基于当归多糖的酶敏肿瘤靶向纳米递药体系的研究

Study of enzymes sensitive tumor targeting nano drug delivery system based on Angelica sinensis polysaccharide

目的构建基于当归多糖(AP)的酶敏肿瘤靶向纳米递药体系:当归多糖-基质金属蛋白酶敏感肽-阿霉素(AP-PP-DOX),研究其理化性质及体外抗肿瘤效果。方法首先将AP用马来酸酐(MA)修饰后得到马来酰化当归多糖(AP-MA),再将APMA和基质金属蛋白酶敏感肽(PP)结合生成当归多糖-基质金属蛋白酶敏感肽(AP-PP),最后将AP-PP和阿霉素(DOX)结合生成当归多糖-基质金属蛋白酶敏感肽-阿霉素(AP-PP-DOX)聚合物。利用FT-IR和1 H-NMR表征各步反应产物;透析法自组装形成纳米粒;粒度分析仪测定纳米粒粒径和电位;TEM观察纳米粒大小及外观形态;紫外分光光度计测定纳米粒载药量;体外模拟释药实验研究纳米粒在MMP-2作用下的酶解释药情况;MTT法研究纳米粒对A549细胞的毒性作用。结果 (1)FT-IR和1 H-NMR表征各步反应产物成功合成;(2)透析法成功制备了AP-PP-DOX纳米粒;(3)测定纳米粒的平均粒径和电位分别是139.00±3.32nm和-28.45±0.22mV;(4)该纳米粒结构圆整,平均粒径为100nm;(5)计算纳米粒载药量为17.00%±1.72%;(6)体外模拟释药结果表明,纳米粒在MMP-2下作用24h累积释药率最高达74.5%;(7)MTT实验表明,当药物质量浓度为9μg·mL-1时,纳米粒对A549细胞的存活率极显著,高于游离DOX(P<0.01),而含有MMP-2的纳米粒对A549细胞存活率极显著,低于不含MMP-2的纳米粒(P<0.01)。结论制备基于AP的酶敏肿瘤靶向纳米递药体系AP-PP-DOX,能够有效实现在MMP-2作用下的酶敏释药及抗肿瘤效果,值得进一步研究。

Objective To construct enzymes sensitive tumor targeting nano drug delivery system based on Angelica sinensis polysaccharide(AP)-matrix metalloproteinases sensitive peptide(MMPs)-doxorubicin(AP-PP-DOX)and to study the physical and chemical properties and antitumor activity in vitro.Methods First,AP was modified by maleic anhydride(MA)and Angelica sinensis polysaccharide-maleic anhydride(AP-MA)was synthesized.Second,the AP-MA was reacted with polypeptide(PP)and Angelica sinensis polysaccharide-matrix metalloproteinases sensitive peptide(AP-PP)was synthetized.Third,the AP-PP was combined with doxorubicin(DOX)and the AP-PP-DOX polymer was synthetized.The structure of this conjugate was confirmed by FT-IR and1H-NMR.The nanoparticles were prepared with the self-assembly dialysis method.Analysis of the particle size and potential of the nanoparticles were analyzed with the particle size analyzer.TEM was used to observe the morphology of the nanoparticles.The drug-loading content efficiency of the nanoparticles were measured by UV-Vis.The drug release profiles of nanoparticles was studied at the presence of MMP-2with the experiment of drug release in vitro.The cytotoxicity of the nanoparticles to the A549cells was studied with MTT method.Results①The conjugates were prepared successfully;②the AP-PP-DOX nanoparticles were prepared successfully by the methods of dialysis;③the particle size and potential of the nanoparticles were139.00±3.32nm and-28.45±0.22mV;④the nanoparticales were proved to be spherical and the average particle size was about100nm;⑤the drug-loading content of the nanoparticles were17.00%±1.72%;⑥the results of drug release study in vitro suggested that the DOX release rate of the nanoparticles were74.5%after24h at the presence of MMP-2;⑦the results of MTT assay suggest that the cell survival rate of nanoparticles was significantly higher than free DOX(P<0.01)and the nanoparticles with MMP-2was significantly lower than nanoparticles without MMP-2(P<0.01)to A549cells for24h at the concentration of DOX9μg·mL-1.Conclusion The preparation of enzymes sensitive tumor targeting nano drug delivery system AP-PP-DOX could achieve the purpose of antitumor effect with MMP-2and expected to be further studied.