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Polycations as gene carriers have attracted considerable attention over the past decade. Generally, polyplexes between polycations and deoxyribonucleic acid (DNA) are formed at low N/P ratios (the ratios of the numbers of nitrogen atoms in a polycation to the numbers of phosphorus atoms in DNA), but high transfection efficiency can only be obtained at much higher N/P ratios. Thus, many polycationic chains are still free in solution. In this study, we investigated the detailed functions of free polyethylenimine chains (PEI-F) and free N,N,N-trimethyl chitosan chains (TMC-F) using the same polyplex, ie, TMC polyplex (TMC-P), which has high stability in Dulbecco's Modified Eagle's Medium (DMEM). Meanwhile, PEI polyplex (PEI-P)/PEI-F was also evaluated rather than PEI-P/TMC-F because the stability of PEI-P is low in DMEM and, in the latter case, the TMC-F may replace the bound PEI chain in PEI-P to form TMC-P. The transfection results show that both TMC-F and PEI-F can significantly increase the transfection efficiency of TMC-P; however, PEI-F can upregulate the gene expression of TMC-P more efficiently than TMC-F. Further investigations on the endocytosis and intracellular trafficking show that PEI-P/PEI-F, TMC-P/PEI-F, and TMC-P/TMC-F exhibit similar cellular uptake efficiency. However, by shutting down the clathrin-mediated endocytosis or vacuolar proton pump, the transfection efficiency decreases in the order of PEI-P/PEI-F &gt; TMC-P/PEI-F &gt; TMC-P/TMC-F. These findings indicate that PEI-F and TMC-F may promote the transfection efficiency of the polyplex by affecting its cellular uptake pathway and intracellular trafficking.

international journal of nanomedicine

Elucidating the role of free polycationic chains in polycation gene carriers by free chains of polyethylenimine or N,N,N-trimethyl chitosan plus a certain polyplex