Éva Kiss

Protein adsorption on functionalized and ESCA-characterized polymer films studied by ellipsometry

The adsorption of bovine serum albumin (BSA), immunoglobulin G (IgG), fibrinogen, and poly-l-lysine (PLL) on polyvinylchloride (PVC), a copolymer of methacrylic acid/methacrylate (PMA) and surface-grafted polyethylene oxide (PEO) films, has been studied by means of ellipsometry. The films were spin-cast on silicon wafers covered with silicon oxide (SiOx). Under physiological conditions, most proteins, but not the polyelectrolyte PLL, adsorbed to a larger extent to the hydrophobic PVC than to the hydrophilic PMA or PEO surfaces. For PLL extensive adsorption to PMA occurred as a result of strong electrostatic interaction. A correlation was found between the amount of protein adsorbed as measured by ellipsometry, and the relative surface content of nitrogen as recorded using ESCA. None of the proteins adsorbed to a constant plateau level. Instead, a monotonically increasing degree of adsorption was found in the concentration range studied, 0–1000 g/m3. For the hydrophobic PVC, the isotherms were similar in shape to those theoretically obtained by I. Lundstrom (Prog. Colloid Polym. Sci. 70, 76–82 (1985)) upon assuming a dynamic adsorption process which takes into account several modes of adsorption characterized by different affinities to the surface. Generally, a low degree of protein adsorption, Γ < 0.5 mg/m2, was observed for surfaces covered with PEO chains (mol wt 1900) which were covalently linked, by means of terminal CHO groups, to surface amino groups.

Interfacial behavior of proteins in three-phase partitioning using salt-containing water/tert-butanol systems

Abstract Three-phase partitioning (TPP) was used to investigate the partitioning of four model proteins (bovine serum albumin, ovalbumin, lysozyme and gelatine) in a system containing water, tert -butanol and an inorganic electrolyte ((NH 4 ) 2 SO 4 ). Phase diagrams of the ternary (protein-free) system and interfacial tensions between the equilibrium upper and lower liquid phases have been determined. The amount of protein precipitated in the middle layer was delineated as a function of the composition of the partitioning system as well as the initial protein concentration. No dependence of the relative amount of protein accumulated in the midlayer on the total amount of protein was observed. The constant partitioning ratio found in TPP emphasizes the partitioning character of the process. The interfacial adsorption of a single protein (indicated by the decreasing interfacial tension between the immiscible liquid phases) shows a good correlation with the accumulation of the same protein in TPP as a middle layer for the various proteins investigated.

Interfacial shear rheological behaviour of proteins in three-phase partitioning systems

Abstract Three-phase partitioning process (TPP) is a relatively recent, but fast developing technique, which is used for the accumulation of proteins from multicomponent extracts. In this process a coherent, protein-rich middle layer is formed between two immiscible liquid phases. The two liquid phases are developed by phase separation of systems consisting of tert-butanol, ammonium sulphate and water, and the system is characterised by low interfacial tension. Interfacial rheological properties of four different proteins (bovine serum albumin, ovalbumin, β-lactoglobulin and lysozyme) were investigated in model systems of TPP by means of a two-dimensional Couette-type interfacial rheometer used in the oscillatory mode. Ovalbumin and β-lactoglobulin showed the highest interfacial shear elasticity and viscosity, whilst the rheological parameters of lysozyme were hardly measurable. Shear viscoelastic behaviour of protein film adsorbed at the interface and of thin emulsion layer obtained by slight mixing of the liquid phases were compared as a function of time. The interfacial rheological results and also the microscopic observation of the middle layer support the crucial role of emulsification and emulsion stability related to viscoelastic character of the interfacial film in the protein separation during TPP.

