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/cm²: 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.     

Study of cytotoxicity of fullerene C60 derivatives

We investigated the cytotoxicity of the fullerene C₆₀ derivatives. We showed that complexes of C₆₀ fullerene with polyvinylpyrrolidone (m.w. of polyvinylpyrrolidone 10000 and 25000), C₆₀-NO₂-proline and C₆₀-alanine had no toxic effect on HEp-2 cells. Sodium salt of polycarboxylic derivative of fullerene C₆₀ exerted a pronounced toxic effect on this cell culture.     

Protection of cells from nitric oxide-mediated apoptotic death by glutathione C₆₀ derivative

The influence of the glutathione C₆₀ derivative on the cytotoxicity of a highly reactive free radical NO (nitric oxide) has been investigated. Consistent with its cytoprotective abilities, the derivative scavenges ROS (reactive oxygen species) and RNS (reactive nitrogen species) both in vitro and under cell‐free conditions. Moreover, the glutathione C₆₀ derivative protected PC12 cells from the cytotoxic effect of the NO‐releasing compound, SNP (sodium nitroprusside). Addition of glutathione C₆₀ derivative alone did not induce apoptosis and necrosis. The results suggest that the glutathione C₆₀ derivative has the potential to prevent NO‐mediated cell death without evident toxicity.     

Effect of different water-soluble forms of fullerene C<Subscript>60</Subscript> on the metabolic activity and ultrastructure of cells in culture

In view of contradictory data on the toxicity of fullerenes for living organisms we studied the effect of water-soluble complexes of C₆₀ with N-polyvinylpyrrolidone (C₆₀/PVP) and γ-cyclodextrin (C₆₀/γ-CD) on MA-104 cells in culture. Both complexes proved to be nontoxic for cultured cells in the dark in a wide range of concentrations. Both complexes provoke changes of cell ultrastructure that reflect enhancement of metabolic activity. At the same time only the exposition with C₆₀/PVP leads to substantial growth of the number and size of mitochondria. However, the effect of two studied water-soluble forms of C₆₀ under intense UV irradiation of cells proved to be opposite: C₆₀/PVP had a cytoprotective action while C₆₀/γ-CD caused a significant growth of phototoxicity. Possible reasons of the differences in the action of different forms of C₆₀ on living organisms are discussed.     

Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C60/Ti Films

Thin films of binary C₆₀/Ti composites, with various concentrations of Ti ranging from ~ 25% to ~ 70%, were deposited on microscopic glass coverslips and were tested for their potential use in bone tissue engineering as substrates for the adhesion and growth of bone cells. The novelty of this approach lies in the combination of Ti atoms (i.e., widely used biocompatible material for the construction of stomatological and orthopedic implants) with atoms of fullerene C₆₀, which can act as very efficient radical scavengers. However, fullerenes and their derivatives are able to generate harmful reactive oxygen species and to have cytotoxic effects. In order to stabilize C₆₀ molecules and to prevent their possible cytotoxic effects, deposition in the compact form of Ti/C₆₀ composites (with various Ti concentrations) was chosen. The reactivity of C₆₀/Ti composites may change in time due to the physicochemical changes of molecules in an air atmosphere. In this study, we therefore tested the dependence between the age of C₆₀/Ti films (from one week to one year) and the adhesion, morphology, proliferation, viability, metabolic activity and potential DNA damage to human osteosarcoma cells (lines MG-63 and U-2 OS). After 7 days of cultivation, we did not observe any negative influence of fresh or aged C₆₀/Ti layers on cell behavior, including the DNA damage response. The presence of Ti atoms resulted in improved properties of the C₆₀ layers, which became more suitable for cell cultivation.     

Induction of prolactin synthesis in rat pituitary tumor cells by 5-bromodeoxyuridine.

