Ma Cristina L. Martins

Albumin and fibrinogen adsorption on PU-PHEMA surfaces

Materials that adsorb specific proteins may find a variety of applications in the biomedical field. The aim of this study was the preparation of a hydrophilic surface, with low protein adsorption, to be used in the future as a support for the immobilisation of several species, e.g. Cibacron Blue F3G-A, which has been described to induce specific albumin adsorption. Poly(hydroxyethylmethacrylate) (PHEMA) and poly(hydroxyethylacrylate) (PHEA) were chosen as the hydrophilic surface because they can be easily polymerised and possess hydroxyl groups that can be used for the immobilisation of different compounds. Thin films of PHEMA and PHEA were successfully graft polymerised onto the surface of a commercial poly(etherurethane) (PU) using ceric ion as initiator. Grafting polymerisations were followed by mass gain and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Since stability tests demonstrated that only PU-PHEMA was stable in alkaline solutions, a necessary condition to future immobilisations, the investigation was focused on the coating of PU with PHEMA. PU-PHEMA films were characterised in detail using several techniques as mass gain, ATR-FTIR, contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Protein adsorption was evaluated using radiolabelled albumin and fibrinogen from pure solutions and from mixtures of both proteins. PU surfaces modified with PHEMA have demonstrated low protein adsorption, showing their potential use as substrates. This opens the possibly of exploring the advantages of selective adsorption by appropriate immobilisation of specific molecules.

Albumin adsorption on alkanethiols self-assembled monolayers on gold electrodes studied by chronopotentiometry

Chronopotentiometry was used to study the adsorption of human serum albumin (HSA) to self-assembled monolayers with the following terminal functional groups: CH(3), COOH and OH. Surfaces were characterized by X-ray photoelectron spectroscopy, water contact angle measurements and cyclic voltammetry. HSA coverage of the different SAMs was investigated by chronopotentiometry and the total amount of adsorbed protein was determined using radiolabelled albumin. Both techniques have demonstrated that HSA adsorption to the different SAM-modified electrodes increases in the following order: OH<COOH<CH(3)-terminated SAMs. A good correlation between coverage and total amount of HSA adsorbed was observed for long adsorption times (900s).

Albumin adsorption on cibacron blue F3G-A immobilized onto oligo(ethylene glycol)-terminated self-assembled monolayers.

Self-assembled monolayers can be tailored with specific ligands to a certain protein and at the same time prevent the non-specific adsorption of other proteins. Cibacron Blue F3G-A (CB-thiol) was successfully immobilized onto tetra(ethylene glycol)-terminated alkanethiol (CB-thiol). The affinity of human serum albumin (HSA) to immobilized Cibacron Blue F3G-A was studied using mixed thiolate self-assembled monolayers on gold with different n-alkyl chain lengths and functional terminal groups (CH3-; OH- and tetra(ethylene glycol)). Surfaces were characterized using X-ray photoelectron spectroscopy and water contact angle measurements. Albumin adsorption and exchangeability of the adsorbed albumin molecules with other albumin molecules in solution were evaluated using 125I-radiolabeled HSA. Competitive adsorption between albumin and fibrinogen to the different self-assembled monolayers (SAMs) was also investigated. Results showed that the incorporation of CB-thiol on the monolayers does not increase the HSA adsorption and reversibility on the SAMs. However, although specific adsorption of HSA to the immobilized Cibacron Blue F3G-A was not demonstrated, the presence of CB-thiol decreases the affinity of fibrinogen to the OH-terminated SAMs.

Adsorbed fibrinogen leads to improved bone regeneration and correlates with differences in the systemic immune response

Designing new biomaterials that can modulate the inflammatory response instead of attempting just to reduce it constitutes a paradigm change in regenerative medicine. This work aimed to investigate the capacity of an immunomodulatory biomaterial to enhance bone regeneration. For that purpose we incorporated a molecule with well-established pro-inflammatory and pro-healing roles, fibrinogen, in chitosan scaffolds. Two different incorporation strategies were tested, leading to concentrations of 0.54±0.10mg fibrinogen g(-1) scaffold immediately upon adsorption (Fg-Sol), and 0.34±0.04mg fibrinogen g(-1) scaffold after washing (Fg-Ads). These materials were implanted in a critical size bone defect in rats. At two months post-implantation the extent of bone regeneration was examined by histology and the systemic immune response triggered was evaluated by determining the percentages of myeloid cells, T and B lymphocytes in the draining lymph nodes. The results obtained indicate that the fibrinogen incorporation strategy conditioned the osteogenic capacity of biomaterials. Fg-Ads scaffolds led to more bone formation, and the presence of Fg stimulated angiogenesis. Furthermore, animals implanted with Fg-Ads scaffolds showed significant increases in the percentages of B lymphocytes and myeloid cells in the draining lymph nodes, while levels of T lymphocytes were not significantly different. Finally, a significant increase in TGF-β1 was detected in the plasma of animals implanted with Fg-Ads. Taken together the results presented suggest a potential correlation between the elicited immune response and biomaterial osteogenic performance.

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation.

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin-thrombin complex to minimize the biotinylation of the residues involved in thrombin-boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²⁵I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilins potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.

