Nanomedicine, Volume IIA: Biocompatibility
© 2003 Robert A. Freitas Jr. All Rights Reserved.
Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003
188.8.131.52 Fullerene-Based Pharmaceuticals
By 2002, several major classes of fullerene-based pharmaceutical applications were under active investigation, most notably by the biotech company C Sixty , including:
(1) Antivirals. Fullerenes have shown activity against HIV [693, 732-735, 1096-1098]. In 1993, MSAD-C60 was found effective against HIV-1 and HIV-2 with 50% effective concentration (EC50) averaging 6 x 10-6 M in acutely or chronically infected human lymphocytes, and with selective activity against HIV-1 protease . MSAD-C60 was noncytotoxic up to 10-4 M in peripheral blood mononuclear cells and in H9, Vero, and CEM cells . Following intravenous administration at 15 mg/kg of body weight, pharmacokinetic studies showed a half-life of 6.8 hours in the blood with distribution into the tissues. Binding studies showed the compound was >99% bound to plasma proteins . MSAD-C60 is well tolerated in mice after IV administration up to 15 mg/kg, but a higher dose of 25 mg/kg produces shortness of breath and violent movement of rats, followed by death with 5 minutes of dosing . By 1998, computational models for optimizing the binding of fullerene inhibitors of the HIV-1 protease led to the synthesis and testing of two C60-derived ligands for the HIV protease active site that displayed ~50-fold increase in affinity compared to previously tested fullerene compounds .
By the late 1990s , photodynamic reactions induced by singlet oxygen-generating agents were known to inactivate enveloped viruses . Pure water-insoluble photosensitizer C60 could be used to mediate the inactivation of enveloped viruses. Buffered solutions containing C60 and Semliki Forest virus (SFV, Togaviridae) or vesicular stomatitis virus (VSV, Rhabdoviridae), when illuminated with visible light for up to 5 hours, resulted in a significant loss of infectivity. Viral inactivation was oxygen-dependent and equally efficient in solutions containing protein. C60 fulleropyrrolidone was also known to have antiviral activity . A C60 molecule covalently linked to peptide T, like peptide T, displays potent human monocyte chemotaxis while weakly inhibiting HIV-1 protease . C Sixty’s anti-HIV fullerene compound CSDF1 exhibits high water solubility (200 gm/liter), complete renal excretion, and a highly nontoxic LD50 of 800 mg/kg in rodents [4630, 5235]. The drug appears effective even against highly resistant strains of the virus. The binding constant is ~nM for C Sixty’s anti-HIV protease inhibitor ; the company’s anti-HIV drugs apparently have about one-tenth the toxicity of current HIV cocktails, and human trials start in 2003 .
(2) Antibacterials. A water-soluble malonic acid derivative of C60 (carboxyfullerene) was protective in mice against E. coli-induced meningitis death in a dose-dependent manner, even when administered intraperitoneally as late as 9 hours after E. coli injection . Fullerene-treated mice had less tumor necrosis factor alpha and less interleukin-1beta production compared to the production levels for nontreated mice. E. coli-induced increases in blood-brain barrier permeability and inflammatory neutrophilic infiltration were also inhibited , suggesting that the C60 compound could be a useful therapeutic agent in some cases of bacterial meningitis . Other fullerene-based inhibitors of E. coli growth have been investigated . Positively-charged water-soluble fullerene derivatives inhibit growth of Mycobacterium tuberculosis at ~0.005 mg/cm3 concentrations . Carboxyfullerene directly inhibits in vitro growth of Streptococcus pyogenes and enhances bactericidal activity of neutrophils in mice in vivo , suggesting that the fullerene derivative “can be considered an antimicrobial agent for group A streptococcus infection.” Subsequent work  by this research group found that the antibacterial action of carboxyfullerene on Gram-positive bacteria is achieved by insertion into the cell wall and destruction of membrane integrity. Other studies  have also found antibacterial activity of fullerene derivatives, and even of carbon nanotubes [U. Sagman, personal communication, December 2002].
(3) Tumor/Anti-Cancer Therapy. A C60-PEG conjugate irradiated by light strongly induced tumor necrosis without any damage to the overlying normal tissue [684, 2576], with complete cure achieved by a C60-PEG dose of 0.424 mg/kg and irradiation power of 1011 J/m2, making this and similar materials  excellent candidates for photodynamic tumor therapy. In vitro cytotoxicity against the HeLa S3 cell line has been evaluated by studying the inhibited growth rate , and some C60 derivatives have shown promise as anti-cancer agents [1090-1092]. Photodynamic activity of PEG-modified fullerene is reported against fibrosarcoma tumors in mice and on erythrocyte membrane [2577, 2578]. Water-soluble C60(OH)24 has been shown (1) to strongly block microtubule assembly, (2) to inhibit cell growth via inhibition of mitotic spindle formation much like taxol, and (3) to affect the growth kinetics of human lymphocyte cultures and HEP-2 epidermal carcinoma cell cultures . Liposomes containing ~0.1 mM of solubilized C60 are reported to have anticancer effects on human cervical cancer cells . Other fullerene-based inhibitors of cancer cell  and tumor  growth have been investigated, and the first fullerene-based clinical treatment of a human patient with rectal adenocarcinoma was attempted by Andrievsky et al . Chemotherapeutic agents are also being attached to larger fullerene structures to be carried inside liposomes, that C Sixty calls “buckysomes.”
