Passive Recording of Bioelectrical Signals from Non-Excitable Cells by Fluorescent Mirroring.

Bioelectrical variations trigger different cell responses, including migration, mitosis, and mutation. At the tissue level, these actions result in phenomena such as wound healing, proliferation, and pathogenesis. Monitoring these mechanisms dynamically is highly desirable in diagnostics and drug testing. However, existing technologies are invasive: either they require physical access to the intracellular compartments, or they imply direct contact with the cellular medium. Here, we present a novel approach for the passive recording of electrical signals from non-excitable cells adhering to 3D microelectrodes, based on optical mirroring. Preliminary results yielded a fluorescence intensity output increase of the 5,8% in the presence of a HEK-293 cell on the electrode compared to bare microelectrodes. At present, this technology may be employed to evaluate cell-substrate adhesion and monitor cell proliferation. Further refinements could allow extrapolating quantitative data on surface charges and resting potential to investigate the electrical phenomena involved in cell migration and cancer progression.

The Central Noradrenergic System in Neurodevelopmental Disorders: Merging Experimental and Clinical Evidence.

The aim of this article is to highlight the potential role of the locus-coeruleus-noradrenergic (LC-NA) system in neurodevelopmental disorders (NdDs). The LC is the main brain noradrenergic nucleus, key in the regulation of arousal, attention, and stress response, and its early maturation and sensitivity to perinatal damage make it an interesting target for translational research. Clinical data shows the involvement of the LC-NA system in several NdDs, suggesting a pathogenetic role in the development of such disorders. In this context, a new neuroimaging tool, LC Magnetic Resonance Imaging (MRI), has been developed to visualize the LC in vivo and assess its integrity, which could be a valuable tool for exploring morphological alterations in NdD in vivo in humans. New animal models may be used to test the contribution of the LC-NA system to the pathogenic pathways of NdD and to evaluate the efficacy of NA-targeting drugs. In this narrative review, we provide an overview of how the LC-NA system may represent a common pathophysiological and pathogenic mechanism in NdD and a reliable target for symptomatic and disease-modifying drugs. Further research is needed to fully understand the interplay between the LC-NA system and NdD.

Zebrafish as an Innovative Tool for Epilepsy Modeling: State of the Art and Potential Future Directions.

This article discusses the potential of Zebrafish (ZF) (Danio Rerio), as a model for epilepsy research. Epilepsy is a neurological disorder affecting both children and adults, and many aspects of this disease are still poorly understood. In vivo and in vitro models derived from rodents are the most widely used for studying both epilepsy pathophysiology and novel drug treatments. However, researchers have recently obtained several valuable insights into these two fields of investigation by studying ZF. Despite the relatively simple brain structure of these animals, researchers can collect large amounts of data in a much shorter period and at lower costs compared to classical rodent models. This is particularly useful when a large number of candidate antiseizure drugs need to be screened, and ethical issues are minimized. In ZF, seizures have been induced through a variety of chemoconvulsants, primarily pentylenetetrazol (PTZ), kainic acid (KA), and pilocarpine. Furthermore, ZF can be easily genetically modified to test specific aspects of monogenic forms of human epilepsy, as well as to discover potential convulsive phenotypes in monogenic mutants. The article reports on the state-of-the-art and potential new fields of application of ZF research, including its potential role in revealing epileptogenic mechanisms, rather than merely assessing iatrogenic acute seizure modulation.

Toxicological Profile of Plasmonic Nanoparticles in Zebrafish Model.

Plasmonic nanoparticles are increasingly employed in several fields, thanks to their unique, promising properties. In particular, these particles exhibit a surface plasmon resonance combined with outstanding absorption and scattering properties. They are also easy to synthesize and functionalize, making them ideal for nanotechnology applications. However, the physicochemical properties of these nanoparticles can make them potentially toxic, even if their bulk metallic forms are almost inert. In this review, we aim to provide a more comprehensive understanding of the potential adverse effects of plasmonic nanoparticles in zebrafish (Danio rerio) during both development and adulthood, focusing our attention on the most common materials used, i.e., gold and silver.

