Bile conjugation and its effect on in vitro lipolysis of emulsions.

Bile Salts (BS) are responsible for stimulating lipid digestion in our organism. Gut microbiota are responsible for the deconjugation process of primary conjugated to secondary unconjugated BS. We use two structurally distinct BS and characterize the rate of lipolysis as a compound parameter. A static in-vitro digestion model as well as meta-analysis of literature data has been performed to determine the most influential factors affecting the lipid digestion process. The results demonstrate that lipolysis of emulsions using conjugated BS (NaTC, FFA = 60.0 %, CMC in SIF = 5.58 mM, MSR of linoleic acid = 0.21, rate of adsorption = -0.057 mN/m.s) enhances the release of FFA compared to deconjugated BS (NaDC, FFA = 49.5 %, CMC in SIF = 2.49 mM, MSR of linoleic acid = 0.16 rate of adsorption = -0.064 mN/m.s). These results indicate that conjugation plays an important role in controlling the rate of lipolysis in our organism which can be in turn, tuned by the microflora composition of our gut, ultimately controlling the rate of deconjugation of the BS.

Autotitrator based on an Arduino Open Source Pump.

Acid-base titration is a quantitative analysis that enables knowing the quantity of acidic or basic groups present in a solution sample. It consists in the addition of base or acid to the solution sample while monitoring the pH to reach a neutral pH. The titration can be automated and here we present a low cost Arduino based Open Source Pump (OSPump) modified to act as an automated titrator with an obsolete but reliable Metrohm 713 pH meter. Our device is 50 times less expensive than second hand units from the pH meter manufacturer and inherently open to customization. We present two validation cases of study, including the lipolysis of a vegetable olive oil in water emulsion, characterized by the OSPump Titrator.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids.

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

Interactions between curcumin and cell membrane models by Langmuir monolayers.

Studying interactions between potential anticancer drugs and cell membrane models is of great interest to explore the capability of novel drugs in the development of anticancer treatments. Lipid membrane models are useful to understand cellular interactions and to discern drug mechanism action. Here, the interactions of curcumin, as a bioactive natural compound with anti-cancer properties, with both healthy and cancerous or tumor cell membrane models, based on Langmuir monolayers, have been studied. The healthy-cell membrane model is composed of cholesterol 67%, and saturated lipid dipalmitoylphosphatidylcholine 33%. The cancerous-cell-membrane-model is composed of a lower proportion of cholesterol, 25%, and unsaturated lipid sphingomyelin 75%. To compare their interaction with curcumin we report the compression isotherms registered for both lipid membrane models and curcumin in different proportions, their compression moduli and the thermodynamic interaction parameters. From this analysis, we evidence a destabilizing interaction between curcumin and the cancerous cell membrane model in comparison with the healthy one. This interaction is further visualized by micro-Brewster Angle and Atomic Force Microscopies. Our experiments show that the drug enhances cohesion in the healthy membrane model whereas it fluidifies the cancerous cell membrane model causing thermodynamic destabilization. These are useful results to improve the selectivity of the drug avoiding adverse side effects of most current anticancer therapies.

Complexation of DNA with Thermoresponsive Charged Microgels: Role of Swelling State and Electrostatics.

Micro- and nanogels are being increasingly used to encapsulate bioactive compounds. Their soft structure allows large loading capacity while their stimuli responsiveness makes them extremely versatile. In this work, the complexation of DNA with thermoresponsive microgels is presented. To this end, PEGylated charged microgels based on poly-N-isopropylacrylamide have been synthesized, allowing one to explore the electrostatics of the complexation. Cationic microgels complexate spontaneously by electrostatic attraction to oppositely charged DNA as demonstrated by electrophoretic mobility of the complexes. Then, Langmuir monolayers reveal an increased interaction of DNA with swollen microgels (20 °C). Anionic microgels require the presence of multivalent cations (Ca2+) to promote the complexation, overcoming the electrostatic repulsion with negatively charged DNA. Then again, Langmuir monolayers evidence their complexation at the surface. However, the presence of Ca2+ seems to induce profound changes in the interaction and surface conformation of anionic microgels. These alterations are further explored by measuring adsorbed films with the pendant drop technique. Conformational changes induced by Ca2+ on the structure of the microgel can ultimately affect the complexation with DNA and should be considered in the design. The combination of microstructural and surface properties for microgels offers a new perspective into complexation of DNA with soft particles with biomedical applications.

Hyaluronic acid and human/bovine serum albumin shelled nanocapsules: Interaction with mucins and in vitro digestibility of interfacial films.

