U.S. patent application number 14/059960 was filed with the patent office on 2014-02-13 for melanin nanoshells for protection against radiation and electronic pulses.
This patent application is currently assigned to Albert Einstein College of Medicine of Yeshiva University. The applicant listed for this patent is Arturo Casadevall, Ekaterina Dadachova. Invention is credited to Arturo Casadevall, Ekaterina Dadachova.
Application Number | 20140044789 14/059960 |
Document ID | / |
Family ID | 38575596 |
Filed Date | 2014-02-13 |
United States Patent
Application |
20140044789 |
Kind Code |
A1 |
Dadachova; Ekaterina ; et
al. |
February 13, 2014 |
MELANIN NANOSHELLS FOR PROTECTION AGAINST RADIATION AND ELECTRONIC
PULSES
Abstract
This invention provides melanin nanoshells and their use for
protection against radiation, particularly ionizing radiation, and
electronic pulses, and methods of making materials comprising
melanin nanoshells.
Inventors: |
Dadachova; Ekaterina;
(Mahopac, NY) ; Casadevall; Arturo; (New Rochelle,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dadachova; Ekaterina
Casadevall; Arturo |
Mahopac
New Rochelle |
NY
NY |
US
US |
|
|
Assignee: |
Albert Einstein College of Medicine
of Yeshiva University
Bronk
NY
|
Family ID: |
38575596 |
Appl. No.: |
14/059960 |
Filed: |
October 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11732130 |
Apr 2, 2007 |
8586090 |
|
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14059960 |
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PCT/US05/35707 |
Oct 3, 2005 |
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11732130 |
|
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60819992 |
Jul 10, 2006 |
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60616056 |
Oct 5, 2004 |
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Current U.S.
Class: |
424/489 ;
250/515.1; 252/478; 424/547; 514/224.5; 514/414; 544/34;
548/455 |
Current CPC
Class: |
A61K 9/51 20130101; A61K
31/405 20130101; A61K 35/618 20130101; G21F 1/10 20130101; A61K
9/0019 20130101; A61K 31/546 20130101; A61K 9/20 20130101 |
Class at
Publication: |
424/489 ;
424/547; 514/224.5; 514/414; 252/478; 544/34; 548/455;
250/515.1 |
International
Class: |
A61K 9/20 20060101
A61K009/20; G21F 1/10 20060101 G21F001/10; A61K 31/405 20060101
A61K031/405; A61K 35/56 20060101 A61K035/56; A61K 31/546 20060101
A61K031/546 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The invention disclosed herein was made with U.S. Government
support under grant number R21AI52042 from the National Institutes
of Health, U.S. Department of Health and Human Services.
Accordingly, the U.S. Government has certain rights in this
invention.
Claims
1. A nanoshell comprising melanin.
2. The nanoshell of claim 1, wherein the nanoshell comprises
polymerized L-dopa, epinephrine, methyldopa and/or a phenolic
derivative that polymerizes into melanin.
3. The nanoshell of claim 1, wherein the nanoshell comprises
synthetic melanin.
4. The nanoshell of claim 1, wherein the nanoshell comprises
melanin isolated or derived from a biological source or generated
by chemical synthetic process.
5. The nanoshell of claim 4, wherein the biological source is a
plant, an animal, a fungus and/or a microorganism.
6. The nanoshell of claim 4, wherein the biological source is a
melanin-containing fungus, bacterium, or cell.
7. The nanoshell of claim 6, wherein the fungus is Cryptococcus
neoformans and/or Histoplasma capsulatum.
8. The nanoshell of claim 1 comprising allomelanin, pheomelanin
and/or eumelanin.
9. The nanoshell of claim 1 comprising pheomelanin and eumelanin,
wherein the ratio of pheomelanin to eumelanin is at least 1:1.
10. The nanoshell of claim 1, wherein the melanin contains divalent
sulphur.
11. The nanoshell of claim 1, wherein the nanoshell comprises a
nanosphere, a nanotube, a nanoellipsoid, a nanoball and/or a
nanorod.
12. The nanoshell of claim 1, wherein the nanoshell has a thickness
of about 10 nm to about 1,000 nm.
13. The nanoshell of claim 1, wherein the nanoshell has a thickness
of about 100 nm.
14. The nanoshell of claim 1, wherein the nanoshell has a linear
attenuation coefficient for radiation that is at least 100-fold
higher than that provided by powdered melanin that is not formed as
a nanoshell.
15. The nanoshell of claim 1, wherein the nanoshell has a linear
attenuation coefficient for radiation that is at least 1,000-fold
higher than that provided by powdered melanin that is not formed as
a nanoshell.
16. The nanoshell of claim 1, wherein the nanoshell has a linear
attenuation coefficient for radiation that is at least 10,000-fold
higher than that provided by powdered melanin that is not formed as
a nanoshell.
17. The nanoshell of claim 1, wherein the nanoshell has a linear
attenuation coefficient for radiation that is at least 10-fold
higher than that provided by lead.
18. The nanoshell of claim 1, wherein the nanoshell has a linear
attenuation coefficient for radiation that is at least 100-fold
higher than that provided by lead.
19. The nanoshell of claim 1, wherein the nanoshell has a linear
attenuation coefficient for radiation that is at least 500-fold
higher than that provided by lead.
20. A method of protecting an object or a subject from radiation
and/or from electronic pulses, where the method comprises providing
melanin nanoshells between the object or subject to be protected
and a source of the radiation and/or electronic pulses.
21-79. (canceled)
80. A method of making a material comprising melanin nanoshells,
where the method comprises fabricating melanin nanoshells into or
onto the material.
81-109. (canceled)
110. A material comprising melanin nanoshells of claim 1 fabricated
in or on the material.
111-135. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
priority of U.S. Provisional Patent Application No. 60/819,992,
filed Jul. 10, 2006, and PCT International Patent Application No.
PCT/US2005/035707, filed Oct. 3, 2005, which designates the United
States of America and claims priority of U.S. Provisional Patent
Application No. 60/616,056, filed Oct. 5, 2004, the contents of all
of which are hereby incorporated by reference in their entirety
into the subject application.
FIELD OF THE INVENTION
[0003] The present invention relates to melanin-based nanoshells
and their use for protection against radiation, particularly
ionizing radiation, and electronic pulses, and to methods of making
materials comprising the melanin nanoshells.
BACKGROUND OF THE INVENTION
[0004] Throughout this application various publications are
referred to in parenthesis. Full citations for these references may
be found at the end of the specification immediately preceding the
claims. The disclosures of these publications are hereby
incorporated by reference in their entireties into the subject
application to more fully describe the art to which the subject
application pertains.
[0005] Melanin is a high molecular weight pigment that is
ubiquitous in nature and has a variety of biological functions (1).
Melanins are found in all biological kingdoms. These pigments are
among the most stable, insoluble, and resistant of biological
materials (30). Melanins can have different structures depending on
the biosynthetic pathway and precursor molecules. Some definitions
of melanin have focused on chemical and physical properties of
melanins instead of defined structures (29). Melanins can be
synthesized in the laboratory by chemical means or by many living
organisms. Melanins formed by the oxidative polymerization of
phenolic compounds are usually dark brown or black (30). However,
melanins may have other colors as illustrated by the finding that
dopamine-derived melanin is reddish-brown. Fungi can make melanins
from at least two major biosynthetic pathways, employing the
precursor 1,8-dihydroxynapthalene (DHN melanin) or the oxidation of
suitable tyrosine derivatives like dihydroxyphenylalanine
(DOPA-melanin) (30). The fungus C. neoformans can make melanins
from a wide variety of phenolic compounds which are oxidized by a
laccase enzyme (31-33).
[0006] Melanins protect against UV light by absorbing a broad range
of the electromagnetic radiation (1), and the melanin pigment is
used in photo-protective creams (10). The presence of melanin is
implicated in the resistance of human malignant pigmented melanoma
to radiation therapy (9). Many fungi constitutively synthesize
melanin (2). The ability of free-living microorganisms to make
melanin may be associated with a survival advantage in the
environment (3) that includes protection against solar radiation
(reviewed in 4). Melanized fungi are also resistant to ionizing
radiation (5). An example of such radiation resistance is provided
by reports that melanized fungi colonize the walls of the damaged
nuclear reactor in Chernobyl (6). The soils around the damaged
reactor have blackened as the resident flora changes to include
disproportionately more melanotic fungi (7). Water in nuclear
reactor cooling pools is sometimes contaminated with melanized
microorganisms (8). However, despite the finding of melanotic
organisms in such harsh environments, the contribution of melanin
to the radiation resistance of these organisms is uncertain.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to nanoshells comprising
melanin.
