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Long-term stability and function are key challenges for optical nanosensors operating in complex biological environments. While much focus is rightly placed on issues related to specificity, sensitivity, reversibility, and response time, many nanosensors are not capable of transducing accurate results over prolonged time periods. Sensors could fail over time due to the degradation of scaffold material, degradation of signaling dyes and components, or a combination of both. It is critical to investigate how such degradative processes affect sensor output, as the consequences could be severe. Herein, we used fluorescent core-shell organosilica pH nanosensors as a model system, incubating them in a range of common aqueous solutions over time at different temperatures, and then searched for changes in fluorescence signal, particle size, and evidence of silica degradation. We found that these ratiometric nanosensors produced stable optical signals after aging for 30 days at 37 °C in standard saline buffers with and without 10% fetal bovine serum, and without any evidence of material degradation. Next, we evaluated their performance as real-time pH nanosensors in bacterial suspension cultures, observing a close agreement with a pH electrode for control nanosensors, yet observing obvious deviations in signal based on the aging conditions. The results show that while the organosilica scaffold does not degrade appreciably over time, careful selection of dyes and further systematic investigations into the effects of salt and protein levels are required to realize long-term stable nanosensors.

Thin polymeric coatings are commonly used for altering surface properties and modulating the interfacial performance of materials. Possible contributions from the substrate to the interfacial forces and effects are, however, usually ignored and are not well understood, nor is it established how the coating thickness modulates and eventually eliminates contributions from substrates to the van der Waals (vdW) interfacial force. In this study we quantified, by colloid-probe atomic force microscope (AFM) and by theoretical calculations, the interfacial vdW contributions from substrates acting through ethanol plasma polymer (EtOHpp) coatings of a range of thicknesses on Au and Si bulk materials. In approach force curves against EtOHpp-coated Au substrates the magnitude of the vdW force decreased as the EtOHpp coating thickness increased to 18 nm and then plateaued with further increases in coating thickness, providing direct evidence for a contribution to the total interfacial vdW force from the Au substrate acting through thin coatings. The experimental observations accord with theoretical calculations of the thickness dependence of Hamaker coefficients derived from rigorous simulation using the Lifshitz theory. In addition, the measured forces agree well with theoretical predictions including correction for finite roughness. Thus, our experimental and theoretical results establish how the thickness of polymer thin film coatings modulates the total interfacial vdW force and how this can be used to tune the net vdW force so as to either contain a large substrate contribution or arise predominantly from the polymeric overlayer. Our findings enable rational design of coating thickness to tailor interfacial interactions and material performance. Refereed/Peer-reviewed

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.

3D bioprinting is a recent technique that can create complex cell seeded scaffolds and therefore holds great promise to revolutionize the biomedical sector by combining materials and structures that more closely mimic the 3D cell environment in tissues. The most commonly used biomaterials for printing are hydrogels, however, many of the hydrogels used still present issues of printability, stability, or poor cell-material interactions. We propose that bioinks with intrinsic self-assembling and shear thinning properties, such as xanthan gum, can be methacrylated (XGMA) and combined with a bio-functional material such as gelatin methacryloyl (GelMa) to create a stable, cell-interactive bioink with improved properties for 3D bioprinting. These biomaterials have reduced viscosity under high shear and recover their viscosity rapidly after the shear is removed, retaining their shape, which translates to easier extrusion whilst maintaining accurate fidelity after printing. This was confirmed in printing studies, with measured normalized strand widths of 1.2 obtained for high gel concentrations (5+5 % XGMA-GelMA). Furthermore, the introduction of a secondary photo-cross-linking method allowed tuning of the mechanical properties of the hydrogel with stiffness between 15 and 30 kPa, as well as improving the stability of the hydrogel with retention of 75 % of its mass after 90 d. The hydrogel was shown to be biocompatible and bio-active with 97 % cell viability, and cell spreading after 7 d of culture for low gel concentrations (3+3 % XGMA-GelMA). Shear stresses were relatively low while printing (1 kPa) as a result of the shear thinning property of the material, which supported cell viability during extrusion. Finally, printed hydrogels retained high cell viability for lower gel concentrations, and showed improved cell viability for more concentrated hydrogels when compared to cells cultured in bulk hydrogels, presumably due to improved nutrient/oxygen diffusion and cell migration. In conclusion, stability and formulation of a XGMA-GelMA shear thinning composite hydrogel has been optimized to create a bio-functional bioink, with improved printability, and in vitro culture stability via secondary photo-induced cross-linking, making this composite a promising bioink for 3D bioprinting.

