Modified exosomes for targeted delivery of doxorubicin and carvedilol to mitochondria in breast cancer cells

Figures

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  Fig. 1. Synergy assessment using CI and DRI indexes The Chou-Talalay method was applied to evaluate drug synergy in MCF-7 and MDA-MB-231 cells. The CI-Dose plots (a) show the synergistic interaction (CI < 1) between DOX and CAR across different doses for 1:1 and 2:1 combination ratio, with lower CI values indicating stronger synergy. The logarithmic DRI plots for 1:1 (b) and 2:1 (c) drug ratios demonstrate the extent to which drug doses can be reduced while maintaining efficacy.

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  Fig. 2. Intracellular distribution of DOX in free form and exosomal formulations Intracellular distribution of free DOX (a), Exo-DOX (b), and TPP-Exo-DOX (c) after 8 h incubation with MCF-7 and MDA-MB-231 cells. Green fluorescence indicates mitochondria stained with MitoTracker Green, red fluorescence represents DOX emission, and yellow spots in the merged images demonstrate co-localization. The enhanced yellow signal in TPP-Exo-DOX treated cells confirms successful mitochondrial targeting of the TPP-conjugated formulation.

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  Fig. 3. Cell viability of MCF-7 and MDA-MB-231 cells after 48 h treatment (a) Cell viability after treatment with blank carriers and CAR alone. (b) Cell viability of various DOX containing formulations. Results are shown as mean ± SD (n = 3). Statistical significance is indicated by: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Fig. 4. Effects of different formulations on ROS production and MMP in cell lines MCF-7 and MDA-MB-231 (a) ROS level is measured by DCF fluorescence intensity, where increased fluorescence indicates elevated ROS production. (b) Mitochondrial function is assessed by Rh123 fluorescence, where decreased fluorescence represents mitochondrial depolarization. CAR induced ROS production and mitochondrial dysfunction when delivered as free DOX-CAR and Exo-DOX-CAR, while the TPP-Exo-DOX-CAR formulation demonstrated protective effects on mitochondrial integrity. Results are shown as mean ± SD (n = 3). Statistical significance is indicated by: *P < 0.05, **P < 0.01.

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  Fig. 5. Flow cytometric apoptosis analysis of MCF-7 and MDA-MB-231 cell after 48 h treatment Representative flow cytometry dot plots showing Annexin V-FITC/PI staining of cell lines after treatment with DOX-CAR containing formulations. Quadrant analysis identifies viable cells (lower left), early apoptotic cells (lower right), late apoptotic cells (upper right), and necrotic cells (upper left).

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  Fig. 6. Wound healing assay illustrates the migration potential of MCF-7 and MDA-MB-231 cells after 24 h treatment Representative microscopic images (40x magnification) showing the wounded area 24 h after treatment with various formulations.