Vasiliu Tudor, Cojocaru Corneliu, Rotaru Alexandru, Pricope Gabriela, Pinteala Mariana, Clima Lilia
Center of Advanced Research in Bionanocojugates and biopolymers, "Petru Poni" Institute of Macromolecular Chemistry, Iasi, Romania Aleea Grigore Ghica Voda 41A, 70487 Iasi, Romania.
Department of Inorganic Polymers, "Petru Poni" Institute of Macromolecular Chemistry, Iasi, Romania Aleea Grigore Ghica Voda 41A, 70487 Iasi, Romania.
Int J Mol Sci. 2017 Jun 17;18(6):1291. doi: 10.3390/ijms18061291.
The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(ʟ-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4.
由核酸和聚阳离子形成的多聚体因其在基因治疗中的潜在应用而备受关注。在我们的研究中，我们报告了关于双链DNA（dsDNA）和聚（L-赖氨酸）（PLL）之间多聚体形成优化的实验结果和建模结果。通过处理从凝胶电泳分析中捕获的图像来定量多聚体形成过程中的结合效率。采用实验设计（DoE）和响应面方法（RSM）来研究影响结合效率的关键因素（pH和N/P比）的耦合效应。根据实验观察和响应面分析，与pH相比 N/P比对结合效率有主要影响。基于模型的优化计算以及实验验证运行揭示了在pH 5.4和N/P比125时达到的最大结合效率（99.4%）。为了支持实验数据并深入了解dsDNA和PLL之间多聚体形成的分子机制，在pH 5.4和7.4下进行了分子动力学模拟。
