U.S. patent application number 17/026523 was filed with the patent office on 2021-01-07 for engineered metal nanoparticles and methods thereof.
The applicant listed for this patent is AUBURN UNIVERSITY. Invention is credited to Ludmila GLOBA, Mahmoud MANSOUR, Oleg PUSTOVYY, Iryna SOROKULOVA, Vitaly VODYANOY.
Application Number | 20210000868 17/026523 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
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United States Patent
Application |
20210000868 |
Kind Code |
A1 |
VODYANOY; Vitaly ; et
al. |
January 7, 2021 |
ENGINEERED METAL NANOPARTICLES AND METHODS THEREOF
Abstract
A pharmaceutical composition comprises a metal nanoparticle
having an average diameter of about 0.5 nm to about 5 nm. The
composition may be used to treat cancer or an anosmia-related
disease.
Inventors: |
VODYANOY; Vitaly; (Auburn,
AL) ; PUSTOVYY; Oleg; (Auburn, AL) ; GLOBA;
Ludmila; (Auburn, AL) ; MANSOUR; Mahmoud;
(Auburn, AL) ; SOROKULOVA; Iryna; (Auburn,
AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AUBURN UNIVERSITY |
Auburn |
AL |
US |
|
|
Appl. No.: |
17/026523 |
Filed: |
September 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15927616 |
Mar 21, 2018 |
10806754 |
|
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17026523 |
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62474140 |
Mar 21, 2017 |
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62501348 |
May 4, 2017 |
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Current U.S.
Class: |
1/1 |
International
Class: |
A61K 33/34 20060101
A61K033/34; A61K 33/30 20060101 A61K033/30; A61K 9/50 20060101
A61K009/50; A61P 35/00 20060101 A61P035/00; A61K 9/00 20060101
A61K009/00; A61K 47/60 20060101 A61K047/60; A61K 9/14 20060101
A61K009/14 |
Claims
1.-20. (canceled)
21. A pharmaceutical composition comprising a therapeutically
effective amount of a metal nanoparticle.
22. The pharmaceutical composition of claim 21, wherein the metal
nanoparticle has an average diameter of about 0.5 nm to about 5
nm.
23. The pharmaceutical composition of claim 21, wherein the metal
nanoparticle has an average diameter of about 1 nm to about 3
nm.
24. The pharmaceutical composition of claim 21, wherein the metal
nanoparticle has an average diameter of about 1 nm to about 2.5
nm.
25. The pharmaceutical composition of claim 21, wherein the metal
nanoparticle comprises about 25 atoms to about 100 atoms.
26. The pharmaceutical composition of claim 21, wherein the metal
nanoparticle comprises about 30 atoms to about 60 atoms.
27. The pharmaceutical composition of claim 21, wherein the metal
nanoparticle further comprises a coating.
28. The pharmaceutical composition of claim 27, wherein the coating
surrounds the periphery of the metal nanoparticle.
29. The pharmaceutical composition of claim 27, wherein the coating
comprises a polypeptide or a polyether.
30. The pharmaceutical composition of claim 27, wherein the coating
comprises polyethylene glycol.
31. The pharmaceutical composition of claim 30, wherein the
polyethylene glycol has a molecular weight of about 100 g/mol to
about 1000 g/mol.
32. The pharmaceutical composition of claim 30, wherein the
polyethylene glycol has a molecular weight of about 300 g/mol to
about 750 g/mol.
33. The pharmaceutical composition of claim 30, wherein the
polyethylene glycol has a molecular weight of about 400 g/mol.
34. The pharmaceutical composition of claim 27, wherein coating is
covalently bonded to the metal nanoparticle.
35. The pharmaceutical composition of claim 27, wherein the coating
withdraws electron charge from the metal nanoparticle and increases
binding energy to the metal nanoparticle.
36. The pharmaceutical composition of claim 27, wherein the coating
donates electron charge to the metal nanoparticle and decreases
binding energy to the metal nanoparticle.
37. The pharmaceutical composition of claim 27, wherein the coating
and the metal nanoparticle form a conjugated electron system.
38. A method of treating cancer in a patient, said method
comprising the step of administering to the patient a
therapeutically effective amount of a pharmaceutical composition
comprising a therapeutically effective amount of a metal
nanoparticle.
39. The method of claim 38, wherein the metal nanoparticle
selectively causes apoptosis in cancerous cells compared to
noncancerous cells.
40. The method of claim 39, wherein the noncancerous cells are
noncancerous astrocytes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application Nos. 62/501,348, filed May
4, 2017, and 62/474,140, filed Mar. 21, 2017, which are expressly
incorporated by reference herein.
TECHNICAL FIELD
[0002] The invention relates to engineered metal nanoparticles for
treating diseases. The invention includes compositions and methods
related to engineered metal nanoparticles, including methods and
compositions for treating cancer and anosmia-related diseases.
BACKGROUND
[0003] Metal nanoparticles are present in and have been isolated
from the blood of several vertebrate species. These metal
nanoparticles have been obtained as part of protein nucleating
centers (PNCs) isolated from animal blood.
[0004] Investigations into PNCs have revealed that these
compositions may be toxic to cultured cancer cells at low
concentrations, such as nanomolar concentrations. Lethal
concentrations of synthetic metal nanoparticles reported in the
literature are orders of magnitude higher than the natural,
blood-isolated metal nanoparticles. Additionally, olfactory
responses have been enhanced with the addition of endogenous zinc
nanoparticles.
SUMMARY
[0005] The present disclosure describes engineered nanoparticles
for use in treating diseases. The nanoparticles are effective at
lower concentrations than nanoparticles that have been used in the
art. Advantageously, the engineered nanoparticles are formed
without the need for animal sources, such as animal blood.
[0006] The following numbered embodiments are contemplated and are
non-limiting:
[0007] 1. A pharmaceutical composition comprising a therapeutically
effective amount of a metal nanoparticle.
[0008] 2. The pharmaceutical composition of clause 1, wherein the
metal nanoparticle has an average diameter of about 0.5 nm to about
5 nm.
[0009] 3. The pharmaceutical composition of clause 1 or 2, wherein
the metal nanoparticle has an average diameter of about 1 nm to
about 3 nm.
[0010] 4. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle has an average diameter of
about 1 nm to about 2.5 nm.
[0011] 5. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle has an average diameter of
about 1.2 nm.
[0012] 6. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle has an average diameter of
about 2.1 nm.
[0013] 7. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle comprises about 25 atoms to
about 100 atoms.
[0014] 8. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle comprises about 30 atoms to
about 60 atoms.
[0015] 9. The pharmaceutical composition of any of the preceding
clauses, further comprising an excipient.
[0016] 10. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle comprises a metal selected
from the group consisting of copper, iron, zinc, silver, gold,
platinum, and magnesium.
[0017] 11. The pharmaceutical composition of any of the preceding
clauses, wherein less than about 50% of the metal nanoparticle is
oxidized.
[0018] 12. The pharmaceutical composition of any of the preceding
clauses, wherein less than about 15% of the metal nanoparticle is
oxidized.
[0019] 13. The pharmaceutical composition of any of the preceding
clauses, wherein less than about 5% of the metal nanoparticle is
oxidized.
[0020] 14. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle has a purity of at least 95
wt. %.
[0021] 15. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle has a purity of at least 99
wt. %.
[0022] 16. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle is formed by an underwater
high-voltage discharge method.
[0023] 17. The pharmaceutical composition of clause 16, wherein the
underwater high-voltage discharge method comprises applying a
voltage of about 10,000 volts to about 20,000 volts to metal
electrodes and creating an electric discharge.
[0024] 18. The pharmaceutical composition of clause 16 or 17,
wherein the underwater high-voltage discharge method comprises
applying a voltage of about 15,000 volts and creating an electric
discharge.
[0025] 19. The pharmaceutical composition of clause 17 or 18,
wherein the metal nanoparticle isolated by centrifugation.
[0026] 20. The pharmaceutical composition of any of the preceding
clauses, wherein the metal nanoparticle further comprises a
coating.
[0027] 21. The pharmaceutical composition of clause 20, wherein the
coating surrounds the periphery of the metal nanoparticle.
[0028] 22. The pharmaceutical composition of clause 20 or 21,
wherein the coating comprises a polypeptide or a polyether.
[0029] 23. The pharmaceutical composition of any of clauses 20 to
22, wherein the coating comprises polyethylene glycol.
[0030] 24. The pharmaceutical composition of clause 23, wherein the
polyethylene glycol has a molecular weight of about 200 g/mol to
about 1000 g/mol.
