StemCell Therapy

By Vince Giuliano

Image source

The popular life extension blogs have been lit up recently with exchanges related to a recent publication that reports that a homogenized solution of olive oil and C60 carbon buckyballs fed to middle age rats extends their lifespans by an average of 90%. If this result stands up it is truly amazing. Compared with other longevity interventions such as rapamycin feeding or calorie restriction which at best extend lifespans by 15-20%, the 90% figure is off the scale. So I decided to delve into the research literature to clarify what is known and what is not known about C60 carbon fullerenes as related to biological impacts and health. I report on this expedition here. I also chime in with my own hypotheses about the mechanisms through which the C60-olive oil cocktail extends rats lifespans, assuming it really does. The main points I will be documenting are:

Image source

C60 buckyball Image sourceBuckminister Fuller

What is a C60 fullerene?

From wickipedia: A fullerene is any molecule composed entirely of carbon, in the form of a hollow sphere, ellipsoid or tube. Spherical fullerenes are also called buckyballs, and they resemble the balls used in football (association football). Cylindrical ones are called carbon nanotubes or buckytubes. Fullerenes are similar in structure to graphite, which is composed of stacked graphene sheets of linked hexagonal rings; but they may also contain pentagonal (or sometimes heptagonal) rings.[1] The first fullerene molecule to be discovered, and the familys namesake, buckminsterfullerene (C60), was prepared in 1985 by Richard Smalley, Robert Curl, James Heath, Sean OBrien, and Harold Kroto at Rice University. The name was an homage to Buckminster Fuller, whose geodesic domes it resembles. The structure was also identified some five years earlier by Sumio Iijima, from an electron microscope image, where it formed the core of a bucky onion.[2] Fullerenes have since been found to occur in nature.[3] More recently, fullerenes have been detected in outer space.[4] According to astronomer Letizia Stanghellini, Its possible that buckyballs from outer space provided seeds for life on Earth.[5]

C60 buckyballs and longevity of rats, mice and other lower species

I start out with the publication that has initiated the current buz, The prolongation of the lifespan of rats by repeated oral administration of [60] fullerene published in August 2011. The study reported was designed to assess the toxicity of C60 in an olive oil suspension, not to assess impact on lifespans of rats. The result that not only was the suspension not toxic but radically increased the livespans of the rats was a surprise to the researchers. Countless studies showed that [60]fullerene (C60) and derivatives could have many potential biomedical applications. However, while several independent research groups showed that C60 has no acute or subacute toxicity in various experimental models, more than 25 years after its discovery the in vivo fate and the chronic effects of this fullerene remain unknown. If the potential of C60 and derivatives in the biomedical eld have to be fullled these issues must be addressed. Here we show that oral administration of C60 dissolved in olive oil (0.8 mg/ml) at reiterated doses (1.7 mg/kg of body weight) to rats not only does not entail chronic toxicity but it almost doubles their lifespan. The effects of C60-olive oil solutions in an experimental model of CCl4 intoxication in rat strongly suggest that the effect on lifespan is mainly due to the attenuation of age-associated increases in oxidative stress. Pharmacokinetic studies show that dissolved C60 is absorbed by the gastro-intestinal tract and eliminated in a few tens of hours. These results of importance in the elds of medicine and toxicology should open the way for the many possible -and waited for- biomedical applications of C60 including cancer therapy, neurodegenerative disorders, and ageing.

A nice thing about this publication is that it describes the experimental conditions in meticulous detail. For example, getting a good solvent vector for administration of C60 to animals has been a serious problem. Unlike many other studies which employed water-based solutions of C60 with poor or uncertain bioavailability and toxic effects, this study used an olive oil brew. Fifty mg of C60 were dissolved in 10 ml of olive oil by stirring for 2 weeks at was increased to 60% for 10 min and then hold constant for the remaining 7 min of ambient temperature in the dark. The resulting mixturewas centrifugedat 5.000gfor each sample run. At least 10 column volumes of the initial composition were ushed 1 h and the supernatant was ltered through a Millipore lter with 0.25 mmporosity.

There were several sub-studies reported in this paper. In the chronic toxicity and longevity sub-study, only 18 rats middle-aged were involved divided into three cohorts of six rats each: a) a control cohort fed normal rat chow, b) a cohort fed food plus olive oil by gavage, and c) a cohort feed the C60-olive oil brew by gavage (forced feeding). The rats were housed three per cage and acclimated for 14 days, before dosing. Three groups of 6 rats (10 months old, weighing 465.31(10 months old,were administered daily for one week, then weekly until the end of the second month and then every two weeks until the end of the 7th month, by gavages with 1 ml of water or olive oil or C60 dissolved in olive oil (0.8 mg/ml), respectively. All rats in cohort (a) were alive until week 18 of the experiment and all were dead by week 38. All rats in cohort (b). were alive until week 36 and all were dead by week 58. In cohort (c). all rats were alive until week 60 and all dead by week 66 (the last one being sacrificed at week 66). Between weeks 38 and 60 all the control rats were dead and all the C60-fed rats were alive and well. Olive oil alone produced a weighted average of 18% life extension while the weighted average for the C60-olive oil brew was 90%. Remarkably, no rats in cohort (c) contracted cancers.

In the sub-study of oxidative stress, the C60-olive oil mix almost completely protected against carbon tetrachloride oxidative liver damage. Sixty rats randomly divided into 10 groups of 6 rats were pre-treated daily for 7 days by oral gavages (og groups) or by i.p. injection (ip groups). Groups A (GAog and Groups B and C (GBog, GCog and GBip, GCip) were pre-treated with 1 ml of olive oil while groups D and E (GDog, GEog and GDip, GEip) were pre-treated with 1 ml of C60-olive oil Twenty-four hours before sacrice, groups GA, GC and GE were i.p. injected with a single dose of CCl4 (1 ml/kg bw) while GB and GD, used as controls, were administered with a 0.9% NaCl aqueous solution under the same conditions. The animals were subsequently sacrificed and their livers examined. the liver sections of GA and GC animals co-treated with water and CCl4 or with olive respectively, showed important damage including many inammatory areas as well as large necrotic areas with ballooning necrotic cells associated with an important steatosis (Fig. 4). In contrast, microscopic examination of the liver sections of GE animals co-treated with C60-olive oil and CCl4, revealed few necrotic areas with some ballooning cells without apoptosis limited to some cords of hepatocytes (Fig. 4).

