Hyaluronic acid-coated PEI-PLGA nanoparticles mediated co-delivery of doxorubicin and miR-542-3p for triple negative breast cancer therapy Shengpeng.

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Hyaluronic acid-coated PEI-PLGA nanoparticles mediated co-delivery of doxorubicin and miR-542-3p for triple negative breast cancer therapy Shengpeng Wang, MS, Jinming Zhang, MS, Yitao Wang, MS, Meiwan Chen, PhD Nanomedicine: Nanotechnology, Biology and Medicine Volume 12, Issue 2, Pages 411-420 (February 2016) DOI: 10.1016/j.nano.2015.09.014 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Synthesis pathway and characterization of PEI-PLGA copolymer. (A) Diagram of the synthesis of the PEI-PLGA copolymer by the coupling reaction. Characterization of the PEI-PLGA copolymer using FT-IR spectra (B), 1HNMR spectra (C), and molecular weight was determination by GPC (D). Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Characterization of DOX and miR-542-3p co-loaded HA/PPNPs. (A) The encapsulation of miR-542-3p in HA/PPNPs at different N/P ratios was determined using gel electrophoresis. (B) The size distribution change of PPNPs with or without HA coating. (C) The zeta potential change of PPNPs with or without HA coating. (D) Size distribution, TEM and appearance of DOX/miR-542-3p-HA/PPNPs. (E) DOX release profiles in vitro from free DOX, DOX/PPNPs and DOX-HA/PPNPs. Data were expressed as the mean±SEM of three independent experiments. Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Stability and intracellular levels of miR-542-3p. Stability of different formulations of miR-542-3p in FBS (1:1, v/v) as measured by agarose gel retardation assay (A) and quantified using Image Lab software (B). (C) Intracellular uptake of miR-542-3p was detected by real-time PCR. Data were expressed as the mean±SEM of three independent experiments. *P<0.05 and ***P<0.001. Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Intracellular accumulation of HA/PPNPs. (A) CD44 expression in MCF-7 and MDA-MB-231 cells as measured by flow cytometry. (B) MCF-7 and MDA-MB-231 cells were incubated with DOX/FAM-miR-542-3p-HA/PPNPs with or without the pretreatment of 1mg/ml of HA for 4h, and the intracellular accumulation of DOX and FAM-miR-542-3p was observed by the Incell Analyzer 2000 (GE Healthcare Life Sciences, USA). Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Intracellular uptake of DOX and FAM-miR-542-3p in MDA-MB-231 cells. (A) Intracellular levels of DOX in MDA-MB-231 cells after incubation with DOX-loaded NPs (2μg/ml) with or without the pretreatment of 1mg/ml of HA for 4h. (B) Intracellular levels of FAM-miR-542-3p in MDA-MB-231 cells after treatment with FAM-miR-542-3p-loaded NPs (100nM) with or without the pretreatment of 1mg/ml of HA for 4h. Data were expressed as the mean±SEM of three independent experiments. *P<0.05 and ***P<0.001. Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Cell viability of MDA-MB-231 (A) and MCF-7 (B) cells after treatment with different formulations of DOX and miR-542-3p. Data were expressed as the mean±SEM of three independent experiments. Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Cell apoptosis assay in MDA-MB-231 cells. (A) Cell apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI double staining. (B) Involvement of caspase-3/7 activation after treatment with for 24h. (C) Western blot analysis of protein expression using indicated antibodies. Each value represents the mean±SEM from triplicate determinations. *P<0.05. Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 8 Intracellular delivery of miR-542-3p upregulated the expression of p53 and downregulated survivin expression. Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions

Nanomedicine: Nanotechnology, Biology and Medicine 2016 12, 411-420DOI: (10.1016/j.nano.2015.09.014) Copyright © 2015 Elsevier Inc. Terms and Conditions