Mitigating Doxorubicin‐Induced Cardiotoxicity and Enhancing Anti‐Tumor Efficacy with a Metformin‐Integrated Self‐Assembled Nanomedicine

Figures

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  Scheme 1. Design and mechanism of PMDDH. A) Schematic representation of metformin enhancing the cytotoxic effect of Dox on 4T1 cells and mitigating its cardiotoxicity on H9c2 cells. B) Synthesis of PMD, PMDD, and PMDDH. C) Illustration of the targeted delivery, enhanced therapeutic efficacy, and reduced cardiotoxicity of PMDDH in vivo.

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  Figure 1. Cytotoxicity of A) MCF‐7 and B) H9c2 cells treated with Dox and metformin (n = 5). C) Schematic illustration of PMet synthesis. D) The particle size of PMD at varying N/P ratios. E) DNA loading efficiency of PMD assessed by agarose gel electrophoresis. F) Drug loading efficiency (LE%) and encapsulation efficiency (EE%) of PMDDH at different Dox concentrations (n = 3). G) Size distribution of PMD, PMDD, and PMDDH. H) ζ‐potential of PMet, PMD, PMDD, and PMDDH (n = 3). I) TEM micrograph of PMDDH (negative staining with 2% phosphotungstic acid). J) UV–vis spectra of PMet, HA, Dox, PMDD, and PMDDH. K) FTIR spectra of PMet, Dox, HA, PMDH, and PMDDH. L) Dox release profiles of PMDDH under different pH conditions (n = 3). Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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  Figure 2. A) Visualization of cellular uptake of Dox, free FAM‐DNA, and PMDDH by CLSM. Scale bar = 20 µm. B,C) Flow cytometry analysis and corresponding quantitative results of PMDDH uptake in 4T1 cells (n = 3). D) Visualization of CD44 expression (green) and PMDDH uptake (red) in 4T1, MDA‐MB‐231, and MCF‐7 cells. Scale bar = 20 µm. E) Dose‐dependent cytotoxicity of Dox, PMDH, and PMDDH in 4T1 cells (n = 5). F) IC50 values of Dox and PMDDH in 4T1 cells (n = 5). G) Crystal violet staining of treated 4T1 cells showing cell density and distribution changes. H) CRT expression (red) in 4T1 cells post‐treatment. Scale bar = 50 µm. I) Flow cytometry analysis of CRT expression and J) corresponding quantitative results (n = 3). K) HMGB1 levels in the supernatant of 4T1 cells following treatment (n = 3). L) ATP release from 4T1 cells after 24 h of different treatments (n = 3). Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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  Figure 3. A) Schematic illustration of cGAS‐STING and AMPK signaling activation in 4T1 cells. B) Western blot analysis of p‐IRF3 and p‐STING levels in 4T1 cells after different treatments. RT‐qPCR analysis of cytokine and chemokine expression: C) IFN‐β, D) CXCL10, E) OAS1, F) ISG15, and G) TNF‐α in 4T1 cells. H) Western blot results showing p‐AMPK and p‐mTOR levels in 4T1 cells post‐treatment. I) Flow cytometry analysis and J) immunofluorescence imaging of PD‐L1 expression in 4T1 cells following various treatments. Scale bar = 20 µm. Data are presented as mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 4. A) Dose‐dependent cytotoxicity of Dox, PMDH, and PMDDH on H9c2 cells (n = 5). B) Annexin V‐PI flow cytometry results of apoptosis in treated H9c2 cells. C) MitoSOX Red staining showing mitochondrial ROS levels in H9c2 cells after various treatments. Scale bar = 50 µm. D) JC‐1 staining images depicting mitochondrial membrane potential in H9c2 cells. Scale bar = 50 µm. E) Western blot analysis of apoptosis‐related proteins BAX, Bcl‐2, cytochrome C, and Caspase‐3 in H9c2 cells, and F–H) corresponding quantification (n = 3). I) Western blot analysis of AMPK and mTOR, and J,K) corresponding quantification (n = 3). L) Western blot of autophagy markers LC3‐I, LC3‐II, and Beclin‐1, and M,N) corresponding quantification (n = 3). O) Schematic illustration of the cardioprotective mechanisms of PMDDH in H9c2 cells. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 5. A) Pharmacokinetics of free Dox and PMDDH in rats, with inset showing the drug‐time curve of the distribution phase (n = 3). B) In vivo distribution of free Cy5‐DNA compared to Cy5‐labeled PMDDH. C) Ex vivo fluorescence imaging of tumors and major organs in tumor‐bearing mice following intravenous administration of free Cy5‐DNA and PMDDH. D) Schematic of treatment regimen for 4T1 tumor‐bearing mice. E) Tumor growth curves after various treatments over 16 days (n = 5). F) Tumor inhibitory rates and G) tumor weights at day 16 (n = 5). H) Histological evaluation of tumor tissues using H&E staining, Ki67 immunohistochemistry, and TUNEL immunofluorescence. Scale bars = 100 µm for H&E staining; 200 µm for Ki67 immunohistochemistry and TUNEL immunofluorescence. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 6. A) Immunofluorescence staining for p‐STING and p‐AMPK in tumors from different treatment groups. Scale bar = 100 µm. B) Quantification of p‐STING and p‐AMPK fluorescence intensity (n = 3). C) Immunohistochemical staining of PD‐L1 in tumor tissue. Scale bar = 100 µm. D) Western blot analysis of PD‐L1 expression in tumor tissues from various treatment groups (n = 3). E) Flow cytometry analysis of M1 and M2 macrophages, activated dendritic cells (DCs), and cytotoxic T lymphocytes (CTLs) in tumors, with F–I) corresponding quantitative analysis (n = 3). J–M) Levels of cytokines IFN‐β, IL‐1β, IL‐6, and IL‐12 p40 in tumor tissues from treated mice (n = 5). N–Q) Serum concentrations of IFN‐β, IL‐1β, IL‐6, and IL‐12 p40 following different treatments (n = 5). Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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  Figure 7. A) Schematic representation of the in vivo cardioprotection experiment. Histograms of B) ejection fraction (EF) and C) fractional shortening (FS) parameters in mice (n = 3). D) M‐mode echocardiography images of indicated groups. Serum levels of E) CK, F) CK‐MB, G) LDH, and H) cTnT in treated mice (n = 3). I) H&E staining and J) TUNEL staining of myocardial tissue, scale bar = 100 µm. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).