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
18.104.22.168 Derivatized Carbon Fullerenes and Nanotubes
Most biocompatibility studies focus not on pure (insoluble) fullerenes but rather on solubilized C60 derivatives which may have potential utility as pharmaceutical agents. Tests have been devised to simplify fullerene biocompatibility testing, as for example methods of quantitative analysis of C60 in blood and tissues using high-performance liquid chromatographic assay .
In general, water-soluble fullerene derivatives  (and possibly their simple metabolites) are not acutely toxic even at 200-500 mg/kg doses in mice . For instance, LD50 acute toxicity of fullerenol-1 for intraperitoneally-treated mice is ~1200 mg/kg . In one study , highly water-soluble polyalkylsulfonated C60 (FC4S) in 50 mg/cm3 concentration was administered to rats. FC4S was completely nontoxic if given orally, but rats died within 30 hours after intraperitoneal injection with an enormous LD50 dose of 600 mg/kg of body weight – i.e., the drug is highly nontoxic at therapeutic concentrations. Intravenous or intraperitoneal injections in rats prompted immediate elimination through the kidney (the primary target organ in this study ), and induced phagolysosomal nephropathy in acute exposure rats and in surviving rats receiving 500 mg/kg intraperitoneally or 100 mg/kg intravenously (~0.1% Nct) – again, highly nontoxic. Toxicity of MSAD-C60 in rats is somewhat higher: 25 mg/kg administered by bolus intravenous injection in rats caused shortness of breath and violent movement, followed by death in 5 minutes , though no toxicity was reported after intraperitoneal administration of 50 mg/kg-day for 6 days to mice . These compounds are believed not to be hemolytic [734, 921].
C60- and C70-derived fullerene carboxylic acids showed no photocytotoxicity toward Raji cells (B lymphocytes) , and intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system . Nor are the derivatized fullerenes particularly mutagenic. One experiment  found no mutagenic effect in fullerol concentrations up to 2.46 mg/cm3.
Small-molecule fullerenes are not normally recognized by the immune system and do not trigger the natural production of antibodies by themselves [719, 2516, 4630], solubilized fullerenes can induce the production of specific antibodies [724, 725, 2164, 2387-2390], usually by interaction with the combining sites of IgG , or can enhance IgG production as adjuvants . Immunization of mice with a water-soluble C60 derivative conjugated to bovine thyroglobulin yielded a population of fullerene-specific antibodies of the IgG isotype. This showed that the immune repertoire was diverse enough to recognize and process fullerenes as protein conjugates . The antibody population also included a subpopulation that crossreacted with a C70 fullerene as determined by immune precipitation and ELISA (enzyme-linked immunoabsorbent assay) procedures. C60 conjugated with BSA produces polyclonal response in rabbits and monoclonal response in rats . It is speculated  that highly hydrophobic fullerenes would be recognized by antibodies with hydrophobic amino acids in their binding sites, as has been reported for the combining site of an Fab’ fragment of a monoclonal antibody specific for progesterone [912, 913], which is a highly apolar molecule of similar size to C60. C60 and other fullerenes can also interact with donor -NH2  and -SH  groups.
Antibodies raised to C60 in mice strongly bind to single-walled carbon nanotubes . There are several reports of antibodies being raised to single-walled carbon nanotubes [2164, 2385-2387], as for example a mutant of 1-10F-8A that targets single-walled carbon nanotubes . Computer simulations suggest that it may be possible to build antibodies which selectively bind to nanotubes of a specific diameter or chirality .
There are reports of fullerene compound interaction directly with biological receptors . For example, the Wilson group  has prepared a fullerene-estrone hybrid compound that has estrogenic activity, binding to cytosolic estradiol receptor with Kd ~ 40 µM. Toniolo et al  has prepared a hydrophilic fullerene-based analog of peptide T which exhibits potent activity in a CD4 receptor-mediated human monocyte chemotaxis assay. Computer models have been used to assess the interaction of fullerenes with HIV protease [735, 2568], glutathione-S-transferase , DNA , and a peptide helix .
Solubilized fullerenes are bioactive  in tests with many different types of living cells. For instance, C60 fullerenol-1 inhibits the proliferative responses (transduction signals) of a number of cells, including rat aortic smooth muscle cells (at 10-6 - 10-2 M concentration), human coronary artery smooth muscle cells, and human CEM lymphocytes – possibly mediated through the inhibition of protein tyrosine kinase . In another experiment, fullerenol-1 applied to rodent liver microsomes reduced monooxygenase activity and decreased cytochrome P450 and b5 contents at 500-1000 mg/kg doses, but had no effect at 10-100 mg/kg doses . Added to rat liver mitochondria, fullerenol-1 decreases mitochondrial oxidative phosphorylation in vitro, producing a dose-dependent inhibition of ADP-induced uncoupling and significantly inhibiting mitochondrial Mg++-ATPase activity with an IC50 level at 7.1 µM . Highly water-soluble polyalkylsulfonated C60 (FC4S) in 50 mg/cm3 concentration administered to rats suppresses liver cytochrome P-450-dependent monooxygenase activities but increases kidney cytochrome P-450-dependent monooxygenase activities . C60 solubilized with polyvinylpyrrolidone (PVP) in water and incubated with mouse embryos in vitro potently inhibits cell differentiation and proliferation .
