Photo-reactive polyvinylalcohol for photo-immobilized microarray

A new photo-reactive polymer, polyvinylalcohol modified with phenylazido groups, was synthesized as a microarray matrix. The polymer is soluble in water and spin-coated onto glass plate. Aqueous solutions of proteins were micro-spotted onto the coated glass and were fixed by ultraviolet light irradiation. Subsequently, cell adhesion on the photo-immobilized protein microarray was investigated. Non-specific adhesion of cells onto non-protein-spotted regions was reduced in comparison with the previously prepared microarray chip (Biomaterials 24 (2003) 3021). The adhesion behavior of cells depended on the kind of immobilized proteins and the type of cells. The microarray will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.     

Photo-immobilization of a phospholipid polymer for surface modification

A photo-reactive polymer having a phospholipid polar group was prepared, and the polymer was photo-immobilized on polymeric surfaces, where its interactions with biocomponents were investigated. By using a photo-immobilization method, the polymer was used for surface modification of polyethylene and polypropylene, polymers whose surfaces were not treated in our previous development of the phosphorylcholine-derived polymer. The photo-reactive polymer was synthesized by a coupling reaction involving copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid with 4-azidoaniline. When the polymer was unpattern immobilized on the surface, X-ray photo-electron spectroscopic analysis and static contact angle measurements were performed. It was shown that the surface was covered with phospholipid polar groups. Micropattern immobilization was carried out using a micropatterned photo-mask. Measurements using atomic force microscopy showed that the swelled micropatterned polymer was five times as thick as the dried one. Protein adsorption and platelet adhesion were reduced on the polymer-immobilized regions. Mammalian cells did not adhere, and formed aggregates on the immobilized regions. In conclusion, the photo-reactive phospholipid polymer was covalently immobilized on the conventional polymer surfaces and it tended to reduce interactions with proteins and cells.     

Gradient micropattern immobilization of a thermo-responsive polymer to investigate its effect on cell behavior

A gradient micropattern immobilization technique using a photomask was developed to investigate by microscopic observation the effect of the surface concentration of an immobilized thermo-responsive polymer. Poly(N-isopropylacrylamide-co-acrylic acid) was chosen as the thermo-responsive polymer, and was conjugated with 4-azidoaniline to form a photo-reactive thermo-responsive polymer (PIA-Az). The PIA-Az was coated onto a polystyrene plate, and immobilized using UV irradiation in the presence of a gradient micropattern photomask. The immobilization was performed with and without gelatin. Mouse fibroblast STO cells cultured on the plate did not adhere to the surface when PIA-Az had a high surface density, and no cell detachment was observed in any region when the temperature was lowered. However, on the gelatin coimmobilized surfaces, the cells adhered to all surfaces independent of the PIA-Az density, and detached from the high PIA-Az surface density areas when the temperature was lowered. The present technique demonstrates the effect of the surface concentration-dependent immobilization of the molecules. We show that cell detachment can be regulated by perturbating a small part of the cell-immobilized polymer interface.     

Immobilization of erythropoietin to culture erythropoietin-dependent human leukemia cell line

To investigate the effect of immobilized cytokine, erythropoietin (Epo) was immobilized on a culture plate and the Epo-dependent human leukemia cell line UT-7/Epo then was cultured upon the plate. A photo-reactive gelatin was mixed with Epo and the mixture was cast on a plate. The plate was then irradiated with ultraviolet light in the presence or absence of a photo-mask. After washing with water, a micropatterned or unpatterned surface was formed. A leukemia cell line dependent on Epo, UT-7/Epo, was cultured on the sample plate. On the micropatterned surface, apoptosis of cells was induced on the surface without Epo, but was not observed on the Epo-immobilized surface. This result demonstrated that Epo stimulated the cells even after immobilization. Although the activity of immobilized Epo was low, the activity was slightly higher than that achieved by soluble Epo at higher concentration. In addition, the immobilized Epo could be repeatedly used for culture of UT-7/Epo cell. The present study provided a convenient immobilization method and indicated that immobilization of cytokines will be useful for creating an artificial cell culture device.     

