Endocytic Pathways Involved In The Uptake Of TAT-LHRH Modified Chitosan/DNA Nanoparticles By HepG2Cells

With the development of molecular biology, cell biology and genetic engineering techniques, gene therapy becomes a new manner in the treatment of genetic diseases, malignant tumors, cardiovascular disease, AIDS and other diseases, and there is a broad application prospects in it. The choice of the gene therapy vector is the key to the success of gene therapy. Gene therapy vectors include viral vectors, nonviral vectors and bacterial vectors. Non-viral vectors exhibit low immunogenicity, low cytotoxicity, a large gene capacity and simple to prepare, these showing their superiority as gene therapy vectors.Our previous research showed that TAT-LHRH modified chitosan/DNA nanoparticle (TLCDN) exhibits high transfection efficiency and targeting to HepG2, but the endocytic pathways of TLCDN entering into HepG2cells is not clear. In this study, we performed uptake inhibition experiments, co-localization experiments and detecting the effects on transffection efficency caused by blocking different endocytic pathways to explore the endocytic pathways of TLCDN.The methods were shown below, plasmid DNA was labeled with fluorescein, and the resulting fluorescent DNA was complexed with TAT-LHRH modified chitosan or unmodified chitosan to form TLCDN and chitosan/DNA nanoparticle (CDN) by the complex coacervation method. Internalization of TLCDN or CDN by HepG2cells were measured in the presence of three kinds of endocytic pathway inhibitors, chlorpromazine (CPM), filipin (FP) or dynasore (DS), and detected gene transfection efficiencies after uptake inhibition. In addition, the TLCDN and CDN were co-incubated with three kinds of endocytosis pathway markers, transferring (TF), cholera toxin B (CTB) or dextran (DX), and calculated the co-localization rates.The results showed that clathrin-dependent endocytic pathway inhibitor CPM led to less decreased uptake of TLCDN than that of CDN (the inhibition rates were12.0%and47.7%, respectively), caveolin-mediated endocytic pathway inhibitor FP demonstrated significant inhibitory effect on the uptake of TLCDN(the inhibition rate was55.4%) while promoted the uptake of CDN (the promotion rate was29.6%), dual-endocytic pathway inhibitor DS resulted in a similar decrease in the uptake of both nanoparticles (the inhibition rates were64.0%and62.6%, respectively). The co-localization rate of clathrin-dependent endocytic pathway marker TF with TLCDN (15.2%) was lower than that with CDN (20.7%), but the difference was not significant. The co-localization rate of caveolin-mediated endocytic pathway marker CTB with TLCDN (48.1%) was significantly higher than that with CDN (9.1%). And the co-localization rate of maropinocytosis pathway marker DX with TLCDN (28.5%) was significantly lower than that with CDN (48.0%). With the treatment of CPM resulted in a less decrease on the TLCDN-mediated gene expression (15.1%) than that of CDN-mediated gene expression (47.0%), whereas treatment with FP led to a decrease on the TLCDN-mediated gene expression (58.7%) and an increase on the CDN-mediated gene expression (26.6%). With the treatment of DS resulted in similar levels of suppression on the TLCDN-mediated gene expression (65.6%) and the CDN-mediated gene gene expression (58.3%)It was demonstrated that CDN was taken up by HepG2cells mainly through maropinocytosis although the clathrin-dependent endocytic pathway and the caveolin-mediated endocytic pathway was involved, and TLCDN was more likely to be internalized by HepG2cells through the caveolin-mediated endocytic pathway although the clathrin-dependent endocytic pathway and maropinocytosis were also involved.