Liver-specific Gene Delivery Using Engineered Virus-Like Particles of Hepatitis E Virus

Figures

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  Figure 1. Schematic diagram of stages to create HEV-LPs. (a) Strategy for pMam-Nt-ORF2 construction. (b) Schematic diagram of HEV-LPs generation.

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  Figure 2. Expression of HEV Nt-ORF2 and purification of HEV-LPs in Huh7 cells. (a) Immunofluorescence staining of Nt-ORF2 in Huh7 cells after transduction with Bac-Nt-ORF2. Red fluorescence represents Nt-ORF2 expression. Nuclei were counterstained with DAPI. Scale bar = 20 μm. (b) Formation of HEV-LPs was detected by TEM in Huh7 cells after transduction with Bac-Nt-ORF2. Red arrows denote HEV-LPs in the cytosol of Huh7 cells. Scale bar = 100 nm. (c) Purified HEV-LPs after CsCl-density gradient centrifugation were verified by CBB staining (upper panel) and western blot assay (lower panel). (d) Purified HEV-LPs were observed by TEM. Red arrows denote purified HEV-LPs. Scale bar = 50 nm.

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  Figure 3. Specific penetration of HEV-LPs in liver-derived cell lines. Intracellular penetration of purified HEV-LPs in various mammalian cells was assessed by flow cytometry and confocal microscopy. (a) The percentage of the cells presenting green fluorescence was determined by flow cytometry after treatment with FITC-HEV-LPs. The black line indicates the fluorescence intensity of cells treated with free FITC. The red line indicates the fluorescence intensity of cells treated with FITC-HEV-LPs. (b) FITC-HEV-LPs were observed as green fluorescence in liver-derived cell lines by confocal microscopy. Nuclei were counterstained with DAPI. Scale bar = 20 μm. (c) Z-stack images were collected at 1.19 μm intervals from 0 to 8.33 μm. Green fluorescence represents FITC-HEV-LPs, Nuclei were counterstained with DAPI and red fluorescence represents cell membranes. Scale bar = 10 μm. (d) The percentage of HEV-LPs in Huh7 cells was determined by flow cytometry. The cells were treated with proteinase K. Huh7 cells treated with HEV-LPs with permeabilization and without permeabilization are shown separately. Huh7 cells treated with HEV-LPs were additionally treated with 0.45 M sucrose and 10 mM acetic…

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  Figure 4. Encapsulation of GFP-encoding plasmids. (a) Schematic diagram of the disassembly/reassembly HEV-LPs process. Electron micrographs of HEV-LPs were obtained under different concentrations of cations. Left) Purified HEV-LPs, Middle) Disrupted HEV-LPs, Right) Reassembled HEV-LPs. Red arrows denote HEV-LPs. Scale Bar = 100 nm. (b) DNA gel electrophoresis of plasmids encapsulated by HEV-LPs. GFP-encoding plasmids (lane 1) and GFP-encoding plasmids within GFP-HEV-LPs (lane 2) were detected an agarose gel. GFP-encoding plasmids digested by 5 U and 10 U DNase I after heating GFP-HEV-LPs (lane 3 and 4). HEV-LPs were also co-electrophoresed to confirm the absence of DNA contamination (lane 5). (c) CsCl-density gradient centrifugation of various types HEV-LPs. The total protein in each fraction (400 μl) was determined by a Bradford assay. Black line indicates empty HEV-LPs. Blue line indicates disassembled and reassembled HEV-LPs. And, Red line indicates GFP-HEV-LPs. (d) To prove protection of encapsulated foreign genes, Huh7 cells treated with both GFP-HEV-LPs and different doses of DNase I and then were observed by a confocal microscopy. Green fluorescence indicates expression of GFP.…

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  Figure 5. Foreign gene delivery. (a) Huh7 cells treated with various doses of GFP-HEV-LPs or GFP-encoding plasmids mixed HEV-LPs were observed by confocal microscopy. Green fluorescence indicates expression of GFP. Nuclei were counterstained with DAPI. Scale bar = 20 μm. (b) Expression of GFP-encoding plasmids was confirmed by green fluorescence in a liver-derived cell line, Huh7, by confocal microscopy. Nuclei were counterstained with DAPI. Scale bar = 20 μm. (c) Huh7 cells were treated with empty HEV-LPs, Bax-encoding plasmids mixed with empty HEV-LPs, or Bax-HEV-LPs, or transfected with Bax-encoding plasmids using FuGENE HD. Apoptosis of the cells was quantified by Annexin V-PI/flow cytometry. (d) Expression of apoptotic-related proteins, cleaved-caspase3, and cleaved-PARP after treatment with empty HEV-LPs, HEV-LPs mixed with Bax-encoding plasmids, and Bac-Nt-ORF2 was evaluated by western blot analysis. Also, Huh7 cells transfected with Bax-encoding plasmids were added as a positive control for Bax activity. β-actin was used as an internal control for loading.