the liquid 3, resulting in, for example,
particle size and size range control and/or particle shape and
shape range control.
[0449] Further, strong electric and magnetic field concentrations
will also affect the interaction of the plasma 4 with the liquid 3
as well as affect the interaction of the electrode 5 with the
liquid 3. Some important aspects of these important interactions
are discussed in greater detail elsewhere herein. Further, a trough
member 30 may comprise more than one cross-sectional shape along
its entire longitudinal length. The incorporation of multiple
cross-sectional shapes along the longitudinal length of a trough
member 30 can result in, for example, varying the field or
concentration or reaction effects (e.g., crystal growth/nucleation
effects) being produced by the inventive embodiments disclosed
herein (discussed in greater detail elsewhere herein). Further, a
trough member 30 may not be linear or "I-shaped", but rather may be
"Y-shaped" or ".PSI.-shaped", with each portion of the "Y" (or
".PSI.") having a different (or similar) cross-sectional shape
and/or set of dimensions and/or set of reaction conditions
occurring therein.
[0450] Also, the initial temperature of the liquid 3 input into the
trough member 30 can also affect a variety of properties of
products produced according to the disclosure herein. For example,
different temperatures of the liquid 3 can affect nanocrystal
size(s) and nanocrystal shape(s), concentration or amounts of
various formed constituents (e.g., transient, semi-permanent or
permanent constituents), pH, zeta potential, etc. Likewise,
temperature controls along at least a portion of, or substantially
all of, the trough member 30 can have desirable effects. For
example, by providing localized cooling, resultant properties of
products formed (e.g., nanocrystal size(s) and/or nanocrystal
shape(s)) can be controlled. Preferable liquid 3 temperatures
during the processing thereof are between freezing and boiling
points, more typically, between room temperature and boiling
points, and even more typically, between about 40-98 degrees C.,
and more typically, between about 50-98 degrees C. Such temperature
can be controlled by, for example, conventional means for cooling
located at or near various portions of the processing
apparatus.
[0451] Further, certain processing enhancers may also be added to
or mixed with the liquid(s) 3. The processing enhancers include
both solids and liquids (and gases in some cases). The processing
enhancer(s) may provide certain processing advantages and/or
desirable final product characteristics. Some portion of the
processing enhancer(s) may function, influence as or become part
of, for example, desirable seed crystals (or promote desirable seed
crystals, or be involved in the creation of a nucleation site)
and/or crystal plane growth promoters/preventers in the
electrochemical growth processes of the invention; or may simply
function as a current or power regulator in the electrochemical
processes of the invention. Such processing enhancers may also
desirably affect current and/or voltage conditions between
electrodes 1/5 and/or 5/5.
[0452] A preferred processing enhancer is sodium bicarbonate.
Examples of other process enhancers are sodium carbonate, potassium
bicarbonate, potassium carbonate, trisodium phosphate, disodium
phosphate, monosodium phosphate, potassium phosphates or other
salts of carbonic acid or the like. Further process enhancers may
be salts, including sodium or potassium, of bisulfite or sulfite.
Still other process enhancers to make gold nanocrystals for medical
applications under certain conditions may be other salts, including
sodium or potassium, or any material that assists in the
electrochemical growth processes described herein; and any material
is not substantially incorporated into or onto the surface of the
gold nanocrystals; and does not impart toxicity to the nanocrystals
or to the suspension containing the nanocrystals. Processing
enhancers may assist in 10 one or more of the electrochemical
reactions disclosed herein; and/or may assist in achieving one or
more desirable properties in products formed according to the
teachings herein.
[0453] For example, certain processing enhancers may dissociate
into positive ions (cations) and negative ions (anions). The anions
and/or cations, depending on a variety of factors including liquid
composition, concentration of ions, applied fields, frequency of
applied fields, waveform of the applied filed, temperature, pH,
zeta potential, etc., will navigate or move toward oppositely
charged electrodes. When said ions are located at or near such
electrodes, the ions may take part in one or more reactions with
the electrode(s) and/or other constituent(s) located at or near
such electrode(s). Sometimes ions may react with one or more
materials in the electrode (e.g., when NaCl is used as a processing
enhancer, various metal chloride (MCl, MCl.sub.2, etc.) may form).
Such reactions may be desirable in some cases or undesirable in
others. Further, sometimes ions present in a solution between
electrodes may not react to form a product such as MCl, MCl.sub.2,
etc., but rather may influence material in the electrode (or near
the electrode) to form metallic nano-crystals that are "grown" from
material provided by the electrode. For example, certain metal ions
may enter the liquid 3 from the electrode 5 and be caused to come
together (e.g., nucleate) to form constituents (e.g., ions,
nanocrystals, etc.) within the liquid 3.
[0454] In the case of gold, a variety of extended surface planes
from which crystal growth can occur are available, so long as
impurities (such as, for example organic impurities) do not inhibit
or prevent such growth. While gold is known to have a face centered
cubic (fcc) structure, gold nanocrystals which are grown according
to the methods of the present invention, are not single crystals
and are typically twinned to result in a variety of desirable and
highly reactive nanocrystalline shapes or shape distributions. For
example, single crystal surfaces {111}, {100} and {110} are among
the most frequently studied and well understood surfaces. The
presence of certain species such as ions (e.g., added to or being
donated by electrode 5) in an electrochemical crystal
nucleation/growth process can influence (e.g., nucleate and/or
promote growth of specifically-shaped nanocrystals or nanocrystal
shape distributions) the presence or absence of one or more of such
extended surfaces. A certain ion (e.g., anion) under certain field
conditions may assist in the presence of more {111} extended
surfaces or planes relative to other crystal surfaces which can
result in the presence of certain nanocrystalline shapes relative
to other shapes (e.g., more decahedron shapes relative to other
shapes such as tetrahedrons, icosahedrons, octahedrons; or the
combination(s) of certain crystalline shapes relative to other
crystalline shapes, etc.). By controlling the presence or absence
(e.g., relative amounts) of such faces, crystal shapes (e.g.,
hexagonal plates, octahedrons, tetrahedrons and pentagonal
bipyramids (i.e., decahedrons)) and/or crystal sizes or extended
crystal planes which contain such faces, nanocrystal shapes, can
thus be relatively controlled. Control of the size and shape of
nanocrystals (as well as the surface properties of nanocrystals)
can control their function(s) in a variety of systems, including
biological systems.
[0455] Specifically, the presence of certain nanocrystalline shapes
(or shape distributions) containing specific spatially extended low
index crystal planes can cause different reactions (e.g., different
biocatalytic and/or biophysical reactions and/or cause different
biological signaling pathways to be active/inactive relative to the
absence of such shaped nanoparticles) and/or different reactions
selectively to occur under substantially identical conditions. One
crystalline shape of a gold nanoparticle (e.g., a pentagonal
by-pyramidal structure, or decahedron, or tetrahedron containing
{111} planes) can result in one set of reactions to occur (e.g.,
binding to a particular protein or homologue and/or affecting a
particular biological signaling pathway of a protein or a cytokine)
whereas a different crystal shape (e.g., a octahedron containing
the same or different crystal planes such as {111} or {100}) can
result in a different reaction endpoint (i.e., a different
biocatalytic or signaling pathway effect). More dramatically, the
lack of any extended crystal growth plane results in a
spherical-shaped nanoparticle (e.g., such as those made by
classical homogenous chemical reduction processes) significantly
affects the performance of the nanoparticle (e.g., relative to an
extended plane nanocrystal). Such differences in performance may be
due to differing surface plasmon resonances and/or intensity of
such resonances. Thus, by controlling amount (e.g., concentration),
nanocrystal sizes, the presence or absence of certain extended
growth crystal planes, and/or nanocrystalline shapes or shape
distribution(s), certain reactions (e.g., biological reactions
and/or biological signaling pathways) can be desirably influenced
and/or controlled. Such control can result in the prevention and/or
treatment of a variety of different diseases or indications that
are a function of certain biologic reactions and/or signaling
pathways (discussed later herein).
[0456] Further, certain processing enhancers may also include
materials that may function as charge carriers, but may themselves
not be ions. Specifically, metallic-based particles, either
introduced or formed in situ (e.g., heterogeneous or homogenous
nucleation/growth) by the electrochemical processing techniques
disclosed herein, can also function as charge carriers, crystal
nucleators and/or growth promoters, which may result in the
formation of a variety of different crystalline shapes (e.g.,
hexagonal plates, octahedrons, tetrahedrons, pentagonal bi-pyramids
(decahedrons), etc.). Once again, the presence of particular
particle crystal sizes, extended crystal planes and/or shapes or
shape distributions of such crystals, can desirably influence
certain reactions (e.g., binding to a particular protein or protein
homologue and/or affecting a particular biological signaling
pathway such as an inflammatory pathway or a proteasomal pathway)
to occur. Further, since the processing enhancers of the present
invention do not contemplate those traditional organic-based
molecules used in traditional reduction chemistry techniques, the
lack of such chemical reductant (or added surfactant) means that
the surfaces of the grown nanocrystals on the invention are very
"clean" relative to nanoparticles that are formed by traditional
reduction chemistry approaches. It should be understood that when
the term "clean" is used with regard to nanocrystal surfaces or
when the phrase "substantially free from organic impurities or
films" (or a similar phrase) is used, what is meant is that the
formed nanocrystals do not have chemical constituents adhered or
attached to their surfaces which (1) alter the functioning of the
nanocrystal and/or (2) form a layer, surface or film which covers a
significant portion (e.g., at least 25% of the crystal, or more
typically, at least 50% of the crystal). In preferred embodiments,
the nanocrystal surfaces are completely free of any organic
contaminants which materially change their functionality. It should
be further understood that incidental components that are caused to
adhere to nanocrystals of the invention and do not adversely or
materially affect the functioning of the inventive nanocrystals,
should still be considered to be within the metes and bounds of the
invention. One example of a nanocrystal surface that is completely
free from organic impurities or films is shown in Example 5
herein.
[0457] The lack of added chemicals (e.g., organics) permits the
growth of the gold atoms into the extended crystal planes resulting
in the novel crystalline shape distributions and also affects the
performance of the nanocrystals in vivo (e.g., affects the protein
corona formed around the nanoparticles/nanocrystals in, for
example, serum). For example, but without wishing to be bound by
any particular theory or explanation, protein corona formation can
control location of a nanoparticle/nanocrystal in vivo, as well as
control protein folding of proteins at or near the
nanoparticle/nanocrystal surfaces. Such differences in performance
may be due to such factors including, but not limited to, surface
charge, surface plasmon resonance, epitaxial effects, surface
double layers, zones of influence, and others.
[0458] Still further, once a seed crystal occurs in the process
and/or a set of extended crystal planes begins to grow (e.g.,
homogenous nucleation) or a seed crystal is separately provided
(e.g., heterogenous nucleation) the amount of time that a formed
particle (e.g., a metal atom) is permitted to dwell at or near one
or more electrodes in an electrochemical process can result in the
size of such nanocrystals increasing as a function of time (e.g.,
metal atoms can assemble into metal nanocrystals and, if unimpeded
by certain organic constituents in the liquid, they can grow into a
variety of shapes and sizes). The amount of time that crystal
nucleation/growth conditions are present can control the shape(s)
and sizes(s) of grown nanocrystals. Accordingly, dwell time
at/around electrodes, liquid flow rate(s), trough cross-sectional
shape(s), etc, all contribute to nanocrystal growth conditions, as
discussed elsewhere herein.
[0459] In a preferred embodiment the percent of pentagonal
bipyramids is at least about 5%, or is in a range of about 5%-35%,
and more typically at least about 10%, or is in a range of about
10%-35%, and even more typically, at least about 15%, or is in a
range of about 15%-35%, and still more typically, at least about
25%, and in some cases at least about 30%.
[0460] In another preferred embodiment the percent of tetrahedrons
is at least 5%, or is in a range of about 5%-35%, and more
typically at least about 10%, or is in a range of about 10%-35%,
and even more typically, at least about 15%, or is in a range of
about 15%-35%, and still more typically, at least about 25%, and in
some cases at least about 30%.
[0461] Still further, the combination of pentagonal bipyramids and
tetrahedrons is at least about 15%, or is in a range of about
15%-50%, and more typically at least about 20%, or is in a range of
about 20%-50%, and even more typically, at least about 30%, or is
in a range of about 30%-50%, and still more typically, at least
about 35%, and in some cases at least about 45%.
[0462] Still further, the combination of pentagonal bipyramids,
tetrahedrons, octahedrons and hexagonal is at least about 50%, or
is in a range of about 50%-85%, and more typically at least about
60%, or is in a range of about 60%-85%, and even more typically, at
least about 70%, or is in a range of about 70%-85%, and still more
typically, at least about 70%, and in some cases at least about
80%.
[0463] In many of the preferred embodiments herein, one or more AC
sources are utilized. The rate of change from "+" polarity on one
electrode to "-" polarity on the same electrode is known as Hertz,
Hz, frequency, or cycles per second. In the United States, the
standard output frequency is 60 Hz, while in Europe it is
predominantly 50 Hz. As shown in the Examples herein, the frequency
can also influence size and/or shape of nanocrystals formed
according to the electrochemical techniques disclosed herein.
Preferable frequencies are 5-1000 Hz, more typically, 20-500 Hz,
even more typically, 40-200 Hz, and even more typically, 50-100 Hz.
For example, and without wishing to be bound by any particular
theory or explanation, nucleated or growing crystals can first have
attractive forces exerted on them (or on crystal growth
constituents, such as ions or atoms, taking part in forming the
crystal(s)) due to, for example, unlike charges attracting and then
repulsive forces being exerted on such constituents (e.g., due to
like charges repelling). These factors also clearly play a large
role in nucleation and/or crystal growth of the novel nanocrystals
formed by affecting particle size and/or shapes; as well as
permitting the crystals to be formed without the need for
reductants or surfactants (i.e., that needed to be added to take
part in the prior art reduction chemistry techniques) causing the
nanocrystal surfaces to be free of such added chemical species. The
lack of organic-based coatings on the surface of grown nanocrystals
alters (and in some cases controls) their biological function.
[0464] Moreover, the particular waveform that is used for a
specific frequency also affects nanocrystal growth conditions, and
thus effects nanocrystal size(s) and/or shape(s). While the U.S.
uses a standard AC frequency of 60 Hz, it also uses a standard
waveform of a "sine" wave. As shown in the Examples herein,
changing the waveform from a sine wave to a square wave or a
triangular wave also affects nanocrystal crystallization conditions
and thus affects resultant nanocrystal size(s) and shape(s).
Preferred waveforms include sine waves, square waves and triangular
waves; however hybrid waveforms should be considered to be within
the metes and bounds of the invention.
[0465] Still further, the voltage applied in the novel
electrochemical techniques disclosed herein can also affect
nanocrystalline size(s) and shape(s). A preferred voltage range is
20-2000 Volts, a more preferred voltage range is 50-1000 Volts and
an even more preferred voltage range is 100-300 Volts. In addition
to voltage, the amperages used with these voltages typically are
0.1-10 Amps, a more preferred amperage range is 0.1-5 Amps and an
even more preferred amperage range is 0.4-1 Amps.
[0466] Still further, the "duty cycle" used for each waveform
applied in the novel electrochemical techniques disclosed herein
can also affect nanocrystalline size(s) and shape(s). In this
regard, without wishing to be bound by any particular theory or
explanation, the amount of time that an electrode is positively
biased can result in a first set of reactions, while a different
set of reactions can occur when the electrode is negatively biased.
By adjusting the amount of time that the electrodes are positively
or negatively biased, size(s) and/or shape(s) of grown nanocrystals
can be controlled. Further, the rate at which an electrode converts
to + or - is also a function of waveform shape and also influences
nanocrystal size(s) and/or shape(s).
[0467] Temperature can also play an important role. In some of the
preferred embodiments disclosed herein, the boiling point
temperature of the water is approached in at least a portion of the
processing vessel where gold nanocrystals are nucleated and grown.
For example, output water temperature in the continuous processing
Examples herein ranges from about 60.degree. C.-99.degree. C.
However, as discussed elsewhere herein, different temperature
ranges are also desirable. Temperature can influence resultant
product (e.g., size and/or shape of nanocrystals) as well as the
amount of resultant product (i.e., ppm level of nanocrystals in the
suspension or colloid). For example, while it is possible to cool
the liquid 3 in the trough member 30 by a variety of known
techniques (as disclosed in some of the Examples herein), many of
the Examples herein do not cool the liquid 3, resulting in
evaporation of a portion of the liquid 3 during processing
thereof.
[0468] FIG. 11a shows a perspective view of one embodiment of
substantially all of one trough member 30 shown in FIG. 10b
including an inlet portion or inlet end 31 and an outlet portion or
outlet end 32. The flow direction "F" discussed in other figures
herein corresponds to a liquid entering at or near the end 31
(e.g., utilizing an appropriate means for delivering fluid into the
trough member 30 at or near the inlet portion 31) and exiting the
trough member 30 through the end 32. FIG. 11b shows the trough
member 30 of FIG. 11a containing three control devices 20a, 20b and
20c removably attached to the trough member 30. The interaction and
operations of the control devices 20a, 20b and 20c containing the
electrodes 1 and/or 5 are discussed in greater detail later herein.
However, in a preferred embodiment of the invention, the control
devices 20, can be removably attached to a top portion of the
trough member 30 so that the control devices 20 are capable of
being positioned at different positions along the trough member 30,
thereby affecting certain processing parameters, constituents
produced (e.g., sizes and shapes of nanocrystals), reactivity of
constituents produced, as well as nanocrystal(s)/fluid(s) produced
therefrom.
[0469] FIG. 11c shows a perspective view of an atmosphere control
device cover 35'. The atmosphere control device or cover 35' has
attached thereto a plurality of control devices 20a, 20b and 20c
controllably attached to electrode(s) 1 and/or 5. The cover 35' is
intended to provide the ability to control the atmosphere within
and/or along a substantial portion of (e.g., greater than 50% of)
the longitudinal direction of the trough member 30, such that any
adjustable plasma(s) 4 created between any electrode(s) 1 and
surface 2 of the liquid 3 can be a function of the previously
discussed parameters of voltage, current, current density,
polarity, etc. (as discussed in more detail elsewhere herein) as
well as a controlled atmosphere (also discussed in more detail
elsewhere herein).
[0470] FIG. 11d shows the apparatus of FIG. 11c including an
additional support means 34 for supporting the trough member 30
(e.g., on an exterior portion thereof), as well as supporting (at
least partially) the control devices 20 (not shown in FIG. 11d). It
should be understood by the reader that various details can be
changed regarding, for example, the cross-sectional shapes shown
for the trough member 30, atmosphere control(s) (e.g., the cover
35') and external support means (e.g., the support means 34) which
are within the metes and bounds of this disclosure, some of which
are discussed in greater detail later herein.
[0471] FIG. 11e shows an alternative configuration for the trough
member 30. Specifically, the trough member 30 is shown in
perspective view and is "Y-shaped". Specifically, the trough member
30 comprises top portions 30a and 30b and a bottom portion 30o.
Likewise, inlets 31a and 31b are provided along with an outlet 32.
A portion 30d corresponds to the point where 30a and 30b meet
30o.
[0472] FIG. 11f shows the same "Y-shaped" trough member shown in
FIG. 11e, except that the portion 30d of FIG. 11e is now shown as a
more definite mixing section 30d'. In this regard, certain
constituents manufactured or produced in the liquid 3 in one or all
of, for example, the portions 30a, 30b and/or 30c, may be desirable
to be mixed together at the point 30d (or 30d'). Such mixing may
occur naturally at the intersection 30d shown in FIG. 11e (i.e., no
specific or special section 30d' may be needed), or may be more
specifically controlled at the portion 30d'. It should be
understood that the portion 30d' could be shaped into any effective
shape, such as square, circular, rectangular, etc., and be of the
same or different depth relative to other portions of the trough
member 30. In this regard, the area 30d could be a mixing zone or
subsequent reaction zone or a zone where a processing enhancer may
be added. More details of the interactions 30d and 30d' are
discussed later herein.
[0473] FIGS. 11g and 11h show a ".PSI.-shaped" trough member 30.
Specifically, a new portion 30c has been added. Other features of
FIGS. 11g and 11h are similar to those features shown in 11e and
11f.
[0474] It should be understood that a variety of different shapes
and/or cross-sections can exist for the trough member 30, any one
of which can produce desirable results as a function of a variety
of design and production considerations. For example, one or more
constituents produced in the portion(s) 30a, 30b and/or 30c could
be transient (e.g., a seed crystal or nucleation point) and/or
semi-permanent (e.g., grown nanocrystals present in a colloid). If
such constituent(s) produced, for example, in portion 30a is to be
desirably and controllably reacted with one or more constituents
produced in, for example, portion 30b, then a final product (e.g.,
properties of a final product) which results from such mixing could
be a function of when constituents formed in the portions 30a and
30b are mixed together. Also, the temperature of liquids entering
the section 30d (or 30d') can be monitored/controlled to maximize
certain desirable processing conditions and/or desirable properties
of final products and/or minimize certain undesirable products.
Still further, processing enhancers may be selectively utilized in
one or more of the portions 30a, 30b, 30c, 30d (30d') and/or 30o
(or at any selected point or portion in the trough member 30).
[0475] FIG. 12a shows a perspective view of a local atmosphere
control apparatus 35 which functions as a means for controlling a
local atmosphere around the electrode sets 1 and/or 5 so that
various localized gases can be utilized to, for example, control
and/or effect certain components in the adjustable plasma 4 between
electrode 1 and surface 2 of the liquid 3, as well as influence
adjustable electrochemical reactions at and/or around the
electrode(s) 5. The through-holes 36 and 37 shown in the atmosphere
control apparatus 35 are provided to permit external communication
in and through a portion of the apparatus 35. In particular, the
hole or inlet 37 is provided as an inlet connection for any gaseous
species to be introduced to the inside of the apparatus 35. The
hole 36 is provided as a communication port for the electrodes 1
and/or 5 extending therethrough which electrodes are connected to,
for example, the control device 20 located above the apparatus 35.
Gasses introduced through the inlet 37 can simply be provided at a
positive pressure relative to the local external atmosphere and may
be allowed to escape by any suitable means or pathway including,
but not limited to, bubbling out around the portions 39a and/or 39b
of the apparatus 35, when such portions are caused, for example, to
be at least partially submerged beneath the surface 2 of the liquid
3. Alternatively, a second hole or outlet (not shown) can be
provided elsewhere in the atmosphere control apparatus 35.
Generally, the portions 39a and 39b can break the surface 2 of the
liquid 3 effectively causing the surface 2 to act as part of the
seal to form a localized atmosphere around electrode sets 1 and/or
5. When a positive pressure of a desired gas enters through the
inlet port 37, small bubbles can be caused to bubble past, for
example, the portions 39a and/or 39b. Alternatively, gas may exit
through an appropriate outlet in the atmosphere control apparatus
35, such as through the hole 36.
[0476] FIG. 12b shows a perspective view of first atmosphere
control apparatus 35a in the foreground of the trough member 30
contained within the support housing 34. A second atmosphere
control apparatus 35b is included and shows a control device 20
located thereon. "F" denotes the longitudinal direction of flow of
liquid through the trough member 30. IF desired, locally controlled
atmosphere(s) (e.g., of substantially the same chemical
constituents, such as air or nitrogen, or substantially different
chemical constituents, such as helium and nitrogen) around
different electrode sets 1 and/or 5 can be achieved.
[0477] FIG. 13 shows a perspective view of an alternative
atmosphere control apparatus 38 wherein the entire trough member 30
and support means 34 are contained within the atmosphere control
apparatus 38. In this case, for example, gas inlet 37 (37') can be
provided along with a gas outlet(s) 37a (37a'). The exact
positioning of the gas inlet(s) 37 (37') and gas outlet(s) 37a
(37a') on the atmosphere control apparatus 38 is a matter of
convenience, as well as a matter of the composition of the
atmosphere contained therein. In this regard, if the gas is heavier
than air or lighter than air, inlet and outlet locations can be
adjusted accordingly. Aspects of these factors are discussed in
greater detail later herein.
[0478] FIG. 14 shows a schematic view of the general apparatus
utilized in accordance with the teachings of some of the preferred
embodiments of the present invention. In particular, this FIG. 14
shows a side schematic view of the trough member 30 containing a
liquid 3 therein. On the top of the trough member 30 rests a
plurality of control devices 20a-20d which are, in this embodiment,
removably attached thereto. The control devices 20a-20d may of
course be permanently fixed in position when practicing various
embodiments of the invention. The precise number of control devices
20 (and corresponding electrode(s) 1 and/or 5 as well as the
configuration(s) of such electrodes) and the positioning or
location of the control devices 20 (and corresponding electrodes 1
and/or 5) are a function of various preferred embodiments of the
invention discussed in greater detail elsewhere herein. However, in
general, an input liquid 3 (for example water or purified water) is
provided to a liquid transport means 40 (e.g., a liquid pump,
gravity or liquid pumping means for pumping the liquid 3) such as a
peristaltic pump 40 for pumping the liquid 3 into the trough member
30 at a first-end 31 thereof. Exactly how the liquid 3 is
introduced is discussed in greater detail elsewhere herein. The
liquid transport means 40 may include any means for moving liquids
3 including, but not limited to a gravity-fed or hydrostatic means,
a pumping means, a regulating or valve means, etc. However, the
liquid transport means 40 should be capable of reliably and/or
controllably introducing known amounts of the liquid 3 into the
trough member 30. The amount of time that the liquid 3 is contained
within the trough member 30 (e.g., at or around one or more
electrode(s) 1/5) also influences the products produced (e.g., the
sizes(s) and/or shapes(s) of the grown nanocrystals).
[0479] Once the liquid 3 is provided into the trough member 30,
means for continually moving the liquid 3 within the trough member
30 may or may not be required. However, a simple means for
continually moving the liquid 3 includes the trough member 30 being
situated on a slight angle .theta. (e.g., less than a degree to a
few degrees for a low viscosity fluid 3 such as water) relative to
the support surface upon which the trough member 30 is located. For
example, a difference in vertical height of less than one inch
between an inlet portion 31 and an outlet portion 32, spaced apart
by about 6 feet (about 1.8 meters) relative to the support surface
may be all that is required, so long as the viscosity of the liquid
3 is not too high (e.g., any viscosity around the viscosity of
water can be controlled by gravity flow once such fluids are
contained or located within the trough member 30). In this regard,
FIGS. 15a and 15b show two acceptable angles .theta..sub.1 and
.theta..sub.2, respectively, for trough member 30 that can process
various viscosities, including low viscosity fluids such as water.
The need for a greater angle .theta. could be a result of
processing a liquid 3 having a viscosity higher than water; the
need for the liquid 3 to transit the trough 30 at a faster rate,
etc. Further, when viscosities of the liquid 3 increase such that
gravity alone is insufficient, other phenomena such as specific
uses of hydrostatic head pressure or hydrostatic pressure can also
be utilized to achieve desirable fluid flow. Further, additional
means for moving the liquid 3 along the trough member 30 could also
be provided inside the trough member 30. Such means for moving the
fluid include mechanical means such as paddles, fans, propellers,
augers, etc., acoustic means such as transducers, thermal means
such as heaters and/or chillers (which may have additional
processing benefits), etc., are also desirable for use with the
present invention.
[0480] FIG. 14 also shows a storage tank or storage vessel 41 at
the end 32 of the trough member 30. Such storage vessel 41 can be
any acceptable vessel and/or pumping means made of one or more
materials which, for example, do not negatively interact with the
liquid 3 (or constituents contained therein) produced within the
trough member 30. Acceptable materials include, but are not limited
to, plastics such as high-density polyethylene (HDPE), glass,
metal(s) (such a certain grades of stainless steel), etc. Moreover,
while a storage tank 41 is shown in this embodiment, the tank 41
should be understood as including a means for distributing or
directly bottling or packaging the fluid 3 processed in the trough
member 30.
[0481] FIGS. 16a, 16b and 16c show a perspective view of one
preferred embodiment of the invention. In these FIGS. 16a, 16b and
16c, eight separate control devices 20a-h are shown in more detail.
Such control devices 20 can utilize one or more of the electrode
configurations shown in, for example, FIGS. 8a, 8b, 8c and 8d. The
precise positioning and operation of the control devices 20 (and
the corresponding electrodes 1 and/or 5) are discussed in greater
detail elsewhere herein. FIG. 16b includes use of two air
distributing or air handling devices (e.g., fans 342a and 342b).
These air handling devices can assist in removing, for example,
humid air produced around the electrodes 1/5. Specifically, in some
cases certain amounts of humidity are desirable, but in other
cases, excessive localized humidity could be undesirable.
Similarly, FIG. 16c includes the use of two alternative air
distributing or air handling devices 342c and 342d.
[0482] The electrode control devices shown generally in, for
example, FIGS. 2, 3, 14 and 16 are shown in greater detail in FIGS.
17d, 17e, 17f, 17m and 17n. In particular, these FIGS. 17d, 17e,
17f, 17m and 17n show a perspective view of various embodiments of
the inventive control devices 20.
[0483] First, specific reference is made to FIGS. 17d, 17e and 17f.
In each of these three Figures, a base portion 25 is provided, said
base portion having a top portion 25' and a bottom portion 25''.
The base portion 25 is made of a suitable rigid plastic material
including, but not limited to, materials made from structural
plastics, resins, polyurethane, polypropylene, nylon, Teflon,
polyvinyl, etc. A dividing wall 27 is provided between two
electrode adjustment assemblies. The dividing wall 27 can be made
of similar or different material from that material comprising the
base portion 25. Two servo-step motors 21a and 21b are fixed to the
surface 25' of the base portion 25. The step motors 21a, 21b could
be any step motor capable of slightly moving (e.g., on a 360-degree
basis, slightly less than or slightly more than 1 degree) such that
a circumferential movement of the step motors 21a/21b results in a
vertical raising or lowering of an electrode 1 or 5 communicating
therewith. In this regard, a first wheel-shaped component 23a is
the drive wheel connected to the output shaft 231a of the drive
motor 21a such that when the drive shaft 231a rotates,
circumferential movement of the wheel 23a is created. Further, a
slave wheel 24a is caused to press against and toward the drive
wheel 23a such that frictional contact exists therebetween. The
drive wheel 23a and/or slave wheel 24a may include a notch or
groove on an outer portion thereof to assist in accommodating the
electrodes 1,5. The slave wheel 24a is caused to be pressed toward
the drive wheel 23a by a spring 285 located between the portions
241a and 261a attached to the slave wheel 24a. In particular, a
coiled spring 285 can be located around the portion of the axis
262a that extends out from the block 261a. Springs should be of
sufficient tension so as to result in a reasonable frictional force
between the drive wheel 24a and the slave wheel 24a such that when
the shaft 231a rotates a determined amount, the electrode
assemblies 5a, 5b, 1a, 1b, etc., will move in a vertical direction
relative to the base portion 25. Such rotational or circumferential
movement of the drive wheel 23a results in a direct transfer of
vertical directional changes in the electrodes 1,5 shown herein. At
least a portion of the drive wheel 23a should be made from an
electrically insulating material; whereas the slave wheel 24a can
be made from an electrically conductive material or an electrically
insulating material, but typically, an electrically insulating
material.
[0484] The drive motors 21a/21b can be any suitable drive motor
which is capable of small rotations (e.g., slightly below
1.degree./360.degree. or slightly above 1.degree./360.degree.) such
that small rotational changes in the drive shaft 231a are
translated into small vertical changes in the electrode assemblies.
A preferred drive motor includes a drive motor manufactured by RMS
Technologies model 1MC17-S04 step motor, which is a DC-powered step
motor. This step motors 21a/21b include an RS-232 connection
22a/22b, respectively, which permits the step motors to be driven
by a remote-control apparatus such as a computer or a
controller.
[0485] The portions 271, 272 and 273 are primarily height
adjustments which adjust the height of the base portion 25 relative
to the trough member 30. The portions 271, 272 and 273 can be made
of same, similar or different materials from the base portion 25.
The portions 274a/274b and 275a/275b can also be made of the same,
similar or different material from the base portion 25. However,
these portions should be electrically insulating in that they house
various wire components associated with delivering voltage and
current to the electrode assemblies 1a/1b, 5a/5b, etc.
[0486] The electrode assembly specifically shown in FIG. 17d
comprises electrodes 5a and 5b (corresponding to, for example, the
electrode assembly shown in FIG. 3c). However, that electrode
assembly could comprise electrode(s) 1 only, electrode(s) 1 and 5,
electrode(s) 5 and 1, or electrode(s) 5 only. In this regard, FIG.
17e shows an assembly where two electrodes 1a/5a are provided
instead of the two electrode(s) 5a/5b shown in FIG. 17d. All other
elements shown in FIG. 17e are similar to those shown in FIG.
17d.
[0487] With regard to the size of the control device 20 shown in
FIGS. 17d, 17e and 17f, the dimensions "L" and "W" can be any
dimension which accommodates the size of the step motors 21a/21b,
and the width of the trough member 30. In this regard, the
dimension "L" shown in FIG. 17f needs to be sufficient such that
the dimension "L" is at least as long as the trough member 30 is
wide, and typically slightly longer (e.g., 10-30%). The dimension
"W" shown in FIG. 17f needs to be wide enough to house the step
motors 21a/21b and not be so wide as to unnecessarily underutilize
longitudinal space along the length of the trough member 30. In one
preferred embodiment of the invention, the dimension "L" is about 7
inches (about 19 millimeters) and the dimension "W" is about 4
inches (about 10.5 millimeters). The thickness "H" of the base
member 25 is any thickness sufficient which provides structural,
electrical and mechanical rigidity for the base member 25 and
should be of the order of about 1/4''-3/4'' (about 6 mm-19 mm).
While these dimensions are not critical, the dimensions give an
understanding of size generally of certain components of one
preferred embodiment of the invention.
[0488] Further, in each of the embodiments of the invention shown
in FIGS. 17d, 17e and 17f, the base member 25 (and the components
mounted thereto), can be covered by a suitable cover 290 (shown in
FIG. 17f) to insulate electrically, as well as creating a local
protective environment for all of the components attached to the
base member 25. Such cover 290 can be made of any suitable material
which provides appropriate safety and operational flexibility.
Exemplary materials include plastics similar to that used for other
portions of the trough member 30 and/or the control device 20 and
is typically transparent. This cover member 290 can also be made of
the same type of materials used to make the base portion 25. The
cover 290 is also shown as having 2 through-holes 291 and 292
therein. Specifically, these through-holes can, for example, be
aligned with excess portions of, for example, electrodes 5, which
can be connected to, for example, a spool of electrode wire (not
shown in these drawings).
[0489] FIGS. 17m and 17n show an alternative configuration for the
control device 20. In these devices, similarly numbered components
are essentially the same as those components shown in FIGS. 17d,
17e and 17f. The primary differences between the control devices 20
shown in FIGS. 17m and 17n is that while a similar master or
drive-pulley 23a is provided, rather than providing a slave wheel
24a or 241 as shown in the embodiments of FIGS. 17d, 17e and 17f, a
resilient electrical contact device 242 is provided as shown in
FIG. 17m and as 242a/242b in FIG. 17n. In this regard, the portions
242, 242a and 242b provide resilient tension for the wire 5a or 5b
to be provided therebetween. Additionally, this control device
design causes there to be an electrical connection between the
power sources 50/60 and the electrodes 1/5. The servo-motor 21a
functions as discussed above, but a single electrode (FIG. 17m) or
two electrodes (FIG. 17n) are driven by a single servo drive motor
21a. Accordingly, a single drive motor 21a can replace two drive
motors in the case of the embodiment shown in FIG. 17n. Further, by
providing the electrical contact between the wires 1/5 and the
power sources 50/60, all electrical connections are provided on a
top surface of (i.e., the surface further away from the liquid 3,
resulting in certain design and production advantages.
[0490] FIGS. 17d and 17e show a refractory material component 29.
The component 29 is made of, for example, suitable refractory
component, including, for example, aluminum oxide or the like. The
refractory component 29 may have a transverse through-hole therein
which provides for electrical connections to the electrode(s) 1
and/or 5. Further a longitudinal through-hole is present along the
length of the refractory component 29 such that electrode
assemblies 1/5 can extend therethrough.
[0491] FIG. 17e shows a perspective view of the bottom portion of
the control device 20. In this FIG. 17e, one electrode(s) 1a is
shown as extending through a first refractory portion 29a and one
electrode(s) 5a is shown as extending through a second refractory
portion 29b. Accordingly, each of the electrode assemblies
expressly disclosed herein, as well as those referred to herein,
can be utilized in combination with the preferred embodiments of
the control device shown herein.
