Antonio Martín-Rodríguez

Physicochemical properties and digestibility of emulsified lipids in simulated intestinal fluids: influence of interfacial characteristics

An improved understanding of the behaviour of lipids within the gastrointestinal tract will facilitate the structural design of foods that provide specific physiological responses. In this work, we studied the influence of interfacial characteristics (emulsifier type and surface area) on the behaviour of emulsified lipids within a simulated small intestine (duodenum). Pluronic F68-stabilized emulsions were more resistant to lipid digestion than lecithin-stabilized emulsions. Interfacial tension, droplet charge (ζ-potential), and microstructure measurements were used to identify the physicochemical origin of this effect. Pluronic F68 was shown to be more difficult than lecithin to displace from lipid droplet surfaces by bile salts and lipase arising from the duodenal juice. Specifically, duodenal components decreased the interfacial tension of a Pluronic-covered interface to a lesser extent, as compared to the interface covered by lecithin. These results demonstrate that the properties of the interfacial layer surrounding lipid droplets can be designed to modulate the lipid digestion process. This knowledge is critical for the rational development of delivery systems for food and pharmaceutical applications that can control the uptake of lipids and lipid-soluble components under physiological conditions.

Controlling lipolysis through steric surfactants: new insights on the controlled degradation of submicron emulsions after oral and intravenous administration.

In this work we have investigated how steric surfactants influence the metabolic degradation of emulsions (lipolysis). To do so, we have prepared submicron emulsions stabilized with Pluronic F68, Pluronic F127, Myrj 52 or Myrj 59, four non-ionic surfactants with key differences on their structure. Submicron emulsions have been prepared also with mixtures of these surfactants with different proportions between them. Then, in vitro methods have been applied to analyze the lipolysis of these emulsions, both under duodenal and intravenous conditions, to simulate lipolysis after oral and intravenous administration. Our results show that the properties of the surfactant influence dramatically the lipolysis rates observed both under duodenal and intravenous conditions, e.g., intravenous lipolysis was completely blocked when Pluronic F127 was used, while it was almost complete within 6h when using Myrj 52. The reason for this seems to be the steric hindrance that the surfactant produces around the droplet and at the interface. As a result, we can modify the lipolysis patterns by changing some characteristics of the surfactant, or by varying the proportion between two surfactants in a mixture. These findings may be applied in the development of novel strategies to rationally design submicron emulsions as lipophilic drug carriers.

Block copolymers at interfaces: interactions with physiological media.

Triblock copolymers (also known as Pluronics or poloxamers) are biocompatible molecules composed of hydrophobic and hydrophilic blocks with different lengths. They have received much attention recently owing to their applicability for targeted delivery of hydrophobic compounds. Their unique molecular structure facilitates the formation of dynamic aggregates which are able to transport lipid soluble compounds. However, these structures can be unstable and tend to solubilize within the blood stream. The use of nanoemulsions as carriers for the lipid soluble compounds appears as a new alternative with improved protection against physiological media. The interfacial behavior of block copolymers is directly related to their peculiar molecular structure and further knowledge could provide a rational use in the design of poloxamer-stabilized nanoemulsions. This review aims to combine the new insights gained recently into the interfacial properties of block copolymers and their performance in nanoemulsions. Direct studies dealing with the interactions with physiological media are also reviewed in order to address issues relating metabolism degradation profiles. A better understanding of the physico-chemical and interfacial properties of block copolymers will allow their manipulation to modulate lipolysis, hence allowing the rational design of nanocarriers with efficient controlled release.

Adsorption of antibody onto Pluronic F68-covered nanoparticles: link with surface properties

