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Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome

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Science Advances  22 Jul 2020:
Vol. 6, no. 30, eaba5379
DOI: 10.1126/sciadv.aba5379
  • Fig. 1 Physicochemical characterization of liposomal formulation.

    (A) CP20 cells were transfected with difference concentrations of CBS-siRNA or control siRNA using HF transfection reagent for 48 hours; CBS silencing was determined by Western blot (WB). (B) Liposome screening: CP20 were treated with CBS-siRNA-LPs (F1-F10) or control (CTL)–siRNA-LPs (F0) for 48 hours; CBS silencing was determined by WB. (C) Schematic representation of siRNA-AuroLPs. (D and E) The size and charge of liposomal formulation with/without CBS-siRNA were measured by Zetasizer. (F) Entrapment of CBS-siRNA into AuroLPs. (G) AuNP content in cLPs analyzed by INAA. (H) Gene silencing efficacy by AuroLPs having various CBS-siRNA/20-nm AuNP ratio (w/w); silencing was determined by WB 48 hours after treatment. (I) Silencing efficacy by AuroLPs. Cells were treated with different groups for 48 hours; CBS silencing was determined by WB. Data are represented as means ± SD, *P < 0.05, n = 3, with Student’s t test. ImageJ was used for intensity quantitation of CBS protein, normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control.

  • Fig. 2 Effect of nanoparticle size, shape, and core material in gene silencing efficacy AuroLPs.

    (A to C) WB demonstrating effect on MICU1 protein expression by treatment with nanoparticles of different sizes, shapes, and core materials containing MICU1-siRNA entrapped in traditional liposome or along with inorganic nanoparticles. (C) MICU1-siRNA-20 nm Fe3O4LPs effect on MICU1 protein expression as observed by WB. (D) Analysis of MICU1 silencing at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR) via the treatment of cells with CTL-siRNA-AuroLPs, 20-nm AuNP, CTL-siRNA-HF, MICU1-siRNA-AuroLPs, MICU1-siRNA-cLPs, complex (1) (25 nM) or complex (2) (100 nM), and MICU1-siRNA-20 nm AuNP. (E) WB analysis demonstrating silencing efficacy of MICU1-siRNA in CTL-siRNA-HF complex (100 nM), complex (1) and complex-2 or MICU1-siRNA-cLPs, MICU1-siRNA-AuroLPs, MICU1-siRNA-Lipofectamine 3000, and MICU1-siRNA-RNAiMax complex (25 nM) by treating the cells for 72 hours. (F) Stability of MICU1-siRNA, MICU1-siRNA-cLPs, MICU1-siRNA-AuroLPs, MICU1-siRNA-HF complex, and MICU1-siRNA-20 nm AuNP for 96 hours by electrophoresis. (G) Inhibition of clonal growth by treatment with CTL-siRNA-AuroLPs, 20-nm AuNP, CTL-siRNA complex, MICU1-siRNA-AuroLPs, MICU1-siRNA-cLPs, complex (1) or complex (2), and conjugate (25 nM) for 12-day through crystal violet staining. Data were expressed as means ± SD. ImageJ was used for quantitation of MICU1 protein, normalized by GAPDH as loading control.

  • Fig. 3 Tumor accumulation of AuroLPs and its effect on tumor growth.

    (A) Quantified fluorescence intensity (Fl. Int.) of accumulated nanoparticles from images and lysates of tumor-bearing mice (n = 4) after intravenous injection of Cy5-siRNA-cLPs and Cy5-siRNA-AuroLPs at 24 hours in terms of normalized Fl. Int. au, arbitrary units. (B) Percent injected dose (%ID) per gram tumor as measured above. (C to K) Reduction in tumor growth by intravenous injections of AuroLPs and cLPs containing MICU1-siRNA or CTL-siRNA-AuroLPs through the 12-day treatment period. Tumor size (C), representative images of tumor (D), tumor mass (E), MICU1 expression at protein and mRNA level with GAPDH as control (F and G), images and its quantification of MICU1 immunostained tumor (H), Ki67-stained (left) and TUNEL-stained (right) sections of tumors (I), the quantification of Ki67-stained proliferating cells (top), and TUNEL-stained apoptotic cells (bottom) (J) (n = 6). H&E-stained sections of tissues showing hepatitis in liver for MICU1-siRNA-cLPs (arrow head) (K). Scale bar, 50 μm. Data are expressed as means ± SD and were analyzed by using Student’s t test (A and B) and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. a.u, arbitrary unit. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.000. ns, not significant. Photo credit: M. N. Hossen (OUHSC).