[0492] In order for the control devices 20 to be actuated, two
general processes need to occur. A first process involves
electrically activating the electrode(s) 1 and/or 5 (e.g., applying
power thereto from a preferred power source 10), and the second
general process occurrence involves determining, for example, how
much power is applied to the electrode(s) and appropriately
adjusting electrode 1/5 height in response to such determinations
(e.g., manually and/or automatically adjusting the height of the
electrodes 1/5); or adjusting the electrode height or simply moving
the electrode into (e.g., progressively advancing the electrode(s)
5 through the liquid 3) or out of contact with the liquid 3, as a
function of time. In the case of utilizing a control device 20,
suitable instructions are communicated to the step motor 21 through
the RS-232 ports 22a and 22b. Important embodiments of components
of the control device 20, as well as the electrode activation
process, are discussed herein.
[0493] A preferred embodiment of the invention utilizes the
automatic control devices 20 shown in various figures herein. The
step motors 21a and 21b shown in, for example, FIGS. 17d-17f, and
17m-17n are controlled either by the electrical circuit diagrammed
in each of FIGS. 17g-17j (e.g., for electrode sets 1/5 that make a
plasma 4 or for electrode sets 5/5); or are controlled by the
electrical circuit diagrammed in each of FIGS. 17k and 17l for
electrode sets 5/5, in some embodiments herein.
[0494] In particular, in this embodiment, the electrical circuit of
FIG. 17j is a voltage monitoring circuit. Specifically, voltage
output from each of the output legs of the secondary coil 603 in
the transformer 60 are monitored over the points "P-Q" and the
points "P'-Q'". Specifically, the resistor denoted by "RL"
corresponds to the internal resistance of the multi-meter measuring
device (not shown). The output voltages measured between the points
"P-Q" and "P'-Q'" typically, for several preferred embodiments
shown in the Examples later herein, range between about 200 volts
and about 4,500 volts. However, higher and lower voltages can work
with many of the embodiments disclosed herein. In Examples 1-4
later herein, desirable target voltages have been determined for
each electrode set 1 and/or 5 at each position along a trough
member 30. Such desirable target voltages are achieved as actual
applied voltages by, utilizing, for example, the circuit control
shown in FIGS. 17g, 17h and 17i. These FIGS. 17g and 17h refer to
sets of relays controlled by a Velleman K8056 circuit assembly
(having a micro-chip PIC16F630-I/P). In particular, a voltage is
detected across either the "P-Q" or the "P'-Q'" locations and such
voltage is compared to a predetermined reference voltage (actually
compared to a target voltage range). If a measured voltage across,
for example, the points "P-Q" is approaching a high-end of a
pre-determined voltage target range, then, for example, the
Velleman K8056 circuit assembly causes a servo-motor 21 (with
specific reference to FIG. 17f) to rotate in a clockwise direction
so as to lower the electrode 5a toward and/or into the fluid 3. In
contrast, should a measured voltage across either of the points
"P-Q" or "P'-Q'" be approaching a lower end of a target voltage,
then, for example, again with reference to FIG. 17f, the server
motor 21a will cause the drive-wheel 23a to rotate in a
counter-clockwise position thereby raising the electrode 5a
relative to the fluid 3.
[0495] Each set of electrodes in Examples 1-4 of the invention has
an established target voltage range. The size or magnitude of
acceptable range varies by an amount between about 1% and about
10%-15% of the target voltage. Some embodiments of the invention
are more sensitive to voltage changes and these embodiments should
have, typically, smaller acceptable voltage ranges; whereas other
embodiments of the invention are less sensitive to voltage and
should have, typically, larger acceptable ranges. Accordingly, by
utilizing the circuit diagram shown in FIG. 17j, actual voltages
output from the secondary coil 603 of the transformer 60 are
measured at "RL" (across the terminals "P-Q" and "P'-Q'"), and are
then compared to the predetermined voltage ranges. The servo-motor
21 responds by rotating a predetermined amount in either a
clockwise direction or a counter-clockwise direction, as needed.
Moreover, with specific reference to FIGS. 17g-17j, it should be
noted that an interrogation procedure occurs sequentially by
determining the voltage of each electrode, adjusting height (if
needed) and then proceeding to the next electrode. In other words,
each transformer 60 is connected electrically in a manner shown in
FIG. 17j. Each transformer 60 and associated measuring points "P-Q"
and "P'-Q'" are connected to an individual relay. For example, the
points "P-Q" correspond to relay number 501 in FIG. 17g and the
points "P'-Q'" correspond to the relay 502 in FIG. 17g.
Accordingly, two relays are required for each transformer 60. Each
relay, 501, 502, etc., sequentially interrogates a first output
voltage from a first leg of a secondary coil 603 and then a second
output voltage from a second leg of the secondary coil 603; and
such interrogation continues onto a first output voltage from a
second transformer 60b on a first leg of its secondary coil 603,
and then on to a second leg of the secondary coil 603, and so
on.
[0496] The computer or logic control for the disclosed
interrogation voltage adjustment techniques are achieved by any
conventional program or controller, including, for example, in a
preferred embodiment, standard visual basic programming steps
utilized in a PC. Such programming steps include interrogating,
reading, comparing, and sending an appropriate actuation symbol to
increase or decrease voltage (e.g., raise or lower an electrode
relative to the surface 2 of the liquid 3). Such techniques should
be understood by an artisan of ordinary skill.
[0497] Further, in another preferred embodiment of the invention
utilized in Example 16 for the electrode sets 5/5', the automatic
control devices 20 are controlled by the electrical circuits of
FIGS. 17h, 17i, 17k and 17l. In particular, the electrical circuit
of FIG. 17l is a voltage monitoring circuit used to measure
current. In this case, voltage and current are the same numerical
value due to choice of a resistor (discussed later herein).
Specifically, voltage output from each of the transformers 50 are
monitored over the points "P-Q" and the points "P'-Q'".
Specifically, the resistor denoted by "RL" corresponds to the
internal resistance of the multi-meter measuring device (not
shown). The output voltages measured between the points "P-Q" and
"P'-Q'" typically, for several preferred embodiments shown in the
Examples later herein, range between about 0.05 volts and about 5
volts. However, higher and lower voltages can work with many of the
embodiments disclosed herein. Desirable target voltages have been
determined for each electrode set 5/5' at each position along a
trough member 30b'. Such desirable target voltages are achieved as
actual applied voltages by, utilizing, for example, the circuit
control shown in FIGS. 17h, 17i, 17k and 17l. These FIG. 17 refer
to sets of relays controlled by a Velleman K8056 circuit assembly
(having a micro-chip PIC16F630-I/P).
[0498] In particular, in the Example 16 embodiments the servo-motor
21 is caused to rotate at a specific predetermined time in order to
maintain a desirable electrode 5 profile. The servo-motor 21
responds by rotating a predetermined amount in a clockwise
direction. Specifically the servo-motor 21 rotates a sufficient
amount such that about 0.009 inches (0.229 mm) of the electrode 5
is advanced toward and into the female receiver portion o5 (shown,
for example in some of FIGS. 20 and 21). Thus, the electrode 5 is
progressively advanced through the liquid 3. In one preferred
embodiment discussed herein, such electrode 5 movement occurs about
every 5.8 minutes. Accordingly, the rate of vertical movement of
each electrode 5 into the female receiver portion o5 is about 3/4
inches (about 1.9 cm) every 8 hours. Accordingly, a substantially
constant electrode 5 shape or profile is maintained by its constant
or progressive advance into and through the liquid 3. Further, once
the advancing end of the electrode 5 reaches the longitudinal end
of the female receiver portion o5, the electrode 5 can be removed
from the processing apparatus. Alternatively, an electrode
collecting means for collecting the "used" portion of the electrode
can be provided. Such means for collecting the electrode(s) 5
include, but are not limited to, a winding or spooling device, and
extended portion o5, a wire clipping or cutting device, etc.
However, in order to achieve different current/voltage profiles
(and thus a variety of different nanocrystal size(s) and/or
shapes(s), other rates of electrode movement are also within the
metes and bounds of this invention.
[0499] Moreover, with specific reference to FIGS. 17h, 17i, 17k and
17l, it should be noted that an interrogation procedure occurs
sequentially by determining the voltage of each electrode, which in
the embodiments of Example 16, are equivalent to the amps because
in FIG. 17l the resistors Ra and Rb are approximately 1 ohm,
accordingly, V=I. In other words, each transformer 50 is connected
electrically in a manner shown in 17h, 17i, 17k and 17l. Each
transformer 50 and associated measuring points "P-Q" and "P'-Q'"
are connected to two individual relays. For example, the points
"P-Q" correspond to relay number 501 and 501' in FIG. 17k and the
points "P'-Q'" correspond to the relay 502, 502' in FIG. 17k.
Accordingly, relays are required for each electrode set 5/5. Each
relay, 501/501' and 502/502', etc., sequentially interrogates the
output voltage from the transformer 50 and then a second voltage
from the same transformer 50, and so on.
[0500] The computer or logic control for the disclosed electrode
height adjustment techniques are achieved by any conventional
program or controller, including, for example, in a preferred
embodiment, standard visual basic programming steps utilized in a
PC. Such programming steps include reading and sending an
appropriate actuation symbol to lower an electrode relative to the
surface 2 of the liquid 3. Such techniques should be understood by
an artisan of ordinary skill.
Definitions
[0501] For purposes of the present invention, the terms and
expressions below, appearing in the Specification and Claims, are
intended to have the following meanings:
[0502] "Carbomer", as used herein in Example 23, means a class of
synthetically derived cross-linked polyacrylic acid polymers that
provide efficient rheology modification with enhanced self-wetting
for ease of use. In general, a carbomer/solvent mixture is
neutralized with a base such as triethanolamine or sodium hydroxide
to fully open the polymer to achieve the desired thickening,
suspending, and emulsion stabilization properties to make creams or
gels.
[0503] "Substantially clean", as used herein should be understood
when used to describe nanocrystal surfaces means that the
nanocrystals do not have chemical constituents adhered or attached
to their surfaces in such an amount that would materially alter the
functioning of the nanocrystal in at least one of its significant
properties of the gold nanocrystals set forth in the Examples
herein. Alternatively, the gold nanocrystal does not have a layer,
surface or film which covers a significant portion (e.g., at least
25% of the crystal, or in another embodiment at least 50% of the
crystal). It also can mean that the nanocrystal surfaces are
completely free of any organic contaminants which materially change
their functionality over bare gold crystal surfaces. It should be
understood that incidental components that are caused to adhere to
nanocrystals of the invention and do not adversely or materially
affect the functioning of the inventive nanocrystals, should still
be considered to be within the metes and bounds of the invention.
The term should also be understood to be a relative term
referencing the lack of traditional organic-based molecules (i.e.,
those used in traditional reduction chemistry techniques) on the
surfaces of the grown nanocrystals of the invention.
[0504] A "diagnostic effective amount", as used herein, means an
amount sufficient to bind to MIF to enable detection of the
MIF-compound complex such that diagnosis of a disease or condition
is possible.
[0505] An "effective amount", as used herein, means a certain
amount of solution or compound which, when administered according
to, for example, a desired dosing regimen, provides the desired MIF
cytokine inhibiting or treatment or therapeutic activity, or
disease/condition prevention or MIF signaling pathway(s). Dosing
may occur at intervals of minutes, hours, days, weeks, months or
years or continuously over any one of these periods.
[0506] As used herein, "immune privilege" refers to an area or site
within a living system (e.g., a body) which tolerates the presence
of an antigen that would normally elicit a response from the immune
system (e.g., an inflammatory immune response).
[0507] The term "operably coating" a stent means coating a stent in
a way that permits the timely release of the inventive
metallic-based nanocrystals (e.g., comprising aqueous gold-based
metal and/or mixtures of gold and other metal(s) and/or alloys of
gold with other metal(s)) into the surrounding tissue to be treated
once the coated stent is administered.
[0508] As used herein, the term "processing-enhancer" or
"processing-enhanced" or "process enhancer" means at least one
material (e.g., solid, liquid and/or gas) and typically means an
inorganic material, which material does not significantly bind to
the formed nanocrystals, but rather facilitates nucleation/growth
during an electrochemical-stimulated growth process. The material
serves important roles in the process including providing charged
ions in the electrochemical solution to permit the crystals to be
grown. The process enhancer is critically a compound(s) which
remains in solution, and/or does not form a coating (in one
embodiment an organic coating), and/or does not adversely affect
the formed nanocrystals or the formed suspension(s), and/or is
destroyed, evaporated, or is otherwise lost during the
electrochemical crystal growth process.
[0509] The term "Steroid-sparing", as used herein, means providing
a material other than a steroid in a combination therapy which
reduces the amount of steroid required to be effective for
treating/preventing an indication.
[0510] The phrase "trough member" as used herein should be
understood as meaning a large variety of fluid handling devices
including, pipes, half pipes, channels or grooves existing in
materials or objects, conduits, ducts, tubes, chutes, hoses and/or
spouts, so long as such are compatible with the electrochemical
processes disclosed herein.
[0511] The following Examples serve to illustrate certain
embodiments of the invention but should not to be construed as
limiting the scope of the disclosure as defined in the appended
claims.
Examples 1-4
Manufacturing Gold-Based Nanoparticles/Nanoparticle Solutions
GT032, GT031, GT019 and GT033
[0512] In general, each of Examples 1-4 utilizes certain
embodiments of the invention associated with the apparatuses
generally shown in FIGS. 16b, 16c and 16g. Specific differences in
processing and apparatus will be apparent in each Example. The
trough member 30 was made from plexiglass, all of which had a
thickness of about 3 mm-4 mm (about 1/8''). The support structure
34 was also made from plexiglass which was about 1/4'' thick (about
6-7 mm thick). The cross-sectional shape of the trough member 30
corresponds to that shape shown in FIG. 10b (i.e., a truncated
"V"). The base portion "R" of the truncated "V" measured about
0.5'' (about lcm), and each side portion "S", "S'" measured about
1.5'' (about 3.75 cm). The distance "M" separating the side
portions "S", "S'" of the V-shaped trough member 30 was about
21/4-2 5/16'' (about 5.9 cm) (measured from inside to inside). The
thickness of each portion also measured about 1/8'' (about 3 mm)
thick. The longitudinal length "L.sub.T" (refer to FIG. 11a) of the
V-shaped trough member 30 measured about 6 feet (about 2 meters)
long from point 31 to point 32. The difference in vertical height
from the end 31 of the trough member 30 to the end 32 was about
1/4-1/2'' (about 6-12.7 mm) over its 6 feet length (about 2 meters)
(i.e., less than 1.degree.).
[0513] Purified water (discussed later herein) was used as the
input liquid 3 in Example 1. In Examples 2-4, a processing enhancer
was added to the liquid 3 being input into the trough member 30.
The specific processing enhancer added, as well as the specific
amounts of the same, were effective in these examples. However,
other processing enhancer(s) and amounts of same, should be viewed
as being within the metes and bounds of this disclosure and these
specific examples should not be viewed as limiting the scope of the
invention. The depth "d" (refer to FIG. 10b) of the water 3 in the
V-shaped trough member 30 was about 7/16'' to about 1/2'' (about 11
mm to about 13 mm) at various points along the trough member 30.
The depth "d" was partially controlled through use of the dam 80
(shown in FIGS. 15a and 15b). Specifically, the dam 80 was provided
near the end 32 and assisted in creating the depth "d" (shown in
FIG. 10b) to be about 7/6''-1/2'' (about 11-13 mm) in depth. The
height "j" of the dam 80 measured about 1/4'' (about 6 mm) and the
longitudinal length "k" measured about 1/2'' (about 13 mm). The
width (not shown) was completely across the bottom dimension "R" of
the trough member 30. Accordingly, the total volume of water 3 in
the V-shaped trough member 30 during operation thereof was about 26
in.sup.3 (about 430 ml).
[0514] The rate of flow of the water 3 into the trough member 30
was about 90 ml/minute. Due to some evaporation within the trough
member 30, the flow out of the trough member 30 was slightly less,
about 60-70 ml/minute. Such flow of water 3 into the trough member
30 was obtained by utilizing a Masterflex.RTM. L/S pump drive 40
rated at 0.1 horsepower, 10-600 rpm. The model number of the
Masterflex.RTM. pump 40 was 77300-40. The pump drive had a pump
head also made by Masterflex.RTM. known as Easy-Load Model No.
7518-10. In general terms, the head for the pump 40 is known as a
peristaltic head. The pump 40 and head were controlled by a
Masterflex.RTM. LS Digital Modular Drive. The model number for the
Digital Modular Drive is 77300-80. The precise settings on the
Digital Modular Drive were, for example, 90 milliliters per minute.
Tygon.RTM. tubing having a diameter of 1/4'' (i.e., size 06419-25)
was placed into the peristaltic head. The tubing was made by Saint
Gobain for Masterflex.RTM.. One end of the tubing was delivered to
a first end 31 of the trough member 30 by a flow diffusion means
located therein. The flow diffusion means tended to minimize
disturbance and bubbles in water 3 introduced into the trough
member 30 as well as any pulsing condition generated by the
peristaltic pump 40. In this regard, a small reservoir served as
the diffusion means and was provided at a point vertically above
the end 31 of the trough member 30 such that when the reservoir
overflowed, a relatively steady flow of water 3 into the end 31 of
the V-shaped trough member 30 occurred.
[0515] With regard to FIGS. 16b and 16c, 8 separate electrode sets
(Set 1, Set 2, Set 3, -Set 8) were attached to 8 separate control
devices 20. Each of Tables 1a-1d refers to each of the 8 electrode
sets by "Set #". Further, within any Set #, electrodes 1 and 5,
similar to the electrode assemblies shown in FIGS. 3a and 3c were
utilized. Each electrode of the 8 electrode sets was set to operate
within specific target voltage range. Actual target voltages are
listed in each of Tables 1a-1d. The distance "c-c" (with reference
to FIG. 14) from the centerline of each electrode set to the
adjacent electrode set is also represented. Further, the distance
"x" associated with any electrode(s) 1 utilized is also reported.
For any electrode 5's, no distance "x" is reported. Other relevant
distances are reported, for example, in each of Tables 1a-1d.
[0516] The power source for each electrode set was an AC
transformer 60. Specifically, FIG. 16d shows a source of AC power
62 connected to a transformer 60. In addition, a capacitor 61 is
provided so that, for example, loss factors in the circuit can be
adjusted. The output of the transformer 60 is connected to the
electrode(s) 1/5 through the control device 20. A preferred
transformer for use with the present invention is one that uses
alternating current flowing in a primary coil 601 to establish an
alternating magnetic flux in a core 602 that easily conducts the
flux.
[0517] When a secondary coil 603 is positioned near the primary
coil 601 and core 602, this flux will link the secondary coil 603
with the primary coil 601. This linking of the secondary coil 603
induces a voltage across the secondary terminals. The magnitude of
the voltage at the secondary terminals is related directly to the
ratio of the secondary coil turns to the primary coil turns. More
turns on the secondary coil 603 than the primary coil 601 results
in a step up in voltage, while fewer turns results in a step down
in voltage.
[0518] Preferred transformer(s) 60 for use in these Examples have
deliberately poor output voltage regulation made possible by the
use of magnetic shunts in the transformer 60. These transformers 60
are known as neon sign transformers. This configuration limits
current flow into the electrode(s) 1/5. With a large change in
output load voltage, the transformer 60 maintains output load
current within a relatively narrow range.
[0519] The transformer 60 is rated for its secondary open circuit
voltage and secondary short circuit current. Open circuit voltage
(OCV) appears at the output terminals of the transformer 60 only
when no electrical connection is present. Likewise, short circuit
current is only drawn from the output terminals if a short is
placed across those terminals (in which case the output voltage
equals zero). However, when a load is connected across these same
terminals, the output voltage of the transformer 60 should fall
somewhere between zero and the rated OCV. In fact, if the
transformer 60 is loaded properly, that voltage will be about half
the rated OCV.
[0520] The transformer 60 is known as a Balanced Mid-Point
Referenced Design (e.g., also formerly known as balanced midpoint
grounded). This is most commonly found in mid to higher voltage
rated transformers and most 60 mA transformers. This is the only
type transformer acceptable in a "mid-point return wired" system.
The "balanced" transformer 60 has one primary coil 601 with two
secondary coils 603, one on each side of the primary coil 601 (as
shown generally in the schematic view in FIG. 16g). This
transformer 60 can in many ways perform like two transformers. Just
as the unbalanced midpoint referenced core and coil, one end of
each secondary coil 603 is attached to the core 602 and
subsequently to the transformer enclosure and the other end of each
secondary coil 603 is attached to an output lead or terminal. Thus,
with no connector present, an unloaded 15,000-volt transformer of
this type, will measure about 7,500 volts from each secondary
terminal to the transformer enclosure but will measure about 15,000
volts between the two output terminals.
[0521] In alternating current (AC) circuits possessing a line power
factor or 1 (or 100%), the voltage and current each start at zero,
rise to a crest, fall to zero, go to a negative crest and back up
to zero. This completes one cycle of a typical sine wave. This
happens 60 times per second in a typical US application. Thus, such
a voltage or current has a characteristic "frequency" of 60 cycles
per second (or 60 Hertz) power. Power factor relates to the
position of the voltage waveform relative to the current waveform.
When both waveforms pass through zero together and their crests are
together, they are in phase and the power factor is 1, or 100%.
FIG. 16h shows two waveforms "V" (voltage) and "C" (current) that
are in phase with each other and have a power factor of 1 or 100%;
whereas FIG. 16i shows two waveforms "V" (voltage) and "C"
(current) that are out of phase with each other and have a power
factor of about 60%; both waveforms do not pass through zero at the
same time, etc. The waveforms are out of phase and their power
factor is less than 100%.
[0522] The normal power factor of most such transformers 60 is
largely due to the effect of the magnetic shunts 604 and the
secondary coil 603, which effectively add an inductor into the
output of the transformer's 60 circuit to limit current to the
electrodes 1/5. The power factor can be increased to a higher power
factor by the use of capacitor(s) 61 placed across the primary coil
601 of the transformer, 60 which brings the input voltage and
current waves more into phase.
[0523] The unloaded voltage of any transformer 60 to be used in the
present invention is important, as well as the internal structure
thereof. Desirable unloaded transformers for use in the present
invention include those that are around 9,000 volts, 10,000 volts,
12,000 volts and 15,000 volts. However, these particular unloaded
volt transformer measurements should not be viewed as limiting the
scope acceptable power sources as additional embodiments. A
specific desirable transformer for use in these Examples is made by
Franceformer, Catalog No. 9060-P-E which operates at: primarily 120
volts, 60 Hz; and secondary 9,000 volts, 60 mA.
[0524] FIGS. 16e and 16f show an alternative embodiment of the
invention (i.e., not used in this Example), wherein the output of
the transformer 60 that is input into the electrode assemblies 1/5
has been rectified by a diode assembly 63 or 63'. The result, in
general, is that an AC wave becomes substantially similar to a DC
wave. In other words, an almost flat line DC output results
(actually a slight 120 Hz pulse can sometimes be obtained). This
particular assembly results in two additional preferred embodiments
of the invention (e.g., regarding electrode orientation). In this
regard, a substantially positive terminal or output and
substantially negative terminal or output is generated from the
diode assembly 63. An opposite polarity is achieved by the diode
assembly 63'. Such positive and negative outputs can be input into
either of the electrode(s) 1 and/or 5. Accordingly, an electrode 1
can be substantially negative or substantially positive; and/or an
electrode 5 can be substantially negative and/or substantially
positive.
[0525] FIG. 16j shows 8 separate transformer assemblies 60a-60h
each of which is connected to a corresponding control device
20a-20h, respectively. This set of transformers 60 and control
devices 20 are utilized in these Examples 1-4.
[0526] FIG. 16k shows 8 separate transformers 60a'-60h', each of
which corresponds to the rectified transformer diagram shown in
FIG. 16e. This transformer assembly also communicates with a set of
control devices 20a-20h and can be used as a preferred embodiment
of the invention, although was not used in these Examples.
[0527] FIG. 16l shows 8 separate transformers 60a''-60h'', each of
which corresponds to the rectified transformer diagram shown in
FIG. 16f. This transformer assembly also communicates with a set of
control devices 20a-20h and can be used as a preferred embodiment
of the invention, although was not used in these Examples.
[0528] Accordingly, each transformer assembly 60a-60h (and/or
60a'-60h'; and/or 60a''-60h'') can be the same transformer, or can
be a combination of different transformers (as well as different
polarities). The choice of transformer, power factor, capacitor(s)
61, polarity, electrode designs, electrode location, electrode
composition, cross-sectional shape(s) of the trough member 30,
local or global electrode composition, atmosphere(s), local or
global liquid 3 flow rate(s), liquid 3 local components, volume of
liquid 3 locally subjected to various fields in the trough member
30, neighboring (e.g., both upstream and downstream) electrode
sets, local field concentrations, the use and/or position and/or
composition of any membrane used in the trough member, etc., are
all factors which influence processing conditions as well as
composition and/or volume of constituents produced in the liquid 3,
nanocrystals and nanocrystal/suspensions or colloids made according
to the various embodiments disclosed herein. Accordingly, a
plethora of embodiments can be practiced according to the detailed
disclosure presented herein.
[0529] The size and shape of each electrode 1 utilized was about
the same. The shape of each electrode 1 was that of a right
triangle with measurements of about 14 mm.times.23 mm.times.27 mm.
The thickness of each electrode 1 was about 1 mm. Each
triangular-shaped electrode 1 also had a hole therethrough at a
base portion thereof, which permitted the point formed by the 23 mm
and 27 mm sides to point toward the surface 2 of the water 3. The
material comprising each electrode 1 was 99.95% pure (i.e., 3N5)
unless otherwise stated herein. When gold was used for each
electrode 1, the weight of each electrode was about 9 grams.
[0530] The wires used to attach the triangular-shaped electrode 1
to the transformer 60 were, for Examples 1-3, 99.95% (3N5) platinum
wire, having a diameter of about 1 mm.
[0531] The wires used for each electrode 5 comprised 99.95% pure
(3N5) gold each having a diameter of about 0.5 mm. All materials
for the electrodes 1/5 were obtained from ESPI having an address of
1050 Benson Way, Ashland, Oreg. 97520.
[0532] The water 3 used in Example 1 as an input into the trough
member 30 (and used in Examples 2-4 in combination with a
processing enhancer) was produced by a Reverse Osmosis process and
deionization process. In essence, Reverse Osmosis (RO) is a
pressure driven membrane separation process that separates species
that are dissolved and/or suspended substances from the ground
water. It is called "reverse" osmosis because pressure is applied
to reverse the natural flow of osmosis (which seeks to balance the
concentration of materials on both sides of the membrane). The
applied pressure forces the water through the membrane leaving the
contaminants on one side of the membrane and the purified water on
the other. The reverse osmosis membrane utilized several thin
layers or sheets of film that are bonded together and rolled in a
spiral configuration around a plastic tube. (This is also known as
a thin film composite or TFC membrane.) In addition to the removal
of dissolved species, the RO membrane also separates out suspended
materials including microorganisms that may be present in the
water. After RO processing a mixed bed deionization filter was
used. The total dissolved solvents ("TDS") after both treatments
was about 0.2 ppm, as measured by an Accumet.RTM. AR20
pH/conductivity meter.
[0533] These examples use gold electrodes for the 8 electrode sets.
In this regard, Tables 1a-1d set forth pertinent operating
parameters associated with each of the 16 electrodes in the 8
electrode sets utilized to make gold-based nanocrystals/nanocrystal
suspensions.
TABLE-US-00003 TABLE 1a Cold Input Water (Au) Run ID: GT032 Flow
Rate: 90 ml/min Wire Dia.: .5 mm Configuration: Straight/Straight
PPM: 0.4 Zeta: n/a Target Distance Distance Average Elec- Voltage
"c-c" "x" Voltage Set # trode # (kV) in/mm in/mm (kV) 7/177.8* 1 1a
1.6113 0.22/5.59 1.65 5a 0.8621 N/A 0.84 8/203.2 2 5b 0.4137 N/A
0.39 .sup. 5b' 0.7679 N/A 0.76 8/203.2 3 5c 0.491 N/A 0.49 .sup.
5c' 0.4816 N/A 0.48 8/203.2 4 1d 0.4579 N/A 0.45 5d 0.6435 N/A 0.6
9/228.6 5 5e 0.6893 N/A 0.67 .sup. 5e' 0.2718 N/A 0.26 8/203.2 6 5f
0.4327 N/A 0.43 .sup. 5f' 0.2993 N/A 0.3 8/203.2 7 5g 0.4691 N/A
0.43 .sup. 5g' 0.4644 N/A 0.46 8/203.2 8 5h 0.3494 N/A 0.33 .sup.
5h' 0.6302 N/A 0.61 8/203.2** Output Water Temperature 65 C.
*Distance from water inlet to center of first electrode set
**Distance from center of last electrode set to water outlet
TABLE-US-00004 TABLE 1b .0383 mg/mL of NaHCO.sub.3 (Au) Run ID:
GT031 Flow Rate: 90 ml/min NaHCO.sub.3: 0.038 mg/ml Wire Dia.: .5
mm Configuration: Straight/Straight PPM: 1.5 Zeta: n/a Target
Distance Distance Average Elec- Voltage "c-c" "x" Voltage Set #
trode # (kV) in/mm in/mm (kV) 7/177.8* 1 1a 1.7053 0.22/5.59 1.69
5a 1.1484 N/A 1.13 8/203.2 2 5b 0.6364 N/A 0.63 .sup. 5b' 0.9287
N/A 0.92 8/203.2 3 5c 0.7018 N/A 0.71 .sup. 5c' 0.6275 N/A 0.62
8/203.2 4 5d 0.6798 N/A 0.68 5d 0.7497 N/A 0.75 9/228.6 5 5e 0.8364
N/A 0.85 .sup. 5e' 0.4474 N/A 0.45 8/203.2 6 5f 0.5823 N/A 0.59
.sup. 5f' 0.4693 N/A 0.47 8/203.2 7 5g 0.609 N/A 0.61 .sup. 5g'
0.5861 N/A 0.59 8/203.2 8 5h 0.4756 N/A 0.48 .sup. 5h' 0.7564 N/A
0.76 8/203.2** Output Water Temperature 64 C. *Distance from water
inlet to center of first electrode set **Distance from center of
last electrode set to water outlet
TABLE-US-00005 TABLE 1c .045 mg/ml of NaCl (Au) Run ID: GT019 Flow
Rate: 90 ml/min NaCl: .045 mg/ml Wire Dia.: .5 mm Configuration:
Straight/Straight PPM: 6.1 Zeta: n/a Target Distance Distance
Average Elec- Voltage "c-c" "x" Voltage Set # trode # (kV) in/mm
in/mm (kV) 7/177.8* 1 1a 1.4105 0.22/5.59 1.41 5a 0.8372 N/A 0.87
8/203.2 2 5b 0.3244 N/A 0.36 .sup. 5b' 0.4856 N/A 0.65 8/203.2 3 5c
0.3504 N/A 0.37 .sup. 5c' 0.3147 N/A 0.36 8/203.2 4 5d 0.3526 N/A
0.37 5d 0.4539 N/A 0.5 9/228.6 5 5e 0.5811 N/A 0.6 .sup. 5e' 0.2471
N/A 0.27 8/203.2 6 5f 0.3624 N/A 0.38 .sup. 5f' 0.2905 N/A 0.31
8/203.2 7 5g 0.3387 N/A 0.36 .sup. 5g' 0.3015 N/A 0.33 8/203.2 8 5h
0.2995 N/A 0.33 .sup. 5h' 0.5442 N/A 0.57 8/203.2** Output Water
Temperature 77 C. *Distance from water inlet to center of first
electrode set **Distance from center of last electrode set to water
outlet
TABLE-US-00006 TABLE 1d .038 mg/mL of NaHCO.sub.3 (Au) Run ID:
GT033 Flow Rate: 90 ml/min NaHCO.sub.3: 0.038 mg/ml Wire Dia.: .5
mm Configuration: Straight/Straight PPM: 2.0 Zeta: n/a Target
Distance Distance Average Elec- Voltage "c-c" "x" Voltage Set #
trode # (kV) in/mm in/mm (kV) 7/177.8* 1 1a 1.6033 0.22/5.59
1.641826 5a 1.1759 N/A 1.190259 8/203.2 2 5b 0.6978 N/A 0.727213
.sup. 5b' 0.8918 N/A 0.946323 8/203.2 3 5c 0.6329 N/A 0.795378
.sup. 5c' 0.526 N/A 0.609542 8/203.2 4 5d 0.609 N/A 0.613669 5d
0.6978 N/A 0.719777 9/228.6 5 5e 0.9551 N/A 0.920594 .sup. 5e'
0.5594 N/A 0.547233 8/203.2 6 5f 0.6905 N/A 0.657295 .sup. 5f'
0.5516 N/A 0.521984 8/203.2 7 5g 0.5741 N/A 0.588502 .sup. 5g'
0.5791 N/A 0.541565 8/203.2 8 5h 0.4661 N/A 0.46091 .sup. 5h'
0.7329 N/A 0.741009 8/203.2** Output Water Temperature 83 C.
*Distance from water inlet to center of first electrode set
**Distance from center of last electrode set to water outlet
[0534] Table 1a shows that a "1/5" electrode configuration was
utilized for Electrode Set #1 and for Electrode Set #4, and all
other sets were of the 5/5 configuration; whereas Tables 1b, 1c and
1d show that Electrode Set #1 was the only electrode set utilizing
the 1/5 configuration, and all other sets were of the 5/5
configuration.
[0535] Additionally, the following differences in manufacturing
set-up were also utilized:
[0536] Example 1: GT032: The input water 3 into the trough member
30 was chilled in a refrigerator unit until it reached a
temperature of about 2.degree. C. and was then pumped into the
trough member 30;
[0537] Example 2: GT031: A processing enhancer was added to the
input water 3 prior to the water 3 being input into the trough
member 30. Specifically, about 0.145 grams/gallon (i.e., about 38.3
mg/liter) of sodium hydrogen carbonate ("soda"), having a chemical
formula of NaHCO.sub.3, was added to and mixed with the water 3.
The soda was obtained from Alfa Aesar and the soda had a formula
weight of 84.01 and a density of about 2.159 g/cm.sup.3 (i.e.,
stock #14707, lot D15T043).
[0538] Example 3: GT019: A processing enhancer was added to the
input water 3 prior to the water 3 being input into the trough
member 30. Specifically, about 0.17 grams/gallon (i.e., about 45
mg/liter) of sodium chloride ("salt"), having a chemical formula of
NaCl, was added to and mixed with the water 3.
[0539] Example 4: GT033: A processing enhancer was added to the
input water 3 prior to the water 3 being input into the trough
member 30. Specifically, about 0.145 grams/gallon (i.e., about 38.3
mg/liter) of sodium hydrogen carbonate ("soda"), having a chemical
formula of NaHCO.sub.3, was added to and mixed with the water 3.
The soda was obtained from Alfa Aesar and the soda had a formula
weight of 84.01 and a density of about 2.159 g/cm.sup.3 (i.e.,
stock #14707, lot D15T043). A representative TEM photomicrograph of
dried solution GT033 is shown in FIG. 32a. Also, FIG. 32b shows
dynamic light scattering data (i.e., hydrodynamic radii) of
suspension GT033.
[0540] The salt used in Example 3 was obtained from Fisher
Scientific (lot #080787) and the salt had a formula weight of 58.44
and an actual analysis as follows:
TABLE-US-00007 Assay .sup. 100% Barium (BA) Pass Test Bromide
<0.010% Calcium 0.0002% Chlorate & Nitrate <0.0003% Heavy
Metals (AS PB) <5.0 ppm Identification Pass Test Insoluble Water
<0.001% Iodide 0.0020% Iron (FE) <2.0 ppm Magnesium
<0.0005% Ph 5% Soln @ 25 Deg C. 5.9 Phosphate (PO4) <5.0 ppm
Potassium (K) <0.003% Sulfate (SO4) <0.0040%
[0541] Table 1e summarizes the physical characteristics results for
each of the three suspensions GT032, GT031 and GT019. Full
characterization of GT019 was not completed, however, it is clear
that under the processing conditions discussed herein, both
processing enhancers (i.e., soda and salt) increase the measured
ppm of gold in the suspensions GT031 and GT019 relative to
GT032.
