Fibrinogen Adsorption Research Articles

Hemocompatible interface control via thermal-activated bio-inspired surface PEGylation

ABSTRACTA facile thermal-activated self-copolymerization coating of antifouling layers was designed. PEGylated poly(ethyleneimine) (PEI-PEGMA) was first synthesized, mixed with an alkaline dopamine solution, and then concurrently codeposited onto versatile substrates of both organic and inorganic materials. Thermal-activated codeposition of dopamine/PEI-PEGMA could then proceed, giving rise to good hemocompatible performance in respect to low fibrinogen adsorption, ultra-low blood cell adhesion and activation, and almost undetectable hemolytic activities. This work also revealed that the hemocompatible performance was contributed by PEGylated PEI but not dopamine, as the poly(dopamine)-coating was shown to induce a high level of fibrinogen adsorption as well as vigorous platelet activation.

Antifouling PVDF membrane prepared by VIPS for microalgae harvesting

Provided the use of a water-insoluble modifier, in-situ modification of PVDF membranes is an ideal method for preparing low-biofouling membranes. We report the formation, characterization and low-biofouling performances of modified PVDF membranes prepared by vapor-induced phase separation for microalgae harvesting. Poly(styrene)-b-poly(ethylene glycol) methacrylate (PS-b-PEGMA) is used as antifouling material. After characterizing the physico-chemical properties of membranes by SEM, AFM, FT-IR, XPS, and tensile tester, their hydrophilicity was assessed. Hydration capability was importantly enhanced with copolymer content. Adsorption of bovine serum albumin (BSA), lysozyme (LY) and fibrinogen (FN) was tested to investigate the resistance of membranes to nano-biofouling. Best results were obtained with membrane prepared from a casting solution containing 4wt% copolymer (PS-b-PEGMA-4). Bacterial attachment tests proved that membranes could also resist micro-biofouling. Flux recovery ratio after filtration of BSA with PS-b-PEGMA-4 membrane was higher than with a commercial hydrophilic PVDF membrane. Applied in microalgae harvesting, it was found that membranes could efficiently resist biofouling by microalgae (FRR=76.9% with PS-b-PEGMA-4), still enabling a rejection ratio over 99.7%.

ChemInform Abstract: Mechanisms of Fibrinogen Adsorption at Solid Substrates

AbstractReview: 100 refs.

Fibrinogen adsorption mechanisms at the gold substrate revealed by QCM-D measurements and RSA modeling

Adsorption kinetics of fibrinogen at a gold substrate at various pHs was thoroughly studied using the QCM-D method. The experimental were interpreted in terms of theoretical calculations performed according to the random sequential adsorption model (RSA). In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated at various pHs. It was revealed that for the lower range of fibrinogen coverage the hydration function were considerably lower than previously obtained for the silica sensor [33]. The lower hydration of fibrinogen monolayers on the gold sensor was attributed to its higher roughness. However, for higher fibrinogen coverage the hydration functions for both sensors became identical exhibiting an universal behavior. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γd vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.6mgm−2 at pH 3.5 and 4.5mgm−2 at pH 7.4 (for ionic strength of 0.15M). These results agree with theoretical eRSA modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data. These results allow one to develop a method for preparing fibrinogen monolayers of well-controlled coverage and molecule orientation.

Controllable Nonspecific Protein Adsorption by Charged Hyperbranched Polyglycerol Thin Films

Antifouling thin films derived from charged hyperbranched polyglycerol (hbPG) layers were fabricated and evaluated. The anionic hbPG (a-hbPG) monolayers and cationic hbPG/anionic hbPG (c/a-hbPG) bilayers were adsorbed on the underlying self-assembled monolayers (SAMs) of cysteamine and 3-mercaptopropionic acid (3-MPA) by electrostatic interaction, respectively, and their procession was monitored by surface plasmon resonance spectroscopy (SPR). The adsorption of bovine serum albumin (BSA) and fibrinogen on the premade a-hbPG and c/a-hbPG thin films was measured and the capability of these thin films to resist nonspecific protein adsorption was evaluated by SPR as well. It is observed that the c/a-hbPG bilayer films possessed good antifouling properties. With c/a-hbPG bilayers consisting of higher molecular weight a-hbPG, the adsorption of BSA and fibrinogen were as low as 0.015 ng/mm(-2) and 0.0076 ng/mm(-2), respectively, comparable to the traditionally ultralow antifouling surfaces (<0.05 ng/mm(-2) of nonspecific protein adsorption). This work proved that the charged hbPG thin films can strongly reduce the nonspecific protein adsorption and have the promise for the antifouling coatings with improved performance.