Electrospun fibres represent a realistic implantable scaffold containing most of the structural three-dimensional (3D) characteristics of the extracellular matrix. However, as a result of their often synthetic nature, surface energy and chemistry, these scaffolds may adsorb a layer of non-specific proteins which can evoke a foreign body response. The precise surface modification of the scaffolds is challenging due to the complex geometrical and structural organization of the fibre meshes, that may limit the efficacy and completeness of approaches used. One flexible strategy that has gained attention is the use of reversible deactivation radical polymerisation (RDRP) techniques, which allow the creation of polymer brushes with controlled molecular weight, whilst retaining fibre morphology. In this study, protein adsorption was reduced with grafting of poly(N,N-dimethylacrylamide) (PDMA), poly(N-(2-hydroxypropyl)acrylamide) (PHPA) and poly(N-acryloylmorpholine) (PNAM) via surface-initiated (SI)-Cu(0) mediated radical polymerisation, from the surface of electrospun fibres prepared using a blend of bromine terminated poly(l-lactide) (PLA-Br) and poly(d,l-lactide-co-glycolide) (PLGA). Optimisation of the levels of Cu(i)Br, Me6TREN and the presence and concentration of a sacrificial initiator facilitated the grafting of well-controlled polymers brushes in less than one hour. Surface characterisation of the grafted scaffolds using X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS), and direct analysis of the molecular weight and polydispersity of polymer formed in solution during the reaction as well as the grafted polymer layer confirmed successful, controlled modification. Finally, protein adsorption experiments demonstrated the low adsorption properties of all polymer coatings with PDMA showing superior performance.

Amphiphilic polymers bearing cationic moieties are an emerging alternative to traditional antibiotics given their broad-spectrum activity and low susceptibility to the development of resistance. To date, however, much remains unclear regarding their mechanism of action. Using functional assays (ATP leakage, cell viability, DNA binding) and super-high resolution structured illumination microscopy (OMX-SR) of fluorescently tagged polymers, we present evidence for a complex mechanism, involving membrane permeation as well as cellular uptake, interaction with intracellular targets and possible complexation with bacterial DNA. Statement of Significance: This manuscript details the first study to systematically and directly investigate the mechanism of action of antimicrobial polymers, using super-resolution fluorescence imaging as well as functional assays. While many in the field cite membrane permeation as the sole mechanism underlying the activity of such polymers, we present evidence for multimodal actions including high cellular uptake and interaction with intracellular targets. It is also the first report to show competitive binding of antimicrobial polymers with bacterial DNA in a dose-dependent manner. Refereed/Peer-reviewed

usc Manipulating the surface properties of materials via the application of coatings is a widely used strategy to achieve desired interfacial interactions, implicitly assuming that the interfacial forces of coated samples are determined exclusively by the surface properties of the coatings. However, interfacial interactions between materials and their environments operate over finite length scales. Thus, the question addressed in this study is whether interactions associated with bulk substrate materials could act through thin coatings or, conversely, how thick a coating needs to be to completely screen subsurface forces contributed by underlying substrates. Plasma polymer layers were deposited on silicon wafer substrates from ethanol vapor, with identical chemical composition, ultrasmooth surfaces, and varying thicknesses. Using colloid-probe atomic force microscopy, electrical double-layer forces were determined in solutions of various ionic strengths and fitted using the Derjaguin-Landau-Verwey-Overbeek theory. For the thicker ethanol plasma polymers, the fitted surface potentials reflected the presence of surface carboxylate groups and were invariant with thickness. In contrast, for coatings

A computational fluid dynamics model for the flow of culture in a Corning™ spinner-flask stirred bioreactor has been used to characterise stresses experienced by microcarriers immersed in the fluid. Validation of the turbulent flow using experimental Particle Image Velocimetry (PIV) found advanced Large Eddy Simulation (LES) to be superior to Unsteady Reynolds averaging (URANS) modelling as a computational strategy for accurately capturing instantaneous velocity fluctuations of the types observed in the experiments. The simulations demonstrated that stress exposures experienced by microcarriers were highest during impeller start-up. After start-up, microcarriers experienced elevated levels of fluctuating stress of magnitudes known to cause cell differentiation, potentially compromising expansion of a homogeneous population with multi-lineage potential. Decreasing the impeller speed from 70 RPM to 50 RPM in the Corning™ flask was not found to necessarily reduce microcarrier stress exposure, because such a measure does not control the spatio-temporal coincidence of the microcarrier population with high regions of stress within the bioreactor. Modifications to bioreactor geometries and operational protocols are identified that can be pursued if such issues with dynamic stem cell culture arise.

Self-initiated photografting polymerization is used to couple the polymerizable initiator monomer 2-(2-chloropropanoyloxy)ethyl acrylate to a range of polymeric substrates. The technique requires only UV light to couple the initiator to surfaces. The initiator surface density can be varied by inclusion of a diluent monomer or via selection of initiator and irradiation parameters. The functionality of the initiator surface is demonstrated by subsequent surface-initiated atom transfer radical polymerization. Surfaces are characterized by x-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM), and UV-induced changes to the initiator are assessed by (1) H NMR and gel permeation chromatography (GPC). This is the first time this one-reactant one-step technique has been demonstrated for creating an initiator surface of variable density.

