Fine tuning of mixed ionic and hydrogen bond interactions for plasmid delivery using lipoplexes

Highlights

• Mixed lipoplexes were formed between cationic lipids, lipothiourea lipids and DNA.

• Lipoplexes involved hydrostatic and hydrogen bonds to condense DNA.

• Hydrogen bonds allowed to reduce the overall cationic content of the lipoplexes.

• This mixture increased DNA transfection at lower cationic charge content.

• The cytotoxicity was reduced as compared to cationic lipoplexes.

Abstract

Non viral gene transfection has been mostly reached via cationic polymer and lipid, required for DNA complexation and cell internalisation. However, cationic charges often induce cytotoxicity and limit the efficacy of the lipoplexes in vivo due to their fast elimination from the blood stream. Few years ago, we had developed noncationic lipid interacting with DNA via hydrogen bond interactions. To take advantage of both the internalisation efficacy of cationic complexes and the higher DNA release efficacy of non cationic lipids, we chose to mix both ionic and hydrogen bond interactions within one lipoplex. The idea behind this strategy would be to reduce the overall charge while maintaining a high level of transfection. Four mixed formulations of cationic lipid and thiourea lipid were prepared. We found that decreasing ionic interactions and increasing hydrogen bond interactions improved cationic lipoplexes properties. Indeed, we showed that replacement of net positive charges by hydrogen bond interactions with DNA phosphates led to efficient lipoplexes for in vitro DNA transfection at lower cationic charge content, which consequently reduced lipoplex cytotoxicity.

Introduction

Nonviral gene transfection could be of high interest to combine safe and efficient nanotechnologies. However, this is not fully achieved yet, mainly because of their lower efficiency as compared to viral vectors. Nevertheless, improvement is still achievable, and few nonviral gene therapy entered clinical trial recently [1]. Few years ago, we had hypothesised that the cationic charge used for DNA interaction beared by either lipids or polymers was the tree which hid bits. Indeed, in vitro transfections were highly efficient for all cationic complexes performed without serum, but led to inefficient in vivo transfections. The reason for this was the extremely fast elimination of complexes from the blood stream. In this context, lipofectamine, PEI, DOGS [2] and our own lipids amongst which DMAPAP were developed [3]. Various strategies were then proposed to reduce the overall charges, such as PEG shielding [4], PEG shielding with anionic PEG [5] and postgrafting [6]. Moreover, interest of tunable lipids neutral at physiological pH and becoming charged at lower pH fulfilled this hypothesis that complexes should present a lower cationic overall charge [7], [8]. Our choice was to develop noncationic lipids able to interact with DNA phosphates via hydrogen bonds [9]. The shape of the so-called lipothiourea lipids is strictly copied from the shape of cationic lipids, in which amines were replaced by thiourea functions [10]. This family of lipids evolved towards the years to yield a lipid quite efficient to transfect in vitro despite its low internalisation into the cells [11]. The ability of lipothiourea to transfect cells efficiently was attributed to the fact that DNA was released very efficiently into the cells thanks to the low binding forces between thiourea and phosphates functions. Based on the very high efficiency of cationic lipids to transfect cells and the capacity of lipothiourea to interact less tightly with DNA, we thought that a mixture of cationic lipids and lipothiourea could be of great interest to reduce the lipoplexes overall charge while providing a nanovector able to transfect efficiently. Noteworthy, combining cationic and hydrogen bonds for DNA interaction via thiourea function have been proposed on two other nanosystems recently reported in the literature. First, lipidic cyclodextrins combining on its hydrophilic face both cationic and thiourea functions have shown higher transfection efficiency [12], [13], [14]. More recently, polyethylene imine whose charges have been masked by methylthiourea have been proposed [15].

The aim of this work was to compare various combinations of cationic and lipothiourea lipids, starting from the more cationic to the less cationic, to evaluate the interest of this combination in terms of nanoparticle surface charge, DNA interaction, in vitro gene transfection and cytotoxicity.