Nanocarbons for Biology and Medicine: Sensing, Imaging, and Drug Delivery.

Nanocarbons with different dimensions (e.g., 0D fullerenes and carbon nanodots, 1D carbon nanotubes and graphene nanoribbons, 2D graphene and graphene oxides, and 3D nanodiamonds) have attracted enormous interest for applications ranging from electronics, optoelectronics, and photovoltaics to sensing, bioimaging, and therapeutics due to their unique physical and chemical properties. Among them, nanocarbon-based theranostics (i.e., therapeutics and diagnostics) is one of the most intensively studied applications, as these nanocarbon materials serve as excellent biosensors, versatile drug/gene carriers for specific targeting in vivo, effective photothermal nanoagents for cancer therapy, and promising fluorescent nanolabels for cell and tissue imaging. This review provides a systematic overview of the latest theranostic applications of nanocarbon materials with a comprehensive comparison of the characteristics of different nanocarbon materials and their influences on theranostic applications. We first introduce the different carbon allotropes that can be used for theranostic applications with their respective preparation and surface functionalization approaches as well as their physical and chemical properties. Theranostic applications are described separately for both in vitro and in vivo systems by highlighting the protocols and the studied biosystems, followed by the toxicity and biodegradability implications. Finally, this review outlines the design considerations for nanocarbon materials as the key unifying themes that will serve as a foundational first principle for researchers to study, investigate, and generate effective, biocompatible, and nontoxic nanocarbon materials-based models for cancer theranostics applications. Finally, we summarize the review with an outlook on the challenges and novel theranostic protocols using nanocarbon materials for hard-to-treat cancers and other diseases. This review intends to present a comprehensive guideline for researchers in nanotechnology and biomedicine on the selection strategy of nanocarbon materials according to their specific requirements.

Nanoparticles in dermatologic surgery.

Nanotechnology is an emerging branch of science that involves the engineering of functional systems on the nanoscale (1-100 nm). Nanotechnology has been used in biomedical and therapeutic agents with the aim of providing novel treatment solutions where small molecule size may be beneficial for modulation of biologic function. Recent investigation in nanomedicine has become increasingly important to cutaneous pathophysiology, such as functional designs directed towards skin cancers and wound healing. This review outlines the application of nanoparticles relevant to dermatologic surgery.

Disposition of fullerene C60 in rats following intratracheal or intravenous administration.

Fullerene C60 is used in a variety of industrial and consumer capacities. As part of a comprehensive evaluation of the toxicity of fullerene C60 by the National Toxicology Program, the disposition following intratracheal (IT) instillation and intravenous (IV) administration of 1 or 5 mg/kg b.wt. fullerene C60 was investigated in male Fischer 344 rats. Following IT instillation, fullerene C60 was detected in the lung as early as 0.5 h post-exposure with minimal clearance over the 168 h period; the concentration increased ≥20-fold with a 5-fold increase in the dose. Fullerene C60 was not detected in extrapulmonary tissues. Following IV administration, fullerene C60 was rapidly eliminated from the blood and was undetectable after 0.5 h post-administration. The highest tissue concentrations of fullerene C60 occurred in the liver, followed by the spleen, lung and kidney. Fullerene C60 was cleared slowly from the kidney and the lung with estimated half-lives of 24 and 139 h, respectively. The liver concentration of fullerene C60 did not change much with time; over 90% of the fullerene C60 remained there over the study duration up to 168 h. Fullerene C60 was also not detected in urine or feces. These data support the hypothesis that fullerene C60 accumulates in the body and therefore has the potential to induce detrimental health effects following exposure.

Fullerenol C60(OH)36 at relatively high concentrations impairs hippocampal theta oscillations (in vivo and in vitro) and triggers epilepsy (in vitro) - A dose response study.

