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Purpose: To investigate the potential of new chitosan-based physical gels, alone or loaded with plant extract or growth factors, for wound healing. Methods: Chitosan (CHI) was autoclaved and then dissolved at 2% w/w in acetic acid 0.1 M; the resulting dispersion was sonicated and then placed under vacuum for 48h. CHI gels were differently loaded depending of the considered active. In particular, gels loaded with PDGF and FGF-?? were prepared by co-dissolution of the growth factors with CHI in acetic acid 0.1 M. Alternatively, Centella asiatica (CEN) or Echinacea angustifolia (ECH) were added to the preformed gel. Thermal and rheological analysis were carried out to highlight the differences due to the heat treatment on the viscoelastic properties of gels. In addition, the effects of the gels on human fetal foreskin fibroblasts (HFFF-2) viability, proliferation and migration were investigated. Finally, the effect of the different gels on wound healing was investigated on C57BL/6J mice using an experimental model of pressure ulcer. Results: The differential scanning calorimeter (DSC) analysis showed that, the thermal treatment modifies the chemical and physical characteristics of the CHI powder. Furthermore, the formulations with non-autoclaved CHI showed a rheological behavior typical of an entangled network, while gels prepared from the autoclaved CHI have a typical gel behavior. The loading of actives into the gel did not affect the rheological properties of the formulations. In vitro tests on HFFF-2 showed that CHI gels were not toxic and led to increased cell proliferation and migration. Finally, the presence of PDGF ,FGF-??, CEN or ECH in the gels shows also to enhance the migration, the proliferation without affecting the vitality of fibroblasts. In vivo, CHI gel enhanced wound healing compared to untreated mice. This effect was higher when using CHI gels containing PDGF, FGF-??, CEN or ECH. Studies are in progress to determine the gel with the highest effect on wound healing, as well as the mechanism driving the tissue regeneration. Conclusions: The rheological properties of the developed CHI gels as well as in vitro and in vivo results, demonstrated a high potential as new products for the medication of wound, i.e. pressure ulcers.

L???obiettivo di questo lavoro è stato lo sviluppo e la caratterizzazione di nuovi gel fisici sterili a base di chitosano, solo o in associazione a estratti vegetali, quali Centella asiatica (CEN) ed Echinacea angustifolia (ECH), e/o fattori di crescita, quali PDGF e FGF-????????? per il trattamento di piaghe da decubito. Per tutte le formulazioni sono stati determinate le proprietà viscoelastiche e termiche, mediante analisi reologica e Calorimetria a Scansione Differenziale (DSC). Infine è stato saggiato l???effetto delle diverse formulazioni su fibroblasti umani di prepuzio (HFFF-2) e sono stati effettuati studi in vivo su modelli sperimentali di piaghe da decubito indotte in topi C57BL/6J. L???analisi DSC ha evidenziato che la sterilizzazione in autoclave della polvere di CHI ne modifica le caratteristiche fisico-chimiche e ne cambia il comportamento reologico, che diventa quello tipico di un gel. Le sostanze attive caricate nei gel non influenzano in maniera significativa le proprietà reologiche delle formulazioni. Gli studi condotti su cellule HFFF-2 hanno evidenziato che i gel a base di CHI non sono tossici e inducono proliferazione e migrazione cellulare. L???impiego di sostanze attive caricate nei gel comporta un significativo aumento della migrazione e della proliferazione, soprattutto quando in miscela tra loro. Gli studi in vivo hanno confermato che i gel a base di CHI, soprattutto quando caricati con sostanze attive, aumentano la velocità di rigenerazione di tessuti lesi.

Autophagy principally serves an adaptive function to protect organisms against diverse human pathologies, including cancer and neurodegeneration. Recent developments using in vitro, ex vivo and in vivo models show the involvement of the autophagy pathway in immunity and inflammation. Moreover, direct interactions between autophagy proteins and immune signalling molecules have also been demonstrated. Defects in autophagy - similar to cancer, neurodegenerative diseases and aging - through autophagy gene mutation and/or microbial antagonism, may underlie the pathogenesis of many infectious diseases and inflammatory syndromes. In spite of the increasing awareness of the importance of autophagy in these pathophysiological conditions, this process remains underestimated and is often overlooked. As a consequence, its role in the initiation, stability, maintenance, and progression of these diseases are still poorly understood. This book reviews the recent advances regarding the functions of the autophagy pathway and autophagy proteins in immunity and inflammation, focusing on their role in self-nonself distinction, their implications in innate and adaptive immune responses and their dysregulation in the pathology of certain inflammatory and autoimmune diseases.

