Synergistic Therapeutic Effect And Mechanism Of A Stem Cell Membrane-coated Nanoparticles Co-loaded Curcumin And SiSTAT3 In Osteosarcoma

Osteosarcoma is a common primary malignant bone tumor that is highly metastatic and locally invasive,often involving the metaphysis of long bones.Unfortunately,osteosarcoma has poor drug resistance and selectivity to chemotherapy,and in recent decades,chemotherapy has not significantly improved its treatment effect.However,as research on the mechanism of osteosarcoma deepens,a new consensus has formed on the important role of key genes in its occurrence and development,turning the treatment strategy to chemotherapy combined with gene therapy.Curcumin（CUR）,a natural phenolic substance,has demonstrated significant anticancer effects in various tumors such as breast cancer,prostate cancer,osteosarcoma,and lung cancer.Despite this,its hydrophobicity and instability lead to low bioavailability.Meanwhile,signal transducer and activator of transcription 3（STAT3）is continuously activated and highly expressed in a variety of malignant tumor tissues.It has been confirmed that STAT3 can regulate epithelial-mesenchymal transition-related proteins（such as E-cadherin protein,N-cadherin protein,and vimentin protein）to control the migration and invasion of osteosarcoma cells.Nevertheless,the clinical application of si RNA,a commonly used gene silencing tool,is limited due to its poor stability in circulation and easy degradation by endogenous ribonucleic acid.Polydopamine nanoparticles（PDA）are widely used biomaterials that exhibit excellent adhesion,hydrophilicity,stability,and biocompatibility.With diverse morphologies and containing various catechol and amino functional groups,PDA can deliver chemotherapy drugs and unstable si RNAs into solid tumor cells,evade degradation by intracellular lysosomes,and alleviate chemotherapy drug side effects.Moreover,PDA can prolong the drug’s half-life,resulting in significant improvement in its anticancer efficacy.However,low diffusion efficiency of PDA in tumor tissue and clearance by the reticuloendothelial system（RES）often leads to immune rejection.To address these issues,bio-coatings are employed to modify the surface of PDA nanoparticles,thereby improving their biocompatibility and tumor targeting.Human umbilical cord mesenchymal stem cells（h UC-MSCs）possess low immunogenicity,strong targeting ability,and receptor integration,and can migrate to tumor tissues through rolling,capturing,adhering,and crossing epithelial cells.During the extraction process,the stem cell membrane（SCM）retains its low immunogenicity and tumor homing-related surface marker proteins.By camouflaging PDA nanoparticles with the SCM,the resulting nanoparticles combine the advantages and characteristics of both cell membranes and nanoparticles,further enhancing their therapeutic efficacy in tumors.The objective of this study is to develop and synthesize a stem cell membrane-coated PDA nanoparticles co-loaded curcumin and si STAT3 for chemical combined gene therapy in osteosarcoma,induce apoptosis of osteosarcoma cells,and inhibit the progress of its epithelial-mesenchymal transition,offering a more efficient treatment approach for patients with osteosarcoma.This research is divided into the following two parts:（1）Firstly,dopamine hydrochloride was used to self-polymerize into PDA nanoparticles in a weakly alkaline aqueous phase,and CUR and si STAT3 were successfully loaded.Through experimental verification including ultraviolet-visible absorption spectrum,infrared absorption spectrum,and fluorescent microplate detection,it was confirmed that CPDA/^{si STAT3} nanoparticles were successfully synthesized with an average drug loading rate of 9.48±0.73%.Secondly,human umbilical cord mesenchymal stem cells were extracted and stem cell membranes were obtained using ultrasound and extrusion methods,and CPDA/^{si STAT3} nanoparticles were disguised to form CPDA/^{si STAT3}@SCM nanoparticles.The hydrated particle size of CPDA/^{si STAT3}@SCM was detected by transmission electron microscope and Nano Sight to be 117±9 nm.Cell colocalization experiments confirmed that osteosarcoma cells could recognize and phagocytize CPDA/^{si STAT3}@SCM nanoparticles,and its structure remained intact for a period of time after being taken up by cells.The mesenchymal stem cell membrane protein was almost not structurally damaged during the preparation process,and the protein on the surface of the mesenchymal stem cell membrane was retained.CPDA/^{si STAT3}@SCM nanoparticles also demonstrated good p H-dependent cumulative drug release ability,which can effectively increase the circulating drug concentration at the tumor site.In vitro targeting experiments proved that CPDA/^{si STAT3}@SCM nanoparticles retained the tumor homing ability of MSC surface membrane and enhanced the tumor targeting ability of nanoparticles.In vitro safety evaluation demonstrated that CPDA/^{si STAT3}@SCM nanoparticles have immune escape ability,can escape the phagocytosis of macrophages,and have less nanotoxicity,good blood compatibility,and biological safety in blood.