Surface properties of copolymers of alkyl methacrylates with methoxy (polyethylene oxide) methacrylates and their application as protein-resistant coatings

New polymeric surfactants, copolymers of alkyl methacrylates with methoxy (polyethylene oxide) methacrylates, were synthesized and characterized by gel permeation chromatography. They were studied as possible means to produce polyethylene oxide-rich surfaces by a simple coating treatment on common hydrophobic medical materials. They were further studied as cleaners for the removal of proteins preadsorbed on hydrophobic surfaces. The surface properties of the copolymers such as the adsorption properties of the copolymer on a hydrophobic surface, low density polyethylene, the protein-resistant character of the prepared polyethylene oxide surfaces and the effectiveness of the copolymers for removal of proteins pre-adsorbed on the surface, were investigated by X-ray photoelectron spectroscopy and by using 125I-labelled copolymers and 125I-labelled proteins. The surface properties of the synthesized copolymers were compared with those of commercially available polyethylene oxide containing block copolymer surfactants.     

Protein adsorption on thin films of carbon and carbon nitride monitored with in situ ellipsometry

Thin films of amorphous carbon and amorphous, graphitic and fullerene-like carbon nitride were deposited by reactive magnetron sputtering and optically characterized with spectroscopic ellipsometry. Complementary studies using scanning electron microscopy and atomic force microscopy were performed. The films were exposed to human serum albumin (HSA) and the adsorption was monitored in situ using dynamic ellipsometry. From the ellipsometric data the adsorbed amount of proteins was quantified in terms of surface mass density using de Feijter's model. The results indicate larger adsorption of proteins onto the amorphous films compared to the films with a more textured structure. Complementary studies with 125I-labeled HSA showed an apparent protein adsorption up to six times larger compared to the ellipsometry measurement. In addition, the four types of films were incubated in blood plasma followed by exposure to anti-fibrinogen, anti-HMWK or anti-C3c, revealing the materials' response to complement and contact activation. The amorphous and graphitic carbon nitride exhibit rather high immune activity compared to a titanium reference, whereas the amorphous carbon and the fullerene-like CNx show less immune complement deposition. Compared to the reference, all films exhibit indications of a stronger ability to initiate the intrinsic pathway of coagulation. Finally, the surfaces' bone-bonding ability was investigated by examination of their ability to form calcium phosphate crystals in a simulated body fluid, with a-CNx depositing most calcium phosphate after 21 days of incubation.     

Protein-Resistant Materials via Surface-Initiated Atom Transfer Radical Polymerization of 2-Methacryloyloxyethyl Phosphorylcholine

Poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) was grafted from various polymeric substrates to prepare protein-resistant materials. The poly(MPC) chain length was adjusted via the ratio of monomer to sacrificial initiator in solution. The surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). The protein-resistant properties of the poly(MPC)-grafted surfaces were evaluated by single adsorption experiments with fibrinogen and lysozyme. It was shown that the simple three-step grafting method could be applied to modify various biomaterial surfaces including polyurethane and silicones. The adsorption of fibrinogen and lysozyme to the modified surfaces was greatly reduced compared to the unmodified surfaces, and adsorption decreased with increasing poly(MPC) chain length. On polyurethane film grafted with poly(MPC) of chain length 100, the reduction in adsorption was approx. 96% for lysozyme and approx. 99% for fibrinogen.     

Biocompatible Mn2+-doped carbonated hydroxyapatite thin films grown by pulsed laser deposition

Mn(2+)-doped carbonated hydroxyapatite (Mn-CHA) thin films were obtained by pulsed laser deposition on Ti substrates. The results of the performed complementary diagnostic techniques, X-ray diffraction, infrared spectroscopy, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy investigations indicate that the films are crystalline with a Ca/P ratio of about 1.64-1.66. The optimum conditions, when nearly stoichiometric crystalline thin films were deposited, were found to be 10 Pa oxygen pressure, 400 degrees C substrate temperature, and postdeposition heat treatment in water vapors at the same substrate temperature. The films were seeded with L929 fibroblast and hFOB1.19 osteoblast cells and subjected to in vitro tests. Both fibroblast and osteoblast cells have a good adherence on the Mn-CHA film and on the Ti or polystyrene references. Proliferation and viability tests showed that osteoblast cells growth on Mn-CHA-coated Ti was enhanced as compared to uncoated pure Ti surfaces. Caspase-1 activity was not affected significantly by the material, showing that Mn-CHA does not induce apoptosis of cultured cells. These results demonstrate that Mn-CHA films on Ti should provoke a faster osteointegration of the coated implants as compared to pure Ti. (c) 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 353-358, 2004.     

