The Preparation Of Fullerene Derivatives Of Lysine And Its Biological Effectsof Cell Irradiated By γ-ray

In recent decades, the extensive utilization of atom energy in many fields such as military, civil industry, agriculture, medicine etc brings about great benefits for people, but in the meantime, how to protect human health and safety from severe nuclear radiation become a critical problem in the world. The continuous development and application of nuclear weapons and increase of nuclear weapon-possessing countries have increased the risk of regional nuclear warfare. China is one of the great nuclear countries, possessing strategic nuclear forces such as nuclear weapons, nuclear submarines and a large number of nuclear power stations. Nuclear radiation protection thus is of special significance to protect the health of employees. And strengthening protection of nuclear radiation damage has an important role in the future nuclear war, anti-nuclear terrorist attacks in peacetime, nuclear power plant accident and emergency rescue tasks and there is still a long way to go for our country. Nowadays, with the development of modern biomedicine, the radiation therapy has become an important therapeutic method for malignant diseases, but the damages to some normal tissues induced by radiation limit the application of radiotherapy. It is a novel scientific topic for radiologists to look for high-effective radiation protectants with low toxicity to protect normal tissues from radiation induced damages. In fact, exploitation the ideal radiation protectants has always been emphasis and obstacles in the field of radiation protection research.As we know, the damage of radiation mainly comes from the y ray and neutron ray, and the biological effects are divided into two ways:the first is direct effect and the second is indirect effect, which accounts for about 70%-80% of the damages. Since the indirect effect mainly due to the abundant free radicals caused from radiation, so blocking and scavenging of free radicals become our most important protecting strategy.Fullerene and its derivatives show good effects on anti-HIV virus, the protection of injury on nerve cells, lung epithelial cells caused by ischemia reperfusion and the protection of injury on Human Epidermal Keratinocytes caused by ultraviolet radiation.But the low water-solubility of fullerene, hamper the application of it in biology. There are three methods to figure out this problem:①Preparation of water-soluble fullerene inclusion complex, Such as the use of liposomes, cyclization dextrin etc. to make fullerene molecules wrapped in hydrophilic compounds. Although such an approach increase water-solubility of fullerene to some extend, it is still severely restricted in bio-medical applications for the following reseaons. Firstly, the efficient of parceling fullerene is low. Secondly, many of the complexes that parcel fullerene are toxic materials. Thirdly, the procedures are complex and it is difficult to apply to reality, so it's mainly used in cell-free system of fundamental research.②Preparation of fullerenes hydrosol:In the hydrosol prepared by this method, fullerenes are in the form of clusters in aqueous solution. The process of the preparation relies mainly on the role of strong acid and strong base. The concentration of the hydrosol is difficult to control, which severely limits their biological applications.③Add a water-soluble group to the fullerene carbon cage to synthetise water-soluble fullerene derivatives. This method can improve water-solubility of fullerenes, also the water-soluble group added may retain its own activity. Adjusting its derived group can reduce or even avoid its toxic effects. Also, this method can be used in cell-free system, as well as for the general experimental study. Currently, there are many ways to enhance water-solubility of fullerenes by adding water-soluble groups, such as hydroxyl, carboxyl, amino and sulfonamide-based ect. Domestic and foreign reports and studies of our laboratory have found fullerols plus hydroxyl and C3 plus carboxyl basically preserved the powerful free radical scavenging capacity of fullerenes, which also show high anti-oxidant activity at the same time.There are some reports about the synthetic of fullerene Derivatives Lysine, Light-induced singlet oxygen and its anti-tumor ability. However, there are few reports of its free radical scavenging capacity. This topic is based on this. Fullerenes direct react with lysine, producing a highly water-soluble fullerene derivative C60-lys. We preliminary identify its characterization and study of the free radical scavenging capacity of the derivatives and its laws. Also, we assessed its the toxic effects to normal cells and tumor cells to select the safe concentration of fullerenes C3.We observed and analyzed the effects of carboxylic acid fullerene C60-lys on the changes of cell survival after y-ray irradiation, and its effects on early apoptotic cells, cell cycle and DNA. We aim to clarify the initial radioprotective effects of C60-lys on cells to provide an initial basic research in exploring a new radioprotective agent.Contents of study:1. Synthesizing and Structure Elucidation of C60-lys:Reference to the literature at home and abroad, we choose to