PEGylated gold nanorods as optical trackers for biomedical applications: an in vivo and in vitro comparative study.

Gold nanorods (AuNRs) are eligible for a variety of biological applications including cell imaging, sensing, and photothermal therapy thanks to their optical properties. The aim of this work is to show how AuNRs could be employed as non-photobleachable optical contrast agents for biomedical applications. In order to demonstrate the feasibility of their use as optical trackers, we employed two-photon emission confocal microscopy on cells incubated with PEGylated AuNRs. Remarkably, AuNRs were localized mostly in the perinuclear zone and microscopy characterization showed the presence of a considerable number of rods inside cell nuclei. Furthermore, we estimated the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs as a function of administered dose on HeLa/3T3 cell lines and on zebrafish during development, employed as an in vivo model. Eventually, we observed good agreement between in vivo and in vitro experiments. The employed AuNRs were prepared through a photochemical protocol here improved by tuning the amount of the cationic surfactant cetyltrimethylammonium bromide for the achievement of AuNRs at two different aspect ratios. Furthermore we also investigated if the AuNR aspect ratio influenced the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs in HeLa/3T3 cell lines and in zebrafish embryos.

Gated CW-STED microscopy: a versatile tool for biological nanometer scale investigation.

Stimulation emission depletion (STED) microscopy breaks the spatial resolution limit of conventional light microscopy while retaining its major advantages, such as working under physiological conditions. These properties make STED microscopy a perfect tool for investigating dynamic sub-cellular processes in living organisms. However, up to now, the massive dissemination of STED microscopy has been hindered by the complexity and cost of its implementation. Gated CW-STED (gCW-STED) substantially helps solve this problem without sacrificing spatial resolution. Here, we describe a versatile gCW-STED microscope able to speedily image the specimen, at a resolution below 50 nm, with light intensities comparable to the more complicated all-pulsed STED system. We show this ability on calibration samples as well as on biological samples.

New insights into the neuroanatomical distribution and phylogeny of opioids and POMC-derived peptides in fish.

This review re-evaluates the use of immunological probes to map enkephalinergic, dynorphinergic, and endorphinergic circuits in the CNS of lobe-finned fishes, ray-finned fishes, and cartilaginous fishes in light of the characterization of proenkephalin, prodynorphin, and POMC sequences from representatives of these groups of fish over the past 20 years. The use of α-MSH specific antisera is a reliable method for detecting POMC immunopositive cell bodies and fibers. Since α-MSH and ß-endorphin are co-localized in the same neurons, these studies also reveal the distribution of endorphinergic networks. Met-enkephalin specific antisera can be used to detect enkephalinergic circuits in the CNS of gnathostomes because of the ubiquitous presence of this pentapeptide in the proenkephalin sequences of gnathostomes. However, the use of leu-enkephalin specific antisera to detect enkephalinergic networks is more problematic. While this immunological probe is appropriate for analyzing enkephalinergic networks in mammals and perhaps teleosts, for the lungfishes and cartilaginous fishes this probe is more likely able to detect dynorphinergic circuits. In this regard, there is a need to re-examine dynorphinergic networks in non-mammalian gnathostomes by using species specific antisera directed against dynorphin end-products.

Effects of Metal Oxide Nanoparticles in Zebrafish.

Metal oxide nanoparticles (MO NPs) are increasingly employed in many fields with a wide range of applications from industries to drug delivery. Due to their semiconducting properties, metal oxide nanoparticles are commonly used in the manufacturing of several commercial products available in the market, including cosmetics, food additives, textile, paint, and antibacterial ointments. The use of metallic oxide nanoparticles for medical and cosmetic purposes leads to unavoidable human exposure, requiring a proper knowledge of their potentially harmful effects. This review offers a comprehensive overview of the possible toxicity of metallic oxide nanoparticles in zebrafish during both adulthood and growth stages, with an emphasis on the role of oxidative stress.

