Chrysin-based supramolecular cyclodextrin-calixarene drug delivery system: a novel approach for attenuating cardiac fibrosis in chronic diabetes

Figures

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  FIGURE 1. In vivo experimental design. This figure was created with BioRender.com.

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  FIGURE 2. CHR-based supramolecular drug delivery system preserved H9c2 morphology in high glucose. (A) Representative optic microscope observations of H9c2 cells cultured in normal glucose (NG) for 48 h and exposed for 6 days to the highest dose (2.5 µM) of OTX008 alone or in the different combinations considered, or (B) cultured in high glucose (HG) for 48 h and treated for 6 days with 3 doses (0.75–1.25–2.5 µM) of OTX008 alone or in the different combinations considered. Magnification = ×20; scale bar = 10 µm. N = 3 per group (three independent experiments, each performed in triplicate). NG: 5.5 mM D-glucose; M: NG + 27.5 mM mannitol; DMSO: dimethyl sulfoxide 2.5%; CHR: 5,7-Dihydroxyflavone 0.399 mg/mL; SBECD: Sulfobutylated β-cyclodextrin sodium salt 7.3 m/m%; SBECD + CHR: SBECD + 0.095 mg/mL CHR; OTX: calixarene OTX008—Calixarene 0118—(0.75–1.25–2.5): OTX008 (0.75–1.25–2.5 µM); SBECD + OTX: SBECD-OTX008 (2.5–1.25–0.75 µM); SBECD + OTX + CHR: SBECD-OTX008 (2.5–1.25–0.75 µM)-CHR; HG: 33 mM D-glucose.

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  FIGURE 3. CHR-based supramolecular drug delivery system does not affect TGF-β signaling and MAPKs in H9c2 cardiomyocytes cultured in normal glucose. TGF-β1, TGFβR1, TGFβR2, Erk1 and Erk2 mRNA levels (2^−ΔΔCt ± SD) were determined by qRT-PCR, using GAPDH as gene control. N = 3 per group (three independent experiments, each performed in triplicate). NG: 5.5 mM D-glucose; M: NG + 27.5 mM mannitol; DMSO: dimethyl sulfoxide 2.5%; CHR: 5,7-Dihydroxyflavone 0.399 mg/mL; SBECD: Sulfobutylated β-cyclodextrin sodium salt 7.3 m/m%; SBECD + CHR: SBECD + 0.095 mg/mL CHR; OTX: calixarene OTX008—Calixarene 0118—(2.5 µM); SBECD + OTX: SBECD-OTX008 (2.5 µM); SBECD + OTX + CHR: SBECD-OTX008 (2.5 µM)-CHR.

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  FIGURE 4. CHR-based supramolecular drug delivery system downregulates TGF-β signaling in H9c2 cardiomyocytes exposed to high glucose. TGF-β1, TGFβR1 and TGFβR2 mRNA levels (2^−ΔΔCt ± SD) were determined by qRT-PCR, using GAPDH as gene control. N = 3 per group (three independent experiments, each performed in triplicate). HG: 33 mM D-glucose; M: NG + 27.5 mM mannitol; DMSO: dimethyl sulfoxide 2.5%; CHR: 5,7-Dihydroxyflavone 0.399 mg/mL; SBECD: Sulfobutylated β-cyclodextrin sodium salt 7.3 m/m%; SBECD + CHR: SBECD + 0.095 mg/mL CHR; OTX (0.75–1.25–2.5): calixarene OTX008—Calixarene 0118—(0.75–1.25–2.5 µM); SBECD + OTX (0.75–1.25–2.5): SBECD-OTX008 (0.75–1.25–2.5 µM); SBECD + OTX (0.75–1.25–2.5)+ CHR: SBECD-OTX008 (0.75–1.25–2.5 µM)-CHR. ****p < 0.0001 vs. NG; # p < 0.5, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 vs. HG; ^ p < 0.05 and ^ ^ p < 0.01 vs. OTX; § p < 0.05 vs. SBECD + OTX (same dose).

