Sphingosine 1-phosphate receptors: do they have a therapeutic potential in cardiac fibrosis?

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is characterized by a peculiar mechanism of action. In fact, S1P, which is produced inside the cell, can act as an intracellular mediator, whereas after its export outside the cell, it can act as ligand of specific G-protein coupled receptors, which were initially named endothelial differentiation gene (Edg) and eventually renamed sphingosine 1-phosphate receptors (S1PRs). Among the five S1PR subtypes, S1PR1, S1PR2 and S1PR3 isoforms show broad tissue gene expression, while S1PR4 is primarily expressed in immune system cells, and S1PR5 is expressed in the central nervous system. There is accumulating evidence for the important role of S1P as a mediator of many processes, such as angiogenesis, carcinogenesis and immunity, and, ultimately, fibrosis. After a tissue injury, the imbalance between the production of extracellular matrix (ECM) and its degradation, which occurs due to chronic inflammatory conditions, leads to an accumulation of ECM and, consequential, organ dysfunction. In these pathological conditions, many factors have been described to act as pro- and anti-fibrotic agents, including S1P. This bioactive lipid exhibits both pro- and anti-fibrotic effects, depending on its site of action. In this review, after a brief description of sphingolipid metabolism and signaling, we emphasize the involvement of the S1P/S1PR axis and the downstream signaling pathways in the development of fibrosis. The current knowledge of the therapeutic potential of S1PR subtype modulators in the treatment of the cardiac functions and fibrinogenesis are also examined

Sphingosine 1-phosphate receptor 1 is required for MMP-2 function in bone marrow mesenchymal stromal cells: implications for cytoskeleton assembly and proliferation

Bone marrow-derived mesenchymal stromal cell- (BM-MSC-) based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP-) 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P) and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers) and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia), S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs' ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair

Surface plasmon resonance based on molecularly imprinted nanoparticles for the picomolar detection of the iron regulating hormone Hepcidin-25

Molecular modelling

Sphingosine 1-Phosphate Receptors: Do They Have a Therapeutic Potential in Cardiac Fibrosis?

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is characterized by a peculiar mechanism of action. In fact, S1P, which is produced inside the cell, can act as an intracellular mediator, whereas after its export outside the cell, it can act as ligand of specific G-protein coupled receptors, which were initially named endothelial differentiation gene (Edg) and eventually renamed sphingosine 1-phosphate receptors (S1PRs). Among the five S1PR subtypes, S1PR1, S1PR2 and S1PR3 isoforms show broad tissue gene expression, while S1PR4 is primarily expressed in immune system cells, and S1PR5 is expressed in the central nervous system. There is accumulating evidence for the important role of S1P as a mediator of many processes, such as angiogenesis, carcinogenesis and immunity, and, ultimately, fibrosis. After a tissue injury, the imbalance between the production of extracellular matrix (ECM) and its degradation, which occurs due to chronic inflammatory conditions, leads to an accumulation of ECM and, consequential, organ dysfunction. In these pathological conditions, many factors have been described to act as pro- and anti-fibrotic agents, including S1P. This bioactive lipid exhibits both pro- and anti-fibrotic effects, depending on its site of action. In this review, after a brief description of sphingolipid metabolism and signaling, we emphasize the involvement of the S1P/S1PR axis and the downstream signaling pathways in the development of fibrosis. The current knowledge of the therapeutic potential of S1PR subtype modulators in the treatment of the cardiac functions and fibrinogenesis are also examined