The clonal strain of pituitary tumor cells GH₁2C₁ does not produce detectable amounts of prolactin (<5 ng/mg cell protein per 24 hr), although it does synthesize growth hormone. When GH₁2C₁ cells were grown in the presence of 5-bromodeoxyuridine (BrdU, 3 μg/ml), the cells did produce prolactin as determined by quantitative microcomplement fixation and incorporation of ³H-leucine into ³H-prolactin. BGH₁2C₁ and F₁BGH₁2C₁, two BrdU-resistant (r) substrains derived from GH₁2C₁ which grow in the presence of 30 μg/ml BrdU, also synthesized prolactin (100–500 ng/mg cell protein per 24 hr). Growth of BrdU^r strains was not dependent upon on the presence of the drug in the medium; however, the continued production of prolactin by F₁BGH₁2C₁ cells was dependent upon the presence of BrdU. Growth hormone production in both BrdU^s and BrdU^r strains was not affected by BrdU. Resistance of F₁BGH₁2C₁ cells to BrdU was not due to a defect in BrdU uptake. Thymidine inhibited the incorporation of ³H-BrdU into DNA in both sensitive and resistant strains, and also reduced BrdU-induced prolactin synthesis in F₁BGH₁2C₁. We postulate that induction of prolactin synthesis by BrdU in GH₁2C₁ and F₁BGH₁2C₁ cells is mediated by the incorporation of the drug into cellular DNA. Furthermore, the lack of measurable prolactin synthesis by the parent strain GH₁2C₁ is not due to deletion of the gene for prolactin, but is probably the result of regulatory mechanisms which do not permit expression of this gene.     

Photochemical Transformations in Fullerene and Molybdenum Oxide Affect the Stability of Bilayer Organic Solar Cells

Thin films of fullerene C₆₀ and molybdenum oxide (MoO₃) are ubiquitously used as the electron acceptor material and hole extraction interfacial layer for the fabrication of organic photovoltaic (OPV) cells. It is well known that light exposure induces color changes in MoO₃ (photochromism) and the formation of intermolecular bonds between C₆₀ molecules (photopolymerization). The influence of these photoinduced reactions on the long‐term stability of OPV cells, however, has not previously been studied in detail. Here, a study and discussion of the early (<5 days) aging mechanisms occurring in illuminated ITO/MoO₃/organic cyanine dye/C₆₀/Alq₃/Ag bilayer solar cells under nitrogen atmosphere is presented. A degradation process at the organic heterojunction is identified and the formation of Mo⁵⁺ species during illumination is found to adversely affect cell behavior. For these widely used materials, the results suggest that light processing is a first necessary step before OPV characteristics can be meaningfully rated.     

Optimized 4‐V Spinel Cathode Material with High Energy Density for Li‐Ion Cells Operating at 60 °C

Spinel cathodes comprising 16‐μm, AlPO₄‐coated Li_1.09Mn_1.83Al_0.08O₄ with a high energy density of 1.2 W h cm^‐3 are synthesized via a conventional solid‐state reaction using MnO₂ and Li₂CO₃ at 770 °C for 10 h and using a solution‐based coating method in bulk scale (>20 kg). The cathodes are coated by aluminum phosphate at a thickness of <10 nm. The coated cathodes exhibit a first discharge capacity of 108 mA·h g^‐1 and a coulombic efficiency of >99.8%, and their capacity retention is 78% after 200 cycles at a 0.5C rate in a Li‐ion cell under 60 °C. More importantly, a Li‐ion cell containing the coated cathode does not exhibit a swelling problem after 200 cycles at 60 °C. Transmission and scanning electron microscopy suggest that the uniformly distributed AlPO₄ coating and the possible formation of a solid solution phase along the surface play key roles in enhancing the electrochemical performance of the LiMn₂O₄ spinel at 60 °C.     

Quantitative analysis of C60 fullerene in blood and tissues by high-performance liquid chromatography with photodiode-array and mass spectrometric detection

A high-performance liquid chromatographic (HPLC) assay method for C₆₀ fullerene, in blood, liver and spleen using photodiode-array detection or mass spectrometric detection (LC–MS) and C₇₀ fullerene, as the internal standard, is described. The recovery from mouse blood and tissues spiked with micronized C₆₀ exceeds 90%. The method is linear from 0.05 to 200 mg of C₆₀ per liter of blood and from 0.05 to 5.00% of C₆₀ per tissue weight. The limit of detection of the method is 0.1 ng of C₆₀ per injection. This method was applied to mouse blood and tissue samples after intraperitoneal administration of a micronized C₆₀ suspension.     

