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. 2016 Nov;5(22):2882-2895.
doi: 10.1002/adhm.201600677. Epub 2016 Oct 10.

Targeted Dual pH-Sensitive Lipid ECO/siRNA Self-Assembly Nanoparticles Facilitate In Vivo Cytosolic sieIF4E Delivery and Overcome Paclitaxel Resistance in Breast Cancer Therapy

Maneesh Gujrati  1 , Amita M Vaidya  1 , Margaret Mack  1 , Dayton Snyder  1 , Anthony Malamas  1 , Zheng-Rong Lu  1
Affiliations

Targeted Dual pH-Sensitive Lipid ECO/siRNA Self-Assembly Nanoparticles Facilitate In Vivo Cytosolic sieIF4E Delivery and Overcome Paclitaxel Resistance in Breast Cancer Therapy

Maneesh Gujrati et al. Adv Healthc Mater. 2016 Nov.

Abstract

RNAi-mediated knockdown of oncogenes associated with drug resistance can potentially enhance the efficacy of chemotherapy. Here, we have designed and developed targeted dual pH-sensitive lipid-siRNA self-assembly nanoparticles, RGD-PEG(HZ)-ECO/siRNA, which can efficiently silence the oncogene, eukaryotic translation initiation factor 4E (eIF4E), and consequently resensitize triple-negative breast tumors to paclitaxel. The dual pH-sensitive function of these nanoparticles facilitates effective cytosolic siRNA delivery in cancer cells, both in vitro and in vivo. Intravenous injection of RGD-PEG(HZ)-ECO/siRNA nanoparticles (1.0 mg-siRNA/kg) results in effective gene silencing for at least one week in MDA-MB-231 tumors. In addition, treatment of athymic nude mice with RGD-PEG(HZ)-ECO/sieIF4E every 6 days for 6 weeks down-regulates the overexpression of eIF4E and resensitizes paclitaxel-resistant MDA-MB-231 tumors to paclitaxel, resulting in significant tumor regression at a low dose, with negligible side effects. Moreover, repeated injections of the RGD-PEG(HZ)-ECO/siRNA nanoparticles in immunocompetent mice result in minimal immunogenicity, demonstrating their safety and low toxicity. These multifunctional lipid/siRNA nanoparticles constitute a versatile platform of delivery of therapeutic siRNA for treating cancer and other human diseases.

Keywords: ECO; cytosolic siRNA delivery; drug resistance; dual pH-sensitive; eIF4E.

