Study On Synthesis And Antitumour Activity Of Water-Soluble Fullerence Derivatives

Due to unique chemical and physical features of C₆₀, the biological activities of fullerenes and their derivatives have been recognized in the recent years. In this thesis, using the methods of macromolecules, organic chemistry and biology the cross topic about the biological activities of fullerene derivatives was studied.The relation between the structures of water-soluble derivatives and their photo-induced cytotoxicity against tumor cells and the mechanism about the photo-induced cytotoxicity against tumor cells of water-soluble fullerene derivatives were studied by means of MTT(3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)etc. Three water-soluble fullerene copolymer: fullerene –vinyl pyrrolidone copolymer, fullerene –maleic anhydride copolymer and fullerene –acrylamide copolymer have been prepared by means of radical pdymerization and molecular design.Three water-soluble fullerene derivatives: fullerene-lysine derivative, fullerene-glycine derivative and fullerene-aspartic acid derivative have been prepared by means of organic chemistry. They were synthesized for the first time. All the six fullerene derivatives can be dissolved in water. Reaction mechanism and reaction condition of six water-soluble fullerene derivatives were investigated for the first time. The structure of the water-soluble fullerene derivatives were characterized by means of GPC, UV, FTIR, 1HNMR etc.TEM analysis shows that the shapes of synthesized water-soluble fullerene derivatives in water are ideal spherical with an average diameter less than 50nm, by comparison, their corresponding homopolymers in water are lamellar in shape. To prove water-soluble fullerene derivatives photodynamic effect on tumor cell,i.e. the photodynamic effect on mouse bone tumor in vivo we injected the fullerene –maleic anhydride copolymer into the mouse bone tumor body.We found that with I-W lamp(500W) illumination the mouse bone tumor body was strongly damaged. Under the intensity of 500W and the concentration of C60 (50ug/ml),mouse average survival time extended 6 days and the turmor size reduced 0.5cm and tumor weight decreased 0.7g. In the other way bone turmor cells was co-cultured in vitro with fullerene –maleic anhydride copolymer for three days.The result of the killing was examed by MTT method after illuminating the fullerene –maleic anhydride copolymer with I-W lamp.Bone tumor cell in vitro were obviously damaged. Fullerene –maleic anhydride copolymer is a kind of strong hotosensitizer. It can kill mouse bone tumor cells both in vivo and in vitro. The in vitro antitumour activity six kinds of water-soluble fullerene derivatives designed specifically were systematically investigated for the first time. The results showed that the water-soluble fullerene derivatives exhibits better antitumour activity such as in vitro against bone tumour cells etc. The in vitro antitumour activity of water-soluble fullerene derivatives were dependent on the power of the light irradiation, and were also related to the side chain structure.The in vitro antitumour activity of water-soluble fullerene derivatives were related to the fullerene derivatives concentration.Among the six kinds of water-soluble fullerene derivatives, fullerene-glycine derivative displayed outstanding antitumour activity possibly because of its better water-soluble.Due to complexity of the biological system ,the influence of reactive oxygen species was one of the important factors on the in vitro antitumour activity of the water-soluble fullerene derivatives,,but the structures and the properties of the addition groups had great influence. By using confocal laser scanning microscope, the photo-induced damage to cell membrane of bone tumour cells by water-soluble fullerene-vinyl pyrrolidone copolymer was investigated in the presence of low powerful laser source (p≤1mW), as well as the change of intracellular calcium concentration during the damage. It was found that the photo-induced cell membrane damage occurred even at the presence of low concentration of water-soluble fullerene-vinyl pyrrolidone copolymer(c≥5ug/ml) and the damage was dose-dependent. It was found that the transient increase of intracellular calcium concentration took place before the damage of cell membrane. These results demonstrated that cell membrane was a target of photo-induced action produced by water-soluble fullerene derivatives, and implied that calcium signaling was probably involved in the photo-induced action. By using the method of flow cytometry, the influence of the water-soluble fullerene derivatives on the bone tumour cell was examed. It was found the fullerene-glycine derivative and fullerene-aspartic acid derivative could induce apoptosison bone tumour cell under irradiation, indicating there possibly existed direct interaction between the water-soluble fullerene derivative and DNA. The antitumour mechanism of six water-soluble fullerene derivative were investigated for the first time. We believe that cell death is mainly due to phospholipid and protein damage within the cell membrane. When irradiated with light, the fullerene derivativer became excited to a singlet excited state and through intersystem crossing passed to an excited triplet state. Subsequently, they could be quenched by O2 to produce singlet oxygen, a species that reacts readily with amino acids, nucleic acids and membrane phospholipids, leading to cell damage and death.