A novel method to fabricate thermoresponsive microstructures with improved cell attachment/detachment properties

A novel, simple, and rapid method to fabricate thermoresponsive micropatterned substrate for cell adhesion, growth, and thermally induced detachment was developed. Thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), was grafted onto the surface of polystyrene (PS) film with microstructure by plasma-induced graft polymerization technique. The thermoresponsive micropatterned films were characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, hydrogen nuclear magnetic resonance ((1) H NMR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). These results indicated that the grafting ratio of PNIPAAm increased with increasing roughness of PS film. However, the microstructures on the substrate were not affected by grafted PNIPAAm. The optimal grafting conditions, such as plasma treatment time, monomer concentration, graft polymerization time, and graft medium were investigated. The thermoresponsive micropatterned films were investigated with the fibroblast cell (L929) adhesion, proliferation, and thermally induced detachment assay. The microstructure on the thermoresponsive micropatterned substrate facilitated cell adhesion above the lower critical solution temperature (LCST) of PNIPAAm and cell detachment below the LCST. Moreover, it can be used to regulate cell organization and tissue growth.     

Resisting protein adsorption on biodegradable polyester brushes

The protein adsorption and degradation behaviors of poly(lactic acid), poly(glycolic acid) (PGA) and poly(ε-caprolactone) (PCL) brushes and their co-polymer brushes with oligo(ethylene glycol) (OEG) were studied. Both brush structure and relative amount of OEG and polyester were found to be important to the protein resistance of the brushes. A protein-resisting surface can be fabricated either by using OEG as the top layer of a copolymer brush or by increasing the amount of OEG relative to polyester when using a hydroxyl terminated OEG (OEG-OH) and a methoxy terminated OEG (OEG-OMe) mixture as the substrate layer. The degradation of single polyester brushes and their co-polymer brushes using OEG-OH as a substrate layer or using OEG as a top layer was hindered. This phenomenon was rationalized by the inhibition of the proposed back-biting process as the hydroxy end groups of polyester were blocked by OEG molecules. Among these brushes tested, PGA co-polymer brushes using the methoxy/hydroxyl OEG mixture as the substrate layer proved to be both protein-resistant and degradable due to the relatively large amount of OEG moieties and the good biodegradability of PGA.     

Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films: characterization and human smooth muscle cell growth on grafted films.

Graft polymerization of acrylic acid onto plasma treated poly(ethylene terephthalate) (PET) films was carried out to develop surfaces for protein immobilization and smooth muscle cell seeding. Films with various graft densities were characterized by contact angle measurements, attenuated total reflectance infrared spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The contact angle was observed to decrease from 72.9 degrees for the virgin PET films to between 26 degrees and 33 degrees depending on the graft density. Storage of grafted films led to an increase in the contact angle, suggesting molecular rearrangement at the surface. However, films with the lowest graft levels showed maximum enhancement in the contact angle up on storage. XPS confirmed the presence of the polyacrylic acid grafts at the film surface and AFM showed a marked increase in the wavelength of the surface roughness as the graft density increased. The amount of collagen immobilized at the surface of the grafted films also increased as the graft density increased. The collagen immobilized films provided an excellent substrate for the growth of human smooth muscle cells.     

Evanescent-imaging-ellipsometry-based microarray reader

We describe the development of a label-less ellipsometric imaging microarray reader. The ability of the ellipsometric microarray reader to measure binding of sample to microarray surface is verified using oligonucleotide complementary DNA (cDNA) microarrays. Polarized light illuminates the microarray surface through a glass substrate at an angle beyond the critical angle and changes in the polarization of totally internally reflected light resulting from binding events on the microarray surface are measured. This polarization change is used to measure the thickness of biomolecules bound to the microarray. A prototype ellipsometric imaging microarray reader is constructed and calibrated, and the performance is evaluated with cDNA microarrays. The microarray reader measures changes in refractive index changes as small as 0.0024 and thickness changes as small as 0.28 nm. The optimization of angle of incidence and substrate refractive index necessary to achieve high sensitivity is also described. This ellipsometric technique offers an attractive alternative to fluorescence-microarray readers in some genomic, proteomic, diagnostic, and sensing applications.     

