Polymer nanoparticles mediated codelivery of antimiR-10b and antimiR-21 for achieving triple negative breast cancer therapy - PubMed (original) (raw)

. 2015 Mar 24;9(3):2290-302.

doi: 10.1021/nn507465d. Epub 2015 Feb 23.

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Polymer nanoparticles mediated codelivery of antimiR-10b and antimiR-21 for achieving triple negative breast cancer therapy

Rammohan Devulapally et al. ACS Nano. 2015.

Abstract

The current study shows the therapeutic outcome achieved in triple negative breast cancer (TNBC) by simultaneously antagonizing miR-21-induced antiapoptosis and miR-10b-induced metastasis, using antisense-miR-21-PS and antisense-miR-10b-PS delivered by polymer nanoparticles (NPs). We synthesized the antisense-miR-21 and antisense-miR-10b loaded PLGA-b-PEG polymer NPs and evaluated their cellular uptake, serum stability, release profile, and the subsequent synchronous blocking of endogenous miR-21 and miR-10b function in TNBC cells in culture, and tumor xenografts in living animals using molecular imaging. Results show that multitarget antagonization of endogenous miRNAs could be an efficient strategy for targeting metastasis and antiapoptosis in the treatment of metastatic cancer. Targeted delivery of antisense-miR-21 and antisense-miR-10b coloaded urokinase plasminogen activator receptor (uPAR) targeted polymer NPs treated mice showed substantial reduction in tumor growth at very low dose of 0.15 mg/kg, compared to the control NPs treated mice and 40% reduction in tumor growth compared to scramble peptide conjugated NPs treated mice, thus demonstrating a potential new therapeutic option for TNBC.

Keywords: PLGA; anti-miRs; antisense-miRNAs; bioluminescence; cancer therapy; in vivo molecular imaging; microRNA; nanoparticles; targeted delivery.

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Figures

Figure 1

Figure 1

Nanoparticle preparation and characterization. (A) Schematic illustration of nanoparticle formulation. (B) Hydrodynamic size of antisense-miRNA encapsulated PLGA-_b_-PEG NPs measured by dynamic light scattering (DLS). (C) TEM image of antisense-miRNA encapsulated PLGA-_b_-PEG NPs taken after staining with 1% phosphotungstic acid (scale bar, 100 nm). (D) Evaluation of coloaded Cy5-labeled antisense-miR-21 (10%) from the encapsulated PLGA-_b_-PEG NPs after being resolved in 3% agarose gel electrophoresis by optical CCD-camera imaging with the excitation of 570 nm and emission filter at 660 nm. (E,F) PLGA-_b_-PEG NPs loaded miRNA-21 release profile evaluated after seeding the coloaded NPs in PBS at physiological pH for 8 days of incubation at 37 °C by qRT-PCR analysis: (E) miR-21 fraction present in NPs different time points after incubation. (F) miR-21 fraction released from NPs different time points after incubation.

Figure 2

Figure 2

(A–G) Sense- and antisense-miRNA loaded PLGA-_b_-PEG NPs delivery in MDA-MB-231 cells, and the PLGA-_b_-PEG NPs loaded miRNAs stability in serum studied after incubation for various time points (0–48 h) at 37 °C by Taqman-qRT-PCR. (A) Evaluation of miR-21 levels in MDA-MB-231 cells delivered by control NP and PLGA-_b_-PEG NPs loaded with miR-21 in different concentrations (10 and 50 pmols) by qRT-PCR analysis. (B) Endogenous expression level of miR-21, miR-10b, and RNU66 in MDA-MB-231 cells (relative expression fold of various miRNAs compared to miR-10b). (C) Ct values measured for miRNAs expressions in (B) (Relative fluorescent intensity by Taqman probe). (D,E) Confocal fluorescent microscope images of MDA-MB-231-Fluc-eGFP cells treated with control-PLGA-_b_-PEG NPs and PLGA-_b_-PEG NPs coloaded with Cy5-antisense-miR-21 (0.5 nmols), antisense-miR-21 (9.5 nmols) and antisense-miR-10b (10 nmols), for 24 h at 37 °C. (F–H) Serum stability of naked miR-21 and miR-21 loaded in PLGA-_b_-PEG NPs evaluated at different time points after initial spiking (0, 12, 24, and 48 h). (F) Fluorescence intensity graph used for measuring Ct-values for serum spiked with naked miR-21. (G) Fluorescence intensity graph used for measuring Ct-values for serum spiked with miR-21 loaded PLGA-_b_-PEG NPs. (H) Relative miR-21 levels measured from serum spiked with naked and PLGA-_b_-PEG NPs loaded miR-21 over time.

