Optimization of DNA delivery by three classes of hybrid nanoparticle/DNA complexes

Qiu Zhong,Tarun K Mandal,Ronald B Luftig,Dakshina Chinta,Haifan Wang,Sarala Pamujula,Xin Yao

doi:10.1186/1477-3155-8-6

Abstract

Plasmid DNA encoding a luciferase reporter gene was complexed with each of six different hybrid nanoparticles (NPs) synthesized from mixtures of poly (D, L-lactide-co-glycolide acid) (PLGA 50:50) and the cationic lipids DOTAP (1, 2-Dioleoyl-3-Trimethyammonium-Propane) or DC-Chol {3β-[N-(N', N'-Dimethylaminoethane)-carbamyl] Cholesterol}. Particles were 100-400 nm in diameter and the resulting complexes had DNA adsorbed on the surface (out), encapsulated (in), or DNA adsorbed and encapsulated (both). A luciferase reporter assay was used to quantify DNA expression in 293 cells for the uptake of six different NP/DNA complexes. Optimal DNA delivery occurred for 105 cells over a range of 500 ng - 10 μg of NPs containing 20-30 μg DNA per 1 mg of NPs. Uptake of DNA from NP/DNA complexes was found to be 500-600 times as efficient as unbound DNA. Regression analysis was performed and lines were drawn for DNA uptake over a four week interval. NP/DNA complexes with adsorbed NPs (out) showed a large initial uptake followed by a steep slope of DNA decline and large angle of declination; lines from uptake of adsorbed and encapsulated NPs (both) also exhibited a large initial uptake but was followed by a gradual slope of DNA decline and small angle of declination, indicating longer times of luciferase expression in 293 cells. NPs with encapsulated DNA only (in), gave an intermediate activity. The latter two effects were best seen with DOTAP-NPs while the former was best seen with DC-Chol-NPs. These results provide optimal conditions for using different hybrid NP/DNA complexes in vitro and in the future, will be tested in vivo.

Highlights

The purpose of this study is to develop a new biodegradable non-viral vector system for the effective transfer of genes to cells and animals
After the adverse events which occurred in clinical trials using an RV vector that induced a lymphoproliferative disorder in 2002-2003 [7] due to insertional mutagenesis [8,9,10], concerns were raised about gene transfer with such a vector
Characterization of hybrid nanoparticle/DNA complexes PLGA based NPs prepared by the solvent evaporation method (Figure 1), with either DOTAP or DC-Chol showed a similar particle size distribution (Figure 3)

Summary

Introduction

The purpose of this study is to develop a new biodegradable non-viral vector system for the effective transfer of genes to cells and animals. Efforts have been directed to develop non-viral gene delivery systems, which include liposome nanoparticles [17,18], the “ballistic” gene gun [19,20], electroporation [21,22,23] and cationic lipid complexes with DNA [24,25,26,27,28] in vitro and in vivo All of these have been beset with issues of cytotoxicity, stability in serum or tissues and like viral vectors, in the duration of gene expression [29,30]. Particle size and safety issues with animals remain potential problems with these approaches

Objectives

Methods

Results

Conclusion