In Vitro and In Vivo Biocompatibility of Boron/Nitrogen Co-Doped Carbon Nano-Onions.

Boron/nitrogen, co-doped, carbon nano-onions (BN-CNOs) have recently shown great promise as catalysts for the oxygen reduction reaction, due to the improved electronic properties imparted by the dopant atoms; however, the interactions of BN-CNOs with biological systems have not yet been explored. In this study, we examined the toxicological profiles of BN-CNOs and oxidized BN-CNOs (oxi-BN-CNOs) in vitro in both healthy and cancer cell lines, as well as on the embryonic stages of zebrafish (Danio rerio) in vivo. The cell viabilities of both cell lines cells were not affected after treatment with different concentrations of both doped CNO derivatives. On the other hand, the analysis of BN-CNOs and oxidized BN-CNO interactions with zebrafish embryos did not report any kind of perturbations, in agreement with the in vitro results. Our results show that both doped CNO derivatives possess a high biocompatibility and biosafety in cells and more complex systems.

Graphene-Like Layers from Carbon Black: In Vivo Toxicity Assessment.

Graphene-like (GL) layers, a new graphene-related material (GRM), possess peculiar chemical, colloidal, optical and transport properties. Considering the very recent promising application of GL layers in biomedical and bioelectronic fields, it is of utmost importance to investigate the toxicological profile of these nanomaterials. This study represents an important first report of a complete in vivo toxicity assessment of GL layers on embryonic zebrafish (Danio rerio). Our results show that GL layers do not lead to any perturbations in the different biological parameters evaluated, indicating their good biocompatibility on a vertebrate model. The new insight into the biosafety of GL layers will expand their applications in nanomedicine.

Modulation of Efficient Diiodo-BODIPY in vitro Phototoxicity to Cancer Cells by Carbon Nano-Onions.

Photodynamic therapy is currently one of the most promising approaches for targeted cancer treatment. It is based on responses of vital physiological signals, namely, reactive oxygen species (ROS), which are associated with diseased condition development, such as tumors. This study presents the synthesis, incorporation, and application of a diiodo-BODIPY-based photosensitizer, based on a non-covalent functionalization of carbon nano-onions (CNOs). In vitro assays demonstrate that HeLa cells internalize the diiodo-BODIPY molecules and their CNO nanohybrids. Upon cell internalization and light exposure, the pyrene-diiodo-BODIPY molecules induce an increase of the ROS level of HeLa cells, resulting in remarkable photomediated cytotoxicity and apoptosis. Conversely, when HeLa cells internalize the diiodo-BODIPY/CNO nanohybrids, no significant cytotoxicity or ROS basal level increase can be detected. These results define a first step toward the understanding of carbon nanomaterials that function as molecular shuttles for photodynamic therapeutics, boosting the modulation of the photosensitizer.

Supramolecular functionalization of carbon nano-onions with hyaluronic acid-phospholipid conjugates for selective targeting of cancer cells.

Carbon nano-onions (CNOs) are promising materials for biomedical applications due to their low cytotoxicity and excellent biocompatibility. Supramolecular functionalization with biocompatible polymers is an effective strategy to develop engineered drug carriers for targeted delivery applications. In this study, we report the use of a hyaluronic acid-phospholipid (HA-DMPE) conjugate to target CD44 overexpressing cancer cells, while enhancing solubility of the nanoconstruct. Non-covalently functionalized CNOs with HA-DMPE show excellent in vitro cell viability in human breast carcinoma cells overexpressing CD44 and are uptaken to a greater extent compared to human ovarian carcinoma cells with an undetectable amount of CD44. In addition, they possess high in vivo biocompatibility in zebrafish (Danio Rerio) during the different stages of development and they prevalently localize in the digestive tract of the zebrafish larvae.

Toxicological profile of calcium carbonate nanoparticles for industrial applications.

