US20080213352A1

US20080213352A1 - Liposome carriers for in vivo delivery of fullerenes

Info

Publication number: US20080213352A1
Application number: US12/073,232
Authority: US
Inventors: Robert P. Lenk; Alexandru Dragos Petru Papoiu; Darren MacFarland; Stephen R. Wilson
Original assignee: Luna Innovations Inc
Current assignee: Luna Innovations Inc
Priority date: 2007-03-02
Filing date: 2008-03-03
Publication date: 2008-09-04
Also published as: WO2008109031A8; WO2008109032A2; WO2008109031A3; WO2008109031A2; US8241648B2; US20080213324A1

Abstract

Described herein are pharmaceutically acceptable compositions comprising fullerene molecules dispersed in vesicles comprising phosphatidylcholine (PC) phospholipid molecules and non-PC phospholipid molecules suspended in aqueous solution. In preferred embodiments, the phospholipid molecules are substantially uniformly organized into vesicles composed of one or more lipid bilayers and the fullerene molecules are substantially uniformly distributed within the lipid bilayers of the vesicles. Methods of forming these fullerene containing liposomes are also described. Such fullerene containing liposomes provide carriers for delivery of fullerenes for cosmetic, therapeutic, and imaging applications among other uses.

Description

RELATED APPLICATION

This application claims priority to U.S. application Ser. No. 60/904,401, filed Mar. 2, 2007, which is hereby incorporated by reference in its entirety for all purposes

FIELD

The invention relates to pharmaceutically compatible lipid vesicles comprising fullerene molecules and their use as therapeutic, diagnostic, or cosmetic compositions.

BACKGROUND

Incorporation of fullerenes into lipid vesicles has been studied. Bensasson et al. (Journal of Physical Chemistry, 98:3492-3500, 1994) described preparing vesicles incorporating C₆₀in L-α-phosphatidyl-choline purified from egg yolk (Egg-PC). However, the authors reported that they were not able to incorporate more than 3% by weight C₆₀in Egg-PC liposomes and the preparation was not uniformly reproducible. The preparation was not designed to be stable and would not have been suitable for administration as a pharmaceutical, diagnostic, cosmetic, or excipient composition.
Further, fullerenes have a tendency to form clusters. For example, Williams et al. (Recueil des Travaux des Pays-Bas, 1:72-6, 1996) incorporated C₆₀into L-α-phosphatidyl-ethanolamine from E. coli (PE). Their procedure intentionally initiated formation of C₆₀clusters, which the authors reported as essential for reproducible preparations. The authors further reported that incorporation of C₆₀in PE was limited to 7% with C₆₀adducts being limited to 3%. However, the presence of clusters is undesirable for in vivo delivery, because of the significant risk of toxicity and lack of uniformity.

SUMMARY

For fullerene containing liposome compositions to be safely and effectively utilized in therapeutic applications, there remains a need for compositions having higher stabilities that incorporate higher concentrations of fullerene molecules substantially uniformly and that minimize or prevent fullerene crystallization in vivo.
Described herein are pharmaceutically acceptable compositions comprising fullerene molecules dispersed in vesicles comprising phosphatidylcholine (PC) phospholipid molecules and non-PC phospholipid molecules suspended in aqueous solution. In preferred embodiments, the phospholipid molecules are substantially uniformly organized into vesicles composed of one or more lipid bilayers and the fullerene molecules are substantially uniformly distributed within the lipid bilayers of the vesicles. Methods of forming these fullerene containing liposomes are also described. Such fullerene containing liposomes provide carriers for delivery of fullerenes for cosmetic, therapeutic, and imaging applications among other uses.

