Mitochondria-Targeted Mesoporous Titanium Dioxide Nanoplatform for Synergistic Nitric Oxide Gas-Sonodynamic Therapy of Breast Cancer

Figures & data

Scheme 1 Schematic illustration of mitochondria-targeted mesoporous titanium dioxide nanoparticles with L-arginine loading for synergistic nitric oxide gas-sonodynamic therapy.

Figure 1 Characterization of T-mTNPs: (A) TEM images of mTNPs, scale bar=30 nm, (B) DLS analysis of mTNPs, (C) N₂ adsorption/desorption isotherms of mTNPs, (D) Pore size distribution of mTNPs, (E) UV−vis absorption spectra, (F) L-Arg release profiles of T-mTNPs@L-Arg under different pH value, (G) Cumulative NO generation at different pH at the presence or absence of H₂O₂, (H) ROS generation detected by DPBF probe, (I) fluorescence spectrum of ONOO⁻ detected by DHR probe.

Figure 2 Mitochondria targeting and hypoxia relief: (A) Confocal microscope images of MCF-7 cells after incubated with FITC-labeled T-mTNPs@L-Arg and mTNPs@L-Arg and stained with Hoechst 33258 and MitoTracker, scale bar=10 nm (B) FITC fluorescence intensity of isolated mitochondria from MCF-7 cells after incubated with FITC-labeled T-mTNPs@L-Arg and mTNPs@L-Arg. The data are presented as the mean ± S.D. (n = 4). *p < 0.05 versus the mTNPs@L-Arg (C) Activity of Cco after various treatments. The data are presented as the mean ± S.D. (n = 4). *p < 0.05 versus the control groups, ^#p < 0.05 versus mTNPs@L-Arg. (D) Relative dissolved oxygen content in the cell medium of various groups. (E) Hypoxic status of MCF-7 cells detected by hypoxia red detection reagent l, scale bar=10 nm.

Figure 3 In Vitro SDT efficacy of T-mTNPs@L-Arg. (A) The destruction of mitochondria in MCF-7 cells after various treatments detected by CLSM, scale bar=10 nm. Cytotoxicity of different nanoparticles towards MCF-7 cells in the absence (B) or presence (C) of US stimulus. (D) Cell viability of hypoxic MCF-7 cells after treated with different nanoparticles in the presence of US. (E) CLSM images of MCF-7 cells stained with DCFH-DA in various groups, scale bar=10 nm. (F) ROS generation in various groups detected by FACS (“H” represents hypoxia). The data are presented as the mean ± S.D. (n = 4).

Figure 4 In Vivo SDT efficacy of T-mTNPs@L-Arg. (A) Biodistribution of T-mTNPs@L-Arg in MCF-7 tumor-bearing mice after intravenous injection at different time points. (B) Immunofluorescence images of tumors in various treatments on day 6. Tumor hypoxia was detected by Pimonidazole binding of HIF-1α antibody staining, scale bar=50 nm. (C) FACS assay of hypoxic area in tumor tissues. *p < 0.05 versus the control groups, ^#p < 0.05 versus mTNPs@L-Arg. (D) Tumor photographs. (E) Tumor volume. *p < 0.05 versus mTNPs@L-Arg. (F) Tumor weight. *p < 0.05 versus the control groups, #p < 0.05 versus mTNPs@L-Arg. All the data are presented as the mean ± S.D. (n = 4).

Figure 5 Evaluation of the systemic toxicity: (A) body weight. (B–E) blood biochemistry indicators include (B) alanine aminotransferase (ALT), (C) aspartate aminotransferase (AST), (D) blood urea nitrogen (BUN), and (E) creatinine (CRE). Data are presented as the mean ± S.D. (n = 4).

Figure 6 H&E staining images of the heart, liver, spleen, lung, and kidney from mice in various treatments, scale bar=100 nm.