Maslinic acid conjugate with 7-amino-4-methylcoumarin as probe to monitor the temperature dependent conformational changes of human serum albumin by FRET.

Synthesis, characterization and spectroscopic investigation of maslinic acid labeled with fluorescent 7-amino-4-methylcoumarin is reported. It was found that the coumarin-maslinic derivative (MaCo) forms an excellent fluorescence resonance energy transfer (FRET) pair with the tryptophan (Trp) residue of human serum albumin (HSA). This feature allowed for monitoring HSA conformational alterations by measuring the distance between donor (Trp) and acceptor (MaCo) through Förster energy transfer mechanism. Displacement experiments confirmed that MaCo binds to subdomain IIA of HSA with independence of temperature. It was observed that, in the temperature range 35-45 °C, the fluorescence emission maximum of HSA-MaCo complex decreased, whereas in the range 45 °C-65 °C, an increment was detected. The concomitant change in the polarity of environment surrounding Trp was confirmed by red edge excitation shift experiments. Thermal denaturation of HSA was followed by time-resolved fluorescence spectroscopy. Average lifetime of Trp residue decreased with temperature due to the increment of solvent collisions and changes in the solvent exposure of Trp. To discriminate the importance of each effect, lifetime of N-Acetyl-L-tryptophanamide (NATA) at different temperatures was measured. Circular dichroism (CD) studies confirmed the loss of secondary structure of HSA with increasing temperature and showed a different trend in the conformational transformation below and above 45 °C, in agreement with steady-state and time-resolved fluorescence experiments.

Albumin-covered lipid nanocapsules exhibit enhanced uptake performance by breast-tumor cells.

Liquid lipid nanocapsules (LLN) represent a promising new generation of drug-delivery systems. They can carry hydrophobic drugs in their oily core, but the composition and structure of the surrounding protective shell determine their capacity to survive in the circulatory system and to achieve their goal: penetrate tumor cells. Here, we present a study of LLN covered by the protein human serum albumin (HSA) and loaded with curcumin as a hydrophobic model drug. A cross-linking procedure was performed to further strengthen the protective protein layer. Physicochemical properties and release kinetics of the nanocapsules were investigated, and cellular uptake and killing capacity were evaluated on the human breast-cancer line MCF-7. The nanocapsules exhibited a half maximal inhibitory concentration (IC50) capacity similar to that of free curcumin, but avoiding problems associated with excipients, and displayed an outstanding uptake performance, entering cells massively in less than 1 min.

Systematic study on the preparation of BSA nanoparticles.

Albumins, in the form of nanoparticles, are increasingly used as drug carriers in the medical field, and the size effect of these nanomaterials is of major importance since it may affect their bioavailability and the in vivo behaviour after intravenous injection. This research provides a comprehensive study on the preparation of BSA nanoparticles, based on a simple coacervation method, with suitable size, size distribution, and surface charge for drug-delivery applications. Numerous experimental variables were examined in order to characterize their impact on nanoparticle size, distribution, electrophoretic mobility, and yield. Particle size was controlled by adjusting self-assembly phenomena of the protein molecules, which was affected by preparation conditions including BSA content, pH, and ionic strength (a parameter that strongly influences nanoparticle formation but surprisingly has not been previously studied in detail). Small particles with a narrow size distribution were obtained under experimental conditions where the repulsion between BSA molecules was high, i.e. at pH values far from the isoelectric point of the protein and low salt concentration. Changes in temperature, volume, and rate of addition of the dehydrating agent (ethanol) also affect nanoparticle characteristics, as they influence the nucleation rate and particle growth. The effect of these experimental conditions on the quantity of protein still dissolved in the aqueous phase after desolvation (i.e. the yield of BSA nanoparticles) was also studied. Nanoparticles surface charge was modulated with the extension of cross-linking. Finally, long-term colloidal stability of samples was evaluated after 2 months of storage.

Adhesion forces between protein layers studied by means of atomic force microscopy.

Adhesion forces between different protein layers adsorbed on different substrates in aqueous media have been measured by means of an atomic force microscope using the colloid probe technique. The effects of the loading force, the salt concentration and pH of the medium, and the electrolyte type on the strength, the pull-off distance, and the separation energy of such adhesion forces have been analyzed in depth. Two very different proteins (bovine serum albumin and apoferritin) and two dissimilar substrates (silica and polystyrene) were used in the experiments. The results clearly point out a very important contribution of the electrostatic interactions in the adhesion between protein layers.

Existence of hydration forces in the interaction between apoferritin molecules adsorbed on silica surfaces.