TABLE-US-00008 TABLE 1e Zeta Predominant Poten- DLS DLS Mass Color
tial % Trans- Distribution Peak of Sus- PPM (Avg) pH mission
(Radius in nm) pension GT032 0.4 -19.30 3.29 11.7% 3.80 Clear GT031
1.5 -29.00 5.66 17.0% 0.78 Purple GT019 6.1 ** ** ** ** Pink GT033
2.0 ** ** .sup. 30% ** Pink ** Values not measured
Examples 5-7
Manufacturing Gold-Based Nanocrystals/Nanocrystal Suspensions
GD-007, GD-016 and GD-015
[0542] In general, each of Examples 5-7 utilize certain embodiments
of the invention associated with the apparatuses generally shown in
FIGS. 17b, 18a, 19a and 21a. Specific differences in processing and
apparatus will be apparent in each Example. The trough members 30a
and 30b were made from 1/8'' (about 3 mm) thick plexiglass, and
1/4'' (about 6 mm) thick polycarbonate, respectively. The support
structure 34 was also made from plexiglass which was about 1/4''
thick (about 6-7 mm thick). The cross-sectional shape of the trough
member 30a shown in FIG. 18a corresponds to that shape shown in
FIG. 10b (i.e., a truncated "V"). The base portion "R" of the
truncated "V" measured about 0.5'' (about lcm), and each side
portion "S", "S'" measured about 1.5'' (about 3.75 cm). The
distance "M" separating the side portions "S", "S'" of the V-shaped
trough member 30a was about 21/4''-2 5/16'' (about 5.9 cm)
(measured from inside to inside). The thickness of each portion
also measured about 1/8'' (about 3 mm) thick. The longitudinal
length "L.sub.T" (refer to FIG. 11a) of the V-shaped trough member
30a measured about 3 feet (about 1 meter) long from point 31 to
point 32.
[0543] Purified water (discussed elsewhere herein) was mixed with
about 0.396 g/L of NaHCO.sub.3 and was used as the liquid 3 input
into trough member 30a. While the amount of NaHCO.sub.3 used was
effective, this amount should not be viewed as limiting the metes
and bounds of the invention, and other amounts are within the metes
and bounds of this disclosure. The depth "d" (refer to FIG. 10b) of
the water 3 in the V-shaped trough member 30a was about 7/16'' to
about 1/2'' (about 11 mm to about 13 mm) at various points along
the trough member 30a. The depth "d" was partially controlled
through use of the dam 80 (shown in FIG. 18a). Specifically, the
dam 80 was provided near the end 32 and assisted in creating the
depth "d" (shown in FIG. 10b) to be about 7/6''-1/2'' (about 11-13
mm) in depth. The height "j" of the dam 80 measured about 1/4''
(about 6 mm) and the longitudinal length "k" measured about 1/2''
(about 13 mm). The width (not shown) was completely across the
bottom dimension "R" of the trough member 30a. Accordingly, the
total volume of water 3 in the V-shaped trough member 30a during
operation thereof was about 6.4 in.sup.3 (about 105 ml).
[0544] The rate of flow of the water 3 into the trough member 30a
was about 150 ml/minute (note: there was minimal evaporation in the
trough member 30a). Such flow of water 3 into the trough member 30a
was obtained by utilizing a Masterflex.RTM. L/S pump drive 40 rated
at 0.1 horsepower, 10-600 rpm. The model number of the
Masterflex.RTM. pump 40 was 77300-40. The pump drive had a pump
head also made by Masterflex.RTM. known as Easy-Load Model No.
7518-10. In general terms, the head for the pump 40 is known as a
peristaltic head. The pump 40 and head were controlled by a
Masterflex.RTM. LS Digital Modular Drive. The model number for the
Digital Modular Drive is 77300-80. The precise settings on the
Digital Modular Drive were, for example, 150 milliliters per
minute. Tygon.RTM. tubing having a diameter of 1/4'' (i.e., size
06419-25) was placed into the peristaltic head. The tubing was made
by Saint Gobain for Masterflex.RTM.. One end of the tubing was
delivered to a first end 31 of the trough member 30a by a flow
diffusion means located therein. The flow diffusion means tended to
minimize disturbance and bubbles in water 3 introduced into the
trough member 30a as well as any pulsing condition generated by the
peristaltic pump 40. In this regard, a small reservoir served as
the diffusion means and was provided at a point vertically above
the end 31 of the trough member 30a such that when the reservoir
overflowed, a relatively steady flow of water 3 into the end 31 of
the V-shaped trough member 30a occurred.
[0545] There were 5 electrode sets used in Examples 5-7 and one set
was a single electrode set 1a/5a located in trough member 30a. The
plasma 4 in trough member 30a from electrode 1a was created with an
electrode 1a similar in shape to that shown in FIG. 5e, and weighed
about 9.2 grams. This electrode was 99.95% pure gold. The other
electrode 5a comprised a right-triangular shaped platinum plate
measuring about 14 mm.times.23 mm.times.27 mm and about 1 mm thick
and having about 9 mm submerged in the liquid 3'. The AC
transformer used to create the plasma 4 was that transformer 60
shown in FIG. 16d and discussed elsewhere herein. AC transformers
50 (discussed below) were connected to the other electrode sets
5/5. All other pertinent run conditions are shown in Tables 2a, 2b
and 2c.
[0546] The output of the processing-enhanced, conditioned water 3'
was collected into a reservoir 41 and subsequently pumped by
another pump 40' into a second trough member 30b, at substantially
the same rate as pump 40 (e.g., minimal evaporation occurred in
trough member 30a). The second trough member 30b measured about 30
inches long by 1.5 inches wide by 5.75 inches high and contained
about 2500 ml of water 3'' therein. Each of four electrode sets 5b,
5b'-5e, 5e' comprised 99.95% pure gold wire measuring about 0.5 mm
in diameter and about 5 inches (about 12 cm) in length and was
substantially straight. About 4.25 inches (about 11 cm) of wire was
submerged in the water 3'' which was about 4.5 inches (about 11 cm)
deep.
[0547] With regard to FIGS. 19a and 21a, 4 separate electrode sets
(Set 2, Set 3, Set 4 and Set 5) were attached to 2 separate
transformer devices 50 and 50a, as shown in FIG. 19a. Specifically,
transformers 50 and 50a were electrically connected to each
electrode set, according to the wiring diagram show in FIG. 19a.
Each transformer device 50, 50a was connected to a separate AC
input line that was 120.degree. out of phase relative to each
other. The transformers 50 and 50a were electrically connected in a
manner so as not to overload a single electrical circuit and cause,
for example, an upstream circuit breaker to disengage (e.g., when
utilized under these conditions, a single transformer 50/50a could
draw sufficient current to cause upstream electrical problems).
Each transformer 50/50a was a variable AC transformer constructed
of a single coil/winding of wire. This winding acts as part of both
the primary and secondary winding. The input voltage is applied
across a fixed portion of the winding. The output voltage is taken
between one end of the winding and another connection along the
winding. By exposing part of the winding and making the secondary
connection using a sliding brush, a continuously variable ratio can
be obtained. The ratio of output to input voltages is equal to the
ratio of the number of turns of the winding they connect to.
Specifically, each transformer was a Mastech TDGC2-5kVA, 10A
Voltage Regulator, Output 0-250V.
[0548] Each of Tables 2a-2c contains processing information
relating to each of the 4 electrode sets in trough 30b by "Set #".
Each electrode of the 4 electrode sets in trough 30b was set to
operate at a specific target voltage. Actual operating voltages of
about 255 volts, as listed in each of Tables 2a-2c, were applied
across the electrode sets. The distance "c-c" (with reference to
FIG. 14) from the centerline of each electrode set to the adjacent
electrode set is also represented. Further, the distance "x"
associated with the electrode 1 utilized in trough 30a is also
reported. For the electrode 5's, no distance "x" is reported. Other
relevant parameters are also reported in each of Tables 2a-2c.
[0549] All materials for the electrodes 1/5 were obtained from ESPI
having an address of 1050 Benson Way, Ashland, Oreg. 97520.
[0550] The water 3 used in Examples 5-7 was produced by a Reverse
Osmosis process and deionization process and was mixed with the
NaHCO.sub.3 processing-enhancer and together was input into the
trough member 30a. In essence, Reverse Osmosis (RO) is a pressure
driven membrane separation process that separates species that are
dissolved and/or suspended substances from the ground water. It is
called "reverse" osmosis because pressure is applied to reverse the
natural flow of osmosis (which seeks to balance the concentration
of materials on both sides of the membrane). The applied pressure
forces the water through the membrane leaving the contaminants on
one side of the membrane and the purified water on the other. The
reverse osmosis membrane utilized several thin layers or sheets of
film that are bonded together and rolled in a spiral configuration
around a plastic tube. (This is also known as a thin film composite
or TFC membrane.) In addition to the removal of dissolved species,
the RO membrane also separates out suspended materials including
microorganisms that may be present in the water. After RO
processing a mixed bed deionization filter was used. The total
dissolved solvents ("TDS") after both treatments was about 0.2 ppm,
as measured by an Accumet.RTM. AR20 pH/conductivity meter.
TABLE-US-00009 TABLE 2a 0.396 mg/ml of NaHCO.sub.3 (Au) Run ID:
GD-007 Flow Rate: 150 ml/min Voltage: 255 V NaHCO.sub.3: 0.396
mg/ml Wire Dia.: .5 mm Configuration: Straight/Straight PPM: 14.8
Zeta: n/a Distance Distance Elec- "c-c" "x" Volt- cross Set# trode#
in/mm in/mm age section 4.5/114.3* 1 1a 0.25 750 V 5a N/A 750
23/584.2** 2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 8.5/215.9 3 5c N/A
255 Rectangle .sup. 5c' N/A 5.25'' 8.5/215.9 Deep 4 5d N/A 255
.sup. 5d' N/A .sup. 8/203.2 5 5e N/A 255 .sup. 5e' N/A 2/50.8**
Output Water Temperature 96 C. *Distance from water inlet to center
of first electrode set **Distance from center of last electrode set
to water oulet
TABLE-US-00010 TABLE 2b 0.396 mg/ml of NaHCO.sub.3 (Au) Run ID:
GD-016 Flow Rate: 150 ml/min Voltage: 255 V NaHCO.sub.3: 0.396
mg/ml Wire Dia.: .5 mm Configuration: Straight/Straight PPM: 12.5
Zeta: -56.12 Distance Distance Elec- "c-c" "x" Volt- cross Set#
trode# in/mm in/mm age section 4.5/114.3* 1 1a 0.25 750 V 5a N/A
750 23/584.2** 2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 8.5/215.9 3 5c
N/A 255 Rectangle .sup. 5c' N/A 5.25'' 8.5/215.9 Deep 4 5d N/A 255
.sup. 5d' N/A .sup. 8/203.2 5 5e N/A 255 .sup. 5e' N/A 2/50.8**
Output Water Temperature 97 C. *Distance from water inlet to center
of first electrode set **Distance from center of last electrode set
to water oulet
TABLE-US-00011 TABLE 2c 0.396 mg/ml of NaHCO3 (Au) Run ID: GD-015
Flow Rate: 150 ml/min Voltage: 255 V NaHCO.sub.3: 0.396 mg/ml Wire
Dia.: .5 mm Configuration: Straight/Straight PPM: 14.5 Zeta: -69.1
Distance Distance Elec- "c-c" "x" Volt- cross Set# trode# in/mm
in/mm age section 4.5/114.3* 1 1a 0.25 750 V 5a N/A 750 23/584.2**
2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 8.5/215.9 3 5c N/A 255
Rectangle .sup. 5c' N/A 5.25'' 8.5/215.9 Deep 4 5d N/A 255 .sup.
5d' N/A .sup. 8/203.2 5 5e N/A 255 .sup. 5e' N/A 2/50.8** Output
Water Temperature 96 C. *Distance from water inlet to center of
first electrode set **Distance from center of last electrode set to
water oulet
[0551] Representative Transmission Electron Microscopy (TEM)
photomicrographs (FIGS. 25a, 26a and 27a) were taken of each dried
suspension made according to each of these Examples 5-7.
Transmission Electron Microscopy
[0552] Specifically, TEM samples were prepared by utilizing a
Formvar coated grid stabilized with carbon having a mesh size of
200. The grids were first pretreated by a plasma treatment under
vacuum. The grids were placed on a microscope slide lined with a
rectangular piece of filter paper and then placed into a Denton
Vacuum apparatus with the necessary plasma generator accessory
installed. The vacuum was maintained at 75 mTorr and the plasma was
initiated and run for about 30 seconds. Upon completion, the system
was vented, and the grids removed. The grids were stable up to 7-10
days depending upon humidity conditions, but in all instances were
used within 12 hours.
[0553] Approximately 1 .mu.L of each inventive nanocrystal
suspension was placed onto each grid and was allowed to air dry at
room temperature for 20-30 minutes, or until the droplet
evaporated. Upon complete evaporation, the grids were placed onto a
holder plate until TEM analysis was performed.
[0554] A Philips/FEI Tecnai 12 Transmission Electron Microscope was
used to interrogate all prepared samples. The instrument was run at
an accelerating voltage of 100 keV. After alignment of the beam,
the samples were examined at various magnifications up to and
including 630,000.times.. Images were collected via the attached
Olympus Megaview III side-mounted camera that transmitted the
images directly to a PC equipped with iTEM and Tecnai User
Interface software which provided for both control over the camera
and the TEM instrument, respectively.
[0555] Within the iTEM software, it was possible to randomly move
around the grid by adjusting the position of a crosshair on a
circular reference plane. By selecting and moving the cross-hairs,
one could navigate around the grid. Using this function, the
samples were analyzed at four quadrants of the circular reference,
allowing for an unbiased representation of the sample. The images
were later analyzed with ImageJ 1.42 software. Another similar
software program which measured the number of pixels across each
particle relative to a known number of pixels in a spacer bar was
used to streamline the particle counting process. The particles
were measured using the scale bar on the image as a method to
calibrate the software prior to measuring each individual particle.
Once calibrated, particles were measured based upon the following
parameters: Tetrahedral particles were measured from the triangle's
apex to the base. Pentagonal bipyramids were measured from either
apex to apex of the diamond or apex of the pentagon to the base of
the pentagon depending upon the particle orientation on the grid.
Icosahedrons were measured using the longest distance between two
faces of a hexagonal particle. Spherical or irregular shaped
particles were measured along the longest axis. The data collected
from each sample set was exported to Excel, and using a simple
histogram function with 50 bins with a minimum of 5 nm and maximum
of 50 nm, a histogram was generated. Subsequently, the data
generated within Excel was exported to Prism (GraphPad.TM.) and fit
to one of two models, a normal distribution or log normal
distribution, each having a unique probability density function
(PDF). Within Prism, it was possible to analyze the histogram data
by performing a non-linear fit to the data which generates a
distribution known as a normal distribution. Moreover, it was
possible to perform a logarithmic transformation on the non-linear
data set to generate a data set that is then fit to a non-linear
model and then transformed via an exponential transformation to
generate a log-normal fit of the data. The two models were then
visually compared to the histogram and the model that fit the data
to a better degree was chosen. The particle diameter noted above,
and reported in the many Histogram Figures and Tables herein, is
the mode of the PDF, which is defined as the maximum value of the
log-normal or normal PDF curve. This PDF curve is overlaid on all
histogram figures wherein the mode value is displayed directly
above and is referenced in text as the TEM average diameter.
[0556] For example, FIGS. 25b, 26b and 27b are crystal size
distribution histograms measured from TEM photomicrographs
corresponding to dried solutions GD-007, GD-016 and GD-015
corresponding to Examples 5, 6 and 7, respectively. Each of the
numbers reported on these histograms corresponds to the discussion
above.
[0557] FIGS. 25a, 26a and 27a are representative TEM
photomicrographs corresponding to dried solutions GD-007, GD-016
and GD-015 corresponding to Examples 5, 6 and 7, respectively.
[0558] The results shown in FIGS. 25d and 25e were obtained using a
Philips 420ST transmission electron microscope equipped with an
Energy Dispersive X-ray Spectroscopy detector (EDS). The microscope
was located in the Electron Microbeam Analytical Facility at Johns
Hopkins University and operated under the guidance of a trained
operator. Briefly, approximately 1 .mu.L of GD-007 nanocrystalline
suspension was placed onto a Formvar carbon-coated 200 square mesh
nickel grid and was allowed to air dry at room temperature for
about 20-30 minutes, or until the droplet evaporated. Upon complete
evaporation, the grids were placed into the TEM sample holder and
interrogated at an accelerating voltage of 120 keV. The
microscope's EDS system was comprised of the following components:
Oxford light electron detector, Oxford XP3 pulse processor, and a 4
pi multi-channel analyzer connected to a Macintosh computer.
Particle composition was determined via energy dispersive x-ray
spectroscopy wherein a high energy beam of electrons was directed
at the surface of the nanocrystal resulting in the ejection of an
electron within the inner shell, thereby creating an available site
for an outer electron to "fall" into, thus emitting a
characteristic x-ray. The x-ray is then detected by the detector
having a resolution of 173.00 eV.
[0559] FIG. 25d shows one of the gold nanocrystals grown according
to Example 5 (i.e., GD-007). The nanocrystal was interrogated with
the electron beam as discussed herein.
[0560] FIG. 25e shows the energy dispersive x-ray pattern of the
interrogation beam point of the nanocrystal from solution GD-007.
Because this measuring technique is accurate to about a mono-layer
of atoms, the lack of a pattern corresponding to a sodium peak
shows that no sodium-based mono-layer was present on the crystal's
surface. Likewise, no significant carbon-based peak is observable
either, indicative of the lack of any carbon-based monolayer. Note
is made of the presence of the oxygen peak, which corresponds to
the underlying nickel grid. Accordingly, these FIGS. 25d and 25e
show: 1) no organics are present on these molecules and 2) that the
nanocrystals contain a relatively clean surface devoid of adverse
molecules or coatings.
[0561] Further, dynamic light scattering techniques were also
utilized to obtain an indication of crystal sizes (e.g.,
hydrodynamic radii) produced according to the Examples herein.
FIGS. 25c, 26c and 27c show the graphical result of the separate
dynamic light scattering data sets.
Dynamic Light Scattering
[0562] Specifically, dynamic light scattering (DLS) measurements
were performed on Viscotek 802 DLS instrument. In DLS, as the laser
light hits small particles and/or organized water structures around
the small particles (smaller than the wavelength), the light
scatters in all directions, resulting in a time-dependent
fluctuation in the scattering intensity. Intensity fluctuations are
due to the Brownian motion of the scattering particles/water
structure combination and contain information about the crystal
size distribution.
[0563] The instrument was allowed to warm up for at least 30 min
prior to the experiments. The measurements were made using 12 .mu.l
quartz cell. The following procedure was used: [0564] 1. First, 1
ml of DI water was added into the cell using 1 ml micropipette,
then water was poured out of the cell to a waste beaker and the
rest of the water was shaken off the cell measuring cavity. This
step was repeated two more times to thoroughly rinse the cell.
[0565] 2. 100 .mu.l of the sample was added into the cell using 200
.mu.l micropipette. After that all liquid was removed out of the
cell with the same pipette using the same pipette tip and expelled
into the waste beaker. 100 .mu.l of the sample was added again
using the same tip. [0566] 3. The cell with the sample was placed
into a temperature-controlled cell block of the Viscotek instrument
with frosted side of the cell facing left. A new experiment in
Viscotek OmniSIZE software was opened. The measurement was started
Imin after the temperature equilibrated and the laser power
attenuated to the proper value. The results were saved after all
runs were over. [0567] 4. The cell was taken out of the instrument
and the sample was removed out of the cell using the same pipette
and the tip used if step 2. [0568] 5. Steps 2 to 4 were repeated
two more times for each sample. [0569] 6. For a new sample, a new
pipette tip for 200 .mu.l pipette was taken to avoid contamination
with previous sample and steps 1 through 5 were repeated.
[0570] Data collection and processing was performed with OmniSIZE
software, version 3,0,0,291. The following parameters were used for
all the experiments: Run Duration--3s; Experiments--100;
Solvent--water, 0 mmol; Viscosity--1 cP; Refractive Index--1.333;
Spike Tolerance--20%; Baseline Drift--15%; Target Attenuation--300k
Counts; block temperature--+40.degree. C. After data for each
experiment were saved, the results were viewed on "Results" page of
the software. Particle size distribution (i.e., hydrodynamic radii)
was analyzed in "Intensity distribution" graph. On that graph any
peaks outside of 0.1 nm-10 .mu.m range were regarded as artifacts.
Particularly, clean water (no particles) results no peaks within
0.1 nm-10 .mu.m range and a broad peak below 0.1 nm. This peak is
taken as a noise peak (noise flow) of the instrument. Samples with
very low concentration or very small size of suspended nanocrystals
or nanoparticles may exhibit measurable noise peak in "Intensity
distribution" graph. If the peaks within 0.1 nm-10 .mu.m range have
higher intensity than the noise peak, those peaks considered being
real, otherwise the peaks are questionable and may represent
artifacts of data processing.
[0571] FIG. 25c shows graphical data corresponding to
representative Viscotek output data sets for Example 5 (i.e.,
GD-007); FIG. 26c shows graphical data corresponding to
representative Viscotek output data sets for Example 6 (i.e.,
GD-016); and FIG. 27c shows graphical data corresponding to
representative Viscotek output data sets for Example 7 (i.e.,
GD-015). The numbers reported at the tops of the peaks in each of
FIGS. 25c, 26c and 27c correspond to the average hydrodynamic radii
of nanocrystals, and light scattered around such nanocrystals,
detected in each solution. It should be noted that multiple (e.g.,
hundreds) of data-points were examined to give the numbers reported
in each data set, as represented by the "s-shaped" curves (i.e.,
each curve represents a series of collected data points). The
reported "% transmission" in each data set corresponds to the
intensity of the interrogation beam required in order to achieve
the dynamic light scattering data. In general, but not always, when
the reported "% transmission" is below 50%, very strong particle
and/or particle/ordered water structures are present. Also, when
the "% transmission" approaches 100%, often ions and/or very small
particles (e.g., pico-sized particles) are present and the reported
hydrodynamic radii may comprise more ordered or structured water
then actual solid particles.
[0572] It should be noted that the dynamic light scattering
particle size information is different from the TEM measured
histograms because dynamic light scattering uses algorithms that
assume the nanocrystals are all spheres (which they are not) as
well as measures the hydrodynamic radius (e.g., the nanocrystal's
influence on the water is also detected and reported in addition to
the actual physical radii of the particles). Accordingly, it is not
surprising that there is a difference in the reported particle
sizes between those reported in the TEM histogram data and those
reported in the dynamic light scattering data, just as in the other
Examples included herein.
Atomic Absorption Spectroscopy
[0573] The AAS values were obtained from a Perkin Elmer Analyst 400
Spectrometer system.
I) Principle
[0574] The technique of flame atomic absorption spectroscopy
requires a liquid sample to be aspirated, aerosolized and mixed
with combustible gases, such as acetylene and air. The mixture is
ignited in a flame whose temperature ranges from about 2100 to
about 2400 degrees C. During combustion, atoms of the element of
interest in the sample are reduced to free, unexcited ground state
atoms, which absorb light at characteristic wavelengths. The
characteristic wavelengths are element specific and are accurate to
0.01-0.1 nm. To provide element specific wavelengths, a light beam
from a hollow cathode lamp (HCL), whose cathode is made of the
element being determined, is passed through the flame. A
photodetector detects the amount of reduction of the light
intensity due to absorption by the analyte. A monochromator is used
in front of the photodetector to reduce background ambient light
and to select the specific wavelength from the HCL required for
detection. In addition, a deuterium arc lamp corrects for
background absorbance caused by non-atomic species in the atom
cloud.
II) Sample Preparation
[0574] [0575] 10 mL of sample, 0.6 mL of 36% v/v hydrochloric acid
and 0.15 mL of 50% v/v nitric acid are mixed together in a glass
vial and incubated for about 10 minutes in 70 degree C. water bath.
If gold concentration in the suspension is expected to be above 10
ppm a sample is diluted with DI water before addition of the acids
to bring final gold concentration in the range of 1 to 10 ppm. For
example, for a gold concentration around 100 ppm, 0.5 mL of sample
is diluted with 9.5 mL of DI water before the addition of acids.
Aliquoting is performed with adjustable micropipettes and the exact
amount of sample, DI water and acids is measured by an Ohaus PA313
microbalance. The weights of components are used to correct
measured concentration for dilution by DI water and acids. [0576]
Each sample is prepared in triplicate and after incubation in water
bath is allowed to cool down to room temperature before
measurements are made.
III) Instrument Setup
[0576] [0577] The following settings are used for Perkin Elmer
Analyst 400 Spectrometer system: [0578] a) Burner head: 10 cm
single-slot type, aligned in three axes according to the
manufacture procedure to obtain maximum absorbance with a 2 ppm Cu
standard. [0579] b) Nebulizer: plastic with a spacer in front of
the impact bead. [0580] c) Gas flow: oxidant (air) flow rate about
12 L/min, fuel (acetylene) flow rate about 1.9 mL/min. [0581] d)
Lamp/monochromator: Au hollow cathode lamp, 10 mA operating
current, 1.8/1.35 mm slits, 242.8 nm wavelength, background
correction (deuterium lamp) is on.
IV) Analysis Procedure
[0581] [0582] a) Run the Au lamp and the flame for approximately 30
minutes to warm up the system. [0583] b) Calibrate the instrument
with 1 ppm, 4 ppm and 10 ppm Au standards in a matrix of 3.7% v/v
hydrochloric acid. Use 3.7% v/v hydrochloric acid as a blank.
[0584] c) Verify calibration scale by measuring 4 ppm standard as a
sample. The measured concentration should be between 3.88 ppm and
4.12 ppm. Repeat step b) if outside that range. [0585] d) Measure
three replicas of a sample. If the standard deviation between
replicas is higher than 5%, repeat measurement, otherwise proceed
to the next sample. [0586] e) Perform verification step c) after
measuring six samples or more often. If verification fails, perform
steps b) and c) and remeasure all the samples measured after the
last successful verification.
V) Data Analysis
[0586] [0587] Measured concentration value for each replica is
corrected for dilution by water and acid to calculate actual sample
concentration. The reported Au ppm value is the average of three
corrected values for individual replica.
Plasma Irradiance and Characterization
[0588] This Example provides a spectrographic analysis of the
adjustable plasmas 4, utilizing a gold electrode 1, all of which
were utilized in the Examples herein. Three different spectrometers
with high sensitivities were used to collect spectral information
about the plasmas 4. Specifically, spectrographic analysis was
conducted on several gold electrode plasmon. The species in the
plasmas 4, as well as different intensities of some of the species,
were observed. The presence/absence of such species can affect
(e.g., positively and negatively) processing parameters and
products made according to the teachings herein.
[0589] In this regard, FIG. 25f shows a schematic view, in
perspective, of the experimental setup used to collect emission
spectroscopy information from the adjustable plasmas 4 utilized
herein.
[0590] Specifically, the experimental setup for collecting plasma
emission data (e.g., irradiance) is depicted in FIG. 25f. In
general, three spectrometers 520, 521 and 522 receive emission
spectroscopy data through a UV optical fiber 523 which transmits
collimated spectral emissions collected by the assembly 524, along
the path 527. The assembly 524 can be vertically positioned to
collect spectral emissions at different vertical locations within
the adjustable plasma 4 by moving the assembly 524 with the X-Z
stage 525. Accordingly, the presence/absence and intensity of
plasma species can be determined as a function of interrogation
location within the plasma 4. The output of the spectrometers 520,
521 and 522 was analyzed by appropriate software installed in the
computer 528. All irradiance data was collected through the hole
531 which was positioned to be approximately opposite to the
non-reflective material 530. The bottom of the hole 531 was located
at the top surface of the liquid 3. More details of the apparatus
for collecting emission radiance follows below.
[0591] The assembly 524 contained one UV collimator (LC-10U) with a
refocusing assembly (LF-10U100) for the 170-2400 nm range. The
assembly 524 also included an SMA female connector made by
Multimode Fiber Optics, Inc. Each LC-10U and LF-10U100 had one UV
fused silica lens associated therewith. Adjustable focusing was
provided by LF-10U100 at about 100 mm from the vortex of the lens
in LF-10U100 also contained in the assembly 524.
[0592] The collimator field of view at both ends of the adjustable
plasma 4 was about 1.5 mm in diameter as determined by a 455 .mu.m
fiber core diameter comprising the solarization resistant UV
optical fiber 523 (180-900 nm range and made by Mitsubishi). The UV
optical fiber 523 was terminated at each end by an SMA male
connector (sold by Ocean Optics; QP450-1-XSR).
[0593] The UV collimator-fiber system 523 and 524 provided 180-900
nm range of sensitivity for plasma irradiance coming from the 1.5
mm diameter plasma cylinder horizontally oriented in different
locations in the adjustable plasma 4.
[0594] The X-Z stage 525 comprised two linear stages (PT1) made by
Thorlabs Inc., that hold and control movement of the UV collimator
524 along the X and Z axes. It is thus possible to scan the
adjustable plasma 4 horizontally and vertically, respectively.
[0595] Emission of plasma radiation collected by UV
collimator-fiber system 523, 524 was delivered to either of three
fiber coupled spectrometers 520, 521 or 522 made by StellarNet,
Inc. (i.e., EPP2000-HR for 180-295 nm, 2400 g/mm grating,
EPP2000-HR for 290-400 nm, 1800 g/mm grating, and EPP2000-HR for
395-505 nm, 1200 g/mm grating). Each spectrometer 520, 521 and 522
had a 7 .mu.m entrance slit, 0.1 nm optical resolution and a
2048-pixel CCD detector. Measured instrumental spectral line
broadening is 0.13 nm at 313.1 nm.
[0596] Spectral data acquisition was controlled by SpectraWiz
software for Windows/XP made by StellarNet. All three EPP2000-HR
spectrometers 520, 521 and 522 were interfaced with one personal
computer 528 equipped with 4 USB ports. The integration times and
number of averages for various spectral ranges and plasma
discharges were set appropriately to provide unsaturated signal
intensities with the best possible signal to noise ratios.
Typically, spectral integration time was order of 1 second and
number averaged spectra was in range 1 to 10. All recorded spectra
were acquired with subtracted optical background. Optical
background was acquired before the beginning of the acquisition of
a corresponding set of measurements each with identical data
acquisition parameters.
[0597] Each UV fiber-spectrometer system (i.e., 523/520, 523/521
and 523/522) was calibrated with an AvaLight-DH-CAL Irradiance
Calibrated Light Source, made by Avantes (not shown). After the
calibration, all acquired spectral intensities were expressed in
(absolute) units of spectral irradiance (mW/m.sup.2/nm), as well as
corrected for the nonlinear response of the UV-fiber-spectrometer.
The relative error of the AvaLight-DH-CAL Irradiance Calibrated
Light Source in 200-1100 nm range is not higher than 10%.
[0598] Alignment of the field of view of the UV collimator assembly
524 relative to the tip 9 of the metal electrode 1 was performed
before each set of measurements. The center of the UV collimator
assembly 524 field of view was placed at the tip 9 by the alignment
of two linear stages and by sending a light through the UV
collimator-fiber system 523, 524 to the center of each metal
electrode 1.
[0599] The X-Z stage 525 was utilized to move the assembly 524 into
roughly a horizontal, center portion of the adjustable plasma 4,
while being able to move the assembly 524 vertically such that
analysis of the spectral emissions occurring at different vertical
heights in the adjustable plasma 4 could be made. In this regard,
the assembly 524 was positioned at different heights, the first of
which was located as close as possible of the tip 9 of the
electrode 1, and thereafter moved away from the tip 9 in specific
amounts. The emission spectroscopy of the plasma often did change
as a function of interrogation position.
[0600] For example, FIGS. 25g-25j show the irradiance data
associated with a gold (Au) electrode 1 utilized to form the
adjustable plasma 4. Each of the aforementioned FIGS. 25g-25j show
emission data associated with three different vertical
interrogation locations within the adjustable plasma 4. The
vertical position "0" (0 nm) corresponds to emission spectroscopy
data collected immediately adjacent to the tip 9 of the electrode
1; the vertical position "1/40" (0.635 nm) corresponds to emission
spectroscopy data 0.635 mm away from the tip 9 and toward the
surface of the water 3; and the vertical position "3/20" (3.81 mm)
corresponds to emission spectroscopy data 3.81 mm away from the tip
9 and toward the surface of the water 3.
[0601] Table 2d shows specifically each of the spectral lines
identified in the adjustable plasma 4 when a gold electrode 1 was
utilized to create the plasma 4.
TABLE-US-00012 TABLE 2d .lamda. meas. - .lamda. tab. .lamda. meas.
.lamda. tab. En Em Amn Transition (nm) (nm) (nm) (1/cm) (1/cm) gn
gm (1/s) NO A.sup.2.SIGMA..sup.+ - X.sup.2.PI. .gamma.-system:
(1-0) 214.7 214.7000 0.0000 NO A.sup.2.SIGMA..sup.+ - X.sup.2.PI.
.gamma.-system: (0-0) 226.9 226.8300 -0.0700 NO
A.sup.2.SIGMA..sup.+ - X.sup.2.PI. .gamma.-system: (0-1) 236.3
236.2100 -0.0900 Au | 5d.sup.106s .sup.2S.sub.1/2 - 5d.sup.106p
.sup.2P.sup.0.sub.3/2 242.795 242.7900 -0.0050 0 41174.613 2 4
1.99E+8 NO A.sup.2.SIGMA..sup.+ - X.sup.2.PI. .gamma.-system: (0-2)
247.1 246.9300 -0.1700 NO A.sup.2.SIGMA..sup.+ - X.sup.2.PI.
.gamma.-system: (0-3) 258.3 258.5300 0.2300 NO A.sup.2.SIGMA..sup.+
- X.sup.2.PI. .gamma.-system: (1-1) 267.1 267.0600 -0.0400 Au |
5d.sup.106s .sup.2S.sub.1/2 - 5d.sup.106p .sup.2P.sup.0.sub.1/2
267.595 267.59 -0.0050 0 37358.991 2 2 1.64E+8 NO
A.sup.2.SIGMA..sup.+ - X.sup.2.PI. .gamma.-system: (0-4) 271
271.1400 0.1400 Au | 5d.sup.96s.sup.2 .sup.2D.sub.5/2 -
5d.sup.9(.sup.2D.sub.5/2)6s6p .sup.24.sup.0.sub.7/2 274.825 274.82
-0.0050 9161.177 45537.195 6 8 OH A.sup.2.SIGMA. - X.sup.2.PI.