Since the first identification of fullerenes (C60) and their synthesis in 1985, those compounds have been extensively studied in the biomedical field. In particular, their water-soluble derivatives, fullerenols (C60(OH)n, n = 2-48), have recently been the subject of numerous investigations concerning their antioxidant and prooxidant properties in biological systems. A small fraction of that research has focused on the possible use of C60 and C60(OH)n in neuroscience and the therapy of pathologies such as dementia, amyloid-ß (Aß) formation, and Parkinson's disease. However, only a few studies have focused on their direct effects on neuronal network viability and excitability, especially with the use of electrophysiological and electrochemical approaches. Therefore, we addressed the issue of the direct effect of hydroxylated fullerene nanoparticles C60(OH)36 on local field potentials at the hippocampal formation (HPC) level. With the use of in vitro hippocampal formation slices as a stable model of inducing theta oscillations, and an in vivo model of an anesthetized rat, herein we provide the first convergent electropharmacological evidence that C60(OH)36 at relatively high concentrations (60 µM and 80 µM in vitro; 0.2 µg/µl in vivo) is capable of attenuating the amplitude, power, and frequency of theta oscillations in the HPC neuronal network. At the same time, lower concentrations did not induce any apparent changes. Theta band oscillations constitute a key physiological phenotypic property, which served here as a sensitive assay enabling the study of neural network excitability. Moreover, we report that C60(OH)36 at the concentrations of 60 µM and 80 µM is capable of producing epilepsy in the HPC in vitro, which suggests that C60(OH)n, when applied at higher doses, may have a deleterious effect on the functioning of neuronal networks.

Small size fullerenol nanoparticles inhibit thrombosis and blood coagulation through inhibiting activities of thrombin and FXa.

Thrombus is one of main causes of death in the world and also a vital trouble of biomaterials application in vivo. Recently, effect of fullerenol nanomaterials on anticoagulation was found in our research through extension of bleeding times in treated Sprague-Dawley rats via intravenous injection. Inhibiting of fullerenols on thrombosis was ascertained further by thromboembolism model. Effects of fullerenols on intrinsic and extrinsic pathway were distinct in prolonging activated partial thromboplastin time and prothrombin time, which supported that fullerenols induced defects in both pathways. Inhibited activities of activated coagulation factor X (FXa) and thrombin were verified by experiments in vitro and AutoDock Vina. The results suggest that fullerenols depending on small size and certainly surface property occupied the active domain of FXa and thrombin to block their activity; further, thrombosis was inhibited. This putative mechanism offers an insight into how fullerenol NPs were utilized further in biomedical applications.

Inhalable gadofullerenol/[70] fullerenol as high-efficiency ROS scavengers for pulmonary fibrosis therapy.

Pulmonary fibrosis has become a fatal disease for its high incidence and few effective drugs available in clinic. In this study, gadofullerenol (GF-OH) and [70] fullerenol (C70-OH) nanoparticles (NPs) prepared by a one-pot reaction were designed as nanomedicines to treat this fatal disease. It was revealed that the inhalation of gadofullerenols and [70] fullerenols substantially alleviates the collagen deposition induced by acute lung injury. Based on detailed studies of oxidative stress parameters and transforming growth factor-ß1 (TGF-ß1), we demonstrated they owned the antioxidant and anti-inflammatory functions for the modulation of ROS-mediated inflammation process. Thus the therapeutic effect may be associated with synergistic mechanism of scavenging free radicals and indirectly modulating TGF-ß1 expression. Moreover, GF-OH NPs were observed to show the superiority to C70-OH NPs both in vitro and in vivo due to the structural distinction. These results suggest the inhalable fullerenols are highly potential for clinical therapy of pulmonary fibrosis.

Diet-sourced carbon-based nanoparticles induce lipid alterations in tissues of zebrafish (Danio rerio) with genomic hypermethylation changes in brain.