The atomic force microscope, together with the colloid probe technique, has become a very useful instrument to measure interaction forces between two surfaces. Its potential has been exploited in this work to study the interaction between protein (apoferritin) layers adsorbed on silica surfaces and to analyze the effect of the medium conditions (pH, salt concentration, salt type) on such interactions. It has been observed that the interaction at low salt concentrations is dominated by electrical double layer (at large distances) and steric forces (at short distances), the latter being due to compression of the protein layers. The DLVO theory fits these experimental data quite well. However, a non-DLVO repulsive interaction, prior to contact of the protein layers, is observed at high salt concentration above the isoelectric point of the protein. This behavior could be explained if the presence of hydration forces in the system is assumed. The inclusion of a hydration term in the DLVO theory (extended DLVO theory) gives rise to a better agreement between the theoretical fits and the experimental results. These results seem to suggest that the hydration forces play a very important role in the stability of the proteins in the physiological media.

Hydration forces between silica surfaces: experimental data and predictions from different theories.

Silica is a very interesting system that has been thoroughly studied in the last decades. One of the most outstanding characteristics of silica suspensions is their stability in solutions at high salt concentrations. In addition to that, measurements of direct-interaction forces between silica surfaces, obtained by different authors by means of surface force apparatus or atomic force microscope (AFM), reveal the existence of a strong repulsive interaction at short distances (below 2 nm) that decays exponentially. These results cannot be explained in terms of the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory, which only considers two types of forces: the electrical double-layer repulsion and the London-van der Waals attraction. Although there is a controversy about the origin of the short-range repulsive force, the existence of a structured layer of water molecules at the silica surface is the most accepted explanation for it. The overlap of structured water layers of different surfaces leads to repulsive forces, which are known as hydration forces. This assumption is based on the very hydrophilic nature of silica. Different theories have been developed in order to reproduce the exponentially decaying behavior (as a function of the separation distance) of the hydration forces. Different mechanisms for the formation of the structured water layer around the silica surfaces are considered by each theory. By the aid of an AFM and the colloid probe technique, the interaction forces between silica surfaces have been measured directly at different pH values and salt concentrations. The results confirm the presence of the short-range repulsion at any experimental condition (even at high salt concentration). A comparison between the experimental data and theoretical fits obtained from different theories has been performed in order to elucidate the nature of this non-DLVO repulsive force.

Amino, chloromethyl and acetal-functionalized latex particles for immunoassays: a comparative study.

Latex particles with different functionalized surface groups (amino, acetal and chloromethyl) for the covalent linking of protein molecules were synthesized and characterized. Immunopurified anti-ferritin antibodies were then covalently coupled with a mean efficiency rate (protein covalently bound to latex particles with respect to the total amount of protein added) of 60%. The reagents developed were applied to the measurement of serum ferritin concentration in a turbidimetric procedure, showing a good measuring range and a lowest detection limit of 3.5 ng/ml in the case of the amino-modified particles. These immunological reagents were compared with a commercial nephelometric method, showing a good linear correlation in all cases but no transferability in the acetal and chloromethyl latex with additional carboxyl groups, probably due to interference with other serum components. The differences among latex found in this study indicate that it would be necessary to optimize the assay conditions for each type of particle, in order to achieve a maximum immunoreactivity.

Development of a new flow-through immunoassay to detect human IgM class antibodies to Brucella melitensis 16 M S-lipopolysaccharide and comparison with IgM-ELISA / Desarrollo de un nuevo inmunoensayo tipo f low-trough para la detección de anticuerpos igm humanos contra s-lipopolisacárido de brucella melitensis 16m y comparación con elisa para igm

Brucellosis is a zoonosis of world-wide distribution, caused by several species of the genus Brucella. The infection causes non-specific symptoms, and differential diagnosis is very important in the early stages of the disease. Lipopolysaccharide (LPS) of the cell wall is a dominant antigen, and specific anti-LPS immunoglobulins of the IgM class are the first antibodies whose concentration increases during an acute infection. The tests most often used to detect specific antiLPS IgM antibodies are a serum agglutination test in the presence of dithiothreitol (SAT-DTT), and an IgM-based enzyme-linked immunosorbent assay (IgM-ELISA). However, both these methods have the disadvantage that they require skilled personnel and well-equipped laboratories to perform them. Here we describe a new rapid test for anti-Brucella L P S IgM, which can be useful even in circumstances where few laboratory facilities are available. The basis of this test was the binding of IgM onto a nitrocellulose membrane previously coated with rabbit anti-human IgM antibodies as the solid phase. A monodispersed, stable conjugate of LPS attached to deeply blue-dyed latex particles was used to visualise the reaction . With this assay, results for a specimen were obtained in about ten minutes (AU)

