Experimental And Mechanistic Study On Multifunctional Bioactive Chondrocyte-targeted Nanozyme System For Osteoarthritis

ObjectiveOsteoarthritis(OA)is a common chronic disease associated with joint degeneration,with a high incidence and no effective treatment.Intra-articular injection(IA)model based on nanoparticles has been widely used in the treatment of osteoarthritis,and is an effective way to achieve controlled release and targeted treatment of osteoarthritis.This study opens up a new way for the treatment of osteoarthritis and provides theoretical basis for clinical transformation by synthesizing inorganic multifunctional nanoplatforms with large specific surface area,which can efficiently load drugs,specifically target collagen to lesions,and have good biocompatibility and self-degradation.Methods and Results:Chapter 1: Synthesis and characterization of C-178@m SMWMethods: Mesoporous Silica nanoparticles(MSNs)were synthesized by oil-water synthesis method,and manganese dioxide(m SM)was grown on the surface of Mesoporous silica nanoparticles.The morphology and size of MSNs and m SM nanoparticles were observed by Scanning Electron Microscope(SEM)and Transmission Electron Microscope(TEM).The structure and phase of MSNs and m SM nanoparticles were determined by X-ray Polycrystalline Diffraction(XRD).The pore size distribution and specific surface area of MSNs and m SM nanoparticles were determined by nano-particle size and Zeta potential analyzer(DLS).Element mapping was used to analyze the distribution of O and Mn elements.The specific surface area and pore size distribution of MSNs and m SM nanoparticles were measured by BET analyzer.Then,the surface modified carboxy-COOH of manganese oxide connected with the amino group of Collagen II targeting peptide(WYRGRL),and C-178,the inhibitor of STING,was further loaded into the mesopore of MSNs by ultrasound.The particle size and surface charge of m SMW and C-178@m SMW nanoparticles were measured by nano-particle size and Zeta potential analyzer(DLS).The modification and loading success of WYRGRL and C-178 were characterized by IR.The morphology and structure of m SMW nanoparticles were detected by SEM.MSMW nanoparticles were placed in different solutions [dd H2 O,PBS buffer and Cell medium] for different time(12,48 and 168 h),and the stability of m SMW nanoparticles was detected by DLS particle size analyzer.Detection of ROS elimination ability of MSNs and m SM nanoparticles by ROS response;The loading rate of C-178 supported by m SMW nanomaterial was measured by uv absorption spectrum.The cumulative release of C-178 from m SMW nanoparticles was detected by dialysis.Degradability of m SMW nanoparticles was examined by TEM.Results: The diameter of MSNs synthesized by oil-water synthesis method is about 100 nm,the particle size is uniform,the dispersion is good,and the MSNs is mesopporous and spherical,the pore channel is uniform(~7 nm),the specific surface area is large(760 m2/g),and the Zeta potential is-31.1 m V.SEM and TEM were used to monitor the growth of manganese oxide on the surface of MSNs,showing a powdery structure with a particle size of about 130 nm and a Zeta potential of 17.4 m V.Mapping elements showed that O and Mn elements were evenly distributed on the surface of MSNs.Compared with MSNs nanoparticles,the specific surface area(680 m2/g)and pore size(~ 9 nm)did not change.Potential,particle size and INFRARED spectrum showed that WYRGRL and C-178 were successfully modified,and the morphology did not change.DLS showed that the particle size of m SMW nanoparticles did not change significantly up to 72 h in dd H2 O,PBS buffer and cell culture medium,indicating that the nanoparticles had good stability.MSM nanoparticles can eliminate ROS more than MSNs.The loading content of C-178 for MSNs reached 41.61 mg/g,while that of C-178 for m SMW was 38.32 mg/g.The surface modification of MSNs did not affect the loading capacity of C-178,and the m SMW slowly releases C-178.Chapter 2: In vitro experimental study on biological properties and treatment of osteoarthritis of C-178@m