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International Journal of Nanomedicine Volume 14, 2019 - Issue
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Original Research

Cationic Antheraea pernyi Silk Fibroin-Modified Adenovirus-Mediated ING4 and IL-24 Dual Gene Coexpression Vector Suppresses the Growth of Hepatoma Carcinoma Cells

Jing Qu1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou215123, People′s Republic of ChinaORCID Iconhttps://orcid.org/0000-0003-4085-8274
ORCID Icon,
Weiwei Wang1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou215123, People′s Republic of China
,
Yanfei Feng1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou215123, People′s Republic of China
,
Longxing Niu1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou215123, People′s Republic of China
,
Mingzhong Li1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou215123, People′s Republic of ChinaCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-3353-9868
ORCID Icon,
Jicheng Yang2 Cell and Molecular Biology Institute, College of Medicine, Soochow University, Suzhou215123, People′s Republic of China
&
Yufeng Xie3 Department of Oncology, First Affiliated Hospital of Soochow University, Suzhou215006, People′s Republic of ChinaCorrespondence[email protected]
 show all
Pages 9745-9761 | Published online: 10 Dec 2019

Figures & data

Figure 1 Identification of the RGD.Ad-ING4-IL-24 vector. (A) Gel electrophoresis of homologous recombinant adenovirus clones: Line 1, pAdTrack-CMV-ING4-polyA+promoter-IL-24 plasmid; Line 2, pAdEasy-1 adenoviral backbone plasmid; Line 3~8, the 6 selected clones. (B) Gel electrophoresis of genes ING4, IL-24 and poly+promoter amplified by PCR: Line 1, ING4 cDNA fragments amplified by PCR; Line 2, negative control group compared to ING4 cDNA fragments amplified by PCR; Line 3, polyA+promoter cDNA fragments amplified by PCR; Line 4, negative control group compared to polyA+promoter cDNA fragments amplified by PCR; Line 5, IL-24 cDNA fragments amplified by PCR; Line 6, negative control group compared to IL-24 cDNA fragments amplified by PCR; M, DL2000 marker. (C) The ING4 expression in QBI-293A cells analyzed by Western blotting. (D) The IL-24 secretion from QBI-293A cells detected by ELISA. Statistically significant in comparison with PBS, *p<0.05.

Abbreviations: RGD, Arg-Gly-Asp; Ad, adenovirus; ING4, inhibitor of growth 4; IL-24, interleukin-24; CMV, cytomegalovirus; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

Figure 1 Identification of the RGD.Ad-ING4-IL-24 vector. (A) Gel electrophoresis of homologous recombinant adenovirus clones: Line 1, pAdTrack-CMV-ING4-polyA+promoter-IL-24 plasmid; Line 2, pAdEasy-1 adenoviral backbone plasmid; Line 3~8, the 6 selected clones. (B) Gel electrophoresis of genes ING4, IL-24 and poly+promoter amplified by PCR: Line 1, ING4 cDNA fragments amplified by PCR; Line 2, negative control group compared to ING4 cDNA fragments amplified by PCR; Line 3, polyA+promoter cDNA fragments amplified by PCR; Line 4, negative control group compared to polyA+promoter cDNA fragments amplified by PCR; Line 5, IL-24 cDNA fragments amplified by PCR; Line 6, negative control group compared to IL-24 cDNA fragments amplified by PCR; M, DL2000 marker. (C) The ING4 expression in QBI-293A cells analyzed by Western blotting. (D) The IL-24 secretion from QBI-293A cells detected by ELISA. Statistically significant in comparison with PBS, *p<0.05.Abbreviations: RGD, Arg-Gly-Asp; Ad, adenovirus; ING4, inhibitor of growth 4; IL-24, interleukin-24; CMV, cytomegalovirus; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

Figure 2 Synthesis and characteristics of CASF. (A) Schematic diagram of CASF synthesis. (B) Zeta potential of CASF at PEI/ASF weight ratios of 0%, 0.1%, 0.5%, 1%, 2%, 4%, 6% and 8%. (C) 1H-NMR spectra of PEI, ASF and CASF in D2O.

Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; PEI, polyethylenimine; ASF, Antheraea pernyi silk fibroin; NMR, nuclear magnetic resonance.

Figure 2 Synthesis and characteristics of CASF. (A) Schematic diagram of CASF synthesis. (B) Zeta potential of CASF at PEI/ASF weight ratios of 0%, 0.1%, 0.5%, 1%, 2%, 4%, 6% and 8%. (C) 1H-NMR spectra of PEI, ASF and CASF in D2O.Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; PEI, polyethylenimine; ASF, Antheraea pernyi silk fibroin; NMR, nuclear magnetic resonance.

Figure 3 The laser confocal fluorescence images of SMMC-7721 and L-02 cells infected with Ad at MOIs of 0, 5, 10, 20, 50, 100, 150 and 200, respectively. (A) Fluorescence field and (B) bright-field images of SMMC-7721 cells; (C) fluorescence field and (D) bright-field images of L-02 cells. Scale bar: 100 μm.

Abbreviations: Ad, adenovirus; MOI, multiplicity of infection.

Figure 3 The laser confocal fluorescence images of SMMC-7721 and L-02 cells infected with Ad at MOIs of 0, 5, 10, 20, 50, 100, 150 and 200, respectively. (A) Fluorescence field and (B) bright-field images of SMMC-7721 cells; (C) fluorescence field and (D) bright-field images of L-02 cells. Scale bar: 100 μm.Abbreviations: Ad, adenovirus; MOI, multiplicity of infection.

Figure 4 Preparation schematic diagram and stability of the CASF/Ad complex. (A) Schematic diagram of the preparation of CASF/Ad complex. (B) Scanning electron microscopy images, (C) particle diameter and (D) zeta potential of the CASF/Ad complex formed by coating Ad with CASF at a concentration of 50 μg/mL at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h after the preparation. Scale bar: 2.00 μm.

Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 4 Preparation schematic diagram and stability of the CASF/Ad complex. (A) Schematic diagram of the preparation of CASF/Ad complex. (B) Scanning electron microscopy images, (C) particle diameter and (D) zeta potential of the CASF/Ad complex formed by coating Ad with CASF at a concentration of 50 μg/mL at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h after the preparation. Scale bar: 2.00 μm.Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 5 Characteristics of the CASF/Ad complex. (A) Scanning electron microscopy images, (B) particle diameter and (C) zeta potential of the Ad and the CASF/Ad complex. Scale bar: 2.00 μm. CASF 10 to CASF 150 represented the CASF/Ad complexes were formed by coating Ad with CASF at concentrations of 10, 50, 100 and 150 μg/mL, respectively.

Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 5 Characteristics of the CASF/Ad complex. (A) Scanning electron microscopy images, (B) particle diameter and (C) zeta potential of the Ad and the CASF/Ad complex. Scale bar: 2.00 μm. CASF 10 to CASF 150 represented the CASF/Ad complexes were formed by coating Ad with CASF at concentrations of 10, 50, 100 and 150 μg/mL, respectively.Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 6 The laser confocal fluorescence images of SMMC-7721 and L-02 cells cultured with DMEM, Ad and CASF/Ad complex for 24 h. (A) Fluorescence field and (B) bright-field images of SMMC-7721 cells; (C) fluorescence field and (D) bright-field images of L-02 cells. Scale bar: 100 μm. CASF 10 to CASF 150 represented cells were infected with the CASF/Ad complexes formed by coating Ad with CASF at concentrations of 10, 50, 100 and 150 μg/mL, respectively.

