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In Vitro Investigations of the Efficacy of Cyclodextrin-siRNA Complexes Modified with Lipid-PEG-Octaarginine: Towards a Formulation Strategy for Non-viral Neuronal siRNA Delivery

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Abstract

Purpose

Development of RNA interference based therapeutics for neurological and neurodegenerative diseases is hindered by a lack of non-viral vectors with suitable properties for systemic administration. Amphiphilic and cationic cyclodextrins (CD) offer potential for neuronal siRNA delivery. We aimed to improve our CD-based siRNA formulation through incorporation of a polyethyleneglycol (PEG) shielding layer and a cell penetrating peptide, octaarginine (R8).

Methods

CD.siRNA complexes were modified by addition of an R8-PEG-lipid conjugate. Physical properties including size, charge and stability were assessed. Flow cytometry was used to determine uptake levels in a neuronal cell model. Knockdown of an exogenous gene and an endogenous housekeeping gene were used to assess gene silencing abilities.

Results

CD.siRNA complexes modified with R8-PEG-lipid exhibited a lower surface charge and greater stability to a salt-containing environment. Neuronal uptake was increased and significant reductions in the levels of two target genes were achieved with the new formulation. However, the PEG layer was not sufficient to protect against serum-induced aggregation.

Conclusions

The R8-PEG-lipid-CD.siRNA formulation displayed enhanced salt-stability due to the PEG component, while the R8 component facilitated transfection of neuronal cells and efficient gene silencing. Further improvements will be investigated in the future in order to optimise stability in serum and enhance neuronal specificity.

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Abbreviations

CD:

cyclodextrin

CNS:

central nervous system

DIW:

deionised water

DLS:

dynamic light scattering

DMSO:

dimethylsulphoxide

DSPE:

1,2-distearoyl-sn-glycero-3-phosphoethanolamine

GAPDH:

glyceraldehyde phosphate dehydrogenase

MR:

mass ratio

mRNA:

messenger RNA

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

ns:

non-silencing

PAMAM:

polyamidoamine

PEG:

polyethyleneglycol

PLL:

poly-L-lysine

R8:

octaarginine

RLU:

relative luminescence units

shRNA:

short hairpin RNA

siRNA:

small interfering RNA

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Acknowledgments AND DISCLOSURES

The authors wish to acknowledge Science Foundation Ireland (Strategic Research Cluster grant no. 07/SRC/B1154), the Irish Drug Delivery Network and the Irish Research Council for Science, Engineering and Technology (scholarship to A.O’Mahony) for research funding. We also wish to acknowledge Dr. Matt Gooding for assistance with the manuscript.

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Authors and Affiliations

  1. Pharmacodelivery Group, School of Pharmacy, University College Cork, Cavanagh Pharmacy Building College Rd.,, Cork, Ireland

    Aoife M. O’Mahony, Aoife Quinlan & Caitriona M. O’Driscoll

  2. Centre for Synthesis and Chemical Biology, UCD Conway Institute University College Dublin, Dublin, Ireland

    Stephane Desgranges, Julien Ogier & Raphael Darcy

  3. Department of Pharmaceutical & Medicinal Chemistry, Royal College of Surgeons in Ireland, Dublin 2, Ireland

    Marc Devocelle

  4. Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland

    John F. Cryan

Authors
  1. Aoife M. O’Mahony
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  2. Stephane Desgranges
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  8. Caitriona M. O’Driscoll
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Correspondence to Caitriona M. O’Driscoll.

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Supplementary Figure 1

Size intensity profiles of (a) MR 20 0% R8 and (b) MR 20 20% R8 after incubation in FBS for 0, 2, 24 and 72 h. (JPEG 44 kb)

High resolution image (TIFF 1623 kb)

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O’Mahony, A.M., Desgranges, S., Ogier, J. et al. In Vitro Investigations of the Efficacy of Cyclodextrin-siRNA Complexes Modified with Lipid-PEG-Octaarginine: Towards a Formulation Strategy for Non-viral Neuronal siRNA Delivery. Pharm Res 30, 1086–1098 (2013). https://doi.org/10.1007/s11095-012-0945-8

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