Protein-resistant Surface Research Articles

Copper-mediated synergistic catalytic titanium dioxide nanofilm with nitric oxide generation and anti-protein fouling for enhanced hemocompatibility and inflammatory modulation

Nitric oxide (NO) has become a highly compelling therapeutic gas for treating vascular diseases due to its versatile functions to vascular responses. Currently, NO biomimetic materials are successfully established through loading catalyst (i.e., Se, Cu) with the vehicle to decompose endogenous NO donors in blood continuously. However, current NO biomimetic materials are susceptible to the non-specific protein fouling, resulting in undesired therapeutic inefficiency with the observation of attenuated or even blocked NO catalytic activities. Herein, we produced a multifunctional nanofilm with dual catalysis based on the vascular stent platform via simply doping one kind of metal (Cu) as cocatalyst into anatase TiO2 crystal. Beneficial synergistic interactions between two kinds of catalysis are reported in this work. The nanofilm surface is endowed with photoinduced super hydrophilic conversion and controlled NO catalytic release simultaneously. The super hydrophilic surface shows the excellent self-cleaning ability to resist protein fouling. Importantly, the maintenance of the protein-resistant surface can effectively improve the NO catalytic release and reduce inflammatory stimuli, contributing to enhanced hemocompatibility. The optimum doping amount of Cu (TiO2@Cu1, 0.77 wt.%) is determined through the characterization of adjustable photoinduced hydrophilic conversion and appropriate NO catalytic generation within the effective physiological concentration. After vascular implantation in rats, the TiO2@Cu1 nanofilms achieved elevated performances on antithrombosis, reducing the intima hyperplasia area and promoting rapid reendothelialization at 4 weeks. This study provides a valuable guideline for NO-biomimetic materials used for blood contact devices and paves the way for the surface modification for vascular implants.

Carboxybetaine functionalized nanosilicas as protein resistant surface coatings.

Materials with protein resistant properties are increasingly sought after for their potential application as low-fouling surface coatings. Hydrophilic coatings with improved resistance to protein fouling have been prepared from zwitterionic carboxybetaine (CB) functionalized silica nanoparticles (SiNPs). The authors report three methods of coating preparation via direct tethering of CB to predeposited particle films, a two-step surface functionalization process, and deposition of CB functionalized particle dispersions. The pH at which aqueous CB solutions were prepared and reacted to SiNPs was found to drastically influence the mechanism of CB attachment and affect the protein resistance of the resultant coatings. Depending on the method of coating preparation, protein binding to functionalized particle coatings was reduced by up to 94% compared to unfunctionalized SiNP control surfaces. As a result, all three methods offer simple and scalable fabrication routes for the generation of hydrophilic, zwitterionic interfaces with improved inhibition to protein fouling.

Protein-resistant surface based on zwitterion-functionalized nanoparticles for marine antifouling applications

A functional nanocomposite (PSBMA@VTMO@silica/TA-PEG) coating was successfully developed through a facile deposition method, and was able to effectively block the adhesion of proteins and subsequent biofouling deposition.

Ceramic membrane with protein-resistant surface via dopamine/diglycolamine co-deposition

Protein separation and purification is important in biotechnology and food processing industries. Ceramic membranes have shown great potentials for this task. But membrane fouling remains a barrier to their widespread application due to the irreversible adsorption of protein onto the membrane. In this work, one-step co-deposition of dopamine (DA) and diglycolamine (DGA) was conducted on the ceramic membranes for fabricating protein-resistant surfaces. CuSO4/H2O2 was used as a trigger to shorten the deposition time and to improve the deposition efficiency. The effect of DA/DGA mass ratio on the membrane structures and properties was studied by FESEM, FTIR, XPS and contact angle measurements. The results indicate that the optimal DA/DGA mass ratio in the co-deposition solution is 1:4. The modified membranes show improved hydrophilicity and negatively charged property. In dynamic protein filtration, these ceramic membranes exhibit a higher permeate flux, a higher water flux recovery, and a lower water flux reduction compared to the original membrane. Moreover, the antifouling property of the modified membrane is preserved after the membranes are treated with high speed water scouring. As a result, this one-step co-deposition method is a simple and effective way to prepare durable fouling-resistant ceramic membranes for protein removal.