Albumin and fibrinogen adsorption on cibacron blue F3G-A immobilised onto PU-PHEMA (polyurethane-poly(hydroxyethylmethacrylate)) surfaces.

In the present work, it is intended to study the effect of Cibacron blue F3G-A (CB) immobilised onto PU-PHEMA (polyurethane-poly(hydroxyethylmethacrylate)) surfaces on protein adsorption and bacterial adhesion. CB immobilisation was carried out by covalent binding between its triazine ring and the hydroxyl groups of the polymer. Characterisation of the films was carried out by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR), contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). CB efficiency was evaluated using radiolabelled albumin and fibrinogen from pure solutions, mixtures of both and plasma. Bacterial adhesion tests before and after albumin pre-coating were also performed. The presence of CB increases albumin and fibrinogen adsorption to PU-PHEMA surfaces. The incorporation of CB onto the PU-PHEMA surface also increases bacterial adhesion. Although albumin pre-coating decreases bacterial adhesion onto PU (67% decrease) and PU-PHEMA-CB (80%), bacterial adhesion is always lower on PU and PU-PHEMA surfaces than on PU-PHEMA-CB. These results demonstrate that, in contrast to what has been described for CB bound to dextran, CB immobilisation on PU-PHEMA surfaces presents low selectivity to albumin and increased bacterial adhesion relatively to PU and PU-PHEMA surfaces.

Bioengineered surfaces promote specific protein–glycan mediated binding of the gastric pathogen Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the worlds population and persistent infection is related with an increase in the risk of gastric cancer. Adhesion of H. pylori to the gastric epithelium, which is essential for infection, is mediated by bacterial adhesin proteins that recognize specific glycan structures (Gly-R) expressed in the gastric mucosa. The blood group antigen binding adhesin (BabA) recognizes difucosylated antigens such as Lewis B (Leb), while the sialic acid binding adhesin (SabA) recognizes sialylated glycoproteins and glycolipids, such as sialyl-Lewis x (sLex). This work aimed to investigate whether these Gly-Rs (Leb and sLex) can attract and specifically bind H. pylori after immobilization on synthetic surfaces (self-assembled monolayers (SAMs) of alkanethiols on gold). Functional bacterial adhesion assays for (Gly-R)-SAMs were performed using H. pylori strains with different adhesin protein profiles. The results demonstrate that H. pylori binding to surfaces occurs via interaction between its adhesins and cognate (Gly-R)-SAMs and bound H. pylori maintains its characteristic rod-shaped morphology only during conditions of specific adhesin-glycan binding. These results offer new insights into innovative strategies against H. pylori infection based on the scavenging of bacteria from the stomach using specific H. pylori chelating biomaterials.

Adhesion of human leukocytes on mixtures of hydroxyl‐ and methyl‐terminated self‐assembled monolayers: Effect of blood protein adsorption

The adhesion of human leukocytes to nanostructured surfaces with different chemical properties and the effect of protein adsorption were investigated. Self-assembled monolayers (SAMs) prepared with mixtures of methyl- and hydroxyl-terminated alkanethiols in different percentages on gold were used. The surfaces were pre-immersed in distinct protein solutions (human serum albumin, human fibrinogen, and autologous plasma). Adherent leukocytes were analyzed both by light and SEM. SAMs submitted to pre-immersion in plasma presented higher numbers of adherent leukocytes in the pure OH-terminated SAM, whereas methyl-terminated surfaces accounted for the lowest number of adherent cells. We observed a general increase in the number of adherent human leukocytes as the percentage of OH groups on the surface of the SAMs increased for all the pre-immersion conditions investigated. The number of adherent human leukocytes is highly influenced by the pre-immersion conditions used, and this observation is particularly relevant in the case of the methyl-terminated SAMs. The results obtained demonstrate that surface chemistry has a major influence in leukocyte adhesion to biomaterials, and that pre-immersion in protein solutions has a determinant effect in leukocyte adhesion.

Atomic force microscopy measurements reveal multiple bonds between Helicobacter pylori blood group antigen binding adhesin and Lewis b ligand.

The strength of binding between the Helicobacter pylori blood group antigen-binding adhesin (BabA) and its cognate glycan receptor, the Lewis b blood group antigen (Leb), was measured by means of atomic force microscopy. High-resolution measurements of rupture forces between single receptor–ligand pairs were performed between the purified BabA and immobilized Leb structures on self-assembled monolayers. Dynamic force spectroscopy revealed two similar but statistically different bond populations. These findings suggest that the BabA may form different adhesive attachments to the gastric mucosa in ways that enhance the efficiency and stability of bacterial adhesion.

4 – Cellular response to the surface chemistry of nanostructured biomaterials

Cell–biomaterial interactions are largely governed by the chemical composition of the surface. Molecular design of substrates allows the development of biomaterials that lead to specific and desirable biological interactions with the surrounding tissues. Self-assembly in nature has inspired the development of a new generation of biomaterials. After implantation, biomaterials are rapidly covered with proteins. Adsorbed proteins are key mediators of cell behaviour. In this chapter the process of self-assembly – in nature, in synthetic analogues of the extracellular matrix and in model surfaces (self-assembled monolayers, SAMs) – is overviewed. The application of SAMs as model surfaces to investigate cell–biomaterial interactions is discussed in detail.