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

ABSTRACT

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-1ß and 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.