Protein adsorption on polymer surfaces: calculation of adsorption energies

In an attempt to understand the mechanisms of protein adsorption at the solid-liquid interface, we have calculated the interaction potential energy between the protein and the polymer surface by a computer simulation approach. The adsorption of four proteins--lysozyme, trypsin, immunoglobulin Fab, and hemoglobin--on five polymer surfaces was examined. The model polymers used for the calculation were polystyrene, polyethylene, polypropylene, poly(hydroxyethyl methacrylate), and poly(vinyl alcohol). All possible orientations of the protein on the polymer surfaces were simulated and the corresponding interaction energies for the initial contact stage of protein adsorption were calculated. In the calculation of interaction energies, the hydrophobic interaction was not treated explicitly owing to the difficulty in the theoretical treatment. The results showed that the interaction energy was dependent on the orientation of the protein on the polymer surfaces. The energy varied from -850 to +600 kJ/mol with an average of about -155 kJ/mol. The interaction energy was also dependent on the type of polymer. The average interaction energies of the four proteins with poly(vinyl alcohol) were always lower than those with the other polymers. The interaction energy was not dependent on the protein size. It was found that the dispersion attraction played the major role in protein adsorption on neutral polymer surfaces.     

Protein adsorption on polymer surfaces: calculation of adsorption energies

In an attempt to understand the mechanisms of protein adsorption at the solid-liquid interface, we have calculated the interaction potential energy between the protein and the polymer surface by a computer simulation approach. The adsorption of four proteins-lysozyme, trypsin, immunoglobulin F_ab, and hemoglobin-on five polymer surfaces was examined. The model polymers used for the calculation were polystyrene, polyethylene, polypropylene, poly(hydroxyethyl methacrylate), and poly(vinyl alcohol). All possible orientations of the protein on the polymer surfaces were simulated and the corresponding interaction energies for the initial contact stage of protein adsorption were calculated. In the calculation of interaction energies, the hydrophobic interaction was not treated explicitly owing to the difficulty in the theoretical treatment. The results showed that the interaction energy was dependent on the orientation of the protein on the polymer surfaces. The energy varied from - 850 to + 600 kJ/mol with an average of about - 155 kJ/mol. The interaction energy was also dependent on the type of polymer. The average interaction energies of the four proteins with poly(vinyl alcohol) were always lower than those with the other polymers. The interaction energy was not dependent on the protein size. It was found that the dispersion attraction played the major role in protein adsorption on neutral polymer surfaces.     

Polymer versus monomer as displacer in immobilized metal affinity chromatography

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.     

Calculation of solvation interaction energies for protein adsorption on polymer surfaces.

Of the interactions that govern protein adsorption on polymer surfaces, solvation interactions (repulsive hydration and attractive hydrophobic interactions) are thought to be among the most important. The solvation interactions in protein adsorption, however, have not been dealt with in theoretical calculation of the adsorption energy owing to the difficulties in modelling such interactions. We have evaluated the solvation interaction energies using the fragment constant method of calculating the partition coefficients of amino acids. The fundamental assumption of this approach is that the partition coefficients of amino acids between water and organic solvent phases are related to the free energies of transfer from bulk water to the polymer surface. The X-ray crystallographic protein structures of lysozyme, trypsin, immunoglobulin Fab, and hemoglobin from the Brookhaven Protein Data Bank were used. The model polymer surfaces were polystyrene, polypropylene, polyethylene, poly(hydroxyethyl methacrylate) [poly(HEMA)], and poly(vinyl alcohol). All possible adsorption orientations of the proteins were simulated to study the effect of protein orientation on the solvation interactions. Protein adsorption on either hydrophobic or hydrophilic polymer surfaces was examined by considering the sum of solvation and other interaction energies. The results showed that the contribution of the solvation interaction to the total protein adsorption energy was significant. The average solvation interaction energy ranged from -259.1 to -74.1 kJ/mol for the four proteins on the hydrophobic polymer surfaces, such as polystyrene, polypropylene, and polyethylene. On the other hand, the average solvation interaction energies on hydrophilic surfaces such as poly(HEMA) and poly(vinyl alcohol) were larger than zero. This indicates that repulsive hydration interactions are in effect for protein adsorption on hydrophilic polymer surfaces. The total interaction energies of the proteins with hydrophobic surfaces were always lower than those with more hydrophilic surfaces. This trend is in agreement with the experimental observations in the literature. This study suggests that consideration of the solvation interaction energies is necessary for accurate calculation of the protein adsorption energies.     