[0008] The invention also provides methods of protecting an object
or a subject from radiation and/or from electronic pulses, where
the methods comprise providing a material comprising melanin
nanoshells between the object or subject to be protected and a
source of the radiation and/or electronic pulses.
[0009] The invention further provides methods of protecting
internal organs of a subject from radiation and/or from electronic
pulses, where the methods comprise administering to the subject
particles comprising melanin nanoshells.
[0010] The invention further provides methods of making a material
comprising melanin nanoshells, where the method comprises
fabricating melanin nanoshells into and/or onto the material; and
materials comprising melanin nanoshells fabricated in and/or on the
material.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1A-1C. Microscopic images of C. neoformans (Cn) cells.
A) a transmission electron microscopy (TEM) image of non-melanized
Cn cells; B) TEM image of melanized Cn cells; C) light microscopy
image of melanin nanosize spheres. Melanized Cn cells were grown in
Sabouraud dextrose broth medium with 1 mM 3,4-dihydroxyphenylalanin
(L-dopa) for 5 days. Melanin spheres were generated by boiling
melanized Cn cells in 6 M HCl.
[0012] FIG. 2A-2D. Survival of non-melanized and melanized Cn and
H. capsulatum (Hc) cells following exposure to external gamma rays:
A) Cn in PBS up to 220 Gy at 14 Gy/min and up to 8,000 Gy at 30
Gy/min; B) Hc in PBS up to 220 Gy at 14 Gy/min and up to 8,000 Gy
at 30 Gy/min; C) melanized and non-melanized Cn on Sabouraud plates
irradiated at 14 Gy/min up to 440 Gy in air; D) in N.sub.2.
[0013] FIG. 3A-3F. High-pressure liquid chromatography (HPLC) of
permanganate-oxidized melanins: A) structure of eumelanin oligomer;
B) structure of pheomelanin oligomer (adapted from ref. 17); C)
visual appearance of oxidized melanin samples, from left to right:
Cn, Hc; D) chromatogram of background solution; E) Cn melanin; F)
Hc melanin.
[0014] FIG. 4. Diagram illustrating multiple interactions of a
photon passing through matter. Energy is transferred to electrons
in a sequence of photon-energy degrading interactions (adapted from
(22)).
[0015] FIG. 5. Electron spin resonance spectroscopy (ESR) spectrum
of melanized Hc cells showing characteristic spectrum of
melanin.
[0016] FIG. 6A-6B. Survival of non-melanized and melanized fungal
cells following exposure to external gamma rays. A) melanized and
non-melanized C. neoformans irradiated at 14 Gy/min up to 400 Gy,
0.02 or 0.4 mg of S. officinalis melanin was added to non-melanized
cells. B) melanized and non-melanized C. neoformans irradiated at
14 Gy/min up to 200 Gy, 0.01 or 0.1 mg of intact or crushed C.
neoformans melanin "ghosts" was added to samples. Cn--C.
neoformans, Hc--H. capsulatum.
[0017] FIG. 7. Poly-lysine-precoated wells in a 96-well ELISA plate
were filled with the suspensions of the following substances in
poly-lysine solution: A--Sepia melanin; B--C. neoformans ghosts;
C--charcoal. For control lead foil was used (D).
[0018] FIG. 8. The image of the radiographic film showing different
exposures of the film depending on the substance in the wells.
A--Sepia melanin; B--C. neoformans ghosts; C--charcoal. For control
lead foil was used (D).
[0019] FIG. 9A-9B. Biodistribution of .sup.188Re-labeled melanized
20 nm silica nanoparticles in BALB/c mice. A) Biodistribution
following administration of nanoshell particles only. B)
Biodistribution following pre-treatment with pluronic acid followed
by administration of nanoparticles. Mice were injected IV with
melanized particles. Pluronic acid (0.13 mg/kg body weight) was
injected IV 3 hours earlier.
[0020] FIG. 10A-10D. Platelet (A, B) and white blood cell (WBC) (C,
D) counts in mice pre-treated with melanized nanoparticles and
irradiated with 1.25 Gy of gamma radiation.
[0021] FIG. 11A-11D. Platelet (A, B) and WBC (C, D) counts in mice
pre-treated with melanized nanoparticles and irradiated with 2.5 Gy
of gamma radiation.
[0022] FIG. 12. Body weights of the control CD-1 mice or mice fed
with 15 mg/kg body weight synthetic melanin or C. neoformans
ghosts.
[0023] FIG. 13A-13C. Histology of GI organs from CD-1 mice fed with
24067 C. neoformans ghosts and sacrificed 24 hr later: A) stomach;
B) small intestine; C) colon.
[0024] FIG. 14. Cumulative weight loss in CD-1 mice fed with either
synthetic melanin of C. neoformans ghosts of water (15 mg/kg body
weight) and irradiated with 9 Gy whole body dose at 3 Gy/min.
[0025] FIG. 15. Survival of mice fed with either synthetic melanin
or C. neoformans ghosts or water (15 mg/kg body weight) and
irradiated with 9 Gy whole body dose at 3 Gy/min.
[0026] FIG. 16A-16F. Histology of GI track tissues obtained from
CD-1 mice irradiated with 9 Gy at 3 Gy/min 4 hr (A-C) and 24 hr
(D-F) post-irradiation: A) stomach, synthetic melanin group; B)
stomach, ghosts melanin group; C) stomach, water; D) colon,
synthetic melanin group; E) colon, ghosts melanin group; F) colon,
water control group.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The subject invention is directed to a nanoshell comprising
melanin. Melanins are high-molecular weight pigments, arising in
the course of oxidation and polymerization of phenols. The
nanoshell can comprise polymerized L-dopa, epinephrine, methyldopa,
a substituted phenol derivative and/or a phenolic derivative that
polymerizes into melanin.
[0028] The nanoshell can comprise synthetic melanin and/or melanin
isolated or derived from a biological source, such as a plant, an
animal, a microorganism, and/or a melanin-containing cell, or
generated by chemical synthetic process. Suitable animals include,
but are not limited to, helminthes, cuttlefish and squids. The
microorganism can be, e.g., a bacterium or preferably a fungus.
Suitable fungi include, but are not limited to, Cryptococcus
neoformans and/or Histoplasma capsulatum.
[0029] The melanin can comprise allomelanin, pheomelanin and/or
eumelanin. Eumelanins are derived from the precursor tyrosine.
Pheomelanin is derived from the precursors tyrosine and cysteine.
Allomelanins are formed from nitrogen-free precursors such as
catechol and 1,8-dihydroxynaphthalenes. In one embodiment, the
nanoshell comprises pheomelanin and eumelanin, wherein the ratio of
pheomelanin to eumelanin is at least 1:1. Preferably, the melanin
contains divalent sulphur.
[0030] The nanoshell can comprises a nanosphere, a nanotube, a
nanoellipsoid, a nanorod, a nanoball, or other suitable shape. The
nanoshells can be hollow or filled with the same type of melanin as
used in the shell or with a different type of melanin or with
another material.
[0031] The nanoshell can have a thickness of about 10 nm to about
1,000 nm. In one embodiment, the nanoshell has a thickness of about
100 nm.
[0032] Preferably, the nanoshell has a linear attenuation
coefficient for radiation that is at least 100-fold higher than
that provided by powdered melanin that is not formed as a
nanoparticle. More preferably, the nanoshell has a linear
attenuation coefficient for radiation that is at least 1,000-fold
higher than that provided by powdered melanin that is not formed as
a nanoparticle. Most preferably, the nanoshell has a linear
attenuation coefficient for radiation that is at least 10,000-fold
higher than that provided by powdered melanin that is not formed as
a nanoparticle.
[0033] Preferably, the nanoshell has a linear attenuation
coefficient for radiation that is at least 10-fold higher than that
provided by lead. More preferably, the nanoshell has a linear
attenuation coefficient for radiation that is at least 100-fold
higher than that provided by lead. Most preferably, the nanoshell
has a linear attenuation coefficient for radiation that is at least
500-fold higher than that provided by lead.
[0034] The invention also provides a method of protecting an object
or a subject from radiation and/or from electronic pulses, where
the method comprises providing melanin nanoshells between the
object or subject to be protected and a source of the radiation
and/or electronic pulses. The melanin nanoshells can be fabricated
in or on the source of the radiation and/or electronic pulses,
and/or the melanin nanoshells can be fabricated in or on the object
or subject to be protected from radiation and/or from electronic
pulses.