Films based on human hair keratin as substrates for cell culture and tissue engineering

Keratin from hair or wool has been proposed as an appropriate material for producing films or cell cultivation scaffolds. The current study was performed to characterize two different approaches involving substrate coating based on keratin from human hair. Our goal was to evaluate cell growth behavior in these systems in comparison with a standard polystyrene substrate. The coating was made in two different ways: (i) by trichloroacetic acid precipitation or (ii) by casting a keratin nanosuspension. The resulting films were characterized using SDS-PAGE, SEM, and X-ray studies. The growth behaviors of twelve cell lines on the keratin films and on polystyrene were estimated using proliferation studies. Furthermore, we assessed the cell detachment behavior during trypsinization and the seeding efficiency. For epithelial cell lines with tight junction proteins, the transepithelial electrical resistance was measured and compared with values achieved using common coating materials. Both of the keratin coatings exhibited similar protein patterns and X-ray diffraction profiles, but we also detected differences in the transparency and ultrastructural surface morphologies. Culture dishes coated with keratin nanoparticles were used to create a transparent substrate that supports cell adherence and improves cell growth as compared with uncoated polystyrene or coatings that use trichloroacetic acid precipitation. We conclude that this coating method may be a new promising substrate for standard cell cultivation.     

Chain-length dependence of the protein and cell resistance of oligo(ethylene glycol)-terminated self-assembled monolayers on gold

Oligo(ethylene glycol) (O-EG(n))-terminated alkanethiol surface-assembled monolayers (SAMs) have been reported to demonstrate protein-resistant properties similar to those of poly(ethylene glycol) (PEG). In this study, we compared the relative protein resistance of short and long ethylene oxide chains, SAMs of PEG 5000, PEG 2000, O-EG(3) (molecular weight = 120), and O-EG(6) (molecular weight = 240), on gold surfaces. Surface plasmon resonance showed that these monolayers were all protein-resistant within the uncertainty of the measurement. However, they exhibited different adhesive properties toward 3T3 mouse fibroblast adhesion in supplemented Dulbecco's modified Eagles medium. The results show that the cell adhesion was sensitive to the concentration of proteins supplemented in the culture medium and to the length of PEG chains.     

Changes in protein adsorption on polycarbonate due to L-ascorbic acid

When a synthetic material is introduced into blood, plasma proteins are rapidly adsorbed on to its surface, followed by the attachment of formed elements of blood, leading to thrombus formation. Previous research⁸ reported that vitamin C (L-ascorbic acid) prolongs the clotting time due to the formation of vitamin C-calcium complexes, which reduce the availability of Ca²⁺ ions for the clotting mechanism in in vitro conditions. Contact angle and platelet adhesion studies have indicated that vitamin C modiRes the surface-protein interaction and surface-platelet binding at the interface. In this paper an increased thickness of protein layer deposited on the polycarbonate substrate has been observed in the presence of vitamin C (˜ 50 Å) using ellipsometric measurements. Further polyacrylamide gel electrophoresis (PAG) and infrared attenuated total reflection spectroscopy (IR-ATR) techniques have provided a better understanding of the interfacial phenomena. It seems, that the adsorption of albumin is increased, relatively, in the presence of vitamin C, when compared with that of fibrinogen and γ-globulin from an equal amount of protein mixture.     

