在Mn(OAc)3•2H2O 催化下, C60分别和亚甲基二膦酸四乙酯、氰基亚甲基膦酸二乙酯或乙氧羰基亚甲基膦酸二乙酯在氯苯中回流, 生成3个单加成环丙烷富勒烯膦酸衍生物C60C(R)PO(OEt)2 [1, R＝PO(OEt)2; 2, R＝COOEt; 3, R＝CN]. 与以前报道的Bingel反应法相比, 该方法副产物少并且缩短了反应时间. 采用循环伏安法发现1, 2的还原电位相对于C60发生负移, 而3的还原电位相对于C60却正移40 mV, 表明引入象氰基一样具有很强吸电子能力的取代基团, 可以改善富勒烯球的电化学性能, 合成电子接受能力较强的富勒烯衍生物.

Collagen extracted from pigskin was coronacharged negatively by a specifically designed device. Different charging voltages, temperatures and times were applied to prepare collagen bioelectret. The decline of the surface potential of the bioelectret under different treatment was then determined. The data showed that the surface potential was markedly varied with the charging conditions. The optimal values of three parameters for charging collagen coatings were defined as follows: voltage, 8 KV; temperature, 40◦C; time, 25 min. Treatment of the bioelectret with distillated water, or saline solution (0.9%) or culture medium induced a sharp decrease of the surface potential. In addition, we investigated the effects of the charged collagen on cell growth and intracellular calcium level of three types of cultured mammalian cell lines, including Chinese hamster ovary CHO cells, human cervix uteri tumor HeLa cells and human promyelocytic HL-60 cells. Cell growth and the intracellular calcium level were determined by MTT reagent-based assay and a fluorescent probe Fura-2, respectively. The results showed that negatively charged collagen stimulated the growth of CHO or HL-60 cell line but inhibited the growth of HeLa cell line. Furthermore, after attaching to the charged collagen, the intracellular calcium level of CHO cells increased, while that of HeLa cells decreased.

Influence of fullerene (C60) derivatives on M-MuLV reverse transcriptase activity is investigated. Two water-soluble fullerene derivatives, fullerol (C60(OH)23-24) and trimalonic acid C60 (TMA C60, C63(COOH)6) are used in the experiments and their effects on in vitro reverse transcription-polymerase chain reaction (RT-PCR) of β-actin mRNA in HeLa cells are determined. PCR products are detected by agarose gel electrophoresis. It is found that the amounts of PCR products decrease with addition of either of two fullerene derivatives to RT reaction mixture. The inhibition of fullerene derivatives is dose-dependent, and IC50 values of fullerol and TMA C60 are 0.089 and 0.039 mmol/L, respectively. The amount of PCR products obtained by direct addition of fullerol or TMA C60 to PCR are greater than those obtained by addition of the equivalent amount of fullerol or TMA C60 to RT, indicating an inhibitory effect of fullerol or TMA C60 on RT. The compensative experiment of M-MuLV reverse transcriptase shows that increasing enzyme amounts can antagonize the activity of fullerol or TMA C60. These results imply that fullerenes can inhibit M-MuLV reverse transcriptase activity, with the inhibition of TMA C60 slightly stronger than fullerol, and that their potential in treatment of diseases induced by RNA viruses such as leukemia virus needs further investigation.

Activities of trimalonic acid fullerene (TMA C60) on DNA restrictive enzymatic reaction were investigated by using two restrictive endonucleases Hind III and EcoR I and plasmid pEGFP-N1 with single restrictive site for both enzymes. Meanwhile, TMA C60 was also tested to clarify its effects on polymerase chain reaction (PCR) with the catalyst of Taq DNA polymerase and the template of plasmid pEGFP-N1. The products from restrictive reactions or PCR were detected by agarose gel electrophoresis. It was found that the product amounts from restrictive reactions or PCR decreased significantly with addition of TMA C60. The inhibition by TMA C60 was dose-dependent and IC50 values for reactions of Hind III, EcoR I and PCR were 16.3, 6.0 and 6.0 μmol/L, respectively. Addition of two scavengers of reactive oxygen species (ROS), L-ascorbic acid-2-phosphate ester magnesium and sodium azide at the concentrations of 2―10 mmol/L did not antagonize the activities of TMA C60 against PCR and two restrictive reactions. However, increase of Taq DNA polymerase amounts in PCR system antagonized the activities of TMA C60. These data implied that TMA C60 was able to inhibit the activities of the three above-mentioned enzymes involved in DNA metabolism, and that this inhibition probably did not correlate to ROS.

The biological activities of fullerene derivatives have attracted much attention in the last decade. In this paper, effects of dimalonic acid C60 (DMA C60) on cytoplasmic membrane, intracellular calcium concentration ([Ca2+]i), and mitochondrial membrane in HeLa cells were studied by using laser scanning confocal microscopy together with fluorescent probes propidium iodide (PI), fluo-3 acetoxymethyl ester (fluo-3 AM), and tetramethyl rhodamine methyl ester (TMRM). The data showed that under laser irradiation produced by a Kr/Ar laser source with a low power less than 1 mW, DMA C60 might induce damages against both cytoplasmic and mitochondrial membranes in a time- and dose-dependent manner. Prior to leakage of cytoplasmic membrane, a transient increase in [Ca2+]i occurred due to influx of calcium from the culture medium. These data provided some novel clues to explain the mechanisms involved in the photo-induced cytotoxicity of fullerene derivatives.