Abbreviations: DMEM, Dulbecco’s modified Eagle medium; CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 6 The laser confocal fluorescence images of SMMC-7721 and L-02 cells cultured with DMEM, Ad and CASF/Ad complex for 24 h. (A) Fluorescence field and (B) bright-field images of SMMC-7721 cells; (C) fluorescence field and (D) bright-field images of L-02 cells. Scale bar: 100 μm. CASF 10 to CASF 150 represented cells were infected with the CASF/Ad complexes formed by coating Ad with CASF at concentrations of 10, 50, 100 and 150 μg/mL, respectively.Abbreviations: DMEM, Dulbecco’s modified Eagle medium; CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 7 The infection efficiency of cells infected with Ad and CASF/Ad complex for 24 h. The flow cytometry images of (A) SMMC-7721 and (B) L-02 cells, and the corresponding histogram of infection efficiency of (C) SMMC-7721 and (D) L-02 cells. CASF 10 to CASF 150 represented cells were infected with the CASF/Ad complexes formed by coating Ad with CASF at concentrations of 10, 50, 100 and 150 μg/mL, respectively. Statistically significant in comparison with naked Ad, **p<0.01.

Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 7 The infection efficiency of cells infected with Ad and CASF/Ad complex for 24 h. The flow cytometry images of (A) SMMC-7721 and (B) L-02 cells, and the corresponding histogram of infection efficiency of (C) SMMC-7721 and (D) L-02 cells. CASF 10 to CASF 150 represented cells were infected with the CASF/Ad complexes formed by coating Ad with CASF at concentrations of 10, 50, 100 and 150 μg/mL, respectively. Statistically significant in comparison with naked Ad, **p<0.01.Abbreviations: CASF, cationic Antheraea pernyi silk fibroin; Ad, adenovirus.

Figure 8 Target gene expression and cell apoptosis. SMMC-7721 cells were cultured with DMEM, Ad and CASF/Ad complex for 48 h. (A) The ING4 expression in SMMC-7721 cells analyzed by Western blotting. (B) The IL-24 secretion from SMMC-7721 cells detected by ELISA. (C) The flow cytometry images and (D) the corresponding histogram of apoptosis rates of SMMC-7721 cells. Statistically significant in comparison of CASF/Ad with Ad, DMEM and Ad with DMEM, *p<0.05, **p<0.01.

Abbreviations: DMEM, Dulbecco’s modified Eagle medium; Ad, adenovirus; CASF, cationic Antheraea pernyi silk fibroin; ING4, inhibitor of growth 4; IL-24, interleukin-24; ELISA, enzyme-linked immunosorbent assay.

Figure 8 Target gene expression and cell apoptosis. SMMC-7721 cells were cultured with DMEM, Ad and CASF/Ad complex for 48 h. (A) The ING4 expression in SMMC-7721 cells analyzed by Western blotting. (B) The IL-24 secretion from SMMC-7721 cells detected by ELISA. (C) The flow cytometry images and (D) the corresponding histogram of apoptosis rates of SMMC-7721 cells. Statistically significant in comparison of CASF/Ad with Ad, DMEM and Ad with DMEM, *p<0.05, **p<0.01.Abbreviations: DMEM, Dulbecco’s modified Eagle medium; Ad, adenovirus; CASF, cationic Antheraea pernyi silk fibroin; ING4, inhibitor of growth 4; IL-24, interleukin-24; ELISA, enzyme-linked immunosorbent assay.

Figure 9 Proliferation of SMMC-7721 and L-02 cells. The cell viability of (A) SMMC-7721 and (B) L-02 cells cultured with DMEM, Ad and CASF/Ad complex for 1, 3, 5 and 7 days. Statistically significant in comparison of CASF/Ad with Ad, DMEM and Ad with DMEM, *p<0.05.

Abbreviations: DMEM, Dulbecco’s modified Eagle medium; Ad, adenovirus; CASF, cationic Antheraea pernyi silk fibroin.

Figure 9 Proliferation of SMMC-7721 and L-02 cells. The cell viability of (A) SMMC-7721 and (B) L-02 cells cultured with DMEM, Ad and CASF/Ad complex for 1, 3, 5 and 7 days. Statistically significant in comparison of CASF/Ad with Ad, DMEM and Ad with DMEM, *p<0.05.Abbreviations: DMEM, Dulbecco’s modified Eagle medium; Ad, adenovirus; CASF, cationic Antheraea pernyi silk fibroin.

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