DETAILED DESCRIPTION

Fullerene molecules comprise closed cages of 60 to 200 carbon atoms and may also include chemical moieties attached to the exterior or incorporated within the cage. In a general notation, C_mrepresents a fullerene cage having m carbon atoms and X@C_mrepresents such a fullerene cage having a chemical group X within the cage. Fullerenes such as C₆₀and C₇₀have the capacity to act like sponges soaking up free radicals, such as reactive oxygen species (ROS), that are produced during normal metabolic processes, but that can cause extensive damage if not controlled. However, the use of fullerenes as treatments of oxidative damage mediated pathologies has not been widely adopted as a therapy to date because of difficulties that arise from the inherent properties of fullerenes. In other embodiments, fullerenes, preferably endohedral metallofullerenes containing one or more caged metal atoms, can be used as contrast agents in biological imaging applications or as radiotherapeutics.
Aerobic organisms depend on harnessing chemical energy from the oxidation/reduction cycle to live and thus are continuously exposed to oxidative stress. Oxidative phosphorylation, the production of ATP, is controlled through the transfer of electrons, which generates highly reactive intermediates: “free radicals.”
“Free radicals” are molecules that have an unpaired electron that may make them highly reactive. The term “free radicals” is commonly used to refer to reactive oxygen species (“ROS”), but other molecules can also be free radicals. Reactive oxygen species include: free hydroxyl radicals (OH⁻), superoxide anions (O₂ ⁻), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), organic peroxides (R—OOH), nitric oxide (NO), and peroxynitrite (ONOO—). Certain active pharmaceutical ingredients may also comprise reactive groups.
Free radicals are potentially damaging, even lethal, if permitted to uncontrollably oxidize macromolecules such as lipids, DNA and proteins. Tissues, cells and organelles where oxidation is prevalent normally maintain significant amounts of anti-oxidant molecules to mitigate oxidative damage. Failure of this system and uncontrolled oxidation is associated with many diseases.
These reactive species can react with, cross link and damage many macromolecules. Reaction products whose presence is indicative of ROS activity, include 8-hydroxy guanosine (an oxidized component of DNA), O-tyrosine or dityrosine (components indicative of protein oxidation), and malondialdehyde (an indicator of peroxidation damage to phospholipids). These reactive biological macromolecules can attack whole families of related macromolecules and thereby affect a number of biological reactions. The consequences can include induction of cell death, mutation of DNA to cause cancer, inflammation and tissue degeneration. Several disease pathologies are caused by metabolic deregulated processes, flooding mitochondria, cells and tissues with highly reactive chemical species such as ROS.
Oxidative damage is a weapon of the immune system in fighting foreign pathogens. Neutrophils and other white blood cells contain specialized lipid vesicles, called peroxisomes, filled with activated oxygen species whose purpose is to damage and/or kill invading organisms. Under the proper set of circumstances, white blood cells self-destruct, spilling a local abundance of free radicals to overwhelm pathogens. Under normal control, these are effective tools for protection, but overaggressive deployment can overwhelm the modulating systems and provoke serious damage that can even be fatal to the host.
The immune system is one example of how loss of control of oxidative processes can have life threatening consequences but there are many others. Accumulated oxidative damage caused by smoking, chemical vapors or radiation therapy can induce destruction of the cells that produce elastic tissue in the lungs, replacing them with inelastic fibroblasts in a debilitating condition known as pulmonary fibrosis.
Degenerative diseases of neural tissue can be initiated by oxidative damage. (Beal, M. F., Lang, A. E. & Ludolph, A. “Neurodegenerative Diseases” (Cambridge University Press 2005)). The brain must process large amounts of oxygen. 20% of the oxygen we breathe is consumed in this organ, because of the level of energy produced by oxidation, there is a serious hazard for spontaneous oxidative damage. Amyotrophic lateral sclerosis (ALS) is a fatal disease in which motor neurons of the cortex, brain stem and spinal cord degenerate. The cause of ALS is unknown, but about 10% of cases are inherited. Familial ALS has been traced to a genetic defect in superoxide dismutase genes (SOD 1), implicating the central role of the free radical superoxide anion in this devastating neurodegenerative disease.
Parkinson's disease results from depletion of neurons in the substantia nigra of the brain that produce the neurotransmitter dopamine. Animals treated with MPTP (methyl phenyl tetrahydropyridine) develop a disease that closely resembles Parkinsonism. Close study of this phenomenon shows that MPTP is metabolized to a compound that concentrates in mitochondria of neurons in the substantia nigra where it interferes with oxidative phosphorylation and provokes excessive free radical release and an increase in superoxides which results in apoptosis of the neurons. (Beal, M. F., Ann. N. Y Acad. Sci., 991:120-31, 2003).
Alzheimer's disease is characterized by progressive cognitive and memory loss associated with deposits of inert plaque and neurofibrils which replace dead cells in the hippocampus, amygdala, and cerebral cortex. Amyloid-β peptide, which is thought to be the agent responsible for neurotoxicity has been shown recently to associate with heme groups to form peroxidases in mitochondria. (Atamna, H. & Boyle, K., Proc. Nat. Acad. Sci., 103:3381-86, 2006). There are many converging lines of evidence to suggest that mitochondria in nerve cells, where the bulk of the oxidative activity takes place, are a target for new therapies in several neurodegenerative diseases.
Diabetes mellitus is caused by dysfunction of the regulatory system that manages blood sugar levels. When the pancreas is functioning normally the level of insulin in the blood is titrated with great precision. Some people lose the ability to produce insulin due to the death of the specialized cells in the pancreas that synthesize the hormone in a disease known as Type 1 diabetes, which used to be called juvenile onset diabetes. The most common form of diabetes, Type 2, results from insensitivity to insulin, either because the cells lose their insulin receptors or their receptors fail to function properly.
Hyperglycemia can cause repeated oxidative stresses, especially on endothelial cells, particularly those in capillaries of the retina, mesangial cells in the kidney and neurons. Oxidative damage accumulated over time can cause a number of pathologies, including damage to the retina that often leads to blindness, heart disease, damage to peripheral nerves, kidney failure and loss of circulation in the extremities. Of particular concern is the deterioration of endothelial cells that causes weakening of the blood vessels and cardiovascular disease. Most, if not all of these side effects can be attributed to oxidative damage to the cells. When blood glucose levels are high many cell types are programmed to load themselves up with sugar, which is fuel for their oxidation/reduction furnaces. If the internal concentration is excessive it stresses the control system and causes damage occasionally. The accumulated effect over many decades of insults leads to the deterioration of subject tissues.
A problem that has impeded the use of fullerenes as neutralizers of ROS and other free radicals in vivo has been the difficulty of stable, safe and effective carrier compositions. Fullerenes are insoluble in many common organic solvents and are not soluble in aqueous solution. Thus, it would not be desirable to inject unmodified fullerenes directly into living organisms. Chemical modifications to the fullerene that add charged and/or polar groups can increase aqueous solubility. However, even with chemical modification, these fullerenes may aggregate into complexes large enough to block capillaries. Addition of multiple polar groups to each fullerene may solve the aqueous solubility problem, but can introduce new complexities. For example, because there are multiple sites on the fullerene to which reactive polar groups can bind, the reaction products are typically mixed isomers, which may be undesirable for pharmaceutical formulations. The addition groups can be subject to metabolism and decomposition, producing intermediates that may cause toxicity. Furthermore, each addition of a functional group reduces the number of available reactive sites available for neutralizing reactive molecules in vivo.
It has now been discovered that spheres formed of phospholipid molecules, for example spheres made up of one or more lamella of phospholipid molecules organized into bilayer structures can be made comprising a substantially uniform distribution of fullerene molecules. Such compositions possess characteristics that make them suitable for use as pharmaceuticals, and can be reproducibly and stably manufactured. Useful fullerenes are preferably C₆₀or C₇₀or fullerene adducts, but also include fullerene molecules having between about 60 to 200 carbon atoms in the fullerene sphere, preferably between about 60 and 92 carbon atoms, and adducts thereof. Adducts may be desirable to achieve disposition at the target where the fullerene pharmaceutical activity is most effective. Adducts may also be desirable to optimize the fullerene activity, such as by tuning the electron affinity of the fullerene such that it reacts with undesirable free radicals yet does not interfere with vital electron transfer. Other fullerenes that may be used include endohedral metallofullerenes with the formula Z_i@C_m, where Z is a metallic element and i=1 or 2 and m is between about 60 and about 200. A preferred embodiment for contrast agents is a tri-metallic nitride endohedral fullerene having a general formula A_3−nX_nN@C_mwhere n ranges from 0 to 3, A and X may be trivalent metals and may be either rare earth metal or group IIIB metals, m is between about 60 and about 200. It can also be advantageous to form adducts C_m(R_q), Z_i@C_m(R_q), or A_3−nX_n@C_m(R_q), where R_qrepresents one or more substituents preferably comprising organic group(s). Functional groups may provide higher and more uniform incorporation into liposomes, targeting to tissue or cellular compartments, or other functionalities.
