Patternable Gelatin Methacrylate/PEDOT/Polystyrene Sulfonate Microelectrode Coatings for Neuronal Recording.

This manuscript addresses the need for new soft biomaterials that can be fabricated on the surface of microelectrodes to reduce the mechanical mismatch between biological tissues and electrodes and improve the performance at the neural interface. By electrochemical polymerization of poly(3,4-dioxythiophene) (PEDOT)/polystyrene sulfonate (PSS) through a gelatin methacrylate (GelMA) hydrogel, we demonstrate the synthesis of a conducting polymer hydrogel (CPH) to meet the performance criteria of bioelectrodes. The hybrid material can be photolithographically patterned and covalently attached to gold microelectrodes, forming an interpenetrating network, as confirmed by infrared spectroscopy. The GelMA/PEDOT/PSS coatings were found to be reversibly electroactive by cyclic voltammetry and had low impedance compared to bare gold and GelMA-coated microelectrodes. The CPH coatings showed impedance at levels similar to conventional PEDOT/PSS coatings at a frequency of 1000 Hz. CPH exhibited electrochemical stability over 1000 CV cycles, and its performance was maintained over 14 days. Biocompatibility of the CPH coatings was confirmed by primary hippocampal neuronal cultures via a neuronal viability assay. The CPH-coated microelectrode arrays (MEAs) successfully recorded neuronal activity from primary hippocampal neuronal cells. The CPH GelMA/PEDOT/PSS is a highly promising coating material to enhance microelectrode performance at the neural interface.

A Subdural Bioelectronic Implant to Record Electrical Activity from the Spinal Cord in Freely Moving Rats.

Bioelectronic devices have found use at the interface with neural tissue to investigate and treat nervous system disorders. Here, the development and characterization of a very thin flexible bioelectronic implant inserted along the thoracic spinal cord in rats directly in contact with and conformable to the dorsal surface of the spinal cord are presented. There is no negative impact on hind-limb functionality nor any change in the volume or shape of the spinal cord. The bioelectronic implant is maintained in rats for a period of 12 weeks. The first subdural recordings of spinal cord activity in freely moving animals are presented; rats are plugged in via a recording cable and allowed to freely behave and move around on a raised platform. Recordings contained multiple distinct voltage waveforms spatially localize to individual electrodes. This device has great potential to monitor electrical signaling in the spinal cord after an injury and in the future, this implant will facilitate the identification of biomarkers in spinal cord injury and recovery, while enabling the delivery of localized electroceutical and chemical treatments.

An interpenetrating and patternable conducting polymer hydrogel for electrically stimulated release of glutamate.

Recent advances in drug delivery have made it possible to release bioactive agents from neural implants specifically to local tissues. Conducting polymer coatings have been explored as a delivery platform in bioelectronics, however, their utility is restricted by their limited loading capacity and stability. This study presents the fabrication of a stable conducting polymer hydrogel (CPH), comprising the hydrogel gelatin methacrylate (GelMA), and conducting polymer polypyrrole (PPy) for the electrically controlled delivery of glutamate (Glu). The hybrid GelMA/PPy/Glu can be photolithographically patterned and covalently bonded to an electrode. Fourier-transform infrared (FTIR) analysis confirmed the interpenetrating nature of PPy through the GelMA hydrogels. Electrochemical polymerisation of PPy/Glu through the GelMA hydrogels resulted in a significant increase in the charge storage capacity as determined by cyclic voltammetry (CV). Long-term electrochemical and mechanical stability was demonstrated over 1000 CV cycles and extracts of the materials were cytocompatible with SH-SY5Y neuroblastoma cell lines. Release of Glu from the CPH was responsive to electrical stimulation with almost five times the amount of Glu released upon constant reduction (-0.6 V) compared to when no stimulus was applied. Notably, GelMA/PPy/Glu was able to deliver almost 14 times higher amounts of Glu compared to conventional PPy/Glu films. The described CPH coatings are well suited in implantable drug delivery applications and compared to conducting polymer films can deliver higher quantities of drug in response to mild electrical stimulus. STATEMENT OF SIGNIFICANCE: Conducting polymer hydrogels (CPH) have been explored for the electrically controlled release of bioactives from implantable devices. Typically, the conducting polymer component does not fully penetrate the hydrogel. We report, for the first time, a completely interpenetrating CPH allowing for the full benefits of the composite material to be realised, the hydrogels provide a reservoir for drug delivery, and conducting polymer renders the material responsive to electrical stimulation for drug release. We report a CPH for the electrically controlled delivery of glutamate (excitatory neurotransmitter) where several-fold more glutamate can be delivered compared to conducting polymer films. The described CPH coatings are well suited for use in bioelectronic devices to deliver large quantities of drug in response to mild electrical stimulus.

