Sialic Acid-based Targeted Drug Delivery Systems For Rheumatoid Arthritis Therapy

Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by multi-joint synovial inflammation.It is often accompanied by involvement of extraarticular organs and positive serum rheumatoid factor,which eventually leads to joint deformity and functional loss.The incidence of RA is increasing year by year in women aged 30-50.At present,the treatment of RA is mainly based on drug therapy and surgical treatment,but surgical treatment is liable to cause secondary injury of joint cavity.The removal of synovium makes the joint lose the basic function of synthesizing synovial fluid,which further aggravates the damage of joint function.Methotrexate is a clinical first-line anti-rheumatic drug with immunosuppressive and anti-inflammatory effects.It can relieve pain,relieve symptoms and control the development of the disease.However,due to its non-specific distribution in the body,the number of drug molecules entering the inflammatory joints is limited.High-dose methotrexate often produces side effects such as ulcerative stomatitis,diarrhea and even fatal intestinal perforation,which seriously affects the life quality of patients,while low-dose methotrexate often restricts the efficacy of treating RA.And long-term application of low-dose methotrexate can only delay the progress of bone erosion rather than prevent the occurrence of osteoporosis caused by long-term inflammatory response,or promote the repair and regeneration of damaged bone.Adopting new nano-drug delivery system is an effective means to improve drug distribution in vivo and reduce side effects.It can realize multi-target therapy of RA by functionalization of carrier materials.Sialic acid(S A)is a kind of nine-carbohydrate located in the outermost layer of cell membrane,which can specifically bind to E-selectin.Sialic acid glycoconjugates can inhibit osteoclast maturation,promote osteoblast differentiation,and then improve bone repair and regeneration.In this study,sialic acid-dextran-octadecanoic acid(SA-Dex-OA,SDO)were synthesized.SDO can self-assemble into micelles in aqueous media with a critical micelle concentration of 55.06 ug/mL and a particle size of 75.20 ± 11.91nm.The micelles can effectively encapsulate anti-rheumatoid drug MTX.The encapsulation efficiency was 89.39 ± 0.26%.The particle size of drag-loaded micelles(SDM)was 117.33 ± 5.77 nm.SDM showed a significant sustained release effect on the drug,and the drug release lasted for more than 48 hours.Human umbilical vein endothelial cells(HUVEC)were used as model cells to investigate the cytotoxicity.The results showed that the grafts had no effect on cell proliferation in the concentration range of 1 and 1000?g/mL.An in vitro inflammatory cell model with high expression of E-selectin receptor stimulated by lipopolysaccharide was constructed to investigate the active targeting ability of SDO.Dex-OA(DO)were used as control.The results showed that inflammatory HUVEC had better uptake behavior on SDO.After blocking the E-selectin receptor on the cell surface,the uptake of SDO by inflammatory cells decreased.The co-localization of E-selectin and SDO micelle confirmed that the active transport of SDO to inflammatory vascular endothelial cells was mediated by the specific binding of SA and E-selectin.Using mouse embryonic osteoblast precursor cells(MC3T3-E1)as model cells,the bone repair and regeneration effect of micelles were investigated.The results showed that SDO could promote the differentiation and mineralization of osteoblasts by promoting the activity of intracellular alkaline phosphatase and the formation of extracellular mineralized nodules.The targeting efficiency of SDO in vivo was evaluated in a rat model of collagen induced arthritis(CIA)The results showed that more SDO/ICG micelles were accumulated in the arthritic paws by the specific combination of sialic acid and E-selectin overexpressed on the surface of vascular endothelial cells around inflammatory joints.After treatment for 14 days,the paw thickness and arthritis score index of SDM group were significantly reduced.Synovial tissue proliferation and intracellular mitochondrial swelling were effectively inhibited.And cell ischemia and hypoxia were significantly improved.At the same time,inflammatory cell infiltration was significantly reduced,and secretion of TNF-alpha and IL-6 decreased.In addition,SDM also up-regulate expression of OPG,down-regulate expression of RANKL,increasing bone mineral density,so as to regulate bone repair effect mediated by sialic acid.After treatment for 14 days,the serum concentrations of alanine transaminase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN)and serum creatininealanine aminotransferase(Scr)were significantly increased.However,the indexes of SDM group were similar to those of normal group,indicating the safety of SDM.Hydroxyapatite(HAP),as one of the natural components of bone,has the characteristics of strong biological adhesion,excellent capacity for large amount of drugs adsorbtion,acid sensitivity,bone induction and so on.It is an excellent bionic regeneration material for human bone.On the basis of previous studies,sialic acid-chitooligosaccharide-deoxycholic acid(SA-CSO-DA,SD)were synthesized.SD could self-assemble into micelles in aqueous media,CMC was 38.45?g/mL.The sialic acid modified chitosan oligosaccharide/hydroxyapatite(SH)nanoparticles were prepared by hydrothermal method using micelles as the stabilizer.The size of the rod-like SH nanoparticles was uniform,with the average size of 30.03±6.54 nm.The nanoparticles could effectively adsorb MTX,and the drug-loaded nanoparticles(SHM)were rod-like in appearance,uniform in distribution.And the size of SHM was 46.08±7.88 nm,the encapsulation efficiency was 68.67 ± 2.80%.The results of in vitro release showed that SHM exhibited obvious acid-sensitive release characteristics.The release was slow in neutral medium at pH 7.4,but accelerated in acidic medium at pH 5.5.The cumulative release was about 87.95%within 48 hours.Using primary rat chondrocytes as model cells,the effect of SH nanoparticles on cartilage repair was investigated.The results showed that SH nanoparticles had low cytotoxicity.After SH treatment of chondrocytes stimulated by IL-1?,the proportion of apoptotic cells decreased significantly,the content of glycosaminoglycan increased significantly,and the expression of type II collagen increased.However,HAP pretreatment did not affect the percentage of apoptotic cells,the content of glycosaminoglycan and the expression of type II collagen.Using primary osteoblasts as model cells,the effect of SH nanoparticles on bone repair and regeneration was investigated.The results showed that after co-incubation with SH,the activity of alkaline phosphatase in cells increased significantly,the number of mineralized nodules increased,the expression of type ?collagen and OPG enhanced,which promoted the differentiation and mineralization process.RA model was established by collagen-induced arthritis.After intravenous administration,SH nanoparticles could effectively distribute to the target site and accumulate in inflammatory joints.After treatment of SHM nanoparticles for 28 days,the paw thickness,the arthritis score index,the spleen index,thymus index,inflammatory cytokines(TNF-alpha and IL-6)were significantly reduced.It could also exert cartilage repair effect by supplementing the content of glycosaminoglycan,increasing the expression of type ? collagen and inhibiting the activity of matrix metalloproteinase.SHM can significantly accelerate the formation of new bone by up-regulating expression of OPG,down-regulating expression of RANKL.After treatment of MTX for 28 days,the ratio of ALT and AST and the concentration of BUN increased significantly.However,the biochemical indexes of SHM were close to those of normal group,and there were no obvious lesion areas in liver and kidney,indicating the safety of SDM.In conclusion,the two functional drug delivery systems designed in the study could effectively inhibit the inflammatory response through the targeted delivery of MTX to the arthritic paws of CIA rats.Simultaneously,the residual chitosan oligosaccharides could promote cartilage repair and the residual nano-hydroxyapatite cooperating with sialic acid could exert bone repair and regeneration effect,thus safe and effective therapy of RA was achieved through the synergistic therapy between drug and the delivery systems.Our findings might provide new insights and strategies for RA treatment.