Hans Jörg Griesser

Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels.

A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups (charge) on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.

Method of immobilization of carboxymethyl-dextran affects resistance to tissue and cell colonization

Coatings from carboxymethylated dextrans (CMDs) were fabricated, analyzed by XPS, and investigated for their ability to inhibit corneal epithelial tissue outgrowth and bovine corneal epithelial cell attachment and growth. CMDs with differing degrees of carboxymethyl substitution and various molecular weights were synthesized by the solution reaction of dextrans with bromoacetic acid under different reactant ratios. The CMD compounds thus obtained were attached onto aminated surfaces produced in two ways: by the plasma deposition of a coating from n-heptylamine vapour, and by the plasma deposition of an acetaldehyde coating onto whose surface aldehyde groups the polyamine compounds polylysine, polyethyleneimine and polyallylamine were immobilized to provide platforms for CMD immobilization. XPS spectra showed that the latter route produced thicker coatings than the former approach. CMD molecules attached directly onto the plasma-fabricated amine surface supported some tissue migration; the extent of carboxymethyl substitution and the molecular weight of the CMDs had little influence. For CMDs immobilized via polyamine spacers, on the other hand, tissue outgrowth was completely inhibited, and again there were no discernible effects from the extent of carboxymethyl substitution and the molecular weight of the CMDs. In assays involving cell attachment and growth, analogous observations were found. Thus, the mode of immobilization of these polysaccharide coatings is the dominant factor in their anti-fouling performance, suggesting that optimization of the architecture of polysaccharide coatings may be an important factor for maximizing their cell-repellent abilities.

Irreversible adsorption of human serum albumin to hydrogel contact lenses: a study using electron spin resonance spectroscopy

Human serum albumin (HSA) was specifically spin labelled with 4-maleimido-tempo (MSL) at its cysteine 34 residue (HSA-MSL). The irreversible adsorption of HSA-MSL to hydrogel contact lenses (etafilcon A, tefilcon and vifilcon A) was investigated using electron spin resonance (ESR) spectroscopy. Changes in ESR spectral characteristics of adsorbed HSA-MSL as compared to HSA-MSL in solution displayed an additional immobilisation of the spin label due to the adsorption. This immobilisation of MSL corresponds to a large conformational alteration of the HSA-MSL near the modified Cys 34 residue. For both etafilcon A and tefilcon, the rate of irreversible adsorption was relatively slow compared with that of vifilcon A where the maximum state of immobilisation and hence conformational change occurred within the first hour of adsorption. Furthermore, tefilcon produced markedly different ESR spectra where a strong conformational change to a less mobile protein was apparent. This supported a model where the direct irreversible adsorption of HSA from solution dominated on tefilcon as opposed to conversion of the adsorbed protein from the reversible to the irreversible state on both etafilcon A and vifilcon A. HSA-MSL adsorption onto hydrophobic poly(methylmethacrylate) (PMMA) and hydrophilic poly(N-ter-butylacrylamide) (PTBAM) latex beads was also investigated. The spin label MSL was found to be less mobile when HSA was adsorbed onto PMMA compared with PTBAM beads. It was also found that the rate of irreversible adsorption of HSA is far higher onto PMMA surfaces than onto PTBAM surfaces.

Interactions between adsorbed lactoferrin layers measured directly with the atomic force microscope

Abstract Lactoferrin is an important protein found in tear fluid and has been implicated in the fouling of contact lenses. The interaction forces between lactoferrin layers, adsorbed onto hydrophobic model substrates, as a function of electrolyte concentration, solution pH and protein concentration were investigated using an atomic force microscope (AFM) where the geometry of the scanning tip was modified by attaching a colloid particle. Bovine lactoferrin adsorbed strongly and modified the interaction forces to include both a long-range electrostatic repulsive force, which could be described using the DLVO theory, and a short-range additional repulsion due to compression of the adsorbed protein layers. The adsorption of lactoferrin was verified by XPS analysis of the surfaces and the adsorbed layer was determined to be present as a full monolayer by AFM imaging. At smaller separation distances, deviations from theoretical predictions indicated that there was an adsorbed layer of at least 5 nm on both surfaces. DLVO theory could be used successfully to describe the longer ranged interaction forces in background electrolyte concentrations of 0.0002 and 0.001 M KNO 3 , at various solution pH values. This analysis yielded effective diffuse electrical double layer potentials, which when plotted against the solution pH, suggested that the point of zero charge (pzc) of the protein coated surfaces was around pH 8, in agreement with literature values of the pI for lactoferrin. At higher electrolyte concentrations, 0.15 M KNO 3 , the interaction forces could no longer be described using DLVO theory. The interaction forces between the adsorbed lactoferrin layers were purely repulsive, and the range and magnitude varied as a function of solution pH. Variations in the forces as a function of solution pH reflected conformational changes that depend on short ranged electrostatic interactions between the peptide residues within the protein molecules and the peptide residues and the substrate as well as van der Waals and hydrophobic interactions. The range of the interaction forces and the high compressibility of the adsorbed layers indicated that a significant number were present in an end-on orientation.

The ocular surface, the tear film, and the wettability of contact lenses.

The tear film is the interface between the ocular surface and the external environment and, as such, plays several important roles.1 (i) It forms a refracting thin film that smooths out the irregular corneal surface topography. (ii) It maintains an extracellular environment for the epithelial cells of the cornea and the conjunctiva that is fairly constant in terms of pH, oxygen and carbon dioxide levels, and nutrient and growth factor concentrations. (iii) Tears dilute and wash away noxious stimuli, including bacteria, which are also combated by an elaborate and effective antibacterial system. (iv) The tear film changes its composition in response to physiological stimuli.