A Simple Method for Patterning Nanoparticles on Planar Surfaces

This paper describes a simple method to pattern nanoparticles on planar surfaces using the antifouling property of poly(ethylene glycol) monolayers deposited from a solution on the native oxide of titanium. Atomic force microcopy was used to pattern the poly(ethylene glycol) monolayers producing protein active sites on the protein-resistant surface. Patterns with different sizes have been generated by shaving the monolayers with different repetitions. Friction force microscopy was used to image the patterns. The smallest patterns are 50 nm and the largest patterns are 500 nm at full width half maximum. The smallest pattern was produced with one shave, whereas the largest pattern was produced by shaving the monolayers 112 times. Protein-coated nanoparticles were immobilised on the shaved (protein active) part of the monolayers by dipping the patterned samples into a solution that contains 2% by volume protein-functionalized nanoparticles with a nominal diameter of 40 nm. Atomic force microscopy was used to take a topographic image of the samples. The topographic image showed that the protein-functionalized nanoparticles were attached onto the shaved part of the substrate but not on the poly(ethylene glycol)-covered part of the substrate. The level of aggregation of the nanoparticles was also investigated from the topographic image. The section analysis of the topographic image of the nanoparticle patterns showed a height of 40 nm which proved that only a monolayer of particles were deposited on the shaved part of the monolayer.

Quantitative design rules for protein-resistant surface coatings using machine learning

Preventing biological contamination (biofouling) is key to successful development of novel surface and nanoparticle-based technologies in the manufacturing industry and biomedicine. Protein adsorption is a crucial mediator of the interactions at the bio – nano -materials interface but is not well understood. Although general, empirical rules have been developed to guide the design of protein-resistant surface coatings, they are still largely qualitative. Herein we demonstrate that this knowledge gap can be addressed by using machine learning approaches to extract quantitative relationships between the material surface chemistry and the protein adsorption characteristics. We illustrate how robust linear and non-linear models can be constructed to accurately predict the percentage of protein adsorbed onto these surfaces using lysozyme or fibrinogen as prototype common contaminants. Our computational models could recapitulate the adsorption of proteins on functionalised surfaces in a test set with an r2 of 0.82 and standard error of prediction of 13%. Using the same data set that enabled the development of the Whitesides rules, we discovered an extension to the original rules. We describe a workflow that can be applied to large, consistently obtained data sets covering a broad range of surface functional groups and protein types.

Facile protein-resistant and anti-biofilm surface coating based on catechol-conjugated poly(N-vinylpyrrolidone)

A facile biofouling-resistant surface coating was developed based on catechol-conjugated poly(N-vinylpyrrolidone) (CA-PVP). CA-PVP can be securely and effectively coated on glass, gold, and tissue culture polystyrene (TCPS) surfaces under a mild condition as characterized by ATR-FTIR and PVP-complexation with iodine and hydrogen peroxide. PVP itself without catechol conjugation, on the other hand, cannot achieve well-coating on surfaces. The coated surfaces became more hydrophilic and protein-resistant due to the presence of PVP as measured by the reduced contact angles and surface plasmon resonance (SPR) responses. The significantly enhanced protein resistance created by CA-PVP coatings leads to its significant anti-biofilm and fibroblast cell adhesion resistance as observed by fluorescent microscope. CA-PVP coating also demonstrated its high biocompatibility that ≥ 95% cell viability of mouse fibroblast cells still can be maintained after 48 h cultivation.

