"Hearing Loss" in QCM Measurement of Protein Adsorption to Protein Resistant Polymer Brush Layers.

Accurate quantification of nonspecific protein adsorption on biomaterial surfaces is essential for evaluation of their antifouling properties. The quartz crystal microbalance (QCM) is an acoustic sensor widely used for the measurement of protein adsorption. However, although the QCM is highly sensitive, it does have performance limitations when working with surfaces modified with thick viscous layers. In the case of polymer brush surfaces, factors such as the thickness and viscosity of the brush may bring such limitations. In the present work, three types of antifouling molecules were used to explore the applicability of QCM for the evaluation of the protein resistance of hydrophilic polymer brush surfaces. Adsorption was also measured by surface plasmon resonance (SPR) as a reference. It was shown that the detection of adsorbed protein requires that protein be located within a critical distance from the QCM chip surface, determined by the viscosity of polymer brush. For larger proteins like fibrinogen, adsorption is expected to occur mainly "on top" of the polymer brush, and brush thickness determines whether protein is located in the "detectable zone". For smaller proteins like lysozyme, adsorption is expected to occur mainly at the chip surface and within the polymer brush layer and to be detectable by QCM. However, the quantity of adsorbed lysozyme may be underestimated when secondary adsorption also occurred. It is concluded that QCM data suggesting very low protein adsorption on polymer brush surfaces should take account of these considerations and should be treated generally with caution.

A versatile, fast, and efficient method of visible-light-induced surface grafting polymerization.

To overcome the problem caused by the lability of the Au-S bond, we demonstrate the first use of Mn2(CO)10 for visible-light-induced surface grafting polymerization on Au surfaces in this paper. The visible-light-induced surface grafting of poly(N-isopropylacrylamide) (PNIPAAm) has the features of a "controlled" polymerization, which is characterized by a linear relationship between the thickness of the grafting layer and the monomer concentration. Ellipsometry indicated the formation of PNIPAAm films of up to ∼200 nm in thickness after only 10 min of polymerization at room temperature, demonstrating that this is a very fast process in comparison with traditional grafting polymerization techniques. Moreover, to demonstrate the potential applications of our approach, different substrates grafted by PNIPAAm and the covalent immobilization of a range of polymers on Au surfaces were also demonstrated. Considering the advantages of simplicity, efficiency, and mild reaction conditions as well as the ability of catecholic derivatives to bind to a large variety of substrates, this visible-light-induced grafting method is expected to be useful in designing functional interfaces.

125I-radiolabeling, surface plasmon resonance, and quartz crystal microbalance with dissipation: three tools to compare protein adsorption on surfaces of different wettability.

The extent of protein adsorption is an important consideration in the biocompatibility of biomaterials. Various experimental methods can be used to determine the quantity of protein adsorbed, but the results usually differ. In the present work, self-assembled monolayers (SAMs) were used to prepare a series of model gold surfaces varying systematically in water wettability, from hydrophilic to hydrophobic. Three commonly used methods, namely, surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), and (125)I-radiolabeling, were employed to quantify fibrinogen (Fg) adsorption on these surfaces. This approach allows a direct comparison of the mass of Fg adsorbed using these three techniques. The results from all three methods showed that protein adsorption increases with increasing surface hydrophobicity. The increase in the mass of Fg adsorbed with increasing surface hydrophobicity in the SPR data was parallel to that from (125)I-radiolabeling, but the absolute values were different and there does not seem to be a "universally congruent" relationship between the two methods for surfaces with varying wettability. For QCM-D, the variation in protein adsorption with wettability was different from that for SPR and radiolabeling. On the more hydrophobic surfaces, QCM-D gave an adsorbed mass much higher than from the two other methods, possibly because QCM-D measures both the adsorbed Fg and its associated water. However, on the more hydrophilic surfaces, the adsorbed mass from QCM-D was slightly greater than that from SPR, and both were smaller than from (125)I-radiolabeling; this was true no matter whether the Sauerbrey equation or the Voigt model was used to convert QCM-D data to adsorbed mass.

Control of lysozyme adsorption by pH on surfaces modified with polyampholyte brushes.