Starch/Poly(glycerol-adipate) Nanocomposites: A Novel Oral Drug Delivery Device

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Biocompatible and bio-based materials are an appealing resource for the pharmaceutical industry. Poly(glycerol-adipate) (PGA) is a biocompatible and biodegradable polymer that can be used to produce self-assembled nanoparticles (NPs) able to encapsulate active ingredients, with encouraging perspectives for drug delivery purposes. Starch is a versatile, inexpensive, and abundant polysaccharide that can be effectively applied as a bio-scaffold for other molecules in order to enrich it with new appealing properties. In this work, the combination of PGA NPs and starch films proved to be a suitable biopolymeric matrix carrier for the controlled release preparation of hydrophobic drugs. Dynamic Light Scattering (DLS) was used to determine the size of drug-loaded PGA NPs, while the improvement of the apparent drug water solubility was assessed by UV-vis spectroscopy. In vitro biological assays were performed against cancer cell lines and bacteria strains to confirm that drug-loaded PGA NPs maintained the effective activity of the therapeutic agents. Dye-conjugated PGA was then exploited to track the NP release profile during the starch/PGA nanocomposite film digestion, which was assessed using digestion models mimicking physiological conditions. The collected data provide a clear indication of the suitability of our biodegradable carrier system for oral drug delivery.Peer reviewedFinal Published versio

Modulation of MMP-2 function in bone marrow mesenchymal stromal cells requires sphingosine 1-phopsphate receptor 1 mediated signaling: implications for cytoskeletal assembly and proliferation

Bone-marrow-derived mesenchymal stromal cells (BM-MSCs)–based therapy represents a promising option in the field of regenerative medicine. Their therapeutic potential is mainly dependent on paracrine secretion, proliferation and ECM remodeling abilities whose modulation involves Matrix Metalloproteinase (MMP)-2 functionality. Thus, the identification of paracrine/autocrine factors regulating MMP-2 expression/activity may be of great biological relevance for potentiating BM-MSC theraputic efficacy. Our research group has demonstrated that BM-MSCs release the bioactive lipid sphingosine-1-phosphate (S1P). Here we demonstrated : i) the requirement for BM-MSC of S1P production to synthesize functional gelatinases; ii) an impairment of gelatinolytic activity and MMP-2 expression/release when the S1P receptor subtype 1 (S1PR1) is blocked. Notably, in these experimental conditions BM-MSCs did not exhibit the formation of plasmamembrane-associated F-actin structures (lamellipodia, filopodia, microspikes) and, in turn, showed a reduction of the proliferation rate. Moreover, S1P1-mediated signaling is required for HIF-1alpha expression and MMP-2 expression/activity, reduction of vinculin expression and stress fiber formation and proliferation in hypoxia, an experimental condition mimicking the injured/regenerating tissue microenvironment. In conclusion, our findings, demonstrating the trophic role exerted by the autocrine S1P/S1PR1 signaling in maintaining BM-MSC ability to modulate MMP-2 function, required for ECM remodeling, cytoskeleton assembly and cell proliferation may provide perspectives for considering S1P/S1PR1 as a pharmacological target to preserve BM-MSCs properties and improve their efficacy in tissue repair