Figure 3

Figure 3

Cytotoxicity evaluation of various antisense-miRNAs loaded PLGA-_b_-PEG-NPs in MDA-MB-231-Fluc-eGFP cells by MTT assay. (A–D) Cells treated with 0 to 25 pmol/mL miRNA equivalent of control NPs, antisense-miR-21 and antisense-miR-10b individually-and coloaded NPs for 24 h and assessed for cytotoxicity by MTT assay. (E–H) Cells treated with 12.5 and 25 pmol/mL miRNA equivalent of control NPs, antisense-miR-21 and antisense-miR-10b individually-and coloaded NPs for various time points (24–72 h) and assessed for cytotoxicity by MTT assay. Error bars are SEM of three determinants (*p < 0.05).

Figure 4

Figure 4

(A–C) Effect of antisense-miRNAs delivered by PLGA-_b_-PEG-NPs in blocking the function of endogenous miR-21 and miR-10b, and subsequent downstream regulation of target gene (miR-21: PTEN and PDCD4; miR-10b: HoxD10) expression in MDA-MB-231-Fluc-EGFP cells. (A) RT-PCR analysis for the expression of target genes of miR-21 and miR-10b (PTEN, PDCD4 and HoxD10) in MDA-MB 231 cells after delivering antisense-miRNAs by PLGA-_b_-PEG-NPs. (B) Immunoblot analysis for the expression of miR-21 and miR-10b (PTEN, PDCD4 and HoxD10) target proteins in MDA-MB-231 cells after treating with antisense-miRNAs delivered by PLGA-_b_-PEG-NPs (GAPDH as internal control). (C) Immunofluorescent staining for PDCD4 expression in MDA-MB 231 cells after treating with antisense-miRNAs delivered by PLGA-_b_-PEG-NPs. (D–G) Evaluation of metastatic properties of MDA-MB-231-Fluc-eGFP cells after treatment with control-NPs, and antisense-miR-21 and antisense-miR-10b coloaded NPs by bioluminescence imaging in mice. (D) Bioluminescence imaging of animals tail vein injected with MDA-MB-231-Fluc-eGFP cells after pretreatment by control-NPs, and NPs coloaded with antisense-miR-21 and antisense-miR-10b combination, for the identification of metastatic tumor growth. (E) Quantitation of bioluminescence signal in animals tail-vein injected with MDA-MB-231-Fluc-eGFP cells pretreated by control-NPs (red), and NPs coloaded with antisense-miR-21 and antisense-miR-10b combination (blue). Error bars are SEM of three determinants (* p < 0.05). (F) Ex-vivo bioluminescence imaging of lung tissues excised from the animals 6 weeks after the initial injection of MDA-MB-231-Fluc-eGFP cells pretreated by control-NPs (A1 and A2) and NPs coloaded with antisense-miR-21 and antisense-10b combination (A3 and A4). Each bioluminescent spot represents one metastatic tumor nodule. (G) H&E staining analysis of lung tissues (A2: section of lung tissue excised from animal injected with MDA-MB-231-Fluc-eGFP cells pretreated by control-NPs; A3: section of lung tissue excised from animal injected with MDA-MB-231-Fluc-eGFP cells pretreated by NPs coloaded with antisense-miR-21 and antisense-miR-10b combination).

Figure 5

Figure 5

In vivo tumor growth analysis and bioluminescence imaging of mice (n = 25) bearing MDA-MB-231 tumors stably expressing Fluc-eGFP that are treated with antisense-miR-21 and antisense-miR-10b loaded or coloaded with uPA–PLGA-_b_-PEG and Sc-uPA–PLGA-_b_-PEG NPs. (A) Tumors growth volume (mm3) measured in different treatment groups over time. (B) Optical bioluminescence images of animals (n = 10, 5 animals bearing two tumors each for each treatment group) treated with different NPs over time. (C) Quantitative graph showing the bioluminescence signals quantitated from animals shown in (B). (D) TUNEL staining of tumor tissues of animals treated with different NPs.

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