Such compositions can be used wherever it is desirable to neutralize free radicals and ROS, for example in vivo, topically, as an ingredient in a cosmetic composition, or where delivery of metallofullerenes for therapeutic or imaging applications (e.g., in diagnostic or experimental imaging). Fullerenes can be incorporated into liposomes that contain, in addition, molecules that have biological activity, e.g., in combination therapy or combined with agents that affect the biodistribution of the liposomes to target specific tissues or cellular compartments. Liposomes incorporating fullerene molecules may also be used to encapsulate highly reactive molecules so that the reactive molecule is kept passive until the liposome reaches its target and the contents are separated from the liposome.
Phospholipids are molecules having a glycerol backbone to which two hydrophobic fatty acids and a hydrophilic polar head group are esterified. In the presence of aqueous solutions these phospholipids self-organize such that the hydrophobic fatty acid tails mix with each other and the polar head groups face the aqueous solvent. When organized in this manner into a configuration having two hydrophilic faces and a hydrophobic interior, the structure is known as a bimolecular layer, or bilayer. Various phospholipid-like molecules (such as synthetic molecules) may also be used that use non-ester linkages.
Such bilayers mimic the configuration of lipids in cell membranes. Spheres made of lipid bilayers may be called vesicles or liposomes. Liposomes (lipid vesicles) are formed when thin lipid films or lipid cakes are hydrated and stacks of liquid crystalline bilayers become fluid and swell. The hydrated lipid sheets detach during agitation and self-close to form large, multilamellar vesicles (MLV) which prevents interaction of water with the hydrocarbon core of the bilayer at the edges. Once these particles have formed, reducing the size of the particle requires energy, for example sonic energy (sonication) or mechanical energy (extrusion).
Previous attempts to incorporate fullerene molecules in liposomes have not produced satisfactory results for use in therapeutic compositions. Being hydrophobic, fullerene molecules may be expected to associate with the alkyl chains of fatty acid groups in lipids when presented to phospholipids. However, fullerene molecules are mobile within the bilayers and can migrate and bind to other fullerene molecules until the aggregate is sufficiently large to form a precipitate that separates from the liposomes.
Some phospholipid mixtures have been discovered to be unacceptable. For example, liposomes prepared with the saturated phospholipid dimyristoyl phosphatidyl choline alone or those prepared with dimyristoyl phosphatidyl glycerol alone did not produce liposomes with a uniform distribution of C₆₀through the bilayer. Rather such preparations produce aggregates of liposomes intertwined with large brown crystals of fullerenes. It appeared that the fullerenes and the phospholipids largely separated from each other during or shortly after exposure to aqueous solution. This is in agreement with previous attempts to incorporate C₆₀into liposomes for research purposes, which found that C₆₀was limited to 3% by weight in PC and 7% by weight in PE liposomes, and found C₆₀aggregating in the bilayers of these preparations.
However, it has been surprisingly discovered that liposomes prepared principally comprising phosphatidylcholine (PC) in combination with a non-PC phospholipid such as phosphatidylethanolamine (PE) were capable of incorporating significantly higher concentrations of C₆₀without the appearance of C₆₀crystals, appearing in the microscope to be substantially uniform, characterized by a yellow-green tinting of the liposomes that indicated that C₆₀, was more or less uniformly distributed through most of the liposomes. It has also been discovered that the addition of an adduct that facilitates dispersion of the fullerene within the lipid bilayer, for example cholesterol or dodecylamine can enhance the ability of fullerenes to incorporate into liposomes and improve the performance. Combinations of different tethers and adducts can be used to manipulate where in the lipid portion of the bilayer the fullerene is positioned, which may be useful in optimizing its performance as a therapeutic.
“Principally comprising” means that the majority or plurality component of a mixture is the principal component and is intended to account for the intentional and unintentional inclusion of other minor components. “Substantially pure” means that a composition contains only the named components and such other components as may be unintentionally present as byproducts of production, due to imperfect purification, chemical reaction or degradation of named components, or other processes. No recitation of any composition in this document is meant to be interpreted as perfectly pure; rather composition purities are understood to correspond to what would be reasonably expected by practitioners in the field and tolerated for the intended use of a composition.
Accordingly, liposomes comprising fullerene molecules for pharmaceutical administration preferentially comprise a mixture of PC and non-PC phospholipids. In further preferred embodiments, liposomes include anionic phospholipids present in sufficient concentration to minimize liposome aggregation. In some compositions, the liposomes may comprise additional components that can affect the stability and physical characteristics of the lipids, for example fatty acid molecules; cholesterol, other lipids or lipid-like molecules including triglycerides, amphipathic molecules (e.g., surfactants and bile acids), and the like, or components introduced to target the liposomes to a particular site in the organism or to a cellular compartment, for example antibody or antigen fragments derivatized to lipophilic chains, glycolipids, and the like.
Lipids which can be employed in making liposome formulations comprising fullerenes include lipids having head groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM), and the like, preferably in combinations comprising PC as a principle component. The phospholipids can be synthetic or derived from natural sources such as egg or soy. Saturated phospholipids such as hydrogenated soy PC may also be used. In the preferred embodiments, the phospholipids are mixtures of natural extracts of PC and PE or synthetic mixtures such as dimyristoyl PC (DMPC) and dimyristoyl PE (DMPE), used in combination in any mole ratio, from 99:1 to 1:99 PC:PE. Lipids principally having myristoyl chains may be chosen because the Tc of di-myristoyl lipids falls in a preferred range below or near physiological temperatures depending on the head group and the presence of other components. Fullerene containing liposomes preferably comprise PC mixed with sufficient non-PC lipid such as PE to make a 19:1mole ratio, or about a 9:1 or 7:3 mole ratio, up to about 2:8 or 1:9 mole ratio of PC to non-PC lipids. PG, PS, PI, SPM and PA may also be used in combination with a lipid mixture comprising principally PC and PE in amounts sufficient to confer a charge distribution to the lipid vesicles.
The liposomes can also contain a steroid component as part of the lipid phase, such steroids may be cholesterol, coprostanol, cholestanol, cholestane, steroids derived from plants, protists or fungi, such as ergosterol, steroid derivatives, such as polyethylene glycol derivatives of cholesterol (PEG-cholesterols), organic acid derivatives of sterols such as cholesterol hemisuccinate (CHS), desoxycholate, and the like. Further components of suitable mixtures can include fatty acids such as myristic acid, isopropyl myristate, isostearic acid, sucrose disrestate, propylene glycol monostearate, and cetylated monoglyceride. Other substances that can be employed include lipids such as trimyristin, the fatty alcohols such as cetyl alcohol and myristyl alcohol, and fatty esters such as myristic acid ethyl ester. The addition of surfactants such as those commonly used in the drug and cosmetic composition formulations can also enhance the uptake and uniform distribution of fullerenes in liposomes.
Organic acid derivatives of tocopherols may also be used as liposome-forming ingredients, such as alpha-tocopherol hemisuccinate (THS). Both CHS- and THS-containing complexes and their tris salt forms may generally be prepared by any method known in the art for preparing liposomes containing these sterols.
Lipids from biological sources (e.g., egg, porcine, bovine, or soybean) typically contain significant levels of polyunsaturated fatty acids and therefore are inherently more susceptible to oxidation than saturated synthetic lipids. While saturated lipids offer greater stability in terms of oxidation, they also have much higher transition temperatures and thus present other difficulties in formulation. It has been found that the presence of unsaturated lipids may enhance fullerene incorporation and stability. Monounsaturated lipids are preferred, for example lipids containing oleic acid (18:1, cisΔ9) provide unsaturation, and can be much more stable than polyunsaturated compounds. Cis unsaturated bonds impart different properties to lipid acyl chains, typically increasing the local disorder in acyl chain packing, which can lower phase transition temperatures and energetically favor the accommodation of heterogeneous molecules among the acyl chains. Hydrogenated lipids have trans unsaturated bonds.
Fullerene containing liposomes or lipid complexes for use as therapeutic agents will preferably stably incorporate at least about 5% by weight of fullerene molecules out of the total weight of the fullerene and lipid molecules, more preferably at least about 10% and most preferably about 15% to 30% fullerene molecules by weight, but may be up to about 50% or 60% fullerene molecules by weight. In terms of molar ratios, the molecular weights of fullerenes such as C₆₀(MW 720) are comparable to the molecular weights of typical lipids such as DMPC (MW 670), POPC (MW 760), DMPE (MW 635) and POPE (MW 718). Thus, 5% by weight of fullerenes can correspond to about a 1:19 molar ratio, depending on the mixture. Accordingly, fullerene containing liposomes or lipid complexes for use as therapeutic agents will preferably stably incorporate at least about 1:19 molar ratio of fullerene to lipid, more preferably at least about 1:9, and most preferably about 6:1 or 4:1 to about 2:3, but may be up to about 1:1 or 2:1.
