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Complexes for gene delivery

par Guillaume Tresset - publié le

 

 

COMPLEXES for GENE DELIVERY


Guillaume Tresset
  • 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.


Nanoparticle made of nucleic acids complexed with lipids. (Left) Cryo-electron micrograph showing the hexagonal arrangement of nucleic acids within the nanoparticle. (Right) Monte Carlo simulation illustrating the local crystallization of flexibles tubes as a model of nanoparticle. [J. Phys. Chem. Lett. 2 (2011) 41]

  • 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".


Self-assembly of a hexagonal lipid-DNA complex by Monte Carlo simulations. The DNA rods, represented in blue, are fixed on a hexagonal lattice during the simulation. Zwitterionic lipids and divalent cations (yellow spheres) are randomly distributed at the initial stage (inset). [J. Phys. Chem. B 111 (2007) 14233]


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.


Transfection of U87 with DOPA and spermine (spr4+). (A) Relative transfection efficiency as a function of spermine concentration. Inset is the efficiency obtained in the absence of lipids. (B) Fluorescence image of U87 cells transfected with plasmid DNA encoding GFP, by DOTAP/DOPE (top) and DOPA/spr4+ (bottom), after 3 days of culture. [Biophys. J. 93 (2007) 637]

REFERENCES



B. MAURY, C. GONCALVES, G. TRESSET, M. ZEGHAL, H. CHERADAME, P. GUEGAN, C. PICHON, P. MIDOUX (2014) Influence of pDNA availability on transfection efficiency of polyplexes in non-proliferative cells. Biomaterials 35 5977-5985.


G. TRESSET, Y. LANSAC (2011) Long-range architecture of single lipid-based complex nanoparticles with local hexagonal packing. J. Phys. Chem. Lett. 2 41-46.


G. TRESSET, W.C.D. CHEONG, Y.M. LAM (2007) Role of multivalent cations in the self-assembly of phospholipid-DNA complexes. J. Phys. Chem. B 111 14233-14238.


G. TRESSET, W.C.D. CHEONG, Y.L.S. TAN, J. BOULAIRE, Y.M. LAM (2007) Phospholipid-based artificial viruses assembled by multivalent cations. Biophys. J. 93 637-644.