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

Devulapally R, Sekar NM, Sekar TV, Foygel K, Massoud TF, Willmann JK, Paulmurugan R - ACS Nano (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Department of Radiology, Stanford University School of Medicine, Stanford University, 3155 Porter Drive, Palo Alto, California 94304, United States.

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.

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(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.
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fig2: (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.

Mentions: Mitchell et al. reported that synthetic naked miRNAs are rapidly degraded in plasma compared to various endogenous miRNAs isolated from human samples.38 It could be possibly due to protein complexes associated with the endogenous microRNAs, which are in general lacking for the delivered synthetic therapeutic miRNAs. We tested the stability of PLGA-b-PEG-NPs encapsulated miR-21 delivered in MDA-MB-231 cells and spiked in mouse serum by TaqMan-qRT-PCR analysis (Figure 2). MDA-MB-231 cells were treated with miR-21 loaded PLGA-b-PEG-NPs for 24 h, and the total microRNAs extracted from the cells after a thorough wash to remove the extracellular NPs were quantitatively measured for the presence of intact miR-21 by qRT-PCR. The cells without any NP treatment, and treated with control NPs were used as controls. The results show that in cells treated with 10 and 50 pmols of miR-21-NPs, the intracellular miR-21 level was ∼836 ± 188 and ∼11322 ± 1486 fold higher, respectively, compared to controls (Figure 2A). The cells treated with control NPs showed a minor but not significant drop in the endogenous miR-21 level compared to untreated control.


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

Devulapally R, Sekar NM, Sekar TV, Foygel K, Massoud TF, Willmann JK, Paulmurugan R - ACS Nano (2015)

(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.
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fig2: (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.
Mentions: Mitchell et al. reported that synthetic naked miRNAs are rapidly degraded in plasma compared to various endogenous miRNAs isolated from human samples.38 It could be possibly due to protein complexes associated with the endogenous microRNAs, which are in general lacking for the delivered synthetic therapeutic miRNAs. We tested the stability of PLGA-b-PEG-NPs encapsulated miR-21 delivered in MDA-MB-231 cells and spiked in mouse serum by TaqMan-qRT-PCR analysis (Figure 2). MDA-MB-231 cells were treated with miR-21 loaded PLGA-b-PEG-NPs for 24 h, and the total microRNAs extracted from the cells after a thorough wash to remove the extracellular NPs were quantitatively measured for the presence of intact miR-21 by qRT-PCR. The cells without any NP treatment, and treated with control NPs were used as controls. The results show that in cells treated with 10 and 50 pmols of miR-21-NPs, the intracellular miR-21 level was ∼836 ± 188 and ∼11322 ± 1486 fold higher, respectively, compared to controls (Figure 2A). The cells treated with control NPs showed a minor but not significant drop in the endogenous miR-21 level compared to untreated control.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Department of Radiology, Stanford University School of Medicine, Stanford University, 3155 Porter Drive, Palo Alto, California 94304, United States.

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.

Show MeSH
Related in: MedlinePlus