[0031] 25. The pharmaceutical composition of clause 23 to 24,
wherein the polyethylene glycol has a molecular weight of about 400
g/mol.
[0032] 26. The pharmaceutical composition of any one of clauses 20
to 25, wherein coating is covalently bonded to the metal
nanoparticle.
[0033] 27. The pharmaceutical composition of any one of clauses 20
to 26, wherein the coating withdraws electron charge from the metal
nanoparticle and increases binding energy to the metal
nanoparticle.
[0034] 28. The pharmaceutical composition of any one of clauses 20
to 26, wherein the coating donates electron charge to the metal
nanoparticle and decreases binding energy to the metal
nanoparticle.
[0035] 29. The pharmaceutical composition of any one of clauses 20
to 28, wherein the coating and the metal nanoparticle form a
conjugated electron system.
[0036] 30. A method of treating cancer in a patient, comprising
administering to the patient a therapeutically effective amount of
a metal nanoparticle of any one of the preceding clauses.
[0037] 31. The method of clause 30, wherein the viability of a
cancerous cell line decreases by at least about 75%.
[0038] 32. The method of clause 30 or 31, wherein the viability of
a cancerous cell line decreases by at least about 90%.
[0039] 33. The method of clause 31 or 32, wherein the viability of
the cancerous cell line is reduced in a dose-dependent manner.
[0040] 34. The method of any of clauses 30 to 33, wherein the metal
nanoparticle selectively causes apoptosis in cancerous cells
compared to noncancerous cells.
[0041] 35. The method of clause 34, wherein the noncancerous cells
are noncancerous astrocytes.
[0042] 36. The method of clause 35, wherein at least about 50% of
the noncancerous astrocytes remain viable.
[0043] 37. The method of clause 35 or 36, wherein at least about
75% of the noncancerous astrocytes remain viable.
[0044] 38. The method of any of clauses 30 to 37, wherein cell
adherence is decreased.
[0045] 39. The method of any of clauses 30 to 38, wherein cell
shrinking occurs.
[0046] 40. The method of any of clauses 30 to 39, wherein cell
rounding occurs.
[0047] 41. The method of any of clauses 30 to 40, wherein nuclear
condensation occurs.
[0048] 42. The method of any of clauses 30 to 41, wherein the
cancer is brain cancer or prostate cancer.
[0049] 43. The method of any of clauses 30 to 42, wherein the
cancer comprises a cancerous cell line selected from the group
consisting of Hs683 cells, SVGp12 cells, PC3 cells, U937 cells, and
HeLa cells.
[0050] 44. The method of any of clauses 30 to 43, wherein the metal
nanoparticle is applied at a dose of about 0.01 nM to about 1.0
nM.
[0051] 45. The method of any of clauses 30 to 44, wherein the metal
nanoparticle is applied at a dose of about 0.05 nM to about 0.3
nM.
[0052] 45. The method of any of clauses 30 to 44, wherein the metal
nanoparticle is applied at a dose of about 0.01 nM to about 0.1
nM.
[0053] 46. A method of treating an anosmia-related disease in a
patient, comprising administering to the patient a therapeutically
effective amount of a metal nanoparticle of any one of the
preceding clauses.
[0054] 47. The method of clause 46, wherein the anosmia-related
disease is Alzheimer's disease.
[0055] 48. The method of clause 46, wherein the anosmia-related
disease is Parkinson's disease.
[0056] 49. The method of any one of clauses 46 to 48, wherein
olfactory response is enhanced for about 10 days.
[0057] 50. The method of any one of clauses 46 to 49, wherein
olfactory response is enhanced for about 100 days.
[0058] 51. The method of any one of clauses 46 to 50, wherein
olfactory response is enhanced for about 300 days.
[0059] 52. The method of any of clauses 46 to 51, wherein the metal
nanoparticle is applied at a dose of about 0.01 nM to about 1.0
nM.
[0060] 53. The method of any of clauses 46 to 52, wherein the metal
nanoparticle is applied at a dose of about 0.05 nM to about 0.3
nM.
[0061] 54. The method of any of clauses 46 to 52, wherein the metal
nanoparticle is applied at a dose of about 0.01 nM to about 0.1
nM.
[0062] Additional features of the present disclosure will become
apparent to those skilled in the art upon consideration of
illustrative embodiments exemplifying the best mode of carrying out
the disclosure as presently perceived.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0063] The detailed description particularly refers to the
accompanying figures in which:
[0064] FIG. 1A shows an atomic force microscope (AFM) image of
0.01% zinc nanoparticles on mica.
[0065] FIG. 1B shows an AFM image of 0.01% copper nanoparticles on
mica.
[0066] FIG. 1C shows a size distribution of the zinc nanoparticles
of FIG. 1A.
[0067] FIG. 1D shows a size distribution of the copper
nanoparticles of FIG. 1B.
[0068] FIG. 1E shows an X-ray photoelectron spectroscopy (XPS)
spectrum of the zinc nanoparticles of FIG. 1A.
[0069] FIG. 1F shows an XPS spectrum of the copper nanoparticles of
FIG. 1B.
[0070] FIG. 2A shows viability of glioma cells at six experimental
conditions related to treatment with zinc nanoparticles.
[0071] FIG. 2B shows a Hill presentation of glioma cell mortality
based on the results shown in FIG. 2A.
[0072] FIG. 2C shows viability of glioma cells at six experimental
conditions related to treatment with copper nanoparticles.
[0073] FIG. 2D shows a Hill presentation of glioma cell mortality
based on the results shown in FIG. 2C.
[0074] FIG. 2E shows viability of astrocytes at six experimental
conditions related to treatment with zinc nanoparticles.
[0075] FIG. 2F shows viability of astrocytes at six experimental
conditions related to treatment with copper nanoparticles.
[0076] FIG. 3A shows viability of PC3 cells at five experimental
conditions related to treatment with copper nanoparticles.
[0077] FIG. 3B shows viability of RWPE-1 cells at five experimental
conditions related to treatment with copper nanoparticles.
[0078] FIG. 4A shows fluorescent labeling of Annexin V in
staurosporine-treated PC3 prostate cancer cells.
[0079] FIG. 4B shows fluorescent labeling of Annexin V in copper
nanoparticle-treated PC3 prostate cancer cells.
[0080] FIG. 4C shows fluorescent labeling of Annexin V in untreated
PC3 prostate cancer cells.
[0081] FIG. 5A shows a light darkfield image of RG2 glioma cells
before exposure to zinc nanoparticles.
[0082] FIG. 5B shows a light darkfield image of RG2 glioma cells
after exposure to 5 nmol/L of zinc nanoparticles.
[0083] FIG. 6A shows an AFM image of uncoated zinc
nanoparticles.
[0084] FIG. 6B shows a size distribution of the zinc nanoparticles
of FIG. 6A.
[0085] FIG. 6C shows an AFM image of ZnPEG400 nanoparticles.
[0086] FIG. 6D shows a size distribution of the zinc nanoparticles
of FIG. 6C.
[0087] FIG. 6E shows an AFM image of ZnPEG1000 nanoparticles.
[0088] FIG. 6F shows a size distribution of the zinc nanoparticles
of FIG. 6E.
[0089] FIG. 7A shows a transmission electron microscope (TEM) image
of non-PEGylated Zn nanoparticles at a first magnification.
[0090] FIG. 7B shows a TEM image of ZnPEG400 nanoparticles.
[0091] FIG. 7C shows a TEM image of the non-PEGylated Zn
nanoparticles of FIG. 7A at a second magnification.
[0092] FIG. 7D shows a TEM image of the non-PEGylated Zn
nanoparticles of FIG. 7A at a third magnification.
[0093] FIG. 7E shows a TEM image of ZnPEG1000 nanoparticles at a
first magnification.
[0094] FIG. 7F shows a TEM image of the ZnPEG1000 nanoparticles of
FIG. 7E at a second magnification.
[0095] FIG. 8A show XPS spectra showing the C is core lines for
uncoated zinc nanoparticles, ZnPEG400 nanoparticles, and ZnPEG1000
stored 1 day at 278 K (5.degree. C.), with the spectra offset to
facilitate viewing. The solid curves indicate the experimentally
obtained spectra, with the dotted curves underneath indicating
their best-fit chemical components.
[0096] FIG. 8B shows XPS spectra showing the Zn 2p3/2 core lines
for the nanoparticles of FIG. 8A, shown in log scale and with the
spectra offset to facilitate viewing. The solid curves indicate the
experimentally obtained spectra, with the dotted curves underneath
indicating their best-fit chemical components.