Stability issues due to hydrolytic degradation is a general problem with lipid products. However, it has been discovered that the presence of fullerene molecules in liposomes can provide a buffer against oxidation and degradation of the lipids. It has also been found that the addition of adducts to fullerenes can provide stability benefits. For example, the presence of a cholesterol group attached to a fullerene can stabilize the lipid membranes to hydrolysis by reducing the extent of water permeation into the lipid bilayers This ability of fullerenes, or fullerene adducts to stabilize the liposomes can also be advantageous for use where the liposomes include an additional therapeutic agent which is subject to oxidative damage. These fullerene or fullerene adduct stabilized liposome formulations enable delivery of active pharmaceutical ingredients with readily oxidized or reduced functional groups.
A procedure for preparing liposomes generally comprises preparation of the lipid for hydration, hydration with agitation, and sizing to a homogeneous distribution of vesicles. When preparing liposomes with a mixed lipid composition, the lipids can be first dissolved and mixed in an organic solvent to assure a homogeneous mixture of lipids. This process is carried out using chloroform or chloroform:methanol mixtures, toluene, tertiary butanol or cyclohexane, dichloromethane, or other suitable solvents with chloroform being most common. Typically, lipid solutions are prepared at 10-20 mg lipid/ml organic solvent, although higher concentrations may be used if the lipid solubility and mixing are acceptable. A pre-selected amount of fullerene, such as C₆₀, to be incorporated into the lipid bilayers of liposomes in a suitable solvent can be mixed with the lipids at this point.
Once the liposome components are thoroughly mixed in the organic solvent, the solvent can be removed to yield a lipid film. For small volumes of organic solvent (<1 mL), the solvent may be evaporated using a dry nitrogen or argon stream in a fume hood. For larger volumes, the organic solvent can be removed by rotary evaporation yielding a thin lipid film on the sides of a round bottom flask. The lipid film can be thoroughly dried to remove residual organic solvent by placing the vial or flask under vacuum for about 8-12 hours.
To remove less volatile solvents, the lipid solution can be transferred to containers and frozen, for example by swirling the container in a dry ice-acetone or alcohol (ethanol or methanol) bath. After freezing completely, the frozen lipid cake is placed under vacuum and lyophilized until dry (which may take 1-3 days depending on volume). Dry lipid films or cakes can be removed from vacuum and stored frozen and sealed until ready to hydrate.
Hydration of the dry lipid film/cake can be accomplished simply by adding an aqueous medium to the container of dry lipid and agitating. The temperature of the hydrating medium is preferably above the gel-liquid crystal transition temperature (Tc) of the lipid with the highest Tc before adding to the dry lipid. After addition of the hydrating medium, the lipid suspension is preferably maintained above the Tc during the hydration period. A hydration time of at least about 1 hour with vigorous shaking, mixing, or stirring is preferable, although the appropriate time can depend on the composition. Allowing the suspension to stand for about 8-12 hours prior to sizing the liposomes may makes the sizing process easier and improve the homogeneity of the size distribution.
Suitable hydration media include pharmaceutically acceptable aqueous buffers or distilled water and may include nonelectrolytes such as sugar solutions. Physiological osmolality (290 mOsm/kg) is recommended for in vivo applications, examples include 0.9% saline, 5% dextrose, and 10% sucrose solutions. Additional pharmaceutically acceptable solutions and components commonly used in pharmaceutical formulations are well known to the skilled practitioner. See, for example, “Remington: The Science and Practice of Pharmacy,” (Lippincott Williams & Wilkins; 21st edition, 2005).
During hydration some lipids form complexes unique to their structure. Highly charged lipids have been observed to form a viscous gel when hydrated with low ionic strength solutions. The problem can be alleviated by addition of salt or by downsizing the lipid suspension. Poorly hydrating lipids such as phosphatidylethanolamine present in molar ratios greater than 60% have a tendency to self aggregate upon hydration.
The product of hydration in this manner is a large, multilamellar vesicle (MLV) analogous in structure to an onion, with each lipid bilayer separated by a water layer. The spacing between lipid layers is dictated by composition with poly hydrating layers being closer together than highly charged layers which separate based on electrostatic repulsion. Once a stable, hydrated MLV suspension has been produced, the particles can be downsized by a variety of techniques, including sonication or extrusion.
Disruption of MLV suspensions using sonication typically produces small, unilamellar vesicles (SUV) with diameters in the range of 15-50 nm. Bath sonicators can be used for preparation of SUV. Sonication of an MLV dispersion can be accomplished in a bath sonicator by placing a container of the suspension in the bath sonicator for 5-10 minutes at a temperature above the gel-to liquid crystal phase transition temperature (Tc) of the lipid.
Under sonication, the MLV suspension will begin to clarify to yield a slightly hazy transparent solution. The haze is due to light scattering induced by residual large particles remaining in the suspension. These particles can be removed by centrifugation, filtration, or other means to yield a clear suspension of SUV. Mean size and distribution is influenced by composition and concentration, temperature, sonication time and power, volume, and sonicator tuning. SUV are inherently unstable and will spontaneously fuse to form larger vesicles when stored below their phase transition temperature.
Lipid extrusion is a technique in which a lipid MLV suspension is forced through a polycarbonate filter with a defined pore size to yield particles having a diameter near the pore size of the filter used. Prior to extrusion through the final pore size, MLV suspensions can be disrupted either by several freeze-thaw cycles or by prefiltering the suspension through a larger pore size (typically 0.2 μm-1.0 μm). This method helps prevent the membranes from fouling and improves the homogeneity of the size distribution of the final suspension. As with all procedures for sizing MLV dispersions, the extrusion is preferably done at a temperature above the Tc of the lipid. Attempts to extrude below the Tc will be unsuccessful as the membrane has a tendency to foul with rigid membranes which cannot pass through the pores. Extrusion through filters with 100 nm pores typically yields large unilamellar vesicles (LUV) with a mean diameter of 120-140 nm. Mean particle size also depends on lipid composition and is quite reproducible from batch to batch.
Analogous to extrusion through a filter, a mixture of MLV can be reduced to a uniform size of liposomes by passing one or more times through a small nozzle. As an example, the mixture of hydrated liposomes may be passed through a milk homogenizing system adapted to produce uniformly sized small vesicles.
Various other techniques for introducing phospholipids to water have been utilized, including ethanol injection, detergent dialysis and reverse phase evaporation. (Szoka, F. & Papahadjopoulos, D., Proc. Nat. Acad. Sci., 75:4194-4198, 1978). The latter method was originally developed to make large unilamellar vesicles, but was subsequently adapted for making other types of liposomes and lipid particles. (Gruner, S., Lenk, R., Janoff, A. & Ostro, M. Biochemistry, 24:2833, 1985; U.S. Pat. No. 5,616,334).
The reverse phase method comprises providing two immiscible liquids, water and an organic solvent. The organic phase is initially present in excess, has a relatively low boiling point, is volatile and is one in which phospholipids are highly soluble. The aqueous phase contains solutes such as salts to establish osmolarity, buffer to control pH and can include water soluble drugs. The solvent mixture is dispersed by ultrasonic vibration using a bath sonicator and the organic solvent is evaporated either by vacuum or sparging with an inert gas such as N₂. At the solvent:water interface the phospholipids form into bilayers, although those far from the interface arrange randomly. As the volume of organic solvent reduces the mixture becomes frothy, as the surface tension of the lipids at the interface stabilizes the emulsion. When the mixture switches from a water in solvent emulsion to a solvent in water emulsion the liposomes begin forming. Ultrasonic dispersion energy maximizes the interface and thorough mixing of water solutes is the result.
A preferred method for preparing fullerene liposomes is a reverse-phase method. The reverse phase evaporation method is based on creating, first, a water in oil phase system which is then converted into an oil in water system. Surprisingly, fullerene molecules remain associated with the lipids during the transition from a water in oil system to an oil in water system.
Phospholipids are typically identified by the trivial names of the acyl chains and the trivial name of the head group. The most common phospholipids are frequently abbreviated by four letter acronyms, di-myristoyl phosphatidyl choline may be abbreviated DMPC, while palmitoyl oleoyl phophatidyl ethanolamine may be abbreviated POPE. The trivial names of several fatty acid moieties are given below together with the IUPAC names of the unsaturated fatty acids moieties, which explicitly name the location and type of double bonds in the acyl chain.


