Some insights about 1,6-diphenyl-1,3,5-hexatriene-lipid supramolecular assemblies by steady-state fluorescence measurements.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is the most widely proposed molecular probe for the post-column fluorescence derivatization of lipids after liquid chromatography separation. This kind of detection consists of a supramolecular combination of DPH and eluted lipids. The detection is optimally performed in a mainly aqueous environment (over 80% v/v) because the weak fluorescence of DPH in water is drastically enhanced upon formation of supramolecular assemblies with lipids. In the present study, and in order to obtain better spectroscopic insights into the nature of these supramolecular assemblies, two different lipids were tested, 1,2,3-tridodecanoylglycerol (LLL) as a model triglyceride (nonpolar lipid) and dimyristoylphosphatidylcholine (DMPC) as a model phosphatidylcholine (charged amphiphilic lipid). Stoichiometry and association constants were determined on the basis of the variation of fluorescence intensity in the presence of various concentrations of lipids. LLL(60)-DPH(2) and DMPC(200)-DPH(2) complexes were identified with association constants as high as K(2) = (5.8 +/- 0.5) x 10(13) M(-2) and (17.3 +/- 2.0) x 10(13) M(-2) for LLL and DMPC, respectively. The fluorescence intensity of DPH in the presence of LLL is greater than in the presence of DMPC. An attempt to characterize the insertion mode of DPH in the lipidic supramolecular assemblies is also made.

Liposomal formulation of a glycerolipidic prodrug for lymphatic delivery of didanosine via oral route.

Didanosine is a polar drug with poor membrane absorption and high hepatic first pass metabolism. This study aimed at developing a lipidic formulation of a glycerolipidic prodrug of didanosine in order to improve its bioavailability. In the course of a preformulation study, the glycerolipidic prodrug of didanosine was characterized by microscopy, DSC and XRDT. In anhydrous conditions, the prodrug displayed a polymorphic behaviour similar to that of triglycerides. Then, we evaluated three types of lipidic formulations (emulsions, mixed micelles and liposomes) in order to encapsulate the prodrug. Solubilities in water - even in the presence of taurocholate micelles - but also in some oils were very low (max 244 microg/mL) as the prodrug was found to be amphiphilic (log P=2). On the contrary, the prodrug was found to be perfectly incorporated in dipalmitoylphosphatidylcholine (DPPC) multilamellar liposomes up to a ratio of 1:5 (mol:mol) prodrug:DPPC as suggested by HPLC-UV and DSC experiments. Moreover, these liposomes could be freeze-dried whereas the chemical integrity of the prodrug was preserved. Then, the freeze-dried liposomal preparation could be formulated as gastro-resistant capsules to prevent didanosine from acidic degradation. Further experiments are on the way to evaluate in vitro the absorption of prodrug incorporated in liposomes by enterocytes.

Structural and thermal characterization of glyceryl behenate by X-ray diffraction coupled to differential calorimetry and infrared spectroscopy.

Physical and thermal properties of glyceryl behenate (Compritol 888 ATO) used as sustained-release matrix in pharmaceutical applications are studied by coupled time-resolved synchrotron X-ray diffraction and Differential Scanning Calorimetry combined with Infrared Spectroscopy. With these techniques, all polymorphs formed in glyceryl behenate, analyzed as received and after various thermal treatments from quenching to slow crystallization, are characterized. By using different well-controlled mixtures of mono-, di- and tribehenate, we identify each lamellar phase observed in the glyceryl behenate. Finally the influence of the crystallization rate on the formation of preferential conformations was also analyzed in order to bring insights into the polymorphism of glyceryl behenate. By changing the crystallization rate of the sample, it was shown that one can favor the formation of preferential polymorphs in the sample. In particular the crystallization at 10 degrees C/min seems to be well adapted for producing a single lamellar phase with a period of 60.9 A while a crystallization rate of 0.4 degrees C/min produces three different lamellar phases.