（2）We used CCK-8,cloning experiments,live/dead staining,flow cytometry,cell scratch experiments,Transwell experiments,and other methods to evaluate the effects of PBS,free CUR,CPDA,PDA/^{si STAT3},CPDA/^{si STAT3},and CPDA/^{si STAT3}@SCM nanoparticles on the proliferation,apoptosis,migration,and invasion of osteosarcoma MG63 and HOS cells.Results indicated that compared to the other groups,CPDA/^{si STAT3}@SCM nanoparticles significantly inhibited the proliferation,migration,and invasion of osteosarcoma cells while promoting their apoptosis.Moreover,CPDA/^{si STAT3}@SCM nanoparticles successfully transfected STAT3 si RNA into osteosarcoma MG63 and HOS cells with better transfection efficiency than current liposome transfection reagents.Apoptosis-related proteins showed that CPDA/^{si STAT3}@SCM nanoparticles had significant promoting abilities of Bax,Bad,and Cleaved-caspase3 proteins while inhibiting Bcl-2 proteins compared to other groups.EMT-related proteins confirmed that CPDA/^{si STAT3}@SCM nanoparticles induced the down-regulation of the expression of the interstitial-associated marker proteins N-Cadherin and Vimentin and the protein expression of the intercellular matrix metalloproteinase MMP9 in osteosarcoma cells while up-regulating the expression of the epithelial-associated protein E-Cadherin.Thus,CPDA/^{si STAT3}@SCM nanoparticles effectively inhibited the progression of epithelial-mesenchymal transition of osteosarcoma cells.Furthermore,we established a subcutaneous xenograft model of MG63 tumor-bearing mice to evaluate the therapeutic effect of CPDA/^{si STAT3}@SCM nanoparticles in vivo.H&E,Ki67,and TUNEL section staining results showed that the CPDA/^{si STAT3}@SCM nanoparticle group had the largest area of necrotic tissue,significantly inhibited the proliferation of tumor tissue,and promoted the apoptosis of tumor tissue.STAT3 immunohistochemical section results showed that the CPDA/@SCM nanoparticle group had the lowest expression level of STAT3.Biodistribution experiments in mice also showed that CPDA/^{si STAT3}@SCM nanoparticles accumulated the most in tumor sites because SCM camouflage enhances the ability of nanoparticles to target tumor sites.The CPDA/^{si STAT3}@SCM nanoparticle treatment group had the highest expression of BAX protein and the lowest expression of Bcl-2 protein in the tumor tissues of mice,indicating that it can promote the apoptosis process of tumor tissues of osteosarcoma-bearing mice.The expression of E-Cadherin protein was the highest,and the expression of N-Cadherin protein was the lowest in the CPDA/^{si STAT3}@SCM nanoparticle treatment group,suggesting that it inhibits the migration and invasion progression in tumor tissues of osteosarcoma-bearing mice through the EMT signaling pathway.Finally,we conducted H&E staining on the major organ tissues of the mice in each group and verified the relevant serum biochemical indicators.Results showed that the synthetic CUR and STAT3 si RNA co-delivery system CPDA/^{si STAT3}@SCM nanoparticles had good biosafety during the treatment of osteosarcoma tumor-bearing mice.In this study,we successfully prepared polydopamine nanoparticles(CPDA/^{si STAT3}@SCM)loaded with curcumin and STAT3 si RNA,and coated with the membrane of umbilical cord mesenchymal stem cells(CPDA/^{si STAT3}@SCM).A series of characterization tests were performed,confirming that the nanoparticles have good biological safety,high gene knockout efficiency,tumor targeting ability,and immune evasion capability.Furthermore,CPDA/^{si STAT3}@SCM nanoparticles were found to significantly inhibit the proliferation,migration,invasion,and epithelial-mesenchymal transition（EMT）progression of osteosarcoma cells,while promoting their apoptosis.This promising nano drug delivery platform using mesenchymal stem cell membrane-coated polydopamine nanoparticles,loaded with curcumin and STAT3 si RNA,provides a potential treatment option for osteosarcoma patients.