[0097] FIG. 9 shows electroolfactogram (EOG) recordings from rat
olfactory epithelium produced by 0.25 s pulses of (1) odorant
mixture, (2) odorant mixture+1.2 nm zinc nanoparticles, (3) odorant
mixture+0.25 mmol/L PEG400, (4) odorant mixture+0.10 mmol/L
PEG1000, (5) water vapor+PEG400, (6) water vapor+PEG1000, and (7)
water vapor. The representative set of traces was obtained from 50
EOG traces.
[0098] FIG. 10 is a chart showing the relative enhancement of EOG
signals as a function of time of storage of zinc nanoparticles.
[0099] FIG. 11A is a chart showing the relative enhancement of EOG
signals evoked by non-PEGylated zinc nanoparticles as a function of
ZnO concentration.
[0100] FIG. 11B is a chart showing the relative enhancement of EOG
signals evoked by ZnPEG400 nanoparticles as a function of ZnO
concentration.
[0101] FIG. 11C is a chart showing the relative enhancement of EOG
signals evoked by ZnPEG1000 nanoparticles as a function of ZnO
concentration.
DETAILED DESCRIPTION
[0102] Various embodiments of the invention are described herein as
follows. In one embodiment, metal nanoparticles are provided. The
metal nanoparticles are engineered rather than being obtained from
biological sources. For example, the nanoparticles do not require
isolation or purification from animal sources. In some embodiments,
the nanoparticles described herein may be obtained at lower costs
than those obtained from biological sources.
[0103] Additionally, the metal nanoparticles of the present
disclosure are effective at low concentrations, such as nanomolar
and subnanomolar concentrations. Furthermore, the nanoparticles
described herein may mimic properties of endogenous metal
nanoparticles and may cause limited side effects compared to other
therapeutics.
[0104] In some embodiments, the metal nanoparticles of the present
disclosure comprise or consist essentially of a plurality of
non-oxidized metal atoms. The metal atoms may be selected from the
group consisting of zinc, copper, iron, gold, silver, platinum, and
magnesium. In some embodiments, the nanoparticles comprise or
consist essentially of only one type of atom rather than a mixture
of atoms.
[0105] In some embodiments, the metal nanoparticles are of high
purity and free from contamination. As used herein, purity refers
to the amount of the particle's constituent metal atoms compared to
all atoms. The purity, as determined based on weight, of the metal
nanoparticles may be at least about 50%, at least about 75%, at
least about 85%, at least about 90%, at least about 95%, at least
about 99%, from about 50% to about 99.9%, from about 75% to about
99.9%, from about 85% to about 99.9%, from about 90% to about
99.9%, from about 95% to about 99.9%, or from about 99% to about
99.9%.
[0106] In some embodiments, the nanoparticles are primarily in a
nonoxidized, elemental state rather than being oxidized. In some
embodiments, the percentage of metal nanoparticles, based on molar
ratio, that are nonoxidized is least about 50%, at least about 75%,
at least about 85%, at least about 90%, at least about 95%, or at
least about 99%. In some embodiments purity of the nanoparticles
and/or the amount of oxidation in the particles is determined by
XPS.
[0107] In some embodiments, the metal nanoparticles are in
crystalline form. The crystal structure of the metal nanoparticles
is the same or substantially the same as the most stable bulk
crystal structure of the nanoparticle's constituent atoms.
[0108] In some embodiments, the nanoparticles are a plurality of
small particles. Without intending to be bound by theory, this
small size might facilitate with cellular uptake or excretion. For
example, it is believed that small, uncharged nanoparticles of can
efficiently penetrate through the external and nuclear membranes
and create aggregates. The average diameter of the particles may be
less than about 50 nm, less than about 40 nm, less than about 30
nm, less than about 20 nm, less than about 10 nm, less than about 5
nm, less than about 4 nm, less than about 3 nm, less than about 2
nm, from about 0.5 nm to about 50 nm, from about 0.5 nm to about 40
nm, from about 0.5 nm to about 30 nm, from about 0.5 nm to about 20
nm, from about 0.5 nm to about 10 nm, from about 0.5 nm to about 5
nm, from about 0.5 nm to about 4 nm, from about 0.5 nm to about 3
nm, from about 0.5 nm to about 2 nm, from about 1 nm to about 50
nm, from about 1 nm to about 40 nm, from about 1 nm to about 30 nm,
from about 1 nm to about 20 nm, from about 1 nm to about 10 nm,
from about 1 nm to about 5 nm, from about 1 nm to about 4 nm, from
about 1 nm to about 3 nm, from about 1 nm to about 2 nm, about 1
nm, or about 2 nm.
[0109] In some embodiments, the nanoparticles of the present
disclosure are made of fewer than about 300 atoms, fewer than about
200 atoms, fewer than about 100 atoms, fewer than about 75 atoms,
fewer than about 65 atoms, fewer than about 50 atoms, about 10 to
about 100 atoms, about 10 to about 75 atoms, about 10 to about 65
atoms, about 10 to about 50 atoms, about 25 to about 300 atoms,
about 25 to about 200 atoms, about 25 to about 100 atoms, about 25
to about 75 atoms, about 25 to about 65 atoms, about 25 to about 50
atoms, about 40 to about 100 atoms, about 40 to about 75 atoms,
about 40 to about 65 atoms, about 40 to about 50 atoms, about 40 to
about 200 atoms, about 40 to about 300 atoms, about 100 to about
200 atoms, about 100 to about 300 atoms, about 200 to about 300
atoms, or about 60 atoms.
[0110] In some embodiments, zinc nanoparticles may be engineered
using a high-voltage electrical discharge method. The high-voltage
electrical discharge method comprises applying an electrical
potential to two bulk metal rods that are submerged in water. The
bulk metal rods produce a discharge that produces metal
nanoparticles.
[0111] In some embodiments, the metal rods used in the electrical
discharge method are of high purity. The purity of the metal may be
greater than about 99%, greater than about 99.9%, greater than
about 99.99%, greater than about 99.999%, or greater than about
99.9999%. In some embodiments, the metal rods are submerged in high
purity water. For example, the water may be LC-MS grade water.
[0112] The distance between the electrodes may be controlled to
create a fine dispersion of metal nanoparticles. In some
embodiments, the distance between the electrodes is about 0.25 cm
to about 2 cm, about 0.25 cm to about 1 cm, about 0.25 to about 0.5
cm, about 0.5 cm to about 2 cm, about 0.5 cm to about 1 cm, or
about 0.5 cm.
[0113] The potential applied to the electrodes may be controlled to
create a fine dispersion of metal nanoparticles. In some
embodiments, the voltage is an AC voltage. In some embodiments, the
voltage is about 1,000 volts to about 50,000 volts, about 5,000
volts to about 50,000 volts, about 10,000 volts to about 50,000
volts, about 15,000 volts to about 50,000 volts, 1,000 volts to
about 25,000 volts, about 5,000 volts to about 25,000 volts, about
10,000 volts to about 25,000 volts, or about 15,000 volts to about
25,000 volts. In some embodiments, the potential is sustained for
at least about 1 hour.
[0114] After the voltage is applied, the suspension of
nanoparticles may be allowed to rest such that large particles
sediment and smaller particles remain in suspension. The suspended
particles may be separated from the sediment and subjected to
centrifugation. The speed and time of centrifugation, along with
the number of centrifugations, may be configured to separate
nanoparticles of a desired size.
[0115] In some embodiments, the metal nanoparticles described
herein may be coated with one or more coating materials. The
coating material may be selected from the group consisting of
thiols such as thioctic acid or cysteine, phosphonic acids such as
6-phosphonohexanoic acid or neridronate, silanes such as
(3-aminopropyl)triethoxysilane, peptides such as short peptides
having 2 to 10 amino acids, and ethers such as polyethylene glycol
or polypropylene glycol. In some embodiments, the coating is
covalently bonded to the metal nanoparticle.
[0116] In some embodiments, the coating material is polyethylene
glycol (PEG). The PEG coating may be provided by PEGylation. As
used herein, PEGylation refers to a chemical process comprising
contacting the surface of a nanoparticle with PEG. The molecular
weight of the PEG coating may be about 100 g/mol to about 1000
g/mol, about 200 g/mol to about 1000 g/mol, about 300 g/mol to
about 1000 g/mol, about 400 g/mol to about 1000 g/mol, about 100
g/mol to about 750 g/mol, about 200 g/mol to about 750 g/mol, about
300 g/mol to about 750 g/mol, about 400 g/mol to about 750 g/mol,
about 100 g/mol to about 500 g/mol, about 200 g/mol to about 500
g/mol, about 300 g/mol to about 500 g/mol, or about 400 g/mol to
about 500 g/mol.
