Design,Synthesis And Biological Evaluation Of New Drug Delivery System LTV-HR And Acid-sensitive Cell Penetrating Peptides LH-1?LH-2

The traditional tumor treatment of chemotherapy has serious side effects,for its poor permeability and nonspecial biological distribution.In order to overcome these shortages,it is desirable to designed and synthesized new kind of tumor carries which have high permeability,tumor cells targeting,and safety.Now,the cell penetrating peptide is one of the most promising drug carriers,which has the characteristics of rapidly entry into cells and low toxicity.However,nature cell penetrating peptides can't distinguish between normal tissue and tumor tissue.At present,there are two kinds of targeted modification strategies: One is targeted to the tumor microenvironment,because its p H,matrix metalloproteinase,hypoxia and vascular system are different from normal tissue.The other one is receptor and ligand targeting strategies.The researchers discovered that many tumor cells have over expressed receptors,while the normal cells don't have or low expression.The first part of the thesis was to design a new drug delivery system.The research group has designed an acid-sensitive membrane penetrating peptide(HR-1),which has the characteristics of low membrane permeability under physiological conditions and high membrane penetrating activity in acid condition.But the targeting effect of HR-1 was passive targeting.In order to enhance the ability of HR-1 to carry more anticancer drugs into cells,we decided to combine the acid sensitive peptide HR-1 with active targeting peptide LTV which can active targeting to HER2 receptor over expressed in breast cancer cells through cysteine to get a new drug delivery system LTV-HR.LTV-HR was covalently bonded with camptothecin via two disulfide bonds to form LTV-CPT-HR.LTV-HR has acid sensitivity shows in the laser scanning confocal microscope,cell apoptosis assay and anti-tumor activity experiments.We found that for it can carry more camptothecin into cancer cells in the pH6.0 condition,but in the antagonistic experiment we found that the targeting peptide LTV of LTV-HR showed no obvious active targeting.LTV-HR shows good safety and low toxicity in vitro experiments,whether it was incubated with the cells for a long time or under different pH conditions or the lactate dehydrogenase release test.In thehemolytic activity test,although the high concentration of 200 ?mol/L,the hemolysis rate reached to 13.35%.But the hemolysis rate of 100 ?mol/L was about 3.44%,which was about 2.5 times of the maximum concentration 40?mol/L in the test,which indicated that LTV-HR was safe in the range of test concentration.In conclusion,LTV-HR has good acid-sensitivity and low toxicity,which is expected to be one of the carriers for tumor targeted therapy.The second part of the study was to design new acid-sensitive penetrating peptides.The LH-1 and LH-2 were obtained by amino acid substitution of LK(LKKLLKLLKKLLKL-NH2).In the cellular uptake experiment we see that LH-2 showed obvious acid sensitivity,under the condition of pH6.0 its fluorescence intensity was significantly higher than that of pH7.4,indicating that a large amount of LH-2 were uptook by cell under acidic conditions,but LH-1 in the two pH conditions showed no significant difference.LH-1 and LH-2 show significantly lower toxicity than LK in all vitro toxicity experiments.In toxicity test and LDH release test at different p H,the LH-2 display very weak toxicity but the toxicity of LH-1 was differences.And the cell survival rate and LDH release rate of LH-1 at p H7.4 were significantly lower than that of pH6.0,In p H7.4 the positive charge of LH-1 is blocked so that the toxicity is very low.But in pH6.0,the histidine is protonated with a large number of positive charges,which shows a similar toxicity to the mother peptide.Through the toxicity test and cell uptake experiment results,we chose LH-2which has lower toxicity and better acid-sensitive to combine with camptothecin.The MTT result shows that LH-2 has stronger penetrating effect in pH6.0,and can carry more camptothecin into cells to kill cancer cells.LH-2 can not only significantly reduce the toxicity but also has the ability of targeting the tumor microenvironment in comparing with the mother peptide LK.