In vitro assessment of the impact of CS on skin

Il fumo di sigaretta è uno degli inquinanti ambientali più tossici ed è composto da migliaia di sostanze chimiche, compresi gli idrocarburi policiclici aromatici (PAHs). Nonostante il divieto di fumare in ambienti interni o anche in alcuni luoghi all’aperto, il rischio che i non fumatori siano esposti al fumo di sigaretta non è ancora stato eliminato. Oltre ai ben noti effetti del fumo di sigaretta sul sistema respiratorio e cardiovascolare, negli ultimi decenni un crescente numero di lavori scientifici ha dimostrato i suoi effetti tossici anche sui tessuti cutanei. Essendo la pelle l'organo più grande del nostro organismo e trovandosi all'interfaccia tra l'ambiente esterno ed interno, agisce come uno scudo naturale che è continuamente esposto ad agenti esogeni dannosi. Quindi, un’esposizione prolungata e/o reiterata a livelli significativi di fumo di sigaretta può avere effetti deleteri sul tessuto cutaneo alterando la sua principale funzione di barriera e aggravando preesistenti patologie infiammatorie (ad es. psoriasi, dermatite atopica). Attraverso lo sviluppo di modelli di tessuti cutanei sempre più sofisticati e di specifici sistemi di esposizione al fumo di sigaretta è aumentata la necessità di comprendere nel dettaglio i pathways intracellulari attivati dall’esposizione ai diversi inquinanti contenuti nel fumo di sigaretta in condizioni di laboratorio sempre più vicine a quelle realistiche, con lo scopo di trovare soluzioni per contrastare tali effetti tossici. Questa tesi di dottorato fornisce innanzitutto una dettagliata analisi sullo stato dell'arte riguardo ai modelli di pelle attualmente disponibili per studiare l'esposizione al fumo di sigaretta, agli effetti deleteri sulla pelle indotti dal fumo di sigaretta, nonché riguardo alle patologie infiammatorie del tessuto cutaneo potenzialmente indotte e/o aggravate dall'esposizione a questo agente tossico. Questo studio fornisce importanti e innovative informazioni sui meccanismi coinvolti nella tossicità indotta da fumo di sigaretta in un modello di pelle 3D: gli RHE, epidermide umana ricostruita in vitro. Durante la prima fase dello studio, il modello epidermico umano ricostruito (RHE) sarà caratterizzato e convalidato come il principale bersaglio biologico in vitro dell’azione dell’agente tossico; successivamente, lo studio si concentrerà sulla realizzazione e l'ottimizzazione di un sistema di esposizione al fumo di sigaretta ben controllato e che rappresenti quanto più possibile la situazione reale delle condizioni alle quali normalmente la pelle è esposta. Una volta convalidati sia il modello sperimentale che le condizioni di esposizione, si passerà all’analisi dei marcatori di stress ossidativo e infiammazione per cercare di mettere in evidenza le vie attivate dalle cellule epidermiche come strategia di difesa contro l'esposizione al fumo di sigaretta. Attraverso queste conoscenze, sarà possibile ipotizzare specifiche soluzioni terapeutiche mirate al controllo dei pathways coinvolti, con lo scopo ultimo di ripristinare l’alterata omeostasi cutanea.Cigarette smoke stands among the most toxic environmental pollutants and is composed of thousands of chemicals including polycyclic aromatic hydrocarbons (PAHs). Despite restrict cigarette smoking ban in indoor or some outdoor locations, the risk of non-smokers to be exposed to environmental cigarette smoke is not yet eliminated. Beside the well-known effects of cigarette smoke to the respiratory and cardiovascular systems, a growing literature has shown during the last three decades its noxious effects also on cutaneous tissues. Being the largest organ as well as the interface between the outer environment and the body, human skin acts as a natural shield which is continuously exposed to harmful exogenous agents. Thus, a prolonged and/or repetitive exposure to significant levels of toxic smoke pollutants may have detrimental effects on the cutaneous tissue by disrupting the epidermal barrier function and by exacerbating inflammatory skin disorders (i.e. psoriasis, atopic dermatitis). With the development of very complex skin tissue models and sophisticated cigarette smoke exposure systems it has become important to better understand the toxicity pathways induced by smoke pollutants in more realistic laboratory conditions to find solutions for counteracting their effects. This doctoral thesis provides first a state of the art on the skin models currently available to study cigarette smoke exposure, the reported deleterious effects induced by CS in skin, as well as the inflammatory skin pathologies potentially induced and/or exacerbated by cigarette smoke exposure. This study consists in investigating and providing further insight in the mechanical pathways involved in CS-induced toxicity in a 3D in vitro skin models, a well-characterized reconstructed human epidermis (RHE). As a first stage of the study, the reconstructed human epidermal model (RHE) will be fully characterized and validated as the main in vitro biological target, secondly it will focus on the implementation and optimization of the exposure conditions of cigarette smoke. Once both the skin model and the exposure conditions have been validated, markers of oxidative stress and inflammation can be assessed to relate pathways activated by the skin epithelial cells as a defense strategy against CS exposure. With this knowledge, therapeutic solutions may be developed targeting the altered markers, hence counteracting the detrimental impact. Finally, the reconstructed skin model was used to screen protective solutions against air pollutants such as cigarette smoke

