Quantitative analysis of biomolecule release from polystyrene-block-polyethylene oxide thin films.

Block copolymers have garnered recent attention due to their ability to contain molecular cargo within nanoscale domains and release said cargo in aqueous environments. However, the release kinetics of cargo from these thin-films has not yet been reported. Knowledge of the release quantities and release profiles of these systems is paramount for applications of these systems. Here, Polystyrene-block-poly(ethylene oxide) (PS-b-PEO) was co-assembled with fluorescein isothiocyanate isomer I-lysozyme (FITC-LZ) and fluorescein isothiocyanate isomer I-TAT (FITC-TAT), such that these molecular cargos arrange within the PEO domains of the thin films. We show that high loading ratios of cargo/PS-b-PEO do not significantly impact the nanostructure of the films; however, a loading limit appears to be present with aggregates of protein forming at the microscale with higher loading ratios. The presence of lysozyme (LZ) within the films was confirmed qualitatively after aqueous exposure through photo-induced force microscopy (PiFM) imaging at the Amide I characteristic peak (∼1650 cm-1). Furthermore, we demonstrate that LZ maintains activity and structure after exposure to the polymer solvent (benzene/methanol/water mix). Finally, we demonstrate quantitatively 20-80 ng cm-2 of cargo is released from these films, depending on the cargo incorporated. We show that the larger molecule lysozyme is released over a longer time than the smaller TAT peptide. Finally, we demonstrate the ability to tune the quantity of cargo released by altering the thickness of the PS-b-PEO thin-films during fabrication.

Conducting polymer based electrochemical biosensors.

Conducting polymer (CP)-based electrochemical biosensors have gained great attention as such biosensor platforms are easy and cost-effective to fabricate, and provide a direct electrical readout for the presence of biological analytes with high sensitivity and selectivity. CP materials themselves are both sensing elements and transducers of the biological recognition event at the same time, simplifying sensor designs. This review summarizes the advances in electrochemical biosensors based on CPs. Recognition probe immobilisation techniques, transduction mechanisms and detection of various target biomolecules have been discussed in detail. Efforts to miniaturize CP-based electrochemical biosensors and fabrication of sensor arrays are also briefly reviewed.

The nanoscale geometrical maturation of focal adhesions controls stem cell differentiation and mechanotransduction.

We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation.

Dynamic composite hydrogels of gelatin methacryloyl (GelMA) with supramolecular fibers for tissue engineering applications.

In the field of tissue engineering, there is a growing need for biomaterials with structural properties that replicate the native characteristics of the extracellular matrix (ECM). It is important to include fibrous structures into ECM mimics, especially when constructing scar models. Additionally, including a dynamic aspect to cell-laden biomaterials is particularly interesting, since native ECM is constantly reshaped by cells. Composite hydrogels are developed to bring different combinations of structures and properties to a scaffold by using different types and sources of materials. In this work, we aimed to combine gelatin methacryloyl (GelMA) with biocompatible supramolecular fibers made of a small self-assembling sugar-derived molecule (N-heptyl-D-galactonamide, GalC7). The GalC7 fibers were directly grown in the GelMA through a thermal process, and it was shown that the presence of the fibrous network increased the Young's modulus of GelMA. Due to the non-covalent interactions that govern the self-assembly, these fibers were observed to dissolve over time, leading to a dynamic softening of the composite gels. Cardiac fibroblast cells were successfully encapsulated into composite gels for 7 days, showing excellent biocompatibility and fibroblasts extending in an elongated morphology, most likely in the channels left by the fibers after their degradation. These novel composite hydrogels present unique properties and could be used as tools to study biological processes such as fibrosis, vascularization and invasion.

Electrically responsive release of proteins from conducting polymer hydrogels.