SMWPart 1: Study on biological properties of C-178@m SMWMethods: Chondrocytes of newborn rat knee joint were extracted by one-step enzyme digestion method(collagenase II).The morphology of chondrocytes was observed by light microscope and identified by toluidine blue staining.The toxic effects of m SMW nanoparticles at different concentrations(5,10,25,50,100 and 200 μg/m L)on primary chondrocytes were detected by CCK-8 and dead cell staining kits.Blood compatibility of m SMW nanoparticles was tested by hemolytic activity assay.Using the Confocal laser scanning microscope(CLSM)detected the uptake of m SM and m SMW nanoparticles(MODIFIED by FITC probe)at different concentrations(0,25,50,100 μg/m L)after incubation with chondrocytes for 6 h.Results: The primary chondrocytes of newborn rats were successfully extracted under optical microscope,and the chondrocytes suspended in the medium were observed under inverted phase contrast microscope with small and round shapes.After standing for 24 h,most chondrocytes adhered to the wall and grew,enlarged and formed protuberance.Early chondrocytes are round and polygonal,with oval nuclei located in the middle of the inclusion.Toluidine blue staining showed that the cultured chondrocytes were monolayer and polygonal in shape with blue-purple cytoplasm.CCK8 results showed that when the m SMW concentration reached 200 μg/m L,the cell viability remained above 80 % after 72 hours of incubation.The dead cell staining kit also showed that the concentration of m SMW material reached 200 μg/m L,but still almost no dead cells appeared(green living cells,red dead cells).Hemolysis test also showed that the concentration of m SMW material reached 200 μg/m L,still has no significant effect on hemolysis rate.It further indicates that m SMW material has no obvious cytotoxicity when the concentration reaches 200 μg/m L,which further indicates that it has good biocompatibility.CLSM showed that compared with m SM nanoparticles,the uptake of m SMW materials by chondrocytes increased with increasing concentration.Part 2: In vitro experimental study on the treatment of osteoarthritis by C-178@m SMWMethods: Chondrocytes were treated with 10 ng/m L INTERleukin-1β(IL-1β)for 24 h to construct osteoarthritis model in vitro,and then inflammatory cytokines IL-1,IL-6 and TNF-α were detected by enzyme Linked immunosorbent assay(ELISA),confirming the successful establishment of osteoarthritis model in vitro.The m RNA and protein expression levels of STING in normal chondrocytes and osteoarthritis model cells were detected by RT-PCR and Western Blot.si-STING and overexpressed plasmid were constructed and transfected into osteoarthritis model cells.STING knockdown and overexpressed cell models were constructed,and the transfection efficiency was verified by RT-PCR.The expression levels of inflammatory cytokines IL-1,IL-6 and TNF-α were detected by ELISA and the protein expression levels of apoptosis-related factors were detected by Western Blot.The m RNA expression levels of Aggrecan,Col2a1,MMP13 and ADAMTS5 of chondrocyte matrix molecules in normal chondrocytes,osteoarthritis model cells,osteoarthritis model STING overexpression cells and osteoarthritis model STING overexpression cells + C-178 treatment group were detected by RT-PCR.Reactive oxygen species assay kit was used to detect the ROS elimination ability of MSNs and m SMW nanoparticles(50 μg/m L)at cell level.The chondrocyte apoptosis of blank group,in vitro osteoarthritis modeling group,modeling + m SMW group,modeling + C-178 group,modeling + C-178@m SMW groupwere detected by Flow cytometry and Western Blot.The effects of the expression of chondrocyte inflammatory factors IL-1β,IL-6 and TNF-αon these groups were assayed by RTPCR and ELISA.The effects of Aggrecan,Col2a1,MMP13 and ADAMTS5 on the expression of chondrocyte matrix molecules were detected by RT-PCR and ELISA.Results: The high expression of IL-1,IL-6 and TNF-α indicated that il-1β induced osteoarthritis cell model was successfully constructed in vitro.RT-PCR and Western Blot results showed that STING was