Needle-like morphology of H2K4b polyplexes associated with increases in transfection in vitro.

summarySeveral synthetic histidine-lysine (HK) polymers have been screened for their efficacy as carriers of nucleic acids in vitro. One branched HK polymer, H2K4b (and its derivatives), has been particularly effective as an in vitro carrier of plasmids. In this study, we investigated whether various salt conditions during formation of the H2K4b/plasmid DNA polyplex affected transfection. We compared the transfection ability of H2K4b polyplexes prepared under three conditions: 1) water, 2) water and then Opti-MEM (or 300 mM NaCl), or 3) Opti-MEM (or 150 mM NaCl). The milieu in which the H2K4b polyplexes were prepared significantly affected in vitro transfection, and conditions that resulted in highest to lowest transfection levels were as follows: water and then Opti-MEM > Opti-MEM (or 150 mM NaCl)>> water. Several biophysical properties (size and shape of polyplex, surface charge, stability) were examined for their correlation with the level of transfection by the HK carrier. Strikingly, electron micrographs showed that HK polyplexes, first formed in water and then in salt, had a needle-like morphology with a mean length of 170 nm and a width of 53 nm; these needle-like polyplexes were observed intracellularly and absorbed to the cell surface, which was in marked contrast to the spherical HK polyplexes formed in water or in Opti-MEM. Notably, these needle-like HK polyplexes entered the cell through clathrin-mediated endocytosis, in contrast to spherical polyplexes, which entered primarily through non clathrin-mediated endocytosis.