Electrically modulated delivery of proteins provides an avenue to target local tissues specifically and tune the dose to the application. This approach prolongs and enhances activity at the target site whilst reducing off-target effects associated with systemic drug delivery. The work presented here explores an electrically active composite material comprising of a biocompatible hydrogel, gelatin methacryloyl (GelMA) and a conducting polymer, poly(3,4-ethylenedioxythiophene), generating a conducting polymer hydrogel. In this paper, the key characteristics of electroactivity, mechanical properties, and morphology are characterized using electrochemistry techniques, atomic force, and scanning electron microscopy. Cytocompatibility is established through exposure of human cells to the materials. By applying different electrical-stimuli, the short-term release profiles of a model protein can be controlled over 4 h, demonstrating tunable delivery patterns. This is followed by extended-release studies over 21 days which reveal a bimodal delivery mechanism influenced by both GelMA degradation and electrical stimulation events. This data demonstrates an electroactive and cytocompatible material suitable for the delivery of protein payloads over 3 weeks. This material is well suited for use as a treatment delivery platform in tissue engineering applications where targeted and spatio-temporal controlled delivery of therapeutic proteins is required. STATEMENT OF SIGNIFICANCE: Growth factor use in tissue engineering typically requires sustained and tunable delivery to generate optimal outcomes. While conducting polymer hydrogels (CPH) have been explored for the electrically responsive release of small bioactives, we report on a CPH capable of releasing a protein payload in response to electrical stimulus. The composite material combines the benefits of soft hydrogels acting as a drug reservoir and redox-active properties from the conducting polymer enabling electrical responsiveness. The CPH is able to sustain protein delivery over 3 weeks, with electrical stimulus used to modulate release. The described material is well suited as a treatment delivery platform to deliver large quantities of proteins in applications where spatio-temporal delivery patterns are paramount.

Conducting polymer hydrogels with electrically-tuneable mechanical properties as dynamic cell culture substrates.

Hydrogels are a popular substrate for cell culture due to their mechanical properties closely resembling natural tissue. Stimuli-responsive hydrogels are a good platform for studying cell response to dynamic stimuli. Poly(N-isopropylacrylamide) (pNIPAM) is a thermo-responsive polymer that undergoes a volume-phase transition when heated to 32 °C. Conducting polymers can be incorporated into hydrogels to introduce electrically responsive properties. The conducting polymer, polypyrrole (PPy), has been widely studied as electrochemical actuators due to its electrochemical stability, fast actuation and high strains. We determine the volume-phase transition temperature of pNIPAM hydrogels with PPy electropolymerised with different salts as a film within the hydrogel network. We also investigate the electro-mechanical properties at the transition temperature (32 °C) and physiological temperature (37 °C). We show statistically significant differences in the Young's modulus of the hybrid hydrogel at elevated temperatures upon electrochemical stimulation, with a 5 kPa difference at the transition temperature. Furthermore, we show a three-fold increase in actuation at transition temperature compared to room temperature and physiological temperature, attributed to the movement of ions in/out of the PPy film that induce the volume-phase transition of the pNIPAM hydrogel. Furthermore, cell adhesion to the hybrid hydrogel was demonstrated with mouse articular chondrocytes.

Further structural characterization of ovine forestomach matrix and multi-layered extracellular matrix composites for soft tissue repair.

Decellularized extracellular matrix (dECM)-based biomaterials are of great clinical utility in soft tissue repair applications due to their regenerative properties. Multi-layered dECM devices have been developed for clinical indications where additional thickness and biomechanical performance are required. However, traditional approaches to the fabrication of multi-layered dECM devices introduce additional laminating materials or chemical modifications of the dECM that may impair the biological functionality of the material. Using an established dECM biomaterial, ovine forestomach matrix, a novel method for the fabrication of multi-layered dECM constructs has been developed, where layers are bonded via a physical interlocking process without the need for additional bonding materials or detrimental chemical modification of the dECM. The versatility of the interlocking process has been demonstrated by incorporating a layer of hyaluronic acid to create a composite material with additional biological functionality. Interlocked composite devices including hyaluronic acid showed improved in vitro bioactivity and moisture retention properties.

