Nanoscale Magnetic Arrays through Block Copolymer Templating of Polyoxometalates.

Magnetic nanoarrays promise to enable new energy-efficient computations based on spintronics or magnonics. In this work, we present a block copolymer-assisted strategy for fabricating ordered magnetic nanostructures on silicon and permalloy substrates. Block copolymer micelle-like structures were used as a template in which polyoxometalate (POM) clusters could assemble in an opal-like structure. A combination of microscopy and scattering techniques was used to confirm the structural and organizational features of the fabricated materials. The magnetic properties of these materials were investigated by polarized neutron reflectometry, nuclear magnetic resonance, and magnetometry measurements. The data show that a magnetic structural design was achieved and that a thin layer of patterned POMs strongly influenced an underlying permalloy layer. This work demonstrates that the bottom-up pathway is a potentially viable method for patterning magnetic substrates on a sub-100 nm scale, toward the magnetic nanostructures needed for spintronic or magnonic crystal devices.

White light and colour-tunable emission from a single component europium-1,8-naphthalimide thin film.

The synthesis and fabrication of spin coated films of a new Eu3+ complex [Eu(1)3] derived from the 1,8-naphthalimide containing ligand 1H is presented. The complex is multi-emissive displaying blue emission from the 1,8-naphthalimide fluorophore and red emission from the Eu3+ centre in both solution-state and solid-state. This allows the overall emission to be tuned by changing the excitaton wavelength, where varing degrees of red and blue emission intensity alter the overall emission colour from blue, to red and including white-light emission. The complex was spin-coated onto quartz slides giving 134 nm thick coatings that retained the multi-emissive and colour tunable properties. Overall, resulting in a colour-tunable system which in solution, solid, and thin film states can alter the overall colour from deep red to dark blue.

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.

Hydrogen bonding dissipating hydrogels: The influence of network structure design on structure-property relationships.

Hydrogels made with semi-interpenetrating networks of the oligomerized polyphenol tannic acid, and poly(acrylamide), exhibit high stiffness and toughness. However, the structure property relationships that give rise to enhanced mechanical properties is not well understood. Herein, we systematically investigate the hydrogels using small angle X-ray scattering and small and Ultra-small angle neutron scattering within a wide length scale range (1 nm to 20 µm), polarized optical microscopy, and rheology. Small angle X-ray and neutron scattering reveal the presence of micron sized hydrogen bonded clusters in the hydrogels. Breaking of hydrogen bonded clusters above a critical solution temperature was clearly observed in the small angle neutron scattering data. Polarized optical microscopy show enhanced anisotropy for the gels with oligomerized tannic acid incorporated - when compared to gels with monomeric tannic acid. Rheological studies at varying temperatures nicely corroborate the structural changes observed at high temperatures and reveal a self-healing behavior of the gels. The knowledge gained from this study will aid in rational design of hydrogels for biomedical applications.

Visible-Light Stiffness Patterning of GelMA Hydrogels Towards In Vitro Scar Tissue Models.

Variations in mechanical properties of the extracellular matrix occurs in various processes, such as tissue fibrosis. The impact of changes in tissue stiffness on cell behaviour are studied in vitro using various types of biomaterials and methods. Stiffness patterning of hydrogel scaffolds, through the use of stiffness gradients for instance, allows the modelling and studying of cellular responses to fibrotic mechanisms. Gelatine methacryloyl (GelMA) has been used extensively in tissue engineering for its inherent biocompatibility and the ability to precisely tune its mechanical properties. Visible light is now increasingly employed for crosslinking GelMA hydrogels as it enables improved cell survival when performing cell encapsulation. We report here, the photopatterning of mechanical properties of GelMA hydrogels with visible light and eosin Y as the photoinitiator using physical photomasks and projection with a digital micromirror device. Using both methods, binary hydrogels with areas of different stiffnesses and hydrogels with stiffness gradients were fabricated. Their mechanical properties were characterised using force indentation with atomic force microscopy, which showed the efficiency of both methods to spatially pattern the elastic modulus of GelMA according to the photomask or the projected pattern. Crosslinking through projection was also used to build constructs with complex shapes. Overall, this work shows the feasibility of patterning the stiffness of GelMA scaffolds, in the range from healthy to pathological stiffness, with visible light. Consequently, this method could be used to build in vitro models of healthy and fibrotic tissue and study the cellular behaviours involved at the interface between the two.