The water vapor permeability of polycrystalline fat barrier films.

The water vapor permeability of fat barrier films has been associated with structural characteristics such as polymorphism, crystal size, and chemical composition, among others. However, no mechanistic models have been proposed to describe this relationship. In this study, we have determined the effects of processing conditions on the structure and physicochemical characteristics of four fats and their relationship to water vapor permeability. Results suggest that the solids' volume fraction and the domain size of the fat crystals seem to be the most important factors controlling water vapor migration. Moreover, materials with relatively large crystalline domains will yield malleable films with relatively low storage and loss moduli and strain/stress at the limit of linearity high tan delta values. The structural effects on the permeability of fat films are related to the nanoscale of the material.

Properties of ternary phospholipid/dimethyl sulfoxide/water systems at low temperatures.

X-ray diffraction, neutron diffraction and differential scanning calorimetry were used to investigate phase transitions in the ternary system phospholipid/dimethyl sulfoxide (DMSO)/water under cooling for three homologous phospholipids: dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). Below the temperature of ice formation from -40 to -113 degrees C, a new lamellar phase of DPPC and DSPC was found at and above a DMSO molar fraction of X(DMSO) = 0.05. Below X(DMSO) = 0.05 only a single dehydrated Lc-phase exists after ice formation. The new phase has an increased membrane repeat distance and coexists with a dehydrated Lc-phase. DPPC with a DMSO molar fraction of X(DMSO) = 0.07 shows a membrane repeat distance of the new phase of d = 6.61 +/- 0.03 nm. The value of d increases at the increase of X(DMSO). The new phase was not observed in the ternary system with DMPC. No correlation between the new phase and the glass transition of bound water in the intermembrane space was detected. The new phase was detected only in the systems with excess of water. The creation of the new phase demonstrates the specific DMSO interaction with hydrocarbon chains.

Thermal and structural behavior of anhydrous milk fat. 3. Influence of cooling rate.

The crystallization behavior of anhydrous milk fat has been examined with a new instrument coupling time-resolved synchrotron x-ray diffraction as a function of temperature (XRDT) at both small and wide angles and high-sensitivity differential scanning calorimetry. Crystallizations were monitored at cooling rates of 3 and 1 degrees C/ min from 60 to -10 degrees C to determine the triacylglycerol organizations formed. Simultaneous thermal analysis permitted the correlation of the formation/melting of the different crystalline species monitored by XRDT to the thermal events recorded by differential scanning calorimetry. At intermediate cooling rates, milk fat triacylglycerols sequentially crystallize in 3 different lamellar structures with double-chain length of 46 and 38.5 A and a triple-chain length of 72 A stackings of alpha type, which are correlated to 2 exothermic peaks at 17.2 and 13.7 degrees C, respectively. A time-dependent slow sub-alpha <--> alpha reversible transition is observed at -10 degrees C. Subsequent heating at 2 degrees C/min has shown numerous structural rearrangements of the alpha varieties into a single beta' form before final melting. This polymorphic evolution on heating, as well as the final melting point observed (approximately 39 degrees C), confirmed that cooling at 3 degrees C/min leads to the formation of crystalline varieties that are not at equilibrium. An overall comparison of the thermal and structural properties of the crystalline species formed as a function of the cooling rate and stabilization time is presented. The influence on crystal size of the cooling rates applied in situ using temperature-controlled polarized microscopy is also determined for comparison.

Interaction of the water-soluble protein aprotinin with liposomes: gel-filtration, turbidity studies, and 31P NMR studies.

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; 31P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV-aprotinin complexes was determined. Lipid-protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. 31P NMR spectroscopy of the MLV-aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.

Effect of membranotropic and mucoadhesive formulations of protein proteinase inhibitors on bovine herpes virus-1 reproduction.

