Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Jiwaji, Meesbah, Sandison, Mairi E., Reboud, Julien, Stevenson, Ross, Daly, Rónán, Barkess, Gráinne, Faulds, Karen, Kolch, Walter, Graham, Duncan, Girolami, Mark A., Cooper, Jonathan M. and Pitt, Andrew (2014). Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells. PLoS ONE, 9 (6),

Abstract

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.

Publication DOI: https://doi.org/10.1371/journal.pone.0099458
Divisions: Life & Health Sciences > Pharmacy
Life & Health Sciences > Biosciences
Life & Health Sciences
Life & Health Sciences > Cellular and Molecular Biomedicine
Additional Information: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: EPSRC (Basic Technology Research Grant, grant number EP/E032745/1).
Uncontrolled Keywords: Agricultural and Biological Sciences(all),Biochemistry, Genetics and Molecular Biology(all),Medicine(all)
Full Text Link: http://www.plos ... al.pone.0099458
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Date: 2014-06-13
Authors: Jiwaji, Meesbah
Sandison, Mairi E.
Reboud, Julien
Stevenson, Ross
Daly, Rónán
Barkess, Gráinne
Faulds, Karen
Kolch, Walter
Graham, Duncan
Girolami, Mark A.
Cooper, Jonathan M.
Pitt, Andrew ( 0000-0003-3619-6503)

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License: Creative Commons Attribution


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