Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology

The development of novel approaches to stimulate neuronal circuits is crucial to understand the physiology of neuronal networks, and to provide new strategies to treat neurological disorders. Nowadays, chemical, electrical or optical approaches are the main exploited strategies to interrogate and dissect neuronal circuit functions. However, although all these methods have contributed to achieve important insights into neuroscience research field, they all present relevant limitations for their use in in-vivo studies or clinical applications. For example, while chemical stimulation does not require invasive surgical procedures, it is difficult to control the pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation provides high temporal bandwidth, but it has low spatial resolution and it requires implantation of electrodes; optical stimulation provides subcellular resolution but the low depth penetration in dense tissue still requires the invasive insertion of stimulating probes. Due to all these drawbacks, there is still a strong need to develop new stimulation strategies to remotely activate neuronal circuits as deep as possible. The development of remote stimulation techniques would allow the combination of functional and behavioral studies, and the design of novel and minimally invasive prosthetic approaches. Alternative approaches to circumvent surgical implantation of probes include transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among v these methods, the use of magnetic and ultrasound (US) fields represents the most promising vector to remotely convey information to the brain tissue. Both magnetic and low-intensity US fields provide an efficient mean for delicate and reversible alteration of cells and tissues through the generation of local mechanical perturbations. In this regard, advances in the mechanobiology research field have led to the discovery, design and engineering of cellular transduction pathways to perform stimulation of cellular activity. Furthermore, the use of US pressure fields is attracting considerable interest due to its potential for the development of miniaturized, portable and implantation-free US stimulation devices. The purpose of my PhD research activity was the establishment of a novel neuronal stimulation paradigm adding a cellular selectivity to the US stimulation technology through the selective mechano-sensitization of neuronal cells, in analogy to the well-established optogenetic approach. In order to achieve the above mentioned goal, we propose the cellular overexpression of mechanosensitive (MS) ion channels, which could then be gated upon the application of an US generated pressure field. Therefore, we selected the bacterial large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL with its extensive characterization represents a malleable nano-valve that could be further engineered with respect to channel sensitivity, conductance and gating mechanism, in order to obtain the desired biophysical properties to achieve reliable and efficient remote mechanical stimulation of neuronal activity. In the first part of the work, we report the development of an engineered MscL construct, called eMscL, to induce the heterologous expression of the bacterial protein in rodent primary neuronal cultures. Furthermore, we report the structural and functional characterization of neuronal cells expressing the eMscL channel, at both single-cell and network levels, in order to show that the functional expression of the engineered MscL channel induces an effective vi neuronal sensitization to mechanical stimulation, which does not affect the physiological development of the neuronal itself. In the second part of the work, we report the design and development of a water tank-free ultrasound delivery system integrated to a custom inverted fluorescence microscope, which allows the simultaneous US stimulation and monitoring of neuronal network activity at single resolution. Overall, this work represents the first development of a genetically mechanosensitized neuronal in-vitro model. Moreover, the developed US delivery system provides the platform to perform high-throughput and reliable investigation, testing and calibration of the stimulation protocols. In this respect, we propose, and envisage in the near future, the exploitation of the engineered MscL ion channel as a mature tool for novel neuro-technological applications

Optogenetic Brain Interfaces

The brain is a large network of interconnected neurons where each cell functions as a nonlinear processing element. Unraveling the mysteries of information processing in the complex networks of the brain requires versatile neurostimulation and imaging techniques. Optogenetics is a new stimulation method which allows the activity of neurons to be modulated by light. For this purpose, the cell-types of interest are genetically targeted to produce light-sensitive proteins. Once these proteins are expressed, neural activity can be controlled by exposing the cells to light of appropriate wavelengths. Optogenetics provides a unique combination of features, including multimodal control over neural function and genetic targeting of specific cell-types. Together, these versatile features combine to a powerful experimental approach, suitable for the study of the circuitry of psychiatric and neurological disorders. The advent of optogenetics was followed by extensive research aimed to produce new lines of light-sensitive proteins and to develop new technologies: for example, to control the distribution of light inside the brain tissue or to combine optogenetics with other modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance imaging. In this paper, the authors review some of the recent advances in the field of optogenetics and related technologies and provide their vision for the future of the field.United States. Defense Advanced Research Projects Agency (Space and Naval Warfare Systems Center, Pacific Grant/Contract No. N66001-12-C-4025)University of Wisconsin--Madison (Research growth initiative; grant 101X254)University of Wisconsin--Madison (Research growth initiative; grant 101X172)University of Wisconsin--Madison (Research growth initiative; grant 101X213)National Science Foundation (U.S.) (MRSEC DMR-0819762)National Science Foundation (U.S.) (NSF CAREER CBET-1253890)National Institutes of Health (U.S.) (NIH/NIBIB R00 Award (4R00EB008738)National Institutes of Health (U.S.) (NIH Director’s New Innovator award (1-DP2-OD002989))Okawa Foundation (Research Grant Award)National Institutes of Health (U.S.) (NIH Director’s New Innovator Award (1DP2OD007265))National Science Foundation (U.S.) (NSF CAREER Award (1056008)Alfred P. Sloan Foundation (Fellowship)Human Frontier Science Program (Strasbourg, France) (Grant No. 1351/12)Israeli Centers of Research Excellence (I-CORE grant, program 51/11)MINERVA Foundation (Germany