With rising environmental levels of carbon-based nanoparticles (CBNs), there is an urgent need to develop an understanding of their biological effects in order to generate appropriate risk assessment strategies. Herein, we exposed zebrafish via their diet to one of four different CBNs: C60 fullerene (C60), single-walled carbon nanotubes (SWCNT), short multi-walled carbon nanotubes (MWCNTs) or long MWCNTs. Lipid alterations in male and female zebrafish were explored post-exposure in three target tissues (brain, gonads and gastrointestinal tract) using 'omic' procedures based in liquid chromatography coupled with mass spectrometry (LC-MS) data files. These tissues were chosen as they are often target tissues following environmental exposure. Marked alterations in lipid species are noted in all three tissues. To further explore CBN-induced brain alterations, Raman microspectroscopy analysis of lipid extracts was conducted. Marked lipid alterations are observed with males responding differently to females; in addition, there also appears to be consistent elevations in global genomic methylation. This latter observation is most profound in female zebrafish brain tissues post-exposure to short MWCNTs or SWCNTs (P < 0.05). This study demonstrates that even at low levels, CBNs are capable of inducing significant cellular and genomic modifications in a range of tissues. Such alterations could result in modified susceptibility to other influences such as environmental exposures, pathology and, in the case of brain, developmental alterations.

C60 fullerene as promising therapeutic agent for correcting and preventing skeletal muscle fatigue.

BACKGROUND: Bioactive soluble carbon nanostructures, such as the C60 fullerene can bond with up to six electrons, thus serving by a powerful scavenger of reactive oxygen species similarly to many natural antioxidants, widely used to decrease the muscle fatigue effects. The aim of the study is to define action of the pristine C60 fullerene aqueous colloid solution (C60FAS), on the post-fatigue recovering of m. triceps surae in anaesthetized rats. RESULTS: During fatigue development, we observed decrease in the muscle effort level before C60FAS administration. After the application of C60FAS, a slower effort decrease, followed by the prolonged retention of a certain level, was recorded. An analysis of the metabolic process changes accompanying muscle fatigue showed an increase in the oxidative stress markers H 2 O 2 (hydrogen peroxide) and TBARS (thiobarbituric acid reactive substances) in relation to the intact muscles. After C60FAS administration, the TBARS content and H 2 O 2 level were decreased. The endogenous antioxidant system demonstrated a similar effect because the GSH (reduced glutathione) in the muscles and the CAT (catalase) enzyme activity were increased during fatigue. CONCLUSIONS: C60FAS leads to reduction in the recovery time of the muscle contraction force and to increase in the time of active muscle functioning before appearance of steady fatigue effects. Therefore, it is possible that C60FAS affects the prooxidant-antioxidant muscle tissue homeostasis, subsequently increasing muscle endurance.

[Fullerene Doxorubicin Nanotransporter for Target Interaction with mutated gene BRCA2]. / Fullerenový nanotransportér doxorubicinu pro cílenou interakci s mutovaným genem BRCA2.

BACKGROUND: Malignant breast tumors are in developed countries, the most common cancer affecting mainly women. It is estimated that approximately 5-10% of breast cancers are conditioned by genetic family burdens, caused by mutation in the BRCA2 gene. In the course of the treatment doxorubicin is frequently used therapeutics. Despite its therapeutic efficacy, however, it shows high cardiotoxicity. Possibility to increase the therapeutic window, represent nanotransporters. Fullerenes are nanoparticles composed of carbon atoms whose physical-chemical properties indicate high stability. The complex of fullerene and doxorubicin enables the targeted method for the treatment. The aim of this work is to develop a nanotransporter system with an expected cytostatic effect without significant toxic effects. MATERIAL AND METHODS: To 5mg of fullerene 0.5ml of distilled water was added and solution was subsequently placed for 30 min in an ultrasonic bath (50 W). Fullerenes with bound doxorubicin (DOXO) were purified from unbound DOXO by centrifugation (16,000g). For DOXO analysis acetate buffer was used. Fe2O3-NPs were prepared by reduction with borohydride and ammonia. Thereafter Fe2O3-AuNPs were prepared by thermal synthesis. RESULTS: Carbon nanotransporter (fullerene) for binding of doxorubicin (FULLER-DOXO) was designed and subsequently studied by biophysical methods. We have found that FULLER-DOXO size is larger than 100 nm and the zeta potential is around 24 mV. DOXO, interacts with FULLER by the electrostatic interaction, and its volume increases with the applied concentration (R2 = 0.96). In the following experiment FULLER-DOXO was modified with oligodeoxynucleotide (ODN; 10 µg/ml), and this way was FULLER-DOXO-ODN1 complex prepared. Bound ODN represents a specific sequence for targeting the complex to a point mutation in the BRCA2 gene. In order to prove the interaction magnetic gold nanoparticle modified with the complementary sequence to the test nanotransporter was designed (ODN2-Fe2O3-AuNPs). Formed complex (FULLER-DOXO-ODN1-ODN2-Fe2O3-AuNPs) was subsequently confirmed by several independent techniques. CONCLUSION: We assume that the proposed nanoconstruct will be able to use for genetic targeting of anticancer drug.Key words: doxorubicin - breast cancer - fullerenes - magnetic gold nanoparticles The work was realized with the support of the project NANO LPR 2017 Liga proti rakovine Praha and The European Technology Platform for Nanomedicine. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 3. 2017Accepted: 26. 3. 2017.