[0117] In some embodiments, the coating material may improve the
stability and avoid degradation of the metal nanoparticle. For
example, the coating material may allow the metal nanoparticle to
remain in the elemental state, i.e., not be oxidized, for a longer
period of time than an uncoated nanoparticle. Additionally, the
coating material may be configured to improve the binding
properties of the metal nanoparticle. The protection of metal
nanoparticles from rapid oxidation and improvement of their
biophysical and biochemical properties may provide for better
interactions with receptors, compatibility of the particle-cell
interface, and/or reduced toxicity. In some embodiments, the
coating may offer antibacterial activity.
[0118] Without intending to be bound by theory, in some
embodiments, the coating may interact with the atoms of the metal
nanoparticles in such a way that the electronic structure of the
nanoparticles is affected, and a conjugated electron system is
produced. For example, the coating may withdraw electron charge and
increase the binding energy to the particle. Conversely, the
coating may act as a passivation layer that leads to a reduction in
binding energy because of the fact that the zinc nanoparticles are
acting as electron donors. It is contemplated that the length,
viscosity, and/or rigidity of the coating may be adjusted, such as
to increase electron transmission probability.
[0119] For example, in some embodiments, PEGylation may decrease
the rate of oxidation of the nanoparticles. Additionally,
PEGylation may provide an increased affinity to a target protein
and reduced cytotoxicity of nanoparticles relative to nanoparticles
that are not PEGylated.
[0120] In some embodiments, the metal nanoparticles described
herein specifically interact with proteins, peptides, and/or
nucleic acids. In some embodiments, the metal nanoparticles are
part of nanoparticle-based drugs, which may cross biological
barriers.
[0121] It is contemplated that the metal nanoparticles are part of
a pharmaceutical composition. As used herein "pharmaceutical
composition" or "composition" refers to a mixture of one or more of
the metal nanoparticles described herein, which may comprise other
chemical components, such as pharmaceutically acceptable
excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a metal nanoparticle to a subject.
Pharmaceutical compositions suitable for the delivery of metal
nanoparticles described and methods for their preparation will be
readily apparent to those skilled in the art. A "pharmaceutically
acceptable excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of
a conjugate such as a diluent or a carrier. In some embodiments,
the pharmaceutical composition comprises metal nanoparticles in a
solution without other chemical components.
[0122] In some embodiments, the nanoparticles are effective in
treating the diseases described herein in a dose-dependent manner
at nanomolar or sub-nanomolar concentrations. The metal
nanoparticles and pharmaceutical compositions thereof may be used
at a dose of about 0.01 nM to about 1.0 nM, about 0.01 nM to about
0.5 nM, about 0.01 nM to about 0.3 nM, about 0.01 nM to about 0.1
nM, about 0.05 nM to about 1.0 nM, about 0.05 nM to about 0.5 nM,
about 0.05 nM to about 0.3 nM, or about 0.05 nM to about 0.1
nM.
[0123] In some embodiments, the metal nanoparticles and
pharmaceutical compositions are administered to a patient to treat
cancer. In preferred embodiments, the metal nanoparticle used to
treat cancer is made of platinum, gold, zinc, copper, iron, or
silver.
[0124] It is contemplated that the nanoparticles described herein
may be administered to treat cancer selected from the group
consisting of lung cancer, bone cancer, pancreatic cancer, skin
cancer, cancer of the head, cancer of the neck, cutaneous melanoma,
intraocular melanoma uterine cancer, ovarian cancer, endometrial
cancer, rectal cancer, stomach cancer, colon cancer, breast cancer,
triple negative breast cancer, metastatic breast cancer, carcinoma
of the fallopian tubes, carcinoma of the endometrium, carcinoma of
the cervix, carcinoma of the vagina, carcinoma of the vulva,
Hodgkin's Disease, cancer of the esophagus, cancer of the small
intestine, cancer of the endocrine system, cancer of the thyroid
gland, cancer of the parathyroid gland, non-small cell lung cancer,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, chronic leukemia,
acute leukemia, lymphocytic lymphomas, pleural mesothelioma, cancer
of the bladder, Burkitt's lymphoma, cancer of the ureter, cancer of
the kidney, renal cell carcinoma, carcinoma of the renal pelvis,
neoplasms of the central nervous system (CNS), primary CNS
lymphoma, spinal axis tumors, glioma, brain stem glioma, pituitary
adenoma, and adenocarcinoma of the gastroesophageal junction. In
some aspects of these embodiments, the cancer is a primary or
secondary brain cancer.
[0125] In some embodiments, the cancer comprises a cancerous cell
line selected from the group consisting of Hs683 cells, SVGp12
cells, PC3 cells, U937 cells, and HeLa cells.
[0126] In some embodiments, the metal nanoparticle selectively
causes apoptosis in cancerous cells compared to noncancerous cells.
In some embodiments, after incubation with a metal nanoparticle of
the present disclosure, the viability of a cell line may decrease
in a dose-dependent manner. The viability of a cell line may
decrease by at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or at least about 95%.
In some embodiments, the viability of noncancerous cells may be
reduced by less than about 50%, less than about 40%, less than
about 30%, less than about 20%, or less than about 10%.
[0127] In some embodiments, exposure to the nanoparticles results
in cell morphological changes, including but not limited to
decreased cell adherence, shrinking/rounding, nuclear condensation,
and budding from cell bodies. The metal-induced cell injuries may
be similar to the effects of staurosporine, an active apoptotic
reagent.
[0128] Without intending to be bound by theory, it is believed that
metal nanoparticles may aggregate inside cancer cells. Large
aggregates may be recognized as foreign particles and expelled by
exocytosis. It is believed that when many aggregate particles leave
the cell, the external cell membrane may deplete causing nucleus
budding and cell death.
[0129] It is contemplated that the nanoparticles described herein
may be administered to treat an anosmia-related disease in a
patient. In preferred embodiments, the metal nanoparticle used to
treat the anosmia-related disease is made of zinc. The metal
nanoparticles of the present disclosure demonstrated a significant
enhancement of olfactory responses to odorant stimulation. In some
embodiments, the olfactory response is enhanced for about 10 days,
about 50 days, about 100 days, about 150 days, about 200 days,
about 250 days, or about 300 days.
[0130] Without intending to be bound by theory, it is believed
that, when nanoparticles are coated, the coating may increase
binding affinity to olfactory receptors in part due to a reduction
of the surface charge and/or because of an increase of
hydrophobicity. The nanoparticles may increase the concentration of
receptor dimers and may serve as electron donors for tunnel
electron transport. Additionally, while oxidation of metal
nanoparticles may eliminate the observed enhancement of olfactory
responses, the coatings described herein may limit oxidization such
that the enhancement is retained.
[0131] In some embodiments, the engineered zinc nanoparticles added
to an odorant cause a significant rise of olfactory associated
brain activity. In some embodiments, the metal nanoparticles and
compositions thereof are administered to a patient suffering from
Alzheimer's disease or Parkinson's disease.
EXAMPLES
Optical Microscopy
[0132] An illumination optical system with a high-aperture cardioid
annular condenser was used in this work. The system produces a
highly oblique hollow cone of light (numerical aperture
[NA]=1.2-1.4). The illumination system was positioned in an Olympus
BX51 microscope by replacing a regular condenser. The illumination
system was connected with a light source (EXFO120; Photonic
Solution Ltd, Edinburgh, UK) by a liquid light guide. The objective
used for this work was an infinity-corrected objective HCX PL APO
100/1.40-0.70, oil, iris from Leica. The image was magnified by a
zoom intermediate lens (2.times.-U-CA, Olympus Corporation,
Shinjuku, Tokyo City, Tokyo, Japan), a homebuilt 40.times. relay
lens, and captured by a Sony MCC-500MD video and a Dimension 8200
Dell computer. The microscope was placed on a vibration-isolated
platform (manufactured by TMC, Peabody, Mass., USA).
Atomic Force Microscopic
[0133] Images of metal nanoparticles were taken by Bruker MultiMode
8 (Santa Barbara, Calif., USA) atomic force microscope in
Tapping.RTM. (intermittent-contact) mode, using PointProbe.RTM.
Plus SEIKO microscopes--Non-Contact/Tapping Mode High Force
Constant (PPP-SEIH) made by Nanosensors.TM. (Neuchatel,
Switzerland) AFM probes. The nominal values specified by the vendor
for the force constant and resonance frequency of these probes were
15 N/m and 130 kHz, respectively.
[0134] Monolayers of nanoparticles were prepared on a mica
substrate for all measurements by adding small amount of 0.01%
nanoparticles solution on freshly cleaved mica surfaces. AFM
imaging was used to measure the size distribution of particles.
