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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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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.
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fig1: 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.

Mentions: PLGA-b-PEG and uPA-peptide conjugated PLGA-b-PEG-NPs loaded with antisense-miRNAs were formulated using water-in-oil-in-water (w/o/w) double emulsion method (Supporting Information (SI) Scheme S1, Figures S1–S2). Two different emulsifiers are crucial for w/o/w multiple emulsion stabilization, one with a low hydrophile–lipophile balance (HLB) for the w/o interface, and the second one with a high HLB for the o/w interface. Tween 80 (HLB = 15) is often used in combination with Span 80 (HLB = 4.3) in multiple w/o/w emulsions because of similarity in their chemical structure.34 We used spermidine as a counterion35,36 for encapsulating various sense- and antisense-miRNAs (SI Table S1), lipophilic surfactant Span 80 for stabilizing the first emulsion (w/o), and hydrophilic surfactant Tween 80 for stabilizing the second emulsion (Figure 1A). Dynamic light scattering (DLS) of prepared NPs showed a size range of 100 to 200 nm (Figure 1B) with a polydispersity index (PDI) of 0.09–0.264. The zeta potential of NPs was in the range of −22 to −46 mV in ultrapure water (SI Tables S2–S3). The highly anionic nature of antisense-miRNAs should increase the negative zeta potential of NPs. As anticipated, the antisense-miRNAs loaded NPs showed much higher negative zeta potential compare to control NPs (SI Tables S2–S3). Morphology and size of NPs were further confirmed by transmission electron microscopy after staining with 1% phosphotungstic acid (Figure 1C). The entrapment efficiency of various antisense-miRNAs loaded in NPs was calculated using Quant-iT RNA Assay kit, as well as by optical CCD camera imaging based quantification for the coloaded Cy5-antisense-miR-21 after resolving the NPs by agarose gel electrophoresis (Figure 1D). The average number of antisense-miRNAs encapsulated in various NP formulations was estimated to be in the range of 400 to 1000 molecules/NP (SI Tables S2–S3). Moreover, we also evaluated the distribution of antisense-miR-21 and antisense-miR-10b in the coloaded nanoparticles by qRT-PCR analysis. The results indicate that coloading of antisense-miR-21 and antisense-miR-10b in PLGA-b-PEG-NPs is found almost at equimolar concentration in NPs prepared in different batches (SI Figure S3). The antisense-miRNAs extracted from the equimolar mixture of NPs formulated with each antisense-miRNAs (antisense-miR-21 and antisense-miR-10b) separately was used as 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)

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.
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fig1: 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.
Mentions: PLGA-b-PEG and uPA-peptide conjugated PLGA-b-PEG-NPs loaded with antisense-miRNAs were formulated using water-in-oil-in-water (w/o/w) double emulsion method (Supporting Information (SI) Scheme S1, Figures S1–S2). Two different emulsifiers are crucial for w/o/w multiple emulsion stabilization, one with a low hydrophile–lipophile balance (HLB) for the w/o interface, and the second one with a high HLB for the o/w interface. Tween 80 (HLB = 15) is often used in combination with Span 80 (HLB = 4.3) in multiple w/o/w emulsions because of similarity in their chemical structure.34 We used spermidine as a counterion35,36 for encapsulating various sense- and antisense-miRNAs (SI Table S1), lipophilic surfactant Span 80 for stabilizing the first emulsion (w/o), and hydrophilic surfactant Tween 80 for stabilizing the second emulsion (Figure 1A). Dynamic light scattering (DLS) of prepared NPs showed a size range of 100 to 200 nm (Figure 1B) with a polydispersity index (PDI) of 0.09–0.264. The zeta potential of NPs was in the range of −22 to −46 mV in ultrapure water (SI Tables S2–S3). The highly anionic nature of antisense-miRNAs should increase the negative zeta potential of NPs. As anticipated, the antisense-miRNAs loaded NPs showed much higher negative zeta potential compare to control NPs (SI Tables S2–S3). Morphology and size of NPs were further confirmed by transmission electron microscopy after staining with 1% phosphotungstic acid (Figure 1C). The entrapment efficiency of various antisense-miRNAs loaded in NPs was calculated using Quant-iT RNA Assay kit, as well as by optical CCD camera imaging based quantification for the coloaded Cy5-antisense-miR-21 after resolving the NPs by agarose gel electrophoresis (Figure 1D). The average number of antisense-miRNAs encapsulated in various NP formulations was estimated to be in the range of 400 to 1000 molecules/NP (SI Tables S2–S3). Moreover, we also evaluated the distribution of antisense-miR-21 and antisense-miR-10b in the coloaded nanoparticles by qRT-PCR analysis. The results indicate that coloading of antisense-miR-21 and antisense-miR-10b in PLGA-b-PEG-NPs is found almost at equimolar concentration in NPs prepared in different batches (SI Figure S3). The antisense-miRNAs extracted from the equimolar mixture of NPs formulated with each antisense-miRNAs (antisense-miR-21 and antisense-miR-10b) separately was used as 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.

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Related in: MedlinePlus