Polymer characterization by temperature gradient interaction chromatography

Thermodynamic principles and some applications of the temperature gradient interaction chromatography (TGIC) recently developed for the characterization of synthetic polymers are described. TGIC is a form of high performance liquid chromatography (HPLC) that varies column temperature in a programmed manner to control the retention of polymeric species during isocratic elution. The retention of polymers strongly depends on their molecular weights, and the polymers are well separated by TGIC in terms of their molecular weights. TGIC is superior to size exclusion chromatography (SEC) in resolution and sample loading capacity, and has higher sensitivity to molecular weight in the analysis of nonlinear polymers. TGIC has an advantage over solvent gradient HPLC because it permits the use of refractive index sensitive detection method such as differential refractometry and light scattering due to the isocratic elution. In addition, temperature provides finer and more reproducible retention control than the solvent composition, which is important in determining the molecular weight distribution by secondary calibration methods. With TGIC analysis, we found that the molecular weight distribution of anionically polymerized polymers is much narrower than has been generally accepted from SEC analysis. We also found the TGIC separation conditions for polystyrene, polyisoprene, poly(methyl methacrylate), poly(vinyl chloride), and poly(vinyl acetate) over wide molecular weight range. Because of its sensitivity to the molecular weight alone, TGIC was successfully applied to the characterization of star shaped polystyrene, and the detailed linking kinetics between living polystyrene anions and a chlorosilane linking agent was investigated.     

Interactions of human serum albumin with a modified poly(vinyl alcohol) gel packing for high-performance liquid chromatography

—The interactions of human serum albumin (HSA) with a poly(vinyl alcohol) gel packing (Asahipak GS-520) for high-performance liquid chromatography of proteins were investigated. Under certain conditions, the elution of HSA from the GS-520 column was retarded and its chromatogram was split into two peaks, indicating weak adsorption of HSA onto the gels and also the existence of two subfractions, i.e. human mercapto-albumin (HMA) and human non-mercapto-albumin (HNA). The chromatograms were confirmed to be greatly influenced by the salt composition, the pH, and the temperature of the isocratic mobile phase. It is characteristic for the adsorption of HSA onto the gels to be suppressed at a pH near its isoelectric point. The HSA-gel interaction parameters calculated using an adsorption chromatography theory demonstrate that the adsorption of HSA is caused by enthalpy-driven interactions, which are depressed by lowering the pH, in addition to hydrophobic interactions. Under the recommended chromatographic conditions for high resolution of HMA/HNA, it was found that the HSA samples possessed some subfractions besides HMA and HNA fractions.     

Interactions of human serum albumin with a modified poly(vinyl alcohol) gel packing for high-performance liquid chromatography

The interactions of human serum albumin (HSA) with a poly(vinyl alcohol) gel packing (Asahipak GS-520) for high-performance liquid chromatography of proteins were investigated. Under certain conditions, the elution of HSA from the GS-520 column was retarded and its chromatogram was split into two peaks, indicating weak adsorption of HSA onto the gels and also the existence of two subfractions, i.e. human mercapto-albumin (HMA) and human non-mercapto-albumin (HNA). The chromatograms were confirmed to be greatly influenced by the salt composition, the pH, and the temperature of the isocratic mobile phase. It is characteristic for the adsorption of HSA onto the gels to be suppressed at a pH near its isoelectric point. The HSA-gel interaction parameters calculated using an adsorption chromatography theory demonstrate that the adsorption of HSA is caused by enthalpy-driven interactions, which are depressed by lowering the pH, in addition to hydrophobic interactions. Under the recommended chromatographic conditions for high resolution of HMA/HNA, it was found that the HSA samples possessed some subfractions besides HMA and HNA fractions.     