[0035] As used herein, to protect against radiation and electronic
pulses means to reduce the amount of radiation or electronic pulses
reaching the object or subject to be protected, compared to the
amount of radiation and electronic pulses that would reach the
object or subject in the absence of the melanin nanoshells. The
melanin can be internal and/or external to the object or subject.
The radiation can comprise ionizing radiation. The radiation can
be, for example, one or more of gamma radiation, x-ray radiation,
solar radiation, cosmic radiation, electromagnetic radiation,
bremsstrahlung radiation, ultraviolet radiation, and particulate
radiation (e.g., .alpha.-radiation and .beta.-radiation). The
source of the radiation can be a medical isotope.
[0036] The melanin nanoshells can be, for example fabricated in a
material, mixed in a material, layered in a material, or coated
onto a material.
[0037] The object that is protected can be, for example, a
computer, an electronic component or circuit, a printed circuit
board, a cell phone, an avionic system and/or a satellite
component. The subject that is protected can be an animal, a human,
and/or a plant. For a human or animal subject, one or more internal
organs can be protected, for example bone marrow, liver, spleen,
kidneys, lungs, and/or portions or all of the gastrointestinal
tract.
[0038] The melanin nanoshells can also be used to contain radiation
and/or electronic pulses.
[0039] The invention further provides a method of protecting
internal organs of a subject from radiation and/or from electronic
pulses, where the method comprises administering to the subject
particles comprising any of the melanin nanoshells described
herein. The subject can be a human or an animal. The organ that is
protected can be, for example, one or more of bone marrow, liver,
spleen, kidneys, lungs, and gastrointestinal tract, e.g. the
intestines. Preferably, bone marrow is protected. The method can
further comprise administering to the subject a co-polymer of the
poloxamer series, which can increase bone marrow uptake of the
melanin particles. Preferably, the co-polymer of the poloxamer
series is administered to the subject prior to administering the
particles comprising the melanin nanoshell. Co-polymers of the
poloxamer series include, for example, pluronic acid F-68,
poloxamer-407 (PEG (polyethylene glycol)/PEO (polyethylene oxide),
MW 13,310) (24), and poloxamine 908 (25, 28). The class of
polyoxypropylene/polyoxyethylene copolymer nonionic surfactant
compounds is reviewed in (27). Preferably, the particles comprising
the melanin nanoshell have a diameter of about 10 nm to about 1,000
nm. The particles may be silica particles. Preferably, systemic
administration such as e.g. intravenous administration is used to
administer the melanin nanoshell particles and the poloxamer series
co-polymer to the subject.
[0040] The invention further provides a method of making a material
comprising the any of the melanin nanoshells disclosed herein,
where the method comprises fabricating melanin nanoshells into
and/or onto the material. The method can comprise polymerizing
melanin or melanin nanoparticles onto a surface. The method can
further comprise growing melanized fungi and extracting melanin
nanoshells from the fungi. The fungi can be encapsulated in melanin
nanospheres. The fungi can include, but are not limited to,
Cryptococcus neoformans (Cn) and/or Histoplasma capsulatum (Hc).
The fungi can be grown in the presence of a melanin precursor,
where the melanin precursor is one or more of L-dopa
(3,4-dihydroxyphenylalanin), D-dopa, catechol, 5-hydroxyindole,
tyramine, dopamine, tyrosine, cysteine, m-aminophenol,
o-aminophenol, p-aminophenol, 4-aminocatechol,
2-hydroxyl-1,4-naphthaquinone, 4-metholcatechol,
3,4-dihydroxynaphthalene, gallic acid, resorcinol, 2-chloroaniline,
p-chloroanisole, 2-amino-p-cresol, 4,5-dihydroxynaphthalene,
1,8-dihydroxynaphthalene, 2,7-disulfonic acid, o-cresol, m-cresol,
and p-cresol.
[0041] The invention also provides materials comprising melanin
nanoshells fabricated in and/or on the material.
[0042] Since melanin nanoshells are negatively charged, they can be
attracted or held in place with positively charged substances, or
repelled using negatively charged substances.
[0043] The material, for example, can be coated with melanin
nanoshells and/or encased in melanin nanoshells. The melanin
nanoshells can be incorporated into the material. The material can
be a plastic that is impregnated with melanin nanoshells or a
surface where melanin is polymerized and/or melanin nanoshells are
attached. The melanin nanoshells can be in a binder between two
layers of material.
[0044] The material comprising the melanin nanoshells can be used,
for example, as clothing, a protective gear, a object worn by a
subject, or a packaging material. The material can be, or can be
incorporated into, a wall, floor and/or ceiling of a room,
building, vehicle, aircraft, ship, spacecraft, and/or
submarine.
Experimental Details
Materials and Methods
[0045] C. neoformans (Cn) and H. capsulatum (Hc).
[0046] American Type Culture Collection (ATCC, Rockville, Md.)
strains Cn 24067 (serotype D) and Hc (CIB strain 1980, a gift from
A. Restrepo, Medellin, Colombia) were used in all experiments. Cn
was grown in Sabouraud dextrose broth (Difco laboratories, Detroit,
Mich.) for 24 hrs at 30.degree. C. with constant shaking at 150
rpm. Hc was grown with shaking at 37.degree. C. in defined media
consisting of 29.4 mM KH.sub.2PO.sub.4, 10 mM
MgSO.sub.4.times.7H.sub.2O, 13 mM glycine, 15 mM D-glucose, 3 .mu.M
thiamine. Melanized Cn and Hc cells were generated by growing the
fungi in their respective media with 1 mM 3,4-dihydroxyphenylalanin
(L-dopa) for 5 days. The cells were collected by centrifugation and
washed three times with PBS, pH 7.2 before radiation exposure.
[0047] Susceptibility of Cn and Hc to External Gamma Radiation.
[0048] Approximately 10.sup.5 melanized or non-melanized Cn or Hc
cells were placed in microcentrifuge tubes in 0.5 mL PBS and
irradiated with a .sup.137Cs source at a dose rate of 14 Gy/min.
The cells were exposed to doses of up to 220 Gy. The exposures of
1,000-8,000 Gy were given by irradiating the cells at 30 Gy/min.
Following radiation exposure, 10.sup.3 cells from each tube were
plated to determine viability as measured by colony forming units
(CFU's). Alternatively, melanized or non-melanized Cn cells were
plated on Sabouraud plates in air or under the nitrogen gas. The
plates were irradiated at a dose rate of 14 Gy/min followed by
determination of viability as measured by CFU's.
[0049] Other Sources of Melanin.
[0050] Melanin from cuttlefish Sepia officinalis was purchased from
Sigma Chemical Co.
[0051] Measurement of Radiation Absorption Properties of Bulk
Melanin.
[0052] A pellet of 13 mm diameter and 4 mm height with the mass of
0.71 g and density of 1.33 g/cm.sup.3 was made from Sepia melanin
by applying a pressure of 6 tonn/cm.sup.2. The measuring of gamma
radiation shielding properties of the pellet was performed by
placing the pellet on the 3 mm in diameter opening in a
lead-shielded castle inside which radioactive sources were placed.
The dose rate in mrad/h at the surface of the opening was measured
with and without the melanin pellet. Absorption of .alpha.- and
.beta.-radiation was evaluated by placing the melanin pellet on the
point sources of 210-Polonium and 32-Phosphorus, respectively.
[0053] Transmission Electron Microscopy (TEM).
[0054] Melanized and non-melanized Cn and Hc were frozen under high
pressure using a Leica EMpact High Pressure Freezer (Leica
Microsystems, Austria). Frozen samples were transferred to a Leica
EM AFS Freeze Substitution Unit and freeze substituted in 1% osmium
tetroxide in acetone. They were brought from -90.degree. C. to room
temperature over 2-3 days, rinsed in acetone and embedded in Spurrs
epoxy resin (Polysciences, Warrington, Pa.). Ultrathin sections of
70-80 nm were cut on a Reichert Ultracut UCT, stained with uranyl
acetate followed by lead citrate and viewed on a JEOL (Tokyo,
Japan) 1200EX transmission electron microscope at 80 kV.
[0055] Isolation and Purification of Melanins.
