| Backgroud:Spermicide is an effective means of birth-control and contraception, in which nonoxynol-9(N-9), because of its strong spermicidal effect become a widely accepted class of surfactant spermicide, has been in the United States and some other countries used more than30years. As an effective topical vaginal spermicidal contraceptive biologics, in addition to have a fast and effective contraceptive spermicide, safe and low toxicity is also very important. But recent studies have shown that, N-9security risk, long-term repeated use of vaginal epithelial cells might be caused by injury, so as soon as possible to develop new security spermicidal contraceptive imminent.Antimicrobial peptides are widely present in vivo resistance to a class of exogenous pathogenic microorganisms pathogenic role of defensive small peptides (molecular weight<10ku). Antimicrobial peptides is an important part of the host innate immune system. Each has its specific antimicrobial peptides antibacterial spectrum, some antimicrobial peptides also possesses spermicidal effect. Amphibian frog skin secretions are rich in antimicrobial peptides, they show out the anti-bacterial, anti-tumor, anti-viral and other aspects of spermicidal powerful, and they have been applied in areas such as agriculture and clinical.Currently, foreign reports about spermicidal antimicrobial peptides such as Magainin, Dermaseptins, Nisin and Subtilosin and so on, those antimicrobial peptides have demonstrated strong in vitro spermicidal effects, for example Magainin,800-1000mg/L Magainin20s can be completely immobilization monkey semen (concentration of60×106/mL-80×108/mL). Notably,When Magainin spermicide in the minimum concentration, the red blood cells and the vaginal mucosa are not toxic, it shows a high level of safety.Therefore, antimicrobial peptides become a hot research topic in recent years Maximin, Which is a linear cationic peptides, is proprietary lead compound by LAI Ren research team who first isolated extracted from Chinese Bombina toad in the skin especially Maximin is similar to Magainin, because of they have the same concentration in spermicidal activity, so it becomes the preferred antimicrobial peptide in our experiments. Through Our experiments are based on spermicidal and cytotoxicity evaluation in vitro to the19kinds of antimicrobial peptides Maximin derivative (M1-M19), screening some new safe and effective spermicide candidates in vitro.Objective:1. By spermicidal experiment in vitro and cytotoxicity assay to Maximin’s19kinds of derivatives (M1-M19), screening some antimicrobial peptides with strong spermicidal effect and low cytotoxicity.2. Through rabbits anti-fertility experiment, observing the isolated antimicrobial peptides who has a strong effect spermicidal effect in vitro and low cytotoxicity are anti-fertility and the preliminary evaluation of its safety.3. Preliminary exploration spermicide mechanisms of antimicrobial peptides and their antibacterial activity.Circuit and method1. Semen collection and analysis:Semen samples from Beijing sperm bank. Selecting childbearing age, health, and have normal reproductive history of48cases of male volunteers, aged25to35years old, abstinence72h and then masturbation method to obtain semen (semen volume>2mL), incubated30min for liquefaction at37℃. After liquefaction, routine analysis conducted in strict accordance with the "WHO human semen and Processing Laboratory Manual " the fifth edition of the recommended standards, of which the total number of sperm60×106-100×106/mL, sperm concentration>20×106/mL, sperm activity rate>60%.2. Spermicidal in vitro experiments:According to WHO recommended Sander-Cramer method, semen mixed with antimicrobial peptides rapidly1:5,37℃water-bath20s, random observation under a microscope counting five horizons, then eosin-nigrosin staining technique to observe sperm cell survival, screening some antimicrobial peptides derivatives who have strong spermicidal effect. Simultaneous determination of spermicidal antimicrobial peptides and N-9’s EC100-The last, Determination the spermicidal antimicrobial peptides’Time-Concentration dependence.3. Cell toxicity studies:the use of CCK-8kit to detect cell toxicity of antimicrobial peptide derivatives and N-9’s cytotoxicity, to evaluate the cytotoxicity of N-9and filter out lower cytotoxic antimicrobial peptide derivatives, and then calculate their IC50values. 4. Rabbits anti-fertility experiment:The method of administration to the vagina in the female reproductive Zealand white rabbits injected the spermicide dose antimicrobial peptide solution to vagina, then observation and statistics rabbit pregnancy after mating. Saline as a negative control, N-9as a positive control. Rabbit in the same components are observed the effect of erythrocyte hemolysis after vaginal administration.5. Preliminary experiments on spermicide mechanism:using HOS-EG experimental methods to understand whether or not and where destruction sperm membrane.6. Antibacterial experiments:according to the microdilution assay to detect the candidates of spermicidal antimicrobial peptide derivatives’antibacterial effect to Escherichia coli and Candida albicans.Results:1.19kinds of antimicrobial peptides Maximin derivative (M1-M19), which in M1、 M7、M11、M15and M17, whose five antimicrobial peptides’s concentration2000mg/L was able to completely immobilize sperm in20s. The five antimicrobial peptides’s ECioo are:M7=M11=2000mg/L, M1=M15=M17=1500mg/L. The N-9’s EC100=100mg/L.2. When using Hela-229analyse cell cytotoxicity, M11’s cytotoxicity is significantly lower than the other four peptides, exhibiting higher safety. N-9’s cytotoxicity is very strong that IC50=3.01mg/L.3. When compared with the negative and positive control group, M11drug group’s number of live births and the contraceptive rate are not statistically significant.4. HOS-EG experiment’s result shows spermicidal antimicrobial peptide and N-9are completely destroyed sperm membrane, A-type are all0; M11major damage to the sperm tail, and M1and M17are substantially damage the sperm head and tail together, this mode of action is similar to the N-9.5. Microdilution analysis spermicidal antimicrobial peptide derivative M11that whether inhibit E. coli and Candida albicans or not, and the results M11can not exhibit inhibitory effects on both in the50mg/L.Conclusions:1.19kinds of antimicrobial peptides Maximin derivative (M1-M19), which in M1、 M7、M11、M15and M17, these five antimicrobial peptides have strong spermicidal effect in vitro.2. Cytotoxicity experiments showed cytotoxicity size in the five antimicrobial peptides is M11<M15<M17<M7<M1. In which that the antimicrobial peptide M11showed almost no cytotoxic characteristics, M11have the highest security.3. Spermicidal antimicrobial peptides (M11, M15and M17) have obviously spermicidal time-concentration-dependent features;HOS-EG experiments show that antimicrobial peptides spermicidal effect by disrupting the cell membrane of sperm.4. Spermicidal antimicrobial peptide derivatives M11fails to achieve the purpose of rabbit anti-fertility experiments.5. Spermicidal antimicrobial peptide derivatives M11’s inhibition of E. coli and Candida albicans experiment failed. |
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