Polymorphism of glyceryl behenates: from the individual compounds to the pharmaceutical excipient.

The present paper deals with the crystallization behavior of glyceryl behenate mixtures that are extensively used in the field of drug delivery. The aim of the study was to understand the structural and thermal behaviors of Compritol(®) by considering first the individual polymorphism of the main components constituting this excipient and then their mixtures. This excipient mainly contains dibehenin (∼50%), tribehenin (∼30%) and monobehenin (20%). It appeared clearly that the mixture polymorphism did not result from a simple addition of the individual behavior. Indeed, the solid state organization of this excipient strongly depended on the presence of the third main component, monobehenin, into the mixture. Furthermore, a threshold ratio of monobehenin, at least 10%, must be reach in order to obtain the typical structural organization (co-existence of α/sub-α subcells) and thermal behavior (solid-solid transition and melting) of Compritol(®). This underlines that special attention is required when mixing Compritol(®) with other pharmaceutical ingredients that could trap monoglycerides and modify the equilibrium present in the pure excipient.

Controlled release of a highly hydrophilic API from lipid microspheres obtained by prilling: analysis of drug and water diffusion processes with X-ray-based methods.

This study deals with the development of an oral controlled-release dosage form of a highly water-soluble antiepileptic drug. In this respect, drug-loaded spheroid particles close to 380 µm in diameter and composed of lipid binders were prepared by prilling. The purpose here was to thoroughly characterize the controlled-release mechanism of the drug in aqueous pH-6.8 buffered dissolution medium. Water and drug diffusion pathways as well as related kinetic parameters were determined by theoretical analysis of experimental data. Conventional in-vitro experiments performed by analytical high performance liquid chromatography showed that the released fraction reaches 90 wt.% only after a 24-hour immersion in the dissolution medium, pointing out an effective sustained release mechanism. Interpretation of these data was strengthened by the implementation of an innovative methodology involving X-ray diffraction and microtomography to follow the structural evolution of the drug-loaded microspheres at molecular and microscopic scales. This approach allowed to explicit that water and drug transports obey to Fickian diffusion behaviours in good agreement with Crank's and non-simplified Higuchi's equations, respectively. In the latter case, independent modelling of drug release assimilating the microspheres to a variable-geometry reservoir was considered to refine the kinetic analysis of the diffusion process. The water diffusion coefficient D(w) was found equal to 6.3 × 10(-9) cm(2)/s and the API apparent diffusion coefficient reduced to the tortuosity of the matrix D(API)/τ equal to 2 × 10(-9) cm(2)/s. This study ranks among the rare examples of monolithic dispersion device constituted by a highly soluble drug incorporated inside a perfectly inert lipid matrix. The dissolution liquid penetrates the particles through channels progressively created by the solubilization of the drug itself which occurs instantaneously at the inner front of the liquid.

The viscoelastic properties of processed cheeses depend on their thermal history and fat polymorphism.

Both the composition and the thermal kinetics that are applied to processed cheeses can affect their texture. This study investigated the effect of the storage conditions and thermal history on the viscoelastic properties of processed cheese and the physical properties of the fat phase. The microstructure of processed cheese has been characterized. Using a combination of physical techniques such as rheometry, differential scanning calorimetry, and X-ray diffraction, the partial crystallization of fat and the polymorphism of triacylglycerols (TG; main constituents of milk fat) were related to changes in the elastic modulus and tan δ as a function of temperature. In the small emulsion droplets (<1 µm) dispersed in processed cheeses, the solid fat phase was studied at a molecular level and showed differences as a function of the thermal history. Storage of processed cheese at 4 °C and its equilibration at 25 °C lead to partial crystallization of the fat phase, with the formation of a ß' 2 L (40.9 Å) structure; on cooling at 2 °C min(-1), the formation of an α 3 L (65.8 Å) structure was characterized. The cooling of processed cheese from 60 to -10 °C leads to the formation of a single type of crystal: α 3 L (72 Å). Structural reorganizations of the solid fat phase characterized on heating allowed the interpretation of the elastic modulus evolution of processed cheese. This study evidenced polymorphism of TG in a complex food product such as processed cheese and allowed a better understanding of the viscoelastic properties as a function of the thermal history.