While polycaprolactone (PCL) and similar polyesters are commonly used as degradable scaffold materials in tissue engineering and related applications, non-specific adsorption of environmental proteins typically precludes any control over the signalling pathways that are activated during cell adhesion to these materials. Here we describe the preparation of PCL-based fibres that facilitate cell adhesion through well-defined pathways while preventing adhesion via adsorbed proteins. Surface-initiated atom transfer radical polymerisation (SI-ATRP) was used to graft a protein-resistant polymer brush coating from the surface of fibres, which had been electrospun from a brominated PCL macroinitiator. This coating also provided alkyne functional groups for the attachment of specific signalling molecules via the copper-mediated azide-alkyne click reaction; in this case, a cyclic RGD peptide with high affinity for αvβ3 integrins. Mesenchymal stem cells were shown to attach to the fibres via the peptide, but did not attach in its absence, nor when blocked with soluble peptide, demonstrating the effective control of cell adhesion pathways.

Materials with 1st order antiferromagnetic (AFM) to high-magnetization (MM) phase transition known for their inverse magnetocaloric effect, abrupt rise in magnetization and magnetoelastic coupling, are promising for application in combined simultaneous diagnosis and targeted cancer therapy. A therapy that combines alternating-current (ac) and direct-current (dc) magnetic fields for simultaneous magnetic hyperthermia therapy (MHT) and magnetic resonance imaging (MRI), using same magnetic particles for heating and as contrast agents. We report a proof-of-concept study on the induction heating ability of 1st order metamagnetic material with moderate specific absorption rates (SAR) and no tendency for agglomeration, for potential MHT and MRI cancer therapy. CoMnSi, a metamagnetic antiferromagnet (MM) was used in this study because of its desirable ability to rapidly switch from a low to high magnetization state in an applied dc bias field condition without particle agglomeration on field removal. The results showed that the magnetization switched from < 20 Am2kg-1 at 0.75 T to about 53.31 Am2kg-1 at 1.0 T applied dc field, a field large enough for magnetic resonance imaging. An SAR value of 10.7 Wg-1 was obtained under an ac field of 31.0 kAm-1 at 212.0 kHz. When combined with a dc bias field of 1.0 T, SAR values of 9.83 Wg-1 and 6.65 Wg-1 were obtained in the directions 45° and 90° away from the dc bias field direction respectively. These SAR values obtained from CoMnSi particles in the presence of simultaneous ac and dc magnetic field bias are in comparison, at least 25 times greater than those obtained from 2nd order magnetic phase transition Fe3O4 suspension. It is observed that Fe3O4 particles showed large suppression of SAR, and agglomeration under the same experimental conditions. This study shows the great potential of 1st order phase transition metamagnets for simultaneous MHT and MRI cancer therapy using MRI equipment.

Electrospun ultrafine fibers prepared using a blend of poly(lactide-co-glycolide) (PLGA) and bromine terminated poly(l-lactide) (PLA-Br), were surface modified using surface-initiated (SI) Cu(0) mediated polymerization. Copolymers based on N-acryloxysuccinimide (NAS) and a low fouling monomer (either N,N-dimethylacrylamide (DMA), N-(2-hydroxypropyl)acrylamide (HPA), or N-acryloylmorpholine (NAM)) were grafted from the fiber surface to impart surface functionality and to reduce nonspecific protein adsorption. Inclusion of the functional NAS monomer facilitated the conjugation of a nonbioactive cyclic RAD peptide and a bioactive cyclic RGD peptide, the latter expected to facilitate cell adhesion through its affinity for the αvβ3 integrin receptor. A detailed analysis of the surface of the electrospun fiber scaffolds in nongrafted form compared to the surface functionalized state is presented. Characteristic amino acid peaks are observed for both conjugated RGD and RAD peptides. Cell culture experiments conf...

Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.

A.E.R. was supported in this work by an Australian Postgraduate Award and by the Cooperative Research Centre for Polymers. D.R.N. was supported by an NHMRC CDA Fellowship.

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.

Host-defense antimicrobial peptides (AMPs) are a promising lead in the search for novel antibiotics. Many of these peptides exhibit broad-spectrum antibacterial ability, low toxicity toward human cells, and little susceptibility to induction of bacterial resistance. Our research focuses on the development of synthetic polymers that are able to mimic the amphiphilic and cation-rich characteristics of AMPs. This derives bioactive polymers that retain the activity profile of AMPs while utilizing a construct that is less expensive and easier to produce and manipulate chemically. This review details structure–activity relationships (SARs) of a new class of arginine-rich, synthetic AMP mimicking polymers (SAMPs), the guanylated polymethacrylates. These are contrasted with those of amine-based polymers that are mimics of lysine-rich AMPs. The ideal composition for candidates for practical applications was identified as those containing guanidines as a cation source, having a low molecular weight and a low level of lipophilicity. This gave polymers with high potency against Gram-positive strains of bacteria (e.g., Staphylococcus epidermidis MIC = 10 μg/mL) and low toxicity towards human red blood cells (

