Aurora Pérez-Gramatges

Physico-chemical studies of molecular interactions between non-ionic surfactants and bovine serum albumin.

Surfactants, particularly non-ionic types, are often added to prevent and/or minimize protein aggregation during fermentation, purification, freeze-drying, shipping, and/or storage. In this work we have investigated the interactions between two non-ionic surfactants (Tween 20 and Tween 80) and bovine serum albumin (BSA), as model protein, using surface tension, fluorescence measurements and computational analysis. The results showed that, in both cases, the surface tension profile of the surfactants curve is modified upon addition of the protein, and the CMC values of Tween 20 and Tween 80 in the presence of protein are higher than the CMC values of the pure surfactants. The results indicate that although Tween 20 and Tween 80 do not greatly differ in their chemical structures, their interactions with BSA are of different nature, with distinct binding sites. Measurements at different protein concentrations showed that the interactions are also dependent on the protein aggregation state in solution. It was found from fluorescence studies that changes observed in both the intensity and wavelength of the tryptophan emission are probably caused by modifications of tryptophan environment due to surfactant binding, rather than by direct interaction. Based on a computational analysis of a BSA three-dimensional model, we hypothesize about the binding mechanism of non-ionic surfactant to globular protein, which allowed us to explain surface tension profiles and fluorescence results.

Influence of organic additives on the cloud point of PONPE-7.5

The effect of the addition of different organic compounds on the critical temperature (cloud point) of micellar solutions of a non-ionic surfactant was studied. In particular, the addition of aliphatic alcohols and some hydrocarbons to polyoxyethylene(7.5) nonylphenyl ether (PONPE-7.5) solutions was investigated. It was found that the short-chain alcohols (methanol, ethanol, propanol and butanol) increased the cloud point temperature, while for the longer-chain alcohols, the temperature decreased. The effect of the first group can be explained by considering that these species tend to remain in solution and promote the formation of expanded water structures, favoring micelle hydration and, in consequence, increasing the cloud point. This influence is more significant as the concentration of the additive increases, and can be negligible at very low concentrations. The effect of longer-chain alcohols suggests a preferential solubilization of these compounds in the inner core of the aggregate, inducing changes to the shape and size of the micelles. Since the volume-to-area ratio in a micelle increases with these additions, there is a general increase of the micelle size and the cloud point decreases. The effects of branching and cyclization were studied using two series: n-butanol, isobutanol and tert-butanol, and n-hexane, cyclohexane and benzene, respectively. For both series, the addition of the compound provoked a marked decrease in the cloud point temperature. These effects can be explained in view of the site of solubilization inside the micelle. Benzene molecules seem to interact preferentially with the ethylene groups, leading to strong dehydration, while the aliphatic compounds are solubilized in the inner core.

Thermodynamics of complex formation of polyacrylic acid with poly(N-vinyl-2-pyrrolidone) and chitosan

SummaryInterpolymer complexing of poly(acrylic acid) (PAA) with poly(N-vinyl-2-pyrrolidone) (PVP) as well as with chitosans (CHI) of various chain lengths was studied at different temperatures by pH measurements. The degree of conversion (θ), the complex stability constant, K, and the thermodynamic parameters of these reactions were evaluated. Complexing proceeds cooperatively in both systems and values of θ near unity are obtained for the higher molecular weight samples. The variation of K with θ for the PAA-CHI system indicates that this complex comprises long sequences of bound pairs of monomer units.

Interactions and film formation in polyethylenimine–cetyltrimethylammonium bromide aqueous mixtures at low surfactant concentration

The interactions involved in aqueous mixtures of polyethyleneimine (PEI) and cetyltrimethylammonium bromide (CTAB) were studied under dilute conditions. The phase diagram of this polyelectrolyte-surfactant system of similar charge was constructed by determining the CAC and CMC* values at different PEI concentrations, using surface-tension and conductivity measurements, respectively. Formation of thin films at the air-solution interface was detected at concentrations belonging to the interaction region of the phase diagram, using Brewster angle microscopy. These films were formed at low polymer and surfactant concentrations, 0.01% w/v PEI and 0.1 mmol dm CTAB. Results from SAXS determinations indicate that, under these conditions, mesostructure formation occurs exclusively at the surface. The effect of PEI on surfactant micellisation is determined by the polyelectrolyte nature of the polymer. The presence of the polymer in the aggregate significantly affects free micelle formation, even when hydrophobic interactions are mainly determined by the surfactant structure. The films obtained at low surfactant concentration are mesostructured, composed of five layers, each one 49.7 Å thick, as was determined using specular X-ray reflectometry. These results indicate that mesostructured film formation is achievable under more economical and environmentally friendly conditions, suggesting novel routes for surfactant templating in mixed polyelectrolyte systems of similar charge.

Unraveling the binding mechanism of polyoxyethylene sorbitan esters with bovine serum albumin: A novel theoretical model based on molecular dynamic simulations

To gain a better understanding of the interactions governing the binding mechanism of proteins with non-ionic surfactants, the association processes of Tween 20 and Tween 80 with the bovine serum albumin (BSA) protein were investigated using molecular dynamics (MD) simulations. Protein:surfactant molar ratios were chosen according to the critical micelle concentration (CMC) of each surfactant in the presence of BSA. It was found that both the hydrophilic and the hydrophobic groups of the BSA equally contribute to the surface area of interaction with the non-ionic surfactants. A novel theoretical model for the interactions between BSA and these surfactants at the atomic level is proposed, where both surfactants bind to non-specific domains of the BSA three-dimensional structure mainly through their polyoxyethylene groups, by hydrogen bonds and van der Waals interactions. This is well supported by the strong electrostatic and van der Waals interaction energies obtained in the calculations involving surfactant polyoxyethylene groups and different protein regions. The results obtained from the MD simulations suggest that the formation of surfactant clusters over the BSA structure, due to further cooperative self-assembly of Tween molecules, could increase the protein conformational stability. These results extend the current knowledge on molecular interactions between globular proteins and non-ionic surfactants, and contribute to the fine-tuning design of protein formulations using polysorbates as excipients for minimizing the undesirable effects of protein adsorption and aggregation.