Pharmacological effects of fullerenes on various tissues have been noted. For example, monomalonic acid C60 (MMA-C60) was applied to endothelium-containing or denuded aorta of rabbit, trachea and ileum of guinea pig, and stomach (fundus), vas deferens and uterus of rat. At 10-5 M concentration, MMA-C60 was found to significantly reduce the endothelium-dependent relaxation induced by acetylcholine, but not to affect the agonist-induced contractile response of smooth muscle . Dimalonic acid C60 at 10-5 M concentration inhibited endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide .
The biodistribution of fullerenes throughout body tissues, after they are introduced in vivo, has been studied. In one experiment , a 14C-labeled trimethylenemethane-based water-solubilized C60 fullerene was administered orally to rats. The compound was not efficiently absorbed and was excreted primarily in the feces. When injected intravenously, however, the compound distributed rapidly to various tissues with most of the material still retained in the body after one week, and with retention mostly in the liver (90%) . The substance also penetrated the blood-brain barrier (Section 22.214.171.124). When administered intraperitoneally to pregnant mice at 50 mg/kg, PVP-solubilized C60 was clearly distributed into the yolk sac and embryos . Microscopic evaluation revealed a harmful effect on conceptuses , although the effects of underivatized C60 on embryogenesis were not reported. A biodistribution study of underivatized C60 in Swiss mice (4-5 mg/kg doses) found that >95% of the fullerene material was retained, mostly in the liver, probably unmetabolized .
In 1999, Gonzalez and Wilson  tested a C60 fullerol (containing 16 hydroxyls) functionalized with an amide bis-phosphonate chemical group. This compound showed selective binding to the hydroxyapatite of bone (thus altering the mineral’s usual crystal growth) which suggests that a rationally-designed molecule could be used to target bone tissue, possibly as an agent to address osteoporosis. Another study found that C60-PEG conjugate injected intravenously into mice carrying a tumor mass in the back subcutis exhibited higher accumulation and more prolonged retention in the tumor tissue than in normal tissue . However, the conjugate was excreted without being accumulated in any specific organ . In vivo fullerene biodistribution studies of insoluble C60 and La@C60 suspensions* [719, 1093] and water-soluble C60 derivatives [720, 734, 751] indicate a short residency in the blood pool with rapid localization and long-term residency in the liver (<1% clearance). One of these studies  demonstrated that fullerenes are not metabolized rapidly in vivo, although fullerene oxidation of C60 derivatives has been observed in vivo , followed by selective absorption by liver cells [732, 1097]. Although their acute toxicity is low at the ~mg/kg dose level , water-soluble fullerenes are retained in the body for long periods which raises concerns about chronic toxic effects.
* The nomenclature “X@C60” is commonly used in fullerene chemistry to indicate that atom X is endohedrally trapped inside the closed C60 molecular cage.
However, another biodistribution experiment by Wilson et al [705, 730, 1094] at Rice University found that solubilized C82 endohedral metallofullerenes – each containing a trapped radioactive holmium atom (Hox@C82(OH)y) – when introduced intravenously remain in the blood for about an hour with nearly total clearance from the blood shortly thereafter. These endohedral metallofullerenes localize in bone, spleen, kidneys, and liver, but with slow and steady clearance from all tissue (~20% after 5 days in rats) except bone, where fullerene concentration steadily increases with time. After 48 hours, metallofullerene concentration falls to 15% of injected dose (ID) in the liver, only slightly lower than the maximum of 24% ID for liver. Concentrations are only 3.6% ID (down from 7.6% ID max) in the kidney and 0.36% ID (down from 5.1% ID max) in the blood pool . Accumulation in the brain is negligible . After the first day, when 88.4% ID remains in rats, clearance is ~1.5% ID per day, with nearly equal amounts eliminated in the feces and urine . Another biodistribution experiment  involved a suspension of insoluble metallofullerene (La@C82) injected directly into the heart of anesthetized rats. After 24 hours, >80% of the material still present in the body was located in the liver and blood pool, with some retention also in the brain .
Most recently, B.F. Erlanger’s group  injected carboxyfullerene and fluorescent-labeled antibodies targeted to naked fullerene to observe possible targeting to specific intracellular compartments of the fullerene-based agent in an animal model. They directly observed via fluorescence that the fullerene derivative had crossed the external cellular membrane and localized preferentially to the intracellular mitochondria. This seems to support “the potential use of fullerenes as drug delivery agents as their structure mimics that of clathrin known to mediate endocytosis.”
While fullerene molecules can exhibit a wide range of interactions, many of these interactions will not take place at the surfaces of medical nanorobots with graphene exteriors. E. Pinkhassik notes that “high mobility of relatively small buckyballs is responsible for many physiological actions observed by different researchers, and since the larger nanodevices will not be able to cross the membranes or easily get to active sites of proteins, they should be even more inert than their smaller counterparts.”
Last updated on 30 April 2004