The study also investigated the pharmacodynamics and pharmacokinetics of C60 administration. The results of this pharmacokinetic study show for the rst time that C60 is absorbed by the gastro-intestinal tract (Fig. 1). In the case of highly hydrophobic drugs (Log P > 5) it is well known that the absorption of the molecules by the gastro-intestinal tract occurs via the mesenteric lymphatic system after association with developing lipoproteins in the enterocytes rather than via the portal blood [40]. Therefore, as the octanol/water partition coefcient of C60 is estimated to be 6.67 [41], the absorption of C60 occursvia the mesenteric lymphatic system rather than via the portal blood. The elimination half-lives indicate that C60 is completely eliminated from blood 97 h after administration irrespective of the route of administration. The elimination process follows a non-urinary route because unmodied C60 was not detected in urine samples taken up 48 h after administration. Previous investigations showed that C60 is mainly eliminated through the bile ducts [21] .

Conclusion: The effect of pristine C60 on lifespan emphasizes the absence of chronic toxicity. These results obtained with a small sample of animals with an exploratory protocol ask for a more extensive studies to optimize the intestinal absorption of C60 as well as the different parameters of the administration protocol: dose, posology, and treatment duration. In the present case, the treatment was stopped when a control rat died at M17, which proves that the effects of the C60 treatment are long-lasting as the estimated median lifespan for C60-treated rats is 42 months. It can be thought that a longer treatment could have generated even longer lifespans. Anyway, this work should open the road towards the development of the considerable potential of C60 in the biomedical eld, including cancer therapy, neurodegenerative disorders and ageing. Furthermore, in the eld of ageing, as C60 can be administered orally and as it is now produced in tons, it is no longer necessary to resort to its water-soluble derivatives, which are difficult to purify and in contrast to pristine C60 may be toxic

A 2008 publication also indicated that a fullerene is capable of extending the lifespans of mice: A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice. In lower organisms, such as Caenorhabditis elegans and Drosophila, many genes identified as key regulators of aging are involved in either detoxification of reactive oxygen species or the cellular response to oxidatively-damaged macromolecules. Transgenic mice have been generated to study these genes in mammalian aging, but have not in general exhibited the expected lifespan extension or beneficial behavioral effects, possibly reflecting compensatory changes during development. We administered a small-molecule synthetic enzyme superoxide dismutase (SOD) mimetic to wild-type (i.e. non-transgenic, non-senescence accelerated) mice starting at middle age. Chronic treatment not only reduced age-associated oxidative stress and mitochondrial radical production, but significantly extended lifespan. Treated mice also exhibited improved performance on the Morris water maze learning and memory task. This is to our knowledge the first demonstration that an administered antioxidant with mitochondrial activity and nervous system penetration not only increases lifespan, but rescues age-related cognitive impairment in mammals. SOD mimetics with such characteristics may provide unique complements to genetic strategies to study the contribution of oxidative processes to nervous system aging.

Another 2011 publication Polyhydroxy Fullerenes (Fullerols or Fullerenols): Beneficial Effects on Growth and Lifespan in Diverse Biological Models indicates that fullerenes can extend the lifespans of certain more primitive organisms. The publication reports Recent toxicological studies on carbon nanomaterials, including fullerenes, have led to concerns about their safety. Functionalized fullerenes, such as polyhydroxy fullerenes (PHF, fullerols, or fullerenols), have attracted particular attention due to their water solubility and toxicity. Here, we report surprisingly beneficial and/or specific effects of PHF on model organisms representing four kingdoms, including the green algae Pseudokirchneriella subcapitata, the plant Arabidopsis thaliana, the fungus Aspergillus niger, and the invertebrate Ceriodaphnia dubia. The results showed that PHF had no acute or chronic negative effects on the freshwater organisms. Conversely, PHF could surprisingly increase the algal culture density over controls at higher concentrations (i.e., 72% increase by 1 and 5 mg/L of PHF) and extend the lifespan and stimulate the reproduction of Daphnia (e.g. about 38% by 20 mg/L of PHF). We also show that at certain PHF concentrations fungal growth can be enhanced and Arabidopsis thaliana seedlings exhibit longer hypocotyls, while other complex physiological processes remain unaffected. These findings may open new research fields in the potential applications of PHF, e.g., in biofuel production and aquaculture. These results will form the basis of further research into the mechanisms of growth stimulation and life extension by PHF.

C60 is a powerful antioxidant

This point is long known and confirmed in a number of studies. From (2007) Medicinal applications of fullerenes: Results published in 1999 have shown that fullerenes have a potential as biological antioxidants. The antioxidant property is based on the fact that fullerenes possess large amount of conjugated double bonds and low lying lowest unoccupied molecular orbital (LUMO) which can easily take up an electron, making an attack of radical species highly possible. It has been reported that up to 34 methyl radicals have been added onto a single C60 molecule. This quenching process appears to be catalytic. In other words the fullerene can react with many superoxides without being consumed. Due to this feature fullerenes are considered to be the worlds most efficient radical scavenger and are described as radical sponges (Krusic et al 1991). The major advantage of using fullerenes as medical antioxidant is their ability to localize within the cell to mitochondria and other cell compartment sites, where in diseased states, the production of free radicals takes place. Experiments on rats done by Najla Gharbi and coworkers proved this remarkable trait. They showed that aqueous C60 suspensions prepared without using any polar organic solvent, not only have no acute or sub acute toxicity in rodents, but also protect their livers against free-radical damage (Gharbi et al 2005).

The 2005 publication [60]fullerene is a powerful antioxidant in vivo with no acute or subacute toxicity confirms both this and another point made in the recent rat study: C60 does not engender toxicity in rodents. In the present work, we report the effects of C(60)-pretreatments on acute carbon tetrachloride intoxication in rats, a classical model for studying free-radical-mediated liver injury. Our results show that aqueous C(60) suspensions prepared without using any polar organic solvent not only have no acute or subacute toxicity in rodents but they also protect their livers in a dose-dependent manner against free-radical damage. To be sure, according to histopathological examinations and biological tests, pristine C(60) can be considered as a powerful liver-protective agent.

