Quantitative evaluation of interaction force of fibrinogen at well-defined surfaces with various structures

<div><p>The effects of functional groups and structures at the surface of biomaterials on protein adsorption were examined using direct interaction force measurements. Three kinds of surface structures were evaluated: polymer brushes, self-assembled monolayers with low molecular weight compounds, and surfaces with conventional polymer coatings. These surfaces had various functional groups including phosphorylcholine (PC) group. The surface characterization demonstrated that surface wettability and flexibility depended on both the structure of the surface and the functional groups at the surface. The interactions of protein with these surfaces were evaluated by a force vs. distance curve using an atomic force microscope (AFM). We used fibrinogen as the protein, and the fibrinogen was immobilized on the surface of the AFM cantilever by a conventional technique. It was observed that the interaction force of fibrinogen was strongly related to surface hydrophobic nature and flexibility. That is, the interaction force increased with the increasing hydrophobic nature of the surface. The relationship between the amount of fibrinogen adsorbed on the surface and the interaction force showed good correlation in the range of fibrinogen adsorption from 0 to 250 ng/cm², that is, in a monolayered adsorption region. The interaction force decreased with increasing surface viscoelasticity. The most effective surface for preventing fibrinogen adsorption was the polymer brush surface with phosphorylcholine (PC) groups, that is, poly(2-methacryloyloxyethyl phosphorylcholine) brush. The interaction force of this sample was less than 0.1 nN and the amount of fibrinogen adsorbed on the surface was minimal. It was found that the evaluation of protein adsorption based on the interaction force measurement is useful for low-protein adsorption surfaces. It was demonstrated that an extremely hydrophilic and flexible surface could weaken the protein interactions at the surface, resulting in greater resistance to protein adsorption.</p></div

Quantitative Evaluation of Interaction Force between Functional Groups in Protein and Polymer Brush Surfaces

To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and <i>n</i>-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be a suitable parameter for understanding protein adsorption

Amphiphilic Triblock Phospholipid Copolymers Bearing Phenylboronic Acid Groups for Spontaneous Formation of Hydrogels with Tunable Mechanical Properties

ABA-type triblock copolymers composed of poly­(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly­(glycidyl methacrylate) (PGMA) segments (PGMA-<i>block</i>-PMPC-<i>block</i>-PGMA (PM<i>b</i>G)) were synthesized by activator-regenerated by electron transfer atom transfer radical polymerization method. The PMPC segment provided water-solubility and the PGMA segments could react with nucleophile reagents for chemical functionalization. Phenylboronic acid (PBA) derivatives were bonded to PM<i>b</i>G via epoxide group to obtain PBA-connecting phospholipid polymer (PM<i>b</i>B). The PM<i>b</i>B formed hydrogels in aqueous medium spontaneously when it was mixed with poly­(vinyl alcohol) (PVA) aqueous solution due to reaction between PBA groups in PM<i>b</i>B and hydroxyl groups in PVA. The mechanical properties of PM<i>b</i>B/PVA hydrogel were dependent on the length of PMPC segment and pH of polymer solution. According to control of both chemical structure and mechanical properties of the hydrogel, the PM<i>b</i>B is a good candidate for applying in biomedical fields as polymer matrices for cell encapsulation or protein immobilization

Initial Cell Adhesion onto a Phospholipid Polymer Brush Surface Modified with a Terminal Cell Adhesion Peptide

Dynamic changes in the properties of adsorbed protein layers at material surfaces make it difficult to analyze a cell adhesion behavior. Adhesion is affected by the ligand molecules in the adsorbed protein layers on the material’s surface. This study aimed to quantitatively analyze the initial cell adhesion onto a polymeric surface modified with immobilized cell adhesion molecules with a well-defined structure. Peptides containing an arginine–glycine–aspartic acid (RGD) sequence were introduced at almost all the termini of the grafted poly­(2-methacryloyloxyethyl phosphorylcholine) [poly­(MPC)] chains using a click reaction at a highly protein-resistant poly­(MPC) brush layer. Thus, the surface could bind to the cell membrane proteins only through the immobilized RGD. Furthermore, the degree of polymerization of the grafted poly­(MPC) chains could control the hydrated poly­(MPC) brush layer softness, as determined by measuring the dissipation energy loss using a quartz crystal microbalance. At the initial stage of cell adhesion, the density of cells adhering to the RGD-immobilized poly­(MPC) brush layers did not depend on the poly­(MPC) brush layer softness. However, spreading of the adherent cells was inhibited on the RGD-immobilized poly­(MPC) brush layers with a higher softness. Hence, the results suggested that the layer softness did not affect the binding number between the RGD and cell membrane protein during initial cell adhesion; however, the intracellular signaling triggered by the RGD-receptor interaction was inhibited. The poly­(MPC) brush surface carrying immobilized cell adhesion molecules has the potential to analyze precisely the effect of the properties of cell adhesion molecules on initial cell adhesion