Free hemoglobin increases von Willebrand factor–mediated platelet adhesion in vitro: implications for circulatory devices

AbstractIntravascular hemolysis occurs in patients on extracorporeal membrane oxygenation. High levels of free acellular adult hemoglobin (free HbA) are associated with clotting in this mechanical device that can result in thrombotic complications. Adsorption of fibrinogen onto the surface of biomaterial correlates with platelet adhesion, which is mediated by von Willebrand factor (VWF). Because free Hb interacts with VWF, we studied the effect of hemoglobin (Hb) on platelet adhesion to fibrin(ogen) under conditions of different hydrodynamic forces. This effect was investigated using purified human HbA and fibrinogen, extracellular matrix, collagen, or purified plasma VWF as surface-coated substrates to examine flow-dependent platelet adhesion. Antibodies and VWF-deficient plasma were also used. Free Hb (≥50 mg/dL) effectively augmented platelet adhesion, and microthrombi formation on fibrin(ogen), extracellular matrix, and collagen at high shear stress. The effect of free Hb was effectively blocked by anti-glycoprotein Ibα (GPIbα) antibodies or depletion of VWF. Unexpectedly, free Hb also promoted firm platelet adhesion and stable microthrombi on VWF. Lastly, we determined that Hb interacts directly with the A1 domain. This study is the first to demonstrate that extracellular Hb directly affects the GPIbα-VWF interaction in thrombosis, and describes another mechanism by which hemolysis is connected to thrombotic events.

Impact of starch content on protein adsorption characteristics in amphiphilic hybrid graft copolymers

Amphiphilic hybrid graft copolymers were synthesized using a graft-to methodology and their protein adsorption profiles studied. Three different hydrophilic side chains were studied: hydroxypropylated high amylose starch, maltodextrin, and polyethylene glycol (PEG). In the high amylose starch compositions, there was a pronounced decrease in protein adsorption with increasing polysaccharide content. As the starch content in the graft copolymers increased from 10wt% to 53wt%, BSA protein adsorption decreased by 83% whereas fibrinogen adsorption was reduced by 40%. Comparisons between the starch-containing hybrid polymers and their respective hydrophobic urethane-linked polyesters were also made. Hybrid 53, containing 53wt% starch, showed a 85% reduction in BSA adsorption and 51% reduction in fibrinogen relative to their urethane-linked polyester backbone controls. Grafting branched high amylopectin-derived maltodextrin to the synthetic polymer backbones also conferred modest protein resistance to the hydrophobic backbone polymer. Lastly, it was found that a high amylose graft structure provided comparable, if not slightly more effective, protein resistance compared to a similarly constructed PEG-containing amphiphilic copolymer.

Interfacial Self-Assembly of Heparin-Mimetic Multilayer on Membrane Substrate as Effective Antithrombotic, Endothelialization, and Antibacterial Coating.

In this study, we design the interfacial self-assembly of heparin-mimetic multilayer on poly(ether sulfone) (PES) membrane, which can endow the substrate with excellent cytocompatibility, highly hemocompatibility and enhanced antibacterial properties. The coated 3D sponge-like multilayer was fabricated by surface engineered layer by layer assembly of sulfonic amino polyether sulfone (SNPES) and quaternized chitosan (QC). The cell morphology observation and viability evaluation suggested that the assembled multilayer coating had remarkable cytocompatibility with endothelial cells due to the synergistic promotion of bovine serum albumin adsorption and heparin-mimetic groups; which further indicated that surface endothelialization could be achieved on the heparin-mimetic multilayer. The systematical tests of antithrombotic and blood activation indicated that the heparin-mimetic multilayer-coated membrane owned significantly suppressed adsorption of bovine serum fibrinogen, platelet adhesion and activation, prolonged clotting times, as well as lower activation of blood complement. Furthermore, the antibacterial test suggested the multilayer coated substrates exhibited obvious inhibition capability for both Escherichia coli and Staphylococcus aureus. Therefore, we believe that the developed SNPES/QC multilayer on PES membrane show great potential as a multifunctional coating toward versatile biomedical applications due to the integrated and highly effective antithrombotic, endothelialization, and antibacterial properties.

Deposition of fibrinogen on the surface of in vitro thrombi prevents platelet adhesion

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion.

Structural Reorganization and Fibrinogen Adsorption Behaviors on the Polyrotaxane Surfaces Investigated by Sum Frequency Generation Spectroscopy.

