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Co-delivery of IKBKE siRNA and cabazitaxel by hybrid nanocomplex inhibits invasiveness and growth of triple-negative breast cancer

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Science Advances  15 Jul 2020:
Vol. 6, no. 29, eabb0616
DOI: 10.1126/sciadv.abb0616
  • Fig. 1 Biological activities of the IKBKE siRNA in MDA-MB-231 cells.

    (A) Silencing effect of the IKBKE siRNA at the mRNA level. (B) Silencing activity of the IKBKE siRNA at the protein level. Inhibitory effect of the IKBKE siRNA on the migration (C and D), invasion (E and F), and proliferation (G) of MDA-MB-231 cells. Cell migration and invasion were evaluated at 6 and 24 hours after transfection, respectively. Quantitative analysis was performed by counting the number of migrated cells in six random microscope fields from three independent samples. Cell proliferation was determined at 48 and 96 hours after transfection using the CellTiter-Glo Luminescent Cell Viability Assay Kit. All results are presented as the means ± SD (n = 3). (H) Effect of the IKBKE siRNA on the apoptosis and necrosis of MDA-MB-231 cells. All results are presented as the means ± SD (n = 3). (I and J) In vivo antitumor efficacy of the IKBKE siRNA in a subcutaneous xenograft TNBC mouse model. The IKBKE siRNA was peritumorally injected at a dose of 0.3 mg/kg. All results are presented as the means ± SEM (n = 7). *P < 0.05 and **P < 0.01. ns, not significant. Photo credit: Z.Z., School of Pharmacy, University of Missouri-Kansas City.

  • Fig. 2 Schematics, characterization, and silencing activity of the HA-modified siRNA nanocomplex.

    (A) Cholesterol-peptide (CP) is self-assembled into a micelle-like structure, followed by loading with cabazitaxel, condensing with the IKBKE siRNA, and coating with cholesteryl-HA (CHA) to form the hybrid siRNA nanocomplex. The nanocomplex will accumulate in TNBC cells by recognizing CD44 on the tumor cells. The nanocomplex will dissociate inside the tumor cells to release the IKBKE siRNA and cabazitaxel to exert antitumor activities. IV, intravenous. (B) Gel retardation assay of the siRNA nanocomplexes at different N/P ratios. Zeta potential (C), particle size (D), silencing activity at the mRNA level (E), and silencing activity at the protein level (F) of the siRNA nanocomplexes at different N/P ratios. Gel retardation assay (G) and zeta potential (H) of the HA-modified siRNA nanocomplexes at different percentages of cholesteryl-HA (CHA) in the carrier. All results are presented as means ± SD (n = 3).

  • Fig. 3 Cellular uptake of the HA-modified siRNA nanocomplex.

    The IKBKE siRNA was labeled with Cy5 for fluorescence analysis using flow cytometry and confocal microscopy. (A) Cellular uptake of free siRNA, CP/siRNA nanocomplex, and CHA/CP/siRNA nanocomplex after 2, 4, and 6 hours of transfection. (B) Percent of the labeled cells. (C) Fluorescence intensity of the labeled cells. Representative confocal images of the cells treated with the siRNA nanocomplexes after 2 hours (D) and 6 hours (E) of transfection. HA (5 mg/ml) was used to pretreat the cells to block the cellular uptake of the nanocomplexes. **P < 0.01. Scale bars, 20 μm. CP, cholesterol-peptide; CHA, cholesteryl-HA.

  • Fig. 4 Characterization of the cabazitaxel-loaded siRNA nanocomplexes.

    (A) Critical micelle concentrations of CP, CP/siRNA nanocomplex, and CHA/CP/siRNA nanocomplex. Particle size (B) and TEM images (C) of the CHA/CP/siRNA and CHA/CP/siRNA/CTX nanocomplexes. (D) Silencing activity of the nanocomplexes at the protein level. Serum stability of the CHA/CP/siRNA/CTX nanocomplex in 50% human serum (E) and mouse serum (F). Heparin was used to dissociate the siRNA from the nanocomplex after incubation with the serum. All results are presented as means ± SD (n = 3). CP, cholesterol-peptide; CHA, cholesteryl-HA; cabazitaxel, CTX.

  • Fig. 5 3D tumor spheroid penetration, invasion, and apoptosis study of the HA-modified siRNA nanocomplex.

    Representative z-stack confocal images of the spheroids with a z-step of 25 μm for CP/Cy5-siRNA/Coumarin 6 and CHA/CP/Cy5-siRNA/Coumarin 6 nanocomplexes after 1 hour (A) and 2 hours (B) of incubation. Red for Cy5-siRNA, green for Coumarin 6, and yellow for merged area. Scale bars, 200 μm. Mean fluorescence intensity of Cy5-siRNA after 1 hour (C) and 2 hours (E) of incubation and Coumarin 6 after 1 hour (D) and 2 hours (F) of incubation in the z-stacked confocal images versus the distance from the periphery of the spheroids (n = 3). (G) Representative images of 3D spheroid invasion of MDA-MB-231 cells (n = 3). Scale bars, 500 μm. (H) Quantification of spheroid invasion based on the change of the total areas of the spheroids. The spheroid areas of each day were normalized to the areas measured at day 1. The data are presented as means ± SD (n = 3). (I) Apoptosis study of the cells using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit and flow cytometry. (J) Percentage of apoptotic and necrotic cells after 48 hours of transfection. All results are presented as means ± SD (n = 3). *P < 0.05; **P < 0.01; ns, not significant. CP, cholesterol-peptide; CHA, cholesteryl-HA; cabazitaxel, CTX.

  • Fig. 6 Biodistribution and antitumor activity of the HA-modified siRNA nanocomplex in an orthotopic TNBC mouse model.

    For biodistribution study, the siRNA was labeled with Cy5 and injected via tail vein at a dose of 1.5 mg siRNA/kg. (A) Live animal imaging of the mice at different time intervals. n = 3. (B) Fluorescence images of the liver, spleen, tumor, lung, kidney, heart, and muscle were taken at 48 hours after injection. (C) Mean fluorescence intensity of each organ was normalized to that of the heart. The results are presented as means ± SD (n = 3). **P < 0.01. For antitumor activity study, saline, CHA/CP/IKBKE siRNA nanocomplex, CTX, CHA/CP/IKBKE siRNA nanocomplex plus free CTX, and CHA/CP/IKBKE siRNA/CTX nanocomplex were administered via the tail vein every 4 days for a total of five times at a dose of 1.5 mg siRNA/kg and 5 mg cabazitaxel/kg. (D) Tumor volumes are presented as means ± SEM. (E) Images of harvested tumors. Photo credit: Z.Z., School of Pharmacy, University of Missouri-Kansas City). (F) Tumor weight. The results are presented as means ± SD (n = 7). (G) Expression of IKBKE protein in tumor tissues (n = 6). Fluorescence images (H) and quantitative analysis (I) of apoptotic cells in tumor specimen using a TUNEL assay. Scale bars, 100 μm. Quantitative analysis was performed by counting the fluorescence intensity of apoptotic cells in three random microscope fields from three independent samples. The results are presented as means ± SD. **P < 0.01. ns, not significant. CP, ns, cholesterol-peptide; CHA, cholesteryl-HA; CTX, cabazitaxel.

Supplementary Materials

  • Supplementary Materials

    Co-delivery of IKBKE siRNA and cabazitaxel by hybrid nanocomplex inhibits invasiveness and growth of triple-negative breast cancer

    Zhen Zhao, Yuanke Li, Hao Liu, Akshay Jain, Pratikkumar Vinodchandra Patel, Kun Cheng

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