Synergistic Treatment Of Osteosarcoma With Biomimetic Nanoparticles Transporting Doxorubicin And SiRNA

Background:Osteosarcoma is the most common primary malignant bone tumor,mostly growing at the stem end of the long bones,and is more common in adolescents and children,and could only be treated by amputation before the 1970 s,with a low 5-year survival rate after surgery.With the progress and development of modern medicine,nowadays clinical treatment usually takes the form of surgical removal of cancerous tissues and combined chemotherapy with various drugs,however,the prognosis of some patients is not satisfactory because they cannot completely remove the tumor tissues or recur after surgery.Therefore,there are still limitations and challenges in the clinical treatment of osteosarcoma.For chemotherapy of osteosarcoma,the majority of regimens are based on the combination of various drugs such as adriamycin,methotrexate,and cisplatin,but these chemotherapeutic drugs have certain drug toxicity after entering the human body because they cannot target the tumor.In recent years,with the in-depth research on the molecular biology of tumors,small interfering RNA(si RNA)has been used as a novel nucleic acid drug for the treatment of cancer and other diseases,and combining traditional chemotherapeutic drugs with genetic drugs to treat tumors has gained wide attention,however,nucleic acid drugs do not easily penetrate cell membranes.Therefore,a suitable drug delivery platform is needed to carry both drugs and si RNAs to increase drug targeting and stability.To avoid the capture and clearance of nano drugs by the reticuloendothelial system,nanoparticles(DOX/si SUR-PLGA@MSCM NPs)camouflaged by mesenchymal stem cell membranes(MSCM)were synthesized to combine conventional chemotherapy and gene therapy to co-deliver doxorubicin hydrochloride(DOX)and small interfering RNA(si RNA)to promote apoptosis of osteosarcoma cells.Purpose:This study aims to use biomimetic nanoparticles to co-deliver DOX and si RNA targeting the apoptosis inhibitor gene survivin to synergistically treat osteosarcoma,improve the therapeutic effect of chemotherapeutic drugs on osteosarcoma,and reduce drug toxicity.Methods:1.Preparation and characterization of DOX/si SUR-PLGA@MSCM NPs: poly(lactic acid)-hydroxyacetic acid copolymer(PLGA)co-loaded DOX and si RNA-survivin(si SUR)nanoparticles were prepared by complex emulsion volatilization method.Mesenchymal stem cells were isolated,cultured,and identified,and mesenchymal stem cell membranes(MSCM)were extracted to prepare DOX/si SURPLGA@MSCM NPs using the co-extrusion method.the loading and encapsulation rates of DOX and size were examined.The DOX/si SUR-PLGA@MSCM NPs were characterized by transmission electron microscopy(TEM)detection,nanoparticle hydration particle size distribution analysis,zeta potential detection,SDS-PAGE gel electrophoresis by Komas Brilliant Blue staining,laser confocal co-localization experiments,and in vitro release experiments.2.in vitro experiments: fluorescence co-localization assay,cell phagocytosis assay,and flow cytometry assay to assess the uptake of nanoparticles by MG63 cells.CCK-8assay and scratch assay were performed to detect nanoparticles’ in vitro killing effect on tumor cells.Real-time fluorescence quantitative PCR and immunoprotein blotting assay assessed the silencing effect of nanoparticles on target genes.In vitro biosafety of nanoparticles was assessed by CCK-8 assay and hemolysis assay.3.In vivo experiments: MG63 subcutaneous transplantation tumor nude mice model was constructed,tumor-bearing nude mice were treated with nanoparticles,and tumor volume changes were recorded during treatment;the distribution of nanoparticles in the organism was observed by in vivo and in vitro imaging of small animals.The in vivo anti-tumor effect of nanoparticles was evaluated by H&E staining,immunohistochemistry,and TUNEL fluorescence staining experiments.The in vivo biosafety of the nanoparticles was assessed by recording changes in body weight of nude mice during treatment and by H&E staining of major organs of nude mice after treatment.Results:1.Preparation and characterization of DOX/si SUR-PLGA@MSCM NPs:DOX/si SUR-PLGA NPs were successfully prepared by compound emulsion volatilization method,and DOX/si SUR-PLGA@MSCM NPs were successfully synthesized by co-extrusion with MSCM.the particle size distribution of nanoparticles was good,with good stability and dispersion,and the core-shell structure was observed by transmission electron microscopy.shell structure and colocalization experiments can be observed to overlap MSCM and DOX/si SUR-PLGA NPs.The protein structure and properties of MSCM were not significantly changed after the coextrusion of nanoparticles and MSCM by mini-extruder.The results of in vitro release experiments showed that DOX/si SUR-PLGA@MSCM NPs released more drugs at p H 5.0 than at p H 7.4.2.In vitro results: Fluorescence co-localization assay revealed that DOX/si SURPLGA@MSCM NPs were phagocytosed by MG63 cells,and both DOX and si RNA were stably taken up by the cells.The phagocytosis assay showed that tumor cells took up more DOX/si SUR-PLGA@MSCM NPs than DOX/si SUR-PLGA NPs,while macrophages did the opposite.CCK-8 and scratch assays showed that MG63 tumor cells in the DOX/si SUR-PLGA@MSCM NPs group had the most apoptosis and the lowest migration rate.Real-time fluorescence quantitative PCR and immunoprotein blotting assays revealed that DOX/si SUR-PLGA@MSCM NPs significantly decreased survivin gene m RNA and protein expression in MG63 cells.DOX/si SURPLGA@MSCM NPs did not undergo hemolytic reaction.CCK-8 assay confirmed that PLGA and PLGA@MSCM had no significant toxic effects on cells.3.In vivo experimental results: in vivo and in vitro imaging results of small animals showed that MSCM camouflaged nanoparticles had the highest fluorescence intensity at tumor sites.The results of in vivo anti-tumor experiments showed that the DOX/si SUR-PLGA@MSCM NPs group had the smallest tumor volume and weight,the largest tumor necrosis area,the lowest survivin gene expression,and the most apoptotic tumor cells compared with other groups.The body weight of nude mice did not decrease significantly during the in vivo experiments,and there was no significant damage to the major organs of nude mice at the end of the in vivo experiments.Conclusion:Based on the stability,tumor targeting,biocompatibility,and immune escape ability of DOX/si SUR-PLGA@MSCM NPs nanoparticles,combining traditional chemotherapeutic drugs with gene therapy effectively reduces the expression of the survivin gene and the toxic side effects of chemotherapeutic drugs,promotes the apoptosis of osteosarcoma cells through the synergistic effect of two drugs,and provides a new strategy for the treatment of osteosarcoma provide a new strategy for the treatment of osteosarcoma...