Preconcentration of copper by cloud point extraction with 1-(2-pyridylazo)-2-naphthol and determination by neutron activation

A method for the preconcentration of Cu(II) by cloud point extraction (CPE) followed by its determination using neutron activation analysis (NAA) was developed. The method involves the use of a nonionic surfactant, namely PONPE-7.5, and a chelating agent 1-(2-pyridylazo)-2-naphthol (PAN). The phase diagram of the surfactant was constructed and the effects of different additives on the cloud point were investigated. Factors, such as the solution pH, ionic strength, temperature, and concentrations of chelating agent and surfactant, which can influence the extraction efficiency of the metal, were optimized. The copper(II) chelate was extracted into a surfactant-rich phase of small volume with a recovery of nearly 100% and a preconcentration factor of 60. The small volume of the surfactant-rich phase obtained complies with the green chemistry concept, allowing the design of extraction procedures having lower toxicity than those using organic solvents. The method was applied to tap water samples.

Multiple thin film formation from dilute mixtures of polyethyleneimine (PEI) and cetyltrimethylammonium bromide (CTAB)

Dilute mixtures of the water soluble polymer polyethyleneimine (PEI) and the cationic surfactant cetyltrimethylammonium bromide (CTAB) form mesostructured thin films at the air/solution interface. In this paper we show that these films form spontaneously, reaching an equilibrium composition. When the film is removed, a new solid film rapidly reforms, with a similar morphology when inspected by Brewster angle microscopy (BAM). Successive removal of films lead to a series of structurally similar films, until the amount of surfactant in solution approaches the lower limit of film forming concentration. The results obtained using surfactant-selective potentiometry suggest that the incipient polymer:surfactant aggregates are in a position to migrate to the surface rapidly after film removal, prior to mesostructure formation. X-ray reflectivity indicates that films formed at different PEI:CTAB compositions retain mesophase regular structures with the same d-spacing feature, equal to 52.2A. Grazing incidence diffraction measurements indicate that films are composed of small crystallites arranged in a random powder fashion, developing a rough surface morphology evidenced by BAM. The central finding is that PEI:CTAB films form when the amount of CTAB in solution is higher than a critical film formation concentration, very close to the critical aggregation concentration (CAC), allowing the formation of several equilibrated films from the same growing solution.

Self-assembly and phase behaviour of PEI : cationic surfactant aqueous mixtures forming mesostructured films at the air/solution interface

Self-assembly processes and corresponding phase boundaries depend on the structure of interacting molecules. We have studied the effect of surfactant head-group structure and counterion on the self-assembly processes occurring in aqueous mixtures of the cationic polymer polyethylenimine (PEI) and the cationic surfactants cetyltrimethylammonium bromide (CTAB), cetylpyridinium bromide (CPB) and cetylpyridinium chloride (CPC). Surface tension and conductometric methods were employed, as well as X-ray scattering and reflectometry techniques. The results indicate that the phase behaviour of PEI : cationic surfactant mixtures under dilute conditions can be tuned by altering head-group and counterion. The surfactant head-group exerts a greater influence overall, especially over micellization, since the counterion effect is screened at high polymer concentrations. Also, the self-assembly processes occurring at lower surfactant concentrations are not significantly affected by the counterion effect. The variation of surface tension with surfactant concentration shows a particular ‘well-like’ profile with a distinct break-point that can be directly related to the onset of film formation. Phase diagrams constructed using surface tension, conductivity and Brewster angle microscopy (BAM) data show three different regions where initial interactions, film formation and micellization take place, respectively, characterized by the two main self-assembly boundaries. Based on these results, we propose a molecular interpretation of the aggregation and film formation processes occurring in PEI : cationic surfactant mixtures under increasing surfactant concentrations within the dilute regime.

Spherical gold nanoparticles and gold nanorods for the determination of gentamicin.

Gentamicin is an antibiotic indicated to treat mastitis in dairy cattle and for the treatment of bacterial resistance in the context of hospital infections. The effect caused by gentamicin on the optical properties of gold nanoparticles aqueous dispersions were used to develop quantitative methods to determine this antibiotic. Two different aqueous dispersions, one containing spherical Au nanoparticles (AuNPs) and the other containing Au nanorods (AuNRs), had their conditions adjusted to enable a stable and sensitive response towards gentamicin. The use of AuNPs, with measurement at 681nm of the rising coupling plasmon band, enabled a limit of detection (LOD) of 0.4ngmL-1 (0.02ng absolute LOD), ten times lower than the one achieved by measuring the decreasing of the longitudinal surface plasmon resonance band (at 662nm). The linear analytical response of AuNPs measured at 681nm did not require rationing of signal values to correct for linearity. Stability of the analytical response resulted in intermediary precision below 2%. No significant interference was imposed by excipients traditionally present in injectable solutions for veterinary use. Percent recoveries obtained in such formulations were between 94.5 and 98.2% regardless the existence of any difference in the proportion of the compounds known as gentamicin (C1, C1a and C2) in standard and in the samples. The method requires no derivatization with toxic reagents as usually is required in other spectroscopic approaches.