Surface presentation of bioactive ligands in a nonadhesive background using DOPA-tethered biotinylated poly(ethylene glycol)

We have developed surfaces for the selective presentation of biotinylated peptides and proteins in a background that resists nonspecific protein adsorption; controlled amounts of biotinylated poly(ethylene glycol) (MW 3400 Da; PEG3400) anchored to titanium-dioxide-coated surfaces via an adhesive tri-peptide sequence of L-3,4-dihydroxyphenylalanine (DOPA3-PEG3400-biotin; DPB) were incorporated within a DOPA3-PEG2000 background. Using optical waveguide lightmode spectroscopy, we found that the amounts of sequentially adsorbed NeutrAvidin and singly biotinylated molecules increased proportionally with the amount of DPB in the surface. Biotinylated peptides (MW approximately 2000 Da) were able to fill all three of the remaining avidin-binding sites, while only one molecule of biotinylated PEG5000 or stem cell factor bound to each avidin. The resulting biotin-avidin-biotin linkages were stable for prolonged periods under continuous perfusion, even in the presence of excess free biotin. Hematopoietic M07e cells bound to immobilized peptide ligands for alpha5beta1 (cyclic RGD) and alpha4beta1 (cylic LDV) integrins in a DPB-dose-dependent manner, with near-maximal binding to cylic LDV for surfaces containing 1% DPB. Multiple ligands were adsorbed in a controlled manner by incubating NeutrAvidin with the respective ligands in the desired molar ratio and then adding the resulting complexes to DPB-containing surfaces. Cell adhesion to surfaces containing both cylic LDV and cyclic RGD increased in an additive manner compared to that for the individual ligands. The bioactivity of adsorbed biotinylated stem cell factor was retained, as demonstrated by DPB-dose-dependent M07e cell adhesion and ERK1/2 activation.     

Photocontrol of cell adhesion on amino-bearing surfaces by reversible conjugation of poly(ethylene glycol) via a photocleavable linker

Dynamic control of cell adhesion on substrates is a useful technology in tissue engineering and basic biology. This paper describes a method for the control of cell adhesion on amino-bearing surfaces by reversible conjugation of an anti-fouling polymer, poly(ethylene glycol) (PEG), via a newly developed photocleavable linker, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (1). This molecule has alkyne and succinimidyl carbonate at each end, which are connected by photocleavable 2-nitrobenzyl ester. Under this molecular design, the molecule crosslinked azides and amines, whose linkage cleaved upon application of near-UV light. By using aminosilanised glass and silicon as model substrates, we studied their reversible surface modification with PEG-azide (M(w) = 5000) based on contact angle measurements, ellipsometry, and AFM morphological observations. Protein adsorption and cell adhesion dramatically changed by PEGylation and the following irradiation, which can be used for cellular patterning. Also, the capability of the substrate to change cell adhesiveness by photoirradiation during cell cultivation was demonstrated by inducing cell migration. We believe this method will be useful for dynamic patterning of cells on protein-based scaffolds.     

Platelet and bacterial repellence on sulfonated poly(ethylene glycol)-acrylate copolymer surfaces

Novel poly(ethylene glycol) (PEG) and sulfonated PEG (PEG-SO₃) acrylate copolymers have been prepared and characterized to apply as coating and blending materials for biomedical applications. The modified surfaces using acrylate copolymers demonstrated increased hydrophilicity, possibly due to the hypothesized reorientation of PEG/PEG-SO₃ chains into water phase. All copolymer surfaces demonstrated less platelet adhesion than control. In addition, platelet adhesion on copolymer surfaces decreased as the chain length of PEG and sulfonated PEG in copolymers increases. All copolymer surfaces reduced bacterial adhesion significantly and the adhesion level differs depending on surfaces as well as media. The obtained results attest to the usefulness of these copolymers as a coating or additive material to improve the blood compatibility of blood contacting devices.     

