Optical Control of Tissue Regeneration through Photostimulation of Organic Semiconducting Nanoparticles.

Next generation bioengineering strives to identify crucial cues that trigger regeneration of damaged tissues, and to control the cells that execute these programs with biomaterials and devices. Molecular and biophysical mechanisms driving embryogenesis may inspire novel tools to reactivate developmental programs in situ. Here nanoparticles based on conjugated polymers are employed for optical control of regenerating tissues by using an animal with unlimited regenerative potential, the polyp Hydra, as in vivo model, and human keratinocytes as an in vitro model to investigate skin repair. By integrating animal, cellular, molecular, and biochemical approaches, nanoparticles based on poly-3-hexylthiophene (P3HT) are shown able to enhance regeneration kinetics, stem cell proliferation, and biomolecule oxidation levels. Opposite outputs are obtained with PCPDTBT-NPs (Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b'] dithiophene)-alt-4,7(2,1,3-benzothiadiazole)], causing a beneficial effect on Hydra regeneration but not on the migratory capability of keratinocytes. These results suggest that the artificial modulation of the redox potential in injured tissues may represent a powerful modality to control their regenerative potential. Importantly, the possibility to fine-tuning materials' photocatalytic efficiency may enable a biphasic modulation over a wide dynamic range, which can be exploited to augment the tissue regenerative capacity or inhibit the unlimited potential of cancerous cells in pathological contexts.

The Aquatic Invertebrate Hydra vulgaris Releases Molecular Messages Through Extracellular Vesicles.

Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offers protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e., mediating the population's response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype, from structural proteins to stress induced messages promoting cell survival. Moreover, positive and negative regulators of the Wnt/ß-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to modulate both head and foot regeneration, suggesting bioactivity of the EV cargo and opening new perspectives on the mechanisms of developmental signalling. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems.

In Vivo Bioengineering of Fluorescent Conductive Protein-Dye Microfibers.

Engineering protein-based biomaterials is extremely challenging in bioelectronics, medicine, and materials science, as mechanical, electrical, and optical properties need to be merged to biocompatibility and resistance to biodegradation. An effective strategy is the engineering of physiological processes in situ, by addition of new properties to endogenous components. Here we show that a green fluorescent semiconducting thiophene dye, DTTO, promotes, in vivo, the biogenesis of fluorescent conductive protein microfibers via metabolic pathways. By challenging the simple freshwater polyp Hydra vulgaris with DTTO, we demonstrate the stable incorporation of the dye into supramolecular protein-dye co-assembled microfibers without signs of toxicity. An integrated multilevel analysis including morphological, optical, spectroscopical, and electrical characterization shows electrical conductivity of biofibers, opening the door to new opportunities for augmenting electronic functionalities within living tissue, which may be exploited for the regulation of cell and animal physiology, or in pathological contexts to enhance bioelectrical signaling.

Gold Nanorods and Nanoprisms Mediate Different Photothermal Cell Death Mechanisms In Vitro and In Vivo.

Photothermal therapy (PTT) is an efficient method of inducing localized hyperthermia and can be achieved using gold nanoparticles as photothermal agents. However, there are many hurdles to get over before this therapy can safely reach the clinics, including nanoparticles' optimal shape and the accurate prediction of cellular responses. Here, we describe the synthesis of gold nanorods and nanoprisms with similar surface plasmon resonances in the near-infrared (NIR) and comparable photothermal conversion efficiencies and characterize the response to NIR irradiation in two biological systems, melanoma cells and the small invertebrate Hydra vulgaris. By integrating animal, cellular, and molecular biology approaches, we show a diverse outcome of nanorods and nanoprisms on the two systems, sustained by the elicitation of different pathways, from necrosis to programmed cell death mechanisms (apoptosis and necroptosis). The comparative multilevel analysis shows great accuracy of in vivo invertebrate models to predict overall responses to photothermal challenging and superior photothermal performance of nanoprisms. Understanding the molecular pathways of these responses may help develop optimized nanoheaters that, safe by design, may improve PTT efficacy for clinical purposes.

An Integrated Multilevel Analysis Profiling Biosafety and Toxicity Induced by Indium- and Cadmium-Based Quantum Dots in Vivo.