Probing the weak interaction of proteins with neutral and zwitterionic antifouling polymers

Protein–polymer interactions are of great interest in a wide range of scientific and technological applications. Neutral poly(ethylene glycol) (PEG) and zwitterionic poly(sulfobetaine methacrylate) (pSBMA) are two well-known nonfouling materials that exhibit strong surface resistance to proteins. However, it still remains unclear or unexplored how PEG and pSBMA interact with proteins in solution. In this work, we examine the interactions between two model proteins (bovine serum albumin and lysozyme) and two typical antifouling polymers of PEG and pSBMA in aqueous solution using fluorescence spectroscopy, atomic force microscopy and nuclear magnetic resonance. The effect of protein:polymer mass ratios on the interactions is also examined. Collective data clearly demonstrate the existence of weak hydrophobic interactions between PEG and proteins, while there are no detectable interactions between pSBMA and proteins. The elimination of protein interaction with pSBMA could be due to an enhanced surface hydration of zwitterionic groups in pSBMA. New evidence is given to demonstrate the interactions between PEG and proteins, which are often neglected in the literature because the PEG–protein interactions are weak and reversible, as well as the structural change caused by hydrophobic interaction. This work provides a better fundamental understanding of the intrinsic structure–activity relationship of polymers underlying polymer–protein interactions, which are important for designing new biomaterials for biosensor, medical diagnostics and drug delivery applications.     

Chemically crosslinkable thermosensitive polyphosphazene gels as injectable materials for biomedical applications

Chemically crosslinkable and thermosensitive poly(organophosphazenes) containing multiple thiol (–SH) groups along with hydrophobic isoleucine ethyl ester and hydrophilic α-amino-ω-methoxy-poly(ethylene glycol) of the molecular weight 550 have been synthesized and characterized as an injectable biomaterial. The aqueous solutions of these polymers were transformed into hydrogel with desired gel strength at body temperature via hydrophobic interactions, and the gel strength was further improved by the cross-linking of thiol groups with crosslinkers, divinyl sulfone (VS) and PEG divinyl sulfone (PEGVS) under physiological conditions. The kinetics of cross-linking behavior of polymer thiol groups with crosslinkers was studied in both in vitro and in vivo conditions. Field Emission-Scanning Electron Microscopy (FE-SEM), swelling experiments, and rheology study of present polymers revealed that the inner three-dimensional hydrogel networks depended on the degree of thiol units in the polymer network. From the in vivo (in mice) degradation studies, the dual cross-linked gels showed to have a controlled degradation. These results demonstrate that the inner network of the hydrogels can be tuned, gel strength and degradation rate can be controlled, and the chemically crosslinkable and thermosensitive poly(organophosphazenes) hold promises for uses as injectable systems for biomedical applications including tissue engineering and protein delivery.     

Why degradable polymers undergo surface erosion or bulk erosion

A theoretical model was developed that allows to predict the erosion mechanism of water insoluble biodegradable polymer matrices. The model shows that all degradable polymers can undergo surface erosion or bulk erosion. Which way a polymer matrix erodes after all depends on the diffusivity of water inside the matrix, the degradation rate of the polymer's functional groups and the matrix dimensions. From these parameters the model allows to calculate for an individual polymer matrix a dimensionless 'erosion number' epsilon. The value of epsilon indicates the mode of erosion. Based on epsilon, a critical device dimension Lcritical can be calculated. If a matrix is larger than Lcritical it will undergo surface erosion, if not it will be bulk eroding. Lcritical values for polymers were estimated based on literature data. Polyanhydrides were found to be surface eroding down to a size of approximately Lcritical = 10(-4) m while poly(alpha-hydroxy esters) matrices need to be larger than Lcritical = 10(-1) m to lose their bulk erosion properties. To support our theoretical findings it was shown experimentally that poly(alpha-hydroxy ester) matrices, which are considered classical bulk eroding materials, can also undergo surface erosion.     

Polymer fractionation by gel permeation chromatography

The molecular weight distributions of polystyrene, poly(vinyl chloride), polychloro-prene and polyethylene have been determined by gel permeation chromatography using silica gel as the stationary phase. The factors affecting fractionation efficiency, such as column dimensions and polymer loading are discussed. Fractionation efficiency for polystyrene, poly(vinyl chloride) and polychloroprene is comparable to that obtained from such established methods as fractional precipitation and fractional elution. For polyethylene, however, the gel permeation technique using silica gel as the stationary phase, does not appear to have the fractionation efficiency of the column elution method of FRANCIS, COOKE, and ELLIOTT.     

