Critical steps during the prilling process of molten lipids: Main stumbling blocks due to pharmaceutical excipient properties.

Prilling by ultrasonic jet break-up is an efficient process to produce perfectly spherical microparticles homogeneous in size. However, the material properties could affect the manufacturability and the final product properties especially with lipid-based excipients which often exhibit complex structural properties. This work presents the characterisation of six lipid-based excipients differing by their melting point and polymorphic behaviour which were used to produce microspheres using a pilot-scale prilling equipment. The experimental results were compared to theoretical calculations, especially the droplet solidification time which is a key-parameter for this process. This work highlighted that monotropic polymorphism of excipients and supercooling effect have a significant impact on process parameters which should be considered with care during formulation design.

Superparamagnetic lipid-based hybrid nanosystems for drug delivery.

INTRODUCTION: The development of multifunctional drug carriers provides many opportunities in the field of drug delivery. Among them, carriers loaded with both drug and superparamagnetic iron oxide nanoparticles would allow the combination of chemotherapy with the possibility of monitoring or controlling the distribution of the nanocarrier in the body, triggering drug release and/or applying a synergistic hyperthermia treatment. AREAS COVERED: The present review covers biocompatible lipid-based nanotechnologies that have been employed to co-encapsulate drug and iron oxide. Depending on their physico-chemical properties, lipids are able to generate monophasic lipophilic nanodispersions or more complex structures containing both lipidic and aqueous domains. This review describes the rationale behind these nanoobjects and how they can be prepared. EXPERT OPINION: This review focuses on the co-encapsulation aspects of these hybrid systems and discusses in particular the possible heterogeneities in drug-to-iron oxide ratio and the difficulties that could be encountered in the construction of these biocompatible multifunctional drug carriers.

[Health and access to health care of 18-25 years old people not in employment, education or training (NEETs) attending the French 'missions locales']. / Santé et recours aux soins des jeunes en insertion âgés de 18 à 25 ans suivis en mission locale.

BACKGROUND: In France, "missions locales" are public assistance units for young people aged 16-25 years not in employment, education or training (NEET). The health status of the more than 1.5 million young adults attending these units annually is unknown. The purpose of this study was to describe the health status and health care use of this population in comparison with the general population of the same age. METHODS: The Presaje survey was conducted in 2011 on a randomized sample of 1453 young adults aged 18-25 years who attended five "missions locales" in mainland France. Data were analyzed and compared with those of participants of the same age interviewed in a French national health survey (Baromètre Santé 2010, n=2899) and in a regional cohort (SIRS, n=204) conducted in the Greater Paris area, both in 2010. RESULTS: The overall social profiles of this NEET population was diverse, but with globally more difficult living conditions than in the general population. Health-related vulnerability factors identified were: insufficient health insurance; low educational level; numerous adverse experiences during childhood and social isolation. Some of their health indicators were remarkably poor in comparison with participants in the Baromètre santé study: 19.2 % (CI95 %=[17.2-21.3]) had a chronic disease-versus 8.2 % (CI95 %=[7.0-9.4]) (P<0.001); 31.9 % (CI95 %=[26.8-37.4]) were overweight or obese-versus 17.9 % (CI95 %=[16.3-19.6]) (P<0.001) and 19.6 % (CI95 %=[15.2-23.9]) were depressed-versus 7.3 %(CI95 %=[3.8-10.9]) (P<0.001). Compared with participants of the SIRS survey, fewer (70.4 %) (CI95 %=[68.0-72.7]) had a family doctor-versus 79.7 % (CI95 %=[73.1-86.2]) (P<0.05). Among the women in the Presaje survey, 38.0 % [30.5-45.4] had a regular gynecological follow-up-versus 80.9 % (CI95 %=[71.5-90.4]) in the SIRS survey (P<0.001). CONCLUSION: Careful attention must be given to the young NEET population. Many of these youths are not familiar with health care services despite their important health care needs. Integrating health services into the "missions locales" may help detect health problems in this population, facilitating links to care.

Critical parameters involved in producing microspheres by prilling of molten lipids: from theoretical prediction of particle size to practice.

