Progettazione e studio delle caratteristiche di autoorganizzazione di corti oligopeptidi

Il principale obiettivo di questa tesi è rappresentato da progettazione, e valutazione delle caratteristiche di autoorganizzazione di nuovi corti oligopeptidi potenzialmente idonei per la preparazione di sistemi nanostrutturati da impiegare per applicazioni biomediche Esso sarà realizzato attraverso un approccio che prevede l’uso combinato di metodologie sperimentali, quali light scattering, spettroscopia UV ed IR, viscosimetria, calorimetria isoterma di titolazione, calorimetria differenziale a scansione, microscopia ottica, microscopia a forza atomica e di tecniche di “molecular modeling”. Sulla base dei risultati ottenuti si tenterà di stabilire una correlazione tra caratteristiche strutturali e capacità di autoorganizzazione. I diversi oligopeptidi studiati saranno inoltre caratterizzati mediante prove in vitro di tossicità ed almeno nei casi più promettenti, di capacità di sostenere l’adesione e la proliferazione cellulare. L’acquisizione di conoscenze che rendano possibile la progettazione di nuovi materiali capaci di autoorganizzarsi in strutture nanometriche predefinite avrebbe un’enorme importanza sia a livello concettuale, che soprattutto a livello applicativo. Vale la pena di ricordare a questo proposito l’intensa attività di ricerca in corso a livello mondiale per la formulazione di oggetti nanoscopici da impiegare nel rilascio mirato di farmaci, nell’ingegneria tissutale e nella realizzazione di biosensori e di biochip

Liposomes loaded with polyphenol-rich grape pomace extracts protect from neurodegeneration in a rotenone-based in vitro model of Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disease with no satisfactory therapy options. Similar to other neurodegenerative conditions, such as Alzheimer's and Huntington's diseases, oxidative stress plays a key factor in the neurodegeneration process. To counteract the uncontrolled increase of reactive oxygen species (ROS) and oxidative stress-dependent cell death, several preclinical and clinical tests exploit natural-derived organic antioxidants, such as polyphenols. Despite some promising results, free antioxidants show scarce brain accumulation and may exhaust their scavenging activity before reaching the brain. In this work, we developed an antioxidant therapeutic nanoplatform consisting of nano-sized functionalized liposomes loaded with selected polyphenol-rich vegetal extracts with high blood-brain barrier crossing capabilities. The antioxidant extracts were obtained from the grape seeds and skins as a byproduct of wine production (i.e., pomace), following a sustainable circular approach with reduced environmental impact. The antioxidant nanoplatform was successfully tested in a relevant in vitro model of PD, where it completely rescued the ROS levels, prevented the aggregation of alpha-synuclein fibrils, and restored cell viability, paving the way for preclinical translation of the approach

Biocompatibility of a Conjugated Polymer Retinal Prosthesis in the Domestic Pig

The progressive degeneration of retinal photoreceptors is one of the most significant causes of blindness in humans. Conjugated polymers represent an attractive solution to the field of retinal prostheses, and a multi-layer fully organic prosthesis implanted subretinally in dystrophic Royal College of Surgeons (RCS) rats was able to rescue visual functions. As a step toward human translation, we report here the fabrication and in vivo testing of a similar device engineered to adapt to the human-like size of the eye of the domestic pig, an excellent animal paradigm to test therapeutic strategies for photoreceptors degeneration. The active conjugated polymers were layered onto two distinct passive substrates, namely electro-spun silk fibroin (ESF) and polyethylene terephthalate (PET). Naive pigs were implanted subretinally with the active device in one eye, while the contralateral eye was sham implanted with substrate only. Retinal morphology and functionality were assessed before and after surgery by means of in vivo optical coherence tomography and full-field electroretinogram (ff-ERG) analysis. After the sacrifice, the retina morphology and inflammatory markers were analyzed by immunohistochemistry of the excised retinas. Surprisingly, ESF-based prostheses caused a proliferative vitreoretinopathy with disappearance of the ff-ERG b-wave in the implanted eyes. In contrast, PET-based active devices did not evoke significant inflammatory responses. As expected, the subretinal implantation of both PET only and the PET-based prosthesis locally decreased the thickness of the outer nuclear layer due to local photoreceptor loss. However, while the implantation of the PET only substrate decreased the ff-ERG b-wave amplitude with respect to the pre-implant ERG, the eyes implanted with the active device fully preserved the ERG responses, indicating an active compensation of the surgery-induced photoreceptor loss. Our findings highlight the possibility of developing a new generation of conjugated polymer/PET-based prosthetic devices that are highly biocompatible and potentially suitable for subretinal implantation in patients suffering from degenerative blindnes

PMMA/Polysaccharides Nanofilm Loaded with Adenosine Deaminase Inhibitor for Targeted Anti-inflammatory Drug Delivery

