Electrically Controlled Vasodilator Delivery from PEDOT/Silica Nanoparticle Modulates Vessel Diameter in Mouse Brain.

Vascular damage and reduced tissue perfusion are expected to majorly contribute to the loss of neurons or neural signals around implanted electrodes. However, there are limited methods of controlling the vascular dynamics in tissues surrounding these implants. This work utilizes conducting polymer poly(ethylenedioxythiophene) and sulfonated silica nanoparticle composite (PEDOT/SNP) to load and release a vasodilator, sodium nitroprusside, to controllably dilate the vasculature around carbon fiber electrodes (CFEs) implanted in the mouse cortex. The vasodilator release is triggered via electrical stimulation and the amount of release increases with increasing electrical pulses. The vascular dynamics are monitored in real-time using two-photon microscopy, with changes in vessel diameters quantified before, during, and after the release of the vasodilator into the tissues. This work observes significant increases in vessel diameters when the vasodilator is electrically triggered to release, and differential effects of the drug release on vessels of different sizes. In conclusion, the use of nanoparticle reservoirs in conducting polymer-based drug delivery platforms enables the controlled delivery of vasodilator into the implant environment, effectively altering the local vascular dynamics on demand. With further optimization, this technology could be a powerful tool to improve the neural electrode-tissue interface and study neurovascular coupling.

Nanotopography-enhanced biomimetic coating maintains bioactivity after weeks of dry storage and improves chronic neural recording.

We developed a nanoparticle base layer technology capable of maintaining the bioactivity of protein-based neural probe coating intended to improve neural recording quality. When covalently bound on thiolated nanoparticle (TNP) modified surfaces, neural adhesion molecule L1 maintained bioactivity throughout 8 weeks of dry storage at room temperature, while those bound to unmodified surfaces lost 66% bioactivity within 3 days. We tested the TNP + L1 coating in mouse brains on two different neural electrode arrays after two different dry storage durations (3 and 28 days). The results show that dry-stored coating is as good as the freshly prepared, and even after 28 days of storage, the number of single units per channel and signal-to-noise ratio of the TNP + L1 coated arrays were significantly higher by 32% and 40% respectively than uncoated controls over 16 weeks. This nanoparticle base layer approach enables the dissemination of biomolecule-functionalized neural probes to users worldwide and may also benefit a broad range of applications that rely on surface-bound biomolecules.

Melatonin Decreases Acute Inflammatory Response to Neural Probe Insertion.

Neural electrode insertion trauma impedes the recording and stimulation capabilities of numerous diagnostic and treatment avenues. Implantation leads to the activation of inflammatory markers and cell types, which is detrimental to neural tissue health and recording capabilities. Oxidative stress and inflammation at the implant site have been shown to decrease with chronic administration of antioxidant melatonin at week 16, but its effects on the acute landscape have not been studied. To assess the effect of melatonin administration in the acute phase, specifically the first week post-implantation, we utilized histological and q-PCR methods to quantify cellular and molecular indicators of inflammation and oxidative stress in the tissue surrounding implanted probes in C57BL/6 mice as well as two-photon microscopy to track the microglial responses to the probes in real-time in transgenic mice expressing GFP with CX3CR1 promotor. Histological results indicate that melatonin effectively maintained neuron density surrounding the electrode, inhibited accumulation and activation of microglia and astrocytes, and reduced oxidative tissue damage. The expression of the pro-inflammatory cytokines, TNF-α and IL-6, were significantly reduced in melatonin-treated animals. Additionally, microglial encapsulation of the implant surface was inhibited by melatonin as compared to control animals following implantation. Our results combined with previous research suggest that melatonin is a particularly suitable drug for modulating inflammatory activity around neural electrode implants both acutely and chronically, translating to more stable and reliable interfaces.

Effects of neuronal cell adhesion molecule L1 and nanoparticle surface modification on microglia.

