Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 386, Issue 4, pp 287–302 | Cite as

MicroRNA 199b-5p delivery through stable nucleic acid lipid particles (SNALPs) in tumorigenic cell lines

  • Pasqualino de Antonellis
  • Lucia Liguori
  • Annarita Falanga
  • Marianeve Carotenuto
  • Veronica Ferrucci
  • Immacolata Andolfo
  • Federica Marinaro
  • Immacolata Scognamiglio
  • Antonella Virgilio
  • Giuseppe De Rosa
  • Aldo Galeone
  • Stefania Galdiero
  • Massimo ZolloEmail author
Original Article


MicroRNA (miR)-199b-5p has been shown to regulate Hes-1, a downstream effector of the canonical Notch and noncanonical SHH pathways, whereby it impairs medulloblastoma (MB) cancer stem cells (CSCs) through a decrease in the CD133+/CD15+ cell population. Here, we have developed stable nucleic acid lipid particles (SNALPs) that encapsulate miR-199b-5p. The efficacy of the miR-199b-5p delivery by these SNALPs is demonstrated by significant impairment of Hes-1 levels and CSC markers in a range of different tumorigenic cell lines: colon (HT-29, CaCo-2, and SW480), breast (MDA-MB231T and MCF-7), prostate (PC-3), glioblastoma (U-87), and MB (Daoy, ONS-76, and UW-228). After treatment with SNALP miR-199b-5p, there is also impairment of cell proliferation and no signs of apoptosis, as measured by caspases 3/7 activity and annexin V fluorescence cell sorter analyses. These data strengthen the importance of such carriers for miRNA delivery, which show no cytotoxic effects and provide optimal uptake into cells. Thus, efficient target downregulation in different tumorigenic cell lines will be the basis for future preclinical studies.


MiR-199b-5p SNALP Hes-1 Cancer stem cells 



Cancer stem cells


1,2-Dioleyl-3-dimethylammonium propane










Phosphate-buffered saline


N-Palmitoyl-sphingosine-1-succinyl [methoxy(polyethylene glycol)2000]


RNA-induced silencing complex


RNA interference


Small interfering RNAs


Stable nucleic acid lipid particles



The authors thank Carlo Pedone, Michele Saviano, and Maurizio Mariani for valuable discussions and comments on the manuscript. The authors also thank the CEINGE Service Facility platforms (the Oligo Synthesis Facility; the FACS Core Laboratory Dr. Marica Gemei, and the Head of the facility Prof. Luigi Del Vecchio; the Tissue Culture Facility headed by Prof. Rosella di Noto) and Advanced Accelerator Applications (AAA) for valuable observations related to the preindustrial development of miR-199b-5p.


This study was financed by FP7-Tumic HEALTH-F2-2008-201662 (MZ), Associazione Italiana per la Ricerca sul Cancro AIRC (MZ), Fondazione Italiana alla lotta del Neuroblastoma (MZ), and Progetto di Ricerca e Formazione PON01_2388: “Verso la medicina personalizzata: nuovi sistemi molecolari per la diagnosi e la terapia di patologie oncologiche ad alto impatto sociale”.

Conflict of interest


Supplementary material

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Supplementary Fig. 1

Luciferase assay in HEK-293T cells treated with SNALP miR-199b-5p and SNALP scramble. Effects of “naked” miR-199b-5p and SNALP miR-199b-5p (25 μg/ml) in HT-29 cells. a Relative luciferase activities were assayed in Hek-293T cells at 48 h and 72 h after transfection with the luciferase reporter plasmid. Schematic representation of the Tk/Ren 3′-UTR Hes-1 construct used. b MiR-199b-5p expression using the TaqMan PCR assay, and proliferation rate and Hes-1 protein levels in HT-29 cells treated with SNALP miR-199b-5p and SNALP scramble (25 μg/ml). c MiR-199b-5p expression using the TaqMan PCR assay and Hes-1 protein levels in HT-29 cells treated with the “naked” miR-199b-5p. de MiR-199b-5p expression at 6 h and 12 h using the TaqMan PCR assay, and proliferation rate and Hes-1 protein in HT-29 cells treated with SNALP miR-199b-5p and SNALP scramble for 24 h (JPEG 90 kb)

