Reduced Protein Adsorption Research Articles

Grafted ionomer complexes and their effect on protein adsorption on silica and polysulfone surfaces

We have studied the formation and the stability of ionomer complexes from grafted copolymers (GICs) in solution and the influence of GIC coatings on the adsorption of the proteins β-lactoglobulin (β-lac), bovine serum albumin (BSA), and lysozyme (Lsz) on silica and polysulfone. The GICs consist of the grafted copolymer PAA28-co-PAPEO22 {poly(acrylic acid)-co-poly[acrylate methoxy poly(ethylene oxide)]} with negatively charged AA and neutral APEO groups, and the positively charged homopolymers: P2MVPI43 [poly(N-methyl 2-vinyl pyridinium iodide)] and PAH∙HCl160 [poly(allylamine hydrochloride)]. In solution, these aggregates are characterized by means of dynamic and static light scattering. They appear to be assemblies with hydrodynamic radii of 8 nm (GIC-PAPEO22/P2MVPI43) and 22 nm (GIC-PAPEO22/PAH∙HCl160), respectively. The GICs partly disintegrate in solution at salt concentrations above 10 mM NaCl. Adsorption of GICs and proteins has been studied with fixed angle optical reflectometry at salt concentrations ranging from 1 to 50 mM NaCl. Adsorption of GICs results in high density PEO side chains on the surface. Higher densities were obtained for GICs consisting of PAH∙HCl160 (1.6 ÷ 1.9 chains/nm2) than of P2MVPI43 (0.6 ÷ 1.5 chains/nm2). Both GIC coatings strongly suppress adsorption of all proteins on silica (>90%); however, reduction of protein adsorption on polysulfone depends on the composition of the coating and the type of protein. We observed a moderate reduction of β-lac and Lsz adsorption (>60%). Adsorption of BSA on the GIC-PAPEO22/P2MVPI43 coating is moderately reduced, but on the GIC-PAPEO22/PAH∙HCl160 coating it is enhanced.

Thermal Oxidation for Controlling Protein Interactions with Porous Silicon

Thermal oxidation of porous silicon (pSi) has been used to control interactions with three proteins; lysozyme, papain, and human serum albumin (HSA) enabling the influences of protein structure, molecular weight, and charge to be elucidated. Adsorption behavior was assessed via adsorption isotherms while the structures of adsorbed proteins were investigated using a bioactivity assay, FTIR, and zeta potential. Time-of-flight secondary ion mass spectrometry was used to examine protein pore penetration. High protein adsorption onto unoxidized pSi (240-610 microg/m(2)) was attributed to predominately hydrophobic interactions which resulted in structural changes of the adsorbed proteins and significant loss of bioactivity. Thermal oxidation at 400 and 800 degrees C significantly reduced protein adsorption (80-485 microg/m(2)) by reducing hydrophobicity. Oxidation of pSi modified the protein adsorption mechanisms to solely electrostatic attraction for positively charged proteins and structural rearrangement for negatively charged proteins. Adsorption via electrostatic attraction preserved protein bioactivity and zeta potential, thus inferring a retention of their native structure. In contrast, the negative charge and globular structure of HSA resulted in a loss of structure. We have demonstrated that thermal oxidation of pSi can be used to control protein interactions, adsorbed structure, and bioactivity.

Reduction of protein adsorption on well-characterized polymer brush layers with varying chemical structures