Size distributions of nanoparticles were measured in five
independent scans over different areas.
X-Ray Photoelectron Spectroscopy
[0135] The Kratos Axis Ultra delay-line detector instrument in
hybrid mode using a monochromatic A1 K.alpha.1,2 X-ray source
(h.nu.=1,486.6 eV) was used for X-ray photoelectron spectroscopy
(XPS). High-resolution spectra of Zn 2p (1,017-1,057 eV) and Cu 2p
(925-965 eV) were acquired using a pass energy of 40 eV with an
energy resolution of 0.1 eV.
[0136] Nanoparticles were analyzed in a water suspension on
gold-coated silicon wafers and evaporated during evacuation of the
system. XPS was used to make quantitative spectroscopic
measurements of the elemental composition of the nanoparticles'
surfaces.
Zeta Potential
[0137] Zeta potentials of the nanoparticles in water suspension
were determined with a Zetasizer Nano ZSP (Malvern Instruments,
Worcestershire, UK) using the laser Doppler velocimetry technique.
The results of six sequential runs were averaged.
[0138] Zeta potentials were calculated with Henry's equation:
.zeta.=3.eta..mu./2.epsilon.F(k.alpha.)
[0139] in which .zeta. is the zeta potential, .eta. is the
viscosity, .mu. is the electrophoretic mobility, .epsilon. is the
dielectric constant of the medium and F(k.alpha.) is Henry's
function, which equals 1.5 using the Smoluchowski
approximation.
Transmission Electron Microscopy
[0140] Transmission electron microscopy (TEM) was carried out
utilizing an FEI Titan operated at 80 kV and 300 kV. Small drops of
nanoparticle water suspension were deposited onto a QUANTIFOIL.RTM.
Holey Carbon Film on copper TEM grids. Widths of the interference
fringes were measured and the Miller-Bravais indices of the
crystalline structures were estimated by the Crystallography lab
software.
Example 1
Preparation of Metal Nanoparticles
[0141] Engineered zinc and copper nanoparticles were prepared using
a high-voltage discharge method from bulk metal rods. By
controlling the voltage and distance between the electrodes, the
plasma created under water produced a fine dispersion of metal
nanoparticles.
[0142] Specifically, two metal electrodes (99.9999%; Alfa Aesar,
Haverhill, Mass., USA) of 2 mm diameter were positioned in a large
Pyrex jar 1 mm below the gas-water interface. The distance between
the rods was about 0.5 cm. 750 mL of double distilled water that
was autoclaved (23 psi, 120.degree. C.) (Omnisolv, Charlotte, N.C.,
USA) was used. The water was chilled to 25.degree. C. and
percolated with nitrogen gas for 20 min. The jar was in the water
bath with running water to prevent overheating. An AC voltage of
15,000 volts was applied to electrodes and the electric discharge
was sustained for 1 hour. Next, the water suspension was collected
in 1 L glass beaker and placed in a refrigerator for 12 hours to
allow large metal particles to sediment. Then, the suspended
particles were separated from the sediment and subjected to
centrifugation at 15,000.times.g for 2 hours at 25.degree. C. After
centrifugation, the pellet was discarded and the supernatant was
subjected to further centrifugations to produce fractions of
nanoparticles enriched in particles of particular sizes. The
centrifuge speed and time to separate nanoparticles by size was
estimated with a Stock's equation. The total concentration of metal
in the suspension was measured by atomic absorption spectra (GTW
Analytical Services, Memphis, Tenn., USA), and the size and the
number of particles were determined by atomic force microscopy.
Example 2
Properties of Metal Nanoparticles
[0143] Atomic force microscopy revealed the size distributions of
Zn and Cu nanoparticles prepared according to Example 1. As shown
in FIG. 1A, a total of 270 nanoparticles with a height of above 1
nm were detected over a 2.5 .mu.m.sup.2 area, and Zn nanoparticles
had an average diameter of 1.2.+-.0.3 nm. As shown in FIG. 1B, 257
nanoparticles with a height of above 0.75 nm were detected over a
2.5 .mu.m.sup.2 area, and Cu nanoparticles had an average diameter
of 2.1.+-.0.6 nm.
[0144] FIGS. 1C and 1D show histograms for Zn and Cu nanoparticles,
respectively, based on the atomic force microscopy data. The
histograms represent distributions with relatively high peaks
around average particle diameter values and tails up to 6 nm.
Standard deviations (SDs) were calculated from the bell
distributions around the peaks. The nanoparticles were homogenously
distributed with polydispersity indexes of 0.062 and 0.082 for zinc
and copper, respectively.
[0145] FIGS. 1E and 1F shows high-resolution XPS of Zn 2p3/2 and Cu
2p3/2, respectively. As shown in FIG. 1E, the spectra for metal and
metal oxide species show 93.9%.+-.3.4% and 5.9%.+-.3.7% of Zn and
ZnO, respectively. As shown in FIG. 1F, the spectra for metal and
metal oxide species show 85.8%.+-.4.1% and 14.2%.+-.4.1% of Cu and
CuO, respectively. These data indicate that, in both instances,
more than 85% of metal atoms were not oxidized.
[0146] Zeta potentials for zinc and copper nanoparticles in water
suspension were .zeta..sub.Zn=-15.4.+-.0.8 (SD) mV and
.zeta..sub.Cu=-17.4.+-.1.7 (SD) mV.
Example 3
Cell Viability Assay
[0147] Two rat glioma cell lines (RG2 and F98) and rat astrocytes
(CTX TNA2) transfected with SV40 were obtained from the American
Type Culture Collection (ATCC) and maintained as recommended by
ATCC.
[0148] A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (MTT) assay was used to measure the effect of metal
nanoparticles on cell viability. Eight experiments with zinc
nanoparticles and five experiments with copper nanoparticles and
RG2 cells were performed. Sixteen control experiments with zinc and
copper nanoparticles and CTX astrocytes were performed. Experiments
tested cells in six conditions: untreated cells, metal
nanoparticles at four different sub-nanomolar concentrations, or 1
.mu.mol/L of staurosporine added to cells. Each condition was
repeated six times. Results are presented as mean values for each
condition.+-.standard error (SE).
[0149] Cells were plated in D5648 media (Sigma 96-well polystyrene
plates) at a density of 3.times.103 cells/well. At 24 hours after
plating, the medium was replaced with Dulbecco's Modified Eagle's
Medium (100 .mu.L/well) containing 1 .mu.mol/L staurosporine, or
zinc or copper nanoparticles with various concentrations (0.05-0.3
nm). At 20 hours after treatment, a 20 .mu.L aliquot of MTT (5
mg/mL in phosphate buffer solution [PBS]) was added to each well,
and the cells were incubated for 4 hours at 37.degree. C. MTT was
reduced in metabolically active cells to form purple formazan
crystals that were subsequently dissolved in dimethyl sulfoxide and
quantified by a plate reader (Bio-Rad, Hercules, Calif., USA). The
dye was converted to a colored product by the activity of
NAD(P)H-dependent dehydrogenase enzymes, which indicated the level
of energy metabolism in cells. The color development from yellow to
blue was proportional to the number of metabolically active cells.
The analysis was carried out with Origin: Data Analysis and
Graphing Software (OriginLab).
[0150] As shown in FIG. 2A, cell death due to treatment with zinc
nanoparticles was confirmed by MTT assay. The relative viability of
RG2 glioma cells was reduced in a dose-dependent manner when added
zinc nanoparticle concentrations increased from 0.05 nmol/L to 0.3
nmol/L, reducing the cell viability by about 60%. Treatments 1-4
correspond to zinc nanoparticles at concentrations of 0.053 nmol/L,
0.106 nmol/L, 0.212 nmol/L, and 0.318 nmol/L, respectively. This
reduction in viability compared well to that caused by 1 .mu.mol/L
staurosporine (treatment 5).
[0151] As shown in FIG. 2E, noncancerous astrocytes were not
affected, within the margin of error, by adding zinc nanoparticles
at concentrations of 0.053 nmol/L, 0.106 nmol/L, 0.212 nmol/L, and
0.318 nmol/L (labeled treatments 1-4, respectively).
