【摘要】 目的:本研究诣在对壳聚糖进行修饰,以解决其水溶性问题和基因释放困难的问题。方法:本研究通过2,3-环氧丙基三甲基氯化铵和N-乙酰-L-半胱氨酸对壳聚糖进行修饰,得到巯基化壳聚糖季铵盐(TMC-SH),使其在生理条件下带正电并含有一定量的游离巯基。以TMC-SH为基因载体,形成基因复合物。通过琼脂糖凝胶电泳考察其稳定性,并测定其粒径和ζ-电位。通过DTT条件下的粒径测定,考察基因复合物的还原响应性。结果:核磁结果表明合成TMC-SH的季铵盐取代度为22%,游离巯基-SH含量为79.22μmol/g;琼脂糖凝胶电泳结果表明以TMC-SH为载体形成的二硫键交联的基因复合物TMC-SS/p DNA具有较好的稳定性;而且,二硫键交联以后基因复合物粒径较小,结构更为密实;在还原条件下粒径变大,表明二硫键交联的基因复合物变得疏松,说明其粒径具有还原响应性。结论:对壳聚糖进行低取代度的季铵盐修饰和一定量的巯基化修饰后,其具有较好的包载p DNA能力和还原响应性的基因释放能力。更多还原

【Abstract】 Objective: Chitosan has been widely used as gene carrier, as it is biocompatible and biodegradable. We develop a solution here, to solve the problem of chitosan’s solubility at neutral p H and gene release in cytosol at the same time. Methods: Thiolated trimethylated chitosan(TMC-SH) was synthesized through trimethylation of chitosan and subsequent thiolation of TMC. Then complexes of TMC-SS/p DNA were formed through initially electrostatic interaction between positively-charged TMC-SH and negatively-charged p DNA and the following cross-linking due to the formation of disulfide bonds. The complexes were characterized in terms of size, ζ-potential, agarose gel electrophoresis, size change in reductive environment. Results: It was determined that 22 % glucose units in TMC were trimethylated according to 1H NMR data and Ellman’s reagent assay showed that there was about 79.22 μmol/g free thiol groups in TMC-SH. Agarose gel electrophoresis assay showed that complexes of TMC-SS/p DNA was more stable than TMC/p DNA which could be ascribed to oxidation of thiol groups into disulfide bonds generating cross-linked complexes. And the size of complexes TMC-SS/p DNA was about 170 nm which was obviously smaller than TMC/p DNA with the size of about 220 nm. After incubated with DTT, the complexes of TMC-SS/p DNA became looser with size of about 320 nm. Conclusions: After modification of chitosan, the product TMC-SH is with low trimethylated substitution and with free thoil. The complexes of TMC-SS/p DNA possess good stability and redox-responsive gene release.更多还原