Stefan Salentinig

Self-assembled structures and pKa value of oleic acid in systems of biological relevance.

In the human digestion process, triglycerides are hydrolyzed by lipases to monoglycerides and the corresponding fatty acids. Here we report the self-assembly of structures in biologically relevant, emulsified oleic acid-monoolein mixtures at various pH values and oleic acid concentrations. Small-angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering were used to investigate the structures formed, and to follow their transitions while these factors were varied. The addition of oleic acid to monoolein-based cubosomes was found to increase the critical packing parameter in the system. Structural transitions from bicontinuous cubosomes through hexosomes and micellar cubosomes (Fd3m symmetry) to emulsified microemulsions occur with increasing oleic acid concentration. At sufficiently high oleic acid concentration, the internal particle structure was also found to strongly depend on the pH of the aqueous phase: transformations from emulsified microemulsion through micellar cubosomes, hexosomes, and bicontinuous cubosomes to vesicles can be observed as a function of increasing pH. The reversible transition from liquid crystals to vesicles occurs at intestinal pH values (between pH 7 and 8). The hydrodynamic radius of the particles decreases from around 120 nm for internally structured particles to around 60 nm for vesicles. All transitions with pH are reversible. Finally, the apparent pK(a) for oleic acid in monoolein could be determined from the change of structure with pH. This value is within the physiological pH range of the intestine and depends somewhat on composition.

Transitions in the internal structure of lipid droplets during fat digestion

This work focuses on colloidal transformations during the digestion of dietary triglyceride-lipids under physiological conditions present in the intestine. In vitrodigestion experiments with triglyceride emulsions and pancreatic lipase are monitored by time-resolved small-angle X-ray scattering, polarized and depolarized dynamic light scattering and cryogenic transmission electron microscopy as complementary methods. In the intestine, the interface-active pancreatic lipase–colipase complex adsorbs to the interface of the emulsified lipids and quantitatively hydrolyses the tri- and diglyceride to 2-monoglycerides and fatty acids as the final digestion products. Both products are amphiphilic under intestinal conditions and form a great variety of self-assembled structures, depending on composition and pH. With time of lipase action, the interior of the emulsion particles was found to self-assemble to oil continuous structures with increasing hydrophilicity of the interface. A transition from oil emulsion to emulsified microemulsion, micellar cubic, inverse hexagonal, and bicontinuous cubic liquid-crystalline droplets was found. Self-assembled structures formed during the in vitro lipid digestion process were investigated as a function of bile-juice concentration, pH and hydrophobic additives. Significant differences in structure formation and transition times (kinetics) could be observed when varying these crucial parameters. Vesicles were found to be the dominating final structures of the triglyceride digestion process.

Preparation of highly concentrated nanostructured dispersions of controlled size

This article presents the use of a shearing procedure for the preparation of stable nanostructured dispersions of lipid mesophases. This new application of the shearing technique is compared with the well-established ultrasonication method for the emulsification of these mesophases in water in terms of particle size, particle size distribution and available concentration range. With a laboratory-built shear device based on a Couette cell, it was possible to produce high quantities of internally self-assembled emulsion particles of controlled size at concentrated hydrophobic phase contents (phi(o)) of up to 70 wt%. The concentration limit of 70 wt% could be reached however, the maximum attainable concentration depended on the internal structure type of the particles. The limit was thus easily attained for emulsified microemulsions (EME) as well as for the emulsified inverse hexagonal phase (H(2)), whereas it was found to be lower for emulsified discontinuous (Fd3m) and bicontinuous (Pn3m) cubic phases. Moreover, by shearing, it was possible to keep the size of the particles relatively constant when increasing phi(o), whereas the particle size significantly increased with phi(o) when ultrasonication was employed. By means of ultrasonication, the hydrodynamic radius of the particles could be tuned linearly between 85 to 180 nm as a function of phi(o) up to a maximum of 20 to 30 wt%. Below the maximum concentration limit, particles displayed a well-controlled size.

