Conducting polymer hydrogels for the release of glutamate and sensing of extracellular action potentials

Abstract

Introduction: Neurological disorders are a significant global health issue affecting almost 6% of the population and often involve abnormal neuronal action potential firing and chemical signalling in the brain. Conventional treatment options including oral and systemic administration of drugs have limited ability to cross the blood brain barrier and hence require frequent administration of drugs in higher doses leading to unwanted side effects. Recent advances in the field of drug delivery have made it possible to release drugs locally from the implantable devices by applications of an external trigger but these are not able to respond quickly to fluctuations of symptoms or isolated episodes of dysfunction Alternative treatment options such as deep brain stimulation have enabled the treatment of various neurological conditions including epilepsy, Parkinson’s disease and deafness. However, a clear need exists for a closed loop delivery system, which can respond quickly to fluctuations in the symptoms by delivering the drugs while recording the neuronal activity. Closed loop systems have been explored for various neurological conditions such as epilepsy and movement disorders. These systems can deliver neurotransmitters or other bioactives with precise spatio-temporal control while simultaneously recording the neural activity. Most of the closed loop delivery systems are based on organic ion pump or microfluidic pump and their widespread use is limited by complicated set ups and technical challenges. A clear need exists for fabrication of new devices based on the idea of a closed loop feedback system which can delivery drugs on-demand while monitoring neuronal action potentials. Conducting polymer (CP) coatings such as polypyrrole (PPy) and poly(3,4- ethylenedioxythiophene) (PEDOT) have been explored as a delivery platform in bioelectronics, however, their utility is restricted by their limited loading capacity and stability. In addition, PEDOT have been widely explored for neural interface applications and have shown high fidelity recordings. However, new biomaterials that are capable to functionalize electrode surfaces for improved neural interface and drug delivery applications are needed. Conducting polymer hydrogels (CPH) are hybrid material consisting of a CP grown within the hydrogel network have the potential to extend the range and amount of drug delivered, thereby creating new opportunities to achieve real-world benefit. Aim: This research aims to fabricate the components of a closed-loop feedback delivery device suitable for the delivery of glutamate (Glu) in response to native cellular signalling, namely a CPH for drug delivery, and a CPH for coating microelectrodes. Methods: A stability indicating high performance liquid chromatography (HPLC) method was developed for the quantification of Glu from forced degradation samples and following release from CPs and CPHs. This analytical method was validated and used as a tool throughout the thesis. Two different CPHs are reported in this thesis, one for drug delivery and one for microelectrode coatings. The first CPH developed comprised the hydrogel gelatin methacrylate (GelMA) and the CP PPy, was fabricated for the electrically controlled delivery of Glu. The CPH material was characterised for surface morphology, interpenetrated network, electrochemical properties, biocompatibility. The Glu release studies were carried out in comparison to the convention PPy/Glu coating. The second CPH consisting of GelMA and PEDOT/polystyrene sulfonate (PSS) was fabricated as a material for microelectrode coating intended for recording and stimulating neurons. The hybrid material was also characterised for electrochemical properties using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), voltage transient measurements (VTM). Furthermore, we investigated the biocompatibility of these coatings and ability of the described CPH coatings to record the neuronal signals. Results and discussion An isocratic quantification method with a short runtime was developed and validated to quantify Glu from the forced degradation samples and drug release samples from CP and CPH coatings. GelMA/PPy/Glu fabricated for drug delivery 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 CP films can deliver higher quantities of drug in response to mild electrical stimulus. GelMA/PEDOT/PSS was fabricated for microelectrode coatings and was demonstrated to be reversibly electroactive, had low impedance and a high charge injection limit (CIL) compared to the bare gold electrodes. The CPH coatings had impedance values similar to conventional PEDOT/PSS coatings at a frequency of 1000 Hz, a key frequency for neuronal action potential activity. The CPH was confirmed to be electrochemical stabile over 1000 CV cycles and long-term performance was maintained over a period of 14 days. Biocompatibility of the CPH coatings was confirmed on primary hippocampal neuronal cultures via neuronal viability assay. The characterisation of the CPH coating was compared with conventional PEDOT/PSS coatings. Microelectrodes with CPH coatings were able to record neuronal activity from in-vitro primary hippocampal neuronal cultures. Conclusion This thesis details fabrication and characterisation of components of a closed loop delivery system for the delivery of Glu triggered by neuronal action potentials. A simple, isocratic HPLC method was developed and validated for the quantification of Glu released from the CP and CPH coated electrodes. A fully interpenetrating, selectively patternable and covalently bonded CPH coatings comprising of GelMA/PPy/Glu was fabricated. The material showed cytocompatibility with undifferentiated human neuroblastoma cell lines SH-SY5Y. We have demonstrated that the GelMA/PPy/Glu system was responsive to electrical stimulation with almost five times the amount of Glu released upon constant reduction (-0.6 V) compared to when no electrical stimulation was applied. The GelMA/PPy/Glu was able to deliver fourteen times higher amount of Glu compared to PPy/Glu films. In addition, a custom MEA devices containing both microelectrodes and drug delivery electrodes compatible with the commercial MCS headstage was fabricated. The MEA was coated with a CPH consisting of an interpenetrating network of PEDOT chains with in the GelMA hydrogel. The CPH coating GelMA/PEDOT/PSS was fully interpenetrating and could be selectively photolithographically patterned and covalently bonded on the gold microelectrodes. The GelMA/PEDOT/PSS coatings was reversibly electroactive, had low impedance, and high CIL compared to the bare gold microelectrodes. The biocompatibility of the CPH coatings was verified on primary hippocampal neuronal cultures. Microelectrodes with CPH coatings were able to record neuronal activity from in-vitro neuronal cultures. These materials, and the experimental setup will support future studies towards the development of a closed loop system to determine if the delivery of Glu can triggered by neuronal action potentials.