Mattias Berglin

Fibrinogen Adsorption and Conformational Change on Model Polymers: Novel Aspects of Mutual Molecular Rearrangement

By combining quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR), the organic mass, water content, and corresponding protein film structure of fibrinogen adsorbed to acrylic polymeric substrates with varying polymer chain flexibility was investigated. Albumin and immunoglobulin G were included as reference proteins. For fibrinogen, the QCM-D model resulted in decreased adsorbed mass with increased polymer chain flexibility. This stands in contrast to the SPR model, in which the adsorbed mass increased with increased polymer chain flexibility. As the QCM-D model includes the hydrodynamically coupled water, we propose that on the nonflexible polymer significant protein conformational change with water incorporation in the protein film takes place. Fibrinogen maintained a more native conformation on the flexible polymer, probably due to polymer chain rearrangement rather than protein conformational change. In comparison with immunoglobulin G and albumin, polymer chain flexibility had only minor impact on adsorbed mass and protein structure. Understanding the adsorption and corresponding conformational change of a protein together with the mutual rearrangement of the polymer chain upon adsorption not only has implications in biomaterial science but could also increase the efficacy of molecular imprinted polymers (MIPs).

Immune complement activation is attenuated by surface nanotopography.

The immune complement (IC) is a cell-free protein cascade system, and the first part of the innate immune system to recognize foreign objects that enter the body. Elevated activation of the system from, for example, biomaterials or medical devices can result in both local and systemic adverse effects and eventually loss of function or rejection of the biomaterial. Here, the researchers have studied the effect of surface nanotopography on the activation of the IC system. By a simple nonlithographic process, gold nanoparticles with an average size of 58 nm were immobilized on a smooth gold substrate, creating surfaces where a nanostructure is introduced without changing the surface chemistry. The activation of the IC on smooth and nanostructured surfaces was viewed with fluorescence microscopy and quantified with quartz crystal microbalance with dissipation monitoring in human serum. Additionally, the ability of pre-adsorbed human immunoglobulin G (IgG) (a potent activator of the IC) to activate the IC after a change in surface hydrophobicity was studied. It was found that the activation of the IC was significantly attenuated on nanostructured surfaces with nearly a 50% reduction, even after pre-adsorption with IgG. An increase in surface hydrophobicity blunted this effect. The possible role of the curvature of the nanoparticles for the orientation of adsorbed IgG molecules, and how this can affect the subsequent activation of the IC, are discussed. The present findings are important for further understanding of how surface nanotopography affects complex protein adsorption, and for the future development of biomaterials and blood-contacting devices.

Gradients in surface nanotopography used to study platelet adhesion and activation

Gradients in surface nanotopography were prepared by adsorbing gold nanoparticles on smooth gold substrates using diffusion technique. Following a sintering procedure the particle binding chemistry was removed, and integration of the particles into the underlying gold substrate was achieved, leaving a nanostructured surface with uniform surface chemistry. After pre-adsorption of human fibrinogen, the effect of surface nanotopography on platelets was studied. The use of a gradient in nanotopography allowed for platelet adhesion and activation to be studied as a function of nanoparticle coverage on one single substrate. A peak in platelet adhesion was found at 23% nanoparticle surface coverage. The highest number of activated platelets was found on the smooth control part of the surface, and did not coincide with the number of adhered platelets. Activation correlated inversely with particle coverage, hence the lowest fraction of activated platelets was found at high particle coverage. Hydrophobization of the gradient surface lowered the total number of adhering cells, but not the ratio of activated cells. Little or no effect was seen on gradients with 36nm particles, suggesting the existence of a lower limit for sensing of surface nano-roughness in platelets. These results demonstrate that parameters such as ratio between size and inter-particle distance can be more relevant for cell response than wettability on nanostructured surfaces. The minor effect of hydrophobicity, the generally reduced activation on nanostructured surfaces and the presence of a cut-off in activation of human platelets as a function of nanoparticle size could have implications for the design of future blood-contacting biomaterials.

Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings

Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization by barnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leaching rate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7 ng cm−2 day−1. This low leaching rate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to the matrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersed in the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the mean barnacle base plate area on the controls was 13 mm2, while on the ivermectin coating it was 3 mm2. After 388 days, no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a mean of 60 mm2. In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycol and glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacle performance of the coatings. An increased release rate (3 ng cm−2 day−1) and dispersion of ivermectin, determined by fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixture in the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in further studies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxication occurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for the mode of action and this is under investigation.

