Construction Of Multifunctional Nanocarrier DSF-CuO₂@DMSNs And Verification Of Its Antibacterial And Antitumor Effects

Objective:Based on the property of the Fenton-like reaction that can release reactive oxygen species（ROS）,Copper Peroxide Nanoparticles（Cu O₂）and Disulfiram（DSF）are loaded onto Dendritic Mesoporous Silica Microspheres（DMSN）,constructing a multifunctional nanomaterial,DSF-Cu O₂@DMSN,activated by sonodynamic power.The effectiveness of this material in promoting wound healing and antibacterial action,and treating malignant peripheral nerve sheath tumors,is then validated.Methods:Dendritic mesoporous silica microspheres（DMSN）were synthesized and used as carriers to co-load copper oxide（Cu O₂）and disulfiram（DSF）to form DSF-Cu O₂@DMSN.The morphology of the materials was evaluated using transmission electron microscopy and scanning electron microscopy.X-ray photoelectron spectroscopy was used to analyze the characteristic valence states of the elements,and energy-dispersive X-ray spectroscopy was used to obtain mapping spectra of different elements.The production of in vitro ROS hydroxyl radicals（·OH）and singlet oxygen（¹O₂）were detected by UV-visible absorption spectrometry and electron spin resonance spectrometry.The antibacterial and wound-healing effects of the materials in vitro were evaluated by colony-forming experiments and scratch experiments.A mouse model of skin wound infection caused by Staphylococcus aureus was established,and the wound size and mouse weight were measured and recorded the day after treatment with DSF-Cu O₂@DMSN under ultrasound guidance.The wound healing area chart and growth curve chart were plotted,and the wound and surrounding healing tissues were collected after euthanizing the mice for H&E staining,Masson staining,and CD31 immunohistochemical staining to analyze the in vivo antibacterial and wound-healing effects of the materials.The MPNST cell lines S462TY and STS26T were cultured,and the DSF-Cu O₂@DMSN were used for treatment under ultrasound treatment.The cell viability was detected by CCK-8,and flow cytometry was used to detect cell apoptosis and necrosis.Western blotting was used to evaluate the protein expression levels of Caspase-3 and Cl.caspase-3 in the cell experiment.The production of ROS was detected by DCFH-DA fluorescence staining,and the number of living and death cells was detected by living/dead（AM/PI）cell double staining.TUNEL staining was used to detect cell apoptosis,and confocal laser scanning microscopy was used to collect images for fluorescence staining analysis.Finally,high-throughput sequencing was used to detect the differential expression of RNA in STS26T cells and for bioinformatics analysis.Results:1.A composite nanomaterial was constructed,and the size of Cu O₂ nanoparticles was determined to be approximately 14.27±12.17 nm by transmission electron microscopy.Different binding energies of Cu,O,N,C,and Si were confirmed by X-ray photoelectron spectroscopy,and the element mapping spectra of O,Si,N,Cu,and S were confirmed by X-ray energy dispersive spectroscopy,which indicated the random distribution of Cu on the surface of DMSN mesoporous materials.The results of UV-visible spectroscopy showed that high concentrations of hydroxyl radicals（·OH）and singlet oxygen（¹O₂）can be produced under weak acid conditions by US irradiation.Electron spin resonance spectroscopy showed that high concentrations of·OH and ¹O₂ signals were detected in DSF-Cu O₂@DMSN under US irradiation at p H 5.5.The UV-visible-near-infrared spectroscopy results showed that two obvious absorption peaks appeared at approximately 260 nm and 400 nm in the DSF-Cu O₂@DMSN group under US irradiation,confirming the generation of Cu（DETC）^II.2.The results of colony formation experiments showed that the bacterial survival rates of DSF-Cu O₂@DMSN in Staphylococcus aureus and Escherichia coli were 3.26%±4.31%and2.74%±1.98%,respectively.The results of cell scratch experiments showed that the healing rate reached 92.14%±2.89%after 24 hours.The animal wound healing effect map and healing area statistics chart showed that the relative wound area was only 3.90%±2.98%on the 11th day after treatment.H&E staining,Masson staining,and CD31 immunohistochemical staining of mice wound tissues suggested that DSF-Cu O₂@DMSN had a good promoting effect on wound healing.3.The CCK-8 experiment results showed that the cell viability of STS26T was 57.24%±2.66%,and that of S462TY was 67.65%±4.46%after treatment with the multifunctional nanocarrier DSF-Cu O₂@DMSN under ultrasound guidance,indicating a significant inhibition of the activity of S462TY and STS26T cell lines by DSF-Cu O₂@DMSN.Flow cytometry results showed that the cell necrosis rate and apoptosis rate of STS26T were 23.34%±5.51%and 24.54%±5.61%,respectively,while those of S462TY were 12.37%±1.91%and 58.59%±1.03%,respectively,indicating a significant increase in the ratio of cell apoptosis and cell necrosis.Western blotting results showed an increase in the expression levels of Caspase-3 and Cl.caspase-3 apoptosis-related proteins.Confocal laser scanning microscopy（CLSM）showed an increase in ROS production and dead cell ratio in both S462TY and STS26T cell lines,and an increase in both types of cell apoptosis.RNA sequencing results of STS26T tumor cells showed 1918 upregulated genes and 4748 downregulated genes.Bioinformatics analysis results suggested that the differentially expressed genes were associated with growth inhibition,autophagy,and apoptosis-related processes.4.In the animal experiment,a nude mouse MPNST tumor model was successfully constructed.After treatment with the multifunctional nanocarrier DSF-Cu O₂@DMSN under ultrasound guidance,the tumor tissue volume decreased significantly to approximately 304.29±101.94 mm³,and the tumor weight was 0.15±0.06 g,indicating a significant inhibition of tumor growth.The growth curve of nude mouse body weight was slowly increased,and there was no significant difference among the groups（P>0.05）.H&E staining of the heart,liver,spleen,lung,and kidney tissues showed no significant differences among the groups,indicating that the multifunctional nanocarrier DSF-Cu O₂@DMSN had good biocompatibility.Histological analysis of the tumor tissue showed a decrease in Ki67-positive signals,and an increase in Caspase 3,ROS,and TUNEL staining positive signals,indicating that DSF-Cu O₂@DMSN could increase ROS production and induce tumor cell necrosis and apoptosis.RNA sequencing results of the tumor tissue showed 1172 upregulated differentially expressed genes and 188 downregulated differentially expressed genes,and bioinformatics analysis showed that the autophagy and apoptosis signaling pathways were activated.Conclusion:1.The multifunctional nanocarrier DSF-Cu O₂@DMSN was successfully constructed.2.In vivo and in vitro experiments confirmed the antibacterial and wound healing effects of the multifunctional nanocarrier DSF-Cu O₂@DMSN.3.Cell experiments confirmed the good biocompatibility and antitumor effects of the multifunctional nanocarrier DSF-Cu O₂@DMSN.4.Animal experiments confirmed that the multifunctional nanocarrier DSF-Cu O₂@DMSN is a safe and effective sonosensitizer with good biocompatibility and antitumor effects.