The 2011 report Antioxidant activity of fullerene C60 against OH free radicals: A Quantum Chemistry and Computational Kinetics Studyreports Fullerenes are considered to be the worlds most efficient radical scavenger, and represents an attractive tool for biological applications. Indeed, it have been demonstrated in vivo and in vitro, that fullerenes and related structures reduce the toxicity of free radical assault on neuronal tissue, reacting readily and at a high rate with free radicals, which are often the cause of cell damage or death. Although there is strong evidence that antioxidant activity is an intrinsec property of fullerenes, the mechanism of radical scavenging and neuroprotection are still unclear. In this work, we have studied the reaction between fullerene C60 and hydroxyl radicals, using high level quantum chemistry and computational kinetics methods. Energy profiles are calculated using different basis sets, and reaction rate constant are reported for the first time. The presence of nonpolar environments seems to enhance the reactivity of fullerene molecule toward OH radicals, compared to the gas phase. Energetic considerations show that, once a first radical is attached to the fullerene cage, further additions are increasingly feasible, suggesting that fullerene can act as OH radical sponges. They also protect their livers in a dose-dependent manner against free-radical damage. To be sure, according to histopathological examinations and biological tests, pristine C(60) can be considered as a powerful liver-protective agent.

C60 has low toxicity and can cross the blood-brain barrier and may lead to many medical applications.

The 2012 publication C60 fullerene derivatized nanoparticles and their application to therapeutics reports Fullerenes can be formed into many new materials and devices. They have a wide range of applications in medicine, electronics, biomaterials, and energy production. An overview of the nanostructure and the physical and chemical characteristics of fullerene-drug derivatives is given. The biological behavior of fullerene derivatives shows their potential to medical application fields because C(60) is rapidly absorbed by tissues and is excreted through urinary tract and enterons, which reveals low toxicity in vitro and in vivo studies. Nanomedicine has become one of the most promising areas of nanotechnology, while many have claimed its therapeutic use against cancer, human immunodeficiency virus (HIV), and neurodegenerative disorders. Water-soluble C(60) fullerene derivatives that come from chemical modification largely enhance the biological efficacy. The blood-brain barrier (BBB) is a physical barrier composed of endothelial tight junctions that restrict the paracellular permeability. A major challenge facing neuropharmacology is to find compounds that can be delivered into the brain through the bloodstream. Fullerene C(60) was demonstratively able to cross the BBB by hybridizing a biologically active moiety dyad, which provides a promising clue as a pharmacological therapy of neural disorders.

Fullerene C60 is neuroprotective

The 2001 publication Fullerene-based antioxidants and neurodegenerative disorders reports: Water-soluble derivatives of buckminsterfullerene (C60) derivatives are a unique class of compounds with potent antioxidant properties. Studies on one class of these compounds, the malonic acid C60 derivatives (carboxyfullerenes), indicated that they are capable of eliminating both superoxide anion and H2O2, and were effective inhibitors of lipid peroxidation, as well. Carboxyfullerenes demonstrated robust neuroprotection against excitotoxic, apoptotic and metabolic insults in cortical cell cultures. They were also capable of rescuing mesence-phalic dopaminergic neurons from both MPP1 and 6-hydroxydopamine-induced degeneration. Although there is limited in vivo data on these compounds to date, we have previously reported that systemic administration of the C3 carboxyfullerene isomer delayed motor deterioration and death in a mouse model of familial amyotrophic lateral sclerosis (FALS). Ongoing studies in other animal models of CNS disease states suggest that these novel antioxidants are potential neuroprotective agents for other neurodegenerative disorders, including Parkinsons disease.

C60 derivative and hybrid structure compounds are also being studied for their neurprotective as well as other medical properties. See for example [Study of the neuroprotective action of hybrid structures based on fullerene C60]. The neuroprotective action of hybrid structures based on fullerene C60 with attached proline amino acid has been studied. Hybrid structures contained natural antioxidant carnosine or addends with one or two nitrate groups. It has been shown that all studied compounds had antioxidant activity and decreased the concentration of malondialdehyde in homogenates of the rat brain.

Fullerene C60 might be useful for the treatment of Alzheimers disease.

The 2012 publication Fullerene C60 prevents neurotoxicity induced by intrahippocampal microinjection of amyloid-beta peptide reports: The dynamics of the state of hippocampal pyramidal neurons after intrahippocampal microinjections of (1) amyloid-beta25-35 (1.6 nmol/1 microl), (2) an aqueous molecule-colloidal solution of C60 (0.46 nmol/1 microl) and (3) an aqueous molecule-colloidal solution of C60 before amyloid-beta25-35 administration were analysed in rats. This model allowed us to study the role of amyloid-beta25-35 in the pathogenesis of Alzheimers disease and to test anti-amyloid substances. Methods of fluorescent (acridine orange) and brightfield (cresyl violet and immunohistochemistry) microscopy were used. Acridine orange staining indicated changes in protein synthesis intensity due to alterations in the rRNA state of neuron ribosomes. One day after administration of amyloid-beta25-35, the intensity of protein synthesis in the population of morphologically intact cells decreased by 45%. By day 14, degeneration occurred in the majority of pyramidal cells, and amyloid-beta25-35 deposits were observed in the neuronal cytoplasm. In necrotic cells, acridine orange staining of the cytoplasm was drastically increased as a result of RNA degradation rather than due to an increase in protein synthesis. Because amyloid-beta25-35 administration provoked oxidative stress, we assumed that an aqueous molecule-colloidal solution of C60 administered before amyloid-beta25-35 prevented protein synthesis changes on day 1, while acting as an antioxidant, and by day 14 it inhibited neurodegeneration and amyloid-beta25-35 accumulation. Based on the data that an aqueous molecule-colloidal solution of C60 prevented amyloid-beta25-35 aggregation in in vitro experiments and based on our present evidence on the antitoxicity of an aqueous molecule-colloidal solution of C60, we suggest that functionalised C60 prevents/diminishes amyloid-beta25-35 aggregation in vivo as well. Thus, an aqueous molecule-colloidal solution of C60 administered at a low concentration before amyloid-beta2-35, prevented disturbances in protein synthesis, neurodegeneration and formation amyloid-beta25-35 deposits in hippocampal pyramidal neurons in vivo. This evidence gives promise that functionalised C60 can be used to develop anti-amyloid drugs combining antioxidant and anti-aggregative properties.