Surface modification-mediated biodistribution of ¹³C-fullerene C60 in vivo.

BACKGROUND: Functionalization is believed to have a considerable impact on the biodistribution of fullerene in vivo. However, a direct comparison of differently functionalized fullerenes is required to prove the hypothesis. The purpose of this study was to investigate the influences of surface modification on the biodistribution of fullerene following its exposure via several routs of administration. METHODS: (13)C skeleton-labeled fullerene C60 ((13)C-C60) was functionalized with carboxyl groups ((13)C-C60-COOH) or hydroxyl groups ((13)C-C60-OH). Male ICR mice (~25 g) were exposed to a single dose of 400 µg of (13)C-C60-COOH or (13)C-C60-OH in 200 µL of aqueous 0.9% NaCl solution by three different exposure pathways, including tail vein injection, gavage and intraperitoneal exposure. Tissue samples, including blood, heart, liver, spleen, stomach, kidneys, lungs, brain, large intestine, small intestine, muscle, bone and skin were subsequently collected, dissected, homogenized, lyophilized, and analyzed by isotope ratio mass spectrometry. RESULTS: The liver, bone, muscle and skin were found to be the major target organs for C60-COOH and C60-OH after their intravenous injection, whereas unmodified C60 was mainly found in the liver, spleen and lung. The total uptakes in liver and spleen followed the order: C60 > > C60-COOH > C60-OH. The distribution rate over 24 h followed the order: C60 > C60-OH > C60-COOH. C60-COOH and C60-OH were both cleared from the body at 7 d post exposure. C60-COOH was absorbed in the gastrointestinal tract following gavage exposure and distributed into the heart, liver, spleen, stomach, lungs, intestine and bone tissues. The translocation of C60-OH was more widespread than that of C60-COOH after intraperitoneal injection. CONCLUSIONS: The surface modification of fullerene C60 led to a decreased in its accumulation level and distribution rate, as well as altering its target organs. These results therefore demonstrate that the chemical functionalization of fullerene had a significant impact on its translocation and biodistribution properties. Further surface modifications could therefore be used to reduce the toxicity of C60 and improve its biocompatibility, which would be beneficial for biomedical applications.

Polyhydroxylated fullerenols regulate macrophage for cancer adoptive immunotherapy and greatly inhibit the tumor metastasis.

Adoptive immunotherapy is a highly effective approach for cancer treatment. Several potential adoptive immunotherapies have high (though reversible) toxicities with disappointing results. Polyhydroxylated fullerenols have been demonstrated as promising antitumor drugs with low toxicities. In this study, we investigate whether polyhydroxylated fullerenols (C60(OH)22 and Gd@C82(OH)22) contribute to cancer immunotherapy by regulating macrophages. Our results show that fullerenols treatment enhances mitochondrial metabolism, phagocytosis and cytokine secretion. Moreover, activated macrophages inhibit the growth of several cancer cell types. It is likely that this inhibition is dependent on an NF-κB-mediated release of multiple cytokines. Using a lung metastasis model, we also show that autologous macrophages greatly suppress cancer cell metastasis to lung when they are activated by C60(OH)22 and Gd@C82(OH)22. More importantly, Gd@C82(OH)22 are shown to have stronger ability than C60(OH)22 to improve the macrophage function, which shed light on the rational design for nanomedicine and clinical application. FROM THE CLINICAL EDITOR: The interest in the use of immunotherapy in cancer has rekindled recently. However, many approaches have shown disappointing results. In this study, the authors investigated the effects of polyhydroxylated fullerenol nanoparticles on regulating macrophages for immunotherapy. These positive findings may point a novel way to cancer treatment.