Membrane Affinity and Antibacterial Properties of Cationic Polyelectrolytes With Different Hydrophobicity

The antibacterial behavior of cationic polyelectrolytes is studied using model membrane experiments and in vitro bacterial investigations. The molecular interaction with lipid films is evaluated by the degree of penetration of the polymers into Langmuir monolayers of neutral or negatively charged lipids. The polymer/lipid interaction results in structural changes of the penetrated lipid layer visualized using AFM. The polymers are found to be effective in inhibiting the proliferation of E. coli, B. subtilis and S. aureus. The influence of the chemical structure on the functional behavior is related to the conformational properties. An optimum structure is identified on the basis of antibacterial and hemolytic tests as well as membrane-destroying efficacy of the antimicrobial polymers.

Protein interaction with a Pluronic-modified poly(lactic acid) Langmuir monolayer

Interaction of bovine serum albumin (BSA) with poly(lactic acid) (PLA) layers mixed with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers (Pluronic) at air/solution interfaces was studied by the Langmuir balance technique. Wettability of the mixed PLA-Pluronic system was characterized in the form of a transferred one-layer Langmuir-Blodgett film, and considerable hydrophilization was obtained for all of the Pluronics (6400, 6800, 10500, and 12700) applied here. The density of PEO chains in the monolayer and hence the coverage of PLA was controlled by the composition and the compression of the mixed monolayers. Tensiometric investigations revealed that a significant reduction of BSA adsorption/penetration was achieved by applying the Pluronic 6800 and 12700 with long PEO blocks for hydrophilization of PLA. Interaction of BSA with the modified PLA monolayer depended on the density and length of the PEO chains. The surface morphological characteristics of the films determined by atomic force microscopy were in good correlation with the results of BSA interaction. The average roughness of the polymer LB layer was high due to BSA penetration into the PLA film, while smooth surfaces with small roughness were obtained when the PLA layer was modified by Pluronic 6800.

Novel method to characterize the hydrolytic decomposition of biopolymer surfaces

Abstract Present pharmaceutical research is focused on the development, modification and characterisation of new drug delivery systems. Among the many different substances, biodegradable polymers and copolymers are of practical importance, especially if their degradation byproducts are non-toxic. The polymeric drug carriers are not easily wettable by water or aqueous solutions, i.e. they are hydrophobic. This surface hydrophobicity is unfavourable for keeping drug carriers circulating in the blood long enough to release the drug so that it reaches its target. Therefore, copolymers with components of different hydrophobicity were introduced, to make them less hydrophobic and hence more suitable for drug delivery in the human body. Exploratory experiments with one homopolymer, d , l -poly(lactic acid), d , l -PLA and two of its copolymers, d , l -poly(lactic/glycolic acid), and d , l -PLGA with 85/15 and 50/50 copolymer ratios were carried out. Films of these substances were prepared by dip coating onto hydrophobic and hydrophilic substrates. The changes in wettability of the polymer layers, caused by the direct contact with an aqueous environment (soaking the samples in distilled water), have been studied to model the hydrolytic decomposition of polymer surfaces and to follow the changes in their wettability by dynamic contact angle measurements in a non-destructive manner. It was found that each polymer film became less hydrophobic (dynamic contact angles decreased) and more heterogeneous as the decomposition progressed with time. Increasingly significant decreases in contact angles were observed for the copolymer films containing 15 and 50% glycolic acid, during the 50–80-day-long study. These findings were supported by gel chromatographic analysis of the soaking liquids. It was concluded that the homopolymer layer of d , l -PLA was the most resistant to hydrolysis and the stability of copolymer films decreased with increasing glycolic acid ratio in the copolymers. This is accordance with the fact that the less crystalline poly(glycolic acid) is more hydrophilic and hence less resistant to hydrolytic decomposition, than the poly(lactic acid). The effect of pH on the rate of hydrolysis of polymer films was also established; the influence of pH on the decomposition was best demonstrated, again, for the copolymer with 50/50 component ratio. The outcome of these experiments showed that the contact angle measuring method enables us to detect, follow and interpret the hydrolytic decomposition of biopolymer substances in a non-invasive manner.