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  FIGURE 5. CHR-based supramolecular drug delivery system downregulates MAPKs in H9c2 cardiomyocytes exposed to high glucose. Erk1 and Erk2 mRNA levels (2^−ΔΔCt ± SD) were determined by qRT-PCR, using GAPDH as gene control. N = 3 per group (three independent experiments, each performed in triplicate). HG: 33 mM D-glucose; M: NG + 27.5 mM mannitol; DMSO: dimethyl sulfoxide 2.5%; CHR: 5,7-Dihydroxyflavone 0.399 mg/mL; SBECD: Sulfobutylated β-cyclodextrin sodium salt 7.3 m/m%; SBECD + CHR: SBECD + 0.095 mg/mL CHR; OTX (0.75–1.25–2.5): calixarene OTX008—Calixarene 0118—(0.75–1.25–2.5 µM); SBECD + OTX (0.75–1.25–2.5): SBECD-OTX008 (0.75–1.25–2.5 µM); SBECD + OTX (0.75–1.25–2.5)+ CHR: SBECD-OTX008 (0.75–1.25–2.5 µM)-CHR. ****p < 0.0001 vs. NG; # p < 0.5, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 vs. HG; ^p < 0.05 vs. OTX (same dose).

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  FIGURE 6. CHR-based supramolecular drug delivery system downregulates the main pro-fibrotic signaling pathways in cardiac tissues. (A) Immunohistochemical expression of α-SMA, Scale bar: 20 μm; (B) RT-PCR analysis of α-SMA gene expression. (C) RT-PCR analysis of Smad2 gene expression; (D) RT-PCR analysis of Smad3 gene expression; (E) RT-PCR analysis of Smad7 gene expression; (F) Immunohistochemical expression of TGF-β1, Scale bar: 20 μm; (G) RT-PCR analysis of TGF-β1 gene expression. N = 10 mice per group. Control: non-diabetic mice; Diabetes: diabetic mice; SBECD: sulfobutylated β-cyclodextrin; OTX: calixarene OTX008 - Calixarene 0118; CHR: 5,7-Dihydroxyflavone; ***p < 0.001 vs. Control; ### p < 0.001 and ## p < 0.01 vs. Diabetes.

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  FIGURE 7. CHR-based supramolecular drug delivery system suppresses the secretion and deposition of collagen in cardiac tissues. (A) Collagen staining with Gomori’s Trichrome kit; green—collagen deposition; (B) RT-PCR analysis of Col-I gene expression; (C) RT-PCR analysis of Timp-1 gene expression; (D) RT-PCR analysis of MMP1 gene expression; (E) RT-PCR analysis of MMP2 gene expression; (F) RT-PCR analysis of MMP3 gene expression; (G) RT-PCR analysis of MMP9 gene expression. N = 10 mice per group. Control: non-diabetic mice; Diabetes: diabetic mice; SBECD: sulfobutylated β-cyclodextrin; OTX: calixarene OTX008—Calixarene 0118; CHR: 5,7-Dihydroxyflavone; ***p < 0.001 compared to control; # p < 0.05 and ### p < 0.001 vs. Diabetes.

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  FIGURE 8. CHR-based supramolecular drug delivery system reduces Gal-1 in H9c2 cells exposed to high glucose and in cardiac tissues. (A) Gal-1 protein levels (pg/mL ± SD), determined by ELISA, in H9c2 cells cultured in normal glucose (NG) or high glucose (HG) for 48 h and exposed to CHR, SBECD + CHR and SBECD for 6 days in HG; (B) Gal-1 protein levels (pg/mL ± SD) in H9c2 cells cultured in HG for 48 h and treated with OTX008 (0.75–1.25–2.5 µM) alone or combined with SBECD/SBECD + CHR; N = 3 per group (three independent experiments, each performed in triplicate). NG: 5.5 mM D-glucose; HG: 33 mM D-glucose; CHR: 5,7-Dihydroxyflavone 0.399 mg/mL; SBECD: Sulfobutylated β-cyclodextrin sodium salt 7.3 m/m%; SBECD + CHR: SBECD + 0.095 mg/mL CHR; OTX: calixarene OTX008—Calixarene 0118—(0.75–1.25–2.5): OTX008 (0.75–1.25–2.5 µM); SBECD + OTX: SBECD-OTX008 (2.5–1.25–0.75 µM); SBECD + OTX + CHR: SBECD-OTX008 (2.5–1.25–0.75 µM)-CHR; **p < 0.01 vs. NG; ^ ^ ^ p < 0.001 vs. HG; § p < 0.05 vs. OTX; (C) Gal-1 protein levels (pg/mL ± SD) determined by ELISA in cardiac tissues. N = 10 mice per group. Control: non-diabetic mice; Diabetes: diabetic mice; SBECD: sulfobutylated β-cyclodextrin; OTX: calixarene…