Quantitative Measurement of Cationic Polymer Vector and Polymer鈥損DNA Polyplex Intercalation into the Cell Plasma Membrane
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- 作者：Sriram Vaidyanathan ; Kevin B. Anderson ; Rachel L. Merzel ; Binyamin Jacobovitz ; Milan P. Kaushik ; Christina N. Kelly ; Mallory A. van Dongen ; Casey A. Dougherty ; Bradford G. Orr ; Mark M. Banaszak Holl
- 刊名：ACS Nano
- 出版年：2015
- 出版时间：June 23, 2015
- 年：2015
- 卷：9
- 期：6
- 页码：6097-6109
- 全文大小：837K
- ISSN：1936-086X
文摘
Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1脳 to 100脳 the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30鈥?0 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.