| Gene therapy for coronary artery disease is a hotspot in clinical medicine field,and micro RNA(mi RNA)is a very promising class of genes for clinical application.It has been found that mi RNA-1 play a key role in the progression of myocardial ischemic injury through post-transcriptional regulation.Mi RNA-1 inhibitor can effectively reduce the apoptosis of cardiomyocytes in vitro.However,the theraputic efficacy of mi RNA-1 inhibitor was greatly limited due to its poor stability and non-myocardium targeting in vivo.Targeted nanoparticle-mediated delivery system may provide an attractive approach to overcome these limitations.Objective To develop a ischemic myocardium-targeted nanovector for delivering mi RNA-1 inhibitor to enhence its myocardium-targeted and theraputic efficacy in moue myocardial infarction(MI)model.In addition,to explore the cardioprotective mechanism of the myocardium-targeted nanolized mi RNA-1 inhibitor via mi RNA-1 inhibition in ischemic myocardium.Methods PEGylated dendrigraft poly-L-lysine(DGL)anchored with myocardium-targeted peptide(AT1)was tailor-made(AT1-PEG-DGL),and it simultaneously armed with specific micro RNA-1 inhibitor(AMO-1),yeiding AT1-PEG-DGL@AMO-1 nanoparticles.Size distribution,zeta potential and bio-stability were detected on in vitro tests.Flow cytometry and confocal microscopy were used to detect and observe the uptake,bio-distribution and the targeting binding in cardiomyocytes.Immunofluorescence staining was performed to evaluate the pathological expression of AT1 R in the myocardium tissue and define optimal timing of administration.In vivo imaging was performed to observe the targeting capacity in MI heart.TUNEL assay,TTC staining and masson staining were conducted to evaluate the therapeutic efficacy with respect to apoptotic cell death in the infarct border zone,MI size and degree of fibrosis.Furthermore,The expression of mi R-1,Bcl-2 and PKCε was assayed by real-time PCR and Western blot 24 h after intravenous administration in MI mice for the mechanistic studies.Results AT1-PEG-DGL showed excellent gene compact capacity and could assemble AMO-1 into 200 nm nanoparticles(AT1-PEG-DGL @AMO-1)with a low positive charge of 4.3 ± 0.34 m V.In vitro study shows that AT1 conjugated PEG-DGL(AT1-PEG-DGL)could not only specifically target injured cardiac cells but also efficiently deliver AMO-1 to the cytoplasm(site of action).Most importantly,in vivo imaging of intravenous administration demonstrated that AT1-PEG-DGL would fast accumulate in the MI heart at desired early period,significantly outperforming the control group without AT1 targeting.Subsequently,once carrying AMO-1,a promisingly enhanced anti-apoptosis effects in vivo was observed upon a single intravenous injection.The apoptotic cell death in the infarct border zone and and the degree of fibrosis was obviously decreased(P<0.05),the myocardial infarct size was significantly reduced about 2.8-fold lower than that of the MI control group(P<0.05).Mechanistic studies reveal micro RNA-1 expression was significantly down-regulated(P<0.05)while relevant anti-apoptotic protein Bcl-2 and PKCε expression were increased(P<0.05)after administration.Conclusion In this study,we successfully encapsulated mi RNA-1 inhibitor into the dendrimer-based nanovector conjugated with a myocardium-targeted AT1 peptide to down-regulate mi R-1 in the ischemic cardiac tissue,so as to exert the anti-apoptotic cardioprotective efficacy.We demonstrated that the conjugation of AT1 peptide was able to enhance the early myocardium-targeting efficiency effectively and quickly,thereby induced obviously attenuation of cardiomyocyte apoptosis and significant reduction of infracted size in MI mice.It suggest that therapeutic nano-agent capable of targeting infarcted heart may prove to be benefit to effective and emergency treatment of MI in future. |
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