Elsevier

Biomaterials

Volume 31, Issue 1, January 2010, Pages 124-132
Biomaterials

Non-viral gene delivery of DNA polyplexed with nanoparticles transfected into human mesenchymal stem cells

https://doi.org/10.1016/j.biomaterials.2009.09.023Get rights and content

Abstract

Human mesenchymal stem cells (hMSCs) represent a potent target for gene delivery for both stem cell differentiation applications and clinical therapies. However, it has, thus far, proven difficult to develop delivery vehicles that increase the efficiency of gene delivery to hMSCs, due to several problematic issues. We have evaluated different vehicles with regard to the efficiency with which they deliver hMSCs and enhance the ability to deliver a reporter gene. In this study, a non-viral gene delivery system using nanoparticles was designed, with emphasis placed on the ability of the system to mediate high levels of gene expression into stem cells. Via polyplexing with polyethylenimine (PEI), the cell-uptake ability of the nanoparticles was enhanced for both in vitro and in vivo culture systems. In experiments with PEI/pNDA polyplexed with nanoparticles, the expression of green fluorescent protein (GFP) with this vehicle was noted in up to 75% of hMSCs 2 days after transfection, and GFP gene expression was detected via Western blotting, flow cytometric analysis, and immunofluorescence using a confocal laser microscope after transfection.

Introduction

Successful gene therapy challenge relies heavily on the fabrication of suitable delivery carriers that can efficiently deliver specific genes to the desired cells with minimum cytotoxicity to the target cells [1]. Viral vector systems have generally been identified as being useful in this regard, owing to their superior ability to deliver and express genes to target cells [2], [3]. However, the use of such viruses as gene delivery carriers is limited by several problematic issues; such systems are expensive to produce, generally of limited quality, frequently cause infection-related cell damage, and can also induce adverse immune issues [4]. In order to overcome these problems, non-viral vector systems have been designed to yield the same positive results, but avoid the aforementioned problems [5], [6], [7]. Therefore, much of the effort in the non-viral field has been devoted to the optimization and improvement of the transfection efficiency of these non-viral vectors [8].

In the field of stem cell biology, multipotent differentiation ability has remained an unsolved problem, and has limited the use of stem cells for cell therapy and tissue engineering protocols [9]. One promising approach would involve the use of genetically modified stem cells as promising vehicles for the targets of a variety of therapeutic molecules [10], [11]. Such stem cell-based gene therapy applications will depend principally on the efficient delivery of the specified gene into the targeted cells [12], [13].

Recently, nanoparticles have attracted a great deal of attention as potential candidates for non-viral gene delivery vehicles [14], [15], [16]. In gene delivery systems, nanoparticles are considered to be a desirable vehicle for the delivery of specific genes, as they can escape the EPR under in vivo conditions. Therefore, gene delivery for target tissues and cells using nanoparticles can be conducted using methods for coatings or conjugating genes that carry specific ligands that are recognized by cell membrane receptors [17]. When using nanoparticles, this unsolved problem has persisted, owing to the potential toxicity of the particles. In order to overcome this issue, the coating of biocompatible materials onto the nanoparticle surfaces with silica (SiO2) is an essential step in overcoming these issues limiting the use of nanoparticles in gene/drug delivery systems [18].

In order to increase the uptake and protection of the intact plasmid DNA (pDNA) into cells, DNA molecules carrying a negative charge have been complexed via electrostatic interaction with positively charged transfection materials. Polyethylenimine (PEI), one of a number of cationic polymers, has been utilized previously for complexation with pDNA, and the results observed with a polyplex including PEI and pDNA have suggested the promotion of gene expression in both in vitro and in vivo studies [19], [20], [21], [22]. Nevertheless, the use of PEI as in vitro and in vivo transfection reagent is limited severely by its cytotoxic effects. Although PEI evidenced a high potential for transfection efficiency, the cytotoxicity remained an unsolved problem [23].

Herein, we report the preparation of a system based on a modified nanoparticle vehicle with a PEI/DNA coating for specific gene delivery into human mesenchymal stem cells (hMSCs). This PEI/DNA coating approach to the nanoparticle vehicle enabled the fabrication of readily tunable non-viral gene carriers via the manipulation of coating conditions and various PEI:DNA ratios as desired for specific needs. The toxicity of PEI by the polyplexed PEI moiety coated on nanoparticles was reduced as the result of the shielding of the high positive charges to reduce toxicity. Several tools, including TEM, DLS, FACS, confocal laser microscopy, and bioimaging analysis evidenced high-efficacy transfection into hMSCs.

Section snippets

Nanoparticle characterization

Silica nanoparticles were purchased from Biterials (Korea). Nanoparticle size measurements were conducted using the Zetasizer Nano ZS apparatus (Malvern, Southborough, MA). In brief, the nanoparticles were suspended in deionized water at a concentration of 0.1 mg/ml. The mean hydrodynamic diameter was determined via cumulative analysis. The zeta potential determinations were predicated on the electrophoretic mobility of the nanoparticles in the aqueous medium, which were evaluated using folded

Results and discussion

Fig. 2A shows the particle sizes of the free-nanoparticles (a), PEI-coated nanoparticles (b), and PEI/pDNA-coated nanoparticles (c) measured via dynamic light scattering (DLS). The PEI/pDNA-coated nanoparticles were observed in an effort to gain a better understanding of the behavior of the polyplexes. The hydrodynamic diameter of the polyplexed PEI/pDNA nanoparticles was 102 nm, whereas the diameters of the nanoparticles and PEI-coated nanoparticles were 72 and 86 nm, respectively.

Fig. 2B showed

Conclusion

The results of this study show that nanoparticles polyplexed with plasmid DNA offer the potential ability to deliver genes for non-viral vectors. These polyplexes may prove to be a successful technique, as the cell-uptake ability of the nanoparticles was shown to enhance transfection efficiency into human mesenchymal stem cells.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0072002) & Pioneer Research Program for Converging Technology through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (M10711060001-08M1106-00110).

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