Bioadhesive control of plasma proteins and blood cells from umbilical cord blood onto the interface grafted with zwitterionic polymer brushes

In this work, bioadhesive behavior of plasma proteins and blood cells from umbilical cord blood (UCB) onto zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymer brushes was studied. The surface coverage of polySBMA brushes on a hydrophobic polystyrene (PS) well plate with surface grafting weights ranging from 0.02 mg/cm(2) to 0.69 mg/cm(2) can be effectively controlled using the ozone pretreatment and thermal-induced radical graft-polymerization. The chemical composition, grafting structure, surface hydrophilicity, and hydration capability of prepared polySBMA brushes were determined to illustrate the correlations between grafting properties and blood compatibility of zwitterionic-grafted surfaces in contact with human UCB. The protein adsorption of fibrinogen in single-protein solutions and at complex medium of 100% UCB plasma onto different polySBMA brushes with different grafting coverage was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The grafting density of the zwitterionic brushes greatly affects the PS surface, thus controlling the adsorption of fibrinogen, the adhesion of platelets, and the preservation of hematopoietic stem and progenitor cells (HSPCs) in UCB. The results showed that PS surfaces grafted with polySBMA brushes possess controllable hydration properties through the binding of water molecules, regulating the bioadhesive and bioinert characteristics of plasma proteins and blood platelets in UCB. Interestingly, it was found that the polySBMA brushes with an optimized grafting weight of approximately 0.1 mg/cm(2) at physiologic temperatures show significant hydrated chain flexibility and balanced hydrophilicity to provide the best preservation capacity for HSPCs stored in 100% UCB solution for 2 weeks. This work suggests that, through controlling grafting structures, the hemocompatible nature of grafted zwitterionic polymer brushes makes them well suited to the molecular design of regulated bioadhesive interfaces for use in the preservation of HSPCs from human UCB.     

A highly stable nonbiofouling surface with well-packed grafted zwitterionic polysulfobetaine for plasma protein repulsion

An ideal nonbiofouling surface for biomedical applications requires both high-efficient antifouling characteristics in relation to biological components and long-term material stability from biological systems. In this study we demonstrate the performance and stability of an antifouling surface with grafted zwitterionic sulfobetaine methacrylate (SBMA). The SBMA was grafted from a bromide-covered gold surface via surface-initiated atom transfer radical polymerization to form well-packed polymer brushes. Plasma protein adsorption on poly(sulfobetaine methacrylate) (polySBMA) grafted surfaces was measured with a surface plasmon resonance sensor. It is revealed that an excellent stable nonbiofouling surface with grafted polySBMA can be performed with a cycling test of the adsorption of three model proteins in a wide range of various salt types, buffer compositions, solution pH levels, and temperatures. This work also demonstrates the adsorption of plasma proteins and the adhesion of platelets from human blood plasma on the polySBMA grafted surface. It was found that the polySBMA grafted surface effectively reduces the plasma protein adsorption from platelet-poor plasma solution to a level superior to that of adsorption on a surface terminated with tetra(ethylene glycol). The adhesion and activation of platelets from platelet-rich plasma solution were not observed on the polySBMA grafted surface. This work further concludes that a surface with good hemocompatibility can be achieved by the well-packed surface-grafted polySBMA brushes.     

Tunable blood compatibility of polysulfobetaine from controllable molecular-weight dependence of zwitterionic nonfouling nature in aqueous solution

This work describes a tunable blood compatibility of zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymers at a wide range of high molecular weights from 50 kDa to 300 kDa controlled with a similar polydispersity via homogeneous free-radical polymerization. The control of molecular weights of polySBMA highly regulates the zwitterionic nonfouling nature to resist the adsorption of plasma proteins, the coagulant of human plasma, and the hemolysis of red blood cells. In this study, the upper critical solution temperatures (UCSTs) and hydrodynamic size of prepared polymers are determined to illustrate the correlations between intermolecular zwitterionic associations and blood compatibility of polySBMA suspension in human blood. The polySBMA exhibited clear shifts of UCSTs in the stimuli-responsive control of solution pH and ionic strength, which were strongly associated with the molecular weights of the prepared polymers. Plasma-protein adsorption onto the polySBMA polymers from single-protein solutions and the complex medium of 100% human plasma were measured by dynamic light scattering to determine the nonfouling stability of polySBMA suspension. It was found that the nonfouling nature as well as hydration capability of polySBMA can be effectively controlled via regulated molecular weights of zwitterionic polymers. This work shows that the polySBMA polymer with an optimized molecular weight of about 135 kDa at physiologic temperature is presented high hydration capability to function the best nonfouling character of anticoagulant activity and antihemolytic activity in human blood. The excellent blood compatibility of zwitterionic polySBMA along with their stimuli-responsive phase behavior in aqueous solution suggests their potential for use in blood-contacting targeted delivery and diagnostic applications.     

