Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
Pharmacy College,, University of Mosul,, Mosul, Iraq.
Pharm Res. 2018 Mar 7;35(4):86. doi: 10.1007/s11095-017-2328-7.
This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity.
The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10 kDa or 25 kDa branched polyethyleneimine (BPEI) was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transgene expression was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential.
The luciferase activity was ~3-fold lower after transfection with PAA polyplexes than with BPEI complexes at the optimal polymer to nucleotide ratio (RU:Nt). However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt.
PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH.
本研究旨在进一步探讨某些线性聚酰胺胺（PAA）在低细胞毒性下转染细胞的能力背后的机制。
通过荧光素酶报告基因检测，比较了分子量超过 10 kDa 或 25 kDa 的支化聚乙烯亚胺（BPEI）的 DNA 复合物与 PAA 的转染效率。通过在转染细胞时添加巴弗洛霉素 A1 或氯喹来研究内体/溶酶体逃逸对转基因表达的影响。通过测量细胞代谢活性、乳酸脱氢酶释放、活性氧形成和线粒体膜电位变化来评估载体引起的细胞毒性。
在最佳聚合物与核苷酸比（RU:Nt）下，与 BPEI 复合物相比，PAA 多聚物转染后荧光素酶活性降低了约 3 倍。然而，与 BPEI 载体不同，PAA 多聚物几乎没有引起细胞毒性。在存在巴弗洛霉素 A1 的情况下，PAA 多聚物的转染效率显著降低，而氯喹则根据 RU:Nt 增强或降低转基因表达。
与 BPEI 多聚物相比，PAA 多聚物显示出 pH 依赖性的内体/溶酶体逃逸，这与观察到的 BPEI 多聚物的细胞毒性事件无关。这可能是由于它们在酸性条件下与生物膜的相互作用大于在中性条件下。
