Synthesis And Radiobiological Study Of Water-soluble Fullerene Derivatives

With the utilization of atom energy in the field of military, civil industry, agriculture, medicine etc, more and more people become concerned with the risk of radiation. As a big country with great nuclear ability, much efforts should be paid on this affairs. But we are facing this serious situation, more need to be done. Now, the most important work for us to do is to find the effective radiation protectant. But for the time being, there are no optimal radiation protectant occured. The main problem we encountered are the low protection effect and the high toxicity. And the former protectants were designed to protect γ rays with low effect on high LET radiation, not to mention the complex injury of both γ rays and neutrons.The biological effects induced by radiation are divided into two ways: the first is direct effect and the second is indirect effect. The indirect effect plays the most important role. So blocking the indirect effect induced by radiation and scavenging of free radicals become our protecting strategy.Fullerene, the third allotrope of carbon, is a hollow cage, composed of 60 carbon atoms. Experiments show that the superoxide anion scavenging rate of polyhydroxylated fullerene can reach to 85%. Through scavenging of free radical fullerene and its derivatives, Human neuronal cells and pulmonary epidermal cells were protected from ischemia and reperfusion injuries and protect the human keratinocyte from the injuries of UV.Although fullerene is called "the radical sponge" , the low solubility in water became a big obstacle of its biological uses. To solve this problem, there are three main methods. And the most promising one is to synthesis water soluble fullerene derivatives.For this project, first, fullerene derivertive was synthesized, its ability of scavenging free radicals was evaluated. After that assessment of the toxicity of the derivertive was evaluated. the survival rate test of the cells after the irradiation of γ rays with different fullerene derivative concentration by trypan blue and PI staining were performed. These are followed by cell cycle analysis and cell apoptosis experiments on the biological effects of fullerene derivative. Thus we found the basis for the study of newly typed radiation protectants. Content of study: 1) synthesis of functional water-soluble fullerene derivative:The fullerene derivative was attained by addingN¹,N³-bis(3-(dimethylamino)propyl)malonamide through fullerene Bingle reaction.Further efforts will be paid on the synthesis of other fullerene derivatives gracefully designed based on this reaction. And we synthesized carboxyfullerene C₃ with good solubility and some free radical scavenging effects.2) Study on scavenging reactivity of hydroxyl radicals by water soluble carboxyfullerene derivative C3.Scavenging reactivity of hydroxyl radicals produced by Fenton system with different concentration of carboxyful lerene derivative C3was analyzed. DMPO was taken as the spin-trapping agents and ESR spectrophy was used.3) Toxicity of the carboxyfullerene derivatives C3 was evaluatedToxicity in vitro using different concentration of C3 was fully investigated.4) Study on the protection effect of C3Choose the proper cell line AHH-1, treated with different dose of y rays, made out the cell survival curve. By determination of SF2, the proper dose of radiation was attained. Treatment of the cells with different concentration of C3, effect of the protection effect was observed.Results:1. Synthesis of the newly typed fullerene derivativesThe low solubility of fullerene was a big obstacle of its biological uses. In this project, the double bonds of the carbon cage was opened, and water soluble derivatives was added through Bingle reaction. Three schemes were tried. Aminolysis reaction between Diethyl Malonate and N,N,-Dimethyl-l,3-propanediamine was chosenfor synthesis of Arl^3-bis(3-(dimethylamino)propyl)malonamide The compoundwascharacterized by 1H-NMR, 13ONMR,ESI ,and UV-vis.2. ESR study on free radical scavenge activity of C3The hydroxyl radical was of great activity in the biological system. Through study of the scavenging effects of hydroxyl radical, protection effects were mirrored. We use Fenton system for producing hydroxyl radical. DMPO was chosen for spin-traping agent. The study got a 93% scavenging rate using lmg/ml carboxyfullerene in 3mmol ferrous fenton system.3. radiobiological study of C31). Antiproliferation effect of carboxyfullerene C3 and low effect on cell survival percents.Cell survival rate was studied by Trypan blue rejection. No significant decreasingeffects was detected observed when cocultured with l-400ug/ml C3 for 48h (P>0.05).Cell apoptos is percent was analyzed by PI staining detected by FACS. No significantchanges were observed. These results showed that C3 is of low toxicity to AHH-1.Coculture AHH-1 with different concentration of C3, dose dependantantiproliferation effects were observed with concentration ranging between 0-100ug/ml. No increasing antiproliferation effects was observed of C3 when theconcentration was between 100 u g/ml and 400 ug/ml. there may be threshold ofantiproliferation effects.2) cell survival and apoptosis study of AHH-1 cocultured with C3 treated with C3.Coculture AHH-1 with different concentration of C3 2h before 4 Gy yi'ays. Cellgrowth fraction and eel 1 survival percent were recorded. Cell apoptosis was detectedby PI staining. Results showed eel 1 growth fraction was improved at the concentrationof 10 u g/ml.The cell survival percent of AHH-1 after irradiation was significantly improved. High protection effects was observed at concentration ranging between 200-400u g/ml. And the cell survival percent was almost the same as the group without irradiation. PI staining confirmed the Trypan blue rejection. The cell apoptosis rate showed a dose dependant effects when the C3 concentration ranged between 10-200 ug/ml. the cell apoptosis rate decreased to 10.49±0.53% at C3 concentration of 200 p g/ml, approximately to the negative control (8.6±0.75%, without C3 and radiation) .Thegroup simply treated with irradiation showed a eel 1 apoptosis rate of 26.30±0.74%, which is much higher than the group pretreated with 200pg/ml C3. The obvious difference between them demonstrated that carboxyfullerene is good protectants of AHH-1.Further investigation showed coculture AHH-1 with 200ug/ml 2h before or after irradiation. Cell death and cell apoptosis were decreased great ly. The cell survival rate of the group singly treated with irradiation was 80%. And the group adding C3 before irradiation was 89-91%.3. DiscussionThe radiobiological effects and injuries to human body by radiation was mainly caused by the indirect effects. The indirect effects was mediated by free radicals induced by irradiation. Radioprotection study was focused on free radical scavenging agent s.6-6 bonds of fullerene are the active sites for chemical modification. Since there are no other atoms except for the carbon ones, no replacement reaction can take place. The physiochemical properties make fullerene a good free radical scavenger. And the low solubility of fullerene is a big obstacle of its biological uses. Synthesis of water-soluble fullerene derivatives, scavenging free radical, and use it for protection from irradiation.In our project, FD was synthesized by Bingle reaction of fullerene. C3 was also synthesized following the articles. Method for fullerene monoadduct was founded. Carboxyfullerene C3 is a fullerene derivative of good solubility in PBS, Dugan LL etc. reported its good radical scavenging activity. Our study also demonstrated its good scavenging effects of hydroxyl radicals. Clues was proposed for good protection effects of radio protection. And our results confirmed this hypothesis. Cell survival percent was greatly improved and apoptosis rate was significantly decreasing by coculturing with C3 before radiation. Dose dependent antiproliferation effects were observed, but the high concentration C3 did not show cell survival percent decreasing effects. So we concluded that C3 is of low toxicity to AHH-1 in vitro. Our study also demonstrated fullerene derivative C3 was a good free radical scavenging compound. Cel Is were protected from injuries by irradiation. Newly typed fullerene derivative of low toxicity, high water-solubility and good free radical scavenging activity should be further investigated. And the fullerene derivatives is a promising radiation protectant deserved to be deeply investigated.