Lysozyme-coupled poly(poly(ethylene glycol) methacrylate)-stainless steel hybrids and their antifouling and antibacterial surfaces.

An environmentally benign approach to impart stainless steel (SS) surfaces with antifouling and antibacterial functionalities was described. Surface-initiated atom transfer radical polymerization (ATRP) of poly(ethylene glycol) monomethacrylate) (PEGMA) from the SS surface-coupled catecholic L-3,4-dihydroxyphenylalanine (DOPA) with terminal alkyl halide initiator was first carried out, followed by the immobilization of lysozyme at the chain ends of poly(ethylene glycol) branches of the grafted PEGMA polymer brushes. The functionalized SS surfaces were shown to be effective in preventing bovine serum albumin (BSA) adsorption and in reducing bacterial adhesion and biofilm formation. The surfaces also exhibited good bactericidal effects against Escherichia coli and Staphylococcus aureus. The concomitant incorporation of antifouling hydrophilic brushes and antibacterial enzymes or peptides onto metal surfaces via catecholic anchors should be readily adaptable to other metal substrates, and is potentially useful for biomedical and biomaterial applications.

Bifunctional Eu(3+)-doped Gd(2)O(3) nanoparticles as a luminescent and T(1) contrast agent for stem cell labeling.

Magnetic resonance tracking of stem cells has recently become an emerging application for investigating cell-tissue interactions and guiding the development of effective stem cell therapies for regeneration of damaged tissues and organs. In this work, anionic Eu(3+)-doped Gd(2)O(3) hybrid nanoparticles were applied as a contrast agent both for fluorescence microscopy and T(1)-weighted MRI. The nanoparticles were synthesized through the polyol method and further modified with citric acid to obtain anionic nanoparticles. These nanoparticles were internalized into human mesenchymal stem cells (hMSCs) as confirmed by confocal laser scanning microscopy and quantified by inductively coupled plasma-mass spectrometry. MTT assay of the labeled cells showed that the nanoparticles did not possess significant cytotoxicity. In addition, the osteogenic, adipogenic and chondrogenic differentiation of the hMSCs was not influenced by the labeling process. With MRI, the in vitro detection threshold of cells after incubation with nanoparticles at a Gd concentration of 0.5 mM for 2 h was estimated to be about 10 000 cells. The results from this study indicate that the biocompatible anionic Gd(2)O(3) nanoparticles doped with Eu(3+) show promise both as a luminescent and T(1) contrast agent for use in visualizing hMSCs.

Antibacterial inorganic-organic hybrid coatings on stainless steel via consecutive surface-initiated atom transfer radical polymerization for biocorrosion prevention.

To enhance the corrosion resistance of stainless steel (SS) and to impart its surface with antibacterial functionality for inhibiting biofilm formation and biocorrosion, well-defined inorganic-organic hybrid coatings, consisting of a polysilsesquioxane inner layer and quaternized poly(2-(dimethyamino)ethyl methacrylate) (P(DMAEMA)) outer blocks, were prepared via successive surface-initiated atom transfer radical polymerization (ATRP) of 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). The cross-linked P(TMASPMA), or polysilsesquioxane, inner layer provided a durable and resistant coating to electrolytes. The pendant tertiary amino groups of the P(DMAEMA) outer block were quaternized with alkyl halide to produce a high concentration of quaternary ammonium groups with biocidal functionality. The so-synthesized inorganic-organic hybrid coatings on the SS substrates exhibited good anticorrosion and antibacterial effects and inhibited biocorrosion induced by sulfate-reducing bacteria (SRB) in seawater media, as revealed by antibacterial assay and electrochemical analyses, and they are potentially useful to steel-based equipment under harsh industrial and marine environments.

Active protein-functionalized poly(poly(ethylene glycol) monomethacrylate)-Si(100) hybrids from surface-initiated atom transfer radical polymerization for potential biological applications.

