Reengineering glycyrrhetinic acid into a therapeutic oligomer for targeted tumor therapy with cardioprotection

Schematic illustration of the fabrication of DOX@PGA-PEG-GA NDs and its role in enhancing the anti-tumor efficacy and cardioprotective function. The therapeutic mechanism of the micellar system involves GA-mediated cellular endocytosis, where subsequent esterase-triggered degradation under acidic pH conditions enables synchronized release of DOX and GA. This dual-drug delivery system demonstrates synergistic antitumor efficacy through complementary pharmacological actions, while the co-released GA effectively attenuates DOX-induced cardiotoxicity by modulating oxidative stress pathways. The integrated process thereby achieves concurrent tumor suppression and cardioprotective intervention through a single administration protocol.

Fig. 1. Schematic illustration of the synthesis of DOX@PGA-PEG-GA NDs and its role in enhancing the anti-tumor efficacy and cardioprotective function

Fig. 2. Characterization of PGA-PEG-GA Copolymer and Its Nanodrugs. (A) 1H-NMR spectra of GA, PGA, and PGA-PEG-GA. (B) FT-IR spectra of GA, PGA, and PGA-PEG-GA. (C) GPC profile of PGA-PEG-GA. (D) CMC of PGA-PEG-GA. (E) SEM images of DOX@PGA-PEG-GA NDs. Scale bar: 100 nm. (F) Fluorescence spectra of DOX, PGA-PEG-GA NDs, and DOX@PGA-PEG-GA NDs (excitation at 480 nm). (G-H) In vitro DOX release from DOX@PGA-PEG-GA NDs at various pH values: G) without esterase and H) with esterase. (I) In vitro hemolysis assay of DOX@PGA-PEG-GA NDs. Data are presented as mean ± SD (n = 3)

Fig. 3. Molecular Dynamics Simulation of PGA-PEG-GA and DOX Interaction. (A-B) Root mean square deviation (RMSD) of PGA-PEG-GA during the (A) first and (B) second simulation periods. (C) Solvent-accessible surface area (SASA) of PGA-PEG-GA over the simulation time. (D) Snapshots of PGA-PEG-GA conformation at different simulation times (0 ns, 20 ns, 50 ns, 80 ns, 100 ns) from various perspectives (Top, Front, Bottom, Side). (E) Number of hydrogen bonds between PGA-PEG-GA and DOX. (F) Coulombic component of binding energy (Coul-SR) between PGA-PEG-GA and DOX. (G) Van der Waals component of binding energy (LJ-SR) between PGA-PEG-GA and DOX

Fig. 4. Targeted Drug Delivery and Biodistribution of Nanodrugs. (A) Confocal microscopy images of HepG2 cells treated with DOX and DOX@PGA-PEG-GA NDs at 1 h, 2 h, and 4 h. The cells were stained with DOX (green), LysoTracker (red), and Hoechst (blue). Scale bars: 10 μm. (B-C) Line scan analysis of the fluorescence intensity of green (DOX) and red (LysoTracker) channels in the confocal images with B) DOX and C) DOX@PGA-PEG-GA NDs. (D) Flow cytometric analysis of DOX fluorescence intensity in HepG2 cells treated with free DOX and DOX@PGA-PEG-GA NDs at 1 h, 2 h, and 4 h. (E) In vivo fluorescence imaging of mice administered with RhB@PGA-PEG NDs and RhB@PGA-PEG-GA NDs at various time points (0 h, 0.5 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h). (F) Time-dependent curve of fluorescence intensity at the tumor site. (G) Ex vivo fluorescence imaging of tumor and major organs (heart, liver, spleen, lung, kidney) from mice treated with RhB@PGA-PEG NDs and RhB@PGA-PEG-GA NDs at 24 h, and 48 h. Data are presented as mean ± SD (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, *** p < 0.001

