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Lipoic Acid Modified Low Molecular Weight Polyethylenimine Mediates Nontoxic and Highly Potent In Vitro Gene Transfection
作者:M. Zheng, Y.N. Zhong, F.H. Meng, R. Peng, and Z.Y. Zhong*
關鍵字:polyethylenimine; hydrophobic modification; reduction-sensitive; plasmid DNA; polyplexes; gene delivery
論文來源:期刊
具體來源:Mol. Pharmaceutics, 2011, 8, 2434-2443.
發表時間:2011年

The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI–LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4–6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI–LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI–LA conjugates condensed DNA into much smaller sizes (183–84 nm) than unmodified 1.8 kDa PEI (444–139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa PEI–LA2 polyplexes formed at an N/P ratio of 10/1 were stable against exchange with 12-fold excess of negatively charged dextran sodium sulfate (DSS) relative to DNA phosphate groups while 1.8 kDa PEI controls dissociated at 6-fold excess of DSS, indicating that lipoylation of 1.8 kDa PEI resulted in stronger binding with DNA. Importantly, DNA was released from 1.8 kDa PEI–LA2 polyplexes upon addition of 10 mM dithiothreitol (DTT). Reduction-triggered unpacking of 1.8 kDa PEI–LA2 polyplexes was also confirmed by DLS. MTT assays demonstrated that all PEI–LA conjugates and polyplexes were essentially nontoxic to HeLa and 293T cells up to a tested concentration of 50 μg/mL and an N/P ratio of 80/1, respectively. The in vitro gene transfection studies in HeLa and 293T cells showed that lipoylation of 1.8 kDa PEI markedly boosted its transfection activity. For example, 1.8 kDa PEI–LA2 polyplexes displayed 400-fold and 500-fold higher levels of gene expression than unmodified 1.8 kDa PEI controls, which were ca. 2-fold and 3-fold higher than 25 kDa PEI controls, in serum-free and 10% serum media, respectively. The transfection efficiency decreased with increasing DS, following an order of 1.8 kDa PEI–LA2 > 1.8 kDa PEI–LA4 > 1.8 kDa PEI–LA6 1.8 kDa PEI. Confocal laser scanning microscopy (CLSM) studies corroborated that 1.8 kDa PEI–LA2 delivered and released DNA into the nuclei of HeLa cells more efficiently than 25 kDa PEI. These nontoxic 1.8 kDa PEI–LA conjugates form a superb basis for the development of targeting, biocompatible and highly efficient carriers of gene delivery.

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