A PH-Responsive Metal-Organic Framework For The Co-delivery Of HIF-2α SiRNA And Curcumin For Enhanced Therapy Of Osteoarthritis And Imaging Evaluation

The occurrence of osteoarthritis(OA)is highly correlated with the reduction of joint lubrication performance,in which persistent excessive inflammation and irreversible destruction of cartilage dominate the mechanism.The incidence and disability rate of OA increased year by year.And the inadequate response to monotherapy methods,suboptimal efficacy caused by undesirable bioavailability,short retention,and lack of stimulus-responsiveness,are few unresolved issues.Herein,we report a pH-responsive metal-organic framework(MOF),namely,MIL-101-NH2,for the co-delivery of anti-inflammatory drug curcumin(CCM)and small interfering RNA(siRNA)for hypoxia inducible factor(HIF-2α).CCM and siRNA were loaded via encapsulation and surface coordination ability of MIL-101-NH2.In order to achieve long-term drug retention and lubrication of joints,nanocomposites were dispersed in hyaluronic acid solution(HA).Local Intra-articular(IA)injection is a promising treatment option for OA with minimal extra-articular symptoms.Our vitro tests showed that MIL-101-NH2 protected siRNA from nuclease degradation by lysosomal escape.The pH-responsive MIL-101-NH2 gradually collapsed in an acidic OA microenvironment to release the CCM payloads to down-regulate the level of pro-inflammatory cytokines,and to release the siRNA payloads to cleave the target HIF-2α mRNA for gene-silencing therapy,ultimately exhibiting the synergetic therapeutic efficacy by silencing HIF-2α genes accompanied by inhibiting the inflammation response and cartilage degeneration of OA.Above all,the low bioavailability and poor solubility of CCM,the poor stability and lack of targeting ability of siRNA were all significantly improved after binding to the material.The hybrid material reported herein exhibited promising potential performance for OA therapy as supported by both in vitro and in vivo studies and may offer an efficacious therapeutic strategy for OA utilizing MOFs as host materials.The main research contents are described as follows:Firstly,MIL-101-NH2 nanocomposites were constructed by hydrothermal method,and CCM and siHIF-2α were further encapsulated under stirring and ultra-sonication conditions to form MIL-101-NH2@CCM-siRNA(denoted as MCS).Transmission electron microscopy(TEM),scanning electron microscopy(SEM),nano-particle potential analyzer(Zetasizer Nano ZS),X-ray diffraction(XRD),Fourier Transform Ioncyclotron Resonance and UV-vis spectroscopy(UV-vis)were used to systematically characterize the nanocomposites.The siHIF-2a loading affinity of MIL-101-NH2@CCM(denoted as MC)was investigated by fluorescence detection and agarose gel electrophoresis.Based on the standard curves of CCM and siRNA,the drug loading capacity(DLC)and drug loading efficiency(DLE)were calculated by testing the absorbance peaks of CCM and siCy5 in solution.Secondly,the primary articular chondrocytes were harvest from one-week-old C57BL/6 mice,and the IL-β-induced chondrocytes were applied as inflammatory cell model.Cell uptake experiments demonstrated that the uptake of MCS by chondrocytes reached saturation at 6 h,and MCS entered chondrocytes through the lysosomal pathway.MTT experiments showed that MCS had no obvious cytotoxicity against chondrocytes even at high concentration(400 μg/mL),and the CCM encapsulation in MIL-101-NH2,significantly improved the biocompatibility of CCM(200 μg/mL).According to the lysosome escape assay,the majority of the siCy5(red)and lysosome tracker(green)fluorescence in the cytoplasm were separated,indicating that the siCy5 escaped from the entrapment of lysosome and then accumulated in the cytoplasm.When the high concentration of phosphate ion in lysosomes triggered the internalized MIL-101-NH2 collapse,the lysosome structure turned to be unstable because of the high binding between the released Fe3+and the phosphate group on the lysosome membrane,so that the siRNA could reduce enzyme degermation and successfully escape.In safranin O staining assay,the MCS group demonstrated the highest abundance and homogeneity of glycosaminoglycan(GAG)after 24 h incubation,which further confirmed by qRT-PCR results.Particularly,these results ultimately supported that MCS remarkably down-regulated the expression of OA-related catabolic markers(including MMP3,MMP13,HIF-2α,IL-6,ADAMTs-5,and COX-2)and slightly up-regulated the expression of cartilage-specific markers(including Acan,Col2a1,and SOX-9).Finally,a mice model of OA was established by destabilized medial meniscus(DMM)surgery.According to the CT images,the width of the articular space and the volumes of osteophytes were both analyzed.All groups displayed an increase in the osteophyte volume,but the MCS group had a lower value in comparison with the other groups,indicating that it had an improved therapeutic effect in reducing DMM-induced osteophyte formation.Besides,histopathological sections and immunohistochemical assays further indicated that the structure of bone and joint has improved more effectively in the MCS group,compared with the other groups.These results further indicated that the synergistic therapy,which simultaneously improves intra-articular lubrication and inhibits the up-regulation of pro-inflammatory cytokines,efficiently obstructed the development of OA.This work successfully constructed MCS NPs for the co-delivery of CCM and siHIF-2α to establish a pH-responsive system for OA therapy.Multi-modal therapeutic system could improve the long-term stability and bioavailability of CCM and siHIF-2α in the joint cavity.Altogether,the MCS NPs exhibit remarkable effects on anti-inflammation and cartilage regeneration by combining drug therapy and gene therapy.Because the pathogenesis of OA is complicated,diverse actually,and sometimes related to microenvironmental factors,this multi-modal therapeutic system may provide a promising strategy for OA treatment.