Liquid lipid nanocapsules are oil droplets surrounded by a protective shell, which enable high load and allow controlled delivery of lipophilic compounds. However, their use in food formulations requires analysing their digestibility and interaction with mucin. Here, serum albumins and hyaluronic acid shelled olive oil nanocapsules are analysed to discern differences between human and bovine variants, the latter usually used as model system. Interfacial interaction of albumins and hyaluronic acid reveals that human albumin presents limited conformational changes upon adsorption, which increase by complexation with the polysaccharide present at the interface. The latter also promotes hydrophobic interactions with mucin, especially at pH 3 and protects albumin interfacial layer under in vitro gastric digestion. The interfacial unfolding induced in human albumin by hyaluronic acid facilitates in vitro lipolysis while its limited conformational changes provide the largest protection against in vitro lipolysis.

Applications of serum albumins in delivery systems: Differences in interfacial behaviour and interacting abilities with polysaccharides.

One of the major applications of Serum Albumins is their use as delivery systems for lipophilic compounds in biomedicine. Their biomedical application is based on the similarity with Human Serum Albumin (HSA), as a fully biocompatible protein. In general, Bovine Serum Albumin (BSA) is treated as comparable to its human homologue and used as a model protein for fundamental studies since it is available in high amounts and well understood. This protein can act as a carrier for lipophilic compounds or as protective shell in an emulsion-based vehicle. Polysaccharides are generally included in these formulations in order to increase the stability and/or applicability of the carrier. In this review, the main biomedical applications of Albumins as drug delivery systems are first presented. Secondly, the differences between BSA and HSA are highlighted, exploring the similarities and differences between these proteins and their interaction with polysaccharides, both in solution and adsorbed at interfaces. Finally, the use of Albumins as emulsifiers for emulsion-based delivery systems, concretely as Liquid Lipid Nanocapsules (LLNs), is revised and discussed in terms of the differences encountered in the molecular structure and in the interfacial properties. The specific case of Hyaluronic Acid is considered as a promising additive with important applications in biomedicine. The literature works are thoroughly discussed highlighting similarities and differences between BSA and HSA and their interaction with polysaccharides encountered at different structural levels, hence providing routes to control the optimal design of delivery systems.

Investigating the role of hyaluronic acid in improving curcumin bioaccessibility from nanoemulsions.

A major challenge in delivering curcumin effectively to the gut is its low solubility. One interesting approach to increase curcumin bioaccessibility is its emulsification. Here, we present curcumin-loaded liquid lipid nanocapsules (LLNs), obtained through olive oil emulsification, in which LLNs are coated by a protective shell composed of Bovine Serum Albumin (BSA) and hyaluronic acid (HA). Bioaccessibility of curcumin is evaluated following a standard in vitro digestion protocol. The presence of HA in the shell increases the amount of curcumin retained in the LLNs after in vitro gastric digestion from ~25% to ~85%. This protective effect occurs when HA binds to BSA in the shell. Moreover, this binding appears to be reinforced under gastric conditions, hence evidencing the crucial role of interfacial composition in protecting encapsulated curcumin. Interfacial engineering of nanoemulsions provides a route to improve the bioaccessibility of encapsulated curcumin at different stages in the gut.

Microgels at interfaces, from mickering emulsions to flat interfaces and back.

In this review, we cover the topic of p(NIPAM) based microgels at interfaces, revisiting classical studies in light of the newest ones. In particular, we focus on their use as emulsifiers in the so-called mickering emulsions, i.e. Pickering emulsion stabilized by soft particles. Given the complexity of the experimental characterization and simulation of these soft particles at interfaces, the review is structured in progressive complexity levels, until we reach the highly interesting and promising responsiveness to stimuli of mickering emulsions. We start from the lowest level of complexity, the current understanding of the behavior of single microgels confined at a flat interface. Then, we discuss their collective behavior upon crowding, their responsiveness at interfaces, and their macroscopic properties as microgel films. Once we have the necessary characterization tools, we proceed to discuss the complex and convoluted picture of responsive mickering emulsions. The way is rough, with current controversial and contradicting studies, but it holds promising results as well. We state open questions worth of being tackled by the Soft Matter community, and we conclude that it is worth the trouble of continuing after the master theory of microgel interfacial activity, as it will pave the way to widely adopt responsive mickering emulsions as the worthy Pickering emulsion successors.

Effect of Hyaluronic Acid and Pluronic-F68 on the Surface Properties of Foam as a Delivery System for Polidocanol in Sclerotherapy.

