Injectable alginate composite hydrogel with spatiotemporal codelivery of pro-angiogenic and anti-fibrotic agents for synergistic myocardial repair

Fig. 1. Effect of UCL-TRO-1938 on HUVECs under hypoxic conditions. (a) Representative bright-field images (Scale bar: 200 μm) and (c) quantitative analysis of HUVECs migration. (b) Representative images (Scale bar: 200 μm) and (d) quantitative analysis of total tube length in HUVECs tube formation assays. (e) qRT-PCR analysis of mRNA expression levels of angiogenesis-related genes (VEGFA, PECAM1, eNOS, PIK3CA, AKT1, S6KB1) in HUVECs under hypoxia. Data are mean ± SD (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Fig. 2. UCL-TRO-1938 protects HUVECs under hypoxia via PI3K/AKT pathway activation. (a) Volcano plot of DEGs between Hypoxia+1938 and Hypoxia groups (|log2FC| > 1, p < 0.05). (b) Venn diagram showing the overlap between DEGs and AGGs. (c) KEGG pathway enrichment analysis identified 19 significantly enriched pathways. Among them, six were directly associated with angiogenesis, including Ras, PI3K-Akt, TNF, MAPK, HIF-1, and Apelin signaling pathways. (d) GSEA confirmed significant activation of the PI3K-Akt signaling pathway. (e) Western blot analysis showing increased AKT phosphorylation following UCL-TRO-1938 treatment. (f) Quantification of Western blot results shown in (e). (g) Schematic illustration of UCL-TRO-1938 promoting HUVECs migration and tube formation via activation of the PI3K/AKT pathway.

Fig. 3. Synthesis, characterization, and responsive release of EMB NPs. (a) Schematic illustration of EMB nanoparticle fabrication. (b) SEM images (Scale bar: 200 nm), (c) TEM images (Scale bar: 50 nm), (c) Size distribution, and (d) Zeta potential of MSN, MB, EMB NPs. (f) EDS elemental mapping of EMB NPs (Scale bar: 0.2 μm). (g) Cumulative BMP-9 release from EMB NPs under different pH conditions. (h) Schematic of pH-responsive BMP-9 release from EMB NPs. Data are mean ± SD (n = 3).

Fig. 4. In vitro assessment of antifibrotic activity of EMB NPs. (a) Representative EdU staining images of untreated and treated NIH3T3 (scale bar: 200 μm) and (b) Quantitative analysis of EdU+ cells. (c) Scratch assay images showing migration of untreated and treated NIH3T3 over 24 h (scale bar: 500 μm) and (d) Corresponding migration rates. (e) Immunofluorescence images of α-SMA in untreated and treated primary CFs (scale bar: 50 μm) and (f) Quantitative analysis of α-SMA expression. Data are mean ± SD (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Fig. 5. Characterization and therapeutic release behavior of Alg/1938@EMB hydrogels. (a) Schematic workflow for Alg/1938@EMB hydrogel fabrication. (b) Representative SEM images of Alg and Alg/1938@EMB (scale bars: 100 μm; 20 μm for enlarged images). (c) Viscosity profiles of Alg and Alg/1938@EMB hydrogels under varying shear rates. (d) Time-dependent storage (G′) and loss (G″) moduli of Alg and Alg/1938@EMB hydrogels. (e) Proliferation of HUVECs cultured on different hydrogels after different time periods. (f) Cumulative release of UCL-TRO-1938 and BMP-9 from Alg/1938@EMB at pH 5.5. Data are mean ± SD (n = 3).

Fig. 6. Therapeutic effects of Alg/1938@EMB on cardiomyocyte apoptosis and inflammation post-MI. (a) TUNEL staining showing cardiomyocyte apoptosis in infarcted myocardium (day 3 post-MI; scale bar: 100 μm). (b) H&E-stained sections of infarcted myocardium. (day 7 post-MI; scale bar: 50 μm), black arrow indicates the location of immune cells.

Fig. 7. The effects of Alg/1938@EMB on angiogenesis post-MI. (a, b) Representative immunofluorescence images of CD31-stained border zone (a) and infarcted region (b) areas at Day 28 post-MI (scale bar: 50 μm). (c, d) Quantification of vessel density in the corresponding regions. Data are mean ± SD (n = 5; ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Fig. 8. The effects of Alg/1938@EMB hydrogel on infarction size and fibrosis at Day 28 post-MI. (a) Representative Masson's trichrome-stained sections at corresponding levels (scale bar: 2 mm) and Quantification of (b) fibrosis area and (c) LV wall thickness (c). (d) TTC-stained sequential heart sections (scale bar: 2 mm). (e) Infarct size expressed as a percentage of the LV area in hydrogel-treated versus MI control mice, based on TTC staining. # indicates no detectable signal. Data are mean ± SD (n = 5; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Fig. 9. Effect of Alg/1938@EMB hydrogel on the cardiac function evaluated by echocardiography. (a) Representative images of M-mode echocardiography at Day 3, Day 14, and Day 28 post MI; (b, c) Quantitative analysis of (b) EF, (c) FS, (d) LVIDd, and (e) LVIDs at the indicated time points. Data are mean ± SD (n = 5; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Fig. 10. Biocompatibility evaluation of Alg/1938@EMB hydrogel. (a–d) Serum biochemical analysis of liver (ALT, AST) and kidney function markers (BUN, CREA); (e) H&E staining of major organs (scale bar: 100 μm). Data are mean ± SD (n = 5).