The Preparation and Analysis of Nanoparticle-Encapsulated Small-Molecule TNF-α Inhibitor C87 Using In Vitro and In Vivo Models
KONG Xinyao, XIONG Qingqing, MA Li, JIANG Shan, LÜ Mengnan, MA Runzhi, ZHANG Shiyue, SUN Lu, WANG Tong, XU Yuanfu*
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™ 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 (IC50=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.
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