Surface analysis of PEGylated nano-shields on nanoparticles installed by hydrophobic anchors.

PURPOSE: This work describes a method for functionalisation of nanoparticle surfaces with hydrophilic "nano-shields" and the application of advanced surface characterisation to determine PEG amount and accumulation at the outmost 10 nm surface that is the predominant factor in determining protein and cellular interactions. METHODS: Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared with a hydrophilic PEGylated "nano-shield" inserted at different levels by hydrophobic anchoring using either a phospholipid-PEG conjugate or the copolymer PLGA-block-PEG by an emulsification/diffusion method. Surface and bulk analysis was performed including X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR) and zeta potential. Cellular uptake was investigated in RAW 264.7 macrophages by flow cytometry. RESULTS: Sub-micron nanoparticles were formed and the combination of (NMR) and XPS revealed increasing PEG levels at the particle surface at higher PLGA-b-PEG copolymer levels. Reduced cellular interaction with RAW 264.7 cells was demonstrated that correlated with greater surface presentation of PEG. CONCLUSION: This work demonstrates a versatile procedure for decorating nanoparticle surfaces with hydrophilic "nano-shields". XPS in combination with NMR enabled precise determination of PEG at the outmost surface to predict and optimize the biological performance of nanoparticle-based drug delivery.

Physicochemical characterization of fish protein adlayers with bacteria repelling properties.

Materials coated with aqueous fish protein extracts can reduce bacterial adhesion, but the mechanism behind the observed effect is not fully understood. In this study we explore the physicochemical properties of fish muscle protein adlayers on four substrates: gold, stainless steel, polystyrene and silicon dioxide. The aims were (i) to determine if the anti-adhesive effect is independent of the underlying substrate chemistry, (ii) to link the physicochemical properties of the adlayer to its ability to repel bacteria, and (iii) to elucidate the mechanism behind this effect. The main proteins on all surfaces were the muscle proteins troponin, tropomyosin, and myosin, and the lipid binding protein apolipoprotein. The quantity, viscoelasticity, and hydration of the protein adlayers varied greatly on the different substrates, but this variation did not affect the bacterial repelling properties. Our results imply that these proteins adsorb to all substrates and provide a steric barrier towards bacterial adhesion, potentially providing a universal antifouling solution.

Electrostatic and capillary force directed tunable 3D binary micro- and nanoparticle assemblies on surfaces.

We report a simple, rapid and cost-effective method based on evaporation induced assembly to grow 3D binary colloidal assemblies on a hydrophobic/hydrophilic substrate by simple drop casting. The evaporation of a mixed colloidal drop results in ring-like or uniform area deposition depending on the concentration of particles, and thus assembly occurs at the periphery of a ring or uniformly all over the drop area. Binary colloidal assemblies of different crystal structure are successfully prepared over a wide range of size ratios (γ = small/large) from 0.06 to 0.30 by tuning the γ of the micro- and nanoparticles used during assembly. The growth mechanism of 3D binary colloidal assemblies is investigated and it is found that electrostatic forces facilitate assembly formation until the end of the evaporation process, with capillary forces also playing a role. In addition, the effects of solvent type, humidity, and salt concentration on crystal formation and ordering behaviour are also examined. Furthermore, long range, highly ordered binary colloidal assemblies can be fabricated by the choice of a low conducting solvent combined with evaporation induced assembly.

Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS.

Protein adsorption onto calcium phosphate (Ca-P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation mass spectrometry (Surface-MALDI-MS) as a technique for the direct detection of foetal bovine serum (FBS) proteins adsorbed to hybrid calcium phosphate/titanium dioxide surfaces produced by a novel radio frequency (RF) magnetron sputtering method incorporating in situ annealing between 500°C and 700°C during deposition. XRD and XPS analysis indicated that the coatings produced at 700°C were hybrid in nature, with the presence of Ca-P and titanium dioxide clearly observed in the outer surface layer. In addition to this, the Ca/P ratio was seen to increase with increasing annealing temperature, with values of between 2.0 and 2.26 obtained for the 700°C samples. After exposure to FBS solution, surface-MALDI-MS indicated that there were significant differences in the protein patterns as shown by unique peaks detected at masses below 23.1 kDa for the different surfaces. These adsorbates were assigned to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role in subsequent bioactivity of the materials.