(1-0) 281.2 281.2000 0.0000 OH A.sup.2.SIGMA. - X.sup.2.PI. (1-0)
282 281.9600 -0.0400 N.sub.2 (C.sup.3.PI..sub.u -
B.sup.3.PI..sub.g) 2.sup.+-system (4-2) 295.32 295.3300 0.0100
N.sub.2 (C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system
(3-1) 296.2 296.1900 -0.0100 N.sub.2 (C.sup.3.PI..sub.u -
B.sup.3.PI..sub.g) 2.sup.+-system (2-0) 297.7 297.7000 0.0000 OH
A.sup.2.SIGMA. - X.sup.2.PI.: (0-0) 306.537 306.4600 -0.0770 OH
A.sup.2.SIGMA. - X.sup.2.PI.: (0-0) 306.776 306.8400 0.0640 OH
A.sup.2.SIGMA. - X.sup.2.PI.: (0-0) 307.844 307.8700 0.0260 OH
A.sup.2.SIGMA. - X.sup.2.PI.: (0-0) 308.986 309.0700 0.0840 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (2-1) 313.57
313.5800 0.0100 N.sub.2 (C.sup.3.PI..sub.u - B.sup.3.PI..sub.g)
2.sup.+-system (1-0) 316 315.9200 -0.0800 O.sub.2
(B.sup.3.SIGMA..sup.-.sub.u - X.sup.3.SIGMA..sup.-.sub.g) (0-14)
337 337.0800 0.0800 N.sub.2 (C.sup.3.PI..sub.u - B.sup.3.PI..sub.g)
2.sup.+-system (0-0) 337.1 337.1400 0.0400 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (2-3) 350.05
349.9700 -0.0800 N.sub.2 (C.sup.3.PI..sub.u - B.sup.3.PI..sub.g)
2.sup.+-system (1-2) 353.67 353.6400 -0.0300 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (0-1) 357.69
357.6500 -0.0400 N.sub.2.sup.+ (B.sup.2.SIGMA..sup.+.sub.u -
X.sup.2+.sub.g) 1.sup.--system (1-0) 358.2 358.2000 0.0000 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (2-4) 371
370.9500 -0.0500 N.sub.2 (C.sup.3.PI..sub.u - B.sup.3.PI..sub.g)
2.sup.+-system (1-3) 375.54 375.4500 -0.0900 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (0-2) 380.49
380.4000 -0.0900 N.sub.2.sup.+ (B.sup.2.SIGMA..sup.+.sub.u -
X.sup.2+.sub.g) 1.sup.--system (1-1) 388.4 388.4200 0.0200
N.sub.2.sup.+ (B.sup.2.SIGMA..sup.+.sub.u - X.sup.2+.sub.g)
1.sup.--system (0-0) 391.4 391.3700 -0.0300 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (1-4) 399.8
399.7100 -0.0900 N.sub.2 (C.sup.3.PI..sub.u - B.sup.3.PI..sub.g)
2.sup.+-system (0-3) 405.94 405.8100 -0.1300 N.sub.2
(C.sup.3.PI..sub.u - B.sup.3.PI..sub.g) 2.sup.+-system (4-8) 409.48
409.4900 0.0100 N.sub.2.sup.+ (B.sup.2.SIGMA..sup.+.sub.u -
X.sup.2+.sub.g) 1.sup.--system (2-3) 419.96 420.0000 0.0400
N.sub.2.sup.+ (B.sup.2.SIGMA..sup.+.sub.u - X.sup.2+.sub.g)
1.sup.--system (1-2) 423.65 423.6400 -0.0100 N.sub.2.sup.+
(B.sup.2.SIGMA..sup.+.sub.u - X.sup.2+.sub.g) 1.sup.--system (0-1)
427.785 427.7700 -0.0150 N.sub.2 (C.sup.3.PI..sub.u -
B.sup.3.PI..sub.g) 2.sup.+-system (3-8) 441.67 441.6200 -0.0500 Au
| 5d.sup.9(.sup.2D.sub.5/2)6s6p .sup.24.sup.0.sub.7/2 -
5d.sup.9(.sup.2D.sub.5/2)6s6p 10.sub.7/2 448.8263 448.7500 -0.0763
45537.195 67811.329 8 8 N.sub.2.sup.+ (B.sup.2.PI..sup.+.sub.u -
X.sup.2+.sub.g) 1.sup.--system (1-3) 465.1 465.1300 0.0300
N.sub.2.sup.+ (B.sup.2.PI..sup.+.sub.u - X.sup.2+.sub.g)
1.sup.--system (0-2) 470.9 470.8400 -0.0600 Na | 3s .sup.2S.sub.1/2
- 3p .sup.2P.sup.0.sub.3/2 588.99 588.995 0.0050 H | 2p
.sup.2P.sub.3/2 - 3d .sup.2D.sub.5/2 656.2852 655.8447 -0.4405
82259.287 97492.357 4 6 6.47E+7 N | 3s .sup.4P.sub.5/2 - 3p
.sup.4S.sub.3/2 746.8312 746.8815 0.0503 83364.62 96750.84 6 4
1.93E+7 N.sub.2 (B.sup.3.PI..sub.g - A.sup.3.SIGMA..sup.-.sub.u)
1.sup.+ -system 750 749.9618 -0.0382 O | 3s .sup.5S.sub.2 -
3p.sup.5P.sub.3 777.1944 776.8659 -0.3285 73768.2 86631.454 5 7
3.69E+7 O | 3s .sup.3S.sub.1 - 3p .sup.3P.sub.2 844.6359 844.2905
-0.3454 76794.978 88631.146 3 5 3.22E+7 N | 3s .sup.4P.sub.5/2 - 3p
.sup.4D.sub.7/2 868.0282 868.2219 0.1937 83364.62 94881.82 6 8
2.46E+7 O | 3p .sup.5P.sub.3 - 3d .sup.5D.sub.4 926.6006 926.3226
-0.2780 86631.454 97420.63 7 9 4.45E+7
[0602] A variety of species associated with the gold metallic
electrode 1 are identified in Table 2d. These species include, for
example, gold from the electrodes 1, as well as common species
including, NO, OH, N2, etc. It is interesting to note that some
species' existence and/or intensity (e.g., amount) is a function of
location within the adjustable plasma. Accordingly, this suggests
that various species can be caused to occur as a function of a
variety of processing conditions (e.g., power, location,
composition of electrode 1, etc.) of the invention.
Examples 8-10
Manufacturing Gold-Based Nanocrystal Suspensions GB-018, GB-019 and
GB-020
[0603] In general, each of Examples 8-10 utilize certain
embodiments of the invention associated with the apparatuses
generally shown in FIGS. 17a, 18a, 19b and 22a (e.g., a tapered
trough member 30b). Specific differences in processing and
apparatus will be apparent in each Example. The trough members 30a
and 30b were made from 1/8'' (about 3 mm) thick plexiglass, and
1/4'' (about 6 mm) thick polycarbonate, respectively. The support
structure 34 was also made from plexiglass which was about 1/4''
thick (about 6-7 mm thick). The cross-sectional shape of the trough
member 30a shown in FIG. 18a corresponds to that shape shown in
FIG. 10b (i.e., a truncated "V"). The base portion "R" of the
truncated "V" measured about 0.5'' (about lcm), and each side
portion "S", "S'" measured about 1.5'' (about 3.75 cm). The
distance "M" separating the side portions "S", "S'" of the V-shaped
trough member 30a was about 21/4-2 5/16'' (about 5.9 cm) (measured
from inside to inside). The thickness of each portion also measured
about 1/8'' (about 3 mm) thick. The longitudinal length "L.sub.T"
(refer to FIG. 11a) of the V-shaped trough member 30a measured
about 3 feet (about 1 meter) long from point 31 to point 32.
[0604] Purified water (discussed elsewhere herein) was mixed with
NaHCO.sub.3 in a range of about 0.396 to 0.528 g/L of NaHCO.sub.3
and was used as the liquid 3 input into trough member 30a. While
this range of NaHCO.sub.3 utilized was effective, it should not be
viewed as limiting the metes and bounds of the invention. The depth
"d" (refer to FIG. 10b) of the water 3 in the V-shaped trough
member 30a was about 7/16'' to about 1/2'' (about 11 mm to about 13
mm) at various points along the trough member 30a. The depth "d"
was partially controlled through use of the dam 80 (shown in FIG.
18a). Specifically, the dam 80 was provided near the end 32 and
assisted in creating the depth "d" (shown in FIG. 10b) to be about
7/6''-1/2'' (about 11-13 mm) in depth. The height "j" of the dam 80
measured about 1/4'' (about 6 mm) and the longitudinal length "k"
measured about 1/2'' (about 13 mm). The width (not shown) was
completely across the bottom dimension "R" of the trough member
30a. Accordingly, the total volume of water 3 in the V-shaped
trough member 30a during operation thereof was about 6.4 in.sup.3
(about 105 ml).
[0605] The rate of flow of the water 3 into the trough member 30a
ranged from about 150 ml/minute to at least 280 ml/minute. Such
flow of water 3 was obtained by utilizing a Masterflex.RTM. L/S
pump drive 40 rated at 0.1 horsepower, 10-600 rpm. The model number
of the Masterflex.RTM. pump 40 was 77300-40. The pump drive had a
pump head also made by Masterflex.RTM. known as Easy-Load Model No.
7518-10. In general terms, the head for the pump 40 is known as a
peristaltic head. The pump 40 and head were controlled by a
Masterflex.RTM. LS Digital Modular Drive. The model number for the
Digital Modular Drive is 77300-80. The precise settings on the
Digital Modular Drive were, for example, 150 milliliters per
minute. Tygon.RTM. tubing having a diameter of 1/4'' (i.e., size
06419-25) was placed into the peristaltic head. The tubing was made
by Saint Gobain for Masterflex.RTM.. One end of the tubing was
delivered to a first end 31 of the trough member 30a by a flow
diffusion means located therein. The flow diffusion means tended to
minimize disturbance and bubbles in water 3 introduced into the
trough member 30a as well as any pulsing condition generated by the
peristaltic pump 40. In this regard, a small reservoir served as
the diffusion means and was provided at a point vertically above
the end 31 of the trough member 30a such that when the reservoir
overflowed, a relatively steady flow of water 3 into the end 31 of
the V-shaped trough member 30a occurred.
[0606] There were 5 electrode sets used in Examples 8-10 and one
electrode set was a single electrode set 1a/5a located in the
trough member 30a. The plasma 4 from electrode 1a in trough member
30a was created with an electrode 1 similar in shape to that shown
in FIG. 5e, and weighed about 9.2 grams. This electrode was 99.95%
pure gold. The other electrode 5a comprised a right-triangular
shaped platinum plate measuring about 14 mm.times.23 mm.times.27 mm
and about 1 mm thick and having about 9 mm submerged in the liquid
3'. The AC transformer used to create the plasma 4 was that
transformer 60 shown in FIG. 16d and discussed elsewhere herein. AC
transformers 50 (discussed elsewhere herein) were connected to the
other electrode sets 5/5. All other pertinent run conditions are
shown in Tables 3a, 3b and 3c.
[0607] The output of the processing-enhanced, conditioned water 3'
was collected into a reservoir 41 and subsequently pumped by
another pump 40' into a second trough member 30b, at substantially
the same rate as pump 40 (e.g., there was minimal evaporation in
trough member 30a). The second trough member 30b shown in FIG. 22a
was tapered and measured about 3.75 inches high, about 3.75 inches
wide at the end 32 thereof, and about 1 inch wide at the end 31
thereof, thus forming a tapered shape. This trough member 30b
contained about 1450 ml of liquid 3'' therein which was about 2.5
inches deep. Each of four electrode sets 5b, 5b'-5e, 5e' comprised
99.95% pure gold wire which measured about 5 inches (about 13 cm)
in length, and about 0.5 mm in diameter in Examples 8 and 9, and
about 1.0 mm in diameter in Example 10. In each of Examples 8-10,
approximately 4.25 inches (about 11 cm) of the wire was submerged
within the water 3'', which had a depth of about 2.5 inches (about
6 cm). Each electrode set 5a, 5a'-5d, 5d' was shaped like a "J", as
shown in FIG. 17a. The distance "g" shown in FIG. 17a measured
about 1-8 mm.
[0608] With regard to FIGS. 19b and 22a, 4 separate electrode sets
(Set 2, Set 3, Set 4 and Set 5) were attached to a single
transformer device 50. Specifically, transformer 50 was the same
transformer used in Examples 5-7, but was electrically connected to
each electrode set according to the wiring diagram shown in FIG.
19b. In contrast, this wiring configuration was different than that
used in Examples 5-7, discussed above, only a single transformer 50
was required due to the lower amperage requirements (e.g., less
wire was in contact with the liquid 3) of this inventive trough 30b
design.
[0609] Each of Tables 3a-3c contains processing information
relative to each of the 4 electrode sets by "Set #". Each electrode
of the 4 electrode sets in trough 30b was set to operate at a
specific target voltage. Actual operating voltages of about 255
volts, as listed in each of Tables 3a-3c, were applied to the four
electrode sets. The distance "c-c" (with reference to FIG. 14) from
the centerline of each electrode set to the adjacent electrode set
is also represented. Further, the distance "x" associated with the
electrode 1 utilized in trough 30a is also reported. For the
electrode 5's, no distance "x" is reported. Other relevant
parameters are reported in each of Tables 3a-3c. All materials for
the electrodes 1/5 were obtained from ESPI having an address of
1050 Benson Way, Ashland, Oreg. 97520.
[0610] The water 3 used in Examples 8-10 was produced by a Reverse
Osmosis process and deionization process and was mixed with the
NaHCO.sub.3 processing-enhancer and together was input into the
trough member 30a. In essence, Reverse Osmosis (RO) is a pressure
driven membrane separation process that separates species that are
dissolved and/or suspended substances from the ground water. It is
called "reverse" osmosis because pressure is applied to reverse the
natural flow of osmosis (which seeks to balance the concentration
of materials on both sides of the membrane). The applied pressure
forces the water through the membrane leaving the contaminants on
one side of the membrane and the purified water on the other. The
reverse osmosis membrane utilized several thin layers or sheets of
film that are bonded together and rolled in a spiral configuration
around a plastic tube. (This is also known as a thin film composite
or TFC membrane.) In addition to the removal of dissolved species,
the RO membrane also separates out suspended materials including
microorganisms that may be present in the water. After RO
processing a mixed bed deionization filter was used. The total
dissolved solvents ("TDS") after both treatments was about 0.2 ppm,
as measured by an Accumet.RTM. AR20 pH/conductivity meter.
TABLE-US-00013 TABLE 3a 0.528 mg/ml of NaHCO.sub.3 (Au) Run ID:
GB-018 Flow Rate: 280 ml/min Voltage: 255 V NaHCO.sub.3: 0.528
mg/ml Wire Dia.: .5 mm Configuration: J/J PPM: 2.9 Zeta: -98.84
Distance Distance Elec- "c-c" "x" Volt- cross Set# trode# in/mm
in/mm age section 4.5/114.3* 1 1a 0.25 750 V 5a N/A 750 23/584.2**
2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 3.5/88.9 3 5c N/A 255 Tapered
.sup. 5c' N/A 3''Deep 3.5/88.9 4 5d N/A 255 .sup. 5d' N/A 3.5/88.9
5 5e N/A 255 .sup. 5e' N/A 376.2** Output Water Temperature 80 C.
*Distance from water inlet to center of first electrode set
**Distance from center of last electrode set to water oulet
TABLE-US-00014 TABLE 3b 0.396 mg/ml of NaHCO.sub.3 (Au) Run ID:
GB-019 Flow Rate: 150 ml/min Voltage: 255 V NaHCO.sub.3: 0.396
mg/ml Wire Dia.: 1 mm Configuration: J/J PPM: 23.6 Zeta: -56.6
Distance Distance Elec- "c-c" "x" Volt- cross Set# trode# in/mm
in/mm age section 4.5/114.3* 1 1a 0.25/6.35 750 V 5a N/A 750
23/584.2** 2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 3.5/88.9 3 5c N/A
255 Tapered .sup. 5c' N/A 3''Deep 3.5/88.9 4 5d N/A 255 .sup. 5d'
N/A 3.5/88.9 5 5e N/A 255 .sup. 5e' N/A 376.2** Output Water
Temperature 97 C. *Distance from water inlet to center of first
electrode set **Distance from center of last electrode set to water
oulet
TABLE-US-00015 TABLE 3c 0.396 mg/ml of NaHCO.sub.3 (Au) Run ID:
GB-020 Flow Rate: 250 ml/min Voltage: 255 V NaHCO.sub.3: 0.396
mg/ml Wire Dia.: 1 mm Configuration: J/J PPM: 4.9 Zeta: -58.01
Distance Distance Elec- "c-c" "x" Volt- cross Set# trode# in/mm
in/mm age section 4.5/114.3* 1 1a 0.25 750 V 5a N/A 750 23/584.2**
2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 3.5/88.9 3 5c N/A 255 Tapered
.sup. 5c' N/A 3''Deep 3.5/88.9 4 5d N/A 255 .sup. 5d' N/A 3.5/88.9
5 5e N/A 255 .sup. 5e' N/A 376.2** Output Water Temperature 86 C.
*Distance from water inlet to center of first electrode set
**Distance from center of last electrode set to water oulet
[0611] FIGS. 28a, 29a and 30a are representative TEM
photomicrographs corresponding to dried suspensions GB-018, GB-019
and GB-020, respectively, showing gold crystals grown in each of
Examples 8, 9 and 10.
[0612] FIGS. 28b, 29b and 30b are particle size distribution
histograms measured from the TEM photomicrographs (i.e., using the
software described earlier in Examples 5-7) corresponding to dried
suspensions taken from Examples 8, 9 and 10, respectively.
[0613] FIGS. 28c, 29c, and 30c show dynamic light scattering data
(i.e., hydrodynamic radii) of the gold nanocrystal suspensions made
in each of Examples 8, 9 and 10, respectively. Each of these
Figures shows the graphical results of dynamic light scattering
data sets.
[0614] It should be noted that the dynamic light scattering
particle size information is different from the TEM measured
histograms because dynamic light scattering uses algorithms that
assume the crystals are all spheres (which they are not) as well as
measures the hydrodynamic radius (e.g., the crystal's influence on
the water is also detected and reported in addition to the actual
physical radii of the crystals). Accordingly, it is not surprising
that there is a difference in the reported crystal sizes between
those reported in the TEM histogram data and those reported in the
dynamic light scattering data, just as in the other Examples
included herein.
Example 11
Manufacturing Gold-Based Nanoparticles/Nanoparticle Solutions or
Colloids IAC-202-7 by a Batch Process
[0615] This Example utilizes a batch process according to the
present invention. FIG. 24a shows the apparatus used to condition
the liquid 3. Once conditioned, the liquid 3' was processed in the
apparatus shown in FIG. 24b.
[0616] Table 4a shows a matrix where the amount of processing
enhancer baking soda (i.e., NaHCO.sub.3) varies from about 1
gram/gallon to about 2 grams/gallon (i.e., about 0.264 g/L to about
0.528 g/L); and the dwell time reflected in Table 4a in the
apparatus of FIG. 24a (i.e., the amount of time that the water 3
with processing enhancer was exposed to the plasma 4) was varied
from about 20 minutes to about 60 minutes, prior to subsequent
processing in the apparatus shown in FIG. 24c. The applied voltage
for each plasma 4 made by electrode 1 was about 750 volts. This
voltage was achieved by a transformer 60 (i.e., the Balanced
Mid-Point Referenced Design) discussed elsewhere herein. A second
and different transformer was electrically connected to the
electrodes 5a/5b shown in FIG. 24c. This transformer was an hy AC
power source having a voltage range of 0-300V, a frequency range of
47-400 Hz and a maximum power rating of 1 kVA. The applied voltage
for each identified run in Tables 4a and 4b was about 250 volts.
The current changed as a function of time with minimum and maximum
amps reported in Table 4b. All other process variables remained
constant.
[0617] Accordingly, Table 4a shows that a number of variables
(e.g., processing enhancer and predetermined dwell time) influence
both the amount or concentration of gold nanocrystals in water, and
the size distribution of the gold nanocrystals. In general, as the
concentration of the processing enhancer increases from about 1
g/gallon (0.264 g/L) to about 2 g/gallon (0.528 g/L), the
concentration (i.e., "ppm") more or less increases under a given
set of processing conditions. However, in some cases the particle
size distribution ("psd") unfavorably increases such that the
formed nanocrystals were no longer stable, and they "settled", as a
function of time (e.g., an unstable suspension was made). These
settling conditions were not immediate thus suggesting that this
suspension of nanocrystals in water could be processed immediately
into a useful product, such as, for example, a gel or cream. This
Example shows clearly various important effects of multiple
processing variables which can be translated, at least
directionally, to the inventive continuous processes disclosed
elsewhere herein. These data are illustrative and should not be
viewed as limiting the metes and bounds of the present invention.
Moreover, these illustrative data should provide an artisan of
ordinary skill with excellent operational directions to pursue.
[0618] As a specific example, Table 4c shows that a first electrode
Set #1 (i.e., FIG. 24a) was operating at a voltage of about 750
volts, to form the plasma 4. This is similar to the other plasmas 4
reported elsewhere herein. However, electrode Set #2 (i.e., FIG.
24c) was powered by the hy-AC source discussed above.
TABLE-US-00016 TABLE 4a Pretreatment Dwell (minutes) 20 40 60
1AC-201 1AC-202 1AC-201 1AC-202 1AC-201 1AC-202 NaHCO.sub.3 .264
ppm 1AC- 11.8 1AC- 11.1 1AC- 13.5 1AC- 11.4 1AC- 14.3 1AC- 12.2
(mg/ml) psd 201-9 18.4 202-1 19.1 201-8 19.5 202-2 18.4 201-7 16.8
202-3 19.6 .396 ppm 1AC- 20.1 1AC- 16.1 1AC- 21.4 1AC- settled 1AC-
23.3 1AC- settled psd 201-6 21.4 202-7 32.3 201-5 126 202-8 84.8
201-4 36.3 202-9 23.8 .528 ppm 1AC- 27.4 1AC- 23 1AC- 31.1 1AC-
24.9 1AC- settled 1AC- settled psd 201-1 17.1 202-4 43.8 201-2 21.6
202-5 21.4 201-3 190 202-6 settled
TABLE-US-00017 TABLE 4b Pretreatment Dwell (minutes) Current 20 40
60 Amps 1AC-201 1AC-202 1AC-201 1AC-202 1AC-201 1AC-202 NaHCO.sub.3
.264 min 1AC- 0.405 1AC- 0.382 1AC- 0.41 1AC- 0.411 1AC- 0.432 1AC-
0.461 (mg/ml) max 201-9 1.1 202-1 1 201-8 1 202-2 1.06 201-7 1
202-3 1.13 .396 min 1AC- 0.554 1AC- 0.548 1AC- 0.591 1AC- 0.598
1AC- 0.617 1AC- 0.681 max 201-6 1.6 202-7 1.35 201-5 1.6 202-8 1.43
201-4 1.6 202-9 1.43 .528 min 1AC- 0.686 1AC- 0.735 1AC- 0.843 1AC-
0.769 1AC- 0.799 1AC- 0.865 max 201-1 1.82 202-4 1.6 201-2 2.06
202-5 2 201-3 2.01 202-6 2.1
TABLE-US-00018 TABLE 4c 1.5 g/Gal of NaHCO.sub.3 (Au) Run ID:
1AC-202-7 Pretreatment: 20 min GZA in 3600 ml Volume: 800 ml Run
time: 35 minutes Voltage: 250 V NaHCO.sub.3: 0.396 mg/ml Wire Dia.:
.5 mm Configuration: J/J PPM: 16.1 Zeta: n/a Distance "x" Set#
Electrode# in/mm Voltage 1 1a 0.25/6.35 750 5a N/A 750 2 5b N/A 250
.sup. 5b' N/A
[0619] FIG. 31a shows a representative TEM Photomicrograph of gold
crystals, dried from solution, made according to this Example
11.
[0620] FIG. 31b shows the particle size distribution histogram
based on TEM measurements of the dried gold nanocrystals made
according to Example 11.
[0621] FIG. 31c shows graphical dynamic light scattering particle
size data (i.e., hydrodynamic radii) from this Example 11.
Specifically, a representative Viscotek data set is set forth in
this Figure, similar to what is reported elsewhere herein.
[0622] It should be noted that the dynamic light scattering
particle size information is different from the TEM measured
histograms because dynamic light scattering uses algorithms that
assume the nanocrystals are all spheres (which they are not) as
well as measures the hydrodynamic radius (e.g., the nanocrystal's
influence on the water is also detected and reported in addition to
the actual physical radii of the nanocrystals). Accordingly, it is
not surprising that there is a difference in the reported
nanocrystals sizes between those reported in the TEM histogram data
and those reported in the dynamic light scattering data, just as in
the other Examples included herein.
Example 12
Manufacturing Gold-Based Nanoparticles/Nanoparticle Solutions or
Colloids IAC-261 by a Batch Process
[0623] This Example utilizes a batch process according to the
present invention. FIG. 24a shows the apparatus used to condition
the liquid 3. Once conditioned, the liquid 3' was processed in the
apparatus shown in FIG. 24c
[0624] The amount of processing enhancer baking soda (i.e.,
NaHCO.sub.3) was about 1.5 grams/gallon (i.e., about 0.396 g/L).
The amount of time that the water 3 with processing enhancer was
exposed to the plasma 4 was about 60 minutes, prior to subsequent
processing in the apparatus shown in FIG. 24c.
[0625] The applied voltage for each plasma 4 made by electrode 1
was about 750 volts. This voltage was achieved by a transformer 60
(i.e., the Balanced Mid-Point Referenced Design) discussed
elsewhere herein.
[0626] A second and different transformer was electrically
connected to the electrodes 5a/5b shown in FIG. 24c. This
transformer was an hy AC power source having a voltage range of
0-300V, a frequency range of 47-400 Hz and a maximum power rating
of 1 kVA. The applied voltage was about 300 volts. The current
changed as a function of time with minimum amps being 0.390 and
maximum amps being 0.420 amps over a 60-minute operating time. The
diameter of the gold wire electrodes was 1 mm.
[0627] The amount of gold nanoparticles produced in the suspension
was about 13.7 ppm as measured by the atomic absorption
spectroscopy techniques discussed elsewhere herein. The sizes and
shapes of the nanoparticles made according to this Example are
fully discussed in Table 12 herein
[0628] FIG. 33a shows a representative TEM Photomicrograph of gold
crystals, dried from suspension 1AC-261, made according to this
Example 12.
[0629] FIG. 33b shows the particle size distribution histogram
based on TEM measurements of the dried gold nanoparticles made
according to Example 12.
Example 13
Manufacturing Gold-Based Nanocrystals/Nanocrystal Suspensions
GB-154-20 Hz, GB-157-40 Hz, GB-159-60 Hz, GB-161-80 Hz, GB-173-100
Hz and GB-156-300 Hz)
[0630] In general, this Example used the same manufacturing set-up
used for making GB-134 in Example 16, and for the sake of brevity,
the specifics of the trough apparatus used are discussed in detail
in that Example. The primary difference in making the suspensions
or colloids in this Example is that different sine waveform
frequencies from a programmable AC source were used as electrical
inputs to the electrodes 5a/5b.
[0631] In particular, sine wave AC frequencies as low as 20 Hz and
as high as 300 Hz were utilized to make nanocrystal suspensions or
colloids, in accordance with the teachings herein. The AC power
source 501AC utilized a Chroma 61604 programmable AC source. The
applied voltage was 300 volts. The waveform was a sine wave at six
different frequencies-20, 40, 60, 80, 100 and 300 Hz. The applied
current varied between 4.2 amps and 4.8 amps.
[0632] FIG. 34a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-154; and FIG. 34b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-154.
[0633] FIG. 35a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-157; and FIG. 35b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-157.
[0634] FIG. 36a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-159; and FIG. 36b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-159.
[0635] FIG. 37a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-161; and FIG. 37b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-161.
[0636] FIG. 38a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-173; and FIG. 38b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-173.
[0637] FIG. 39a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-156; and FIG. 39b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-156.
[0638] It is clear form this Example that particle size "mode" and
particle size distribution both increased as a function of
increasing the frequency AC sine waveform under the conditions of
this Example.
Example 14
Manufacturing Gold-Based Nanocrystals/Nanocrystal Suspensions
(GB-166-Sine, GB-165-Square and GB-162-Triangle)
[0639] In general, this Example used the same manufacturing set-up
used for making GB-134 in Example 16, and for the sake of brevity,
the specifics of the trough apparatus used are discussed in detail
in that Example. The primary difference in making the suspensions
or colloids in this Example was three different types of waveforms
(i.e., sine, square, and triangular waves) were generated by a BK
Precision 4040 20 MHz function generator, 501FG. The waveform
output was input into a chroma 61604 programmable AC source, 501AC.
The applied voltage for the sine waves ("SI") and square waves
("SQ") was 300 volts, while the applied voltage for the
triangular-shaped waveforms ("TR") was 250 volts. Each of these
waveforms is shown in FIG. 41. Specifically, GB-166 utilized a sine
wave; GB-165 utilized a square wave; and GB-162 utilized a
triangular wave as electrical inputs to the electrodes 5a/5b.
[0640] FIG. 42a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-166; and FIG. 42b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-166.
[0641] FIG. 43a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-165; and FIG. 43b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-165.
[0642] FIG. 44a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-162; and FIG. 44b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-162.
Example 15
Manufacturing Gold-Based Nanoparticles/Nanoparticle Suspensions
(GB-163 and GB-164)
[0643] In general, this Example used the same manufacturing set-up
used for making GB-134 in Example 16, and for the sake of brevity,
the specifics of the trough apparatus used are discussed in detail
in that Example. The primary difference in making the suspensions
or colloids in this Example was that two different duty cycles for
the triangular waveforms from the signal wave generator 501FG and
programmable AC power source 501AC (i.e., discusses in Example 14)
were used. The applied voltage for each triangular waveform was 250
volts. Specifically, each of GB-166 and GB-164 utilized the
triangular-shaped waveforms TR-1, TR-2 and TR-3 shown in FIG. 45 as
electrical inputs to the electrodes 5a/5b. Waveform TR-2 was the
maximum duty cycle, while TR-3 was the minimum duty cycle.
[0644] FIG. 46a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-163; and FIG. 46b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-163.
[0645] FIG. 47a shows a representative TEM Photomicrograph of gold
nanocrystals, dried from suspension GB-164; and FIG. 47b shows the
particle size distribution histogram based on TEM measurements of
the dried gold nanocrystals from suspension GB-164.
Example 16
Manufacturing Gold-Based Nanocrystals/Nanocrystal Suspensions
(GB-134); (GB-098, GB-113 and GB-118); (GB-120 and GB-123);
(GB-139); (GB-141 and GB-144); (GB-079, GB-089 and GB-062); and
(GB-076 and GB-077)
[0646] In general, this Example 16 utilizes certain embodiments of
the invention associated with the apparatuses generally shown in
FIGS. 20c-h, 21b-g and 22b. Additionally, Table 5 summarizes key
processing parameters used in conjunction with FIGS. 20c-h, 21b-g
and 22b. Also, Table 5 discloses: 1) resultant "ppm" (i.e., gold
nanoparticle concentrations), 2) a single number for "Hydrodynamic
Radii" taken from the average of the three highest amplitude peaks
shown in each of FIGS. 49c-61 (discussed later herein) and 3) "TEM
Average Diameter" which is the mode, corresponding to the particle
diameter that occurs most frequently, determined by TEM histogram
graphs shown in FIGS. 49b-61b. These physical characterizations
were performed as discussed elsewhere herein.
TABLE-US-00019 TABLE 5 Run ID: GB-134 GB-098 GB-113 GB-118 GB-120
GB-123 GB-139 Flow In (ml/min) 150 150 150 150 150 150 150 Rate:
Out (ml/min) 110 110 110 110 110 110 110 Set # 1 750 750 750 750
750 750 750 Volts: Set # 2 300 297 300 300 300 300 300 Set #'s 3-9
300 297 300 300 300 300 300 PE: NaHCO3 (mg/ml) 0.53 0.40 0.53 0.53
0.53 0.53 0.53 Wire Diameter (mm) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Contact "W.sub.L" (in/mm) .75/19 1/25 0.5/13 0.5/13 0.5/13 0.5/13
0.75/19 Electrode Config. FIG. 17b 17b 17b 17c 17b 17b 17d Produced
Au PPM 8.9 8.0 10.3 9.3 10.4 10.1 10.0 Output Temp .degree. C. at
32 85 93 88 86 84 93 87 Dimensions Plasma 4 FIGS. 18a 18a 18a 18a
18a 18a 18a Process FIGS. 20h, 21e 20f, 21b 20f, 21b 20f, 21b 20g,
21d 20g, 21d 20c, 20h 21e, 21f, 21g M1 (in/mm) 2/51 1/25 2/51 2/51
3.5/89 2/51 2/51 M2 (in/mm) n/a n/a n/a n/a n/a n/a n/a L.sub.T
(in/mm) 36/914 48/1219 36/914 36/914 36/914 36/914 36/914 d (in/mm)
.75/19 1/25 0.5/13 0.5/13 0.5/13 0.5/13 0.75/19 S (in/mm) 1.5/38.1
3/76.2 2.5/63.5 2.5/63.5 2.5/63.5 2.5/63.5 1.5/38.1 Electrode Curr.
(A) 0.56 0.53 0.53 0.52 0.51 0.48 FIG. 54d Total Curr. Draw (A) n/a
n/a n/a n/a n/a n/a n/a Hydrodynamic r (nm) 16.2 20.02 12.8 12.3
12.8 12.8 15.9 TEM Avg. Dia. (nm) 17.48 20.03 13.02 12.06 13.34
13.65 13.97 "c-c" (mm) 76 83 83 83 83 83 83 Set electrode # 1a 1a
1a 1a 1a 1a 1a 1 "x" (in/mm) 0.25/6.4 0.25/6.4 0.25/6.4 0.25/6.4
0.25/6.4 0.25/6.4 0.25/6.4 electrode # 5a 5a 5a 5a 5a 5a 5a "c-c"
(mm) 89 83 89 89 89 89 83 Set electrode # 5b 5b 5b 5b 5b 5b 5b 2
"x" (in/mm) n/a n/a n/a n/a n/a n/a n/a electrode # .sup. 5b' .sup.
5b' .sup. 5b' .sup. 5b' .sup. 5b' .sup. 5b' .sup. 5b' "c-c" (mm) 38
76 59 56 57 38 76 Set electrode # 5c 5c 5c 5c 5c 5c 5c 3 electrode
# .sup. 5c' .sup. 5c' .sup. 5c' .sup. 5c' .sup. 5c' .sup. 5c' .sup.
5c' "c-c" (mm) 38 105 60 59 64 38 76 Set electrode # 5d 5d 5d 5d 5d
5d 5d 4 electrode # .sup. 5d' .sup. 5d' .sup. 5d' .sup. 5d' .sup.
5d' .sup. 5d' .sup. 5d' "c-c" (mm) 89 143 70 68 70 44 127 Set
electrode # 5e 5e 5e 5e 5e 5e 5e 5 electrode # .sup. 5e' .sup. 5e'
.sup. 5e' .sup. 5e' .sup. 5e' .sup. 5e' .sup. 5e' "c-c" (mm) 89 165
84 103 70 51 127 Set electrode # 5f.sup. 5f.sup. 5f.sup. 5f.sup.
5f.sup. 5f.sup. 5f.sup. 6 electrode # 5f' 5f' 5f' 5f' 5f' 5f' 5f'
"c-c" (mm) 89 178 108 102 64 54 127 Set electrode # 5g 5g 5g 5g 5g
5g 5g 7 electrode # .sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g' .sup.
5g' .sup. 5g' .sup. 5g' "c-c" (mm) 178 178 100 100 76 54 216 Set
electrode # 5h 5h 5h 5h 5h 5h 5h 8 electrode # .sup. 5h' .sup. 5h'
.sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' "c-c" (mm) 89 216
127 135 76 57 83 Set electrode # n/a 5i.sup. 5i.sup. 5i.sup.
5i.sup. 5i.sup. n/a 9 electrode # n/a 5i' 5i' 5i' 5i' 5i' n/a "c-c"
(mm) n/a 76 191 178 324 464 n/a Run ID: GB-141 GB-144 GB-079 GB-089
GB-062 GB-076 GB-077 Flow In (ml/min) 150 110 150 150 150 150 150
Rate: Out (ml/min) 110 62 110 110 110 110 110 Set # 1 750 750 750
750 750 750 750 Volts: Set # 2 299 299 255 255 750 750 750 Set #'s
3-9 299 299 255 255 249 306 313 PE: NaHCO3 (mg/ml) 0.53 0.53 0.40
0.40 0.40 0.53 0.40 Wire Diameter (mm) 1.0 1.0 0.5 0.5 0.5 0.5 0.5
Contact "W.sub.L" (in/mm) 0.5/13 0.5/13 2/51 2/51 2/51 1/25 1/25
Electrode Config. FIG. 17d 17d 17b 17b 17b 17b 17b Produced Au PPM
10.1 20.2 10.8 12.4 16.7 7.8 7.5 Output Temp .degree. C. at 32 86
89 94 99 95 98 97 Dimensions Plasma 4 FIGS. 18a 18a 18a 18a 18b 18b
18b Process FIGS. 20c, 20h 20c, 20h 20d, 21c 20d, 21c 20e, 21c 20e,
22b 20e, 22b 21e, 21f, 21e, 21f, 21g 21g M1 (in/mm) 2/51 2/51 1/25
0.75/19 1/25 2.7/68.6 2.7/686 M2 (in/mm) n/a n/a n/a n/a n/a 0.5/13
0.5/13 L.sub.T (in/mm) 36/914 36/914 24/610 24/610 24/610 24/610
24/610 d (in/mm) 0.5/13 0.5/13 2/51 2/51 2/51 1/25 1/25 S (in/mm)
1.5/38.1 1.5/38.1 3.3/83.8 3.3/83.8 3.3/83.8 3.5/88.9 3.5/88.9
Electrode Curr. (A) FIG. 55d FIG. 56d 0.66 n/a 0.7 0.51 0.48 Total
Curr. Draw (A) n/a n/a 11.94 8.98 12.48 13.62 12.47 Hydrodynamic r
(nm) 26.2 16.4 14.9 17.2 17.0 9.7 11.5 TEM Avg. Dia. (nm) 11.42
18.12 10.63 15.89 11.75 11.07 8.69 "c-c" (mm) n/a 83 n/m n/m n/m
n/m n/m Set electrode # n/a 1a 1a 1a 1a 1a 1a 1 "x" (in/mm) n/a
0.25/6.4 0.25/6.4 0.25/6.4 0.25/6.4 0.25/6.4 0.25/6.4 electrode #
n/a 5a 5a 5a 5a 5a 5a "c-c" (mm) 83 83 n/m n/m n/m n/m n/m Set
electrode # 5b 5b 5b 5b 1b 1b 1b 2 "x" (in/mm) n/a n/a n/a n/a
0.25/6.4 0.25/6.4 0.25/6.4 electrode # .sup. 5b' .sup. 5b' .sup.