Interaction of a new anticancer prodrug, gemcitabine-squalene, with a model membrane: coupled DSC and XRD study.

Gemcitabine is an anticancer nucleoside analogue active against a wide variety of solid tumors. However it is rapidly deaminated to an inactive metabolite, leading to short biological half-life and induction of resistance. A new prodrug of gemcitabine, coupling squalene to gemcitabine (GemSq), has been designed to overcome the above drawbacks. It has been previously shown that this prodrug displays significantly higher anticancer activity than gemcitabine against leukemia. In the present study the structural modifications of dipalmitoylphosphatidylcholine (DPPC) model membranes induced by increasing concentrations of GemSQ have been investigated using small and wide angle X-ray scattering (SWAXS) and differential scanning calorimetry (DSC). At room temperature an unusual inverse bicontinuous cubic phase formed over a broad composition range. The basic bilayer structure displayed an intermediate order between those of the gel and fluid phases of DPPC. A reversible transition to a fluid lamellar phase occurred upon heating. The transitions between these two phases were governed by different mechanisms depending on the GemSq concentration in the membrane. Finally, the biological relevance of these observations for the cytotoxic activity of GemSq has been discussed.

Self-organization of synthetic cholesteryl oligoethyleneglycol glycosides in water.

Lectin-sugar recognition systems are of interest in the pharmaceutical field, especially for the development of drug carriers, tailored for selective delivery. This paper deals with the anhydrous and aqueous self-organization properties of a synthetic cholesteryl oligoethyleneglycol glycoside with the aim of their incorporation in liposomes. Successive phases (lamellar, R3m, Im3m, micelles) have been described depending on water content and temperature. As a result of the presence of sugar residues and their hydration ability, this glycolipid shows a large range of packing parameter with increasing water content. However, because of oligoethyleneglycol spacer, a slight dehydration has been observed with increasing temperature from 20 to 60 degrees C.

Triglycerides obtained by dry fractionation of milk fat: 2. Thermal properties and polymorphic evolutions on heating.

The crystallographic and thermal properties of milk fat and fractions were investigated on heating using the coupling of synchrotron X-ray diffraction with differential scanning calorimetry. We showed that re-crystallisations occurred during the heating of the stearin and the olein fractions with the formation of a beta' 2L (41.1-42.6A) structure and a beta' 3L (66A) structure, respectively. By creating a quantified solid-liquid phase behaviour versus temperature diagram, the amount of the solid and liquid phases and the relative proportion of each of the crystalline structures within the solid phase were determined.

Crystallization and polymorphism of triacylglycerols contribute to the rheological properties of processed cheese.

The thermal, rheological, and structural behaviors of a spreadable processed cheese were studied by complementary techniques including differential scanning calorimetry (DSC), rheology, and X-ray diffraction as a function of temperature. In this product, fat is present as a dispersed phase. Thermal and rheological properties were studied at different cooling rates between 0.5 and 10 degrees C min(-1) from 60 to 3 degrees C. Crystallization properties of fat were monitored at a cooling rate of -2 degrees C min(-1) from 60 to -10 degrees C. Fat triacylglycerols (TGs) crystallized at 15 degrees C in a triple-chain length 3Lalpha (72 A) structure correlated to exothermic events and to the sudden increase in the rheological moduli G' and G''. Upon heating at 2 degrees C min(-1), the polymorphic transition of TGs evidence the melting of the 3Lalpha structure and the formation of a 2Lbeta' (36.7-41.5 A) structure. Melting of the latter follows. These transformations coincide with thermal events observed by DSC and the decrease in two steps of the rheological moduli. The influence of fat crystallization, melting, and polymorphism upon the viscoelastic properties is clearly demonstrated upon both heating and cooling.