Surface modification of chromatography adsorbents by low temperature low pressure plasma.

In this study we show how low temperature glow discharge plasma can be used to prepare bi-layered chromatography adsorbents with non-adsorptive exteriors. The commercial strong anion exchange expanded bed chromatography matrix, Q HyperZ, was treated with plasmas in one of two general ways. Using a purpose-designed rotating reactor, plasmas were employed to either: (i) remove anion exchange ligands at or close to the exterior surface of Q HyperZ, and replace them with polar oxygen containing functions ('plasma etching and oxidation'); or (ii) bury the same surface exposed ligands beneath thin polymer coatings ('plasma polymerization coating') using appropriate monomers (vinyl acetate, vinyl pyrrolidone, safrole) and argon as the carrier gas. X-ray photoelectron spectroscopy analysis (first ∼10 nm depth) of Q HyperZ before and after the various plasma treatments confirmed that substantial changes to the elemental composition of Q HyperZ's exterior had been inflicted in all cases. The atomic percent changes in carbon, nitrogen, oxygen, yttrium and zirconium observed after being exposed to air plasma etching were entirely consistent with: the removal of pendant Q (trimethylammonium) functions; increased exposure of the underlying yttrium-stabilised zirconia shell; and introduction of hydroxyl and carbonyl functions. Following plasma polymerization treatments (with all three monomers tested), the increased atomic percent levels of carbon and parallel drops in nitrogen, yttrium and zirconium provided clear evidence that thin polymer coats had been created at the exteriors of Q HyperZ adsorbent particles. No changes in adsorbent size and surface morphology, nor any evidence of plasma-induced damage could be discerned from scanning electron micrographs, light micrographs and measurements of particle size distributions following 3 h exposure to air (220 V; 35.8 W L(-1)) or 'vinyl acetate/argon' (170 V; 16.5 W L(-1)) plasmas. Losses in bulk chloride exchange capacity before and after exposure to plasmas enabled effective modification depths within hydrated Q HyperZ adsorbent particles to be calculated as 0.2-1.2 µm, depending on the conditions applied. The depth of plasma induced alteration was strongly influenced by the power input and size of the treated batch, i.e. dropping the power or increasing the batch size resulted in reduced plasma penetration and therefore shallower modification. The selectivity of 'surface vs. core' modification imparted to Q HyperZ by the various plasma treatments was evaluated in static and dynamic binding studies employing appropriate probes, i.e. plasmid DNA, sonicated calf thymus DNA and bovine serum albumin. In static binding studies performed with adsorbents that had been exposed to plasmas at the 5 g scale (25 g L(-1) of plasma reactor), the highest 'surface/core' modification selectivity was observed for Q HyperZ that had been subjected to 3 h of air plasma etching at 220 V (35.8 W L(-1)). This treatment removed ∼53% of 'surface' DNA binding at the expense of a 9.3% loss in 'core' protein binding. Even more impressive results were obtained in dynamic expanded bed adsorption studies conducted with Q HyperZ adsorbents that had been treated with air (220 V, 3 h) and 'vinyl acetate/argon' (170 V, 3 h) plasmas at 10.5 g scale (52.5 g L(-1) of plasma reactor). Following both plasma treatments: the 10% breakthrough capacities of the modified Q HyperZ adsorbents towards 'surface' binding DNA probes dropped very significantly (30-85%); the DNA induced inter-particle cross-linking and contraction of expanded beds observed during application of sonicated DNA on native Q HyperZ was completely eradicated; but the 'core' protein binding performance remained unchanged cf. that of the native Q HyperZ starting material.

Tunable 3D and 2D polystyrene nanoparticle assemblies using surface wettability, low volume fraction and surfactant effects.

Polymer-based nanopatterning on metal surfaces is of increasing importance to a number of applications, including biosensors, bioelectronic devices and medical implants. Here we show that polycrystalline gold surfaces can be functionalized with monocomponent nanoparticle (NP) assemblies by a simple drop deposition method. Ordered 3D hexagonal close-packed structures consisting of 350 nm polystyrene (PS) NPs on hydrophobically modified gold surfaces from solutions of very low volume fraction (varphi = 0.0006) were obtained as a result of capillary force induced self-assembly, whilst 2D self-assembly of PS NPs was generated over large area on hydrophilic gold and TiO(2) surfaces by spin coating. Furthermore, we show that when Triton X-100 is added to the PS NP suspending medium longer range ordering is obtained. Our observations may initiate interesting applications in the areas of nanoengineering of metal-based sensors and as a means to design new nanostructures for biocompatible implant surfaces.

Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films.

AIMS: Preconditioning of stainless steel with aqueous cod muscle extract significantly impedes subsequent bacterial adhesion most likely due to repelling effects of fish tropomyosin. The purpose of this study was to determine if other food conditioning films decrease or enhance bacterial adhesion to stainless steel. METHODS AND RESULTS: Attachment of Pseudomonas fluorescens AH2 to stainless steel coated with water-soluble coatings of animal origin was significantly reduced as compared with noncoated stainless steel or stainless steel coated with laboratory substrate or extracts of plant origin. Coating with animal extracts also decreases adhesion of other food-relevant bacteria. The manipulation of adhesion was not attributable to growth inhibitory effects. Chemical analysis revealed that the stainless steels were covered by homogenous layers of adsorbed proteins. The presence of tropomyocin was indicated by appearance of proteins with similar molecular weight based in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in several extracts that reduced adhesion but also extracts not containing this protein reduced bacterial adhesion, indicating that several molecular species may be involved in the phenomenon. CONCLUSIONS: It is a common perception that food materials facilitate bacterial adhesion to surfaces; however, this study demonstrates that aqueous coatings of food origin may actually reduce bacterial adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: Compounds from food extracts may potentially be used as nontoxic coatings to reduce bacterial attachment to inert surfaces.

Effect of nitinol wire surface properties on albumin adsorption.

The superelastic, shape memory alloy nitinol ( approximately 50% nickel and approximately 50% titanium) is an important medical device material used for stent applications. However, the role specific surfaces properties have in protein adsorption remain controversial. In this study the effects of nitinol wire surface roughness, hydrophobicity and elemental composition upon albumin adsorption are investigated. In particular, we demonstrate that the technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the so-called surface mode can be used for the direct detection of albumin on the wire surfaces. In addition, albumin adsorbing to the wires was determined by using (125)I-labelled albumin. Albumin was detected on all wire samples. Surface roughness and hydrophobicity appeared to have no effect on albumin adsorption. There was however a clear correlation between the surface nickel and oxygen concentration and the amount of albumin adsorbed. Samples with higher levels of nickel and less oxygen in the surface oxide layer of the wires showed increased albumin adsorption, which could lead to improved biocompatibility. However, nickel is a toxic substance and can cause many adverse effects on humans, and thus nitinol with a slightly enriched surface nickel concentration that does not exhibit nickel release may have potential as a medical device material.

The method of surface PEGylation influences leukocyte adhesion and activation.

The influence of different surface modifications with poly(ethyleneglycol) (PEG) layers on the adsorption of fibrinogen and the adhesion and activation of macrophage-like human leukocytes was investigated. Poly(ethylene terephthalate) (PET) was modified using pulsed AC plasma polymerization with two types of starting monomers to generate: 1) a reactive acid surface using maleic anhydride (MAH) as monomer, and 2) a PEG-like surface using diethyleneglycol methyl vinyl ether (DEGVE) as monomer. The MAH surface was used as a reactive platform to graft linear chains of non-fouling mPEG via an intermediate layer of poly(ethyleneimine) (PEI) under lower critical solution temperature (LCST) conditions of the mPEG. The DEGVE monomer is used to create PEG-like layers by use of low power plasma conditions. The ability of the surfaces to resist protein adsorption was investigated quantitatively using (125)I-radiolabeled human fibrinogen, and the conformation of the adsorbed protein was tested using an anti-fibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. The results showed that PEGylated surfaces adsorbed significantly less (up to 90% less) fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mPEG layers than on the PEG-like plasma polymer surfaces. Adhesion of in-vitro differentiated macrophage-like U937 cells was reduced on both the PEG-like plasma polymer surfaces and the linear mPEG layers compared to the unmodified PET surface, but cells adhering to the PEG-like plasma polymer surfaces secreted less tumor necrosis factor-alpha (TNF-alpha) than cells adhering to the linear mPEG layers. In conclusion, the method for preparing non-fouling surfaces for long-term implanted devices influence surface-induced cellular responses of the host.

Minimization of protein adsorption on poly(vinylidene fluoride).

