Valentina Marchesano

Mechanisms underlying toxicity induced by CdTe quantum dots determined in an invertebrate model organism

A systematic and thorough quantitative analysis of the in vivo effects of inorganic nanoparticles is extremely important for the design of functional nanomaterials for diagnostic and therapeutic applications, better understanding of their non-specificity toward tissues and cell types, and for assessments of their toxicity. This study was undertaken to examine the impact of CdTe quantum dots (QDs) on an invertebrate freshwater model organism, Hydra vulgaris, for assessment of long term toxicity effects. The continuous exposure of living polyps to sub-lethal doses of QDs caused time and dose dependent morphological damages more severe than Cd(2+) ions at the same concentrations, impaired both reproductive and regenerative capability, activated biochemical and molecular responses. Of remarkable interest, low QD doses, apparently not effective, caused early changes in the expression of general stress responsive and apoptotic genes. The occurrence of subtle genetic variations, in the absence of morphological damages, indicates the importance of genotoxicity studies for nanoparticle risk assessment. The versatility in morphological, cellular, biochemical and molecular responses renders Hydra a perfect model system for high-throughput screening of toxicological and ecotoxicological impact of nanomaterials on human and environmental health.

Imaging Inward and Outward Trafficking of Gold Nanoparticles in Whole Animals

Gold nanoparticles have emerged as novel safe and biocompatible tools for manifold applications, including biological imaging, clinical diagnostics, and therapeutics. The understanding of the mechanisms governing their interaction with living systems may help the design and development of new platforms for nanomedicine. Here we characterized the dynamics and kinetics of the events underlying the interaction of gold nanoparticles with a living organism, from the first interaction nanoparticle/cell membrane, to the intracellular trafficking and final extracellular clearance. By treating a simple water invertebrate (the cnidarian Hydra polyp) with functionalized gold nanoparticles, multiple inward and outward routes were imaged by ultrastructural analyses, including exosomes as novel undescribed carriers to shuttle the nanoparticles in and out the cells. From the time course imaging a highly dynamic picture emerged in which nanoparticles are rapidly internalized (from 30 min onward), recruited into vacuoles/endosome (24 h onward), which then fuse, compact and sort out the internalized material either to storage vacuoles or to late-endosome/lysosomes, determining almost complete clearance within 48 h from challenging. Beside classical routes, new portals of entry/exit were captured, including exosome-like structures as novel undescribed nanoparticle shuttles. The conservation of the endocytic/secretory machinery through evolution extends the value of our finding to mammalian systems providing dynamics and kinetics clues to take into account when designing nanomaterials to interface with biological entities.

Hymyc1 Downregulation Promotes Stem Cell Proliferation in Hydra vulgaris

Hydra is a unique model for studying the mechanisms underlying stem cell biology. The activity of the three stem cell lineages structuring its body constantly replenishes mature cells lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. In vertebrates, one of many genes that participate in regulating stem cell homeostasis is the protooncogene c-myc, which has been recently identified also in Hydra, and found expressed in the interstitial stem cell lineage. In the present paper, by developing a novel strategy of RNA interference-mediated gene silencing (RNAi) based on an enhanced uptake of small interfering RNAi (siRNA), we provide molecular and biological evidence for an unexpected function of the Hydra myc gene (Hymyc1) in the homeostasis of the interstitial stem cell lineage. We found that Hymyc1 inhibition impairs the balance between stem cell self renewal/differentiation, as shown by the accumulation of stem cell intermediate and terminal differentiation products in genetically interfered animals. The identical phenotype induced by the 10058-F4 inhibitor, a disruptor of c-Myc/Max dimerization, demonstrates the specificity of the RNAi approach. We show the kinetic and the reversible feature of Hymyc1 RNAi, together with the effects displayed on regenerating animals. Our results show the involvement of Hymyc1 in the control of interstitial stem cell dynamics, provide new clues to decipher the molecular control of the cell and tissue plasticity in Hydra, and also provide further insights into the complex myc network in higher organisms. The ability of Hydra cells to uptake double stranded RNA and to trigger a RNAi response lays the foundations of a comprehensive analysis of the RNAi response in Hydra allowing us to track back in the evolution and the origin of this process.

Effects of Lithium Niobate Polarization on Cell Adhesion and Morphology

Understanding how the interfacial effects influence cell adhesion and morphology is of fundamental interest for controlling function, growth, and movement of cells in vitro and in vivo. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells is investigated. The spontaneous polarization of LN has one of the largest known magnitudes at room temperature (∼78 μC/cm(2)), and its orientation can be patterned easily by an external voltage, this motivating highly the investigation of its interaction with cells. Immunofluorescence and migration assays show strong evidence that the surface polarity regulates the adhesion functions, with enhanced spreading of the cytoskeleton on the negative face. The results suggest the potential of LN as a platform for investigating the role of charges on cellular processes, thus favoring new strategies in fabricating those biocompatible constructs used for tissue engineering. In fact, the orientation of the high-magnitude polarization can be patterned easily and, in combination with piezoelectric, pyroelectric, and photorefractive properties, may open the route to more sophisticated charge templates for modulating the cell response.

Gold nanoprisms for photothermal cell ablation in vivo

AIM To develop new methodologies for selective cell ablation in a temporally and spatially precise fashion in model organisms. MATERIALS & METHODS living polyps (Hydra vulgaris) treated with gold nanoprisms were near-infrared (NIR) irradiated and the photothermal effects evaluated at whole-animal, cellular and molecular levels. RESULTS Nanoprisms showed good efficiency of internalization in living specimens, with no sign of toxicity; under NIR irradiation they induced cell death and the overexpression of the hsp70 gene. CONCLUSION gold nanoprisms could be employed as efficient heat mediators in model organisms, and NIR-triggered cell ablation may represent a new advanced tool to study cell function. Solving bioethical and economical issues, invertebrates may provide alternative models bridging the gap between cell research and preclinical studies of photothermal therapy.