The 2012 publication [Antiamyloid properties of fullerene C60 derivatives]reports A comparative estimation of the ability of complexes of fullerene C60 with polyvinylpyrrolidone and fullerene C60 derivatives (the sodium salt of the polycarboxylic derivative of fullerene C60, sodium fullerenolate), has been carried out. The fullerenes destroyed amyloid fibrils of the Abeta(1-42) peptide of the brain and the muscle X-protein. A study of the effect of fullerenes on muscle actin showed that complexes of fullerene C60 with polyvinylpyrrolidone and sodium fullerenolate did not prevent the filament formation of actin, nor did they destroy its filaments in vitro. Conversely, sodium salt of the polycarboxylic derivative of fullerene C60 destroyed actin filaments and prevented their formation. It was concluded that sodium fullerenolate and complexes of fullerene C60 with polyvinylpyrrolidone are the most effective antiamyloid compounds among the fullerenes examined.

Fullerenes may enable new anticancer therapies via various mechanisms: one is as a carrier for conventional anticancer drugs; another is enhancing cytotoxic effects of chemotherapy drugs; another yet is based on the anti-cancer activities of the fullerene molecules themselves.

With respect to the first role, as a potential carrier of conventional anti-cancer drugs, the new (November 2012) publication Water-Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo- and Photodynamic Therapeutic Actions reports Prodrug therapy is one strategy to deliver anticancer drugs in a less reactive manner to reduce nonspecific cytotoxicity. A new multifunctional anticancer prodrug system based on water-dispersible fullerene (C60) aggregates is introduced; this prodrug system demonstrates active targeting, pH-responsive chemotherapy, and photodynamic therapeutic (PDT) properties. Incorporating (via a cleavable bond) an anticancer drug, which is doxorubicin (DOX) in this study, and a targeting ligand (folic acid) onto fullerene while maintaining an overall size of approximately 135 nm produces a more specific anticancer prodrug. This prodrug can enter folate receptor (FR)-positive cancer cells and kill the cells via intracellular release of the active drug form. Moreover, the fullerene aggregate carrier exhibits PDT action; the cytotoxicity of the system towards FR-positive cancer cells is increased in response to light irradiation. As the DOX drug molecules are conjugated onto fullerene, the DOX fluorescence is significantly quenched by the strong electron-accepting capability of fullerene. The fluorescence restores upon release from fullerene, so this fluorescence quenching-restoring feature can be used to track intracellular DOX release. The combined effect of chemotherapy and PDT increases the therapeutic efficacy of the DOX-fullerene aggregate prodrug. This study provides useful insights into designing and improving the applicability of fullerene for other targeted cancer prodrug systems.

Another publication, dated 2013, related to use of fullerenes for anti-cancer drug delivery is PEI-derivatized fullerene drug delivery using folate as a homing device targeting to tumor.You can also see (1007) Nanotubes, Nanorods, Nanofibers and Fullerenes for Nanoscale Drug Delivery.

C60 compounds are also promising as delivery vehicles for drugs.

For example, related to myocardial treatments the 2010 publicationThe C60-fullerene porphyrin adducts for prevention of the doxorubicin-induced acute cardiotoxicity in rat myocardial cellsreports: This is a fullerene-based low toxic nanocationite designed for targeted delivery of the paramagnetic stable isotope of magnesium to the doxorubicin (DXR)-induced damaged heart muscle providing a prominent effect close to about 80% recovery of the tissue hypoxia symptoms in less than 24 hrs after a single injection (0.03 0.1 LD50). Magnesium magnetic isotope effect selectively stimulates the ATP formation in the oxygen-depleted cells due to a creatine kinase (CK) and mitochondrial respiratory chain-focusing attack of 25Mg2+ released by nanoparticles. These smart nanoparticles with membranotropic properties release the overactivating cations only in response to the intracellular acidosis. The resulting positive changes in the energy metabolism of heart cell may help to prevent local myocardial hypoxic (ischemic) disorders and, hence, to protect the heart muscle from a serious damage in a vast variety of the hypoxia-induced clinical situations including DXR side effects.

C60 can enhance the cytotoxic action of chemotherapeutic agents against cancer through autophagy.

The 2009 publication Autophagy-mediated chemosensitization in cancer cells by fullerene C60 nanocrystalreports: Autophagy may represent a common cellular response to nanomaterials, and modulation of autophagy holds great promise for improving the efficacy of cancer therapy. Fullerene C60 possesses potent anti-cancer activities, but its considerable toxicity towards normal cells may hinder its practical applications. It has been reported that fullerene C60 induces certain hallmarks of autophagy in cancer cells. Here we show that the water-dispersed nanocrystal of underivatized fullerene C60 (Nano-C60) at noncytotoxic concentrations caused authentic autophagy and sensitized chemotherapeutic killing of both normal and drug-resistant cancer cells in a reactive oxygen species (ROS)-dependent and photo-enhanced fashion. We further demonstrated that the chemosensitization effect of Nano-C60 was autophagy-mediated and required a functional Atg5, a key gene in the autophagy signaling pathway. Our results revealed a novel biological function for Nano-C60 in enhancing the cytotoxic action of chemotherapeutic agents through autophagy modulation and may point to the potential application of Nano-C60 in adjunct chemotherapy.

