Étude de propriétés physico-chimiques des membranes lipidiques chargées d’acide palmitique/stérol et de stéarylamine/cholestérol

Sterosomes are artificial vesicles that are composed of monoalkylated amphiphiles and a large percentage of sterols. They are promising in areas such as pharmaceutical and food industries. Sterosomes can be found in anionic, cationic and neutral form. The work of this master’s thesis focuses on gaining additional knowledge on the physicochemical properties of charged sterosomes such as palmitic acid (PA)/sterol and stearlyamine (SA)/cholesterol (Chol). Our first aim was to find an approach to form liquid-ordered (lo) bilayers that can orient in a magnetic field with the PA/sterol (30/70 mol/mol) mixture. This will allow us to study the lateral diffusion of PA. It has been demonstrated that mixing 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), a short chain lipid, with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) results in bilayers that orient with their normal (n) perpendicular to the magnetic field. Therefore, perdeuterated PA/hexanoic acid (HA)/Chol (25/18/57) mixture was studied at different temperatures; however, results showed no bilayer orientation. It was suggested that this could be due to the partial solubility of HA in aqueous phase. Consequently, an insufficient quantity of HA was available to induce bilayer orientation. Furthermore, perdeuterated PA/DHPC/Chol mixture was studied at different temperatures and lipid concentrations, which also led to no bilayer orientation. While studying DMPC/DHPC/Chol (67/17/14) formulation, it was understood that Chol inhibits bilayer orientation. On the other hand, two different molar proportions of DMPC/DHPC/stigmasterol (SS) (67/19/14 and 72/21/7) led to bilayers that orient with their n perpendicular to the magnetic field at 40 °C et 50 °C. These results suggest that by adding a short chain lipid such as HA and DHPC to PA/SS mixture could lead to oriented bilayers. Another interesting track would be to work with PA/short chain lipid/Chol mixture in the presence of lanthanides. Our second aim was to find different formulations of vesicles, containing at least PA and a sterol, with distinctive permeability. It has already been established that PA/Chol (30/70) bilayers are in lo phase as long as PA is deprotonated (pH ≥ 7,5) and have a very limited permeability. The ability to modulate permeability would allow control over the release time of an encapsulated product, which is an important parameter in the development of novel liposomal drug delivery systems. It was proven that oleic acid (OA)/Chol (30/70) mixture is able to form bilayers, which has not been shown previously. However, OA/Chol (30/70) bilayers were not much more permeable than PA/Chol (30/70) bilayers. Adding 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in PA/Chol system did not lead to higher permeability either. Results of calcein release kinetics from PA/POPC/Chol (15/27.5/57.5), POPC/Chol (40/60) and POPC vesicles were not very different from the one found for PA/Chol (30/70) vesicles. It was noted that contradictory results were found in the literature regarding calcein permeability from phosphatidylcholine (PC) membranes. For the moment, explanations related to these divergences have yet to be given. Furthermore, no increase in membrane permeability was found after substituting half of the molar proportion of Chol by sodium cholate (SC) in PA/Chol (30/70) mixture. One suggestion would be that SC insertion in the bilayer does not induce a reduction in the packing of lipids. Another suggestion would be that SC does not insert in PA/Chol bilayers due to its considerable hydrophilic character and forms micelles on its own. Highly permeable membrane was found when Chol was completely replaced by cholesterol sulfate (SChol), a negatively charged sterol, and vesicle preparation was done at low pH. The pH-triggered release method was used. Our work suggests that by varying the molar proportion of PA/SChol/Chol mixture between 30/63/7 and 30/70/0, it would be possible to obtain different vesicle formulations with distinctive permeability. In between these molar proportions, the pH-composition diagram of PA/SChol/Chol shows the coexistence of solid and lo phases in different proportions at pH 7.4, which could modulate the permeability. Some ambiguous calcein release results and struggles arising from the extrusion of calcein-encapsulated liposomes led us to wonder if this dye interacts with charged bilayers. The impact of some anions, including calcein, was examined on SA/Chol (50/50) charged bilayers. Thermodynamic studies were done by differential scanning calorimetry (DSC). SA/Chol (50/50) mixture showed no transition between 25 and 90 °C at pH 7.4. In the presence of sodium chloride (375 mM), there was no indication of aggregation or the appearance of a transition on the thermogram. Macroscopic aggregates were instantly observed after addition sodium hydrogenphosphate (125 mM), sodium sulphate (125 mM) and calcein (3 mM). Furthermore, a phase transition was also noticed on the thermograms in the presence of these salts. It is suggested that the appearance of the transition can be associated with the formation of aggregates. The effect of anions on the transition temperature and enthalpy follows the Hofmeister series: sulfate > hydrogenphosphate > chloride (no peak). Calcein had the highest impact on the formation of aggregates. This indicates that calcein is not a good candidate to be used as a fluorescent dye with SA/Chol mixture. It was suggested that calcein could be a large chaotrope anion. In contrast to PC-calcein interactions, SA-calcein interactions led to the aggregation of vesicles probably due to stronger interactions in the presence of positively charged SA.

Keyphrases: Permeability, Bilayers oriented under magnetic field, Palmitic acid, Oleic acid, Cholesterol, Stearylamine, Calcein, Hofmeister series