ROS Responsive Hydrogel Microspheres For Targeted Therapy Of Osteoarthritis

Objective To focus on the goal of functional microspheres for targeted therapy of osteoarthritis(OA),herein,we designed and constructed the dual-drug delivery hydrogel microspheres(KGN/Dex-TSPBA@WHMs)with cartilage-targeting and ROS-responsive ability.Moreover,we validated the therapeutic efficacy of the hydrogel microsphere.Methods(1)Inspired by the unique biological phenomenon that bees track the scent of flowers for collecting nectar,we initially used the polyethylene glycol-polycaprolactone-N1(4-boronobenzyl)-N3-(4-boronobenzyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diaminium(PEG-PCL-TSPBA)to load kartogenin(KGN)and dexamethasone(Dex),and fabricated KGN/Dex-TSPBA micelles with ROS-responsive property through dialysis method.Then,the"bees",hydrogel microspheres,were prepared via combining the microfluidic method and photopolymerization processes to integrate cartilage-targeting peptides WYRGRL and ROSresponsive nanoparticles in the hydrogel matrix.The nanoparticles and hydrogel microspheres were characterized using a transmission electron microscope(TEM),a scanning electron microscope(SEM),a bright-field microscope,and a microplate reader,respectively,while the drug release behaviors of different biomaterials were investigated by the microplate reader.(2)ATDC5 cells were used as the chondrocytes to assess the cytotoxicity and cellular uptake of different biomaterials.(3)The ROS-responsiveness of hydrogel microspheres was evaluated by dihydroethidium(DHE)staining,while the anti-inflammatory abilities and chondrogenic differentiation effect of hydrogel microspheres were evaluated by enzyme-linked immunosorbent assay(ELISA)and immunofluorescence staining,respectively.(4)Rat models of osteoarthritis were established for in vivo experiments.The cartilage-targeting property of hydrogel microspheres was further evaluated by an in vivo imaging system(IVIS).(5)To further confirmed the in vivo therapeutic efficacy of "bees" hydrogel microspheres for OA,rat models of osteoarthritis were established,followed by X-ray radiography analysis,histopathological assessment,and immunohistochemical evaluation.Results(1)In this study,we synthesized KGN/Dex-TSPBA micelles with a particle size of 66.32 ± 10.32 nm.The TEM and dynamic light scattering(DLS)results indicated that monodisperse nanoparticles had a favorable ROS-responsive ability.Subsequently,the cartilage-targeting peptide and ROS-responsive nanoparticles were immobilized in hydrogel matrix by combining the microfluidic method and photopolymerization processes.The brightfield microscope and SEM images showed the uniform size of hydrogel microspheres,while a superior performance in sustained release was observed in KGN/Dex-TSPBA@WHM group compared with the KGN/Dex-TSPBA group or free drug group.(2)The results of Live/Dead and MTT assays revealed the favorable biocompatibility of the KGN/Dex-TSPBA@WHMs.Compared with the hydrogel microsphere group without WYRGRL loaded,the KGN/DexTSPBA@WHM group was observed an enhanced the cellular uptake capacity of indocyanine green(ICG)-loaded nanoparticles,suggesting the cartilage-targeting property of KGN/DexTSPBA@WHM.(3)The favorable efficacy of the "bees" KGN/Dex-TSPBA@WHMs on the massive consumption of intracellular ROS and alleviating inflammation were,respectively,verified by the DHE staining results and ELISA analyses.Additionally,the immunofluorescence staining determined that "bees" KGN/Dex-TSPBA@WHMs could effectively promote the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)was determined.(4)The IVIS images further validated the favorable targeting therapeutic potential of KGN/Dex-TSPBA@WHM in vivo compared with the hydrogel microspheres without WYRGRL encapsulated.(5)In vivo experiments showed that "bees"hydrogel microspheres could effectively reduce osteophyte formation and cartilage matrix loss in OA model,and significantly promote the repair of osteoarthritis.Conclusion In the present study,inspired by the phenomenon that bees track the scent of flowers for collecting nectar,injectable hydrogel microspheres encapsulated with cartilagetargeting peptides WYRGRL and ROS-responsive nanoparticles KGN/Dex-TSPBA were developed via microfluidic technology.The "bees" KGN/Dex-TSPBA@WHMs achieved the targeted therapy and enhanced the cellular uptake capacity of nanoparticles.And the KGN/DexTSPBA nanoparticles diffused from the hydrogel microspheres simultaneously reacted with OA-induced intracellular ROS and resulted in the responsive release of KGN and Dex,realizing a combined effect of long-term treatment,ROS scavenging,down-regulation of inflammatory factors and chondrogenic differentiation of endogenous stem cells.Thus,the injectable hydrogel microspheres exerted a favorable potential to repair OA.