The adsorption of lysozyme is difficult to control by pH because of the relatively high isoelectric point of this protein (11.1). In this article, we demonstrate good control of lysozyme adsorption by pH in the range of 4-10 on silicon surfaces through modification with poly(2-(dimethylamino ethyl) methacrylate)-block-poly(methacrylic acid) (PDMAEMA-b-PMAA) diblock copolymer brushes. We show that the thickness of the outer PMAA block (lPMAA) is critical to the adsorption. When lPMAA was less than 10 nm, adsorption increased with increasing pH, and the difference in adsorption between high and low pH increased with lPMAA. The ratio of adsorption at pH 10 and pH 4 reached values as high as 16.4. When lPMAA was more than 10 nm, the adsorption tendency on the PDMAEMA-b-PMAA diblock copolymer brushes was similar to that on PMAA homopolymer brushes. These results indicate that the combination of PDMAEMA and PMAA gives adsorption behavior reflecting the properties of both polymers. However, if the outer PMAA block is thicker than a critical value, then the protein-resistant effect of the inner PDMAEMA block is screened.

Unidirectional rotating molecular motors dynamically interact with adsorbed proteins to direct the fate of mesenchymal stem cells.

Artificial rotary molecular motors convert energy into controlled motion and drive a system out of equilibrium with molecular precision. The molecular motion is harnessed to mediate the adsorbed protein layer and then ultimately to direct the fate of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). When influenced by the rotary motion of light-driven molecular motors grafted on surfaces, the adsorbed protein layer primes hBM-MSCs to differentiate into osteoblasts, while without rotation, multipotency is better maintained. We have shown that the signaling effects of the molecular motion are mediated by the adsorbed cell-instructing protein layer, influencing the focal adhesion-cytoskeleton actin transduction pathway and regulating the protein and gene expression of hBM-MSCs. This unique molecular-based platform paves the way for implementation of dynamic interfaces for stem cell control and provides an opportunity for novel dynamic biomaterial engineering for clinical applications.

Polarization of Macrophages, Cellular Adhesion, and Spreading on Bacterially Contaminated Gold Nanoparticle-Coatings in Vitro.

Biomaterial-associated infections often arise from contaminating bacteria adhering to an implant surface that are introduced during surgical implantation and not effectively eradicated by antibiotic treatment. Whether or not infection develops from contaminating bacteria depends on an interplay between bacteria contaminating the biomaterial surface and tissue cells trying to integrate the surface with the aid of immune cells. The biomaterial surface plays a crucial role in defining the outcome of this race for the surface. Tissue integration is considered the best protection of a biomaterial implant against infectious bacteria. This paper aims to determine whether and how macrophages aid osteoblasts and human mesenchymal stem cells to adhere and spread over gold nanoparticle (GNP)-coatings with different hydrophilicity and roughness in the absence or presence of contaminating, adhering bacteria. All GNP-coatings had identical chemical surface composition, and water contact angles decreased with increasing roughness. Upon increasing the roughness of the GNP-coatings, the presence of contaminating Staphylococcus epidermidis in biculture with cells gradually decreased surface coverage by adhering and spreading cells, as in the absence of staphylococci. More virulent Staphylococcus aureus fully impeded cellular adhesion and spreading on smooth gold- or GNP-coatings, while Escherichia coli allowed minor cellular interaction. Murine macrophages in monoculture tended toward their pro-inflammatory "fighting" M1-phenotype on all coatings to combat the biomaterial, but in bicultures with contaminating, adhering bacteria, macrophages demonstrated Ym1 expression, indicative of polarization toward their anti-inflammatory "fix-and-repair" M2-phenotype. Damage repair of cells by macrophages improved cellular interactions on intermediately hydrophilic/rough (water contact angle 30 deg/surface roughness 118 nm) GNP-coatings in the presence of contaminating, adhering Gram-positive staphylococci but provided little aid in the presence of Gram-negative E. coli. Thus, the merits on GNP-coatings to influence the race for the surface and prevent biomaterial-associated infection critically depend on their hydrophilicity/roughness and the bacterial strain involved in contaminating the biomaterial surface.

Using γ-Ray Polymerization-Induced Assemblies to Synthesize Polydopamine Nanocapsules.

