Biomedical Polymers Laboratory and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering, and Materials Science, Soochow University , Suzhou, 215123, People's Republic of China.
Biomacromolecules. 2012 Mar 12;13(3):881-8. doi: 10.1021/bm2017965. Epub 2012 Feb 27.
Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80-245 nm) particles with moderate positive surface charges (+7.2 ∼ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications.
聚（环氧乙烷）接枝 1.8 kDa 支化聚乙烯亚胺（PEO-g-PEI）共聚物，具有不同的组成，即 PEO（13k）-g-10PEI、PEO（24k）-g-10PEI 和 PEO（13k）-g-22PEI，被制备并用于体外非病毒基因转染。凝胶电泳分析表明，PEO（13k）-g-10PEI、PEO（24k）-g-10PEI 和 PEO（13k）-g-22PEI 在 N/P 比为 4/1、4/1 和 3/1 时，均能完全抑制 DNA 的迁移。动态光散射（DLS）和zeta 电位测量表明，所有三种接枝共聚物都能在 N/P 比为 5/1 至 40/1 时，将 DNA 有效地浓缩成小尺寸（80-245nm）的颗粒，具有适度的正表面电荷（+7.2∼+24.1mV）。聚阳离子的粒径和zeta 电位与 PEO 分子量和 PEI 接枝密度密切相关。值得注意的是，与 25kDa PEI 对照物不同，由于 PEO 的屏蔽作用，PEO-g-PEI 聚阳离子在生理盐和 20%血清条件下不易聚集。293T 细胞的 MTT 测定表明，随着 PEO 分子量的增加和 PEI 接枝密度的降低，PEO-g-PEI 聚阳离子的细胞毒性降低，其中 PEO（13k）-g-22PEI、PEO（13k）-g-10PEI 和 PEO（24k）-g-10PEI 的聚阳离子在 N/P 比为 20/1、30/1 和 40/1 时，细胞活力均大于 80%，细胞毒性较低。有趣的是，体外转染结果表明，PEO（13k）-g-10PEI 聚阳离子具有最佳的转染活性。例如，在 N/P 比为 20/1 时形成的 PEO（13k）-g-10PEI 聚阳离子基本无毒（细胞活力 100%），在无血清和 10%血清条件下，与 25kDa PEI 标准相比，转染效率分别提高了 3 倍和 4 倍。在 10%血清条件下，转染 4 小时后，用 Cy5 标记的 DNA 的共聚焦激光扫描显微镜（CLSM）研究证实，这些 PEO-g-PEI 共聚物在 N/P 比为 20/1 时，能够有效地将 DNA 递送至 293T 细胞的核周区和核内。具有优异胶体稳定性、低细胞毒性和高效转染的 PEO-g-PEI 聚阳离子，有望在安全有效的体外和体内基因转染应用中得到广泛应用。
