基于牛乳外泌体与脂质体的杂化外泌体制备及其负载青藤碱治疗类风湿性关节炎的药效学研究

Preparation of hybrid exosomes based on bovine milk exosomes and liposomes and pharmacodynamic study of sinomenine loaded exosomes in treatment of rheumatoid arthritis

研究乳源性外泌体与脂质体进行膜融合得到的杂化外泌体装载青藤碱(sinomenine,SIN)后对胶原诱导性关节炎(CIA)大鼠的治疗效果。通过蔗糖密度梯度离心法从鲜牛乳中提取外泌体,采用乳化蒸发-低温固化法制备脂质体,共孵育法进行膜融合后对杂化外泌体进行表征:透射电镜检测形貌,纳米粒度电位仪检测粒径与电位,蛋白免疫印迹法(Western blot,WB)检测膜融合前后外泌体膜表面特征蛋白CD63和TSG101的表达。超声法装载青藤碱后,酶标仪检测其载药量与包封率。胶原抗体诱导法建立CIA大鼠模型,药效实验设对照组、模型组、SIN组、SIN-脂质体组、SIN-乳外泌体组、SIN-杂化外泌体组及阳性药(地塞米松)组。记录给药期间大鼠体质量变化,以足肿胀度、免疫器官指数、关节炎指数、微循环指标变化、滑膜组织病理学检查、血清炎症因子水平变化为指标进行药效学研究。透射电镜结果显示,杂化外泌体与乳外泌体外观均呈茶托状,共孵育后外泌体粒径由(97.92±3.42)nm增长到(132.70±4.07)nm,Zeta电位由(-2.01±0.33)mV变为(-17.90±2.13)mV,WB结果显示CD63与TSG101蛋白在乳外泌体及杂化外泌体中均正常表达。酶标仪测定乳外泌体包封率31.64%±2.48%、载药量2.35%±0.52%,杂化外泌体包封率48.21%±3.12%、载药量3.17%±0.36%。药效学结果显示,与模型组相比,各给药组一般情况及足肿胀度、关节炎指数及免疫器官指数均得到显著改善(P<0.05,P<0.01),微血管综合评分及血管阻力显著下降(P<0.05,P<0.01),血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)炎症因子水平显著降低(P<0.01),滑膜组织的病变得到一定程度的改善。同时与SIN组、SIN-脂质体组及SIN-乳外泌体组相比,SIN-杂化外泌体组具备更平稳持久的药效。该研究将乳源性外泌体与脂质体共孵育得到的杂化外泌体成功改善了外泌体载药量小与脂质体生物相容性差等问题,负载青藤碱的杂化外泌体对CIA大鼠有着良好的疗效,可有效解决青风藤等疗效佳但生物半衰期短的问题,为传统中药的新发展及类风湿性关节炎等疾病的治疗提供了新思路。

This study investigates the therapeutic effect of hybrid exosomes loaded with sinomenine(SIN)obtained by membrane fusion of milk exosomes with liposomes in collagen-induced arthritis(CIA)rats.Exosomes were isolated from fresh bovine milk by sucrose density gradient centrifugation,while liposomes were prepared using the emulsion solvent evaporation-low temperature curing method.Hybrid exosomes were characterized after membrane fusion through co-incubation:The morphology was detected by transmission electron microscopy,the particle size and potential by nanoparticle size potentiostat,and the expressions of surface characteristic proteins CD63 and TSG101 before and after fusion by Western blot(WB).The drug loading capacity and encapsulation rate of sinomenine were measured after the loading of sinomenine on exosomes by ultrasonic method.The CIA rat model was induced by collagen antibody.The efficacy experiment consisted of the control group,model group,SIN group,SIN-liposome group,SIN-milk exosome group,SIN-hybrid exosome group and positive drug(dexamethasone)group.The changes in body mass of rats during administration were recorded.Besides,the foot swelling,immune organ index,arthritis index,microcirculation index,synovial histopathology,and serum inflammatory factor levels detected by enzyme-linked immunosorbent assay were observed for pharmacodynamical study.Under transmission electron microscopy,both hybrid exosomes and milk exosomes showed saucer-like appearance.After co-incubation,the exosome particle size increased from(97.92±3.42)nm to(132.70±4.07)nm,and the Zeta potential changed from(-2.01±0.33)mV to(-17.90±2.13)mV.WB assay showed that CD63 and TSG101 proteins were normally expressed in milk exosomes and hybrid exosomes.The encapsulation rate of milk exosomes was 31.64%±2.48%,with a drug loading of 2.35%±0.52%,while the hybrid exosomes exhibited an encapsulation rate of 48.21%±3.12%and drug loading of 3.17%±0.36%,as determined by the microplate reader.Pharmacodynamic results showed that compared with the model group,the general condition,swelling degree of foot,arthritis index and immune organ index of all drug administration groups were significantly improved(P<0.05,P<0.01);microvascular comprehensive score and vascular resistance were significantly decreased(P<0.05,P<0.01);serum levels of TNF-α,IL-1βand IL-6 inflammatory factors were significantly decreased(P<0.01);and the lesions of synovial tissue were improved to some extent.Meanwhile,compared with the SIN group,SIN-liposome group and SIN-milk exosome group,the SIN-hybrid exosome group had a more stable and durable drug effect.The hybrid exosomes obtained by co-incubation of milk-derived exosomes with liposomes successfully improved the drug carrying capacity of exosomes and biocompatibility of liposomes.The hybrid exosomes loaded with sinomenine have good efficacy on CIA model rats,and can effectively solve the problems of TCM such as sinomenine,which have good efficacy but short biological half-life.The study provides new insights for the development of TCM and the treatment of diseases such as rheumatoid arthritis.