Preparation Of Sinomenine Hydrochloride Biomimetic Nanosystem And Its Targeted Treatment For Rheumatoid Arthriti

Background:Sinomenine is a bioactive alkaloid isolated from the Chinese medicinal plant Sinomenium acutum（Thunb.）Rehd.et Wils.（family Menispermaceae）,which exhibits various bioactivities of anti-inflammation,analgesic,and immunosuppression.Currently,conventional and sustained-releasing tablets of sinomenine hydrochloride（SIN）are applied for treatment of RA and other rheumatic diseases.Despite its definitive effects in RA,the clinical application of SIN is seriously limited by its short half-life and low bioavailability.Overcoming these drawbacks and accordingly improving its efficacy were essential to improve the clinical application of SIN.Objective:A biomimetic SIN-loaded nanocomplex is developed to prolong the drug half-life,target accumulation of SIN within the arthritic sites,and investigate its effects for treat-to-target therapy of RA.Methods:（1）In this study,a biomimetic nanocomplex（HA@M@PB@SIN NPs）based on Prussian blue nanoparticles（PB NPs）was developed for overcoming clinical limitations of SIN and accordingly improving its efficacy.After loading SIN into PB NPs（PB@SIN NPs）,macrophage membrane hybridized with red blood cell membrane（M）were adopted to camouflage PB@SIN NPs with aims of both prolonging circulation time and increasing the recruitment of the NPs to affected joints.Lastly,DSPE-PEG₂₀₀₀-hyaluronic acid（HA）was inserted into the membrane to improve the targeting ability of the nanodrugs to the inflammatory macrophages and fibroblast-like synoviocytes（FLSs）in the rheumatoid synovial joints.The transmission electron microscopic（TEM）,X-ray photoelectron spectroscopic（XPS）,Fourier transform infrared spectroscopy（FT-IR）,X-ray diffraction（XRD）,UV-Vis spectra assay,and Dynamic light scattering analysis were used to demonstrate the successful synthesis of NPs.The F（?）rster resonance energy transfer（FRET）study was employed to characterize the fusion efficiency of macrophage membrane（M（?）m）and red blood cell membrane（RBCm）.Components of membrane coated on NPs were characterized by the SDS-PAGE and western blot.Hemolysis,coagulation,and cytotoxicity assays were performed for evaluating biosafety of NPs.（2）PB NPs labeled with Rhodamine B were used to investigate the mechanism underlying the cellular uptake of HA@M@PB@SIN NPs.The effects of HA@M@PB@SIN NPs on the viability of activated macrophages or HFLS-RA as inflammatory cell models were investigated.The levels of cytokines were quantified using the Bio-Plex Pro^TM Rat Cytokine 23-Plex Assay kits.The effects of different NPs on the intracellular ROS levels were monitored using the ROS kits.The ability of HA@M@PB@SIN NPs to kill HFLS-RA in vitro was determined using the calcein-AM//PI assay.（3）Pharmacokinetics and target distribution of HA@M@PB@SIN NPs in rats were analysed by fluorescence imaging.Adjuvant induced arthritis（AIA）rats,a widely-used model for RA,was established to investigate the efficacy of HA@M@PB@SIN NPs on RA.The severity of arthritis was evaluated using a system of arthritic scoring,measurement of bi-hind paw volumes every three days after arthritis induction.To further investigate whether HA@M@PB@SIN NPs suppress joint inflammation by targeting inflammatory macrophages and FLSs in rheumatoid synovial joints,the ankle joints of rats were immunohistochemically stained.Serum levels of cytokines were quantified using the Bio-Plex Pro^TM Rat Cytokine 23-Plex Assay kits detected in the Bio-Plex 200 System.The biosafety of HA@M@PB@SIN NPs in rats were evaluated by routine blood examination,liver and renal function,and H&E staining.Results:（1）The transmission electron microscopic（TEM）images showed that the uniform PB NPs with a hollow mesoporous structure were surrounded by an outer film.Dynamic light scattering analysis showed that the average diameters of HA@M@PB@SIN NPs were 110.2±12.35 nm.The surface zeta