[0152] Similarly to the results with zinc, as shown in FIG. 2C,
cell death due to treatment with copper nanoparticles was confirmed
by MTT assay. The relative viability of RG2 glioma cells was
reduced in a dose-dependent manner when added copper nanoparticle
concentrations increased. Treatments 1-4 correspond to copper
nanoparticles at concentrations of 0.017 nmol/L, 0.033 nmol/L,
0.066 nmol/L, and 0.1 nmol/L, respectively.
[0153] As shown in FIG. 2F, noncancerous astrocytes were not
affected, within the margin of error, by adding copper
nanoparticles at concentrations of 0.017 nmol/L, 0.033 nmol/L,
0.066 nmol/L, and 0.1 nmol/L (labeled treatments 1-4,
respectively).
[0154] Without intending to be bound by theory, assuming that cell
death is proportional to the number of bound metal nanoparticles,
the ratio of relative number of dead (Y) and living (1-Y) cells can
be determined according to the Hill equation. FIG. 2B shows a plot
of Y/1-Y vs. the concentration zinc nanoparticles for glioma cell
mortality. The values of K.sub.d and n obtained for zinc by
viability experiments were 0.22.+-.0.08 nmol/L (SE) and
1.45.+-.0.17 (SE), respectively. IC.sub.50 values for RG2 cells
were 0.27.+-.0.1 nmol/L (SE) for Zn. FIG. 2D shows a plot of Y/1-Y
vs. the concentration of copper nanoparticles for glioma cell
mortality. The values of K.sub.d and n obtained for copper
nanoparticles were 0.12.+-.0.02 nmol/L (SE) and 1.09.+-.0.1 (SE),
respectively. IC.sub.50 values for RG2 cells were 0.26.+-.0.1
nmol/L (SE) for Cu.
[0155] Androgen dependent LNCaP, androgen independent PC3 cells,
and normal prostate epithelium-derived RWPE-1 cells were also
cultured with copper, zinc, silver, gold, or platinum nanoclusters.
MTT cell proliferation assays showed a significant decrease in
LNCaP and PC3 cell viability after treatment with copper or zinc
compared to RWPE-1 cells, as shown in FIGS. 3A and 3B. Gold and
platinum nanoparticles killed PC3 cells, but not LNCaP cells
(androgen dependent) or RWPE-1 cells. Staurosporine, an apoptotic
reagent, was used as a positive control. Cells were cultured for 48
hours. Data points are average of three experiments and eight
replicas. The metal nanoparticles had estimated diameters of 1 nm-2
nm and were more than 85% non-oxidized.
[0156] DNA fragmentation in PC3 cell samples of either untreated
negative control, cells treated with 1 .mu.M staurosporine, or
cells treated with copper visually confirmed apoptosis, as shown in
FIG. 4A-4C. Copper nanocluster-induced apoptosis was visually
confirmed with Alexa 488-conjugated Annexin V, which binds
apoptotic cells. As shown in FIG. 4B, increased fluorescent
labeling of Annexin V in copper nanoparticles-treated PC3 prostate
cancer cells suggests cell death by apoptosis. FIG. 4A shows a
positive control with staurosporine and FIG. 4C shows a negative
control.
Light Darkfield Images
[0157] Following incubation with zinc nanoparticles for 20 hours,
cells displayed morphological features consistent with cell death,
including a decrease in adherence, shrinking/rounding, nuclear
condensation, and budding from cell bodies. FIG. 5A shows a light
darkfield image of RG2 glioma cells before exposure to zinc
nanoparticles. FIG. 5B shows a light darkfield image of the RG2
glioma cells after exposure to 5 nmol/L of zinc nanoparticles.
Zn-induced cell injury was similar to the effects of 1 .mu.mol/L
staurosporine, an effective apoptotic reagent.
Example 4
PEGylation of Zinc Nanoparticles
[0158] Zinc nanoparticles of about 1.2 nm in size were prepared by
a high-voltage electrical discharge method, as described in Example
1. Polyethylene glycol solutions of molecular weight 1000 g/mol or
400 g/mol (Sigma-Aldrich), PEG1000 or PEG400, respectively, were
added to a suspension of the zinc nanoparticles to make 1% w/v of
PEGs. Zinc nanoparticles PEGylated with 400 g/mol or 1000 g/mol
molecular weight polyethylene glycol are referred to as ZnPEG400
and ZnPEG1000, respectively. The suspensions were heated to
40.degree. C., purged with N2 obtained from liquid nitrogen, and
maintained at these conditions for 20 minutes. The suspensions were
sonicated at 19 W and 40 kHz for 20 min.
Example 5
Properties of PEGylated Zinc Nanoparticles
[0159] Properties of the PEG coatings on the nanoparticle surface
are shown in Table 1.
TABLE-US-00001 TABLE 1 Properties of PEG on the surface of zinc
nanoparticles. Property ZnPEG400 ZnPEG1000 Area per PEG single
chain (nm.sup.2) 1.3 2.91 Number of monomers in PEG 9 23 Thickness
of PEG layer (nm) 1.3 2.3
[0160] The PEGylated nanoparticles obtained according to Example 4
were characterized using transmission electron microscopy (TEM) and
atomic force microscopy (AFM).
[0161] As shown in FIGS. 6A-6F, the physical properties of
non-PEGylated zinc nanoparticles, ZnPEG400 nanoparticles, and
ZnPEG1000 nanoparticles were compared using AFM. The diameter
distribution of non-PEGylated zinc nanoparticles is shown in FIG.
6B. The non-PEGylated zinc nanoparticles had a mean size of
1.2.+-.0.3 nm (SD). The diameter distributions of ZnPEG400 and
ZnPEG1000 nanoparticles are shown in FIGS. 6D and 6F, respectively.
The ZnPEG400 and ZnPEG1000 nanoparticles each had a mean size of
1.2.+-.0.3 nm (SD).
[0162] As shown in FIGS. 7A-7F, TEM images were obtained for
non-PEGylated zinc, ZnPEG400, and ZnPEG1000 nanoparticles. FIGS.
7A, 7C, and 7D show TEM micrographs of non-PEGylated Zn
nanoparticles at different magnifications. FIG. 7B shows a TEM
micrograph of ZnPEG400 showing the metal core and the PEG
passivation layer. FIGS. 7E and 7F show TEM micrographs of
ZnPEG1000 nanoparticles at different magnifications, where the
labels 1, 2, 3 point to the nanoparticles with visible lattice
fringe patterns, indicating their crystallinity. Arrows show layers
surrounding nanoparticles that are presumed to be coatings of
PEG1000. The characteristic fringes of 0.21 nm and 0.17 nm found in
both non-PEGylated and PEGylated zinc nanoparticles match the (011)
and (012) vectors for the hcp crystal lattice of zinc. Larger than
average size particles are shown to emphasize the crystal
fringes.
[0163] The crystalline structure of zinc nanoparticles revealed in
this work is consistent with those obtained for zinc nanowires and
nanorods. The extent of the similarities of the zinc lattices and
core lattices of PEGylated zinc nanoparticles indicates that
PEGylating maintains the crystalline nature of the metal
particles.
Example 6
Time and Temperature Studies of PEGylated Zinc Nanoparticles
[0164] The PEGylated nanoparticles obtained according to Example 4
were also characterized using X-ray photoelectron spectroscopy
(XPS). For the XPS experiments, the non-PEGylated and PEGylated
zinc nanoparticles were subjected to four experimental
conditions:
[0165] 1. Particles were stored for 1 day at of 278 K (5.degree.
C.);
[0166] 2. Particles were stored for 317 days at 278 K (5.degree.
C.);
[0167] 3. Particles were stored for 2 days at the temperature of
303 K (30.degree. C.); or
[0168] 4. Particles were stored for 2 days at 323 K (50.degree.
C.).
[0169] Physical analyses of all samples were performed immediately
after the end of each of the exposure conditions to minimize any
changes in chemistry.
[0170] FIGS. 8A and 8B show representative XPS spectra obtained for
freshly prepared non-PEGylated zinc and PEGylated zinc
nanoparticles stored 1 day at 278 K (5.degree. C.). For the C is
spectra, the solid vertical line represents the position of the
C--C peaks, to which all spectra were calibrated to, with the
dotted lines showing the spectral shift of the C--O peaks, which
were 286 eV, 286.4 eV, and 286.2 eV for the Zn, ZnPEG400, and
ZnPEG1000 systems, respectively. Each spectrum represents an
average of six spectral runs. For the Zn 2p plots, the solid line
represents the position of the Zn peak for the bare Zn system,
while the dotted lines show the spectral shift of the ZnO peaks,
which were 1024.6 eV, 1025.6 eV, and 1026.0 eV for the Zn,
ZnPEG400, and ZnPEG1000 systems, respectively. Each spectrum
represents an average of six spectral runs.