C60 protects against radiation-induced cell damage

The 2010 publication Dendro[C(60)]fullerene DF-1 provides radioprotection to radiosensitive mammalian cells reports: In this study, the ability of the C(60) fullerene derivative DF-1 to protect radiosensitive cells from the effects of high doses of gamma irradiation was examined. Earlier reports of DF-1s lack of toxicity in these cells were confirmed, and DF-1 was also observed to protect both human lymphocytes and rat intestinal crypt cells against radiation-induced cell death. We determined that DF-1 protected both cell types against radiation-induced DNA damage, as measured by inhibition of micronucleus formation. DF-1 also reduced the levels of reactive oxygen species in the crypt cells, a unique capability of fullerenes because of their enhanced reactivity toward electron-rich species. The ability of DF-1 to protect against the cytotoxic effects of radiation was comparable to that of amifostine, another ROS-scavenging radioprotector. Interestingly, localization of fluorescently labeled DF-1 in fibroblast was observed throughout the cell. Taken together, these results suggest that DF-1 provides powerful protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, and cell death.

See also the 2010 publication The polyhydroxylated fullerene derivative C60(OH)24 protects mice from ionizing-radiation-induced immune and mitochondrial dysfunction.

C60 fullerenes have anti-viral properties and might be useful for preventing or delaying the onset of AIDS.

From (2007) Medicinal applications of fullerenes:Compounds with antiviral activity are generally of great medical interest and different modes of pharmaceutical actions have been described. Replication of the human immunodeficiency virus (HIV) can be suppressed by several antiviral compounds, which are effective in preventing or delaying the onset of acquired immunodeficiency syndrome (AIDS). Fullerenes (C60) and their derivatives have potential antiviral activity, which has strong implications on the treatment of HIV-infection. The antiviral activity of fullerene derivatives is based on several biological properties including their unique molecular architecture and antioxidant activity. It has been shown that fullerenes derivatives can inhibit and make complex with HIV protease (HIV-P) (Friedman et al 1993; Sijbesma et al 1993). Dendrofullerene 1 (Figure 1) has shown the highest anti-protease activity (Brettreich and Hirsch 1998; Schuster et al 2000). Derivative 2, the trans-2 isomer (Figure 1), is a strong inhibitor of HIV-1 replication. The study suggests that relative position (trans-2) of substituents on fullerenes and positive charges near to fullerenes cage provide an antiviral structural activity. Also Amino acid derivatives of fullerene C60 (ADF) are found to inhibit HIV and human cytomegalovirus replication (Kotelnikova et al 2003).

Fullerenes inhibit the allergic response

The 2007 publication Fullerene nanomaterials inhibit the allergic response reports Fullerenes are a class of novel carbon allotropes that may have practical applications in biotechnology and medicine. Human mast cells (MC) and peripheral blood basophils are critical cells involved in the initiation and propagation of several inflammatory conditions, mainly type I hypersensitivity. We report an unanticipated role of fullerenes as a negative regulator of allergic mediator release that suppresses Ag-driven type I hypersensitivity. Human MC and peripheral blood basophils exhibited a significant inhibition of IgE dependent mediator release when preincubated with C(60) fullerenes. Protein microarray demonstrated that inhibition of mediator release involves profound reductions in the activation of signaling molecules involved in mediator release and oxidative stress. Follow-up studies demonstrated that the tyrosine phosphorylation of Syk was dramatically inhibited in Ag-challenged cells first incubated with fullerenes. In addition, fullerene preincubation significantly inhibited IgE-induced elevation in cytoplasmic reactive oxygen species levels. Furthermore, fullerenes prevented the in vivo release of histamine and drop in core body temperature in vivo using a MC-dependent model of anaphylaxis. These findings identify a new biological function for fullerenes and may represent a novel way to control MC-dependent diseases including asthma, inflammatory arthritis, heart disease, and multiple sclerosis.

C60 fullerenes exercise immunomodulary effects.

The 2012 publication [The condition of lipid peroxidation in mice and the effect of fullerene C60 during immune response] reports: The aim of this study was to assess the influence of fullerene C60 on lipid peroxidation (POL) and antioxidant protection during the induction of the immune response to heteroantigen. Balb/c mice were immunized intraperitoneal (i.p.) with sheep erythrocytes for the primary immunization. Water dispersion of fullerene C60 was injected i.p. once at the dose 50 ng to mice on first, third and sixth days after immunization. During immune response, the increment ofmalonic dialdehide (MDA) was enhanced in liver, kidneys and heart tissues. Fullerene C60 induced POL during the latent phase of immune response, but inhibited this process during progression of immune response. Activities of superoxide dismutase (SOD) and catalase in liver and spleen tissues were induced after injection of fullerene C60 to intact mice. After immunization, high level of activity of antioxidant enzymes and low level of organs mass factor were determined. Injection of fullerene C60 reduced the activities of SOD and catalase in spleen tissues. The results of our study indicate that fullerene C60 can display positive effect on POL processes and antioxidant enzymes activity which is probably due to membranes stabilization action or the ability of fullerene C60 to bind free radicals independently.

Another 2012 publication that demonstrates anti-arthritis immunomodulatory activity in rats is [Fullerene C60 exhibits immunomodulatory activity during adjuvant-induced arthritis in rats].The effect of fullerene C60 (FC60) on the immune processes during experimental adjuvant-induced arthritis (AA) in rats has been studied. The results indicate the inhibitory action of FN60 during AA on cellular splenocyte proliferation, neutrophil phagocytic and oxygen-stimulatory activities in the NBT test, and humoral immune mechanisms involved in the production of antinuclear antibodies, formation of circulating immune complexes, and restoration of morphological structure of spleen. Taken together, these results allow FC60 to be considered as a new potential pharmacological agent that can realize its effects mainly through anti-inflammatory and immunomodulatory activity.