Saturated Acyl Chain	Trivial Name

3:0	Propionoyl
4:0	Butanoyl
5:0	Pentanoyl
6:0	Caproyl
7:0	Heptanoyl
8:0	Capryloyl
9:0	Nonanoyl
10:0	Capryl
11:0	Undecanoyl
12:0	Lauroyl
13:0	Tridecanoyl
14:0	Myristoyl
15:0	Pentadecanoyl
16:0	Palmitoyl
16:0 [(CH₃)₄]	Phytanoyl
17:0	Heptadecanoyl
18:0	Stearoyl
19:0	Nonadecanoyl
20:0	Arachidoyl


Unsaturated Acyl
Chain
Carbons:alkenes	Trivial Name	IUPAC Name

14:1	Myristoleoyl	9-cis-tetradecenoic
14:1	Myristelaidoyl	9-trans-tetradecenoic
16:1	Palmitoleoyl	9-cis-hexadecenoic
16:1	Palmitelaidoyl	9-trans-hexadecenoic
18:1	Petroselinoyl	6-cis-octadecenoic
18:1	Oleoyl	9-cis-octadecenoic
18:1	Elaidoyl	9-trans-octadecenoic
18:2	Linoleoyl	9-cis-12-cis-
		octadecadienoic
18:3	Linolenoyl	9-cis-12-cis-15-
		cisoctadecatrienoic
20:1	Eicosenoyl	11-cis-eicosenoic
20:4	Arachidonoyl	5,8,11,14(all-cis)
		eicosatetraenoic
22:1	Erucoyl	13-cis-docosenoic
22:6	DHA	4,7,10,13,16,19 (all -cis)
		docosahexaenoic
24:1	Nervonoyl	15-cis-tetracosenoic

Liposomes may be used in multilamellar vesicle form (MLV), large unilamellar vesicle form (LUV) having between about 500 nm and 50 nm diameters, preferably about 100 nm diameters, or as small unilamellar vesicles (SUV) having diameters of about 50 nm or less. 100 nm LUVs are desirable for a number of reasons. The distribution of C₆₀is more homogeneous compared to a preparation containing MLVs. This size liposome can persist in circulation for longer because it will avoid uptake by the reticuloendothelial system, and 100 nm vesicles can escape the vasculature to reach interstitial space. Generally, preferred fullerene containing liposomes for pharmaceutical use will be principally in the range of 0.1 to 0.5 microns, with compositions comprising liposomes principally in the range 0.1 to 0.3 microns being more preferred.
Liposomes may be frozen or dried, for example by lyophilization to increase stability for longer term storage. Lipid preparations can be stabilized for freezing and drying by including carbohydrates in the composition. Stabilizing agents may be included in a fullerene containing liposome composition. Suitable agents include one or more sugars selected from the group consisting of glucose, sucrose, maltose, lactose, galactose, trehalose, and raffinose.
Many biological membranes carry a net negative charge on their surface. The charge is generally imparted by the presence of anionic phospholipid species in the membrane. The major naturally occurring anionic phospholipids are phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Some bacterial systems also contain phosphatidylglycerol. Having a charged surface can minimize the extent of liposome aggregation and model native membranes. Native brain tissue extracts can be modeled by a blend of unsaturated synthetic lipids (e.g., dioleoyl) in a ratio of about 5:3:2 (wt %), PE:PS:PC. This models the general phospholipid composition of most brain tissues.
Cholesterol is a membrane constituent widely found in biological systems which serves a unique purpose of modulating membrane fluidity, elasticity, and permeability. Cholesterol can fill in gaps created by imperfect packing of other lipid species when non-lipid molecules are embedded in the membrane.
The phase transition temperature Tc of a lipid is defined as the temperature required to induce a change in the lipid physical state from the ordered gel phase, where the hydrocarbon chains are fully extended and closely packed, to the disordered liquid crystalline phase, where the hydrocarbon chains are randomly oriented and fluid. There are several factors which directly affect the phase transition temperature including hydrocarbon length, unsaturation, charge, and headgroup species. As the hydrocarbon length is increased, van der Waals interactions become stronger requiring more energy to disrupt the ordered packing, thus the phase transition temperature increases. Likewise, introducing cis a double bond into the acyl group puts a kink in the chain which requires much lower temperatures to induce an ordered packing arrangement. Packing of headgroups can greatly influence Tc. The transition temperature of DMPE is 50° C. while the transition temperature of DMPC is 23° C. The PC head group is sterically larger than the PE head group.


