ToF-SIMS analysis of poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) ultrathin adlayers.

Understanding of the interfacial chemistry of ultrathin polymeric adlayers is fundamentally important in the context of establishing quantitative design rules for the fabrication of nonfouling surfaces in various applications such as biomaterials and medical devices. In this study, seven poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-PMOXA) copolymers with grafting density (number of PMOXA chains per lysine residue) 0.09, 0.14, 0.19, 0.33, 0.43, 0.56, and 0.77, respectively, were synthesized and characterized by means of nuclear magnetic resonance spectroscopy (NMR). The copolymers were then adsorbed on Nb2O5 surfaces. Optical waveguide lightmode spectroscopy method was used to monitor the surface adsorption in situ of these copolymers and provide information on adlayer masses that were then converted into PLL and PMOXA surface densities. To investigate the relationship between copolymer bulk architecture (as shown by NMR data) and surface coverage as well as surface architecture, time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed. Furthermore, ToF-SIMS method combined with principal component analysis (PCA) was used to verify the protein resistant properties of PLL-PMOXA adlayers, by thorough characterization before and after adlayer exposure to human serum. ToF-SIMS analysis revealed that the chemical composition as well as the architecture of the different PLL-PMOXA adlayers indeed reflects the copolymer bulk composition. ToF-SIMS results also indicated a heterogeneous surface coverage of PLL-PMOXA adlayers with high grafting densities higher than 0.33. In the case of protein resistant surface, PCA results showed clear differences between protein resistant and nonprotein-resistant surfaces. Therefore, ToF-SIMS results combined with PCA confirmed that the PLL-PMOXA adlayer with brush architecture resists protein adsorption. However, low increases of some amino acid signals in ToF-SIMS spectra were detected after the adlayer has been exposed to human serum.

Antimicrobial properties of 8-hydroxyserrulat-14-en-19-oic acid for treatment of implant-associated infections.

Treatment options are limited for implant-associated infections (IAI) that are mainly caused by biofilm-forming staphylococci. We report here on the activity of the serrulatane compound 8-hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. EN4 elicited antimicrobial activity toward various Gram-positive bacteria but not to Gram-negative bacteria. It showed a similar bactericidal effect against logarithmic-phase, stationary-phase, and adherent Staphylococcus epidermidis, as well as against methicillin-susceptible and methicillin-resistant S. aureus with MICs of 25 to 50 µg/ml and MBCs of 50 to 100 µg/ml. The bactericidal activity of EN4 was similar against S. epidermidis and its Δica mutant, which is unable to produce polysaccharide intercellular adhesin-mediated biofilm. In time-kill studies, EN4 exhibited a rapid and concentration-dependent killing of staphylococci, reducing bacterial counts by >3 log(10) CFU/ml within 5 min at concentrations of >50 µg/ml. Investigation of the mode of action of EN4 revealed membranolytic properties and a general inhibition of macromolecular biosynthesis, suggesting a multitarget activity. In vitro-tested cytotoxicity on eukaryotic cells was time and concentration dependent in the range of the MBCs. EN4 was then tested in a mouse tissue cage model, where it showed neither bactericidal nor cytotoxic effects, indicating an inhibition of its activity. Inhibition assays revealed that this was caused by interactions with albumin. Overall, these findings suggest that, upon structural changes, EN4 might be a promising pharmacophore for the development of new antimicrobials to treat IAI.

Supported lipopolysaccharide bilayers.

In this report, the formation of supported lipopolysaccharide bilayers (LPS-SLBs) is studied with extracted native and glycoengineered LPS from Escherichia coli ( E. coli ) and Salmonella enterica sv typhimurium ( S. typhimurium ) to assemble a platform that allows measurement of LPS membrane structure and the detection of membrane tethered saccharide-protein interactions. We present quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP) characterization of LPS-SLBs with different LPS species, having, for example, different molecular weights, that show successful formation of SLBs through vesicle fusion on SiO(2) surfaces with LPS fractions up to 50 wt %. The thickness of the LPS bilayers were investigated with AFM force-distance measurements which showed only a slight thickness increase compared to pure POPC SLBs. The E. coli LPS were chosen to study the saccharide-protein interaction between the Htype II glycan epitope and the Ralstonia solanacearum lectin (RSL). RSL specifically recognizes fucose sugars, which are present in the used Htype II glycan epitope and absent in the epitopes LPS1 and EY2. We show via fluorescence microscopy that the specific, but weak and multivalent interaction can be detected and discriminated on the LPS-SLB platform.

