Protein resistance of titanium oxide surfaces modified by biologically inspired mPEG-DOPA

In the present study, we have utilized X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry (ELM), and optical waveguide lightmode spectroscopy (OWLS) to examine the surface adsorption and protein resistance behavior of bio-inspired polymers consisting of poly(ethylene glycol) (PEG) conjugated to peptide mimics of mussel adhesive proteins. Peptides containing up to three residues of 3,4-dihydroxyphenylalanine (DOPA), a key component of mussel adhesive proteins, were conjugated to monomethoxy-terminated PEG polymers. These mPEG-DOPA polymers were found to be highly adhesive to TiO2 surfaces, with quantitative XPS analysis providing useful insight into the binding mechanism. Additionally, the antifouling properties of immobilized PEG were reflected in the excellent resistance of mPEG-DOPA-modified TiO2 surfaces to protein adsorption. Measurements of mPEG-DOPA and human serum adsorption were related in terms of ethylene glycol (EG) surface density and serum mass adsorbed and demonstrated a threshold of approximately 15-20 EG/nm2, above which substantially little protein adsorbs. With respect to surface density of adsorbed PEG and the associated nonfouling behavior of the adlayers, strong parallels exist between the nonfouling properties of the surface-bound mPEG-DOPA polymers and PEG polymers immobilized to surfaces using other approaches. Peptide anchors containing three DOPA residues resulted in PEG surface densities higher than those achieved using several existing PEG immobilization strategies, suggesting that peptide mimics of mussel adhesive proteins may be useful for achieving high densities of protein-resistant polymers on surfaces.     

Protein resistant polyurethane surfaces by chemical grafting of PEO: amino-terminated PEO as grafting reagent

The objective of this work was to gain a better understanding of the mechanism of resistance to protein adsorption of surfaces grafted with poly(ethylene oxide) (PEO). A polyurethane-urea was used as a substrate to which PEO was grafted. Grafting was carried out by introducing isocyanate groups into the surface followed by reaction with amino-terminated PEO. Surfaces grafted with PEO of various chain lengths (PUU-NPEO) were prepared and characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). XPS data indicated higher graft densities on the PUU-NPEO surfaces than on analogous surfaces prepared using hydroxy-PEO (PUU-OPEO) as reported previously [J.G. Archambault, J.L. Brash, Colloids Surf. B: Biointerf. 33 (2004) 111-120]. Protein adsorption experiments using radiolabeled myoglobin, concanavalin A, albumin, fibrinogen and ferritin as single proteins in buffer showed that adsorption was reduced on the PEO-grafted surfaces by up to 95% compared to the control. Adsorption decreased with increasing PEO chain length and reached a minimum at a PEO MW of 2000. Adsorption levels on surfaces with 5000 and 2000 MW grafts were similar. There was no clear effect of protein size on resistance to protein adsorption. Adsorption on the PUU-NPEO surfaces was significantly lower than on the corresponding PUU-OPEO surfaces, again suggesting higher graft densities on the former. Adsorption of fibrinogen from plasma was also greatly reduced on the grafted surfaces. From analysis (SDS-PAGE, immunoblotting) of the proteins eluted after plasma exposure, it was found that the grafted surfaces and the unmodified substrate adsorbed the same proteins in roughly the same proportions, suggesting that adsorption to the PEO surfaces occurs on patches of bare substrate. The PEO grafts did not apparently cause differential access to the substrate based on protein size.     

Size-selective protein adsorption to polystyrene surfaces by self-assembled grafted poly(ethylene glycols) with varied chain lengths