Cells respond to their environment in complex and sometimes poorly understood ways. Protein, peptide and synthetic peptidomimetic ligands may all be used to stimulate cells via receptor signaling, using interactions that are often highly specific. Polymer substrates that present these ligands provide a promising way to control cell development, both for applications in biotechnology and for fundamental studies of cell biology. Here we review a large range of techniques that have been employed to create and characterize ligand-functionalized substrates, with a particular focus on techniques that allow specific and consistent stimulation.

The controlled ring-opening polymerisation (ROP) of an e-caprolactone derivative that contains an ATRP initiator pendant to the ring, γ-(2-bromo-2-methyl propionyl)-e-caprolactone (γ-BMPCL), and its copolymerisation with e-caprolactone (CL) is reported. Functional PCL copolymers that contained pendant ATRP initiators were obtained with higher than previously reported molecular weights using diphenyl phosphate (DPP) as the catalyst at room temperature. Surface-initiated ATRP grafting of oligo(ethylene glycol) methacrylate was successfully carried out on the surface of two dimensional (2D) substrates comprising thin films of a functional PCL copolymer.

Several growth factors feature prominently in the control of hematopoiesis. Thrombopoietin, a class I hematopoietic cytokine, plays critical roles in regulating hematopoietic stem cell numbers and also stimulates the production and differentiation of megakaryocytes, the bone marrow cells that ultimately produce platelets. Thrombopoietin interacts with the c-Mpl cell-surface receptor. Recently, several peptide and small-molecule agonists and antagonists of c-Mpl have been reported. We conducted a bioinformatics and molecular modeling study aimed at understanding the agonist activities of peptides that bind to c-Mpl, and developed new potent peptide agonists with low nanomolar activity. These agonists also show very high activity in human CD34(+) primary cell cultures, and doubled the mean blood platelet counts when injected into mice.

The development of electrospun ultrafine fibres from biodegradable and biocompatible polymers has created exciting opportunities for biomedical applications. Fibre meshes with high surface area, suitable porosity and stiffness have been produced. Despite desirable structural and topographical properties, for most synthetic and some naturally occurring materials, the nature of the fibre surface chemistry has inhibited development. Hydrophobicity, undesirable non-specific protein adsorption and bacterial attachment and growth, coupled with a lack of surface functionality in many cases and an incomplete understanding of the myriad of interactions between cells and extracellular matrix (ECM) proteins have impeded the application of these systems. Chemical and physical treatments have been applied in order to modify or control the surface properties of electrospun fibres, with some success. Chemical modification using controlled radical polymerization, referred to here as reversible-deactivation radical polymerization (RDRP), has successfully introduced advanced surface functionality in some fibre systems. Atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT) are the most widely investigated techniques. This review analyses the practical applications of electrospinning for the fabrication of high quality ultrafine fibres and evaluates the techniques available for the surface modification of electrospun ultrafine fibres and includes a detailed focus on RDRP approaches.

Traditionally, most antibiotics are relatively low molecular weight chemical compounds. Bacteria have shown the ability to acquire resistance to many antibiotics. In nature, on the other hand, there are examples of antibiotics to which resistance has not been developed. This is particularly the case for naturally occurring antimicrobial peptides. In this chapter we discuss antimicrobial peptides and their postulated mechanisms of action, followed by a review of synthetic polymers with structures inspired by biological molecules, particularly antimicrobial peptides. We also review the grafting of polymers onto biomaterials and biomedical devices, so as to generate polymeric antimicrobial coatings.

A scalable, solution-phase synthesis of the selectively protected non-peptide RGD (arginine–glycine–aspartic acid) mimetic 6 is described. This synthesis serves as an alternative to the previously described solid-phase synthesis of this compound, thereby making this important integrin-binding mimetic readily accessible. The free carboxylic acid of 6 was conjugated to a protected diamine, followed by global deprotection to give a derivative 27 , suitable for immobilization onto amine-reactive surfaces. The RGD mimetic 28 demonstrated superior biological activity in comparison to a native linear RGD peptide and the semi-synthetic cyclic cRGDfK peptide in a cell attachment inhibition assay.

In this novel platform, a micropatterned polymer brush was obtained by grafting poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMA)) from a thin macroinitiator film using atom transfer radical polymerization (ATRP). A pattern of holes was formed in the macroinitiator film by taking advantage of its spontaneous dewetting above the glass transition temperature from a bottom polystyrene film, driven by unfavorable intermolecular forces. Patterning by dewetting can be achieved at length-scales from a few hundred nanometers to several tens of micrometers, by simply thermally annealing the bilayer above the glass transition temperature of the polymer. This approach is substrate-independent, as polymer films can be cast onto surfaces of different size, shape, or material. As a demonstration of its potential, proteins, and individual cells were attached on targeted bioadhesive polystyrene areas of the micropatterns within poly(PEGMA) protein-repellent brushes. We anticipate this approach will be suitable for the patterning of brushes, especially for biomedical applications such as in the study of single cells and of cell cocultures.