The use of foams to deliver bioactive agents and drugs is increasing in pharmaceutics. One example is the use of foam as a delivery system for polidocanol (POL) in sclerotherapy, with the addition of bioactive compounds to improve the delivery system being a current subject of study. This work shows the influence of two bioactive additives on the structure and stability of POL foam: hyaluronic acid (HA) and Pluronic-F68 (F68). HA is a natural non-surface-active biopolymer present in the extracellular matrix while F68 is a surface-active poloxamer that is biocompatible with plasma-derived fluids. Both additives increase the bulk viscosity of the sample, improving foam stability. However, HA doubled and F68 quadruplicated the foam half lifetime of POL. HA reduced the size and polydispersity of the bubble size distribution and increased the surface elasticity with respect to POL. Both facts have a positive impact in terms of foam stability. F68 also altered bubble structure and increased surface elasticity, again contributing to the enhancement of foam stability. The surface characterization of these systems is important, as in foam sclerotherapy it is crucial to assure the presence of POL at the surface of the bubbles in order to deliver the sclerosant agent in the target vein.

Foamy oysters: vesicular microstructure production in the Gryphaeidae via emulsification.

The vesicular microstructure is a very distinctive arrangement of calcite, consisting of hollow cavities (vesicles) of diverse sizes and shapes, usually elongated in the direction of shell thickening. It is uniquely found among living bivalves in a single oyster family, Gryphaeidae. The vesicles are distributed in lenses interleaved with compact foliated layers. We have studied the morphology and distribution of vesicles within the lenses using optical and electron microscopy, and micro-computed tomography. At a small scale, vesicles do not follow a classical von Neumann-Mullins route typical of ideal foams. At a larger scale, the initiation and evolution of a vesicular layer statistically proceed like a foam, with vesicles becoming more numerous, larger and more even in size. In summary, the vesicular material follows a foam-like coarsening to reduce the number of energetically costly interfaces. However, a steady state is never reached because the animal permanently introduces energy in the system by creating new vesicles. The fabrication of the vesicular material is mediated by the production of an emulsion between the extrapallial fluid and the precursor PILP of the calcitic walls within the thin extrapallial space. For this mechanism to proceed, the mantle cells must perform highly sophisticated behaviours of contact recognition and secretion. Accordingly, the vesicular material is under mixed physical-biological control.

Bile salts in digestion and transport of lipids.

Because of their unusual chemical structure, bile salts (BS) play a fundamental role in intestinal lipid digestion and transport. BS have a planar arrangement of hydrophobic and hydrophilic moieties, which enables the BS molecules to form peculiar self-assembled structures in aqueous solutions. This molecular arrangement also has an influence on specific interactions of BS with lipid molecules and other compounds of ingested food and digestive media. Those comprise the complex scenario in which lipolysis occurs. In this review, we discuss the BS synthesis, composition, bulk interactions and mode of action during lipid digestion and transport. We look specifically into surfactant-related functions of BS that affect lipolysis, such as interactions with dietary fibre and emulsifiers, the interfacial activity in facilitating lipase and colipase anchoring to the lipid substrate interface, and finally the role of BS in the intestinal transport of lipids. Unravelling the roles of BS in the processing of lipids in the gastrointestinal tract requires a detailed analysis of their interactions with different compounds. We provide an update on the most recent findings concerning two areas of BS involvement: lipolysis and intestinal transport. We first explore the interactions of BS with various dietary fibres and food emulsifiers in bulk and at interfaces, as these appear to be key aspects for understanding interactions with digestive media. Next, we explore the interactions of BS with components of the intestinal digestion environment, and the role of BS in displacing material from the oil-water interface and facilitating adsorption of lipase. We look into the process of desorption, solubilisation of lipolysis, products and formation of mixed micelles. Finally, the BS-driven interactions of colloidal particles with the small intestinal mucus layer are considered, providing new findings for the overall assessment of the role of BS in lipid digestion and intestinal transport. This review offers a unique compilation of well-established and most recent studies dealing with the interactions of BS with food emulsifiers, nanoparticles and dietary fibre, as well as with the luminal compounds of the gut, such as lipase-colipase, triglycerides and intestinal mucus. The combined analysis of these complex interactions may provide crucial information on the pattern and extent of lipid digestion. Such knowledge is important for controlling the uptake of dietary lipids or lipophilic pharmaceuticals in the gastrointestinal tract through the engineering of novel food structures or colloidal drug-delivery systems.

Interaction of DNA with likely-charged lipid monolayers: An experimental study.

