Polyplexes assembled from
poly(aspartamide) derivatives bearing
1,2-diaminoethane side chains, [
PAsp(DET)] display amplified in vitro and in vivo transfection activity with minimal cytotoxicity. To elucidate the molecular mechanisms involved in this unique function of
PAsp(DET) polyplexes, the physicochemical and biological properties of
PAsp(DET) were thoroughly evaluated with a control bearing
1,3-diaminopropane side chains,
PAsp(DPT). Between
PAsp(DET) and
PAsp(DPT) polyplexes, we observed negligible physicochemical differences in particle size and zeta-potential. However, the one methylene variation between
1,2-diaminoethane and
1,3-diaminopropane drastically altered the transfection profiles. In sharp contrast to the constantly high transfection efficacy of
PAsp(DET) polyplexes, even in regions of excess polycation to plasmid
DNA (pDNA) (high N/P ratio),
PAsp(DPT) polyplexes showed a significant drop in the transfection efficacy at high N/P ratios due to the progressively increased cytotoxicity with N/P ratio. The high cytotoxicity of
PAsp(DPT) was closely correlated to its strong destabilization effect on cellular membrane estimated by
hemolysis, leakage assay of cytoplasmic
enzyme (LDH assay), and confocal
laser scanning microscopic observation. Interestingly,
PAsp(DET) revealed minimal membrane destabilization at physiological pH, yet there was significant enhancement in the membrane destabilization at the acidic pH mimicking the late endosomal compartment (pH approximately 5). Apparently, the pH-selective membrane destabilization profile of
PAsp(DET) corresponded to a protonation change in the flanking
diamine unit, i.e., the monoprotonated gauche form at physiological pH and diprotonated anti form at acidic pH. These significant results suggest that the protonated charge state of
1,2-diaminoethane may play a substantial role in the endosomal disruption. Moreover, this novel approach for endosomal disruption neither perturbs the membranes of cytoplasmic vesicles nor organelles at physiological pH. Thus,
PAsp(DET) polyplexes, residing in late endosomal or lysosomal states, smoothly exit into the cytoplasm for successful transfection without compromising cell viability.