Introduction: The trans-resveratrol (t-res) is a non flavonoid polyphenol with promising properties for the treatment or prevention of different diseases. In particular, topical application of resveratrol could be advantageous in the prevention of skin cancer or in the treatment of inflammation-based diseases such as psoriasis. However, different studies demonstrated the importance of the formulation to enhance t-res delivery into or trough the skin. Nanocarriers, such as ethosomes and transferosomes, have been successfully used to deliver drugs with different characteristics into or through the skin. Purpose: The aim of the present work was to develop a nanocarrier-based formulations for skin delivery of t-res. Methods: Ethosomes or transferosomes containing t-res were prepared by a modified hand-shaking method followed by extrusion or sonication. All the formulations were characterised in terms of mean diameter, size distribution (I.P.), t-res loading, t-res stability upon encapsulation during storage at 4°C. The inhibition of reactive oxygen species (ROS) and lipids peroxidation in human keratinocyte (HaCaT) cell line stimulated with H2O2 for 24 h and then incubated with the t-res containing nanocarriers was investigated. Moreover, for all the formulations, ex vivo permeation studies on porcine skin was carried out by means of Franz diffusion cells. At appropriate intervals (0.5, 1, 2, 4, 6, 24 h), an aliquots of the receptor medium were withdrawn and the t-res concentration was determined by HPLC. Results: All the t-res containing carriers were characterised by a very high (between 70 to 100%) encapsulation efficiency, a limited t-res release at 4°C during the storage and stability of resveratrol in its trans form. Nanocarriers encapsulating t-res reduced, in a concentration-dependent manner, ROS production induced by H2O2, with the higher effect observed in the case of ethosomes. Permeation studies showed that only ethosomes were able to promote t-res permeation through porcine skin and this effect was affected by lipid composition of the carrier. Conclusions: In this work, transferosomes showed the highest encapsulation efficiency and the lowest t-res release. On the other hand, only ethosomes were able to promote t-res permeation through the skin.

NF-kB blockade by decoy oligonucleotides (ODNs) able to interfere with NF-kB transcriptional activity may be of great help in limiting the progression of CF lung chronic inflammation. Although data proving the validity of this approach are just emerging, early studies on CF airway epithelial cell lines are encouraging. Major concerns arise from the need of appropriate systems for ODN inhalation, that are able to efficiently target the desired region of the lungs, protect the ODN in the biological environment and release it in a sustained manner. The technological approaches currently adopted for lung delivery of ODNs reliy on the use of lipidic nanoparticles or cationic liposomes, providing new formulation options for dispersed liquid droplet dosage forms (i.e., bulky, expensive and more time-consuming nebulizers) with poor control over ODN release rate. Prompted by this, we are currently developing respirable dry powders, for prolonged pulmonary delivery of a decoy ODN to NF-kB (dec-ODN), consisting of biodegradable large porous particles (LPP) based on poly(lactic-co-glycolic) acid (PLGA). The effects of dec-ODN LPP on NF-kB/DNA binding activity as well as IL-6 and IL-8 mRNA levels were studied on DF508 CFTR-mutated human bronchial epithelial cells stimulated with LPS from P. aeruginosa for 24 and 72 h. Finally, the effects of intratracheally administered naked dec-ODN or dec-ODN LPP on neutrophil lung recruitment induced by LPS from P. aeruginosa at 6, 24 and 72 h were investigated in a rat model of airway inflammation.

Purpose: Aim of this work was to engineer injectable polymeric platform, able to undergo reversal thermal gelation at body temperature, to fill the cavities of wounds and release active ingredients in situ with a controlled kinetic. Methods: Polymeric platforms made up of chitosan (CHI) and blends of methylcellulose (MC) with low (~150 kDa) or high (~1*106 Da) molecular weight hyaluronic acid (HA), at different concentration, were prepared. The gelation temperature and viscoelastic properties of the platforms were investigated and optimized by means of rheological analysis. In particular, small amplitude frequency sweep tests were performed to evaluate the dependence of dynamic moduli on frequency. In order to evaluate the gelation temperatures, the viscoelastic parameters were monitored as a function of the temperature from 20°C up to 40°C at a fixed oscillation frequency of 0.01 Hz. In addition, we investigated the effects of the gels on human foreskin foetal fibroblasts (HFFF-2) viability. Results: By varying polymers molecular weight and concentration into the blends, it was possible to obtain an optimized formulation showing a rheological behaviour typical of a viscous solution at 25°C and of a gel at 37°C. In vitro studies showed a good biocompatibility of MC and low molecular weight HA based gels since they did not affect HFFF-2 cells viability. In contrast CHI based as well as high molecular weight HA formulations decreased cell viability. Conclusions: Thermosensitive and biocompatible gels were formulated, with a phase transition temperature near to body temperature, potentially useful for wound healing applications.