Toward the development of biomimetic polymers by protein immobilization: PEGylation of insulin as a model reaction

Many current tissue-engineering investigations aim at the rational control of cell adhesion and tailored composition of biomaterial surfaces by immobilizing various protein and peptide components, such as growth factors. As a step on the way to develop polymers that allow for such surface modifications, water-soluble polymers were used as model substances to examine reactions with proteins containing amine groups. Consequently, the uncommon PEGylation of insulin in aqueous buffers was used to characterize reaction products and simulate the intended immobilization step for surface modification. Amine reactive poly(ethylene glycol)s were synthesized and characterized by (1)H nuclear magnetic resonance and gel-permeation chromatography. Furthermore, the model protein insulin was characterized concerning its accessible amino groups, using a fluorescent dye (TAMRA-SE). The resulting reaction products were identified by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. After PEGylation with hydrolytically stable poly(ethylene glycol) succinimidyl ester, the obtained PEGylated insulin was investigated by gel filtration chromatography, indicating successful attachment of the hydrophilic polymer chains. Application of an aqueous PEGylation scheme opens the door to the immediate investigation of various growth factors in cell culture, allowing for direct assessment of biological activity after forming the polymer-protein constructs with regard to later immobilization on surfaces.     

Surface-immobilized dextran limits cell adhesion and spreading

Dextran has recently been investigated as an alternative to polyethylene glycol (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although anti-fouling properties of surface-grafted dextran and PEG are quite similar, the multivalent properties of dextran are advantageous when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired. The preferred methods of dextran immobilization for biomaterial applications should be simple with minimal toxicity. In this report, a method is described for covalent immobilization of dextran to material surfaces which involves low residual toxicity reagents in mild aqueous reaction conditions. 70 kDa MW dextran was immobilized on glass and polyethylene terephthalate (PET) surfaces. 3T3 fibroblast cell adhesion was compared on untreated, aminated, and dextran-coated materials. Dextran coatings effectively limited cell adhesion and spreading on glass and PET surfaces in the presence of serum-borne cell adhesion proteins. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

Quantification of protein immobilization on substrates for cellular microarray applications

Cellular microarray developments and its applications are the next step after DNA and protein microarrays. The choice of the surface chemistry of the substrates used for the implementation of this technique, that must favor proper protein immobilization while avoiding cell adhesion on the nonspotted areas, presents a complex challenge. This is a key issue since usually the best nonfouling surfaces are also the ones that retain immobilized the smallest amounts of printed protein. To quantitatively assess the amount of protein immobilization, in this study several combinations of fluorescently labeled fibronectin (Fn*) and streptavidin (SA*) were microspotted, with and without glycerol addition in the printing buffer, on several substrates suitable for cellular microarrays. The substrates assayed included chemically activated surfaces as well as Poly ethylene oxide (PEO) films that are nonfouling in solution but accept adhesion of proteins in dry conditions. The results showed that the spotted Fn* was retained by all the surfaces, although the PEO surface did show smaller amounts of immobilization. The SA*, on the other hand, was only retained by the chemically activated surfaces. The inclusion of glycerol in the printing buffer significantly reduced the immobilization of both proteins. The results presented in this article provide quantitative evidence of the convenience of using a chemically activated surface to immobilize proteins relevant for cellular microarray applications, particularly when ECM proteins are cospotted with smaller factors which are more difficult to be retained by the surfaces.     

Acrylic acid grafting and collagen immobilization on poly(ethylene terephthalate) surfaces for adherence and growth of human bladder smooth muscle cells