Micropatterning of functional conductive polymers with multiple surface chemistries in register.

A versatile procedure is presented for fast and efficient micropatterning of multiple types of covalently bound surface chemistry in perfect register on and between conductive polymer microcircuits. The micropatterning principle is applied to several types of native and functionalized PEDOT (poly(3,4-ethylenedioxythiophene)) thin films. The method is based on contacting PEDOT-type thin films with a micropatterned agarose stamp containing an oxidant (aqueous hypochlorite) and applying a nonionic detergent. Where contacted, PEDOT not only loses its conductance but is entirely removed, thereby locally revealing the underlying substrate. Surface analysis showed that the substrate surface chemistry was fully exposed and not affected by the treatment. Click chemistry could thus be applied to selectively modify re-exposed alkyne and azide functional groups of functionalized polystyrene substrates. The versatility of the method is illustrated by micropatterning cell-binding RGD-functionalized PEDOT on low cell-binding PMOXA (poly(2-methyl-2-oxazoline)) to produce cell-capturing microelectrodes on a cell nonadhesive background in a few simple steps. The method should be applicable to a wide range of native and chemically functionalized conjugated polymer systems.

Polyoxazolines for nonfouling surface coatings--a direct comparison to the gold standard PEG.

The prevention of surface fouling is becoming increasingly important for the development of anti-infective medical implants, biosensors with improved signal-to-noise ratios, and low-fouling membranes to name a few examples. We review a direct comparison of poly(ethylene glycol), the gold standard polymer to impart surfaces with nonfouling properties, to an alternative polymer, poly(2-methyl-2-oxazoline) (PMOXA), and show that both polymers are equally excellent in rendering surfaces nonfouling while PMOXA coatings are more stable in oxidative environments. We discuss prerequisites for the fabrication of nonfouling surface coatings and implications for the polymer choice according to application requirements.

Triggered release from liposomes through magnetic actuation of iron oxide nanoparticle containing membranes.

The ideal nanoscale drug delivery vehicle allows control over the released dose in space and time. We demonstrate that this can be achieved by stealth liposomes comprising self-assembled superparamagnetic iron oxide nanoparticles (NPs) individually stabilized with palmityl-nitroDOPA incorporated in the lipid membrane. Alternating magnetic fields were used to control timing and dose of repeatedly released cargo from such vesicles by locally heating the membrane, which changed its permeability without major effects on the environment.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties.

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

Pleckstrin homology-phospholipase C-δ1 interaction with phosphatidylinositol 4,5-bisphosphate containing supported lipid bilayers monitored in situ with dual polarization interferometry.

We have determined the kinetics and affinity of binding of PH-PLCδ(1) to the PIP(2) headgroup lipids using an optical surface-sensitive technique in a time-resolved manner. The use of dual polarization interferometry to probe supported lipid bilayers (SLBs) of different compositions allowed determination of accurate affinity constants and a layer structure of the peptide binding to the model membrane platform. In addition, the platform enabled us to monitor the detailed adsorption kinetics characterized by a strong initial electrostatic attraction of the peptide to the SLB surface followed by rearrangement and loss of possibly clustered peptides upon specific binding to the phosphoinositide headgroup. These kinetics differed substantially from adsorption kinetics for nonspecific binding to similarly charged control SLBs.

Formation of nanopore-spanning lipid bilayers through liposome fusion.