Large area protein patterning reveals nanoscale control of focal adhesion development.

Focal adhesion development in cells adherent to surface bound fibronectin presented as 200, 500, or 1000 nm diameter circular patches or as homogeneous controls is studied by fluorescence and scanning electron microscopy. Fundamental cellular processes such as adhesion, spreading, focal adhesion and stress fiber formation are shown to be dependent on the spatial distribution of ligands at this scale. Large area samples enable the study of whole cell populations and opens for new potential applications.

Modulation of hydrogel stiffness by external stimuli: soft materials for mechanotransduction studies.

Mechanotransduction is an important process in determining cell survival, proliferation, migration and differentiation. The extracellular matrix (ECM) is the component of natural tissue that provides structural support and biochemical signals to adhering cells. The ECM is dynamic and undergoes physical and biochemical changes in response to various stimuli and there is an interest in understanding the effect of dynamic changes in stiffness on cell behaviour and fate. Therefore, stimuli-responsive hydrogels have been developed to mimic the cells' microenvironment in a controlled fashion. Herein, we review strategies for dynamic modulation of stiffness using various stimuli, such as light, temperature and pH. Special emphasis is placed on conducting polymer (CP) hydrogels and their fabrication procedures. We believe that the redox properties of CPs and hydrogels' biological properties make CPs hydrogels a promising substrate to investigate the effect of dynamic stiffness changes and mechanical actuation on cell fate in future studies.

Polystyrene-block-polyethylene oxide thin films: In vitro cytocompatibility and protein adsorption testing.

Polystyrene-block-polyethylene oxide (PS-b-PEO) coated surfaces have been explored as cell culture substrates in the past decade. However, their cytocompatibility has not been extensively assessed. In this study, the in vitro cytocompatibility of PS-b-PEO was investigated. Cellular morphology, metabolic activity, and viability were evaluated at 1, 3, and 5 days after cell seeding. Viability was greater than 90% throughout the 5 days culture, with abundant cell spreading evident by the formation of prominent F-actin stress fibres. The cytocompatibility study was complemented by the analysis of adsorption of a range of extracellular matrix proteins on PS-b-PEO thin films by quartz crystal microbalance with dissipation. Protein adsorption tests revealed that there was no significant difference in protein adhesion between surfaces with a PEO domain coverage of ≈28%, compared to the homogeneous polystyrene control. The findings demonstrate that PS-b-PEO thin films are cytocompatible and are a favourable surface coating for cell culture studies.

Surfactant protein SP-B strongly modifies surface collapse of phospholipid vesicles: insights from a quartz crystal microbalance with dissipation.

Pulmonary surfactant protein B (SP-B) facilitates the rapid transfer of phospholipids from bilayer stores into air-liquid interfacial films along the breathing cycle, and contributes to the formation of a surface-associated multilayer reservoir of surfactant to optimize the stability of the respiratory interface. To obtain more insights into the mechanisms underlying this transfer and multilayer formation, we established a simple model system that captures different features of SP-B action. We monitored the formation of supported planar bilayers from the collapse of intact phospholipid vesicles on a silica surface using a technique called quartz crystal microbalance with dissipation, which provides information on changes in membrane thickness and viscosity. At physiologically relevant concentrations, SP-B dramatically alters vesicle collapse. This manifests itself as a reduced buildup of intact vesicles on the surface before collapse, and allows the stepwise buildup of multilayered deposits. Accumulation of lipids in these multilayer deposits requires the presence of SP-B in both the receptor and the arriving membranes, surrounded by a comparable phospholipid charge. Thus, the quartz crystal microbalance with dissipation system provides a useful, simplified way to mimic the effect of surfactant protein on vesicle dynamics and permits a detailed characterization of the parameters governing reorganization of surfactant layers.

An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants.