In tissue engineering, degradable or non-degradable polymer matrices can act as cell-carrier-scaffolds. Cell adhesion and growth on these scaffolds can be promoted by immobilizing extracellular matrix proteins. Therefore, in this study, polymer poly(ethylene terephthalate) (PET) films were surface modified by graft polymerization of acrylic acid, to subsequently allow collagen (types I and III) immobilization and human smooth muscle cell expansion. The surfaces of PET were activated by plasma, followed by acrylic acid graft polymerization, resulting in covalently bound brushes, containing an average of either 0.22+/-0.1 or 5.93+/-0.87 microg/cm2 of poly(acrylic acid) (PAA). Subsequent electrostatic adsorption of collagen gave a surface concentration of 4.96 and 17.2 microg/cm2, respectively, as determined using radiolabelled 125I collagen. Both PET films grafted with 0.22 microg/cm2 of PAA with or without adsorbed collagen were apt for smooth muscle cell adhesion and proliferation. However, films grafted with 5.93 microg/cm2 were not. PAA-grafted PET films, onto which serum proteins of the culture medium adsorbed spontaneously, proved to be better matrices than films on which collagen has been immobilized. It, therefore, can be speculated that other serum proteins are more important than collagen for the human smooth muscle cell adhesion and growth on surface-modified polymer matrices.     

Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) was immobilized on substrata in photoreactive gelatin to control the adhesion and growth of vascular endothelial cells. The gelatin and VEGF were mixed in water and cast on a polystyrene dish or a silane-coated glass plate. The surface was then photoirradiated in the presence or absence of a photomask and washed. Toughness of the immobilized material was confirmed by ethanol treatment. Human umbilical vein endothelial cells (HUVECs) grew on the immobilized VEGF but not on a nontreated surface. Growth of HUVEC increased significantly with an increase in the amount of immobilized VEGF, and the effects were inhibited by treatment with anti-VEGF antibody. Thus, immobilized VEGF specifically interacted with HUVECs to permit growth in culture. Micropatterning of HUVEC cultures was also achieved using micropattern-immobilized VEGF. This patterning technique may be useful for the formation of blood vessel networks in vitro.     

Fibronectin immobilized by a novel surface treatment regulates fibroblast attachment and spreading

In order to understand the influence of cell-adhesive molecules on anchorage-dependent cell behavior on biomaterial surfaces, a model system is required where these molecules can be applied to surfaces with controlled surface ligand density and resistance to the adsorption of additional proteins present in the medium. This study asked whether fibronectin could be immobilized in a controlled manner to a hydrophobic surface with a chemically modified triblock surfactant. ELISA studies indicated that variation of the soluble fibronectin concentration used for immobilization could be used to control the amount of fibronectin immobilized to the surface. Furthermore, fibroblasts seeded on these surfaces in 10% serum-containing medium attached and spread as a function of the amount of immobilized fibronectin. Surfaces treated with unmodified surfactant did not support cell attachment, suggesting that cell attachment and spreading were primarily regulated by the immobilized fibronectin with minimal interference from adsorption of serum proteins. Together, these results suggest that covalent immobilization to Pluronic F108 provides a method for studying cellular responses to cell adhesive proteins with little interference from competing adsorbates, even in the presence of complex biological fluids such as serum. This technique may be applicable to a variety of existing hydrophobic biomedical polymers as a basic science tool as well as for influencing cell behavior at implant interfaces.     

Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge

The blood compatibility of poly(ethylene oxide) (PEO)-grafted and heparin (Hep) immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on heparin-immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on heparin-immobilized PUs, and the amount of interleukin-6 (IL-6) released from PBMCs stimulated with surface-modified PUs decreased with a decrease in PBMC adhesion.     

Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge

The blood compatibility of poly(ethylene oxide) (PEO)-grafted and heparin (Hep) immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on heparin-immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on heparin-immobilized PUs, and the amount of interleukin-6 (IL-6) released from PBMCs stimulated with surface-modified PUs decreased with a decrease in PBMC adhesion.     

The effect of self-designed bifunctional RGD-containing fusion protein on the behavior of human keratinocytes and dermal fibroblasts