Formation of Highly Organized Nanostructures during the Digestion of Milk

Natures own emulsion, milk, consists of nutrients such as proteins, vitamins, salts, and milk fat with primarily triglycerides. The digestion of milk fats is the key to the survival of mammal species, yet it is surprising how little we understand this process. The lipase-catalyzed hydrolysis of dietary fats into fatty acids and monoglyceride is essential for efficient absorption of the fat by the enterocytes. Here we report the discovery of highly ordered geometric nanostructures during the digestion of dairy milk. Transitions from normal emulsion through a variety of differently ordered nanostructures were observed using time-resolved small-angle X-ray scattering on a high-intensity synchrotron source and visualized by cryogenic transmission electron microscopy. Water and hydrophilic molecules are transferred into the lipid phase of the milk particle, turning the lipid core gradually into a more hydrophilic environment. The formation of highly ordered lipid particles with substantial internal surface area, particularly in low-bile conditions, may indicate a compensating mechanism for maintenance of lipid absorption under compromised lipolysis conditions.

Influence of the Stabilizer Concentration on the Internal Liquid Crystalline Order and the Size of Oil-Loaded Monolinolein-Based Dispersions

The internal phase of monolinolein-based dispersions loaded with tetradecane or (R)-(+)-limonene was investigated as a function of the stabilizer content by small-angle X-ray scattering. Phase transitions at the colloidal scale were found in some of nanostructured aqueous dispersions by increasing the stabilizer content. For particles containing a bicontinuous cubic phase, a large increase of the stabilizer concentration promoted a liquid crystalline phase transition from the Pn3m to the Im3m cubic symmetry. The coexistence of both phases is observed in an intermediate stabilizer concentration range. For particles with an internal micellar cubic Fd3m symmetry, the internal structure changes in the isotropic fluid L(2) phase. In case of particles with an internal hexagonal phase (H(2) symmetry), the increasing amount of stabilizer did not alter the lattice parameter but decreased the size of the nanostructured domain. Moreover, we showed for hexagonal and emulsified micellar phase particles that the increase of the stabilizer content induced a strong decrease of the mean hydrodynamic size of the particles, allowing producing nanostructured lipid-based liquid crystalline particles down to a radius of 70 nm at the same energy input.

A novel approach to enhance the mucoadhesion of lipid drug nanocarriers for improved drug delivery to the buccal mucosa

Targeted drug delivery to the buccal mucosa offers distinct advantages over oral delivery to the gastrointestinal tract including by-passing hepatic first-pass metabolism. However, the buccal route is often limited by low bioavailability, low drug loading and reduced residence time due to salivary excretion and clearance. To overcome these limitations, a novel mucoadhesive formulation based on liquid crystalline nanoparticles was designed. Utilising a pH induced in situ transition from a stable vesicle formulation to dispersed inverse hexagonal phase nanoparticles (hexosomes) enhanced adsorption onto the mucosal surface was enabled. Firstly, the phase behaviour of the amphiphilic lipid phytantriol (PHY) and oleic acid (OA) was assessed from pH 2-9 using small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (cryo-TEM) to determine the appropriate composition for the vesicle to hexosome transition. The colloidal stability of the formulation was determined using turbidity studies. Dispersions comprising 30% w/w OA in PHY were able to form stable vesicles at pH 8 and transition to hexosomes when exposed to pH<7 (as encountered on the buccal mucosal surface). Subsequent ex vivo studies utilising excised porcine buccal tissue indicated significant retention of the in situ-formed PHY/OA hexosomes when compared to control DOPC vesicles (p<0.005), confirmed independently using confocal fluorescence microscopy, radioactive scintillation counting and HPLC analysis for incorporated drug. Thus, a novel approach providing a stable vesicle formulation, with in situ transformation to mucoadhesive hexosomes has been identified with the potential to enhance drug delivery to mucosal surfaces.