Anionic lipids are increasingly being used in lipoplexes for synthetic gene vectors as an alternative to cationic lipids. This is primarily due to their lower toxicity, which makes them biocompatible and adaptable to be tissue specific. However, anionic lipoplexes require the presence of multivalent cations to promote the electrostatic attraction between DNA and anionic lipid mono- and bilayers. In this work we provide for the first time experimental results of the adsorption of linear DNA onto anionic/zwitterionic lipid monolayers without any addition of cations. This is demonstrated experimentally by means of Langmuir monolayers of DOPE/DOPG (1:1) lipids spread on a water subphase that contains calf thymus DNA. The adsorption of DNA onto anionic/zwitterionic lipid monolayers is discussed in terms of the surface pressure-molecular area isotherms recorded in the absence and in the presence of different electrolytes. Measurements of the surface potential provide additional evidence of the different interaction of DNA anionic/zwitterionic lipid monolayers depending on the presence and nature of electrolyte. These experimental results are further analysed in terms of the overall dipole moment normal to the monolayers providing new insight into the behaviour of anionic lipoplexes and the role of zwitterionic lipids.

Interaction of surfactant and protein at the O/W interface and its effect on colloidal and biological properties of polymeric nanocarriers.

HYPOTHESIS: The use of polymer-based surfactants in the double-emulsion (water/oil/water, W/O/W) solvent-evaporation technique is becoming a widespread strategy for preparing biocompatible and biodegradable polymeric nanoparticles (NPs) loaded with biomolecules of interest in biomedicine, or biotechnology. This approach enhances the stability of the NPs, reduces their size and recognition by the mononuclear phagocytic system, and protects the encapsulated biomolecule against losing biological activity. Different protocols to add the surfactant during the synthesis lead to different NP colloidal properties and biological activity. EXPERIMENTS: We develop an in vitro model to mimic the first step of the W/O/W NP synthesis method, which enables us to analyze the surfactant-biomolecule interaction at the O/W interface. We compare the interfacial properties when the surfactant is added from the aqueous or the organic phase, and the effect of pH of the biomolecule solution. We work with a widely used biocompatible surfactant (Pluronic F68), and lysozyme, reported as a protein model. FINDINGS: The surfactant, when added from the water phase, displaces the protein from the interface, hence protecting the biomolecule. This could explain the improved colloidal stability of NPs, and the higher biological activity of the lysozyme released from nanoparticles found with the counterpart preparation.

Assessing in vitro digestibility of food biopreservative AS-48.

AS-48 is a bacteriocin with potential application as food biopreservative. In order to optimize its use for oral consumption, we assess the impact of gastrointestinal digestion, both in bulk and adsorbed at the air-water interface. Analysis of AS-48 digestion fragments in bulk by SDS-PAGE, RP-HPLC, and MALDI-TOF proves that the previous pepsin exposition promotes digestion by trypsin/chymotrypsin by exposing new cleavage sites. Regarding adsorbed AS-48, the in vitro digestion profile shows that the conformational change undergone by AS-48 upon adsorption affects its digestibility. Gastrointestinal enzymes cleave only susceptible residues, which are oriented into the aqueous phase, while hydrophobic susceptible residues remain undigested. Evaluation of the elasticity of the adsorbed layer confirms also the presence of undigested AS-48. These results are important towards the use of AS-48 in food formulations; assuring that some intact AS-48 resists digestion guarantees its antibacterial activity throughout the gastrointestinal tract.

Comparative interfacial in vitro digestion of protein and polysaccharide oil/water films.

The behaviour of proteins (ß-lactoglobulin (ßlg) and soy protein isolate (SPI)) and a surface active polysaccharide (hydroxypropylmethylcellulose, HPMC) o/w interfacial films under simulated gastrointestinal conditions using the interfacial tensiometer Octopus were compared and related to the performance of the emulsions (using the same emulsifiers) under in vitro digestion. The evolution of interfacial tension (γ) was used to investigate the effect of gastrointestinal fluids on o/w interfacial films. Clear differences were observed among these emulsifiers. During the gastric phase, HPMC showed the lowest change in γ values as compared to protein films. The most important changes occurred during the intestinal stage where it was observed an important decrease of γ associated with the rapid penetration of BS, followed by a lower rate of decrease attributable to the accumulation of FFA at the interface. In the last stage, the subphase was exchanged by buffer alone, to remove the reversibly adsorbed digestion products. SPI formed the most resistant interface to the remotion of digestion products, followed by HPMC and finally by ßlg. The results agree with the degree of lipolysis reported for the emulsions stabilized by these emulsifiers, which suggest that lipid digestion could be modulated by the ability of emulsifiers to prevent the BS activity (to adsorb at the O/W interface or remove the inhibitory digestion products from the interface). Thus, emulsifiers-BS interactions appears as a key factor in controlling the lipolysis.