[0171] A summary of the XPS data for non-PEGylated and PEGylated
zinc nanoparticles for all four experimental conditions is shown in
Table 2.
TABLE-US-00002 TABLE 2 XPS properties of uncoated and PEGylated
zinc nanoparticles, stored at various durations and temperatures.
Survey High Resolution Zn 2p T Time O 1s C 1s Zn2p BE Zn BE ZnO
Sample (K) (days) (%) (%) (%) (eV) (%) (eV) (%) Zn 278 1 27.8 .+-.
70.5 .+-. 1.8 .+-. 1021.8 97.0 .+-. 1024.6 3.0 .+-. 2.3 1.7 0.1 0.8
0.8 ZnPEG400 278 1 32.3 .+-. 67.4 .+-. 0.3 .+-. 1022.0 89.5 .+-.
1023.8 10.5 .+-. 0.5 0.7 0.1 4.0 4.0 ZnPEG1000 278 1 30.3 .+-. 69.6
.+-. 0.1 .+-. 1021.9 88.0 .+-. 1023.5 12.0 .+-. 0.9 2.1 0.0 3.8 3.8
Zn 278 317 38.4 .+-. 60.2 .+-. 1.4 .+-. 1021.9 96.0 .+-. 1023.7 4.0
.+-. 1.0 1.5 0.1 1.0 1.0 ZnPEG400 278 317 31.5 .+-. 68.4 .+-. 0.1
.+-. 1022.2 75.9 .+-. 1023.9 24.1 .+-. 0.1 0.1 0.0 8.2 8.2
ZnPEG1000 278 317 31.1 .+-. 68.0 .+-. 0.9 .+-. 1021.9 86.1 .+-.
1024.2 13.9 .+-. 0.3 0.4 0.2 0.5 0.5 Zn 303 2 24.5 .+-. 75.1 .+-.
0.4 .+-. 1021.7 92.2 .+-. 1024.1 7.8 .+-. 4.1 0.6 0.1 3.5 3.5
ZnPEG400 303 2 23.2 .+-. 76.6 .+-. 0.2 .+-. 1022.0 93.0 .+-. 1023.7
7.0 .+-. 4.4 3.0 0.0 2.8 2.8 ZnPEG1000 303 2 24.6 .+-. 75.2 .+-.
0.2 .+-. 1021.8 94.2 .+-. 1023.4 5.8 .+-. 5.4 5.2 0.1 5.6 5.6 Zn
323 2 38.3 .+-. 60.8 .+-. 0.9 .+-. 1021.0 92.9 .+-. 1023.3 7.1 .+-.
5.3 3.5 0.3 3.3 3.3 ZnPEG400 323 2 27.7 .+-. 72.1 .+-. 0.2 .+-.
1021.9 88.6 .+-. 1023.7 11.4 .+-. 4.6 6.8 0.1 2.6 2.6 ZnPEG1000 323
2 21.8 .+-. 77.9 .+-. 0.3 .+-. 1021.7 92.7 .+-. 1024.0 7.3 .+-. 3.5
2.7 0.1 1.6 1.6 High Resolution C 1s BE C--C BE C--O BE C.dbd.O(OH)
Sample (eV) (%) (eV) (%) (eV) (%) Zn 284.3 66.2 .+-. 286.2 22.6
.+-. 288.5 11.2 .+-. 2.9 1.5 1.7 ZnPEG400 285.0 3.7 .+-. 286.9 92.0
.+-. 288.5 4.3 .+-. 2.1 3.0 1.0 ZnPEG1000 283.2 3.0 .+-. 286.7 94.5
.+-. 290.2 2.6 .+-. 0.6 0.6 0.0 Zn 284.4 60.2 .+-. 285.8 18.6 .+-.
288.1 21.2 .+-. 0.9 0.7 0.5 ZnPEG400 285.0 0.9 .+-. 287.0 92.8 .+-.
288.0 6.3 .+-. 0.2 0.9 0.8 ZnPEG1000 285.1 11.5 .+-. 286.7 83.5
.+-. 288.0 5.0 .+-. 2.4 1.9 0.8 Zn 284.4 60.8 .+-. 286.5 31.5 .+-.
289.2 7.7 .+-. 6.8 4.0 4.0 ZnPEG400 284.4 38.9 .+-. 286.8 56.9 .+-.
288.8 4.2 .+-. 15.0 15.3 0.5 ZnPEG1000 284.4 39.2 .+-. 286.6 55.7
.+-. 288.6 5.1 .+-. 14.9 14.9 1.1 Zn 284.2 65.6 .+-. 285.9 20.6
.+-. 288.2 13.8 .+-. 14.7 8.8 6.1 ZnPEG400 284.4 35.2 .+-. 286.9
62.6 .+-. 283.7 2.2 .+-. 10.7 12.7 0.1 ZnPEG1000 284.4 50.5 .+-.
286.5 43.7 .+-. 288.6 5.8 .+-. 15.7 15.4 0.8
[0172] The spectra were calibrated using the adventitious carbon C
is peak at a binding energy of 284.6 eV, which allowed the
identification of the following chemical species at their
respective binding energies: hydrocarbon (C--C, about 285 eV),
ether (C--O, about 286.1 eV), carboxyl (C.dbd.O(OH), about 288.1
eV), zinc (Zn 2p.sub.3/2, about 1021.8 eV) and ZnO (Zn 2p.sub.3/2,
about 1024 eV). The rise in the amplitude of C--O peak and the
reduction in the binding energy of Zn 2p.sub.3/2 peaks in the
ZnPEG400 and PEG1000 samples confirmed the successful PEGylation of
zinc nanoparticles. The Zn 2p.sub.3/2 band showed that metallic
zinc nanoparticles were oxidized only slightly.
[0173] The interaction of the PEG molecules and zinc nanoparticles
affected the binding energies of carbon and zinc. Bonding of the
PEG molecules onto the nanoparticles was associated with an
increase in the binding energy of the C--O bond, and a
corresponding decrease in the ZnO binding energy. The highest shift
in binding energy was observed for ZnPEG400 nanoparticles stored at
278 K for 317 days. Similar shifts in binding energy were also
observed for PEGylated zinc nanoparticles exposed to elevated
temperatures of 303 K (30.degree. C.) and 323 K (50.degree. C.).
These observations point to the covalent binding of PEG to the
engineered zinc nanoparticles.
[0174] The XPS spectra of ZnPEG400 subjected to elevated
temperature showed similar features to the nanoparticles stored for
longer times. Without intending to be bound by theory, this
observation provides evidence for the large number of covalently
bound species and production of a conjugated electron system of
zinc nanoparticles and PEG400 molecules.
[0175] Using an apparent concentration of primarily-elemental zinc
as a function of time and storage temperature, the Arrhenius
activation energies of oxidation were estimated by equation as
shown in Table 3. After PEGylation, zinc nanoparticles showed
reduction of the activation energy.
TABLE-US-00003 TABLE 3 Thermodynamic analysis of oxidation of Zn,
ZnPEG400, and ZnPEG1000 nanoparticles. T Estimated activation
Particle (.degree. C.) energy (kj/mol) Zn, 1.2 nm 5-50 113 Zn foil,
0.126 mm 300-400 119 ZnO powder, oxygen desorption 86-97 96 Molten
Zn 600-700 104 ZnPEG400, 1.4 nm 5-50 15.5 ZnPEG1000, 1.4 nm 5-50
34.9 Pd/Fe nanoparticles, 60-100 nm 20-35 39.47 Pd/FePEG200 20-35
38.66
[0176] The change in the physical properties of zinc nanoparticles
after PEGylation was also confirmed by determining zeta potential.
The PEG binding resulted in a sharp reduction of zeta potential in
water. Zeta potential was -42.4.+-.4.8 (SE) mV, -26.1.+-.2.5 (SE)
mV (t(7)=7.96, p=0.0001), and -27.5.+-.2.5 (SE) mV (t(7)=7.28,
p=0.0001) for Zn, ZnPEG400, and ZnPEG1000 nanoparticles,
respectively.
Example 7
Olfactory Studies of PEGylated Zinc Nanoparticles
[0177] To characterize the olfactory response properties of zinc
nanoparticles under varying conditions, two sets of experimental
settings were utilized:
[0178] (1) Non-PEGylated and PEGylated zinc nanoparticles were
stored at 283 K (5.degree. C.) and then employed, along with a
standard odorant mixture in electroolfactogram (EOG) olfactory
experiments over a storage period of 317 days; or
[0179] (2) Non-PEGylated and PEGylated zinc nanoparticles were
subjected to accelerated aging at temperatures of 303 K (30.degree.