5b' .sup. 5b' 5b 5b 5b "c-c" (mm) 76 76 n/m n/m n/m n/m n/m Set
electrode # 5c 5c 5c 5c 5c 5c 5c 3 electrode # .sup. 5c' .sup. 5c'
.sup. 5c' .sup. 5c' .sup. 5c' .sup. 5c' .sup. 5c' "c-c" (mm) 76 76
n/m n/m n/m n/m n/m Set electrode # 5d 5d 5d 5d 5d 5d 5d 4
electrode # .sup. 5d' .sup. 5d' .sup. 5d' .sup. 5d' .sup. 5d' .sup.
5d' .sup. 5d' "c-c" (mm) 127 127 n/m n/m n/m n/m n/m Set electrode
# 5e 5e 5e 5e 5e 5e 5e 5 electrode # .sup. 5e' .sup. 5e' .sup. 5e'
.sup. 5e' .sup. 5e' .sup. 5e' .sup. 5e' "c-c" (mm) 127 127 n/m n/m
n/m n/m n/m Set electrode # 5f.sup. 5f.sup. 5f.sup. 5f.sup. 5f.sup.
5f.sup. 5f.sup. 6 electrode # 5f' 5f' 5f' 5f' 5f' 5f' 5f' "c-c"
(mm) 127 127 n/m n/m n/m n/m n/m Set electrode # 5g 5g 5g 5g 5g 5g
5g 7 electrode # .sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g'
.sup. 5g' .sup. 5g' "c-c" (mm) 216 216 n/m n/m n/m n/m n/m Set
electrode # 5h 5h 5h 5h 5h 5h 5h 8 electrode # .sup. 5h' .sup. 5h'
.sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' "c-c" (mm) 83 83
n/m n/m n/m n/m n/m Set electrode # n/a n/a n/a n/a 5i.sup. 5i.sup.
5i.sup. 9 electrode # n/a n/a n/a n/a 5i' 5i' 5i' "c-c" (mm) n/a
n/a n/a n/a n/m n/m n/m
[0647] All trough members 30a' and 30b' in the aforementioned
Figures were made from 1/8'' (about 3 mm) thick plexiglass, and
1/4'' (about 6 mm) thick polycarbonate, respectively. The support
structure 34 (not shown in many of the Figures but discussed
elsewhere herein) was also made from plexiglass which was about
1/4'' thick (about 6-7 mm thick). In contrast to the embodiments
shown in FIGS. 19a and 19b, each trough member 30a was integral
with trough member 30b' and was thus designated 30a' (e.g., no
separate pumping means was provided after trough member 30a, as in
certain previous examples). The cross-sectional shape of each
trough member 30a' used in this Example corresponded to that shape
shown in FIG. 10b (i.e., was a trapezoidal-shaped cross-section).
Relevant dimensions for each trough member portion 30b' are
reported in Table 5 as "M1" (i.e., inside width of the trough at
the entrance portion of the trough member 30b'), "M2" (i.e., inside
width of the trough at the exit portion of the trough member 30b'),
"L.sub.T" (i.e., transverse length or flow length of the trough
member 30b'), "S" (i.e., the height of the trough member 30b'), and
"d" (i.e., depth of the liquid 3'' within the trough member 30b').
In some embodiments, the distance "M" separating the side portions
"S", "S'" (refer to FIG. 10a) of the trough member 30b' were the
same. In these cases, Table 5 represents a value dimension for only
"M1" and the entry for "M2" is represented as "N/A". In other
words, some trough members 30b' were tapered along their
longitudinal length and in other cases, the trough members 30b'
were substantially straight along their longitudinal length. The
thickness of each sidewall portion also measured about 1/4'' (about
6 mm) thick. Three different longitudinal lengths "L.sub.T" are
reported for the trough members 30b' (i.e., either 610 mm, 914 mm
or 1219 mm) however, other lengths L.sub.T should be considered to
be within the metes and bounds of the inventive trough.
[0648] Table 5 shows that the processing enhancer NaHCO.sub.3 was
added to purified water (discussed elsewhere herein) in amounts of
either about 0.4 mg/ml or 0.53 mg/ml. It should be understood that
other amounts of this processing enhancer also function within the
metes and bounds of the invention. The purified water/NaHCO.sub.3
mixture was used as the liquid 3 input into trough member 30a'. The
depth "d" of the liquid 3' in the trough member 30a' (i.e., where
the plasma(s) 4 is/are formed) was about 7/16'' to about 1/2''
(about 11 mm to about 13 mm) at various points along the trough
member 30a'. The depth "d'" was partially controlled through use of
the dam 80 (shown in FIGS. 18a and 18b). Specifically, the dam 80
was provided near the output end 32 of the trough member 30a' and
assisted in creating the depth "d" (shown in FIG. 10b as "d") to be
about 7/6''-1/2'' (about 11-13 mm) in depth. The height "j" of the
dam 80 measured about 1/4'' (about 6 mm) and the longitudinal
length "k" measured about 1/2'' (about 13 mm). The width (not
shown) was completely across the bottom dimension "R" of the trough
member 30a'. Accordingly, the total volume of liquid 3' in the
trough member 30a' during operation thereof was about 2.14 in.sup.3
(about 35 ml) to about 0.89 in.sup.3 (about 14.58 ml).
[0649] The rate of flow of the liquid 3' into the trough member
30a' as well as into trough member 30b', was about 150 ml/minute
for all but one of the formed samples (i.e., GB-144 which was about
110 ml/minute) and the rate of flow out of the trough member 30b'
at the point 32 was about 110 ml/minute (i.e., due to evaporation)
for all samples except GB-144, which was about 62 ml/minute. The
amount of evaporation that occurred in GB-144 was a greater percent
than the other samples because the dwell time of the liquid 3'' in
the trough member 30b' was longer relative to the other samples
made according to this embodiment. Other acceptable flow rates
should be considered to be within the metes and bounds of the
invention.
[0650] Such flow of liquid 3' was obtained by utilizing a
Masterflex.RTM. L/S pump drive 40 rated at 0.1 horsepower, 10-600
rpm. The model number of the Masterflex.RTM. pump 40 was 77300-40.
The pump drive had a pump head also made by Masterflex.RTM. known
as Easy-Load Model No. 7518-10. In general terms, the head for the
pump 40 is known as a peristaltic head. The pump 40 and head were
controlled by a Masterflex.RTM. LS Digital Modular Drive. The model
number for the Digital Modular Drive is 77300-80. The precise
settings on the Digital Modular Drive were, for example, 150
milliliters per minute for all samples except GB-144 which was, for
example, 110 ml/minute. Tygon.RTM. tubing having a diameter of
1/4'' (i.e., size 06419-25) was placed into the peristaltic head.
The tubing was made by Saint Gobain for Masterflex.RTM.. One end of
the tubing was delivered to a first end 31 of the trough member
30'a by a flow diffusion means located therein. The flow diffusion
means tended to minimize disturbance and bubbles in water 3
introduced into the trough member 30a' as well as any pulsing
condition generated by the peristaltic pump 40. In this regard, a
small reservoir served as the diffusion means and was provided at a
point vertically above the end 31 of the trough member 30a' such
that when the reservoir overflowed, a relatively steady flow of
liquid 3' into the end 31 of the V-shaped trough member 30a'
occurred.
[0651] Table 5 shows that there was a single electrode set 1a/5a,
or two electrode sets 1a/5a, utilized in this Example 18. The
plasma(s) 4 was/were created with an electrode 1 similar in shape
to that shown in FIG. 5e, and weighed about 9.2 grams. This
electrode was 99.95% pure gold. The other electrode 5a comprised a
right-triangular shaped platinum plate measuring about 14
mm.times.23 mm.times.27 mm and about 1 mm thick and having about 9
mm submerged in the liquid 3'. All other pertinent run conditions
are shown in Table 5.
[0652] As shown in FIGS. 20c-h, the output from the trough member
30a' was the conditioned liquid 3' and this conditioned liquid 3'
flowed directly into a second trough member 30b'. The second trough
member 30b', shown in FIGS. 21b-g and 22b had measurements as
reported in Table 5. This trough member 30b' contained from about
600 ml of liquid 3'' therein to about 1100 ml depending on the
dimensions of the trough and the depth "d''" of the liquid 3''
therein. Table 5, in connection with FIGS. 20c-h, 21b-g and 22b,
show a variety of different electrode configurations. For example,
previous examples herein disclosed the use of four sets of
electrodes 5/5, with one electrode set 1/5. In this Example, either
eight or nine electrode sets were used (e.g., one 1/5 set with
seven or eight 5/5' sets; or two 1/5 sets with seven 5/5' sets).
Each of the electrode sets 5/5' comprised 99.99% pure gold wire
measuring either about 0.5 mm in diameter or 1.0 mm in diameter, as
reported in Table 5. The length of each wire electrode 5 that was
in contact with the liquid 3'' (reported as "W.sub.L" in Table 5)
measured from about 0.5 inches (about 13 mm) long to about 2.0
inches (about 51 mm) long. Two different electrode set
configurations 5/5' were utilized. FIGS. 21b, 21c, 21e, 21f, 21g
and 22b all show electrode sets 5/5' oriented along a plane (e.g.,
arranged in line form along the flow direction of the liquid 3'').
Whereas FIG. 21d shows that the electrode sets 5/5' were rotated
about 90.degree. relative to the aforementioned electrode sets
5/5'. Further, the embodiments shown in FIGS. 20a-20h show the
electrode sets 1/5 and 5/5' were all located along the same plane.
However, it should be understood that the imaginary plane created
between the electrodes in each electrode set 1/5 and/or 5/5' can be
parallel to the flow direction of the liquid 3'' or perpendicular
to the flow direction of the liquid 3'' or at an angle relative to
the flow direction of the liquid 3.''
[0653] With regard to FIGS. 20c-h, 21b-g and 22b, each separate
electrode set 5/5' (e.g., Set 2, Set 3-Set 8 or Set 9) were
electrically connected to the transformer devices, 50 and 50a, as
shown therein. Specifically, transformers 50 and 50a were
electrically connected to each electrode set, according to the
wiring diagram show in FIGS. 20c-h. The exact wiring varied between
examples and reference should be made to the FIGS. 20c-20g for
specific electrical connection information. In most cases, each
transformer device 50, 50a was connected to a separate AC input
line that was 1200 out of phase relative to each other. The
transformers 50 and 50a were electrically connected in a manner so
as not to overload a single electrical circuit and cause, for
example, an upstream circuit breaker to disengage (e.g., when
utilized under these conditions, a single transformer 50/50a could
draw sufficient current to cause upstream electrical problems).
Each transformer 50/50a was a variable AC transformer constructed
of a single coil/winding of wire. This winding acts as part of both
the primary and secondary winding. The input voltage is applied
across a fixed portion of the winding. The output voltage is taken
between one end of the winding and another connection along the
winding. By exposing part of the winding and making the secondary
connection using a sliding brush, a continuously variable ratio can
be obtained. The ratio of output to input voltages is equal to the
ratio of the number of turns of the winding they connect to.
Specifically, each transformer was a Mastech TDGC2-5 kVA, 10A
Voltage Regulator, Output 0-250V.
[0654] Table 5 refers to each of the electrode sets by "Set #"
(e.g., "Set 1" through "Set 9"). Each electrode of the 1/5 or 5/5
electrode sets was set to operate within a specific voltage range.
The voltages listed in Table 5 are the voltages used for each
electrode set. The distance "c-c" (with reference to FIG. 14) from
the centerline of each electrode set to the adjacent electrode set
is also reported. Further, the distance "x" associated with each
electrode 1 utilized is also reported. For the electrode 5, no
distance "x" is reported. Sample GB-118 had a slightly different
electrode 5a/5b arrangement from the other examples herein.
Specifically, tips or ends 5t and 5t' of the electrodes 5a/5b,
respectively, were located closer to each other than other portions
of the electrodes 5a/5b. The distance "dt" between the tips 5t and
5t' varied between about 7/16 inches (about 1.2 cm) and about 2
inches (about 5 cm). Other relevant parameters are also reported in
Table 5.
[0655] All materials for the electrodes 1/5 were obtained from
ESPI, having an address of 1050 Benson Way, Ashland, Oreg. 97520.
All materials for the electrodes 5/5 in runs GB-139, GB-141,
GB-144, GB-076, GB-077, GB-079, GB-089, GB-098, GB-113, GB-118,
GB-120 and GB-123 were obtained from Alfa Aesar, having an address
of 26 Parkridge Road, Ward Hill, Mass. 01835. All materials for the
electrodes 5/5 in run GB-062 were obtained from ESPI, 1050 Benson
Way, Ashland, Oreg. 97520.
[0656] FIGS. 49a-61a show two representative TEM photomicrographs
for each of the gold nanocrystals, dried from each suspension or
colloid referenced in Table 5, and formed according to Example
16.
[0657] FIGS. 49b-61b show the measured size distribution of the
gold nanocrystals measured by using the TEM instrument/software
discussed earlier in Examples 5-7 for each dried solution or
colloid referenced in Table 5 and formed according to Example
16.
[0658] FIGS. 49c-61c show graphically dynamic light scattering data
measurement sets for the nanocrystals (i.e., the hydrodynamic
radii) made according to each suspension or colloid referenced in
Table 5 and formed according to Example 16. It should be noted that
the dynamic light scattering particle size information is different
from the TEM measured histograms because dynamic light scattering
uses algorithms that assume the particles are all spheres (which
they are not) as well as measures the hydrodynamic radius (e.g.,
the particle's influence on the water is also detected and reported
in addition to the actual physical radii of the particles).
Accordingly, it is not surprising that there is a difference in the
reported particle sizes between those reported in the TEM histogram
data of those reported in the dynamic light scattering data just as
in the other Examples included herein.
[0659] Reference is now made to FIGS. 20c, 20h, 21e, 21f and 20g
which are representative of structures that were used to make
samples GB-139, GB-141 and GB-144. The trough member 30b' used to
make these samples was different from the other trough members 30b'
used this Example 16 because: 1) the eight electrode sets 1/5 and
5/5 were all connected to control devices 20 and 20a-20g (i.e., see
FIG. 20h) which automatically adjusted the height of, for example,
each electrode 1/5 or 5/5 in each electrode set 1/5; and 2) female
receiver tubes o5a/o5a'-o5g/o5g' which were connected to a bottom
portion of the trough member 30b' such that the electrodes in each
electrode set 5/5 could be removably inserted into each female
receiver tube o5 when, and if, desired. Each female receiver tube
o5 was made of polycarbonate and had an inside diameter of about
1/8 inch (about 3.2 mm) and was fixed in place by a solvent
adhesive to the bottom portion of the trough member 30b'. Holes in
the bottom of the trough member 30b' permitted the outside diameter
of each tube o5 to be fixed therein such that one end of the tube
o5 was flush with the surface of the bottom portion of the trough
30b'. The inside diameters of the tubes o5 effectively prevented
any significant quantities of liquid 3'' from entering into the
female receiver tube o5. However, some liquid may flow into the
inside of one or more of the female receiver tubes o5. The length
or vertical height of each female receiver tube o5 used in this
Example was about 6 inches (about 15.24 cm) however, shorter or
longer lengths fall within the metes and bounds of this disclosure.
Further, while the female receiver tubes o5 are shown as being
subsequently straight, such tubes could be curved in a J-shaped or
U-shaped manner such that their openings away from the trough
member 30b' could be above the top surface of the liquid 3,'' if
desired.
[0660] With reference to FIGS. 21e, f and g, each electrode 5/5'
was first placed into contact with the liquid 3'' such that it just
entered the female receiver tube o5. After a certain amount of
process time, gold metal was removed from each wire electrode 5
which caused the electrode 5 to thin (i.e., become smaller in
diameter) which changed, for example, current density and/or the
rate at which gold nanoparticles were formed. Accordingly, the
electrodes 5 were moved toward the female receiver tubes o5
resulting in fresh and thicker electrodes 5 entering the liquid 3''
at a top surface portion thereof. In essence, an erosion profile or
tapering effect was formed on the electrodes 5 after some amount of
processing time has passed (i.e., portions of the wire near the
surface of the liquid 3'' were typically thicker than portions near
the female receiver tubes o5), and such wire electrode profile or
tapering can remain essentially constant throughout a production
process, if desired, resulting in essentially identical product
being produced at any point in time after an initial
pre-equilibrium phase during a production run allowing, for
example, the process to be cGMP under current FDA guidelines and/or
be ISO 9000 compliant as well.
[0661] The movement of the electrodes 5 into the female receiver
tubes o5 can occur by monitoring a variety of specific process
parameters which change as a function of time (e.g., current, amps,
nanocrystals concentration, optical density or color, conductivity,
pH, etc.) or can be moved a predetermined amount at various time
intervals to result in a fixed movement rate, whichever may be more
convenient under the totality of the processing circumstances. In
this regard, FIGS. 54d, 55d and 56d show that current was
monitored/controlled as a function of time for each of the 16
electrodes used to make samples GB-139, GB-141 and GB-144,
respectively, causing a vertical movement of the electrodes 5 into
the female receiver tubes o5. Under these processing conditions,
each electrode 5 was moved at a rate of about 3/4 inch every 8
hours (about 2.4 mm/hour) to maintain the currents reported in
FIGS. 54d, 55d and 56d. FIGS. 55d and 56d show a typical ramp-up or
pre-equilibrium phase where the current starts around 0.2-0.4 amps
and increases to about 0.4-0.75 after about 20-30 minutes. Samples
were collected only from the equilibrium phase. The pre-equilibrium
phase occurs because, for example, the concentration of
nanocrystals produced in the liquid 3'' increases as a function of
time until the concentration reaches equilibrium conditions (e.g.,
substantially constant nucleation and growth conditions within the
apparatus), which equilibrium conditions remain substantially
constant through the remainder of the processing due to the control
processes disclosed herein.
[0662] Energy absorption spectra were obtained for the samples in
Example 16 by using UV-VIS spectroscopy. This information was
acquired using a dual beam scanning monochromator system capable of
scanning the wavelength range of 190 nm to 1100 nm. The Jasco V-530
UV-Vis spectrometer was used to collect absorption spectroscopy.
Instrumentation was setup to support measurement of
low-concentration liquid samples using one of a number of
fused-quartz sample holders or "cuvettes". The various cuvettes
allow data to be collected at 10 mm, Imm or 0.1 mm optic path of
sample. Data was acquired over the wavelength range using between
250-900 nm detector with the following parameters; bandwidth of 2
nm, with data pitch of 0.5 nm, a silicon photodiode with a water
baseline background. Both deuterium (D2) and halogen (WI) scan
speed of 400 nm/mm sources were used as the primary energy sources.
Optical paths of these spectrometers were setup to allow the energy
beam to pass through the center of the sample cuvette. Sample
preparation was limited to filling and capping the cuvettes and
then physically placing the samples into the cuvette holder, within
the fully enclosed sample compartment. Optical absorption of energy
by the materials of interest was determined. Data output was
measured and displayed as Absorbance Units (per Beer-Lambert's Law)
versus wavelength.
[0663] Spectral patterns in a UV-Visible range were obtained for
each of the solutions/colloids produced in Example 16.
[0664] Specifically, FIG. 61d shows UV-Vis spectral patterns of
each of the 14 suspensions/colloids, (GB-134) (GB-098, GB-113 and
GB-118); (GB-120 and GB-123); (GB-139); (GB-141 and GB-144);
(GB-079, GB-089 and GB-062); and (GB-076 and GB-077) within a
wavelength range of about 250 nm-750 nm.
[0665] FIG. 61e shows the UV-Vis spectral pattern for each of the
14 suspensions/colloids over a wavelength range of about 435 nm-635
nm.
[0666] In general, UV-Vis spectroscopy is the measurement of the
wavelength and intensity of absorption of near-ultraviolet and
visible light by a sample. Ultraviolet and visible light are
energetic enough to promote outer electrons to higher energy
levels. UV-Vis spectroscopy can be applied to molecules and
inorganic ions or complexes in solution or suspension.
[0667] The UV-Vis spectra have broad features that can be used for
sample identification but are also useful for quantitative
measurements. The concentration of an analyte in solution can be
determined by measuring the absorbance at some wavelength and
applying the Beer-Lambert Law.
Example 17
Manufacturing Gold-Based Nanocrystals/Nanocrystal Suspension
GB-056
[0668] In general, Example 17 utilizes certain embodiments of the
invention associated with the apparatuses generally shown in FIGS.
17a, 18a, 20b and 22a. The trough members 30a (30a') and 30b were
made from 1/4'' (about 6 mm) thick plexiglass, and 1/8'' (about 3
mm) thick polycarbonate, respectively. The support structure 34 was
also made from plexiglass which was about 1/4'' thick (about 6-7 mm
thick). As shown in FIG. 20b, the trough member 30a was integrated
with trough member 30b' and was designated 30a' (e.g., no separate
pumping means was provided after trough member 30a, as in certain
previous examples). The cross-sectional shape of the trough member
30a' as shown in FIGS. 18a and 20b corresponds to that shape shown
in FIG. 10b (i.e., a truncated "V"). The base portion "R" of the
truncated "V" measured about 0.5'' (about 1 cm), and each side
portion "S", "S'" measured about 1.5'' (about 3.75 cm). The
distance "M" separating the side portions "S", "S'" of the V-shaped
trough member 30a was about 21/4''-2 5/16'' (about 5.9 cm)
(measured from inside to inside). The thickness of each sidewall
portion also measured about 1/8'' (about 3 mm) thick. The
longitudinal length "L.sub.T" (refer to FIG. 11a) of the V-shaped
trough member 30a' measured about 1 foot (about 30 cm) long from
point 31 to point 32.
[0669] Purified water (discussed elsewhere herein) was mixed with
about 0.396 g/L of NaHCO.sub.3 and was used as the liquid 3 input
into trough member 30a'. The depth "d" (refer to FIG. 10b) of the
liquid 3' in the V-shaped trough member 30a' was about 7/16'' to
about 1/2'' (about 11 mm to about 13 mm) at various points along
the trough member 30a'. The depth "d" was partially controlled
through use of the dam 80 (shown in FIG. 18a). Specifically, the
dam 80 was provided near the end 32 and assisted in creating the
depth "d" (shown in FIG. 10b) to be about 7/6''-1/2'' (about 11-13
mm) in depth. The height "j" of the dam 80 measured about 1/4''
(about 6 mm) and the longitudinal length "k" measured about 1/2''
(about 13 mm). The width (not shown) was completely across the
bottom dimension "R" of the trough member 30a'. Accordingly, the
total volume of liquid 3' in the V-shaped trough member 30a' during
operation thereof was about 2.14 in.sup.3 (about 35 ml).
[0670] The rate of flow of the liquid 3' into the trough member
30a' was about 150 ml/minute and the rate of flow out of the trough
member 30b' at the point 32 was about 110 ml/minute (i.e., due to
evaporation). Such flow of liquid 3' was obtained by utilizing a
Masterflex.RTM. L/S pump drive 40 rated at 0.1 horsepower, 10-600
rpm. The model number of the Masterflex.RTM. pump 40 was 77300-40.
The pump drive had a pump head also made by Masterflex.RTM. known
as Easy-Load Model No. 7518-10. In general terms, the head for the
pump 40 is known as a peristaltic head. The pump 40 and head were
controlled by a Masterflex.RTM. LS Digital Modular Drive. The model
number for the Digital Modular Drive is 77300-80. The precise
settings on the Digital Modular Drive were, for example, 150
milliliters per minute. Tygon.RTM. tubing having a diameter of
1/4'' (i.e., size 06419-25) was placed into the peristaltic head.
The tubing was made by Saint Gobain for Masterflex.RTM.. One end of
the tubing was delivered to a first end 31 of the trough member
30'a by a flow diffusion means located therein. The flow diffusion
means tended to minimize disturbance and bubbles in water 3
introduced into the trough member 30a' as well as any pulsing
condition generated by the peristaltic pump 40. In this regard, a
small reservoir served as the diffusion means and was provided at a
point vertically above the end 31 of the trough member 30a' such
that when the reservoir overflowed, a relatively steady flow of
liquid 3' into the end 31 of the V-shaped trough member 30a'
occurred.
[0671] There was a single electrode set 1a/5a utilized in this
Example 17. The plasma 4 was created with an electrode 1 similar in
shape to that shown in FIG. 5e, and weighed about 9.2 grams. This
electrode was 99.95% pure gold. The other electrode 5a comprised a
right-triangular shaped platinum plate measuring about 14
mm.times.23 mm.times.27 mm and about 1 mm thick and having about 9
mm submerged in the liquid 3'. All other pertinent run conditions
are shown in Table 10.
[0672] As shown in FIG. 20b, the output from the trough member 30a'
was the conditioned liquid 3' and this conditioned liquid 3' flowed
directly into a second trough member 30b'. The second trough member
30b', shown in FIG. 22a measured about 3.75 inches high, about 3.75
inches wide at the end 32 thereof, and about 1 inch wide at the end
31 thereof. This trough member 30b' contained about 1450 ml of
liquid 3'' therein which was about 2.5 inches deep. In this
Example, each of four electrode sets 5a, 5a'-5d, 5d' comprised
99.95% pure gold wire measuring about 0.5 mm in diameter. The
length of each wire 5 measured about 5 inches (about 12 cm) long.
The liquid 3'' was about 2.5 inches deep (about 6 cm) with about
4.25 inches (about 11 cm) of the j-shaped wire being submerged
therein. Each electrode set 5b, 5b'-5e, 5e' was shaped like a "J",
as shown in FIG. 17a. The distance "g" shown in FIG. 17a measured
about 1-8 mm.
[0673] With regard to FIGS. 20b and 22a, 4 separate electrode sets
(Set 2, Set 3, Set 4 and Set 5) were attached to 2 separate
transformer devices, 50 and 50a as shown in FIG. 20b. Specifically,
transformers 50 and 50a were electrically connected to each
electrode set, according to the wiring diagram show in FIG. 19a.
Each transformer device 50, 50a was connected to a separate AC
input line that was 1200 out of phase relative to each other. The
transformers 50 and 50a were electrically connected in a manner so
as not to overload a single electrical circuit and cause, for
example, an upstream circuit breaker to disengage (e.g., when
utilized under these conditions, a single transformer 50/50a could
draw sufficient current to cause upstream electrical problems).
Each transformer 50/50a was a variable AC transformer constructed
of a single coil/winding of wire. This winding acts as part of both
the primary and secondary winding. The input voltage was applied
across a fixed portion of the winding. The output voltage was taken
between one end of the winding and another connection along the
winding. By exposing part of the winding and making the secondary
connection using a sliding brush, a continuously variable ratio was
obtained. The ratio of output to input voltages is equal to the
ratio of the number of turns of the winding they connect to.
Specifically, each transformer was a Mastech TDGC2-5 kVA, 10A
Voltage Regulator, Output 0-250V.
[0674] Table 6 refers to each of the 4 electrode sets by "Set #".
Each electrode of the 4 electrode sets was set to operate within a
specific voltage range. The actual voltages, listed in Table 10,
were about 255 volts. The distance "c-c" (with reference to FIG.
14) from the centerline of each electrode set to the adjacent
electrode set is also represented. Further, the distance "x"
associated with the electrode 1 utilized is also reported. For the
electrode 5, no distance "x" is reported. Other relevant parameters
are reported in Table 6.
[0675] All materials for the electrodes 1/5 were obtained from ESPI
having an address of 1050 Benson Way, Ashland, Oreg. 97520.
TABLE-US-00020 TABLE 6 0.396 mg/ml of NaHCO.sub.3 (Au) Run ID:
GB-056 Flow Rate: 150 ml/min Voltage: 255 V NaHCO.sub.3: 0.396
mg/ml Wire Dia.: .5 mm Configuration: J/J PPM: 12 Distance Distance
Elec- "c-c" "x" Volt- cross Set# trode# in/mm in/mm age section
4.5/114.3* 1 1a 0.25/6.35 750 V 5a N/A 750 23/584.2** 2.5/63.5* 2
5b N/A 255 .sup. 5b' N/A 3.5/88.9 3 5c N/A 255 Tapered .sup. 5c'
N/A 3''Deep 3.5/88.9 4 5d N/A 255 .sup. 5d' N/A 3.5/88.9 5 5e N/A
255 .sup. 5e' N/A 376.2** Output Water Temperature 98 C. *Distance
from water inlet to center of first electrode set **Distance from
center of last electrode set to water outlet
[0676] FIGS. 100a-e show five representative TEM photomicrographs
of the gold nanocrystals, dried from the solution/colloid GB-056,
formed according to Example 16.
[0677] FIG. 101a shows the measured size distribution of the gold
nanocrystals dried from the suspension/colloid measured by using
the TEM instrument/software discussed earlier in Examples 5-7.
[0678] FIG. 101b shows graphically three dynamic light scattering
data measurement sets for the nanocrystals (i.e., the hydrodynamic
radii) made according to this Example 17. It should be noted that
the dynamic light scattering particle size information is different
from the TEM measured histograms because dynamic light scattering
uses algorithms that assume the nanocrystals are all spheres (which
they are not) as well as measures the hydrodynamic radius (e.g.,
the nanocrystal's influence on the water is also detected and
reported in addition to the actual physical radii of the
nanocrystals). Accordingly, it is not surprising that there is a
difference in the reported nanocrystal sizes between those reported
in the TEM histogram data of those reported in the dynamic light
scattering data just as in the other Examples included herein.
[0679] FIGS. 102a[-], 102b, 102c and 102d show additional
representative TEM photomicrographs of the same suspension/colloid
GB-056 made according to Example 17, however, this
suspension/colloid was exposed to the mice via their water bottles
in Treatment Group B discussed in Example 26. It should be noted
that these representative TEM nanocrystal images are of the dried
solution GB-056 so certain drying conditions can affect the images.
It is clear that some clustering together of the gold nanocrystals
occurred, for example, during drying. However, FIG. 103a shows
nanocrystal size distributions which are substantially similar to
those that are shown in FIG. 101a. In this regard, the data shown
in FIGS. 102 and 103 correspond to suspensions that were in the
mouse drinking bottles for a 24-hour time period between day 2 and
day 3 of the Example 26 EAE study. Of interest, is the comparison
of FIG. 103b to FIG. 101b. In this regard, the dynamic light
scattering data has changed. Specifically, the largest hydrodynamic
radius shown in FIG. 101b is about 16.8 nm, whereas in FIG. 103b,
it is about 20.2 nm. Clearly, the dynamic light scattering data is
recognizing some type of the clustering of nanocrystals in
suspension which is also represented by the dried suspension/gold
nanocrystal TEM photomicrographs shown in FIGS. 102a[-], 102b, 102c
and 102d.
[0680] Likewise, FIGS. 104a-104c; FIG. 105a; and FIG. 105c all
correspond to suspension/colloid GB-056 that was in the drinking
bottles for a 24-hour time period between day 4 and day 5 of the
EAE study discussed in Example 26. Once again, it is evident that
some type of clumping together of the nanocrystals was
occurring.
[0681] While FIGS. 101a, 103a and 105a are all substantially
similar for TEM measured nanocrystal sizes, it is clear that the
dynamic light scattering radii (e.g., the hydrodynamic radii) of
the nanocrystals has enlarged, as shown in FIG. 105b, just as it
enlarged in FIG. 103b, both relative to the smaller hydrodynamic
radii reported in FIG. 101b.
[0682] Taken together, these data suggest that exposure of the
inventive compositions disclosed herein to certain constituents in,
for example, mouse saliva, can cause a clustering or clumping
together of the nanocrystals suspended in the liquid. Accordingly,
prolonged exposure to certain proteins may have a "denaturing"
effect on these inventive compositions. This "denaturing" effect is
manageable, and without wishing to be bound by any particular
theory or explanation, may be very desirable in that such
reactivity due to very "clean" surfaces may support desirable in
vivo activity (e.g., certain protein-binding mechanisms).
Example 18
Manufacturing Gold-Based Nanocrystals/Nanocrystal Suspensions
(GB-151, GB-188, GB-175, GB-177, GB-176, GB-189, GB-194, GB-195,
GB-196, GB-198 and GB-199)
[0683] In general, this Example utilizes certain embodiments of the
invention associated with the apparatuses generally shown in FIGS.
18a and 21d. Control devices 20 (not shown in FIG. 21d) were
connected to the electrodes 1/5 and 5/5, however, due to the short
run times in each "Run ID," there was no need to actuate the
control devices 20. Accordingly, in reference to FIGS. 3c and 9c,
the ends 9' of the electrodes 5a and 5b were juxtaposed with the
bottom of the trough member 30b'. Additionally, Table 7 summarizes
key processing parameters used in conjunction with FIGS. 18a and
21d. Also, Table 7 discloses: 1) resultant "ppm" (i.e., gold
nanocrystal concentrations) and 2) "TEM Average Diameter" which is
the mode, corresponding to the crystal diameter that occurs most
frequently, determined by the TEM histograms shown in FIGS.
62b-72b. These physical characterizations were performed as
discussed elsewhere herein.
TABLE-US-00021 TABLE 7 Run ID: GB-151 GB-188 GB-175 GB-177 GB-176
Flow In (ml/min) 220 230 230 230 230 Rate: Out (ml/min) 175 184 184
184 184 Volts: Set # 1 750 750 750 750 n/a Set #'s 2-8 230 198 210
208 210 PE: NaHCO3 (mg/ml) 0.53 0.53 0.53 0.53 0.53 Wire Diameter
(mm) 1.0 1.0 2.0 1.1 3.0 Contact "W.sub.L" (in/mm) 1/25 1/25 1/25
1/25 1/25 Electrode Separation "y" (in/mm) .25/6.4 .25/6.4 .25/6.4
.25/6.4 .25/6.4 Electrode Config. FIG. 17b 17b 17b 17b 17b Produced
Au PPM 8.3 8.4 10.5 9.5 10.1 Output Temp .degree. C. at 32 89 84 89
88 86 Dimensions Plasma 4 FIGS. 18a 18a 18a 18a n/a Process FIGS.
21d 21d 21d 21d 21d M1 (in/mm) 2/51 1.5/38 1.5/38 1.5/38 1.5/38
L.sub.T (in/mm) 30/762 36/914 36/914 36/914 36/914 d (in/mm) 1/25
1/25 1/25 1/25 1/25 S (in/mm) 1.5/38 1.5/38 2/51 2/51 2/51
Electrode Curr. (A) 0.89 .85 .93 .80 .88 Total Curr. Draw (A) n/m
6.06 7.02 6.84 6.82 Hydrodynamic r (nm) 11.6 12 14 13.1 13.2 TEM
Avg. Dia. (nm) 10.85 10.63 11.76 10.85 10.42 "c-c" (mm) 152 76 76
76 n/a Set electrode # 1a 1a 1a 1a n/a 1 "x" (in/mm) 0.25/6.4
0.25/6.4 0.25/6.4 0.25/6.4 n/a electrode # 5a 5a 5a 5a n/a "c-c"
(mm) 63 102 102 102 102 Set electrode # 5b 5b 5b 5b 5b 2 "x"
(in/mm) n/a n/a n/a n/a n/a electrode # .sup. 5b' .sup. 5b' .sup.
5b' .sup. 5b' .sup. 5b' "c-c" (mm) 76 76 76 76 76 Set electrode #
5c 5c 5c 5c 5c 3 electrode # .sup. 5c' .sup. 5c' .sup. 5c' .sup.
5c' .sup. 5c' "c-c" (mm) 76 76 76 76 76 Set electrode # 5d 5d 5d 5d
5d 4 electrode # .sup. 5d' .sup. 5d' .sup. 5d' .sup. 5d' .sup. 5d'
"c-c" (mm) 114 127 127 127 127 Set electrode # 5e 5e 5e 5e 5e 5
electrode # .sup. 5e' .sup. 5e' .sup. 5e' .sup. 5e' .sup. 5e' "c-c"
(mm) 114 127 127 127 127 Set electrode # 5f.sup. 5f.sup. 5f.sup.
5f.sup. 5f.sup. 6 electrode # 5f' 5f' 5f' 5f' 5f' "c-c" (mm) 114
152 152 152 152 Set electrode # 5g 5g 5g 5g 5g 7 electrode # .sup.