[0056] The cells were suspended in 1.0 M sorbitol-0.1 M sodium
citrate (pH 5.5). Lysing enzymes (Sigma Chemical Co.) were added to
suspension at 10 mg/mL and the suspensions were incubated overnight
at 30.degree. C. Protoplasts were collected by centrifugation and
incubated in 4.0 M guanidine thiocyanate overnight at room
temperature and were frequently vortexed. The resulting particulate
material was collected by centrifugation, and the reaction buffer
(10.0 mM tris, 1.0 mM CaCl.sub.2, 0.5% SDS) was added to the
particles. Proteinase K was added to suspension at 1.0 mg/mL
followed by overnight incubation at 37 (Hc) or 65.degree. C. (Cn).
The particles were boiled in 6.0 M HCl for 1 hour. Finally,
resulting material was washed with PBS, dialyzed against deionized
water overnight and dried in the air at 65.degree. C. for 2 days.
Approximately 1.5.times.10.sup.10 Cn cells and 2.2.times.10.sup.10
Hc cells were used. The isolation procedure yielded approximately
2.0 mg melanin per 10.sup.10 cells for Cn, and 2.3 mg per 10.sup.10
cells for Hc. The yield of melanized cells per 1 liter of medium is
2 g and the yield of purified ghosts is 0.3 g. Hollow melanin
shells that remain after the treatment of melanized cells with
enzymes, guanidinium isothiocyanate and 6 M HCl were dubbed
"ghosts" because they preserved the shape of the cells.
[0057] Quantitative Elemental Analysis of Melanins.
[0058] Elemental analysis for carbon, hydrogen, and nitrogen was
performed by Quantitative Technologies Inc. (Whitehouse, N.J.).
[0059] Oxidation of Melanins and HPLC of Oxidized Melanins.
[0060] Cn and He melanin underwent acidic permanganate oxidation
using the procedure described by Ito and Fujita (16).
Pyrrole-2,3,5-tricarboxylic acid (PTCA) and
1,3-thiazole-4,5-dicarboxylic acid (TDCA) were used as standard
compounds. The oxidation products were analyzed by HPLC using a
Shimadzu LC-600 liquid chromatograph, Hamilton PRP-1 C.sub.18
column (250.times.4.1 mm dimensions, 7 .mu.m particle size), and
Shimadzu SPD-6AV UV detector. The mobile phase was 0.1%
trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic
acid in acetonitrile (solvent B). At 1.0 mL/min, the elution
gradient was (min, % B): 0, 0; 1, 0; 12, 25; 14, 25; 16, 0. The UV
detector was set at a 255 nm absorbance.
[0061] MALDI Mass Spectrometry.
[0062] The major peaks generated during chromatography of oxidized
melanins were collected and analyzed by MALDI-TOF mass spectrometry
in positive pressure mode on PE-Biosystems Mariner ESI TOF mass
spectrometer. Peptide mixture with molecular weights of 1059.56,
1296.68 and 1672.95 in 2,5-dihydroxybenzoic acid matrix was used
for calibration.
[0063] Electron Spin Resonance Spectroscopy (ESR).
[0064] The ESR of purified melanins from Cn and He cells was
performed on ER 200D EPR/ENDOR spectrometer with ESP 300 upgrade
(Brucker Instruments, Inc. Billerica, Mass.).
[0065] Statistical Analysis.
[0066] The slopes of the survival curves were determined by linear
regression (GraphPad PRISM software, San Diego, Calif.) and a
Student's test for unpaired data was performed to analyze the
differences in survival. Differences were considered statistically
significant when P values were <0.05.
Results and Discussion
[0067] As described herein, the ability of melanin to protect
against ionizing radiation was demonstrated in two fungi capable of
melanogenesis, Cryptococcus neoformans (Cn) and Histoplasma
capsulatum (Hc). These fungi were chosen as model organisms because
they can be grown in either melanized or non-melanized states,
while fungi found in Chernobyl are constitutively melanized. Cn and
He cells became encapsulated in melanin when grown with L-dopa
(3,4-dihydroxyphenylalanin). Previous work (2) as well as this
study showed that all melanin in the cells is concentrated in the
cell wall (FIG. 1B). The melanin forms coherent and robust spheres
capable of withstanding boiling in concentrated hydrochloric acid
(FIG. 1C). From transmission electron microscopy (TEM) of melanized
Cn and Hc, the thickness of the melanin layer was estimated to be
100 nm. Analysis of `ghost` particles recovered from C. neoformans
grown with different precursors reveals that the pigments and
melanins made from different precursors have different properties
with regards to color, charge, and electron spin resonance.
[0068] Melanized and non-melanized Cn and Hc cells in phosphate
buffered saline (PBS) were subjected to extremely high doses of
radiation--up to 8,000 Gy. For comparison, a dose of just 5 Gy is
lethal to humans. The radioprotective effect of melanin was more
readily demonstrable at the higher radiation doses, as the
LD.sub.90 for these organisms in non-melanized form is around 50 Gy
(Cn) or 100 (Hc) Gy (11). Melanized Cn cells demonstrated reduced
susceptibility to external gamma radiation (P=0.01) in the dose
range of 0-220 Gy (FIG. 2A). At the dose range of 1,000-8,000 Gy
some protective effects were also seen for melanized Cn cells (FIG.
2A); however, the difference in survival of irradiated cells was
not statistically significant (P=0.4). For Hc cells melanin
provided protection against gamma radiation up to 8,000 Gy
(P<0.01) (FIG. 2B). Since some of the cytocidal effects of
radiation are mediated by radiolysis of water and are significantly
more pronounced in the presence of O.sub.2 (12), the effects of
radiation on Cn cells were compared in air and in N.sub.2
atmospheres. When Cn cells were irradiated directly on agar plates
either in air (FIG. 2C) or in N.sub.2 (FIG. 2D) with the doses of
up to 440 Gy, less killing was observed in N.sub.2 than in air
(P<0.02) for both melanized and non-melanized Cn cells in the
150-300 Gy region. In both air and N.sub.2, melanization conferred
a greater survival advantage for Cn (P<0.01).
[0069] To compare the radioprotective properties of melanin with
other materials such as lead, the linear attenuation coefficient
and half value layer were calculated according to the
equations:
I=I.sub.oe.sup.-.mu.x (1)
HVL=0.693/.mu. (2),
where I.sub.o and I are the radiation intensity before and after
shielding, respectively; .mu. is the linear attenuation coefficient
in cm.sup.-1, x is the thickness of the shield in cm, and half
value layer (HVL) is the thickness of shielding necessary to reduce
the intensity of radiation to half of its original value. The
reduction in radiation intensity was calculated from the linear
parts of survival curves assuming that a 10% increase in survival
is equivalent to a 10% decrease in radiation intensity. Linear
attenuation coefficient and HVL for Hc melanin were calculated to
be 1.4.times.10.sup.4 cm.sup.-1 and 0.5 .mu.m, respectively. This
melanin linear attenuation coefficient is several orders of
magnitude higher than that of lead (27.1 cm.sup.-1) (13),
indicating that fungal melanin in nanosphere form is a much more
efficient radioprotector than lead.
[0070] To gain insights into the unusual radioprotective properties
of melanin, high-pressure liquid chromatography (HPLC), matrix
assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF) and elemental analysis of the fungal
melanins were performed. Unlike synthetic melanins (10, 14, 15),
the structures of natural melanins including fungal melanin are
poorly understood. These pigments are amorphous and insoluble,
characteristics that preclude a structural solution of melanins
given currently available analytical tools, and have to be
converted into low molecular weight fragments prior to analysis.
Consequently, acidic permanganate oxidation of fungal melanins was
carried out before HPLC. Two major types of melanin have been
described. Eumelanin is a dark-brown to black pigment with 6-9%
nitrogen and 0-1% sulphur, and is composed of 5,6-dihydroxyindole
(DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) monomer
units (16, 17) (FIG. 3A). In contrast, pheomelanin is a
reddish-brown pigment with 8-11% nitrogen and 9-12% sulfur,
composed of benzothiazine monomer units (16, 17) (FIG. 3B). Acidic
permanganate oxidation yields pyrrole-2,3,5-tricarboxylic acid
(PTCA) from DHICA-derived structures, and
1,3-thiazole-4,5-dicarboxylic acid (TDCA) from benzothiazole
subunits (16, 17). Hence, the presence of PTCA in oxidation
products indicates eumelanin and the presence of TDCA indicates
pheomelanin. The appearance of solutions following acidic
permanganate oxidation of melanins is shown in FIG. 3C.