Surface modification of thermoplastic polyurethane in order to enhance reactivity and avoid cell adhesion

Recently, the definition of coating procedures which leads to strong cell repellent surfaces has been an extremely important issue. In the present study, the cell repellency of thermoplastic polyurethane material (Elastollan®1180A50) surfaces was achieved by chemical treatment. Samples of Elastollan®1180A50 processed by injection molding, were oxidized with hydrogen peroxide (H₂O₂) and then impregnated with poly(ethylene glycol). The oxidation time was evaluated as were the effects of the PEG impregnation. Of all the evaluated modifications, a surface oxidation for 2 h, followed by impregnation with poly(ethylene glycol) resulted in the best cell repellent surface.     

Investigating How Organic Solvents Affect Tissue Culture Polystyrene Surfaces through Responses of Mesenchymal Stem Cells

Polymer coating of tissue culture polystyrene (TCPS) surfaces promotes their biofunctionality, which can aid manipulation of cellular functions. However, the effect of the solvent used for polymer coating is yet to be elucidated. In this study, solvent‐treated TCPS surfaces using water, methanol, ethanol, 2‐propanol, and dimethyl sulfoxide are fabricated. Solvent treatment of TCPS surfaces is performed by spreading solvents onto the surfaces and allowing them to dry. Solvent treatment changes the surface roughness and wettability, depending on the kind of solvents. In addition, these surface property changes affected the extension, proliferation, and differentiation of human bone marrow–derived mesenchymal stem cells. These results suggest that solvent selection for polymer coating is crucial in the regulation of cell responses. Further, treatment with an appropriate solvent can result in a more suitable culture environment for modulating cellular functions.     

Adsorption and conformation behavior of biotinylated fibronectin on streptavidin-modified TiO(X) surfaces studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.     

Design of poly(ethylene glycol)/streptavidin coimmobilized upconversion nanophosphors and their application to fluorescence biolabeling

Infrared-to-visible upconversion phosphors (i.e., rare earth ion-doped Y2O3 nanoparticles (UNPs)) were synthesized by the homogeneous precipitation method. Because the charge on the erbium (Er) ion-doped Y2O3 (Y2O3:Er) NP (UNP1) surface is positive under neutral conditions, the UNP1 surface was electrostatically PEGylated using negatively charged poly(ethylene glycol)- b-poly(acrylic acid) (PEG- b-PAAc). The adsorption of PEG- b-PAAc was confirmed by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The surface charge of the PEGylated UNP1s (PEG-UNP1s) was effectively shielded by the PEGylation. The dispersion stability of the UNP1s was also significantly improved by the PEGylation. The PEG-UNP1s were dispersed over 1 week under physiological conditions as a result of the steric repulsion between the PEG chains on the UNP1 surface. The upconversion emission spectrum of PEG-UNP1s was observed under physiological conditions and was confirmed by near-infrared excited fluorescence microscope observation. Streptavidin (SA)-installed ytterbium (Yb) and Er ion-codoped Y2O3 (Y2O3:Yb,Er) NPs (UNP2s) were prepared by the coimmobilization of PEG- b-PAAc and streptavidin. The PEG/SA coimmobilized UNP2s (PEG/SA-UNP2s) specifically recognized biotinylated antibodies and emitted strong upconversion luminescence upon near-infrared excitation. The obtained PEG/streptavidin coimmobilized UNPs are promising as high-performance near-infrared biolabeling materials.     

Linear Poly(methyl glycerol) and Linear Polyglycerol as Potent Protein and Cell Resistant Alternatives to Poly(ethylene glycol)

The nonspecific interaction of proteins with surfaces in contact with biofluids leads to adverse problems and is prevented by a biocompatible surface coating. The current benchmark material among such coatings is poly(ethylene glycol) (PEG). Herein, we report on the synthesis of linear polyglycerol derivatives as promising alternatives to PEG. Therefore, gold surfaces as a model system are functionalized with a self‐assembled monolayer (SAM) by a two‐step anhydride coupling and a direct thiol immobilization of linear poly(methyl glycerol) and polyglycerol. Surface plasmon resonance (SPR) spectroscopy reveals both types of functionalized surfaces to be as resistant as PEG towards the adsorption of the test proteins fibrinogen, pepsin, albumin, and lysozyme. Moreover, linear polyglycerols adsorb even less proteins from human plasma than a PEG‐modified surface. Additional cell adhesion experiments on linear poly(methyl glycerol) and polyglycerol‐modified surfaces show comparable cell resistance as for a PEG‐modified surface. Also, in the case of long‐term stability, high cell resistance is observed for all samples in medium. Additional in vitro cell‐toxicity tests add to the argument that linear poly(methyl glycerol) and polyglycerol are strong candidates for promising alternatives to PEG, which can easily be modified for biocompatible functionalization of other surfaces.     