significantly up-regulated in osteoarthritis.STING knockdown and overexpression cell models were successfully constructed.Si RNA doubled the expression of STING,and overexpression increased the expression of STING by 15 times.In osteoarthritis,siSTING could inhibit the content of inflammatory factors and the level of apoptosis,and overexpression of STING further promoted the content of inflammatory factors and the level of apoptosis in osteoarthritis.The overexpression of STING promoted the secretion of ECM matrix,while the secretion of ECM matrix was inhibited after the addition of STING inhibitor C-178,suggesting that C-178 could treat osteoarthritis by inhibiting the expression of STING.According to the results of the first part,we selected m SMW concentration of 50 μg/m L for in vitro osteoarthritis treatment study.Compared with MSNs nanoparticles,m SMW nanoparticles can significantly remove IL-1β-induced ROS.Flow cytometry showed that IL-1β promoted chondrocyte apoptosis,m SMW and C-178 alone could partially inhibit apoptosis,while C-178@m SMW inhibited almost all apoptosis induced by IL-1β.Western Blot also confirmed that C-178@m SMW was the most significant antiapoptotic.RT-PCR and ELISA showed that after IL-1β treatment alone,the expressions of IL-1β,TNF-α,IL-6,MMP13 and ADAMTS5 were significantly upregulated,while the m RNA expressions of Aggrecan and Col2a1 were significantly down-regulated.After adding m SMW,C-178 and C-178@m SMW,the expression of IL-1β,TNF-α,IL-6,MMP13 and ADAMTS decreased significantly,while the expression of Aggrecan and Col2a1 increased gradually.The anti-catabolic ability and anti-inflammation of chondrocytes in C-178@m SMW group are more prominent,indicating that C-178@m SMW has a good effect on osteoarthritis in vitro.Chapter 3: In vivo experimental study on the treatment of osteoarthritis by C-178@m SMWMethods: 6-8 weeks old SD rats were divided into 5 groups(3 rats in each group).1)Sham group;2)DMM group;3)DMM + m SMW group;4)DMM + C-178 group;5)DMM + C-178@m SMW group.The medial meniscus tibial ligament was resected by DMM(Destabilization of the medial meniscus)to induce osteoarthritis.One and a half months later,corresponding drugs(20 μL,50 μg/m L)were injected into the bone and joint in situ every 4 days for 6 weeks.The body weight of SD rats was measured after injection at 0,2,4 and 6 weeks.And the rats were killed at the end of the treatment at week 6,Knee tissues were collected for micro-CT scanning,bone histomorphology,hematoxylin-eosin staining(H&E staining),phrosin-solid green staining,toluidine blue staining,OARSI histological score,COL-I and COLII immunohistochemistry,ELISA and RT-PCR detection of inflammatory factors(IL-1β,TNF-α,IL-6)and matrix molecules(MMP13,ADAMTS5,Aggrecan and Col2a1)to evaluate the therapeutic effect of each group on osteoarthritis.Orbital blood samples were collected for routine blood and biochemical tests(AST,ALT,RBC,WBC,Plateles,Hemogobin),and H&E staining of vital organs to determine the in vivo biosafety of each group of materials.SPSS16.0 was used for data management and statistical analysis of the appeal groups.Results: Compared with the Sham group,the DMM group showed obvious pathological phenomena of osteoarthritis,including bone fracture,bone fibrillation,increased tissue cells,matrix loss,etc after 6 weeks of injection.Among all the treatment groups,the DMM + C-178@m SMW group showed the most effective recovery of cartilage surface injury,the most complete cartilage structure,and the strongest ferro O staining(red).The expression of COL I and COL II in each group was detected by immunohistochemistry.The surface of cartilage in DMM group showed strong positive staining(dark brown).In contrast,the intensity of positive staining decreased after treatment,followed by DMM + C-178@m SMW group,DMM + C-178 group and DMM + m SMW group.Micro-CT analysis showed that bone volume fraction(BV/TV)in DMM group were significantly decreased compared with Sham group,as well as bone mineral density(BMD)and bone trabecular thickness(TB.Th),then trabecular