Miyata Kanjiro, Oba Makoto, Nakanishi Masataka, Fukushima Shigeto, Yamasaki Yuichi, Koyama Hiroyuki, Nishiyama Nobuhiro, Kataoka Kazunori
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
J Am Chem Soc. 2008 Dec 3;130(48):16287-94. doi: 10.1021/ja804561g.
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.
由带有1,2 - 二氨基乙烷侧链的聚（天冬酰胺）衍生物组装而成的多聚体[PAsp(DET)]在体外和体内均表现出增强的转染活性，且细胞毒性极小。为了阐明PAsp(DET)多聚体这种独特功能所涉及的分子机制，我们用带有1,3 - 二氨基丙烷侧链的对照物PAsp(DPT)对PAsp(DET)的物理化学和生物学性质进行了全面评估。在PAsp(DET)和PAsp(DPT)多聚体之间，我们观察到粒径和zeta电位的物理化学差异可忽略不计。然而，1,2 - 二氨基乙烷和1,3 - 二氨基丙烷之间的一个亚甲基变化极大地改变了转染情况。与PAsp(DET)多聚体持续的高转染效率形成鲜明对比，即使在聚阳离子相对于质粒DNA（pDNA）过量的区域（高N/P比），PAsp(DPT)多聚体在高N/P比时由于细胞毒性随N/P比逐渐增加，其转染效率也显著下降。PAsp(DPT)的高细胞毒性与其通过溶血、细胞质酶泄漏测定（LDH测定）和共聚焦激光扫描显微镜观察估计的对细胞膜的强烈去稳定作用密切相关。有趣的是，PAsp(DET)在生理pH下显示出最小的膜去稳定作用，但在模拟晚期内体区室的酸性pH（约5）下膜去稳定作用显著增强。显然，PAsp(DET)的pH选择性膜去稳定情况对应于侧翼二胺单元的质子化变化，即在生理pH下为单质子化的gauche形式，在酸性pH下为双质子化的anti形式。这些重要结果表明1,2 - 二氨基乙烷的质子化电荷状态可能在内体破坏中起重要作用。此外，这种用于内体破坏的新方法在生理pH下既不干扰细胞质囊泡也不干扰细胞器的膜。因此，处于晚期内体或溶酶体状态的PAsp(DET)多聚体能够顺利进入细胞质以成功转染，而不会损害细胞活力。