C.) and 323 K (50.degree. C.) for 2 days before the EOG experiments
that followed directly after storage.
[0180] Non-PEGylated and PEGylated zinc nanoparticles mixed with
eugenol, ethyl butyrate, or (.+-.) carvone after storage and/or
heating and odorant responses were tested by EOG with isolated rat
olfactory epithelium (OE). An odorant mixture of 1.6 mmol/L each of
ethyl butyrate, eugenol, and (+) and (.+-.) carvone in water was
prepared with a vortex mixer and stored in a dark glass container
at 283K (5.degree. C.). Odorants were obtained from
Sigma-Aldrich.
[0181] The EOG instrumentation included Axon Instrument MultiClamp
700A amplifier and 1322A DigiData acquisition system. Rat OE was
dissected out and positioned in a perfusion chamber such that the
basal parts were immersed in physiological solution, while the
olfactory cilia ware positioned in the water/air interface. Adult
male Sprague.+-.Dawley rats (Envigo, Dublin, Va.) weighing about
300 g were used. All olfactory experiments were performed ex vivo.
Glass electrodes of about 24 .mu.m tip openings were linked to the
amplifier to record signals from the OE.
[0182] After the connection between the electrode and the OE was
made, an air pulse of the odorant mixture was applied and a
continuous EOG signal was recorded as function of time. A 0.25 s
pulse of the odorant mixture at 55158 N/m.sup.2 (8 psi) was
generated by a computer-controlled Pneumatic PicoPump PV800 (World
Precision Instruments, Sarasota, Fla.). A pulse of positive
pressure pushed the odorant into a glass nozzle toward the OE. The
computer controlled odorant delivery was comprised of 0.25 s pulses
at 20 s and 60 s times for EOG data collection. The single EOG
recording took 200 s and contained 10 response traces. A
nanoparticle suspension was combined with odorant solutions to
reach a final nanoparticle concentration of 0.02 nmol/L. During the
pulse of delivery, the vapor of odorant with metal nanoparticles
was transferred to the OE surface.
[0183] Electroolfactograms are shown in FIG. 9. The EOG recordings
from rat olfactory epithelium were produced by 0.25 s pulses of (1)
odorant mixture, (2) odorant mixture+1.2 nm zinc nanoparticles, (3)
odorant mixture+0.25 mmole/L PEG400, (4) odorant mixture+0.10
mmole/L PEG1000, (5) water vapor+PEG400, (6) water vapor+PEG1000,
and (7) water vapor. The concentration of zinc nanoparticles and
odorant mixture were 0.02 nmole/L and 1.6 mmole/L, respectively.
The representative set of traces was obtained from 50 EOG
traces.
[0184] The effects of PEG400 and PEG1000 without zinc nanoparticles
were recorded from the rat OE, as depicted by traces 5 and 6. The
EOG responses evoked by PEG400 and PEG1000 without the odorant
mixture are small and nearly indistinguishable from the EOG signal
of water, as depicted by trace 7. This result is consistent with
polyethylene glycol being odorless. The EOG evoked by the odorant
mixture with zinc nanoparticles (trace 2) showed a strong
enhancement compared to the signal induced by the odorant mixture
alone (trace 1). Combining odorants with ZnPEG400 and ZnPEG1000
resulted in the lower signals depicted by the traces 3 and 4,
respectively.
[0185] The mean values of seven measurements of the relative
responses to the odorant mixtures with Zn, ZnPEG400, and ZnPEG1000
were 2.69.+-.0.11, 0.821.+-.0.05, and 0.310.+-.0.04, respectively.
An analysis of variance showed that at the 0.05 level, the means
were significantly different, F(2, 18)=1900, p=0.000.
[0186] The relative enhancement of olfactory responses by zinc
nanoparticles was calculated as (EOG.sub.O+Zn-EOG.sub.O)/EOG.sub.0,
where EOG.sub.O was the peak of electrical response evoked by the
odorant alone, and EOG.sub.O+Zn was the peak response stimulated by
the mixture of the odorant and Zn nanoparticles. Similarly, the
relative enhancements by ZnPEG400 and ZnPEG1000 were determined as
(EOG.sub.O+ZnPEG400-EOG.sub.O)/EOG.sub.O and
(EOG.sub.O+ZnPEG1000-EOG.sub.O)/EOG.sub.O, respectively.
[0187] As shown in FIG. 10, the olfactory responses to odorant
mixed with PEG400 or PEG1000 were tested systematically over the
period of 317 days. The difference between peak values of EOG
evoked by odorant and by non-PEGylated and PEGylated zinc
nanoparticles was normalized by the EOG peak evoked by an odorant
alone. As analysis of variance showed, at the level of 0.05, there
was no significant difference between the relative olfactory
responses to Zn, ZnPEG400, and ZnPEG1000 nanoparticles ranging from
3 days to 64 days of storage. After 280 days and 317 days of
storage a significant difference between the relative olfactory
responses to Zn, ZnPEG400, and ZnPEG1000 nanoparticles was
observed.
[0188] Data indicate that stored ZnPEG400 nanoparticles maintain
physiologically-consistent olfactory enhancement for over 300 days.
Following 280 days of storage, ZnPEG400 provided the highest
enhancement followed by Zn and then ZnPEG1000.
[0189] By these ex vivo experiments, enhancement was found to be
dose-dependent, specific, and reversible. In conjunction with ex
vivo analyses of sensory neurons in the rodent OE, in vivo
cognitive effects on the brain regions associated with olfaction of
non-anesthetized dogs were noninvasively analyzed using functional
magnetic resonance imaging (fMRI). These studies indicated that
engineered zinc nanoparticles added to odorant caused a significant
rise of olfactory associated brain activity.
[0190] To further characterize effects of thermally-enhanced
formation of ZnO on the odorant responses, the effects of
non-PEGylated and PEGylated zinc nanoparticles stored for two days
at an elevated temperature were analyzed. FIGS. 11A-11C show the
relative enhancement of responses to odorants as the function ZnO
concentration determined by XPS and temperature.
[0191] Referring to FIG. 11A, the difference between peak values of
EOG evoked by odorant and by zinc nanoparticles was normalized by
the EOG peak evoked by an odorant alone as a function of ZnO
concentration that was determined by XPS. The first bar at 3% of
ZnO, corresponds to zinc nanoparticles stored one day at 278 K
(5.degree. C.). The second and third bars reflect zinc
nanoparticles stored for two days at 303 K (30.degree. C.) and 323
K (50.degree. C.), at 7.1% and 7.8% respectively. The forth bar at
11.5% of ZnO corresponds to the negative enhancement (inhibition)
that was observed with zinc nanoparticles oxidized by percolating
air.
[0192] Referring to FIG. 11B, the relative EOG enhancement produced
by ZnPEG400 nanoparticles after they were stored for one day at 278
K (5.degree. C.), two days at 303 K (30.degree. C.) and 323 K
(50.degree. C.), respectively, is shown.
[0193] Referring to FIG. 11B, the relative EOG enhancement produced
by ZnPEG1000 nanoparticles after they were stored for one day at
278 K (5.degree. C.), two days at 303 K (30.degree. C.) and 323 K
(50.degree. C.), respectively, is shown.
[0194] The relative olfactory enhancement by the zinc nanoparticles
declined from 55% to -7%, when the ZnO concentration increased from
3.0% to 11.5%, as shown in FIG. 11A. The enhancement with ZnPEG1000
declined with the temperature, as shown in FIG. 11C. The
enhancement with ZnPEG400, shown in FIG. 11B was less sensitive to
zinc oxidation and temperature. The addition of the ZnPEG400
nanoparticles to odorant at the end of 317 days of storage at 278 K
(5.degree. C.) resulted in the highest relative EOG enhancement
that was larger than enhancement by the un-PEGylated zinc
nanoparticles.
[0195] The olfactory enhancement of approximately 140% using
ZnPEG400 after 317 days of storage was not an anticipated result
based on the higher level of ZnO of 24.1% ZnO compared to 10.5% for
the freshly prepared ZnPEG400, as shown in Table 2. Although no
significant change in the C--O concentration was observed, the C--C
concentration decreased from 3.7% to 0.9%. Without intending to be
bound by theory, the increase in the observed level of ZnO in this
case does not imply a high level of oxidized zinc atoms, but
suggests that a conjugated electron system between the zinc
nanoparticles and PEG400 molecules forms that may be contribute to
the increase in olfactory response.
* * * * *