目的 制备负载毛蕊异黄酮（calycosin，Cal）的叶酸（folic acid，FA）修饰膜荚黄芪Astragalusmembranaceus外泌体样纳米颗粒（FA-Exos@Cal），并考察其在体外对巨噬细胞M1极化抑制作用。方法 采用差速离心结合蔗糖密度梯度离心法提取黄芪外泌体样纳米颗粒（exosome-like vesicles，Exos）；通过薄膜分散-孵育法制备FA-Exos@Cal；利用透射电子显微镜（transmission electron microscopy，TEM）、二辛可宁酸法（bicinchoninic acid，BCA）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）、动态光散射（dynamic light scattering，DLS）、HPLC法及透析袋法，表征颗粒形态、蛋白特征、粒径、ζ电位、载药特性及体外释放行为；采用荧光显微和流式细胞技术评价其对M1型RAW264.7细胞的靶向性；采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）法检测其对巨噬细胞增殖抑制作用，采用流式细胞技术考察其对M1标志物CD86表达水平的影响，通过酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）等测定炎症相关因子白细胞介素-1β（interleukin-1β，IL-1β）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、IL-6、一氧化氮（nitric oxide，NO）的表达水平。结果 成功制备FA-Exos@Cal，其呈典型囊泡样结构，蛋白质量浓度、粒径、ζ电位、载药量、包封率分别为（3.52±0.26）mg/mL、（127.4±2.6）nm、（-11.14±0.30）mV、（12.14±0.18）%、（27.21±1.04）%，修饰前后蛋白特征无明显差异，药物体外缓释效果良好。叶酸修饰能显著提升M1型巨噬细胞对FA-Exos@Cal颗粒的摄取效率，并降低毛蕊异黄酮对巨噬细胞的毒性；FA-Exos@Cal可显著降低M1型巨噬细胞CD86阳性率，并显著下调IL-1β、TNF-α、IL-6及NO的表达水平；与Exos、毛蕊异黄酮及Exos&Cal物理混合物的对比分析显示，Exos与毛蕊异黄酮联用具有协同增效效应。结论 成功制备工程化靶向修饰的仿生纳米递送系统FA-Exos@Cal，证实其具备优异的M1型巨噬细胞靶向性，可有效抑制M1极化及炎症因子释放，有望为炎症性疾病治疗提供新策略。

Objective To prepare folic acid (FA)-modified Mojiahuangqi (Astragalus membranaceus, AM) exosome-like nanoparticles loaded with calycosin (Cal) (FA-Exos@Cal) and investigate their inhibitory effect on M1 polarization of macrophages in vitro. Methods AM exosome-like nanoparticles (Exos) were isolated by differential centrifugation combined with sucrose density gradient centrifugation. FA-Exos@Cal was prepared by the thin-film dispersion-incubation method. Transmission electron microscopy (TEM), bicinchoninic acid (BCA) assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering, HPLC, and dialysis bag method were used to characterize the particle morphology, protein properties, particle size, ζ potential, drug-loading characteristics, and in vitro release behavior. Fluorescence microscopy and flow cytometry were employed to evaluate the targeting ability of FA-Exos@Cal to M1-type RAW264.7 cells. Cell counting kit-8 (CCK-8) assay was used to detect the effect of FA-Exos@Cal on macrophage proliferation. Flow cytometry was applied to investigate its influence on the expression level of M1 marker CD86. Enzyme-linked immunosorbent assay (ELISA) and other methods were used to determine the expression levels of inflammatory factors including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, and nitric oxide (NO). Results FA-Exos@Cal was successfully prepared. TEM showed a typical vesicular structure. The protein concentration, particle size, ζ potential, drug loading capacity, and entrapment efficiency were (3.52 ± 0.26) mg/mL, (127.4 ± 2.6) nm, (-11.14 ± 0.30) mV, (12.14 ± 0.18)%, and (27.21±1.04)%, respectively. There was no significant difference in protein properties before and after modification, and the drug exhibited good sustained-release effect in vitro. FA modification significantly enhanced the cellular uptake efficiency of FA-Exos by M1-polarized macrophages while reducing the cytotoxicity of Cal toward macrophages. Moreover, FA-Exos@Cal could significantly reduce the CD86-positive rate of M1-type macrophages and down-regulate the expression levels of IL-1β, TNF-α, IL-6, and NO. Comparative analysis with Exos, Cal, and the physical mixture of Exos and Cal revealed that the combined use of Exos and Cal exerted a synergistic effect. Conclusion FA-Exos@Cal, an engineered targeted biomimetic nano-delivery system, was successfully prepared. It was confirmed to have excellent targeting ability to M1-type macrophages, and could effectively inhibit M1 polarization and the release of inflammatory factors, which is expected to provide a new strategy for the treatment of inflammatory diseases.

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国家自然科学基金青年科学基金项目（81903813）；山西省基础研究计划项目（202303021211171）；山西省中医药管理局资助项目（2022ZYYC087）；山西中医药大学科技创新项目（2024PY-NS-009）；山西中医药大学科技创新项目（2022PY-TH-12）