Chromatograms of PTCA and TDCA standards yielded peaks at 11 and
6.1 min, respectively. The chromatograms of both Cn and Hc melanins
revealed PTCA and TDCA (FIG. 3E, 3F). The MALDI-TOF analysis of
these peaks confirmed the presence of PTCA (MW 199) and TDCA (MW
173). Importantly, the PTCA to TDCA ratio was 0.90 for Hc melanin,
whereas for Cn melanin the ratio was 47.7, as calculated from the
chromatographic data. Although these ratios do not reflect
quantitative measure of eumelanic/pheomelanic character, they
indicate that benzothiazine subunits predominate in Hc melanin
while DHICA subunits predominate in Cn melanin. The low TDCA
content in Cn is consistent with very low levels of
aminohydroxyphenylalanine in Cn, which is a specific indicator of
cysteinyldopa (18). In contrast, the relatively high levels of TDCA
in Hc melanin suggest a significant higher content of pheomelanin.
This observation is also consistent with the fact that Hc colonies
often display some measure of pigmentation, and red He colonies
have been described (19). The results of elemental analyses of
various melanins performed in this study as well as reported in the
literature (20) are given in Table 1. The C:N ratio for Cn melanin
was 11.4:1, while that for He melanin was significantly
higher--18.6:1 (20).
TABLE-US-00001 TABLE 1 Elemental composition of various melanins
C/N Type of Melanin Ratio Reference l-dopa-melanin 6.65 16
Copolymer of l-dopa and of 6.75 16 5-S-Cysteinyl-dopamine
5-S-Cysteinyldopa-melanin 5.02 16 Pheomelanin from l-dopa and
cysteine 4.95 16 Dopamine-melanin 7.02 16
5-S-Cysteinyldopamine-melanin 4.41 16 C. neoformans 24067 black
particulate 12.5 2 Sigma melanin 7.5 2 Synthetic l-dopa-melanin 9.0
20 Dopa-melanin from S. officinalis 7.0 20 Dopa-melanin from C.
neoformans 24067 8.0 20 Melanin from A. niger J9901 conidia 14.5 20
Melanin from MNT1 melanoma tumor 6.3 23 Melanin from C. neoformans
24067 11.41 This work Melanin from H. capsulatum CIB 1980 18.63
This work C = carbon; N = nitrogen.
[0071] Since the density of melanin is only slightly greater than
that of water, it cannot contribute significantly to its remarkable
radioprotective properties. However, the number of electrons per
gram could make a significant contribution to melanin protective
properties. The number of electrons is an especially important
contributor to the attenuation properties of a material at the
energy levels where the Compton effect predominates (13). Thus, the
higher number of electrons in oligomers of pheomelanin in
comparison with eumelanin--388 versus 287, and the structure
composed of electron-rich covalently linked aromatic motifs could
account for better scattering properties of Hc melanin rich in
pheomelanin oligomers in comparison with Cn. Secondly, pheomelanin
contains divalent sulfur (FIG. 3B) which may also contribute to
superior radioprotective properties of Hc melanin, as compounds
containing divalent sulfur are efficient radioprotectors (12).
[0072] Efficient Compton scattering by melanin alone is unlikely to
explain the radioprotective properties of melanin. The transfer of
radiation (photon) energy to living matter occurs in a series of
interactions, where energy is transferred to high-energy electrons,
and then to secondary photons of progressively less energy (FIG.
4). These high-energy electrons are ultimately responsible for the
radiobiologic effects caused by gamma-ray, x-ray or bremsstrahlung
radiation by either direct "hits" of DNA or through radiolysis of
water in the cells which results in formation of reactive
short-lived free radicals such as hydroxyl OH. or perhydroxyl
HO.sub.2., both capable of damaging DNA. Hence, melanin may trap
these high-energy electrons thus preventing them from entering a
cell and triggering radiolysis of water. Consistent with this
hypothesis, electron spin resonance spectroscopy (ESR) of fungal
melanins revealed strong signals for melanized Hc (FIG. 5) and Cn
(results not shown) indicative of a stable radical population (21).
Thus, these stable free radicals may act as efficient traps of
Compton and photoelectrons and short-lived free radicals.
[0073] In the macroscale experiment, the 4 mm thick melanin pellet
made of Sepia (bulk) melanin completely absorbed .alpha.- and
.beta.-radiation from 210-Po and 32-P sources, respectively. This
is better than plastic, since to stop a .beta.-particle 7 mm of
plastic (e.g., Lucite) are needed and the density of Lucite is
higher than the density of the 1.33 g/cm.sup.3 melanin pellet made
of Sepia melanin.
[0074] Measurement of the bulk melanin shielding effect towards
gamma radiation of 122-140 keV energies showed that 4 mm of melanin
cut the dose by .about.33%. Using these data, the linear
attenuation coefficient (.mu.) for bulk melanin was calculated to
be 1.01 cm.sup.-1. For comparison, at 140 keV lead has a higher
.mu.=27.1 cm.sup.-1 but its density is 11.34 g/cm.sup.3; and
aluminum has .mu.=0.386 cm.sup.-1 and a density of 2.7 g/cm.sup.3.
It is obvious from these measurements, that melanin nanoparticles
possess several orders of magnitude better radiation shielding
properties than bulk melanin. Since the absorbance of radiation by
matter also depends on the geometric arrangement of the photon
source and the absorber, an important factor contributing to the
radioprotective properties of fungal melanin can be the spatial
arrangement of melanin in fungal cells. The location of melanin in
the fungal cell wall outside of the plasma membrane (FIG. 1) where
it forms a sphere of nanosize thickness places it in a position
such that incident radiation must unavoidably pass thorough the
melanin layer which scatters and/or absorbs it.
[0075] To prove the contribution of the nanospherical arrangement
of melanin in fungal cells to radioprotection, non-melanized C.
neoformans cells were irradiated with doses of up to 400 Gy in the
presence of melanin from Sepia officinalis (cuttlefish), which is
not arranged in hollow spheres, in amounts equal or 20 times higher
than the amount of melanin in the same number of melanized C.
neoformans cells. S. officinalis melanin conferred no protection at
any dose (FIG. 6A), suggesting that the spatial arrangement of
melanin particles in the `ghosts` was important in radioprotection.
To exclude the possibility that differences in chemical composition
of fungal and S. officinalis melanins accounted for the lack of
radioprotection by the latter, the experiment was modified by
irradiating non-melanized C. neoformans cells with the same amounts
of intact and powder-crushed melanin "ghosts" (FIG. 6B). 0.1 mg
intact "ghosts" protected the cells up to 120 Gy in the same way as
melanization, while crushed "ghosts" afforded only slight
protection. Hence, when melanin is arranged in nanospheres, it
scatters/absorbs radiation more efficiently than powdered melanin
of the same chemical composition.
[0076] To further assess the shielding properties of C. neoformans
melanin "ghosts", a 96 well ELISA plate was coated with a solution
of poly-lysine to make the surface of the wells positively charged
in order to counteract the negative charge carried by melanin
"ghosts". The ghosts were mixed with poly-lysine solution to
prepare a homogeneous suspension, and 3 different concentrations of
ghosts were placed in the wells. For control different
concentrations of Sepia melanin and charcoal suspensions in
poly-lysine solution were used as well as lead foil of similar
weights (FIG. 7). The ELISA plate was exposed to diagnostic X-rays
(40 kVp at 10 mA) and the shielding effect was detected with
Hi-speed Kodak x-omat (xb-1) radiographic film placed under the
plate during exposure to X-rays (FIG. 8). The radiographic film was
analyzed for corresponding global intensities by subtraction method
(Biorad Quantity One software) and internal pixel density (IPD) per
mm.sup.2 was calculated. The attenuation of X-rays by substances in
the wells was calculated as the ratio of IPD in the wells to IPD of
the background wells (Table 2).
TABLE-US-00002 TABLE 2 Attenuation of diagnostic X-ray by
suspensions of C. neoformans nd Sepia melanin in comparison with
non-melanin substances. Internal pixel density (IPD) per mm.sup.2
mean Amount, 2nd IPD/IPD Attenuation mg 1st (repeat) (bkrd) (%) A
Sepia melanin 50 1370 1370 1.12 12 25 1280 1280 1.05 5 B C.
neoformans 100 1720 1720 1.41 41 50 1470 1470 1.21 21 30 1346 1346
1.11 11 C Charcoal 40 1329 1329 1.09 9 20 1248 1248 1.02 2 15 1244
1244 1.02 2 D Lead foil 100 2202 2217 1.81 81 200 2269 2277 1.87 87
Background -- 1218 1218 1 0
[0077] These results demonstrate that C. neoformans ghosts possess
superior radiation shielding properties in comparison with other
types of melanin (Sepia) or non-melanin carbon-based compounds
(charcoal). The shielding properties of the ghosts were comparable
to those of lead when one takes into consideration that the ghosts
were used in the form of a suspension in poly-lysine solution,
which has a lot of "gaps" between the ghosts for X-rays to
penetrate without being scattered, while lead foil is a material
with continuous close packing of lead atoms.