C60 fullerenes appear to affect the innate immune system

An august 2012 publication Effect of buckminsterfullerenes o cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cellsreported: C60 nanoparticles, the so-called buckminsterfullerenes, have attracted great attention for medical applications as carriers, enzyme inhibitors or radical scavengers. However, publications evaluating their immunological mechanisms are still rather limited. Therefore, we aimed to analyze systematically the in vitro influence of polyhydroxy-C60 (poly-C60) and N-ethyl-polyamino-C60 (nepo-C60) on peripheral blood mononuclear cells (PBMC) from healthy individuals, angling their effect on proliferation, expression of surface markers, and cytokine production. We isolated PBMC from 20 healthy subjects and incubated them in a first step only with poly-C60 or nepo-C60, and in a second step together with recall antigens (purified protein derivative, tetanus toxoid, bacillus Calmette-Gurin). Proliferation was determined by (3)H-thymidine incorporation, activation of PBMC-subpopulations by flow cytometry by measurement of the activation marker CD69, and secretion of T helper cell type 1 (TH1)- (interferon-gamma [IFN-], tumor necrosis factor beta [TNF-]), TH2- (interleukin-5 [IL-5], -13, -10) and macrophage/monocyte-related cytokines (IL-1, IL-6, TNF-) into the supernatants by enzyme-linked immunosorbent assay. Both fullerenes did not influence T cell reactivity, with no enhanced expression of CD69 and production of T cell cytokines observed, the CD4/CD8 ratio remaining unaffected. In contrast, they significantly enhanced the release of IL-6 and CD69-expression by CD56 positive natural killer cells. PBMC, which had been cultured together with the three recall antigens were not affected by both fullerenes at all. These data indicate that fullerenes do not interact with T cell reactivity but may activate cells of the innate immune system. Furthermore, they seem to act only on nave cells, which have not been prestimulated with recall antigens, there are however, large inter individual differences.

C60 may affect platelet aggregation

A 2012 Russian publicationEffects of fullerene C60 nanocomposites on human platelet aggregationREPORTS: The effects of fullerene C(60) nanocomposites on human platelet aggregation induced by ADP, ristocetin, and collagen were studied. The nanocomposite containing fullerene C(60) in polyvinyl pyrrolidone solution did not change platelet aggregation, while fullerene C(60) in crown ether and Twin-80 solutions inhibited ADP-induced platelet aggregation by 20 and 30%, respectively. I do not know if the study was controlled to take account the effects of the solvents used.

Fullerenes can potentiate hair growth

The 2009 publicationFullerene nanomaterials potentiate hair growthreports Hair loss is a common symptom resulting from a wide range of disease processes and can lead to stress in affected individuals. The purpose of this study was to examine the effect of fullerene nanomaterials on hair growth. We used shaved mice as well as SKH-1 bald mice to determine if fullerene-based compounds could affect hair growth and hair follicle numbers. In shaved mice, fullerenes increase the rate of hair growth as compared with mice receiving vehicle only. In SKH-1 hairless mice fullerene derivatives given topically or subdermally markedly increased hair growth. This was paralleled by a significant increase in the number of hair follicles in fullerene-treated mice as compared with those mice treated with vehicle only. The fullerenes also increased hair growth in human skin sections maintained in culture. These studies have wide-ranging implications for those conditions leading to hair loss, including alopecia, chemotherapy, and reactions to various chemicals.

Less perspective be lost, it is important to keep in mind that the major interests in C60 relate to developing new structural materials and electronic applications.

For these reasons C60 is currently being manufactured in industrial quantities measured in tons and there has been considerable concern about the biological impact of C60 and other fullerenes being released into the environment.

Literature related to the toxicity of C60 comes to mixed conclusions. One the one hand, there has been much general concern about toxicities and long-term biological impacts of fullerenes. And theoretical studies strongly suggest toxic actions of C60 against DNA and other cell components. On the other hand, specific studies of C60 show few or no toxic effects on whole animals. Researchers caution against possible yet-unobserved long-term effects.

The rat longevity study mentioned earlier was basically conducted to measure C60 toxicity, and found little or none. Another 2012 study Sub-acute oral toxicity study with fullerene C60 in ratsreports: To obtain initial information on the possible repeated-dose oral toxicity of fullerene C60, Crl:CD(SD) rats were administered fullerene C60 by gavage once daily at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days, followed by a 14-day recovery period. No deaths occurred in any groups, and there were no changes from controls in detailed clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined at the end of the administration period and at the end of the recovery period. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. There were no changes from controls in the liver and spleen weights at the end of the administration period, but those weights in males in the 1,000 mg/kg/day group increased at the end of the recovery period. Using liquid chromatography-tandem mass spectrometry, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60; however, the slight increases in liver and spleen weights after the 14-day recovery period may be because of the influence of fullerene C60 oral administration. In the future, it will be necessary to conduct a long-term examination because the effects of fullerene C60 cannot be ruled out.

More on the cautious side is the 2009 book chapter Cytotoxicity and Genotoxicity of Carbon Nanomaterials: With the recent development in nanoscience and nanotechnology, there is a pressing demand for assessment of the potential hazards of carbon nanomaterials to humans and other biological systems. This chapter summarizes our recent in vitro cytotoxicity and genotoxicity studies on carbon nanomaterials with an emphasis on carbon nanotubes and nanodiamonds. The studies summarized in this chapter demonstrate that carbon nanomaterials exhibit material-specific and cell-specific cytotoxicity with the general trend for biocompatibility: nanodiamonds > carbon black powders > multiwalled carbon nanotubes > single-walled carbon nanotubes, with macrophages being much more sensitive to the cytotoxicity of these carbon nanomaterials than neuroblastoma cells. However, the cytotoxicity to carbon nanomaterials could be tuned by functionalizing the nanomaterials with different surface groups. Multiwalled carbon nanotubes and nanodiamonds, albeit to a less extend, can accumulate in mouse embryonic stem (ES) cells to cause DNA damage through reactive oxygen species (ROS) generation and to increase the mutation frequency in mouse ES cells. These results point out the great need for careful scrutiny of the toxicity of nanomaterials at the molecular level, or genotoxicity, even for those materials like multiwalled carbon nanotubes and nanodiamonds that have been demonstrated to cause limited or no toxicity at the cellular level.

Despite its apparent benevolence when ingested by rats, C60 and its derivatives solutions when photo-activated can produce singlet oxygen radicals which are biologically damaging.

For example, see Photo-Induced Damages of Cytoplasmic and Mitochondrial Membranes by a [C60]Fullerene Malonic Acid Derivative. On the one hand, the photo-activation properties of C60 appear to make it toxic and dangerous for some aquatic species(ref)(ref)(ref). So, there is serious concern about release of manufactured C60 into natural aquatic environments. On the other hand, there has been thought of exploiting these properties in photo-based anticancer therapies(ref). fullerenes can effectively photoinactivate either or both pathogenic microbial cells and malignant cancer cells. The mechanism appears to involve superoxide anion as well as singlet oxygen, and under the right conditions fullerenes may have advantages over clinically applied photosensitizers for mediating photodynamic therapy of certain diseases(ref). Photo-responsiveness of cells exposed to C60 can be fairly complex(ref).

