Cécile Grabielle-Madelmont

Vesicle reconstitution from lipid-detergent mixed micelles.

The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and reconstitution of liposomes participated to our knowledge of the structure and properties of mixed lipid-detergent systems. The mechanisms involved in both the lipid self assembly and the micelle-vesicle transition are first reviewed. The simplistic three step minimum scheme is described and criticized in relation with isothermal as well as a function of the [det]/[lip] ratio, phase diagram explorations. The techniques of detergent elimination are reviewed and criticized for advantages and disadvantages. New methods inducing micelle-vesicle transition using enzymatic reaction and T-jump are also described and compared to more classical ones. Future developments of these techniques and improvements resulting of their combinations are also considered. Proper reconstitution of membrane constituents such as proteins and drugs into liposomes are examined in the light of our actual understanding of the micelle-vesicle transition.

Characterization of loaded liposomes by size exclusion chromatography.

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.

Partition coefficient of a surfactant between aggregates and solution: application to the micelle-vesicle transition of egg phosphatidylcholine and octyl beta-D-glucopyranoside.

The mechanism of the solubilization of egg phosphatidylcholine containing 10% (M/M) of egg phosphatidic acid unilamellar vesicles by the nonionic detergent, octyl beta-D-glucopyranoside, has been investigated at both molecular and supramolecular levels by using fluorescence and turbidity measurements. In the lamellar region of the transition, the solubilization process has been shown to be first a function of the initial size before reaching an equilibrium aggregation state at the end of this region (the onset of the micellization process). The analysis during the solubilization process of the evolution of both the fluorescence energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rho-PE) and the fluorescence of 6-dodecanoyl-2-dimethylaminoaphtalene (Laurdan) has allowed us to determine the evolution of the detergent partitioning between the aqueous and the lipidic phases, i.e., the evolution of the molar fraction of OG in the aggregates (XOG/Lip) with its monomeric detergent concentration in equilibrium ([OG]H2O), throughout the vesicle-to-micelle transition without isolating the aqueous medium from the aggregates. The curve described by XOG/Lip versus [OG]H2O shows that the partition coefficient of OG is changing throughout the solubilization process. From this curve, which tends to a value of 1/(critical micellar concentration), five different domains have been delimited: two in the lamellar part of the transition (for 0 < [OG]H2O < 15.6 mM), one in the micellization part, and finally two in the pure micellar region (for 16.5 < [OG]H2O < 21 mM). The first domain in the lamellar part of the transition is characterized by a continuous variation of the partition coefficient. In the second domain, a linear relation relates XOG/Lip and [OG]H2O, indicating the existence of a biphasic domain for which the detergent presents a constant partition coefficient of 18.2 M-1. From the onset to the end of the solubilization process (domain 3), the evolution of (XOG/Lip) with [OG]H2O can be fitted by a model corresponding to the coexistence of detergent-saturated lamellar phase with lipid-saturated mixed micelles, both in equilibrium with an aqueous phase, i.e., a three-phase domain. The micellar region is characterized first by a small two-phase domain (domain 4) with a constant partition coefficient of 21 M-1, followed by a one-phase mixed-micellar domain for which XOG/Lip no longer linearly depends on [OG]H2O. The results are discussed in terms of a phase diagram.

Study of size distribution and stability of liposomes by high performance gel exclusion chromatography

Abstract Applications of high performance liquid chromatography on a gel exclusion column (HPLC-GEC) for the characterization of liposomes are presented following a review of the conventional uses of gel exclusion chromatography (GEC). The practical aspects of HPLC-GEC concerning the choice of the gel and the column operation are examined. A procedure for column calibration with vesicles is proposed, in order to obtain the selectivity curve of the TSK-G6000 PW gel; this appears to be the most convenient gel for sizing of liposomes. The mechanism of elution is discussed as a function of the particle size and composition. Methods involving this type of column and different detection modes (light scattering, refractive index, fluorescence and radioactivity) or their combination, were investigated in detail either to analyse the polydispersity of liposomes or their size stability in terms of aggregation and/or fusion.

