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Delivery of RIPK4 small interfering RNA for bladder cancer therapy using natural halloysite nanotubes

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Science Advances  25 Sep 2019:
Vol. 5, no. 9, eaaw6499
DOI: 10.1126/sciadv.aaw6499
  • Fig. 1 Characterization of the siRNA-loaded HNTs.

    (A) Transmission electron microscopy images of HNTs and HNTs/siRNA. (B) Schematic diagram of HNTs combined with siRNA. (C) siRNA cumulative release from HNTs/siRNA under neutral conditions. (D) Zeta potential of HNTs and Al2O3.

  • Fig. 2 The transfection efficiency and distribution of HNT-delivered siRIPK4 in T24 bladder cancer cells.

    (A) Bright-field and fluorescence microscopy images of HNTs/FAM-siRIPK4 fluorescence at 6 hours after transfection. (B) Flow cytometry analysis of fluorescent cells: representative histograms (upper panels) and means ± SD (lower panels, from three independent experiments). FAM-siRIPK4, fluorescein-labeled siRNA targeting RIPK4. (C) Confocal laser scanning microscopy analysis of the distribution of HNTs/FAM-siRIPK4 in T24 bladder cancer. Fluorescein (green)–labeled siRIPK4. DAPI (4′,6-diamidino-2-phenylindole; blue) was used to stain cell nuclei.

  • Fig. 3 Tumor transfection in vivo and inhibition of tumor growth by HNTs/siRIPK4.

    (A) In vivo imaging of T24 xenograft–bearing mice after intratumoral injection of HNTs/FAM-siRIPK4, free FAM-siRIPK4, HNTs, or PBS (n = 3 mice in each group). (B) In vivo imaging of T24 xenograft–bearing mice after tail vein injection of HNTs/FAM-siRIPK4, free FAM-siRIPK4, HNTs, or PBS (n = 3 mice in each group). (C) Timeline for the assessment of the antitumor activities of the HNTs/siRIPK4 complexes in the in vivo subcutaneous xenograft model. (D) Images of the tumors dissected from tumor-bearing mice receiving various treatments (n = 5 mice in each group). (E) Relative tumor volume in BALB/c nude mice with HNTs/siRIPK4 xenografts over time. (F) Weight of tumors dissected from mice after treatment. ***P < 0.001.

  • Fig. 4 In vivo siRIPK4 delivered by HNTs inhibits tumor promotion of T24 bladder cancer cells in an in situ model of bladder cancer (n = 12 rats in each group).

    (A) Representative excised bladders and H&E-stained tissue slices (muscle-invasive bladder cancer: ≥stage pT2) treated with PBS. (B) Representative excised bladders and H&E-stained tissue slices (muscle-invasive bladder cancer: ≥stage pT2) treated with siRIPK4. (C) Representative excised bladders and H&E-stained tissue slices (muscle-invasive bladder cancer: ≥stage pT2) treated with HNTs. (D) Representative excised bladders and H&E-stained tissue slices (noninvasive papillary carcinoma: stage pTa) treated with HNTs/siRIPK4. (Photo credit: Jianye Liu and Ke Cao, The Third Xiangya Hospital of Central South University).

  • Fig. 5 In vivo inhibits tumor metastasis and toxicity of HNTs/siRIPK4 by tail vein injection of HNTs/siRIPK4 (n = 5 mice in each group).

    (A) In vivo siRIPK4 delivered by HNTs inhibits tumor metastasis in the tail vein injection of the lung metastasis model. N, normal lung; M, lung metastatic tumor. Separated lungs from mice receiving PBS, siRIPK4, HNTs, or HNTs/siRIPK4 were stained with H&E (left). Quantitative assessment of pulmonary metastatic nodules in the T24 tumor–bearing mice (right). **P < 0.01. (B) Twenty-four hours after the final intravenous injection of PBS, siRIPK4, HNTs, and HNTs/siRIPK4, H&E staining of tissue sections for histopathological analysis.