Microelectrode arrays for neural recording suffer from low yield and stability partly due to the inflammatory host responses. A neuronal cell adhesion molecule L1 coating has been shown to promote electrode-neuron integration, reduce microglia activation and improve recording. Coupling L1 to surface via a nanoparticle (NP) base layer further increased the protein surface density and stability. However, the exact L1-microglia interaction in these coatings has not been studied. Here we cultured primary microglia on L1 modified surfaces (with and without NP) and characterized microglia activation upon phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) stimulation. Results showed L1 coatings reduced microglia's superoxide production in response to PMA and presented intrinsic antioxidant properties. Meanwhile, L1 decreased iNOS, NO, and pro-inflammatory cytokines (TNF alpha, IL-6, IL-1 beta), while increased anti-inflammatory cytokines (TGF beta 1, IL-10) in LPS stimulated microglia. Furthermore, L1 increased Arg-1 expression and phagocytosis upon LPS stimulation. Rougher NP surface showed lower number of microglia attached per area than their smooth counterpart, lower IL-6 release and superoxide production, and higher intrinsic reducing potential. Finally, we examined the effect of L1 and nanoparticle modifications on microglia response in vivo over 8 weeks with 2-photon imaging. Microglial coverage on the implant surface was found to be lower on the L1 modified substrates relative to unmodified, consistent with the in vitro observation. Our results indicate L1 significantly reduces superoxide production and inflammatory response of microglia and promotes wound healing, while L1 immobilization via a nanoparticle base layer brings added benefit without adverse effects. STATEMENT OF SIGNIFICANCE: Surface modification of microelectrode arrays with L1 has been shown to reduce microglia coverage on neural probe surface in vivo and improves neural recording, but the specific mechanism of action is not fully understood. The results in this study show that surface bound L1 reduces superoxide production from cultured microglia via direct reduction reaction and signaling pathways, increases anti-inflammatory cytokine release and phagocytosis in response to PMA or LPS stimulation. Additionally, roughening the surface with nanoparticles prior to L1 immobilization further increased the benefit of L1 in reducing microglia activation and oxidative stress. Together, our findings shed light on the mechanisms of action of nanotextured and neuroadhesive neural implant coatings and guide future development of seamless tissue interface.

Surface Area and Local Curvature: Why Roughness Improves the Bioactivity of Neural Implants.

While roughening the surface of neural implants has been shown to significantly improve their performance, the mechanism for this improvement is not understood, preventing systematic optimization of surfaces. Specifically, prior work has shown that the cellular response to a surface can be significantly enhanced by coating the implant surface with inorganic nanoparticles and neuroadhesion protein L1, and this improvement occurs even when the surface chemistry is identical between the nanoparticle-coated and uncoated electrodes, suggesting the critical importance of surface topography. Here, we use transmission electron microscopy to characterize the topography of bare and nanoparticle-coated implants across 7 orders of magnitude in size, from the device scale to the atomic scale. The results reveal multiscale roughness, which cannot be adequately described using conventional roughness parameters. Indeed, the topography is nearly identical between the two samples at the smallest scales and also at the largest scales but vastly different in the intermediate scales, especially in the range of 5-100 nm. Using a multiscale topography analysis, we show that the coating causes a 76% increase in the available surface area for contact and an order-of-magnitude increase in local surface curvature at characteristic sizes corresponding to specific biological structures. These are correlated with a 75% increase in bound proteins on the surface and a 134% increase in neurite outgrowth. The present investigation presents a framework for analyzing the scale-dependent topography of medical device-relevant surfaces, and suggests the most critical size scales that determine the biological response to implanted materials.

Explant Analysis of Utah Electrode Arrays Implanted in Human Cortex for Brain-Computer-Interfaces.

Brain-computer interfaces are being developed to restore movement for people living with paralysis due to injury or disease. Although the therapeutic potential is great, long-term stability of the interface is critical for widespread clinical implementation. While many factors can affect recording and stimulation performance including electrode material stability and host tissue reaction, these factors have not been investigated in human implants. In this clinical study, we sought to characterize the material integrity and biological tissue encapsulation via explant analysis in an effort to identify factors that influence electrophysiological performance. We examined a total of six Utah arrays explanted from two human participants involved in intracortical BCI studies. Two platinum (Pt) arrays were implanted for 980 days in one participant (P1) and two Pt and two iridium oxide (IrOx) arrays were implanted for 182 days in the second participant (P2). We observed that the recording quality followed a similar trend in all six arrays with an initial increase in peak-to-peak voltage during the first 30-40 days and gradual decline thereafter in P1. Using optical and two-photon microscopy we observed a higher degree of tissue encapsulation on both arrays implanted for longer durations in participant P1. We then used scanning electron microscopy and energy dispersive X-ray spectroscopy to assess material degradation. All measures of material degradation for the Pt arrays were found to be more prominent in the participant with a longer implantation time. Two IrOx arrays were subjected to brief survey stimulations, and one of these arrays showed loss of iridium from most of the stimulated sites. Recording performance appeared to be unaffected by this loss of iridium, suggesting that the adhesion of IrOx coating may have been compromised by the stimulation, but the metal layer did not detach until or after array removal. In summary, both tissue encapsulation and material degradation were more pronounced in the arrays that were implanted for a longer duration. Additionally, these arrays also had lower signal amplitude and impedance. New biomaterial strategies that minimize fibrotic encapsulation and enhance material stability should be developed to achieve high quality recording and stimulation for longer implantation periods.