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Supplementary Fig. 2

MiR-199b-5p delivery by SNALP and impairment of proliferation rate in HT-29, Daoy, UW-228, and MDA-MB231T cells. Basal levels of Hes-1 protein in all cells analyzed. a MiR-199b-5p expression after 72 h of treatment, using the miRNA TaqMan PCR assay and the proliferation assay using MTS in Daoy, UW-228, HT-29, and MDA-MB231T cells. b MiR-199b-5p expression after 72 h in all of the cell lines analyzed: colon cancer CaCo-2, SW480, and HT-29 cells; breast cancer MCF-7 and MDA-MB231T cells; prostate cancer PC-3 cells; glioblastoma U-87 cells; and MB ONS-76, Daoy, and UW-228 cells. c Basal level of the Hes-1 protein in all of the cells analyzed: colon cancer CaCo-2, SW480, and HT-29 cells; breast cancer MCF-7 and MDA-MB231T cells; prostate cancer PC-3 cells; glioblastoma U-87 cells; and MB ONS-76, Daoy, and UW-228 cells (JPEG 86 kb)

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Supplementary Fig. 3

Expression of the Hes-1 protein and cytotoxicity effects of the SNALP miR-199b-5p and SNALP scramble treatments. a Densitometry of Hes-1 protein expression after treatments with SNALP miR-199b-5p and SNALP scramble for 72 h in colon cancer CaCo-2, SW480, and HT-29 cells; breast cancer MCF-7 and MDA-MB231T cells; prostate cancer PC-3 cells; glioblastoma U-87 cells; and MB ONS-76, Daoy, and UW-228 cells. Green line treatment with SNALP scramble, red line treatment with SNALP miR-199b-5p. b, c FACS analysis after 24 h of treatment with SNALP miR-199b-5p and SNALP scramble for the relative levels of annexin V in HT-29, Daoy, UW-228, and MDA-MB231T cells. d Caspase 3/7 activity after 72 h of treatment with 50 μg/ml SNALP miR-199b-5p and SNALP scramble in colon cancer CaCo-2, SW480, and HT-29 cells; breast cancer MCF-7 and MDA-MB231T cells; prostate cancer PC-3 cells; glioblastoma U-87 cells; and MB ONS-76, Daoy, and UW-228 cells. Green line treatment with SNALP scramble, red line treatment with SNALP miR-199b-5p, black line untreated cells (JPEG 139 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Pasqualino de Antonellis
    • 1
    • 2
  • Lucia Liguori
    • 1
    • 2
  • Annarita Falanga
    • 3
  • Marianeve Carotenuto
    • 2
  • Veronica Ferrucci
    • 2
  • Immacolata Andolfo
    • 2
  • Federica Marinaro
    • 2
  • Immacolata Scognamiglio
    • 4
  • Antonella Virgilio
    • 5
  • Giuseppe De Rosa
    • 4
  • Aldo Galeone
    • 5
  • Stefania Galdiero
    • 3
  • Massimo Zollo
    • 1
    • 2
    Email author
  1. 1.Dipartimento di Medicina Molecolare e Biotecnologie Mediche“Federico II” University of NaplesNaplesItaly
  2. 2.Biotecnologie AvanzateCEINGENaplesItaly
  3. 3.Dipartimento di FarmaciaFederico II University of NaplesNaplesItaly
  4. 4.Dipartimento di Chimica Farmaceutica e Tossicologica, Facoltà di FarmaciaFederico II University of NaplesNaplesItaly
  5. 5.Dipartimento di Chimica delle Sostanze Naturali, Facoltà di FarmaciaFederico II University of NaplesNaplesItaly

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