Functional nano-bio interfaces for cell modulation

Interacting cellular systems with nano-interfaces has shown great promise in promoting differentiation, regeneration, and stimulation. Functionalized nanostructures can serve as topological cues to mimic the extracellular matrix network to support cellular growth. Nanostructures can also generate signals, such as thermal, electrical, and mechanical stimulus, to trigger cellular stimulation. At this stage, the main challenges of applying nanostructures with biological systems are: (1) how to mimic the hierarchical structure of the ECM network in a 3D format and (2) how to improve the efficiency of the nanostructures while decreasing its invasiveness. To enable functional neuron regeneration after injuries, we have developed a 2D nanoladder scaffold, composed of micron size fibers and nanoscale protrusions, to mimic the ECM in the spinal cord. We have demonstrated that directional guidance during neuronal regeneration is critical for functional reconnection. We further transferred the nanoladder pattern onto biocompatible silk films. We established a self-folding strategy to fabricate 3D silk rolls, which is an even closer system to mimic the ECM of the spinal cord. As demonstrated by in vitro and in vivo experiments, such a scaffold can serve as a grafting bridge to guide axonal regeneration to desired targets for functional reconnection after spinal cord injuries. Benefited from the robust self-folding techniques, silk rolls can also be used for heterogeneous cell culture, providing a potential therapeutic approach for multiple tissue regeneration directions, such as bones, muscles, and tendons. For achieving neurostimulation, we have developed photoacoustic nanotransducers (PANs), which generate ultrasound upon excitation of NIR II nanosecond laser light. By surface functionalize PAN to bind to neurons, we have achieved an optoacoustic neuron stimulation process with a high spatial and temporal resolution, proved by in-vitro and in-vivo experiments. Such an application can enable non-invasive, optogenetics free and MRI compatible neurostimulation, which provides a new direction of gene-transfection free neuromodulation. Collectively, in this thesis, we have developed two systems to promote functional regeneration after injuries and stimulate neurons in a minimally invasive manner. By integrating those two functions, a potential new generation of the bioengineered scaffold can be investigated to enable functional and programmable control during the regeneration process

Anisotropic scaffolds for peripheral nerve and spinal cord regeneration

The treatment of long-gap (\u3e10 mm) peripheral nerve injury (PNI) and spinal cord injury (SCI) remains a continuous challenge due to limited native tissue regeneration capabilities. The current clinical strategy of using autografts for PNI suffers from a source shortage, while the pharmacological treatment for SCI presents dissatisfactory results. Tissue engineering, as an alternative, is a promising approach for regenerating peripheral nerves and spinal cords. Through providing a beneficial environment, a scaffold is the primary element in tissue engineering. In particular, scaffolds with anisotropic structures resembling the native extracellular matrix (ECM) can effectively guide neural outgrowth and reconnection. In this review, the anatomy of peripheral nerves and spinal cords, as well as current clinical treatments for PNI and SCI, is first summarized. An overview of the critical components in peripheral nerve and spinal cord tissue engineering and the current status of regeneration approaches are also discussed. Recent advances in the fabrication of anisotropic surface patterns, aligned fibrous substrates, and 3D hydrogel scaffolds, as well as their in vitro and in vivo effects are highlighted. Finally, we summarize potential mechanisms underlying the anisotropic architectures in orienting axonal and glial cell growth, along with their challenges and prospects

Multifunctional Polydopamine Nanomaterials for Biomedical and Environmental Applications