The use of nanoparticles as drug delivery systems is an emerging application to improve intravenous therapy. Controlling the biocompatibility of the nanoparticles is a crucial step towards the optimal implementation of these systems. Adsorption of serum components onto the nanoparticles is driven mainly by hydrophobic forces. Thus, incorporation of hydrophilic polymers such as polyethylene oxide (PEO) derivatives to the nanostructure surface reduces the interaction of nanoparticles with blood stream components (IgG). The effectiveness of the poloxamer for reducing protein adsorption depends on the resistance of this coating layer. A fundamental understanding of the properties of this surface coating is crucial towards the rational design of these systems. Here, we have used an innovative combination of experimental techniques to evaluate the properties of the nanoparticles and more specifically, the mechanical properties of the coating. Electrophoretic mobility and colloidal stability data suggest similar surface characteristics between IgG–Pluronic–polystyrene (PS) and IgG–PS complexes, indicating that the protein adsorption is just slightly reduced by the presence of poloxamer. Nevertheless, the biological activity of the adhered antibodies suggests that the Pluronic F68 significantly altered their immunoactivity. The decrease in the activity might indicate a partial denaturation of the protein and/or changes in the preferential orientation when adsorbing caused by the surfactant–protein interactions. The surface characterisation of the IgG layers adsorbed onto a Pluronic covered surface importantly provides evidence of the conformational change undergone by the protein, supporting the partial protein denaturation suggested by the loss of immunoreactivity in the IgG–Pluronic–PS particles. The use of surface tension to obtain structural and mechanical information about the coating procedure is a novel approach to understand generic features of the biocompatibility of colloidal systems. These results may help to understand why drug nanocarriers coated by poloxamers improve their long-circulating properties in comparison with uncoated particles.

A comparative study on the electrokinetic behavior of bovine serum albumin molecules adsorbed onto different polymer colloids

Abstract The adsorption of protein onto polymer surfaces is mainly due to the dehydration of hydrophobic patches on the polymers. Hence the degree of hydrophobicity of the polymer surfaces plays an important role in the adsorption of bovine serum albumin (BSA) molecules onto polystyrene beads. The main purpose of the present work is to study the electrokinetic behavior of BSA molecules adsorbed onto polystyrene beads of different superficial hydrophobicities. Four different samples of polystyrene (PS) beads were used in this study. Two samples showed a marked hydrophobic character ([PS(−)] and [PS (+)]), and were prepared by the conventional emulsion polymerization of styrene, using potassium persulfate and ADMBA as initiators. Both latex samples, although charged differently (−5.17 μC cm −2 and +11.0 μC cm −2 respectively), showed a lower occupancy area per adsorbed surfactant molecule at saturation ( A m ), indicating a strong hydrophobic character. However, the other latex samples prepared by copolymerization of styrene and 2-hydroxyethyl methacrylate (HEMA) ([PS—H]) and acrylic acid ([PS—A]) showed a high A m , indicating a pronounced hydrophilic character. Adsorption isotherms at low ionic strength and different pH were obtained. In all cases the adsorption isotherms showed well-defined plateaus. The maximum adsorbed amount on the hydrophobic latices occurred at around the isoelectric point of the dissolved BSA (i.e. pH 5), whereas it occurred at around pH 4 on the hydrophilic latices. The dependence of electrophoretic mobility on the adsorbed amount seems to indicate that ions of small molecular weight are adsorbed on the protein—polymer interface. This ionic adsorption effect depends mainly on the surface charge density of the latex particles.

Adsorption of monoclonal IgG on polystyrene microspheres

In this paper the adsorption of a monoclonal antibody IgG-1 isotype against HBsAg onto positively and negatively charged polystyrene beads has been studied. To determine the role played by electrostatic forces in the adsorption process different pH values were used. It was confirmed that the affinity of adsorption isotherms depends on the electrostatic interaction between protein and polymer surface. The maximum adsorption amount is located around the i.e.p. of the dissolved protein, and decreases markedly as pH moves away. Thus, the major driving force for adsorption of monoclonal antibodies on polystyrene beads comes from the hydrophobic interaction between the antibody molecules and the adsorbent surface. Desorption of preadsorbed IgG molecules by increasing ionic strength has shown that the positively charged polystyrene is also more hydrophobic in character than the negatively charged one. Finally, electrokinetic experiments have determined that the electric double layer (e.d.l.) of monoclonal antibody changes as the consequence of adsorbing on charged polymer surfaces.