Rheological and Morphological Properties of Various Blends Containing Liquid Crystalline Polyesters

This contribution is concerned with the unique rheological properties of blends of liquid crystalline polymers (LCP) dispersed in thermoplastic matrices. The materials under investigation were rigid rod polyesters solution blended with poly(ε‐caprolactone), polypropene, polystyrene or poly(methyl methacrylate) as matrix polymers. Oscillatory measurements of these melted blends revealed that LCP contents of only 1 wt.‐% cause the dynamic shear moduli to increase at low frequencies by a factor of several magnitudes compared to the neat matrices. The extraordinary effect is primarily governed by a spatial arrangement of the LCP molecules forming a network as shown by microscopy. The different strength of this effect in different matrices is explained by the interaction between matrix and LCP.     

Functionalized Biocompatible Poly(N‐vinyl‐2‐caprolactam) With pH‐Dependent Lower Critical Solution Temperature Behaviors

Functionalized temperature‐ and pH‐sensitive poly(N‐vinyl‐2‐caprolactam) (PVCL) polymers are prepared by copolymerizing monomers of N‐vinyl‐2‐caprolactam (VCL) and a VCL derivative, 3‐(tert‐butoxycarbonyl)‐N‐vinyl‐2‐caprolactam (TBVCL). Different molar compositions are studied, with the functional monomer at 9 and 14 mol%, respectively, (COOH‐PVCL9 and COOH‐PVCL14). Sharp, complete, and reversible phase transitions of the copolymers with little hysteresis are shown to be pH‐dependent, with cloud points ranging from 35 to 44 °C for COOH‐PVCL9, and 29 to 64 °C for COOH‐PVCL14, upon pH change from 2.0 to 7.4. Cytotoxicity assay demonstrates that the functionalized PVCL copolymers are biocompatible with NIH/3T3 up to 2 mg mL^?1. Such new PVCL‐based water soluble copolymers with tunable properties could be useful in a variety of biomedical applications.     

The effect of the processing and formulation parameters on the size of nanoparticles based on block copolymers of poly(ethylene glycol) and poly(N-isopropylacrylamide) with and without hydrolytically sensitive groups

Block copolymers of poly(ethylene glycol) (PEG) as a hydrophilic block and N-isopropylacrylamide (PNIPAAm) or poly (NIPAAm-co-N-(2-hydroxypropyl) methacrylamide-dilactate) (poly(NIPAAm-co-HPMAm-dilactate)) as a thermosensitive block, are able to self-assemble in water into nanoparticles above the cloud point (CP) of the thermosensitive block. The influence of processing and the formulation parameters on the size of the nanoparticles was studied using dynamic light scattering. PNIPAAm-b-PEG 2000 polymers were not suitable for the formation of small and stable particles. Block copolymers with PEG 5000 and 10000 formed relatively small and stable particles in aqueous solutions at temperatures above the CP of the thermosensitive block. Their size decreased with increasing molecular weight of the thermosensitive block, decreasing polymer concentration and using water instead of phosphate buffered saline as solvent. Extrusion and ultrasonication were inefficient methods to size down the polymeric nanoparticles. The heating rate of the polymer solutions was a dominant factor for the size of the nanoparticles. When an aqueous polymer solution was slowly heated through the CP, rather large particles (> or = 200 nm) were formed. Regardless the polymer composition, small nanoparticles (50-70 nm) with a narrow size distribution were formed, when a small volume of an aqueous polymer solution below the CP was added to a large volume of heated water. In this way the thermosensitive block copolymers rapidly pass their CP ('heat shock' procedure), resulting in small and stable nanoparticles.     