Indium phosphide quantum dots (QDs) have emerged as a new class of fluorescent nanocrystals for manifold applications, from biophotonics to nanomedicine. Recent efforts in improving the photoluminescence quantum yield, the chemical stability and the biocompatibility turned them into a valid alternative to well established Cd-based nanocrystals. In vitro studies provided first evidence for the lower toxicity of In-based QDs. Nonetheless, an urgent need exists for further assessment of the potential toxic effects in vivo. Here we use the freshwater polyp Hydra vulgaris, a well-established model previously adopted to assess the toxicity of CdSe/CdS nanorods and CdTe QDs. A systematic multilevel analysis was carried out in vivo, ex vivo, and in vitro comparing toxicity end points of CdSe- and InP-based QDs, passivated by ZnSe/ZnS shells and surface functionalized with penicillamine. Final results demonstrate that both the chemical composition of the QD core (InP vs CdSe) and the shell play a crucial role for final outcomes. Remarkably, in absence of in vivo alterations, cell and molecular alterations revealed hidden toxicity aspects, highlighting the biosafety of InP-based nanocrystals and outlining the importance of integrated multilevel analyses for proper QDs risk assessment.

In Vivo Toxicity Assessment of Hybrid Diatomite Nanovectors Using Hydra vulgaris as a Model System.

Drug nanocarriers based on nanostructured materials are very promising for precision and personalized medicine applications. Diatomite porous biosilica has been recently proposed as a novel and effective material in formulations of drug systems for oral and systemic delivery. In this paper, the cytotoxicity of hybrid diatomite silica functionalized nanovectors is assessed in vivo in a living model organism, the cnidarian freshwater polyp Hydra vulgaris. Hydra specimens are exposed to modified diatomite nanoparticles by prolonged incubation within their medium. Uptake and toxicological effects on Hydra are examined from viability and genetic points of view. High concentrations, up to 3.5 g L-1 for 72 h, of diatomite modified nanoparticles do not affect Hydra morphology nor do growth rate and the genetic analysis confirm the biosafety of this material, opening the way to new applications in nanomedicine.

Real time dynamics of ß-catenin expression during Hydra development, regeneration and Wnt signalling activation.

Understanding the dynamic cellular behaviours driving morphogenesis and regeneration is a long-standing challenge in biology. Live imaging, together with genetically encoded reporters, may provide the necessary tool to address this issue, permitting the in vivo monitoring of the spatial and temporal expression dynamics of a gene of interest during a variety of developmental processes. Canonical Wnt/ß-catenin signalling controls a plethora of cellular activities during development, regeneration and adulthood throughout the animal kingdom. Several reporters have been produced in animal models to reveal sites of active Wnt signalling. In order to monitor in vivo Wnt/ß-catenin signalling activity in the freshwater polyp Hydra vulgaris, we generated a ß-cat-eGFP transgenic Hydra, in which eGFP is driven by the Hydra ß-catenin promoter. We characterized the expression dynamics during budding, regeneration and chemical activation of the Wnt/ß-cat signalling pathway using light sheet fluorescence microscopy. Live imaging of the ß-cat-eGFP lines recapitulated the previously reported endogenous expression pattern of ß-catenin and revealed the dynamic appearance of novel sites of Wnt/ß-catenin signalling, that earlier evaded detection by mean of in situ hybridization. By combining the Wnt activity read-out efficiency of the ß-catenin promoter with advanced imaging, we have created a novel model system to monitor in real time the activity of Hydra ß-cat regulatory sequences in vivo, and open the path to reveal ß-catenin modulation in many other physiological contexts.

Dissecting common and divergent molecular pathways elicited by CdSe/ZnS quantum dots in freshwater and marine sentinel invertebrates.

Water ecosystems represent main targets of unintentional contamination of nanomaterials, due to industrial waste or other anthropogenic activities. Nanoparticle insult to living organisms may occur in a sequential way, first by chemical interactions of the material with the target membrane, then by progressive internalisation and interaction with cellular structures and organelles. These events trigger a signal transduction, through which cells modulate molecular pathway in order to respond and survive to the external elicitation. Therefore, the analysis of the global changes of the molecular machinery, possibly induced in an organism upon exposure to a given nanomaterial, may provide unique clues for proper and exhaustive risk assessment. Here, we tested the impact of core/shell CdSe/ZnS QDs coated by a positively charged polymer on two aquatic species, the polyp Hydra vulgaris and the coral S. pistillata, representative of freshwater and sea habitats, respectively. By using reliable approaches based on animal behaviour and physiology together with a whole transcriptomic profiling, we determined several toxicity endpoints. Despite the difference in the efficiency of uptake, both species were severely affected by QD treatment, resulting in dramatic morphological damages and tissue bleaching. Global transcriptional changes were also detected in both organisms, but presenting different temporal dynamics, suggesting both common and divergent functional responses in the two sentinel organisms. Due to the striking conservation of structure and genomic organisation among animals throughout evolution, our expression profiling offers new clues to identify novel molecular markers and pathways for comparative transcriptomics of nanotoxicity.

