M.M.M. Bilek

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3

Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin αVβ3 antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin αVβ3 as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion.

Covalent immobilisation of tropoelastin on a plasma deposited interface for enhancement of endothelialisation on metal surfaces

Currently available endovascular metallic implants such as stents exhibit suboptimal biocompatibility in that they re-endothelialise poorly leaving them susceptible to thrombosis. To improve the interaction of these implants with endothelial cells we developed a surface coating technology, enabling the covalent attachment of biomolecules to previously inert metal surfaces. Using horseradish peroxidase as a probe, we demonstrate that the polymerised surface can retain the presentation and activity of an immobilised protein. We further demonstrated the attachment of tropoelastin, an extracellular matrix protein critical to the correct arrangement and function of vasculature. Not only it is structurally important, but it plays a major role in supporting endothelial cell growth, while modulating smooth muscle cell infiltration. Tropoelastin was shown to bind to the surface in a covalent monolayer, supplemented with additional physisorbed multilayers on extended incubation. The physisorbed tropoelastin layers can be washed away in buffer or SDS while the first layer of tropoelastin remains tightly bound. The plasma coated stainless steel surface with immobilised tropoelastin was subsequently found to have improved biocompatibility by promoting endothelial cell attachment and proliferation relative to uncoated stainless steel controls. Tropoelastin coatings applied to otherwise inert substrates using this technology could thus have broad applications to a range of non-polymeric vascular devices.

A comprehensive survey of M2AX phase elastic properties

M(2)AX phases are a family of nanolaminate, ternary alloys that are composed of slabs of transition metal carbide or nitride (M(2)X) separated by single atomic layers of a main group element. In this combination, they manifest many of the beneficial properties of both ceramic and metallic compounds, making them attractive for many technological applications. We report here the results of a large scale computational survey of the elastic properties of all 240 elemental combinations using first-principles density functional theory calculations. We found correlations revealing the governing role of the A element and its interaction with the M element on the c axis compressibility and shearability of the material. The role of the X element is relatively minor, with the strongest effect seen in the in-plane constants C(11) and C(12). We identify several elemental compositions with extremal properties such as W(2)SnC, which has by far the lowest value of C(44), suggesting potential applications as a high-temperature dry lubricant.

Plasma modified surfaces for covalent immobilization of functional biomolecules in the absence of chemical linkers: towards better biosensors and a new generation of medical implants

Plasma modification and plasma polymer deposition are valuable technologies for the preparation of surfaces for the covalent binding of biomolecules for applications such as biosensors, medical prostheses, and diagnostic devices as well as surfaces for enzyme-mediated reactions. Covalency is conveniently tested by the ability of the surface to retain the attached molecules after vigorous washing with sodium dodecyl sulphate (SDS). Covalency is indicated if the fraction of protein retained lies above the curve characteristic of physisorption. Confidence in covalency is strengthened when the washing protocol is aggressive enough to remove all adsorbed protein from a control significantly more hydrophobic than the test surface. The use of linker chemistry to space the molecules from the surface is in some cases beneficial. However, the use of linker chemistry is not necessary to retain molecular function for long periods when the polymer surface is modified by energetic bombardment. The energetic bombardment retains hydrophilicity of the surface by crosslinking the subsurface, and this appears to facilitate retention of protein function. Energetic bombardment also increases the functional life of molecules immobilized and then freeze dried on plasma-modified surfaces. Analysis of the surfaces shows that the covalent binding mechanism is related to the presence of free radicals on the surface and in the subsurface regions. The unpaired electrons associated with the radicals appear to be mobile within the modified region and can diffuse to the surface to take part in binding interactions. Proactive implantable devices can make use of these principles of covalent attachment by seeding the surface of an implant with a biomolecule that elicits the desired interaction with cells and prevents undesirable responses.