Fabrication of Self-Cleaning, Reusable Titania Templates for Nanometer and Micrometer Scale Protein Patterning

The photocatalytic self-cleaning characteristics of titania facilitate the fabrication of reuseable templates for protein nanopatterning. Titania nanostructures were fabricated over square centimeter areas by interferometric lithography (IL) and nanoimprint lithography (NIL). With the use of a Lloyd's mirror two-beam interferometer, self-assembled monolayers of alkylphosphonates adsorbed on the native oxide of a Ti film were patterned by photocatalytic nanolithography. In regions exposed to a maximum in the interferogram, the monolayer was removed by photocatalytic oxidation. In regions exposed to an intensity minimum, the monolayer remained intact. After exposure, the sample was etched in piranha solution to yield Ti nanostructures with widths as small as 30 nm. NIL was performed by using a silicon stamp to imprint a spin-cast film of titanium dioxide resin; after calcination and reactive ion etching, TiO2 nanopillars were formed. For both fabrication techniques, subsequent adsorption of an oligo(ethylene glycol) functionalized trichlorosilane yielded an entirely passive, protein-resistant surface. Near-UV exposure caused removal of this protein-resistant film from the titania regions by photocatalytic degradation, leaving the passivating silane film intact on the silicon dioxide regions. Proteins labeled with fluorescent dyes were adsorbed to the titanium dioxide regions, yielding nanopatterns with bright fluorescence. Subsequent near-UV irradiation of the samples removed the protein from the titanium dioxide nanostructures by photocatalytic degradation facilitating the adsorption of a different protein. The process was repeated multiple times. These simple methods appear to yield durable, reuseable samples that may be of value to laboratories that require nanostructured biological interfaces but do not have access to the infrastructure required for nanofabrication.

Choline phosphate functionalized surface: protein-resistant but cell-adhesive zwitterionic surface potential for tissue engineering.

A choline phosphate (CP) modified surface is designed to resist protein adsorption due to its zwitterionic properties and simultaneously promote cell adhesion though its universal interaction with phosphate choline (PC) headgroups of the cell membrane. This work provides a new approach to obtain a cell-adhesive surface with a non-biofouling 'background', which has a potential for tissue engineering.

Phosphonate based organosilane modification of a simultaneously protein resistant and bioconjugable silica surface.

A facile method to coat silica surfaces with THPMP is introduced, forming simultaneously a protein resistant and bioconjugable surface. The coating is experimentally identified and its anti-fouling and bioconjugable characteristics are demonstrated.

Grafting hyaluronic acid onto gold surface to achieve low protein fouling in surface plasmon resonance biosensors.

Antifouling surfaces capable of reducing nonspecific protein adsorption from natural complex media are highly desirable in surface plasmon resonance (SPR) biosensors. A new protein-resistant surface made through the chemical grafting of easily available hyaluronic acid (HA) onto gold (Au) substrate demonstrates excellent antifouling performance against protein adsorption. AFM images showed the uniform HA layer with a thickness of ∼10.5 nm on the Au surface. The water contact angles of Au surfaces decreased from 103° to 12° with the covalent attachment of a carboxylated HA matrix, indicating its high hydrophilicity mainly resulted from carboxyl and amide groups in the HA chains. Using SPR spectroscopy to investigate nonspecific adsorption from single protein solutions (bovine serum albumin (BSA), lysozyme) and complex media (soybean milk, cow milk, orange juice) to an HA matrix, it was found that ultralow or low protein adsorptions of 0.6-16.1 ng/cm(2) (e.g., soybean milk: 0.6 ng/cm(2)) were achieved on HA-Au surfaces. Moreover, anti-BSA was chosen as a model recognition molecule to characterize the immobilization capacity and the antifouling performance of anti-BSA/HA surfaces. The results showed that anti-BSA/HA sensor surfaces have a high anti-BSA loading of 780 ng/cm(2), together with achieving the ultralow (<3 ng/cm(2) for lysozyme and soybean milk) or low (<17 ng/cm(2) for cow milk and 10% blood serum) protein adsorptions. Additionally, the sensor chips also exhibited a high sensitivity to BSA over a wide range of concentrations from 15 to 700 nM. Our results demonstrate a promising antifouling surface using extremely hydrophilic HA as matrix to resist nonspecific adsorption from complex media in SPR biosensors.

