Mozley Olivia L, Thompson Ben C, Fernandez-Martell Alejandro, James David C
Dept. of Chemical and Biological Engineering, ChELSI Inst., University of Sheffield, Mappin St., Sheffield, S1 3JD, U.K.
Biotechnol Prog. 2014 Sep-Oct;30(5):1161-70. doi: 10.1002/btpr.1932. Epub 2014 Jun 18.
In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl-PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process.
在本研究中，我们研究了支撑中国仓鼠卵巢（CHO）细胞对聚乙烯亚胺（PEI）:DNA复合物（多聚体）进行有效内化和利用的分子与细胞相互作用。细胞表面多聚体的结合和内化是一个双相过程，包括一个初始的快速阶段（阶段I），持续约15分钟，随后是一个较慢的第二阶段（阶段II），在转染后约240分钟达到饱和。第二阶段占多聚体内化的大部分（60 - 70%）。虽然在阶段I期间细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）与多聚体迅速共同内化，但细胞表面多聚体的结合并不依赖于HSPG。然而，阶段II多聚体的内化和HSPG在胰蛋白酶处理细胞表面的再生以相似的速率发生，这表明含HSPG的膜循环回到质膜的速率限制了阶段II的内化速率。在最佳转染条件下，多聚体具有接近中性的表面电荷（ζ电位），细胞表面结合依赖于疏水相互作用，从质膜化学螯合胆固醇和添加非离子表面活性剂均能显著抑制这种相互作用。使用柠檬酸铁降低表面电荷以及改变PEI:DNA比例增加表面电荷来诱导多聚体ζ电位的改变，分别用于抑制多聚体结合或降低分泌型碱性磷酸酶报告基因的表达以及细胞活力。为了增加多聚体的疏水性和内化，化学合成了PEI的烷基化衍生物丙基-PEI。利用实验设计-响应面建模来优化转染过程，将丙基-PEI的功能与未修饰的PEI在亲本CHO-S细胞和一个亚克隆（克隆4）中的功能进行了比较，克隆4通过对多聚体细胞毒性的抗性增加而表现出卓越的转基因表达。丙基-PEI和克隆4的组合使重组DNA利用效率和报告蛋白产量提高了一倍。这些数据表明，为了达到最大功效，增加多聚体进入细胞内化的策略必须与抵消这一过程固有细胞毒性的策略协同使用。