The lipidized derivatives of Bowman-Birk soybean protease inhibitor (BBI) containing one to three oleoyl groups were synthesized and characterized. The (ole)(1)- and (ole)(2)BBI were demonstrated to have 200- and 100-fold higher uptake into Caco-2 cell monolayers compared to native BBI. The acylated BBI had increased affinity to elastase-like proteases. Aprotinin-loaded starch/bovine serum albumin microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Various formulations of protein proteinase inhibitors were tested for their influence on BHV-1 reproduction in cell cultures. Native aprotinin possessed palpable dose-dependent effect inhibiting the virus reproduction up to 4.0 lg (10,000-fold). The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective decreasing virus infectious titer up to 4.0 lg and delaying the cytopathic effect up to 144 h in lesser doses of aprotinin. The lipophilic derivative (ole)(1)BBI was shown to exhibit effective inhibition (>100-fold) of BHV-1 reproduction unlike native BBI.

Does sucrose influence the properties of DMPC vesicles?

Small-angle neutron and X-ray scattering, dynamic light scattering, X-ray diffraction coupled with differential scanning calorimetry, and Raman spectroscopy were applied to investigate unilamellar (ULVs) and multilamellar (MLVs) dimyristoylphosphatidylcholine (DMPC) vesicles in aqueous sucrose solutions with sucrose concentrations from 0 to 60% w/w. In case of ULVs, the addition of sucrose decreases the polydispersity of vesicle population. A minimum value of polydispersity was found at 20% sucrose. For sucrose concentration from 0 to 35% oligolamellar vesicles in the ULV population have a minimum presence. Vesicles with 5-10% sucrose exhibit the best stability in time. For the case of MLVs, sucrose influences the temperature of the phase transitions, but the internal membrane structure remains unchanged.

Thermal and structural behavior of milk fat. 1. Unstable species of anhydrous milk fat.

The thermal and structural behavior of anhydrous milk fat (AMF) was studied by a technique that allowed simultaneous time-resolved synchrotron X-ray diffraction as a function of temperature (XRDT) and high sensitivity differential scanning calorimetry (DSC) to be carried out in the same apparatus from the same sample. In this paper (the first of a series), the less stable crystalline structures made by triacylglycerols (TG) of bulk AMF after its liquid quenching down to -8 degrees C are addressed The coexistence of two lamellar structures characterized by sharp long spacing reflections corresponding to well-defined 3L (70 A) and 2L (47 A) longitudinal stackings but broad short spacing lines related to poorly ordered hexagonal (alpha) lateral packing is shown for the first time, The bilayered structure was very unstable, since it disappeared during a 20-min isothermal recording. Simultaneous DSC and X-ray monitoring of AMF heating in the range -8, +50 degrees C at a rate of 2 degrees C/min allows the same sample to be followed on the evolution of these unstable forms to more stable varieties. The 3L stacking transforms into a new 2L crystalline structure characterized by broad LS reflections corresponding to a ill-defined 2L (37 A) longitudinal stacking but a more compact orthorhombic (beta') lateral packing. A delimitation of the domains of existence of the crystalline structures resulted from the comparison of detailed analysis of the evolutions of positions, intensities, and widths of X-ray peaks as a function of temperature to microcalorimetry recording.

Thermal and structural behavior of anhydrous milk fat. 2. Crystalline forms obtained by slow cooling.