Atomic force microscopy as a tool to study the adsorption of DNA onto lipid interfaces.

The Atomic Force Microscopy (AFM) technique appears as a central tool for the characterization of DNA adsorption onto lipid interfaces. Regardless of the huge number of surveys devoted to this issue, there are still fascinating phenomena in this field that have not been explored in detail by AFM. For instance, adsorption of DNA onto like-charged lipid surfaces mediated by cations is still not fully understood even though it is gaining popularity nowadays in gene therapy and nanotechnology. Studies related to the complexation of DNA with anionic lipids as a non-viral gene delivery vehicle as well as the formation of self-assembled nanoscale DNA constructs (DNA origami) are two of the most attractive systems. Unfortunately, molecular mechanisms underlying the adsorption of DNA onto anionic lipid interfaces remain unclear so far. In view of that, AFM becomes an appropriate technique to provide valuable information to understand the adsorption of DNA to anionic lipid surfaces. As a second part of this review we provide an illustrative example of application of the AFM technique to probe the DNA adsorption onto a model lipid monolayer negatively charged. Microsc. Res. Tech. 80:11-17, 2017. © 2016 Wiley Periodicals, Inc.

Effect of cross-linker glutaraldehyde on gastric digestion of emulsified albumin.

Human serum albumin (HSA) has been shown to be an ideal protein for nanoparticle preparation. These are usually prepared by using cross linker agents such as glutaraldehyde (GAD). Liquid lipid nanocapsules (LLN) constitute a new generation of nanoparticles more biocompatible and versatile for oral delivery of lipophylic drugs. The first barrier that an orally administered formulation must cross is the gastrointestinal tract. Hence, it is crucial to address the impact of gastrointestinal digestion on these structures in order to achieve an optimal formulation. This study evaluates the effect of gastric digestion on HSA emulsions structured with GAD as a model substrate for the preparation of LLN. This is done by SDS-PAGE, emulsion microstructure, and interfacial tension techniques. Our results demonstrate that the cross- linking procedure with GAD strongly inhibits pepsin digestion by formation of inter- and/or intramolecular covalent bonds between substrate amino acids. Emulsification of HSA also protects from gastric digestion probably by the orientation of the HSA molecule, which exposes the majority of pepsin cleaving sites preferably to the hydrophobic part of the oil-water interface. In this emulsified HSA, cross-linking with GAD at the interface promotes structural modifications on the HSA interfacial layer, restricting the access of pepsin to cleavage sites. We identify interfacial aspects underlying enzymatic hydrolysis of the protein. Assuring that HSA-GAD structures resist passage through the gastric compartment is crucial is important towards the rational design of oral delivery systems and the first step to get the complete digestion profile.

Specific Ion Effects in Cholesterol Monolayers.

The interaction of ions with interfaces and, in particular, the high specificity of these interactions to the particular ions considered, are central questions in the field of surface forces. Here we study the effect of different salts (NaI, NaCl, CaCl2 and MgCl2) on monolayers made of cholesterol molecules, both experimentally (surface area vs. lateral pressure isotherms measured by a Langmuir Film Balance) and theoretically (molecular dynamics (MD) all-atomic simulations). We found that surface isotherms depend, both quantitatively and qualitatively, on the nature of the ions by altering the shape and features of the isotherm. In line with the experiments, MD simulations show clear evidences of specific ionic effects and also provide molecular level details on ion specific interactions with cholesterol. More importantly, MD simulations show that the interaction of a particular ion with the surface depends strongly on its counterion, a feature ignored so far in most theories of specific ionic effects in surface forces.

Natural Inhibitors of Lipase: Examining Lipolysis in a Single Droplet.

Inhibition of lipase activity is one of the approaches to reduced fat intake with nutritional prevention promoting healthier diet. The food industry is very interested in the use of natural extracts, hence reducing the side effects of commercial drugs inhibiting lipolysis. In this work we propose a novel methodology to rapidly assess lipolysis/inhibition in a single droplet by interfacial tension and dilatational elasticity. The evolution of the interfacial tension of lipase in simplified duodenal fluid in the absence and that in the presence of the pharmaceutical drug Xenical are the negative (5 ± 1 mN/m) and positive (9 ± 1 mN/m) controls of the inhibition of lipolysis, respectively. Then, we correlate the inhibition with the reduction of the interfacial activity of lipase and further identify the mode of action of the inhibition based on dilatational response (conformational changes induced in the molecule/blocking of adsorption sites). This work provides new insight into the lipase inhibition mechanism and a rapid methodology to identify the potential of new natural inhibitors.