Drug-loaded, bivalent-bottle-brush polymers by graft-through ROMP

Graft-through ring-opening metathesis polymerization (ROMP) using ruthenium N-heterocyclic carbene catalysts has enabled the synthesis of bottle-brush polymers with unprecedented ease and control. Here we report the first bivalent-brush polymers; these materials were prepared by graft-through ROMP of drug-loaded polyethylene-glycol (PEG) based macromonomers (MMs). Anticancer drugs doxorubicin (DOX) and camptothecin (CT) were attached to a norbornene-alkyne-PEG MM via a photocleavable linker. ROMP of either or both drug-loaded MMs generated brush homo- and co-polymers with low polydispersities and defined molecular weights. Release of free DOX and CT from these materials was initiated by exposure to 365 nm light. All of the CT and DOX polymers were at least 10-fold more toxic to human cancer cells after photoinitiated drug release while a copolymer carrying both CT and DOX displayed 30-fold increased toxicity upon irradiation. Graft-through ROMP of drug-loaded macromonomers provides a general method for the systematic study of structure-function relationships for stimuli-responsive polymers in biological systems.     

A novel method to fabricate patterned bilayer lipid membranes

We introduce a new method for forming tethered bilayer lipid membranes on surfaces patterned using a photocleavable self-assembled monolayer (SAM). A SAM terminated with a hydrophobic fluorocarbon residue was bound to a gold surface through a link containing a photocleavable ortho-nitrobenzyl moiety. Hydrophilic regions were produced by irradiation with soft UV (365 nm) through a photomask. The patterned surface was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. Tethered bilayer lipid membranes with well-defined bilayer and monolayer regions were then formed by exposure to egg PC vesicles. The membranes had resistance and capacitance values of 0.52 MOmega.cm2 and 0.83 microF.cm-2, respectively.     

Efficient One-Step Passivation of Polyurethane Using Transurethanization

The uncontrolled accumulation of biological materials on the surface of medical devices through protein adsorption or cell adhesion causes adverse biological reactions in the living host system, leading to complications. In this study, poly(ethylene glycol) (PEG) is successfully grafted onto polyurethane (PU) surfaces by using a new strategy through a simple and efficient transurethanization reaction. The PEG hydroxyl group is deprotonated and then reacted with the PU surface to provide antiadhesive hydrophilic surfaces in a single step. Surface analysis techniques proved the grafting to be efficient and the formation of a hydrophilic polymeric layer at the surface of PU. Biological assays showed that the surface modification induced lower protein adsorption, cell, platelet, and bacterial adhesion than untreated surfaces, showing a potential for biomedical applications.     

Formation of antifouling functional coating from deposition of a zwitterionic-co-nonionic polymer via “grafting to” approach

We develop a new process for the preparation of synergistic antifouling functional coatings on gold surfaces via a “grafting to” approach. The strategy includes a synthetic step of polymer brushes that consist of poly (ethylene glycol) (PEG) and zwitterionic side chains via a typical reversible-addition fragmentation chain transfer (RAFT) polymerization process, and a subsequent deposition of the polymer brushes onto a gold substrate. The presence of PEG and zwitterion chains on these polymer brush-coated gold surfaces has been proved to have a synergistic effect on the final antifouling property of the coating. PEG chains lower the electrostatic repulsion between zwitterionic polymer chains and increase their graft density on gold surfaces, while zwitterionic polymer effectively improves the antifouling property that is offered by PEG chains alone. Protein adsorption and cell attachment assays tests are conducted to confirm that this copolymer layer on gold surface has a pronounced resistance against proteins such as Bovine serum albumin and Lysozyme. Importantly, the antifouling property can be systematically adjusted by varying the molar ratio of PEG to zwitterionic chains in the final coating copolymer.     