		Transition
	Carbons:	Temperature	Net Charge
Lipids	Unsaturation	Tc (° C.)	at pH 7.4

DLPC	12:0	−1	0
DMPC	14:0	23	0
DPPC	16:0	41	0
DSPC	18:0	55	0
DOPC	18:1	−20	0
DMPE	14:0	50	0
DPPE	16:0	63	0
DOPE	18:1	−16	0
DMPA.Na	14:0	50	−1.3
DPPA.Na	16:0	67	−1.3
DOPA.Na	18:1	−8	−1.3
DMPG.Na	14:0	23	−1
DPPG.Na	16:0	41	−1
DOPG.Na	18:1	−18	−1
DMPS.Na	14:0	35	−1
DPPS.Na	16:0	54	−1
DOPS.Na	18:1	−11	−1

Phospholipids are not uniformly miscible with each other in binary mixtures. Differences in chain length and chain flexibility (such as resulting from cis-unsaturated bonds) can create voids in the lipid bilayer interior that are energetically unfavorable, resulting in segregation of lipid types and flat phase transition temperature curves. Mixtures of lipids with different head groups but similar chain compositions tend to be more miscible. The presence of fullerene molecules in the interior of the bilayer can alter the usual behavior by occupying voids, so that combinations of lipids that are not normally miscible may be surprisingly capable of incorporating and uniformly distributing fullerenes. Neutral phospholipid head groups such as phosphatidyl choline and phosphatidyl ethanolamine are also strongly dipolar. These head groups possess an intrinsic charge separation with a negative charge appearing at the phosphate group and a positive charge centered at the nitrogen on the exposed end of the head group. The head groups typically do not lay horizontal in the plane of the lipid bilayer, but are rather tilted with the positive ends directed somewhat towards the water exposed surfaces. The degree of tilt, and the electrostatic profile can be influenced by the composition of the head-group region of the bilayer. This, combined with the local structure of water molecules, the glycerol backbone of the lipid and other structural factors results in an electrostatic charge profile in which there is a significant electrostatic voltage drop across the polar aqueous interface of the bilayer extending a few carbons into the non-polar acyl chain interior of the bilayer.
In addition, atoms in the lipid bilayer are more or less rigidly ordered as a function of position in the bilayer depending on several factors. Other things being equal, at temperatures above the liquid-crystal phase temperature, the rigidity of the atomic packing is highest among carbon atoms of the acyl chains near the level of the glycerol backbone. Closer to the ends of the acyl chains at the center of a bilayers, the carbon atoms are much less rigidly packed, approximating a fluid oil. At the polar aqueous interface of the bilayer, the degree of order is affected by how well the acyl chains are able to pack as well as the uniformity of the head groups and the degree of hydration associated with the head-groups that comprise the aqueous interface region.
Molecules that are incorporated into the lipid bilayer can interact with and be affected by the electrostatic profile and order profile of a lipid bilayer. C₆₀is a symmetric molecule about 1 nm in diameter. The size and shape of fullerenes suggest a preference for less rigid, more fluid potions of the lipid bilayer. The use of mono-unsaturated lipids in liposomes can increase the relative degree of disorder at positions in the bilayer closer to the aqueous interface thereby potentially increasing the capacity of a lipid bilayer to accommodate fullerenes.
The symmetry of an un-derivatized fullerene suggests the lack of an intrinsic electrostatic dipole. This intuitively suggests a preference for the least polar central regions of a bilayer. However, the network of resonance structures that comprise a fullerene molecular structure can provide for substantial transient polarization. This transient polarization allows the fullerenes to be incorporated nearer to the aqueous interface region than similarly sized and shaped non-polarizable molecules. This surprising result is beneficial for a variety of applications such as MRI contrast agents and therapeutics. Furthermore, asymmetrically derivatized fullerenes can possess an intrinsic dipole that makes localization nearer to the aqueous interface of a bilayer even more favorable. The practitioner may consider these and other factors in preparing an optimal composition for a specific fullerene and an intended application.
“Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease or condition, is sufficient to effect a desirable treatment for the disease or condition. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated. A “therapeutically effective amount” need not result in a complete cure, but may provide partial relief of one or more symptoms or retard the progression of a disease or condition.
Generally, the capacity of fullerene molecules to neutralize reactive molecules, such a free radicals and reactive oxygen species, means that compositions comprising fullerene molecules can be used to treat diseases and conditions in which reactive species and/or oxidative damage causes tissue damage. When incorporated into an effective delivery system, non-water soluble fullerene compositions may be used in such therapeutic applications. In particular, the fullerene containing liposome compositions may be used in methods to treat diseases or conditions associated with oxidative damage. Conditions include oxidative damage to skin, which may be treated by topically administering a therapeutically effective amount of a composition comprising non-water soluble fullerene, for example liposomes comprising fullerene molecules, to the affected tissue. Alternatively, a composition comprising fullerene containing liposomes may be administered by any appropriate route to deliver the composition to tissues affected by oxidative damage or damage caused by reactive molecules.
Thus, formulations of non-water soluble fullerenes (whether derivatized or not) such as the liposome containing fullerene formulations described herein may be used in a method of treatment of diseases caused by free radicals comprising administering a composition comprising non water soluble fullerenes or fullerene derivatives to an individual so as to neutralize free radicals within cell membranes. The non water soluble fullerenes can be modified such that they neutralize very highly reactive radicals but do not interfere with less reactive radicals, for example by derivatizing the fullerene to reduce the affinity particular classes of reactive molecules. The fullerenes may be derivatized with one or more lipophilic groups to enhance their ability to stably associate with lipid membranes. Liposomes comprising fullerenes may incorporate an amphiphilic moiety that enhances their delivery to a specific target tissue, for example the fullerene bearing liposomes may be targeted to endothelial cells, thrombi, or inflammatory cells.
A fullerene containing liposome composition may be administered in vivo by any suitable route including but not limited to: inoculation or injection (e.g., intravenous, intra-peritoneal, intramuscular, subcutaneous, intra-aural, intra-articular, intra-mammary, and the like). A fullerene containing liposome composition may be incorporated into a broad range of materials including but not limited to gels, oils, emulsions and the like for administration as a topical therapeutic or incorporated into cosmetic formulations to provide an antioxidant capacity to the cosmetic formulation. Such uses include topical application (e.g., on areas such as eyes, skin, in ears or on afflictions such as wounds and bums), and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like).
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. The following examples are illustrative of the preparation and analysis of fullerene containing liposome compositions including favorable and comparative unfavorable results and should not be considered as limiting of the foregoing disclosure in any way.

EXAMPLES

Example 1

Phosphatidyl choline purified from eggs was added to toluene. To this solution C₆₀was added such that the molar ratio of lipids to C₆₀was about 86:1. The mixture was dried in a rotary evaporator to form a thin film. To this film, 5 cc of phosphate buffered saline was added and the solution swirled over the lipid film to slough off the lipids from the round bottom flask. The flask was exposed to a bath type sonicator for 5 sec to facilitate sloughing of the film. The resultant cloudy suspension contained flocculent material visible to the eye as well as a cloudy suspension of a yellow green hue. An aliquot was exposed to 18,000 G for 20 min to collect the denser material in a pellet. A second aliquot was layered on top of a 20% sucrose cushion and then exposed to 18,000 G for 20 min. In the first tube most of the visible material collected in a pellet, although a small amount of cloudy tinted material remained in solution. In the second tube a very small amount of brown material was dense enough to form a pellet, while the majority of the sample formed a cushion buoyed on top of the sucrose solution. Since liposomes have a buoyant density less than that of the sucrose solution only material that is denser, that is, crystalline C₆₀, collected in the bottom of the tube.