To clarify protein adsorption behavior on polymer brush layers, surface characteristics and protein adsorption repellency on polymer brush layers should be precisely determined. Here, we clearly delineated the chemical structure of the polymer brush layers containing various hydrophilic groups, namely, phosphorylcholine, sulfoxybetaine, carboxybetaine (zwitterionic), and hydroxyl group (nonionic) and examined the effects of the chemical structure on initial protein adsorption behavior. Kinetic analysis performed during surface-initiated atom transfer radical polymerization revealed that graft polymerization proceeded in a living manner. The graft density of each type of polymer chain and its surface coverage were high enough to form dense polymer brush structures. The hydroxyl group-bearing polymer brush structure exhibited the highest graft density. Among the zwitterionic polymer brush structures, the graft density and surface coverage of sulfoxybetaine- and carboxybetaine-bearing polymer chains were higher than those of the phosphorylcholine-bearing polymer chains. The amount of protein relative to 100% serum adsorbed on polymer brush layers was quantified using quartz crystal microbalance with dissipation (QCM-D). Protein adsorption on all zwitterionic polymer brush layers apparently decreased with increasing thickness of the grafted polymer layers. Protein adsorption was highly suppressed on thick polymer brush layers bearing phosphorylcholine or sulfoxybetaine groups. However, the amount of proteins adsorbed on thick polymer brush layers bearing hydroxyl groups was 10 times more than that adsorbed on polymer brush layers bearing phosphorylcholine groups. Thus, we concluded that the chemical structure of the polymer brush layer is a significant factor affecting resistance to protein adsorption even for dense polymer brush structures.

Protein interactions with bottle-brush polymer layers: Effect of side chain and charge density ratio probed by QCM-D and AFM

Silica surfaces were coated with a range of cationic bottle-brush polymers with 45 units long poly(ethylene oxide) side chains, and their efficiency in reducing protein adsorption was probed by QCM-D, reflectometry and AFM. Preadsorbed layers formed by bottle-brush polymers with different side chain to charge ratio was exposed to two proteins with different net charge, lysozyme and BSA. The reduction in protein adsorption was found to depend on both the type of protein and on the nature of the polyelectrolyte layer. The most pronounced reduction in protein adsorption was achieved when the fraction of charged backbone segments was in the range 0.25–0.5 equivalent to a fraction of poly(ethylene oxide) side chains of 0.75–0.5. It was concluded that these polymers have enough electrostatic attachment points to ensure a strong binding to the surface, and at the same time a sufficient amount of poly(ethylene oxide) side chains to counteract protein adsorption. In contrast, a layer formed by a highly charged polyelectrolyte without side chains was unable to resists protein adsorption. On such a layer the adsorption of negatively charged BSA was strongly enhanced, and positively charged lysozyme adsorbed to a similar extent as to bare silica. AFM colloidal probe force measurement between silica surfaces with preadsorbed layers of bottle-brush polymers were conducted before and after exposure to BSA and lysozyme to gain insight into how proteins were incorporated in the bottle-brush polymer layers.

Characterization and cell material interactions of PEGylated PNIPAAM nanoparticles

The aim of this study was to investigate the haemocompatibility of poly(N-isopropylacrylamide)-co-poly(ethylene glycol), PNIPAAM-PEG based nanoparticles and the influence of poly(ethylene glycol), PEG on the interactions of nanoparticles with cells. To achieve this purpose, thermosensitive PNIPAAM-PEG nanoparticles were synthesized by free radical dispersion polymerization method. Optimized nanosystems had particle sizes less than 200 nm, low polydispersity and LCST of 40–41 °C. The nanoparticles also showed nearly 83% encapsulation efficiency for doxorubicin HCl with temperature dependent release. Presence of PEG resulted in reduced protein adsorption by more than 50% in comparison to non-PEG containing nanoparticles. Protein adsorption was noted to be dependent on PEG chain length and was the least with M n = 4000. The particles up to a concentration of 2 mg/ml did not show any toxicity on J774 and L929 cell lines. No interactions were observed when NIPAAM-PEG nanoparticles were incubated with blood cells viz. RBCs, neutrophils, platelets and the coagulation system suggesting their haemocompatibility.

