Synthesis Of RGD Peptide Targeting Lipids And Its Research On Drug Delivery And Gene Therapy For Tumors

Cancer is one of the major diseases threatening human life and health.The morbidity and mortality of cancer in China are the highest in the world.Therefore,the development of safe and efficient cancer treatment methods has become a major strategic demand of the country.Among a variety of tumor treatment methods,drug therapy has become an important method for clinical treatment of cancer,including chemotherapy and gene therapy.However,chemical drugs have some problems such as poor targeting,low bioavailability and high toxicity.And the low efficiency of gene delivery has been restricting the development of gene therapy.A delivery carrier is needed to change the way that drugs enter the cells and its distribution in the body,control the release rate of drugs,prolong the action time of drugs,so as to improve the utilization of drugs.Therefore,delivery vector is the key for the development of drug therapy.In this study,the integrin αvβ3 overexpressed in tumors was used as a therapeutic target.Based on the specific bingding characteristics of the targeting ligand RGD peptide and integrin αvβ3,a targeted liposome carrier system was constructed for the delivery of chemical drugs and genes to realize targeted chemotherapy and gene therapy of tumors.Firstly,3-amino-1,2-propanediol,carbonyl diimidazole(CDI)and tetradecylamine were used as raw materials to synthesize targeted lipid intermediates by activated ester method.Linear and cyclic RGD peptide-based lipids were prepared by linking an RGD peptide to lipid intermediates via amide condensation reaction.The structure of RGD-lipid and cRGD-lipid was confirmed by mass spectrometry(MS),infrared spectroscopy(IR)and nuclear magnetic resonance spectroscopy(1H NMR).Their puritites were 95%and 97%detected by high performance liquid chromatography(HPLC),respectively.Then RGD peptide doxorubicin liposomes,using RGD-lipid and cRGD-lipid as targeted components,lipid CDO14 and co-lipid DOPE,were obtained by thin-film ultrasonic dispersion method to deliver chemotherapy drug DOX for tumor targeted chemotherapy.RGD peptide doxorubicin liposomes were spherical or ellipsoidal in shape,with particle size ranging from 90-130 nm and Zeta potential ranging from 40-60 mV.RGD peptide doxorubicin liposomes had higher drug loading(6.25%)and encapsulation efficiency(80%-98%)at the lipid-drug mass ratio of 10:1.In vitro drug release results showed that RGD peptide doxorubicin liposomes exhibted better sustained drug release and were pH-sensitive.RGD peptide doxorubicin liposomes improved cellular uptake,selectively inhibited the growth of tumor cells and had no significant effect on normal cells.In vivo imaging of mouse organs showed that the modified liposomes could effectively aggregate at the tumor site.Therefore,the RGD peptide doxorubicin liposomes had good tumor suppression effects,reduced the damage of drugs to normal tissues and organs,improved the bioavailability of DOX and greatly reduced the toxicity of DOX in vivo.The tumor volume of cRGD(5%)/CD-DOX group was 2.17 times smaller than that of Saline group.In order to achieve targeted gene delivery,a series of RGD peptides targeting liposomes,using RGD-lipid and cRGD-lipid as the targeted components,were prepared by adjusting the targeting modification density.These liposomes could bind genes through electrostatic action for tumor targeted gene therapy.The particle size of RGD peptide targeting liposomes ranged from 50 to 100 nm,and the Zeta potential was between 40-60 mV.The results of protein adsorption experiments showed that RGD peptide targeting liposomes had less non-specific protein adsorption,which could extend the blood circulation time of liposomes,and the targeted modification density of liposomes had no significant effect on its protein adsorption.Gel electrophoresis experiments,in vitro transfection experiments and gene silencing showed that RGD peptide targeting liposomes havd good ability to bind and deliver genes.The highest cell uptake rate was up to 94%,and the highest gene silencing efficiency was up to 50%.The results of CCK-8 cytotoxicity test,cell apoptosis and anti-tumor experiment in mice proved that RGD peptide targeting liposomes loaded with IGF-1R siRNA could actively target tumor cells and induce apoptosis,thereby significantly enhancing the antitumor efficacy of genes in vivo and in vitro.Among them,cRGD(5%)/CD@IGF-1R siRNA had the best anti-tumor effect in vivo.Its tumor volume was 6.94 times smaller than that of the Saline group,and its anti-tumor effect was better than the commercial reagent LipoRNAiMAX.Serum biochemical indicators and H&E staining results showed that IGF-1R siRNA loaded RGD peptide targeting liposomes had no obvious side effects on normal tissues and organs of mice.In summary,two RGD peptide cationic targeting lipids were designed and synthesized for the first time.The targeting liposome carrier systems constructed by these lipids had the advantages of good targeting ability,drug and gene carrying capacity and biocompatibility.This paper showed that RGD peptide targeting liposome carrier systems had a good application prospect in the targeted therapy of tumors.