Glycosylated liposomes against Helicobacter pylori: behavior in acidic conditions.

Helicobacter pylori was isolated in 1982 and confirmed as a gastric pathogenic agent at the end of the 1980s. The present work deals with liposomes formulations in which are incorporated cholesteryl tetraethylene glycol oside as model ligands for H. pylori adhesins. This study is devoted to the behavior of liposomes in gastric conditions. The glycosylated vesicles are stable and the pH of the internal aqueous compartment remains close to 4 even through more acidic conditions are imposed to the external phase (pH 1.2-2). Such a pH gradient depends essentially on the nature of phospholipids used and is not extensively affected by the incorporation of the targeting agent. These aspects are particularly important to the development of liposome formulations against H. pylori, bacteria sensitive to antibiotics which are unstable in very acidic conditions.

Supramolecular organization of S12363-liposomes prepared with two different remote loading processes.

The S12363 anticancer drug was encapsulated into liposomes in an attempt to increase its therapeutic index. Loading of S12363 was achieved using two different processes based on the formation of either a pH gradient or an ammonium gradient between the acidic inner liposomal compartment and the basic outer phase. High encapsulation yields (>90%) were obtained using both processes for sphingomyelin/cholesterol/cholesterol-PEG vesicles. Spectrofluorimetry measurements have shown that liposomes were characterized by an internal pH around 4 for both loading processes. This internal pH was stable over a period of at least 20 days. Differential scanning calorimetry coupled with time-resolved synchrotron X-ray diffraction was used to study the drug/carrier supramolecular organization. In ammonium sulfate, S12363 was inserted into the bilayer in the vicinity of the polar headgroup. In citrate buffer, S12363 was mainly adsorbed at the water-lipid interface. The drug partitioning into the membrane was inhomogeneous and led to the formation of drug-rich and drug-poor domains. This effect was enhanced in the presence of cholesterol, especially in ammonium sulfate. To conclude, for both processes, the encapsulated drug was found inside the liposome aqueous core but strongly interacting with the membrane.

Solubilisation of dipalmitoylphosphatidylcholine bilayers by sodium taurocholate: a model to study the stability of liposomes in the gastrointestinal tract and their mechanism of interaction with a model bile salt.

In order to better understand the mechanism of destabilization of liposomes used as drug carriers for oral administration by bile salts, the insertion and partition of sodium taurocholate (TC) into small unilamellar vesicles (SUV) and multilayers (ML) of dipalmitoylphosphatidylcholine (DPPC) were examined by continuous turbidity analysis and DSC. Optical density was recorded during the progressive solubilisation of DPPC SUV and ML into DPPC/TC mixed micelles by varying the rate of TC addition and the temperature. The results show that the insertion and diffusion of TC in the DPPC membrane is a slow process influenced by the polymorphism of the lipid, independently of its organisation. This dynamic study mimics physiological phenomena of the digestion of liposomes. In the gastrointestinal tract, DPPC SUV would be more resistant to TC than egg phosphatidylcholine (EPC) SUV [K. Andrieux, L. Forte, S. Lesieur, M. Paternostre, M. Ollivon, C. Grabielle-Madelmont, Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles, Pharm. Res. 21 (2004) 1505-1516] because of the lower insertion of TC into DPPC bilayer at 37 degrees C at low TC concentration in the medium (fasted conditions). At high TC concentration (postprandially or after lipid absorption), the use of DPPC to prepare liposomes will delay or reduce the liberation of a drug encapsulated into liposomes in the gastrointestinal tract. As a conclusion, the addition of DPPC appears an attractive strategy to formulate orally administered liposomes.

Crystallization properties and polymorphism of triacylglycerols in goat's milk fat globules.