Enhanced brain penetration of hexamethonium in complexes with derivatives of fullerene C60.

The present report describes development of hexamethonium complexes based on fullerene C60. Hexamethonium has a limited penetration into CNS and therefore can antagonize central effects of nicotine only when given at high doses. In the present studies conducted in laboratory rodents, intraperitoneal administration of hexamethonium-fullerene complexes blocked effects of nicotine (convulsions and locomotor stimulation). When compared to equimolar doses of hexamethonium, complexes of hexamethonium with derivatives of fullerene C60 were 40 times more potent indicating an enhanced ability to interact with central nicotine receptors. Thus, fullerene C60 derivatives should be explored further as potential carrier systems for polar drug delivery into CNS.

Synthesis and evaluation of anticancer activity in cells of novel stoichiometric pegylated fullerene-doxorubicin conjugates.

PURPOSE: To synthesize pegylated stoichiometrically and structurally well-defined conjugates of fullerene (C60) with doxorubicin (DOX) and investigate their antiproliferative effect against cancer cell lines. METHODS: Stoichiometric (1:1 and 1:2) pegylated conjugates of C60 with DOX were synthesized using the Prato reaction to create fulleropyrrolidines equipped with a carboxyl function for anchoring a polyethylene glycol (PEG) moiety and either a hydroxyl group for attaching one molecule of DOX or a terminal alkyne group for attaching two molecules of DOX through a click reaction. In both conjugates, the DOX moieties are held through a urethane-type bond. Drug release was studied in phosphate buffer (PBS, pH 7.4) and MCF-7 cancer cells lysate. The uptake of the conjugates by MCF-7 cancer cells and their intracellular localization were studied with fluorescence microscopy. The antiproliferative activity of the conjugates was investigated using the WST-1 test. RESULTS: One or two DOX molecules were anchored on pegylated C60 particles to form DOX-C60-PEG conjugates. Drug liberation from the conjugates was significantly accelerated in the presence of tumor cell lysate compared to PBS. The conjugates could be internalized by MCF-7 cells. DOX from the conjugates exhibited much delayed, compared to free DOX, localization in the nucleus and antiproliferative activity. CONCLUSION: Pegylated DOX-C60 conjugates (1:1) and (2:1) with well-defined structure were successfully synthesized and found to exhibit comparable, but with a delayed onset, antiproliferative activity with free DOX against MCF-7 cancer cells. The results obtained justify further investigation of the potential of these conjugates as anticancer nanomedicines.

Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies.

A family of N-methylpyrrolidinium fullerene iodide salts has been intensively studied to determine their applicability in antimicrobial photodynamic therapy (APDT). This study examined in vitro the efficacy of a C60 fullerene functionalized with one methylpyrrolidinium group to kill upon irradiation with white light gram-negative and gram-positive bacteria, as well as fungal cells, and the corresponding mechanism of the fullerene bactericidal action. The in vitro studies revealed that the high antistaphylococcal efficacy of functionalized fullerene could be linked to their ability to photogenerate singlet oxygen and superoxide anion. Following Staphylococcus aureus photoinactivation, no modifications of its genomic DNA were detected. In contrast, photodamage of the cell envelope seemed to be a dominant mechanism of bactericidal action. In in vivo studies, a 2 log10 reduction in the average bioluminescent radiance between treated and non-treated mice was reached. One day post APDT treatment, moist and abundant growth of bacteria could be observed on wounds of non-fulleropyrrolidine and dark control mice. APDT-treated wounds stayed visibly clear up to the third day. Moreover, cytotoxicity test on human dermal keratinocytes revealed great safety of using the sensitizer toward eukaryotic cells. These data indicate potential application of functionalized fullerene as antistaphylococcal sensitizer for superficial infections.

