Résumé :
|
Communication n° 659 The success of gene therapy largely relies on the availability of vectors that would deliver the genetic material efficiently to the target cells with a minimal toxicity. Although many nonviral vectors have already been proposed, none of them fully satisfies these requirements. This is largely because the relationship between the physicochemical properties of the DNA/carrier complexes, their intracellular pathway and their transfection efficiency is largely unknown. In this context, our purpose was to evaluate as possible vectors a series of newly synthesized low molecular weight (5 kDa) chitosan derivatives grafted with dodecenoyl (DDC) groups at different percentages (3, 9, 16 and 25 %). In the absence of DNA, the Critical Micellar Concentration (CMC) of these derivatives in 20 mM MES buffer pH 6.5 was found to be strongly dependent on the percentage of DDC but not on pH or salt concentrations. This indicates that the DDC groups confer to the chitosan derivatives the potency to self-assemble probably in micellar structures: a property that may dictate the formation and the structure of their complexes with DNA. Moreover, from phase transition experiments with large unilamellar vesicles (LUV) taken as model membranes, it was found that the DDC-chitosan derivatives interact with negatively charged DMPG vesicles but not with neutral DMPC vesicles. This suggests that these derivatives may preferentially interact with the internal leaflet of the plasma membrane. Next, we investigated by quasielastic light scattering the size and the surface charge of complexes of plasmid DNA with these derivatives at different pH, salt concentrations and N/P ratios (expressed in charged units of chitosan amines to DNA phosphates). We found the smallest and more positively charged complexes were obtained at pH 5.8 and N/P=5 in the absence of salt: a condition where the chitosan derivatives were fully protonated and in excess over the DNA phosphate groups. Additional studies are currently performed to further investigate the physicochemical properties of these complexes as well as their transfection efficiency and intracellular pathway.
|