The crystallization behavior of milk fat has been examined on slow cooling at 0.1 degrees C/min from 50 to -15 degrees C, to determine the variations of triacylglycerol organizations as a function of temperature. The experiments have been conducted with an instrument allowing coupled X-ray diffraction (XRD) at both small and wide angles and high-sensitivity differential scanning calorimetry (DSC) recordings from the same sample by taking advantage of the high-energy flux of a synchrotron. On slow cooling, milk fat triacylglycerols sequentially crystallize in four different lamellar structures with double-chain length of 41.5, 48.3, and 39.2 A and a triple-chain length of 62.2 A stackings. Simultaneous wide-angle XRD has shown that initial nucleation occurs in a packing of beta' type at about 24 degrees C. For temperature < 13 degrees C, triacylglycerols crystallize in an hexagonal subcell of alpha type, leading to the coexistence of the beta' + alpha polymorphic forms, which is recorded until -15 degrees C. Thermal analysis allowed to correlate the formation of the different crystalline species monitored by XRDT (XRD as a function of temperature) to the exothermal events recorded simultaneously by differential scanning calorimetry. The evolution of the species formed during crystallization was also monitored on heating at 2 degrees C/min. The absence of polymorphic evolution on heating, as well as the high final melting point observed, about 40 to 41 degrees C, confirmed that cooling at 0.1 degrees C/min leads to quasi equilibrium.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in excess water.

The cytoplasmic delivery of drugs encapsulated into pH-sensitive liposomes is under the control of a lamellar-to-hexagonal transition. In a previous study, under anhydrous conditions, oligonucleotides (ODN) encapsulated in pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) were shown to modify the phase behaviour of DOPE. In the present study, the lipid/ODN interactions were evaluated in fully hydrated samples by surface tension measurements, differential scanning calorimetry, X-ray diffraction and turbidimetry. Concerning the lipids, it was shown that OA provoked a disorganisation of DOPE lamellar phases and led to the complete disappearance of hexagonal transition along with heating. The addition of CHOL further decreased the lipid packing in the bilayers. Concerning ODN, these molecules provoked an increase in the surface pressure of a DOPE/OA/CHOL monolayer, indicating the existence of molecular interactions with the lipids. At a supramolecular level, ODN induced a more ordered organisation of DOPE molecules in the lamellar and hexagonal phases, and completely abolished the disorganisational effect of OA and CHOL.

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.

Thermal and Structural Behavior of Milk Fat.

The thermal and structural properties of unstable varieties of triacylglycerols (TGs) crystallizing in milk fat globules of cream are examined in the range -8- +50 degrees C using a new instrument allowing simultaneously time-resolved synchrotron X-ray diffraction at both wide and small angles as a function of temperature (XRDT) and high sensitivity differential scanning calorimetry (DSC). Small angle X-ray diffraction shows that the unstable alpha form first formed by cream quenching to -8 degrees C corresponds in fact to two different lamellar phases corresponding to 2L (47 Å) and 3L (70.4 Å) arrangements of TGs. The bilayered structure is very unstable since it disappears during the course of a 20-min isothermal conditioning at -8 degrees C. On fast heating, the crystalline evolution of cream TGs demonstrates the monotropic character of their polymorphism. The structural and thermal behaviors of cream which are compared to that of its anhydrous milk fat isolated from the cream (C. Lopez et al., J. Dairy Sci., submitted) show that the crystallization occurring in emulsion droplets is similar to bulk. However, the comparison of XRD peak widths indicates that the TG crystallization is more disordered in emulsion. This disorder is attributed to the constraints due to the interface curvature in emulsion droplets. Copyright 2000 Academic Press.

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.

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.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples.

pH-sensitive liposomes made of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) mixtures were shown to be very promising carriers for oligonucleotides (ON). However, it appeared necessary to clarify the structural consequence of the interactions of ON with the liposome, and especially on DOPE, the lipid responsible for the pH sensitivity. The present study was carried out by differential scanning calorimetry and X-ray diffraction, at low hydration. In such a case, DOPE generally adopt a hexagonal phase. It could be shown that ON increased DOPE transition temperature and increased v/al, as a result of electrostatic interactions between ON and DOPE headgroups. OA was found to have exactly opposite effects, its presence between DOPE molecules inhibiting the formation of hydrogen bonds. The presence of both ON and OA allowed the system to organize in a lamellar phase below the solid/liquid transition, whereas above this temperature ON preferably interacted with DOPE in a hexagonal phase and led OA to separate.

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.

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.