de Wolf Holger K, de Raad Markus, Snel Cor, van Steenbergen Mies J, Fens Marcel H A M, Storm Gert, Hennink Wim E
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 80082, 3508 TB, Utrecht, The Netherlands.
Pharm Res. 2007 Aug;24(8):1572-80. doi: 10.1007/s11095-007-9299-z. Epub 2007 Apr 11.
Previously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity.
p(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity.
Four p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25-29 mV), and had a size of 80-90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz's (Mw 130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression.
Polymer molecular weight fractionation enabled us to optimize the transfection efficiency/toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection.
此前，我们已经证明，质粒DNA与可生物降解聚合物聚（2-二甲基氨基乙氨基）磷腈（p(DMAEA)-ppz）形成的复合物在小鼠静脉注射后介导肿瘤选择性基因表达。在本研究中，我们研究了p(DMAEA)-ppz分子量对体外和体内肿瘤转染以及复合物诱导毒性的影响。
通过制备型尺寸排阻色谱法对具有宽摩尔质量分布的p(DMAEA)-ppz进行分级分离。测试了由质粒DNA和收集的聚合物级分组成的多聚体的生物物理性质、（细胞）毒性和转染活性。
收集到四个p(DMAEA)-ppz级分，重均分子量范围为130至950 kDa，分子量分布窄（Mw/Mn为1.1至1.3）。在聚合物与DNA（N/P）比率高于6时，基于这些聚合物的多聚体均带正电（ζ电位为25-29 mV），大小为80-90 nm。多聚体的体外细胞毒性与聚合物分子量呈正相关。不同多聚体的体外转染活性取决于其N/P比率，并受细胞毒性程度以及不同多聚体的胶体稳定性影响。基于高分子量聚合物的多聚体静脉注射导致明显毒性，这是多聚体诱导红细胞聚集的结果。另一方面，基于低分子量p(DMAEA)-ppz（Mw 130 kDa）的多聚体给药未显示毒性迹象，并导致肿瘤选择性基因表达。
聚合物分子量分级分离使我们能够优化p(DMAEA)-ppz多聚体用于体外和体内肿瘤转染时的转染效率/毒性比。