The aim of this work was to identify the key parameters which influence the running of the prilling process with lipid material from the initial melting to the formation of solid microspheres. The microsphere size would essentially result from break-up at the Rayleigh-Weber's wavelength which mostly depends on the liquid properties (mass density, surface tension and dynamic viscosity). After molten liquid extrusion through the nozzle, the cooling rate is very fast and the instantaneous temperature of the liquid jet decreases rapidly of 0.2-0.3 °C during the duration of the droplet formation (1-2 ms). This leads to no significant modification of the physical characteristics of the lipids and only a very slight change in the dynamic viscosity. Consequently, no significant effect on the optimal wavelength λ(W) and on droplet formation can occur. However, coalescence of liquid droplets has been observed during their fall, probably caused by turbulence into the air column, leading to a minor population of larger microspheres.

[Migration, health and access to care in Mayotte Island in 2007: lessons learned from a representative survey]. / Migration, santé et recours aux soins à Mayotte en 2007: enseignements d'une enquête représentative en population générale.

BACKGROUND: Mayotte Island, located in the Indian Ocean, is a French overseas departmental community with certain specificities: recent development of sanitary institutions, significant immigration, free access to care for legal residents but with co-payments for irregular residents, the absence of many of the social benefits which exist in mainland France and poor or non-existent health information systems. We report here the first population-based survey describing the links between health, migration and healthcare utilization in this territory. METHODS: Cross sectional population-based study using a three-stage random sample (geographic areas, households, individuals). In all, 2105 individuals were interviewed either in French, Shimaore or Kibushi (response rate=96%), using a questionnaire adapted to the context of Mayotte Island after a preliminary qualitative survey. Descriptive analyses and logistic regression models were performed. RESULTS: Foreigners make up 40% of the Mayotte population (total 186,452 inhabitants), of which one-quarter are children born in Mayotte and 80% have no regular residence status. The median length of residence of migrant foreigners is 10 years. Foreigners represent a majority of the female population, of the 20 to 35 years old population and of the urban areas. Main determinants for migration were economical (50%) or family-related (26%). Health was stated as a cause of migration by 11% of migrants. The social situation of foreigners is more precarious and their perceived health poorer than those of the French. Their access to care is also perceived as more difficult. We did not observe any notable difference in terms of frequency of healthcare attendance over the last 12 months between the two groups, but foreigners have consulted less often private GPs and more often traditional practitioners than French. CONCLUSION: In this overseas French island, the migrant population is numerous and resident for a long time. Their main motivations to immigrate are economic and family-related. They report hurdles to healthcare related with their precarious living conditions, including their illegal residence status.

Exposure to interparental violence and psychosocial maladjustment in the adult life course: advocacy for early prevention.

BACKGROUND: Early family-level and social-level stressors are both assumed to be the components of two main path models explaining the association between exposure to interparental violence in childhood and its long-term consequences on mental health explored through life-course epidemiological studies. AIMS: To investigate the association between exposure to interparental violence in childhood and mental health outcomes in adulthood when taking into account early family and social stressors. METHODS: A retrospective French cohort study of 3023 adults representative of the general population in the Paris metropolitan area was conducted in 2005 through at-home, face-to-face interviews. The outcomes measures were current depression and lifetime suicide attempt, intimate partner violence, violence against children and alcohol dependence. RESULTS: The adults exposed to interparental violence during childhood had a higher risk of psychosocial maladjustment. After adjusting for family- and social-level stressors in childhood, this risk was, respectively, 1.44 (95% CI 1.03 to 2.00) for depression, 3.17 (1.75 to 5.73) for conjugal violence, 4.75 (1.60 to 14.14) for child maltreatment and 1.75 (1.19 to 2.57) for alcohol dependence. CONCLUSIONS: The adult consequences of parental violence in childhood-and this independently of the other forms of domestic violence and the related psychosocial risks-should lead to intensifying the prevention of and screening for this form of maltreatment of children.

Sterically stabilized superparamagnetic liposomes for MR imaging and cancer therapy: pharmacokinetics and biodistribution.