Protein-resistant poly(poly(ethylene glycol)monomethacrylate)-graft-Si(100), or Si-g-P(PEGMA) hybrids, were prepared via surface-initiated atom transfer radical polymerization (ATRP) of the poly(ethylene glycol)monomethacrylate (PEGMA) macromonomer from the hydrogen-terminated Si(100) surface (Si-H surface). The resultant robust Si-C bonded P(PEGMA) brushes can be further functionalized by the immobilization of human immunoglobulin (IgG) protein via different strategies, namely, the direct use of the alkyl halide chain ends preserved throughout the ATRP process and the postmodification of the hydroxyl side chains with by 1,1'-carbonyldiimidazole (CDI) or succinic anhydride (SA). The CDI exhibited a higher efficiency in activating the hydroxyl groups for coupling proteins. The surface density of the immobilized protein above 2.5 microg/cm(2) could be readily achieved. The distribution of active protein-docking sites on the Si-C bonded P(PEGMA) brushes can be also controlled by controlling the brush length. The resulting IgG-coupled Si-g-P(PEGMA) hybrid surface interacts only and specifically with the anti-IgG protein, while the dense P(PEGMA) brushes effectively prevent nonspecific protein binding and fouling. The simple concomitant incorporation of protein-resistant P(PEGMA) brushes and highly specific and active protein onto silicon surfaces via robust Si-C bonding should readily endow the silicon substrates with new and interesting properties for applications in silicon-based protein sensors or microarrays.

Grafting of antibacterial polymers on stainless steel via surface-initiated atom transfer radical polymerization for inhibiting biocorrosion by Desulfovibrio desulfuricans.

To enhance the biocorrosion resistance of stainless steel (SS) and to impart its surface with bactericidal function for inhibiting bacterial adhesion and biofilm formation, well-defined functional polymer brushes were grafted via surface-initiated atom transfer radical polymerization (ATRP) from SS substrates. The trichlorosilane coupling agent, containing the alkyl halide ATRP initiator, was first immobilized on the hydroxylated SS (SS-OH) substrates for surface-initiated ATRP of (2-dimethylamino)ethyl methacrylate (DMAEMA). The tertiary amino groups of covalently immobilized DMAEMA polymer or P(DMAEMA), brushes on the SS substrates were quaternized with benzyl halide to produce the biocidal functionality. Alternatively, covalent coupling of viologen moieties to the tertiary amino groups of P(DMAEMA) brushes on the SS surface resulted in an increase in surface concentration of quaternary ammonium groups, accompanied by substantially enhanced antibacterial and anticorrosion capabilities against Desulfovibrio desulfuricans in anaerobic seawater, as revealed by antibacterial assay and electrochemical studies. With the inherent advantages of high corrosion resistance of SS, and the good antibacterial and anticorrosion capabilities of the viologen-quaternized P(DMAEMA) brushes, the functionalized SS is potentially useful in harsh seawater environments and for desalination plants.

(Carboxymethyl)chitosan-modified superparamagnetic iron oxide nanoparticles for magnetic resonance imaging of stem cells.

Magnetic resonance imaging (MRI) is emerging as a powerful tool for in vivo noninvasive tracking of magnetically labeled stem cells. In this work, we present an efficient cell-labeling approach using (carboxymethyl)chitosan-modified superparamagnetic iron oxide nanoparticles (CMCS-SPIONs) as contrast agent in MRI. The CMCS-SPIONs were prepared by conjugating (carboxymethyl)chitosan to (3-aminopropyl)trimethoxysilane-treated SPIONs. These nanoparticles were internalized into human mesenchymal stem cells (hMSCs) via endocytosis as confirmed by Prussian Blue staining and electron microscopy investigation and quantified by inductively coupled plasma mass spectrometry. A MTT assay of the labeled cells showed that CMCS-SPIONs did not possess significant cytotoxicity. In addition, the osteogenic and adipogenic differentiations of the hMSCs were not influenced by the labeling process. The in vitro detection threshold of cells after incubation with 0.05 mg/mL of CMCS-SPIONs for 24 h was estimated to be about 40 cells. The results from this study indicate that the biocompatible CMCS-SPIONs show promise for use with MRI in visualizing hMSCs.

Inorganic-organic hybrid coatings on stainless steel by layer-by-layer deposition and surface-initiated atom-transfer-radical polymerization for combating biocorrosion.