We report about the surface modification of polystyrene (PSt) with photoreactive alpha-4-azidobenzoyl-omega-methoxy poly(ethylene glycol)s (ABMPEG) of three different molecular weights (MWs of approximately 2, approximately 5, and approximately 10 kg/mol) and with two poly(ethylene glycol)/poly(propylene glycol) triblock copolymers (PEG-PPG-PEG) of about identical PEG/PPG ratio (80/20, w/w) and MW(PEG) of approximately 3 and approximately 6 kg/mol, all via adsorption from aqueous solutions. For ABMPEGs, an additional UV irradiation was used for photografting to the PSt. Contact angle (CA) and atomic force microscopy data revealed pronounced differences of the hydrophilicity/hydrophobicity and topography of the surfaces as a function of PEG type and concentration used for the modification. In all cases, an incomplete coverage of the PSt was observed even after modification at the highest solution concentrations (10 g/L). However, clear differences were seen between PEG-PPG-PEGs and ABMPEGs; only for the latter was a nanoscale-ordered interphase structure with an influence of MW(PEG) on the PEG density observed; after modification at the same solution concentrations, the density was significantly higher for lower MW(PEG). The adsorption of three proteins, myoglobin (Mgb), bovine serum albumin (BSA), and fibrinogen to the various surfaces was analyzed by surface plasmon resonance. Pronounced differences between the two PEG types with respect to the reduction of protein adsorption were found. At high, but still incomplete, surface coverage and similar CA, the shielding of ABMPEG layers toward the adsorption of Mgb and BSA was much more efficient; e.g., the adsorbed Mgb mass relative to that of unmodified PSt was reduced to 10% for ABMPEG 2 kg/mol while for both PEG-PPG-PEGs the Mgb mass was still around 100%. In addition, for the ABMPEG layers an effect of MW(PEG) on adsorbed protein mass-decrease with decreasing MW-could be confirmed; and the highest Mgb/BSA selectivities were also observed. A "two-dimensional molecular sieving", based on PEG molecules having a nanoscale order at the hydrophobic substrate polymer surface has been proposed, and the main prerequisites were the use of PEG conjugates which are suitable for an "end-on" grafting (e.g., ABMPEGs), the use of suitable (not too high) concentrations for the surface modification via adsorption/self-assembly, optionally the photografting on the substrate (possible only for ABMPEG), and presumably, a washing step to remove the excess of unbound PEGs. The results of this study also strongly support the hypothesis that the biocompatibility of hydrophobic materials can be very much improved by PEG modifications at surface coverages that are incomplete but have an ordered layer structure controlled by the size and steric interactions of surface-bound PEGs.     

Characterization of insulin adsorption in the presence of albumin by time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.     

Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.     

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.

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.     

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.     

Protein-resistant and fibrinolytic polyurethane surfaces

Surfaces with resistance to non-specific protein adsorption and a high capacity to bind plasminogen from plasma are developed for application as fibrinolytic surfaces in blood contact. A new method is reported for grafting poly(OEGMA-co-HEMA) copolymers on polyurethane surfaces. The OEGMA provides effective protein resistance due to the PEG side chains and the HEMA provides a high density of OH groups for attachment of lysine. Adsorption of fibrinogen from buffer and plasma to these surfaces is low, indicating significant protein resistance. Plasminogen binding from plasma is high, and clot dissolution on surfaces where plasminogen adsorbed from plasma is converted to plasmin is rapid.     

Fibrinogen adsorption on three silica-based surfaces: conformation and kinetics

Using AFM (atomic force microscopy) to probe protein conformation and arrangement, and TIRF (total internal reflectance fluorescence) to monitor kinetics, fibrinogen adsorption on three different silica-based surfaces was studied: the native oxide on silicon, acid-etched microscope slides, and acid-etched polished glass. The three are chemically similar, but the microscope slide is rougher and induces AFM tip instabilities that appear as high spots on the bare surface. Fibrinogen's conformation and transport-limited adsorption kinetics are found to be quantitatively similar on all three surfaces. Further, the number of adsorbed proteins in progressive AFM micrographs quantitatively match the coverages measured by TIRF during early adsorption. Surfaces appear full, via AFM, when adsorbed amounts are about an order of magnitude below their true saturation levels (via TIRF) because, above about 0.26 mg/m(2), individual proteins cannot be discerned. The results demonstrate how the appearance of AFM micrographs can be misleading regarding surface saturation. On all three surfaces, fibrinogen is, at most, slightly aggregated, showing limited, if any, surface mobility. The complexities of the microscope slide's surface landscape minimally impact adsorption.     

Fibrinolytic poly(dimethyl siloxane) surfaces

PDMS surfaces have been modified to confer both resistance to non-specific protein adsorption and clot lyzing properties. The properties and chemical compositions of the surfaces have been investigated using water contact angle measurements, ATR FT-IR spectroscopy, and XPS. The ability of the PEG component to suppress non-specific protein adsorption was assessed by measurement of radiolabeled fibrinogen uptake from buffer. The adsorption of plasminogen from human plasma to the various surfaces was studied. In vitro experiments demonstrated that lysine-immobilized surfaces with free epsilon-amino groups were able to dissolve fibrin clots, following exposure to plasma and tissue plasminogen activator. [Figure: see text].     