Superlow fouling sulfobetaine and carboxybetaine polymers on glass slides

This work describes the superlow fouling properties of glass slides grafted with zwitterionic polymers to highly resist the adsorption of proteins and the adhesion of mammalian cells. Glass slides were first silanized using 2-bromo-2-methyl-N-3-[(triethoxysilyl)propyl]propanamide (BrTMOS). Two zwitterionic polymers, poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA), were then grafted from the silanized glass substrates using the atom-transfer radical polymerization (ATRP) method. X-ray photoelectron spectroscopy (XPS) was used to analyze the surfaces of the silanized glass substrates and the substrates grafted with the polymers. An enzyme-linked immonosobrbent assay (ELISA) using polyclonal antibodies was used to measure fibrinogen adsorption on these surfaces. The surfaces with polySBMA or polyCBMA layers were shown to reduce fibrinogen adsorption to a level comparable with that of adsorption on poly(ethylene glycol)-like films. Bovine aortic endothelial cells (BAECs) were seeded on these surfaces. The attachment and spreading of the cells were observed only on unpolymerized glass surfaces. This work further demonstrates that zwitterionic polymers highly resist nonspecific protein adsorption and cell adhesion and provides an effective method to modify glass slides or other oxide surfaces to achieve superlow fouling.     

Film thickness dependence of protein adsorption from blood serum and plasma onto poly(sulfobetaine)-grafted surfaces

In this work, we investigate protein adsorption from single protein solutions and complex media such as 100% blood serum and plasma onto poly(sulfobetaine methacrylate) (polySBMA)-grafted surfaces via atom transfer radical polymerization (ATRP) at varying film thicknesses. It is interesting to observe that protein adsorption exhibits a minimum at a medium film thickness. Results show that the surface with 62 nm polySBMA brushes presents the best nonfouling character in 100% blood serum and plasma although all of these surfaces are highly resistant to nonspecific protein adsorption from single fibrinogen and lysozyme solutions. Surface resistance to 100% blood serum or plasma is necessary for many applications from blood-contacting devices to drug delivery. This work provides a new in vitro evaluation standard for the application of biomaterials in vivo.     

Formation of antifouling functional coating from deposition of a zwitterionic-co-nonionic polymer via “grafting to” approach

We develop a new process for the preparation of synergistic antifouling functional coatings on gold surfaces via a “grafting to” approach. The strategy includes a synthetic step of polymer brushes that consist of poly (ethylene glycol) (PEG) and zwitterionic side chains via a typical reversible-addition fragmentation chain transfer (RAFT) polymerization process, and a subsequent deposition of the polymer brushes onto a gold substrate. The presence of PEG and zwitterion chains on these polymer brush-coated gold surfaces has been proved to have a synergistic effect on the final antifouling property of the coating. PEG chains lower the electrostatic repulsion between zwitterionic polymer chains and increase their graft density on gold surfaces, while zwitterionic polymer effectively improves the antifouling property that is offered by PEG chains alone. Protein adsorption and cell attachment assays tests are conducted to confirm that this copolymer layer on gold surface has a pronounced resistance against proteins such as Bovine serum albumin and Lysozyme. Importantly, the antifouling property can be systematically adjusted by varying the molar ratio of PEG to zwitterionic chains in the final coating copolymer.     

Influence of brush length of PVP chains immobilized on silicon wafers on their blood compatibility

In this study, the influence of variations in chain length of polyvinylpyrrolidone (PVP) brushes on the blood compatibility of silicon wafer surfaces to which they were chemically attached was studied. Si surfaces were functionalized with various lengths of PVP brush using atom transfer radical polymerization technology and confirmed by elliptical polarized light, atom force microscopy, and X‐ray photoelectron spectroscopy. The blood compatibility of these biointerfaces were systematically investigated by measuring protein adsorption, clotting time, platelet adhesion, contact activation, and complement activation. The results indicate that the hemocompatibility of the surfaces was highly related to PVP brush chain length and hydrophilicity of the surfaces. Our results contribute to rational biointerface design for specific biomedical applications. The results reveal that the PVP brushes with a long chain length have great potential for blood contacting applications due to their reduced protein absorption and cell activation following its contact with blood.     

