WO2006124056A2

WO2006124056A2 - Melanin nanoshells for protection against radiation and electronic pulses

Info

Publication number: WO2006124056A2
Application number: PCT/US2005/035707
Authority: WO
Inventors: Ekaterina Dadachova; Arturo Casadevall
Original assignee: Albert Einstein College Of Medicine Of Yeshiva University
Priority date: 2004-10-05
Filing date: 2005-10-03
Publication date: 2006-11-23
Also published as: WO2006124056A3

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.

Description

MELANIN NANOSHELLS FOR PROTECTION AGAINST RADIATION

AND ELECTRONIC PULSES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority of U.S. Provisional Patent Application No.

60/616,056, filed October 5, 2004, the contents of which are hereby incorporated by reference into the subject application.

STATEMENT OF GOVERNMENT SUPPORT

[0002] The invention disclosed herein was made with U.S. Government support under grant number R21 AI52042 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.

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 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

[0006] The present invention is directed to nanoshells comprising melanin.

[0007] 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.

[0008] 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.

[0009] The invention further provides methods of making a material comprising melanin nanoshells, where the method comprises fabricating melanin nanoshells into or onto the material.

BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure IA- 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.

[0011] Figure 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 N₂.

[0012] Figure 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. [0013] Figure 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)).

[0014] Figure 5. Electron spin resonance spectroscopy (ESR) spectrum of melanized

Hc cells showing characteristic spectrum of melanin.

[0015] Figure 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.

[0016] Figure 7A-7B Biodistribution of 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.

DETAILED DESCRIPTION OF THE INVENTION

[0017] 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. [0018] 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.

[0019] The melanin can comprise allomelanin, plieomelanin 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.

[0020] The nanoshell can comprises a nanosphere, a nanotube, a nanoellipsoid and/or a nanorod.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 a material comprising any of the melanin nanoshells disclosed herein between the object or subject to be protected and a source of the radiation and/or electronic pulses. 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., α-radiation and β-radiation). The source of the radiation can be a medical isotope.

[0025] The object that is protected can be, for example, a computer, an electronic circuit, 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.

[0026] 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.

[0027] 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 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-l,4-naphthaquinone, 4-metholcatechol, 3,4- diliydroxynaphthalene, 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.

[0028] 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.

[0029] The material comprising the melanin nanoshells can be used, for example, as clothing, a protective gear 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

[0030] C. neoformans (Cn) and H. capsulatum (Hc). 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, MI) for 24 hrs at 30°C with constant shaking at 150 rpm. Hc was grown with shaking at 37⁰C in defined media consisting of 29.4 niM KH₂PO₄, 10 mM MgSO₄x7H₂O, 13 mM glycine, 15 mM D-glucose, 3 μM 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. [0031] Susceptibility of Cn and Hc to external gamma radiation. Approximately 10⁵ melanized or non-melanized Cn or Hc cells were placed in microcentrifuge tubes in 0.5 mL PBS and irradiated with a 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, 10³ 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.

[0032] Other sources of melanin. Melanin from cuttlefish Sepia officinalis was purchased from Sigma Chemical Co.

[0033] Measurement of radiation absorption properties of bulk melanin. A pellet of 13 mm diameter and 4 mm height with the mass of 0.71 g and density of 1.33 g/cm³ was made from Sepia melanin by applying a pressure of 6 tonn/cm . 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 α- and β-radiation was evaluated by placing the melanin pellet on the point sources of 210-Polonium and 32-Phosphorus, respectively.

[0034] Transmission electron microscopy (TEM). 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 -9O⁰C 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 IcV. [0035] Isolation and purification of melanins. 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/niL and the suspensions were incubated overnight at 30°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 CaCl₂, 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°C (Cn)I 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°C for 2 days. Approximately 1.5 x 10¹⁰ Cn cells and 2.2 x 10¹⁰ Hc cells were used. The isolation procedure yielded approximately 2.0 mg melanin per 10¹⁰ cells for Cn, and 2.3 mg per 10¹⁰ cells for Hc. [0036] Quantitative elemental analysis of melanins. Elemental analysis for carbon, hydrogen, and nitrogen was performed by Quantitative Technologies Inc. (Whitehouse, NJ). [0037] Oxidation of melanins and HPLC of oxidized melanins. Cn and Hc melanin underwent acidic permanganate oxidation using the procedure described by Ito and Fujita (16). Pyrrole-2,3,5-tricarboxylic acid (PTCA) and l,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-I C₁₈ column (250 x 4.1 mm dimensions, 7 μm 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.

