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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–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).
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fig4: (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).

Mentions: To test bioactivity of antisense-miRNAs delivered by PLGA-b-PEG-NPs, we treated TNBC cells with 25 pmols of antisense-miR-21 and antisense-miR-10b either individually or coloaded NPs for 48 h and measured mRNA (mRNA) levels of target genes PTEN, PDCD4, and HoxD10 by RT-PCR analysis. The expressions were normalized to the house keeping control gene β-Actin. The results show upregulation of PDCD4 expression in cells treated with NPs loaded with antisense-miR-21 and coloaded with antisense-miR-21 and antisense-miR-10b combination. Similarly, upregulation of HoxD10 was observed in cells treated with NPs loaded with antisense-miR-10b and coloaded with antisense-miR-21 and antisense-miR-10b combination (Figure 4A; SI Table S4). The respective protein levels were measured by immunoblot analysis. An increase in the target protein expression was also observed in cells treated with respective antisense-miRNAs loaded NPs (Figure 4B). Interestingly, we observed that PTEN mRNA and protein levels increase when cells were treated with antisense-miR-10b. Similarly, confocal microscopic imaging of cells immunostained for miR-21 target PDCD4 showed higher level of PDCD4, with disintegrated nuclear structure in cells treated with NPs loaded with antisense-miR-21 and coloaded with antisense-miR-10b- and antisense-miR-21 combination, and not with control NPs or NPs loaded with antisense-miR-10b (Figure 4C). To further test the functional role of antisense-miRNAs in blocking the endogenous miRNAs, downregulation of miR-21-target genes maspin and PDCD4 were indirectly monitored in cells by expressing firefly luciferase reporter gene containing the 3′-UTR sequences derived from maspin or PDCD4 gene.41 We found a significant upregulation of luciferase signal in cells treated with NPs loaded with antisense-miR-21 and coloaded with antisense-miR-21 and antisense-miR-10b combination, as compared to cells treated with NPs loaded with the scrambled antisense-miRNA or antisense-miR-10b (SI Figure S11A–D).


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–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).
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fig4: (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).
Mentions: To test bioactivity of antisense-miRNAs delivered by PLGA-b-PEG-NPs, we treated TNBC cells with 25 pmols of antisense-miR-21 and antisense-miR-10b either individually or coloaded NPs for 48 h and measured mRNA (mRNA) levels of target genes PTEN, PDCD4, and HoxD10 by RT-PCR analysis. The expressions were normalized to the house keeping control gene β-Actin. The results show upregulation of PDCD4 expression in cells treated with NPs loaded with antisense-miR-21 and coloaded with antisense-miR-21 and antisense-miR-10b combination. Similarly, upregulation of HoxD10 was observed in cells treated with NPs loaded with antisense-miR-10b and coloaded with antisense-miR-21 and antisense-miR-10b combination (Figure 4A; SI Table S4). The respective protein levels were measured by immunoblot analysis. An increase in the target protein expression was also observed in cells treated with respective antisense-miRNAs loaded NPs (Figure 4B). Interestingly, we observed that PTEN mRNA and protein levels increase when cells were treated with antisense-miR-10b. Similarly, confocal microscopic imaging of cells immunostained for miR-21 target PDCD4 showed higher level of PDCD4, with disintegrated nuclear structure in cells treated with NPs loaded with antisense-miR-21 and coloaded with antisense-miR-10b- and antisense-miR-21 combination, and not with control NPs or NPs loaded with antisense-miR-10b (Figure 4C). To further test the functional role of antisense-miRNAs in blocking the endogenous miRNAs, downregulation of miR-21-target genes maspin and PDCD4 were indirectly monitored in cells by expressing firefly luciferase reporter gene containing the 3′-UTR sequences derived from maspin or PDCD4 gene.41 We found a significant upregulation of luciferase signal in cells treated with NPs loaded with antisense-miR-21 and coloaded with antisense-miR-21 and antisense-miR-10b combination, as compared to cells treated with NPs loaded with the scrambled antisense-miRNA or antisense-miR-10b (SI Figure S11A–D).

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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Related in: MedlinePlus