Nanoenabled Direct Contact Interfacing of Syringe-Injectable Mesh Electronics.

Polymer-based electronics with low bending stiffnesses and high flexibility, including recently reported macroporous syringe-injectable mesh electronics, have shown substantial promise for chronic studies of neural circuitry in the brains of live animals. A central challenge for exploiting these highly flexible materials for in vivo studies has centered on the development of efficient input/output (I/O) connections to an external interface with high yield, low bonding resistance, and long-term stability. Here we report a new paradigm applied to the challenging case of injectable mesh electronics that exploits the high flexibility of nanoscale thickness two-sided metal I/O pads that can deform and contact standard interface cables in high yield with long-term electrical stability. First, we describe the design and facile fabrication of two-sided metal I/O pads that allow for contact without regard to probe orientation. Second, systematic studies of the contact resistance as a function of I/O pad design and mechanical properties demonstrate the key role of the I/O pad bending stiffness in achieving low-resistance stable contacts. Additionally, computational studies provide design rules for achieving high-yield multiplexed contact interfacing in the case of angular misalignment such that adjacent channels are not shorted. Third, the in vitro measurement of 32-channel mesh electronics probes bonded to interface cables using the direct contact method shows a reproducibly high yield of electrical connectivity. Finally, in vivo experiments with 32-channel mesh electronics probes implanted in live mice demonstrate the chronic stability of the direct contact interface, enabling consistent tracking of single-unit neural activity over at least 2 months without a loss of channel recording. The direct contact interfacing methodology paves the way for scalable long-term connections of multiplexed mesh electronics neural probes for neural recording and modulation and moreover could be used to facilitate a scalable interconnection of other flexible electronics in biological studies and therapeutic applications.

Advanced One- and Two-Dimensional Mesh Designs for Injectable Electronics.

The unique structure and mechanical properties of syringe-injectable mesh electronics have enabled seamless tissue integration and stable chronic recording of the activities of the same neurons on a year scale. Here, we report studies of a series of structural and mechanical mesh electronics design variations that allow injection using needles at least 4-fold smaller than those previously reported to minimize the footprint during injection of the electronics in soft matter and tissue. Characterization of new ultraflexible two-dimensional (2D) and one-dimensional (1D) probes has demonstrated reproducible injection of the newly developed mesh electronics designs via needles as small as 100 µm in inner diameter (ID) with reduced injection volumes. In vitro hydrogel and in vivo mouse brain studies have shown that ultraflexible 2D and 1D probes maintain their structural integrity and conformation post-injection after being transferred through the reduced diameter needles. In addition, analysis of the variation of the post-injection mesh cross sections suggests a smaller degree of tissue deformation and relaxation with decreasing needle diameters. The capability to implement rational design for mesh electronic probes that can be delivered via much smaller diameter needles should open up new opportunities for integration of electronics with tissue and soft matter in fundamental and translational studies.

Bioinspired neuron-like electronics.

As an important application of functional biomaterials, neural probes have contributed substantially to studying the brain. Bioinspired and biomimetic strategies have begun to be applied to the development of neural probes, although these and previous generations of probes have had structural and mechanical dissimilarities from their neuron targets that lead to neuronal loss, neuroinflammatory responses and measurement instabilities. Here, we present a bioinspired design for neural probes-neuron-like electronics (NeuE)-where the key building blocks mimic the subcellular structural features and mechanical properties of neurons. Full three-dimensional mapping of implanted NeuE-brain interfaces highlights the structural indistinguishability and intimate interpenetration of NeuE and neurons. Time-dependent histology and electrophysiology studies further reveal a structurally and functionally stable interface with the neuronal and glial networks shortly following implantation, thus opening opportunities for next-generation brain-machine interfaces. Finally, the NeuE subcellular structural features are shown to facilitate migration of endogenous neural progenitor cells, thus holding promise as an electrically active platform for transplantation-free regenerative medicine.

A method for single-neuron chronic recording from the retina in awake mice.

The retina, which processes visual information and sends it to the brain, is an excellent model for studying neural circuitry. It has been probed extensively ex vivo but has been refractory to chronic in vivo electrophysiology. We report a nonsurgical method to achieve chronically stable in vivo recordings from single retinal ganglion cells (RGCs) in awake mice. We developed a noncoaxial intravitreal injection scheme in which injected mesh electronics unrolls inside the eye and conformally coats the highly curved retina without compromising normal eye functions. The method allows 16-channel recordings from multiple types of RGCs with stable responses to visual stimuli for at least 2 weeks, and reveals circadian rhythms in RGC responses over multiple day/night cycles.

Tissue-like Neural Probes for Understanding and Modulating the Brain.