5g' .sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g' "c-c" (mm) 127 178 178
178 178 Set electrode # 5h 5h 5h 5h 5h 8 electrode # .sup. 5h'
.sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' "c-c" (mm) 76 76 76 76 76
Run ID: GB-189 GB-194 GB-195 GB-196 GB-198 GB-199 Flow In (ml/min)
230 250 250 250 150 150 Rate: Out (ml/min) 184 200 200 200 120 120
Volts: Set # 1 750 750 750 750 n/a 750 Set #'s 2-8 208 210 210 210
205 205 PE: NaHCO3 (mg/ml) 0.53 0.53 0.53 0.53 0.26 0.26 Wire
Diameter (mm) 1.2 4.0 1.3 5.0 1.4 6.0 Contact "W.sub.L" (in/mm)
1/25 1/25 1/25 1/25 1/25 1/25 Electrode Separation "y" (in/mm)
.25/6.4 .25/6.4 .25/6.4 .25/6.4 .125/3.18 .125/3.18 Electrode
Config. FIG. 17b 17b 17b 17b 17b 17b Produced Au PPM 8.4 8.7 7.7
8.7 9.9 12.4 Output Temp .degree. C. at 32 85 93 96 89 74 80
Dimensions Plasma 4 FIGS. 18a 18a 18a 18a n/a 18a Process FIGS. 21d
21d 21d 21d 21d 21d M1 (in/mm) 1.5/38 .75/19 .5/13 1/25 1.5/38
1.5/38 L.sub.T (in/mm) 36/914 36/914 36/914 36/914 36/914 36/914 d
(in/mm) 1/25 1/25 1/25 1/25 .75/19 .75/19 S (in/mm) 2/51 2/51 2/51
1.5/38 2/51 2/51 Electrode Curr. (A) .91 n/m n/m n/m n/m n/m Total
Curr. Draw (A) 6.36 6.25 5.59 5.93 3.57 3.71 Hydrodynamic r (nm) 12
16 16 12.5 13.9 14.2 TEM Avg. Dia. (nm) 10.42 12.06 11.11 12.06
11.74 13.02 "c-c" (mm) 76 76 76 76 n/a 76 Set electrode # 1a 1a 1a
1a n/a 1a 1 "x" (in/mm) 0.25/6.4 0.25/6.4 0.25/6.4 0.25/6.4 n/a
0.25/6.4 electrode # 5a 5a 5a 5a n/a 5a "c-c" (mm) 102 102 102 102
102 102 Set electrode # 5b 5b 5b 5b 5b 5b 2 "x" (in/mm) n/a n/a n/a
n/a n/a n/a electrode # .sup. 5b' .sup. 5b' .sup. 5b' .sup. 5b'
.sup. 5b' .sup. 5b' "c-c" (mm) 76 76 76 76 76 76 Set electrode # 5c
5c 5c 5c 5c 5c 3 electrode # .sup. 5c' .sup. 5c' .sup. 5c' .sup.
5c' .sup. 5c' .sup. 5c' "c-c" (mm) 76 76 76 76 76 76 Set electrode
# 5d 5d 5d 5d 5d 5d 4 electrode # .sup. 5d' .sup. 5d' .sup. 5d'
.sup. 5d' .sup. 5d' .sup. 5d' "c-c" (mm) 127 127 127 127 127 127
Set electrode # 5e 5e 5e 5e 5e 5e 5 electrode # .sup. 5e' .sup. 5e'
.sup. 5e' .sup. 5e' .sup. 5e' .sup. 5e' "c-c" (mm) 127 127 127 127
127 127 Set electrode # 5f.sup. 5f.sup. 5f.sup. 5f.sup. 5f.sup.
5f.sup. 6 electrode # 5f' 5f' 5f' 5f' 5f' 5f' "c-c" (mm) 152 152
152 152 152 152 Set electrode # 5g 5g 5g 5g 5g 5g 7 electrode #
.sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g' .sup. 5g' "c-c"
(mm) 178 178 178 178 178 178 Set electrode # 5h 5h 5h 5h 5h 5h 8
electrode # .sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' .sup. 5h' .sup.
5h' "c-c" (mm) 76 76 76 76 76 76
[0684] All trough members 30a' and 30b' in the aforementioned FIGS.
18a and 21d were made from 1/8'' (about 3 mm) thick plexiglass, and
1/4'' (about 6 mm) thick polycarbonate, respectively. The support
structure 34 (not shown in the Figures but discussed elsewhere
herein) was also made from plexiglass which was about 1/4'' thick
(about 6-7 mm thick). In contrast to the embodiments shown in FIGS.
19a and 19b, each trough member 30a was integral with trough member
30b' and was thus designated 30a' (e.g., no separate pumping means
was provided after trough member 30a, as in certain previous
examples). The cross-sectional shape of each trough member 30a'
used in this Example corresponded to that shape shown in FIG. 10b
(i.e., was a trapezoidal-shaped cross-section). Relevant dimensions
for each trough member portion 30b' are reported in Table 7 as "M1"
(i.e., the inside width of the trough at the entrance portion of
the trough member 30b') was the same as the inside width of the
trough at the exit portion of the trough member 30b'), "L.sub.T"
(i.e., transverse length or flow length of the trough member 30b'),
"S" (i.e., the height of the trough member 30b'), and "d" (i.e.,
depth of the liquid 3'' within the trough member 30b'). The
thickness of each sidewall portion also measured about 1/4'' (about
6 mm) thick. Two different longitudinal lengths "L.sub.T" are
reported for the trough members 30b' (i.e., either 762 mm or 914
mm) however, other lengths L.sub.T should be considered to be
within the metes and bounds of the inventive trough.
[0685] Table 7 shows that the processing enhancer NaHCO.sub.3 was
added to purified water (discussed elsewhere herein) in amounts of
either about 0.26 mg/ml or 0.53 mg/ml. It should be understood that
other amounts of this processing enhancer (and other processing
enhancers) also function within the metes and bounds of the
invention. The purified water/NaHCO.sub.3 mixture was used as the
liquid 3 input into trough member 30a'. The depth "d" of the liquid
3' in the trough member 30a' (i.e., where the plasma(s) 4 is/are
formed) was about 7/16'' to about 1/2'' (about 11 mm to about 13
mm) at various points along the trough member 30a'. The depth "d'"
was partially controlled through use of the dam 80 (shown in FIGS.
18a and 18b). Specifically, the dam 80 was provided near the output
end 32 of the trough member 30a' and assisted in creating the depth
"d" (shown in FIG. 10b as "d") to be about 7/6''-1/2'' (about 11-13
mm) in depth. The height "j" of the dam 80 measured about 1/4''
(about 6 mm) and the longitudinal length "k" measured about 1/2''
(about 13 mm). The width (not shown) was completely across the
bottom dimension "R" of the trough member 30a'. Accordingly, the
total volume of liquid 3' in the trough member 30a' during
operation thereof was about 2.14 in.sup.3 (about 35 ml) to about
0.89 in.sup.3 (about 14.58 ml).
[0686] The rate of flow of the liquid 3' into the trough member
30a' as well as into trough member 30b', varied (as shown in Table
7) and the rate of flow out of the trough member 30b' at the point
32 also varied due to different flow rate inputs and evaporation.
Other acceptable flow rates should be considered to be within the
metes and bounds of the invention.
[0687] Such flow of liquid 3' was obtained by utilizing a
Masterflex.RTM. L/S pump drive 40 rated at 0.1 horsepower, 10-600
rpm. The model number of the Masterflex.RTM. pump 40 was 77300-40.
The pump drive had a pump head also made by Masterflex.RTM. known
as Easy-Load Model No. 7518-10. In general terms, the head for the
pump 40 is known as a peristaltic head. The pump 40 and head were
controlled by a Masterflex.RTM. LS Digital Modular Drive. The model
number for the Digital Modular Drive is 77300-80. The precise
settings on the Digital Modular Drive were, for example, 150
milliliters per minute for all samples except GB-144 which was, for
example, 110 ml/minute. Tygon.RTM. tubing having a diameter of
1/4'' (i.e., size 06419-25) was placed into the peristaltic head.
The tubing was made by Saint Gobain for Masterflex.RTM.. One end of
the tubing was delivered to a first end 31 of the trough member
30'a by a flow diffusion means located therein. The flow diffusion
means tended to minimize disturbance and bubbles in water 3
introduced into the trough member 30a' as well as any pulsing
condition generated by the peristaltic pump 40. In this regard, a
small reservoir served as the diffusion means and was provided at a
point vertically above the end 31 of the trough member 30a' such
that when the reservoir overflowed, a relatively steady flow of
liquid 3' into the end 31 of the V-shaped trough member 30a'
occurred.
[0688] Table 7 shows that there was a single electrode set 1a/5a,
utilized in this Example 18. The plasma(s) 4 was/were created with
an electrode 1 similar in shape to that shown in FIG. 5e, and
weighed about 9.2 grams. This electrode was 99.95% pure gold. The
other electrode 5a comprised a 99.95% 1 mm gold wire submerged in
the liquid 3'. All other pertinent run conditions are shown in
Table 7.
[0689] The output from the trough member 30a' was the conditioned
liquid 3' and this conditioned liquid 3' flowed directly into a
second trough member 30b'. The second trough member 30b', shown in
FIG. 21d had measurements as reported in Table 7. This trough
member 30b' contained from about 260 ml of liquid 3'' therein to
about 980 ml depending on the dimensions of the trough and the
depth "d''" of the liquid 3'' therein. Table 7, in connection with
FIG. 21d the electrode configurations used. For example, previous
examples herein disclosed the use of four sets of electrodes 5/5,
with one electrode set 1/5. In this Example, eight electrode sets
were used (e.g., one 1/5 set with seven or eight 5/5' sets). Each
of the electrode sets 5/5' comprised 99.99% pure gold wire
measuring either about 0.5 mm in diameter or 1.0 mm in diameter, as
reported in Table 7. The length of each wire electrode 5 that was
in contact with the liquid 3'' (reported as "W.sub.L" in Table 7)
measured from about 0.75 inches (about 19 mm) long to about 1 inch
(about 25 mm) long. FIG. 21d shows that the electrode sets 5/5'
were arranged as shown in FIG. 5c.
[0690] Each electrode set 5a/5b was connected to a Chroma 61604
programmed AC power source (not shown and as discussed elsewhere
herein). The applied voltages are reported in Table 7.
Specifically, Table 7 refers to each of the electrode sets by "Set
#" (e.g., "Set 1" through "Set 8"). Each electrode of the 1/5 or
5/5 electrode sets was set to operate within a specific voltage
range. The voltages listed in Table 7 are the voltages used for
each electrode set. The distance "c-c" (with reference to FIG. 14)
from the centerline of each electrode set to the adjacent electrode
set is also reported. Further, the distance "x" (e.g., see FIG. 2a)
associated with each electrode 1 utilized is also reported. Other
relevant parameters are also reported in Table 7.
[0691] All materials for the electrodes 1/5 were obtained from
Hi-Rel Alloys having an address of 23. Lewis Street, Fort Erie,
Ontario L2A2P6, Canada.
[0692] FIGS. 62a-72a show two representative TEM photomicrographs
for each of the gold nanoparticles, dried from each solution or
colloid referenced in Table 7, and formed according to Example
18.
[0693] FIGS. 62b-72b show the measured size distribution of the
gold particles measured by using the TEM instrument/software
discussed earlier in Examples 5-7 for each dried solution or
colloid referenced in Table 7 and formed according to Example
18.
[0694] Energy absorption spectra were obtained for the samples in
Example 18 by using UV-VIS spectroscopy. This information was
acquired using a dual beam scanning monochromator system capable of
scanning the wavelength range of 190 nm to 1100 nm. The Jasco V-530
UV-Vis spectrometer was used to collect absorption spectroscopy.
Instrumentation was setup to support measurement of
low-concentration liquid samples using one of a number of
fused-quartz sample holders or "cuvettes." The various cuvettes
allow data to be collected at 10 mm, 1 mm or 0.1 mm optic path of
sample. Data was acquired over the wavelength range using between
250-900 nm detector with the following parameters; bandwidth of 2
nm, with data pitch of 0.5 nm, a silicon photodiode with a water
baseline background. Both deuterium (D2) and halogen (WI) scan
speed of 400 nm/mm sources were used as the primary energy sources.
Optical paths of these spectrometers were setup to allow the energy
beam to pass through the center of the sample cuvette. Sample
preparation was limited to filling and capping the cuvettes and
then physically placing the samples into the cuvette holder, within
the fully enclosed sample compartment. Optical absorption of energy
by the materials of interest was determined. Data output was
measured and displayed as Absorbance Units (per Beer-Lambert's Law)
versus wavelength.
[0695] Spectral patterns in a UV-Visible range were obtained for
each of the solutions/colloids produced in Example 18.
[0696] Specifically, FIG. 72c shows UV-Vis spectral patterns of
each of the 11 suspensions/colloids, (GB-151, GB-188, GB-175,
GB-177, GB-176, GB-189, GB-194, GB-195, GB-196, GB-198 and GB-199)
within a wavelength range of about 250 nm-750 nm.
[0697] FIG. 72d shows the UV-Vis spectral pattern for each of the
11 suspensions/colloids over a wavelength range of about 435 nm-635
nm.
[0698] In general, UV-Vis spectroscopy is the measurement of the
wavelength and intensity of absorption of near-ultraviolet and
visible light by a sample. Ultraviolet and visible light are
energetic enough to promote outer electrons to higher energy
levels. UV-Vis spectroscopy can be applied to molecules and
inorganic ions or complexes in solution.
[0699] The UV-Vis spectra have broad features that can be used for
sample identification but are also useful for quantitative
measurements. The concentration of an analyte in solution can be
determined by measuring the absorbance at some wavelength and
applying the Beer-Lambert Law.
Example 19
Manufacturing Gold-Based Nanoparticles/Nanoparticle Suspensions or
Colloids Aurora-002, Aurora-004, Aurora-006, Aurora-007,
Aurora-009, Aurora-011, Aurora-012, Aurora-013, Aurora-014,
Aurora-016, Aurora-017, Aurora-019, Aurora-020, Aurora-021,
Aurora-022, Aurora-023, Aurora-024, Aurora-025, Aurora-026,
Aurora-027, Aurora-028, Aurora-029 and Aurora-030
[0700] In general, Example 19 utilizes a trough member 30 and
electrode 1/5 combination different from any of the other Examples
disclosed herein. Specifically, this Example utilizes a first set
of four electrodes 1 and a single electrode 5a in a trough member
30a' which create a plurality of plasmas 4, resulting in
conditioned liquid 3'. The conditioned liquid 3' flows into and
through a longitudinal trough member 30b', wherein parallelly
located electrodes 5b/5b' are positioned along substantially the
entire longitudinal or flow length of the trough member 30b'.
Specific reference is made to FIGS. 23a, 23b, 23c and 23d which
show various schematic and perspective views of this embodiment of
the invention. Additionally, Table 8 contains relevant processing
parameters associated with this embodiment of the invention.
TABLE-US-00022 TABLE 8 Aurora- Aurora- Aurora- Aurora- Aurora- Run
ID: 002 004 006 007 009 Flow Rate: In (ml/min) 300 300 150 150 150
Volts: Set # 1 1000 1000 1000 1000 1000 Electrodes 5b 100 120 100
50 100 # of Electrodes 1 4 4 4 4 4 PE: NaHCO3 (mg/ml) 0.396 0.396
0.396 0.396 0.396 Wire Diameter (mm) 0.5 0.5 0.5 0.5 0.5 Electrode
Config. FIG. 23a 23a 23a 23a 23a Produced Au PPM 12.3 15.9 39.6 4.1
17.8 Dimensions Plasma 4 FIGS. 23a 23a 23a 23a 23a Process FIGS.
23a, 23b, 23a, 23b, 23a, 23b, 23a, 23b, 23a, 23b, 23c, 23d 23c, 23d
23c, 23d 23c, 23d 23c, 23d Wire Length (in) 54 54 54 54 54
"W.sub.L" L.sub.T (in/mm) 59/1500 59/1500 59/1500 59/1500 59/1500
wire apart 0.125/3.2 0.125/3.2 0.125/3.2 0.125/3.2 0.125/3.2
(in/mm) "b" Electrode Curr. (A) 10.03 14.2 15.3 5.2 11.9
Hydrodynamic r (nm) 23.2 19.4 23.2 26.2 19.6 TEM Avg. Dia. (nm) n/a
n/a n/a n/a n/a Aurora- Aurora- Aurora- Aurora- Run ID: 011 012 013
014 Flow Rate: In (ml/min) 300 450 60 60 Volts: Set # 1 1000 1000
1000 1000 Electrodes 5b 90 110 50 40 # of Electrodes 1 4 4 4 4 PE:
NaHCO3 (mg/ml) 0.396 0.396 0.396 0.396 Wire Diameter (mm) 0.5 0.5
0.5 0.5 Electrode Config. FIG. 23a 23a 23a 23a Produced Au PPM 17.4
12.7 46.5 65.7 Dimensions Plasma 4 FIGS. 23a 23a 23a 23a Process
FIGS. 23a, 23b, 23a, 23b, 23a, 23b, 23a, 23b, 23c, 23d 23c, 23d
23c, 23d 23c, 23d Wire Length (in) 54 54 54 54 "W.sub.L" L.sub.T
(in/mm) 59/1500 59/1500 59/1500 59/1500 wire apart 0.063/1.6
0.063/1.6 0.063/1.6 0.063/1.6 (in/mm) "b" Electrode Curr. (A) 15.9
19.5 10 7.87 Hydrodynamic r (nm) 16.3 13.1 26.2 22.0 TEM Avg. Dia.
(nm) n/a n/a n/a n/a Aurora- Aurora- Aurora- Aurora- Aurora- Run
ID: 016 017 019 020 021 Flow Rate: In (ml/min) 60 30 30 30 30
Volts: Set # 1 1000 1000 1000 1000 1000 Electrodes 5b 30 30 30 50
50 # of Electrodes 1 4 4 1 1 4 PE: NaHCO3 (mg/ml) 0.396 0.396 0.396
0.396 0.396 Wire Diameter (mm) 0.5 0.5 0.5 0.5 0.5 Electrode
Config. FIG. 23a 23a 23a 23a 23a Produced Au PPM 35.5 24.8 22.5
128.2 67.1 Dimensions Plasma 4 FIGS. 23a 23a 23a 23a 23a Process
FIGS. 23a, 23b, 23a, 23b, 23a, 23b, 23a, 23b, 23a, 23b, 23c, 23d
23c, 23d 23c, 23d 23c, 23d 23c, 23d Wire Length (in) 54 54 54 54 54
"W.sub.L" L.sub.T (in/mm) 59/1500 59/1500 59/1500 59/1500 59/1500
wire apart 0.063/1.6 0.063/1.6 0.063/1.6 0.063/1.6 0.063/1.6
(in/mm) "b" Electrode Curr. (A) 5.18 4.95 4.65 10.7 10 Hydrodynamic
r (nm) 26.6 27.4 26.0 31.0 27.1 TEM Avg. Dia. (nm) n/a n/a n/a
16-40 n/a Aurora- Aurora- Aurora- Aurora- Run ID: 022 023 024 025
Flow Rate: In (ml/min) 60 60 60 60 Volts: Set # 1 1000 1000 1000
1000 Electrodes 5b 50 80 30 30 # of Electrodes 1 4 4 4 4 PE: NaHCO3
(mg/ml) 0.396 0.396 3.963 3.963 Wire Diameter (mm) 0.5 0.5 0.5 0.5
Electrode Config. FIG. 23a 23a 23a 23a Produced Au PPM 64.2 73.8
0.8 0.5 Dimensions Plasma 4 FIGS. 23a 23a 23a 23a Process FIGS.
23a, 23b, 23a, 23b, 23a, 23b, 23a, 23b, 23c, 23d 23c, 23d 23c, 23d
23c, 23d Wire Length (in) 54 50 50 50 "W.sub.L" L.sub.T (in/mm)
59/1500 59/1500 59/1500 59/1500 wire apart 0.063/1.6 0.063/1.6
0.063/1.6 0.063/1.6 (in/mm) "b" Electrode Curr. (A) 9.8 18 17 14.96
Hydrodynamic r (nm) 28.3 27.0 n/a n/a TEM Avg. Dia. (nm) n/a n/a
n/a n/a Aurora- Aurora- Aurora- Aurora- Aurora- Run ID: 026 027 028
029 030 Flow Rate: In (ml/min) 60 60 60 60 60 Volts: Set # 1 1000
1000 1000 1000 1000 Electrodes 5b 30 30 100 130 150 # of Electrodes
1 4 4 4 4 4 PE: NaHCO3 (mg/ml) 3.963 3.963 0.106 0.106 0.106 Wire
Diameter (mm) 0.5 0.5 0.5 0.5 0.5 Electrode Config. FIG. 23a 23a
23a 23a 23a Produced Au PPM 3.7 2.0 8.1 21.6 41.8 Dimensions Plasma
4 FIGS. 23a 23a 23a 23a 23a Process FIGS. 23a, 23b, 23a, 23b, 23a,
23b, 23a, 23b, 23a, 23b, 23c, 23d 23c, 23d 23c, 23d 23c, 23d 23c,
23d Wire Length (in) 50 50 50 50 50 "W.sub.L" L.sub.T (in/mm)
59/1500 59/1500 59/1500 59/1500 59/1500 wire apart 0.063/1.6
0.063/1.6 0.063/1.6 0.063/1.6 0.063/1.6 (in/mm) "b" Electrode Curr.
(A) 13.4 16.32 6.48 10 12 Hydrodynamic r (nm) 33.7 and n/a 26.1
21.9 25.2 77.5 TEM Avg. Dia. (nm) n/a n/a n/a n/a n/a
[0701] With regard to FIG. 23a, two AC power sources 60 and 60a are
electrically connected as shown and create four separate plasmas
4a, 4b, 4c and 4d at four corresponding electrodes 1a, 1b, 1c and
1d, in a first trough member portion 30a'. As shown in FIG. 23a,
only a single electrode 5a is electrically connected to all four
electrodes 1. These power sources 60 and 60a are the same power
sources reported in other Examples herein. Two different amounts of
processing enhancer NaHCO.sub.3 were added to the liquid 3 prior to
the four plasmas 4a-4d conditioning the same as reported in Table
13. The amount and type of processing enhancer reported should not
be construed as limiting the invention. The rate of flow of the
liquid 3/3' into and out of the trough member 30a', as well as into
the trough member 30b' is also reported in Table 8. The rate of
flow out of the trough member 30b' was approximately 5% to 50%
lower due to liquid loss in evaporation, with higher evaporation at
higher power input at electrodes 5b/5b'. Varying flow rates for the
liquid 3/3' can be utilized in accordance with the teachings
herein.
[0702] Only one set of electrodes 5b/5b' was utilized in this
particular embodiment. These electrodes 5b/5b' were connected to an
AC power source 50, as described in the other Examples herein. The
gold wire electrodes 5b/5b' used in this particular Example were
the same gold wires, with dimensions as reported in Table 8, that
were used in the other Examples reported herein. However, a
relatively long length (i.e., relative to the other Examples
herein) of gold wire electrodes was located along the longitudinal
length L.sub.T of the trough member 30b'. The wire length for the
electrodes 5b/5b' is reported in Table 8. Two different wire
lengths either 50 inches (127 cm) or 54 inches (137 cm) were
utilized. Further, different transverse distances between the wires
5b/5b' are also reported. Two separate transverse distances are
reported herein, namely, 0.063 inches (1.6 mm) and 0.125 inches
(3.2 mm). Different electrode 5b/5b' lengths are utilizable as well
as a plurality of different transverse distances between the
electrodes 5b/5b'.
[0703] The wire electrodes 5b/5b' were spatially located within the
liquid 3'' in the trough member 30b' by the devices Gb, Gb', T8,
T8', Tb and Tb' near the input end 31 (refer to FIG. 23c) and
corresponding devices Gb, Gb', Cb, Cb', Cbb and Cb'b' (refer to
FIG. 23d) near the output end 32. It should be understood that a
variety of devices could be utilized to cause the electrodes 5b/5b'
to be contiguously located along the trough member 30b' and those
reported herein are exemplary. Important requirements for locating
the electrodes 5b/5b' include the ability to maintain desired
transverse separation between the electrodes along their entire
lengths which are in contact with the liquid 3'' (e.g., contact of
the electrodes with each other would cause an electrical short
circuit). Specifically, the electrodes 5b/5b' are caused to be
drawn through guide members Gb and Gb' made of polycarbonate near
the input end 31 and the glass near output end 32. The members Gb
and Gb' at each end of the trough member 30b' are adjusted in
location by the compasses Cbb, Cb'b' near an output end 32 of the
trough member 30b' and similar compasses Cb and Cb' at the opposite
end of the trough 30b'. Electrical connection to the electrodes
5b/5b' was made at the output end 32 of the trough member 30b' near
the top of the guide members Gb and Gb'. Tension springs Tb and Tb'
are utilized to keep the electrode wires 5b/5b' taught so as to
maintain the electrodes in a fixed spatial relationship to each
other. In this regard, the electrodes 5b/5b' can be substantially
parallel along their entire length, or they can be closer at one
end thereof relative to the other (e.g., creating different
transverse distances along their entire length). Controlling the
transverse distance(s) between electrode 5b/5b' influences current,
current density concentration, voltages, etc. Of course, other
positioning means will occur to those of ordinary skill in the art
and the same are within the metes and bounds of the present
invention.
[0704] Table 8 shows a variety of relevant processing conditions,
as well as certain results including, for example, "Hydrodynamic r"
(i.e., hydrodynamic radii (reported in nanometers)) and the process
current that was applied across the electrodes 5b/5b'.
Additionally, resultant ppm levels are also reported for a variety
of process conditions with a low of about 0.5 ppm and a high of
about 128 ppm.
[0705] FIG. 73a shows two representative TEM photomicrographs of
the gold nanoparticles, dried from the solution or colloid
Aurora-020, which has a reported 128 ppm concentration of gold
measured next day after synthesis. In two weeks the concentration
of that sample reduced to 107 ppm, after another 5 weeks the
concentration reduced to 72 ppm.
[0706] FIG. 73b shows the measured size distribution of the gold
nanoparticles measured by the TEM instrument/software discussed
earlier in Examples 5-7 corresponding to dried Aurora-020.
[0707] FIG. 73c shows graphically dynamic light scattering data
measurement sets for the nanocrystals (i.e., the hydrodynamic
radii) made according to Aurora-020 referenced in Table 8 and
measured after 7 weeks from the synthesis. The main peak in
intensity distribution graph is around 23 nm. Dynamic light
scattering measurements on fresh Aurora-020 sample (not shown)
resulted in main peak at 31 nm. It should be noted that the dynamic
light scattering particle size information is different from the
TEM measured histograms because dynamic light scattering uses
algorithms that assume the particles are all spheres (which they
are not) as well as measures the hydrodynamic radius (e.g., the
particle's influence on the water is also detected and reported in
addition to the actual physical radii of the particles).
Accordingly, it is not surprising that there is a difference in the
reported particle sizes between those reported in the TEM histogram
data of those reported in the dynamic light scattering data just as
in the other Examples included herein.
[0708] Accordingly, it is clear from this continuous processing
method that a variety of process parameters can influence the
resultant product produced.
Example 20
Manufacturing Gold-Based Nanoparticles/Nanoparticle Suspensions or
Colloids GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013
by a Batch Process
[0709] This Example utilizes a batch process according to the
present invention. FIG. 24a shows the apparatus used to condition
the liquid 3 in this Example. Once conditioned, the liquid 3' was
processed in the apparatus shown in FIG. 24c. A primary goal in
this Example was to show a variety of different processing
enhancers (listed as "PE" in Table 9). Specifically, Table 9 sets
forth voltages used for each of the electrodes 1 and 5, the dwell
time for the liquid 3 being exposed to plasma 4 in the apparatus of
FIG. 24a; the volume of liquid utilized in each of FIGS. 24a and
24c; the voltages used to create the plasma 4 in FIG. 24a, and the
voltages used for the electrodes 5a/5b in FIG. 24c.
TABLE-US-00023 TABLE 9 Run ID: GA-002 GA-003 GA-004 GA-005 GA-009
GA-011 GA-013 Dwell Plasma 4 25 25 25 25 25 25 25 Times Electrodes
42 42 42 42 42 42 42 (min) 5a/5b Volume Plasma 4 3790 3790 3790
3790 3790 3790 3790 H.sub.2O & PE Electrodes 900 900 900 900
900 900 900 (mL) 5a/5b Volts: Plasma 4 750 750 750 750 750 750 750
Electrodes 300 300 300 300 298 205.6 148 5a/5b PE* Type:
Na.sub.2CO.sub.3 K.sub.2CO.sub.3 KHCO.sub.3 NaHCO.sub.3 NaHCO.sub.3
NaHCO.sub.3 NaHCO.sub.3 mg/ml: 0.22 0.29 0.44 0.47 0.52 0.51 0.51
Wire Diameter (mm) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Wire Configuration
FIG. 17b 17b 17b 17b 17b 17b 17b PPM: 7.8 10.0 10.0 11.3 9.7 10.0
7.7 Final Liquid Temp .degree. C. 96 93.5 90.5 89 90.5 74.5 57
Dimensions & Plasma 4 24a 24a 24a 24a 24a 24a 24a Configuration
FIG. Electrodes 24c 24c 24c 24c 24c 24c 24c 5a/5b FIG. Contact
"W.sub.L" 0.75/19 0.75/19 0.75/19 0.75/19 0.75/19 0.75/19 0.75/19
(in/mm) Separation 1.5/38 1.5/38 1.5/38 1.5/38 1.5/38 0.25/6
0.063/1.6 (in/mm) Electrode Current (A) 0.69 0.65 0.64 0.66 0.76
0.78 0.60 Hydrodynamic r (nm) 11.1 12.0 13.9 11.9 17.6 17.1 10.3
TEM Avg. Diameter (nm) 12.24 12.74 14.09 14.38 11.99 11.99 11.76
"c-c" (in/mm) n/m n/m n/m n/m n/m n/m n/m Plasma 4 electrode # 1a
1a 1a 1a 1a 1a 1a "x" (in/mm) 0.25/6.4 0.25/6.4 0.25/6.4 0.25/6.4
0.25/6.4 0.25/6.4 0.25/6.4 electrode # 5a 5a 5a 5a 5a 5a 5a "c-c"
(in/mm) n/m n/m n/m n/m n/m n/m n/m Electrodes electrode # 5a 5a 5a
5a 5a 5a 5a electrode # 5b 5b 5b 5b 5b 5b 5b
[0710] With regard to the reported processing enhancers (PE)
utilized, different mg/ml amounts were utilized in an effort to
have similar conductivity for each solution (e.g., also similar
molar quantities of cations present in the liquid 3/3'). The
electrode wire diameter used in each Example was the same, about
1.0 mm, and was obtained from ESPI, having an address of 1050
Benson Way, Ashland, Oreg. 97520, as reported elsewhere herein.
[0711] The amount of electrode contacting the liquid 3' in the
apparatus shown in FIG. 24c was the same in each case, namely, 0.75
inches (19.05 mm).
[0712] Table 9 also shows the effects of transverse electrode
separation (i.e., the distance "b" between substantially parallel
electrodes 5a/5b shown in FIG. 24c) for the same processing
enhancer, namely, NaHCO.sub.3. It is clear that electrode current
and corresponding final liquid temperature were less for closer
electrode placement (i.e., smaller "b" values).
[0713] A voltage source 60 (discussed elsewhere herein) was used to
create the plasma 4 shown in FIG. 24a. A voltage source 50
(discussed elsewhere herein) was used to create a voltage and
current between the electrodes 5a/5b shown in FIG. 24c.
[0714] Table 9 also reports the measured hydrodynamic radius (i.e.,
a single number for "Hydrodynamic Radii" taken from the average of
the three highest amplitude peaks shown in each of FIGS. 74c-80c
and "TEM Average Diameter" which corresponds to the average
measured gold nanocrystal size calculated from the TEM histogram
graphs shown in FIGS. 74b-80b).
[0715] FIGS. 74a1,a2-80a1,a2 show two representative TEM
photomicrographs each of the gold nanocrystals, dried from each
solution or colloid referenced in Table 9 formed according to this
Example.
[0716] FIGS. 74b-80b show the measured size distribution of the
gold nanocrystals measured by using the TEM instrument/software
discussed earlier in Examples 5-7 for each suspension or colloid
referenced in Table 9 formed according to this Example.
[0717] FIGS. 74c-80c show graphically dynamic light scattering data
measurement sets for the nanocrystals (i.e., the hydrodynamic
radii) made according to each suspension or colloid referenced in
Table 9 formed according to this Example. It should be noted that
the dynamic light scattering particle size information is different
from the TEM measured histograms because dynamic light scattering
uses algorithms that assume the nanocrystals are all spheres (which
they are not) as well as measures the hydrodynamic radius (e.g.,
the nanocrystal's influence on the water is also detected and
reported in addition to the actual physical radii of the
nanocrystals). Accordingly, it is not surprising that there is a
difference in the reported nanocrystal sizes between those reported
in the TEM histogram data of those reported in the dynamic light
scattering data just as in the other Examples included herein.
Comparative Example 21
Manufacturing Gold-Based Nanoparticles/Nanoparticle Suspensions
According to the Bredig/Svedberg Processes
[0718] This Example utilizes an underwater AC plasma created
between two gold electrodes in an attempt to make a gold
nanoparticle suspension similar to those made by Bredig and
Svedberg (discussed in the Background).
[0719] Specifically, FIG. 81a shows a perspective view of an
apparatus designed to function like the AC plasma apparatus of
Svedberg. FIG. 81b shows a cross-sectional view of the same
apparatus. In each of these figures, gold electrodes e1 and e2,
each having a 1 mm diameter, were submerged into the water 3. About
1 gallon of water 3 was contained in a glass vessel. Electrically
insulating sleeve members s1 and s2 prevented electrical arcing
where undesired. The electrodes e1 and e2 were energized with the
same transformer 60 discussed elsewhere herein. The electrode e1
was brought into close proximity of the end of electrode e2 at an
area designated "Sh". The end "ea" of electrode of e1 was pounded
to make it approximately flat. The flat end ea was then brought
into close proximity with the end of the electrode e2 near the
portion Sh. When the electrode end ea approached the portion Sh, an
underwater plasma 4w was created. Once stabilized, the underwater
plasma 4w was allowed to run for about 2.5 hours to make about 1
gallon of colloid. The results of the 2.5-hour run are shown in
FIGS. 82a and 82b.
[0720] FIG. 82a is a representative TEM photomicrograph of the gold
nanoparticles made according to this Example. FIG. 82b is a
particle size distribution histogram from TEM measurements of the
gold nanoparticles made according to this Example. As is clear from
the TEM photomicrograph, no nanocrystals similar to those of the
present invention are present.
Comparative Example 22a
Colloidal-Based Nanoparticle Suspensions Commercially Available
[0721] For comparison purposes, eight commercially available
colloidal gold solutions were obtained. The commercial names and
sources are listed in Table 10 below:
TABLE-US-00024 TABLE 10 Solution Name Manufacturer Description
Utopia Gold Utopia Silver Supplements Colloidal Gold SNG911219
Source Naturals, Inc. Ultra Colloidal Gold Nanopartz Nanopartz
Accurate Spherical Gold Nanoparticles Nanocomposix NanoComposix
Tannic Acid 15 nm NanoXact Gold Nanocomposix NanoComposix Tannic
NanoXact 10 nm Gold Harmonic Gold Harmonic Innerprizes ElectraClear
InSpiral Technologies Colloidal Gold MesoGold Purest Colloids,
Inc.
[0722] FIG. 90c shows the UV-Vis spectral patterns of each of the 7
of the 8 commercially available gold nanoparticle suspensions
discussed in FIG. 22a (Utopia Gold, SNG911219, Nanopartz,
Nanocomposix 15 nm, Nanocomposix 10 nm, Harmonic Gold and MesoGold)
over an interrogating wavelength range of about 250 nm-750 nm.
[0723] FIG. 90d shows the UV-Vis spectral patterns for 7 of the 8
commercially available gold nanoparticle suspensions discussed in
FIG. 22a (Utopia Gold, SNG911219, Nanopartz, Nanocomposix 15 nm,
Nanocomposix 10 nm, Harmonic Gold and MesoGold) over an
interrogating wavelength range of about 435 nm-635 nm.
Particle-Size and Particle-Shape Analysis
[0724] Transmission electron microscope (TEM) images were analyzed
by visual observation with the aid of software referenced in
Examples 5-7. Individual particles/crystals were assigned to one of
five groups according to the two-dimensional projection shown in
the photomicrographs. The five categories are: triangle, pentagon,
hexagon, diamond and other. These categories correspond to
three-dimensional morphologies elucidated in the literature and
prior TEM studies which utilized a tilting sample holder. The 2D/3D
correspondence of the particle/crystal shape categories is listed
in Table 11.