Control over cell-material surface interactions is the key to many new and improved biomedical devices. It can only be achieved if interactions that are mediated by nonspecifically adsorbed serum proteins are minimized and if cells instead respond to specific ligand molecules presented on the surface. Here, we present a simple yet effective surface modification method that allows for the covalent coupling and presentation of specific biological signals on coatings which have significantly reduced nonspecific biointerfacial interactions. To achieve this we synthesized bottle brush type copolymers consisting of poly(ethylene glycol) methyl ether methacrylate and (meth)acrylates providing activated NHS ester groups as well as different spacer lengths between the NHS groups and the polymer backbone. Copolymers containing different molar ratios of these monomers were grafted to amine functionalized polystyrene cell culture substrates, followed by the covalent immobilization of the cyclic peptides cRGDfK and cRADfK using residual NHS groups. Polymers were characterized by GPC and NMR and surface modification steps were analyzed using XPS. The cellular response was evaluated using HeLa cell attachment experiments. The results showed strong correlations between the effectiveness of the control over biointerfacial interactions and the polymer architecture. They also demonstrate that optimized fully synthetic copolymer coatings, which can be applied to a wide range of substrate materials, provide excellent control over biointerfacial interactions. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Silicone hydrogel contact lenses, which have been a major advance in the field of vision correction, require surface modification or coatings for comfort and biocompatibility. While current coatings show adequate clinical performance, advanced coatings may improve the biocompatibility of contact lenses further by reducing biofouling and related adverse clinical events. Here, we have produced coatings on Lotrafilcon A contact lenses by deposition of a thin film of allylamine plasma polymer (ALAPP) as a reactive interlayer for the high density grafting of poly(ethylene oxide) dialdehyde (PEO(ALD) 2 ), which had previously shown complete resistance to protein adsorption in vitro . The performance of these contact lenses was evaluated in a controlled clinical study over 6 h using Focus ® Night and Day™ (also known as Air Optix ® Night & Day ® ) contact lenses as control lenses. Surface modified lenses were characterised by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) before and after wear. Clinical data showed a high level of biocompatibility of the PEO coated lenses equivalent to control lenses. Surface analysis of worn contact lenses demonstrated that the high density PEO coating is effective in reducing biofouling in vivo compared to control lenses, however small amounts of protein deposits were still detected on all worn contact lenses. This study highlights that elimination of biofouling in vivo can be much more demanding than in vitro and discusses issues that are important for the analysis of worn contact lenses as well as the design of improved contact lenses.

Molecules that mimic the cytokine thrombopoietin that act by an atypical mechanism of binding to a receptor transmembrane (TM) domain are widely understood to require zinc for their biological activity. We investigated potent thrombopoietin mimetics from three chemical classes including the recently registered drug Eltrombopag, which operate via this novel mechanism, to determine whether zinc is essential for inducing cell proliferation. Using addition of zinc and a potent metal chelator, we show that the existing paradigm is incorrect and the compounds exhibit excellent thrombopoietin-mimetic activity even in the presence of high concentrations of EDTA. The implications of these findings for the mechanism of action are discussed.

Bio-inspired, mineralized polysaccharide coatings consisting of interspaced alginate–chitosan and calcium phosphate carbonate planar composite films were rapidly fabricated via electrostatic layer-by-layer (LBL) self-assembly and organic-matrix-mediated nucleation and growth. Experimental design, a fractional factorial design (2 8-4 ), was used to identify the optimum LBL film process parameters for fast film growth. Calcium phosphate carbonates rapidly precipitated on the fast growing LBL films from a supersaturated solution in around 15 min. The number of bilayers of LBL films and soaking time in calcium phosphate carbonate solution were found to affect the morphology of calcium phosphate carbonates, indicating a complex, organic matrix mediated mineralization process. The coating protocol in the current study was readily transferable to a variety of template geometries and materials. The resulting planar composite coatings could be applied for the modification of orthopaedic and periodontal implant surfaces and also for controlled growth of hematopoietic or mesenchymal stem cells.