Influence of electrostatic forces on IgG adsorption onto polystyrene beads

Abstract Some electrostatic aspects of the interaction between rabbit immuno gamma globulins (IgG) and positively and negatively charged polystyrene (PS) beads are reported. Adsorption and electrophoresis experiments have been performed as a function of pH and ionic strength. Both polystyrene beads reached a maximum adsorption around pH 6.0. The isoelectric points of the IgG-PS complexes were 6.0 and 8.0 for the anionic and cationic latices, respectively. This difference indicates that the charge of the PS beads at least partially compensates the net charge of the IgG molecules. The influence of electrostatic forces on protein adsorption has been studied by investigating the ability of adsorbed proteins to be displaced by an increase in ionic strength. Finally, the ionic strength effect on electrophoretic mobility of the sensitized cationic polystyrene beads has been analyzed.

Characterization of Different Functionalized Lipidic Nanocapsules as Potential Drug Carriers

Lipid nanocapsules (LNC) based on a core-shell structure consisting of an oil-filled core with a surrounding polymer layer are known to be promising vehicles for the delivery of hydrophobic drugs in the new therapeutic strategies in anti-cancer treatments. The present work has been designed as basic research about different LNC systems. We have synthesized—and physico-chemically characterized—three different LNC systems in which the core was constituted by olive oil and the shell by different phospholipids (phosphatidyl-serine or lecithin) and other biocompatible molecules such as Pluronic® F68 or chitosan. It is notable that the olive-oil-phosphatidyl-serine LCN is a novel formulation presented in this work and was designed to generate an enriched carboxylic surface. This carboxylic layer is meant to link specific antibodies, which could facilitate the specific nanocapsule uptake by cancer cells. This is why nanoparticles with phosphatidyl-serine in their shell have also been used in this work to form immuno-nanocapsules containing a polyclonal IgG against a model antigen (C-reactive protein) covalently bounded by means of a simple and reproducible carbodiimide method. An immunological study was made to verify that these IgG-LNC complexes showed the expected specific immune response. Finally, a preliminary in vitro study was performed by culturing a breast-carcinoma cell line (MCF-7) with Nile-Red-loaded LNC. We found that these cancer cells take up the fluorescent Nile- Red molecule in a process dependent on the surface properties of the nanocarriers.

Surface characterization of latexes with different interfacial properties

In this paper we have studied the effect of copolymerization of a non-ionic but hydrophilic monomer on the interfacial properties of the latex surface. Hydrophilic polymer colloids were prepared by the emulsifier-free emulsion copolymerization of styrene and 2-hydroxyethyl methacrylate (HEMA) with potassium persulfate as initiator. The copolymer compositions were evaluated by 1H nuclear magnetic resonance and compared with the initial concentration of each monomer. Results show very good agreement which is an indication of the absence of secondary nucleation. Also, the presence of the carbonyl group (i.e. a weak acid group) was detected by Fourier transform-infrared and 13C nuclear magnetic resonance analysis, together with conductometric and potentiometric titration experiments. In all cases, a conventional hydrophobic polystyrene latex was used as a reference. The hydrophobic character of the latexes was estimated by using contact angle and viscosity measurements, and adsorption of a non-ionic surfactant. These three methods are compared. As expected, the results showed that as the amount of hydrophilic monomer in the synthesis increases, so does the hydrophilic character of the latex surface.

CHLOROACTIVATED LATEX PARTICLES FOR COVALENT COUPLING OF ANTIBODIES. APPLICATION TO IMMUNOASSAYS

The aim of the present work is to prepare and characterize a functionalized latex with chloromethyl groups on the surface and to perform the covalent coupling of anti-human serum albumin (a-HSA) IgG protein. The chloromethyl-styrene latex (CMS) was synthesized by means of a core-shell emulsion polymerization in a batch reactor. The monodisperse-obtained latex was characterized by determining the diameter (TEM and PCS), the surface charge density (conductometric and potentiometric titration), the amount of chloromethyl groups on the surface (hydrolysis reaction), and the stability vs electrolyte concentration (turbidity measurements). Electrokinetic characterization was also performed (electrophoretic mobility versus pH and ionic strength). IgG was chemically bound to the latex particles under different sensitization and block-stabilization conditions. Colloidal stability of complexes was studied to select an immunolatex suitable for the development of latex immunoassays. The final part of this work consists of a study of the immunoreactivity of the IgG-latex complexes at different pH and ionic strength, in particular under physiological conditions. The results show that chemically bound IgG to chloromethyl latex provides an IgG-latex complex suitable for application in immunodiagnosis tests.