壳聚糖纳米颗粒作为基因载体的初步研究

Chitosan-DNA nanoparticles as gene carriers

目的：采用适当粒径大小的壳聚糖纳米颗粒（CS-NP）连接质粒DNA，观察CS-NP结合DNA的能力及介导基因转染的效率。方法：利用静电吸附作用连接CS-NP与报告基因pEGFP—N1，形成pEGFP-N1-CS-NP复合物。用透射电镜及激光粒度分析仪观察pEGFP-N1-CS—NP的形态、粒径大小、分布及Zeta电位。用琼脂糖凝胶电泳分析CS-NP与DNA结合能力，以比色法检测其包封率；用DNase I消化实验观察pEGFP-N1-CS-NP抗核酸酶降解的能力。体外转染A549细胞观察pEGFP-N1-CS-NP的转染活性。结果：激光粒度分析仪及透射电镜观察到pEGFP-N1-CS-NP呈较均匀的不规则球形，平均粒径约为100～200nm，Ztea电位为16—22．8mV。WCS-NP／WDNA≥4时，能有效地结合DNA，包封率〉90％。CS-NP能有效介导pEGFP-N1转染A549细胞，并在A549细胞中表达绿色荧光蛋白。结论：CS-NP能有效地与质粒连接，并有很高的包封率，能保护DNA免受核酸酶的降解。CS-NP在体外能将报告基因转染入细胞内，并在细胞内表达。这些研究结果为采用CS-NP作为基因载体应用于活细胞及活体成像的研究奠定了基础。

Objective： To load the appropriate chitosan nanoparticle （CS-NP） with plasmid DNA and observe their binding capability and transfection efficiency in vitro. Methods：CS-NP were loaded with pEGFP-N1 plasmid DNA as the report gene through static electricity absorption. The diameter, diametric distribution and morphology of the pEGFP-N1 -CS-NP were measured with the transmission electron microscope and the laser particle size analysis apparatus. The binding capability of pEGFP-N1 was evaluated by agrose gel eleetrophoresis. The encapsulation rate was determined by colorimetry and the resistance ability of the pEGFP-N1-CS-NP to nuclease by DNase I degradation. Transfection in vitro studies were performed with A549 cells. Results：Transmission electron microscopy and laser particle size analysis showed that the morphology of the pEGFP-N1-CS-NP with different WCS-NP/WDNA was almost uniform, irregularly spherical and well distributed with a mean diameter of about 155 -200 nm and Zeta potential of 16 -22.8 mV. The CS-NP were fully loaded with DNA, with an encapsulation rate of 〉 90% under the condition of WCS-NP/WDNA ≥3, and mediated pEGFP-N1 into A549 cells, in which the expression of green fluorescent proteins was observed. Conclusion： CS-NP can be efficiently loaded with plasmid DNA, with a high encapsulation rate, and transfect the pEGFP-N1 report gene into A549 cells in vitro for its expression. These findings have prepared grownd for further application of CS-NP as gene carriers in the stndies of living cells and organism imaging.