Inflammasome involvement in CS-induced damage in HaCaT keratinocytes

Cigarette smoke (CS) alters cutaneous biological processes such as redox homeostasis and inflammation response that might be involved in promoting skin inflammatory conditions. Exposure to CS has also been linked to a destabilization of the NLRP3 inflammasome in pollution target tissues such as the lung epithelium, resulting in a more vulnerable immunological response to several exogenous and endogenous stimuli related to oxidative stress. Thus, CS has an adverse effect on host defense, increasing the susceptibility to develop lung infections and pathologies. In the skin, another direct target of pollution, inflammasome disorders have been linked to an increasing number of diseases such as melanoma, psoriasis, vitiligo, atopic dermatitis, and acne, all conditions that have been connected directly or indirectly to pollution exposure. The inflammasome machinery is an important innate immune sensor in human keratinocytes. However, the role of CS in the NLRP1 and NLRP3 inflammasome in the cutaneous barrier has still not been investigated. In the present study, we were able to determine in keratinocytes exposed to CS an increased oxidative damage evaluated by 4-HNE protein adduct and carbonyl formation. Of note is that, while CS inhibited NLRP3 activation, it was able to activate NLRP1, leading to an increased secretion of the proinflammatory cytokines IL-1 beta and IL-18. This study highlights the importance of the inflammasome machinery in CS that more in general, in pollution, affects cutaneous tissues and the important cross-talk between different members of the NLRP inflammasome family

Ethosomes for Coenzyme Q10 Cutaneous Administration: From Design to 3D Skin Tissue Evaluation

Ethosome represents a smart transdermal vehicle suitable for solubilization and cutaneous application of drugs. Coenzyme Q10 is an endogenous antioxidant whose supplementation can counteract many cutaneous disorders and pathologies. In this respect, the present study describes the production, characterization, and cutaneous protection of phosphatidylcholine based ethosomes as percutaneous delivery systems for coenzyme Q10. CoQ10 entrapment capacity in ethosomes was almost 100%, vesicles showed the typical ‘fingerprint’ structure, while mean diameters were around 270 nm, undergoing an 8% increase after 3 months from production. An ex-vivo study, conducted by transmission electron microscopy, could detect the uptake of ethosomes in human skin fibroblasts and the passage of the vesicles through 3D reconstituted human epidermis. Immunofluorescence analyses were carried on both on fibroblasts and 3D reconstituted human epidermis treated with ethosomes in the presence of H2O2 as oxidative stress challenger, evaluating 4-hydroxynonenal protein adducts which is as a reliable biomarker for oxidative damage. Notably, the pretreatment with CoQ10 loaded in ethosomes exerted a consistent protective effect against oxidative stress, in both models, fibroblasts and in reconstituted human epidermis respectively

Exploring the cellular uptake and localisation of phosphorescent rhenium fac-tricarbonyl metallosurfactants as a function of lipophilicity

A systematic study of the cellular uptake of emissive complexes as a function of their lipophilicity is presented. Here a series of amphiphilic rhenium fac-tricarbonyl bisimine complexes bearing axial substituted imidazole or thiazole ligands, [Re(bpy)(CO)3(ImCnHm)]+ {n = 1 m = 3 (1+), n = 4 m = 9 (2+), n = 8 m = 17 (3+), n = 12 m = 25 (4+), n = 16 m = 33 (5+), n = 2 m = 3 (6+); bpy = 2,2'-bipyridine, Im = imidazole} and [Re(bpy)(CO)3(L)]+ {L = 1-mesitylimidazole, ImMes (7+), 4,5-dimethylthiazole, dmt (8+) and 4-methyl-5-thiazole-ethanol, mte (9+)} is reported. The X-ray crystal structures of 2+, 8+ and 9+ confirm the geometry and expected distribution of ligands and indicated that the plane of the imidazole/thiazole ring is approximately parallel to the long axis of the bipy ligand. Luminescence studies revealed excellent properties for their use in cell imaging with visible excitation and broad emission profiles. Their uptake in two distinct species has been examined by fluorescence imaging of the diplomonad fish parasite Spironucleus vortens (S. vortens) and rod-shaped yeast Schizosaccharomyces pombe (Schiz. pombe) as a function of their lipophilicity. The uptake of the complexes was highest for the more lipophilic 2+-5+ in both S. vortens and Schiz. pombe in which the long alkyl chain aids in crossing bilipid membranes. However, the increased lipophilicity of longer chains also resulted in greater toxicity. Localisation over the whole cell varied with differing alkyl chain lengths with complex 2+ preferentially locating to the nucleus of S. vortens, 3+ showing enhanced nuclear partitioning in Schiz. pombe, and 4+ for the remaining cell wall bound in the case of S. vortens. Interestingly, complexes of intermediate lipophilicity such as 7+ and 8+ showed reasonable uptake, proved to be non-toxic, and were capable of crossing exterior cell walls and localising in the organelles of the cells.</p