The Experimental Study Of RANKL SiRNA Loaded Bone-targeting Nanomaterials In The Treatment Of Osteolysis Induced By Wear Particles

Background and objective: Total hip arthroplasty(THA)is one of the most effective treatments for various end-stage hip diseases.The main reason for revision surgery is periprosthetic osteolysis.Wear particles play a key role in the process of osteolysis,which is caused by local inflammation.The small interfering RNA targeting RANKL gene(si RANKL)has good theoretical feasibility in the treatment of wear particle-induced osteolysis.si RNA needs the assistance of a carrier to efficiently reach the lesion cells.Therefore,the aim of this study is to combine the bone-targeting drug zoledronate(ZOL)with the nano-carrier ZIF-8 to construct a novel nano-drug delivery system that can target RANKL si RNA to the area of particle-induced osteolysis.This study provides new ideas and methods for the treatment of osteolysis induced by wear particles.Methods:1、ZIF-8 nanoparticles with size of about 50 nm were synthesized at room temperature by adjusting the concentration and ratio of the reaction substrate in aqueous solution,and the ZOL was directly added to the reaction system after ZIF-8 was formed.Moreover,polyvinylpyrrolidone(PVP)was added to stabilize the particles surface,then a novel nanoparticles ZZF@PVP was synthesized.Doxorubicin(DOX)and bovine serum albumin(BSA)were used as template cargos to evaluate the loading ability of the ZZF@PVP nanoparitcles.The release performance of ZZF@PVP for different loaded drugs was tested under acidic(p H=5.5)and neutral(p H=7.4)conditions.2、The candidate si RNA targeting RANKL gene was designed and synthesized according to the si RNA design principle by referring to the gene database.The candidate si RNA was transfected into mouse osteocytelike cells MLO-Y4,and the silencing efficiency of RANKL gene was detected by RT-q PCR.Western blotting was used to detect the expression of RANKL protein with the si RNA which has the strongest silencing efficiency to evaluate the inhibitory effect on protein translation and synthesis.MLO-Y4 cells transfected with si RANKL were co-cultured with RAW264.7 cells for 5 days,and TRAP staining was performed to evaluate the inhibitory effect of si RANKL on the osteoclastogenesis of RAW264.7cells induced by MLO-Y4 cells.si RANKL was loaded into ZZF@PVP nanoparticles for basic characterization,and CLSM was used to detect the internalization efficiency of RAW264.7 cells after loading si RANKL into ZZF@PVP.The cytotoxicity of ZZF@PVP nanoparticles at different concentrations on RAW264.7 cells was evaluated by CCK-8 assay.3、A murine air pouch model was constructed,and WB was used to detect RANKL protein content in the pouch tissue after different interventions.The inhibitory effect of si RANKL@ZZF@PVP nanoparticles on RANKL protein synthesis in the air pouch tissues were evaluated.A modified murine calvarial osteolysis model was constructed.The targeting effect of ZZF@PVP nanoparticles on the modeling area was detected by living-imaging using Cy5.5 as a fluorescent tracer.Through Micro-CT scanning and data analysis,TRAP and HE staining of skull slices,the inhibitory effect of si RANKL@ZZF@PVP nanoparticles on osteoclast differentiation and the therapeutic effect on osteolysis were evaluated.Results:1、The ZZF@PVP nanoparticles synthesized by ZOL combined with ZIF-8 found to have a particle size of 40-50 nm by electron microscope scanning,and the hydrated particle size was 142 nm with uniformed dispersion.After two weeks of storaged in aqueous solution,the morphology of the ZZF@PVP nanoparticls was maintained and had good colloidal stability.The results of XRD showed that the ZZF@PVP nanoparticles had an amorphous structure,and FTIR confirmed the successful loading of ZOL in the nanoparticles.Combined with the hollow structure morphology of ZZF@PVP which was significantly different from that of ZIF-8 in transmission electron microscopy,it was confirmed that the addition of ZOL changed the original crystal structure of ZIF-8 rather than simply attaching to the surface.The loading efficiency of DOX and BSA with ZZF@PVP was 6.6wt% and 15.5wt%,respectively.The nanoparticles basically maintained the morphology after loading the two drugs.At the same time,ZZF@PVP inherited the characteristics of ZIF-8nanoparticles,which were relatively stable in neutral conditions but rapidly decomposed and released in acidic environment.2、siRANKL with the best gene silencing efficiency was screened by RT-q PCR.Western blotting verified that it had a significant inhibitory effect on the synthesis of RANKL protein in MLO-Y4 cells.TRAP staining showed that si RANKL inhibited the osteoclastogenesis of RAW264.7 cells induced by MLO-Y4 cells.The ZZF@PVP nanoparticles could effectively loading si RANKL at a high efficency of 0.5OD/mg,and maintained its morphology.Cell transfection assay showed that the encapsulation of si RANKL by ZZF@PVP could effectively transfect it into cells,and the transfection efficiency was similar to that of commercial cationic liposome transfection reagents.CCK-8 assay showed that ZZF@PVP at the concentration of 80μg/ml had no effect on the activity of RAW264.7 cells.3、The results of living fluorescence images showed that ZZF@PVP nanoparticles had a good targeting effect on the murine calvarial osteolysis model,and a high fluorescence signal could still be detected in the model area 48 hours after a single intravenous injection,confirming that ZZF@PVP had a strong binding ability to the osteolysis area.Western blotting verified that si RANKL@ZZF@PVP had a good inhibitory effect on the synthesis of RANKL protein in the air pouch model.In the treatment experiment of murine calvarial osteolysis model,skull TRAP staining showed that the number of TRAP positive cells in the model area of si RANKL@ZZF@PVP group was the least,which proved that it could effectively inhibit the differentiation and activation of osteoclasts in vivo.Micro-CT scanning results showed that the treatment with si RANKL@ZZF@PVP could effectively improve the particle induced osteolysis,the treatment group had a significant improvement in BV/TV and total porosity compared with the control groups.Conclusions:1 、 A novel ZZF@PVP nanoparticles were synthesize by the combination with ZOL and ZIF-8 nanoparticles in aquous solution.The ZZF@PVP with particle size of 40-50 nm,and has good colloidal stability,good drug loading and p H controlled release performance.2、We designed and synthesized si RANKL that can efficiently silence RANKL gene in mice.The ZZF@PVP nanoparticles can effectively encapsulate and increase the cell internalization ablility of si RANKL.3、siRANKL has a inhibitory effect on RANKL protein in mice air pouch model.ZZF@PVP nanoparticles have a good targeting effect on the murine calvarial osteolysis model area.The si RANKL@ZZF@PVP nanoparticles have a good therapeutic effect on the osteolysis model,which can effectively inhibit the differentiation and activation of local osteoclasts and effectively improve bone destruction.