Self-assembly of nanopore-spanning lipid bilayers (npsLBs) paves the way toward chip-based integrated membrane protein biosensing. We present a novel approach to analyze the formation of npsLB at individual nanopores using quantitative analysis of high-resolution microscopy images. From this analysis we derive necessary conditions for the formation of npsLBs on nanopore arrays by liposome fusion and discuss the limitations of the process as a function of nanopore geometry, lipid membrane properties, and surface interaction. Most importantly, applying liposomes with diameters larger than the nanopore is demonstrated to be a necessary but not sufficient condition for npsLB formation. A theoretical model is used to discuss and explain this experimental finding.

"Docking sites": nanometer-scale organization of a reactive, protein-resistant, graft copolymer-based interface for macromolecule immobilization.

The signal-to-noise ratio of a sensor system is determined by the affinity of its active component for the analyte on one hand and its inertness with respect to unrelated stimuli (noise) on the other hand. Nonspecific interactions between the environment and biosensor components (typically constructed from glass, silica, or transition metal oxides) result in nonspecific adsorption onto the latter and constitute a major source of noise. We have previously introduced a polymeric interface for preventing nonspecific adsorption while allowing for high-affinity, specific interactions. It is based on the coassembly of biotinylated and nonbiotinylated poly(l-lysine)-graft-poly(ethylene glycol) from aqueous solutions to negatively charged surfaces, such as Nb(2)O(5). In this study, we investigated by atomic force microscopy the nanoscale organization of this interface for each individual step involved in the preparation of a bioactive interface: polymer adsorption, loading with streptavidin, and binding of biotinylated vesicles.

An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes.

This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC), and whether it enhances their post expansion chondrogenic capacity. HAC/substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS) with a poly(ethylene glycol) (PEG) based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD). As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold), maintained higher type II collagen mRNA expression (4.9-fold) and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10%) and proliferation rate (0.56±0.04 doublings/day), as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio). RDG (bioinactive peptide) and PEG (no peptide ligand) controls yielded drastically reduced attachment efficiency (lower than 11%) and proliferation (lower than 0.20 doublings/day). Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue.

Poly(ethylene glycol) adlayers immobilized to metal oxide substrates through catechol derivatives: influence of assembly conditions on formation and stability.

We have investigated five different poly(ethylene glycol) (PEG, 5 kDa) catechol derivatives in terms of their spontaneous surface assembly from aqueous solution, adlayer stability, and resistance to nonspecific blood serum adsorption as a function of the type of catechol-based anchor, assembly conditions (temperature, pH), and type of substrate (SiO(2), TiO(2), Nb(2)O(5)). Variable-angle spectroscopic ellipsometry (VASE) was used for layer thickness evaluation, X-ray photoelectron spectroscopy (XPS) for layer composition, and ultraviolet-visible optical spectroscopy (UV-vis) for cloud point determination. Polymer surface coverage was influenced by the type of catechol anchor, type of the substrate, as well as pH and temperature (T) of the assembly solution. Furthermore, it was found to be highest for T close to the cloud point (T(CP)) and pH of the assembly solution close to pK(a1) (dissociation constant of the first catechol hydroxy group) of the polymer and to the isoelectric point (IEP) of the substrate. T(CP) turned out to depend on not only the ionic strength of the assembly solution, but also the type of catechol derivative and pH. PEG-coating dry thickness above 10 A correlated with low serum adsorption. We therefore conclude that optimum coating protocols for catechol-based polymer assembly at metal oxide interfaces have to take into account specific physicochemical properties of the polymer, anchor, and substrate.

Self-assembly of poly(ethylene glycol)-poly(alkyl phosphonate) terpolymers on titanium oxide surfaces: synthesis, interface characterization, investigation of nonfouling properties, and long-term stability.

This contribution deals with the self-assembling of a terpolymer on titanium oxide (TiO(2)) surface. The polymer structure was obtained by polymerization of different methacrylates, i.e., alkyl-phosphonated, butyl and PEG methacrylate, in the presence of a chain transfer agent. The resulting PEG-poly(alkyl phosphonate) material, characterized mainly by SEC and NMR, self-organized at the interface of TiO(2). AR-XPS demonstrated the binding of phosphonate groups to TiO(2) substrate and the formation of a PEG-brush layer at the outermost part of the system. The stability of this terpolymer adlayer, after exposure to solutions of pH 2, 7.4, and 9 up to 3 weeks, was evaluated quantitatively by XPS and ellipsometry. We demonstrated an overall stability improvements of this coating against desorption in contact with aqueous solutions in comparison with reference self-assembly systems. Finally, the PEG-terpolymer adlayer proved to impart to TiO(2) substrate antifouling properties when exposed to full blood serum.