A novel drug delivery vector, a free-standing polymeric ultrathin film (nanofilm) composed of PMMA and a polysaccharides multilayer, is presented. Chitosan and sodium alginate are alternatively deposited by spin-assisted LbL assembly onto a plasma-treated PMMA thin film. Hydrophobic anti-inflammatory drugs, an adenosine deaminase inhibitor (APP) and its fluorescent dansyl derivate (APP-Dns), are encapsulated inside the LbL multilayer using a simple casting deposition procedure. The resulting drug loaded nanofilm can be suspended in water upon dissolution of a PVA sacrificial layer. Morphological characterization of the nanofilm shows that PMMA/LbL nanofilms possess nanometric thickness (<200 nm) and very low surface roughness (1−2 nm for drug loaded nanofilms and <1 nm for blank nanofilm). Drug loaded films exhibit a diffusion controlled release mechanism following the Korsmayer−Peppas release model, confirmed by the fit of release data with a characteristic power law. Drug release is impaired through the PMMA layer, which acts effectively as a barrier for drug transport. This ultrathin polymer film can find application as a nanopatch for targeted inflammatory drug delivery to treat localized pathologies as inflammatory bowel disease

Structural color tuning in a Ag/TiO2 nanoparticle one-dimensional photonic crystal induced by electric field

We present the electric field-induced tuning of the light transmission in a photonic crystal device. The device, with alternating layers of Silver and Titanium dioxide nanoparticles, shows a shift of around 10 nm for an applied voltage of 10 V. An accumulation of charges at the Silver/TiO2 interface due to electric field leads to an increase of the number of charges contributing to the plasma frequency in Silver. This results in a blue shift of the Silver plasmon band, with concomitant blue shift of the photonic band gap as a result of the decrease in the Silver dielectric function

Nano-Structured Brownian Surfaces Prepared through Two-Photon Polymerization : Investigation of Stem Cell Response

Non-deterministic phenomena are at the base of plenty of biological processes, that comprise physiological signalling, cellular communications, and biological architectures. Among them, natural surface topographies are often characterized by "chaotic" features, that are not trivial to be re-created in vitro. Recently, some methods have been proposed to resemble the hierarchical organization of the extracellular microenvironment, through the chemical preparation of randomly rough and self-affine fractal surfaces. Notwithstanding, this approach does not allow the fractal dimension to be modulated at a desired value, being moreover the self-affinity maintained just for one decade of spatial frequencies. Here, we propose the replication of in silico generated Brownian surfaces through a two-photon polymerization technique. Thanks to the direct laser writing of the desired patterns, we were able to obtain highly reproducible selfaffine (in a range of two spatial frequency decades) structures characterized by the desired predetermined Hurst exponents. Rat mesenchymal stem cells were moreover cultured on the obtained substrates, highlighting interesting phenomena concerning cell adhesion, cytoskeleton conformation and actin polymerization, strictly depending on the fractal dimension of the surfaces

Rapid and Controllable Digital Microfluidic Heating by Surface Acoustic Waves

Fast and controllable surface acoustic wave (SAW) driven digital microfluidic temperature changes are demonstrated. Within typical operating conditions, the direct acoustic heating effect is shown to lead to a maximum temperature increase of about 10 °C in microliter water droplets. The importance of decoupling droplets from other on‐chip heating sources is demonstrated. Acoustic‐heating‐driven temperature changes reach a highly stable steady‐state value in ≈3 s, which is an order of magnitude faster than previously published. This rise time can even be reduced to ≈150 ms by suitably tailoring the applied SAW‐power excitation profile. Moreover, this fast heating mechanism can lead to significantly higher temperature changes (over 40 °C) with higher viscosity fluids and can be of much interest for on‐chip control of biological and/or chemical reactions

Liquid single crystal elastomer/conducting polymer bilayer composite actuator: Modelling and experiments

In order to integrate electroconductive properties in a Liquid Single Crystal Elastomer (LSCE) and to test direct actuation of the LSCE by Joule heating, we present a new bi-layered all-organic composite actuator based on the coupling of a nematic LSCE with a conductive polymer. The bending actuator is fabricated by depositing a thin conductive polymer layer of poly(3,4- ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) over the surface of a polysiloxane-based monodomain nematic LSCE film. Mechanical properties of PEDOT:PSS, better matched with LSCE ones compared with metals or inorganic nanoparticles used in other approaches, allowed us to develop an all-organic reliable millimetre-scale actuating composite. The thermally induced elongation/compression of the LSCE over 30% is exploited for the fabrication of bending actuators with curvature up to κ = 0.64 mm-1. The LSCE and the composite material are characterized as regards their thermo-mechanical and electrical properties. A model is introduced to describe bending of the composite as a function of the thermo-mechanical properties of the LSCE, and the model is assessed by comparing the model results with the experimental findings. Bending actuation via direct Joule heating of the composite is also assessed by supplying the necessary current (50 mA at 1.3 V) through wires connected to the composite. These results open new possibilities for the application of LCEs in the micro and soft robotics fields, as well as in the biomedical field