Conducting polymer hydrogels for electrically responsive drug delivery.

Electrically responsive drug delivery is an attractive approach for the localised, tuneable release of drugs to meet therapeutic demands. Conducting polymers and hydrogels have been explored individually as materials for drug delivery applications with hybrids of these two materials offering potential to extend the range and amount of drug delivered, thereby creating new opportunities to achieve real-world benefit. Although accurate and long-term on-demand release of drugs through conducting polymer hydrogels still presents challenges, these are promising materials for the next generation of electrically responsive drug delivery devices. Here we review the fabrication methods and properties of conducting polymer hydrogels, relevant to drug delivery. In addition, the mechanisms behind drug loading and release are discussed, and applications for these systems presented. The current state of the field is discussed, alongside future steps required to achieve successful translation of these materials to the clinic.

A simultaneous optical and electrical in-vitro neuronal recording system to evaluate microelectrode performance.

OBJECTIVES: In this paper, we aim to detail the setup of a high spatio-temporal resolution, electrical recording system utilising planar microelectrode arrays with simultaneous optical imaging suitable for evaluating microelectrode performance with a proposed 'performance factor' metric. METHODS: Techniques that would facilitate low noise electrical recordings were coupled with voltage sensitive dyes and neuronal activity was recorded both electrically via a customised amplification system and optically via a high speed CMOS camera. This technique was applied to characterise microelectrode recording performance of gold and poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT/PSS) coated electrodes through traditional signal to noise (SNR) calculations as well as the proposed performance factor. RESULTS: Neuronal activity was simultaneously recorded using both electrical and optical techniques and this activity was confirmed via tetrodotoxin application to inhibit action potential firing. PEDOT/PSS outperformed gold using both measurements, however, the performance factor metric estimated a 3 fold improvement in signal transduction when compared to gold, whereas SNR estimated an 8 fold improvement when compared to gold. CONCLUSION: The design and functionality of a system to record from neurons both electrically, through microelectrode arrays, and optically via voltage sensitive dyes was successfully achieved. SIGNIFICANCE: The high spatiotemporal resolution of both electrical and optical methods will allow for an array of applications such as improved detection of subthreshold synaptic events, validation of spike sorting algorithms and a provides a robust evaluation of extracellular microelectrode performance.

Stretchable Electronics Based on Laser Structured, Vapor Phase Polymerized PEDOT/Tosylate.

The fabrication of stretchable conductive material through vapor phase polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) is presented alongside a method to easily pattern these materials with nanosecond laser structuring. The devices were constructed from sheets of vapor phase polymerized PEDOT doped with tosylate on pre-stretched elastomeric substrates followed by laser structuring to achieve the desired geometrical shape. Devices were characterized for electrical conductivity, morphology, and electrical integrity in response to externally applied strain. Fabricated PEDOT sheets displayed a conductivity of 53.1 ± 1.2 S cm-1; clear buckling in the PEDOT microstructure was observed as a result of pre-stretching the underlying elastomeric substrate; and the final stretchable electronic devices were able to remain electrically conductive with up to 100% of externally applied strain. The described polymerization and fabrication steps achieve highly processable and patternable functional conductive polymer films, which are suitable for stretchable electronics due to their ability to withstand externally applied strains of up to 100%.

