ABSTRACT
Self-assembled DNA delivery systems based on cationic lipids are simple to produce and weakly hazardous in comparison with viral vectors, but possess a significant toxicity at high doses. Phospholipids are in contrast intrinsically safe; yet their association with DNA is problematic because of unfavorable electrostatic interactions. We achieve the phospholipid-DNA complexation through the like-charge attraction induced by cations. Monovalent cations are inappropriate due to their poor binding affinity with lipids as inferred from electrophoretic mobility, whereas x-ray diffractions reveal that with multivalent cations, DNA is complexed within an inverted hexagonal liquid-crystalline phase. Coarse-grained Monte Carlo simulations confirm the self-assembly of a DNA rod wrapped into a lipid layer with cations in between acting as molecular glue. Transfection experiments performed with Ca^sup 2+^ and La^sup 3+^ demonstrate efficiencies surpassing those obtained with optimized cationic DOTAP-based systems, while preserving the viability of cells. Inspired by bacteriophages that resort to polycations to compact their genetic materials, complexes assembled with tetravalent spermine achieve unprecedented transfection efficiencies for phospholipids. Influence of complex growth time, lipid/DNA mass ratio, and ion concentration are examined. These complexes may initiate new developments for nontoxic gene delivery and fundamental studies of biological self-assembly.
INTRODUCTION
Gene therapy suffers from a lack of efficient and nontoxic delivery systems (1). DNA is traditionally delivered by either viral or nonviral vector-mediated systems (2). Viral methods are by far the most efficient in ternis of delivery’ and expression, owing to the highly evolved and specialized com ponents of natural viruses (3,4). They present, however, a number of restrictions due to the induced toxicity and immunogenicily, the limited size of DNA that can be carried, the lack of specificity, and the high cost of production (5). Considerably easier to prepare and saler to use, artificial viruses are made of self-assembling complexes of DNA with positively charged molecules such us lipids. polymers, peplides, or combinations thereof; yet two major difficulties have limited their success so far in clinical applications (5-8): i), low transfeclion efficiencies (TB) in comparison with (hat of their viral counterpart, and ii). problems of carrier toxicity. elimination, and biodegradability, especially occurring at high injected doses when increasing the total amount of delivered DNA.
A particular emphasis has been given to lipids because they are the main constituents of cell and organelle membranes for all living organisms; accordingly, numerous cationic lipids have been synthesized to date for the delivery of nucleic acids into cultured cells (9-11). as well as in clinical trials (12-14). Despite a few successful and promising attempts in vivo, the toxicily arising from these synthetic materials has been hampering their use in the pharmaceutical industry. Strong administered doses and high lipid charges are generally more toxic to a variety of cell types including cancer cell lines. Cell shrinking, inflammatory reactions, and immunoioxicity are among the many harmful effects associated with cationic lipids (15).
Unlike positively charged lipids thai are only found in eMremely small amounts in certain tissues (16), phospholipids, either anionic or zwitterionic, are ubiquitous in cell membranes and may thereby consiitute bet 1er candidates for lipkl-based deliver) systems. The challenge is to achieve the complcxation between nucleic acids and phospholipids, given their low affinity arising from unfavorable electrostatic interactions. Several x-ray diffraction studies have revealed ordered phases in which DNA was complexcd within liquidcrystalline structures of phospholipids through the mediation of divalent cations (17-20). Interestingly, the geometry of these complexes was identical to thai observed with cut ionic lipids, that is. lamellar and inverted hexagonal structures (21 ). These findings support the capability of phospholipids to compact nucleic acids under certain conditions. Very recently, phosphalidylcholine lipids could transfer plasmid DNA inlo mouse librohlasts in the presence of bivalent metal cations. Unfortunately, the transfcction efficiency achieved was loo low for practical applications (22).
In mis work, we report highly efficient phospholipid-DNA complexes assembled by using multivalcnt cations including !rivaient and telravaleni calions. We also attempt to shed light on the mechanisms underlying the self-assembly process, as well as to identify the key paramelers necessary to achieve high delivery performances. We first investigate the binding properties of mullivalent cations wiih phospholipid membranes and correlate them with the complexalion of DNA. X-ray diffractions and Monte Carlo simulations enable us to get some insight into the assembly of complexes at a molecular level. We next monitor lhe transfection efficiencies ol’ complexes imule of various phospholipids and mullivalent calions, and compare llicm Io a cationic mixture of lipids. The cylotoxicity on cultured cells is also assessed. Then, we seek lhe factors affecting lhe formation of complexes, and analyze their influence on the transfcction performances. We finish with a summary on the supratnolecular siruclure of complexes, the key parameters for gene delivery, and on further possible improvements.
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