Research ArticleHEALTH AND MEDICINE

Microneedle-mediated gene delivery for the treatment of ischemic myocardial disease

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Science Advances  17 Jun 2020:
Vol. 6, no. 25, eaaz3621
DOI: 10.1126/sciadv.aaz3621
  • Fig. 1 Schematic showing the overall study design using MN-AAV.

    (A) Ischemic hearts were administered MN-AAV with the assistance of a customized apparatus. The MNs swelled following application; consequently, the therapeutic agents were burst-released into precise regions to ameliorate cardiac dysfunction through angiogenic effects. (B) Diagram of our practice for the application of the MNs to rat heart via endoscopy assisted microthoracotomy surgery. (C) A series of endoscopic images demonstrating the application of MNs for delivery of therapeutic agents to a rat heart. Scale bars, 600 μm. Photo credits: Hongpeng Shi, Department of Cardiac Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

  • Fig. 2 Properties of MNs.

    (A) SEM images of MNs. (B) Representative stress-strain curves between the group of MNs with AAV (MN-AAV) or MNs without AAV. The histograms represent the comparison test of the two groups. n = 4 patches in each group. (C) Transitions between the dried and swollen states of the MNs. The histograms show the fold changes in MN volume between the dried and swollen stages (n = 8 MN tips, randomly selected from three patches). Photo credits: Hongpeng Shi, Department of Cardiac Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. (D) Release kinetics of MN-AAV. (E) Fluorescent images (scale bars, 500 μm) and magnified images (scale bars, 250 μm) indicating MNs surface-coated with FITC-AAV (green) particles and MNs without loading of FITC-AAV. (F) The three-dimensional (3D) construction images of MN-FITC-AAV. All data are reported as the means ± SD. NS, not significant.

  • Fig. 3 Gene transfection and expression of MN-AAV in vitro.

    (A) Schematic of the cell culture experimental procedures performed to investigate the cell infectivity of released AAV. (B) Representative fluorescent images of GFP-positive cells in the MN-AAV-GFP group captured under a confocal microscope. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Qualification and comparison of GFP-positive cells between normal 293 cells and AAV-GFP transfected cells as detected by flow cytometry. SSC-A, side-scatter area; FSC-A, forward-scatter area. (D) Representative images of crystal violet–stained migratory human umbilical vein endothelial cells (HUVECs) on the porous membranes of Transwell inserts among the three groups and histograms of the numbers of migrated cells. Five random fields were selected for the statistical analysis. All data are reported as the means ± SD. **P < 0.01 and ****P < 0.0001.

  • Fig. 4 Successful AAV delivery and gene transfection of MN-AAV in vivo.

    (A) Schematic illustrating the study design, involving the MN application in this section. (B) Confirmed insertion of methylene blue–loaded MNs into the myocardium. The black arrow denotes an area of methylene blue–stained myocardium. Photo credits: Hongpeng Shi, Department of Cardiac Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. (C) Representative fluorescent and hematoxylin and eosin (HE) images of LV walls that received DI of FITC-AAV and MN-FITC-AAV. The LV wall was cryosectioned horizontally (n = 3 animals per group; scale bars, 500 μm) or transversely (scale bars, 400 μm) for the MN-FITC-AAV–treated hearts. The dashed line denotes the shape of MN-FITC-AAV following application. (D) Representative echocardiographic images and left ventricular function parameters between the MN and NC groups. The data are presented as the means ± SD; n = 3 animals per group. (E) Representative images of bioluminescence (n = 5 animals per group) and Western blot (WB) assay results (n = 3 animals per group) 4 weeks following MN application. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Representative fluorescence micrographs showing the spatial distribution of GFP-positive cells (green) in the MN-AAV-GFP and DI-AAV-GFP groups at day 28. Cardiomyocytes were identified by anti-cTnT (cardiac troponin T) antibodies (red); nuclei were stained with DAPI (blue). n = 5 animals per group. Scale bars, 200 μm. Separated and merged distribution data of fluorescent signals between the MN-AAV-GFP and DI-AAV-GFP groups are presented. All data are reported as the means ± SD.

  • Fig. 5 AAV-VEGF ameliorated cardiac dysfunction.

    (A) Schematic illustrating the study design involving MN-AAV-VEGF application and improvement of injured heart function. (B) Representative echocardiographic images of the experimental groups 4 weeks following MN application. Left ventricular function parameters (EF, FS, LVIDs, and LVIDd) and absolute changes in heart function (ΔEF and ΔFS) were also measured and compared among the three groups. n = 6 animals per group. (C) Representative Masson’s trichrome–stained myocardial sections 4 weeks after MN-AAV-VEGF application. The scar areas and infarct sizes were quantified on the basis of Masson’s trichrome–stained images. Scale bars, 1 mm. (D) Identification of collagens via picrosirius red staining among the three groups. Scale bars, 1 mm. Representative polarized light images of the picrosirius red–stained sections were subjected to polarized light microscopy. Scale bars, 100 μm. Histograms showing the comparisons of collagen content and the type I/type III collagen ratios among the three groups. Right: Representative fluorescence micrographs identifying type I collagen (green) and type III collagen (red); the nuclei were stained with DAPI (blue). n = 3 animals per group. Scale bars, 500 μm. All data are reported as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Neovascularization and activation of signals by MN-AAV-VEGF.

    (A) Representative immunofluorescent images of vWF (green) and αSMA (red) in the tissues of the infarction and border region showing increased vessel density in the MN-AAV-VEGF group compared with those in the other two groups. n = 3 animals per group. Scale bars, 50 μm. Vessels are indicated by white triangles. (B) Quantification of capillary density, arterial density, and the mature index among the three groups in the infarction and border regions. (C) VEGF levels were detected by enzyme-linked immunosorbent assay (ELISA) in serum from the MI + MN-VEGF and MI groups. n = 3 animals per group. (D) Representative WB results for VEGF, VEGF receptor (VEGFR), phosphoinositide 3-kinase (PI3K), Akt, phosphorylated Akt (p-Akt), and caspase-9 in heart homogenates from the MI + MN-VEGF and MI groups. n = 3 animals per group. The bar graphs show the quantified protein levels. All data are reported as the means ± SD. *P < 0.05 and ****P < 0.0001.

Supplementary Materials

  • Supplementary Materials

    Microneedle-mediated gene delivery for the treatment of ischemic myocardial disease

    Hongpeng Shi, Tong Xue, Yang Yang, Chenyu Jiang, Shixing Huang, Qi Yang, Dong Lei, Zhengwei You, Tuo Jin, Fei Wu, Qiang Zhao, Xiaofeng Ye

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