septum(Th.Sp)increased(P<0.05).BMD,BV/TV and Tb.Th were all increased in the treatment group(the increasing intensity was DMM + m SMW group,DMM + C-178 group and DMM + C-178@m SMW group,respectively),and Th.Sp instead(P<0.05).RT-PCR and ELISA results showed that the expressions of IL-1β,TNF-α,IL-6,MMP13 and ADAMTS5 were significantly up-regulated in DMM group compared with Sham group,while the expressions of Aggrecan and Col2a1 were significantly downregulated.The expressions of IL-1β,TNF-α,IL-6,MMP13 and ADAMTS5 in the treatment group were significantly inhibited,while the expressions of Aggrecan and Col2a1 were significantly up-regulated.Chapter 4: Experimental study on molecular mechanism of C-178@m SMW in the treatment of osteoarthritisMethods: The phosphorylation levels of NF-κB signaling pathway related proteins(p65 and IκB)in IL-1β-induced chondrocytes were detected by Western Blot.Western Blot was used to detect the effects of si-STING and STING overexpression on the phosphorylation levels of NF-κB signaling pathway p65 and IκB in osteoarthritis model cells.After STING overexpression and c-178 treatment,the phosphorylation levels of p65 and IκB were further detected.p65 si RNA was used to down-regulate the expression of p65 in osteoarthritis model cells.The protein expression levels of Aggrecan,Col2a1,ADAMTS5 and MMP13 were detected by Western Blot to clarify the role of NF-κB in STING mediated ECM regulation.The contents of proinflammatory cytokines(IL-6,IL-1β,TNF-α)in supernatant of osteoarthritis cell model treated with si-p65 were determined by ELISA.Western Blot analysis of the phosphorylation of apoptosis-related proteins(Bax,Bcl-2,cleaved caspase-3/caspase 3)revealed the role of NF-κB signaling pathway in STING induced inflammation and chondrocyte apoptosis.Results: In NF-κB signaling pathway,IL-1β activates phosphorylation of p65 and IκB.Overexpression of STING significantly upregates phosphorylation of p65 and IκB in NF-κB signaling pathway in osteoarthritis cell models.These results indicated that STING was involved in the process of osteoarthritis through activation of NF-κB signaling pathway by phosphorylation of p65 and IκB.Furthermore,the addition of C-178 to the osteoarthritis cell model with overexpression of STING inhibited the phosphorylation levels of p65 and IκB,indicating that C-178 further inhibited the activation of phosphorylation of p65 and IκB by inhibiting the expression of STING,thus treating osteoarthritis.The addition of si-p65 significantly reversed the down-regulation of Aggrecan and Col2a1 protein expression mediated by STING overexpression,as well as the upregulation of ADAMTS5 and MMP13.These results indicated that STING induced ECM degradation through NF-κB signaling pathway.si-p65 significantly reduced the expression of STING-induced inflammatory cytokines(IL-1β,TNF-α,IL-6)and apoptosis-associated proteins(Bax,Cleaved caspase-3/caspase 3).Then si-p65 increased the expression of Bcl-2.The addition of C-178 further inhibited the expression of inflammatory cytokines(IL-1β,TNF-α,IL-6)and apoptosis-related proteins(Bax,cleaved caspase-3/caspase 3),and increased the expression level of Bcl-2.p65 si RNA reversed STING-induced chondrocyte senescence and apoptosis,suggesting that STING regulates inflammatory levels and chondrocyte apoptosis in osteoarthritis through a NF-κB signaling cascade.Conclusion:In this study,we successfully synthesized a novel anti-inflammatory and sustained-release bi-functional nanomaterial(C-178@m SMW)for the treatment of osteoarthritis.The experimental results show that the drug delivery system has good drug release effect.In addition,Mn O2 acts as an anti-inflammatory by scavenging free radicals from cells.Importantly,the nanoplatform inhibited STING expression through highly targeted delivery of C-178,thereby inhibiting the activation of NF-κB signaling pathway to further not only rescue chondrocyte apoptosis and inflammation,but also inhibit cartilage degeneration in vivo.In conclusion,this multifunctional nanoplatform opens a new approach for the treatment of osteoarthritis.