Preparation and properties of branched oligoglycerol modifiers for stabilization of liposomes

We examined the effect on drug delivery of liposomes with surfaces that were modified with branched oligoglycerols (BGLs) and explored possible formulation advantages to increase drug exposure. BGL012 is a branched oligoglycerol derivative with a cascade-like structure of 12 glycerol units, characterized as a widely spread structure in aqueous solution. We prepared BGL-phospholipid derivatives (BGL-PEs), including BGL012, by coupling 1,2-distearoylphosphatidylethanolamine to BGLs. BGL012-PE modification of the liposomes (BGL012L) achieved a long circulation time after intravenous injection in rats. The circulation lifetime of BGL012L was almost the same as that of polyethylene glycol (PEG)-modified liposomes. The surface of BGL012L induced the formation of a fixed aqueous layer and reduced protein adsorption on the liposome surface, without strong interference with the binding reaction on the liposome. Thus, the newly synthesized branched oligoglycerol derivatives are considered to have useful hydrophilic and physical properties for modifying the liposome surface to increase drug exposure.

Super-hydrophilic silicone hydrogels with interpenetrating poly(2-methacryloyloxyethyl phosphorylcholine) networks

We synthesized silicone hydrogels from 2-methacryloyloxyethyl phosphorylcholine (MPC) and bis(trimethylsilyloxy)methylsilylpropyl glycerol methacrylate (SiMA) using two methods: random copolymerization with a small amount of cross-linker (P(SiMA- co-MPC)) and construction of an interpenetration network (IPN) structure composed of cross-linked poly(MPC)(PMPC) chains and cross-linked poly(SiMA)(PSiMA) chains (PSiMA- ipn-PMPC). The polymerization was carried out by photoreaction. The surface hydrophilicity and water absorbability of P(SiMA- co-MPC) increased with an increase in the MPC unit composition. On the other hand, in the case of PSiMA- ipn-PMPC, a super-hydrophilic surface was obtained by the surface enrichment of MPC units. The optical and mechanical properties of PSiMA- ipn-PMPC are suitable for use as a material for preparing contact lenses. In addition, the oxygen permeability of PSiMA- ipn-PMPC remains high because of the PSiMA chains. The MPC units at the surface of the hydrogels reduce protein adsorption effectively. From these results for PSiMA- ipn-PMPC, we confirmed that it has the potential for application to silicone hydrogel contact lenses.

Modification of Poly(lactic/glycolic acid) Surface by Chemical Attachment of Poly(ethylene glycol)

Biodegradable polyesters such poly(lactic acid) and poly(lactic/glycolic acid) (PLGA) copolymers are preferred biomaterials and used among others as drug delivery systems, although their surface hydrophobicity limits their application. In this work, chemical modification of the PLGA surface was developed by coupling of either linear or starlike poly(ethylene glycol) (PEG) molecules via chemical bonds to the PLGA surface following amino functionalization as a first step to improve its biocompatibility. The chemical attachment was followed by detailed X-ray photoelectron spectroscopy (XPS) studies. It was shown that substantial modification can be achieved by linear PEG, but even higher surface coverage with hydrophilic groups can be obtained when the six-armed PEG is applied with the additional advantage of possible further functionalization via free amino groups available on the surface of the latter. As a final goal, a significant increase of water wettability together with reduced protein adsorption was achieved on PEG-coupled PLGA surfaces.

Carboxybetaine–Group Immobilized onto Pore Surface Reduced Protein Adsorption to Porous Membrane

Carboxybetaine–Group Immobilized onto Pore Surface Reduced Protein Adsorption to Porous Membrane

Synthesis and Characterization of Novel Amphiphilic Fluorinated Triblock Copolymer

Atom transfer radical polymerization (ATRP) has been employed for the synthesis of a novel amphiphilic fluorinated triblock copolymer PEG-b-PS-b-PFHEM for anti-fouling coatings. The macroinitiator based on poly(ethylene oxide) monomethyl ether was used to prepare an amphiphilic diblock copolymer PEG-b-PSt-Br, which was then utilized to initiate the ATRP of fluorinated monomer perfluorohexylethyl acrylate (FHEA), resulting in an amphiphilic triblock copolymer. These copolymers were characterized by means of 1H NMR and GPC. The amphiphilic triblock copolymer surface composes of fluorinated and PEGylated blocks, and the fluorinated surface has critical surface energy, while the PEGylated surface is expected to have a relatively low interfacial energy when in contact with water. Microphase-separation of both blocks could take place and result in the reduction of protein adsorption and cell adhesion. The amphiphilic fluoropolymer has the potential application as excellent antifouling coatings and antifouling membranes.