One-step method for generating PEG-like plasma polymer gradients: chemical characterization and analysis of protein interactions.

In this work we report a one-step method for the fabrication of poly(ethylene glycol) PEG-like chemical gradients, which were deposited via continuous wave radio frequency glow discharge plasma polymerization of diethylene glycol dimethyl ether (DG). A knife edge top electrode was used to produce the gradient coatings at plasma load powers of 5 and 30 W. The chemistry across the gradients was analyzed using a number of complementary techniques including spatially resolved synchrotron source grazing incidence FTIR microspectroscopy, X-ray photoelectron spectroscopy (XPS) and synchrotron source near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Gradients deposited at lower load power retained a higher degree of monomer like functionality as did the central region directly underneath the knife edge electrode of each gradient film. Surface derivatization experiments were employed to investigate the concentration of residual ether units in the films. In addition, surface derivatization was used to investigate the reactivity of the gradient films toward primary amine groups in a graft copolymer of poly (L-lysine) and poly(ethylene glycol) (PLL-g-PEG copolymer) which was correlated to residual aldehyde, ketone and carboxylic acid functionalities within the films. The protein adsorption characteristics of the gradients were analyzed using three proteins of varying size and charge. Protein adsorption varied and was dependent on the chemistry and the physical properties (such as size and charge) of the proteins. A correlation between the concentration of ether functionality and the protein fouling characteristics along the gradient films was observed. The gradient coating technique developed in this work allows for the efficient and high-throughput study of biomaterial gradient coating interactions.

Ru(II) glycodendrimers as probes to study lectin-carbohydrate interactions and electrochemically measure monosaccharide and oligosaccharide concentrations.

We describe a novel platform on which to study carbohydrate-protein interactions based on ruthenium(II) glycodendrimers as optical and electrochemical probes. Using the prototypical concanavalin A (ConA)-mannose lectin-carbohydrate interaction as an example, oligosaccharide concentrations were electrochemically monitored. The displacement of the Ru(II) complex from lectin-functionalized gold surfaces was repeatedly regenerated. This new platform presents a method to monitor many different complex sugars in parallel.

Ultrastable iron oxide nanoparticle colloidal suspensions using dispersants with catechol-derived anchor groups.

We have found catechol-derivative anchor groups which possess irreversible binding affinity to iron oxide and thus can optimally disperse superparamagnetic nanoparticles under physiologic conditions. This not only leads to ultrastable iron oxide nanoparticles but also allows close control over the hydrodynamic diameter and interfacial chemistry. The latter is a crucial breakthrough to assemble functionalized magnetic nanoparticles, e.g., as targeted magnetic resonance contrast agents.

Self-assembly of iron oxide-poly(ethylene glycol) core-shell nanoparticles at liquid-liquid interfaces.

Nanoparticles (NPs) play an increasingly important role in the fabrication of functional advanced materials. Two major steps need to be carried out in order to achieve control of the material properties. First of all, the properties of the single NPs have to be under control, especially in relation to colloidal stability; aggregation and corrosion negate all the benefits associated to the nanoscopic dimensions. Secondly, the assembly process has to be controlled to achieve a material with the desired properties. We propose here to use stabilized ceramic NPs consisting of a magnetite core, coated by a poly(ethylene glycol) (PEG) shell and study their assembly at polar/ non-polar liquid interfaces, en route to fabricating functional NP membranes. These NPs show extraordinary stability in aqueous solutions achieved by anchoring linear PEG chains through an end-terminating nitroDOPA group to their surface. Furthermore, the core and shell sizes of these NPs can be independently varied with ease. We first describe the details of the NP synthesis and stabilization in bulk solutions, discussing the PEG molecular weight needed to achieve bulk stability. Subsequently, we demonstrate self-assembly of these particles at liquid-liquid interfaces (SALI) into monolayers of stable properties. SALI has been chosen as path for the assembly given its suitability for fabricating two-dimensional materials. We report here results from pendant drop tensiometry which illustrate the kinetics of NP adsorption at the liquid-liquid interface and highlight the role played by the molecular weight of the PEG shell in the interfacial assembly. In particular we show that the requisites to ensure particle stability at a liquid interface are more stringent compared to the bulk case.