Calcium carbonate nanoparticles (CaCO3NPs) derived from CO2 are promising materials for different industrial applications. It is imperative to understand their toxicological profile in biological systems as the human and environmental exposures to CaCO3NPs increases with growing production. Here, we analyse the cytotoxicity of CaCO3NPs synthesized from a CaO slurry on two cell lines, and in vivo on zebrafish (Danio Rerio). Our results demonstrate the CaCO3NPs in vitro safety as they do not cause cell death or genotoxicity. Moreover, zebrafish treated with CaCO3NPs develop without any abnormalities, confirming the safety and biocompatibility of this nanomaterial.

Laser-Fabricated Fluorescent, Ligand-Free Silicon Nanoparticles: Scale-up, Biosafety, and 3D Live Imaging of Zebrafish under Development.

This work rationalizes the scalable synthesis of ultrasmall, ligand-free silicon nanomaterials via liquid-phase pulsed laser ablation process using picosecond pulses at ultraviolet wavelengths. Results showed that the irradiation time drives hydrodynamic NP size. Isolated, monodisperse Si-NPs are obtained at high yield (72%) using post-treatment process. The obtained Si-NPs have an average size of ∼10 nm (not aggregated) and display photoemission in the green spectral range. We directly characterized the ligand-free Si-NPs in a vertebrate animal (zebrafish) and assessed their toxicity during the development. In vivo assay revealed that Si-NPs are found inside in all the early life stages of embryos and larvae growth, showing that the biosafety of Si-NPs and malformation types are independent of the Si-NP dose. Si-NPs were directly imaged inside developing embryos by spinning disk-imaging technique with optical sectioning capability. We showed that Si-NPs can passively enter inside embryos by the pore canals of chorion, can diffuse in the circulatory system, i.e., blood vessel, and accumulate inside larvae midgut and yolk sac, and in the eye lens, indicating the crossing of the blood barrier.

Carbon Nano-Onions as Non-Cytotoxic Carriers for Cellular Uptake of Glycopeptides and Proteins.

Carbon nano-onions (CNOs) possess favorable properties that make them suitable for biomedical applications, including their small size, ready surface modification, and good biocompatibility. Here, we report the covalent immobilization of a synthetic glycopeptide and the protein bovine serum albumin (BSA) onto the surface of carbon nano-onions using the maleimide-thiol "addition reaction". The glycopeptide and BSA are readily transported inside different cell lines, together with carbon nano-onions, through the endocytosis pathway. Our results show that carbon nano-onions are excellent scaffolds for glycopeptides and proteins immobilization and act as intracellular carriers for these biomolecules. These findings open new perspectives in the application of carbon nano-onions as intracellular transporters in diverse biomedical applications.

Zebrafish structural development in Mueller-matrix scanning microscopy.

Zebrafish are powerful animal models for understanding biological processes and the molecular mechanisms involved in different human diseases. Advanced optical techniques based on fluorescence microscopy have become the main imaging method to characterize the development of these organisms at the microscopic level. However, the need for fluorescence probes and the consequent high light doses required to excite fluorophores can affect the biological process under observation including modification of metabolic function or phototoxicity. Here, without using any labels, we propose an implementation of a Mueller-matrix polarimeter into a commercial optical scanning microscope to characterize the polarimetric transformation of zebrafish preserved at different embryonic developmental stages. By combining the full polarimetric measurements with statistical analysis of the Lu and Chipman mathematical decomposition, we demonstrate that it is possible to quantify the structural changes of the biological organization of fixed zebrafish embryos and larvae at the cellular scale. This convenient implementation, with low light intensity requirement and cheap price, coupled with the quantitative nature of Mueller-matrix formalism, can pave the way for a better understanding of developmental biology, in which label-free techniques become a standard tool to study organisms.

The Utility of Zebrafish as a Model for Screening Developmental Neurotoxicity.