Nanoparticle and Biomolecule Surface Modification Synergistically Increases Neural Electrode Recording Yield and Minimizes Inflammatory Host Response.

Due to their ability to interface with neural tissues, neural electrodes are the key tool used for neurophysiological studies, electrochemical detection, brain computer interfacing, and countless neuromodulation therapies and diagnostic procedures. However, the long-term applications of neural electrodes are limited by the inflammatory host tissue response, decreasing detectable electrical signals, and insulating the device from the native environment. Surface modification methods are proposed to limit these detrimental responses but each has their own limitations. Here, a combinatorial approach is presented toward creating a stable interface between the electrode and host tissues. First, a thiolated nanoparticle (TNP) coating is utilized to increase the surface area and roughness. Next, the neural adhesion molecule L1 is immobilized to the nanoparticle modified substrate. In vitro, the combined nanotopographical and bioactive modifications (TNP+L1) elevate the bioactivity of L1, which is maintained for 28 d. In vivo, TNP+L1 modification improves the recording performance of the neural electrode arrays compared to TNP or L1 modification alone. Postmortem histology reveals greater neural cell density around the TNP+L1 coating while eliminating any inflammatory microglial encapsulation after 4 weeks. These results demonstrate that nanotopographical and bioactive modifications synergistically produce a seamless neural tissue interface for chronic neural implants.

Real-Time Fast Scan Cyclic Voltammetry Detection and Quantification of Exogenously Administered Melatonin in Mice Brain.

Melatonin (MT) has been recently considered an excellent candidate for the treatment of sleep disorders, neural injuries, and neurological diseases. To better investigate the actions of MT in various brain functions, real-time detection of MT concentrations in specific brain regions is much desired. Previously, we have demonstrated detection of exogenously administered MT in anesthetized mouse brain using square wave voltammetry (SWV). Here, for the first time, we show successful detection of exogenous MT in the brain using fast scan cyclic voltammetry (FSCV) on electrochemically pre-activated carbon fiber microelectrodes (CFEs). In vitro evaluation showed the highest sensitivity (28.1 nA/µM) and lowest detection limit (20.2 ± 4.8 nM) ever reported for MT detection at carbon surface. Additionally, an extensive CFE stability and fouling assessment demonstrated that a prolonged CFE pre-conditioning stabilizes the background, in vitro and in vivo, and provides consistent CFE sensitivity over time even in the presence of a high MT concentration. Finally, the stable in vivo background, with minimized CFE fouling, allows us to achieve a drift-free FSCV detection of exogenous administered MT in mouse brain over a period of 3 min, which is significantly longer than the duration limit (usually < 90 s) for traditional in vivo FSCV acquisition. The MT concentration and dynamics measured by FSCV are in good agreement with SWV, while microdialysis further validated the concentration range. These results demonstrated reliable MT detection using FSCV that has the potential to monitor MT in the brain over long periods of time.

Neuroadhesive protein coating improves the chronic performance of neuroelectronics in mouse brain.

Intracortical microelectrodes are being developed to both record and stimulate neurons to understand brain circuitry or restore lost functions. However, the success of these probes is hampered partly due to the inflammatory host tissue responses to implants. To minimize the foreign body reactions, L1, a brain derived neuronal specific cell adhesion molecule, has been covalently bound to the neural electrode array surface. Here we evaluated the chronic recording performance of L1-coated silicon based laminar neural electrode arrays implanted into V1m cortex of mice. The L1 coating enhanced the overall visually evoked single-unit (SU) yield and SU amplitude, as well as signal-to-noise-ratio (SNR) in the mouse brain compared to the uncoated arrays across the 0-1500 µm depth. The improvement in recording is most dramatic in the hippocampus region, where the control group showed severe recording yield decrease after one week, while the L1 implants maintained a high SU yield throughout the 16 weeks. Immunohistological analysis revealed significant increases of axonal and neuronal density along with significantly lowered microglia activation around the L1 probe after 16 weeks. These results collectively confirm the effectiveness of L1 based biomimetic coating on minimizing inflammatory tissue response and improving neural recording quality and longevity. Improving chronic recording will benefit the brain-computer interface technologies and neuroscience studies involving chronic tracking of neural activities.

Electrochemical detection of exogenously administered melatonin in the brain.