Structure of Protein Layers during Competitive Adsorption

The formation of protein layers during competitive adsorption was studied with ellipsometry. Single, binary, and ternary protein solutions of human serum albumin (HSA), IgG, and fibrinogen (Fgn) were investigated at concentrations corresponding to blood plasma diluted 1/100. As a model surface, hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer modified silica was used. By using multiambient media measurements of the bare substrate prior to protein adsorption the adsorbed amount as well as the thickness and refractive index of the adsorbed protein layer could be followedin situand in real time. Under conditions used in these experiments neither IgG nor fibrinogen could fully displace serum albumin from the interface. The buildup of the protein layer occurred via different mechanisms for the different protein systems. Fgn adsorbed in a rather flat orientation at low adsorbed amounts, while at higher surface coverage the protein reoriented to a more upright orientation in order to accommodate more molecules in the adsorbed layer. IgG adsorption proceeded mainly end-on with little reorientation or conformational change on adsorption. Finally, for HSA an adsorbed layer thickness greater than the molecular dimensions was observed at high concentrations (although not at low), indicating that aggregates or multilayers formed on HMDSO plasma polymer surfaces. For all protein mixtures the adsorbed layer structure and buildup indicated that Fgn was the protein dominating the adsorbed layer, although HSA partially blocked the adsorption of this protein. At high surface concentration, HSA/Fgn mixtures show an abrupt change in both adsorbed layer thickness and refractive index suggesting, e.g., an interfacial phase transition of the mixed protein layer. A similar but less pronounced behavior was observed for HSA/IgG. For IgG/Fgn and HSA/IgG/Fgn a buildup of the adsorbed layer similar to that displayed by Fgn alone was observed.     

Ellipsometry studies of fibronectin adsorption

The adsorption of fibronectin on a series of different surfaces was investigated with in situ ellipsometry. For silica and methylated silica, the adsorbed amount (Γ), the adsorbed layer thickness (δ_el) and the mean adsorbed layer refractive index (n_f) were obtained by a procedure involving studies of the bare substrate at two different ambient refractive indices, as well as four-zone averaging. It was found that the adsorbed amount of fibronectin was the same (1.9 ± 0.1 mg m⁻²) on both silica and methylated silica surfaces. However, the adsorbed layers formed on methylated silica were more extended and had a lower average protein concentration than those formed on silica. Furthermore, on both silica and methylated silica, an increasing adsorbed amount is achieved both by a denser packing of the fibronectin molecules and by a growth of the adsorbed layer normal to the surface. Furthermore, the adsorption of fibronectin on lipid surfaces was investigated. It was found that the adsorption of fibronectin on phosphatidic acid was quite significant (2.2 ± 0.2 mg m⁻²), while that on phosphatidylcholine, phosphatidylinositol and phosphatidylserine was much smaller (all 0.1 ± 0.05 mg m⁻²). These results are correlated to findings on the adsorption of fibrinogen on these surfaces, as well as on the opsonization of lipid-stabilized colloidal particles.     

Development of dextran-derivative arrays to identify physicochemical properties involved in biofouling from serum

To control protein adsorption on surfaces, low-fouling polymer coatings such as poly(ethylene oxide) (PEG or PEO) and polysaccharides are used. Their ability to resist protein adsorption is related to the layer structure, hence the immobilization mode. A polymer array technology was developed to study the structural diversity of carboxymethyl dextran (CMD) layers, whose immobilization conditions were varied. CMD arrays were analyzed by X-ray photoelectron spectroscopy (XPS) and by atomic force microscopy (AFM) colloidal probe force measurements. Serum protein adsorption was studied directly on the CMD arrays using surface plasmon resonance (SPR) microscopy. Physicochemical characterization revealed that pinning density regulates surface coverage and the amount of adsorbed molecules, and that salt concentration influences the surface structure of the charged polymer, forming extended or short layers. Protein adsorption experiments from serum showed that repulsive CMD layers are dense, with extended flexible chains. The present study underlines the usefulness of polymer arrays to study structural diversity of thin graft layers and to relate their physicochemical properties to their resistance to nonspecific protein adsorption.     