The developing central nervous system and the blood brain barrier are especially vulnerable and sensitive to different chemicals, including environmental contaminants and drugs. Developmental exposure to these compounds has been involved in several neurological disorders, such as autism spectrum disorders as well as Alzheimer's and Parkinson's diseases. Zebrafish (Danio Rerio) have emerged as powerful toxicological model systems that can speed up chemical hazard assessment and can be used to extrapolate neurotoxic effects that chemicals have on humans. Zebrafish embryos and larvae are convenient for high-throughput screening of chemicals, due to their small size, low-cost, easy husbandry, and transparency. Additionally, zebrafish are homologous to other higher order vertebrates in terms of molecular signaling processes, genetic compositions, and tissue/organ structures as well as neurodevelopment. This mini review underlines the potential of the zebrafish as complementary models for developmental neurotoxicity screening of chemicals and describes the different endpoints utilized for such screening with some studies illustrating their use.

Biodistribution and biocompatibility of passion fruit-like nano-architectures in zebrafish.

Passion fruit-like nano-architectures (NAs) are all-in-one platforms of increasing interest for the translation of metal nanoparticles into clinics. NAs are nature-inspired disassembling inorganic theranostics, which jointly combine most of the appealing behaviors of noble metal nanoparticles with their potential organism excretion. Despite their unique and promising properties, NAs in vivo interactions and potential adverse effects have not yet been investigated. In this study, we employ zebrafish (Danio Rerio) to assess the development toxicity of NAs as well as their uptake and bioaccumulation at different stages of growth. The evaluation of multiple endpoints related to the toxicity clearly indicates that NAs do not induce mortality, developmental defects, or alterations on the hatching rate and behavior of zebrafish. Moreover, the analysis of nanostructures uptake and biodistribution demonstrates that NAs are successfully internalized and present a specific localization. Overall, our results demonstrate that NAs are able to pass through the embryos chorion and accumulate in specific tissues, exhibiting an impressive biocompatibility.

Fluorescent single-digit detonation nanodiamond for biomedical applications.

Detonation nanodiamonds (DNDs) have emerged as promising candidates for a variety of biomedical applications, thanks to different physicochemical and biological properties, such as small size and reactive surfaces. In this study, we propose carbon dot decorated single digit (4-5 nm diameter) primary particles of detonation nanodiamond as promising fluorescent probes. Due to their intrinsic fluorescence originating from tiny (1-2 atomic layer thickness) carbonaceous structures on their surfaces, they exhibit brightness suitable for in vitro imaging. Moreover, this material offers a unique, cost effective alternative to sub-10 nm nanodiamonds containing fluorescent nitrogen-vacancy color centers, which have not yet been produced at large scale. In this paper, carbon dot decorated nanodiamonds are characterized by several analytical techniques. In addition, the efficient cellular uptake and fluorescence of these particles are observed in vitro on MDA-MD-231 breast cancer cells by means of confocal imaging. Finally, the in vivo biocompatibility of carbon dot decorated nanodiamonds is demonstrated in zebrafish during the development. Our results indicate the potential of single-digit detonation nanodiamonds as biocompatible fluorescent probes. This unique material will find application in correlative light and electron microscopy, where small sized NDs can be attached to antibodies to act as a suitable dual marker for intracellular correlative microscopy of biomolecules.

Carbon nano-onions as fluorescent on/off modulated nanoprobes for diagnostics.

Multishell fullerenes, known as carbon nano-onions (CNOs), have emerged as a platform for bioimaging because of their cell-penetration properties and minimal systemic toxicity. Here, we describe the covalent functionalization of CNOs with a π-extended distyryl-substituted boron dipyrromethene (BODIPY) dye with on/off modulated fluorescence emission activated by an acidic environment. The switching properties are linked to the photoinduced electron transfer (PET) characteristics of the dimethylamino functionalities attached to the BODIPY core. The on/off emission of the fluorescent CNOs is fast and reversible both in solution and in vitro, making this nanomaterial suitable as pH-dependent probes for diagnostic applications.