La brucelosis, una zoonosis de distribución mundial, es causada por varias especies del género Brucella. La infección causa síntomas inespecíficos, y su diagnóstico diferencial en los primeros periodos de la enfermedad es muy importante. El lipopolisacárido de la pared celular es un antígeno dominante, y las inmunoglobulinas específicas anti-LPS de la clase IgM son los primeros anticuerpos cuya concentración aumenta durante una infección aguda. Los ensayos que se usan con mayor frecuencia para detectar anticuerpos IgM anti-LPS son: aglutinación del suero en presencia de ditiotreitol (SAT-DTT) y ELISA-IgM. Sin embargo, ambos métodos tienen la desventaja para ser llevados a cabo de que requieren personal entrenado y laboratorios bien equipados. En este trabajo se describe un nuevo ensayo rápido para IgM anti-LPS de Brucella, que puede ser útil aun en laboratorios de bajos recursos. La base de este ensayo es la reacción de la IgM sobre una membrana de nitrocelulosa donde previamente se han adsorbido anticuerpos de conejo anti-IgM humana. Un conjugado monodisperso y estable de LPS ligado a partículas de látex azules se utiliza para visualizar la reacción. Con este ensayo, se puede obtener en aproximadamente 10 minutos el resultado de una muestra (AU)

Interaction of bacterial endotoxine (lipopolysaccharide) with latex particles: application to latex agglutination immunoassays.

The latex agglutination immunoassay technique uses polymer colloids as carriers for antibodies or antigens to enhance the immunological reaction. In this work, the interaction of a lipopolysaccharide (LPS) of Brucella Melitensis with two conventional latexes has been studied. Some experiments on the physical adsorption of the LPS onto these polystyrene beads have been performed and several complexes with different coverage degrees were obtained by modifying the incubation conditions. Regarding the application in the development of diagnostic test systems, it is advisable to study the latex-LPS complexes from an electrokinetic and colloidal stability point of view. The complexes were electrokinetically characterized by measuring the electrophoretic mobility under different redispersion conditions. The colloidal stability was determined by simple turbidity measurements. Experimental and theoretical data have been employed to study the molecular disposition of the LPS in the latex particle surface to compare with the outer membrane of bacterial cells. Latex complexes covered by different LPS amounts showed high colloidal stability and adequate immunoreactivity that remains for a long time period.

Specific cation adsorption on protein-covered particles and its influence on colloidal stability.

Protein coated particles present an anomalous colloidal stability at high ionic strength when the classical theory (DLVO) predicts aggregation. This observed deviation from DLVO behaviour appears for electrolyte concentrations above some critical bulk value. As we have suggested in previous publications the existence of an additional short-range repulsive 'hydration force' due to specific hydrated cation adsorption could explain this anomalous stability. The overlap of the hydration layers when two particles approach should provoke this repulsive force. New evidence of this mechanism has been observed when electrophoretic mobilities of protein-carrying latex particles were measured at various concentrations of sodium and calcium chloride. In the latter case a sign reversal of zeta-potential was found, probably due to the specific adsorption of Ca(2+) ions on protein molecules. The adsorption increases with the medium pH. These results have been analyzed following the treatment proposed by Ohshima and co-workers for large charged colloidal particles coated with a layer of protein. This study shows an increase in the positive fixed-charge density on the protein caused by the adsorption of cations.

Forces acting on particle-enhanced immunoassays.

The aim of the present work is to study the role of the different forces involved in the agglutination of immuno gamma-globulin (IgG) covered latex particles due to antigen-antibody reaction. An experimental investigation on the adsorption of IgG molecules on three latexes with different surface charge densities is described. Photon correlation spectroscopy was used to determine the hydrodynamic layer thickness of the IgG molecules adsorbed on the latexes. In order to get an insight into the forces acting between two antibody-covered particles approaching each other, the colloidal stability and immunoreactivity of these biocomplexes were studied. They can be stabilized by electrostatic or hydration forces. The immunological agglutination of IgG-immobilized latex particles due to the addition of the antigen was quantified through scattered light intensity measurements. The immunoresponse increases with ionic strength of the medium until a maximum value is achieved. Above this maximum, the immunoreactivity decreases.

Cluster Morphology of Protein-Coated Polymer Colloids.

We report measurements on the aggregation processes in a colloidal suspension of polystyrene particles covered with F(ab')2 (immunoglobulin IgG fragment) performed by static and dynamic light scattering. In order to study the cluster morphology of aggregates, the fractal dimension is obtained from the dependence of the scattered intensity on the scattering wave number. The stability domains of bare and protein-coated polystyrene particles were examined by plotting the stability ratio as a function of electrolyte concentration. The stability results have been explained using a modified Derjaguin-Landau-Verwey-Overbeek theory to describe the interparticle interaction. The observed change in the fractal dimension can be explained by the existence of a minimum separation distance between coagulated particles (restructuring). This minimum distance is attributed to the layer of hydrated ions and water molecules adsorbed on the particle surface. Our results are in agreement with the reversible-growth model of W. Y. Shih, I. A. Aksay, and R. Kikuchi (Phys. Rev. A. 36, 5015 (1987)) and they were supported by transmission electron microscopy observation. Copyright 1998 Academic Press.