I strongly suspect that a deeper biological mechanism is involved in the health and longevity-producing effects of C60 despite the prevailing wisdom. As I see it the candidates for these deeper effects of C60 are (1) effects exercised on DNA including impacts on structural configuration, epigenetic gene activation effects, histones and nuclear envelope shape, (2) effects exercised on microtubule structures in cells, (3) effects on mitochondria, and (4) epigenetic impacts such as on histones and DNA methylation.

I cannot prove this suspicion; that will require further research. However I can cite arguments that tend to confirm my suspicion.

(1) C60 is known to bind to and have impact on DNA. While the results of modeling studies indicate toxic effects on DNA, certain effects could possibly be beneficial.

That C60 binds to and deforms DNA has been known for some time. A 2005 publication C60 binds to and deforms nucleotides reported: Atomistic molecular dynamics simulations are performed for up to 20 ns to monitor the formation and the stability of complexes composed of single- or double-strand DNA molecules and C60 in aqueous solution. Despite the hydrophobic nature of C60, our results show that fullerenes strongly bind to nucleotides. The binding energies are in the range -27 to -42 kcal/mol; by contrast, the binding energy of two fullerenes in aqueous solution is only -7.5 kcal/mol. We observe the displacement of water molecules from the region between the nucleotides and the fullerenes and we attribute the large favorable interaction energies to hydrophobic interactions. The features of the DNA-C60 complexes depend on the nature of the nucleotides: C60 binds to double-strand DNA, either at the hydrophobic ends or at the minor groove of the nucleotide. C60 binds to single-strand DNA and deforms the nucleotides significantly. Unexpectedly, when the double-strand DNA is in the A-form, fullerenes penetrate into the double helix from the end, form stable hybrids, and frustrate the hydrogen bonds between end-group basepairs in the nucleotide. When the DNA molecule is damaged (specifically, a gap was created by removing a piece of the nucleotide from one helix), fullerenes can stably occupy the damaged site. We speculate that this strong association may negatively impact the self-repairing process of the double-strand DNA. Our results clearly indicate that the association between C60 and DNA is stronger and more favorable than that between two C60 molecules in water. Therefore, our simulation results suggest that C60 molecules have potentially negative impact on the structure, stability, and biological functions of DNA molecules.

The recent 2012 publicationA large-scale association study for nanoparticle C60 uncovers mechanisms of nanotoxicity disrupting the native conformations of DNA/RNA,a modeling study, reports: Nano-scale particles have attracted a lot of attention for its potential use in medical studies, in particular for the diagnostic and therapeutic purposes. However, the toxicity and other side effects caused by the undesired interaction between nanoparticles and DNA/RNA are not clear. To address this problem, a model to evaluate the general rules governing how nanoparticles interact with DNA/RNA is demanded. Here by, use of an examination of 2254 native nucleotides with molecular dynamics simulation and thermodynamic analysis, we demonstrate how the DNA/RNA native structures are disrupted by the fullerene (C60) in a physiological condition. The nanoparticle was found to bind with the minor grooves of double-stranded DNA and trigger unwinding and disrupting of the DNA helix, which indicates C60 can potentially inhibit the DNA replication and induce potential side effects. In contrast to that of DNA, C60 only binds to the major grooves of RNA helix, which stabilizes the RNA structure or transforms the configuration from stretch to curl. This finding sheds new light on how C60 inhibits reverse transcription as HIV replicates. In addition, the binding of C60 stabilizes the structures of RNA riboswitch, indicating that C60 might regulate the gene expression. The binding energies of C60 with different genomic fragments varies in the range of -56 to -10 kcal mol(-1), which further verifies the role of nanoparticle in DNA/RNA damage. Our findings reveal a general mode by which C60 causes DNA/RNA damage or other toxic effects at a systematic level, suggesting it should be cautious to handle these nanomaterials in various medical applications.

A 2011 publication DNA Exposure to Buckminsterfullerene (C60): Toward DNA Stability, Reactivity, and Replicationconveys a somewhat different story, indicating that fullernols not only have major impacts on the structures and biological properties of DNA, but also that they can contribute remarkably to DNA stability against thermal degredation.

Buckminsterfullerene (C60) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C60 could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNAC60 binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C60 aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 7085 C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C60 on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.

The general topic of nanopartucles binding is covered in a 2012 review publication Prospects of nanoparticleDNA binding and its implications in medical biotechnology. This remains a very new and immature area of research.

Right now it seems fair to conclude that C60 is very likely to bind to and interact with DNA/RNA, but the macroscopic outcomes of such interactions are unknown. There does seem to be contradictions between rodent studies that suggest no overall toxic effects of C60 and the molecular-chemical studies which suggest that C60 could play havoc with DNA.

(2) C60 is known to affect the formation and durability of microtubules.

