Delivery of Short Interfering Ribonucleic Acid-Complexed Magnetic Nanoparticles in an Oscillating Field Occurs via Caveolae-Mediated Endocytosis

Jenson Lim,Michael A Clements,Jon Dobson

doi:10.1371/journal.pone.0051350

Jenson Lim, Michael A Clements + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0051350

Copy DOI

Abstract

Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.

Highlights

Recent decades have seen the rise of gene delivery technologies to introduce foreign genes into highly differentiated cells like neurons or leukocytes, as such cells are known to be resistant to either accepting or expressing exogenous genes
Stably-expressing green fluorescence protein (GFP)-HeLa cells were transfected with short interfering RNA (siRNA) against GFP using Lipofectamine 2000, nucleofection technology and the oscillating magnet array system (Figure 2)
Cells transfected using Lipofectamine 2000, in accordance with manufacturer’s instructions, demonstrated good gene silencing (54.866.3%), though cell rounding was observed which correlated with reduced viability. These results demonstrate the ability of the oscillating magnet array system to achieve gene knock-down in a cell line with an over-expressed reporter gene

Summary

Introduction

Recent decades have seen the rise of gene delivery technologies to introduce foreign genes into highly differentiated cells like neurons or leukocytes, as such cells are known to be resistant to either accepting or expressing exogenous genes. Such technologies range from the relatively inexpensive lipid-based (e.g Lipofectamine) or non-lipid based (e.g. Fugene) reagents to more costly nucleofection (e.g. Amaxa) or gene gun (e.g. Helios) methods (reviewed in [1]). Negatively-charged nucleic acids are electrostatically associated to positively-charged, polymer-coated superparamagnetic iron oxide nanoparticles (SPIONs). These complexes are subjected to a strong high-gradient magnet field produced by arrays of permanent magnets sited underneath the cell culture plate. Our group has found that by introducing a linear oscillating motion to the magnet array, we can regulate the uptake of nanoparticle/ plasmid DNA complexes (Figure 1)

Methods

Results

Conclusion