In this study, self-designed bifunctional RGD-containing fusion protein (BFP) was grafted on the petri dish to evaluate its cytotoxicity and attachment efficiency on primary cultured keratinocytes and dermal fibroblasts. Two lengths of the GRGDS sequences were separately fused to the N-terminus and C-terminus of the Trichoderma koningii cellobiohydrolase I gene cellulose-binding domain, to serve as linking molecule between the cell and the substrate. The grafting procedure was no more labor-intensive and could be done just in aqueous condition itself. The epidermal keratinocytes and dermal fibroblasts, harvested and separated from human foreskin, were cultured in serum-free keratinocyte culture medium and DMEM, respectively. The BFP was dissolved in double-deionized water, and was prepared at different concentrations. The BFP solution was subsequently added into the petri dish for grafting. MTT assay, total DNA measurement, and lactate dehydrogenase analysis were used to evaluate the cell viability, cell proliferation, and cytotoxicity. The immunochemical stain and SEM examination were chosen to make sure that the cultured cells still kept in phenotype. The results showed that the self-designed BFP was successfully coated on the petri dish to improve the cells' adhesion. The whole coating procedure was just done in aqueous solution without any organic solvent being involved. This method was much simpler than the traditional one, and there was no possibility to damage the immobilized biomolecules. From the results of the study, BFP could enhance attachment of keratinocytes and dermal fibroblasts without losing normal cell morphology and keep keratinocytes on the desired differentiation pathway. We believe that coating BFP on petri dish not only enhanced the keratinocyte attachment but also promoted keratinocytes proliferation. We suggest that the self-designed BFP has a great potential to apply on surface modification for the tissue-engineering scaffolds in the future.     

Collagen release kinetics of surface functionalized 45S5 Bioglass-based porous scaffolds

A highly interconnected porous scaffold made from 45S5 Bioglass was fabricated by the polymer replica technique and surface functionalized for protein immobilization. Subsequently rat-tail collagen type I was immobilized on the scaffolds. The protein and ion release rates were determined by UV-vis spectroscopy and ion chromatography, respectively, and the impact on hydroxyapatite (HA) formation on the scaffolds upon immersion in SBF was evaluated. It was discovered that the surface functionalization enhanced the stability of the collagen attachment and stability against the increment of pH in a biological environment, resulting in similar collagen release kinetics in solutions of different pH values. Without the surface modification, collagen release was considerably expedited by the increment of pH in a surrounding solution. It was also found that the collagen immobilization does not effect the formation of carbonated HA on the scaffold surface. The stable collagen attachment to the functionalized scaffold makes this approach potentially suitable for improving cell attachment and thus for enhancing the application potential of the scaffold in tissue engineering.     

Cell growth on immobilized cell-growth factor. II. Adhesion and growth of fibroblast cells on poly(methyl methacrylate) membrane immobilized with proteins of various kinds.

The surface of poly(methyl methacrylate) membrane was partially hydrolysed and the carboxyl groups produced were coupled with various protein molecules with water-soluble carbodiimide. The immobilized proteins were a cell-growth factor insulin, cell adhesion factors fibrinogen and fibronectin, and serum proteins albumin and gamma-globulin. The insulin-immobilized poly(methyl methacrylate) membrane strongly accelerated the growth and slightly accelerated the adhesion of fibroblast cells. The immobilized fibronectin and fibrinogen enhanced the cell adhesion, and the former also accelerated the cell growth. The immobilized albumin and gamma-globulin influenced the adhesion and growth of cells very little. It was found that various proteins specifically influence the adhesion and growth of cells in an immobilized state.     

Synthesis and in vitro evaluation of a novel thiolated chitosan

In order to achieve the same properties as chitosan-4-thio-butyl-amidine and to overcome at the same time its insufficient stability, the aim of this study was to evaluate the imidoester reaction of isopropyl-S-acetylthioacetimidate for the chemical modification of chitosan and to study the properties of the resulting chitosan-thioethylamidine (TEA) derivative. The thioalkylamidine substitute was introduced without the formation of N-substituted non-thiol products. The resulting conjugates exhibited 1.05+/-0.17% or 139.68+/-17.13 micromol immobilized free thiol groups per gram polymer and a total amount of reduced and oxidized thiol groups of 1.81+/-0.65% or 179.46+/-67.95 micromol/g polymer. By the immobilization of thiol groups mucoadhesion was strongly improved due to the formation of disulfide bonds with mucus glycoproteins. Chitosan-TEA was investigated regarding to its mucoadhesive properties via tensile studies and the rotating cylinder method. In tensile studies the total work of adhesion of chitosan-TEA was increased 3.3-fold in comparison to unmodified chitosan. Results from the rotating cylinder method showed an improvement ratio of 8.9 for chitosan-TEA compared with unmodified chitosan. In spite of the immobilization of thiol groups onto chitosan its swelling behavior in aqueous solutions was not significantly altered. Cumulative release studies out of matrix tablets comprising the chitosan-TEA and the model compound fluorescence labeled dextrane (FD(4)) demonstrated a controlled release over 3h with a trend toward a pseudo-zero-order kinetic. Because of these features the new chitosan thioamidine conjugate might represent a promising new polymeric excipient for various drug delivery systems.     