Comparison of different supramolecular architectures for oligonucleotide biosensing

This work describes a comparative study between two biosensing platforms that are commonly used to immobilize capture probes. These platforms refer to thiolated and biotinylated oligonucleotide strands chemisorbed on Au surfaces (DNA SAM) and bioconjugated on streptavidin (SA) monolayers (SA SAM), respectively. Both interfacial architectures were studied using surface acoustic wave (SAW) devices and surface plasmon spectroscopy (SPR). Our studies indicated that DNA SAM platforms enable higher densities of surface-confined oligonucleotide probes. However, their hybridization efficiency is lower when compared to that obtained in SA SAM platforms, thus impacting on a lower detection limit, 5 nM. Furthermore, binding of SA molecules to the biotinylated targets, in an attempt to enhance the signal in both platforms, revealed striking differences between both architectures. The SA underlayer used in the SA SAM configuration confers nonfouling characteristics to the interfacial assembly, thus precluding the nonspecific binding of SA onto the surface. The antifouling behavior of the SA DNA platform is an important feature to be considered in the amplification of hybridization events through the bioconjugation of biotinylated targets with streptavidin-based tags.     

Photosensitive chitosan to control cell attachment

An approach to control cell adhesion using a photocleavable molecule on chitosan has been developed and studied. Photocleavable 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC) was introduced into chitosan to control the surface properties. The two UV illuminations with a photomask controlled the cleavage of NVOC and the presentation of deprotected amines on one chitosan surface spatially and temporally. The following immobilizations of cell repulsive poly(ethylene glycol) after the first illumination and cell adhesive sequence Arg-Gly-Asp-Ser (RGDS) after the second illumination on the surface helped create surface heterogeneity. Fourier transform infrared spectroscopy (FTIR), water contact angle, and UV-visible spectroscopy were used to characterize the surfaces and photoactivation during the process. To study the cell attachment and morphology on our designed surfaces, NIH/3T3 fibroblast cell was used. Cell number and morphology on the surfaces were investigated. The cell study demonstrated the feasibility of the surfaces on the control of cell adhesion and the formation of cell patterns by UV illuminations and the following immobilizations of different biomolecules.     

Hydrophilization of Magnetic Nanoparticles with Modified Alternating Copolymers. Part 1: The Influence of the Grafting

Iron oxide nanoparticles (NPs) with a diameter 21.6 nm were coated with poly(maleic acid-alt-1-octadecene) (PMAcOD) modified with grafted 5,000 Da poly(ethyelene glycol) (PEG) or short ethylene glycol (EG) tails. The coating procedure utilizes hydrophobic interactions of octadecene and oleic acid tails, while the hydrolysis of maleic anhydride moieties as well as the presence of hydrophilic PEG (EG) tails allows the NP hydrophilicity. The success of the NP coating was found to be independent of the degree of grafting which was varied between 20 and 80% of the -MacOD-units, but depended on the length of the grafted tail. The NP coating and hydrophilization did not occur when the modified copolymer contained 750 Da PEG tails independently of the grafting degree. To explain this phenomenon the micellization of the modified PMAcOD copolymers in water was analyzed by small angle x-ray scattering (SAXS). The PMAcOD molecules with the grafted 750 Da PEG tails form compact non-interacting disk-like micelles, whose stability apparently allows for no interactions with the NP hydrophobic shells. The PMAcOD containing the 5,000 Da PEG and EG tails form much larger aggregates capable of an efficient coating of the NPs. The coated NPs were characterized using transmission electron microscopy, dynamic light scattering, ζ-potential measurements, and thermal gravimetry analysis. The latter method demonstrated that the presence of long PEG tails in modified PMAcOD allows the attachment of fewer macromolecules (by a factor of ~20) compared to the case of non-modified or EG modified PMAcOD, emphasizing the importance of PEG tails in NP hydrophilization. The NPs coated with PMAcOD modified with 60% (towards all -MAcOD- units) of the 5,000 PEG tails bear a significant negative charge and display good stability in buffers. Such NPs can be useful as magnetic cores for virus-like particle formation.     