Self-assembled structures formed during lipid digestion: characterization and implications for oral lipid-based drug delivery systems

There is increasing interest in the use of lipid-based formulations for the delivery of poorly water-soluble drugs. After ingestion of the formulation, exposure to the gastrointestinal environment results in dispersion and digestion processes, leading to the production of amphiphilic digestion products that form self-assembled structures in the aqueous environment of the intestine. These structures are crucial for the maintenance of drug in a solubilized state prior to absorption. This review describes the structural techniques used to study such systems, the structures formed in assembled ‘equilibrium’ compositions where components are combined in expected ratios representative of the endpoint of digestion, structures formed using dynamic in vitro ‘non-equilibrium’ digestion models where the composition and hence structures present change over time and observations from ex vivo aspirated samples. Possible future directions towards an improved understanding of the structural aspects of lipid digestion are proposed.

pH-responsive micelles based on caprylic acid.

Free fatty acids play a vital role as fuel for cells and in lipid metabolism. During lipid digestion in the gastrointestinal tract, triglycerides are hydrolyzed, resulting in free fatty acid and monoglyceride amphiphilic products. These components, together with bile salts, are responsible for the transport of lipids and poorly water-soluble nutrients and xenobiotics from the intestine into the circulatory system of the body. In this study, we show that the self-assembly of digestion products from medium-chain triglycerides (tricaprylin) in combination with bile salt and phospholipid is highly pH-responsive. Individual building blocks of caprylic acid within the mixed colloidal structures are mapped using a combination of small-angle X-ray and neutron scattering combined with both solvent contrast variation and selective deuteration. Modeling of the scattering data shows transitions in the size and shape of the micelles in combination with a transfer of the caprylic acid from the core of the micelles to the shell or into the bulk water upon increasing pH. The results help to understand the process of lipid digestion with a focus on colloidal structure formation and transformation for the delivery of triglyceride lipids and other hydrophobic functional molecules.

Material transfer in cubosome-emulsion mixtures: effect of alkane chain length.

We present here results on the transfer kinetics of monoglyceride and n-alkanes in water. Transfer kinetics between cubosomes and emulsion droplets were followed using time-resolved small-angle X-ray scattering measurements, while dynamic light scattering was used to study the changes in the particle radii. The effect of the initial size of cubosomes and emulsion droplets on the final droplet size of the mixed components was investigated. Decane was transferred into the cubosomes with the disappearance of all emulsion droplets, with no detectable transfer of monoglyceride. Cubosomes were even found to absorb bulk decane into the solution from a surface layer, which raises the possibility of using cubosomes to bind hydrophobic molecules from the bulk phase. In mixtures with longer alkanes, transfer of both monoglyceride and oil was observed; while with octadecane the transfer of monoglyceride into emulsion droplets dominated. Calculating the point at which the transfer of monoglyceride becomes dominant over that of oil as the alkane chain length is increased shows that, in compositional ripening, monoglyceride behaves as an alkane with 15.3 carbons. These results further our understanding of the interactions of internally self-assembled particles in various media and suggest possible ways of controlling the size of the particles.

Self-Assembly Structure Formation during the Digestion of Human Breast Milk†

An infants complete diet, human breast milk, is the basis for its survival and development. It contains water-soluble and poorly water-soluble bioactive components, metabolic messages, and energy, all of which are made bioavailable during the digestion process in the infants gastrointestinal tract. Reported is the first discovery of highly geometrically organized structures formed during the digestion of human breast milk under simulated in vivo conditions using small-angle X-ray scattering and cryogenic transmission electron microscopy. Time of digestion, pH, and bile salt concentration were found to have symbiotic effects gradually tuning the oil-based environment inside the breast milk globules to more water-like structures with high internal surface area. The structure formation is necessarily linked to its function as carriers for poorly water-soluble molecules in the digestive tract of the infant.