(4) Anti-Apoptosis Agents. C60 is a free-radical scavenger and can act as antagonist for ceramide-triggered (but not Fas-triggered) apoptosis . Transforming growth factor-beta (TGF-beta) induces apoptosis in normal hepatocytes and hepatoma cells, but carboxyfullerene blocks the apoptotic signaling of TGF-beta in human hepatoma cells . The antiapoptotic activity of C60 carboxyfullerene is correlated with its ability to eliminate TGF-beta-generated reactive oxygen species , and carboxyfullerene protects human keratinocytes from ultraviolet (UVB) damage  “possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria.” Carboxyfullerene also exerts some protection against oxidative stress-induced apoptosis in human peripheral blood mononuclear cells (PBMCs) . Another water-soluble C60 derivative protects epithelial cells from substrate-restriction apoptosis by exerting a trophic effect on actin microfilaments, thus influencing cell adhesion ability .
More interesting are the neuron anti-apoptotic effects. In one experiment , water-soluble C60 fullerenols decreased excitotoxic neuronal death following brief exposure to N-methyl-D-aspartate (NMDA) (by 80%), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (by 65%), or kainate (by 50%). The fullerenols also reduced neuronal apoptosis induced by serum deprivation . (The fullerenols were not NMDA or AMPA/kainate receptor antagonists.) In a related experiment , carboxylic acid C60 derivatives inhibited the excitotoxic death of cultured cortical neurons induced by exposure to NMDA, AMPA, or oxygen-glucose deprivation. One C60 derivative fully blocked even rapidly triggered NMDA receptor-mediated toxicity and reduced apoptotic neuronal death induced by either serum deprivation or exposure to amyloid beta peptide (Abeta1-42), the established cause of Alzheimer’s disease . This suggested that polar carboxylic acid C60 derivatives might have attractive therapeutic properties in several acute or chronic degenerative diseases such as amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease) . In 2002, C Sixty had fullerene-based drugs to combat ALS and Parkinson’s disease under development , with human trials expected to begin in 2003 .
(5) Antioxidants. C60 derivatives [683, 726, 739, 748, 2583] including fullerenols [741-744, 2581], carboxyfullerene [745-747, 5875, 5879], polyalkylsulfonated C60 , hexa(sulphobutyl)fullerene , C60-dimalonic acid  and C62 bis(malonate)  are known or suspected free-radical (oxygen-radical) scavengers. These derivatives often provide potent antioxidative action (e.g., preventing hydrogen peroxide- and cumene hydroperoxide-elicited cellular damage ) without increasing lipid peroxidation . In experiments with mice, Dugan et al [741, 745] found potent antioxidant properties in buckyballs (hundreds of times more powerful than Vitamin E) that could shield nerve cells from free radicals. C60 monomalonate selectively inhibited activity of the neuronal nitric oxide synthase (nNOS) isoform . C60 trisamine adducts also inhibited nNOS, but this was completely reversible by calmodulin, which suggests that these fullerene adducts are potent calmodulin antagonists at ~50 nM . Other fullerene-derived NOS inhibitors are known [4636, 5239]. C60 molecules immobilized at a silicon surface also display anti-oxidant activity . The trimalonic acid derivative of fullerene (carboxyfullerene) is a water-soluble compound that has been found to be an effective antioxidant both in vivo and in vitro .
(6) DNA Cleavage. Water-soluble fullerene carboxylic acid cleaves DNA fragments at guanine residues upon exposure to light [922, 2569, 2574]. Boutorine et al  describe a fullerene-oligonucleotide that can bind single- or double-stranded DNA, and which also cleaves the strand(s) proximal to the fullerene moiety upon exposure to light. A C60 derivative linked to a gold surface and DNA was shown to bind and be cleaved with the gold-linked fullerene . Nakanishi et al  also observed DNA cleavage by functionalized C60.
(7) Other Applications. Fullerenol-1 significantly attenuates noncholinergic (e.g., exsanguination-induced) airway constriction in guinea pigs . It also produces a slight bronchial constricting action at high doses (2 mg/kg) when applied via intratracheal instillation . Fullerene compounds have effects on nitric oxide  and acetylcholine  signaling pathways. Fullerene redox chemistry may be applicable to biosensor technologies [2579, 2580]. In one experiment, a C60-containing bilayer lipid membrane was shown to be a light-sensitive diode potentially useful in electrochemical biosensor devices . Favorable blood contact properties of surface-immobilized C60 have been reported . Paramagnetic malonodiamide C60 derivatives may be useful in making MRI contrast agents . C Sixty is also investigating possible drug-delivery “nanopills” consisting of two closed-end single-walled carbon nanotubes nested mouth-to-mouth, forming a capsule-like container [4630, 5242]. More generally, the company  is investigating the targeted therapeutic delivery of drugs or radioactive atoms enclosed in surface-functionalized fullerenes to specific tissues or diseased cells. In 2002 this research area was quite active and the interested reader should consult the literature for the most current results.
Last updated on 30 April 2004