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 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.

Biomechanical responses of encysted zoospores of the oomycete Achlya bisexualis to hyperosmotic stress are consistent with an ability to turgor regulate.

Zoospores are motile, asexual reproductive propagules that enable oomycete pathogens to locate and infect new host tissue. While motile, they have no cell wall and maintain tonicity with their external media using water expulsion vacuoles. Once they locate host tissue, they encyst and form a cell wall, enabling the generation of turgor pressure that will provide the driving force for germination and invasion of the host. It is not currently known how these spores respond to the osmotic stresses that might arise due to different environments on and around their hosts that have different osmotic strengths. We have made microaspiration (MA) measurements on > 800 encysted zoospores and atomic force microscopy (AFM) measurements on 12 encysted zoospores to determine their mechanical properties and how these change after hyperosmotic stress. Two types of encysted zoospores (Type A and Type B) were produced from the oomycete Achlya bisexualis, that differed in their morphology and response. With a small hyperosmotic stress (using 0.1 and 0.2 M sorbitol to give media osmolality changes of 155.4 and 295.6 mOsmol/kg), Type A zoospores initially became stiffer, with an increase in the Young's modulus (E) over 30 mins from 0.16 MPa to 0.25 and 0.22 MPa respectively. E then returned to its original value after 120 min. With a greater osmotic stress (using 0.3, 0.4 and 0.5 M sorbitol to give media osmolality changes of 438.2, 587.2 and 787.6 mOsmol/kg) the reverse occurred, with an initial decrease in E over 30 - 60 mins to values of 0.1, 0.08 and 0.09 MPa respectively, before recovery to the original value after 120 min. In 0.5 M sorbitol this recovery was only observed with AFM, but not with MA. Type B zoospores, which may be primary/secondary spores about to release secondary/tertiary spores, or else spores that were damaged during encystment, initially stiffened in response to the lower hyperosmotic stresses with a slight increase in E (from 0.077 to 0.1 MPa after 15 min (with both 0.1 and 0.2 M sorbitol) before recovering to the original value after 60 min. These spores showed no change in response to the higher osmotic stresses. The responses of the Type A spores are consistent with rapid changes in cell wall thickness and a turgor regulation mechanism. Turgor regulation is further supported by microscopic observations of the Type A spores showing protoplast retraction from the cell wall followed by deplasmolysis, coupled with measurements of spore volume. As far as we are aware this is the first demonstration of turgor regulation, not just in encysted zoospores, but in oomycetes in general.

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.

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.

Geometric Frustration and Long-Range Ordering Induced by Surface Pressure Oscillation in a Langmuir-Blodgett Monolayer of Magnetic Soft Spheres.

As a step toward the bottom-up construction of magnonic systems, this paper demonstrates the use of a large-amplitude surface-pressure annealing technique to generate 2-D order in a Langmuir-Blodgett monolayer of magnetic soft spheres comprising a surfactant-encapsulated polyoxometalate. The films show a distorted square lattice interpreted as due to geometric frustration caused by 2-D confinement between soft walls, one being the air interface and the other the aqueous subphase. Hysteresis and relaxation phenomena in the 2-D layers are suggested to be due to folding and time-dependent interpenetration of surfactant chains.

A comparative study of tough hydrogen bonding dissipating hydrogels made with different network structures.