An effective method for quantitative evaluation of proteins adsorbed on biomaterial surfaces

An effective method for the quantitative evaluation of proteins adsorbed on biomaterial surfaces has been developed. First, the kinetic behavior of a range of human fibrinogen (Fib) adsorbed onto polystyrene (PS) films was investigated by using a reflectometry interference spectroscopy setup. The specific molecular number of adsorbed proteins, N(p,) was then defined. According to the definition, the numbers of Fib molecules adsorbed on PS films were calculated. An atomic force microscope (AFM) was used to scan the lateral distribution of the Fib molecules adsorbed on the PS films. From the AFM images, the practical specific molecular numbers were obtained by direct counting of the molecules. In order that the adsorbed number of Fib molecules on a unit area of the PS films could be counted easily, the solution concentration of proteins was reduced to 5 ag/mL (10(-18)g/mL). There was good consistency between the numbers calculated with the formula defined by us and the numbers counted from AFM images. Therefore, the results of the present study prove the validity of our definition of the specific molecular number of adsorbed proteins and the effectiveness of the reflectometry interference spectroscopy-based method for quantitative evaluation of adsorptive proteins.     

Hardness and elastic modulus of TiO2 anodic films measured by instrumented indentation

The aim of this work was to investigate the mechanical properties of the titanium anodic films (TiO2) produced by anodic oxidation under galvanostatic conditions, using a 1.4M phosphoric acid electrolyte, with different current densities (J) on commercially pure titanium (cp-Ti). The morphology of the oxide films were observed by scanning electron microscopy (SEM), whereas the composition of the film was determined by Raman spectroscopy. Porosity, average roughness (Ra) and thickness of the TiO2 films increased with the applied J. Hardness and elastic modulus were measured by instrumented indentation technique, and the influence of the substrate was corrected using analytical models. The anodic films presented higher hardness and lower elastic modulus values compared with the cp-Ti.     

Anti-biofouling and functionalizable bioinspired chitosan-based hydrogel coating via surface photo-immobilization

Zwitterionic polymer is a new generation of anti-fouling materials with its good resistance to protein and bacterial adhesion. Constructing the anti-fouling surfaces with zwitterionic polymer has been regarded as an effective approach for improving the biocompatibility and biofunctionality of clinic devices. Herein, we reported a facile approach to construct a biodegradable anti-biofouling and functionalizable hydrogel coating via photo-immobilization using commercial polyethylene terephthalate (PET) films as the substrate, based on zwitterionic glycidyl methacrylate-phosphorylcholine-chitosan (PCCs-GMA). The surface structure and physicochemical properties of zwitterionic PCCs-GMA hydrogel coating were investigated by X-ray photoelectron spectroscopy (XPS), atomic force microscope (AFM) and static water contact angle measurement, and its functionalizable sites were detected by fluorescence labeling. Compared with the pristine PET and cationic chitosan - GMA and hydroxypropyltrimethyl ammonium chloride chitosan (HTCC) - GMA hydrogel coatings, zwitterionic PCCs-GMA hydrogel coating exhibited excellent biocompatibility, and significantly reduced protein adsorption for three model proteins of fibrinogen, immunoglobulin and lysozyme, repelled platelet adhesion, as well as showed a high resistance to bacterial attachment of Escherichia coli and Staphylococcus aureus and superior anti-fouling properties to MRC-5 cells. The results indicated that photo-immobilized zwitterionic PCCs-GMA hydrogel coating has perspective as a dual functional platform with integrated antifouling and further biofunctional properties for various biomedical applications.     

High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces

Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).     

Protein repellant silicone surfaces by covalent immobilization of poly(ethylene oxide)

Polydimethylsiloxane elastomers were surface modified with passivating polyethylene oxide (PEO) polymers of different molecular weights, both monofunctional and bifunctional. Following the introduction of Si-H groups on the surfaces by acid-catalyzed equilibration in the presence of polymethylhydrosiloxane, the PEO was linked by platinum-catalyzed hydrosilylation. ATR-FTIR, X-ray photoelectron spectroscopy (XPS) and water contact angle results confirmed that the PEO was successfully grafted to the silicone rubber. Atomic force microscopy and XPS suggested that surface coverage with PEO was very high on the modified surfaces but not complete. The protein-resistant properties of the PEO-modified surfaces were demonstrated by measuring the adsorption of fibrinogen from both buffer and plasma. Fibrinogen adsorption from buffer to the PEO-modified surfaces was reduced by more than 90% compared with controls.     