To improve the biocorrosion resistance of stainless steel (SS) and to confer the bactericidal function on its surface for inhibiting bacterial adhesion and biofilm formation, well-defined inorganic-organic hybrid coatings, consisting of the inner compact titanium oxide multilayers and outer dense poly(vinyl-N-hexylpyridinium) brushes, were successfully developed. Nanostructured titanium oxide multilayer coatings were first built up on the SS substrates via the layer-by-layer sol-gel deposition process. The trichlorosilane coupling agent, containing the alkyl halide atom-transfer-radical polymerization (ATRP) initiator, was subsequently immobilized on the titanium oxide coatings for surface-initiated ATRP of 4-vinylpyridine (4VP). The pyridium nitrogen moieties of the covalently immobilized 4VP polymer, or P(4VP), brushes were quaternized with hexyl bromide to produce a high concentration of quaternary ammonium salt on the SS surfaces. The excellent antibacterial efficiency of the grafted polycations, poly(vinyl-N-pyridinium bromide), was revealed by viable cell counts and atomic force microscopy images of the surface. The effectiveness of the hybrid coatings in corrosion protection was verified by the Tafel plot and electrochemical impedance spectroscopy measurements.

Bioactive titanium implant surfaces with bacterial inhibition and osteoblast function enhancement properties.

Infection in orthopedic implant surgery is a serious complication and a major cause of implant failure. Upon implant insertion, a contest between microbial colonization and tissue integration of the implant surface ensues. This race for the surface determines the probability of tissue integration or infection, and the surface properties of the substrate have an important role to play in determining the outcome. A number of strategies have been developed for the modification of implant surfaces to promote bone cell (osteoblast) functions and inhibit bacterial adhesion and growth. In this article, a review is given of these surface modification strategies, in particular those which can achieve the dual aim of bacterial inhibition and simultaneous enhancement of osteoblast functions.Surfaces of these types can be expected to have excellent potential for orthopedic applications.

Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces.

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate-co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA-co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(100) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA-co-PEGMEMA) microdomains were activated directly by 1,1'-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.

Bacteria-surface interaction in the presence of proteins and surface attached poly(ethylene glycol) methacrylate chains.

This study analyzes the adhesion behavior of the gram positive bacteria, Staphylococcus aureus (S. aureus), and the gram negative bacteria, Escherichia coli (E. coli), on polypyrrole (PPY) surfaces in the presence of poly(ethylene glycol) methacrylate (PEGMA) chains and plasma proteins (bovine serum albumin and bovine plasma fibrinogen) either preadsorbed on the film surface or in the bacterial suspension. Bacterial adhesion experiments performed in a suspension of bacterial cells and protein may give important insights on the behavior of bacterial adhesion in an in vivo environment. Protein adsorption and bacterial adhesion on PEGMA-grafted PPY films were reduced by about a factor of 2-4 compared with those on the pristine PPY films. In addition, the number of bacterial cells adhering on the substrate is dependent not only on the type of protein present, but also the sequence of exposure to the protein relative to the bacteria. Furthermore, bacteria-surface adhesion force was measured using the atomic force microscopy with increasing lateral force to detach the individual cell. The adhesion force of S. aureus is influenced by PEGMA and plasma protein modification and is significantly higher than that of E. coli for all substrates tested. The number of adherent cells on the substrate is shown to be directly correlated to the bacterial adhesion force.

Preparation of conductive polypyrrole-palladium composite nanospheres by inverse microemulsion polymerization.

Conductive polypyrrole-palladium (PPy-Pd) composite nanospheres of about 50 nm in diameter, containing dispersed Pd metal nanoparticles of about 2-4 nm in size, were prepared in a 1-step oxidative polymerization of pyrrole by Pd(NO3)2. Pyrrole was oxidized by Pd(NO3)2 in an inverse microemulsion polymerization system, yielding PPy nanospheres and elemental Pd nanoparticles simultaneously. Palladium nanoparticles were uniformly dispersed in the nanospheres of PPy chains. The latter also exhibited an enhanced effective conjugation. The chemical composition of the PPy-Pd composite nanospheres was characterized by X-ray photoelectron spectroscopy and FTIR spectroscopy. The crystalline structure of the Pd nanoparticles was deduced from X-ray diffraction patterns. The morphology of the composites was revealed by scanning and transmission electron microscopy.

Synthesis and in vitro anti-cancer evaluation of tamoxifen-loaded magnetite/PLLA composite nanoparticles.