First of all, a little on microtubules for those not familiar with them. Although almost never mentioned in the longevity literature they are critical to health and longevity. According to Wikipedia, Microtubules are a component of the cytoskeleton. These cylindrical polymers of tubulin can grow as long as 25 micrometers and are highly dynamic. The outer diameter of microtubule is about 25 nm. Microtubules are important for maintaining cell structure, providing platforms for intracellular transport, forming the mitotic spindle, as well as other cellular processes.[1] There are many proteins that bind to microtubules, including motor proteins such as kinesin and dynein, severing proteins like katanin, and other proteins important for regulating microtubule dynamics Microtubules are part of a structural network (the cytoskeleton) within the cells cytoplasm. The primary role of the microtubule cytoskeleton is mechanical. However, in addition to structural support, microtubules also take part in many other processes. A microtubule is capable of growing and shrinking in order to generate force, and there are also motor proteins that allow organelles and other cellular factors to be carried along a microtubule. This combination of roles makes microtubules important for organising cell layout. A notable structure involving microtubules is the mitotic spindle used by most eukaryotic cells to segregate their chromosomes correctly during cell division. The process of mitosis is facilitated by a subgroup of microtubules known as astral microtubules, defined as a microtubule originating from the centrosome that does not connect to a kinetochore. Astral microtubules develop in the actin skeleton and interact with the cell cortex to aid in spindle orientation. They are organized into radial arrays around the centrosomes. The turn-over rate of this population of microtubules is higher than that of any other population. Astral microtubules function in concert with specialized dynein motors, which are oriented with the light chain portion attached to the cell membrane and the dynamic portion attached to the microtubule. This allows for dynein contraction to pull the centrosome toward the cell membrane, thus assisting in cytokinesis. Astral microtubules are not required for the progression of mitosis, but they are required to ensure the fidelity of the process; they are required for the correct positioning and orientation of the mitotic spindle apparatus. They are also involved in determination of cell division site based on the geometry and polarity of the cells (ref).[2][3]

Image source

Microtubules and microfiliments

I first discussed microtubules in my blog entry Quantum Biology. There I pointed out how some quantum biologists argue that there is yet-another role for microtubules they are quantum computers possibly exercising command and control functions for cell processes. In fact it is known that microtubules are semiconductors as are certain arrays of fullerenes. However, the quantum computer role for microtubules remains controversial. For now, it is enough to know that microtubules are important for cell structure and are main rail lines for transport of molecules within cells.

The 2004 publication In Vitro and In Vivo Investigation of Collagen C60(OH)24 Interactionargues that fullerole affects intermolecular communications from collegen fibers through integrines and microtubules to cell nucleus.

A 2011 publication In vitro polymerization of microtubules with a fullerene derivative reports that a fullerene C60 derivative inhibits the polymerization of tubulin and therefore inhibits the formation of new microtubules. Fullerene derivative C(60)(OH)(20) inhibited microtubule polymerization at low micromolar concentrations. The inhibition was mainly attributed to the formation of hydrogen bonding between the nanoparticle and the tubulin heterodimer, the building block of the microtubule, as evidenced by docking and molecular dynamics simulations. Our circular dichroism spectroscopy measurement indicated changes in the tubulin secondary structures, while our guanosine-5-triphosphate hydrolysis assay showed hindered release of inorganic phosphate by the nanoparticle. Isothermal titration calorimetry revealed that C(60)(OH)(20) binds to tubulin at a molar ratio of 9:1 and with a binding constant of 1.3 0.16 10(6) M(-1), which was substantiated by the binding site and binding energy analysis using docking and molecular dynamics simulations. Our simulations further suggested that occupancy by the nanoparticles at the longitudinal contacts between tubulin dimers within a protofilament or at the lateral contacts of the M-loop and H5 and H12 helices of neighboring tubulins could also influence the polymerization process. This study offered a new molecular-level insight on how nanoparticles may reshape the assembly of cytoskeletal proteins, a topic of essential importance for illuminating cell response to engineered nanoparticles and for the advancement of nanomedicine. An in-vitro result, it suggests the opposite of a health-producing effect of C60 on microtubules.

Again, the interactions of C60 with cell microtubules and their creation and destruction appear to be not well understood. It seems such interactions do exist. Although modeling studies suggest that the macroscopic results of such interactions may be toxic rather than health-producing, we just dont know for sure.

(3) C60 buckballs cross cell barriers and preferentially localize themselves in mitochondria. There, they exercise powerful antioxidant effects and possibly other effects as well.

When fullerene is derivatized with polar groups, as in case of polyhydroxylated fullerenes (fullerenol) and C60 tris(malonic)acid, they become water soluble enabling them to cross the cell membrane and localize preferentially to mitochondria (Foley et al 2002; Youle and Karbowski 2005), which generate great masses of cellular oxygen free radicals. This phenomenon makes them useful for a variety of medical applications (Tsai et al 1997; Lotharius et al 1999; Bisaglia et al 2000). These radical scavengers have shown to protect cell growth from various toxins that can induce apoptotic injuries in vitro (Lin et al 1999; Lin et al 2002; Chen et al 2004) in different cell types such as neuronal cells (Dugan et al 1997; Bisaglia et al 2000), hepatoma cells (Huang et al 1998), or epithelial cells (Straface et al 1999).(ref)

Does C60 do more in the microchondria than act as a super anti-oxidant? Or does the super antioxidant power of C60 create permanent changes in the mitochondria? If the research literature is indicative, no one has so far grappled with these questions or even asked them for that matter.

Final comments

I could quote and discuss here only a small but hopefully representative sample of the unfolding literature related to C60 and its biological impacts. The rodent longevity studies are tantalizing but tiny and hopefully will be soon followed by much larger ones. There appear to be some basic contradictions and many more basic questions are raised than those answered. For rodents at least, far from being toxic pure C60 appears to be not only benevolent but life-extending. On the other hand, mostly-theoretical studies of the likely impacts of C60 on DNA and on microtubules and cell morphology suggest that C60 may generate all kinds of havoc on the cell level. Without question C60 is a powerful antioxidant. However it tends to generate permanent longevity-enhancing changes and it is not at all clear how an antioxidant could do that? How does it work to so grossly extend longevity? Are there other means through which C60 works its health and longevity benefits, and if so, what are they?

The literature references I have been able to surface seemed to focus on the lipid membrane and antioxidant and other chemical properties of C60 mostly 1990s ways of looking at biological mechanisms which are valid but limited. The research literature so far seems to be remarkably silent on certain issues that could turn out to be key: C60 and DNA methylation, impacts of C60 on histones, C60 and the DNA repair machinery, C60 as related to stem cells, C60 and siRNAs, and C60 as related to key known aging pathways. It the longevity impacts of C60 hold up, there are important layers of knowledge here yet to be revealed. If this were an archeological dig, we have so far only gone down a foot or two.

Here is the original post:
Buckyballs, health and longevity state of knowledge ...

This entry was posted on October 25, 2018 at 9:43 am and is filed under Longevity. You can follow any responses to this entry through the RSS 2.0 feed. Both comments and pings are currently closed. |