Action of octylglucoside on non-ionic monoalkyl amphiphile-cholesterol vesicles : study of the solubilization mechanism

Abstract Non-ionic surfactant vesicles (NSV) were prepared at room temperature, from a mixture of diglycerol hexadecylether (C16G2) and cholesterol (CHOL) with a small amount of dicetylphosphate (DCP) (47.5, 47.5, 5 wt%) by either sonication or detergent dialysis of octylglucoside (OG)-lipids mixed micelles. NSV were characterized by quasielastic light scattering (QLS) and high performance liquid chromatography (HPLC) on gel exclusion column. Respective mean diameters of 72 nm for sonicated NSV and of 287 and 322 nm for NSV prepared by detergent dialysis were found. The continuous dissolution of both small vesicles (SV) and large ones (LV) by a 100-mM or 400-mM OG micellar solution was systematically examined by monitoring turbidity at 350 nm. The molecular composition of aggregates [OG/lip]agg as well as the OG concentration in the continuous phase [OG]bulk were determined at break points observed on the solubilization curves. Initial vesicles and mixed aggregates at each break point were also characterized by plots of optical density (OD) versus total lipid concentration [lip]tot. The solubilization curves of SV and LV exhibit different shapes until [OG]bulk reaches about the critical micellar concentration (cmc) of pure OG; thereafter one single dissolution process occurs involving the same intermediate aggregates for SV and LV. At any stage of the solubilization, [OG/lip]agg and [OG]bulk remained independent of the size of the corresponding aggregates, suggesting that NSV solubilization is governed by molecular processes. By comparison with the OG-egg phosphatidylcholine (EPC) system previously studied, it was found that the mechanism of NSV dissolution by OG is very similar to that of EPC small unilamellar vesicles (EPC SUV) solubilization by the same detergent. However, it has been shown that (i) NSV are impermeable to OG until [OG]tot = 13 mM, (ii) their transformation into mixed micelles is kinetically dependent on the lateral diffusion of the detergent molecules in lipid bilayers. The rather efficient barrier of the NSV membrane to OG suggests a strong cohesion of C16G2 and CHOL.

A differential scanning calorimetry study of the interaction of lasalocid antibiotic with phospholipid bilayers.

Interaction of lasalocid sodium salt (Las-Na) with dipalmitoylphosphatidylcholine (DPPC) as a membrane model was investigated by highly-sensitive differential scanning calorimetry (DSC). The insertion properties of the antibiotic were studied both in multilamellar suspensions and unilamellar vesicles, for Las-Na/DPPC molar ratios (r) ranging from 0.005 to 0.1. The effect of the antibiotic on the lipid thermotropic behavior is concentration dependent and drastically changes at a critical r of 0.04 in both model membranes. Below this ratio, Las-Na molecules interact with DPPC bilayers without disrupting the global organization of the membrane. In the multilamellar systems only the transition cooperativity is affected whereas for the mixed vesicles, a decrease in the enthalpy change suggests a different mode of insertion. Above this ratio, implantation of the antibiotic give rise to lateral phase separation in multilamellar systems. These structural modifications have repercussions on the formation of mixed LAS-Na/DPPC vesicles which seems limited to an r value of 0.04.

Sodium Taurocholate-Induced Lamellar-Micellar Phase Transitions of DPPC

The thermotropic transitions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and the structural changes of its lamellar phases have been studied between 0 and 50°C by both DSC and synchrotron small angle X-ray diffraction/scattering as a function of temperature (XRDT) and sodium taurocholate concentration [TC] in the 0–40 mM range ([DPPC]=50 mM) at pH 7.4. The existence of multiple phase transitions (up to 5 peaks within a 5°C interval) in a narrow domain of temperature between 25 and 42°C depending on the [TC]/[lipid] ratio was observed in the DSC curves. XRDT showed that at low ratios they might correspond to transitions between lamellar phases, the structural characteristics of which are given. At higher ratios a lamellar to micellar transition was observed, and the temperature at which it was observed decreased as a function of the TC content. The relationships with DPPC vesicle bilayer permeabilization and solubilization are discussed.

Methodology for vesicle permeability study by high-performance gel exclusion chromatography.

A methodology based on high-performance gel exclusion chromatography (HPLC-GEC) has been developed to perform permeability studies of vesicles. Encapsulation of two marker isothiocyanate fluorescein (FITC) dextrans of 4400 and 40,500 molecular mass was used as a model system. Combination of two TSK-PW columns, one efficient in vesicle sizing (G6000 PW), the other in that of dextrans (G4000 PW), was required to achieve complete particle separation and to remove entirely the unentrapped dextran after encapsulation into vesicles. Coupling fluorescence and light scattering detection allowed to control the efficiency of the separation, to quantify the vesicle leakage and to follow both the integrity of the vesicles and changes in their size. This methodology can be applied to other fields such as encapsulation of water soluble compounds and drug delivery systems.