In Situ Polymer Analogue Generation of Azlactone Functions at Poly(oxazoline)s for Peptide Conjugation

The physical and chemical stability of peptides for biomedical applications can be greatly enhanced through the conjugation of polymers. A well‐known but rather underemployed selective coupling functionality is the azlactone group, which readily reacts with a number of different nucleophiles without the need for activation and the formation of any by‐products. For example, azlactone functional polymers are used to react with peptides and proteins, rich in amino and thiol groups, to form polymeric beads for affinity‐based column chromatography. So far, side chain functional azlactone polymers have been mainly synthesized by radical polymerization using 2‐vinyl‐4,4‐dimethyl azlactone together with different acrylate monomers. Here, a new azlactone precursor equipped with a functional thiol is presented, which can be attached to any vinyl functional polymer by thiol–ene chemistry. Subsequently, the formation of the reactive azlactone ring can be performed in situ at high conversion rate without the need for illumination. This approach is tested on an azlactone side functional poly(2‐oxazoline) by coupling amine containing molecules including a model peptide and is proven via ¹H NMR spectroscopy, IR spectroscopy, as well as HPLC measurements.     

Toward the development of biomimetic polymers by protein immobilization: PEGylation of insulin as a model reaction

Many current tissue-engineering investigations aim at the rational control of cell adhesion and tailored composition of biomaterial surfaces by immobilizing various protein and peptide components, such as growth factors. As a step on the way to develop polymers that allow for such surface modifications, water-soluble polymers were used as model substances to examine reactions with proteins containing amine groups. Consequently, the uncommon PEGylation of insulin in aqueous buffers was used to characterize reaction products and simulate the intended immobilization step for surface modification. Amine reactive poly(ethylene glycol)s were synthesized and characterized by (1)H nuclear magnetic resonance and gel-permeation chromatography. Furthermore, the model protein insulin was characterized concerning its accessible amino groups, using a fluorescent dye (TAMRA-SE). The resulting reaction products were identified by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. After PEGylation with hydrolytically stable poly(ethylene glycol) succinimidyl ester, the obtained PEGylated insulin was investigated by gel filtration chromatography, indicating successful attachment of the hydrophilic polymer chains. Application of an aqueous PEGylation scheme opens the door to the immediate investigation of various growth factors in cell culture, allowing for direct assessment of biological activity after forming the polymer-protein constructs with regard to later immobilization on surfaces.     

Metachromasia of pinacyanol chloride induced by synthetic polyanions

The metachromasia of the cyanine dye pinacyanol chloride sharply depends on the nature of the polyanions the dye interacts with. The metachromatic spectra of the dye induced by polymethacrylate (only at polymer/dye mole ratios P/D>3), polyacrylate and poly(vinyl sulfate) are broad and multiple-banded; whereas the spectra are sharp and single-banded with poly(methacrylic acid) and polystyrenesulfonate as the chromotropes. Poly(methacrylic acid) and polystyrenesulfonate form compounds with the dye with 2:1 stoichiometry, whereas the polyanion-dye complexes with polyacrylate and poly(vinyl sulfate) as polyanions have 1:1 stoichiometry. This observed difference in the metachromasia of this dye has been discussed in terms of the differences in stoichiometry, flexibility and conformation of the polyanion chains.     

Degradation of poly(ester) microspheres

Biodegradable polymeric microspheres have been prepared by spray drying, precipitation, rotary evaporation and press grinding methods. Erosion of microspheres of poly(lactide), poly(3-hydroxybutyrate), copolymers of lactide and glycolide, and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate at 85°C and 37°C have been studied using ion chromatography, nuclear magnetic resonance, residual mass measurements, viscometry and gel permeation chromatography. Such studies demonstrated that these polyester matrices degraded via (1) random chain scission and (2) release of soluble monomeric and oligomeric products. Protein release from microspheres prepared by these methods indicated that most of the protein is released before the polymer matrix loses weight.     

Reaction of excited oxygen species with polymer films

The reaction of atomic oxygen and singlet oxygen molecules with films from various polymeric substrates was studied. The polymers used were poly(vinyl chloride), polyacrylonitrile, polystyrene, nylon 66, and poly(ethylene terephthalate). Atomic oxygen reacted readily with all of these polymers. The reaction was confined to the surface layers which results in the polymer films becoming more hydrophilic. No change was observed in the contact angle of water with films of poly(vinyl chloride) and polystyrene after they had been exposed to singlet oxygen molecules for several hours.