Electrophysiology tools have contributed substantially to understanding brain function, yet the capabilities of conventional electrophysiology probes have remained limited in key ways because of large structural and mechanical mismatches with respect to neural tissue. In this Perspective, we discuss how the general goal of probe design in biochemistry, that the probe or label have a minimal impact on the properties and function of the system being studied, can be realized by minimizing structural, mechanical, and topological differences between neural probes and brain tissue, thus leading to a new paradigm of tissue-like mesh electronics. The unique properties and capabilities of the tissue-like mesh electronics as well as future opportunities are summarized. First, we discuss the design of an ultraflexible and open mesh structure of electronics that is tissue-like and can be delivered in the brain via minimally invasive syringe injection like molecular and macromolecular pharmaceuticals. Second, we describe the unprecedented tissue healing without chronic immune response that leads to seamless three-dimensional integration with a natural distribution of neurons and other key cells through these tissue-like probes. These unique characteristics lead to unmatched stable long-term, multiplexed mapping and modulation of neural circuits at the single-neuron level on a year time scale. Last, we offer insights on several exciting future directions for the tissue-like electronics paradigm that capitalize on their unique properties to explore biochemical interactions and signaling in a "natural" brain environment.

Highly scalable multichannel mesh electronics for stable chronic brain electrophysiology.

Implantable electrical probes have led to advances in neuroscience, brain-machine interfaces, and treatment of neurological diseases, yet they remain limited in several key aspects. Ideally, an electrical probe should be capable of recording from large numbers of neurons across multiple local circuits and, importantly, allow stable tracking of the evolution of these neurons over the entire course of study. Silicon probes based on microfabrication can yield large-scale, high-density recording but face challenges of chronic gliosis and instability due to mechanical and structural mismatch with the brain. Ultraflexible mesh electronics, on the other hand, have demonstrated negligible chronic immune response and stable long-term brain monitoring at single-neuron level, although, to date, it has been limited to 16 channels. Here, we present a scalable scheme for highly multiplexed mesh electronics probes to bridge the gap between scalability and flexibility, where 32 to 128 channels per probe were implemented while the crucial brain-like structure and mechanics were maintained. Combining this mesh design with multisite injection, we demonstrate stable 128-channel local field potential and single-unit recordings from multiple brain regions in awake restrained mice over 4 mo. In addition, the newly integrated mesh is used to validate stable chronic recordings in freely behaving mice. This scalable scheme for mesh electronics together with demonstrated long-term stability represent important progress toward the realization of ideal implantable electrical probes allowing for mapping and tracking single-neuron level circuit changes associated with learning, aging, and neurodegenerative diseases.

Syringe-injectable mesh electronics integrate seamlessly with minimal chronic immune response in the brain.

Implantation of electrical probes into the brain has been central to both neuroscience research and biomedical applications, although conventional probes induce gliosis in surrounding tissue. We recently reported ultraflexible open mesh electronics implanted into rodent brains by syringe injection that exhibit promising chronic tissue response and recording stability. Here we report time-dependent histology studies of the mesh electronics/brain-tissue interface obtained from sections perpendicular and parallel to probe long axis, as well as studies of conventional flexible thin-film probes. Confocal fluorescence microscopy images of the perpendicular and parallel brain slices containing mesh electronics showed that the distribution of astrocytes, microglia, and neurons became uniform from 2-12 wk, whereas flexible thin-film probes yield a marked accumulation of astrocytes and microglia and decrease of neurons for the same period. Quantitative analyses of 4- and 12-wk data showed that the signals for neurons, axons, astrocytes, and microglia are nearly the same from the mesh electronics surface to the baseline far from the probes, in contrast to flexible polymer probes, which show decreases in neuron and increases in astrocyte and microglia signals. Notably, images of sagittal brain slices containing nearly the entire mesh electronics probe showed that the tissue interface was uniform and neurons and neurofilaments penetrated through the mesh by 3 mo postimplantation. The minimal immune response and seamless interface with brain tissue postimplantation achieved by ultraflexible open mesh electronics probes provide substantial advantages and could enable a wide range of opportunities for in vivo chronic recording and modulation of brain activity in the future.

Assessment of in vivo systemic toxicity and biodistribution of iron-doped silica nanoshells.

Silica nanoparticles are an emerging class of biomaterials which may be used as diagnostic and therapeutic tools for biomedical applications. In particular, hollow silica nanoshells are attractive due to their hollow core. Approximately 70% of a 500 nm nanoshell is hollow, therefore more particles can be administered on a mg/kg basis compared to solid nanoparticles. Additionally, their nanoporous shell permits influx/efflux of gases and small molecules. Since the size, shape, and composition of a nanoparticle can dramatically alter its toxicity and biodistribution, the toxicology of these nanomaterials was assessed. A single dose toxicity study was performed in vivo to assess the toxicity of 500 nm iron-doped silica nanoshells at clinically relevant doses of 10-20 mg/kg. This study showed that only a trace amount of silica was detected in the body 10 weeks post-administration. The hematology, biochemistry and pathological results show that the nanoshells exhibit no acute or chronic toxicity in mice.

Stable long-term chronic brain mapping at the single-neuron level.