Covalently grafted poly(ethylene oxide) coatings have been widely studied for use in biomedical applications,particularly for the reduction of protein and other biomolecule adsorption. However, many of these studies have not characterized the hydrated structure of the coatings. This new study uses a combination of silica colloid probe interaction force measurements using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) in order to determine the grafting density and hydrated layer structure of monomethoxy poly(ethylene oxide) aldehydelayers, covalently grafted onto amine plasma polymer surfaces, and their interactions with silica surfaces. For high grafting densities, purely repulsive interactions were measured as expected for densely grafted polymer brushes. These interactions could be described by theoretical expectations for compression of one polymer brush layer. However, at lower grafting densities, attractive interactions were observed at larger separation distances, originating from bridging interactions due to adsorption of the PEO chains on the surface of the silica colloid probe. This is a new finding indicating that the coupled PEO molecules have sufficient conformational freedom to interact strongly with an adjacent surface or, for example, protein molecules for which there is an affinity. The attractive interactions could be removed by grafting an additional PEO layer onto the silica colloid probe. Protein adsorption measurements confirmed that at highgrafting densities, the amount of adsorbed protein on the PEO grafted surfaces was greatly reduced, to the order of thedetection limit for the XPS technique. Refereed/Peer-reviewed

Liposomes that are surface-bound to a biomaterial such as a contact lens are of interest for localized delivery of therapeutic agents, but it is not known whether such liposome layers are sufficiently robust. The stability of a dense, PEG-functionalized layer of liposomes, affinity-bound onto a multilayer coated surface, was tested under various stress conditions using colloid-probe atomic force miscroscopy (AFM). The different stress effects were generated by varying the applied normal load of the probe and the impinging fluid shear through different approach velocities and by varying the applied lateral forces by scanning under increasing force loads. The effect of applied forces (normal and lateral) was further investigated by coating the probe with a layer of albumin. The liposomes remained intact following the ramping of both protein-coated and uncoated probes under the normal and lateral loads. The low-fouling nature of these liposomes, with respect to nonspecific protein adsorption, was also demonstrated from the interaction force measurements which showed only weak adhesion from the protein layer during the contact period of the albumin-coated probe. The observed adhesive interactions were concluded to be a direct result of the applied load from the probe, during the force measurements, rather than from attraction of the protein molecules for the surface-bound liposomes. The low frictional response of the liposome layer indicated the viscoelastic nature of these molecules, which enabled liposome structure retention during the continuous load application. The demonstrated stability of the liposomes presents a system of viable and localized drug delivery in, for example, ophthalmic applications. Refereed/Peer-reviewed

Low-protein-fouling poly(ethylene glycol) (PEG-like) plasma polymer films were prepared using radio frequency glow discharge polymerization of diethylene glycol dimethyl ether (DGpp) on top of a heptylamine plasma polymer primer layer. By varying the plasma deposition conditions, the chemistry of the DGpp film was influenced, especially in regard to the level of ether content, which in turn influenced the relative levels of bovine serum albumin and lysozyme protein fouling. Surface potential measurements indicated that these surfaces carried a net negative charge. While protein fouling remained low ( approximately 10 ng/cm2), there was a slightly higher level of the positively charged protein adsorbed on these films than the negative protein. The interaction forces measured between a silica spherical surface on both "high"- and "low"-protein-fouling DGpp films were all repulsive and short ranged (2-3 nm). There was no correlation between the surface forces measured for high- and low-protein-fouling DGpp films. Thus, it appears that enthalpic effects are very important in reducing protein adsorption. We therefore conclude that it is the concentration of residual, ethylene glycol containing species that are the crucial parameter determining protein resistance due to a combination of both entropic and enthalpic effects.

Effects of surface oxidation on the properties of amine plasma polymer (pp) films in aqueous media have not been widely studied, despite their use to control bio-interfacial properties. Changes in the surface composition of allylamine and heptylamine pp films, on water exposure, were followed by XPS. The surface potential was monitored via AFM measurements and comparison to DLVO theory. Analysis of XPS data implied high initial oxidation rates (rapid quenching of surface radicals). Oxidation of the pp films followed evolution of carbon-based rather than nitrogen-based species. Analysis of AFM force data showed changes in the sign of the surface potential with aging from positive to negative. Implications, for example for surface immobilization of molecules in aqueous environments, are also discussed.

OBJECTIVES Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. METHODS We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms. RESULTS Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2ΔΔ mutant, we implicate matrix polysaccharide β-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy. CONCLUSIONS Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms.

While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.