Fabrication of Robust Hydrogel Coatings on Polydimethylsiloxane Substrates Using Micropillar Anchor Structures with Chemical Surface Modification

A durable hydrophilic and protein-resistant surface of polydimethylsiloxane (PDMS) based devices is desirable in many biomedical applications such as implantable and microfluidic devices. This paper describes a stable antifouling hydrogel coating on PDMS surfaces. The coating method combines chemical modification and surface microstructure fabrication of PDMS substrates. Three-(trimethoxysilyl)propyl methacrylates containing C═C groups were used to modify PDMS surfaces with micropillar array structures fabricated by a replica molding method. The micropillar structures increase the surface area of PDMS surfaces, which facilitates secure bonding with a hydrogel coating compared to flat PMDS surfaces. The adhesion properties of the hydrogel coating on PDMS substrates were characterized using bending, stretching and water immersion tests. Long-term hydrophilic stability (maintaining a contact angle of 55° for a month) and a low protein adsorption property (35 ng/cm(2) of adsorbed BSA-FITC) of the hydrogel coated PDMS were demonstrated. This coating method is suitable for PDMS modification with most crosslinkable polymers containing C═C groups, which can be useful for improving the anti-biofouling performance of PDMS-based biomedical microdevices.

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.

Cell Adhesion on a POEGMA-Modified Topographical Surface

It is well known that adsorbed proteins play a major role in cell adhesion. However, it has also been reported that cells can adhere to a protein-resistant surface. In this work, the behavior of L02 and BEL-7402 cells on a protein-resistant, 3D topographical surface was investigated. The topographical gold nanoparticle layer (GNPL) surfaces were prepared by chemical gold plating, and the topography was described by roughness parameters acquired from a multiscale analysis. Both smooth Au and GNPL surfaces were modified with POEGMA polymer brushes using surface-initiated ATRP. The dry and hydrated thicknesses of POEGMA brushes on both smooth and rough surfaces were measured by AFM using a nanoindentation method. Protein adsorption experiments using (125)I radiolabeling revealed similarly low levels of protein adsorption on smooth and GNPL surfaces modified with POEGMA, thus allowing an investigation of the effects of topography on cell behavior under conditions of minimal protein adsorption. The roles of VN and FN adsorption in both L02 cells and BEL-7402 cells adhesion were investigated using cell culturing with and without a serum supplement. It was found that initial cell adhesion occurred via proteins adsorbed from the cell culture medium, whereas subsequent durable cell adhesion could be attributed to the topographical structure of the surface. Although cell spreading on protein-resistant surfaces was constrained because of the lack of adsorbed proteins, we found that cells adherent to topographical surfaces were more firmly attached and thus were more durable compared to those on smooth surfaces. In general, however, we conclude that topography is more important for cell adhesion on a protein-resistant surface.

Polymeric material for anti-biofouling

Copolymerization of methyl methacrylate (MMA) and acrylate terminated poly(ethylene oxide-co-ethylene carbonate) (PEOC) macromonomer yields graft copolymer with PEOC side chains. By use of quartz crystal microbalance with dissipation (QCM-D), we have investigated the adsorption of proteins on the surface constructed by the copolymer in seawater. It shows the polymeric surface exhibits protein resistance depending on the content of the side chains. Marine field tests show the protein resistant surface can effectively inhibit marine biofouling for more than 12 weeks.

One step multifunctional micropatterning of surfaces using asymmetric glow discharge plasma polymerization

Micropatterning of surfaces with varying chemical, physical and topographical properties usually requires a number of fabrication steps. Herein, we describe a micropatterning technique based on plasma enhanced chemical vapour deposition (PECVD) that deposits both protein resistant and protein repellent surface chemistries in a single step. The resulting multifunctional, selective surface chemistries are capable of spatially controlled protein adhesion, geometric confinement of cells and the site specific confinement of enzyme mediated peptide self-assembly.