This work reports a simple and robust strategy for synthesis of polydopamine nanocapsules (PDA NCs). First, polymer assemblies were synthesized by a γ-ray-induced liquid-liquid (H2O-acrylate) interface polymerization strategy, in the absence of any surfactants. 1H nuclear magnetic resonance analysis and molecular dynamics simulation reveal that the generation of polymer assemblies largely depends on the hydrophilicity of acrylate and gravity of the oligomers at the interface. By virtue of the spherical structure and mechanic stability of the polymer assemblies, PDA NCs are next prepared by the interfacial polymerization of dopamine onto the assemblies, followed by the removal of templates by using ethanol. The polydopamine nanocapsules are shown to load and release ciprofloxacin (CIP, a model drug), such that the CIP-loaded PDA NCs are able to inhibit the growth of Escherichia coli.

Long-range interactions between protein-coated particles and POEGMA brush layers in a serum environment.

Hydrophilic poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA) brush layers with different thickness and graft densities were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) to construct a model surface to examine protein-surface interactions in a serum environment. The thickness of the POEGMA brush layers could be well controlled by the polymerization time and density of the immobilized initiators. The interactions between these brush-modified surfaces and the protein-coated polystyrene (PS) particles in newborn calf serum (NBCS) environment were then measured by total internal reflection microscopy (TIRM). In addition, protein adsorption properties onto the polymer brush surface layers were examined by atomic force microscopy (AFM). Relatively large amounts of protein adsorbed to short (4nm and 9nm-thick) POEGMA-coated surfaces or surfaces grafted with a low density of polymer chains. It was considered that shorter polymer chains or chains with low grafted density cannot fully cover the surfaces, proteins in serum could directly interact with the material surface and then deposited to form an adsorbed layer. The TIRM measurements showed that such adsorbed protein layer could mediate the interactions between the two surfaces by generating steric or bridging forces, resulting in different interaction potentials. Some particles were freely diffusing, some experienced intermittent diffusion and more than 50% of particles were irreversibly deposited to the surfaces covered by short polymer brushes. However, for longer (17 and 30nm-thick) POEGMA brush layer surfaces, material surface would be sufficiently covered by the dense coating and the first step of protein adsorption on surface was avoided. TIRM measurements showed that around 95% of the protein-coated particles could freely move in the serum and no attractive force between two surfaces was detected. The steric repulsion generated from the long POEGMA brush layer in the swollen state was long-range and strong so that the protein adsorption is very unlikely. These results concluded that the adsorbed protein layer on POEGMA surfaces plays an important role in regulating the interaction between protein-coated particles and POEGMA surfaces which are highly repellent toward protein adsorption.

Comparison of the Responsivity of Solution-Suspended and Surface-Bound Poly(N-isopropylacrylamide)-Based Microgels for Sensing Applications.

In this submission, the phase transition behavior for poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgels and their assemblies was investigated as a function of temperature and pH using UV-vis spectroscopy (to probe light scattering behavior) and quartz crystal microbalance with dissipation (QCM-D) measurements. PNIPAm-co-AAc microgels were "painted" onto Au-coated glass substrates (for UV-vis) and the Au electrode of a QCM crystal to generate monolayers. The subsequent deposition of another Au layer on top of the pNIPAm-co-AAc microgel layer yields what is known as an etalon. UV-vis/QCM-D measurements revealed that the temperature and pH responsivities for the microgel assemblies match well with their solution behavior. UV-vis spectroscopy shows that the transmittance of the microgel monolayers decreased with increasing solution temperature at pH 3.0. At pH 6.5, the AAc groups in the microgels were deprotonated, leading to strong Coulombic repulsive forces inside the microgels that prevented their collapse and lead to minimal change in the transmitted light intensity. However, QCM-D analysis reveals more complex behavior as it is sensitive to the viscosity/viscoelasticity and thickness changes of the microgel layer, which ultimately depends on the microgel chemical composition and the interaction of the etalon's Au layer with the crystal. The maximum sensitivity to temperature is 0.8 × 10-3 °C·Hz-1, which is the most sensitive pNIPAm microgel-based QCM temperature sensor thus far reported in the literature. Finally, we exploit this new understanding to characterize the pH and ionic strength of a solution using pNIPAm-co-XAAc microgel-based etalon coated crystals. The research results and the sensing demonstration can inspire new and improved sensor designs for a variety of analytes.

Surface immobilization of a protease through an inhibitor-derived affinity ligand: a bioactive surface with defensive properties against an inhibitor.

The concept of enzyme immobilization via an inhibitor-derived peptide was developed. This method of immobilization was shown to be advantageous over physical adsorption and covalent bonding in retaining the enzymatic activity. Moreover, the surface-immobilized enzyme exhibited resistance against its inhibitor due to the occupation of an inhibitor binding site on the enzyme.