ToF-SIMS Analysis of Adsorbed Proteins: Principal Component Analysis of the Primary Ion Species Effect on the Protein Fragmentation Patterns

In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs(+), Au(+), Au(3) (+), Bi(+), Bi(3) (+), Bi(3) (++), C(60) (+)) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs(+), Au(+) and Bi(+) primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C(60) primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C(4)H(8)N(+) to K(+) peak intensities. This ratio correlated with the thickness of the protein films and Bi(1) (+) primary ions produced the most surface sensitive spectra.     

Effect of chain density and conformation on protein adsorption at PEG-grafted polyurethane surfaces

Polyurethanes were modified using monobenzyloxy polyethylene glycol (BPEG) which possesses a bulky hydrophobic benzyloxy group at one end and a hydroxyl group at the other end as a preconstructed BPEG layer, and poly(ethylene glycol) (PEG) and monomethoxyl poly(ethylene glycol) (MPEG) with various chain lengths as fillers. Our objective was to investigate the effect of PEG graft density and conformation on protein adsorption at PEGlated surface. The graft density was estimated by a chemical titration method. The combination of ATR-FTIR, AFM and titration results provide evidences that the graft density can be increased by backfilling PEG or MPEG to a BPEG layer. However, fibrinogen and albumin adsorption significantly increased on all surfaces after PEG or MPEG backfilling. We conclude that the conformation of hydrophobic benzyloxy end groups of the BPEG layer plays a key role. The benzyloxy end groups of preconstructed PEG chains stretch to the surface after PEG backfilling, which possibly accounts for the observed increase in protein adsorption. The BPEG conformation change after backfilling with PEG or MPEG was also suggested by contact angles. Additionally, protein adsorption was slightly influenced by the length of filler, suggesting a change in surface morphology.     

'Stealth' corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption

Nanoparticles possessing poly(ethylene glycol) (PEG) chains on their surface have been described as blood persistent drug delivery system with potential applications for intravenous drug administration. Considering the importance of protein interactions with injected colloidal dug carriers with regard to their in vivo fate, we analysed plasma protein adsorption onto biodegradable PEG-coated poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) and poly(-caprolactone) (PCL) nanoparticles employing two-dimensional gel electrophoresis (2-D PAGE). A series of corona/core nanoparticles of sizes 160–270 nm were prepared from diblock PEG-PLA, PEG-PLGA and PEG-PCL and from PEG-PLA:PLA blends. The PEG Mw was varied from 2000–20 000 g/mole and the particles were prepared using different PEG contents. It was thus possible to study the influence of the PEG corona thickness and density, as well as the influence of the nature of the core (PLA, PLGA or PCL), on the competitive plasma protein adsorption, zeta potential and particle uptake by polymorphonuclear (PMN) cells. 2-D PAGE studies showed that plasma protein adsorption on PEG-coated PLA nanospheres strongly depends on the PEG molecular weight (Mw) (i.e. PEG chain length at the particle surface) as well as on the PEG content in the particles (i.e. PEG chain density at the surface of the particles). Whatever the thickness or the density of the corona, the qualitative composition of the plasma protein adsorption patterns was very similar, showing that adsorption was governed by interaction with a PLA surface protected more or less by PEG chains. The main spots on the gels were albumin, fibrinogen, IgG, Ig light chains, and the apolipoproteins apoA-I and apoE. For particles made of PEG-PLA45K with different PEG Mw, a maximal reduction in protein adsorption was found for a PEG Mw of 5000 g/mole. For nanospheres differing in their PEG content from 0.5 to 20 wt %, a PEG content between 2 and 5 wt % was determined as a threshold value for optimal protein resistance. When increasing the PEG content in the nanoparticles above 5 wt % no further reduction in protein adsorption was achieved. Phagocytosis by PMN studied using chemiluminescence and zeta potential data agreed well with these findings: the same PEG surface density threshold was found to ensure simultaneously efficient steric stabilization and to avoid the uptake by PMN cells. Supposing all the PEG chains migrate to the surface, this would correspond to a distance of about 1.5 nm between two terminally attached PEG chains in the covering ‘brush’. Particles from PEG5K-PLA45K, PEG5K-PLGA45K and PEG5K-PCL45K copolymers enabled to study the influence of the core on plasma protein adsorption, all other parameters (corona thickness and density) being kept constant. Adsorption patterns were in good qualitative agreement with each other. Only a few protein species were exclusively present just on one type of nanoparticle. However, the extent of proteins adsorbed differed in a large extent from one particle to another. In vivo studies could help elucidating the role of the type and amount of proteins adsorbed on the fate of the nanoparticles after intraveinous administration, as a function of the nature of their core. These results could be useful in the design of long circulating intravenously injectable biodegradable drug carriers endowed with protein resistant properties and low phagocytic uptake.     