Folacin C60 derivative exerts a protective activity against oxidative stress-induced apoptosis in rat pheochromocytoma cells

In the present study, we describe the synthesis and characterization of a novel folacin C₆₀ derivative. The compound was analyzed by FT-IR, ¹H NMR, ¹³C NMR, LC–MS and elemental analysis. This water soluble fullerene derivative was able to scavenge both superoxide and hydroxyl radical with biocompatibility. Rat pheochromocytoma (PC12) cells treated with hydrogen peroxide underwent cytotoxicity and apoptotic death determined by MTT assay and flow cytometry analysis. As a novel derivative of C₆₀, the folacin C₆₀ derivative self-assembled to form spherical aggregates in H₂O. Because the compound was amphiphilic, it could penetrate the cell membrane and play its distinguished role in protecting PC12 cells against hydrogen peroxide-induced cytotoxicity. The results suggest that folacin C₆₀ derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity.     

Tissue Sites of Uptake ofC-Labeled C60

This paper describes thein vivobehavior and potential metabolism of C₆₀and a more water-soluble quaternary ammonium salt-derivatized C₆₀. In both cases, a¹⁴C-labeled fullerene core was utilized for the target molecules that were intravenously injected into female Sprague–Dawley rats. The¹⁴C-labeled C₆₀(*C₆₀) was rapidly (within 1 min) cleared from the circulation and the majority of the *C₆₀accumulated in the liver (90–95%). *C₆₀was not eliminated from the liver over the 120-h period of this study. Our results also suggest that C₆₀is not metabolized by the typical oxidative patterns characteristic of other polycyclic aromatics. Therefore, although not acutely toxic, use of C₆₀, or its derivatives that could be cleaved back to the parent C₆₀in vivo, would likely lead to long-term fullerene accumulation in the liver. The uptake of *C₆₀and¹⁴C-labeled ammonium salt-derivatized C₆₀(1)by human keratinocytesin vitroshowed that while both *C₆₀and1are readily taken up by cells,1accumulates more slowly. Additionally, while C₆₀, at rather high concentrations (2.0 μM) and over extended periods of time (8 days), is able to inhibit the growth of human keratinocytes by about 50%, this effect showed little, if any, photoinducability.     

Expression of biochemical properties of oligodendrocytes in oligodendrocyte x glioma cell hybrids proliferating in vitro

In order to generate cell lines that grow continuously in tissue culture and that express the biochemical properties of oligodendrocytes (the cells which produce myelin in the central nervous system), we isolated oligodendrocytes from calf brain and fused them with C₆ rat glioma cells. Of the 60 hybrid clones tested, several expressed oligodendroglial properties at levels comparable to isolated oligodendrocytes. In particular, hybrid clone CO-13-7 showed a high level of expression of all six oligodendroglial properties tested: 2′ : 3′-cyclic nucleotide 3′-phosphohydroiase, glycero-3-phosphate dehydrogenase, and induction of GPDH by hydrocortisone, all of which were also found in C₆ cells and in oligodendrocytes; and galactocerebroside, sulfatide, and myelin basic protein, which were found in normal oligodendrocytes but not in C₆ glioma cells. Therefore, the hybrids express a spectrum of oligodendrocyte biochemical properties that is not found in any other cell line that can be maintained continuously in tissue culture.     