Pharmacokinetics of magnetic-fluid-loaded liposomes (MFLs) with mean hydrodynamic diameter of 200 nm sterically stabilized by poly(ethylene glycol) (PEG) and labelled by a fluorescent lipid probe, N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (Rho-PE) was studied. The loading consisted in an aqueous suspension of maghemite nanocrystals close to 8 nm in size at 1.7 Fe(III)mol/mol total lipids ratio. Double tracking of MFL in blood was performed versus time after intravenous administration in mice. Lipids constituting vesicle membrane were followed by Rho-PE fluorescence spectroscopy while iron oxide was determined independently by relaxometry. MFLs circulating in the vascular compartment conserved their vesicle structure and content. The pharmacokinetic profile was characterized by two first-order kinetics of elimination with distinct plasmatic half-lives of 70 min and 12.5 h. Iron biodistribution and organ histology clearly highlighted preferential MFL accumulation within liver and spleen. The pathway in spleen supported that elimination was governed by the mononuclear phagocyte system (MPS). PEG coating was essential to prolong MFL circulation time whereas iron oxide loading tends to favour uptake by the MPS. Despite partial uptake in the earlier times after administration, MFLs exhibited long circulation behaviour over a 24-h period that, coupled to magnetic targeting, encourages further use in drug delivery.

Periodate oxidation of sodium alginate in water and in ethanol-water mixture: a comparative study.

Periodate oxidation of sodium alginate in aqueous solution as well as a dispersion in 1:1 ethanol-water was examined. The oxidation proceeded smoothly in both media, and the kinetics of oxidation was surprisingly similar. Polymer cleavage was observed in both media, but it was extensive in ethanol-water. The weight-average molar mass (Mw) of the oxidized product obtained from aqueous solution showed a gradual decrease with increase in the periodate concentration, whereas, except for very high periodate equivalent, the change in Mw was not reflected with increase in concentration of periodate in ethanol-water. The oxidized alginate obtained from the ethanol-water mixture was found to be more efficient in crosslinking proteins such as gelatin, leading to hydrogels. Oxidation of a dispersion has the advantage of generating large quantities of the oxidized alginate in higher yield with one reaction using less solvent.

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.

Phase behavior of fully hydrated DMPC-amphiphilic cyclodextrin systems.

With the aim of exploring relationships between the chemical structure and the physico-chemical properties of amphiphilic beta-cyclodextrin, a reappraisal of the obtaining of pure heptakis (2,3-di-O-hexanoyl)-beta-cyclodextrin (beta-CDC(6)) was undertaken. In this paper the chemical characterization of the newly synthesized beta-CDC(6) and its ability to form mixed structures with dimyristoylphosphatidylcholine (DMPC) are reported. Miscibility of the two amphiphiles is examined: (i) in monolayers formed at the air-water interface by analyzing the surface pressure-area isotherms; and (ii) in fully hydrated mixtures by differential scanning calorimetry (DSC) and X-ray diffraction at small and wide angles. Results demonstrate that the beta-cyclodextrin derivative is partially miscible to the phospholipid: intimate mixing occurs at beta-CDC(6) molar ratios smaller than 7-15 mol%, depending on the dimensional scale considered, while beyond these compositions phase separation is observed. At the air-water interface, the miscibility region of the two compounds shows non-ideal behavior characterized by the non-additivity of the molecular areas in the mixed monolayers. At the three-dimension level, the formation of a beta-CDC(6)/DMPC mixed lamellar phase occurs except at beta-CDC(6) molar ratios close to 5 mol% at which a highly ordered structure is depicted below the solid-to-liquid state transition of the DMPC hydrocarbon chains. At beta-CDC(6) contents higher than 7 mol%, the mixed assemblies coexist with excess amphiphilic cyclodextrin which then forms a separated hexagonal structure.

Self-evolving microstructured systems upon enzymatic catalysis.