potential of HA@M@PB@SIN NPs slightly increased from-16.55 m V of PB NPs to-12.65 m V.The composition and structural characteristics of HA@M@PB@SIN NPs were determined by X-ray photoelectron spectroscopic（XPS）,Fourier transform infrared spectroscopy（FT-IR）,X-ray diffraction（XRD）,and UV-Vis spectra assay.The release behavior profile was p H-dependent,which favorably contributed to the increased drug release at the arthritic sites.The corresponding cumulative rate for HA@M@PB@SIN NPs was 40.3%within 72 h,indicating that the encapsulation of SIN within the hybrid biomimetic membranes significantly attenuated drug release.Meanwhile,under the camouflage of biomimetic cell membranes,HA@M@PB@SIN NPs exhibited excellent immune evasion ability,homologous targeting uptake ability,and good biocompatibility.（2）Cellular uptake experiments demonstrated the activated macrophages have high uptake efficiency to HA@M@PB@SIN NPs via caveolae-mediated endocytosis and micropinocytosis.The viability of activated macrophages or HFLS-RA incubated with HA@M@PB@SIN NPs was significantly lower than that of cells incubated with same concentration of SIN（P<0.01）.The significant increase of TNF-αand G-CSF in the LPS-activated macrophages exposed to SIN,while HA@M@PB@SIN NPs inhibited the secretion of these cytokines in the activated macrophages,which is comparable to that of normal macrophages（P<0.01）.In addition,the HA@M@PB@SIN NPs have strong intracellular ROS scavenging ability（P<0.01）.（3）In vivo imaging demonstrated that the improved immune-escape properties of the HA@M@PB@SIN NPs resulted in markedly increased half-life of circulation and levels of accumulated drugs at arthritic sites of AIA rats（P<0.01）.The administration of SIN alone did not exhibit obvious anti-arthritic effects on AIA rats;in contrast,treatment with HA@M@PB@SIN NPs administration significantly suppressed joint inflammation as evidenced by the significant reduction of hind paw volume and arthritic score（P<0.01）.Histopathological examination also revealed that AIA rats treated with HA@M@PB@SIN NPs manifested synovial hyperplasia,inflammatory cell infiltration,joint space narrowing,and cartilage destruction and bone erosion to a light degree compared with those in the alone SIN-treated AIA rats（P<0.01）.CT images further demonstrated that rough bone surfaces and severe bone destruction of inflamed joints were reversed by treatment with HA@M@PB@SIN NPs.Notably,the HA@M@PB@SIN NPs significantly suppressed joint inflammation and protected against bone destruction of AIA rats by inhibiting inflammatory cytokine secretion of the synovial macrophages and FLSs（P<0.05）.Meanwhile,HA@M@PB@SIN NPs had no effect on routine blood analyses,hepatotoxicity,and nephrotoxicity.Moreover,H&E staining exhibited that administration of HA@M@PB@SIN NPs caused no damage or toxicity to the important organs such as the heart,liver,spleen,lungs and kidneys.Conclusion:（1）A biomimetic SIN-loaded PB nanocomplex was constructed for treat-to-target therapy of RA.This multiple modification strategy offers references for researches of other drugs.（2）This rationally designed HA@M@PB@SIN NPs exhibit the satisfactory ability of NPs to escape from immune surveillance,highly selective targeting of inflammatory cells,and potent anti-inflammatory activities by scavenging ROS and inhibiting secretion of proinflammatory cytokines.This nanomedicine may offer new candidate therapies for treatments of RA as well as other involved inflammatory diseases.（3）In view of the fact that prolonged circulation half-life and specific arthritis-targeting ability of HA@M@PB@SIN NPs.The nanocomplexes markedly suppress joint inflammation and protect against bone destruction of AIA rats by targeting synovial macrophages and FLSs and inhibiting their production of various inflammatory cytokines.Taken together,the developed HA@M@PB@SIN NPs represent a promising therapeutic strategy to achieve disease remission for RA.