Platelet adhesion onto protein-coated and uncoated polyetherurethaneurea having tertiary amino groups in the substituents and its derivatives

Interactions of platelet with novel polyetherurethaneurea and its heparinized derivative were investigated. Platelet adhesion onto the material and release of serotonin or adenosine phosphate from platelet-rich plasma (PRP) were suppressed by an introduction of amino groups to polyetherurethaneurea, by quaternization of the polymer, and further by heparinization of the polymer. When the material was precoated with one of major plasma proteins and the protein-coated materials were taken to contact with washed platelet suspension (WP), the dependence of platelet adhesion and activation on the properties of polymers was different from that observed for PRP interaction. Platelet adhesion and activation were promoted according to the nature of coating proteins in the order albumin less than gamma-globulin less than fibrinogen and with increasing degree of denaturation of coating proteins. When the polymer materials were coated with proteins by immersing in aqueous solution containing two kinds of plasma proteins, adhesion behaviors of platelet were similar to those observed for PRP-uncoated material interaction. These experimental facts indicate that the selectivity of platelet for protein-coated material cannot be assessed by the interaction of WP with materials coated with a single kind of protein. It was concluded that material surface to which albumin is selectively adsorbed without denaturation does not stimulate adhering platelets for release reactions.     

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin-avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.     

Physical and biological properties of collagen-phospholipid polymer hybrid gels

We successfully developed a novel method for immobilizing poly(2-methacryloyloxyethyl phosphorylcholine) [Poly(MPC)] polymer onto collagen using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linkers. In order to obtain the highest possible molar ratio of immobilized MPC moieties on the collagen gel, a collagen-phospholipid polymer hybrid gel was prepared by repeating the cross-linking process up to three times to create a dense network of collagen and PMA. Network formation by repeating the immobilization process was successful, resulting in decreased free amine group content and a low swelling ratio. The hybrid gel displayed very high stability against degradation by collagenase and possessed high hydrophilicity. Fibrinogen adsorption and cell adhesion were reduced and demonstrated less cell proliferation as compared to that by uncross-linked collagen gel. The collagen-phospholipid polymer hybrid gel did not exhibit toxicity, and the cell morphology remained intact (round); this implies that the interaction between the cell and the collagen-phospholipid polymer hybrid gel is safe and mild.     

Two-Dimensional Protein Micropatterning for Sensor Applications Through Chemical Selectivity Technique

Two-dimensional protein micropatterning with immobil-ization of IgG and poly (ethylene glycol) (PEG) on patterned Au and Si surfaces was performed through a new technique. The technique for micropatterning is based on a chemical selectivity method by creating chemical bonding between protein, self-assembled monolayers (SAMs) and substrates rather than physical means. The substrates used in this study are pre-fabricated with silicon wafer patterned with arrays of gold squares. The silicon regions of the substrate are modified with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. The gold regions on the substrate are first immobilized with bifunctional SAM layers that can covalently bound adhesion proteins for individual cell attachment against a PEG background. The surface coatings are characterized by contact angle measurement, ellips-ometry, and atomic force microscopy (AFM). The patterns of fluorescence-labeled proteins are examined using fluorescence microscopy. Our study demonstrated that the PEG modified silicon region showed an effective protein reduction while the gold regions were successfully covalently bonded with proteins. This technique also demonstrated a combined feature of ensuring the activity, selectivity, and stability of the immobilized proteins. A simple lift-off microfabrication process was introduced in this study to pattern metal on silicon substrates without using expensive metal etching.