TABLE-US-00025 TABLE 11 Two-Dimensional Possible Three-Dimensional
Nanoparticle Projection Morphologies Triangle Tetrahedron Pentagon
Pentagonal Bipyramid (i.e., Decahedron) Hexagon Hexagonal
Bipyramid, Icosahedrons, Octahedron Diamond Octahedron, Various
Elongated Bipyramids, Fused Tetrahedrons, Side View of Bipyramids
Other Icosahedrons, Spheroids, Ellipsoids, Rods, Aggre- gated
Particles, Platelets, Particles of Uncertain Form
[0725] Certain nanocrystal forms can take on multiple
two-dimensional projections. For example, an icosahedron, a
possible shape for gold nanocrystals, can appear as a hexagon, an
irregular heptagon or a spheroid in a TEM micrograph. While care
was taken to discern the hexagonal, octagonal and other shapes when
viewed in the two-dimension projection, conclusive information
regarding the true form of such nanocrystals cannot always be
discerned in the two-dimensional projection. Therefore, only the
tetrahedron and pentagonal bipyramid (i.e., decahedron) categories
can be absolutely discerned. Hexagonal, Diamond and Other
categories are grouped together.
[0726] A pentagonal bipyramid nanocrystal viewed on its side could
be projected as a diamond. This is an unlikely occurrence given the
planar nature of the sample substrate and taking into consideration
the very low number of diamonds counted throughout the analysis.
Those decahedrons counted via the pentagon two-dimensional
projection are distinct from this former group, per se, and their
count was taken as one a figure of merit or method of
distinguishing the inventive crystals from those of the art.
Likewise, triangles or tetrahedrons are also readily
distinguishable and can also be used for comparison purposes.
[0727] Aggregation and agglomeration of particles or nanocrystals
can occur in a colloid or as an artifact of the drying process
required for TEM sample preparation/analysis. Dense agglomerations
and larger aggregations (greater than approximately 50
particles/nanocrystals) were not analyzed due to possible counting
errors. The crystal/particle number and particle/crystal shapes of
smaller aggregates and visually resolvable agglomerations were
analyzed. Additionally, only well resolved images were used for
this investigation.
[0728] In order to be very conservative, during the analysis of TEM
micrographs of all suspensions or colloids produced according of
the invention, any questionable crystals were assigned to the group
labeled "Other". Questionable crystals were those that possibly
belong to a well-defined crystal categories, but some uncertainty
exists (e.g., a small pentagon with one corner obscured by an
adjacent particle). In contrast, when performing the analysis of
the particles in the commercially available colloids, any particle
of questionable shape was given "the benefit of the doubt" and was
assigned to the "category "Hexagonal" despite the uncertainty of
its actual crystal structure. Thus the crystal/particle shape
comparisons are not biased and are very conservative regarding
possible differences between commercially available colloids and
nanocrystalline colloids made according to the invention.
[0729] It is clear from Table 12 that the presence of nanocrystals
corresponding in shape to pentagonal bipyramids and/or tetrahedrons
is/are quite different from the commercially available colloids and
ARCG-05. Moreover, these nanocrystals have substantially "clean"
surfaces, as discussed, shown and defined elsewhere herein.
TABLE-US-00026 TABLE 12 TEM Average Example Pentagonal Other
Diameter Product Number Bipyramid Tertrahedron Octahedron Hexagonal
Shapes PPM (nm) pH GD-007 5 21% 10% 2% 40% 27% 14 14.3 8.9 GB-056
17 34% 13% 6% 30% 17% 12 12.1 9.1 GB-077 16 22% 8% 3% 40% 27% 8 8.7
9.0 GB-134 16 31% 18% 5% 27% 19% 9 17.5 9.2 GB-151 18 32% 8% 5% 36%
19% 8 10.9 9.4 GB-154 13 14% 7% 4% 23% 51% 5 14.1 9.7 GB-156 13 18%
16% 5% 30% 30% 5 19.4 9.2 GB-162 14 15% 32% 1% 16% 37% 8 8.9 9.0
GB-163 15 9% 21% 2% 28% 40% 8 20.6 9.1 GB-164 15 12% 12% 7% 32% 37%
8 20.4 9.3 GB-165 14 22% 19% 5% 24% 30% 7 14.7 9.0 GB-166 14 15%
10% 2% 24% 49% 6 13.0 9.0 GB-175 18 25% 22% 1% 23% 29% 11 11.8 9.3
GB-176 18 23% 20% 1% 35% 21% 10 10.4 9.3 GB-177 18 29% 19% 1% 28%
23% 10 10.9 9.3 GB-188 18 25% 23% 6% 23% 24% 8 10.6 9.1 GB-189 18
26% 21% 0% 23% 30% 8 10.4 9.2 GB-194 18 22% 19% 3% 33% 23% 9 12.1
9.2 GB-195 18 17% 16% 3% 45% 19% 8 11.1 9.2 GB-196 18 21% 16% 1%
31% 30% 9 12.1 9.1 GB-198 18 14% 10% 0% 51% 25% 10 11.7 9.2 GB-199
18 33% 9% 1% 40% 17% 12 13.0 9.1 GA-002 20 30% 23% 5% 24% 18% 11
12.2 10.5 GA-003 20 27% 17% 6% 32% 18% 10 12.7 10.3 GA-004 20 15%
9% 3% 38% 35% 10 14.1 9.0 GA-005 20 14% 13% 4% 31% 37% 11 14.4 9.1
GA-009 20 11% 11% 2% 36% 39% 10 12.0 9.2 GA-011 20 8% 6% 6% 37% 44%
10 12.0 8.9 GA-013 20 8% 13% 5% 28% 48% 8 11.8 8.7 GT-033 1-4 4% 1%
1% 26% 68% 2 11.8 6.7 1AC-261-1 12 12% 12% 2% 37% 37% 14 12.2
AURORA 020 19 15% 14% 1% 31% 39% 128 20.6 9.0 ARCG-05 21 3% 0% 2%
6% 89% 5 13.7 6.3 Utopia Gold 22 5% 2% 1% 5% 89% 9 4.7 5.1
SNG911219 22 2% 0% 0% 11% 87% 13 18.4 6.9 Nanopartz 22 2% 0% 0% 21%
77% 39 21.9 7.6 Nanocomposix 22 3% 4% 2% 10% 81% 49 17.8 5.2 15 nm
Nanocomposix 22 2% 1% 1% 22% 73% 51 13.7 5.1 10 nm Harmonic Gold 22
8% 2% 2% 35% 55% 5 8.9 8.8 ElectraClear 22 6% 2% 2% 20% 71% 3 5.7
6.3 MesoGold 22 5% 1% 2% 15% 78% 20 8.5 5.7
Example 22b
The Zeta Potential Example
[0730] The nature and/or amount of the surface change (i.e.,
positive or negative) on formed nanoparticles can also have a large
influence on the behavior and/or effects of the
nanoparticle/suspension or colloid. For example, a protein corona
can be influenced by surface change on a nanoparticle. Such surface
changes are commonly referred to as "zeta potential". In general,
it is well known that the larger the zeta potential (either
positive or negative), the greater the stability of the
nanoparticles in the solution (i.e., the suspension is more
stable). However, by controlling the nature and/or amount of the
surface charges of formed nanoparticles the performance of such
nanoparticle solutions in a variety of systems can be controlled.
It should be clear to an artisan of ordinary skill that slight
adjustments of chemical composition, reactive atmospheres, power
intensities, temperatures, etc., can cause a variety of different
chemical compounds (both semi-permanent and transient)
nanoparticles (and nanoparticle components) to be formed, as well
as different nanoparticle/solutions (e.g., including modifying the
structures of the liquid 3 (such as water) per se). Accordingly,
this Example measures the zeta potential of several suspensions
made according to the invention, as well as several commonly
available colloidal gold suspensions.
[0731] "Zeta potential" is known as a measure of the electo-kinetic
potential in colloidal systems. Zeta potential is also referred to
as surface charge on particles. Zeta potential is also known as the
potential difference that exists between the stationary layer of
fluid and the fluid within which the particle is dispersed. A zeta
potential is often measured in millivolts (i.e., mV). The zeta
potential value of approximately 25 mV is an arbitrary value that
has been chosen to determine whether or not stability exists
between a dispersed particle in a dispersion medium. Thus, when
reference is made herein to "zeta potential", it should be
understood that the zeta potential referred to is a description or
quantification of the magnitude of the electrical charge present at
the double layer.
[0732] The zeta potential is calculated from the electrophoretic
mobility by the Henry equation:
U E = 2 .times. .times. .times. zf .function. ( ka ) 3 .times.
.eta. ##EQU00003##
where z is the zeta potential, U.sub.E is the electrophoretic
mobility, .epsilon. is a dielectric constant, .eta. is a viscosity,
f(ka) is Henry's function. For Smoluchowski approximation
f(ka)=1.5.
[0733] Electrophoretic mobility is obtained by measuring the
velocity of the particles in an applied electric field using Laser
Doppler Velocimetry ("LDV"). In LDV the incident laser beam is
focused on a particle suspension inside a folded capillary cell and
the light scattered from the particles is combined with the
reference beam. This produces a fluctuating intensity signal where
the rate of fluctuation is proportional to the speed of the
particles (i.e. electrophoretic mobility).
[0734] In this Example, a Zeta-Sizer "Nano-ZS" produced by Malvern
Instruments was utilized to determine zeta potential. For each
measurement a 1 ml sample was filled into clear disposable zeta
cell DTS1060C. Dispersion Technology Software, version 5.10 was
used to run the Zeta-Sizer and to calculate the zeta potential. The
following settings were used: dispersant--water,
temperature--25.degree. C., viscosity--0.8872 cP, refraction
index--1.330, dielectric constant--78.5, approximation
model--Smoluchowski. One run of hundred repetitions was performed
for each sample.
[0735] FIG. 91 shows the Zeta potential of two colloidal
nanocrystal solutions (GB-134 and GB-151) as a function of pH. The
pH was varied by titrating 1 wt % solution of acetic acid. The
measurements were performed on a Malvern Instruments Zeta sizer
Nano-ZS90 in folded capillary cell DTS 1060 at 25.degree. C. 20 and
50 sub runs per measurements were used at low and high pHs,
respectively.
[0736] FIG. 92 shows the conductivity measurements for the same
colloidal solutions tested for Zeta potential. The conductivity
measurements were obtained simultaneously on the Malvern
Instruments Zeta Sizer NanoZS90 when the Zeta potential was
determined.
Example 23a
[0737] This Example 13a utilized a set of processing conditions
similar to those set forth in Examples 5-7. This Example utilized
an apparatus similar to those shown in FIGS. 17b, 18a, 19 and 21.
Table 8 sets forth the specific processing conditions of this
Example which show the differences between the processing
conditions set forth in Examples 5-7. The main differences in this
Example includes more processing enhancer added to the liquid 3 and
a more rapid liquid 3 input flow rate.
TABLE-US-00027 TABLE 13 0.528 mg/ml of NaHCO.sub.3 (Au) Run ID:
GD-006 Flow Rate: 240 ml/min Voltage: 255 V NaHCO.sub.3: 0.528
mg/ml Wire Dia.: .5 mm Configuration: Straight/Straight PPM: 8.7
Distance Distance Elec- "c-c" "x" Volt- cross Set# trode# in/mm
in/mm age section 4.5/114.3* 1 1a 0.25 750 V 5a N/A 750 23/584.2**
2.5/63.5* 2 5b N/A 255 .sup. 5b' N/A 8.5/215.9 3 5c N/A 255
Rectangle .sup. 5c' N/A 5.25'' 8.5/215.9 Deep 4 5d N/A 255 .sup.
5d' N/A .sup. 8/203.2 5 5e N/A 255 .sup. 5e' N/A 2/50.8** Output
Water Temperature 95 C. *Distance from water inlet to center of
first electrode set **Distance from center of last electrode set to
water oulet
[0738] FIG. 93 shows a representative Viscotek output for the
suspension produced in accordance with Example 23a. The numbers
reported correspond to hydrodynamic radii of the nanocrystals in
the suspension.
Example 23b
[0739] This Example 23b utilized the suspension of Example 23a to
manufacture a gel or cream product. Specifically, about 1,300 grams
of the suspension made according to Example 13a was heated to about
60.degree. C. over a period of about 30 minutes. The suspension was
heated in a 1-liter Pyrex.RTM. beaker over a metal hotplate. About
9.5 grams of Carbopol.RTM. (ETD 2020, a carbomer manufactured by
Noveon, Inc., Cleveland, Ohio) was added slowly to the heated
suspension, while constantly stirring using a squirrel rotary
plastic paint mixer. This mixing occurred for about 20 minutes
until large clumps of the Carbopol were dissolved.
[0740] About 15 grams of high purity liquid lanolin (Now Personal
Care, Bloomingdale, Ill.) was added to the suspension and mixed
with the aforementioned stirrer.
[0741] About 16 grams of high purity jojoba oil were then added and
mixed to the suspension.
[0742] About 16 grams of high purity cocoa butter chunks (Soap
Making and Beauty Supplies, North Vancouver, B.C.) were heated in a
separate 500 mL Pyrex.RTM. beaker and placed on a hotplate until
the chunks became liquid and the liquid cocoa butter then was added
and mixed to the aforementioned suspension.
[0743] About 16 grams of potassium hydroxide (18% solution) was
then added and mixed together with the aforementioned ingredients
to cause the suspension to gel. The entire suspension was
thereafter continuously mixed with the plastic squirrel rotating
mixer to result in a cream or gel being formed. During this final
mixing of about 15 minutes, additional scent of "tropical island"
(2 mL) was added. The result was a pinkish, creamy gel.
Example 23c
[0744] This Example 23c utilized the suspension made according to
Example 7. Specifically, this Example utilized the product of
Example 7 (i.e., GD-015) to manufacture a gel or cream product.
Specifically, about 650 grams of the solution made according to
Example 7 was heated to about 60.degree. C. over a period of about
30 minutes. The suspension was heated in a Iliter Pyrex.RTM. beaker
over a metal hotplate. About 9.6 grams of Carbopol.RTM. (ETD 2020,
a carbomer manufactured by Noveon, Inc., Cleveland, Ohio) was added
slowly to the heated suspension, while constantly stirring using a
squirrel rotary plastic paint mixer. This mixing occurred for about
20 minutes until large clumps of the carbopol were dissolved.
[0745] About 7 grams of high purity liquid lanolin (Now Personal
Care, Bloomingdale, Ill.) was added to the solution and mixed with
the aforementioned stirrer.
[0746] About 8 grams of high purity jojoba oil were then added and
mixed to the suspension.
[0747] About 8 grams of high purity cocoa butter chunks (Soap
Making and Beauty Supplies, North Vancouver, B.C.) were heated in a
separate 500 mL Pyrex.RTM. beaker and placed on a hotplate until
the chunks became liquid and the liquid cocoa butter then was added
and mixed to the aforementioned suspension.
[0748] About 45 grams of the liquid contained in Advil.RTM. liquid
gel caps (e.g., liquid ibuprofen and potassium) was added to, and
thoroughly mixed with, the suspension.
[0749] About 8 grams of potassium hydroxide (18% solution) was then
added and mixed in to cause the suspension to gel. The entire
solution was thereafter continuously mixed with the plastic
squirrel rotating mixer to result in a cream or gel being formed.
During this final mixing of about 15 minutes, additional scent of
"tropical island" (2 mL) was added. The result was a pinkish,
creamy gel.
Example 23d
[0750] This Example 23d utilized suspension equivalent to GB-139 to
manufacture a gel or cream product. Specifically, about 650 grams
of the suspension was heated to about 60.degree. C. over a period
of about 30 minutes. The suspension was heated in a 1-liter
Pyrex.RTM. beaker over a metal hotplate. About 6 grams of
Carbopol.RTM. (ULTREZ10, a carbomer manufactured by Noveon, Inc.,
Cleveland, Ohio) was added slowly to the heated suspension, while
constantly stirring using a squirrel rotary plastic paint mixer.
This mixing occurred for about 20 minutes until large clumps of the
Carbopol were dissolved.
[0751] About 7 grams of high purity liquid lanolin (Now Personal
Care, Bloomingdale, Ill.) was added to the suspension and mixed
with the aforementioned stirrer.
[0752] About 8 grams of high purity jojoba oil were then added and
mixed to the suspension.
[0753] About 8 grams of high purity cocoa butter chunks (Soap
Making and Beauty Supplies, North Vancouver, B.C.) were heated in a
separate 500 mL Pyrex.RTM. beaker and placed on a hotplate until
the chunks became liquid and the liquid cocoa butter then was added
and mixed to the aforementioned suspension.
[0754] About 8 grams of potassium hydroxide (18% solution) was then
added and mixed together with the aforementioned ingredients to
cause the suspension to gel. The entire suspension was thereafter
continuously mixed with the plastic squirrel rotating mixer to
result in a cream or gel being formed. The result was a pinkish,
creamy gel.
Example 23e
[0755] This Example 23e utilized a suspension substantially
equivalent to 1AC-261 to manufacture a gel or cream product.
Specifically, about 450 grams of the suspension was heated to about
60.degree. C. over a period of about 30 minutes. The suspension was
heated in a 1-liter Pyrex.RTM. beaker over a metal hotplate. About
4.5 grams of Carbopol.RTM. (ULTREZ10, a carbomer manufactured by
Noveon, Inc., Cleveland, Ohio) was added slowly to the heated
suspension, while constantly stirring using a squirrel rotary
plastic paint mixer. This mixing occurred for about 20 minutes
until large clumps of the Carbopol were dissolved.
[0756] About 6.5 grams of potassium hydroxide (18% solution) was
then added and mixed together with the aforementioned ingredients
to cause the suspension to gel. The entire suspension was
thereafter continuously mixed with the plastic squirrel rotating
mixer to result in a cream or gel being formed. The result was a
pinkish, creamy gel.
Example 24
In Vitro Study of the Effects of Gold Nanocrystalline Formulation
GB-079 on Monocyte Cytokine Production
Summary
[0757] This in vitro Example was designed to determine the effects
of gold nanocrystalline suspension GB-079 on four different
cytokines/chemokines. Specifically in this Example, human
peripheral blood mononuclear cells ("hPBMC") were cultured in the
presence or absence of each of four different concentration or ppm
levels of gold nanocrystalline suspension GB-079 (i.e., a
suspension or colloid made in accordance with the disclosure of one
example herein) in the presence or absence of (as disclosed herein)
bacterial lipopolysaccharide ("LPS").
[0758] It is known that lipopolysaccharide binds to TLR4, a
receptor expressed on a number of different immune system cells,
and such binding typically triggers activation and/or expression of
a series of cytokines, typically in an NFkB-dependent (i.e.,
Nuclear Factor kB-dependent) manner. After about 24 hours of
culture conditions at about 37.degree. C. in about 5% CO.sub.2 and
a humidified atmosphere of about 95% relative humidity,
supernatants were removed and assayed for the presence of a series
of different cytokines/chemokines, including: MIF, TNF.alpha., IL-6
and IL-10. The majority of, but not the only source of, these
cytokines in the hPBMC population would be expected to be
monocytes. Cultures in the absence of LPS indicate whether
treatments induce the production of these cytokines/chemokines,
while those cultures in the presence of LPS will indicate whether
treatments are able to modulate the production of cytokines in
response to an inflammatory stimulus. Cytokine assays were
performed by the Luminex.RTM. Extracellular Assay Protocol. The
Luminex system uses antibody coated microspheres that bind
specifically to the cytokine being assayed. When excited by laser
light the microspheres that have bound the antigen are measured and
this is a direct assessment of the amount of the cytokine being
produced and data were provided as raw data and absolute quantities
of each cytokine/chemokine measured.
Preparation of hPBMC
Materials Used for Cell Preparation:
TABLE-US-00028 [0759] Supplier Cat. No. PBMC Isolation Histopaque
1077 Sigma H8889 RPMI 1640 .times. 10 Sigma R1145 Endotoxin-free
water (EFW) Gibco 15230-170 50 ml falcon tubes Corning 430829
Citrate ACD Sigma C3821-50 ml AB serum National Blood Service
Plastic 24 well plates Costar/Corning 3524 LPS Sigma Media
supplements Penicillin/streptomycin Sigma P0871 HEPES Sigma H0887
Glutamine Gibco 25030-024 Sodium Bicarbonate (7.5%) Gibco 25080
Equipment
[0760] NucleoCounter (i.e., cell number and viability counter made
by Chemometec) Benchtop centrifuge Tissue culture hood
Collection of Human Blood
[0761] Blood from a healthy volunteer was drawn into a syringe and
placed into a 50 ml falcon tube. 3.3 ml citrate anticoagulant (ACD,
Sigma) was added to the 50 ml falcon tubes in a sterile manner.
Tubes were inverted to mix.
Cell Preparation Method
[0762] 1. 10.times.RPMI+supplements (25 ml 10.times.RPMI+2.5 ml
Penstrep+2.5 ml L-glutamine+5 ml HEPES+6.7 ml sodium bicarbonate
solution (7.5%)) were mixed together in a falcon tube, herein
referenced to as the "culture media". [0763] 2. Blood was
resuspended in an equal volume of 1.times.RPMI 1640 (diluted from
10.times.RPMI in EFW--200 ml prepared [20 ml in 180 ml]) and mixed
by inversion in a falcon tube. [0764] 3. The histopaque was
prewarmed to room temperature (RT) and 20 ml was added to a 50 ml
falcon tube. [0765] 4. The histopaque was gently overlayed with 30
ml blood/medium then mixed in. [0766] 5. The histopaque blood mix
sample was spun at 1600 rpm in a benchtop centrifuge for about 25
min at RT (no brake). [0767] 6. PBMC were separated into the
interface layer between the medium and the histopaque, cells were
removed by aspriation into a 50 ml falcon tube and 10 ml of the
culture media was added thereto. [0768] 7. The cell sample was spun
at 1800 rpm for about 10 minutes at RT. [0769] 8. The cell sample
was washed twice with 30 ml RPMI and resuspended in culture medium
(RPMI, supplemented as described above=RPMI/no serum). [0770] 9.
During the spin, RPMI supplemented with 5% AB serum was prepared.
[0771] 10. The cell sample was resuspended in 2 ml
RPMI+supplements+serum. [0772] 11. Cell counts were completed and
viability assessment was performed using the Nucleocounter (i.e., a
cell viability counter). [0773] 12. Cells were resuspended in
1.times.RPMI to give a final concentration of 2.5.times.10.sup.6
cells/ml. [0774] 13. 500 .mu.l of cells were transferred into a
24-well plate. [0775] 14. 10.times.RPMI+supplements (500 .mu.l
PenStrep, 500 .mu.l L-Glutamine, lml HEPES, 2.5 ml AB serum) was
prepared by mixing together in a falcon tube, thereby forming the
"test media". [0776] 15. The inventive GB-079 gold nanocrystal
suspension was added to the wells in the 24 well plate (900 .mu.l
total volume) [0777] 16. 100 .mu.l 10.times.RPMI+supplements were
added to each well of a costar 24 well plate. [0778] 17. The 24
well plates were placed into a humidified incubator set at
37.degree. C./5% CO.sub.2 for 1 hour. [0779] 18. LPS was prepared
at 4.times. final concentration in 1.times.RPMI [0780] 19. 500
.mu.l of LPS was added per well, or 500 .mu.l media to wells not
receiving LPS, bringing the total well volume of material to each
well to 2 ml. [0781] 20. Plates were placed into a humidified
incubator set at 37.degree. C./5% CO.sub.2 for about 24 hours.
[0782] 21. 1800 .mu.l (3.times.600 .mu.l aliquots) of supernatant
were removed for ELISA analysis and Luminex analysis. [0783] 22.
Supernatants were stored at -80.degree. C. until assayed in the
Luminex.RTM. system.
Luminex.RTM. Assay System
[0784] The supernatants were assayed in accordance with the
Luminex.RTM. Extracellular Assay Protocol, accessed on Jan. 11,
2010.
TABLE-US-00029 TABLE 14 Sample Compound EFW 10x RPMI Cells LPS 1x
RPMI Cells + Vehicle 900 .mu.l 100 .mu.l 500 .mu.l 500 .mu.l Cells
+ Vehicle + LPS 900 .mu.l 100 .mu.l 500 .mu.l 500 .mu.l Cells +
[Test].sub.1:5 400 .mu.l 500 .mu.l 100 .mu.l 500 .mu.l 500 .mu.l
Cells + [Test].sub.1:10 200 .mu.l 700 .mu.l 100 .mu.l 500 .mu.l 500
.mu.l Cells + [Test].sub.1:20 100 .mu.l 800 .mu.l 100 .mu.l 500
.mu.l 500 .mu.l Cells + [Test].sub.1:40 50 .mu.l 850 .mu.l 100
.mu.l 500 .mu.l 500 .mu.l Cells + [Test].sub.1:100 20 .mu.l 880
.mu.l 100 .mu.l 500 .mu.l 500 .mu.l Cells + [Test].sub.1:5 + LPS
400 .mu.l 500 .mu.l 100 .mu.l 500 .mu.l 500 .mu.l Cells +
[Test].sub.1:10 + LPS 200 .mu.l 700 .mu.l 100 .mu.l 500 .mu.l 500
.mu.l Cells + [Test].sub.1:20 + LPS 100 .mu.l 800 .mu.l 100 .mu.l
500 .mu.l 500 .mu.l Cells + [Test].sub.1:40 + LPS 50 .mu.l 850
.mu.l 100 .mu.l 500 .mu.l 500 .mu.l Cells + [Test].sub.1:100 + LPS
20 .mu.l 880 .mu.l 100 .mu.l 500 .mu.l 500 .mu.l
[0785] Cells were stimulated with LPS (a high dose of lmg/ml and a
low dose of 10 ng/ml), the supernatants were then collected after
24 hours and analyzed for the amounts present of the 4 cytokines
discussed herein. Control wells contained cells and the inventive
test compound GB-079 but no LPS. Results obtained for each of the
other cytokines/chemokines are shown in FIGS. 94a-94d.
[0786] FIG. 94a shows the effects of GB-079 on IL-6 production by
human peripheral blood mononuclear cells (hPBMCs). It is clear from
FIG. 94a that IL-6 levels were reduced by GB-079 in LPS stimulated
PBMC. Some IL-6 production was also observed with the highest
concentrations of GB-079 in the absence of LPS stimulation at five
different concentration levels.
[0787] FIG. 94b shows the effects of GB-079 on IL-10 production by
hPBMCs. It is clear from FIG. 94b that the levels of IL-10 observed
were unaffected by the addition of GB-079 at all concentration
levels.
[0788] FIG. 94c shows the effects of GB-079 on MIF production by
hPBMCs. Specifically, FIG. 94c shows that following LPS
stimulation, the levels of MIF were reduced in a dose dependent
manner. This reduction was observed at dilution levels of 1:5 and
1:10, while MIF levels returned to that of control samples by the
1:20 concentration of GB-079.
[0789] Further, FIG. 94d shows that GB-079 at the highest
concentrations caused an increase in TNF.alpha. levels (with both
tested doses of LPS) above that of vehicle control stimulated
samples. Some TNF.alpha. production was also observed with the
highest doses of GB-079 in the absence of LPS stimulation.
Example 25
Collagen Induced Arthritis (CIA) Study in Mice
Summary
[0790] This Example demonstrates the efficacy of two of the
inventive gold nanocrystalline compositions (i.e., GT033 and
GD-007) in a mouse CIA model. Specifically, male DBA/l mice (12
weeks old) were given 100 .mu.g Chicken Type II collagen emulsified
into complete Freund's adjuvant ("CII/CFA") on day 0 of the study
by injection at the base of the tail. Clinical joint swelling was
scored three times weekly from day 14 until termination at day 42.
Those results are summarized in FIG. 95. Treatments were given
according to the protocol below. Bleeds were taken on day 0 and day
42. At termination animals were bled, hind legs were removed, and
ankle joints were prepared for histopathology examination.
Histopathology results are set forth in Table 6 and Table 7.
Methodology
Animals
[0791] Species: Mice [0792] Strain: DBA/1 [0793] Source: Harlan
[0794] Gender and number: Male, 30 [0795] Age: About 12 weeks old
at the start of the study. [0796] Identification: Each mouse was
given a unique identity number. [0797] Animal husbandry: On
receipt, all animals were examined for external signs of ill-health
and all unhealthy animals were excluded from further evaluation.
Animals were housed in groups of five under specific pathogen free
(spf) conditions, in a thermostatically monitored room
(22.+-.4.degree. C.) in an animal unit. Animals were equilibrated
under standard animal house conditions for at least 72 hours prior
to use. The health status of the animals was monitored throughout
this period and the suitability of each animal for experimental use
was assessed prior to study start. [0798] Housing Animals were
housed in groups of 10 per cage in a controlled room, to ensure
correct temperature, humidity and 12-hour light/dark cycle for the
duration of the study. [0799] Diet: Irradiated pellet diet and
water was available ad libitum throughout the holding,
acclimatization and post-dose periods.
Compound and Reagents
Chicken Collagen Type II (Sigma, C9301).
Incomplete Freund's Adjuvant ("IFA") (Sigma, FF5506)
[0800] Mycobacterium tuberculosis H37Ra (BD Biosciences, 231141)
Phosphate buffered saline ("PBS") Test compounds gold nanocrystal
formulations GT033 and GD-007.
Vehicle: Water.
Treatment Groups and Dosages
[0801] Control Group 1, First Treatment "Group 2" and Second
Treatment "Group 3" each had 10 animals per group. Group 1: Day 0
CII/CFA, given normal drinking water from day 0-42. Group 2: Day 0
CII/CFA, gold nanocrystal formulation (GT033; Example 4/Table 1d;
gold ppm 2.0) as drinking water from day 0-42.
[0802] Group 3: Day 0 CII/CFA, gold nanocrystal formulation
(GD-007; Example 5/Table 2a; gold ppm 14.8) as the only liquid for
drinking from day 0-42.
Protocol
[0803] 1. On arrival of animals, the health of all animals was
checked and after passing the health test, each was numbered with a
unique ear tag. [0804] 2. The animals were allowed to acclimate for
at least 72 hours. [0805] 3. Chicken Type II collagen was prepared
so as to achieve a suspension with a concentration of about 16
mg/ml in 0.1M acetic acid. After dissolution overnight at 4.degree.
C., the solution was diluted with cold PBS to achieve a suspension
with a concentration of about 8 mg/ml. [0806] 4. Fresh
mycobacterium was prepared by grinding it finely with a mortar and
pestle and adding about 7 ml of IFA, drop-by-drop, to create an
emulsion or suspension of CFA with a final concentration of about 5
mg/ml. [0807] 5. An emulsion of Chicken Type II collagen and CFA
was prepared using approximately equal volumes of each to result in
the injectable suspension of collagen in CFA (i.e., "CII/CFA").
[0808] 6. On Day 0, the animals were injected with 50 .mu.l of the
CII/CFA solution at the base of the tail. [0809] 7. Treatments
using gold nanocrystal formulation GT033 (i.e., Group 2) and gold
nanocrystal formulation GD-007 (i.e., Group 3) were given according
to the schedule above until Day 42. Specifically, each water bottle
containing either normal drinking water, GT033 or GD-007 was
topped-off as needed either every other day or every third day. The
bottles were not specifically cleaned or specifically emptied
during the 42-day trial. [0810] 8. The limb scores were determined
three times per week from Day 14 to the end of the study. Each of
the four limbs was given a score according to the following; [0811]
0=Normal. [0812] 1=Slight swelling of whole joint or individual
digit inflammation. [0813] 2=Intermediate swelling of whole joint
with redness and/or inflammation in more than one digit. [0814]
3=severe joint inflammation and redness spreading to multiple
digits. [0815] 4=severe joint inflammation and redness spreading to
multiple digits; overt signs of bone remodeling. [0816] 9. All
animals were bled on days 0 and day 42 and the retrieved serum was
stored for optional analysis. [0817] 10. The animals were
sacrificed on Day 42 and the ankle joints were removed and placed
in neutral-buffered formalin in preparation for histopathology.
[0818] 11. These sections were processed and stained with
hematoxylin and eosin stain ("H & E") and were scored by a
qualified (and experimentally blinded) histopathologist using a
semi-quantitative measurement of the degree of infiltration and
damage.
[0819] FIG. 95 shows graphically the results of the limb scoring
CIA-test. Clearly the gold nanocrystal formulation GD-007 (Group
3), having a measured gold concentration of about 14.8 ppm,
performed the best, on par (or better), perhaps with a typical
steroid treatment, the results of which have also been placed onto
FIG. 95 (even though not actually measured). The gold nanocrystal
formulation GT033 (Group 2) performed better than Control Group 1
at a concentration of about 2.0 ppm of gold nanocrystals suspended
in water.
[0820] Histopathology was performed on the left and right paws from
each of the 10 mice in Group 1 (control) and Group 3 (GD-007). No
histopathology was performed on Group 2 mice.
[0821] Each pair of paws was assigned a Pathology numerical code
(e.g., R0248-09 for one mouse in Group 1) and the limbs
distinguished as left ("L") or right ("R") from each numbered
animal.
Histopathology/Methodology:
[0822] The skin was dissected from the paw. [0823] The dissected
samples were decalcified to permit sectioning. [0824] The
decalcified samples were routinely processed, sectioned and one H
& E-stained section was prepared for examination. This included
both halves of each specimen being hemi-sectioned. [0825] Each
histopath paw was scored as described below. Samples were scored in
blinded fashion, without knowledge of the experimental protocol or
identity of groups. [0826] Multiple phalangeal and tarsal joints
were generally present on each section. Scoring related to the most
severely affected of these joints in each case.
Scoring System
[0827] In this instance, three aspects of the joint pathology were
scored individually to contribute to a composite score (i.e.,
maximum possible score=9). Thus, the higher the number, the greater
the damage. Representative photomicrographs of joints corresponding
to the aforementioned grades 0-3 are shown in FIGS. 96a-96d,
respectively. Representative compilations of these grades 0-3 are
shown in FIG. 97a (i.e., Grade 0) through FIG. 97e (i.e., Grade
9).
TABLE-US-00030 TABLE 15 Aspect Grade 0 Grade 1 Grade 2 Grade 3
Inflammation Normal joint Mild synovial Synovial hyperplasia
Synovial hyperplasia hyperplasia with with moderate to with marked
inflammation inflammation marked inflammation involving both
neutro- dominated by involving both phils and macrophages.
neutrophils. Low neutrophils and Loss of synoviocyte lining.
numbers of macrophages. Inflammation may extend neutrophils and
Neutrophils and from synovium to macrophages in macrophages in
surrounding tissue joint space. joint space; may be including
muscle. some necrotic tissue Numerous neutrophils and debris.
macrophages in joint space, together with significant necrotic
tissue debris. Articular cartilage Normal joint Articular cartilage
Articular cartilage Significant disruption damage shows only mild
shows moderate and loss of articular degenerative change.
degenerative change cartilage with extensive Early pannus formation
and focal loss. Pannus pannus formation. may be present formation
is present peripherally. focally. Damage to Normal joint No change
to May be focal Disruption or collapse underlying underlying
necrosis or fibrosis of metaphyseal bone. metaphyseal metaphyseal
bone. of metaphyseal Extensive inflammation, bone bone. necrosis or
fibrosis extending to medullary space of the metaphysis.
TABLE-US-00031 TABLE 16 Paw Histopathology Scoring Mouse Pathology
Number Total Number and Limb Inflammation Cartilage Bone score
Comments Control R0248-09 1.1 L 1 0 0 1 Few neutrophils in mildly
thickened synovium ideally. 1.1 R 2 2 2 6 R0249-09 1.2 L 3 2 2 7
1.2 R 1 0 0 1 R0250-09 1.3 L 3 2 2 7 1.3 R 0 0 0 0 R0251-09 1.4 L 3
2 2 7 1.4 R 3 1 1 5 Reaction localized to P1-metatarsal R0252-09
1.5 L 3 2 2 7 1.5 R 3 2 2 7 R0253-09 1.6 L 0 0 0 0 1.6 R 3 1 2 6
R0254-09 1.7 L 3 2 2 7 1.7 R 3 2 2 7 R0255-09 1.8 L 0 0 0 0 1.8 R 3
1 1 5 R0256-09 1.9 L 0 0 0 0 1.9 R 0 0 0 0 R0257-09 1.10 L 0 0 0 0
1.10 R 0 0 0 0 Treatment R0258-09 2.1 L 0 0 0 0 Group3 2.1 R 0 0 0
0 R0259-09 2.2 L 0 0 0 0 2.2 R 0 0 0 0 R0260-09 2.3 L 0 0 0 0 2.3 R
0 0 0 0 R0261-09 2.4 L 0 0 0 0 2.4 R 0 0 0 0 R0262-09 2.5 L 0 0 0 0
2.5 R 0 0 0 0 Has localized subcutaneous inflammatory response;
joints normal. R0263-09 2.6 L 0 0 0 0 2.6 R 0 0 0 0 R0264-09 2.7 L
0 0 0 0 2.7 R 0 0 0 0 R0265-09 2.8 L 0 0 0 0 2.8 R 0 0 0 0 R0266-09
2.9 L 3 1 0 4 2.9 R 0 0 0 0 R0267-09 2.10 L 3 1 2 6 Localized
metatarsal-P1 reaction with marked periosteal new bone and
cartilage formation - probably localized fracture repair rather
than joint disease. 2.10 R 3 2 2 7
TABLE-US-00032 TABLE 17 Mean Group Scores Number [%] of Group Paws
(n=) Mean Score Joints affected 1-Control 20 3.65 14/20 [70%]
2-GD-007 20 0.85 3/20 [15%]
[0828] As is typical for this type of murine CIA model, one animal
in Treatment "Group 3," GD-007, (i.e., R0266-09) exhibited a lack
of correlation between its right and left joints in terms of the
presence/absence of arthritis. Similar discrepancies occur in some
of the control mice, as well as differences in the severity of the
arthritis between different joints in the same mouse (e.g.,
R0250-09).
