﻿Développement de nouvelles formulations d’antifongiques et évaluation de l’activité sur Candida spp. et Aspergillus spp.
The work done presented in this thesis was aimed to develop new formulations of antifungal drugs in order to improve the effectiveness and specificity of treatments. The formulations of polymeric NP encapsulated antifungals were tested on sensitive or resistant strains of Candida spp, Aspergillus spp and strains of Candida albicans that are able to develop biofilm structure. The first part of this work focussed on the synthesis and characterizations of a polyester-co-polyether polymer grafted with poly(ethylene glycol) (PEG-g-PLA). Besides being original and innovative, polymeric nanoparticles composed of PEG-g-PLA co-polymers have been used as drug delivery devices due to their biodegradable and biocompatible nature and controllable release characteristics. Three antifungal drugs (voriconazole, itraconazole and amphotericin B) commonly administered have been selected due to physicochemical properties and pharmacokinetics problems. In fact, these drugs are known to have significant delivery and toxicity issues. Polymeric nanoparticles were prepared by a high pressure homogenization oil-in-water emulsion method. Nanoparticles were designed in order to have an hydrophobic core (PLA) and a hydrophilic corona (PEG). Antifungal drugs were successfully incorporated into nanoparticles with appropiate mean diameters (~ 200 nm). Drug loading efficiency for VRZ was the lowest with 1,3% w/w of loading. A fraction of the VRZ was lost during the manufacturing process because of its solubility in water. However, AMB encapsulated in NP showed better loading efficiency (25,3% w/w). Indeed, the hydrophobic nature of PLA ensured a good affinity with the hydrophobic drugs, particularly for AMB who is the most hydrophobic of the selected drugs. Drug release study from new formulations showed a burst effet during 24 h followed by a prolonged release over several days. Furthermore, new formulation of AMB was tested on a cascade impactor, an in vitro lung model and successfully demonstrated the potential of this formulation to be administered effectively by the pulmonary route. Indeed, the results obtained showed that the majority of the formulation was found on the collection stage corresponding to the bronchial region, where is located predominantly pulmonary fungal infections. The second part of the thesis was aimed at testing the new formulations on planktonic strains of Candida spp and Aspergillus spp. and biofilm structure of Candida albicans according to NCCLS standardized procedures. The selected strains have demonstrated resistance to azoles and polyenes. Studies in vitro have proven that the new formulations offer significantly improved effictiveness compared to free antifungal agent. To evaluate if improved antifungal effectiveness was due to internalization of NP, we evaluated the behavior of NP with fungal cells. We conducted qualitative studies in fluorescence microscopy on NP labeled with rhodamine. As expected, the NP showed intracellular localization. To exclude the possibility of NP absorption on the surface of yeast cells, we also confirmed internalization by confocal fluorescence microscopy. Noteworthy, only a few studies have focused on the development of new formulations of antifungal drug, giving great value and originality of this work. Encouraging results pave the way to a new approach to overcome fungal resistance issues, a problem more acute in the field of fungal diseases.
PEG-g-PLA Candida spp Aspergillus spp biofilms Polymeric nanoparticles PEG-g-PLA antifungal drugs Candida spp Aspergillus spp biofilms Cellular internalization