Stable in vivo mapping and modulation of the same neurons and brain circuits over extended periods is critical to both neuroscience and medicine. Current electrical implants offer single-neuron spatiotemporal resolution but are limited by such factors as relative shear motion and chronic immune responses during long-term recording. To overcome these limitations, we developed a chronic in vivo recording and stimulation platform based on flexible mesh electronics, and we demonstrated stable multiplexed local field potentials and single-unit recordings in mouse brains for at least 8 months without probe repositioning. Properties of acquired signals suggest robust tracking of the same neurons over this period. This recording and stimulation platform allowed us to evoke stable single-neuron responses to chronic electrical stimulation and to carry out longitudinal studies of brain aging in freely behaving mice. Such advantages could open up future studies in mapping and modulating changes associated with learning, aging and neurodegenerative diseases.

Alkaline and ultrasonic dissolution of biological materials for trace silicon determination.

A simple method for trace elemental determination in biological tissue has been developed. Novel nanomaterials with biomedical applications necessitate the determination of the in vivo fate of the materials to understand their toxicological profile. Hollow iron-doped calcined silica nanoshells have been used as a model to demonstrate that potassium hydroxide and bath sonication at 50 °C can extract elements from alkaline-soluble nanomaterials. After alkali digestion, nitric acid is used to adjust the pH into a suitable range for analysis using techniques such as inductively coupled plasma optical emission spectrometry which require neutral or acidic analytes. In chicken liver phantoms injected with the nanoshells, 96% of the expected silicon concentration was detected. This value was in good agreement with the 94% detection efficiency of nanoshells dissolved in aqueous solution as a control for potential sample matrix interference. Nanoshell detection was further confirmed in a mouse 24 h after intravenous administration; the measured silica above baseline was 35 times greater or more than the standard deviations of the measurements. This method provides a simple and accurate means to quantify alkaline-soluble nanomaterials in biological tissue.

Hollow iron-silica nanoshells for enhanced high intensity focused ultrasound.

BACKGROUND: High intensity-focused ultrasound (HIFU) is an alterative ablative technique currently being investigated for local treatment of breast cancer and fibroadenomas. Current HIFU therapies require concurrent magnetic resonance imaging monitoring. Biodegradable 500 nm perfluoropentane-filled iron-silica nanoshells have been synthesized as a sensitizing agent for HIFU therapies, which aid both mechanical and thermal ablation of tissues. In low duty cycle high-intensity applications, rapid tissue damage occurs from mechanical rather than thermal effects, which can be monitored closely by ultrasound obviating the need for concurrent magnetic resonance imaging. MATERIALS AND METHODS: Iron-silica nanoshells were synthesized by a sol-gel method on polystyrene templates and calcined to yield hollow nanoshells. The nanoshells were filled with perfluoropentane and injected directly into excised human breast tumor, and intravenously (IV) into healthy rabbits and Py8119 tumor-bearing athymic nude mice. HIFU was applied at 1.1 MHz and 3.5 MPa at a 2% duty cycle to achieve mechanical ablation. RESULTS: Ex vivo in excised rabbit livers, the time to visually observable damage with HIFU was 20 s without nanoshells and only 2 s with nanoshells administered IV before sacrifice. Nanoshells administered IV into nude mice with xenograft tumors were activated in vivo by HIFU 24 h after administration. In this xenograft model, applied HIFU resulted in a 13.6 ± 6.1 mm(3) bubble cloud with the IV injected particles and no bubble cloud without particles. CONCLUSIONS: Iron-silica nanoshells can reduce the power and time to perform HIFU ablative therapy and can be monitored by ultrasound during low duty cycle operation.

Color Doppler ultrasound and gamma imaging of intratumorally injected 500 nm iron-silica nanoshells.

Perfluoropentane gas filled iron-silica nanoshells have been developed as stationary ultrasound contrast agents for marking tumors to guide surgical resection. It is critical to establish their long-term imaging efficacy, as well as biodistribution. This work shows that 500 nm Fe-SiO2 nanoshells can be imaged by color Doppler ultrasound over the course of 10 days in Py8119 tumor bearing mice. The 500 nm nonbiodegradable SiO2 and biodegradable Fe-SiO2 nanoshells were functionalized with diethylenetriamine pentaacetic acid (DTPA) ligand and radiolabeled with (111)In(3+) for biodistribution studies in nu/nu mice. The majority of radioactivity was detected in the liver and kidneys following intravenous (IV) administration of nanoshells to healthy animals. By contrast, after nanoshells were injected intratumorally, most of the radioactivity remained at the injection site; however, some nanoshells escaped into circulation and were distributed similarly as those given intravenously. For intratumoral delivery of nanoshells and IV delivery to healthy animals, little difference was seen between the biodistribution of SiO2 and biodegradable Fe-SiO2 nanoshells. However, when nanoshells were administered IV to tumor bearing mice, a significant increase was observed in liver accumulation of SiO2 nanoshells relative to biodegradable Fe-SiO2 nanoshells. Both SiO2 and Fe-SiO2 nanoshells accumulate passively in proportion to tumor mass, during intravenous delivery of nanoshells. This is the first report of the biodistribution following intratumoral injection of any biodegradable silica particle, as well as the first report demonstrating the utility of DTPA-(111)In labeling for studying silica nanoparticle biodistributions.