Fine-tuning of POPC liposomal leakage by the use of β-cyclodextrin and several hydrophobic guests

The effect of entrapped β-cyclodextrin (β-CD) on the stability of multilamellar vesicles (MLVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), prepared by the dehydration-rehydration method, was studied by monitoring the release of 5(6)-carboxyfluorescein encapsulated into the liposomes. Different hydrophobic guests, such as Fullerene C₆₀, have been incorporated into the POPC bilayer in order to modify the membrane composition. The kinetic results as well as ESI-MS measurements evidenced that the destabilizing activity of β-CD is due to the formation of β-CD inclusion complexes and the consequent removal of selected bilayer constituents from the liposomal membrane. Hence, when β-CD was added to the liposomes in the form of a strong, water-soluble 2:1 β-CD/C₆₀ inclusion complex, such a destabilizing effect was not observed. However, the same β-CD/C₆₀ inclusion complex does not form as a result of C₆₀ extraction from the bilayer. This may be attributed either to the overwhelming concentration of POPC with respect to C₆₀ and/or to the fact that C₆₀ is largely aggregated in the bilayer. Turbidimetric and fluorimetric determinations of lamellarity and entrapped volume of the studied MLVs provided further evidence of the alteration of the liposomal bilayer as a consequence of the addition of β-CD and/or the presence of the studied guests.     

Syntheses of water-soluble [60]fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells

Water-soluble [60]fullerene (C₆₀) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C₆₀ derivatives together with some reported ones were tested. Among the compounds tested, C₆₀/(γ-CyD)₂, C₆₀-bis(γ-CyD) (5) containing C₆₀-mono(γ-CyD) (5′), and C₆₀/PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.     

Similarity of stomatal index in the C4 plant Salsola kali L. in material Collected in 1843 and in 1987: relevance to changes in atmospheric CO2 content

Summary

Earlier work (by F.I. Woodward) demonstrated that those plants with the C₃ pathway of photosynthesis which were examined, i.e. present day plants and dated herbarium material showed a decreased stomatal index (stomata as a percentage of stomata plus epidermal cells) over the last 200 years. Growth of some of these, and of some other, C₃ plants at different CO₂ partial pressures shows that the fall in stomatal index over the last 200 years is probably a consequence of the increased atmospheric CO₂ partial pressure resulting from the industrial revolution. The work reported here shows that Salsola kali (one of the few plants with the C₄ pathway which are native to Scotland) showed the same stomatal index 144 years ago as it does today. Properties of photosynthetic gas exchange in C₃ and C₄ plants are reviewed. It is concluded that responses to decreased CO₂ partial pressure for growth which involve increased stomatal index in C₃ plants are achieved in C₄ plants without an increase in stomatal index. While C₃ plants growing at low CO₂ appear to sacrifice water use efficiency in partially maintaining net photosynthetic rate, the C₄ plants may emphasize more the maintenance of their already high water use efficiency.     

Fullerene Nucleating Agents: A Route Towards Thermally Stable Photovoltaic Blends

The bulk‐heterojunction nanostructure of non‐crystalline polymer:fullerene blends has the tendency to rapidly coarsen when heated above its glass transition temperature, which represents an important degradation mechanism. We demonstrate that fullerene nucleating agents can be used to thermally arrest the nanostructure of photovoltaic blends that comprise a non‐crystalline thiophene‐quinoxaline copolymer and the widely used fullerene derivative [6,6]‐phenyl‐C₆₁‐butyric acid methyl ester (PCBM). To this end, C₆₀ fullerene is employed to efficiently nucleate PCBM crystallization. Sub‐micrometer‐sized fullerene crystals are formed when as little as 2 wt% C₆₀ with respect to PCBM is added to the blend. These reach an average size of only 200 nanometers upon introduction of more than 8 wt% C₆₀. Solar cells based on C₆₀‐nucleated blends indicate significantly improved thermal stability of the bulk‐heterojunction nanostructure even after annealing at an elevated temperature of 130 °C, which lies above the glass transition temperature of the blend. Moreover, we find that various other compounds, including C₇₀ fullerene, single‐walled carbon nanotubes, and sodium benzoate, as well as a number of commercial nucleating agents—commonly used to clarify isotactic polypropylene—permit to control crystallization of the fullerene phase.     