Polydopamine (PDA), a synthetic and organic material, has emerged as a promising materialplatform for various applications in energy, environmental, and biomedical fields. PDA, formed by self-polymerization of dopamine, is rich in catechol and amine groups, which facilitate covalent conjugation and/or other non-covalent interactions with organic and inorganic materials. It is highly biocompatible, biodegradable, has broadband light absorption spectrum and excellent light-to-heat conversion efficiency. Also, it is easy to synthesize and functionalize. The combination of excellent characteristics of polydopamine-based nanomaterials, make them a promising adsorbent agent for environmental wastewater treatment and photothermal agent for biomedical applications. In the first half of thesis, we utilize the surface chemical functionality of polydopamine nanoparticles and their affinity to heavy metal ions and organic dyes to realize multifunctional filtration membranes that remove heavy metal ions and organic dyes from water through adsorption and catalytic degradation. Polydopamine exhibits high adsorption capacity toward heavy metal ions and organic dyes. Adsorption-based membrane technologies can be ideal for continuous flow water purification and have been extensively employed at industrial scale forxxiii water reclamation. By introducing polydopamine nanoparticles during bacteria-mediated cellulose growth, we fabricated a composite foam and membrane to study the adsorption behavior of the nanocomposites in different environmentally relevant pH and concentrations. The PDA/BNC membrane was used to investigate the removal efficiency of toxic heavy metals ions such as Pb (II) and Cd (II) and organic pollutants such as rhodamine 6G and methylene blue. Furthermore, to improve the range of pH in which the composite membrane is effective for dye removal, we fabricated another novel polydopamine/nanocellulose membrane, which is decorated with palladium (Pd) nanoparticles to remove organic dyes from contaminated water through catalytic dye degradation. In the second part of thesis, we develop polydopamine-based nanomaterials and experimental setups to be used in biomedical applications such as drug delivery and photothermal stimulation of cells. Using mesoporous silica-coated PDA nanoparticles as drug carrier and tetradecanol (TD) as gate keeper, we demonstrated that we could enhance the immune system response toward Melanoma cancer in mouse model through combination of photothermal and immunotherapy. Polydopamine core works as a photothermal agent to cause localized release of gardiquimod and tumor cell death upon NIR laser irradiation, hence, release of tumor associated antigens. Antigen presenting cells (APCs) including the dendritic cells and macrophages uptake these antigens and be activated around tumor site in response to these signals. Furthermore, these activated APCs, present the antigen to CD8+ cytotoxic T cells to actuate anti-tumor immune response. We have shown that this treatment is effective in reducing the tumor size and eliminating it in majority of cases. Also, the treatment created a memory effect in immune system toward melanoma cancer when second cancer event happened in mice that were treated before. Finally, we investigated the possibility of controlling the excitable cells’ activity through nanoheating. This was made possible by using polydopamine nanoparticles to localize the heat on cell membrane. We demonstrated that by using polydopamine nanoparticle and polydopamine/collagen 3D foam, and by applying NIR laser light, we can reversibly modulate the activity of in vitro cultured neurons and cardiomyocytes. A reduction in firing rate of neurons and an increase in beating rate of cardiomyocytes with different degree of inhibition and excitation was observed. Effect of different parameters on the quality of modulation was investigated

Organic bioelectronic devices to control cell signalling

The nervous system consists of a network of specialized cells that coordinate the actions of the body by transmitting information to and from the brain. The communication between the nerve cells is dependent on the interplay of both electrical and chemical signals. As our understanding of nerve cell signalling increases there is a growing need to develop techniques capable of interfacing with the nervous system. One of the major challenges is to translate between the signal carriers of the nervous system (ions and neurotransmitters) and those of conventional electronics (electrons). Organic conjugated polymers represent a unique class of materials that can utilize both electrons and ions as charge carriers. Taking advantage of this combined feature, we have established a novel communication interface between electronic components and biological systems. The organic bioelectronic devices presented in this thesis are based on the organic electronic ion pump (OEIP) made of the conducting organic polymer poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). When electronically addressed, electrochemical redox reactions in the polymer translate electronic signals into electrophoretic migration of ions. We show that the device can transport a range of substances involved in nerve cell signaling. These include positively charged ions, neurotransmitters and cholinergic substances. Since the devices are designed to be easily incorporated in conventional microscopy set-ups, we use Ca2+ imaging as readout to monitor cell responses. We demonstrate how electrophoretic delivery of ions and neurotransmitters with precise, spatiotemporal control can be used to modulate intracellular Ca2+ signaling in neuronal cells in the absence of convective disturbances. The electronic control of delivery enables strict control of dynamic parameters, such as amplitude and frequency of Ca2+ responses, and can be used to generate temporal patterns mimicking naturally occurring Ca2+ oscillations. To enable further control and fine-tuning of the ionic signals we developed the electrophoretic chemical transistor, an analogue of the traditional transistor used to amplify and/or switch electronic signals. We thereby take the first step towards integrated chemical circuits. Finally, we demonstrate the use of the OEIP in a new “machine-to-brain” interface. By encapsulating the OEIP we were able to use it in vivo to modulate brainstem responses in guinea pigs. This was the first successful realization of an organic bioelectronic device capable of modulating mammalian sensory function by precise delivery of neurotransmitters. Our findings highlight the potential of communication interfaces based on conjugated polymers in generating complex, high-resolution, signal patterns to control cell physiology. Such devices will have widespread applications across basic research as well as future applicability in medical devices in multiple therapeutic areas