The biocompatibility of sulfobetaine engineered polymethylmethacrylate by surface entrapment technique

Sulfobetaine-modified polymethylmethacrylate (PMMA) systems were created by physically entrapping the zwitterionic species on the PMMA surface. The presence of the sulfobetaine molecules on these surfaces were verified by ATR-FTIR and SEM-EDAX analysis, while wettability of the films was investigated by dynamic contact angle measurements. The short-term (4 h) adhesion of two bacterial species (gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa) on these surfaces were studied. Mouse RAW 264.7 macrophage cells were used to assess the cell adhesion and inflammatory response by quantifying the expression levels of proinflammatory cytokines namely TNFalpha and IL1beta by measuring their mRNA profiles in the cells using real-time polymerase chain reaction (RT-PCR) normalized to the house keeping gene GAPDH. Whilst mouse L-929 fibroblast cells were used to assess the propensity for the materials to support fibroblast cell adhesion. A decrease in the adhesion of S. aureus by 63% and P. aeruginosa by 49% was observed on sulfobetaine modified PMMA films after 4 h. In all the cases, sulfobetaine modified PMMA films reduced cellular adhesion events (*P < 0.05) with respect to the base materials, which could be linked to the reduced protein adsorption observed on these surfaces. The cellular inflammatory response was suppressed on sulfobetaine modified substrates as expression levels of pro-inflammatory cytokines (TNFalpha and IL1beta) was found to be up regulated on bare PMMA, while it was significantly lower on sulfobetaine modified PMMA (**P < 0.001). Thus the sulfobetaine entrapment process can be applied on polymethylmethacrylate in order to achieve low biointeractions and reduced inflammatory host responses for various biomedical and biotechnological applications.

Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: Chemical characterization and evaluation of the protein adsorption

This work deals with the optimization of argon plasma-induced graft-polymerization of polyethylene glycol acrylate (PEGA) on polypropylene (PP) films in order to obtain surfaces with a reduced protein adsorption for possible biomedical applications. To this end, we examined the protein adsorption on the treated and untreated surfaces. The graft-polymerization process consisted of four steps: (a) plasma pre-activation of the PP substrates; (b) immersion in a PEGA solution; (c) argon plasma-induced graft-polymerization; (d) washing and drying of the samples. The efficiency of these processes was evaluated in terms of the amount of grafted polymer, coverage uniformity and substrates wettability. The process was monitored by contact angle measurements, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray Photoelectron Spectroscopy (XPS) and atomic force microscopy (AFM) analyses. The stability of the obtained thin films was evaluated in water and in Phosphate Buffer Saline (PBS) at 37°C. The adsorption of fibrinogen and green fluorescent protein (GFP) – taken as model proteins – on the differently prepared surfaces was evaluated through a fluorescence approach using laser scanning confocal microscopy with photon counting detection. After plasma treatments of short duration, the protein adsorption decreases by about 60–70% with respect to that of the untreated film, while long plasma exposure resulted in a higher protein adsorption, due to damaging of the grafted polymer.

The Biocompatibility of Sulfobetaine Engineered Poly (Ethylene Terephthalate) by Surface Entrapment Technique