The sensorial, functional, and nutritional properties of goat dairy products result from the specific fatty acid composition of goat's milk fat. However, information on the physical and thermal properties of goat's milk fat is scarce. In this study, crystallization of triacylglycerols (TG) in goat's milk fat globules was investigated using polarized light microscopy and the coupling of time-resolved synchrotron radiation X-ray diffraction (XRD) and high-sensitivity differential scanning calorimetry (DSC). The molecular organization of the solid fat phase was characterized for cooling rates between 3 and 0.1 degrees C/min. Quenching of goat's milk fat globules from 50 to -8 degrees C and 4 degrees C was also examined to identify the most unstable polymorphic forms of TG. Then, the melting behavior of fat crystals was studied on subsequent heating at 1 degrees C/min. Triple chain length (3L: 68.6-70 A) and double chain length (2L: 37-45.4 A) structures were characterized and 5 polymorphic forms, alpha, sub-alpha, beta' 1, beta' 2, and beta were identified. Polymorphic transitions were observed within goat's milk fat globules as a function of time after quenching and as a function of temperature on heating. From a technological point of view, this work will contribute to a better understanding of the rheological properties as well as on the flavor evolutions of goat's milk-based products.

Consequences of ions and pH on the supramolecular organization of sphingomyelin and sphingomyelin/cholesterol bilayers.

For drug delivery purpose the anticancer drug S12363 was loaded into ESM/Chol-liposomes using either a pH or an ammonium gradient. Association between the drug and the liposome depends markedly on the liposome membrane structure. Thus, ESM and ESM/Chol bilayer organization had been characterized by coupled DSC and XRDT as a function of both cholesterol concentration and aqueous medium composition. ESM bilayers exhibited a ripple lamellar gel phase P(beta') below the melting temperature and adopted a L(beta)-like gel phase upon Chol insertion. Supramolecular organization of ESM and ESM/Chol bilayers was not modified by citrate buffer or ammonium sulfate solution whatever the pH (3< or = pH < or =7). Nevertheless, in ESM bilayer, ammonium sulfate salt induced a peculiar organization of head groups, leading to irregular d-spacing and weakly correlated bilayers. Moreover, in the presence of salts, a weakening of van der Waals attraction forces was seen and led to a swelling of the water layer.

Multiscale characterization of the organization of triglycerides and phospholipids in emmental cheese: from the microscopic to the molecular level.

The chemical composition and properties of lipids, both triglycerides and phospholipids, play a major role in the functional and nutritional properties of food products. In this study, the suprastructure of fat, solid fat content, and crystallographic properties of triglycerides were investigated in hard-type cheeses from the microscopic scale to the molecular level using the combination of relevant techniques. Two industrial cheeses with different oiling off properties were compared with experimental cheeses manufactured in the laboratory. Microstructural analysis performed using confocal laser scanning microscopy showed that milk processing led to the disruption of fat globules with the formation of nonglobular fat. For a similar fatty acid composition, oiling off was mainly related to the fat in dry matter content and to the suprastructure of fat in cheese. An exogenous fluorescent phospholipid permitted the localization of milk phospholipids in the cheese matrix, which mainly remain around fat inclusions after disruption of the milk fat globule membrane, and to show heterogeneities. We also showed using differential scanning calorimetry that the suprastructure of fat did not affect the solid fat content in cheese at 4 degrees C: 71.6 +/- 4.9%. The organization of triglyceride molecules in fat crystals, elucidated at a molecular level using X-ray diffraction, corresponded to the coexistence of 2 lamellar structures (2L 40.5 angstroms and 3L 54.6 angstroms) with four polymorphic forms: alpha, two beta' and beta. A schematic representation of the multiscale organization of triglycerides and phospholipids in cheese is proposed.

Conformational and hydrational properties during the L(beta)- to L(alpha)- and L(alpha)- to H(II)-phase transition in phosphatidylethanolamine.