Caged phosphoproteins

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.     

Surface modification of polycarbonate urethane by grafting polyethylene glycol and bivalirudin drug for improving hemocompatibility

As the clinical demand for blood-contacting materials increases, higher requirements are placed on their physicochemical properties, durability and hemocompatibility in vivo. In this work, a multiple functionalized material was developed through a facile modification process. Herein, polycarbonate urethane (PCU) surface was co-modified with polyethylene glycol (PEG) and bivalirudin (BVLD). PCU provides excellent physical and mechanical properties, PEG and BVLD, especially BVLD, enable the surface with outstanding anticoagulant capacity. Specifically, PCU surface was first treated with hexamethylene diisocyanate to introduce active isocyanate groups onto the surface, followed by hydroxy-PEG grafting to improve the hydrophilicity. Finally, BVLD was immobilized on the surface via Michael addition reaction to improve antithrombotic properties. Attenuated total reflection Fourier transforms infrared spectroscopy and UV spectrophotometers were used to confirm the modified surfaces. The hydrophilicity was characterized by static water contact angle measurement, the morphology of the modified surfaces was observed by scanning electron microscopy. Blood compatibility of the modified surfaces was characterized by the hemolysis rate, platelet adhesion assay and cell culture test. The results showed that the BVLD immobilized surface has excellent anticoagulant properties, good fibrin-bound thrombin inhibition, and good resistance against non-specific adhesion of proteins. Hence, the co-modification with PEG and BVLD was proved an encouraging strategy for improving hemocompatibility.     

Electroresponsive Structurally Colored Materials: A Combination of Structural and Electrochromic Effects

Electroresponsive structurally colored materials composed of ordered arrays of polyaniline@poly(methyl methacrylate) (PANI@PMMA) core–shell nanoparticles have been successfully prepared. The core–shell nanoparticles were synthesized by deposition of PANI shells on the surfaces of the PMMA cores by the oxidative polymerization of anilinium chloride. Ordered arrays were then fabricated by using the fluidic cell method. Because the ordered arrays and the PANI shells generate structural and electrochromic colors, respectively, these core–shell colloidal crystals exhibited colors resulting from the combined effects of these materials. The crystal colors depended greatly on the size of PANI@PMMA particles and could also be varied by the application of a voltage. The electrochromic colors of these arrays were found to be quite different from those exhibited by pure PANI films prepared by electrochemical oxidation.     

Stainless steel modified with poly(ethylene glycol) can prevent protein adsorption but not bacterial adhesion

The surface of AISI 316 grade stainless steel (SS) was modified with a layer of poly(ethylene glycol) (PEG) (molecular weight 5000) with the aim of preventing protein adsorption and bacterial adhesion. Model SS substrates were first modified to introduce a very high density of reactive amine groups by the adsorption of branched poly(ethylenimine) (PEI) from water. Methoxy-terminated aldehyde-poly(ethylene glycol) (M-PEG-CHO) was then grafted onto the PEI layers using reductive amination at the lower critical solution temperature (LCST) of the PEG in order to optimize the graft density of the linear PEG chains. The chemical composition and uniformity of the surfaces were determined using X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) in the imaging mode. The effects of PEI concentration and different substrate pre-cleaning methods on the structure and stability of the final PEG layer was examined. Piranha solution proved to be the most effective method for removing adventitious hydrocarbon contamination, compared to cleaning with ultrasonication in organic solvents, and was the SS substrate that produced the most stable and thickest PEI layer. The surface density of PEI was shown to increase with increasing PEI concentration (up to 30 mg/ml), as determined from XPS measurements, and subsequently produced the PEG layer with the highest density of attached chains. In model experiments using β-lactoglobulin no protein adsorption was detected on the optimized PEG surface as determined by XPS and ToF-SSIMS analysis. However, neither the adhesion of a Gram-negative (Pseudomonas sp.) nor a Gram-positive (Listeria monocytogenes) bacterium was affected by the coating as equal numbers adhered to all surfaces tested. Our results show that preventing protein adsorption is not a prerequisite stopping bacterial adhesion, and that other mechanisms most likely play a role.