The Interaction Between Model Biomaterial Coatings and Nylon Microparticles as Measured with a Quartz Crystal Microbalance with Dissipation Monitoring

The interaction between cells and biomaterials has been mimicked using nylon microparticles as pseudo-cells and PLMA and PIBMA as biomaterial model acrylate polymers. The shift of fundamental resonance frequencies was negative for both polymers, indicating mass-coupling to the sensor surface. The shifts of the 3rd, 5th and 7th overtone frequencies were initially positive for both polymers, indicating a particle slip or wobbling on the surface. The QCM technique could discriminate between the two different polymers, showing increased interaction between microparticle and PLMA. The dissipation shift was positive for all overtones on both polymers, but again with faster and more prominent response for PLMA.

Use of Surface-Sensitive Methods for the Study of Adsorption and Cross-Linking of Marine Bioadhesives

ABSTRACT The establishment of the bond of sessile marine organisms such as barnacles, mussels, and algae in the marine environment starts with the secretion and the adsorption of the adhesive biopolymers to the substrate. Subsequently, this is followed by the formation of cohesive interactions with the next layer of adhesive biopolymers that are deposited/adsorbed on top of the first layer. These two fundamental processes for the adhesive plaque buildup have been subjected to several investigations in recent years using model molecules, especially Mefp-1 extracted from the blue mussel Mytilus edulis. With the introduction of optical surface-sensitive methods such as ellipsometry, surface plasmon resonance (SPR), and infrared spectroscopy (IR), it has been possible to elucidate both the kinetics of adsorption and structure of the Mefp-1 film. In contrast to adsorption, the cohesive interactions or the cross-linking are not easily followed with these optical methods and new approaches and techniques are required. One such technique that has been useful is the quartz-crystal microbalance with dissipation monitoring (QCM-D), which has been used for cross-linking studies of a variety of biopolymers including bioadhesives from mussel and algae. One of a collection of papers honoring Manoj K. Chaudhury, the February 2005 recipient of The Adhesion Society Award for Excellence in Adhesion Science, sponsored by 3M.

Templating gold surfaces with function: a self-assembled dendritic monolayer methodology based on monodisperse polyester scaffolds.

The antibiotic resistance developed among several pathogenic bacterial strains has spurred interest in understanding bacterial adhesion down to a molecular level. Consequently, analytical methods that rely on bioactive and multivalent sensor surfaces are sought to detect and suppress infections. To deliver functional sensor surfaces with an optimized degree of molecular packaging, we explore a library of compact and monodisperse dendritic scaffolds based on the nontoxic 2,2-bis(methylol)propionic acid (bis-MPA). A self-assembled dendritic monolayer (SADM) methodology to gold surfaces capitalizes on the design of aqueous soluble dendritic structures that bear sulfur-containing core functionalities. The nature of sulfur (either disulfide or thiol), the size of the dendritic framework (generation 1-3), the distance between the sulfur and the dendritic wedge (4 or 14 Å), and the type of functional end group (hydroxyl or mannose) were key structural elements that were identified to affect the packaging densities assembled on the surfaces. Both surface plasmon resonance (SPR) and resonance-enhanced surface impedance (RESI) experiments revealed rapid formation of homogenously covered SADMs on gold surfaces. The array of dendritic structures enabled the fabrication of functional gold surfaces displaying molecular covering densities of 0.33-2.2 molecules·nm(-2) and functional availability of 0.95-5.5 groups·nm(-2). The cell scavenging ability of these sensor surfaces for Escherichia coli MS7fim+ bacteria revealed 2.5 times enhanced recognition for G3-mannosylated surfaces when compared to G3-hydroxylated SADM surfaces. This promising methodology delivers functional gold sensor surfaces and represents a facile route for probing surface interactions between multivalently presented motifs and cells in a controlled surface setting.

Change of Colloidal and Surface Properties of Mytilus edulis Foot Protein 1 in the Presence of an Oxidation (NaIO4) or a Complex-Binding (Cu2+) Agent

Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to study the viscoelastic properties of the blue mussel, Mytilus edulis, foot protein 1 (Mefp-1) adsorbed on modified hydrophobic gold surfaces. The change in viscoelasticity was studied after addition of Cu2+ and Mn2+, which theoretically could induce metal complex formation with 3,4-dihydroxyphenylalanine (DOPA) moieties. We also used NaIO4, a nonmetal oxidative agent known to induce di-DOPA formation. Reduction in viscoelasticity of adsorbed Mefp-1 followed the order of NaIO4 > Cu2+ > buffer control > Mn2+. We also studied the formation of molecular aggregates of Mefp-1 in solution with the use of dynamic light scattering (DLS). We found that addition of Cu2+, but not Mn2+, induced the formation of larger DLS-detectable aggregates. Minor aggregate formation was found with NaIO4. With the analytical resolution of small angle X-ray scattering (SAXS), we could detect differences in the molecular structure between NaIO4- and Cu2+-treated Mefp-1 aggregates. We concluded from this study that Cu2+ could participate in intermolecular cross-linking of the Mefp-1 molecule via metal complex formation. Metal incorporation in the protein most likely increases the abrasion resistance of the Mefp-1 layer. NaIO4, on the other hand, resulted in mainly intramolecular formation of di-DOPA, but failed to induce larger intermolecular aggregation phenomena. The described methodological combination of surface sensitive methods, like QCM-D, and bulk sensitive methods, like DLS and SAXS, generates high resolution results and is an attractive platform to investigate intra- and intermolecular aspects of assembly and cross-linking of the Mefp proteins.

Self-Assembled Arrays of Dendrimer–Gold-Nanoparticle Hybrids for Functional Cell Studies†

Engineered surfaces with nanoscale features of gold on silicon or glass have recently been used to improve the understanding of adhesion-mediated environmental sensing of cells. Often such surfaces present a cell-binding ligand, such as arginine–glycine–aspartic acid (RGD) peptide motifs, at controlled intramolecular distances on an inert background surface such as polyethylene glycol (PEG). The adhesion mechanism of macromolecular ligands in which direct interaction with cells is nonspecific is not known and the cell response is dictated by the type and the concentration of proteins adsorbed from solution. Dendrimers may increase the availability and multivalency of cell-interacting ligands as a consequence of their branched shape and inherently high concentration of end groups. It is therefore interesting to examine the eventual effect of the macromolecular architecture on the cell viability by the controlled reduction of ligands on a surface. Herein, we demonstrate the fabrication of selfassembledmacromolecular hybrid arrays in which the relative position of two anionic macromolecules of different architectures—a carboxy-functionalized dendrimer and a linear polymer—is straightforwardly controlled on a PEG surface. We also show how the interaction of primary human endothelial cells with these surfaces is modulated by the molecular spacing and how protein binding to the macromolecular arrays can be evaluated by using a standard surface plasmon resonance (SPR) technique. Self-assembled, short-range-ordered Au nanoparticle (NP) arrays were used as a versatile template to arrange polymeric entities at the nanometer scale (Figure 1a). This

Investigation of Adsorption and Cross-Linking of a Mussel Adhesive Protein Using Attenuated Total Internal Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR)

Mytilus edulis foot protein 1 (Mefp-1) contains the redox-functional amino acid 3,4-dihydroxyphenylalanine (DOPA), which is a typical feature of most mefp proteins. We have previously shown, using combined optic (ellipsometry) and acoustic (QCM-D) measurements, that the oxidizing agent sodium periodate (NaIO4) and the transition metal ion Cu2+ promote cross-linking of Mefp-1. However, different chemical reaction mechanisms can not be distinguished using these methods. In the present study, we have complemented our previous investigations using Attenuated Total Internal Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR), allowing a spectroscopic analysis of NaIO4 and Cu2+-induced cross-linking of Mefp-1 adsorbed on a ZnSe surface. In aqueous solution, adsorbed Mefp-1 displays absorption bands at 1570, 1472, 1260, and 973 cm−1. Upon addition of NaIO4 and Cu2+, the absorptions at 1570, 1472, and 973 cm−1 increase by approximately a factor of two. In contrast, the band at 1260 cm−1 disappears upon cross-linking using NaIO4, but remains unchanged upon addition of Cu2+. This demonstrates that the band at 1260 cm−1 is attributed to the C‒O stretching vibration of the side chain hydroxyl groups in DOPA and that Cu2+ forms complexes with DOPA rather than transform it into an o-quinone. Moreover, upon addition of NaIO4 after cross-linking using Cu2+, the band at 1260 cm−1 disappears, indicating that the complex formation between DOPA and Cu2+ is reversed when DOPA is transformed into the o-quinone. These results demonstrate that NaIO4, which initiates a similar reaction to the naturally occurring enzyme catechol oxidase, contributes to the formation of di-DOPA cross-links. In contrast, the dominating contribution to the cross-linking from Cu2+, which is accumulated at high concentrations in the byssus thread of the blue mussel, is via complex formation between the metal and DOPA residues.