BSA adsorption on bimodal PEO brushes

BSA adsorption onto bimodal PEO brushes at a solid surface was measured using optical reflectometry. Bimodal brushes consist of long (N=770) and short (N=48) PEO chains and were prepared on PS surfaces, applying mixtures of PS(29)-PEO(48) and PS(37)-PEO(770) block copolymers and using the Langmuir-Blodgett technique. Pi-A isotherms of (mixtures of) the block copolymers were measured to establish the brush regime. The isotherms of PS(29)-PEO(48) show hysteresis between compression and expansion cycles, indicating aggregation of the PS(29)-PEO(48) upon compression. Mixtures of PS(29)-PEO(48) and PS(37)-PEO(770) demonstrate a similar hysteresis effect, which eventually vanishes when the ratio of PS(37)-PEO(770) to PS(29)-PEO(48) is increased. The adsorption of BSA was determined at brushes for which the grafting density of the long PEO chains was varied, while the total grafting density was kept constant. BSA adsorption onto monomodal PEO(48) and PEO(770) brushes was determined for comparison. The BSA adsorption behavior of the bimodal brushes is similar to the adsorption of BSA at PEO(770) monomodal brushes. The maximum of BSA adsorption at low grafting density of PEO(770) can be explained by ternary adsorption, implying an attraction between BSA and PEO. The contribution of primary adsorption to the total adsorbed amount is negligible.     

In-situ investigation of the adsorption of globular model proteins on stimuli-responsive binary polyelectrolyte brushes

Binary brushes constituted from two incompatible polymers can be used in the form of ultrathin polymeric layers as a versatile tool for surface engineering to tune physicochemical surface characteristics such as wettability, surface charge, chemical composition, and morphology and furthermore to create responsive surface properties. Mixed brushes of oppositely charged weak polyelectrolytes represent a special case of responding surfaces that are sensitive to changes in the pH value of the aqueous environment and therefore represent interesting tools for biosurface engineering. The polyelectrolyte brushes used for this study were composed of two oppositely charged polyelelctrolytes poly(2-vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). The in-situ properties and surface characteristics such as as surface charge, surface tension, and extent of swelling of these brush layers are functions of the pH value of the surrounding aqueous solution. To test the behavior of the mixed polylelctrolyte brushes in contact with biosystems, protein adsorption experiments with globular model proteins were performed at different pH values and salt concentrations (confinement of counterions) of the buffer solutions. The influence of the pH value, buffer salt concentration, and isoelectric points (IEP) of the brush and protein on the adsorbed amount and the interfacial tension during protein adsorption as well as the protein adsorption mechanism postulated in reference to recently developed theories of protein adsorption on polyelectrolyte brushes is discussed. In the salted regime, protein adsorption was found to be similar to the often-described adsorption at hydrophobic surfaces. However, in the osmotic regime the balance of electrostatic repulsion and a strong entropic driving force, "counterion release", was found to be the main influence on protein adsorption.     

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super‐resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA‐Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4–10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.     

Investigation of the hydration of nonfouling material poly(sulfobetaine methacrylate) by low-field nuclear magnetic resonance

The strong surface hydration layer of nonfouling materials plays a key role in their resistance to nonspecific protein adsorption. Poly(sulfobetaine methacrylate) (polySBMA) is an effective material that can resist nonspecific protein adsorption and cell adhesion. About eight water molecules are tightly bound with one sulfobetaine (SB) unit, and additional water molecules over 8:1 ratio mainly swell the polySBMA matrix, which is obtained through the measurement of T(2) relaxation time by low-field nuclear magnetic resonance (LF-NMR). This result was also supported by the endothermic behavior of water/polySBMA mixtures measured by differential scanning calorimetry (DSC). Furthermore, by comparing both results of polySBMA and poly(ethylene glycol) (PEG), it is found that (1) the hydrated water molecules on the SB unit are more tightly bound than on the ethylene glycol (EG) unit before saturation, and (2) the additional water molecules after forming the hydration layer in polySBMA solutions show higher freedom than those in PEG. These results might illustrate the reason for higher resistance of zwitterionic materials to nonspecific protein adsorptions compared to that of PEGs.     

Adsorption and immobilization of cytochrome c on nanodiamonds

Methods have been developed to immobilize proteins onto the surfaces ofnanodiamonds with an average size of 5 +/- 1 nm. The immobilization started with carboxylation/oxidization of diamonds with strong acids, followed by coating the surfaces with poly-L-lysine (PL) for covalent attachment of proteins using heterobifunctional cross-linkers. The feasibility of this approach is proven with fluorescent labeling of the PL-coated diamonds by Alexa Fluor 488 and subsequent detection of the emission using a confocal fluorescence microscope. Immobilization of proteins onto the surfaces is also demonstrated with yeast cytochrome c, which possesses a free SH group for linkage and a characteristic Soret absorption band for observation.     