Sulfobetaine-modified poly(ethylene terephthalate) (PET) systems were created by physically entrapping the zwitterionic species on the PET surface. The presence of the sulfobetiane molecules on these surfaces were verified by ATR-FTIR and SEM-EDAX analysis, while wettability of the films was investigated by water contact angle measurements. The blood compatibility of the modified films was evaluated by platelet adhesion in human platelet-rich plasma (PRP). The adhesion and inflammatory response of Mouse RAW 264.7 macrophage cells were studied. The surface induced cellular inflammatory response was determined by quantifying the expression levels of proinflammatory cytokines namely TNF-alpha and IL-1beta by measuring their mRNA profiles in the cells using real time polymerase chain reaction normalized to the housekeeping gene GAPDH. L-929 fibroblast cells were used to assess the propensity of the materials to support the fibroblast cell adhesion. A lower platelet adhesion and activation were observed on the sulfobetaine-modified PET film incubated in PRP after 2h when compared to control. The modified film reduced cellular adhesion events ( p<0.05) with respect to the base material, which could be linked to the reduced protein adsorption observed on this surface. The cellular inflammatory response was suppressed on sulfobetaine-modified substrate. Expression levels of pro-inflammmatory cytokines (TNF-alpha and IL-1beta) was found to be upregulated on bare PET, while it was significantly lower on modified PET ( p<0.001). Thus the sulfobetaine entrapment process can be applied on PET in order to achieve low biointeractions and reduced inflammatory host response for various biomedical and biotechnological applications.

Amphiphilicity in homopolymer surfaces reduces nonspecific protein adsorption.

Amphiphilic homopolymer films have been immobilized onto substrates to study the interactions of these polymers with proteins. X-ray photoelectron spectroscopy (XPS) was utilized to measure the amount of protein adsorption. Amphiphilic homopolymers have been shown to reduce protein adsorption, despite the high affinity of the hydrophobic or hydrophilic functional groups by themselves toward proteins. This protein-resistant property seems to arise from the unique molecular-scale alternation of incompatible functionalities. The combination of incompatible functionalities with a predefined alternating pattern within a monomer could provide a potential design for nonfouling materials.

The effects of urease immobilization on the transport characteristics and protein adsorption capacity of cellulose acetate based hemodialysis membranes

In this study, cellulose acetate (CA) based hemodialysis membranes were prepared by a dry phase inversion method and the influences of urease immobilization on the clearing performance and protein adsorption capacity of the membranes were investigated. Permeation experiments have shown that modification of CA membranes with urease immobilization not only enhanced the transport rate of urea but also increased the permeation coefficients of uric acid and creatinine by changing the structure of the membrane. Furthermore, the protein adsorption capacity of the CA membranes decreased. On the other hand, the mechanical strength of the modified CA membrane did not change significantly compared with that of the unmodified one. A mathematical model was derived to determine the rate of mass transfer of urea through modified CA membranes. Model predictions along with the experimental data suggest that urease immobilization can be used as an alternative method in preparing CA based hemodialysis membranes with improved transport characteristics and biocompatibility through reduced protein adsorption capacities.

Cell outer membrane mimetic modification of a cross-linked chitosan surface to improve its hemocompatibility

A novel strategy has been developed to improve the hemocompatibility of chitosan surface by cell outer membrane mimetic structure able to reduce protein adsorption and cell adhesion. Phosphorylcholine dichloride was synthesized and grafted onto a glutaraldehyde-cross-linked chitosan (CS-GA) film surface to prepare phosphorylcholine-coated CS-GA film (CS-GA-PC) through a heterogeneous reaction process. The spectroscopic and contact angle characterization show that a cell outer membrane mimetic structure was formed on the cross-linked chitosan surface, and the significantly improved hemocompatibility of the modified surface was shown by a suppression of 94% on platelet adhesion, a suppression of 60–70% for bovine plasma fibrinogen and bovine serum albumin adsorptions. These results demonstrated that this cell outer membrane mimetic surface modification with phosphorylcholine dichloride is a promising strategy to improve the hemocompatibility of chitosan.