Fig. 5. In Vitro Evaluation of Cytotoxicity, Apoptosis, and Dead Cell Analysis of Nanodrugs. (A-B) Cell viability of (A) HepG2 and (B) H22 cells treated with different formulations (DOX, DOX@PGA NDs, DOX@PGA-PEG NDs, DOX@PGA-PEG-GA NDs) at concentrations of 0, 0.5, 1, 2, and 4 µg/mL. (C) Cell viability of LX-2 cells treated with different blank nanodrugs (GA, PGA, PGA-PEG, PGA-PEG-GA) at concentrations of 0, 20, 40, 60, and 80 µg/mL. (D) Flow cytometric analysis of apoptosis in HepG2 cells treated with different formulations. The percentages of cells in each quadrant (Q1–Q4) are indicated, representing different apoptotic stages. (E) Quantitative analysis of cell apoptosis rates. (F) Confocal microscopy images of dead cells (red fluorescence) and live cells (green fluorescence) in HepG2 cells treated with different formulations. Scale bars: 100 μm. (G) Quantitative analysis of dead cell percentages. Data are presented as mean ± SD (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, *** p < 0.001

Fig. 6. In Vivo Antitumor Efficacy and Histological Analysis of Nanodrugs. (A) Schematic timeline of the in vivo antitumor efficacy evaluation. H22 tumor-bearing mice were inoculated with tumor cells, and when the tumor volume reached approximately 70 mm³, different formulations (G1: Saline, G2: PGA-PEG-GA NDs, G3: DOX, G4: DOX@PGA NDs, G5: DOX@PGA-PEG NDs, G6: DOX@PGA-PEG-GA NDs) were intravenously administered. The tumor inhibition effect was assessed at day 14. (B) Tumor volume changes over time in different treatment groups (G1-G6). (C) Representative images of excised tumors from different treatment groups. (D) Tumor inhibition rate (%) calculated from the tumor weights of different groups. Data are presented as mean ± SD (n = 5), with statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001. (E) HE staining of different groups of tumor tissues, scale bar: 100 μm. (F) TUNEL staining of different groups of tumor tissues, scale bar: 100 μm. (G) Quantitative analysis of TUNEL fluorescence intensity. (H) DOX fluorescence of different groups of tumor tissues, scale bar: 100 μm. (I) Quantitative analysis of DOX fluorescence intensity. Data were expressed as mean ± SD (n =…

Fig. 7. Protective Effects of GA and Its Nanocomposites on DOX-Induced Cytotoxicity in HCM Cells. (A) Cell viability of HCM cells treated with different concentrations of DOX. (B-C) Cell viability B) and LDH release C) of HCM cells treated with different formulations (G1: Ctrl, G2: GA, G3: DOX, G4: GA + DOX, and G5: DOX@PGA-PEG-GA NDs). (D) Flow cytometric analysis of apoptosis in HCM cells treated with different formulations. The percentage of cells in each quadrant (Q1-Q4) represents different apoptosis stages. (E) Quantitative analysis of apoptosis rates. (F) LCSM images of HCM cells treated with different formulations to observe ROS expression. Scale bar: 100 μm. (G) Quantitative analysis of green fluorescence intensity corresponding. (H) LCMS images of HCM cells treated with different formulations. Cells were stained with JC-1 (red/green) to observe changes in mitochondrial membrane potential. Scale bar: 10 μm. (I) Quantitative analysis of the JC-1 fluorescence ratio (red/green). Data are expressed as mean ± SD (n = 3), and statistical significance is indicated by *p < 0.05, **p < 0.01, *** p < 0.001

Fig. 8. In Vivo Cardiac Safety Evaluation of Nanodrugs. (A) Schematic timeline of the in vivo cardiac safety evaluation. Mice were adaptively fed for 7 days, randomly grouped, and then intravenously administered with different formulations (G1: Saline, G2: PGA-PEG-GA NDs, G3: DOX, G4: DOX@PGA-PEG-GA NDs). Cardiac function was assessed by ECHO at day 20, and cardiac injury was evaluated at day 21. (B) Echocardiographic images of mice from different groups (G1-G4). (C-D) Quantitative analysis of left ventricular fractional shortening (LVFS, C) and left ventricular ejection fraction (LVEF, D) from echocardiographic images. (E) Body weight changes over time in different treatment groups (G1-G4). (F) Images of heart samples from different groups. (G) Heart weight to body weight ratio in different groups. (H) Levels of cardiac injury markers (CK, CK-MB, LDH, LDH1) in different groups. Data are presented as mean ± SD (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, *** p < 0.001