Single component and selective competitive protein adsorption in a patchy polymer brush: opposition between steric repulsions and electrostatic attractions

This work explores the use of "patchy" polymer brushes to control protein adsorption rates on engineered surfaces and to bind targeted species from protein mixtures with high selectivity but without invoking molecular recognition. The brushes of interest contain embedded cationic "patches" composed of isolated adsorbed poly(l-lysine) coils (PLL) that are about 10 nm in diameter and are randomly arranged on a silica substrate. Around these patches is a protein-resistant poly(ethylene glycol) (PEG) brush that is formed from the adsorption of a PLL-g-PEG graft copolymer on the remaining silica surface. Electrostatic attractions between individual cationic patches and the negative regions of approaching proteins may be energetically insufficient to bind proteins. Furthermore, protein-patch attractions are reduced by steric repulsions between proteins and the PEG brush. We show that protein adsorption, gauged by ultimate short-term coverages and by the initial protein adsorption rate, exhibits an adhesion threshold: pure PEG brushes of the architectures employed here and brushes containing sparse loadings of PLL patches do not adsorb protein. Above a critical PLL patch loading or threshold, protein adsorption proceeds, often dramatically. The PLL patch thresholds are specific to the protein of interest, allowing surfaces to be engineered to adhesively discriminate different proteins within a mixture. The separation achieved is remarkably sharp: one protein adsorbs, but the second is completely rejected from the interface. The surfaces in this study, by virtue of their well-controlled and well-characterized patchy nature, distinguish themselves from multicomponent brushes or brushes used to end-tether peptide sequences and nucleotides.     

Comparative microRNA detection from precursor-microRNA-transfected hepatocellular carcinoma cells by capillary electrophoresis with dual-color laser-induced fluorescence

A dual-LIF (dLIF) setup combined with CE for microRNA (miRNA) detection is proposed in this study. An argon ion laser (488 nm) and a solid state laser (640 nm) were chosen to excite the fluorescent dye-labeled DNA probe after splinted ligation of miRNA. The crosstalk of emission spectrum of Alex Fluor 488 and Alex Fluor 647 is minimized with a zero crosstalk matrix for Alex Fluor 647 to 488 channels. The linear ranges of the device for the fluorescent dye-labeled DNA probe were both from 1.0 nM to 0.1 pM. The limits of detection for Alexa Fluor 488-labeled DNA and Alex Fluor 647-labeled DNA were 9.3 and 31 fM, respectively. The detection of specific miRNA has been accomplished by combining splinted ligation with the fluorescent dye-labeled oligonucleotides. The linear range for the synthetic miRNA is from 1.0 nM to 1.0 pM. Without PCR amplification, CE-dLIF was applied to discriminate a pre-miR-10b*-transfected cells (contains precursor miR-10b*) from hepatocellular carcinoma cell (control cells). Therefore, this result indicates CE-dLIF has great potential to provide a rapid comparative assay for miRNAs detection.     

Structure and dynamics of alpha-lactalbumin adsorbed at a charged brush interface

We have studied the adsorption of alpha-lactalbumin at a planar poly(acrylic acid) (PAA) brush using neutron reflectometry (NR) and total internal reflection fluorescence (TIRF) spectroscopy. The PAA brush has been prepared by spin-coating silicon or quartz plates with a hydrophobic poly(styrene) film and by transferring the copolymer poly(styrene)-poly(acrylic acid) onto the modified surface. In the case of NR, the poly(styrene) film and the poly(styrene) chain ends of the copolymer were perdeuterated in order to generate a high contrast to the non-deuterated PAA brush. alpha-Lactalbumin was chosen as the model protein because it is a relatively small globular protein with a negative net charge at neutral pH-values, as chosen in the experiments. Thus, it is interacting with the PAA brush on the 'wrong' side of its isoelectric point. In addition, the effects of temperature on the volume fraction profile and the reorientational mobility of the protein within the PAA brush were determined. From the analysis of the NR data, it has been found that despite of its negative net charge, alpha-lactalbumin is penetrating into the PAA brush. Its volume fraction profile parallels that of the PAA brush, indicating an exclusive interaction between the protein and the PAA. No protein accumulation is found at the inner poly(styrene) or the outer solution interface of the PAA brush. When increasing the temperature from 20 to 40 degrees C, less protein is adsorbed, suggesting the presence of enthalpic interaction contributions. From the analysis of the TIRF data, a high degree of reorientational mobility of alpha-lactalbumin within a PAA brush can be inferred. The reorientational correlation time of alpha-lactalbumin labeled with the Alexa Fluor 488 dye was found to increase from 5.5 to 32 ns upon adsorption, which can well be explained by the higher viscosity inside the PAA brush. Overall, the results of this study quantify for the first time the molecular details of the unique interaction of a protein on the 'wrong' side of its isoelectric point with a planar charged brush interface. It is concluded that the high mobility of alpha-lactalbumin within a PAA brush can partially be understood by the presence of repulsive electrostatic interactions. There is no 'freezing' of the protein dynamics, which is a precondition for biological activity.     