Impact of Amorphous SiO2 Nanoparticles on a Living Organism: Morphological, Behavioral, and Molecular Biology Implications.

It is generally accepted that silica (SiO2) is not toxic. But the increasing use of silica nanoparticles (SiO2NPs) in many different industrial fields has prompted the careful investigation of their toxicity in biological systems. In this report, we describe the effects elicited by SiO2NPs on animal and cell physiology. Stable and monodisperse amorphous silica nanoparticles, 25 nM in diameter, were administered to living Hydra vulgaris (Cnidaria). The dose-related effects were defined by morphological and behavioral assays. The results revealed an all-or-nothing lethal toxicity with a rather high threshold (35 nM NPs) and a LT50 of 38 h. At sub lethal doses, the morphophysiological effects included: animal morphology alterations, paralysis of the gastric region, disorganization and depletion of tentacle specialized cells, increase of apoptotic and collapsed cells, and reduction of the epithelial cell proliferation rate. Transcriptome analysis (RNAseq) revealed 45 differentially expressed genes, mostly involved in stress response and cuticle renovation. Our results show that Hydra reacts to SiO2NPs, is able to rebalance the animal homeostasis up to a relatively high doses of SiO2NPs, and that the physiological modifications are transduced to gene expression modulation.

A New In Vivo Model System to Assess the Toxicity of Semiconductor Nanocrystals

In the emerging area of nanotechnology, a key issue is related to the potential impacts of the novel nanomaterials on the environment and human health, so that this technology can be used with minimal risk. Specifically designed to combine on a single structure multipurpose tags and properties, smart nanomaterials need a comprehensive characterization of both chemicophysical properties and adequate toxicological evaluation, which is a challenging endeavour; the in vitro toxicity assays that are often employed for nanotoxicity assessments do not accurately predict in vivo response. To overcome these limitations and to evaluate toxicity characteristics of cadmium telluride quantum dots in relation to surface coatings, we have employed the freshwater polyp Hydra vulgaris as a model system. We assessed in vivo acute and sublethal toxicity by scoring for alteration of morphological traits, population growth rates, and influence on the regenerative capabilities providing new investigation clues for nanotoxicology purposes.

Control of Wnt/β-Catenin Signaling Pathway in Vivo via Light Responsive Capsules.

The possibility to remotely manipulate intracellular pathways in single cells is among the current goals of biomedicine, demanding new strategies to control cell function and reprogramming cell fate upon external triggering. Optogenetics is one approach in this direction, allowing specific cell stimulation by external illumination. Here, we developed optical switchers of an ancient and highly conserved system controlling a variety of developmental and adult processes in all metazoans, from Hydra to mammals, the Wnt/β-catenin signaling pathway. An intracellular modulator of the Wnt pathway was enclosed into polyelectrolyte multilayer microcapsules engineered to include self-tracking (i.e., fluorescence labeling) and light mediated heating functionalities (i.e., plasmonic nanoparticles). Capsules were delivered in vivo to Hydra and NIR triggered drug release caused forced activation of the Wnt pathway. The possibility to remotely manipulate the Wnt pathway by optical switchers may be broadly translated to achieve spatiotemporal control of cell fate for new therapeutic strategies.

Impact of Carbon Nano-Onions on Hydra vulgaris as a Model Organism for Nanoecotoxicology

The toxicological effects of pristine and chemically modified carbon nano-onions (CNOs) on the development of the freshwater polyp Hydra vulgaris were investigated in order to elucidate the ecotoxicological effects of CNOs. Chemical modifications of the CNOs were accomplished by surface functionalization with benzoic acid, pyridine and pyridinium moieties. thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy confirmed the covalent surface functionalization of CNOs. Hydra specimens were exposed to the carbon nanomaterials by prolonged incubation within their medium. Uptake was monitored by optical microscopy, and the toxicological effects of the CNOs on Hydra behavior, morphology, as well as the long-term effects on the development and reproductive capability were examined. The obtained data revealed the absence of adverse effects of CNOs (in the range 0.05–0.1 mg/L) in vivo at the whole animal level. Together with previously performed in vitro toxicological analyses, our findings indicate the biosafety of CNOs and the feasibility of employing them as materials for biomedical applications.

Nanotoxicology using the sea anemone Nematostella vectensis: from developmental toxicity to genotoxicology

Abstract Concomitant with the fast-growing advances in the synthesis and engineering of colloidal nanocrystals, an urgent evaluation of their toxicity on human beings and environment is strongly encouraged by public health organisations. Despite the in vitro approaches employed for toxicological screening of hazardous compounds, the use of simple and cost-effective living organisms may enormously contribute to solve unanswered questions related to embryotoxic and teratogenic effects of nanomaterials. Here, the sea anemone Nematostella vectensis (Cnidaria, Anthozoa) is presented as a novel model organism to profile bio/non-bio interactions and to show a comprehensive toxicological analysis performed on embryos, larvae and adults treated with fluorescent cadmium-based nanocrystals. Spanning from in vivo biodistribution to molecular investigations, different behaviours and effects depending on the composition and surface coatings are showed. Rod-shaped cadmium selenide/cadmium sulfide (CdSe/CdS) nanocrystals resulted in excellent imaging probes to track N. vectensis development with negligible adverse effects, while spherical CdTe nanocrystals severely impaired embryogenesis, resulting in aberrant phenotypes and deregulation of developmental genes, which raise severe worries for a safe use of this type of nanoparticles for human purposes and environmental contamination.