Preparation And Antitumor Activity Research Of PH-Responsive Targeted Drug-loading Peptides

Doxorubicin(DOX)as an anticancer drug could damage normal cells and tissues because of lack of tumor cell targeting,and results in serious toxic and negative effects.Therefore,nano-drug carriers with targeting tumor cells were important to reduce drug dosage and improve the efficacy of drugs.Nano-micelles was widely used for nano-drug carriers because exhibits excellent properties of easy preparation,long circulation time in vivo,and long-term drug release.Peptides is a common raw material for nano-drug carriers,which have advantages of good biocompatibility,low cost and rich variety.In this paper,an amphiphilic peptide P14(DGRGHHHFFFAAAA)was designed and prepared using solid-phase synthesis technique for encapsulating DOX to form P14-DOX micelles.The FFFAAAA sequence in the P14 peptide as a hydrophobic fragment could encapsulate hydrophobic DOX in aqueous solution to form the core of the nano-micelles.However,the DGRGHHH is a hydrophilic fragment,and could form the hydrophilic shell of the micelles.Simultaneously,the DGR fragment in P14 peptide could interact with integrins of tumor cells.The HHH sequence in P14 peptide as a pH-sensitive fragment could drive the self-assembly and disassembly of the nano-micelles under different pH conditions to control release of drugs.The main contents of this paper are as follows:1.P14 peptide was synthesized by solid-phase synthesis method,and then purified by high-performance liquid chromatography,the purity of P14 peptide was 96.838%.The critical micelle concentration of the P14 peptide was 1.758 mg/L.The results of acid-base titration experiments showed that P14 peptide had proton buffering ability.The results of IR,UV and fluorescence spectra showed that P14 peptide could encapsulate DOX to form P14-DOX micelles,the encapsulation efficiency and drug loading efficiency of P14-DOX micelles were 28.02%±1.35%and 12.06%±0.59%,respectively.The results of scanning electron microscopy and granulometry measurement showed that the P14-DOX micelles were relatively intact and homogeneous spheres.Simultaneously,the average particle size of the P14-DOX micelles was 121.64 nm,the PDI value was 0.281.2.The in vitro drug release results of P14-DOX micelles at different pH conditions(pH=7.4,6.5 and 4.5)showed that the drug release of P14DOX micelles was related to the pH of the solution and the curves of drug release were following the First-order kinetics model.On this basis,The CCK-8 kit was used to analyze the toxicity of P14 peptide,DOX and P14DOX micelle to MCF-7 tumor cells,and the results showed that P14 peptide was almost non-toxic to MCF-7 cells,while P14-DOX micelles were more toxic than free DOX under the same DOX concentration,and the drug concentration was negatively correlated with the cell survival rate.The flow cytometry results showed that the pro-apoptotic ability of P14DOX micelles was stronger than that of free DOX.Simultaneously,the tumor cell uptake of P14-DOX micelles was better than free DOX due to the presence of targeted RGD peptide in the P14 peptide sequence.3.To further improve the targeting and penetration ability of micelles to tumor cells,the RGD of the targeting hydrophilic terminal of P14 peptide was modified and P15 peptide(RGdRGHHHFFFAAAA,d is aspartic acid of type D)was synthesized.The P15-DOX micelles were characterized by UV spectrophotometer,IR spectrometer,fluorescence spectrophotometer,scanning electron microscopy,and granulometry,respectively.The results showed that the critical micelle concentration of the P15 was 0.0093 mg/mL,the P15 peptide could encapsulate DOX to form P15-DOX micelles,the encapsulation efficiency and drug loading effciency of P15-DOX micelles reached 34.66±0.97%and 14.50%±0.80%,respectively,the average particle size of the P15-DOX micelles was 234.21 nm and the PDI value was 0.213.In vitro drug release results showed that P15-DOX micelles had a significant pH sensitivity.In vitro cellular assays showed that P15-DOX micelles can be effectively taken up by MCF-7 cells,P15-DOX micelles were more toxic to MCF-7 cells than P14-DOX micelles and effectively promoted apoptosis in tumor cells.Finally,the body weight,normal organs and tumor tissues of hairless nude mice were investigated after injection of P15-DOX micelles reagent,and the results showed that micelles had almost no effect on the body weight of mice and low damage to normal organs,but could inhibit the growth of tumour volume in vivo,while the P15-DOX micelles had better in vivo killing effect on tumor tissues than free DOX.