Synthesis Of New Fullerene Derivatives And Studies On Their Cytotoxicity,Radiobiological Cellular Activity

Radiotherapy is the most common used therapy for tumor treatment besides operation and chemotherapy. Radiotherapy will cause severe side effect since it fails to distinguish normal cell with abnormal ones. Increasing radiosensitivity is the most promising way to improve the effectiveness of radiotherapy. Photodynamic therapy and photosensitizers are available for an adjunct way in radiosensitization. There are synergistic effects when radiation combined with photodynamic therapy. Most common used photosensitizers present radiosensitization effects.As a radiosensitizer, CMNa is a Class I New Drug approved by SDA. Except its great radiosensitization effect, CMNa also showed some disadvantages such as insolubility and instability which affect its clinical application. As the precursor of CMNa, metronidazole exhibits excellent radiosensitivity.In 1985, the third allotropic form of elemental carbon named as fullerene was discovered by Kroto et al.. Its distinct properties may be exploited for numerous applications including being used as drugs or carriers for other biological molecules . Due to its biologically stableness and a convenient three-dimensional scaffolding for covalent attachment of drugs ,we intend to create a drug-delivery system of fullerene and metronidazole, and try to understand its photodynamic cellular activity and radiosensitization effects due to metronidazole attachment.Studying of biological effects of C₆₀ was used hindered by its strong hydrophobicity. We are interested in the cytotoxicity of aqueous solutions of C₆₀ and its underlying photo-,radio-activity in cellular level,which is vacant before 2005. Aiming at these, the following were tried in this research: 1 , Synthesis of C₆₀-Metronidazole complex and Nano-C₆₀ water clusterpreparation:By esterization and [1+2] cycloaddition we synthesized a series of fullerene derivatives( Fig.1 B to E, NC2 NC5) in a productive and effective synthetic route;then we prepared the C60-H2O collodial cluster solution which named Nano-C6o (Fjg.1 A, NC1).A:NCiB:NC2C:NC3D:NC4Fig.1 Fullerene and its Derivatives2> We investigated the cytotoxic effect of water-soluble fullerene aggregates, nano-C6o(NCi), on B16 and SMMC-7721 cells in culture and its underlying mechamism. The cell growth inhibition was evaluated for B16 and SMMC-7721 cells using MTT. In the presence of nano-C6o, there appears to be significant cell growth inhibition under certain dose and incubation time. Results show that there is a dose-time dependent cytotoxic effect for both cell lines. It shows SMMC-7721 were more tolerant to NC1 than B16,e.g.by the concentration of 720 ji g/L, some differences in cell cytotoxicity were observed for these two cell lines.By Hoechst33258 and Annexin V/PI dual staining, the cells showed morphological changes and the characterristics of apoptosis in early or late stages. It is concluded that the cytotoxicity induced by NG| were mainly by apoptosis. The apoptotic cells were characterized as shrinkage of cellmembrane, loss cell volume, condensation of cytoplasm, with some cells falling off from the dish. Cells were observed with both fluorescence microscope and light microscopy with Hoechst 33258 staining .Only those cells with a chromatin condensation or nuclear shrinkage were considered morphologic markers of apoptosis.Annexin V/PI assay was then performed using FACS for detecting the differences in affecting cells with scaled concentration of NCi.The appearance of Annexin V7PI' and Annexin V+/PI+ were respective to the NCi concentration and incubation time. For lower concentrations of NC^ will mainly cause early apoptosis which is due to nuclear destroy;of higher concentration of NCi, the cell damage is mainly due to membrane damage as an advanced apoptosis or necrosis. Statistic difference was found between the blank control and different concentrations groups of NCi (P<0.01).By cellular SOD and MDA detection, there were evidences that NCi 72/jg/L would cause SOD increasing while MDA decreasing. During the first 36hrs , SOD was increasing slowly as an intracellular antioxidant action;up to 48hrs,SOD decreased which may be explained by the loss of the imbalance between the intracellular oxidation and antioxidation mechanism. MDA is a criteria of membrane oxidation. By NCi 72/ug/L , the increase of MDA presented the oxidative effect of NCi on B16 cells.ROS have been observed in aqueous solutions of water-soluble fullerenes by ROS kit and Confocal Laser Microscopy with 2,7-dichlorofluorescein diacetate (DCFH-DA) probe respectively. The overexpressed ROS were toxic to cell membrane. Compared with control group, B16 and SMMC-7721 treated with NCi will produce more ROS ,and then this maybe the main reason to induce apoptosis.As compared with NC2> NC3 and fullerenol, the cytotoxicity was relatedwith their changes in the fullerene cage structure. As the number of hydroxyl or carboxyl groups presented on the cage, the cytotoxicity would decrease by a large orders of magnitude. Pristine Ceo was the most toxic to SMMC-7721 cell. 3, Combined treatment is usually applied for tumor therapy. RT and PDT have something ,in common with the biological system, which cou\d produce synergistic effect when combined in a proper way. Sensitizers namely as radiosensitizer or photosensitizer are commonly used with RT and PDT. As presented here, the pristine C^ was an effective photosensitizer, while the NC4 with a branch to the pristine C?) were less phototoxice to B16 cells. By Hoechst 33258 and AnnexinV/Pl dual staining, we found that the phototoxicity of fullerenes was mainly due to the B16 cellular membrane damage which may be due to the singlet oxygen production as others reported.When combined with y irradiation, cellular colony forming assay and MTT were applied to determine the radioeffect of NCi and NC4. It is shown that following different light exposure,NCi and NC4 could enhance the y irradiation effects under certain concentrations. It would be more synergistic when combined with light radiation, and the synergistic effects was related with time of irradiations interval and concentration of chemicals while irradiation. The shorter the interval was, the less of B16 SF. y irradiation with NCi of B16 showed slow-down of SF decreasing up to 8Gy .while no similar effect of NC4 was found under the same condition.Compared with metronidazole,NC4 seemed more effective on B16 by moving down the cellular surviving curve. By Laser Confocal Microscopy specific probe DCFH-DAto ROS it showed that the radio-/photo- sensitization effect of Nd and NC4 may related with the elevatingintracellular ROS production. Comet Assay was used to detect the DNA damages .By Alkaline CometAssay , we found the 5 Gy y irradiation of the cellular suspention in the presence of NCi and NC4 was followed by the increase of DNA damage,namely the single stand breaks (SSB). This could occur as a result of the excited single oxygen interaction with the oligonucleotides. NC4 is more efficient than the pristine NCi. Though the underlying mechanism is not clear, it is assumed that these two chemicals would inhibit the DNA sublethal and potentially lethal damage repair. This inhibition maybe due to the interaction of this nanocarbon molecules with some repair enzymes.Taken together, we have prepared the nano-C6o colloid NC^ and synthesized fullerene-metronidazole conjugate NC4. It was shown that pritine C<3o is cytotoxic to B16 and SMMC-7721 cells. Both NCi and NC4.showed photo/radio sensitization effects. Based on these results, further investigations are expected to reveal the related bioactivities of these fullerene chemicals .