The consequences of cell microstructuration on enzyme functions is discussed in the framework of self-evolving microstructured systems. Molecular assemblies of amphiphiles or lipids are spontaneously formed by self-organisation. Among these different structures, reversed micelles, liquid crystalline mesophases and vesicles are hosts for enzymatic reaction studies. Inside a living cell, phospholipid metabolism is responsible for membrane structural modifications; the catalytic behaviour of lipolytic enzymes, mainly phospholipase (PL) A2, is described in relation with structural aspects of biological membranes. The implication in cellular regulation events of PLC and PLD is discussed in relation with the role of their reaction products as second messengers in membrane fusion processes. The in vitro synthesis of dialkyl phosphatidylcholines, via the enzymatic 'salvage pathway' which leads to the formation of vesicles upon phospholipid formation, is considered in relation with autopoiesis. More recent studies on self-evolving systems based on enzyme-surfactants reactions are detailed. The interactions between amphiphilic aggregates and enzymes allow to explore the OG/octanol/water phase diagram. Enzymatic formation of dipalmitoylphosphatidylcholine (DPPC) liposomes and non-ionic surfactant vesicles (NSV), starting from mixed micelles or open structures, finally sets an example of a biomimetic self-evolving system.

New sterically stabilized vesicles based on nonionic surfactant, cholesterol, and poly(ethylene glycol)-cholesterol conjugates.

Monomethoxypoly(ethylene glycol) cholesteryl carbonates (M-PEG-Chol) with polymer chain molecular weights of 1000 (M-PEG1000-Chol) and 2000 (M-PEG2000-Chol) have been newly synthesized and characterized. Their aggregation behavior in mixture with diglycerol hexadecyl ether (C16G2) and cholesterol has been examined by cryotransmission electron microscopy, high-performance gel exclusion chromatography, and quasielastic light scattering. Nonaggregated, stable, unilamellar vesicles were obtained at low polymer levels with optimal shape and size homogeneity at cholesteryl conjugate/ lipids ratios of 10 mol% M-PEG1000-Chol or 5 mol% M-PEG2000-Chol, corresponding to the theoretically predicted brush conformational state of the PEG chains. At 20 mol% M-PEG1000-Chol or 10 mol% M-PEG2000-Chol, the saturation threshold of the C16G2/cholesterol membrane in polymer is exceeded, and open disk-shaped aggregates are seen in coexistence with closed vesicles. Higher levels up to 30 mol% lead to the complete solubilization of the vesicles into disk-like structures of decreasing size with increasing PEG content. This study underlines the bivalent role of M-PEG-Chol derivatives: while behaving as solubilizing surfactants, they provide an efficient steric barrier, preventing the vesicles from aggregation and fusion over a period of at least 2 weeks.

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.

Evaluation of the hydrophobic drug loading characteristics in nanoprecipitated amphiphilic cyclodextrin nanospheres.

The aim of this work was to evaluate the loading capacity and the association characteristics of the hydrophobic drug progesterone on amphiphilic cyclodextrin nanospheres prepared by the nanoprecipitation method. The colloidal suspensions were prepared in the presence or absence of two different surfactants, Pluronic F68 and Tween 80. The physicochemical characteristics of the nanospheres were assessed using a nanosizer, zetameter, and transmission electron microscope. The physical state of the drug was verified using differential scanning calorimetry (DSC) and x-ray diffraction (XRD) methods. The in vitro progesterone release was investigated at 37 degrees C after dilution of the suspensions in sink conditions. Nanospheres with a mean diameter from 100 to 300 nm and a low degree of polydispersity were prepared from amphiphilic hexanoyl-gamma-cyclodextrin. The progesterone loading capacity was not affected by the formulation parameters tested. The DSC and XRD studies demonstrated the absence of the crystalline domains of progesterone in loaded nanospheres. The DSC studies also demonstrated the presence of interactions between the drug and carrier. The release of the drug from the carrier was extremely rapid and was governed by a partition phenomenon that depends only on the solubility of the drug in the release medium. From these results, we concluded that with this method, the progesterone is molecularly associated at the surface of the cyclodextrin nanospheres, probably through hydrophobic interactions in specific sites. The release profiles obtained can be of value when an improvement in the bioavailability of the drug is desired.

Thermotropic phase behavior of in vivo extracted human stratum corneum lipids.

