载ACE-shRNA pGenesil质粒与壳聚糖纳米粒的结合及释放实验

In vitro chitosan nanoparticle loading and release of ACE-shRNA pGenesil plasmid

目的制备适当粒径的壳聚糖纳米粒,并连接上质粒,研究壳聚糖纳米粒对质粒DNA的结合能力及在体外的释放。方法采用离子交联法制备壳聚糖纳米粒,通过喷金电镜观察其大小、形态及分布;经琼脂糖凝胶电泳分析纳米载体与质粒DNA的结合能力;在4种不同pH值的磷酸盐缓冲液(PBS)中观察壳聚糖质粒纳米粒的释放情况;通过紫外分光光度计检测其包埋率及释放率。结果喷金电镜证实壳聚糖纳米粒分布均匀,呈近似球形,平均粒径约5nm;琼脂糖凝胶电泳结果显示壳聚糖纳米粒能有效地结合质粒,当纳米粒与质粒的比例为10∶10时包埋率达98.7%;壳聚糖质粒纳米粒的性质较稳定,在pH值<7.5的PBS溶液能够平稳释放100h左右。结论制备出适当粒径且分布均匀的壳聚糖纳米粒,能有效地结合质粒,并且能够持续平稳地释放。

Objective To prepare size-compatible chitosan nanoparticles （CS-NP） , load it with plasmid, and explore its loading and release in vitro. Methods CS-NPs were prepared with cocervation process and observed for size, shape and distribution under scanning electron microscope. The binding of pDNA was evaluated by agarose gel electrophoresis analysis; the releasing was observed in phosphate buffers with four different pH levels; the capsulating rate and releasing rate were determined by fluorospectrophotometry. Results Under scanning electron microscope, CS-NPs were shown to be evenly distributing spherical particles with a mean diameter of about 5 nm. Agarose gel electrophoresis showed that pDNA was absorbed to CS-NP effectively. The encapsulation efficiency （nanoparticles： plasmid=10：10） was 98.7% by fluorescent microscopy. Stable release of DNA from nanoparticles lasting about 100 hours in vitro was confirmed in PBS with pH〈7.5. Conclusion Homogeneous in diameter CS-NP was prepared and loaded with plasmid effectively. Moreover, pDNA was released from the complexes continually and stably.