MOLECULARLY IMPRINTED NANOPARTICLES FOR THE MEASUREMENT OF THE IRON REGULATOR HORMONE HEPCIDIN IN SERUM SAMPLES

L\u2019epcidina \ue8 un ormone peptidico secreto principalmente dal fegato, organo di importanza primaria nella regolazione sistemica del ferro. Negli ultimi anni la quantificazione dell\u2019epcidina ha acquisito un\u2019importanza significativa sia per la diagnosi che per la prognosi di malattie umane correlate all\u2019omeostasi del metabolismo del ferro. Nonostante ci\uf2 lo sviluppo di un saggio analitico affidabile e largamente applicabile per la quantificazione dell\u2019epcidina nei fluidi biologici si \ue8 dimostrato estremamente difficoltoso; tanto che non \ue8 stata ancora individuata una soluzione che abbia incontrato il consenso generale. La presente tesi propone un metodo analitico per la quantificazione dell\u2019epcidina 25, basato sull\u2019utilizzo di nanoparticelle sintetiche in grado di riconoscere specificamente l\u2019analita d\u2019interesse. In particolare, questi nano-recettori sono stati sintetizzati sfruttando la tecnologia dei polimeri a stampo molecolare. I polimeri a stampo molecolare (MIP) sono materiali biomimetici capaci di legare un dato composto in modo specifico. Grazie alle loro peculiari caratteristiche, quali la capacit\ue0 di riconoscimento molecolare, l\u2019alta stabilit\ue0 e resistenza e la facilit\ue0 di sintesi, i MIP si sono dimostrati un\u2019interessante alternativa all\u2019uso degli anticorpi. Per questo motivo si \ue8 deciso di sintetizzare MIP in forma di nanoparticella per la \u201ccattura\u201d selettiva dell\u2019epcidina. Nello specifico \ue8 stata prodotta una collezione di nanoparticelle capaci di legare l\u2019epcidina 25. Il metodo base usato per la sintesi \ue8 la polimerizzazione per precipitazione, che ci ha permesso di ottenere nanoparticelle \u201cstampate\u201d in modo non-covalente. La collezione di nanoparticelle \ue8 stata interamente caratterizzata da un punto di vista fisico mediante diverse strategie: con esperimenti di diffusione dinamica della luce (DLS), per raccogliere informazioni sulle dimensioni e la monodispersit\ue0 dei campioni; con esperimenti di diffusione statica della luce (SLS), per determinare il peso molecolare medio delle nanoparticelle; con microscopia a forza atomica (AFM) in aria, per investigare la morfologia dei diversi tipi di nanoparticella. In seguito si \ue8 passati all\u2019analisi delle caratteristiche funzionali mediante risonanza plasmonica di superficie (SPR). Si \ue8 potuto cos\uec constatare che alcune formulazioni di MIP presentavano una buona affinit\ue0 di legame per l\u2019epcidina 25 umana rispetto a nanoparticelle di controllo non stampate molecolarmente (NIP). In particolare, le risposte SPR pi\uf9 significative sono state rilevate per tre differenti formulazioni di MIP caratterizzate da valori di Kd nanomolari, analogamente agli anticorpi monoclonali umani. E\u2019 stato infine sviluppato un saggio per la quantificazione dei livelli di epcidina 25 nel siero basato sull\u2019uso di MIP. Il saggio qui proposto \ue8 del tutto affine ai saggi di tipo immuno-assorbente legati ad un enzima (ELISA), ma con la sostituzione degli anticorpi con nanoparticelle MIP. Per sviluppare il saggio sono state considerate diverse variabili, quali la quantit\ue0 di nanoparticelle da depositare per pozzetto nelle piastre da saggio, le soluzioni di lavaggio pi\uf9 adatte per la soppressione del segnale aspecifico, il tipo di saggio (diretto o competitivo), il tempo di risposta del saggio. Soluzioni di epcidina con concentrazioni tra 1 e 100 nM sono state utilizzate per determinare la risposta del saggio. Al fine di minimizzare il rumore di fondo del saggio \ue8 stato messo a punto un protocollo per il pretrattamento dei campioni reali, volto a ridurre l\u2019interferenza di matrici complesse durante la quantificazione dell\u2019analita. Nello specifico \ue8 stata ottimizzata la strategia di filtrazione del campione, cos\uec da minimizzare la deplezione dell\u2019analita durante tale pretrattamento. Il saggio sviluppato, basato sull\u2019uso di MIP, \ue8 stato infine impiegato per la quantificazione di epcidina 25 in campioni di siero, con risposte rilevabili nell\u2019ambito di concentrazioni di interesse fisiologico (i.e. 48 1-100 nM). L\u2019ampia diffusione di saggi simil-ELISA pu\uf2 potenzialmente essere favorita dall\u2019uso di MIP come \u201canticorpi di plastica\u201d, in quanto caratterizzati da interessanti propriet\ue0, quali l\u2019alta stabilit\ue0, la selettivit\ue0, il basso costo e la semplicit\ue0 procedurale di sintesi.Hepcidin is a peptide hormone, mainly excreted by the liver, that was identified as the central systemic iron-regulator. The quantification of hepcidin gained significant importance in the past years for diagnostic and prognostic purposes concerning human diseases related to iron homeostasis. However the development of a reliable and broadly applicable assay for the assessment of hepcidin levels in biological fluids has proved to be very challenging and a general consensus solution for hepcidin determination is still missing. In the present thesis, a method for hepcidin 25 quantitation based on synthetic recognition nanoparticles (NPs) prepared via Molecularly Imprinted Polymer (MIP) technology is proposed. Molecularly imprinted polymers (MIPs) are biomimetic materials capable of specific recognition towards an analyte. Their molecular recognition properties, combined with their high stability, robustness, and easy synthesis, make MIPs extremely attractive as alternative to antibodies. For this reason, imprinted nanoparticles (MIP NPs) were synthesized for the selective capturing of hepcidin. During my PhD the synthesis of a library of MIP NPs for hepcidin 25 was performed, exploiting a non-covalent imprinting precipitation polymerization strategy. The NPs library was physically characterized using: Dynamic Light Scattering (DLS), to collect information about the size distribution and the sample monodispersity; Static Light Scattering (SLS), for measuring the averaged molecular weight (Mn) of the NPs; and Atomic Force Microscopy (AFM) in air, to investigate the morphology of our NPs batches. In a subsequent part of the work, the NP library was functionally characterized by Surface Plasmon Resonance (SPR). In fact, good affinity for human hepcidin 25 of MIP respect to control non-imprinted (NIP) polymer was estimated for some NP formulations by Surface Plasmon Resonance (SPR) analysis. In particular the most significant SPR responses were found for three MIP batches exhibiting Kd values in the nanomolar range, in similarity with the Kds of natural monoclonal antibodies. In the last part of the PhD thesis, the development of an ELISA like MIP-based assay for the assessment of the hepcidin 25 levels in serum was approached. Variables, such as the quantity of NPs deposited per microtiter plate well, the most suitable solution to quench the aspecific binding, the competitive or direct format of assay, the time of response of the assay, were evaluated. The response of the ELISA like assay with model hepcidin solutions was evaluated in the range 1-100nM. In order to diminish the background noise of the assay, a protocol for real sample pre-treatment was set up. In particular, a filtration strategy to avoid hepcidin depletion from samples after filtration was optimized. This method allowed the clean-up of real samples in order to minimize the interference of complex matrixes during the analyte quantification. Finally, the developed ELISA like MIP-based assay for hepcidin 25 level assessments in serum was approached with resulting responses within the range of concentrations of physiological interest, i.e. 48 1-100 nM. The possibility of using MIPs as plastic antibodies in ELISA-like assays has the advantage of a potential widespread diffusion thanks to their high stability, selectivity, low cost and simple manufacture procedure