Interaction of Cationic Proteins and Polypeptides with Biocompatible Cationically-Anchored PEG Brushes

Polyetheylene glycol (PEG) brushes, for instance, physically tethered to a surface via the adsorbing portion of a PEG-containing copolymer, are a popular protein-resistant surface treatment. Though physisorbed brushes might be displaced by competing species, they continue to dominate diagnostic chips and other applications. In this work, we examine the interactions of two cationic species, poly l-lysine (PLL) and lysozyme, with a popular type of PEG brush, formed by the adsorption of a graft copolymer of PLL–PEG on negative silica. Here, 20K molecular weight (MW) PLL comprises the main backbone that adsorbs to the silica and the PEG side chains (2K or 5K, in different samples) form tethers. This work examines variations in brush heights and densities, still confining the study to brush architectures (near 30% functionalization of the PLL by PEG side chains) that completely prevent the adsorption of blood proteins such as fibrinogen and albumin. It is found that lysozyme adsorbs to interfaces passivated with these PLL–PEG copolymers in amounts that increase with the amount of PEG in the brush. This suggests attractions between the PEG tethers and lysozyme itself. When PLL–PEG brushes are challenged by homopolymer PLL (a random coil at the physiological pH studied here), the PLL–PEG is almost completely displaced from the silica substrate. The rapid displacement kinetics (with complete loss of protein repellence) for all brush architectures suggest the absence of a steric barrier against PLL penetration of the PEG brush. A small overshoot in surface coverage prior to the displacement of the PLL–PEG chains demonstrates the adsorption of PLL on regions of silica at the base of the brush prior to chain displacement, further arguing for the accessibility of the substrate despite the presence of the brush. Differences between the interactions of lysozyme or PLL with the brush suggest an important role of the globular nature of folded proteins compared with random coil polypeptides in protein–brush interactions and brush penetration. The results emphasize the technological challenge of retaining seemingly robust brushes adsorbed to interfaces, and eliminating protein adhesion from the brush itself.

Metody kontrolowanej polimeryzacji rodnikowej (CRP) w syntezie kopolimerow poliuretanowych

This paper is a review covering the methods most often applied in the synthesis of polyurethane copolymers of various structures (e.g. block or star-shaped). New Controlled Radical Polymerization (CRP) methods such as Controlled Radical Polymerization using the iniferter technique, Atom Transfer Radical Polymerization (ATRP) and the Surface Initiated Atom Transfer Radical Polymerization (SI ATRP) have been discussed in detail. There has been progress made in recent years in each of these methods allowing for the synthesis of small or large molecular weight polymers, functionalization with various types of terminal groups and the formation of novel products with unique topology (nanotubes, core-shell particles) as well as block or star-shaped copolymers. Specifically, CRP allows for the development of biomedical materials — antimicrobial coatings of controlled thickness or protein-resistant surface layers.

Direct write tethered protein arrays

Existing methods for fabricating protein arrays and patterns on solid surfaces rely upon multiple-step chemical reaction procedures. A more straightforward approach is described where proteins are written directly onto a protein resistant surface which eliminates the need for back-filling. This is achieved by the spatially controlled delivery of protein-containing activation solutions onto poly(N-acryloylsarcosine methyl ester) protein-resistant surfaces. The surface tethered proteins are shown to retain their biological function whilst the surrounding poly(N-acryloylsarcosine methyl ester) background prohibits non-specific protein binding.

Tween surfactants: Adsorption, self-organization, and protein resistance

Tween surfactants, each containing hydrophilic ethylene glycol head groups and a hydrophobic alkyl tail, are being actively explored as protein-resistant surface coatings, but little is known about how they adsorb on surfaces. We carry out a comparative study of the adsorption of two Tween molecules (same hydrophilic head group, but a shorter dodecyl tail for Tween 20 and a longer octadecyl tail for Tween 40) on Au and polystyrene surfaces. Despite the similarity between these two molecules, there is a drastic difference in their protein resistance: a monolayer of Tween 20 on a hydrophobic surface is repulsive against protein adsorption but that of Tween 40 is not. The difference in protein resistance can be attributed to two distinctly different adsorption mechanisms. While the adsorption of Tween 40 is described by a simple first-order mechanism, that of Tween 20 consists of a fast adsorption step and a slower reorganization process at a high surface coverage. The latter leads to the formation of a high-density and self-organized monolayer, which is responsible for the enhanced stability and resistance against non-specific protein adsorption.