Multifunctional microneedle patch loaded with microfluidic-synthesized hybrid gas-nanozyme for myocardial infarction treatment

Figures

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  Fig. 1. Schematic illustration of preparation and therapeutic mechanism of Gas-nanozyme MN patches. (A) Preparation of hybrid gas-nanozyme via a microfluidic method. (B) MN patches sustainably release H2S to reduce oxidative stress and regulate inflammation for MI treatment

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  Fig. 2. Characteristics of ZnSBS hybrid gas-nanozyme. (A) TEM image of ZnSBS nanoparticles. Scale bar: 20 nm for (I), 200 nm for (II). (B) Average size and (C) Zeta potential of ZnSBS and ZnSB. (D) X-ray diffraction pattern of ZnSBS nanoparticles. (E) XPS spectra related to the valence state of Zn in ZnSBS nanoparticles. (F) EDS element mapping of ZnSBS nanoparticles. Scale bar: 50 nm. (G) EDS patterns of different elements in ZnSBS nanoparticles. (H) Release profile of H2S from ZnSBS in solutions with different pH of 7.4, 6.5, and 6.0. (I) Relative enzyme activity of pure SOD and ZnSBS (n = 3, mean ± SD)

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  Fig. 3. Antioxidant effects of ZnSBS hybrid gas-nanozyme. (A) UV-Vis spectra of superoxide radical elimination. (B) UV-Vis spectra of hydroxyl radical elimination. (C) UV-Vis spectra of ABTS elimination. (D) UV-Vis spectra of DPPH elimination. (E) UV-Vis spectra of DPPH elimination with different concentrations of ZnSBS. (F) UV-Vis spectra of ABTS elimination with different concentrations of ZnSBS

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  Fig. 4. In vitro antioxidant effects of ZnSBS in H9C2 cells. (A) Cell viability of H9C2 cells with different treatments (pH 6.0, n = 3, mean ± SD). (B) Intracellular green fluorescence imaging and (C) fluorescence intensity (MFI) of H2S in H9C2 after incubated for 4 h, determining by fluorescence probe of WSP-5. (D) Cell viability of H2O2-induced oxidative injury in H9C2 cells after various treatments. (E) Intracellular fluorescence imaging of ROS (green) and (F) intracellular fluorescence intensity of DCFH-DA probes. Scale bar: 100 μm. (G) Quantification analysis of LPO levels and (H) MDA levels. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001)

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  Fig. 5. In vitro anti-inflammatory effects of ZnSBS in macrophages. (A) Cell viability of iBMDM cells with different treatments (pH 6.0, n = 3, mean ± SD). (B-D) Polarization of iBMDM cells treated with different nanoparticles, evaluating by expression of CD206 (M2) by flow cytometry. (E) Immunofluorescence staining of CD68, iNOS, and CD206 in iBMDM cells with LPS induction and different treatments. Scale bar: 50 μm. (F-H) Quantification analysis of expression of CD68, iNOS and CD206. (I-K) The expression levels of TNF-α, IL-1β and IL-6 in macrophages treated with different nanoparticles after LPS induction. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001)

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  Fig. 6. Characterization of microneedle patches. (A) Original morphology of MN patches and SEM images of MN patches. Scale bar: 5 mm for (I), 1 mm for (II) and 200 μm for (III). (B-C) Mechanical analysis of MN patches. (D) Photographs of the degradation MN patches in heart on days 0, 3 and 7

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  Fig. 7. Implantation of anisotropic patches after MI and their therapeutic effects on cardiac function recovery. (A) Infarct area in the left ventricle after cardiovascular ligation and postoperative image of myocardial patch implantation to the infarcted myocardium region. (B) Electrocardiographic manifestations before LAD ligation. (C) Bow-back-like elevation of the ST segment on the lead II ECG after LAD ligation (shown by the black arrow). (D) Representative echocardiograms of rats from different treatment groups after 28 days. (E-J) Cardiac function indexes including EF, FS, LVIDd, LVIDs, LV vol; d, and LV vol; s measured in rats from different treatment groups after 28 days. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (n = 6, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001)

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  Fig. 8. Therapeutic efficacy of ZnSBS on cardiac infarction area in rat MI model by regulating inflammation and oxidative stress. (A) Masson’s trichrome staining for cardiac sections in different treatments. Blue: fibrosis tissue; red: myocardium. Scale bar: 2 mm and 500 μm. (B-D) Quantitative analysis of fibrosis area, infarction size and LV wall thickness. (E) Immunofluorescence staining results of TUNEL apoptotic cells at 28 days postoperatively. (F) Immunofluorescence staining of CD163-positive cells 28 days after surgery. (G-K) Levels of TNF-α, IL-1β, IL-6, MDA and LPO in heart tissue homogenates of rats from different treatment groups after 28 days. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (n = 6, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, Scale bar: 50 μm)

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  Fig. 9. Angiogenesis activity of ZnSBS gas-nanozyme on cardiac infarction area in MI Rat. (A-E) Representative immunofluorescence images and quantitative analysis of heart tissues co-stained with vWF, CD31, α-SMA, α-actin, Cx43 and DAPI in 28 days after treated with different MN patches. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (n = 6, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, Scale bar: 50 μm)