Melatonin (MT) is an important electroactive hormone that regulates different physiological actions in the brain, ranging from circadian clock to neurodegeneration. An impressive number of publications have highlighted the effectiveness of MT treatments in different types of sleep and neurological disorders, including Alzheimer's and Parkinson's disease. The ability to detect MT in different regions of the brain would provide further insights into the physiological roles and therapeutic effects of MT. While multiple electrochemical methods have been used to detect MT in biological samples, monitoring MT in the brain of live animals has not been demonstrated. Here, we optimized a square wave voltammetry (SWV) electroanalytical method to evaluate the MT detection performance at CFEs in vitro and in vivo. SWV was able to sensitively detect the MT oxidation peak at 0.7 V, and discriminate MT from most common interferents in vitro. More importantly, using the optimized SWV, CFEs successfully detected and reliably quantified MT concentrations in the visual cortex of anesthetized mice after intraperitoneal injections of different MT doses, offering stable MT signals for up to 40 minutes. To the best of our knowledge, this is the first electrochemical measurement of exogenously administered MT in vivo. This electrochemical MT sensing technique will provide a powerful tool for further understanding MT's action in the brain.

Nanoparticle Doped PEDOT for Enhanced Electrode Coatings and Drug Delivery.

In order to address material limitations of biologically interfacing electrodes, modified silica nanoparticles are utilized as dopants for conducting polymers. Silica precursors are selected to form a thiol modified particle (TNP), following which the particles are oxidized to sulfonate modified nanoparticles (SNPs). The selective inclusion of hexadecyl trimethylammonium bromide allows for synthesis of both porous and nonporous SNPs. Nonporous nanoparticle doped polyethylenedioxythiophene (PEDOT) films possess low interfacial impedance, high charge injection (4.8 mC cm-2 ), and improved stability under stimulation compared to PEDOT/poly(styrenesulfonate). Porous SNP dopants can serve as drug reservoirs and greatly enhance the capability of conducting polymer-based, electrically controlled drug release technology. Using the SNP dopants, drug loading and release is increased up to 16.8 times, in addition to greatly expanding the range of drug candidates to include both cationic and electroactive compounds, all while maintaining their bioactivity. Finally, the PEDOT/SNP composite is capable of precisely modulating neural activity in vivo by timed release of a glutamate receptor antagonist from coated microelectrode sites. Together, this work demonstrates the feasibility and potential of doping conducting polymers with engineered nanoparticles, creating countless options to produce composite materials for enhanced electrical stimulation, neural recording, chemical sensing, and on demand drug delivery.

A superoxide scavenging coating for improving tissue response to neural implants.

The advancement of neural prostheses requires implantable neural electrodes capable of electrically stimulating or recording signals from neurons chronically. Unfortunately, the implantation injury and presence of foreign bodies lead to chronic inflammation, resulting in neuronal death in the vicinity of electrodes. A key mediator of inflammation and neuronal loss are reactive oxygen and nitrogen species (RONS). To mitigate the effect of RONS, a superoxide dismutase mimic compound, manganese(III) meso-tetrakis-(N-(2-aminoethyl)pyridinium-2-yl) porphyrin (iSODm), was synthesized to covalently attach to the neural probe surfaces. This new compound showed high catalytic superoxide scavenging activity. In microglia cell line cultures, the iSODm coating effectively reduced superoxide production and altered expression of iNOS, NADPH oxidase, and arginase. After 1 week of implantation, iSODm coated electrodes showed significantly lower expression of markers for oxidative stress immediately adjacent to the electrode surface, as well as significantly less neurons undergoing apoptosis. STATEMENT OF SIGNIFICANCE: One critical challenge in the translation of neural electrode technology to clinically viable devices for brain computer interface or deep brain stimulation applications is the chronic degradation of the device performance due to neuronal degeneration around the implants. One of the key mediators of inflammation and neuronal degeneration is reactive oxygen and nitrogen species released by injured neurons and inflammatory microglia. This research takes a biomimetic approach to synthesize a compound having similar reactivity as superoxide dismutase, which can catalytically scavenge reactive oxygen and nitrogen species, thereby reducing oxidative stress and decreasing neuronal degeneration. By immobilizing the compound covalently on the surface of neural implants, we show that the neuronal degeneration and oxidative stress around the implants is significantly reduced.

Facile Synthesis of a 3,4-Ethylene-Dioxythiophene (EDOT) Derivative for Ease of Bio-Functionalization of the Conducting Polymer PEDOT.