[0038] MALDI mass spectrometry. 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.

[0039] Electron spin resonance spectroscopy (ESR). The ESR of purified melanins from Cn and Hc cells was performed on ER 200D EPR/ENDOR spectrometer with ESP 300 upgrade (Brucker Instruments, Inc. Billerica, Mass.).

[0040] Statistical analysis. The slopes of the survival curves were determined by linear regression (GraphPad PRISM software, San Diego, CA) 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

[0041] 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 Hc 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. IB). 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.

[0042] 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 LD₉₀ 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=O.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=O.4). For Hc cells melanin provided protection against gamma radiation up to 8,000 Gy (PO.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 O₂ (12), the effects of radiation on Cn cells were compared in air and in N₂ atmospheres. When Cn cells were irradiated directly on agar plates either in air (Fig. 2C) or in N₂ (Fig. 2D) with the doses of up to 440 Gy, less killing was observed in N₂ than in air (PO.02) for both melanized and non-melanized Cn cells in the 150-300 Gy region. In both air and N₂, melanization conferred a greater survival advantage for Cn (PO.01).

[0043] 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_oe-^μx (1)

HVL=0.693/μ (2), where I₀ and I are the radiation intensity before and after shielding, respectively; μ is the linear attenuation coefficient in cm^"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 1.4x10⁴ cm^"1 and 0.5 μm, respectively. This melanin linear attenuation coefficient is several orders of magnitude higher than that of lead (27.1 cm^"1) (13), indicating that fungal melanin in nanosphere form is a much more efficient radioprotector than lead. [0044] 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 l,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. 3 C. 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 Hc 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 Hc melanin was significantly higher - 18.6:1 (20). [0045] 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).

[0046] 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 HO₂ ^', 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. [0047] In the macroscale experiment, the 4 mm thick melanin pellet made of Sepia (bulk) melanin completely absorbed α- and β-radiation from 210-Po and 32-P sources, respectively. This is better than plastic, since to stop a β-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/cm³ melanin pellet made of Sepia melanin.

[0048] 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 -33%. Using these data, the linear attenuation coefficient (μ) for bulk melanin was calculated to be 1.01 cm^"1. For comparison, at 140 keV lead has a higher μ=27.1 cm^"1 but its density is 11.34 g/cm³; and aluminum has μ=0.386 cm^" and a density of 2.7 g/cm . 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) whep 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.

[0049] 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.

[0050] 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

[0051] 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.

[0052] 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 35⁰C 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 (niAb) 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.

[0053] 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 (¹⁸⁸Re) by incubating 16 mg of particles per sample with 40 μL SnCl₂ and Na¹⁸⁸ReO₄ for 2 hr at 37⁰C, separating the particles from unreacted Na¹⁸⁸ReO₄ 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 μL (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 3 hr later were injected IV with the above amount of 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. 7. 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.

[0054] 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

[0055] Preparation of melanin-containing plastics: To make plastics impregnated with fungal 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 rnL of diethylene glycol bis(allyl-carbonate) (CR-39) at 5O⁰C. 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 ⁰C for one day. The mixture will be heated for two additional days at 65⁰C under nitrogen, and then cured in a vacuum oven at 110⁰C for 2 h. [0056] Incorporation of melanin between two layers of material: Purified melanin naiioshells 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.

[0057] Coating surfaces with melanin: 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. [0058] Protection of subjects against radiation. 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. Table 1 Elemental composition of various melanins

Type of Melanin C/N Reference Ratio

1-dopa-melanin 6.65 16

Copolymer of 1-dopa and of 6.75 16 5-S-Cysteinyl-dopamine

5-S-Cysteinyldopa-melanin 5.02 16

Pheomelanin from 1-dopa and cysteine 4.95 16

Dopamine-melanm 7.02 16

5-S-Cysteinyldopamine-melanin 4.41 16

C. neoformans 24067 black particulate 12.5 2

Sigma melanin 7.5 2

Synthetic 1-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 ]990l conidia 14.5 20

Melanin from MNTl 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.

