Galunisertib-Loaded Gelatin Methacryloyl Hydrogel Microneedle Patch for Cardiac Repair after Myocardial Infarction

Figures

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  Figure 1. Characterization of the galunisertib-loaded GelMA MN patch. (a) Schematic diagram of the fabrication of MN patches. (b) Digital photo of the whole MN patch; scale bar: 4 mm. (c–d) Microscopic images of MNs; scale bars: 600 μm in (c) and 260 μm in (d). (e) SEM image of a single MN; scale bar: 100 μm. (f) Uniformity statistics of MNs. (g) Compressive mechanical performance of MN patches. (h–i) Optical and fluorescence microscopic images of the MN patch loaded with Rhodamine B; scale bars: 300 μm. (j) Fluorescence photographs of the release status of MNs; scale bar: 150 μm. (k–l) Accumulative Rhodamine B release percentage from MNs in the first 12 h (k) and next 15 days (l).

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  Figure 2. MN-Gal patch preserved the cardiac structure and function following MI. (a) Schematic showing the overall animal study design used to test the therapeutic benefits of MN-Gal patch in a rat model of MI. (b) Placement of an MN patch on the rat heart. (c) H&E staining indicates the MN pinholes (black arrow) on the infarcted and peri-infarct zone of the infarcted heart (scale bars: 2 mm, left; 100 μm, right). (d) Survival curve of MN-Gal patch-treated MI rats compared with that of blank MN patch-treated MI rats and only MI rats. (e) Representative M-mode echocardiographic image showing the LV wall motion of the hearts 28 days after MI. Diastolic and systolic cycles were analyzed for each image, and three images for each time point were analyzed per rat. (f–k) LVEF, LVFS, LV internal dimension at end-diastole (LVID; d) and end-systole (LVID; s), and LV volume at end-diastole (LV Vol; d) and end-systole (LV Vol; s) were measured by echocardiography before MI surgery (baseline) and 7, 14, and 28 days (endpoint) after MI. n ≥ 5 animals per group. All data were presented as means ± SD. Comparisons between three groups were performed using two-way ANOVA, followed by Tukey’s multiple…

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  Figure 3. MN-Gal patch decreased infarct scar extension and expansion and protected from myocardial hypertrophy in a rat model of MI. (a–c) HW/BW ratio, HW/BW ratio, and lung wet/dry weight ratio on 28 days after MI. (d) Plasma BNP level at the endpoint. (e) Representative images of WGA staining (scale bars: 50 μm (upper) and 10 μm (lower). (f) CAS shows the relative cardiomyocyte size. n ≥ 5 animals per group. (g) H&E-stained and Sirius red-stained images of the heart tissue were collected at the endpoint (scale bar: 2 mm and 100 μm). (h) Schematic image revealed the definition of BZ and infarcted zone of a rat model of MI. The BZ area was defined as the total fibrotic area minus the infarct zone area in which LV thickness became thinner. (i) Infarction wall thickness, (j) fibrosis area to LV area ratio, and (k) BZ area to LV area ratio were measured from Sirius red-stained image. n ≥ 5 animals per group. All data were presented as means ± SD. Comparisons between three groups were performed using one-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between each group and every other group.

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  Figure 4. MN-Gal patch prevented fibroblast activation by inhibiting TGF-β/Smad2 signaling but had no apparent effect on angiogenesis and inflammation. (a) Representative IF images of activated fibroblasts stained for periostin (green, row 1) and αSMA (red, row 2), total fibroblasts stained for vimentin (red, row 3), macrophages stained for CD68 (green, row 4), and neutrophils stained for Ly6G (green, row 5). The nuclei were stained for DAPI (blue), scale bar: 100 μm. (b)TGF-β, Smad2, phosphorylated Smad2 (p-Smad2), and β-actin protein expression in rat myocardium at the endpoint were examined by western blot. Quantification of the relative TGF-β/β-actin ratio and p-Smad2/Smad2 ratio. (c) Representative IHC images of angiogenesis stained for CD31 [scale bars: 2 mm (upper) and 100 μm (lower)]. (d) Representative IHC images of inflammatory cell infiltration stained for CD11b [scale bars: 2 mm (upper) and 100 μm (lower)]. (e–h) Myocardium cytokine levels of TNF-α, IL-1β, IL-6, and IL-10 were examined by enzyme-linked immune sorbent assay. n ≥ 5 animals per group. All data were presented as means ± SD. Comparisons between three groups were performed using one-way ANOVA, followed by Tukey’s…