The present study deals with the synthesis and characterization of tamoxifen-loaded magnetite/poly(l-lactic acid) composite nanoparticles (TMCN), and their in vitro anti-cancer activity against MCF-7 breast cancer cells. The composite nanoparticles with an average size of approximately 200 nm, were synthesized via a solvent evaporation/extraction technique in an oil/water emulsion. The superparamagnetic property (saturation magnetization value of approximately 7 emu/g) of the TMCN is provided by Fe(3)O(4) nanoparticles of approximately 6 nm encapsulated in the poly(l-lactic acid) matrix. The encapsulation efficiency of the Fe(3)O(4) and tamoxifen as a function of the concentration in the organic phase was investigated. The uptake of TMCN and tamoxifen by MCF-7 was estimated from the intracellular iron concentration. After 4h incubation of MCF-7 with TMCN, significant changes in the cell morphology were discernible from phase contrast microscopy. Cytotoxicity assay shows that while the Fe(3)O(4)-loaded poly(l-lactic acid) composite nanoparticles exhibit no significant cytotoxicity against MCF-7, approximately 80% of the these cells were killed after incubation for 4 days with TMCN.

In vitro antibacterial and cytotoxicity assay of multilayered polyelectrolyte-functionalized stainless steel.

Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic and implant surgery. In the present study, a new strategy for confering stainless steel with antibacterial property via the alternate deposition of quaternized polyethylenimine (PEI) or quaternized polyethylenimine-silver complex and poly(acrylic acid) (PAA) was investigated. The success of the deposition of the polyelectrolyte multilayers (PEM) and its chemical nature was investigated by static water contact angle and X-ray photoelectron spectroscopy (XPS), respectively. The antibacterial activity was assessed using Escherichia coli (E. coli, a gram-negative bacterium) and Staphylococcus aureus (S. aureus, a gram-positive bacterium). The inhibition of E. coli and S aureus growth on the surface of functionalized films was clearly shown using the LIVE/DEAD Baclight bacterial viability kits and fluorescence microscopy. The cytotoxicity of the PEM to mammalian cells, evaluated by the MTT assay, was shown to be minimal and long-term antibacterial efficacy can be maintained. These results indicate new possibilities for the use of such easily built and functionalized architectures for the functionalization of surfaces of implanted medical devices.

Porous and electrically conductive polypyrrole-poly(vinyl alcohol) composite and its applications as a biomaterial.

Bulk modification of polypyrrole (PPY) with poly(vinyl alcohol) (PVA) was carried out by the electropolymerization of pyrrole in the presence of PVA in the reaction solution, with tetraethylammonium perchlorate (TEAP) as the electrolyte. The surface morphology of the as-synthesized PPY-TEAP-PVA film was investigated using scanning electron microscopy, and the film was further characterized using X-ray photoelectron spectroscopy, electrical conductivity, the water contact angle, and BET surface area measurements. The PPY-TEAP-PVA composite is electrically conductive, hydrophilic, and microporous with a high surface area. Its potential as a biomaterial was investigated with respect to its blood compatibility and function as a substrate for biosensor fabrication and cell culture. The presence of PVA in the film attenuates blood protein adsorption, and the porous nature of the PPY-TEAP-PVA film results in a 10-fold increase in the amount of glucose oxidase covalently immobilized on the film over that on a nonporous PPY film. PC12 cell attachment and growth on the PPY-TEAP-PVA film was also shown to be enhanced compared with that on tissue culture polystyrene. The attached cells proliferated and formed a monolayer on the film surface after 48 h of seeding.

Heparin-coupled poly(poly(ethylene glycol) monomethacrylate)-Si(111) hybrids and their blood compatible surfaces.

Well-defined (nearly monodispersed) poly(poly(ethylene glycol)monomethacrylate)-Si hybrids were prepared via surface-initiated atom transfer radical polymerization (ATRP) of the poly(ethylene glycol)monomethacrylate (PEGMA) macromonomer on the hydrogen-terminated Si(111) surface (Si-H surface). Both the active chloride groups at the chain ends (from the ATRP process) and the chloride groups converted from some ( approximately 32%) of the -OH groups of the Si-C bonded PEGMA polymer, or P(PEGMA), brushes were used as leaving groups for the covalent coupling of heparin. For the heparinized P(PEGMA)-Si hybrid surfaces, protein adsorption and platelet adhesion were significantly suppressed. The well-defined and dense P(PEGMA) brushes, prepared from surface-initiated ATRP, had allowed the immobilization of a relatively high concentration of heparin (about 14 mug/cm(2)). The resulting silicon surface exhibited significantly improved antithrombogenecity with a plasma recalcification time (PRT) of about 150 min. The persistence of high bioactivity for the immobilized heparin on the hybrid surfaces can be attributed to the biocompatibility of the PEGMA units, as well as their role as spacers in providing the immobilized heparin with a higher degree of conformational freedom in a more hydrophilic environment. Thus, the heparin-coupled P(PEGMA)-Si hybrids with anti-fouling and antithrombogenic surfaces are potentially useful in silicon-based implantable devices and tissue engineering.

