Cationic Helical Polypeptide-Mediated SiRNA Delivery Toward The Treatment Of Myocardial Ischemia Reperfusion Ischemia Reperfusion Injury

Myocardial ischemia reperfusion(IR)injury usually occurs during cardiovascular surgery and the treatment of myocardial infarction,which can cause irreversible tissue damage.During the process of IR injury,overwhelming inflammatory response caused by continuous up-regulation of pro-inflammatory factors will aggravate cell apoptosis and tissue damage.In the meanwhile,the limited proliferation ability of cardiomyocytes restricts the regeneration and repairing of heart tissues,further promotes cell apoptosis and collagen deposition,and ultimately hinders heart repair.Small molecular inhibitors are commonly used for the anti-inflammatory treatment of IR,but the potential side effects and high cost greatly limit their applications in clinical IR treatment.Gene therapy,especially RNA interference technology(RNAi),has been developed and utilized in the treatment of a variety of diseases,owing to the high specificity and high efficiency.Efficient delivery of siRNA can inhibit the excessive inflammatory response through down-regulation of the overexpressed pro-inflammatory factors.On the other side,it can also alleviate cardiac cells apoptosis,promote cell regeneration and repair,and reshape the unbalanced microenvironment of injured myocardium.These two methods can both protect the heart from acute myocardial infarction progressing to heart failure,and thus achieve the purpose of treating IR injury.The transfection efficiency of gene therapy relies on the construction of efficient and biocompatible gene delivery vectors.Cationic helical polypeptides,based on cell membrane penetrating peptides,have strong membrane penetrating activities,which can mediate efficient trans-membrane gene delivery to promote highly effective gene transfection into target cells.Moreover,the serum stability is another challenge for the construction of cationic gene delivery carriers,which can be improved by shielding the positive surface charge of cationic vectors via cell membrane camouflage for the widely ranged bio-applications in vivo.Based on the above understandings,we present a brief literature overview on the ischemia reperfusion injury,gene therapy,barriers of gene delivery,non-viral gene vectors,and cell membrane-coated drug delivery systems in Chapter 1.In Chapter 2,we constructed a series of guanidine and acromatic groups(benzyl,biphenyl,naphthyl,anthryl,and pyrenyl)modified cationic polypeptides with ?-helical structure to investigate the effect of aromatic types on siRNA delivery and transfection efficiency.The optimal one,benzyl group-modified polypeptide(P-Ben),was then used to condense RAGE siRNA(siRAGE)and suppress the inflammatory response toward the treatment of myocardial IR injury in rats.P-Ben could eficiently encapsulate siRNA to form stable and uniform nanocomplexes(NCs,?160 nm),promote cell uptake level,release RAGE siRNA in cardiac cells,and silence the target gene effectively.In hypoxia-induced H9C2 cells,RAGE mRNA expression was inhibited by 72%after P-Ben/siRAGE NCs treatment.In the rat model of myocardial IR injury,RAGE mRNA expression in the injured tissue was reduced by 84%after intracardiac injection of P-Ben/siRAGE NCs(150 ?g siRNA/kg).Significant reduction of the expression levels of pro-inflammatory factors,such as tumor necrosis factor(TNF-?)and interleukin-6(IL-6)were also observed,indicating the effectively suppressed inflammatory response.Moreover,the injection of P-Ben/siRAGE NCs reduced the myocardial infarct area(?7%),tissue fibrosis(?12%)and tissue necrosis,greatly lessened the apoptotic levels of cardiac cells(?13%),and basically restored the cardiac function.In order to improve the in vivo application of this kind of polypeptide,we constructed a hybrid cell membrane-camouflaged nanocomplex(named as BSPC@HM)with outer membrane shedding ability in acidic inflammation environment via acid-responsive charge reversal process in Chapter 3.This kind of BSPC@HM nanocomplex was used to deliver Salvador siRNA(siSavl)for the treatment of myocardial IR injury.The outer platelet-macrophage hybrid membrane(HM)enabled it long circulation and injury targeting ability in vivo.The acid-responsive polymer,polylysine-cis-aconitic anhydride(PC),could undergo charge reverse under acidic inflammation circumstances to trigger the outer membrane shedding specifically.The exposed polypeptide/siSavl(BS)NCs thus promoted the cellular internalization of siRNA molecules,effectively silenced Salvador expression,and motivated cardiac cells regeneration through the inactivation of Hippo signaling pathway.The hybrid membrane-coated BSPC@HM NCs had desired serum stability,and could inhibited Salvador mRNA levels(?90%)in hypoxia-induced H9C2 cells.In the rat model of IR injury,intravenous injection of BSPC@HM NCs(150?g siRNA/kg)could mediate effective uptake of siRNA in cardiac cells in the injured tissue,downregulated the Salvador mRNA expression(?67%),inhibited cardiac cells apoptosis(?16%),promoted the proliferation of injured cardiomyocytes through suppressing Hippo signaling pathway,consequently reduced the infarct area(?16%)and fibrosis(?15%),and promoted the recovery of cardiac function.