The immobilization of recombinant human tropoelastin on metals using a plasma-activated coating to improve the biocompatibility of coronary stents

Current endovascular stents have sub-optimal biocompatibility reducing their clinical efficacy. We previously demonstrated a plasma-activated coating (PAC) that covalently bound recombinant human tropoelastin (TE), a major regulator of vascular cells in vivo, to enhance endothelial cell interactions. We sought to develop this coating to enhance its mechanical properties and hemocompatibility for application onto coronary stents. The plasma vapor composition was altered by incorporating argon, nitrogen, hydrogen or oxygen to modulate coating properties. Coatings were characterized for 1) surface properties, 2) mechanical durability, 3) covalent protein binding, 4) endothelial cell interactions and 5) thrombogenicity. The N(2)/Ar PAC had optimal mechanical properties and did not delaminate after stent expansion. The N(2)/Ar PAC was mildly hydrophilic and covalently bound the highest proportion of TE, which enhanced endothelial cell proliferation. Acute thrombogenicity was assessed in a modified Chandler loop using human blood. Strikingly, the N(2)/Ar PAC alone reduced thrombus weight by ten-fold compared to 316L SS, a finding unaltered with immobilized TE. Serum soluble P-selectin was reduced on N(2)/Ar PAC and N(2)/Ar PAC + TE (p < 0.05), consistent with reduced platelet activation. We have demonstrated a coating for metal alloys with multifaceted biocompatibility that resists delamination and is non-thrombogenic, with implications for improving coronary stent efficacy.

Plasma-based ion implantation utilising a cathodic arc plasma

Plasma-based ion implantation (PBII) is usually carried out with isotropic gaseous plasmas, such as a discharge in nitrogen. More recently, it has been applied using drifting plasmas, such as those produced by cathodic arcs, in order to allow efficient implantation of metallic species. The condensable nature of a cathodic arc plasma allows for the deposition of ion-stitched thin film coatings, as well as surface modification by ion implantation. In this paper the promising results for biomaterial fabrication are discussed in light of current limitations of the technique. The use of PBII to control preferred orientation in titanium nitride films is also discussed, together with implications for the physical mechanisms involved in the development of preferred orientations in thin films.

A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture

This paper reports the first use of a linker-free covalent approach for immobilizing an enzyme mixture. Adsorption from a mixture is difficult to control due to varying kinetics of adsorption, variations in the degree of unfolding and competitive binding effects. We show that surface activation by plasma immersion ion implantation (PIII) produces a mildly hydrophilic surface that covalently couples to protein molecules and avoids these issues, allowing the attachment of a uniform monolayer from a cellulase enzyme mixture. Atomic force microscopy (AFM) showed that the surface layer of the physically adsorbed cellulase layer on the mildly hydrophobic surface (without PIII) consisted of aggregated enzymes that changed conformation with incubation time. The evolution observed is consistent with the existence of transient complexes previously postulated to explain the long time constants for competitive displacement effects in adsorption from enzyme mixtures. AFM indicated that the covalently coupled bound layer to the PIII-treated surface consisted of a stable monolayer without enzyme aggregates, and became a double layer at longer incubation times. Light scattering analysis showed no indication of aggregates in the solution at room temperature, which indicates that the surface without PIII-treatment induced enzyme aggregation. A model for the attachment process of a protein mixture that includes the adsorption kinetics for both surfaces is presented.

Influence of gas pressure and cathode composition on ion energy distributions in filtered cathodic vacuum arcs

We report measurements of ion energy distributions of ionized species in titanium and aluminium filtered cathodic vacuum arcs operating in oxygen and nitrogen gas atmospheres. The ion energy distributions were recorded using a Hiden mass selected ion energy analyzer. The results show that a significant reduction in ion energies and a change in the shape of ion energy distributions occurs as the gas pressure is increased. The degree of the energy reduction depends on both the type of gas and the metal ions making up the arc plasma. This has important implications for the deposition of thin films, such as titanium nitride, commonly produced using vacuum arcs in reactive gas atmospheres. The ion energy distributions of the cathode ion species in the absence of background gas and at low gas pressures are well fitted by shifted Maxwellian distributions. As the gas pressure rises the distributions consist of a progressively increasing thermalized Maxwellian component and a decreasing shifted Maxwellian. An inve...

Attachment of horseradish peroxidase to polytetrafluorethylene (teflon) after plasma immersion ion implantation

The aim of this work was to investigate the potential of polytetrafluorethylene (PTFE) as a surface for biologically active protein attachment. A plasma immersion ion implantation (PIII) treatment was applied to PTFE to produce an activated surface for the functional attachment of the enzyme, horseradish peroxidase (HRP). Fourier transform infrared-attenuated total reflectance spectra show oxidation and carbonization of the surface layer as a function of ion fluence. The PIII treatment increases by threefold the amount of attached HRP and the activity of HRP on the modified surface is about seven times higher than that on an untreated PTFE surface. This result indicates that the PIII surface modification improves both the polymers protein binding capacity and its ability to retain the protein in a bioactive state.