  • Fig. 6 Delivery of HNTs/siRIPK4 complexes for RIPK4 silencing and bladder cancer therapy.

    The HNTs/siRIPK4 complex protects the RIPK4 siRNA from serum degradation by nucleases and clearance through the kidneys, and promotes RIPK4 siRNA accumulation in tumor cells, thereby silencing RIPK4 in bladder tumors. Down-regulated RIPK4 expression inhibits bladder cancer proliferation and progression.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw6499/DC1

    Fig. S1. Efficiency and cytotoxicity of the siRNA binding to HNTs.

    Fig. S2. Lysosomal escape of HNTs/siRIPK4 in T24 bladder cancer cells.

    Fig. S3. Intracellular trafficking and lysosomal escape of HNTs/siRIPK4.

    Fig. S4. qRT-PCR of RIPK4 mRNA and Western blotting analysis of the RIPK4 protein in T24 cells after HNTs/siRIPK4 transfection.

    Fig. S5. HNTs/siRIPK4 inhibited the growth of T24 cells in vitro by MTT assay and LDH assay.

    Fig. S6. HNTs/siRIPK4 inhibited the growth of T24 cells in vitro (colony formation).

    Fig. S7. Annexin V and propidium iodide staining of T24 cells treated with HNTs/siRIPK4 complexes and other formulations, and analysis of apoptosis using flow cytometry.

    Fig. S8. After HNT-mediated RIPK4 knockdown in T24 cells, FACS was used to analyze the cell cycle.

    Fig. S9. Tumor tissues analyzed using Western blotting from the subcutaneous xenograft model.

    Fig. S10. Tumor tissues analyzed using IHC from the subcutaneous xenograft model.

    Fig. S11. HNTs/siRIPK4 reduced Ki-67 expression and induced apoptosis of mice bearing T24 xenografts.

    Fig. S12. Immunotoxicity analysis of blood of mice at 24 hours after PBS, siRIPK4, HNTs, and HNTs/siRIPK4 injection.

    Table S1. Tumor suppression effect of different treatment on bladder weight and histopathologic changes in SD rat bladders of different groups.

    Table S2. Biochemical parameters of blood from the mice at 24 h after last injection of various drugs.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Efficiency and cytotoxicity of the siRNA binding to HNTs.
    • Fig. S2. Lysosomal escape of HNTs/siRIPK4 in T24 bladder cancer cells.
    • Fig. S3. Intracellular trafficking and lysosomal escape of HNTs/siRIPK4.
    • Fig. S4. qRT-PCR of RIPK4 mRNA and Western blotting analysis of the RIPK4 protein in T24 cells after HNTs/siRIPK4 transfection.
    • Fig. S5. HNTs/siRIPK4 inhibited the growth of T24 cells in vitro by MTT assay and LDH assay.
    • Fig. S6. HNTs/siRIPK4 inhibited the growth of T24 cells in vitro (colony formation).
    • Fig. S7. Annexin V and propidium iodide staining of T24 cells treated with HNTs/siRIPK4 complexes and other formulations, and analysis of apoptosis using flow cytometry.
    • Fig. S8. After HNT-mediated RIPK4 knockdown in T24 cells, FACS was used to analyze the cell cycle.
    • Fig. S9. Tumor tissues analyzed using Western blotting from the subcutaneous xenograft model.
    • Fig. S10. Tumor tissues analyzed using IHC from the subcutaneous xenograft model.
    • Fig. S11. HNTs/siRIPK4 reduced Ki-67 expression and induced apoptosis of mice bearing T24 xenografts.
    • Fig. S12. Immunotoxicity analysis of blood of mice at 24 hours after PBS, siRIPK4, HNTs, and HNTs/siRIPK4 injection.
    • Table S1. Tumor suppression effect of different treatment on bladder weight and histopathologic changes in SD rat bladders of different groups.
    • Table S2. Biochemical parameters of blood from the mice at 24 h after last injection of various drugs.

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