TNF-α小分子抑制剂C87的纳米药物制备及其体内外活性研究

The Preparation and Analysis of Nanoparticle-Encapsulated Small-Molecule TNF-α Inhibitor C87 Using In Vitro and In Vivo Models

该研究首先制备并表征包载TNF-α小分子抑制剂C87的纳米粒子,进而评价其在L929细胞和小鼠自身免疫性肝损伤模型中抑制TNF-α细胞毒性的作用。采用纳米沉淀法制备载C87的纳米粒子(C87 NP),并对其理化性质及体外释放行为进行表征;在L929细胞中用MTT法分析C87 NP拮抗TNF-α细胞毒作用的效果;经尾静脉注射刀豆蛋白A(ConA)诱导小鼠发生自身免疫性肝损伤,采用C87 NP预给药方式,防治小鼠自身免疫性肝损伤;使用LEGENDplex^(TM)分析小鼠血清中13种细胞因子的表达水平;流式细胞术分析肝和脾中T细胞及亚群、NK细胞等的分布、比例和数量。制备出的C87 NP具有较高(34.4%)的载药量,且稳定性好,其包封率为48.1%,C87 NP平均粒径为82.57 nm,多分散系数为0.115,呈电中性,透射电镜结果显示其为球形结构;该纳米粒子可在体外持续缓慢释放C87,持续时间不少于8 h;C87 NP可以在体外抑制TNF-α对L929细胞的杀伤能力(IC_(50)=9.13 μmol/L),并呈浓度依赖性;动物实验结果显示,C87 NP治疗组小鼠的存活率为66.7%,进一步的生化和病理分析结果表明,ConA小鼠尾静脉给药12 h后,C87 NP预防性给药能显著降低血清中ALT、AST与部分细胞因子的水平(P<0.01),减轻肝损伤,减少CD4^(+)T、CD8^(+)T、NK细胞等对肝脾的浸润(P<0.01),提高脾脏Treg细胞比例(P<0.05)。该研究成功制备出纳米药物C87 NP,且C87 NP在体内外具有良好的抑制TNF-α细胞毒性的作用,这为小分子药物C87今后在临床上的应用奠定了基础。

In this study,nanoparticles loaded with C87,a small-molecule inhibitor of TNF-α,were prepared and characterized.The effects of the inhibition of TNF-α cytotoxicity were assessed in L929 cells using a mouse model of autoimmune hepatitis.Specifically,C87 NPs were prepared by using the nanoprecipitation method and examined their physicochemical properties and the drug release profile in vitro.For mouse studies,LEGENDplex^(TM) was utilized to determine the levels of 13 cytokines in the mouse serum and flow cytometry was used to analyze the distribution,proportion and number of T cells (including subsets of T cells) and NK cells in the liver and spleen.The C87 NPs exhibited a high loading capacity (34.4%) and stability,with an encapsulation rate of 48.1%,an average particle size of 82.57 nm,and a polydispersity coefficient of 0.115.The C87 NPs were electrically neutral and displayed spherical structures as revealed by transmission electron microscopy.The NPs released C87 in a sustained manner with a duration of no less than 8 h.The C87 NPs inhibited the killing effect of TNF-α on L929 cells in vitro in a concentration-dependent manner (IC_(50)=9.13 μmol/L).Moreover,the results from the mouse studies showed that the C87 NPs increased the survival rate of the mice from 0% to 66.7%.while subsequent biochemical and pathological analyses demonstrated that the tail vein administration of C87 NPs in ConA-treated mice significantly reduced the serum levels of ALT,AST and several cytokines (P<0.01),the liver injury,and the infiltration of CD4^(+)T,CD8^(+)T and NK cells into the liver and spleen (P<0.01) after 12 h,while increasing the proportion of Treg cells in the mouse spleen (P<0.05).Together,this study has successfully established C87 NPs that exert a strong inhibitory effect on the cytotoxicity of TNF-α both in vitro and in vivo.It lays a foundation for future clinical applications of the small-molecule inhibitor C87.