Differential scanning calorimetry (DSC) measurements have been carried out simultaneously with small- and wide-angle X-ray scattering recordings on liposomal dispersions of stearoyl-oleoyl-phosphatidylethanolamine (PE) in a temperature range from 20 to 80 degrees C. The main transition temperature, T(m), was determined at 30.9 degrees C with an enthalpy of 28.5 kJ/mol and the lamellar-to-inverse hexagonal phase transition temperature, T(hex), at 61.6 degrees C with an enthalpy of 3.8 kJ/mol. Additionally highly resolved small angle X-ray diffraction experiments performed at equilibrium conditions allowed a reliable decomposition of the lattice spacings into hydrophobic and hydrophilic structure elements as well as the determination of the lipid interface area of the lamellar gel-phase (L(beta)), the fluid lamellar phase (L(alpha)) and of the inverse hexagonal phase (H(II)). The rearrangement of the lipid matrix and the coincident change of free water per lipid is illustrated for both transitions. Last, possible transition mechanisms are discussed on a molecular level.

Discovery of new hexagonal supramolecular nanostructures formed by squalenoylation of an anticancer nucleoside analogue.

In this study, the dynamically folded conformation of squalene (SQ) is taken advantage of to link this natural compound to the anticancer nucleoside analogue gemcitabine (gem) in order to achieve the spontaneous formation of nanoassemblies (SQgem) in water. Cryogenic transmission electron microscopy examination reveals particles (104 nm) with a hexagonal or multifaceted shape that display an internal structure made of reticular planes, each particle being surrounded by an external shell. X-ray diffraction evidences the hexagonal molecular packing of SQgem, resulting from the stacking of direct or inverse cylinders. The respective volumes of the gem and SQ molecules as well as molecular modeling of SQgem suggest the stacking of inverse hexagonal phases, in which the central aqueous core, consisting of water and gem molecules, is surrounded by SQ moieties. These SQgem nanoassemblies also exhibit impressively greater anticancer activity than gem against a solid subcutaneously grafted tumor, following intravenous administration. To our knowledge, this is the first demonstration of hexagonal phase organization with a SQ derivative.

Small-angle neutron and X-ray scattering from amphiphilic stimuli-responsive diamond-type bicontinuous cubic phase.

The structural evolution of a diamond-type bicontinuous lipid cubic phase upon application of thermal and chemical (hydration agent) stimuli is investigated by means of small-angle neutron (SANS) and X-ray scattering (SAXS). The soft-matter cubic architecture responds by dramatic swelling (DLarge cubic structure) upon incorporation of a hydration-enhancing guest component (octyl glucoside) at low and ambient temperatures, the aqueous channel diameter increasing twice to approximately 7 nm. DLarge appears to be built up from an assembly of cubosomic domains, which may coexist with an amphiphilic lamellae domain at low temperatures. The chemical stimulus concentration can be selected as to tune the hydration of the nanochannels in the DLarge phase and its transformation into a DNormal phase at temperatures above the body temperature. Two-dimensional SANS images recorded upon heating scan reveal growth of spontaneously oriented domains of single-crystal cubic nature. Phase separation and squeezing out the guest-hydrating agent from the higher-curvature regions of the amphiphilic bilayer suggest a possible mechanism for the established transformations. The order-order structural transition, cubic DLarge-cubic DNormal, is found to be reversible upon cooling. The obtained results put forward a structure-based concept for release of encapsulated guest molecules from stimuli-responsive and self-regulated cubosomic nanocarriers.

Anhydrous goat's milk fat: thermal and structural behaviors studied by coupled differential scanning calorimetry and X-ray diffraction. 2. Influence of cooling rate.