Polyrotaxanes, such as supramolecular assemblies with methylated α-cyclodextrins (α-CDs) as host molecules noncovalently threaded on the linear polymer backbone, are promising materials for biomedical applications because they allow adsorbed proteins possessing a high surface flexibility as well as control of the cellular morphology and adhesion. To provide a general design principle for biomedical materials, we examined the surface reorganization behaviors and adsorption conformations of fibrinogen on the polyrotaxane surfaces with comparison to several random copolymers by sum frequency generation (SFG) vibrational spectroscopy. We showed that the polyrotaxane (OMe-PRX-PMB) with methylated α-CDs as the host molecule exhibited unique surface structures in an aqueous environment. The hydrophobic interaction between the methoxy groups of the methylated α-CD molecules and methyl groups of the n-butyl methacrylate (BMA) side chains may dominate the surface restructuring behavior of the OMe-PRX-PMB. The orientation analysis revealed that the orientation of the fibrinogen adsorbed on the OMe-PRX-PMB surface is close to a single distribution, which is different from the adsorption behaviors of fibrinogen on other polyrotaxane or random copolymer surfaces.

New dextran coated activated carbons for medical use

Activated carbon (AC) can be used for blood purification (hemoperfusion) to treat a range of conditions by direct perfusion of blood through an adsorbent column. The clinical effects of hemoperfusion relate to the AC adsorptive capacity, and any inflammatory response generated by direct blood contact. The use of a biocompatible coating minimizes side-effects but at the same time significantly reduces AC adsorption capacity in particular towards substances with large molecular mass. We investigated the adsorption capacity for biological molecules in a wide range of molecular masses and biocompatibility of a dextran (Dx) coated mesoporous AC (HSGD) produced from a polymer precursor. The hemocompatibility of the Dx-coated beads was investigated according to the ISO guidelines. Coating the HSGD with a Dx coating (5–30% weight of AC) decreased the specific surface area and pore volume of the materials and reduced adsorption capacity for methylene blue, vitamin B12, bilirubin and albumin, while minimizing fines production. Even the partially coated materials did not activate the inflammatory response and thus could be considered hemocompatible, with the Dx coating reducing fibrinogen adsorption and complement generation, suggesting the material warrants continued development for hemoperfusion applications.

Two surface gradients of polyethylene glycol for a reduction in protein adsorption.

Polyethylene glycol (PEG) coatings have been commonly used in reducing protein adsorption with the intent of improving a biomaterial's biocompatibility. To elucidate the role of PEG surface density in reducing protein adsorption, two types of grafted PEG surface density gradients were evaluated for the adsorption and desorption of albumin and fibrinogen, two blood proteins. PEG density gradients were characterized using contact angle measurements and X-ray photoelectron spectroscopy. Total internal reflection fluorescence was used to measure protein adsorption kinetics and adsorption profiles on the two types of PEG gradients. The PEG gradient generated by the flow method decreased adsorption of both proteins in proportion to the PEG surface density; however, their desorption by buffer solution from the grafted PEG layer was not complete. In contrast, desorption of two proteins from the grafted PEG layer generated by a UV oxidation method resulted in near-zero adsorbed amount. The difference between the two types of gradients might have originated from counter-diffusion of PEG and water molecules occurring during the flow method procedure.

PEG Functionalization of Whispering Gallery Mode Optical Microresonator Biosensors to Minimize Non-Specific Adsorption during Targeted, Label-Free Sensing.

Whispering Gallery Mode (WGM) optical microresonator biosensors are a powerful tool for targeted detection of analytes at extremely low concentrations. However, in complex environments, non-specific adsorption can significantly reduce their signal to noise ratio, limiting their accuracy. To overcome this, poly(ethylene glycol) (PEG) can be employed in conjunction with appropriate recognition elements to create a nonfouling surface capable of detecting targeted analytes. This paper investigates a general route for the addition of nonfouling elements to WGM optical biosensors to reduce non-specific adsorption, while also retaining high sensitivity. We use the avidin-biotin analyte-recognition element system, in conjunction with PEG nonfouling elements, as a proof-of-concept, and explore the extent of non-specific adsorption of lysozyme and fibrinogen at multiple concentrations, as well as the ability to detect avidin in a concentration-dependent fashion. Ellipsometry, contact angle measurement, fluorescence microscopy, and optical resonator characterization methods were used to study non-specific adsorption, the quality of the functionalized surface, and the biosensor’s performance. Using a recognition element ratio to nonfouling element ratio of 1:1, we showed that non-specific adsorption could be significantly reduced over the controls, and that high sensitivity could be maintained. Due to the frequent use of biotin-avidin-biotin sandwich complexes in functionalizing sensor surfaces with biotin-labeled recognition elements, this chemistry could provide a common basis for creating a non-fouling surface capable of targeted detection. This should improve the ability of WGM optical biosensors to operate in complex environments, extending their application towards real-world detection.