[0078] The properties of materials change dramatically when one
moves from bulk materials to nanomaterials. The superior radiation
shielding properties of fungal melanin nanospheres in comparison
with melanin powder (bulk material) are direct consequence of
principally different mechanism of radiation absorption by melanin
nanoparticles--a gamma photon becomes "trapped" within a melanin
nanoparticle as it is reflected several times by its inner walls
and is unable to escape the particle until it transfers all of its
energy to melanin.
Melanized Nanoparticles for Protection of Bone Marrow and Internal
Organs from Ionizing Radiation
[0079] As bone marrow is the dose-limiting organ for both external
beam radiation therapy and radioimmunotherapy, protection of bone
marrow against radiation would increase safety and efficacy of
these treatments. An investigation was conducted of whether melanin
nanoshells administered before a dose of external radiation protect
bone marrow in mice from radiation damage. It is known that,
following intravenous administration of nanoparticles, 0.5-1% of
the injected dose goes into bone marrow, while the majority of
nanomaterial is sequestered by the mononuclear phagocytes of the
liver and to a lesser degree of the spleen (24). It has been
demonstrated that nanoparticles can be efficiently redirected into
the bone marrow in rats by pre-treatment or co-administration of
block co-polymers of the poloxamer series, for example,
poloxamer-407 (PEG (polyethylene glycol)/PEO (polyethylene oxide),
MW 13,310) (25), which minimizes interaction of nanoparticles with
the reticuloendothelial elements of liver and spleen.
[0080] Silica nanoparticles (20 nm) were utilized in the present
experiments. The surface of unmodified silica particles is covered
with hydroxyl groups. Nanoparticles were melanized overnight at
35.degree. C. in 10 nM L-Dopa solution, precipitated by lowering
the pH to 1, washed from unreacted L-Dopa and transferred into
deionized water. To prove that the dark color of melanized
particles was due to the presence of melanin, immunofluorescence of
these particles was performed with melanin-binding monoclonal
antibody (mAb) 6D2 as previously described (26). 6D2 mAb bound
avidly to the surface of the particles, thus proving that they were
covered with a layer of melanin.
[0081] To measure the uptake of melanized particles in major organs
and bone marrow with and without a co-polymer of the poloxamer
series, melanized particles were radiolabeled with 188-Rhenium
(.sup.188Re) by incubating 16 mg of particles per sample with 40
.mu.L SnCl.sub.2 and Na.sup.188ReO.sub.4 for 2 hr at 37.degree. C.,
separating the particles from unreacted Na.sup.188ReO.sub.4 in
supernatant by centrifugation, and suspending them in Na carbonate
buffer (pH=8.5). Two groups of 4 BALB/c mice were injected IV with
100 .mu.L (1.6 mg, 50 mg/kg body weight) of melanized particles
while other two groups of 4 mice were pre-injected IV with 0.13
mg/kg body weight of pluronic acid (pluronic acid F-68 is a member
of the poloxamer series, and is available from Sigma as 10%
solution) and 12 hr later were injected IV with the above amount of
.sup.188Re-labeled particles. The animals were sacrificed 3 and 24
hr post-injection, their major organs were removed, blotted from
blood if necessary, weighed, and their radioactivity was counted in
a gamma counter. The results of the biodistribution are presented
in FIG. 9. Pre-injection of the animals with pluronic acid
significantly (more than 30-fold) increased the uptake of melanized
nanoparticles in the bone marrow, thus providing the potential for
delivering amounts of nanoparticles sufficient to protect bone
marrow from radiation damage. It also should be noted that although
liver and spleen, which also take up nanoparticles, are not
dose-limiting organs during radiation therapy or
radioimmunotherapy, their protection by melanized nanoparticles
will be also beneficial, especially in case of radioimmunotherapy
when liver and spleen receive significant radiation doses as a
result of antibody concentration and metabolism in these
organs.
[0082] To investigate the radiation protective properties of
melanized nanoparticles (MNs), groups of 3 CD-1 mice were injected
IV with MNs alone or with pluronic acid (PA)+MNs. Control groups
consisted of untreated mice, mice given PA alone, non-melanized
nanoparticles alone, and PA+non-melanized nanoparticles. The mice
were irradiated with either 1.25 or 2.50 Gy of gamma radiation and
their platelet and white blood cell (WBC) counts were monitored for
28 days post-treatment (FIGS. 10-11). The pluronic acid had an
immunomodulatory effect by itself but MNs alone clearly provided
protection in comparison with control groups on Days 2, 5, and 14
post-treatment. The protective effect was more pronounced in mice
irradiated with 1.25 Gy (FIG. 10) than with 2.5 Gy (FIG. 11). Thus,
MNs which deposit themselves in bone marrow due to the body
"sieving" effect can protect bone marrow against high doses of
external gamma radiation, and mouse pre-treatment with PA increases
the MNs protective effect. These results are encouraging for the
development of similar strategies in patients undergoing EBRT or
RIT.
[0083] The protective effect of different types of melanin was
evaluated on the GI tract in mice receiving lethal dose of 9 Gy at
a high dose rate. CD-1 female mice were used in all experiments.
Initially a check was made for potential toxicity of synthetic
melanin and C. neoformans (Cn) melanin "ghosts" made from Cn strain
24067 to the GI track. Mice were fed 15 mg/kg body weight synthetic
melanin or ghosts via gavage needle and their body weight and
condition were monitored for 30 days. Also, 2 mice out of each
group were sacrificed at 24 hr post-feeding with melanin, and their
stomachs, small intestine and colon were removed and fixed in
formaline-buffered PBS. The parafinized tissues were subsequently
cut, stained with H&E and analyzed histologically.
[0084] There was no loss in the body weight of melanin-fed mice
with all mice consistently gaining weight (FIG. 12). The stomachs,
small intestine and colon in mice sacrificed at 24 hr post-feeding
were normal (FIG. 13). However, for the first 10 days post-feeding
mice given Cn ghosts were gaining more weight which might suggest
some inflammation and edema due to immunogenecity of fungal melanin
which resolved by day 10. The conclusion from the preliminary
experiments was that neither synthetic melanin nor 24067 Cn ghosts
were acutely toxic to CD-1 mice in the amount of 15 mg/kg body
weight and can be used in radiation protection experiments in
vivo.
[0085] In the next experiment female CD-1 mice (10 mice per group)
were fed 15 mg/kg body weight synthetic melanin or ghosts or water
via gavage needle and 1 hr post feeding were subjected to whole
body irradiation in 137-Cs irradiator with the total dose of 9 Gy
delivered at 3 Gy per min; their body weight and survival were
monitored for 22 days. At 4 hr and 24 hr 2 mice per group were
sacrificed and their stomachs, small intestine and colon were
removed and fixed in formalin-buffered PBS. The parafinized tissues
were subsequently cut, stained with H&E and analyzed
histologically. For the first 4 days post-irradiation, mice fed
with ghosts were loosing less weight than mice fed with either
water or synthetic melanin (FIG. 14), which might be explained by
some edema accompanying administration of fungal melanin. At day 5
the cumulative weight loss in all groups equalized and for the rest
of the observation period the weight loss was the least pronounced
in mice fed with synthetic melanin. The overall survival on day 11
post-irradiation was 100% in the synthetic melanin group, 66% in
the ghosts group and 33% in control mice fed with water, with the
last mouse in this group dying on day 16 (FIG. 15). Mice fed with
fungal ghosts survived for 21 days and with synthetic melanin for
22 days, which represents a significant advantage in survival in
comparison with the control group. Histological evaluation of GI
tissues obtained from mice 4 hr and 24 hr post-irradiation showed
that at 4 hr in the stomach of mice fed with ghosts there were
fewer apoptotic cells than in stomachs of synthetic melanin or
water fed mice (FIG. 16A-C). At 24 hr this trend continued with
stomach of mice fed with ghosts having glandular cells which were
less vacuolated and attenuated than in the synthetic melanin group)
(results not shown). Simultaneously, there were more mitotic
figures and less apoptotic cells in both melanin groups in
comparison with control water fed mice. In the small intestines
there were no difference between the groups with glands of the
mucosa showing numerous apoptotic cells. In the colonic glands of
mice fed with ghosts there was less cellular reaction and apoptosis
as compared to the other colon samples (FIG. 16D-F).