Example 2

Phosphatidyl choline purified from eggs and phosphatidyl ethanolamine purified from bovine brain were added together in toluene at a molar ratio of 5:1. To this solution C₆₀was added such that the molar ratio of lipids to C₆₀was 86:1 or 43:1. The mixture was dried in a rotary evaporator to form a thin film. To this film, 5 cc of phosphate buffered saline was added and the solution swirled over the lipid film to slough off the lipids from the round bottom flask. The flask was exposed to a bath type sonicator for 5 sec to facilitate sloughing of the film. The resultant cloudy suspension contained flocculent material visible to the eye as well as a cloudy suspension of a yellow green hue. An aliquot was exposed to 18,000 G for 20 min to collect the denser material in a pellet. A second aliquot was layered on top of a 20% sucrose cushion and then exposed to 18,000 G for 20 min. In the first tube most of the visible material collected in a pellet, although a small amount of cloudy tinted material remained in solution. In the second tube there was no brown material visible to form a pellet, while the majority of the sample formed a cushion buoyed on top of the sucrose solution. A small amount of material, presumably vesicles with one or a few lamellae, remained in the solution atop the sucrose cushion. Since liposomes have a buoyant density less than that of the sucrose solution only material that is denser, that is, crystalline C₆₀, collected in the bottom of the tube. The fact that no brown pellet was observed indicates the C₆₀was all associated with the liposomes.

Example 3

Phosphatidyl choline purified from eggs and phosphatidyl ethanolamine purified from bovine brain were added together in toluene at a molar ratio of 5:1. To this solution C₆₀was added such that the molar ratio of lipids to C₆₀was 21:1. The mixture was dried in a rotary evaporator to form a thin film. To this film, 5 cc of phosphate buffered saline was added and the solution swirled over the lipid film to slough off the lipids from the round bottom flask. The flask was exposed to a bath type sonicator for 5 sec to facilitate sloughing of the film. The resultant cloudy suspension contained flocculant material visible to the eye as well as a cloudy suspension of a yellow green hue. An aliquot was exposed to 18,000 G for 20 min to collect the denser material in a pellet. A second aliquot was layered on top of a 20% sucrose cushion and then exposed to 18,000 G for 20 min. In the first tube most of the visible material collected in a pellet, although a small amount of cloudy tinted material remained in solution. In the second tube there was no brown material visible to form a pellet, while the majority of the sample formed a cushion buoyed on top of the sucrose solution. A small amount of material, presumably unilamellar vessicles, remained in the solution atop the sucrose cushion. Since liposomes have a buoyant density less than that of the sucrose solution only material that is denser, that is, crystalline C₆₀, if any, would have collected in the bottom of the tube. The fact that we observed no brown pellet indicates the C₆₀was all associated with the liposomes even at this lower ratio of lipid to C₆₀. An aliquot of the liposome suspension was transferred to microscope slide and the material examined. Small vesicles, barely visible in the light microscope were visible as were large aggregates of vesicles 1 to 10μ in diameter. In no fields were brown crystals visible. Rather there was a yellow green tinge to the lipids, indicating a relatively uniform dispersion of fullerenes and lipids.

Example 4

Dimyristoyl phosphatidyl choline was added to toluene. To this solution C₆₀was added such that the molar ratio of lipids to C₆₀was 86:1. The mixture was dried in a rotary evaporator to form a thin film. To this film, 5 cc of phosphate buffered saline was added and the solution swirled over the lipid film to slough off the lipids from the round bottom flask. The flask was exposed to a bath type sonicator for 5 sec to facilitate sloughing of the film. The resultant cloudy suspension contained flocculant material visible to the eye as well as a brown cloudy suspension. An aliquot was exposed to 18,000 G for 20 min to collect the denser material in a pellet. A second aliquot was layered on top of a 20% sucrose cushion and then exposed to 18,000 G for 20 min. In the first tube most of the visible material collected in a pellet, although a small amount of cloudy tinted material remained in solution. In the second tube a significant amount of brown material was dense enough to form a pellet, while the majority of the sample formed a cushion buoyed on top of the sucrose solution. Since liposomes have a buoyant density less than that of the sucrose solution only material that is denser, that is, crystalline C₆₀, collected in the bottom of the tube. An aliquot of the liposomes were transferred to a microscope slide and examined. Large brown crystals of C₆₀were clearly visible, as were large, multilamellar vesicles that were colorless. These results indicate that the C₆₀either did not associate with the lipids or that is dissociated from the lipids very soon after dispersing.

Example 5

Dimyristoyl phosphatidyl glycerol was added to toluene. To this solution C₆₀was added such that the molar ratio of lipids to C₆₀was 86:1. The mixture was dried in a rotary evaporator to form a thin film. To this film, 5 cc of phosphate buffered saline was added and the solution swirled over the lipid film to slough off the lipids from the round bottom flask. The flask was exposed to a bath type sonicator for 5 sec to facilitate sloughing of the film. The resultant cloudy suspension contained flocculent material visible to the eye as well as a brown cloudy suspension. An aliquot was exposed to 18,000 G for 20 min to collect the denser material in a pellet. A second aliquot was layered on top of a 20% sucrose cushion and then exposed to 18,000 G for 20 min. In the first tube most of the visible material collected in a pellet, although a small amount of cloudy tinted material remained in solution. In the second tube a significant amount of brown material was dense enough to form a pellet, while the majority of the sample formed a cushion buoyed on top of the sucrose solution. Since liposomes have a buoyant density less than that of the sucrose solution only material that is denser, that is, crystalline C₆₀, collected in the bottom of the tube. An aliquot of the liposomes were transferred to a microscope slide and examined. Large brown crystals of C₆₀were clearly visible, as were large, multilamellar vesicles that were colorless. These results indicate that the C₆₀either did not associate with the lipids or that it dissociated from the lipids very soon after dispersing.

Example 6

Egg phosphatidyl choline was dissolved in 10 cc of dichloromethane and C₆₀was dissolved in carbon disulfide and mixed with the egg-PC at a molar ratio of 40:1 ePC:C₆₀. The mixture was a clear solution with a pink hue from the fullerene chromophore. The solvent mixture was removed by rotary evaporation to form a thin film. This film was dissolved in dichloromethane. This second solution was also clear and pink. C₆₀has limited solubility in this solvent, but the association with the lipids appears to help the solubilization.
Phosphate buffered saline was added to the above solution, forming a two phase mixture. The two phases were dispersed using ultrasonic energy and the solvent evaporated by vacuum. The organic phase remained pink until the end of the evaporation, at which point the aqueous solution appeared yellow:brown. Examination of the resultant liposomes revealed minute brown grains dispersed amid the liposomes. Upon centrifugation the liposomes which sedimented formed a pellet that appeared striated. The bottom of the pellet appeared brown on top of which was a yellow layer which was in turn topped by a white layer. This striation indicates the distribution of C₆₀in the lipids is heterogeneous. When the liposome preparation was carefully layered atop a cushion of 40% sucrose and spun the lipids all remained above the sucrose cushion. A black pellet at the bottom of the tube indicated some C₆₀had crystallized out during the evaporation.