The interaction forces between layers of the triblock copolymer Pluronic F108 adsorbed onto hydrophobic radio frequency glow discharge (RFGD) thin film surfaces and hydrophilic silica, in polymer-free 0.15 M NaCl solution, have been measured using the atomic force microscope (AFM) colloid probe technique. Compression of Pluronic F108 layers adsorbed on the hydrophobic RFGD surfaces results in a purely repulsive force due to the steric overlap of the layers, the form of which suggests that the PEO chains adopt a brush conformation. Subsequent fitting of these data to the polymer brush models of Alexander-de Gennes and Milner, Witten, and Cates confirms that the adsorbed Pluronic F108 adsorbs onto hydrophobic surfaces as a polymer brush with a parabolic segment density profile. In comparison, the interaction between Pluronic F108 layers adsorbed on silica exhibits a long ranged shallow attractive force and a weaker steric repulsion. The attractive component is reasonably well described by van der Waals forces, but polymer bridging cannot be ruled out. The weaker steric component of the force suggests that the polymer is less densely packed on the surface and is less extended into solution, existing as polymeric isolated mushrooms. When the surfaces are driven together at high piezo ramp velocities, an additional repulsive force is measured, attributable to hydrodynamic drainage forces between the surfaces. In comparing theoretical predictions of the hydrodynamic force to the experimentally obtained data, agreement could only be obtained if the flow profile of the aqueous solution penetrated significantly into the polymer brush. This finding is in line with the theoretical predictions of Milner and provides further evidence that the segment density profile of the adsorbed polymer brush is parabolic. A velocity dependent additional stepped repulsive force, reminiscent of a solvation oscillatory force, is also observed when the adsorbed layers are compressed under high loads. This additional force is presumably a result of hindered drainage of water due to the presence of a high volume fraction of polymer chains between the surfaces.

This paper critically reviews reports on the interactions of phospholipids and poly(ethylene glycol) (PEG)—also called poly(ethylene oxide) (PEO)—with biological systems (model polypeptides, proteins, macrophages, etc). The review arises from the need to rationalize the widely varying findings reported in the literature. The paper discusses the interaction of simple polypeptides with phospholipids, followed by the interaction of proteins with supported phospholipid layers and liposomes. In addition, the interactions of liposomes with more complex biological systems, in vitro and in vivo, are discussed. Since many proposed liposome formulations contain grafted PEG molecules, a discussion of the interactions of PEG molecules and PEG surface layers with proteins is also provided as well as a substantial section dealing with the mechanistic aspects of these interactions. As well as providing an introduction to the multidisciplinary and complex literature available, we hope that this review article will provide the necessary information for more rational design of systems and devices for in vivo application. In particular, the article stresses the need for much better characterization of the systems and materials used.

Streaming potential measurements and atomic force microscropy (AFM) were used to determine the zeta potentials, diffuse layer potentials and isoelectric points (ieps) of alpha alumina sapphire single crystals for four different crystallographic orientations: c-plane, 0001 ; a-plane, 11 2 0 ; m-plane, 10 1 0 ; and r-plane, 1 1 02 . Both types of measurements indicated that the iep of the sapphire crystals was between about pH 5.0 and 6.0 for all crystals investigated. Unfortunately the techniques utilized could not conclusively differentiate any subtle differences in iep between the four orientations studied. The iep of alpha alumina powder was found to be pH 9.4. The difference in the ieps of the two different types of alpha alumina (single crystal and powder) is attributed to the presence of different types of surface hydroxyl groups on the two different types of surfaces. Powder is likely to have a greater fraction of singly coordinated surface hydroxyl groups (that have a high p K a ), while sapphire single crystals are more likely to have most surface hydroxyls multiply coordinated (which have a low p K a ).

This study reports the covalent immobilization of poly(acrylic acid) (PAAC) onto thin films deposited using radio-frequency glow discharge from a vapor of n-heptylamine (HApp). The successful immobilization of PAAC onto HApp was demonstrated by XPS analyses and AFM colloid probe force measurements. The force profiles obtained between silica particles and the PAAC coating were repulsive in nature, roughly exponentially decaying and of long range. The interaction measurements for the PAAC surfaces were not purely electrostatic in nature but also a result of compression of the covalently attached PAAC layer by the silica surface (i.e., electrosteric). The concentration of EDC/NHS added during the coupling influenced both the range and magnitude of the interaction force profiles between the silica colloid probe and the grafted PAAC layer; i.e., the concentration of EDC/NHS added during coupling of PAAC influenced the structure of the layer formed. This result was in good qualitative agreement with XPS analyse...

When complexed PSS/CTAB is coadsorbed onto silica, a long-range, predominantly electrosteric repulsion is measured which decays exponentially with surface separation. Adsorption is driven by locally hydrophobic, surfactant-rich regions along the PSS chain. These anchor the PSS chain to the surface, resulting in a classical loop and tail adsorbed conformation. An increase in pH results in an increase in electrostatic repulsion between the silica surface and PSS, which drives an initially rapid desorption of some of the PSS/CTAB complex. Any remaining PSS/CTAB desorbs via a slow unravelling, as points of attachment to the surface are broken over time, allowing the PSS to extend further into solution. This continues until desorption is complete after 66 h. In contrast, adsorption of the PSS/CTAB complex onto a preadsorbed CTAB layer leads to a less extended, more compact surface layer since now adsorption is driven not only by the hydrophobic surfactant-rich regions along the PSS chain but also by a hydropho...