Surfaces covered with polyethylene glycol (PEG) have been shown to be biocompatible because PEG yields nonimmunogenicity, nonantigenicity and protein rejection. To produce a biocompatible surface coating, we have developed a method for grafting PEG onto modified poly(vinylidene fluoride) (PVDF) films. The first step was to create carboxy groups on the PVDF surface following covalente coupling of polyethylenimine (PEI) to achieve high density of amino groups. These surface amines were reacted with formyl-terminated PEG's with various molecular weight. The modified PVDF surface was characterized by means of static contact angle measurements, infrared (IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The influence of the chain length on lysozyme repellence was investigated by means of surface-MALDI-Tof mass spectrometry (Surface-MALDI-Tof-MS). Lysozyme adsorption was significantly suppressed on the PEG 5000 modified PVDF surface.

Matrix-assisted laser desorption ionization mass spectrometry detection of proteins adsorbed in vivo onto contact lenses.

Identification of the biomolecules that form the first adsorbed monolayer, which thus effect "interface conversion", in competitive adsorption from multicomponent biological solutions can be challenging because of limitations in mass resolution and sensitivity of established techniques. In this study matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry is developed and applied as a novel surface analytical method to enable analysis of adsorbed multicomponent biomolecule layers directly on the biomaterial surfaces. We show that proteins adsorbed in vivo (on human eyes) on contact lenses can be detected rapidly and conveniently by the diagnostic highly resolved mass signals recorded by MALDI mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers at levels substantially below monolayer coverage. Identification is done by comparison with molecular masses of known proteins. Specifically, it is shown that in addition to lysozyme, other low molecular weight proteins adsorb from human tear fluid onto contact lenses; these proteins had not been detected in earlier studies using other techniques.

Direct detection of proteins adsorbed on synthetic materials by matrix-assisted laser desorption ionization-mass spectrometry.

The irreversible accumulation of biological material on synthetic surfaces ("biofouling") adversely affects for instance contact lenses, implantable biomedical devices, biosensors, water purification, transport and storage systems, and marine structures. It is shown here that proteins adsorbed on contact lenses can be detected directly, rapidly, and conveniently, with high sensitivity, by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers on samples retrieved from clinical usage and in vitro protein adsorption studies, at levels substantially below monolayer coverage. Identification of the detected biological molecules can be done by comparison of the detected mass peaks with known protein molecular masses or with spectra recorded of pure compounds or by separate biochemical assays. The MALDI mass spectra recorded on different contact lenses contain peaks assignable to lysozyme and a number of smaller proteins. Such sensitive characterization of the early stages of biofouling enhances the understanding of protein/materials interactions and assists in designing guided strategies toward control of biological adsorption processes.

Characterization of Ferritin Adsorption onto Gold

The adsorption of ferritin from phosphate-buffered saline (PBS) onto gold has been examined using a quartz crystal microbalance (QCM), surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Application of these techniques allows elucidation of the surface coverage and layer thickness of ferritin on gold. The kinetics of ferritin adsorption onto gold were investigated by monitoring the resonant frequency shift of a QCM. Adsorption isotherms for ferritin in PBS and in air have been measured by QCM. These isotherms suggest less than monolayer coverage of ferritin on gold. A layer thickness of 4.8 ± 0.2 nm was calculated for the dry ferritin film from QCM data. Measurements of the shift in the reflectivity of light at a fixed angle close to the SPR plasmon resonance were also used to follow the kinetics of ferritin adsorption. Full SPR curves were measured in PBS solution and air and were used to determine the effective thickness of the ferritin layer in both environments. The ferritin layer on gold from SPR data was found to be twice as thick when measured in PBS as it was for a dry film. This difference in thickness is attributed to shrinkage of ferritin with drying. Angle-resolved XPS measurements on a dry ferritin film (preadsorbed on gold) yield a ferritin thickness of 4.7 ± 0.5 nm, a value in good agreement with those determined from QCM and SPR. QCM, SPR, and XPS all yield a surface coverage of 6.3 ± 0.7 mg m-2 for dry ferritin layers on gold. AFM enabled examination of the topography of ferritin adsorbed on gold on the nanometer scale and confirmed that ferritin forms an incomplete monolayer. In all cases, ferritin was found to be irreversibly adsorbed to gold and to form a stable protein layer, thus making it well suited as a biological receptor layer for immunosensing applications.