Matrix Metalloproteinase‐Responsive Controlled Release of Self‐Assembly Nanoparticles Accelerates Heart Valve Regeneration In Situ by Orchestrating Immunomodulation

Figures

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  Figure 1. Preparation and Characterization of miR‐93‐PEG‐HSA NPs. A,B) Immunofluorescence staining of the BMDMs. A), (CD68; red), (iNOS; green); B), (CD68; red), (Arg‐1; green). Scale bars = 50 µm. C) Representative graphs of CD206+ M2 macrophages. n = 3. D) Schematic illustration of miR‐93 5p mimics loaded PEG‐HSA nanoparticles prepared using self‐assembly method. E) Representative image of agarose gel electrophoresis of free or wrapped miR‐93 5p mimics. F) Representative SEM image of miR‐93@HSA NPs. Scale bars = 2 µm G) Size distribution of nanoparticles. H) Zeta potential distribution of nanoparticles. I) Representative graphs of CD206+ M2 macrophages. Scale bars = 20 µm. n = 3. J) Immunofluorescence staining of the BMDMs. (iNOS; red), (Arg‐1; green). Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. Construction of the immune‐modulating scaffold. A) Schematic picture of preparing the immune‐modulating scaffolds. B) SEM shows the representative picture of the hydrogels of different oxidation degree. Scale bars = 100 µm. C) General view of the different scaffolds. D) SEM showing the topology of the surface and the cross‐section in different scaffolds. Scale bars = 1 µm. E) MMP‐response release of nanoparticles encapsulated in scaffolds detected by a multimode microplate reader. n = 3. Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001. DVS: decellularized valve scaffolds; miR‐93@HSA@DVS: miR‐93@HSA nanoparticles coated DVS; HS: hydrogel coated DVS; miR‐93@HSA@HS: hydrogel and miR‐93@HSA nanoparticles coated DVS.

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  Figure 3. In vitro hemocompatibility of the immune‐modulating scaffold. A) Photographs of thrombogenesis on different scaffolds after incubating with blood. B) Hemolysis rates of the scaffolds. C) Platelet adhesion of the scaffolds observed by SEM. D) Quantification of the thrombus on the scaffolds. n = 5. E) Quantification of hemolysis rates of the scaffolds. n = 3. F) Quantification of the platelet on the scaffolds. n = 4. Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001. DVS: decellularized valve scaffolds; miR‐93@HSA@DVS: miR‐93@HSA nanoparticles coated DVS; HS: hydrogel coated DVS; miR‐93@HSA@HS: hydrogel and miR‐93@HSA nanoparticles coated DVS; GAV: glutaraldehyde crosslinked aortic valve.

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  Figure 4. In vitro endocytocompatibility and macrophage reprogramming of the immune‐modulating scaffold. A) Live/dead cell staining of scaffolds seeded with HUVECs after 3 days. B) Live/dead cell staining of scaffolds seeded with HUVECs after 7 days. (live; green), (dead; red). Scale bars = 100 µm. C) Immunofluorescence staining of the BMDMs seeded in different scaffolds. (iNOS; red), (CD206; green). Scale bars = 100 µm. DVS: decellularized valve scaffolds; miR‐93@HSA@DVS: miR‐93@HSA nanoparticles coated DVS; HS: hydrogel coated DVS; miR‐93@HSA@HS: hydrogel and miR‐93@HSA nanoparticles coated DVS; GAV: glutaraldehyde crosslinked aortic valve.

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  Figure 5. In vivo histocompatibility and immune remodeling after subcutaneous implantation. A) Immunofluorescence staining of CD3+ T cells in scaffolds after 7‐days, 14‐days, and 28‐days implantation. B) Immunofluorescence staining of CD68+ macrophages in scaffolds after 7‐days, 14‐days, and 28‐days implantation. Scale bars = 50 µm. C) Immunofluorescence staining of macrophage subtypes in scaffolds after 7‐days, 14‐days, and 28‐days implantation. iNOS (red) stain for M1 macrophages; CD206 (green) stain for M2 macrophages. DVS: decellularized valve scaffolds; miR‐93@HSA@HS: hydrogel and miR‐93@HSA nanoparticles coated DVS; GAV: glutaraldehyde crosslinked aortic valve.

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  Figure 6. Constructive remodeling of the immune‐modulating scaffold after abdominal aorta implantation. A) Schematic illustration showing the procedure of scaffolds duct‐abdominal aorta heterotopic transplantation. B) General views and ultrasound pictures of the scaffolds after 7‐days, 14‐days, and 28‐days implantation in the rat abdominal aorta. C) Representative histological images of the scaffolds after 7‐days, 14‐days, and 28‐days transplantation. H&E stain indicating cell infiltration of the scaffolds. Masson stain indicating collagen degradation and remodeling. Scale bars = 20 µm. DVS: decellularized valve scaffolds; miR‐93@HSA@HS: hydrogel and miR‐93@HSA nanoparticles coated DVS; GAV: glutaraldehyde crosslinked aortic valve.

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  Figure 7. Dynamic evolution of immune microenvironment and endothelialization after abdominal aorta implantation. A) Immunofluorescence staining of macrophages in scaffolds after 7‐days, 14‐days, and 28‐days implantation in the rat abdominal aorta. Scale bars = 50 µm. B) Immunofluorescence staining of macrophage subtypes in scaffolds after 7‐days, 14‐days, and 28‐days implantation in the rat abdominal aorta. iNOS (red) stain for M1 macrophages; CD206 (green) stain for M2 macrophages. Scale bars = 50 µm. C) Immunofluorescence staining of CD31+ endothelial cell in scaffolds after 7‐days, 14‐days, and 28‐days implantation in the rat abdominal aorta. Scale bars = 50 µm. DVS: decellularized valve scaffolds; miR‐93@HSA@HS: hydrogel and miR‐93@HSA nanoparticles coated DVS; GAV: glutaraldehyde crosslinked aortic valve.

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  Figure 8. Mechanism of immune‐modulating scaffold accelerating endothelialization. A) The schematic illustration showing the co‐culture of BMDMs and HUVECs. B) HUVECs proliferation detected with CCK‐8. n = 6. C,D) The migration of HUVECs was detected with transwell assay. n = 3. E–G) RNA‐sequence results of scaffolds after 14‐days implantation in the rat abdominal aorta. E) Heatmap of the top twenty differentially expressed genes, sorted by log fold change. F) Gene ontology terms enrich the highly expressed genes of the DVS group versus miR‐93@HSA@HS groups. G) KEGG Pathway enriches group DVS versus group miR‐93@HSA@HS highly expressed genes. H,I) The protein expression level of α7nAChR, ERK, AKT, IκBα, p65. J) HUVECs proliferation detected with CCK‐8. K,L) The migration of HUVECs detected with transwell assay. Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.