Preparation and characterization of brush-like PEGMA-graft-PDA coating and its application for protein separation by CE

In this paper, a novel brush-like copolymer consisting of poly(ethylene glycol) methyl ether methacrylate and 2-aminoethyl methacrylate (AEMA) named as poly(PEGMA₃₀₀-co-AEMA) was synthesized by atom transfer radical polymerization (ATRP), and then, poly(PEGMA₃₀₀-co-AEMA) copolymer was immobilized onto material surfaces through polydopamine (PDA)-anchored coating. The defined copolymer structure was characterized by nuclear magnetic resonance hydrogen spectroscopy (¹H NMR) and gel permeation chromatography (GPC). The chemical component and surface morphology of the brush-like copolymer-graft-PDA coating were studied by using X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM), respectively. The hydrophilicity of the brush-like copolymer-graft-PDA coating was investigated by using static water contact angle. The protein-resistant property of the brush-like copolymer-graft-PDA coating was investigated by using quartz crystal microbalance with dissipation (QCM-D), and finally the coating was applied to capillary inner surface for protein separation by capillary electrophoresis (CE).     

Novel Wheat Protein Films as Substrates for Tissue Engineering

This paper demonstrates that gliadin-free wheat glutenin can be an excellent biomaterial for tissue-engineering applications, better than poly(lactic acid) (PLA). Although plant proteins are more readily available than collagen and silk, limited studies have been conducted on understanding the potential of using plant proteins as biomaterials. Wheat proteins have not been used for tissue engineering due to the cytotoxicity of gliadin. In this research, wheat gluten, glutenin and gliadin were used to develop films and the mechanical properties, water stability and ability of the films to promote the attachment, growth and viability of osteoblast cells was studied in comparison to PLA films. The wheat protein films have good strength ranging from 8 to 30 MPa. Gliadin films experience about 50% weight loss whereas glutenin films have about 90% weight loss after being in water (pH 7.2) for 15 days at 37°C. Gliadin is cytotoxic and the presence of gliadin restricts the cell proliferation on wheat gluten films. However, gliadin-free glutenin films show a higher rate of proliferation of osteoblast cells than the PLA films. Wheat gluten promises to be a potential substrate for tissue engineering and other medical applications.     

Novel wheat protein films as substrates for tissue engineering

This paper demonstrates that gliadin-free wheat glutenin can be an excellent biomaterial for tissue-engineering applications, better than poly(lactic acid) (PLA). Although plant proteins are more readily available than collagen and silk, limited studies have been conducted on understanding the potential of using plant proteins as biomaterials. Wheat proteins have not been used for tissue engineering due to the cytotoxicity of gliadin. In this research, wheat gluten, glutenin and gliadin were used to develop films and the mechanical properties, water stability and ability of the films to promote the attachment, growth and viability of osteoblast cells was studied in comparison to PLA films. The wheat protein films have good strength ranging from 8 to 30 MPa. Gliadin films experience about 50% weight loss whereas glutenin films have about 90% weight loss after being in water (pH 7.2) for 15 days at 37°C. Gliadin is cytotoxic and the presence of gliadin restricts the cell proliferation on wheat gluten films. However, gliadin-free glutenin films show a higher rate of proliferation of osteoblast cells than the PLA films. Wheat gluten promises to be a potential substrate for tissue engineering and other medical applications.     

Grafting of proteins onto polymer surfaces with the use of oxidized starch.

A study has been carried out on the coupling of proteins onto crosslinked poly(vinyl alcohol) hydrogel membranes and ethylene-vinyl alcohol copolymer(EVA) films previously grafted with oxidized starches having many pendant aldehyde groups. The coupling reaction of proteins is based on the Schiff's base formation between the amino groups of proteins and the aldehyde groups of the oxidized starches which have been grafted onto the substrate membrane or film through acetalization of the aldehyde of starch with hydroxyl groups of the substrate polymers. The grafting of oxidized starches onto the EVA films seems to be restricted to the film surface, since no detectable change is observed in the weight and the attenuated total reflection infrared spectrum of the grafted films. The amount of grafted protein, determined by the ninhydrin method, reveals that, at least, plasma proteins such as serum albumin and fibrinogen are grafted to the film surface in a monomolecular layer without undergoing a marked denaturation. The alpha-amylase grafted onto the EVA film showed a distinct enzymatic activity in hydrolysis of amylose and starch, but the activity was very low compared with that of the ungrafted, soluble alpha-amylase.