Development of LCEs with 100% Azobenzene Moieties: Thermo-Mechanical Phenomena and Behaviors

Azobenzene is one of the most investigated photo-responsive liquid crystalline molecules. It can isomerize between two different isoforms, trans (E) and cis (Z) configurations, when stimulated by light. It is used as a molecular engine in photo-mobile materials (PMPs). The use of liquid crystals (LCs) as building blocks enhances the mechanical properties of the PMPs. It is not easy to obtain PMPs with monodomain configurations when the LCs are 100% azobenzene. In this work, we studied three LC mixtures, describing the thermo/mechanical phenomena that regulate the actuation of such materials. The nematic temperature of the LC elastomers was measured and the PMPs carefully characterized for their bending and speed capability. Our finding suggests that the ratio between linear and cross-linker monomer greatly influences the nematic temperature of the mixture. Furthermore, 100% azobenzene materials polymerized using dicumyl peroxide can be useful to design polarization-selective switches

SERS Biosensor Based on Engineered 2D-Aperiodic Nanostructure for In-Situ Detection of Viable Brucella Bacterium in Complex Matrix

Brucella is a foodborne pathogen globally affecting both the economy and healthcare. Surface Enhanced Raman Spectroscopy (SERS) nano-biosensing can be a promising strategy for its detection. We combined high-performance quasi-crystal patterned nanocavities for Raman enhancement with the use of covalently immobilized Tbilisi bacteriophages as high-performing bio-receptors. We coupled our efficient SERS nano-biosensor to a Raman system to develop an on-field phage-based bio-sensing platform capable of monitoring the target bacteria. The developed biosensor allowed us to identify Brucella abortus in milk by our portable SERS device. Upon bacterial capture from samples (104 cells), a signal related to the pathogen recognition was observed, proving the concrete applicability of our system for on-site and in-food detection