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本文拟针对传统巯基化聚合物稳定性差的缺点制备稳定化巯基化聚合物并对其促渗透功能进行研究。以聚丙烯酸(PAA)为模板,加入L-半胱氨酸(Cys)合成巯基化聚合物PAA-Cys;用6,6’-二硫二烟酸与PAACys反应生成PAA-Cys-6MNA,即在PAA-Cys的巯基上接入保护基6-巯基烟酸(6MNA)。生成的PAA-Cys-6MNA命名为稳定化巯基化聚丙烯酸。采用Ellmann’s试剂法测定PAA-Cys中游离巯基的含量,谷胱甘肽还原法测定PAA-Cys-6MNA中6MNA的偶联率;采用Franz扩散池法研究PAA-Cys和PAA-Cys-6MNA在离体小肠上对模型药物异硫氰酸荧光素-右旋糖酐(FD4)的促渗透功能以及保护基的引入对传统巯基化聚合物PAA-Cys稳定性的改善作用;采用共聚焦荧光显微镜观察聚合物对细胞间紧密连接的影响。结果表明,PAA-Cys和PAA-Cys-6MNA均能促进FD4在离体小肠的渗透作用;引入保护基后,聚合物的促渗透功能不再受到贮存pH值及空气氧化的影响;共聚焦荧光显微镜观察到罗丹明-鬼笔环肽标记的Caco-2细胞间紧密连接蛋白微丝的分布受到影响。综上,稳定化巯基化聚丙烯酸维持了传统巯基化聚丙烯酸的促渗透功能,提高了稳定性,其促渗透机制可能与影响细胞间紧密连接蛋白的分布有关。
In this paper, we aimed to prepare stabilized thiolated polymers and improve the penetration-promoting effect of the traditional mercapto-based polymers with poor stability. Polyacrylic acid (PAA) was used as a template to synthesize thiolated polymer PAA-Cys by adding L-cysteine (Cys); PAA-Cys-6MNA was synthesized by reaction of 6,6’-dithiodinicotinic acid with PAACys The protective group 6-mercaptonicotinic acid (6MNA) was attached to the sulfhydryl group of PAA-Cys. The resulting PAA-Cys-6 MNA was named stabilized thiolated polyacrylic acid. The content of free thiol in PAA-Cys was determined by Ellmann’s reagent method, and the coupling rate of 6MNA in PAA-Cys-6MNA was determined by glutathione reduction method. The diffusion of PAA-Cys and PAA-Cys- The promoting effect of model drug-fluorescein isothiocyanate-dextran (FD4) on the small intestine and the protective effect of the protective group on the stability of traditional thiolated polymer PAA-Cys were observed. Confocal fluorescent microscopy The effect of tight junctions between cells. The results showed that both PAA-Cys and PAA-Cys-6MNA could promote the infiltration of FD4 in the isolated small intestine. After the protective group was introduced, the promoting effect of the polymer was no longer affected by the storage pH value and the air oxidation. The confocal fluorescence The distribution of rhodamine-phalloidin-labeled Caco-2 intercellular tight junctions microfilaments was observed microscopically. In summary, the stabilized thiolated polyacrylic acid maintains the traditional penetration-enhancing function and improves the stability of thiolated polyacrylic acid, and its mechanism of promoting penetration may be related to the distribution of tight junction protein.