Liposomes were immobilized onto the surface of perfluorinated polymer tape samples and tissue culture polystyrene well-plates using a multilayer immobilization strategy. In the first step, a thin interfacial bonding layer with surface aldehyde groups was deposited from a glow discharge struck in acetaldehyde vapour. Polyethylenimine was then covalently bound onto the aldehyde groups by reductive amination, followed by covalent binding of NHS-PEG-biotin molecules onto the surface amine groups by carbodiimide chemistry. Next, NeutrAvidin protein molecules were bound onto the PEG-biotin layer. Finally, liposomes containing PEG-biotinylated lipids were docked onto the remaining binding sites of the surface-immobilized NeutrAvidin molecules. AFM was used to image surface-bound liposomes and revealed a density well below close packing. The release characteristics of the surface-bound liposomes were measured by the fluorescence intensity changes of carboxyfluorescein upon release. Liposomes filled with sodium orthovanadate were surface immobilized and used in two in vitro angiogenesis assays. Marked differences compared to various control samples were observed, demonstrating the utility of drug-filled, surface-bound liposomes for evoking localized, controlled biological host responses proximal to an implanted biomedical device.

The interactions between silica and α-alumina surfaces with and without polyelectrolyte and combinations of both polyelectrolyte (sodium poly(styrene sulfonate) or PSS) and surfactant (cetyltrimethylammonium bromide or CTAB) have been measured using the atomic force microscope (AFM) colloid probe technique. In this case, silica particles were glued to the AFM cantilever to give a surface of known geometry. The forces between silica and α-alumina in electrolyte only were well described by the Derjaguin−Landau−Verwey−Overbeek theory at all separation distances. However, introduction of a solution of negatively charged PSS and mixtures of PSS and a positively charged surfactant, CTAB, at the point of zero charge of α-alumina, modified the interaction forces, introducing short-range steric interactions superimposed onto electrostatic interactions. Thus, the adsorption of strongly charged polyelectrolytes onto net neutral surfaces resulted in a flat conformation with few loops and tails. When PSS and CTAB were...

Poly(oligo(ethylene glycol) methacrylate) (pOEGMA) brushes were grafted via surface-initiated atom transfer radical polymerization (SI-ATRP) from a poly(styrene-co-vinylbenzyl chloride) macroinitiator. While bromoisobutyryl initiator groups are most commonly used for this purpose, benzyl chloride initiators may be advantageous for some applications due to superior stability. Water-only graft solutions produced thicker brush coatings with superior low fouling properties (low protein adsorption and cell adhesion) versus mixed water/alcohol solutions. Coatings produced using 475 Da OEGMA (methyl ether terminated) further reduced non-specific interactions compared to 360 Da OEGMA (hydroxyl terminated). Initiator density had minimal effect on low fouling properties.

Effective control over biointerfacial interactions is essential for a broad range of biomedical applications. At this point in time, only a relatively small range of radically polymerizable monomers have been described that are able to generate low fouling polymer materials and surfaces. The most important examples that have been successfully used in the context of the reduction of nonspecific protein adsorption and subsequent cell attachment include PEG-based monomers such as poly(ethylene glycol) methacrylate (PEGMA), zwitterionic monomers such as 2-methacryloyloxyethyl phosphorylcholine and noncharged monomers such as acrylamide and N-(2-hydroxypropyl) methacrylamide (HPMAm). However, issues such as oxidative degradation and poor polymerization characteristics limit the applicability of most of these candidates. Here we have synthesized the monomer N-(2-hydroxypropyl) acrylamide (HPAm), examined its polymerization kinetics and evaluated its suitability for RAFT mediated polymerization in comparison to HPMAm. We also synthesized hydrogels using HPMAm and HPAm and evaluated the ability of HPAm polymers to occlude protein adsorption and cell attachment. In RAFT-controlled polymerization, much faster (8×) polymerization was observed for HPAm relative to HPMAm and better control was achieved over the molecular weight distribution. The performance of hydrogels prepared from HPAm in the prevention of protein adsorption and cellular attachment was equivalent to or better than that observed for materials made from HPMAm and PEG. These results open the door for HPAm based polymers in applications where effective control over biointerfacial interactions is required.

The flow in a New Brunswick Scientific (NBS (now Eppendorf)) 5 L stirred-tank bioreactor (STR) partially filled with 2.2 L of water and agitated at 60 rpm using a pitched-blade impeller is studied in this work, to determine the suitability of the configuration for expanding stem cell lines. Computational Fluid Dynamics (CFD) model development and testing in this work has found Large Eddy Simulation (LES) to be essential for model fidelity and for capturing spatiotemporal stress fluctuations. Stresses were at levels similar to or even higher than those known to damage stem cells or modulate their cellular function to favour differentiation instead of phenotype maintenance. The results raise questions as to the appropriateness of such STRs for stem cell expansion, and motivate better experimental studies to properly quantify the spatiotemporal variability in fluid shear stresses and its effect on stem cell expansion and stem cell fate.Copyright © 2014 by ASME