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

Jiwaji, Meesbah and Sandison, Mairi E. and Reboud, Julien and Stevenson, Ross and Daly, Rónán and Barkess, Gráinne and Faulds, Karen and Kolch, Walter and Graham, Duncan and Girolami, Mark A. and Cooper, Jonathan M. and Pitt, Andrew R. (2014) Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells. PLOS One, 9 (6). e99458. ISSN 1932-6203

[img]
Preview
PDF (journal.pone.0099458)
journal.pone.0099458.pdf
Final Published Version
License: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 logo

Download (751kB)| Preview

    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.