A t-PA/nanoparticle conjugate with fully retained enzymatic activity and prolonged circulation time.

A major issue in the therapeutic use of tissue plasminogen activator (t-PA) for the treatment of thrombotic diseases is its very short half-life in the circulation due to the effects of inhibitors. The present study aims to resolve the issue using a t-PA/gold nanoparticle (t-PA/AuNP) conjugate prepared via bio-affinity ligation under physiological conditions. The ligation is based on the specific interactions between t-PA and ε-lysine (a ligand that has affinity to a specific domain in t-PA) immobilized on the AuNP surface through polyvinyl pyrrolidone (PVP) as a spacer. The conjugate can not only retain almost full enzymatic activity and clot dissolving efficiency, but also protect t-PA from inhibition by PAI-1 to some extent as compared with free t-PA in vitro. Moreover, the conjugate showed prolonged circulation time in vivo.

Surfaces having dual affinity for plasminogen and tissue plasminogen activator: in situ plasmin generation and clot lysis.

Surface modification with affinity ligands capable of capturing bioactive molecules in situ is a widely used strategy for developing biofunctional materials. However, many bioactive molecules, for example zymogens, exist naturally in a "quiescent" state, and become active only when "triggered" by specific activators. In the present study, in situ activation of a surface-integrated zymogen was achieved by introducing affinity ligands for both the zymogen and its activator. Specifically a dual affinity surface was designed for the integration of plasminogen (Plg) and tissue plasminogen activator (t-PA). This surface was expected to have plasmin-generating and, therefore, fibrinolytic properties. A polyurethane surface was modified with a copolymer of 2-hydroxyethyl methacrylate and 1-adamantan-1-ylmethyl methacrylate poly(HEMA-co-AdaMA). The affinity ligands, ARMAPE peptide (for t-PA) and ε-lysine-containing ß-cyclodextrin (ß-CD-(Lys)7) (for Plg), were attached in sequence via covalent bonding and host-guest interactions, respectively. The resulting surfaces were shown to have high binding capacities for both t-PA and Plg while resisting nonspecific protein adsorption. Pre-loading with t-PA followed by Plg uptake from plasma generated plasmin and thus endowed the surface with fibrinolytic activity. In general the incorporation of dual affinity ligands to achieve surface-promoted bioactivity is a promising approach for the development of biofunctional materials. The method reported herein for the sequential attachment of plasminogen and t-PA affinity ligands can be extended to systems of multiple ligands generally.

Conjugation of polymers to proteins through an inhibitor-derived peptide: taking up the inhibitor "berth".

A hexapeptide derived from an enzyme inhibitor was used as an affinity ligand for the conjugation of a hydrophilic polymer to the enzyme. The peptide targeted the polymer to the "berth" of the inhibitor in the enzyme, affording the enzyme resistance to the inhibitor without affecting the enzymatic activity.

Thermoresponsive copolymer decorated surface enables controlling the adsorption of a target protein in plasma.

The control of protein/surface interactions by external stimuli is often required in bioapplications such as bioseparation and biosensors. Although regulation of protein adsorption has been achieved on the surfaces modified with stimuli-responsive polymers, controlled protein adsorption is still challenging for a target protein in a multiprotein system. The present study developed a concept of surface design for the controlled adsorption of a specific protein from plasma by combining a thermoresponsive polymer with an affinity ligand on the surface. In this regard, a polyurethane (PU) surface was modified with the copolymer of N-isopropylacrylamide (NIPAAm) and a ε-lysine-containing monomer (LysMA). ε-Lysine is a specific ligand for plasminogen that was used as the model "target protein" in this study. The PU-P(NIPAAm-co-Lys) surfaces exhibited distinct thermoresponsivity of plasminogen adsorption from plasma with a larger quantity adsorbed at 37 °C than at 23 °C. By contrast, the surfaces showed a low level of adsorption for other plasma proteins at both temperatures. In addition, plasminogen adsorbed on a PU-P(NIPAAm-co-Lys) surface could be partly desorbed by lowering the temperature, and the activity of plasminogen adsorbed was well preserved. We believe that the concept developed in this study can be extended to other proteins by combining PNIPAAm and specific ligands with affinities for the proteins of interest.