Modification of fluorous substrates with oligo(ethylene glycol) via “click” chemistry for long-term resistance of cell adhesion

In this work perfluorinated substrates fabricated from SiO2 glass slides are modified with oligo(ethylene glycol) (OEG) units for long-term resistance of cell adhesion purposes, based on fluorous interactions and click chemistry. Specifically, fluorous substrates, prepared by treatment of glass slides with 1H, 1H, 2H, 2H-perfluorodecyltrimethoxysilane (FAS17), were coated with ethynyl-OEG-C8F17, followed by covalent attachment of an azido-OEG via copper-catalyzed azide-alkyne cycloaddition (CuAAC) “click” reaction. We demonstrate that the resultant surface avoid fibrinogen adsorption and resisted cell adhesion for over 14days. X-ray photoemission spectroscopy (XPS) analysis and contact angle goniometry measurements confirm the presence of the OEG molecules on the fluorous substrates. Bright field optical images show total absence of 3T3 fibroblast cells on the OEG modified fluorinated substrate for 1 and 5days, and a remarkably decrease of cell adhesion at 14days.

Fibrinogen matrix deposited on the surface of biomaterials acts as a natural anti-adhesive coating

Adsorption of fibrinogen on the luminal surface of biomaterials is a critical early event during the interaction of blood with implanted vascular graft prostheses which determines their thrombogenicity. We have recently identified a nanoscale process by which fibrinogen modifies the adhesive properties of various surfaces for platelets and leukocytes. In particular, adsorption of fibrinogen at low density promotes cell adhesion while its adsorption at high density results in the formation of an extensible multilayer matrix, which dramatically reduces cell adhesion. It remains unknown whether deposition of fibrinogen on the surface of vascular graft materials produces this anti-adhesive effect. Using atomic force spectroscopy, single cell force spectroscopy, and standard adhesion assays with platelets and leukocytes, we have characterized the adhesive and physical properties of the contemporary biomaterials, before and after coating with fibrinogen. We found that uncoated PET, PTFE and ePTFE exhibited high adhesion forces developed between the AFM tip or cells and the surfaces. Adsorption of fibrinogen at the increasing concentrations progressively reduced adhesion forces, and at ≥2 μg/ml all surfaces were virtually nonadhesive. Standard adhesion assays performed with platelets and leukocytes confirmed this dependence. These results provide a better understanding of the molecular events underlying thrombogenicity of vascular grafts.

Mechanisms of fibrinogen adsorption at the silica substrate determined by QCM-D measurements

Adsorption kinetics of fibrinogen at a silica substrate was thoroughly studied in situ using the QCM-D method. Because of low dissipation, the Sauerbrey’s equation was used for calculating the wet mass per unit area (wet coverage of the protein). Measurements were done for various bulk suspension concentrations, flow rates and pHs. These experimental data were compared with the theoretical dry coverage data derived from the solution of the mass transfer equation. In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated for various pHs. In the case of pH 7.4 and ionic strength of 0.15M, the hydration function changed from 0.75 to 0.6 for the dry coverage Γd equal to 0 and 4mgm−2, respectively. Interestingly, for pH 7.4 and 4.5 (ionic strength of 10−2M) a minimum of the hydration function appeared at Γd ca. 2mgm−2. Analytical polynomial expressions were formulated for the interpolation of the experimental results. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γd vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.2mgm−2 at pH 3.5 and 4.2mgm−2 at pH 7.4 for ionic strength of 0.15M. These results agree with theoretical modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data whose validity was also confirmed by the dissipation vs. the dry mass relationships. Beside significance to basic science, these results enable to develop a robust technique, based on the QCM-D measurements, suitable for precisely determining the dry mass of protein monolayers adsorbed under various physicochemical conditions.

Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake.

Here we have investigated the effect of enshrouding polymer-coated nanoparticles (NPs) with polyethylene glycol (PEG) on the adsorption of proteins and uptake by cultured cells. PEG was covalently linked to the polymer surface to the maximal grafting density achievable under our experimental conditions. Changes in the effective hydrodynamic radius of the NPs upon adsorption of human serum albumin (HSA) and fibrinogen (FIB) were measured in situ using fluorescence correlation spectroscopy. For NPs without a PEG shell, a thickness increase of around 3 nm, corresponding to HSA monolayer adsorption, was measured at high HSA concentration. Only 50% of this value was found for NPs with PEGylated surfaces. While the size increase clearly reveals formation of a protein corona also for PEGylated NPs, fluorescence lifetime measurements and quenching experiments suggest that the adsorbed HSA molecules are buried within the PEG shell. For FIB adsorption onto PEGylated NPs, even less change in NP diameter was observed. In vitro uptake of the NPs by 3T3 fibroblasts was reduced to around 10% upon PEGylation with PEG chains of 10 kDa. Thus, even though the PEG coatings did not completely prevent protein adsorption, the PEGylated NPs still displayed a pronounced reduction of cellular uptake with respect to bare NPs, which is to be expected if the adsorbed proteins are not exposed on the NP surface.