Example 7

A mixture of egg phosphatidyl choline and bovine brain phosphatidyl ethanolamine was dissolved in 10 cc of dichloromethane and C₆₀was dissolved in carbon disulfide and mixed with the lipids at a molar ratio of 40:1 lipid:C₆₀. The mixture was a clear solution with a pink hue from the fullerene chromophore. The solvent mixture was removed by rotary evaporation to form a thin film. This film was dissolved in dichloromethane. This second solution was also clear and pink. C₆₀has limited solubility in this solvent, but the association with the lipids appears to help the solubilization. Phosphate buffered saline was added to the above solution, forming a two phase mixture. The two phases were dispersed using ultrasonic energy and the solvent evaporated by vacuum. The organic phase remained pink until the end of the evaporation, at which point the aqueous solution appeared yellow:brown. Examination of the resultant liposomes revealed fewer minute brown grains dispersed amid the liposomes than appeared in the above example. Upon centrifugation the liposomes which sedimented formed a pellet that still appeared striated. There was a smaller brown pellet on top of which was a yellow layer which was in turn topped by a small white layer. This striation indicates the distribution of C₆₀in the lipids is heterogeneous, albeit better than in the egg-PC preparation and the preparations made by the thin film method. When the liposome preparation was carefully layered atop a cushion of 40% sucrose and spun the lipids all remained above the sucrose cushion. There was no visible crystal at the bottom of the centrifuge tube, indicating all the C₆₀was associated with the lipids.

Example 8

Arrays of fullerene containing liposome compositions are constructed by mixing aliquots of DMPC, DPPC, hydrogenated soy-PC, PSPC, POPC, and Egg-PC in solution in organic solvent (e.g., toluene or another suitable organic solvent) with DMPE, POPE, SOPE, and brain-PE (porcine) in organic solvent to make an array of samples having molar ratios of each combination of PC:PE at 9:1 and 5:1, to which C₆₀is added to a 9:1 lipid:fullerene ratio to form a matrix of compositions with a uniform total sample size in one or more suitable multiwell plates. Solvent is removed to apparent dryness under a stream of dry nitrogen, followed by further drying under vacuum in the presence of CaCl₂for at least 12 hours. 0.2 ml of phosphate buffered saline solution at pH 7.4 is added to each sample. The tray is covered so as to seal each well and agitated. The plate is suspended in a sonication bath at 35° C. for about 20 minutes.
Each sample is characterized by observation under a microscope immediately following preparation and after 72 hours sealed at room temperature. Liposomes having complete uptake and uniform distribution of the fullerene molecules are characterized by containing uniform yellow-green tinted liposomes and no appearance of brown fullerene crystals.
The procedure is repeated for compositions demonstrating complete uptake and uniform distribution of the fullerene molecules and corresponding compositions with the addition of PS lipids (e.g., 1:19 PS lipids of approximately the same acyl chain composition as the PE moiety of the composition) with the lipid:fullerene ratio decreased to 4:1, 3:1, and 3:2.
The foregoing procedure can be repeated substituting a variety of PG lipids or mixtures of other lipids for PE lipids. Likewise, additional components such as lipid membrane modifies and targeting ligands may be introduced into the matrix of samples to determine optimal conditions for a specific application. Samples demonstrating optimal characteristics of uniformity and stability of maximal fullerene incorporation represent preferred compositions.

Example 9

Dodecylamine endohedral metalofullerene was prepared by reacting dodecylamine with Gd₃N@C₈₀in toluene with butanone hydroperoxide. The result of this reaction is a Gd₃N@C₈₀sphere with dodecylamine attached to the fullerene cage via an amine. A mixture of egg phosphatidyl choline and bovine brain phosphatidyl ethanolamine was dissolved in 10 cc of toluene and dodecylamine Gd₃N@C₈₀was mixed with the lipids at a molar ratio of 10:1 lipid:fullerene. The solvent was removed by rotary evaporation to form a thin film yellow in color. To this film, 5 cc of phosphate buffered saline was added and the solution swirled over the lipid film to slough off the lipids from the round bottom flask. The flask was exposed to a bath type sonicator for 5 sec to facilitate sloughing of the film. Examination of the resultant liposomes in a microscope revealed a uniform distribution. When the liposome preparation was carefully layered atop a cushion of 40% sucrose and spun the lipids all remained above the sucrose cushion. There was no visible crystal at the bottom of the centrifuge tube, indicating all the dodecylamine Gd₃N@C₈₀was associated with the lipids.

Claims

1. A pharmaceutically acceptable composition comprising fullerene molecules dispersed in a mixture comprising phosphatidylcholine (PC) phospholipid molecules and/or non-PC phospholipid molecules suspended in aqueous solution; wherein the phospholipid molecules are substantially uniformly organized into vesicles composed of one or more lipid bilayers and wherein the fullerene molecules are substantially uniformly distributed within the lipid bilayers of the vesicles.

2. The composition of claim 1, wherein the ratio of PC phospholipid molecules to non-PC phospholipid molecules is in the range from about 19:1 to about 3:1.

3. The composition of claim 1, wherein the ratio of PC phospholipid molecules to non-PC phospholipid molecules is in the range from about 10:1 to about 3:1.

4. The composition of claim 1, wherein the ratio of PC phospholipid molecules to non-PC phospholipid molecules is in the range from about 7:1 to about 4:1.

5. The composition of claim 1, wherein the ratio of PC phospholipid molecules to non-PC phospholipid molecules is about 5:1.

6. The composition of claim 1, wherein the ratio of all phospholipid molecules to fullerene molecules is in the range of about 19:1 to about 1:2.

7. The composition of claim 1, wherein ratio of all phospholipid molecules to fullerene molecules is in the range of about 9:1 to about 1:2.

8. The composition of claim 1, wherein ratio of all phospholipid molecules to fullerene molecules is in the range of about 6:1 to about 1:1.

9. The composition of claim 1, wherein ratio of all phospholipid molecules to fullerene molecules is in the range of about 4:1 to about 3:2.

10. The composition of claim 1, wherein the PC phospholipid molecules are principally lipid molecules having a saturated alkyl chain and an unsaturated alkyl chain.

11. The composition of claim 1, wherein the non-PC phosopholipid molecules are principally PE molecules.

12. The composition of claim 1, wherein the non-PC phosopholipid molecules are principally phosphatidyl glycerol molecules.

13. The composition of claim 1, wherein the non-PC phosopholipid molecules include phosphatidyl serine molecules.

14. The composition of claim 1, wherein the fullerene molecules are principally un-derivatized fullerene molecules.

15. The composition of claim 1, wherein the fullerene molecules are principally un-derivatized C₆₀fullerene molecules.

16. The composition of claim 1, wherein the fullerene molecules are principally derivatized fullerene molecules.

17. The composition of claim 1, wherein the fullerene molecules are principally fullerene molecules selected from among mono-, di-, and tri-derivatized fullerene molecules.

18. The composition of claim 1, wherein the composition is substantially free of crystals of fullerene molecules.

19. The composition of claim 1, wherein the vesicles are principally vesicles having a diameter in the range between 0.1 to 0.5 microns.

20. The composition of claim 1, wherein the vesicles are principally vesicles having a diameter in the range between about 0.1 to 0.3 microns.

21. The composition of claim 1, wherein the fullerene molecule has >=70 carbon atoms.