The thermotropic phase behavior of lipids extracted either in vivo from inner forearm (SCLE) or plantar callus (PC) was investigated by differential scanning calorimetry and small angle X-ray diffraction. PC composition was chromatographically modified (MPC) by eliminating the more polar lipids in order to evaluate their role. Analysis of composition confirms the potential use of PC as a source of stratum corneum lipids. MPC and SCLE exhibit similar differential scanning calorimetry (DSC) profiles with a main transition around 50 degrees C attributed to the solid-to-liquid phase transition of the ceramides. The absence of a transition around 50 degrees C for PC suggests the possible perturbation of ceramide packing by the significantly high proportion of phospholipids. X-ray data suggest a high miscibility of sebum components in stratum corneum lipids with possible modification of chain packing. The MPC patterns show a lipid phase separation which underscores the role of polar lipids in cholesterol/free fatty acids/sterol esters/ceramides structural cohesion.

Structure of in-serum transfecting DNA-cationic lipid complexes.

Noticeable modifications of in-serum transfection efficiency of dioctadecylamidoglycyl-spermine (DOGS)-DNA complexes are observed, depending on DNA condensation conditions. The structures of the complexes are studied, keeping in mind the variability of lipid polymorphism, by cryo-transmission electron microscopy and X-ray diffraction. By increasing both pH and ionic strength, well-organised lamellar structures with a period of 65 A replace supramicellar aggregates. A relationship between the structures and their in-vitro transfection activity is established. Efficiency in the presence of serum is maintained when a lamellar arrangement is involved.

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.

Reconstitution of non-ionic monoalkyl amphiphile-cholesterol vesicles by dilution of lipids-octylglucoside mixed micelles.

Kinetic aspects of the formation of non-ionic surfactant vesicles (NSV) using the mixed micelle dilution procedure are examined. Mixed micelles composed of a mixture of lipids, i.e. diglycerol hexadecylether (C16G2), cholesterol (CHOL), dicetylphosphate (DCP) and detergent, octylglucoside (OG), were diluted with detergent-free buffer added either instantaneously or progressively at different rates ranging from 3.47 x 10(-2) to 6.94 x 10(-4) ml/min. The resulting particles were analysed by quasielastic light scattering (QELS), high performance liquid chromatography (HPLC) on gel exclusion column and cryogenic transmission electron microscopy (cryo-TEM). NSV exhibit mean diameters (MD) varying from 100 to 600 nm depending on the kinetics of OG removal. When the dilution of mixed micelles is instantaneous the vesicles are characterized by a spherical shape and MD values close to 100 nm. They show narrow size distribution and stability for 2 months. NSV recovered with progressive micelle dilution, at fast buffer addition rates (3.47 x 10(-2) and 1.39 x 10(-2) ml/min) exhibit MD values of 170-240 nm, elongated shapes, low polydispersities and 2-month stabilities. When the rate of buffer addition is lowered to 6.94 x 10(-4) ml/min, unstable particles with larger MD values and broad size distributions are obtained. Turbidity monitoring at 350 nm and 25 degrees C was used to characterize the lipids-OG mixed aggregate rearrangements either as a function of time when detergent-free buffer was continously added to the mixed micelles or after equilibrium setting when the micelles were instantaneously diluted. In the latter case the intermediate aggregates were also analysed by QELS. For continuous dilutions, the molecular composition of aggregates, [OG/lip]agg, as well as the OG concentration in the aqueous medium, [OG]bulk, were determined at the break points observed on the plots of optical density (OD) versus total OG concentration ([OG]tot). [OG/lip]agg and [OG]bulk values are independent of the rate of buffer addition, suggesting that the micelle to NSV transition is not mainly limited by the kinetics of the molecular processes involved during detergent removal from the mixed aggregates. The examination of the apparent partition coefficient of OG between the aqueous phase and the lipidic aggregates shows, however, that OG depletion from the bilayered structures is more difficult than its elimination from the mixed micelles. QELS analysis of the intermediate lipids-detergent aggregates, performed with time, demonstrates very slow supramolecular rearrangements during the vesicle closure. These rearrangements explain the significant increase in both size and polydisperity of the final vesicles observed with slow rates of buffer addition.

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering.

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.