Distribution and biomarker of carbon-14 labeled fullerene C60 ([(14) C(U)]C60 ) in pregnant and lactating rats and their offspring after maternal intravenous exposure.

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon-14 labeled C60 ([(14) C(U)]C60 ). Rats were administered [(14) C(U)]C60 (~0.2 mg [(14) C(U)]C60 kg(-1) body weight) or 5% polyvinylpyrrolidone (PVP)-saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was < 2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [(14) C(U)]C60 exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [(14) C(U)]C60 exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [(14) C(U)]C60 exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl-tRNA biosynthesis. This study demonstrated that [(14) C(U)]C60 crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk.

Analysis of toxicity biomarkers of fullerene C60 nanoparticles by confocal fluorescent microscopy.

The methods of laser confocal microscopy were employed to study the changes in rat target organs (iliac mucosa and liver) provoked by peroral administration of dispersion of nanosized (31 nm) multimolecular fullerene C60 particles in doses of 0.1, 1.0, and 10 mg/kg body weight over 92 days. The micropreparations were selectively stained with fluorescent dyes to mark the cell nuclei (DAPI), actin microfilaments (fluorescently labeled phalloidin), and the membrane proteins CD106, CD31, and claudins in tight junctions (fluorescently labeled monoclonal antibodies). In rats treated with fullerene in the examined doses, the iliac mucosa demonstrated normal morphology of the villi. There were no signs of inflammation and no alterations in the actin fi laments of cytoskeleton and in enterocytic tight junctions. The count of CD106(+) and CD31(+) cells did not change. The highest examined doses of fullerene (1 and 10 mg/kg body weight) increased population and modified distribution of hepatic CD106(+) cells. They also resulted in accumulation of cytoplasmic granules presumably identified as Kupffer macrophages without any signs of visible inflammation or necrotic areas. This phenomenon can reflect the early stages of toxic reaction being a sensitive bioindicator of the damage produced by administered fullerene C60 in the hepatic tissue.

[EFFECTIVENESS OF FULLERENE-(TRIS-AMINOCAPRONIC ACID) HYDRATE IN THE MODEL OF EXPERIMENTAL VIRAL-BACTERIAL PNEUMONIA OF MICE].

AIM: Study the effectiveness of the substance and various drug formulations of fullerene-(tris-aminocapronic acid) hydrate (FTAAH onwards) in the model of experimental viral-bacterial pneumonia of mice. MATERIALS AND METHODS: BALB/c mice were infected with influenza virus A/California/04/2009 and subsequently infected with Staphylococcus aureus. The animals were treated after viral infection with the substance and various drug forms of FTAAH, as well as comparative preparations--oseltamivir and arbidol. Therapy effectiveness was evaluated by clinical indicators (survival, lifespan, animal mass decrease reduction), virological (virus titer), microbiological (density of bacteria in lungs) parameters, confirmed by pathomorphological characteristics of lungs. RESULTS: FTAAH therapy in injectable form was effective in the model of a combined viral-bacterial pneumonia of mice by all the studied criteria: treatment increased mice survival, reduced the decrease of their body weight, resulted in a reduction of virus titers and density of bacteria in lungs, that correlated with the data from morphological study and signs of bronchopneumonia resolution in mice. FTAAH therapy in rectal form depended on animal infection schemes, as well as preparation dose, increasing with its increase. CONCLUSION: FTAAH substance is effective in the model of experimental viral-bacterial pneumonia of mice.

The effects of hydrated C(60) fullerene on gene expression profile of TRPM2 and TRPM7 in hyperhomocysteinemic mice.