Semiconducting polymers are light nanotransducers in eyeless animals.

Current implant technology uses electrical signals at the electrode-neural interface. This rather invasive approach presents important issues in terms of performance, tolerability, and overall safety of the implants. Inducing light sensitivity in living organisms is an alternative method that provides groundbreaking opportunities in neuroscience. Optogenetics is a spectacular demonstration of this, yet is limited by the viral transfection of exogenous genetic material. We propose a nongenetic approach toward light control of biological functions in living animals. We show that nanoparticles based on poly(3-hexylthiophene) can be internalized in eyeless freshwater polyps and are fully biocompatible. Under light, the nanoparticles modify the light response of the animals, at two different levels: (i) they enhance the contraction events of the animal body, and (ii) they change the transcriptional activation of the opsin3-like gene. This suggests the establishment of a seamless and biomimetic interface between the living organism and the polymer nanoparticles that behave as light nanotransducers, coping with or amplifying the function of primitive photoreceptors.

Deciphering intracellular events triggered by mild magnetic hyperthermia in vitro and in vivo.

AIM: To assess the cell response to magnetic nanoparticles under an alternating magnetic field by molecular quantification of heat responsive transcripts in two model systems. MATERIALS & METHODS: Melanoma cells and Hydra vulgaris treated with magnetic nanoparticles were subjected to an alternating magnetic field or to macroscopic heating. Effect to these treatments were assessed at animal, cellular and molecular levels. RESULTS: By comparing hsp70 expression following both treatments, thermotolerance pathways were found in both systems in absence of cell ablation or global temperature increment. CONCLUSION: Analysis of hsp70 transcriptional activation can be used as molecular thermometer to sense cells' response to magnetic hyperthermia. Similar responses were found in cells and Hydra, suggesting a general mechanism to the delivery of sublethal thermal doses.

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.

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.

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.

Multiple functionalization of fluorescent nanoparticles for specific biolabeling and drug delivery of dopamine.

The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed hydrolysis by esterases, yielding a smart nanotool for specific biolabeling and controlled drug release.

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.

Synthesis and biological assay of GSH functionalized fluorescent quantum dots for staining Hydra vulgaris.

Quantum dots (QDs) have been used extensively as fluorescent markers in several studies on living cells. Here, we report the synthesis of conjugates based on glutathione (GSH) and QDs (GSH-QDs) and we prove how these functionalized fluorescent probes can be used for staining a freshwater invertebrate called Hydra vulgaris. GSH is known to promote Hydra feeding response by inducing mouth opening. We demonstrate that GSH-QDs as well are able to elicit biological activity in such an animal, which results in the fluorescent staining of Hydra. GSH-QDs, once they reach the gastric region, are internalized by endodermal cells. The efficiency of GSH-QD internalization increases significantly when nanoparticles are coadministrated with free GSH. We also compared the behavior of bare QDs to that of GSH-QDs both in the presence and in the absence of free GSH. The conclusions from these series of experiments point to the presence of GSH binding proteins in the endodermal cell layer and uncover a novel role played by glutathione in this organism.

Putative NMDA receptors in Hydra: a biochemical and functional study.

The feeding behaviour of the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) is modulated by a number of molecules acting as neurotransmitters in other nervous systems. Here we present biochemical and functional evidence of the occurrence of putative NMDA receptors in Hydra tissues. Saturation experiments showed the presence of one population of binding sites with nanomolar affinity and low capacity for [3H]MK-801. Before equilibrium, [3H]MK-801 binding was increased by the agonists glutamate and glycine as well as by reduced glutathione (GSH). In vivo the glutamate receptor agonist NMDA markedly decreased the duration of the response to GSH. This effect was linearly related to ligand doses in the nanomolar concentration range and was counteracted by either the NMDAR-specific antagonist D-AP5 or by the d-serine antagonist DCKA. When NMDA concentration was increased to 10 or 100 microm, duration of the response to GSH was no longer affected unless the lectin concanavalin A, which prevents receptor desensitization in other systems, was added to the test medium. Simultaneous administration of ineffective doses of NMDA and strychnine, glycine or d-serine, an agonist at the glycine binding site of the NMDA receptor in vertebrate CNS, resulted in a strong reduction of response duration. Both D-AP5 and DCKA suppressed this effect. These results, together with the decrease in response duration produced by d-serine, support the hypothesis that NMDA-like glutamate receptors may occur in Hydra tissues where they are involved in modulation of the response to GSH with opposite actions to those of GABA and glycine.