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

Abstract

RNA interference (RNAi) technology can specifically silence the expression of a target gene and has emerged as a promising therapeutic method to treat cancer. In the present study, we showed that natural halloysite nanotube (HNT)-assisted delivery of an active small interfering RNA (siRNA) targeting receptor-interacting protein kinase 4 ( RIPK4 ) efficiently silenced its expression to treat bladder cancer. The HNTs/siRNA complex increased the serum stability of the siRNA, increased its circulation lifetime in blood, and promoted the cellular uptake and tumor accumulation of the siRNA. The siRNA markedly down-regulated RIPK4 expression in bladder cancer cells and bladder tumors, thus inhibiting tumorigenesis and progression in three bladder tumor models (a subcutaneous model, an in situ bladder tumor model, and a lung metastasis model), with no adverse effects. Thus, we revealed a simple but effective method to inhibit bladder cancer using RIPK4 silencing, indicating a promising therapeutic method for bladder cancer.

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.