Determining Neurotrophin Gradients in Vitro To Direct Axonal Outgrowth Following Spinal Cord Injury.

A spinal cord injury can damage neuronal connections required for both motor and sensory function. Barriers to regeneration within the central nervous system, including an absence of neurotrophic stimulation, impair the ability of injured neurons to reestablish their original circuitry. Exogenous neurotrophin administration has been shown to promote axonal regeneration and outgrowth following injury. The neurotrophins possess chemotrophic properties that guide axons toward the region of highest concentration. These growth factors have demonstrated potential to be used as a therapeutic intervention for orienting axonal growth beyond the injury lesion, toward denervated targets. However, the success of this approach is dependent on the appropriate spatiotemporal distribution of these molecules to ensure detection and navigation by the axonal growth cone. A number of in vitro gradient-based assays have been employed to investigate axonal response to neurotrophic gradients. Such platforms have helped elucidate the potential of applying a concentration gradient of neurotrophins to promote directed axonal regeneration toward a functionally significant target. Here, we review these techniques and the principles of gradient detection in axonal guidance, with particular focus on the use of neurotrophins to orient the trajectory of regenerating axons.

A Macroscopic Diffusion-Based Gradient Generator to Establish Concentration Gradients of Soluble Molecules Within Hydrogel Scaffolds for Cell Culture.

Concentration gradients of soluble molecules are ubiquitous within the living body and known to govern a number of key biological processes. This has motivated the development of numerous in vitro gradient-generators allowing researchers to study cellular response in a precise, controlled environment. Despite this, there remains a current paucity of simplistic, convenient devices capable of generating biologically relevant concentration gradients for cell culture assays. Here, we present the design and fabrication of a compartmentalized polydimethylsiloxane diffusion-based gradient generator capable of sustaining concentration gradients of soluble molecules within thick (5 mm) and thin (2 mm) agarose and agarose-collagen co-gel matrices. The presence of collagen within the agarose-collagen co-gel increased the mechanical properties of the gel. Our model molecules sodium fluorescein (376 Da) and FITC-Dextran (10 kDa) quickly established a concentration gradient that was maintained out to 96 h, with 24 hourly replenishment of the source and sink reservoirs. FITC-Dextran (40 kDa) took longer to establish in all hydrogel setups. The steepness of gradients generated are within appropriate range to elicit response in certain cell types. The compatibility of our platform with cell culture was demonstrated using a LIVE/DEAD® assay on terminally differentiated SH-SY5Y neurons. We believe this device presents as a convenient and useful tool that can be easily adopted for study of cellular response in gradient-based assays.

Make it simple: long-term stable gradient generation in a microfluidic microdevice.

Microfluidics-based gradient generators have been used for various biological applications, specifically chemotaxis in cell culture. However, the ability to generate and maintain long term gradients alongside the ability to quickly switch solutions is a challenge of the current microfabricated systems. In this study, a simple flow-driven microfluidic system was developed to achieve long-term stable concentration gradients. Computational modelling was performed to highlight the fluid dynamics as well as to verify the ability of maintaining stable gradients over 7 days. Numerical simulation was analysed to evaluate the static pressure, velocity magnitude and wall shear stress distribution in the chamber. A microdevice fabricated with polydimethylsiloxane (PDMS), using a standard soft lithography technique is presented. It consists of eight parallel microchannels (5 µm × 30 µm × 1,800 µm) linking source and sink chambers; a syringe pump drives fluid through the sink chamber, advection/diffusion from source to sink establishes a gradient. A gradient of a fluorescent dye was generated under the low flow control at 1-10 µl/h of a simple syringe pump equipped with a pulsation damper that was comparable to a pulseless microfluidic pump. Concentration gradients were formed in 1 h and stable from 2 h out to 5 days and consuming less than 1.0 ml of solution. This study focuses on a novel solution to achieve a long-term microfluidic gradient generator using simple engineering techniques of biomedical microdevices.