Antifouling properties of poly(methyl methacrylate) films grafted with poly(ethylene glycol) monoacrylate immersed in seawater

Biofouling of all structures immersed in seawater constitutes an important problem, and many strategies are currently being developed to tackle it. In this context, our previous work shows that poly(ethylene glycol) monoacrylate (PEGA) macromonomer grafted on preoxidized poly(methyl methacrylate) (PMMAox) films exhibits an excellent repellency against the bovine serum albumin used as a model protein. This study aims to evaluate the following: (1) the prevention of a marine extract material adsorption by the modified surfaces and (2) the antifouling property of the PEGA-g-PMMAox substrates when immersed in natural seawater during two seasons (season 1: end of April-beginning of May 2007, and season 2: end of October-beginning of November 2007). The antifouling performances of the PEGA-g-PMMAox films are investigated for different PEG chain lengths and macromonomer concentrations into the PEGA-based coatings. These two parameters are followed as a function of the immersion time, which evolves up to 14 days. The influence of the PEGA layer on marine compounds (proteins and phospholipids) adsorption is evidenced by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). It was found that the antifouling efficiency of the PEGA-grafted surfaces increases with both PEGA concentration and PEG chain length.     

Detection of nisin and fibrinogen adsorption on poly(ethylene oxide) coated polyurethane surfaces by time-of-flight secondary ion mass spectrometry (TOF-SIMS)

Stable, pendant polyethylene oxide (PEO) layers were formed on medical-grade Pellethane? and Tygon? polyurethane surfaces, by adsorption and gamma-irradiation of PEO-polybutadiene-PEO triblock surfactants. Coated and uncoated polyurethanes were challenged individually or sequentially with nisin (a small polypeptide with antimicrobial activity) and/or fibrinogen, and then analyzed with time-of-flight secondary ion mass spectrometry (TOF-SIMS). Data reduction by robust principal components analysis (PCA) allowed detection of outliers, and distinguished adsorbed nisin and fibrinogen. Fibrinogen-contacted surfaces, with or without nisin, were very similar on uncoated polymer surfaces, consistent with nearly complete displacement or coverage of previously-adsorbed nisin by fibrinogen. In contrast, nisin-loaded PEO layers remained essentially unchanged upon challenge with fibrinogen, suggesting that the adsorbed nisin is stabilized within the pendant PEO layer, while the peptide-loaded PEO layer retains its ability to repel large proteins. Coatings of PEO loaded with therapeutic polypeptides on medical polymers have the potential to be used to produce anti-fouling and biofunctional surfaces for implantable or blood-contacting devices.     

Synthesis and characterization of poly(ethylene glycol) methyl ether endcapped poly(ethylene terephthalate)

Linear and branched poly(ethylene terephthalate) (PET) copolymers with polyethylene glycol) (PEG) methyl ether (700 or 2000 g/mol) end groups were synthesized using conventional melt polymerization. DSC analysis demonstrated that low levels of PEG end groups accelerated PET crystallization. The incorporated PEG end groups also decreased the crystallization temperature of PET dramatically, and copolymers with a high content of PEG (>17.6 wt%) were able to crystallize at room temperature. Rheological analysis demonstrated that the presence of PEG end groups effectively decreased the melt viscosities and facilitated melt processing. XPS and ATR-FTIR revealed that the PEG end groups tended to aggregate on the surface, and the surface of compression molded films containing 34.0 wt% PEG were PEG rich (85 wt% PEG). PEG end-capped PET (34.0 wt% PEG) and PET films were immersed into a fibrinogen solution (0.7 mg/mL BSA) for 72 h to investigate the propensity for protein adhesion. XPS demonstrated that the concentration of nitrogen (1.05%) on the surface of PEG endcapped PET film was statistically lower than PET (7.67%). SEM analysis was consistent with XPS results, and revealed the presence of adsorbed protein on the surface of PET films.