Polyelectrolyte Multilayers Capped with Polyelectrolytes Bearing Phosphorylcholine and Triethylene Glycol Groups: Parameters Influencing Antifouling Properties

In this paper, we investigate the design of antifouling surfaces by the deposition of polyelectrolytes modified by grafting of antifouling groups onto a (PSS/PAH)n precursor multilayer film [PSS, poly(styrenesulfonate); PAH, poly(allylamine)]. Different polyelectrolytes and different antifouling moieties are investigated, in particular, (EO)3 and (EO)3PC moieties (EO, ethylene oxide; PC, phosphorylcholine group). We find that protein adsorption can strongly be reduced and even practically suppressed through the deposition of only one layer of polyelectrolyte modified with PC and/or (EO)3 groups. We discuss the influence of various parameters such as the nature of the polyelectrolyte backbone, the nature of the antifouling moiety, and the grafting ratio on the reduction of protein adsorption. We find in particular that (EO)3 and (EO)3PC moieties grafted on poly(acrylic acid) (PAA) totally prevent protein adsorption for grafting ratios of 25% or more, at least within the detection limits of the used quartz crystal microbalance and optical waveguide light mode spectroscopy devices. The mechanism that leads to the antifouling property is discussed and compared to that leading to the antifouling properties of ethylene oxide self-assembled monolayers. Finally, by incorporating biotin on top of the precursor film, we show that one layer of PAA-(EO)3PC is not sufficient to prevent interaction with streptavidin but a PAA-(EO)3PC/PAH/PAA-(EO)3PC multilayer largely protects the biotin from interacting with streptavidin.

The effect of polymer surface modification via interfacial polymerization on polymer–protein interaction

AbstractMembrane separation is an important processing technology used for separating food ingredients and fractionating value‐added components from food processing byproducts. Long‐term performance of polymeric membranes in food protein processing is impeded by the formation of fouled layers on the membrane surface as a result of protein adsorption onto the membrane surface. Surface modification of synthetic membranes, i.e., changing surface characteristics to reduce protein adsorption permanently, is one of the innovative ways of reducing the fouling of membrane surfaces. In this study, surface modification of flat‐sheet ultrafiltration membrane, polyethersulfone (PES), was investigated in improving the hydrophilicity of PES surfaces, thereby reducing adsorption of the protein caused by hydrophobic–hydrophobic interaction between the protein and the membrane. Hydrophilic polymer grafting through thin‐film composite using interfacial polymerization was employed to improve the hydrophilicity of the commercial PES membranes. Poly(vinyl alcohol), poly(ethylene glycol), and chitosan were chosen as hydrophilic polymers to graft on PES membrane because of their excellent hydrophilic property. Modified PES membranes were characterized by contact angle, FTIR, XPS, and AFM. Contact angles of modified PES membranes were reduced by 25 to 40% of that of the virgin PES membrane. XPS spectrum supported that the PES membranes were successfully modified by interfacial polymerization. Tapping‐mode AFM was used to examine the changes in surface topography of modified PES membranes. The PES membranes modified by interfacial polymerization showed lower roughness (from 1.2 to 2.0 nm) than that of virgin PES membrane (2.1 nm). The results of these instrumental analyses indicated that the PES membranes were successfully enhanced hydrophilically through interfacial polymerization. The protein adsorption on the modified membranes was reduced by 30 to 35% as a result of surface modification of the PES membranes using interfacial polymerization technique. Published 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Self-assembling polystyrene- block-poly(ethylene oxide) copolymer surface coatings: Resistance to protein and cell adhesion

In this paper we report a method for biomaterial surface modification that utilizes the self-assembly of block copolymers of poly(styrene- block-ethylene oxide) (PS–PEO) to generate micro-phase separated surfaces with varying density PEO domains. These PS–PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control polystyrene surfaces. The adhesion of NIH-3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-domains, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface.

Protein‐Resistant Surfaces through Mild Dopamine Surface Functionalization

The synthesis and evaluation of new dopamine-based catechol anchors coupled to poly(ethylene glycol) (PEG) for surface modification of TiO(2) are reported. Dopamine is modified by dimethylamine-methylene (7) or trimethylammonium-methylene (8) groups, and the preparation of mPEG-Glu didopamine polymer 11 is presented. All these PEG polymers allow stable adlayers on TiO(2) to be generated through mild dip-and-rinse procedures, as evaluated both by variable angle spectroscopic ellipsometry and X-ray photoelectron spectroscopy. The resulting surfaces substantially reduced protein adsorption upon exposure to full human serum.