The Study Of PCM And TAT Co-modified Liposome:Myocardium Targeting Delivery System

Ischemic heart disease is a kind of coronary artery disease,it is one of the most common causes of death in the world.The recognized traditional therapeutic methods at home and abroad include drug therapy,interventional therapy and surgical treatment.Though the drug treatment is medicable and safe,the drugs can not be concentrated in myocardium effectively.So exploring new and more targeted drug carriers has become a research trend in this field.Liposome is one of the most commonly used targeting drug delivery systems.Liposomes can enter cells through receptor mediated pathway or charge mediated endocytosis by modifying with different ligands.Thus liposomes can concentrate the drug at the disease site accurately and effectively to improve the targeting efficiency,so bioavailability can be improved and side effects can be reduced.PCM(WLSEAGPVVTVRALR GTGSW)is a 20-mer peptide obtained by phage display biopanning,it is a specific myocardium targeting peptide.TAT(YGRKKRRQRRR)is a kind of cell penetrating peptide which is widely used in the modification of drugs and drug carriers in recent years.TAT consist of 11 amino acids,drug and drug carriers can be delivered to cells after connecting with TAT through a concentration dependent way efficiently and quickly.In this paper,PCM and TAT modified liposomes were prepared,coumarin-6 and EGFP plasmids were used as a traditional model drug and a gene model drug respectively.Myocardial targeting ability of PCM and TAT co-modified liposome containing coumarin-6 was evaluated by in vitro and in vivo experiments,the results showed that PCM and TAT co-modified liposomes had pretty good myocardial targeting ability.The transfection efficiency of PCM modified liposomes loaded with EGFP plasmid was studied in vitro,the results showed that PCM modified liposome had fairly good myocardial transfection efficiency.In this study,PCM and TAT co-modified liposomes were designed and constructed to improve the myocardial targeting ability.In the first section,the preparation process and prescription of PCM and TAT co-modified liposomes loaded with coumarin-6 were screened.Firstly,the liposomes were prepared by film-ultrasonic method which was simple operation and the preparation process was stable.PCM and TAT were conjugated covalently to the distal end of DSPE-PEG2000-MAL by addition reaction of cysteine sulfhydryl group and double bonds of maleimide,and then the polypeptide is attached to the surface of liposome.The optimal dosage of coumarin-6 and the connection mode of PCM and TAT were optimized by the encapsulation efficiency.The cellular uptake by H9C2 cells was evaluated and the optimal dosage of PCM was 3%,the optimal dosage of TAT was about 1%.The morphology of co-modified liposome was rounded and the particle size of co-modified liposome was about 115 nm.The second section evaluated the myocardial targeting ability of PCM and TAT co-modified liposomes loaded with coumarin-6 in vitro preliminarily.Ordinary liposome(L),PCM modified liposome(3%P-Lip),TAT modified liposome(1%T-Lip)and PCM and TAT co-modified liposome(3%P-1%T-Lip)loaded with or without coumarin-6 were prepared.The toxicity of various liposomes without coumarin-6 were determined by CCK-8,the results showed that there were no obvious toxicity of liposomes when the dosage of various liposome were less than 0.4 mM.The uptake of primary myocardial cells of different liposomes were tested by confocal microscopy and flow cytometry qualitatively and quantitatively,the cell uptake rate of different liposomes were 3%P-1%T-Lip higher than 3%P-Lip higher than 1%T-Lip higher than Lip.At the same time,the effects of free PCM and TAT on the uptake of liposomes were also studied,the results showed that free PCM inhibited the uptake of PCM modified liposomes,while the free TAT had no significant inhibition on the uptake of TAT modified liposomes.In the third section,the in vivo evaluation was carried out to verify the myocardial targeting ability of PCM and TAT co-modified liposomes loaded with coumarin-6 further.Qualitative observation of heart frozen sections of Kunming mice injected with various liposomes by tail vein injection was carried out by confocal laser scanning microscopy,and the content of coumarin-6 in heart,liver,spleen,lung,and kidney were determined by HPLC-FLD quantitatively.The results of qualitative observation and quantitative determination showed that accumulation of various liposomes in the heart were 3%P-1%T-Lip higher than 3%P-Lip higher than 1%T-Lip higher than Lip.The results proved that 3%P-1%T-Lip had fairly good myocardial targeting ability.Though vorious liposomes were also distributed in liver,spleen,lung and kidney,compared with the control group,3%P-1%T-Lip could increase the accumulation in myocardium,there was no significantly increase in the distribution of liver,spleen,lung and kidney.The forth section studied the preparation and in vitro targeting ability of PCM modified liposome loaded with enhanced green fluorescent protein expression plasmid(pEGFP)to improve the transfection efficiency of gene drugs.DOTAP was added in this part to be cationic lipid materials,liposomes were prepared by film-ultrasonic method,and then DSPEPEG2000-PCM was inserted into liposomes in a micellar form.LIP,1%PCM-LIP,3%PCM-LIP and 5%PCM-LIP were prepared.The binding method of PCM was screened by measuring the connection rate of PCM,and the drug loading capacity of liposomes were examined by agarose gel electrophoresis.The transfection efficiency of different PCM modified liposomes were investigated by qualitative observation and quantitative analysis.The optimal dosage of PCM was 3%.The particle size of LIP and 3%P-LIP loaded with pEGFP were 212.4±2.95 nm and 261.9±2.20 nm respectively,the particle size of liposome was increased after PCM modified.In summary,PCM and TAT co-modified liposomes containing coumarin-6 were successfully prepared,in vitro and in vivo experimental study indicated that it is a potential delivery system for the treatment of heart disease.PCM modified liposomes containing pEGFP were also prepared,in vitro experiments showed that PCM modified liposomes had myocardial targeting properties,but the in vivo experiments should be studied further.