Lipoplex nanostructures reveal a general self-organization of nucleic acids

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Abstract

Background

Lipid and plasmid DNA complexes (Lx) were designed for gene transfer and were studied comprehensibly to elucidate their formation and ultrastructure.

Methods

We compared supramolecular self-assembly into stable Lx containing nucleic acids of various types and lengths using Cryo-Electron Microscopy, Small Angle X-ray Scattering and dynamic light scattering.

Results

Analysis of these complexes showed that they reproducibly formed monodisperse and spherical multilamellar particles. The same concentric and lamellar structure with different packing regimes was produced by circular double-stranded DNA, linear double-stranded DNA, single-stranded DNA, oligodeoxynucleotides or RNA. Strikingly, thousands of oligonucleotide molecules seem to align with one another and to behave as longer nucleic acid molecules in forming structurally similar particles. Neither excess cationic lipids nor excess DNA of different forms changed significantly the mean diameter and the size distribution of Lx particles. This suggests a role for Lx formation of steric size in addition to the conventional thermodynamic mechanism. The Lx ultrastructure is highly ordered and crystalline and is in a lamellar and/or hexagonal phase. Increasing NA size led to an increased proportion of Lx in a hexagonal structure phase as in the case of T4 phage virus DNA. These observations were made using two Lx made from different lipids exhibiting negative and positive charged surface. We also demonstrated structural similarities between the supramolecular auto-organization of Lx and that found in some viruses. In particular, both synthetic and viral particles have an ultrastructure that exhibits a phase transition between lamellar and hexagonal phases.

General significance

Taken together, our data point towards the possible existence of a ubiquitous organization of genetic materials, at least with cationic lipids, that has implications for both therapy and the origins of life.

Introduction

Nucleic acids (NA) of very different lengths and compositions are under intense investigation as they constitute new powerful and specific tools for therapy. The most exciting strategies for therapy include plasmid DNA (pDNA) for gene transfer, small interfering RNA (siRNA) for inhibiting expression of a target gene, and antisense oligonucleotides for correction of the splicing alterations encountered in many genetic diseases. In general, NA transport is limited by their lability in vivo. pDNA in particular must be delivered totally intact to the nucleus of the target cell to enable expression of the transgene. The successful implementation of these genetic strategies – especially in the case of “in vivo” strategies – will rely on the development of an efficient cell delivery system. By combining such a delivery system with a synthetic vector as carrier, a safe and durable, drug-like gene therapy could be created. Potential methods of gene delivery that could be employed in this therapy include NA/polymer complexes [1] or NA/cationic lipid complexes (Lipoplex, Lx, [2]) [3], [4], [5], [6].

pDNA/cationic lipid complexes based on electrostatic interactions were first designed in 1987 [3]. Although there are numerous, commercially available, reagents that effectively transfect cells cultured in vitro [3], [4], [8], only a few Lx systems have been used successfully to transfer genes in vivo, especially following systemic administration [5], [6].

To deliver NA using various synthetic vectors, our first priority has been to obtain pharmaceutically suitable vectors in terms of stability and reproducibility. We have designed and developed an efficient lipidic vector termed DLS [6]. The intracellular distribution and uptake of DLS containing different types of oligodeoxynucleotides (ODN), some of which were modified, was studied in several cell culture systems [7], [8]. Our observations were consistent with complete release of the DNA from the endocytic vesicles and are therefore consistent with the complete or partial release of the DNA from the lipid carrier [9]. We showed that systemic administration of plasmid DNA led to widespread and long-lasting reporter gene expression [6]. We further demonstrated increased pDNA half-life in plasma and efficient uptake in blood cells following intravenous administration in mice [9]. The DLS (Delivery Liposomal System) system was developed for transgene expression [9] and used in various experimental therapeutic models for gene transfer such as human MDR1 in vivo expression in mouse bone marrow progenitor cells [10].

ODN delivery by the DLS system in different cell cultures in vitro was used to study, for example, inhibition of HIV [7]. Recently, we used antisense ODN directed against VEGF RNA to treat AIDS-related, Kaposi's Sarcoma, both in vitro and in vivo [11]. Significant effects (39% inhibition) were observed at nanomolar doses (0.010 μM). This result confirms those previously obtained in HIV culture systems showing effects even at subnanomolar concentration. This level of ODN activity is unprecedented and illustrates the potential of the DLS system for ODN delivery. In order to enhance stability in serum following systemic administration we have designed the first globally anionic charged lipoplex which we term Neutraplex (Nx) [11]. Use of the Nx delivery system improved significantly the bioavailability and the ODN pharmacokinetics profile using whole body Positron Emission Tomography (PET) and an enzyme-based competitive hybridization assay [12]. Anionic vectors therefore constitute promising delivery systems for in vivo administration of therapeutic ODN. Previously, we and others observed a highly ordered liquid crystalline structure in Lx composed of different cationic lipids [11], [14], [15], [16], [17], [18], [19], [20], [21]. Here, we compare directly the supramolecular assembly into stable Lx of NA of various types and lengths, and of different lipid composition and global net charge and draw a general conclusion about NA self-organization and condensation.

Section snippets

Nucleic acids

Long linear double strand DNA consisted of the T4 phage DNA (169,372 base pairs). Plasmid DNA (pDNA) is the pBKd1RSV-luc construct (10,400 base pairs) previously designed and used as an episomal/reporter expression vector containing the luciferase gene [6]. Linear single strand DNA (M13rmp8, 7229 bases) is the DNA from M13 bacteriophage derivatives. Linear double strand DNA (M13mp8; 7229 base pairs) is the intracellular replicative form of the M13mp8 phage produced by chronic infection in

Components before complex formation

In attempt to mutually condense lipids with NA the first approach was to prepare liposome-like particles. Several lipids have been used in attempts to prepare liposome-like particles. One such lipid mixture is Lipofectin TM (Invitrogen Corp, Carlsbad, CA) which is formed with the cationic lipid DOTMA, N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride, and DOPE, dioleylphosphatidyl ethanolamine at a 1:1 molar ratio. The lipid particles prepared with this formulation spontaneously

General significance

Self-organization in general, refers to the various mechanisms by which pattern, structure and order emerge spontaneously in complex systems. We propose that the formation of Lx particles results from the universal tendency of NA to self-organize with cationic lipids and that this emergence is one reason to consider them as complex systems [50]. Studying the physico-chemical properties of lipoplexes may help in understanding better such phenomena as nuclear condensation/decondensation,

Acknowledgements

The authors thank Durand D. for performing SAXS analysis, and F. Livolant, F. and Steven A. for helpful discussions. We are grateful to Lepault J. and Steven A. for providing a cryoEM image of T4 and T7 particles, respectively.

Glossary

NA
Nucleic Acids
Lx
Lipoplex
ODN
oligodeoxynucleotides
VEGF
Vascular Endothelial Growth Factor
siRNA
small interfering RNA
miRNA
micro RNA
pDNA
Plasmid DNA
DOPE
dioleoyl Phosphatidyl Ethanolamine
SUV
small unilamellar vesicles
DLS
Delivery Liposomal System
Nx
Neutraplex
cryoEM
Cryo-Electron Microscopy
DOGS
Spermine-5-carboxy-glycinediotade-cylamide
SAXS
Small Angle X-ray Scattering
ssDNA
single-stranded DNA
dsDNA
double-stranded DNA

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