Cardiomyocytes Targeting Peptide Modified PAMAM Derivative Mediated MiR-378 Delivery To Inhibit Pathological Cardiac Hypertrophy

Persistent pressure overload can promote adverse cardiac remodeling,mainly manifested as left ventricular hypertrophy and interstitial fibrosis,which is one of the major causes of heart failure.Previous studies have confirmed that cardiac remodeling is affected by hemodynamic load and neurohormonal activation,but the molecular mechanisms regulating cardiac remodeling remain unclear.Although the current treatments have delayed the occurrence and development of myocardial hypertrophy and fibrosis to a certain extent,they have not delayed the progression of heart failure.Therefore,it is of great significance and value to find drugs that inhibit the development of cardiac remodeling.Gene therapy has become one of the important research topics in the field of cardiology.Gene therapy refers to the artificial introduction of therapeutic genes into target cells or target tissues in order to correct or improve the expression of abnormal genes and achieve the purpose of treatment.Delivery of DNA,miRNA and si RNA is an attractive approach for gene therapy,which regulates gene expression in a specific,efficient,and low-toxic manner.miR-378 has been confirmed to be one of the miRNAs specifically expressed in cardiac tissue.Previousstudies have shown that the expression of miR-378 is down-regulated in pressure overload models,and exogenous supplementation of miR-378 can inhibit the occurrence of cardiac hypertrophy.In addition,miR-378 further inhibits myocardial fibrosis through paracrine manner.miR-378 has received some attention as a gene therapy for cardiovascular disease.However,miRNA,as a small nucleic acid,is not stable enough and can be easily degraded.And most of the small nucleic acid themselves have a negative charge,which make it difficult for them to pass through the cell membrane with the same negative charge.Moreover,non-specific delivery of nucleic acid will greatly reduce the effects of gene therapy,and even produce serious toxic side effects.Therefore,miRNAs must rely on efficient and safe gene delivery carriers to exert their biological functions.Polyamidoamine(PAMAM)dendrimer is a kind of mature non-viral gene delivery system.It is widely used in gene delivery field because of its abundant positive charges,highly branched structure,water solubility,and amino groups which can be further modified.However,PAMAM carriers have high cytotoxicity and their transfection efficiency is not satisfactory,limiting their clinical application.Based on previous studies,we constructed a class of nucleobase-modified PAMAM derivative carriers(AP-PAMAM)with high efficiency and low toxicity to achieve efficient and stable delivery of small nucleic acids in different diseases.However,since the carrier itself does not have cardiac targeting capability,in order to further improve the cardiac targeting capability of the carrier and optimize delivery efficiency,a cardiomyocyte targeting peptide(CMP)modified AP-PAMAM with capable of targeting and recognizing cardiomyocytes was constructed in this study,named MP.We used this carrier to mediate the delivery of miR-378 to verify its role in pathological cardiac hypertrophy.This research is divided into three parts:1.Synthesis and characterization of MP carrier:the 5th generation PAMAM was modified with 2-amino-6-chlorpurine,and AP-PAMAM was successfully constructed,referred to as AP for short.CMP was coupled with AP through the dehydration condensation reaction of amino and carboxyl groups,and a gene delivery carrier MP with cardiac targeting ability was obtained.The structure and relative molecular weight distribution of MP were characterized by nuclear magnetic resonance(~1H NMR)and gel permeation chromatography(GPC),which proved that MP carrier was successfully synthesized.The morphology of MP was characterized by transmission electron microscopy(TEM),and the particle size distribution of nanoparticles was quantitatively analyzed by the Nano measure.Furthermore,through MTT assay,it was confirmed that MP had low cytotoxicity.Through gel retardation experiment,it was confirmed that MP has strong nucleic acid condensation and binding ability.2.In vitro,MP-mediated miR-378 delivery achieved the goal of anti-myocardial hypertrophy and myocardial fibrosis at the cellular level.In this part,we successfully achieved the intracellular delivery of miR-378 in neonatal cardiomyocytes,cardiac fibroblasts,and rat embryonic cardiomyocytes H9c2 by using MP as the gene delivery system.Through flow cytometry,RT-q PCR,and confocal laser microscopy,it was confirmed that MP could targeted delivery miR-378 into cardiomyocytes and successfully achieved lysosome escape.Subsequently,RT-q PCR experiments confirmed that miR-378 could regulate the expression of hypertrophy related genes(Nppa,Nppb,Myh7)and fibrosis related genes(col1α1,col3α1,α-SMA).Western blot proved that MP mediated the delivery of miR-378 reduced the phosphorylation of key proteins in MAPKs pathway ofcardiomyocytes and also reduced the phosphorylation of key proteins in p38 and Smad2/3 pathway.These results suggested that MP can successfully mediate miR-378 delivery to achieve against cardiac remodeling.3.In vivo,MP mediated miR-378 delivery to achieved the goal of cardiac targeted delivery and against myocardial hypertrophy and fibrosis at the animal level.In this part,we established a pressure overload animal model by transverse aortic constriction(TAC)in 8-week-old male C57BL/6 mice,and verified that MP/miR-378 nanoparticles could significantly improve the accumulation capacity of miR-378 in cardiac tissues.In this way,a more significant against myocardial hypertrophy and fibrosis effects could be achieved,and cardiac function could be further improved.In addition,the pathological analysis of the major organs of mice confirmed that the MP/miR-378 nanoparticles had good biological safety.The above studies indicate that MP can be used for cardiac targeted delivery of miR-378,achieving good therapeutic effects against myocardial remodeling at the animal level.In conclusion,this study designed a nucleic acid delivery carrier MP which modified with cardiomyocyte targeting peptide and capable of targeting recognition of cardiomyocytes.MP can deliver small nucleic acids to cardiomyocytes and heart tissues in a targeted,efficient,and safe manner,and exert their biological functions.It provides new evidence for the design of nano drugs based on small nucleic acids and the exploration of precise therapeutic targets.