Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States.
Mol Pharm. 2012 Mar 5;9(3):523-38. doi: 10.1021/mp200368p. Epub 2012 Feb 1.
The aim of this study is to compare the cytotoxicity mechanisms of linear PEI to two analogous polymers synthesized by our group: a hydroxyl-containing poly(l-tartaramidoamine) (T4) and a version containing an alkyl chain spacer poly(adipamidopentaethylenetetramine) (A4) by studying the cellular responses to polymer transfection. We have also synthesized analogues of T4 with different molecular weights (degrees of polymerization of 6, 12, and 43) to examine the role of molecular weight on the cytotoxicity mechanisms. Several mechanisms of polymer-induced cytotoxicity are investigated, including plasma membrane permeabilization, the formation of potentially harmful polymer degradation products during transfection including reactive oxygen species, and nuclear membrane permeabilization. We hypothesized that since cationic polymers are capable of disrupting the plasma membrane, they may also be capable of disrupting the nuclear envelope, which could be a potential mechanism of how the pDNA is delivered into the nucleus (other than nuclear envelope breakdown during mitosis). Using flow cytometry and confocal microscopy, we show that the polycations with the highest amount of protein expression and toxicity, PEI and T4(43), are capable of inducing nuclear membrane permeability. This finding is important for the field of nucleic acid delivery in that direct nucleus permeabilization could be not only a mechanism for pDNA nuclear import but also a potential mechanism of cytotoxicity and cell death. We also show that the production of reactive oxygen species is not a main mechanism of cytotoxicity, and that the presence or absence of hydroxyl groups and polymer length play a role in polyplex size and charge in addition to protein expression efficiency and toxicity.
本研究旨在通过研究细胞对聚合物转染的反应，比较线性聚乙烯亚胺与我们小组合成的两种类似聚合物的细胞毒性机制：一种含羟基的聚（L-酒石酰胺基胺）（T4）和一种含烷基链间隔基的聚（己二酰亚胺五乙撑四胺）（A4）。我们还合成了 T4 的不同分子量类似物（聚合度为 6、12 和 43），以研究分子量对细胞毒性机制的影响。研究了聚合物诱导细胞毒性的几种机制，包括质膜通透性、转染过程中潜在有害聚合物降解产物的形成，包括活性氧和核膜通透性。我们假设，由于阳离子聚合物能够破坏质膜，它们也可能能够破坏核膜，这可能是 pDNA 递送入核的潜在机制（除了有丝分裂期间核膜破裂）。通过流式细胞术和共聚焦显微镜，我们表明具有最高蛋白表达量和毒性的聚阳离子，聚乙烯亚胺和 T4（43），能够诱导核膜通透性。这一发现对于核酸递送领域非常重要，因为直接细胞核通透性不仅可以作为 pDNA 核内导入的机制，也可能是细胞毒性和细胞死亡的潜在机制。我们还表明，活性氧的产生不是细胞毒性的主要机制，并且羟基的存在与否以及聚合物长度除了蛋白表达效率和毒性之外，还在多聚物大小和电荷方面发挥作用。
