Giron-Gonzalez M Dolores, Salto-Gonzalez Rafael, Lopez-Jaramillo F Javier, Salinas-Castillo Alfonso, Jodar-Reyes Ana Belen, Ortega-Muñoz Mariano, Hernandez-Mateo Fernando, Santoyo-Gonzalez Francisco
Department of Biochemistry and Molecular Biology II, School of Pharmacy, and ‡Department of Organic Chemistry, Biotechnology Institute, §Department of Analytical Chemistry, and ⊥Biocolloid and Fluid Physics Group, Department of Applied Physics, Faculty of Sciences, University of Granada , E-18071 Granada, Spain.
Bioconjug Chem. 2016 Mar 16;27(3):549-61. doi: 10.1021/acs.bioconjchem.5b00576. Epub 2016 Feb 18.
Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers.
由阳离子聚合物聚乙烯亚胺（PEI）介导的基因转染被认为是一种标准方法。然而，尽管高度分支的PEI与DNA形成较小的多聚体，表现出高转染效率，但它们具有显著的细胞毒性。相反，具有良好细胞毒性特征的低分子量PEI（LMW-PEI）由于DNA络合和保护不足，表现出最小的转染活性。为了解决这一矛盾，通过将分支的1.8 kDa PEI与柠檬酸（CA）进行离子交联制备了一种新型聚电解质复合物。该系统协同利用了LMW-PEI所具有的良好细胞毒性特征以及高度分支和高分子量PEI所显示的高转染效率。聚电解质复合物（1.8 kDa-PEI@CA）是通过基于对1.8 kDa PEI和CA溶液进行微波辐射的简单合成方案获得的。与DNA络合后，测量所得颗粒的固有性质（大小和表面电荷），并确定它们形成稳定多聚体的能力。与未修饰的PEI相比，新的复合物表现为高效的基因载体，并显示出增强的DNA结合能力，这与促进细胞内DNA释放以及增强DNA对内切核酸酶降解的保护作用相关。此外，虽然LMW-PEI的转染值几乎为零，但在几种培养的细胞系如CHO-k1、FTO2B肝癌细胞、L6成肌细胞或NRK细胞中，新试剂的转染效率是Lipofectamine 2000和25 kDa PEI的2.5至3.8倍，同时显示出可忽略不计的毒性。此外，1.8 kDa-PEI@CA聚电解质复合物在血清存在下保留了转染真核细胞的能力，并表现出在小鼠（作为动物模型）中促进体内转染的能力，与25 kDa PEI相比效率有所提高。结果支持LMW-PEI和CA的聚电解质复合物作为有前景的通用非病毒基因载体。
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