Human Neural Tissues from Neural Stem Cells Using Conductive Biogel and Printed Polymer Microelectrode Arrays for 3D Electrical Stimulation.

Electricity is important in the physiology and development of human tissues such as embryonic and fetal development, and tissue regeneration for wound healing. Accordingly, electrical stimulation (ES) is increasingly being applied to influence cell behavior and function for a biomimetic approach to in vitro cell culture and tissue engineering. Here, the application of conductive polymer (CP) poly(3,4-ethylenedioxythiophene)-polystyrenesulfonate (PEDOT:PSS) pillars is described, direct-write printed in an array format, for 3D ES of maturing neural tissues that are derived from human neural stem cells (NSCs). NSCs are initially encapsulated within a conductive polysaccharide-based biogel interfaced with the CP pillar microelectrode arrays (MEAs), followed by differentiation in situ to neurons and supporting neuroglia during stimulation. Electrochemical properties of the pillar electrodes and the biogel support their electrical performance. Remarkably, stimulated constructs are characterized by widespread tracts of high-density mature neurons and enhanced maturation of functional neural networks. Formation of tissues using the 3D MEAs substantiates the platform for advanced clinically relevant neural tissue induction, with the system likely amendable to diverse cell types to create other neural and non-neural tissues. The platform may be useful for both research and translation, including modeling tissue development, function and dysfunction, electroceuticals, drug screening, and regenerative medicine.

Development of a Low Cost & Low Noise Amplification System For In Vitro Neuronal Recording through Microelectrode Arrays.

In order to effectively record from electrically active cells through microelectrode arrays a low-noise amplification and data acquisition system is required. Although commercially available, these systems can be expensive and lack the freedom to customise hardware and software. In this work we present a low-cost (US$21 for the first channel + US$11 for each additional channel), low-noise amplifier coupled with an analog to digital converter from National Instruments. The amplifier was designed to (i) operate between 0 and 5 V utilising a DC battery power supply, (ii) operate within a bandwidth of 10 kHz, (iii) remove DC voltage created at the electrode/electrolyte interface with a high-pass cut-off frequency of 0.7 Hz and (iv) have a gain of 2000. Strategies to reduce environment electromagnetic interference at the amplifier front end were employed and involved a customised neural interface board connected between the microelectrode array and amplifier. The constructed amplifier achieved an intrinsic noise amplitude of 0.8 µVrms, which facilitated high quality recordings as exemplified by in vitro recordings from primary hippocampal neurons.

Micelle directed chemical polymerization of polypyrrole particles for the electrically triggered release of dexamethasone base and dexamethasone phosphate.

Conducting polymers such as polypyrrole (PPy) can be used as electrically responsive drug delivery systems typically prepared by electrochemical polymerisation, however, the amount of drug that can be delivered is typically low. To increase drug delivery capacity and prepare larger amounts of polymer, PPy nanoparticles were produced by chemical polymerisation over drug-loaded micelles. Two forms of dexamethasone were included to increase total drug loading and to explore the mechanisms of loading and release. The particles produced were approximately 50 nm in size and their conductivity and reversible redox activity were demonstrated. Loading of the hydrophobic dexamethasone base was more efficient than for the more hydrophilic phosphate salt. After pressing the particles into the desired form, electrically-responsive drug release was achieved with a pulsed potential signal being the most effective way to trigger release. Notably, the anionic phosphate salt of the drug was more sensitive to electrically stimulated release than the uncharged base of dexamethasone, highlighting the role of electrostatic forces in driving drug release. This system has potential to be loaded with different drugs widening the scope of application of these smart particles to treat a range of disease states.

Conducting Polymers as Electrode Coatings for Neuronal Multi-electrode Arrays.

In this new era of bioelectronics, refining transduction pathways between biological systems and electrodes is becoming an increasingly important task. This forum provides insights on how conducting polymer electrode coatings provide more sensitive and compatible systems for recording between the nervous system and electronics.