BACKGROUND: Hyperhomocysteinemia (HHcy) is associated with neurodegenerative diseases. Transient receptor potential melastatin (TRPM2) and TRPM7 channels may be activated by oxidative stress. Hydrated C(60) fullerene (C(60)HyFn) have recently gained considerable attention as promising candidates for neurodegenerative states. We aimed to examine the effects on TRPM2 and TRPM7 gene expression of C(60)HyFn due to marked antioxidant activity in HHcy mice. METHODS: C57BL/6 J. mice were divided into four groups: (1) Control group, (2) HHcy, (3) HHcy + C(60)HyFn-treated group and (4) C(60)HyFn-treated group. TRPM2 and TRPM7 gene expression in brains of mice were detected by real-time PCR, Western blotting and immunohistochemistry. Apoptosis in brain were assessed by TUNEL staining. RESULTS: mRNA expression levels of TRPM2 were significantly increased in HHcy group compared to the control group. C(60)HyFn administration significantly decreased serum levels of homocysteine and TRPM2 mRNA levels in HHcy + C(60)HyFn group. Whereas, HHcy-treatment and C(60)HyFn administration did not change the expression of TRPM7. CONCLUSION: Administration of C(60)HyFn in HHcy mice significantly reduces serum homocysteine level, neuronal apoptosis and expression level of TRPM2 gene. Increased expression level of TRPM2 induced by oxidative stress might be involved in the ethiopathogenesis of HHcy related neurologic diseases.

Photodynamic anti-cancer effects of fullerene [C60]-PEG complex on fibrosarcomas preferentially over normal fibroblasts in terms of fullerene uptake and cytotoxicity.

A water-soluble complex of fullerene [C60]:polyethylene glycol (PEG) (1:350 wt/wt) (C60-PEG), but not PEG alone, was found in the present study by ESR/DMPO spin-trap method to generate hydroxyl radicals 6.5-fold as abundant as the non-irradiation level, when irradiated with visible light (400-600 nm, 140 J/cm(2): 450-fold as intense as in average outdoor), but not to generate without irradiation. At 3 h after irradiation with C60-PEG, human fibrosarcoma cells HT1080 were obviously degenerated together with diminished microvilli, cell shrinkage and cell fragmentation as observed by SEM and were shown either for increased cytotoxicity by dual stains with calcein-AM and propidium iodide or for nuclear condensation and fragmentation by Hoechst 33342 stain, any of which were, in contrast, scarcely changed in normal human fibroblastic cells DUMS16 derived from the same connective tissue type as HT1080 cells. Under the conditions, the maximum intracellular uptake amount was more abundant for HT1080 cells than for DUMS16 cells, either by immunostain/fluorography using polyclonal antibody against fullerene [C60], or by HPLC method indicating the 2.4-fold preferential uptake of C60-PEG into HT1080 cells, suggested to greater phagocytotic ability characteristic of cancer cells, over DUMS16 cells being non-macrophage-like normal cells. Thus, C60-PEG is expected as a photosensitizer for photodynamic therapy with scarce side effects to normal cells and preferential reactive oxygen species generation in cancer cells.

Reduction of conspicuous facial pores by topical fullerene: possible role in the suppression of PGE2 production in the skin.

BACKGROUND: Conspicuous facial pores are therapeutic targets for cosmeceuticals. Here we examine the effect of topical fullerene on conspicuous facial pores using a new image analyser called the VISIA® system. Ten healthy Japanese females participated in this study, and they received applications of 1% fullerene lotion to the face twice a day for 8 weeks. FINDINGS: Fullerene lotion significantly decreased conspicuous pores by 17.6% (p < 0.05, Wilcoxon signed-rank test) after an 8-week treatment. A self-administered questionnaire indicated that this reduction achieved cosmetically appreciable effects. In addition, to investigate the mechanism of effect of fullerene, we examined its effect on UVB-induced prostaglandin E2 (PGE2) production in reconstructed human epidermis (RhE). The results showed that irradiation of RhE with 1000 mJ/cm2 increased PGE2 production by 62.3% (p < 0.05, Mann-Whitney U-test) and the addition of 28 µM fullerene significantly suppressed the UVB-induced PGE2 production by 18.3% (p < 0.05). CONCLUSIONS: Fullerene lotion significantly decreases conspicuous facial pores after an 8-week treatment possibly through the suppression of PGE2 production in the epidermis.