ROS-Responsive Berberine Polymeric Micelles Effectively Suppressed the Inflammation of Rheumatoid Arthritis by Targeting Mitochondria

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease, which attacks human joint system and causes lifelong inflammatory condition. To date, no cure is available for RA and even the ratio of achieving remission is very low. Hence, to enhance the efficacy of RA treatment, it is essential to develop novel approaches specifically targeting pathological tissues. In this study, we discovered that RA synovial fibroblasts exhibited higher reactive oxygen species (ROS) and mitochondrial superoxide level, which were adopted to develop ROS-responsive nano-medicines in inflammatory microenvironment for enhanced RA treatment. A selenocystamine-based polymer was synthesized as a ROS-responsive carrier nanoplatform, and berberine serves as a tool drug. By assembling, ROS-responsive berberine polymeric micelles were fabricated, which remarkably increased the uptake of berberine in RA fibroblast and improved in vitro and in vivo efficacy ten times higher. Mechanistically, the anti-RA effect of micelles was blocked by the co-treatment of AMPK inhibitor or palmitic acid, indicating that the mechanism of micelles was carried out through targeting mitochondrial, suppressing lipogenesis and finally inhibiting cellular proliferation. Taken together, our ROS-responsive nano-medicines represent an effective way of preferentially releasing prodrug at the inflammatory microenvironment and improving RA therapeutic efficacy.

Keywords: Berberine; Nanoparticles; Oxygen consumption rate; Reactive oxygen species; Rheumatoid arthritis.

Fig. 1

ROS level is associated with the progress of RA. a, b ROS and mitochondrial superoxide levell were detected and compared in MTX-resistant, RA, OA, and normal synovial fibroblast. c OCR was evaluated in MTX-resistant HFLS-RA, HFLS, and HFLS-RA. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

a Average particle size and zeta potential of BPseP. b A uniform spherical morphology of nanoparticles. c BPseP could maintain stability at 37 °C for 2 weeks. d BPseP micelles could cumulatively release berberine quickly more than 80% because the diselenium bond in the PseP structure is sensitive to the ROS. e BPseP significantly up-regulated the cellular accumulation of berberine. f Compared with HFLS, HFLS-RA induced much berberine intracellular accumulation. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 3

a, b LC–MS results showed that cellular and mitochondrial concentration of berberine was greatly increased by BPseP, when compared with berberine administration. c The efficacy of BPseP and berberine on primary HFLS cells. d BPseP effectively inhibited the growth of cells, and its IC₅₀ value is around 0.6 ug mL⁻¹. For other parallel controls, no significant inhibitory effect was observed. e The efficacy of BPseP among different types of HFLS was detected. f Compared with HFLS-RA, IC₅₀ of BPseP in MTX-resistant HFLS is much lower. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

a BPseP significantly caused G2 arrest, down-regulation of CCNB1 and the increase of p21. b Immunofluorescence detection of berberine and mitochondria. The intracellular location of berberine was mostly overlapped with mitochondria. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

a OCR was remarkably suppressed by BPsep in HFLS-RA. b With the increasing concentration of BPseP, the morphology change of mitochondria became shorter and smaller. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

a, b SREBP1 and FASN were significantly down-regulated by the treatment of BPseP. c The cellular level of TG was decreased. d AMPK inhibitor compound C and palmitic acid (the key intermediator of lipogenesis) partially counteract the suppressive effect of BPseP. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 7

a, b BPseP treatment acquired a significant regression in edema of the hind paw. c BPseP remarkably inhibited the secretions of IL-1 and IL-6. d The results of X-ray imaging showed that BPseP is able to protect the joint bone from destruction induced by AIA. Data were analyzed as the mean ± SEM for triplicate individual experiments (*P < 0.05, **P < 0.01, ***P < 0.001)