C60对体外培养癌细胞的光致作用研究

研究结果表明用C₆₀-磷脂酰胆碱与体外培养的HeLa细胞融合后,以4 000 lx光强度激发C₆₀(C₆₀浓度20 mg／L)对HeLa细胞具有显著的光致杀伤作用.生化测定证实光激发C₆₀导致膜蛋白巯基含量减少、膜脂过氧化、丙二醛含量增高,SDS-PAGE证明膜蛋白交联,荧光偏振显示膜流动性降低,电镜超微膜结构破坏,经MTT法检测,大部分细胞死亡.C₆₀的强烈光致作用证实了Arbogast等认为光激发C₆₀可产生单线态氧的观态.     

Ethoxyzolamide Inhibition of CO(2)-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC7942

Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (C_i) conditions (1% CO₂; pH 8) were found to be photosynthetically dependent on exogenous CO₂. This was judged by the fact that they had a similar photosynthetic affinity for CO₂ (K_0.5[CO₂] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for C_i measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO₂ in high C_i cells. The addition of 200 micromolar EZ to high C_i cells increased K_0.5(CO₂) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low C_i cells (grown at 30 microliters CO₂ per liter of air) were less sensitive to EZ. EZ inhibition in high and low C_i cells was largely relieved by increasing exogenous C_i up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO₂ usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem II activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO₂ usage. High C_i cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO₂ usage in high C_i cells of Synechococcus PCC7942 may be due to inhibition of a `CA-like' function associated with the CO₂ utilizing C_i pump or due to inhibition of an internal CA activity, thus affecting CO₂ supply to ribulose bisphosphate carboxylase-oxygenase.     

Characterization of a Sorghum bicolor gene family encoding putative protein kinases with a high similarity to the yeast SNF1 protein kinase

C₄ photosynthesis is functionally dependent on metabolic interactions between mesophyll and bundle-sheath cells. Although the C₄ cycle is biochemically well understood many aspects of the regulation of enzyme activities, gene expression and cell differentiation are elusive.Protein kinases are likely involved in these regulatory processes providing links to hormonal, metabolic and developmental signal transduction pathways. We have identified several protein kinases that are differentially expressed in mesophyll and bundle-sheath cells of the C₄ plant Sorghum bicolor. Here we describe the characterization of two putative protein kinases that show high similarity to the SNF1/AMPK family of protein serine/threonine kinases. The mRNA of both kinases accumulates to much higher levels in mesophyll cells than in the bundle-sheath and can also be detected in root tissue. Complementation experiments with a snf1 mutant of Saccharomyces cerevisiae indicate that the S. bicolor protein kinase SNFL1 does not represent a functional homologue of the yeast SNF1 protein kinase.     

Cycloheximide inhibitation on hypoxanthine transport in cultured cells

Cycloheximide preincubation inhibits hypoxanthine uptake into the acid-soluble fractions of cultured rat hepatoma cells (MH₁C₁) and human skin epithelial cells (NCTC 2544, HE cells) in a time- and dose-dependent manner 50% inhibition is seen after 4 h preincubation with 10^?4 M cycloheximide of MH₁C₁ cells and after 2.5 h of HE cells. Adenine uptake is much less affected, after 10 h preincubation with 10^?4 M cycloheximide it was reduced to 83% and 67% of controls in MH₁C₁ cells and HE cells respectively. Cycloheximide inhibits hypoxanthine uptake in a dose-dependent manner above 10^?7 M, with 50% inhibition in MH₁C₁ cells at 4 · 10^?7 M after 12 h preincubation and at 10^-6 M in HE cells after 6 h preincubation. Puromycin mimics the action of cycloheximide. The inhibition of hypoxanthine uptke is not caused by reduction of the activity of hypoxanthine phosphoribosyltransferase in the two cell lines. 10^?4 M cycloheximide preincubation for 10 h does not significantly reduce the uptake of the two non-metabolizable amino acids α-aminoisobutyric acid or 1-aminocyclopentane-1-carboxylic acid (cycloleucine). It is suggested that cycloheximide inhibits the synthesis of a rapidly turning over the protein involved in hypoxanthine transport.