Pleiotropic Multi-Drug Co-Assembled Nanocomposites Offer Protection Against Doxorubicin-Induced Cardiotoxicity

Figures

• 

  Figure 1. Schematic illustration of the preparation and therapeutic mechanism of PGPP/NPs.

• 

  Figure 2. Synthesis and characterization. (A) TEM images of GPP/NPs and PGPP/NPs. Scale bars, 200 nm. (B) The particle size and Zeta potential analysis of GPP/NPs and PGPP/NPs (n=3). (C) The particle size of PGPP/NPs in DMEM supplemented with 10% fetal bovine serum (FBS), PBS, or pure water within 7 days (n = 3). (D) Drug release curve of PI3Kγ inhibitor in PGPP/NPs (n=3). (E) Cell viability evaluation of PGPP/NPs in H9C2 cells (n = 6). (F) Hemolysis assays of GPP/NPs and PGPP/NPs (n=6). Data are presented as the mean ± SD. ***p < 0.001; ****p < 0.0001.

• 

  Figure 3. Cellular uptake assays by H9C2 cells in vitro. (A) CLSM images and (B and C) Flow cytometry analysis for internalization ability among of GPP/NPs and PGPP/NPs at 2 h and 4 h after incubation with H9C2 cells (n=3). (D) CLSM images and (E and F) Flow cytometry analysis for internalization ability of PGPP/NPs at various concentrations after incubation with H9C2 cells (n=3). Data are presented as the mean ± SD. *p < 0.05; **p < 0.01; ****p < 0.0001.

• 

  Figure 4. PGPP/NPs conferred protection to normal cells against DOX-induced cytotoxicity in vitro. (A) CLSM images and (B) Flow cytometry analysis of ROS expression in H9C2 cells after treating with PBS, DOX, GPP + DOX, PGPP + DOX, and PI + GI + PB + DOX (n = 3). Scale bars, 100 μm. (C) LIVE–DEAD cell-staining of H9C2 cells after treating with PBS, DOX, DOX + GPP/NPs, and DOX + PGPP/NPs (Green: Live,Red: Dead). Scale bars, 100 μm. (D) Cell viabilities of H9C2 incubated with Dox at IC50 value after preincubation with increasing concentrations of PGPP/NPs (n = 6). (E) CLSM images and (F) fluorescence quantification of LC3 expression in primary cardiomyocytes after treating with PBS, DOX, DOX + GPP/NPs, and DOX + PGPP/NPs (n = 3). Scale bars, 10 μm. Flow cytometry results of the expression of (G and H) CD86 and (I and J) CD206 in RAW 264.7 cells after treating with PBS, LPS, PGPP/NPs, and LPS + PGPP/NPs (n = 3). Data are presented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

• 

  Figure 5. The blood circulation and biodistribution of GPP/NPs and PGPP/NPs in vivo. (A and B) Ex vivo fluorescence imaging and quantitative analysis of fluorescent intensity of heart after injection of GPP/NPs or PGPP/NPs for 24 h (n = 5). (C and D) Ex vivo fluorescence imaging and quantitative analysis of fluorescent intensity of liver after injection of GPP/NPs or PGPP/NPs for 24 h (n = 5). (E) CLSM images showing the distribution of DiO-labeled GPP/NPs and PGPP/NPs. (F) Relative serum signal of GPP/NPs and PGPP/NPs intravenously administered (n = 3). Data are presented as the mean ± SD. *p < 0.05; ****p < 0.0001.

• 

  Figure 6. PGPP/NPs detoxification of Dox in vivo. (A) Representative M-mode echocardiographic images 21 days after treating with PBS, DOX, GPP + DOX, PGPP + DOX, and PI + GI + PB + DOX. (B) Left ventricular ejection fraction (LVEF) and (C) fractional shortening (FS) 21 days after treating as in (A) (n = 3). The content of (D) cTnI and (E) BNP in serum 21 days after treating as in (A) (n = 3). (F and G) Representative images of H&E staining and quantification of cardiomyocyte area treated as in (A) (n = 3). Scale bars, 20 μm. (H and I) Representative images of TUNEL staining and quantification of TUNEL-positive nuclei per field in whole hearts treated as in (A) (n = 3). Scale bars, 50 μm. (J and K) Representative images of PicroSirius Red staining and relative quantification of collagen deposition in heart sections treated as in (A) (n = 3). Scale bars, 100 μm. Data are presented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

• 

  Figure 7. PGPPNPs alleviate oxidative stress, promote M2 macrophage polarization, and enhance cardiac autophagy. (A) Immunofluorescence staining images and (B) Flow cytometry analysis of ROS expression in cardiac tissue after treating with PBS, DOX, GPP + DOX, PGPP + DOX, and PI + GI + PB + DOX (n = 3). Scale bars, 50 μm. (C) Immunohistochemical staining images and (D and E) Histochemistry score of CD86 (M1) and CD206 (M2) after treating as in (A). (n = 3). Scale bars, 50 μm. (F) Immunofluorescence staining images and (G) relative quantification of LC3 puncta in heart sections from animals treated as in (A) (n = 3). Scale bars, 10 μm. Data are presented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.