Controlled release of heparin from polypyrrole-poly(vinyl alcohol) assembly by electrical stimulation.

A surface modification technique was developed for the covalent immobilization of poly(vinyl alcohol) (PVA)-heparin hydrogel onto electrically conductive polypyrrole (PPY) film with the objective of achieving controlled release of heparin. First, aldehyde groups were introduced onto PPY film through poly(ethylene glycol) monomethacrylate graft copolymerization and subsequent oxidation in acetic anhydride and dimethyl sulfoxide mixture. Then, the prepared PVA-heparin hydrogel was cast onto the PPY film and covalently immobilized to the film through the reaction between the aldehyde groups on the PPY film and the hydroxyl groups of PVA. X-ray photoelectron spectroscopy was used to characterize the surface-modified film after each stage. The strong attachment of the PVA-heparin layer on the PPY film was confirmed by peel test and scanning electron microscopy. The release behavior of heparin from the substrate with and without electrical stimulation was studied and the experimental results showed that the heparin release rate from the prepared substrate using an electric current of 3.5 mA is twofold higher than that without current.

Assessment of in vitro bioactivity of hyaluronic acid and sulfated hyaluronic acid functionalized electroactive polymer.

Electrically conductive polypyrrole (PPY) was surface functionalized with hyaluronic acid (HA) and sulfated hyaluronic acid (SHA) to improve its surface biocompatibility. The immobilization of HA on the PPY film was facilitated by the use of a cross-linker having the appropriate functional groups. The biological activity of the HA functionalized PPY film was assessed by means of an in vitro PC12 cell culture. The cell attachment on different substrates was studied and determined by bicinchoninic acid protein analysis. Cell attachment on the HA functionalized PPY film surface was significantly enhanced in the presence of nerve growth factor. The SHA functionalized PPY film was obtained by the sulfonation of the immobilized HA using pyridinesulfonate. The retention of the biological activity of the immobilized HA after sulfonation was evaluated by the in vitro assessment of the plasma recalcification time (PRT) and platelet adhesion on the substrate. The PRT observed from the SHA functionalized PPY film was significantly prolonged compared with the HA functionalized PPY. Some reduction of platelet adhesion was observed for the SHA functionalized PPY film, compared with that of the HA functionalized PPY film.

Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films.

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.

Reactive coupling of 4-vinylaniline with hydrogen-terminated Si(100) surfaces for electroless metal and "synthetic metal" deposition.

Pristine and resist-patterned Si(100) substrates were etched by aqueous HF to produce hydrogen-terminated silicon (H-Si(100)) surfaces. The H-Si(100) surface was then subjected to UV-induced reactive coupling of 4-vinylaniline (VAn) to produce the VAn monolayer-modified silicon (VAn-Si) surface. The VAn-Si surface was first functionalized with a "synthetic metal" by oxidative graft polymerization of aniline with the aniline moieties of the coupled VAn molecules. The composition and topography of the VAn-Si and polyaniline (PAn)-grafted VAn-Si (PAn-VAn-Si) surfaces were characterized by X-ray photoelectron spectroscopy and atomic force microscopy, respectively. The doping-undoping (protonation-deprotonation) and redox-coupling (metal reduction) behavior, as well as the electrical conductivity, of the surface-grafted PAn were found to be similar to those of the aniline homopolymer. The VAn-Si surface was also funtionalized by the electroless plating of copper. Not only did the VAn layer provide chemisorption sites for the palladium catalyst, in the absence of prior sensitization by SnCl2, during the electroless plating process, it also served as an adhesion promotion layer and a low-temperature diffusion barrier for the electrolessly deposited copper. Finally, micropatterning of the grafted PAn and of the electrolessly deposited copper were demonstrated on the resist-patterned VAn-Si surfaces.

Surface functionalization of polypyrrole film with glucose oxidase and viologen.

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).