Comparative assessment of antitumor effects between doxorubicin and mitochondria-targeted doxorubicin in combination with radiotherapy

Figures

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  Figure 1. The synthesis process of TPP-DOX.

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  Figure 2. TPP-DOX targets to mitochondria with high accumulation in the cancer cells. (A) 4T1 cytotoxicity investigation of different drug treatment groups (DOX, TPP-DOX); n = 3, (**p < 0.01); (B) 4T1 cytotoxicity of different drug treatment groups (DOX, TPP-DOX) combined with radiotherapy; (n = 3, ns, p > 0.05,); (C) intracellular drug uptake of breast cancer 4T1 cells incubated with DOX or TPP-DOX for 12 h (n = 3, ****p < 0.0001) detected by confocal microscopy; (D) fluorescence image of 4T1 cells stained with Calcein AM/PI (bar = 200 μm); (E) fluorescence intensity qualified by ImageJ in Fig. S5 (n = 3, *p < 0.05, ***p < 0.001); (F) distribution of DOX (green) in mitochondria (red) or nucleus (blue) of 4T1 cells (bar = 40 μm); (G) distribution of TPP-DOX (green) in 4T1 cells (bar = 40 μm); (H) pearson correlation coefficients of colocalization of Mito-Tracker Red CMX ROS and drug fluorescence (n = 3, *p < 0.05).

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  Figure 3. TPP-DOX induces cell apoptosis in 4T1 cells while decreasing mitochondrial membrane and increasing ROS levels. (A) Detection of apoptosis level of breast cancer 4T1 cells treated with DOX or TPP-DOX by flow cytometry; (B) ATP secretion (n = 3, *p < 0.05, ***p < 0.001); (C and D) Semi-quantitative analysis of mitochondrial membrane potential (bar = 50 μm) and green fluorescence intensity of JC-1 monomer in 4T1 cells before and after treatment with DOX or TPP-DOX combined with radiotherapy (n = 3, ns, p > 0.05, ***p < 0.001); (E) mtDNA quantity and quality (n = 3, **p < 0.01); (F and G) Relative production of ROS in 4T1 cells before and after DOX or TPP-DOX combined with radiotherapy (n = 18, **p < 0.01, ***p < 0.001, ****p < 0.0001); (H) transmission electron microscopy (TEM) of mitochondria (bar = 1 µm). The red arrow indicates the magnified area of the image, where the mitochondrial morphology is prominently displayed.

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  Figure 4. Anti-tumor study of different treatment groups in tumor-bearing grafted nude mice. (A) The time of injection and radiotherapy; (B) the daily body weight change curve of mice during the treatment period (n = 6); (C) the curve of the average relative tumor volume of each group over time, V represents the measured volume of tumor, while V₀ indicates the initial measured volume of tumor before the treatment was applied (n = 6); (D) tumor change curves in each treatment group (n = 6); (E) H&E staining evaluation of tumor tissue (bar = 50 μm).

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  Figure 5. CYT C, Ki67, CD31, TUNEL staining image of mouse tumor tissue after treatment (bar = 50 μm).

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  Figure 6. In vivo biosafety evaluation of tumor-bearing grafted nude mice in different treatment groups. (A) H&E staining to evaluate organ toxicity. The arrow points to the necrotic tissue; (B) blood biochemical evaluation of liver function and myocardial function in blood samples of mice after treatment (n = 3). ALT (Alanine transaminase), AST (Aspartate transaminase), LDH (Lactate dehydrogenase), CK (Creatine kinase); (C) blood routine evaluation of blood samples of mice after treatment (n = 3). RBS (Red blood cell), HGB (Hemoglobin), HCT (Hematocrit), MCV (Mean corpuscular volume), MCH (Mean corpuscular hemoglobin), MCHC (Mean corpuscular hemoglobin concentration).