| Osteosarcoma is the most common primary malignant bone tumor originating from mesenchymal tissue,and approximately 70-80% of patients are children and adolescents.The current standard treatment protocol for osteosarcoma is surgery combined with multi-course,multi-drug sequential neoadjuvant chemotherapy.Although combined with neoadjuvant chemotherapy can improve the limb salvage rate and survival rate of patients,patients are often forced to undergo amputation due to the emergence of chemotherapy resistance.Finally,patients are deprived of life due to the occurrence of tumor metastases.Ferroptosis is defined as a regulated cell death caused by rupture of the cell plasma membrane due to excessive accumulation of ROS and lipid peroxidation.Because it is morphologically,biochemically,and genetically regulated differently from other forms of cell death such as apoptosis,necrosis,autophagy,and pyroptosis,it offers a new therapeutic option for overcoming apoptosis resistance.The use of nanomaterials to induce ferroptosis has emerged as an emerging anti-tumor strategy.Different from previously reported metallic nanomaterials that induce ferroptosis via the Fenton reaction,we confirmed that arsenene nanosheets,which belong to inorganic nonmetallic nanomaterials,can effectively deplete GSH in vitro.Therefore,we hypothesized that arsenene nanosheets could inhibit the activity of GPX4 by depleting intracellular GSH and thus induce tumor cell ferroptosis.In the present study,we designed and reported an arsenene nanosheet(Her2-ANs@CDDP)with the function of targeting Her2 and delivering cisplatin,which is expected to combine ferroptosis and apoptosis for the treatment of osteosarcoma and enable targeted therapy of osteosarcoma.1.The expression of Her2 in osteosarcoma tissues and osteosarcoma cell lines Object: To verify whether Her2 can be used as a target for osteosarcoma-targeted therapy.Methods: The expression of Her2 in tumor tissue derived from patients with osteosarcoma and in osteosarcoma cell lines was detected by single-cell sequencing,immunohistochemistry,WB,and flow cytometry.Results: Immunohistochemical analysis of samples collected from eight patients with osteosarcoma revealed that six of them had positive Her2 expression in tumor tissues,and single-cell sequencing showed that Her2 was only aberrantly expressed in cell populations of malignant origin.Flow cytometry results showed positive Her2 expression in four of the five human-derived osteosarcoma cell lines.Conclusion: Since Her2 is expressed in a variety of osteosarcoma cell lines and osteosarcoma samples from multiple patients,Her2 can be used as a target for osteosarcoma therapy.2.Synthesis and characterization of arsenene nanosheetsObject: The purpose of this experiment is to verify the successful construction of ANs,Her2-ANs and Her2-ANs@CDDP.Methods: Arsenene nanosheets and modified arsenene nanosheets were characterized using SEM,TEM,AFM,EDS,UV spectrophotometer,Raman spectrometer,and Fourier transform infrared spectrometer.Results: The successful exfoliation of ultrathin nanoscale ANs from the arsenic powder was confirmed by comparing the characterization results of arsenene nanosheets and raw arsenic powder.The successful attachment of the Anti-Her2 affibody to the surface of ANs was confirmed by comparing the characterization results of ANs and Her2-ANs.In addition,EDS mapping showed that platinum elements were uniformly distributed in arsenic-based Her2-ANs@CDDP,which indicated the successful drug loading of arsenene nanosheets.Drug release from Her2-ANs@CDDP can be promoted in a simulated tumor microenvironment(low p H and high GSH concentration).Conclusion:The above results indicate that we successfully prepared Her2-ANs@CDDP.3.Arsenene nanosheets overcome cisplatin resistance by inducing ferroptosis Object: Considering that arsenene nanosheets can effectively scavenge GSH in vitro,we further explored whether arsenene nanosheets taken up by cells can induce ferroptosis and overcome cisplatin resistance in tumor cells.Methods: First,we verified whether Anti-Her2 affibody could increase the uptake of Her2-ANs by Her2-positive tumor cells by laser confocal microscopy,flow cytometry,and ICP-MS.Subsequently,the toxic effects of arsenene nanosheets and modified arsenene nanosheets on tumor cells were compared by CCK-8 assay.Cellular immunofluorescence and flow cytometry was used to detect excessive intracellular accumulation of ROS and lipid peroxides.GPX4 activity assay kit and WB to detect changes in key regulatory molecules of the ferroptosis pathway in cells.Cisplatin-resistant osteosarcoma cells(U2OS/CDDP)were constructed by a stepwise drug screening approach.The cytotoxic effect of Her2-ANs@CDDP on U2OS/CDDP cells was used to verify whether arsenene nanosheets could effectively overcome and reverse cisplatin resistance.Results: The results of the cell uptake assay showed that Her2-ANs were taken up by Her2-positive cells significantly more than ANs at the same dose,and the killing effect of Her2-ANs on Her2-positive tumor cells was significantly higher than that of ANs.The levels of ROS and lipid peroxides were significantly increased in Her2-ANs-treated osteosarcoma cells,however,the activity and expression of GPX4 were significantly decreased.When cisplatin had no killing effect,Her2-ANs and Her2-ANs@CDDP were able to inhibit the cell viability of U2OS/CDDP.Both total Pt and DNA-bound Pt were significantly increased in Her2-ANs@CDDP-treated cells compared to the cisplatin-treated group.Conclusion: The above results suggest that Her2-targeted modification can increase the uptake of nanosheets by Her2-positive tumor cells,resulting in better anti-tumor effects.Mechanism analysis confirmed that arsenene nanosheets could kill drug-resistant tumor cells by inducing ferroptosis and restoring the sensitivity of drug-resistant cells to cisplatin by reducing glutathione-mediated cisplatin efflux.Thus,Her2-ANs@CDDP can promote apoptosis and ferroptosis through a reciprocal cascade reaction between cisplatin and carrier,respectively,and can significantly inhibit the activity of drug-resistant cells.4.In vivo antitumor efficacy and biosafety assessment of arsenene nanosheets Object: Because of the excellent in vitro antitumor activity of arsenene nanosheets,we further evaluated the in vivo antitumor effects as well as the biosafety of arsenene nanosheets in a tumor-bearing mouse model.Methods: Then,30 tumor-bearing mice were divided into PBS,CDDP,ANs,Her2-ANs,and Her2-ANs@CDDP groups,and the body weight and tumor volume of each group were recorded during the 14-day treatment period.Tumor tissue,major organs,and blood from mice were collected at the end of the experiment for histopathological analysis and biosafety assessment.Results:In vivo antitumor assay results showed that the Her2-ANs@CDDP group had the smallest tumor volume and the highest tumor suppression rate among all groups and no significant organ damage or hematological abnormalities were observed in the Her2-ANs@CDDP group.Conclusion: These results suggest that Her2-ANs@CDDP can synergistically inhibit tumor progression through ferroptosis and apoptosis,and has excellent biosafety.In summary,the ingeniously designed arsenic-based nanomaterials are capable of targeting drug delivery and exerting intrinsic anti-tumor activity while enhancing chemotherapy and ferroptosis-mediated therapeutic effects through synergistic effects of drugs and carriers,respectively.Therefore,we expect that cisplatin-loaded arsenene nanosheets,which have the characteristics of simple preparation,exerting synergistic effects,overcoming drug resistance,and excellent biosafety,can be applied to the clinical treatment of chemotherapy-insensitive osteosarcoma,thus also expanding the application of arsenic in the treatment of solid tumors. |
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