Advances in Nano Neuroscience: From Nanomaterials to Nanotools

During the last decades, neuroscientists have increasingly exploited a variety of artificial, de-novo synthesized materials with controlled nano-sized features. For instance, a renewed interest in the development of prostheses or neural interfaces was driven by the availability of novel nanomaterials that enabled the fabrication of implantable bioelectronics interfaces with reduced side effects and increased integration with the target biological tissue. The peculiar physical-chemical properties of nanomaterials have also contributed to the engineering of novel imaging devices toward sophisticated experimental settings, to smart fabricated scaffolds and microelectrodes, or other tools ultimately aimed at a better understanding of neural tissue functions. In this review, we focus on nanomaterials and specifically on carbon-based nanomaterials, such as carbon nanotubes (CNTs) and graphene. While these materials raise potential safety concerns, they represent a tremendous technological opportunity for the restoration of neuronal functions. We then describe nanotools such as nanowires and nano-modified MEA for high-performance electrophysiological recording and stimulation of neuronal electrical activity. We finally focus on the fabrication of three-dimensional synthetic nanostructures, used as substrates to interface biological cells and tissues in vitro and in vivo

Electrical characterization of organic monolayers at the nanoscale: a differential scanning conductive tip AFM investigation

2006/2007CT-AFM (Conductive Tip AFM) is commonly used for electrical characterization of organic and inorganic surface systems. Understanding electron transfer at the molecular level may lead to the development of molecular assemblies with unique properties and is of great importance for the advancement of both organic, molecular and bio-electronics. In this work we follow an approach to the study of Metal-molecule-Metal surface junctions that uses a combination of different AFM-based techniques. We first use nanografting to build nanopatches of the molecules of interest into a hosting reference self assembled monolayer (SAM) typically made of alkane and aromatic thiols. After the tip is changed to a conductive one, CT-AFM is used to characterize electrically the whole system recording, at the same time, the system topography. Some of the advantages of this approach are the possibility to build and study a wide range of different monolayers side-by-side on the same sample and the in-situ control of the quality both of the hosting monolayer and that of the grafted patches. Results will be presented on saturated and unsaturated thiols self-assembled and nanografted on Au(111) surfaces. We will also show a clear correlation between the contrast in current images and the quality of molecular packing inside the nanopatches.XX Ciclo197

Sculpting neurotransmission during synaptic development by 2D nanostructured interfaces

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BDNF impact on synaptic dynamics: extra or intracellular long-term release differently regulates cultured hippocampal synapses

Brain Derived Neurotrophic Factor (BDNF) signalling contributes to the formation, maturation and plasticity of Central Nervous System (CNS) synapses. Acute exposure of cultured brain circuits to BDNF leads to up-regulation of glutamatergic neuro-transmission, by the accurate tuning of pre and post synaptic features, leading to structural and functional synaptic changes. Chronic BDNF treatment has been comparatively less investigated, besides it may represent a therapeutic option to obtain rescue of post-injury alterations of synaptic networks. In this study we used a paradigm of BDNF long-term (4 days) incubation to assess in hippocampal post-natal neurons in culture, the ability of such a treatment to alter synapses. By patch clamp recordings we describe the augmented function of excitatory neurotransmission and we further explore by live imaging the presynaptic changes brought about by long-term BDNF. In our study, exogenous long-term BDNF exposure of post-natal neurons did not affect inhibitory neurotransmission. We further compare, by genetic manipulations of cultured neurons and BDNF release, intracellular overexpression of this neurotrophin at the same developmental age. We describe for the first-time differences in synaptic modulation by BDNF with respect to exogenous or intracellular release paradigms. Such a finding holds the potential of influencing the design of future therapeutic strategies

Quantification of Circulating Cancer Biomarkers via Sensitive Topographic Measurements on Single Binder Nanoarrays

Early detection of cancer plays a crucial role in disease prognosis. It requires the recognition and quantification of low amounts of specific molecular biomarkers, either free or transported inside nanovesicles, through the development of novel sensitive diagnostic technologies. In this context, we have developed a nanoarray platform for the noninvasive quantification of cancer biomarkers circulating in the bloodstream. The assay is based on molecular manipulation to create functional spots of surface-immobilized binders and differential topography measurements. It is label-free and requires just a single binder per antigen, and when it is implemented with fluorescence labeling/readout, it can be used for epitope mapping. As a benchmark, we focused on the plasma release of Her2 extracellular domain (ECD), a proposed biomarker for the progression of Her2-positive tumors and response to anticancer therapies. By employing robust, easily engineered camelid nanobodies as binders, we measured ECD-Her2 concentrations in the range of the actual clinical cutoff value for Her2-positive breast cancer. The specificity for Her2 detection was preserved when it was measured in parallel with other potential biomarkers, demonstrating a forthcoming implementation of this approach for multiplexing analysis. Prospectively, this nanorarray platform may be customized to allow for the detection of promising new classes of circulating biomarkers, such as exosomes and microvesicles

Myoblast adhesion, proliferation and differentiation on Human Elastin-Like Polypeptide (HELP) hydrogels

Purpose: The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cells morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimicking some properties of the native matrix protein; when employed as myoblasts adhesion substrates they stimulate in vitro myogenesis. Given the consequences that biophysical properties of extracellular matrix exert on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts viability and functions. Methods: We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa stretch present in the α2 chain of type IV collagen, containing two RGD motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer density as adhesion substrates for C2C12 cells, used as myoblasts model. Results: By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictates cell morphology, spreading, focal adhesions formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low serum-medium, and partially inhibited myogenic differentiation. Conclusions: In the whole, the results underline the potentiality of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis

Bidirectional Modulation of Neuronal Cells Electrical and Mechanical Properties Through Pristine and Functionalized Graphene Substrates

[Abstract] In recent years, the quest for surface modifications to promote neuronal cell interfacing and modulation has risen. This course is justified by the requirements of emerging technological and medical approaches attempting to effectively interact with central nervous system cells, as in the case of brain-machine interfaces or neuroprosthetic. In that regard, the remarkable cytocompatibility and ease of chemical functionalization characterizing surface-immobilized graphene-based nanomaterials (GBNs) make them increasingly appealing for these purposes. Here, we compared the (morpho)mechanical and functional adaptation of rat primary hippocampal neurons when interfaced with surfaces covered with pristine single-layer graphene (pSLG) and phenylacetic acid-functionalized single-layer graphene (fSLG). Our results confirmed the intrinsic ability of glass-supported single-layer graphene to boost neuronal activity highlighting, conversely, the downturn inducible by the surface insertion of phenylacetic acid moieties. fSLG-interfaced neurons showed a significant reduction in spontaneous postsynaptic currents (PSCs), coupled to reduced cell stiffness and altered focal adhesion organization compared to control samples. Overall, we have here demonstrated that graphene substrates, both pristine and functionalized, could be alternatively used to intrinsically promote or depress neuronal activity in primary hippocampal cultures.This work was funded by the European Union’s Horizon 2020 Research and Innovation Program under the Grant Agreements 785219 and 881603 of the Graphene Flagship. DS acknowledges the support of the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement no. 838902. MP as the recipient of the AXA Bionanotechnology Chair, is grateful to the AXA Research Fund for financial support. This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency-grant no. MDM-2017- 0720. AC thanks Xunta de Galicia for his research grant Atracción de Talento (no. ED431H 2020/17). GR acknowledges funding from RYC-2016-21412. HH acknowledges funding from Juan de la Cierva – Incorporación no. IJC-2018-037396-IXunta de Galicia; ED431H 2020/1

Distributed interfacing by nanoscale photodiodes enables single-neuron light activation and sensory enhancement in 3D spinal explants

Among emerging technologies developed to interface neuronal signaling, engineering electrodes at the nanoscale would yield more precise biodevices opening to progress in neural circuit investigations and to new therapeutic potential. Despite remarkable progress in miniature electronics for less invasive neurostimulation, most nano-enabled, optically triggered interfaces are demonstrated in cultured cells, which precludes the studies of natural neural circuits. We exploit here free-standing silicon-based nanoscale photodiodes to optically modulate single, identified neurons in mammalian spinal cord explants. With near-infrared light stimulation, we show that activating single excitatory or inhibitory neurons differently affects sensory circuits processing in the dorsal horn. We successfully functionalize nano-photodiodes to target single molecules, such as glutamate AMPA receptor subunits, thus enabling light activation of specific synaptic pathways. We conclude that nano-enabled neural interfaces can modulate selected sensory networks with low invasiveness. The use of nanoscale photodiodes can thus provide original perspective in linking neural activity to specific behavioral outcome

Three-dimensional printing as a cutting-edge, versatile and personalizable vascular stent manufacturing procedure:Toward tailor-made medical devices

Vascular stents (VS) have revolutionized the treatment of cardiovascular diseases, as evidenced by the fact that the implantation of VS in coronary artery disease (CAD) patients has become a routine, easily approachable surgical intervention for the treatment of stenosed blood vessels. Despite the evolution of VS throughout the years, more efficient approaches are still required to address the medical and scientific challenges, especially when it comes to peripheral artery disease (PAD). In this regard, three-dimensional (3D) printing is envisaged as a promising alternative to upgrade VS by optimizing the shape, dimensions and stent backbone (crucial for optimal mechanical properties), making them customizable for each patient and each stenosed lesion. Moreover, the combination of 3D printing with other methods could also upgrade the final device. This review focuses on the most recent studies using 3D printing techniques to produce VS, both by itself and in combination with other techniques. The final aim is to provide an overview of the possibilities and limitations of 3D printing in the manufacturing of VS. Furthermore, the current situation of CAD and PAD pathologies is also addressed, thus highlighting the main weaknesses of the already existing VS and identifying research gaps, possible market niches and future directions.This work was funded by the Basque Country Government/Eusko Jaurlaritza (Department of Education, University and Research, Consolidated Groups IT448- 22) . Sandra Ruiz-Alonso and Fouad Al -Hakim thank the Basque Country Government for the granted fellowships PRE_2021_2_0153 and PRE_2021_2_0181, respectively. Denis Scaini gratefully acknowledges support from IKERBASQUE, the Basque Foundation of Science

Functional rewiring across spinal injuries via biomimetic nanofiber scaffolds

The regrowth of severed axons is fundamental to reestablish motor control after spinal-cord injury (SCI). Ongoing efforts to promote axonal regeneration after SCI have involved multiple strategies that have been only partially successful. Our study introduces an artificial carbon-nanotube based scaffold that, once implanted in SCI rats, improves motor function recovery. Confocal microscopy analysis plus fiber tracking by magnetic resonance imaging and neurotracer labeling of long-distance corticospinal axons suggest that recovery might be partly attributable to successful crossing of the lesion site by regenerating fibers. Since manipulating SCI microenvironment properties, such as mechanical and electrical ones, may promote biological responses, we propose this artificial scaffold as a prototype to exploit the physics governing spinal regenerative plasticity