Il trans-resveratrolo è un polifenolo non flavonoide di origine naturale particolarmente abbondante nella buccia degli acini d’uva. Esistono due isoforme del resveratrolo, il cis ed il trans resveratrolo. Studi recenti hanno dimostrato che la maggior parte dell’attività farmacologica è da attribuire all’isoforma trans. Numerosi sono gli effetti farmacologici attribuiti al resveratrolo, quali antitumorale, anti-aggregante piastrinico e fluidificante del sangue, antiinfiammatorio, antivirale ed antibatterico soprattutto a livello cutaneo1. Recentemente il nostro gruppo di ricerca ha evidenziato l’utilità del trans-resveratrolo nel trattamento dell’acne volgare2. Lo scopo di questo lavoro è stato lo sviluppo di nanovettori a base lipidica per la veicolazione topica del resveratrolo, con lo scopo di aumentare l’accumulo di tale molecola nella cute, garantendone però la sua stabilità nella forma trans. A tale scopo è stata studiata la possibilità di preparare transferosomi ed etosomi, nanovettori che hanno mostrato poter migliorare la penetrazione e/o la permeazione di numerosi farmaci nella cute3. I vettori sono stati preparati mediante la tecnica del film lipidico seguita da idratazione del film e successivo dimensionamento tramite estrusione o sonicazione. Per quanto riguarda i transferosomi, essi sono stati preparati con tre diversi attivatori di membarana, ed in particolare con Tween 80, sodio colato e sodio deossicolato. I vettori sono poi stati caratterizzati in base a dimensioni, indice di polidispersità, tassi d’incapsulazione, rilascio del resveratrolo. Le analisi sono state effettuate dopo preparazione e dopo conservazione a 4°C. Inoltre sono stati effettuati saggi in vitro per valutare la tossicità dei carrier e la capacità di tali vettori di ridurre la produzione di radicali dell’ossigeno in cheratinociti umani stimolati con H2O2. Sono state ottenute formulazioni in cui il diametro medio è compreso tra 78 e 120 nm, con un indice di polidispersità (IP) molto basso (< 0,1), con eccezione dei transferosomi contenenti Tween80 (IP di 0,3-0,4). Queste differenze sono state attribuite in parte al tipo di tecnica utilizza per il dimensionamento, ed i parte alla possibile aggregazione indotta dal Tween80. Dopo 30 giorni di conservazione non sono state osservate variazioni nel diametro medio. Tutte le formulazioni sono caratterizzare da un alto tasso d’incapsulazione, che non varia in maniera significativa dopo conservazione a 4°C. In studi su colture cellulari, è stato osservato che, nel caso degli etosomi, si osserva una lieve tossicità dovuta probabilmente dovuto alla presenza di etanolo nella formulazione. Nel caso dei transferosomi, è stata osservata riduzione della vitalità solo con la formulazione contenente il Tween80, tossicità che però viene quasi del tutto annullata in presenza di resveratrolo incapsulato. In saggi sull’attività antiossidante, è stato osservato che il solo resveratrolo è in grado di ridurne in maniera significativa la produzione di radicali dell’ossigeno (ROS) in maniera concentrazione-dipendente. Sia gli etosomi che i transferosomi (ad eccezione della preparazione contente Tween80), sono in grado di ridurre la produzione di ROS in maniera simile al solo resveratrolo. Sono attualmente i corso studi di permeazione attraverso la cute di maiale per valutare l’efficienza di permeazione e l’accumulo di resveratrolo attraverso la cute in presenza di etosomi e dei transferosomi.

The aim of this work was to develop large porous particles (LPP) of poly(DL-lactide-co-glycolide) (PLGA) for pulmonary delivery of an oligonucleotide decoy to NF-kB.

Purpose: The aim of the present work was to develop a nanocarrier-based formulation for topical delivery of resveratrol. Methods: Trans-resveratrol (t-res) was encapsulated in liposomes, ethosomes or transferosomes, by a modified hand-shaking method followed by extrusion. All the formulations were characterised in terms of mean diameter, size distribution (I.P.), t-res loading, t-res stability upon encapsulation during storage. The nanocarriers containing t-res were then introduced in cellulose-based gel to allow their final administration on the skin and the viscoelastic properties of the resulting formulation were investigated. Finally, we studied the inhibition of reactive oxygen species (ROS) in human keratinocyte (HaCaT) cell line stimulated with H2O2 for 24 h and then incubated with the t-res containing nanocarriers. Results: All the t-res containing carriers were characterised by a very high (close to 100%) encapsulation efficiency, a negligible t-res release at 4°C and stability of resveratrol in its trans form. The carriers only slightly influenced the viscoelastic characteristics of cellulose-based gels. Nanocarriers encapsulating t-res reduced, in a concentration-dependent manner, ROS production induced by H2O2 and this effect was higher when using t-res-encapsulating nanocarrier, with the higher effect observed in the case of ethosomes. Conclusions: In this work nanocarriers with high encapsulation efficiency, high physical stability and negligible t-res release during storage at 4°C were prepared. To allow their final administration on the skin, the nanocarrier can be easilisy loaded in cellulose-based gels without altering its rheological properties. Moreover, the use of t-res-encapsulating ethosomes led to an efficient antioxidant activity. Further ex vivo and in vivo studies will clarify the role of the different carrier when administered on the skin.