Department of Chemical Engineering, Institute of Biomedical Engineering, Ecole Polytechnique of Montreal, P.O. Box 6079, Station Centre-Ville, Montreal, QC, H3C 3A7, Canada.
Mol Biotechnol. 2010 Oct;46(2):182-96. doi: 10.1007/s12033-010-9286-1.
This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan-DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8-24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24-40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression.
本研究旨在系统评估 pH 值和血清对壳聚糖-DNA 复合物转染过程的影响，以期最大限度地提高其效率。通过动态光散射（DLS）测量复合物的水动力直径发现，其在盐和 pH 值的作用下从水中的 243nm 增加到 PBS 中的 1244nm（pH7.4），并在存在 10%血清时发生聚集。通过流式细胞术和共聚焦荧光成像评估复合物进入 HEK293 细胞的摄取情况发现，在较低 pH 值和血清存在下，复合物的摄取增加。基于这些数据，我们测试了新的方法学，当在 pH6.5 时用 10%血清转染 8-24 小时以最大限度地提高摄取量，然后将培养基更换为 pH7.4 和 10%血清再持续 24-40 小时时，实现了超过 40%的高转染效率 (>40%)。壳聚糖/DNA 复合物的细胞毒性也明显低于 Lipofectamine。我们的研究表明，评估转染方法学中重要参数的影响，可以理解关键的物理化学和生物学机制，从而设计出最大限度提高转基因表达的方法学。
