Studies On NGR Modified Docetaxel Loaded PH-sensitive Long Circulating Lipsomes

Since applicated in clinic, tumor therapy effect of docetaxel is remarkable, the listed agents are used more and more widely in clinic. But the side effects such as gastrointestinal tract and blood toxicity, allergic reactions, influence the following treatment, bring pain to patients. So the study of new drug delivery system, further improving the therapeutic index of drug, lowering the side effects of drugs, is one of the hot spot of research. Due to phagocytosis and clearance of reticuloendothelial cells, conventional liposomes can hardly get into tumor tissue, instead polyethylene glycol modified long circulating liposomes have extended blood circulation time and easily get into the tumor tissue through the capillaries. In the tumor itself and the tumor angiogenesis, some receptors on the endothelial cells can be modified to the surface of liposomes, which can enhance the targeting of the preparation, enhance the therapeutic effect and reduce the side effects of drugs.On the basis of common pH sensitive liposomes, this paper prepared passive and active tumor targeting to NGR modification of combination of docetaxel pH sensitive long circulating liposomes:reduce phagocytosis and clearance of reticuloendothelial system by long circulating of modified polyethylene glycol, improve the stability of the preparation in vivo; increase drug accumulation in tumor tissue through the modification of tumor targeting polypeptide; release drug targetedly by pH-sensitive release mechanism, combination of targeting delivery and controlled release. This paper prepared passive and active tumor targeting NGR-modified docetaxel loaded pH-sensitive long circulation liposomes, examine the pharmacy properties of liposomes and tumor targeting of in vivo and in vitro, provide experimental foundation and theoretical basis for the clinical application of liposomes.The main research methods and the results are as follows:1. Studies on docetaxel loaded pH-sensitive long circulating liposome (DTX/PLL)Using high performance liquid chromatography to determine concentration of docetaxel and establish a method for determination of content and entrapment efficiency of docetaxel in liposomes. Based on docetaxel loaded pH-sensitive liposome prescription, determine modification rate of long circulating material CHEMS-PEG2000 by single factor screening, the optimal prescription for the long circulating material modification rate of PE was 5%, have the highest liposome envelopment rate 71.88±0.99%. Liposomes prepared according to the optimal prescription had good outward appearance, physical and chemical properties, uniform particle size 192.54±7.73 nm, Zeta potential was 27.9±0.9 mV. The results of in vitro release experiments in different pH release medium showed that, the prepared docetaxel pH-sensitive long circulation liposomes is stable in pH7.4 slightly medium, the accumulative release rate at 72 h was 81.88%, and liposome in pH5.0 medium release faster, the accumulative release rate at 48h was 85.5%, indicating that liposomes had pH-sensitive in vitro release characteristics.2. Studies on NGR modified docetaxel loaded pH-sensitive long circulating liposomes (DTX/NGR-PLL)Synthesized peptide GNGRG, targeting new blood vessels endothelial cells, modified CHEMS-PEG2000-GNGRG polymers, the optimal molar ratio of target material is 0.8%, which is determined by with liposome envelopment rate, fluorescence intensity of cell intake; The size of optimal liposome was 202.34±7.40 nm, Zeta potential was-25.7±2.9 mV; The drug release behavior of NGR modified liposomes (DTX/NGR-PLL) was similar to undecorated liposomes (DTX/PLL), the accumulative release rate at 72 h was 81.58%, while the accumulative release rate at 48 h was 89.39%; After stored 3 weeks in the condition of room temperature (25℃),6 weeks in the low temperature of 4℃, appearance, size and the encapsulation rate of liposome solution changed little, mean stability of liposome is good.3. Pharmacokinetic studiesTake Doupafei(?) and ordinary liposome (DTX/PSL) as contrast agents, in vivo pharmacokinetic process of liposomes (DTX/PLL and DTX/NGR-PLL) in rats had high blood concentration and AUC0-∞ of DTX/PLL and DTX/NGR-PLL (AUC0-∞=41.521 and AUC0-∞=46.430) were higher than Duopafei(?)(AUC0-∞=12.290) and DTX-PSL (AUC0-∞=28.749). Compared with ordinary liposomes (DTX/PSL), PEG modification of two kinds of long circulating liposomes (DTX/NGR-PLL> DTX/PLL) had extended MRT in rats, MRT:DTX/NGR-PLL> DTX/PLL> DTX/PSL, can obviously prolong drug retention in the body and delay drug release.4. In vivo and in vitro tumor targeting research of DTX/PLL and DTX/NGR-PLLIn vitro targeting property of liposome was researched in the MCF-7 and HT-1080 cells. The cytotoxicity results determined by MTT method show that the antitumor activity of two kinds of liposomes were higher than free drugs, having lower IC50 alue concentration. Liposome preparation can increase the quantity of fluorescent material into the cell in cell uptake experiment, luorescent absorb experiment of adding free NGR solution further verify the targeting effect of NGR peptide in liposome. Fluorescence slice of liposomes in the tumor tissue tumor-bearing nude mice results show that both liposomes can targeting to the tumor tissue successfully, NGR modified liposome had more obvious accumulation; HE staining slices organization of mice showed that DTX/NGR-PLL has good security, similar to normal organs.In this paper, synthesis NGR modified targeted materials, and prepared DTX/PLL and DTX/NGR-PLL, which had good appearance, uniform particle size, pH-sensitive in vitro release ability, long circulating time in pharmacokinetics experiment in rats, in vivo and in vitro targeting property, all above were proved through experiments.