  • Fig. 4 Antitumor efficacy of MICU1-siRNA-AuroLPs PDX.

    (A) MICU1 protein expression in PDX models of primary high-grade serous ovarian cancer. MICU1 expression in different PDX tumors, compared to normal OSE and CP20 cells (top). Histopathological analysis of human ovarian tumor tissues by using H&E to confirm its tumorigenic characteristics (bottom). Scale bar, 50 μm. (B to E) Assessment of antitumor efficacy of auroLPs in PDX mice. PDX-098 was subcutaneously transplanted into NOD/SCID background mice (n = 80). Tumor-bearing PDX model mice (tumor size, 100 mm3) were intravenously injected with cLPs and AuroLPs containing MICU1-siRNA (0.2 mg/kg/thrice weekly) or in combination with intraperitoneal injection of cisplatin (0.5 mg/kg/twice a week). The treatment was continued for 35 days. The tumor volume (tv) was measured weekly (B), and 35-day tv was shown separately (C). Representative tumor images (D) and tumor masses (E) were shown. (F) MICU1 expression in tumor lysates at 35 days with GAPDH as loading control. (G) The representative Ki67-, TUNEL-, H&E-, and Sirius red–stained sections of corresponding tumors. All statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, n = 5 to 10. Photo credit: M. N. Hossen (OUHSC).

  • Fig. 5 Mechanisms of enhanced silencing efficacy of auroliposomal formulation of siRNA.

    (A) Evaluation of cellular uptake mechanisms of AuroLPs by pretreatment of OV90 cells with several chemical inhibitors for 2 hours followed by a further 4-hour incubation with Cy5-siRNA-cLPs and Cy5-siRNA-AuroLPs at 37°C. Data were represented as % uptake, means ± SD, n = 3. (B to E) Inhibition of cellular uptake of AuroLPs by CAV1 silencing. WB analysis showing expression of CAV1 after treatment of OV90 with CAV1-siRNA + RNAiMAX at 72 hours (B). The qualitative and quantitative uptake of Cy5-siRNA-AuroLPs and Cy5-siRNA-cLPs in CAV1 knockdown cells (OV90-CAV1KD) and OV90 (C to E). Scale bars, 10 μm. (F) WB of MICU1 protein in OV90 and OV90-CAV1KD after transfection with MICU1-siRNA-cLPs and MICU1-siRNA-AuroLPs for 72 hours. (G and H) Study of endosomal and lysosomal escape for Cy5-siRNA-cLPs, Cy5-siRNA-AuroLPs, and treated/nontreated cells for 12 and 24 hours. The endosomes (green), Cy5-siRNA (red), and nuclei (blue) were visualized under a fluorescence microscope (G). Observation of lysosomal colocalization (yellow) of above treatments at 24 hours (H). Scale bars, 10 and 5 μm. (I) Measurement of protein phosphatase 2A (PP2A) enzymes activity using immunoprecipitation with and/or without treating cells with CTL-siRNA-cLPs and CTL-siRNA-AuroLPs for 15 min. The statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparisons test, means ± SD, *P < 0.05, n = 3.

  • Fig. 6 Graphical illustration explaining enhanced gene silencing and antitumor activity of AuroLPs.

    (A) TEM micrographs of cLPs and auroliposomes (AuroLPs) stained with 0.2% uranyl acetate. Scale bar, 100 nm. (B) siRNA-cLPs was internalized into cancer cell using multiple pathways, including macropinocytosis, clathrin-, and caveolae-mediated endocytosis, whereas because of the addition of small amount of 20-nm AuNP into cLPs, the internalization route for AuroLPs was switched to mainly CvME. The concept of the switching of internalization pathway from multiple pathways to a single pathway was proved using three approaches: (i) evaluation of uptake in the presence of small chemical inhibitors, (ii) silencing of pathway-related target protein, and (iii) measuring the PP2A enzymatic activity. The resulting CvME pathway of uptake of AuroLPs resulted in several advantages including a decrease in lysosomal degradation due to a decrease in PP2A activity, enhanced silencing, and its corresponding antitumor efficacy in two ovarian tumor models.

Supplementary Materials

  • Supplementary Materials

    Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome

    Md. Nazir Hossen, Lin Wang, Harisha R. Chinthalapally, Joe D. Robertson, Kar-Ming Fung, Stefan Wilhelm, Magdalena Bieniasz, Resham Bhattacharya, Priyabrata Mukherjee

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