Molecular Interaction Forces Generated during Protein Adsorption to Well-Defined Polymer Brush Surfaces

The molecular interaction forces generated during the adsorption of proteins to surfaces were examined by the force-versus-distance (<i>f</i>–<i>d</i>) curve measurements of atomic force microscopy using probes modified with appropriate molecules. Various substrates with polymer brush layers bearing zwitterionic, cationic, anionic, and hydrophobic groups were systematically prepared by surface-initiated atom transfer radical polymerization. Surface interaction forces on these substrates were analyzed by the <i>f</i>–<i>d</i> curve measurements using probes with the same polymer brush layer as the substrate. Repulsive forces, which decreased depending on the ionic strength, were generated between cationic or anionic polyelectrolyte brush layers; these were considered to be electrostatic interaction forces. A strong adhesive force was detected between hydrophobic polymer brush layers during retraction; this corresponded to the hydrophobic interaction between two hydrophobic polymer layers. In contrast, no significant interaction forces were detected between zwitterionic polymer brush layers. Direct interaction forces between proteins and polymer brush layers were then quantitatively evaluated by the <i>f</i>–<i>d</i> curve measurements using protein-immobilized probes consisting of negatively charged albumin and positively charged lysozyme under physiological conditions. In addition, the amount of protein adsorbed on the polymer brush layer was quantified by surface plasmon resonance measurements. Relatively large amounts of protein adsorbed to the polyelectrolyte brush layers with opposite charges. It was considered that the detachment of the protein after contact with the polymer brush layer hardly occurred due to salt formation at the interface. Both proteins adsorbed significantly on the hydrophobic polymer brush layer, which was due to hydrophobic interactions at the interface. In contrast, the zwitterionic polymer brush layer exhibited no significant interaction force with proteins and suppressed protein adsorption. Taken together, our results suggest that to obtain the protein-repellent surfaces, the surface should not induce direct interaction forces with proteins after contact with them

Effects of molecular architecture of phospholipid polymers on surface modification of segmented polyurethanes

<div><p>To modify the surface properties of segmented polyurethane (SPU), effects of the molecular architecture of the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers on the performance of the SPU/MPC polymer membrane were investigated. We combined the random-type, block-type, and graft-type of the MPC polymers with a typical SPU, Tecoflex^® using double solution casting procedure. The graft-type MPC polymers composed of a poly(MPC) main chain and poly(2-ethylhexyl methacrylate (EHMA)) side chains were synthesized through the combination of two different living radical polymerization techniques to regulate the density and chain length of the side chains. The SPU membranes modified with the MPC polymers were characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The results revealed that the MPC units were located on the SPU surface. Although the breaking strength of the SPU membranes modified with block-type poly(MPC-block-EHMA) and graft-type poly(MPC-graft-EHMA) was lower than that of SPU membranes modified with random-type poly(MPC-random-EHMA), their breaking strengths were adequate for manufacturing medical devices. On the other hand, better stability was observed in the MPC polymer layer on the SPU membrane after immersion in an aqueous medium, wherein the SPU membrane had been modified with the poly(MPC-<i>graft</i>-EHMA). This was because of the intermixing of the hydrophobic poly(EHMA) segments in the domain of the hard segments in the SPU membrane. After this modification, each SPU/MPC polymer membrane showed hydrophilic nature based on the MPC polymers and a dramatic suppression of protein adsorption. From these results, we concluded that the SPU membrane modified with the poly(MPC-<i>graft</i>-EHMA) was one of the promising polymeric biomaterials for making blood-contacting medical devices.</p></div

Durable modification of segmented polyurethane for elastic blood-contacting devices by graft-type 2-methacryloyloxyethyl phosphorylcholine copolymer

<div><p>We propose a novel application of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers for enhancing the performance of modified segmented polyurethane (SPU) surfaces for the development of a small-diameter vascular prosthesis. The SPU membranes were modified by random-type, block-type, and graft-type MPC polymers that were prepared using a double-solution casting procedure on stainless steel substrates. Among these MPC polymers, the graft-type poly(MPC-<i>graft</i>-2-ethylhexyl methacrylate [EHMA]), which is composed of a poly(MPC) segment as the main chain and poly(EHMA) segments as side chains, indicated a higher stability on the SPU membrane after being peeled off from the stainless steel substrate, as well as after immersion in an aqueous medium. This stability was caused by the intermiscibility in the domain of the poly(EHMA) segments and the soft segments of the SPU membrane. Each SPU/MPC polymer membrane exhibited a dramatic suppression of protein adsorption from human plasma and endothelium cell adhesion. Based on these results, the performance of SPU/poly(MPC-<i>graft</i>-EHMA) tubings 2 mm in diameter as vascular prostheses was investigated. Even after blood was passed through the tubings for 2 min, the graft-type MPC polymers effectively protected the blood-contacting surfaces from thrombus formation. In summary, SPU modified by graft-type MPC polymers has the potential for practical application in the form of a non-endothelium, small-diameter vascular prosthesis.</p></div