Hyperbranched Nanoparticle Based Gene Transfection Combined With Skeletal Myoblasts Transplantation For Ischemic Heart Disease

Background:As excellent seed cells and gene-engineering cells, skeletal myoblasts with high purity exhibit promising application in clinic. The fast, effective and economic method for skeletal myoblasts isolation is being investigated.Objective:This study was designed to establish a novel method of isolation and purification for adult mice skeletal myoblasts.Methods:Bupivacaine was used for proliferation of mice skeletal myoblasts. Forty eight hours later, muscles were digested by mixed enzyme and skeletal myoblasts were purified by our novel method. After immunofluorescence cell staining, the novel method was compared with different adhesion time method, repeatedly different adhesion time method, incontinuous density Percoll gradient centrifugation technique by the purity of skeletal myoblasts.Results:Using our novel method, the putity of skeletal myoblasts could achieve92.59±1.29%, which were significantly higher than that of cells purified by traditional methods (p<0.05). And excellent proliferation and differentiation were observed in these primary skeletal myoblasts.Conclusions:The novel method could be used to rapidly isolate and obtain high-purity myoblasts derived from skeletal muscle of adult mice. Background:Gene therapy is a promising strategy for a few diseases. And the most demanding task in gene delivery systems is optimization for gene vectors.Objective:In this study, hyperbranched polyamidoamine (h-PAMAM) was developed as a novel non-viral gene vector for the first time.Methods:The h-PAMAM nanoparticles were synthesized using a modified "one-pot" method. Deoxyribonucleic acid (DNA) was then bound to h-PAMAM nanoparticles at different weight ratios (Wh-PAMAM/WDNA).The particle size and zeta potential was investigated. And deoxyribonuclease I (Dnase I) was used for degrade h-PAMAM-DNA complexes. The efficiency and cytotoxicity of h-PAMAM based gene transfection in COS7and HEK293cell lines were evaluated by flowcytometry. And the gene expression after transfection was tested by immunofluorescence cell staining, enzyme linked immunosorbent assay and realtime polymerase chain reaction.Results:The higher weight ratio could bring larger particle size and higher zeta potential of h-PAMAM-DNA complexes. The encapsulated DNA was protected by h-PAMAM from degradation for over2h. Under the optimal condition, high gene transfection efficiency could be achieved in COS7cells (47.47±1.42%) and HEK293cells (40.8±0.98%) cell lines. And h-PAMAM showed rather minor cytotoxicity in vitro (cell viability=91.38±0.46%in COS7cells and92.38±0.61%in HEK293cells). The h-PAMAM mediated human vascular endothelial growth factor165(hVEGF165) gene transfected cells could express hVEGF165stably for14days, with the peak expression at day2.Conclusions:In conclusion, h-PAMAM based gene delivery was economical, effective and biocompatible, and may serve as a promising non-viral vehicle for gene therapy. BackgroundSkeletal myoblasts (SkMs) transplantation combined with vascular endothelial growth factor (VEGF) gene delivery has been proposed as a promising therapy for cardiac repair. Nevertheless, the defective gene vectors and unregulable VEGF expression in vivo hinder its application. Therefore, sought of an economical, effective, controllable gene delivery system is quite necessary.Obejective:We investigated the feasibility and efficacy of hyperbranched polyamidoamine (h-PAMAM) nanoparticle based hypoxia regulated human vascular endothelial growth factor-165plasmid (pHRE-hVEGF165) transfer combined with SkMs transplantation for cardiac repair.Methods:Deoxyribonucleic acid (DNA) was then bound to h-PAMAM at different weight ratios (Wh-PAMAM/WDNA).The efficiency and cytotoxicity of h-PAMAM based gene transfection in SkMs were evaluated by flowcytometry. And the gene expression after transfection under hypoxia condition was tested by immunofluorescence cell staining, enzyme linked immunosorbent assay and realtime polymerase chain reaction. The h-PAMAM-HRE-hVEGF165transfected SkMs were transplanted in ischemic myocardium. The apoptosis of myocadiocytes, grafted skeletal myoblasts survival, infarct size and interstitial fibrosis, blood vessel density and heart function were investigated on C57/BL6mice models.Results:Transfection efficiency of h-PAMAM in SkMs was43.47±2.22%with cell viability of91.38±0.48%under optimal condition. h-PAMAM-pHRE-hVEGF165transfected SkMs could express hVEGF165stably for18days under hypoxia. After cell transplantation, the overexpressed VEGF could result in reduction of apoptotic myocardiocytes, improvement of grafted cell survival, decreasement of infarct size and interstitial fibrosis, and increasement of blood vessel density, which inhibited left ventricle remodeling and improved heart function at the late phase following infarction.Conclusions:These results indicate that h-PAMAM-based pHRE-hVEGF165gene delivery into SkMs is feasible and effective, and may serve as a novel and promising gene therapy strategy in ischemic heart disease.