Adil Maroof M, Erdman Zachary S, Kokkoli Efrosini
Department of Chemical Engineering and Materials Science, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Langmuir. 2014 Apr 8;30(13):3802-10. doi: 10.1021/la5001396. Epub 2014 Mar 27.
Receptor targeted, PEGylated transfection agents can improve stability and delivery specificity of current cationic lipid and polymer based nonviral gene delivery vehicles, but their mode of transfection is poorly understood. We therefore investigated the transfection mechanisms of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipoplexes, branched polyethylenimine (bPEI) polyplexes, and bPEI encapsulated in either PEGylated (stealth) nontargeted liposomes or PR_b peptide (targeted to α5β1 integrin) functionalized stealth liposomes in DLD-1 colorectal cancer cells in vitro with gene expression assays, flow cytometry and confocal microscopy. DOTAP/DOPE and PR_b functionalized stealth liposomes mediated higher gene expression compared to nontargeted stealth liposomes and bPEI. However DOTAP/DOPE was internalized slowly leading to lower levels of DNA uptake. In contrast, despite high internalization of bPEI polyplexes, gene expression levels were low as DNA was unable to escape from the endosomes. Nontargeted stealth liposomes also mediated low gene expression due to low amounts of DNA internalized and slow internalization kinetics. PR_b functionalized stealth liposomes struck an optimal balance among these transfection agents with efficient transfection arising from fast integrin mediated internalization kinetics, high amounts of DNA uptake, and endosomal escape. We found α5β1 integrin to be a valuable target for gene delivery and that the caveolar endocytic pathway may offer an advantage to receptor targeted PEGylated transfection agents in DLD-1 cells.
受体靶向的聚乙二醇化转染剂可以提高当前基于阳离子脂质和聚合物的非病毒基因递送载体的稳定性和递送特异性，但其转染模式仍知之甚少。因此，我们通过基因表达分析、流式细胞术和共聚焦显微镜，在体外研究了1,2-二油酰基-3-三甲基铵丙烷（DOTAP）/1,2-二油酰基-sn-甘油-3-磷酸乙醇胺（DOPE）脂质体、支链聚乙烯亚胺（bPEI）多聚体，以及封装在聚乙二醇化（隐形）非靶向脂质体或PR_b肽（靶向α5β1整合素）功能化隐形脂质体中的bPEI在DLD-1结肠癌细胞中的转染机制。与非靶向隐形脂质体和bPEI相比，DOTAP/DOPE和PR_b功能化隐形脂质体介导了更高的基因表达。然而，DOTAP/DOPE内化缓慢，导致DNA摄取水平较低。相比之下，尽管bPEI多聚体具有较高的内化率，但由于DNA无法从内体中逃逸，基因表达水平较低。非靶向隐形脂质体也由于内化的DNA量少和内化动力学缓慢而介导低水平的基因表达。PR_b功能化隐形脂质体在这些转染剂中达到了最佳平衡，通过快速的整合素介导的内化动力学、大量的DNA摄取和内体逃逸实现了高效转染。我们发现α5β1整合素是基因递送的一个有价值的靶点，并且小窝内吞途径可能为DLD-1细胞中受体靶向的聚乙二醇化转染剂提供优势。