Latex immunoassays: comparative studies on covalent and physical immobilization of antibodies. I. F(ab')2 fragments.

Colloidal particles coated with antibodies are currently used in diagnostic test systems for the detection of antigens in biological fluids. Immobilization is usually carried out by physical adsorption. Covalent coupling of antibodies to particles, however, offers certain advantages. The present research deals with the study of these possible advantages. A sulphonated polystyrene latex has been used to prepare an immunolatex with physically adsorbed antibodies, while a functionalized latex with chloromethyl groups on the surface has been used for the partly covalent coupling of the antibody (F(ab')2 fragments). The immunoreactivity was studied by measuring the variations in scattered light intensity after mixing a solution of CRP antigen and the sensitized latex. The influence on the immunoresponse of the scattering angle (5, 10, and 20 deg), protein coverage and storage time have been studied for both systems.

Latex immunoassays: comparative studies on covalent and physical immobilization of antibodies. II. IgG.

Latex particles coated with IgG, currently used for immunoassay tests, are not colloidally stable under physiological conditions. Post-treatment of sensitized polystyrene microspheres with different substances (BSA, surfactants) to increase colloidal stability has been often used to solve this problem. We propose the possibility of stabilizing the antibody-latex conjugates by hydration forces at high ionic strength. On the other hand. immobilization of the proteins may be performed either by physical adsorption or by covalent binding to functionalized surface groups. In this second part of these series we have studied the immunoreactivity of IgG antibody partly covalently bound and physically adsorbed, and the results were compared to those obtained with F(ab')2 fragments in Part I.

Anomalous Colloidal Stability of Latex-Protein Systems.

An experimental study on the colloidal stability of latex-F(ab')2 and latex-IgG systems is described. The stability domains were obtained using a low-angle scattering technique to measure the rates of aggregate formation and plotting the stability ratio as a function of electrolyte concentration. The protein-coated particles present an anomalous stability at high ionic strength when the classical theory predicts aggregation. This observed deviation from DLVO behavior appears for electrolyte concentrations above some critical bulk concentration called critical stabilization concentration (csc). As we have suggested in previous publications, the existence of an additional short-range repulsive "hydration force" can explain this anomalous stability. In order to resolve more fully if this anomalous stability at high ionic strength is due to the hydration forces, the effects of pH, cation and anion type, temperature, and polyethylene glycol (a dehydrating polymer) on the experimental stability are investigated. Finally, the immunoreactivity of an anti-CRP F(ab')2-latex conjugate is studied in reaction buffers with high ionic strength. Copyright 1998 Academic Press.

Particle enhanced immunoassays stabilized by hydration forces: a comparative study between IgG and F(ab)2 immunoreactivity.

In previous publications we have discussed the stabilization mechanism of hydration forces as applied to the development of latex agglutination tests. We describe here how we have obtained stable and reactive IgG-latex conjugates in a high-ionic-strength reaction buffer. To this end we have made agglutination tests with polystyrene beads sensitized with IgG, measuring the immunoaggregation reaction with human C-reactive protein in a stopped-flow nephelometer. The results are compared to those obtained with a F(ab')2-latex conjugate with similar antibody molecule coverage. Adsorption isotherms of F(ab')2 and IgG on latex at pH 7.2 were obtained to study the affinity of these antibodies for the surface. The results of the electrokinetic characterization of the antibody-latex conjugates agree satisfactorily with those obtained from stability studies. This research throws light upon the use of hydration forces as a new approach to stabilizing immunoassay reagents that are colloidally unstable in physiological reaction buffers.

On the structure of electrical double layer of IgG immobilized on polystyrene microspheres.

Latex particles used in the development of immunoassays usually present stability problems when the antibody is attached to the surface. This work is an attempt to know the potential distribution parameters around these complexes by electrokinetic measurements. The conversion of mobility data into zeta-potential was carried out by different theoretical approaches developed by Smoluchowski (classical equation), O'Brien and White, and Dukhin and Semenikhin. The zeta-potential calculated with allowance for electrical double layer (e.d.l.) polarization was substantially greater than zeta calculated according to the classical electrokinetic theory. The greater values of zeta D-S in comparison with zeta O-W were readily explained on the basis that in the first theory, the contribution to polarization from all ions of the diffuse layer was taken into account, whereas O'Brien and White accounted for only the ions of the hydrodynamically mobile part of the e.d.l. This is an indication that the anomalous surface conductance of latex particles coated by IgG molecules is much higher than that of bare latex particles.