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负载毛蕊异黄酮的叶酸修饰膜荚黄芪外泌体样纳米颗粒的制备及体外抑制巨噬细胞极化作用评价

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李秀英, 张峰源, 王鑫, 刘彩霞, 杨丽霞, 李钦青, 闫志芳

中草药 | 药剂与工艺 2026,57(8): 3042-3050

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中草药 |药剂与工艺 2026 , 57 (8) : 3042 -3050

负载毛蕊异黄酮的叶酸修饰膜荚黄芪外泌体样纳米颗粒的制备及体外抑制巨噬细胞极化作用评价

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李秀英, 张峰源, 王鑫, 刘彩霞, 杨丽霞, 李钦青, 闫志芳

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通讯作者:

李钦青

作者简介:

李秀英: 李秀英,副教授,研究方向为中药制剂新技术与新剂型。E-mail:lixiuying85@163.com

Preparation of calycosin-loaded folate-modified Astragalus membranaceus exosome-like nanoparticles and in vitro evaluation of inhibitory effect on macrophage M1 polarization

LI Xiuying, ZHANG Fengyuan, WANG Xin, LIU Caixia, YANG Lixia, LI Qinqing, YAN Zhifang

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doi: 10.7501/j.issn.0253-2670.2026.08.017

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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。

关键词

毛蕊异黄酮 / 黄芪外泌体样纳米颗粒 / 叶酸受体靶向 / 巨噬细胞M1极化 / 抗炎

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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.

Key words

calycosin / Astragalus membranaceus exosome-like nanoparticles / folate receptor-mediated targeting / macrophage M1 polarization / anti-inflammation

引用本文

李秀英, 张峰源, 王鑫, 刘彩霞, 杨丽霞, 李钦青, 闫志芳. 负载毛蕊异黄酮的叶酸修饰膜荚黄芪外泌体样纳米颗粒的制备及体外抑制巨噬细胞极化作用评价. 中草药, 2026 , 57 (8) : 3042 -3050 . DOI: 10.7501/j.issn.0253-2670.2026.08.017

LI Xiuying, ZHANG Fengyuan, WANG Xin, LIU Caixia, YANG Lixia, LI Qinqing, YAN Zhifang. Preparation of calycosin-loaded folate-modified Astragalus membranaceus exosome-like nanoparticles and in vitro evaluation of inhibitory effect on macrophage M1 polarization[J]. Chinese Traditional and Herbal Drugs, 2026 , 57 (8) : 3042 -3050 . DOI: 10.7501/j.issn.0253-2670.2026.08.017

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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)

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2026年第57卷第8期

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doi: 10.7501/j.issn.0253-2670.2026.08.017

接收时间：2025-11-04
首发时间：2026-09-09

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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