[0086] Overall, the prolongation in survival of melanin-fed mice in
comparison with control water-fed mice was statistically
significant by log-rank test for both synthetic and fungal melanin
with synthetic melanin being a better radioprotector in the
conditions of experiment. It might be possible that if the ghosts
are eliciting inflammation by themselves it could increase the
amount of damage sustained from radiation and this could account
for less protection with the ghosts than with synthetic melanin. To
avoid administration of immunogenic fungal melanin, but to use the
advantage of hollow sphere shape which contributes to the radiation
scattering by melanin, biodegradable particles of 200-1000 nm
diameter made of poly-DL-lactic/glycolic acid (PLGA) polymer from a
commercial vendor (Corpuscular Inc, Cold Spring, N.Y.) can be
covered with different melanins by incubation in a solution of
melanin pre-cursors during autopolymerization as described above
and then dissolved the PLGA polymer with the hydrochloric acid. As
melanin is extremely resistant to acid, the melanin shell will
remain intact while the biodegradable core of the particle will be
dissolved. The results of this study indicate that it is possible
to utilize melanin for protection of the GI tract from radiation
injury.
[0087] In summary, the results described herein establish that
fungal melanin arranged in nanosize spheres protects against
extremely high levels of ionizing radiation and suggest that the
protective efficacy of this pigment is a function of its chemical
structure, stable free radical presence, and spatial arrangement.
In essence, melanin protects against ionizing radiation by
mechanisms that are different from the radiation shielding
properties of heavy metals, which depend largely on density. These
results demonstrate the feasibility of designing low-density
nanoshells with radiation shielding properties, which could find
uses in a variety of applications by virtue of their low weight.
The term "nanoshells" is used to describe nanoparticles of
different shapes--e.g., nanospheres, nanotubes, nanoellipsoids and
nanorods. Melanin used for manufacturing of nanoshells can be of
synthetic or biological origin.
Prophetic Applications
[0088] Preparation of Additional Melanin Nanoshells:
[0089] Melanin-filled nanoshells can be generated by incubating
melanin such as Sepia melanin in an aerated solution of melanin
precursor such as L-dopa or cysteinyl-dopa to provide the
conditions for oxidative polymerization as described herein in
experiments on generation of melanin-covered nanoparticles for bone
marrow protection. To generate melanin-covered nanoshells filled
with different materials one can use the above approach with
nanoparticles of choice to cover with melanin. For generation of
hollow nanospheres, biodegradable nanoparticles made of materials
such as proteins or biodegradable plastics will be covered with
melanin as above and then treated with concentrated acid which will
dissolve the biodegradable nanoparticles but leave the melanin
nanoshell intact.
[0090] Preparation of Melanin-Containing Plastics:
[0091] To make plastics impregnated with melanin nanoshells, the
melanin nanoshells will be dispersed in a liquid monomer, such as
diethylene glycol bis(allyl-carbonate), otherwise know as CR-39,
styrene, or methylmethacrylate. Polymerization of the plastic
monomer will be initiated with the help of a free-radical
initiator. For example, 400 mg benzoyl peroxide will be dissolved
in 10 mL of diethylene glycol bis(allyl-carbonate) (CR-39) at
50.degree. C. Then, purified melanin nanoshells will be added,
under thorough mixing, in increasing amounts starting from 30 mg
until it is possible to form a homogeneous mixture. The mixture
will be heated at 50.degree. C. for one day. The mixture will be
heated for two additional days at 65.degree. C. under nitrogen, and
then cured in a vacuum oven at 110.degree. C. for 2 h.
[0092] Incorporation of Melanin Between Two Layers of Material:
[0093] Purified melanin nanoshells will be added to a
binder/adhesive in the form of a suspension to achieve dispersion
of melanin in the binder/adhesive. Then, a hardener will be
combined with the binder/adhesive, which will then be immediately
"sandwiched" between two layers of material. For example,
increasing amounts of purified melanin nanoshells starting from 500
mg will be suspended in 10 mL of chloroform. This suspension will
be mixed with 2 mL epoxy resin. The chloroform will then be removed
by evaporation leaving melanin homogeneously dispersed in the epoxy
resin. Epoxy catalyst, or hardener, will be added, and the mixture
will be slowly stirred. Drops of the product will be deposited onto
a material such as a plastic or glass, and an identical material
will be placed on top of the melanin-epoxy suspension.
[0094] Coating Surfaces with Melanin:
[0095] As an example, purified melanin nanoshells in increasing
amounts starting from 1 g will be suspended in 30 mL of water.
Drops of this concentrated melanin suspension will be allowed to
spread on the hydrophilized surface of a plastic or glass. The
water will be allowed to evaporate leaving melanin attached to the
surface of the plastic or glass. As an alternative, a melanin
coating may be made on surfaces by first immobilizing on the
surface the enzyme laccase which catalyzes melanin formation in
fungi. Melanin coated surfaces may also be generated by
autopolymerization of melanin precursors. Enzymatically-mediated
generation of melanin nanoshells in situ could provide an
attractive alternative for coating vulnerable surfaces with this
material. Furthermore, since melanin nanoshells are negatively
charged, they can be attracted to a surface that is positively
charged.
[0096] Treatment of Buildings to Reduce Entry of Radon and
Radiation from Radon:
[0097] Radon is a gas that forms naturally during the decay of
uranium-238. Radon that occurs naturally in soil can seep from the
soil into homes and other buildings. Melanin nanoshells will be
added to paints, coatings, and/or building materials. Melanin-based
paints and coatings applied to the foundations of buildings, and
areas where pipes enter the buildings, can be used for the purpose
of trapping radon when it is emitted from rocks or soil before
radon enters the building. Radon decays over the course of several
days to 210-Pb, which is a beta and gamma emitter and has a
half-life of 22 years. Melanin should absorb 210-Pb even tighter
than radon through chemosorption as melanin is known to bind
two-valent metals and thus should reduce the entry not only of
radon but also of radioactive lead into the environment of the
building.
[0098] Protection of Subjects Against Radiation:
[0099] A sterile preparation of melanin nanospheres will be
injected into an individual at risk for radiation injury. The
melanin nanospheres localize to the bone marrow where they provide
shielding against the cytotoxic effects of radiation on vulnerable
cells. In another application an oral preparation of melanin
particles will be ingested to provide protection for the
gastrointestinal mucosa. Melanin nanoshells can also be used in
protective clothing and gear.
[0100] Containment of Radioactively Contaminated Sites:
[0101] Melanin nanoshells will be used in environmental
bioremediation. The following types of radiation are given off by
radioactive material: alpha particles, beta particles, x-rays, and
gamma rays. The spread of radioactive particles will be reduced by
applying the melanin nanoshells to the radioactive particles.
Melanin is expected to encapsulate the radioactive particles and
thereby reduce their spread. Thus, melanin nanoshells may, for
example, prevent the spread of radioactive contamination to ground
water. Similarly, melanin nanoshells will be used to contain
radiation from radioactive waste and biomedical radioactive
materials. Melanin nanoshells may be used in remediation in
connection with, for example, waste containers, fuel cladding,
packaging containers, transport coverings for all land, air, and
water vessels, and nuclear waste clean-up.
[0102] Containment of Metal Ions:
[0103] Since melanin nanoshells are negatively charged, they may be
used to contain positively charged compounds, for example to act as
metal chelators and to contain mercury.
[0104] Absorption of Radiation:
[0105] Melanin nanoshells may be used to absorb radiation, for
example to absorb radar or radiation generated in association with
NMR systems.
