Formation et caracterisation physico-chimique des complexes ADN/chitosane pour la therapie genique.

摘要

Chitosan is a prominent natural polymer used in nonviral gene delivery, due to its biocompatibility and biodegradability. It can condense DNA through electrostatic interactions to form nanoparticles that can be internalized by cells. In the first part of this thesis, the interaction of chitosan with plasmid DNA was investigated as a function of pH, buffer composition, degree of deacetylation (DDA) and molecular weight of chitosan, using isothermal titration microcalorimetry (ITC). The chitosan-DNA interaction was shown to be coupled with proton transfer from the buffer to chitosan. This proton transfer is induced by the strong polyanionic nature of DNA which facilitates the ionization of glucosamines of chitosan upon binding. The measured enthalpy of binding was almost entirely due to the ionization changes of the buffer and of chitosan. The chitosan-DNA binding constant was found in the range of 109-10 10 M-1. The binding constant was pH-dependent and was greater at lower pH due to increased electrostatic attraction to DNA when chitosan is highly charged. The binding constant between chitosan and plasmid DNA was significantly influenced by molecular weight and by DDA. The electrostatic effects were found to dictate the binding of chitosan to DNA. The results of this study provide insights into previously measured dependence of transfection efficiencies of DNA/chitosan complexes on chitosan DDA and molecular weight, where a balance between complex stability and chitosan-DNA binding strength was suggested to play a critical role.;The subsequent part of this thesis treats the characterization of different preparations of DNA/chitosan complexes by the AF4 combined system. Parameters known to influence the transfection efficiency of DNA/chitosan complexes were investigated, including the DNA concentration at mixing, the ratio of chitosan amine to DNA phosphate (N/P) used in the preparations, the chitosan molecular weight, and its degree of deacetylation. We found that all preparations yielded similar ranges of particle hydrodynamic radii (15 ≤ R H ≤ 160 nm) but that differed in size distribution. Either an increase of the DNA concentration at mixing or an increase of chitosan molecular weight generated the formation of a higher fraction of larger particles ( RH > 60 nm) in the dispersions. The dispersions contained a majority of free chitosan in solution that was separated from the nanoparticles and quantified by the AF4 combined system. The free chitosan content was 53 to 92% in dispersions prepared with N/P ratios from 3 to 15, respectively, corresponding to an N/P ratio in the particles that was almost constant (1.3 to 1.6). The accuracy of the free chitosan determination by AF4 was confirmed by ultracentrifugation of the dispersion and analysis of the supernatant. This study reveals the utility of AF4 in the analysis of DNA/polycation dispersions and the importance of quantifying and understanding the role of the free polycation component in these nonviral gene delivery systems.;In the last part of our work, we assessed the stability of DNA/chitosan complexes upon exposure to hyaluronan (HA), chondroitin sulfate (CS), and heparin (Hp). Fluorescence spectroscopy was used and Picogreen was selected as the probe to quantify the release of DNA. Only the highly charged heparin was found to destabilize the DNA/chitosan complexes and release DNA in solution. The ability of the competing polyanions to release DNA from the DNA/chitosan complexes was related to the binding affinities of chitosan with the different negatively charged polyelectrolytes (including DNA). The stability of the DNA/chitosan complexes exposed to heparin increased with chitosan DDA and molecular weight, in agreement with increasing binding affinities previously determined by ITC. Heparin was unable to dissociate the complexes in dispersions with a significant amount of free chitosan. This amount of free chitosan was found to be sufficient for binding to both DNA and heparin. These findings suggest that free polycation can prevent premature dissociation of DNA/polycation complexes upon interactions with anionic components in the extracellular matrix.;We then report a new approach to characterize DNA/polycation complexes using asymmetrical flow field-flow fractionation (AF4) coupled online with UV/Vis spectroscopy, multi-angle light scattering (MALS), and dynamic light scattering (DLS). We demonstrated that this AF4 combined system can provide in a single measurement, three important physicochemical parameters of the complexes: the amount of unbound polycation, the hydrodynamic size of the complexes, and their size distribution. The accuracy of the particles sizes was confirmed by comparison with data from batch-mode DLS and scanning electron microscopy. Accurate quantification of unbound polycation can provide insight into the contribution of the free polycation in the process of gene delivery.