[0829] It is clear, however, that the most severe pathology
occurred in Control Group 1 (i.e., drinking water) and the least
severe pathology occurred in First "Treatment Group 2" (i.e., gold
nanocrystal formulation GD-007).
[0830] One animal in Treatment Group 3 (i.e., R0267-09) suffered a
broken bone which probably accounted for its higher scores.
Exclusion of this animal resulted in a mean score of 0.22. Further,
the histopathology data suggests no resulting damage at all in 8 of
the 10 mice (i.e., 16 total paw joints examined). Clearly the gold
nanocrystal formulation GD-007 had a significant positive effect in
this CIA test.
[0831] It is clear that the gold nanocrystal formulations produced
according to the invention significantly reduced the negative
induced arthritis effects in the CIA model, relative to the
control. It is known that reduction of excessive IL-6 and/or
reduction of excessive MIF both reduce the negative effects of
arthritic conditions. Accordingly, without wishing to be bound by
any particular theory or explanation, by reducing excessive MIF,
and/or one or more signaling pathways associated with MIF,
arthritic conditions can be reduced. The gold nanocrystalline
formulation GD-007 showed significantly improved results, relative
to the control. These results, along with the results shown in the
in vitro example and the EAE mouse model Example herein, suggest
that the inventive gold nanocrystal compositions may be altering
MIF and/or more signaling pathways associated with MIF, as well as
IL-6.
Example: Doses Comparison
[0832] As stated above, in the gold nanocrystal trial, each mouse
had access to GD-007 solution as the only source of drinking fluid.
To calculate the dose of gold consumed by a mouse per day,
following equation was used:
Dose=Volume consumed (ml).times.Concentration (mg/L) Animal weight
(kg)
[0833] where [0834] Dose is the nanocrystal gold consumed per mouse
per day in mg/kg/day, [0835] Volume is an average amount of GB-134
solution drunk by a mouse per day in mL/day, [0836] Concentration
is the amount of nanocrystal gold in the GD-007 solution in mg/mL,
[0837] Weight is a mouse body weight in kg.
[0838] The following assumptions were used to calculate the
nanocrystal gold dose: [0839] Volume=4 mL [0840]
Concentration=0.0148 mg/mL [0841] Weight=0.025 kg
[0842] This results in a nanocrystal gold dose of 2.4
mg/kg/day.
[0843] Below is the comparison of the gold content in doses
typically used for Auranofin treatment in the type II
collagen-induced arthritis mouse model. Typical Auranofin dose is
40 mg/kg/day (Agata et al., 2000). Since the gold content in
Auranofin is 29%, this results in gold dose of approximately 12
mg/kg/day.
[0844] In the only known human study (Abraham et al. 1997, 2008)
using gold nanoparticles, a 30 mg/day gold nanoparticle dose was
used for patients weighing from 108 to 280 lb. This corresponds to
approximately 0.24 to 0.61 mg/kg/day gold nanoparticle dose.
[0845] A comparison between dose levels of gold content in
Auranofin, gold in gold nanoparticles, and the novel gold
nanocrystals, used in these different efficacy studies, is shown
below in Table 17a, demonstrating that the present novel gold
nanocrystals are fundamentally different from, and perform very
differently and at a much higher level of potency than,
conventional gold, whether in molecular form in Auranofin, or in
nanoparticle form as in Abraham, et. al.
TABLE-US-00033 TABLE 17a Study Type of Gold Product Gold mg/kg/day
Mouse RA CIA Novel Gold Nanocrystals 2.4 Agata/Mouse RA CIA
Auranofin 12 (5X) Estimated Human dose* Novel gold Nanocrystals
0.005 Abraham/human Colloidal gold 0.24 to 0.61 (47X to 122X)
*Using mouse/mouse Auranofin/Nanocrystals potency factor applied to
Auranofin human dose
Example 26
Acute Murine Model of Experimental Auto-Immune Encephalitis
("EAE")
Summary
[0846] This Example demonstrates the efficacy of the inventive gold
nanocrystalline composition GB-056 in a mouse EAE model. Female
Biozzi mice 7-8 weeks old were challenged in the flank with mouse
spinal cord homogenate in CFA on day 0 of the study by injection at
the base of the tail. Ten treatment group mice were orally
administered the gold nanoparticle suspension treatment GB-056
(i.e., as discussed in Example 17) as their only liquid for
drinking by using standard water bottles. Fresh gold
nanocrystalline formulation GB-056 was provided daily along with
clean water bottles. Control group mice were provided ordinary tap
drinking water. Clinical scoring in this EAE test was completed by
a standard scoring system of 0-5.0 scored from day 1 until
termination at day 28. Those results are presented in Tables 9a and
9b, as well as in FIGS. 98-99. Treatments were given according to
the protocol below.
Methodology
Animals
[0847] Species: Mice [0848] Strain: BIOZZI [0849] Source: Harlan
[0850] Gender and number: Female, 20 [0851] Age: About 7-8 weeks
old at the start of the study. [0852] Identification: Each mouse
was given a unique identity number. [0853] Animal husbandry: On
receipt, all animals were examined for external signs of ill-health
and all unhealthy animals were excluded from further evaluation.
Animals were housed in groups of five under specific pathogen free
(spf) conditions, in a thermostatically monitored room
(22.+-.4.degree. C.) in an animal unit. Animals were equilibrated
under standard animal house conditions for at least 72 hours prior
to use. The health status of the animals was monitored throughout
this period and the suitability of each animal for experimental use
was assessed prior to study start. [0854] Housing Animals were
housed in groups of 10 per cage in a controlled room, to ensure
correct temperature, humidity and 12-hour light/dark cycle for the
duration of the study. [0855] Diet: Irradiated pellet diet and
water was available ad libitum throughout the holding,
acclimatization and post-dose periods.
Compound and Reagents
[0856] Mouse and Spinal Cord Homeogenate ("MSCH") produced
in-house.
Incomplete Freund's Adjuvant ("IFA") (Sigma, FF5506)
[0857] Mycobacterium tuberculosis H37Ra (BD Biosciences, 231141)
Phosphate buffered saline ("PBS") in-house. Test compound gold
nanocrystalline suspension GB-056 (discussed elsewhere herein)
Vehicle: Water.
[0858] Treatment Groups and Dosages
Control Group 1 and the Treatment Group 2 each had 10 animals per
group. Group 1: Day 0 a mixture of MSCH/IFA/tuberculosis (see
Protocol below) was injected into each mouse at base of tail and
each was given normal drinking water dispensed from a water bottle,
from day 0 to day 28. Group 2: Day 0 a mixture of
MSCH/CFA/tuberculosis was injected into each mouse at base of tail
and each was given gold nanocrystal formulation (GB-056) dispensed
from a daily-cleaned water bottle with fresh GB-056 provided daily,
as the only liquid for drinking, from day 0 to day 28.
Protocol
[0859] On arrival of animals, the health of all animals was checked
and after passing the health test, each was numbered with a unique
ear tag. [0860] 1. The animals were allowed to acclimate for at
least 72 hours. [0861] 2. The spinal cord was reconstituted in PBS
containing Mycobacterium tuberculosis H37RA. This resulted in 6.6
mg/ml of MSCH and 400 ug/ml of H37RA. An equal volume of Freund's
incomplete adjuvant was added to this mixture to make the final
immunogen (3.3 mg/ml SCH and 200 ug/ml H37RA). This mixture could
not be considered complete Freund's because amount of mycobacterium
was much lower. [0862] 3. On Day 0, the animals were injected with
50 .mu.l of the solution discussed in step 3 at the base of the
tail. [0863] 4. Treatment using gold nanocrystal formulation GB-056
was given according to the schedule above until Day 28. Fresh
GB-056 was provided daily (i.e., replaced approximately every 24
hours). [0864] 5. The scores were determined daily from Day 1 to
the end of the study. Scoring of each mouse occurred according to
the following; [0865] 0: Normal [0866] 0.5: Paretic tail [0867]
1.0: Flaccid tail [0868] 1.5: Slow and/or absent righting reflex
[0869] 2.0: One hind limb paralysis [0870] 2.5: One hind limb
paralysis and unusual gait [0871] 3.0: Two hind limbs paralysis
[0872] 3.5: Two hind limbs paralysis+one front limb paresis [0873]
4.0: Two hind limbs paralysis+one or two front limb paralysis
[0874] 5.0: Moribund [0875] 6. The animals were sacrificed on Day
28 and the brain and spinal cord were removed and placed in
neutral-buffered formalin in preparation for histopathology. [0876]
7. These sections were processed and stained with hematoxylin and
eosin stain ("H & E"). Tables 9a and 9b show the raw scoring
for each of the 20 mice in this EAE study.
TABLE-US-00034 [0876] TABLE 18a Animal Day Day Day Day Day Day Day
Day Day Day Day Day Day Day Day Day Day Day Day # 10 11 12 13 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 Water Control 1 0 0 0 0 0 0
0 0 0 0 1.5 2 2.5 5 5 5 5 5 5 2 0 0 0 0 0 0 0 1.5 1.5 1.5 2.5 2.5 5
5 5 5 5 5 5 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.5 1.5 1.5 0 4 0 0 0 0
0 0 0 1 1 2.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0 5 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 1.5 6 0 0 0 0 0 0 0 0 1.5 1.5 1.5 5 5 5 5 5 5 5 5
7 0 0 0 0 0 0 0 0 0 0 1.5 2 2.5 1.5 1.5 1.5 1.5 1.5 0 8* 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 1 1.5 2 2 2 2 2 1.5
1.5 0 10 0 0 0 0 0 0 0 0 0 0 0 1.5 1.5 3 3 3 3 3 3 GR-056 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 1.5 1.5 2.5 3 3 2 0 0 0 0 0 0 0 0 0 0 0 1.5 2
2.5 3 3 2.5 1.5 1.5 3* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0
0 0 0 0 0 0 0 1.5 1.5 2 5 5 5 5 5 5 5 5* 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 6* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7* 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 8* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 1 1.5
1.5 1.5 1.5 1.5 2.5 3 3 1.5 *= Disease Free
TABLE-US-00035 TABLE 18b Day Day Day Day Day Day Day Day Day Day 10
11 12 13 14 15 16 17 18 19 MEAN Water Control 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.25 0.40 0.65 GR-056 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.25 SEM Water Control 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.17 0.21 0.29 GR-056 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.17 INCIDENCE Water Control 0 0 0 0 0 0 0 20 30 40 GR-056 0 0
0 0 0 0 0 0 0 20 CUM. INCIDENCE Water Control 0 0 0 0 0 0 0 20 30
40 GR-056 0 0 0 0 0 0 0 0 0 20 CUM. DISEASE FREE Water Control 100
100 100 100 100 100 100 80 70 60 GR-056 100 100 100 100 100 100 100
100 100 80 Day Day Day Day Day Day Day Day Day 20 21 22 23 24 25 26
27 28 MEAN Water Control 1.00 1.65 2.00 2.30 2.30 2.45 2.40 2.40
1.95 GR-056 0.30 0.50 0.85 0.90 1.10 1.20 1.30 1.25 1.10 SEM Water
Control 0.29 0.48 0.59 0.66 0.66 0.62 0.63 0.63 0.73 GR-056 0.20
0.26 0.52 0.53 0.54 0.56 0.57 0.57 0.54 INCIDENCE Water Control 60
70 70 70 70 80 80 80 50 GR-056 20 30 30 30 40 40 40 40 40 CUM.
INCIDENCE Water Control 60 70 70 70 70 80 80 80 90 GR-056 20 30 30
30 40 40 40 40 40 CUM. DISEASE FREE Water Control 40 30 30 30 30 20
20 20 10 GR-056 80 70 70 70 60 60 60 60 60
[0877] FIG. 98 shows graphically the percent of animals developing
any symptoms of disease in each of the Control Group 1 and the gold
nanocrystal Treatment Group 2 (i.e., GB-056). Control Group 1
showed that 90% of the mice developed at least some symptoms,
whereas only 40% of the mice in Treatment Group 2 developed some
level of symptoms.
[0878] FIG. 99 shows the EAE scoring averages for each group. Of
note, the onset of any symptoms was delayed by two days in gold
nanocrystal Treatment Group 2 and the overall scoring for Treatment
Group 2 was significantly less than the reported averages in
Control Group 1. Clearly the gold nanocrystal formulation GB-056,
having a measured gold concentration of about 12 ppm, significantly
outperformed the Control Group 1 in this EAE test.
[0879] As is typical for this EAE model, one animal in Treatment
Group 2 (i.e., animal 4) died; whereas 3 animals in Control Group 1
died.
[0880] The most severe pathology occurred in Control Group 1 and
the least severe in Treatment Group 2.
[0881] The one animal in Treatment Group 2 that died (i.e., animal
4) caused the group to have a much higher score. Clearly the
inventive gold nanocrystal suspension GB-056 had a significant
positive effect in this EAE test. Without wishing to be bound by
any particular theory or explanation, the results of this Example,
in combination with the results of the murine CIA model and the in
vitro MIF cytokine analysis, strongly suggest that MIF, and/or MIF
signaling pathways, are being favorably influenced by the inventive
gold nanocrystalline compositions of the present invention.
Example 27
Long Term Exposure of Gold Nanocrystal Suspension GD-013 in
Mice
[0882] The purpose of this Example was to observe if any negative
toxicology effects occurred in mice when the mice drank, ad
libitum, gold nanocrystal suspension GD-013 as their only source of
liquid for an extended period of time.
[0883] A total of 25 female mice were used in this Example, five
(5) in the control group; and ten (10) in each of two treatment
groups. The control group received regular bottled water in their
drinking bottles. The two treatment groups received two different
concentrations of GD-013 as their only drinking liquid. A first
treatment group received a 50% GD-013 crystal suspension (with the
other 50% being purified DI/RO water) while the second treatment
group received 100% GD-013 crystal suspension. All groups were
permitted to drink as much, or as little, as desired; food was
provided ad libitum as well. The weight of each animal and the
average amount of liquid consumed were recorded weekly. At week 23
of the study, 6 mice were sacrificed (3 mice from each of the
GD-013 crystal suspension treatment groups) for necropsy and
pathology. The remaining mice continued to consume the two
treatment suspensions through 46 weeks.
Materials and Methods:
[0884] In this type of exposure study it is acceptable to use only
one sex, females, for the purposes of testing for toxicity. Data
from other studies have shown that there is generally no difference
between the sexes, but when one sex does react more strongly it is
typically the females. Males are only used when there is some form
of evidence indicating that they may have a stronger reaction.
Since, there is no such information indicating that males would be
affected in this way, only females were used. The females used were
adult, nulliparous and non-pregnant. The Swiss Webster strain of
outbred mice was used in this example. This strain was chosen
because of its widespread use in general purpose and toxicology
research. It also is known to not have any detrimental genetic
deficits that could potentially interfere with data collection.
TABLE-US-00036 TABLE 19 Study Information Mode of Adminis- Species
Strain Group tration Doses Duration Mus Swiss Control - 5/F Via
Water Ad 23 weeks musculus Webster 50% GD-013 - Bottle libitum and
46 10/F weeks 100% GD-013 - 10/F
Dose Preparation
[0885] All treatment groups involved in this study received the
referenced GD-013 nanocrystalline suspensions in their water
bottles. The mice were allowed to drink free choice. The control
group received purified, bottled water.
TABLE-US-00037 TABLE 20 GD-013 Treatment Information Treatment
Group Lot Numbers Au Content Control Bottled Water 0.0 ppm Au 50%
GD-013 Au 50/50 GD-013/RO H.sub.2O 7.6 ppm Au 50% RO H.sub.2O 100%
GD-013 Au GD-013 15.2 ppm Au
Housing and Feeding
[0886] All study personnel entering the mouse study area wore
personal protection clothing (i.e. gloves, face mask, and shoe
covers). Mice were purchased from Harlan Laboratories. Upon receipt
of the mice, the mice were given permanent identification in the
form of a tail tattoo (Harvard Apparatus Tattoo). The mice were
then randomly assigned and housed by groups of 5 mice per cage. The
cages were large enough to allow adequate room for 5 individuals
and were not so small as to hinder clear observations of each
animal. The mice were acclimated to the lab environment for a
period of one week. The housing area was maintained at a constant
temperature of 22.degree. C. (f 3.degree. C.), and the relative
humidity was maintained at 30%-50%. Artificial, full spectrum
lighting was used (PureLite 60 w, 120 v bulbs). Timers were used to
achieve a 12-hour light 12-hour dark cycle. Food was provided ad
libitum (Purina Certified Rodent Diet 5002). Standard corncob
bedding was provided in the cages. Cage changes were carried out
once weekly. When an animal was found dead the cage that it was
housed in was changed immediately after the dead animal was
removed.
Procedure and Observation
[0887] After the acclimation period, both treatment groups began
receiving the noted GD-013 nanocrystalline suspensions in their
water bottles. The control group continued to receive purified
drinking water. On the first day of treatment each mouse was
weighed, and their weights were recorded. At the start of each
week, all of the mice were again weighed, and their weights
recorded. Also, the approximate amount of water and GD-013 crystal
suspension consumed, was recorded each week. Throughout the study
the mice were observed for any abnormalities or signs of
distress.
Weight Gain
[0888] When the study began, all of the mice were approximately the
same weight. Each week, each animal was weighed, and its weight was
recorded. The individual weights of each animal in the groups were
then averaged and plotted graphically in FIG. 106 to show the
average weight gain of all the groups over the course of the study.
A vertical line at week 23 is present in FIG. 106 and denotes the
time when histopathology was performed.
Average Daily Consumption
[0889] Every week the amount of: (1) water, (2) 50% GD-013, and (3)
100% GD-013 that each group consumed was measured. Once the amount
of liquid, 50% purified water, that had been consumed during the
previous week had been determined, calculations were made to find
an approximate daily intake per animal over the course of the week.
The liquid consumption data for 46 weeks is shown in FIG. 107.
Results/Conclusions:
Weight Gain
[0890] Statistical analysis of the average weights of the groups
was performed to determine if there was any difference in weight
gain and/or loss between the groups. Each treatment group was
compared to the control group; and the two treatment groups were
also compared to each other. Overall there was a statistically
significant weight loss between the 100% GD-013 Treatment Group and
the Control Group (P<0.05). There was no statistically
significant weight gain/loss between the two Treatment Groups or
between the 50% GD-013 Treatment Group and the Control Group.
Average Weekly Consumption
[0891] All three of the groups consumed what is considered to be
normal amounts of liquid daily, so dehydration was not an issue.
Again, statistical analyses of the consumption values for each
group was performed to determine if there was a significant
difference in consumption. Both Treatment Groups were compared to
the Control Group and both Treatment Groups were compared to each
other. The Control Group consumed significantly less than both
Treatment Groups (P<0.05). There was no statistical difference
between the amounts consumed by the Treatment Groups (P>0.05).
There were no observable differences in health, behavior, or issues
related to dehydration.
Mortality
[0892] There were two recorded deaths in the study, one from each
Treatment Group. The first death occurred in the 50% GD-013 group
at week 20. The second death occurred at week 22 in the 100% GD-013
group. The mouse from the 50% GD-013 treatment group had always
been much smaller than the rest and had not been gaining weight;
the cause of this is unknown. The other mouse had not shown any
indicators of distress or poor health. No pathology was possible
for these two mice.
Pathology
[0893] Three mice from each treatment group were submitted for
pathology at week 23. The following organs were submitted for
histopathological evaluation: heart, thymus, lung, liver, kidney,
spleen, stomach, duodenum, jejunum, ileum, cecum, colon, urinary
bladder, ovary, striated muscle, haired skin, bone marrow
(femur/tibia), pituitary and brain. The pathology findings
concluded that despite some of the abnormalities that were noted,
all were considered incidental findings that were associated with
normal variation between individuals and normal wear and tear. None
of the findings in the pathology report indicated any degree of
toxicity to target organs. The pathologist was completely blind to
what treatment the mice in the study received, nor did the
pathologist have any knowledge of treatment in control mice in
order to eliminate possible bias in the pathology findings.
[0894] All tissues referenced above were grossly examined and only
the spleen and liver were found to have minimal to mild variations
in color. The only specific histopathological findings are reported
in Table 21. The numbers "2-3," "2-5" and "4-7" in the 50% GD-013
row refer to three different mice, to which the "Comments" are
directed. Likewise, the histopathology "Comments" regarding the
spleen are directed to three mice, "3-3," "5-9" and "5-10;" whereas
the "Comments" regarding the liver apply to only one mouse (i.e.,
"5-10"). All gross examinations were consistent with congestion
from euthanasia and/or fat storage and were considered to be within
normal limits. No gross lesions were noted.
TABLE-US-00038 TABLE 21 Pathology Findings Histopathological Group
Findings Comments 50% Spleen: Hematopoiesis, EMH: normally observed
in GD-013 extramedullary, multifocal, minimal to moderate minimal
red pulp (2-3, 2-5, degrees; is considered a 4-7 common, incidental
finding not indicative of toxic change or infection 100% Spleen:
Hematopoiesis, EMH: normally observed in GD-013 extramedullary,
multifocal, minimal to moderate minimal to moderate, red degrees;
is considered a pulp (3-3, 5-9, 5-10) common, incidental finding
Liver: Microgranuloma, not indicative of toxic focal, minimal,
hepatocytes change or infection (5-10 Liver: Condition considered
to be from bacterial showering from the hepatic portal system; not
indicative of infection or toxic change
Example 28
35-Day Uptake and Distribution Acute Toxicity Study
[0895] The purpose of this 35-day study was to determine the uptake
and distribution and acute toxicity (if any) of two crystal
suspensions (GB-134 and GB-151) and compare the results to a
commercially available Mesogold product. Thirteen mice were
involved in this study. Concentrations of gold were determined in
the urine and the feces, as well as in certain vital organs and
blood of the test animals. Additionally, a selection of organs from
some individuals were examined histologically to determine if there
were any abnormalities. Further, all mice were permitted to drink
up to the point that they were sacrificed for this study. This
procedure was followed to insure, for example, that accurate gold
concentrations in the blood could be determined.
Materials and Methods:
TABLE-US-00039 [0896] TABLE 22 Study Information Mode of Adminis-
Species Strain Group tration Doses Duration Mus Swiss Mesogold -
Free Mesogold, 35 days musculus Webster 3/F Choice GB-134, GB-134 -
GB-151 10/F GB-151 - 10/F
Dose Preparation
[0897] All treatment groups involved in this study received their
solutions in their water bottles. The mice were allowed to drink
free choice. Each group received either: (1) Mesogold, (2) GB-134,
or (3) GB-151 (all of which were not diluted) in their drinking
bottles.
TABLE-US-00040 TABLE 23 Au Solution Treatment Information Treatment
Group Lot Numbers Au Content Mesogold Mesogold 19.8 ppm Au GB-134
GB-134 8.9 ppm Au GB-151 GB-151 8.3 ppm Au
Procedure and Observation
[0898] After the animals received their respective treatments for
one day, metabolic cage collections of urine and feces were
initiated. A total of nine animals per week were housed in the
metabolic cages and had their urine and feces collected. While in
the metabolic cages the subject mice continued to receive in their
water bottles the liquid they had been assigned to drink. The
amount of liquid consumed during the 24-hour period was also
measured and recorded. The urine and feces samples were then
collected and tested for Au concentration. The volume of urine
excreted, and the weight of feces collected were also measured and
recorded.
[0899] At the end of the study, all 13 animals were sent to Taconic
Laboratories (Rockville, Md.) for the performance of a gross
necropsy and pathology report or to have organ and blood samples
collected and returned for further analysis (discussed later
herein). Microscopic evaluations were performed on the following
tissues: heart, lung, liver, spleen, kidney, brain, stomach,
duodenum, jejunum, ileum, cecum and colon. Additionally, certain
heart, lung (left and right), liver, spleen, kidney (left and
right), and brain were collected and returned in an empty, sterile
glass vial for further concentration analysis.
Procedure for the Digestion of Feces and Urine Samples
[0900] Specific methods were developed to determine the amount of
gold in the feces and the urine. PTFE sample cups and microwave
digestion bombs were ordered from Fisher Scientific and obtained
from Parr Instrument Company). 23 mL PTFE sample cup (Fisher Cat
No. 0102322A) and Parr 4781 microwave digestion bomb (Fisher Cat
No. 0473155) were used for digestion.
[0901] The microwave used was a Panasonic 1300 Watt. Model No.
NN-SN667 W, Serial No. 6B78090247.
Urine
[0902] 1.5 grams of urine was weighed in a PTFE sample cup. When
urine exceeded that mass, another digestion was prepared. When the
urine sample mass was below 1.5 grams the appropriate amount of
D.I. water was added to bring the mass up to approximately 1.5
grams. 0.24 mL of 50% v/v HNO.sub.3 was added to the sample cup,
followed by 0.48 mL of 36% v/v HCl. The sample cup was sealed and
placed inside a microwave bomb. The microwave bomb was sealed and
placed in the center of a microwave. The sample was irradiated
until the Teflon indicator screw raised up 1 mm from the top of the
bomb. The time the bomb spent in the microwave ranged between 30 to
60 seconds depending on the urine sample. The microwave digestion
bomb was removed from the microwave and cooled for 20-30 minutes,
until the Teflon indicator screw was lowered to its original
position. The sample cup was removed from the microwave digestion
bomb, and the liquid sample was transferred to a vial for
testing.
Feces (1 Pellet Sample):
[0903] A singe fecal pellet was weighed in a PTFE sample cup. 5 mL
of D.I. water was added to the sample cup. 0.8 mL of 50% v/v
HNO.sub.3 was added to the sample cup, followed by 1.6 mL of 36%
v/v HCl. The sample cup was sealed and placed inside a microwave
bomb. The microwave bomb was sealed and placed in the center of the
microwave. The sample was irradiated until the Teflon indicator
screw raised up 1 mm from the top of the bomb. The time the bomb
spent in the microwave ranged between 20 to 30 seconds depending on
the mass of the 1 pellet fecal sample. The microwave digestion bomb
was removed from the microwave and cooled for 20-30 minutes, until
the Teflon indicator screw was lowered to its original position.
The sample cup was removed from the microwave digestion bomb, and
the liquid sample was transferred to a vial for testing.
Bulk Feces Sample
[0904] About 0.300 grams of feces was weighed in a PTFE sample cup.
5 mL of D.I. water was added to the sample cup. 0.8 mL of 50% v/v
HNO.sub.3 was added to the sample cup, followed by 1.6 mL of 36%
v/v HCl. The sample cup was sealed and placed inside a microwave
bomb. The microwave bomb was sealed and placed in the center of a
microwave. The sample was irradiated until the Teflon indicator
screw raised up 1 mm from the top of the bomb. The time the bomb
spent in the microwave ranged between 20 to 40 seconds depending on
the mass of the bulk feces sample. The microwave digestion bomb was
removed from the microwave and cooled for 20-30 minutes, until the
Teflon indicator screw was lowered to its original position. The
sample cup was removed from the microwave digestion bomb, and the
liquid sample was transferred to a vial for testing. Bulk feces
samples may require several digestions to digest all the feces
present in the original sample.
Note: If the sample didn't appear to be fully digested (i.e. solids
still present/discoloration on the PTFE sample cup's side walls) a
second digestion was performed. This required a second addition of
the volumes of D.I. water, 50% v/v HNO.sub.3 and 36% v/v HCl
specified for the appropriate sample. (See above procedures for
correct volumes) The sample was then microwaved again, and allowed
to cool for 20-30 minutes before transferring to a sample vial for
testing. *D.I. water=Deionized water.
*PTFE=polytetrafluoroethylene
[0905] One digested, all samples were analyzed using the atomic
absorption spectroscopy techniques discussed above herein.
[0906] The pathology findings for the 35-day study are shown in
Table 24. All tissues were grossly examined and only the spleen and
liver were found to have minimal to mild variations in color. All
gross examinations were consistent with congestion from euthanasia
and/or fat storage and were considered to be within normal limits.
No gross lesions were noted. The comments were directed to specific
mice and are noted in Table 24. The designation "M-3" refers to one
mouse in the Mesogold group; whereas "GB-134-7" refers to one mouse
in the "GB-134" group; and "G151-9" refers to one mouse in the
"GB-151" group.
TABLE-US-00041 TABLE 24 Histopathological Group Findings Comments
Mesogold Spleen: Hematopoiesis, EMH: normally observed in
extramedullary, multifocal, minimal to moderate minimal to
moderate, red degrees; is considered a pulp (M-3) common,
incidental finding Liver: Microgranuloma, not indicative of toxic
focal, minimal, hepatocytes change or infection (M-3) Liver:
Condition considered to be from bacterial showering from the
hepatic portal system; not indicative of infection or toxic change
GB-134 Spleen: Hematopoiesis, EMH: normally observed in
extramedullary, multifocal, minimal to moderate minimal red pulp
(GB-134- degrees; is considered a 7, GB-134-8) common, incidental
finding Liver: Microgranuloma, not indicative of toxic focal,
minimal, hepatocytes change or infection (GB-134-8) Liver:
Condition considered to be from bacterial showering from the
hepatic portal system; not indicative of infection or toxic change
GB-151 Spleen: Hematopoiesis, EMH: normally observed in
extramedullary, multifocal, minimal to moderate minimal to
moderate, red degrees; is considered a pulp (GB-151-9, GB-151-
common, incidental finding 10) not indicative of toxic change or
infection
[0907] FIG. 108 shows there were no significant difference in
weight gain found between any of the groups (all P>0.05)
[0908] FIG. 109 shows that there were no significant difference in
consumption of fluids found between any of the groups (all
P>0.05)
[0909] FIG. 110 shows that there was a significant difference in
the amount of Au found in the feces between the MesoGold group and
both GB-134 and GB-151 groups (P<0.01). There was no significant
difference found between the GB-134 and GB-151 groups (P>0.05).
Table 25 shows the actual recorded results.
TABLE-US-00042 TABLE 25 Average Weekly Amount of Au Found in Feces
Treatment Groups Week Meso (ppm) GB-134 (ppm) GB-151 (ppm) 0 1.7286
0.5343 0.6871 1 58.8611 24.3989 24.8668 2 59.0330 19.1658 27.4792 3
91.3662 15.9090 19.6045 4 86.5076 18.4982 18.1742 5 65.3942 20.3575
24.9802
[0910] FIG. 111 shows that there was no significant difference in
the average amount of Gold found in the urine between any of the
groups (all P>0.05)
TABLE-US-00043 TABLE 26 Average Weekly Amount of Au Found in Urine
Treatment Groups Week Meso (ppm) GB-134 (ppm) GB-151 (ppm) 0 0.0090
0.0240 0.0330 1 0.1318 0.0821 0.0263 2 0.1004 0.3453 0.0727 3
0.4471 0.1518 0.1264 4 0.1457 0.0920 0.0360 5 0.1953 0.0261
0.0380
Procedure for Neutron Activation Analysis Measurements of Tissue
Samples and Blood
[0911] Certain samples of heart, liver, spleen, kidney, brain and
blood were analyzed for gold content. Specifically, neutron
activation analysis was utilized. Instrumental neutron activation
analysis (NAA) is especially powerful in its sensitivity and its
ability to determine accurately many elements in a single sample.
NAA does not require any chemical treatments or special chemical
preparation of samples, thus minimizing the possibilities of
losses, contamination and any incomplete tissue sample dissolution,
for example.
[0912] The NAA method involves weighing the tissue sample in
polyethylene vials. An inert material is added to each vial to
prevent evaporative loss. Each vial is uniquely identified with a
bar code and a neutron flux monitor affixed to the base of each
vial. These vials are stacked into one-foot long bundles for
irradiation with neutrons from a nuclear reactor. The bundles
contain randomly selected duplicate samples and gold standards (or
known concentrations of gold) are inserted at random positions in
the bundles.
[0913] All bundles are treated in a similar manner. The bundles are
submitted for exposure to a flux of neutrons at a nuclear reactor.
Specifically, the bundles are inserted into the core of a nuclear
reactor for about 45 minutes. The bundles are rotated during
irradiation so that there is no horizontal flux variation. (The
vertical flux variation is monitored with the individual flux
monitors.) This irradiation causes any gold present in the sample
to become radioactive and gold then begins to emit radiation in the
form of penetrating gamma rays whose energies (or wavelengths) are
characteristic of gold (e.g., Au 198, 411.8 keV).
[0914] After a decay period of about six days, the irradiated
samples are loaded onto a counting system. Specifically, each
radiated and partially decayed sample is placed adjacent to a
gamma-ray spectrometer with a high resolution, coaxial germanium
detector. Gamma rays radiate continuously from each sample (so long
as gold is present) and the interaction of the radiated gamma rays
with the detector leads to discrete voltage pulses proportional in
height to the incident gamma-ray energies. A specially developed
multi-channel analyser sorts out the voltage pulses from the
detector according to their size and digitally constructs a
spectrum of gamma-ray energies versus intensities. The counting
time is about 45 minutes per sample. By comparing spectral peak
positions and areas with library standards, gold is both
qualitatively and quantitatively identified. The results of the
analysis are set forth below.
[0915] In conjunction with Table 27 below, FIG. 112 shows a bar
chart, by mouse organ type and the colloid that was orally consumed
by the identified mice. The numbers at the end of each colloid
identification refer to a specific mouse. Specifically, organs from
two mice, GB-151-4 and GB-151-5 were examined. GB-151-4 means that
mouse #4 consumed GB-151. Organs from another mouse, GB-134-3
(i.e., mouse #3 that consumed suspension GB-134) were examined as
well. Organs from another mouse, Mouse #2, (Meso-2) consumed a
commercially available colloidal gold. While the sample size was
relatively small, differences are apparent.
[0916] Gold was not detected in two brain samples, GB-151-6 and
GB-134-3, with the detection limit of 0.35 ppb and 0.25 ppb,
respectively. Blood samples GB-151-5 and GB-134-3 were not analyzed
because of insufficient amount available for analysis.
TABLE-US-00044 TABLE 27 Gold concentration in different tissue
samples and blood measured by NAA. Sample ID Sample mass, g Gold wt
%, ppb GB-151-4, -5 Heart* 0.356 0.89 .+-. 0.187 GB-151-5 Liver
1.536 1.76 .+-. 0.107 GB-151-4, -5 Spleen* 0.213 1.74 .+-. 0.244
GB-151-4, -5, -5 Kidney* 0.661 2.54 .+-. 0.170 GB-151-4, -5 Brain*
0.889 0.73 .+-. 0.102 GB-151-6 Heart 0.129 0.94 .+-. 0.329 GB-151-6
Liver 0.899 2.34 .+-. 0.140 GB-151-6 Spleen 0.093 4.00 .+-. 0.480
GB-151-6 Blood 0.386 1.06 .+-. 0.212 GB-151-6 R& L Kidney 0.476
2.16 .+-. 0.203 GB-151-6 Brain 0.432 <0.35 GB-134-3 Heart 0.158
1.10 .+-. 0.275 GB-134-3 Liver 0.523 0.91 .+-. 0.146 GB-134-3
Spleen 0.118 1.14 .+-. 0.342 GB-134-3 R&L Kidney 0.406 1.59
.+-. 0.191 GB-134-3 Brain 0.455 <0.25 Meso-2 Heart 0.145 1.67
.+-. 0.301 Meso-2 Liver 0.935 6.67 .+-. 0.254 Meso-2 Spleen 0.080
3.01 .+-. 0.572 Meso-2 R&L Kidney 0.415 7.63 .+-. 0.351 Meso-2
Brain 0.400 0.74 .+-. 0.148 Meso-2 Blood 0.268 2.05 .+-. 0.287
*organs from two mice were combined to make one sample
* * * * *