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Claims

What is claimed is:

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 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 nanoparticle.

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 nanoparticle.

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 nanoparticle.

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 a material comprising the melanin nanoshell of claim 1 between the object or subject to be protected and a source of the radiation and/or electronic pulses.

21. The method of claim 20, wherein the material comprising the melanin nanoshell protects the object or subject from electronic pulses. W 2

23

22. The method of claim 20, wherein the material comprising the melanin nanoshell protects the object or subject from radiation.

23. The method of claim 22, wherein the radiation comprises ionizing radiation.

24. The method of claim 22, wherein the radiation comprises one or more of gamma radiation, x-ray radiation, solar radiation, cosmic radiation, electromagnetic radiation, bremsstrahlung radiation, ultraviolet radiation, and particulate radiation.

25. The method of claim 20, wherein the object is a computer, an electronic circuit, and/or a satellite component.

26. The method of claim 20, wherein the subject is an animal, a human, and/or a plant.

27. The method of claim 20, wherein a source of the radiation is a medical isotope.

28. 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 the melanin nanoshell of claim 1.

29. The method of claim 28, wherein the organ that is protected is one or more organ selected from the group consisting of bone marrow, liver, spleen, kidneys, lungs, and gastrointestinal tract.

30. The method of claim 28, wherein bone marrow is protected.

31. The method of claim 28, which further comprises administering to the subject a copolymer of the poloxamer series.

32. The method of claim 31, wherein the co-polymer of the poloxamer series is pluronic acid F-68.

33. The method of claim 31, wherein the co-polymer of the poloxamer series is administered to the subject prior to administering the particles comprising the melanin nanoshell.

34. The method of claim 28, wherein the particles comprising the melanin nanoshell protect the subject from electronic pulses.

35. The method of claim 28, wherein the particles comprising the melanin nanoshell protect the subject from radiation.

36. The method of claim 35, wherein the radiation comprises ionizing radiation.

37. The method of claim 35, wherein the radiation comprises one or more of gamma radiation, x-ray radiation, solar radiation, cosmic radiation, electromagnetic radiation, bremsstrahlung radiation, ultraviolet radiation, and particulate radiation.

38. The method of claim 35, wherein a source of the radiation is a medical isotope.

39. The method of claim 28, wherein the particles comprising the melanin nanoshell have a diameter of about 10 mil to about 1,000 nm.

40. The method of claim 28, wherein the particles comprise silica.

41. A method of making a material comprising the melanin nanoshell of claim 1, where the method comprises fabricating melanin nanoshells into or onto the material.

42. The method of claim 41, wherein the method further comprises growing melanized fungi and extracting melanin nanoshells from the fungi.

43. The method of claim 42, wherein the fungi are grown in the presence of a melanin precursor.

44. The method of claim 43, wherein 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, A- aminocatechol, 2-hydroxyl-l,4-naphthaquinone, 4-metholcatecliol, 3,4- dihydroxynaphthalene, gallic acid, resorcinol, 2-chloroaniline, p-chloroanisole, 2- amino-p-cresol, 4,5-diliydroxynaphthalene, 1,8-dihydroxynaphthalene, 2,7-disulfonic acid, o-cresol, m-cresol, and p-cresol.

45. The method of claim 41, wherein the fungi are encapsulated in melanin nanospheres.

46. The method of claim 42, wherein the fungi are Cryptococcus neoformans (Cn) and/or Histoplasma capsulatum (Hc).

47. The method of claim 41, wherein the material is coated with melanin nanoshells and/or encased in melanin nanoshells.

48. The method of claim 41, wherein melanin nanoshells are incorporated into the material.

49. The method of claim 41, wherein the material is a plastic that is impregnated with melanin nanoshells.

50. The method of claim 41, wherein the melanin nanoshells are in a binder between two layers of material.

51. The method of claim 41, wherein the material is used as clothing, a protective gear or a packaging material.

52. The method of claim 41, wherein the material is, or is incorporated into, a wall, floor and/or ceiling of a room, building, vehicle, aircraft, ship, spacecraft, and/or submarine.