synthetic fullerene derivatives of lysine C6o-lys, which is easy to synthesize, has better water-solubility, have great free radical scavenging ability. We aim at working out a better method of synthesizing and technology.2. Preliminarily studying on the toxic effects of different concentrations of C6o-lys on the AHH-1 cells and K562 cells to find out a concentration of non-toxic or a concentration that almost has no side effects, but also has a strong free radical scavenging ability.3. Exploring radiation biological effects of C60-lys on cells:we selected several strains of cells to make the dose-survival curve of cells irradiated at different doses of irradiation. The cells were incubated with the fullerene derivative C60-lys lysine. We observed the following subjects:①Radioprotective effect of different concentrations of fullerene derivatives of lysine C60-lys on cells irradiated with the same dose of y-ray.②Radioprotective effect of the unique concentration of fullerene derivatives of lysine C60-lys on cells irradiated with different doses ofγ-ray.③Radioprotective effect of fullerene derivatives of lysine C60-lys on different cells.④Radioprotective effect of fullerene derivatives of lysine C60-lys on cells by administrated at different times.4. Preliminarily exploring the mechanism of radioprotective effects of C60-lys on cell lines:Analyze the effects of the compounds on cell cycle, DNA,8-OhdG.Methods:1. Synthesis of fullerene derivatives of lysine C60-lys:Reference to the literature at home and abroad, using the chemical synthesis platform of our laboratory, we optimize the methods of synthesis and synthesis fullerene derivatives of lysine C6o-lys.2. Cells:AHH-1 cells are derived from human lymphoblastoid(Long-term cultured in our laboratory). K562 cells are Human erythroleukemia leukocytes(Long-term cultured in our laboratory). Cell culture conditions:RMPI-1640 medium (Hyclone Corporation), 10%NCS(GIBCO Corporation,37℃,5%CO2, Saturated moisture, incubated in cell incubator.3. Irradiation:different doses ofγ-rays by Co-60.4. toxic effects of Fullerene amino acid derivatives on cells:We assessed survival rate of normal cells and tumor cell with different concentrations of C60-lys by CCK-8 and select the safe concentration. Formula:Viability(%)=OD values of drug group-Background/ control OD values-Background)×100%.5. Determination of cell viability:We assessed the survival rate of different groups of cells by CCK-8. Formula:Viability(%)=OD values of drug group-Background/control OD values-Background)×100%. 6. Apoptosis and cell cycle analysis:The cells were stained by using fluorescence staining and Annexin V/PI double staining, and analysed by flow cytometry. Cell cycle was assessed by PI single-staining.7. Analyse concentrations of 8-OhdG:Detect the effects of different concentrations of cell C60-lys on cells post irradiation by using 8-OhdG elisa kit.8. Cell DNA Detection:To observe the tail under the fluorescence microscope by single-cell gel electrophoresis analysis in each treatment group. The longer the tail is, the more seriously that DNA is damaged.9. Statistical analysis:the difference between the groups using single factor analysis of variance represented as x±sd. P<0.05 was considered to be statistically significant.Results:1. Synthesis and analyze chemical structure of C60-lys:According to related literature, Fullerenes react with lysine directly. Then, purify the combination preliminarily by extraction, chromatography and other methods. We preliminarily analyzed the compounds by NMR, C NMR, IR, elemental analysis.2. Analyze the toxicity Fullerene derivatives of lysine C60-lys:Different concentrations of C60-lys, were incubated respectively with AHH-1 and K562 cells for 48h. The cell viability was detected by CCk-8. The results showed that when the concentrations of C60-lys were less than 1200mg/L, the survival rates of AHH-1 and K562 cells with the control group have no statistical difference. When the concentration was 1600mg/L, the survival rates of AHH-1 and K562 cells were 80.4% and 78.3%, which were significantly lower than the control group(P＜0.01)3. Biological effects of Radiation of C60-lys on AHH-1 cells3.1 Effects of different concentrations of C60-lys on the survival rate of AHH-1 cell at the same dose of irradiation:We detected the changes of survival rate of AHH-1 cells 24h after 4Gy irradiation. We found among the 0-800 mg/L range, with the increase of drug concentration, the cell viability also increased. When the drug concentration was 800 mg/ L, the cell survival rates were 86.5% and 84.8% detected by CASY and CCK-8. There is a significant difference compared with the irradiation group which were 63.7% and 61.3%(P<0.05). The survival rates of the group treated with C60-lys and C3 among the 0-800 mg/L were higher than the irradiated group pretreated without drugs(p<0.05). But there is no significant difference between the groups pretreated with drugs themselves(p>0.05).3.2 The effects of the same concentration of C60-lys on the survival rate of the AHH-1 cells at different doses of irradiation:the AHH-1 cell viability was detected by CCK-8 24h after different doses of irradiation. The cell survival rate dropped to about 55.4% under irradiation of 4Gy. When AHH-1 cells pretreated with