Supramolecular assemblies of lipid-coated polyelectrolytes
By using high-resolution cryo-electron microscopy, we have demonstrated that the internal structure of single nanoparticles made of nucleic acids complexed with lipids is less ordered than commonly recognized from X-ray diffraction. Besides, we have devised a coarse-grained model of self-avoiding flexible tubes mimicking the lipid-coated nucleic acids and interacting via a short-range attractive potential : consistently with cryo-electron microscopy, the resulting clusters exhibit a varying degree of order ranging from weakly organized aggregates to partially organized spooled and straight tubes, depending on the length and the rigidity of the tubes. These findings may help in the design of novel vectors for efficient gene transfection or in the fabrication of lipid-based nanostructured biomaterials.
Non toxic lipid-based gene delivery systems
The compaction of DNA – and more generally, of nucleic acids – is a ubiquitous phenomenon in biological systems carrying genetic information. Because DNA is negatively charged under physiological conditions, a strong electrostatic barrier of repulsion must be overcome to accommodate it into the confined space of a cell nucleus or of a viral capsid. In the particular application of non-viral gene delivery, the tight packing of nucleic acid, indispensable for an efficient uptake by cells, is generally achieved by complexation with a positively charged entity such as polymers, peptides, lipids, or a combination thereof. Cationic lipid-DNA complexes have been intensively studied as promising nonviral gene delivery vectors, but they still suffer from a low efficiency of DNA transfer compared to their viral counterparts and more importantly, a high induced toxicity to cells.
In spite of unfavorable electrostatic interactions, DNA and noncationic lipids such as phospholipids have been complexed into an ordered liquid-crystalline structure upon the mediation of multivalent cations. Coarse-grained Monte Carlo simulations supported the hexagonal arrangement inferred from X-ray diffraction experiments and showed that lipids self-assemble to form inverted micelles around DNA rods with multivalent cations acting as “molecular glue”.
Phospholipid-DNA complexation is not limited to inorganic ions. Tetravalent polycation spermine (spr4+) is found in a wide variety of organisms and tissues, and is in particular exploited by bacteriophage T4 and herpes simplex virion as a compacting agent to accommodate DNA in their tiny viral capsid. In vitro, spermine is also known to precipitate pure DNA or in a mononucleosome core particle state. We assembled phospholipid-DNA complexes that are inspired by these above-mentioned natural viruses. The figure below shows the very high efficiency exhibited by the combination DOPA-spr4+ on U87 cells : an 18-fold higher expression of luciferase than that achieved with cationic DOTAP/DOPE, for just 500 mM of spermine. This outcome was confirmed under fluorescence microscope by expressing green fluorescent protein instead of luciferase in cells. Even in the presence of serum and transfecting directly in supplemented culture medium, the luminescence assay yielded better readings because the complexes were in contact with cells for longer durations. In the absence of DOPA or cations, the level of transfection was negligible.
- G. Tresset, Y. Lansac, G. Romet-Lemonne, Supramolecular assemblies of lipid-coated polyelectrolytes, Langmuir, 28 (2012) 5743-5752
- G. Tresset, Y. Lansac, Long-Range Architecture of Single Lipid-Based Complex Nanoparticles with Local Hexagonal Packing, J. Phys. Chem. Lett., 2 (2011) 41-46
- G. Tresset, W.C.D. Cheong, Y.M. Lam, Role of Multivalent Cations in the Self-Assembly of Phospholipid-DNA Complexes, J. Phys. Chem. B, 111 (2007) 14233-14238
- G. Tresset, W.C.D. Cheong, Y.L.S. Tan, J. Boulaire, Y.M. Lam, Phospholipid-Based Artificial Viruses Assembled by Multivalent Cations, Biophys. J., 93 (2007) 637-644