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Figures

Figure 1
Figure 1
Design and working of the dual pH-sensitive PEG(HZ)-ECO/siRNA nanoparticles. (A) Formation of targeted dual pH-sensitive ECO/siRNA nanoparticles by 1) reaction of peptide-PEG-hydrazone-maleimide (RGD-PEG(HZ)-MAL with one of the thiol groups of small portion of multifunctional lipid ECO and 2) self-assembly with siRNA electrostatic complexation, hydrophobic condensation, and disulfide cross-linking and PEG shedding in acidic endosomes. (B) RGD-PEG(HZ)-ECO/siRNA nanoparticles facilitate receptor-mediated endocytosis, resulting in trafficking of the nanoparticles into endosomes. Within late endosomes, the increasingly acidic environment cleaves the hydrazone linkage to facilitate shedding of the PEG layer and to expose the core ECO/siRNA nanoparticles. Next, pH-sensitive amphiphilicity of ECO promotes endosomal escape by enhanced amphiphilic interaction of the nanoparticles with the lipid bilayer of the endosomes. Once release into the cytosol, endogenous glutathione (GSH) mediates reduction of disulfide bonds within ECO/siRNA nanoparticles to facilitate dissociation of the nanoparticles and siRNA release. Upon release, free siRNA is able to initiate RNAi-induced gene silencing.
Figure 2
Figure 2
pH-sensitive shedding of PEG from PEG(HZ)-ECO/siRNA nanoparticles. MALDI-TOF mass spectra of mPEG (A) and mPEG(HZ) (B) at pH 7.4, 6.5, and 5.4, the pH at different stages of intracellular trafficking. Dynamic changes of zeta potential of ECO/siRNA (C), PEG-ECO/siRNA (D), and PEG(HZ)-ECO/siRNA (E)nanoparticles incubated in PBS solutions at pH 7.4, 6.5, 5.4. Comparison of hemolytic activity of ECO/siRNA, PEG-ECO/siRNA, and PEG(HZ)-ECO/siRNA nanoparticles at pH 7.4, 6.5, 5.4 (F). The pH sensitivity (G) and hemolytic activity (H) of PEGylated ECO/siRNA nanoparticles at a PEGylation degree of 0, 1, 2.5, 5, 10 mol-% measured by changes in zeta potential at pH 7.4, 6.5, and 5.4. Relative hemolytic activity was calculated with respect to the hemolytic activity of 1% Triton-X-100.
Figure 3
Figure 3
Targeted PEG(HZ)-ECO/siRNA nanoparticles induce potent in vitro gene silencing by enhanced endosomal escape. (A) Cellular uptake of unmodified, PEG, PEG(HZ), RGD-PEG, and RGD-PEG(HZ) modified ECO/siRNA nanoparticles quantified with flow cytometry using an AF647-labeled siRNA. (B) Luciferase silencing of unmodified, PEG, PEG(HZ), RGD-PEG, and RGD-PEG(HZ) modified ECO/siRNA nanoparticles in MDA-MB-231-luc TNBC cells compared to no treatment control group. (C) Fluorescence confocal microscopy images of live MDA-MB-231 cells incubated with RGD-PEG, and RGD-PEG(HZ) modified ECO/siRNA nanoparticles at 10 min, 3 hr, and 6 hr. DAPI, cell nucleus (blue); Lysotracker DND-26, late endosomes and lysosomes (green); siRNA, AF-647 (red). (D) Luciferase silencing efficiency of different ECO/siRNA nanoparticles after 48 hours in MDA-MB-231-luc cells transfected with or without the endosomolytic agent chloroquine (100 μM).
Figure 4
Figure 4
RGD-PEG(HZ)-ECO/siRNA nanoparticles mediate potent and sustained tumor gene silencing in vivo. Luciferase silencing efficiency in orthotopic MDA-MB-231-Luc tumors of mice following a single intravenous injection of various surface-modified ECO/siRNA nanoparticles (1.0 mg/kg siRNA dose) compared to PBS-treated control group. (A) Bioluminescence intensity from the tumor at different time points after the treatment (siLuc). (B) Representative bioluminescence images of the different treatment groups (siLuc). (C) Representative FMT images showing tumor accumulation and retention of the surface-modified ECO/siRNA nanoparticles of an AF647-tagged siRNA within 24 hours after intravenous administration. (D) Fluorescence intensity of AF647-tagged siRNA quantifying tumor accumulation and retention of the modified nanoparticles in tumor. Cell suspensions obtained from primary MDA-MB-231 mammary fat pad tumors following intravenous administration of the modified ECO/siRNA nanoparticles were analyzed using flow cytometry and confocal microscopy analysis. (E) FACS analysis following staining for EpCAM expression using a FITC labeled anti-EpCAM antibody in tumor cell suspensions in the FITC channel revealed two populations of EpCAM(+) and EpCAM(−) cells. (F) Gating for the EpCAM(−) cell population and examining in the AF647 channel revealed minimal uptake of both non-targeted and RGD-targeted nanoparticles when compared to PBS control (data shown for the systems with hydrazone). (G) Gating for the EpCAM (+) cell population in the AF647 channel revealed a significant shift for RGD-targeted nanoparticles compared to the non-targeted and PBS control (data shown for the systems with hydrazone). (H) Data represented as a two dimensional contour plot highlights the EpCAM (−) and EpCAM (+) populations along the FITC channel axis. For targeted nanoparticles, fluorescent signal from siRNA in AF647 channel axis is distinctly greater in the EpCAM (+) population. For non-targeted nanoparticles, the AF647 signal is evenly distributed between EpCAM (−) and EpCAM (+) populations. (I) Cell suspensions from the tumors were examined under a confocal microscope. Targeted nanoparticles display a greater amount of siRNA signal (red) in the EpCAM (+) (green) cells.
Figure 5
Figure 5
Silencing eIF4E by RGD-PEG(HZ)ECO/siRNA nanoparticles sensitizes drug-resistant TNBC cells to paclitaxel. Expression of eIF4E at mRNA and protein levels determined by qRT-PCR (A) and western blotting analysis (B) in MDA-MB-231 and MDA-MB-231.DR cells at 5 days following treatment with RGD-PEG(HZ)-ECO/siRNA nanoparticles (N/P=8) delivering sieIF4E or siNS (100 nM) compared to no treatment control group. (C) Cell viability of MDA-MB-231 and drug resistant MDA-MB-231.DR cells treated with varying concentrations of PTX following prior treatment with RGD-PEG(HZ)-ECO/siRNA nanoparticles delivering sieIF4E or siNS. The cells were first treated with RGD-PEG(HZ)-ECO/siRNA nanoparticles for 48 hours followed by treatment with varying concentrations of PTX for an additional 48 hours.
Figure 6
Figure 6
The efficacy of combination therapy of PTX and RGD-PEG(HZ)-ECO/sieIF4E nanoparticles in treating orthotopic luciferase labeled MDA-MB-231.DR TNBC tumors in mice compared to PBS-treated control group. Alternating treatment of the sieIF4E nanoparticles and PTX every 6 days began at 4 weeks once the primary tumors reach an average of 150 mm3. (A) Bioluminescence intensity over the course of the experiment (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01 comparing to the no treatment control) and (B) bioluminescence images at week 10. (C) Tumor growth was monitored using digital caliper measurements (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01). (D) Primary tumors resected at week 10 and E) final tumor weights (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01). (F) Relative eIF4E mRNA expression in the resected primary tumors determined by qRT-PCR (data represents mean ± SE, n=5, *p≤0.05, **p≤0.0). (G) Immunofluorescence staining of eIF4E, VEGF, cyclin D1, and survivin from primary tumors.
Figure 7
Figure 7
(A) Histological evaluation of liver and kidney (10X) from different treatment groups. (B) Immunogenicity of ECO/siRNA and RGD-PEG(HZ)-ECO/siRNA nanoparticles in immunocompetent mice following intravenous administrations. At 2 h and 24 h following the first, third, and fifth injection, blood samples were collected and the plasma was isolated to be used for cytokine ELISA measurements of TNF-α, IL-12, IFN-γ, and IL-6 (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01 comparing to the baseline). Solid and dotted lines indicate the mean ± SE pertaining to baseline levels of each cytokine.
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