the drug at a concentration of 800mg /L, the cell survival rates decreased to varying degrees 24h after irradiation of 1,2,4,8 Gy. However, the survival rate of drug-treated cells were higher than group without drugs. The survival rates of administration group and irradiated group were 64.6% and 41.3% under irradiation of 8Gy.3.3 The effects of the same concentration of C60-lys administered at different times on the survival rate of the AHH-1 cells at the same doses of irradition:the cell survival rate increased at varying degrees when the drugs were administrated before and after the irradiation. Compared with the control group,there is significant difference in the group which was administered before irradiaton(P<0.01),while there is no significant difference in the group which was administered after irradiaton(P>0.05).3.4 The effects of different concentrations of C60-lys on the early apoptosis rate of the AHH-1 cells at the same doses of irradiation:Annexin V/PI double staining can detect early apoptosis in cells. Under the conditions of the same dose, early apoptosis rate of the cells at 4Gy irradiation is 32.63%, while apoptotic rate of the 100mg/L treatment group was 24.86%. As the drug concentration increased, the early apoptosis rate declined in varying degrees. When the drug concentration was 1200mg/L, the apoptosis rate dropped to 14.22% which is significant difference when compared with 39.2%,the apoptosis rate of group irradiated without drugs(P<0.05).4.Biological effects of Radiation of C6o-lys on K562 cells4.1 Effects of different concentrations of C60-lys on the survival rate of K562 cell at the same dose of irradiation:We detected the changes of survival rates of K562 cells 24h after 24Gy irradiation by CCK-8. We found among the 0-800 mg/L range, with the increase of drug concentrations, the cell viability also increased. When the drug concentration was 800 mg/L, the cell survival rates were 74.3%. There is a significant difference compared with the irradiation group without drugs(58.1%)(P<0.05)4.2 The effects of the same concentration of C60-lys on the survival rate of the K562 cells at different doses of irradiation:with the increase of the doses of irradiation, the cell viability decreased. However, the survival rate of drug-treated cells were higher than group without drugs. (P＜0.01)5.The Mechanism of Biological effect of Radiation of C60-lys on AHH-1 cells5.1 The effects of different concentrations of C60-lys on the cell cycle of the AHH-1 cells after irradiation:PI single staining can detect cell cycle. We found that the proportion of G2/M cells is lower in the group treated with different doses drugs compared with the group irradiated without drugs(P<0.05).5.2 The effects of C60-lys on the concentration of 8-OHdG in AHH-1 cells after irradiation: The concentrations of 8-OhdG in the groups irradiated with and without drugs are detected by the 8-OhdG detection kit which reflects the degrees of cell damage. The results showed that 8-OhdG concentrations decreased significantly in the group treated with drugs compared with group treated without drugs in AHH-1 cells. And among concentrations of 100-1200mg/L range, with the rise of the concentrations, the concentrations of 8-OhdG decreased accordingly (P<0.01).5.3 The effects of different concentrations of C60-lys on DNA in AHH-1 cells post irradiation:we detected DNA double-strand break damage of AHH-1 cells through the neutral single cell gel electrophoresis. And we analysis the results by CAPS-1 software. The results showed that normal cells in neutral electrophoresis revealing almost no tailing. Irradiation groups with and without drugs have some degrees of tailing. However, with the increase in drug concentrations, the cell tailing decreased.(P<0.01)Discussion and preliminary conclusions:Radiation damage to the body can be divided into direct damage and indirect damage, The main point of radiation protection is against indirect damage by clearing the radiation-induced free radicals. Fullerene is composed of 60 carbon atoms football-like substance with anti-virus, anti-bacterial and other biological properties. In addition, it has a strong free radical scavenging capabilities, known as "free radical scavenging sponge." However, fullerene is spherical non-polar substances, in itself is difficult to dissolve in water, which greatly affected its biological effects. Currently the primary means of resolving water-soluble is adding water-soluble group in the fullerene carbon cage,preparing water-soluble fullerene derivatives. C60-lys is a water-soluble fullerene derivatives which adding lysine in the fullerene carbon balls,and confirmed by NMR and IR spectra. Cell biology experiments found that C60-lys is low toxicity on the AHH-1 cells and K562 cells, it can improve the cell survival rate and reduce early apoptosis of AHH-1 cells after irradiation.And adding C60-lys before radiation is better then after radiadion in improving the survival rate of AHH-1 cells. We preliminary studied the mechanism of the radiation protection of C60-lys,and found that C60-lys can reduce radiation-induced AHH-1 cells in G2/M phase ratio, at the same time, adding C60-lys before radiation can be effective in reducing the 8-OhdG levels and DNA damage of the AHH-1 cells.