Mixing-sequence-dependent nucleic acid complexation and gene transfer efficiency by polyethylenimine

Polyplexes, complexed nucleic acids by cationic polymers, are the most common forms of nonviral gene delivery vectors. In contrast to a great deal of efforts in synthesizing novel cationic polymers and exploring their extracellular and intracellular delivery pathways, polyplex preparation methods of mixing nucleic acids and cationic polymers are often overlooked. In this study, the mixing sequence, that is adding nucleic acids to polymers or vice versa, was found to greatly affect complexation of both plasmid DNA and siRNA, polyplexes' size, and polyplexes' surface charge, which all collaboratively affected the transfection efficiency and cytotoxicity. Adding polyethylenimine (PEI), the most conventionally used standard in nonviral gene delivery, to plasmid DNA and siRNA resulted in larger polyplexes, higher gene expression and silencing, but higher cytotoxicity than polyplexes prepared in the reverse order. Based on the experimental results, the authors developed a model that gradual addition of cationic polymers (e.g., PEI) to nucleic acids (e.g., plasmid DNA and siRNA) incorporates more copies of nucleic acids in larger polyplexes in a smaller number, results in higher gene expression and silencing levels in transfected cells, and generates higher cytotoxicity by leaving more free polymers upon complete mixing than the other mixing sequence. The proposed model can be explored using a broad range of cationic polymers and nucleic acids, and provide insightful information about how to prepare polyplexed nonviral vectors for efficient and safe gene delivery.