Influence of Surface Charge/Potential of a Gold Electrode on the Adsorptive/Desorptive Behaviour of Fibrinogen

The effect of gold substrate surface charge (potential) on adsorptive/desorptive behaviour of fibrinogen (FG) was studied by employing differential capacitance (DC) and polarization modulated infrared reflection absorption spectroscopy (PM-IRRAS), in terms of FG adsorption thermodynamics, kinetics, and desorption kinetics. The gold substrate surface charge was modulated in-situ within the electrochemical double-layer region by means of electrochemical potentiostatic polarization in a FG-containing electrolyte, thus avoiding the interference of other physico-chemical properties of the gold surface on FG’s interfacial behaviour. The FG adsorption equilibrium was modeled using the Langmuir isotherm. Highly negative values of apparent Gibbs free energy of adsorption (ranging from from −52.1±0.4 to −55.8±0.8kJmol−1, depending on the FG adsorption potential) indicated a highly spontaneous and strong adsorption of FG onto the gold surface. The apparent Gibbs free energy of adsorption was found to be independent of surface charge when the surface was negatively charged. However, when the gold surface was positively charged, the apparent Gibbs free energy of adsorption exhibited a pronounced linear relationship with the surface charge, shifting to more negative values with an increase in positive electrode potential. The adsorption kinetics of FG was also found to be dependent on gold surface charge in a similar manner to the apparent Gibbs free energy of adsorption.It was suggested that the driving force for the adsorption of FG on a negatively charged surface represents a positive gain in the entropy of the system, whereas the adsorption on a positively charged gold surface was found to be controlled by electrostatic forces.FG desorption measurements revealed that when the gold surface is polarized within the electrochemical double-layer region during the desorption process, the protein desorption kinetics is rather slow. However, within the regions of hydrogen and oxygen evolution, the FG desorption kinetics accelerates significantly, due to the physical removal of the adsorbed protein layer by gas bubbles evolving from the substrate surface, which enables a complete removal of the pre-adsorbed FG layer. The latter could potentially be employed for electrochemical cleaning of electrically-conducting surfaces fouled by adsorbed protein layers (heat exchangers, filtration membranes, etc.).

Fast and easily applicable glycerol-based spray coating

This work describes the fabrication and evaluation of a transparent hydrogel based spray coating to reduce marine biofouling on glass surfaces. A glycerol based copolymer was synthesized and covalently immobilized by applying a simple spray coating procedure. To test its nonfouling behavior, modified glass surfaces were exposed to different marine fouling species including bacteria, green algae, and blue mussels. For all tested species the coating could considerably reduce the settlement as compared to pristine glass surfaces. The settlement of blue mussels on coated surfaces was additionally compared to polytetrafluoroethylene (PTFE) substrates. The glycerol based copolymer showed an even better resistance against blue mussel adhesion than PTFE. Furthermore, the nonfouling performance of the coating was tested via fibrinogen adsorption after aging coated silica slides under marine conditions. The major aim of this study is to provide an easy synthesis and application procedure for a polyglycerol based nonfouling coating and the evaluation of its nonfouling properties in marine environments.

Evaluation of Factors To Determine Platelet Compatibility by Using Self-Assembled Monolayers with a Chemical Gradient.

Intercorrelation among surface chemical composition, packing structure of molecules, water contact angles, amounts and structures of adsorbed proteins, and blood compatibility was systematically investigated with self-assembled monolayers (SAMs) with continuous chemical composition gradients. The SAMs were mixtures of two thiols: n-hexanethiol (hydrophobic and protein-adsorbing) and hydroxyl-tri(ethylene glycol)-terminated alkanethiol (hydrophilic and protein-resistant) with continuously changing mixing ratios. From the systematic analyses, we found that protein adsorption is governed both by sizes of proteins and hydrophobic domains of the substrate. Furthermore, we found a clear correlation between adsorption of fibrinogen and adhesion of platelets. Combined with the results of surface force measurements, we found that the interfacial behavior of water molecules is profoundly correlated with protein resistance and antiplatelet adhesion. On the basis of these results, we conclude that the structuring of water at the SAM-water interface is a critical factor in this context.