The possibility to control electrical activity of living cells by optical excitation offers several advantages respect to more traditional electrical excitation methods, such as reduced invasivity, avoidance of crosstalk between the excitation and the recording site, high spatial and temporal resolution. Existing methods however present also some disadvantages: optogenetics requires viral transfer and it is not applicable in vivo at the moment; infrared stimulation requires high excitation density; use of caged compounds is not specific, irreversible and works under UV light, which is detrimental to cell viability. For this reason many efforts are currently focused on the search for new exogenous, light sensitive materials, capable to transduce optical excitation into electrical activity of the living tissues, both for in vitro and in vivo applications. In this context organic semiconductors, and in particular conjugated polymers, are emerging as promising tools, due to their outstanding properties: intrinsic sensitivity to visible light, high biocompatibility, good mechanical properties, easy fabrication technology. Conducting polymer thin films were already reported to reliably and efficiently excite electrical activity in neuronal networks, brain slices, retinal tissues both ex vivo and in vivo. However, aiming at in vivo applications, for instance for deep brain optical stimulation or for replacing damaged photoreceptors in blind patients, it would be preferable to avoid surgical procedure needed to implant a polymer-based device, and dispose of light sensitive, injectable polymer beads. This work investigates for the first time the possibility to use polymer nanoparticles for optical control of living cells. In particular, aiming at in vivo applications, the nanoparticles biocompatibility and efficacy has been tested in a simple invertebrate, Hydra Vulgaris. Hydra represents a valuable animal model for in-vivo testing of new materials. In fact, despite its simplicity, Hydra presents a net of neurons extended throughout the body and tentacles, which makes this animal an ideal candidate for screening light sensitive materials in-vivo on a functionally connected neural net, avoiding the difficulties of investigating a complex vertebrate nervous system. Nanoparticles internalization and toxicity are studied by fluorescence imaging. Subsequently, behavioral analysis is carried out, in different experimental conditions, aiming at identifying the effect of nanoparticles photo-excitation on animal behavior. Molecular biology studies corroborated our findings. Moreover, in this thesis are reported experiments of calcium imaging on Hydra Vulgaris, which, to the best of our knowledge, were never carried out in this animal model. Indeed, these measurements put an high degree of technological complexity, due to the difficulty of immobilizing the animal while studying it in a physiological condition. The fabrication of a microfluidics chip permitted the acquirement of calcium dynamics, the first necessary step to understand the neurotrasmission mechanism in hydra. Overall, the results reported in this thesis demonstrate that polymer nanoparticles do not present specific toxicity in in vivo conditions, and hold the promise to become a valuable tool for optical control of animal behavior.

Study of the impact of poly (3-hexylthiophene) nanoparticles on Hydra Vulgaris

SAMANNI, GIULIA
2014/2015

Abstract

The possibility to control electrical activity of living cells by optical excitation offers several advantages respect to more traditional electrical excitation methods, such as reduced invasivity, avoidance of crosstalk between the excitation and the recording site, high spatial and temporal resolution. Existing methods however present also some disadvantages: optogenetics requires viral transfer and it is not applicable in vivo at the moment; infrared stimulation requires high excitation density; use of caged compounds is not specific, irreversible and works under UV light, which is detrimental to cell viability. For this reason many efforts are currently focused on the search for new exogenous, light sensitive materials, capable to transduce optical excitation into electrical activity of the living tissues, both for in vitro and in vivo applications. In this context organic semiconductors, and in particular conjugated polymers, are emerging as promising tools, due to their outstanding properties: intrinsic sensitivity to visible light, high biocompatibility, good mechanical properties, easy fabrication technology. Conducting polymer thin films were already reported to reliably and efficiently excite electrical activity in neuronal networks, brain slices, retinal tissues both ex vivo and in vivo. However, aiming at in vivo applications, for instance for deep brain optical stimulation or for replacing damaged photoreceptors in blind patients, it would be preferable to avoid surgical procedure needed to implant a polymer-based device, and dispose of light sensitive, injectable polymer beads. This work investigates for the first time the possibility to use polymer nanoparticles for optical control of living cells. In particular, aiming at in vivo applications, the nanoparticles biocompatibility and efficacy has been tested in a simple invertebrate, Hydra Vulgaris. Hydra represents a valuable animal model for in-vivo testing of new materials. In fact, despite its simplicity, Hydra presents a net of neurons extended throughout the body and tentacles, which makes this animal an ideal candidate for screening light sensitive materials in-vivo on a functionally connected neural net, avoiding the difficulties of investigating a complex vertebrate nervous system. Nanoparticles internalization and toxicity are studied by fluorescence imaging. Subsequently, behavioral analysis is carried out, in different experimental conditions, aiming at identifying the effect of nanoparticles photo-excitation on animal behavior. Molecular biology studies corroborated our findings. Moreover, in this thesis are reported experiments of calcium imaging on Hydra Vulgaris, which, to the best of our knowledge, were never carried out in this animal model. Indeed, these measurements put an high degree of technological complexity, due to the difficulty of immobilizing the animal while studying it in a physiological condition. The fabrication of a microfluidics chip permitted the acquirement of calcium dynamics, the first necessary step to understand the neurotrasmission mechanism in hydra. Overall, the results reported in this thesis demonstrate that polymer nanoparticles do not present specific toxicity in in vivo conditions, and hold the promise to become a valuable tool for optical control of animal behavior.
ANTOGNAZZA, MARIA ROSA
ING - Scuola di Ingegneria Industriale e dell'Informazione
18-dic-2015
2014/2015
Tesi di laurea Magistrale
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10589/115845