In the pursuit of conducting polymer based bio-functional devices, a cost-effective and high yield synthesis method for a versatile monomer is desired. We report here a new synthesis strategy for a versatile monomer 2-methylene-2,3-dihydrothieno (3,4-b) (1,4) dioxine, or 3,4-ethylenedioxythiophene with a exomethylene side group (EDOT-EM). Compared to the previously reported synthesis route, the new strategy uses less steps, with faster reaction rate, and higher yield. The presence of EM group opens up endless possibility for derivatization via either hydro-alkoxy addition or thiol-ene click chemistry. EDOT-EM could be polymerized into stable and low impedance PEDOT-EM polymer using electro-polymerization method on different conducting substrates at both macro and micro scales. Facile post-functionalization of PEDOT-EM with molecules of varying size and functionality (from small molecules to DNAs and proteins) was achieved. The new synthetic route of EDOT-EM and the ease of post-functionalization of PEDOT-EM will greatly accelerate the use of conducting polymer in a broad range of organic electronics and bioelectronics applications.

Soft Conducting Elastomer for Peripheral Nerve Interface.

State-of-the-art intraneural electrodes made from silicon or polyimide substrates have shown promise in selectively modulating efferent and afferent activity in the peripheral nervous system. However, when chronically implanted, these devices trigger a multiphase foreign body response ending in device encapsulation. The presence of encapsulation increases the distance between the electrode and the excitable tissue, which not only reduces the recordable signal amplitude but also requires increased current to activate nearby axons. Herein, this study reports a novel conducting polymer based intraneural electrode which has Young's moduli similar to that of nerve tissue. The study first describes material optimization of the soft wire conductive matrix and evaluates their mechanical and electrochemical properties. Second, the study demonstrates 3T3 cell survival when cultured with media eluted from the soft wires. Third, the study presents acute in vivo functionality for stimulation of peripheral nerves to evoke force and compound muscle action potential in a rat model. Furthermore, comprehensive histological analyses show that soft wires elicit significantly less scar tissue encapsulation, less changes to axon size, density and morphology, and reduced macrophage activation compared to polyimide implants in the sciatic nerves at 1 month postimplantation.

ROS responsive resveratrol delivery from LDLR peptide conjugated PLA-coated mesoporous silica nanoparticles across the blood-brain barrier.

BACKGROUND: Oxidative stress acts as a trigger in the course of neurodegenerative diseases and neural injuries. An antioxidant-based therapy can be effective to ameliorate the deleterious effects of oxidative stress. Resveratrol (RSV) has been shown to be effective at removing excess reactive oxygen species (ROS) or reactive nitrogen species generation in the central nervous system (CNS), but the delivery of RSV into the brain through systemic administration is inefficient. Here, we have developed a RSV delivery vehicle based on polylactic acid (PLA)-coated mesoporous silica nanoparticles (MSNPs), conjugated with a ligand peptide of low-density lipoprotein receptor (LDLR) to enhance their transcytosis across the blood-brain barrier (BBB). RESULTS: Resveratrol was loaded into MSNPs (average diameter 200 nm, pore size 4 nm) at 16 µg/mg (w/w). As a gatekeeper, the PLA coating prevented the RSV burst release, while ROS was shown to trigger the drug release by accelerating PLA degradation. An in vitro BBB model with a co-culture of rat brain microvascular endothelial cells (RBECs) and microglia cells using Transwell chambers was established to assess the RSV delivery across BBB. The conjugation of LDLR ligand peptides markedly enhanced the migration of MSNPs across the RBECs monolayer. RSV could be released and effectively reduce the activation of the microglia cells stimulated by phorbol-myristate-acetate or lipopolysaccharide. CONCLUSIONS: These ROS responsive LDLR peptides conjugated PLA-coated MSNPs have great potential for oxidative stress therapy in CNS.

Recent Advances in Neural Electrode-Tissue Interfaces.

Neurotechnology is facing an exponential growth in the recent decades. Neural electrode-tissue interface research has been well recognized as an instrumental component of neurotechnology development. While satisfactory long-term performance was demonstrated in some applications, such as cochlear implants and deep brain stimulators, more advanced neural electrode devices requiring higher resolution for single unit recording or microstimulation still face significant challenges in reliability and longevity. In this article, we review the most recent findings that contribute to our current understanding of the sources of poor reliability and longevity in neural recording or stimulation, including the material failure, biological tissue response and the interplay between the two. The newly developed characterization tools are introduced from electrophysiology models, molecular and biochemical analysis, material characterization to live imaging. The effective strategies that have been applied to improve the interface are also highlighted. Finally, we discuss the challenges and opportunities in improving the interface and achieving seamless integration between the implanted electrodes and neural tissue both anatomically and functionally.