Controlling the motion and placement of micrometer-sized metal particles using patterned polymer brush surfaces

In this paper, we show that silicon surfaces patterned with poly(methacrylic acid) brushes are able to control the Brownian motion of 2-3 μm iron particles, which sediment onto the surface in aqueous solution and experience differences in repulsive force depending upon their position. Differences in repulsion lead to different gravitational potential energies across the surface, which gives bias to the Brownian motion taking place. Three regimes have been identified depending upon the brush height: (i) no control of Brownian motion when the brush height is small, (ii) Brownian motion that is influenced by the polymer brush when the brush 17 height is intermediate, (iii) Brownian motion that is confined by polymer brush barriers when the brush height is greatest. The height of brush found necessary to significantly influence iron particle motion was small at 39 nm or 2% of the particle diameter.     

Primary versus ternary adsorption of proteins onto PEG brushes

Polyethylene glycol (PEG) brushes are used to reduce protein adsorption at surfaces. Their design needs to allow for two leading adsorption modes at the brush-coated surface. One is primary adsorption at the surface itself. The second is ternary adsorption within the brush as a result of weak PEG-protein attraction. We present a scaling theory of the equilibrium adsorption isotherms allowing for concurrent primary and ternary adsorption. The analysis concerns the weak adsorption limit when individual PEG chains do not bind proteins. It also addresses two issues of special relevance to brushes of short PEGs: the consequences of large proteins at the surface protruding out of a shallow brush and the possibility of marginal solvent conditions leading to mean-field behavior. The simple expressions for the adsorption isotherms are in semiquantitative agreement with experiments.     

Selective Adsorption of Functionalized Nanoparticles to Patterned Polymer Brush Surfaces and Its Probing with an Optical Trap

The site‐specific attachment of nanoparticles is of interest for biomaterials or biosensor applications. Polymer brushes can be used to regulate this adsorption, so the conditions for selective adsorption of phosphonate‐functionalized nanoparticles onto micropatterned polymer brushes with different functional groups are optimized. By choosing the strong polyelectrolytes poly(3‐sulfopropyl methacrylate), poly(sulfobetaine methacrylate), and poly[2‐(methacryloyloxy)ethyl trimethylammonium chloride], it is possible to direct the adsorption of nanoparticles to specific regions of the patterned substrates. A pH‐dependent adsorption can be achieved by using the polycarboxylate brush poly(methacrylic acid) (PMAA) as substrate coating. On PMAA brushes, the nanoparticles switch from attachment to the brush regions to attachment to the grooves of a patterned substrate on changing the pH from 3 to 7. In this manner, patterned substrates are realized that assemble nanoparticles in pattern grooves, in polymer brush areas, or substrates that resist the deposition of the nanoparticles. The nanoparticle deposition can be directed in a pH‐dependent manner on a weak polyelectrolyte, or is solely charge‐dependent on strong polyelectrolytes. These results are correlated with surface potential measurements and show that an optical trap is a versatile method to directly probe interactions between nanoparticles and polymer brushes. A model for these interactions is proposed based on the optical trap measurements.     

Image printing on the surface of anti-biofouling zwitterionic polymer brushes by ion beam irradiation

A CMB monomer was polymerized on a glass plate with a surface-confined ATRP initiator containing a 2-bromoisobutyryl group. The glass plate modified with a PCMB brush was highly hydrophilic and showed a strong resistance against non-specific adsorption of proteins and cell adhesion. Upon ion beam irradiation, furthermore, the PCMB brush was ablated and a hollow space with a designed shape could be made to which HEK293 cells (from human embryonic kidney) and Hep G2 (from human hepatoma) cells non-specifically adhered, while no adhesion of these cells to the non-treated area on the brush was observed. The present results clearly indicate the usefulness of ion beam-printed patterns of anti-biofouling zwitterionic polymer brushes in the biomedical field.