Formation of supported lipid bilayers on indium tin oxide for dynamically-patterned membrane-functionalized microelectrode arrays.

Supported lipid bilayers (SLBs) are ideal platforms for the study of membrane proteins and function. Assembly of functional SLBs in an array format would lead to a breakthrough in high-throughput screening of membrane-associated processes, e.g., drugs binding to transmembrane proteins. We report the formation of SLBs from the rupture of anionic vesicles in the presence of Ca(2+) ions on ITO-coated surfaces and characterise the assembly and SLB properties. Furthermore, the formation, manipulation and regeneration of SLBs adsorbed on ITO microelectrode array spots using an electric potential switch are demonstrated. This platform enables addressable assembly and the study of electrochemically mediated membrane processes in a microarray format which can be regenerated in situ.

Furanone at subinhibitory concentrations enhances staphylococcal biofilm formation by luxS repression.

Brominated furanones from marine algae inhibit multicellular behaviors of gram-negative bacteria such as biofilm formation and quorum sensing (QS) without affecting their growth. The interaction of furanone with QS in gram-positive bacteria is unknown. Staphylococci have two QS systems, agr and luxS, which lower biofilm formation by two different pathways, RNAIII upregulation and bacterial detachment, and polysaccharide intercellular adhesin (PIA) reduction, respectively. We synthesized natural furanone compound 2 [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] from Delisea pulchra and three analogues to investigate their effect on biofilm formation in gram-positive bacteria. Compound 2, but not the analogues, enhanced the biofilms of Staphylococcus epidermidis 1457 and 047 and of S. aureus Newman at concentrations between 1.25 and 20 microM. We show the growth inhibition of S. epidermidis and S. aureus by free furanone and demonstrate bactericidal activity. An induction of biofilm occurred at concentrations of 10 to 20% of the MIC and correlated with an increase in PIA. The biofilm effect was agr independent. It was due to interference with luxS, as shown by reduced luxS expression in the presence of compound 2 and independence of the strong biofilm formation in a luxS mutant upon furanone addition. Poly(l-lysine)-grafted/poly(ethylene glycol)-grafted furanone was ineffective on biofilm and not bactericidal, indicating the necessity for free furanone. Free furanone was similarly toxic for murine fibroblasts as for staphylococci, excluding a therapeutic application of this compound. In summary, we observed a biofilm enhancement by furanone in staphylococci at subinhibitory concentrations, which was manifested by an increase in PIA and dependent on luxS.

Surface functionalization of single superparamagnetic iron oxide nanoparticles for targeted magnetic resonance imaging.

Magnetic resonance imaging (MRI), a non-invasive, non-radiative technique, is thought to lead to cellular or even molecular resolution if optimized targeted MR contrast agents are introduced. This would allow diagnosing progressive diseases in early stages. Here, it is shown that the high binding affinity of poly(ethylene glycol)-gallol (PEG-gallol) allows freeze drying and re-dispersion of 9 +/- 2-nm iron oxide cores individually stabilized with approximately 9-nm-thick stealth coatings, yielding particle stability for at least 20 months. Particle size, stability, and magnetic properties of PEGylated particles are compared to Feridex, a commercially available untargeted negative MR contrast agent. Biotin-PEG(3400)-gallol/methoxy-PEG(550)-gallol stabilized nanoparticles are further functionalized with biotinylated human anti-VCAM-1 antibodies using the biotin-neutravidin linkage. Binding kinetics and excellent specificity of these nanoparticles are demonstrated using quartz crystal microbalance with dissipation monitoring (QCM-D). These MR contrast agents can be functionalized with any biotinylated ligand at controlled ligand surface density, rendering them a versatile research tool.