Modification of Poly(lactic/glycolic acid) Surface by Chemical Attachment of Poly(ethylene glycol)

Biodegradable polyesters such poly(lactic acid) and poly(lactic/glycolic acid) (PLGA) copolymers are preferred biomaterials and used among others as drug delivery systems, although their surface hydrophobicity limits their application. In this work, chemical modification of the PLGA surface was developed by coupling of either linear or starlike poly(ethylene glycol) (PEG) molecules via chemical bonds to the PLGA surface following amino functionalization as a first step to improve its biocompatibility. The chemical attachment was followed by detailed X-ray photoelectron spectroscopy (XPS) studies. It was shown that substantial modification can be achieved by linear PEG, but even higher surface coverage with hydrophilic groups can be obtained when the six-armed PEG is applied with the additional advantage of possible further functionalization via free amino groups available on the surface of the latter. As a final goal, a significant increase of water wettability together with reduced protein adsorption was achieved on PEG-coupled PLGA surfaces.

Temperature dependence of bovine serum albumin adsorption onto a poly(ethylene oxide)-grafted surface

Abstract The adsorption of bovine serum albumin (BSA) onto bare polyethylene (PE) and poly(ethylene oxide)-grafted PE particles was studied by the solution depletion method. Adsorption measurements were performed at different temperatures in the range of 25–65°C. The PE surface adsorbs high amounts of protein corresponding to a complete side-on monolayer in the temperature range 25–45°C. The considerable increase in the adsorbed amount and the shape of the isotherms at temperatures above 55°C indicate surface aggregation approaching the denaturation temperature of BSA. A lower degree of protein adsorption was obtained for PEO-grafted surfaces, and the amount of BSA adsorbed increases continuously with the temperature over the whole range studied. This is in accordance with the changing wettability of the PEO-grafted surface with temperature. The different temperature dependences of the adsorption behaviour found for PE and PEO-grafted surfaces show the joint influence of the protein properties in solution and the surface interactions on the adsorption.

Molecular Interaction of a New Antibacterial Polymer with a Supported Lipid Bilayer Measured by an in situ Label-Free Optical Technique

The interaction of the antibacterial polymer–branched poly(ethylene imine) substituted with quaternary ammonium groups, PEO and alkyl chains, PEI25QI5J5A815–with a solid supported lipid bilayer was investigated using surface sensitive optical waveguide spectroscopy. The analysis of the optogeometrical parameters was extended developing a new composite layer model in which the structural and optical anisotropy of the molecular layers was taken into consideration. Following in situ the change of optical birefringence we were able to determine the composition of the lipid/polymer surface layer as well as the displacement of lipid bilayer by the antibacterial polymer without using additional labeling. Comparative assessment of the data of layer thickness and optical anisotropy helps to reveal the molecular mechanism of antibacterial effect of the polymer investigated.

Comparison of spray drying, electroblowing and electrospinning for preparation of Eudragit E and itraconazole solid dispersions

Three solvent based methods: spray drying (SD), electrospinning (ES) and air-assisted electrospinning (electroblowing; EB) were used to prepare solid dispersions of itraconazole and Eudragit E. Samples with the same API/polymer ratios were prepared in order to make the three technologies comparable. The structure and morphology of solid dispersions were identified by scanning electron microscopy and solid phase analytical methods such as, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and Raman chemical mapping. Moreover, the residual organic solvents of the solid products were determined by static headspace-gas chromatography/mass spectroscopy measurements and the wettability of samples was characterized by contact angle measurement. The pharmaceutical performance of the three dispersion type, evaluated by dissolution tests, proved to be very similar. According to XRPD and DSC analyses, made after the production, all the solid dispersions were free of any API crystal clusters but about 10 wt% drug crystallinity was observed after three months of storage in the case of the SD samples in contrast to the samples produced by ES and EB in which the polymer matrix preserved the API in amorphous state.