Crystallization and melting properties of triacylglycerols (TGs) in anhydrous goat's milk fat (AGMF) are investigated by X-ray diffraction as a function of temperature (XRDT) coupled with high-sensitivity differential scanning calorimetry (DSC), using synchrotron radiation and Microcalix. The polymorphic behavior of AGMF was monitored by varying the cooling rates between 5 and 1 degrees C/min from 45 to -20 degrees C with their subsequent melting at 1 degrees C/min. Quenching of AGMF at -20 degrees C was also examined to determine the metastable polymorphic form of AGMF. At intermediate cooling rates, TGs in AGMF crystallize, from about 18 degrees C in two different lamellar structures with triple chain length 3Lalpha stacking of 72 A and a double chain length 2Lalpha stacking of 48 A, which are correlated to two overlapped exothermic peaks recorded by DSC. A reversible transition sub alpha <--> alpha was observed. Subsequent heating at 1 degrees C/min shows numerous structural rearrangements before final melting. At fast cooling of AGMF (5 degrees C/min), similar unstable crystalline varieties are formed while three endotherms are recorded. Several new unstable lamellar structures are observed after quenching. All of these data are compared to those previously reported at slow cooling (0.1 degrees C/min) showing a relative stability of the structures formed. In spite of general similitude, the thermal and structural behavior of the goat's milk is more complex than that of the cow's milk.

Encapsulation-stucturing of edible oil attenuates acute elevation of blood lipids and insulin in humans.

Blood triglyceride, free fatty acid and insulin levels are lower after acute intake of an oil-water-monoglyceride gel versus an oil-water mixture, demonstrating that food matrix nanostructure and microstructure can be engineered to modulate the physiological response. Oil emulsification by the monoglyceride Lα liquid-crystalline lamellar phase, followed by droplet wall crystallization, encapsulates oil and creates a material with the functionality and properties of a fat. This novel phase is devoid of trans fatty acids and can be manufactured with as little as 4% added saturated monoglyceride.

Milk fat and primary fractions obtained by dry fractionation 1. Chemical composition and crystallisation properties.

The chemical composition and crystallisation properties of milk fat and its primary fractions, obtained by dry fractionation at 21 degrees C, were investigated. The solid fraction (stearin) and the liquid fraction (olein) displayed a different triacylglycerol (TG) composition. Stearin fraction was enriched in long-chain fatty acids, whereas olein fraction was enriched in short-chain and unsaturated fatty acids. Crystallisation properties of milk fat, and both the stearin and olein fractions were studied on cooling at |dT/dt|=1 degrees C min(-1) by differential scanning calorimetry and time-resolved synchrotron X-ray diffraction (XRD) at small and wide angles. Two main types of crystals corresponding to double chain length structures were characterised in the stearin fraction: alpha 2L(1) (47.5 Angstrom) and beta' 2L(2) (41.7 Angstrom). A triple chain length structure was formed in the olein fraction: alpha 3L (72.1 Angstrom). Crystallization of milk fat showed the formation of two 2L (47.3 and 41.6 Angstrom) and one 3L (72.1 Angstrom) lamellar structures with an hexagonal packing (alpha form). A schematic representation of the 3L packing of olein fraction was proposed to explain how a wide diversity of TG can accommodate to form a lamellar structure with a thickness of 72 Angstrom. Furthermore, the sharpness of the small-angle XRD lines associated to the alpha form was explained by the formation of liquid crystals of smectic type.

Detailed structure of diamond-type lipid cubic nanoparticles.

Supramolecular three-dimensional self-assembly of nonlamellar lipids with fragments of the protein immunoglobulin results in a bicontinuous cubic phase fragmented into nanoparticles with open water channels (cubosomes). The structure of the diamond-type cubic nanoparticles is characterized experimentally by freeze-fracture electron microscopy, and it is mathematically modeled with nodal surfaces emphasizing the fluid-like undulations of the cubosomic interfaces. Based on scaling-up and scaling-down approaches, we present stable and intermediate-kind nanoparticles resulting from the cubosomic growth. Our results reveal the smallest stable diamond-type cubosomic entity that can serve as a building block of more complex nanostructured fluid drug delivery vehicles of therapeutic proteins. The evidence presented for lipid-bilayer undulations in the surface region of the protein/lipid cubosomes could have important consequences for possible applications of these hierarchically organized porous nanoparticles.