Reducible Poly(amido ethylenimine)s Designed for Triggered Intracellular Gene Delivery

摘要

Poly(amido ethylenimine) polymers,a new type of peptidomimetic polymer,containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery.More specifically,three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers,i.e.,ethylenediamine (EDA),diethylenetriamine (DETA),or triethylenetetramine (TETA).Complete addition reactions were confirmed by ~1H NMR.The molecular weight,buffer capacity,and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC),acid-base titration,and liquid chromatography-mass spectroscopy (LC-MS),respectively.Physicochemical characteristics of polymer/pDNA complexes (polyplexes) were analyzed by gel electrophoresis,particle size,and zeta-potential measurements.All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of approx 32 mV.The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3),primary bovine aortic endothelial cell (BAEC),and rat aortic smooth muscle cell (A7R5) lines.Interestingly,polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k.The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium.Furthermore,confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI,most likely due to environmentally triggered release.Therefore,SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.