Hydrogels are excellent soft materials to interface with biological systems. Precise control and tunability of dissipative properties of gels are particularly interesting in tissue engineering applications. In this work, we produced hydrogels with tunable dissipative properties by photopolymerizing a second polymer within a preformed cross-linked hydrogel network of poly(acrylamide). We explored second networks made with different structures and capacity to hydrogen bond with the first network, namely linear poly(acrylic acid) and branched poly(tannic acid). Gels incorporating a second network made with poly(tannic acid) exhibited excellent stiffness (0.35 ± 0.035 MPa) and toughness (1.64 ± 0.26 MJ m-3) compared to the poly(acrylic acid) counterparts. We also demonstrate a strategy to fabricate hydrogels where the dissipation (loss modulus) can be tuned independently from the elasticity (storage modulus) suitable for cell culture applications. We anticipate that this modular design approach for producing hydrogels will have applications in tailored substrates for cell culture studies and in load bearing tissue engineering applications.

The intrinsic piezoelectric properties of materials - a review with a focus on biological materials.

Piezoelectricity, a linear electromechanical coupling, is of great interest due to its extensive applications including energy harvesters, biomedical, sensors, and automobiles. A growing amount of research has been done to investigate the energy harvesting potential of this phenomenon. Traditional piezoelectric inorganics show high piezoelectric outputs but are often brittle, inflexible and may contain toxic compounds such as lead. On the other hand, biological piezoelectric materials are biodegradable, biocompatible, abundant, low in toxicity and are easy to fabricate. Thus, they are useful for many applications such as tissue engineering, biomedical and energy harvesting. This paper attempts to explain the basis of piezoelectricity in biological and non-biological materials and research involved in those materials as well as applications and limitations of each type of piezoelectric material.

Mechanical Characterization for Cellular Mechanobiology: Current Trends and Future Prospects.

Accurate mechanical characterization of adherent cells and their substrates is important for understanding the influence of mechanical properties on cells themselves. Recent mechanobiology studies outline the importance of mechanical parameters, such as stress relaxation and strain stiffening on the behavior of cells. Numerous techniques exist for probing mechanical properties and it is vital to understand the benefits of each technique and how they relate to each other. This mini review aims to guide the reader through the toolbox of mechanical characterization techniques by presenting well-established and emerging methods currently used to assess mechanical properties of substrates and cells.

Polystyrene-block-poly(ethylene oxide) Thin Films Fabricated from a Solvent Mixture for the Co-Assembly of Polymers and Proteins.

The co-assembly of peptides and proteins in poly(styrene-block-ethylene oxide) (PS-b-PEO) thin films has proven to be a promising method to fabricate polymer-biomolecule functional materials. Contrary to the covalent immobilization of biomolecules on surfaces, co-assembly presents the opportunity to arrange cargo within thin films, which can be released upon exposure to an aqueous environment. The use of a mixed solvent system ensures the solubilization of hydrophobic polymer as well as the solubilization and protection of the biomolecule cargo. However, to produce largely defect-free films of PS-b-PEO from a solvent mixture containing water is challenging due to the narrow range of solvent miscibility and polymer/protein solubility. This work explores the limits of using a benzene/methanol/water solvent mixture for the production of thin PS-b-PEO films and provides a template for the fabrication optimization of block copolymer thin films in different complex solvent systems. The film quality is analyzed using optical microscopy and atomic force microscopy and correlated to the solvent composition. By adjusting the solvent composition to 80/18.8/1.2 vol % benzene/methanol/water, it was possible to reliably fabricate thin films with less than 1% macroscopic defect surface coverage. Using the optimized solvent composition, we also demonstrate the fabrication of ordered PS-b-PEO films containing lysozyme. Furthermore, we show the release of lysozyme into aqueous media, which highlights the potential use of such films for drug delivery applications.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM.

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 µm long was observed.

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.

Atomic Force Microscopy of Proteins.

Atomic force microscopy (AFM) enables imaging of surface-deposited proteins and protein structures under physiological conditions, which is a benefit compared to ultra-high vacuum techniques such as electron microscopy. AFM also has the potential to provide more information from the phase in tapping mode or from functional AFM modes. The sample preparation, probe selection, and imaging conditions are crucial for successful imaging of proteins. Here we give a detailed account of the steps toward imaging of soft samples in both air and liquid along with the basic theory underpinning these details.

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.