A magnetically actuated microrobot for targeted neural cell delivery and selective connection of neural networks

There has been a great deal of interest in the development of technologies for actively manipulating neural networks in vitro, providing natural but simplified environments in a highly reproducible manner in which to study brain function and related diseases. Platforms for these in vitro neural networks require precise and selective neural connections at the target location, with minimal external influences, and measurement of neural activity to determine how neurons communicate. Here, we report a neuron-loaded microrobot for selective connection of neural networks via precise delivery to a gap between two neural clusters by an external magnetic field. In addition, the extracellular action potential was propagated from one cluster to the other through the neurons on the microrobot. The proposed technique shows the potential for use in experiments to understand how neurons communicate in the neural network by actively connecting neural clusters. Copyright © 2020 The Authors, some rights reserved.1

Trends and challenges in neuroengineering: toward "Intelligent" neuroprostheses through brain-"brain inspired systems" communication

Future technologies aiming at restoring and enhancing organs function will intimately rely on near-physiological and energy-efficient communication between living and artificial biomimetic systems. Interfacing brain-inspired devices with the real brain is at the forefront of such emerging field, with the term "neurobiohybrids" indicating all those systems where such interaction is established. We argue that achieving a "high-level" communication and functional synergy between natural and artificial neuronal networks in vivo, will allow the development of a heterogeneous world of neurobiohybrids, which will include "living robots" but will also embrace “intelligent” neuroprostheses for augmentation of brain function. The societal and economical impact of intelligent neuroprostheses is likely to be potentially strong, as they will offer novel therapeutic perspectives for a number of diseases, and going beyond classical pharmaceutical schemes. However, they will unavoidably raise fundamental ethical questions on the intermingling between man and machine and more specifically, on how deeply it should be allowed that brain processing is affected by implanted "intelligent" artificial systems. Following this perspective, we provide the reader with insights on ongoing developments and trends in the field of neurobiohybrids. We address the topic also from a "community building" perspective, showing through a quantitative bibliographic analysis, how scientists working on the engineering of brain-inspired devices and brain-machine interfaces are increasing their interactions. We foresee that such trend preludes to a formidable technological and scientific revolution in brain-machine communication and to the opening of new avenues for restoring or even augmenting brain function for therapeutic purposes

Bipolar Electroactive Conducting Polymers for Wireless Cell Stimulation

Electrochemical stimulation (ES) promotes wound healing and tissue regeneration in biomedical applications and clinical studies and is central to the emerging field of electroceuticals. Traditional ES such as deep brain stimulation for Parkinson’s disease, utilises metal electrodes that are hard wired to a power supply to deliver the stimulation. Bipolar electrochemistry (BPE) introduces an innovative approach to cell stimulation that is wireless. Developing conducting polymers (CPs)-based stimulation platforms wireless powdered by BPE bipolar will provide an exciting new dimension to medical bionics. In this project, Chapter 2 deals with development of a bipolar electrochemical activity testing system and bipolar electrochemical stimulation (BPES) system. Then, bipolar electroactive and biocompatible CPs grown on FTO substrate are successfully synthesised, modified, and characterised in Chapter 3 and Chapter 4 using the above systems prior to using for wireless cell stimulation. Furthermore, free standing and soft CP templates are developed (Chapter 5). More importantly, all these bipolar electroactive CPs have been applied to wireless cell stimulation using BPE (all research Chapters). Significant increase in both cell number and neurite growth has been demonstated, suggesting that the BPES system is highly efficient for stimulation of animal PC 12 cell and human SH-SY5Y cell. More specific information is presented in each chapter as below. In Chapter 3, a CP-based bipolar electrochemical stimulation (BPES) system for cell stimulation was present. Polypyrrole (PPy) films with different dopants have demonstrated reversible and recoverable bipolar electrochemical activity under a low driving DC voltage

Nanotechnology in peripheral nerve repair and reconstruction

The recent progress in biomaterials science and development of tubular conduits (TCs) still fails in solving the current challenges in the treatment of peripheral nerve injuries (PNIs), in particular when disease-related and long-gap defects need to be addressed. Nanotechnology-based therapies that seemed unreachable in the past are now being considered for the repair and reconstruction of PNIs, having the power to deliver bioactive molecules in a controlled manner, to tune cellular behavior, and ultimately guide tissue regeneration in an effective manner. It also offers opportunities in the imaging field, with a degree of precision never achieved before, which is useful for diagnosis, surgery and in the patientâ s follow-up. Nanotechnology approaches applied in PNI regeneration and theranostics, emphasizing the ones that are moving from the lab bench to the clinics, are herein overviewed.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the financial support provided to Joaquim M. Oliveira (IF/01285/2015) and Joana Silva-Correia (IF/00115/2015) under the program “Investigador FCT”.info:eu-repo/semantics/publishedVersio