[0106] Industrial, Physical Buildings and Construction:
[0107] Melanin nanoshells may be used for shielding or containment
in buildings, construction and containment structures in connection
with, for example, concrete, plastics, steel, titanium, composites,
coatings, "wafer" boards or sheeting in walls, roofs, flooring, and
conduits. Melanin nanoshells may be used, for example, in shielding
in connection with industrial radiation shielding; X-Ray rooms and
enclosures (x-rays, gamma radiation); storage and process
equipment; airport detection systems (gamma radiation); hot cells
(gamma radiation); paints and pigments (alpha and beta particles);
glass (alpha particles/radon); power lines (EM radiation/EM field);
conductors (EM radiation/EM field); wiring (EM radiation/EM field),
transformers (EM radiation/EM field); switches (EM radiation/EM
field); meter boxes (EM radiation/EM field); line hardware (EM
radiation/EM field); fuses (EM radiation/EM field); breakers (EM
radiation/EM field); drywall (alpha particles/radon); plywood
(alpha particles/radon); doors (alpha particles/radon); door frames
(alpha particles/radon), window frames (alpha particles/radon);
granite (alpha particles/radon); concrete (alpha particles/radon);
ceramic materials (tile); commercial fertilizers (alpha
particles/radium); angles (alpha particles/radon); pigs (alpha
particles/radon); castings (alpha particles/radon); heat lamps
(infrared radiation, UV light); road construction materials; pipes
and colts (alpha particles/radon); heaters (infrared radiation);
radio wave transmitters (EM radiation/radio waves); industrial
radiographers (x-ray and gamma radiation); roof tiles (alpha
particles/radon); metal (alpha particles/radon); steel (alpha
particles/radon); titanium (alpha particles/radon); stucco (alpha
particles/radon); caulk (alpha particles/radon); plastic (all types
of radiation); mortar (alpha particles/radon); brick (alpha
particles/radon); and VDUs (Vacuum Distillation units) (all types
of radiation). This includes the use of melanin nanoshells in
fossil-fuel power plants, chemical plants, paper plants, etc. and
clean-up/binding of mercury and other toxic release inventory (TRI)
gases/metals emissions.
[0108] Operational Equipment:
[0109] Melanin nanoshells may be used for shielding and containment
in composites, coatings, or inserts in equipment with radiation
exposure; for pressurized water reactors (PWRs), equipment on
primary sides of plant, including, but not limited to, reactor
core, reactor vessel, steam generators, pumps, conduit, electrical
relay boxes; and in boiling water reactors (BWRs), primary side
equipment including boilers, pumps, conduits, and also secondary
side equipment including turbines, condensers, pumps, relays, and
generators where radiation exists.
[0110] Airlines:
[0111] Melanin nanoshells may be used in shielding in air craft in
connection with, for example, airplane materials (windows, cockpit
gauges, mechanical parts, etc.) (cosmic radiation); cabinet X-ray
system (x-rays); human X-ray scanner (x-rays); and blimps (cosmic
radiation).
[0112] Space:
[0113] Melanin nanoshells may be used for shielding and containment
in space craft in connection with, for example, astronaut jumpsuits
(galactic cosmic radiation), spacecraft parts (galactic cosmic
radiation), and rocket parts (engines, turbines, etc.) (galactic
cosmic radiation).
[0114] Vehicles:
[0115] Melanin nanoshells may be used for shielding and containment
in connection with, for example, ship parts (hull, engines, motor,
etc.), vehicle parts, gauges (beta particles/tritium), and
alternate fuel sources (e.g. nuclear energy).
[0116] Defense Application:
[0117] Melanin nanoshells may be used for shielding and containment
in defense applications in connection with, for example, helicopter
materials (cosmic radiation); submarine materials (alpha particles,
beta particles, x-rays, and gamma radiation); navy carrier parts
(alpha particles, beta particles, x-rays, and gamma radiation);
fighter jet parts (cosmic radiation); tank parts (alpha particles,
beta particles, x-rays, and gamma radiation); naval nuclear
propulsion (alpha particles, beta particles, x-rays, and gamma
radiation); nuclear powered vehicles; and weapon night sights
(e.g., night vision goggles) (beta particles/tritium; infrared
radiation). Other applications include use of melanin nanoshells in
radar elusion in manned and unmanned vehicles.
[0118] Nuclear Application:
[0119] Radioactive materials in nuclear applications give off the
following types of radiation: alpha particles, beta particles,
x-rays, gamma rays, neutrons, protons, and heavy ions. Melanin
nanoshells may be used for shielding and containment in nuclear
applications in connection with, for example, power plant building
materials, decay drums, waste containers, power reactors,
pressurized water reactors, plant building materials, reactor core,
reactor vessel, steam generators, steam turbines, pumps, electrical
relay boxes, conduits, boiling water reactors, boilers, pumps,
condensers, relays, respirators, neutron generators, nuclear fuel
reprocessors, master-slave manipulators, nuclear batteries,
radiation fallout material, and tools, gear, and equipment. In
radioactive material or waste storage, including spent fuel
storage, melanin nanoshells may be used for shielding and
containment in building material, equipment, and fuel cladding. In
transport of radioactive material or waste, melanin nanoshells may
be used for shielding and containment in packaging, containers,
trucks/railcars/planes/water vessels, and
covering/coating/composites. In radiation contamination clean-up,
melanin nanoshells may be used for stabilizing radioactive isotopes
in clean-up conditions.
[0120] Homeland Security:
[0121] Melanin nanoshells may be used for shielding and containment
in homeland security applications in connection with, for example,
protection of buildings, equipment, computers, satellites, etc. and
protection of masses of people through clothing applications, house
shielding, etc. from nuclear or "dirty" bombs.
[0122] Medical/Dental:
[0123] Melanin nanoshells may be used for shielding and containment
in medical and dental applications in connection with, for example,
MRI machines (gamma radiation); X-Ray machines (gamma radiation);
mammogram machines (gamma radiation); lasers (infrared radiation);
dental crowns (gamma radiation/uranium); PET Scans (beta
particles); dental porcelains (gamma radiation/uranium/thorium);
external-beam radiation therapy machines (used to target localized
areas of a tumor) (gamma radiation, electron beams, neutron and
heavy ion beams); X-ray Tubes (gamma radiation); lab coats,
coveralls, and head covers; sterilizers (gamma
radiation/cobalt-60); sonogram machines (gamma radiation);
radiopharmaceuticals (injectable radioisotopes) (gamma, alpha, beta
(both positive and negative) and Auger electron radiation); medical
diagnostic imaging; cardiac cath swing lab shielded partitions
(gamma radiation); and nuclear medicine products (gamma, alpha,
beta (both positive and negative) and Auger electron radiation). In
medical radiation therapy, melanin nanoshells may be used in
coatings to protect, for example, the following against x-ray and
gamma radiation: linear accelerator swinging door systems, linear
accelerator sliding door systems, H.D.R. automated swing door
systems, gamma knife door systems, H.V.A.C. shielding systems,
H.D.R. treatment enclosures, treatment room shielding upgrade
systems, square-edge and interlocking bricks, modular vault
systems, and proton therapy shielding systems.
[0124] Science Labs:
[0125] Melanin nanoshells may be used in shielding in science
laboratories in connection with, for example, anodes (x-rays),
atomic particle accelerators (x-rays, UV radiation), X-Ray
diffraction units (x-rays), and electron microscopes (EM radiation,
x-rays, and beta particles).
[0126] Consumer Products:
[0127] Melanin nanoshells may be used in shielding in consumer
products in connection with, for example, protective clothes,
shoes, sunglasses (EM radiation/UV light), eye glasses (EM
radiation/UV light), contacts (EM radiation/UV light), make-up (EM
radiation/UV light), lip gloss (EM radiation/UV light), ovens
(alpha particles), toaster ovens (alpha particles), cell phone and
covers (EM radiation/radio waves), televisions (alpha particles,
extremely low frequency EM fields, x-rays), watches (beta
particles/tritium), glow in the dark products (beta particles),
light bulbs (UV radiation, infrared radiation), fire alarms (alpha
particles), smoke detectors (alpha particles/americium-241, low
energy gamma radiation), emergency exit signs (beta particles,
tritium), tobacco (alpha particles), wireless technology, water
fountains (alpha particles, radon), lantern mantles (alpha, beta,
and gamma particles), lamp starters (beta particles, tritium,
promethium, gamma particles, thorium), static eliminators (alpha
particles/polonium-210), compasses (beta particles), batteries
(beta particles/tritium), pagers (EM radiation), generators,
purses, hats, gloves, shampoo and conditioner (EM radiation/UV
light), hair spray (EM radiation/UV light), CRT (cathode-ray tube)
monitors (x-rays), tanning bed goggles (EM radiation/UV light),
cable tv wires (EM radiation), hair dryers (infrared radiation),
pottery glaze (alpha, beta, and gamma particles), and food
packaging materials.
[0128] Energy Production, Transmission and Distribution:
[0129] Melanin nanoshells may be used in connection with, for
example, coating/composites on conductors and wiring to
reduce/avoid electromagnetic field (EMF) radiation and line current
losses, and coating/composites/inserts into electrical equipment
and electrical working tools including, but not limited to,
transformers (all sizes and types), switches, meter boxes, line
hardware, fuses, and breakers, etc.
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