Herein, we present that insect odorant receptors reconstituted into the lipid bilayers of liposomes can be successfully immobilized onto a gold surface and selectively and sensitively detect odorant molecules. The odorant receptors (OrXs) Or10a, Or22a, and Or71a from the common fruit fly, Drosophila melanogaster, were recombinantly expressed, purified and integrated into nano-liposomes (100-200 nm). These liposomes were covalently attached to the self-assembled monolayers (SAMs) of a 6-mercaptohexanoic acid (MHA)-modified gold surface. X-ray Photo Electron Spectroscopy (XPS) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements confirmed the successful modification of the gold surface and immobilization of liposomes. Atomic Force Microscopy (AFM) revealed that the liposomes were covalently attached to the surface without any disruption of vesicles. The liposomes tethered to the gold sensor surface were then treated with a range of known ligands of various concentrations. We demonstrated by Electrochemical Impedance Spectroscopy (EIS) that an OrX/liposome EIS sensor can sensitively and selectively detect its known ligand to femtomolar concentrations by detecting a change in electrical signal upon binding. Our study is the first step towards using purified insect odorant receptors alone in biosensors to enable the development of novel ultrasensitive volatile sensors for medical diagnostic, air quality, food safety and border security applications.

Conducting electrospun fibres with polyanionic grafts as highly selective, label-free, electrochemical biosensor with a low detection limit for non-Hodgkin lymphoma gene.

A highly selective, label-free sensor for the non-Hodgkin lymphoma gene, with an aM detection limit, utilizing electrochemical impedance spectroscopy (EIS) is presented. The sensor consists of a conducting electrospun fibre mat, surface-grafted with poly(acrylic acid) (PAA) brushes and a conducting polymer sensing element with covalently attached oligonucleotide probes. The sensor was fabricated from electrospun NBR rubber, embedded with poly(3,4-ethylenedioxythiophene) (PEDOT), followed by grafting poly(acrylic acid) brushes and then electrochemically polymerizing a conducting polymer monomer with ssDNA probe sequence pre-attached. The resulting non-Hodgkin lymphoma gene sensor showed a detection limit of 1aM (1 × 10-18mol/L), more than 400 folds lower compared to a thin-film analogue. The sensor presented extraordinary selectivity, with only 1%, 2.7% and 4.6% of the signal recorded for the fully non-complimentary, T-A and G-C base mismatch oligonucleotide sequences, respectively. We suggest that such greatly enhanced selectivity is due to the presence of negatively charged carboxylic acid moieties from PAA grafts that electrostatically repel the non-complementary and mismatch DNA sequences, overcoming the non-specific binding.

Osteopontin presentation affects cell adhesion-Influence of underlying surface chemistry and nanopatterning of osteopontin.

Osteopontin is a promising coating material for biomaterials, being important both in remodeling and formation of mineralized tissue and in immunological responses. We have investigated cell attachment to osteopontin adsorbed at different surface chemistries (-NH(2), -COOH, -CH(3), and bare gold) and to osteopontin presented as a nanopattern of 50 nm protein patches separated by a nonadhesive background. MDA-MB-435 cells adhere well to osteopontin presented at the hydrophilic chemistries (-NH2, -COOH, and gold) suggesting that osteopontin is presented in a functional form on these surfaces. On the amine surface, the cell attachment appears partly driven by electrostatic attraction between the positively charged substrate and the negatively charged cell membrane, whereas the spreading of the cells depends on the specific interaction with osteopontin presented at the surface. Significantly, fewer cells adhere to osteopontin presented at the methyl-terminated hydrophobic surface and the cells are less spread. On the nanopatterned osteopontin, only a very low number of cells adhered and those few attached cells showed an elongated morphology with few adhesion points to the surface. This indicates that the adhesive patches are not large enough to support stable focal contacts. The good cell attachment and spreading on the hydrophilic surfaces holds promise for osteopontin as a future coating for biomaterials.