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Narcisa Mandras,1 Monica Argenziano,2 Mauro Prato,1 Janira Roana,1 Anna Luganini,3 Valeria Allizond,1 Vivian Tullio,1 Nicole Finesso,4 Sara Comini,1 Bruno Emilio Bressan,4 Francesca Pecoraro,4 Giuliana Giribaldi,4 Adriano Troia,5 Roberta Cavalli,2 Anna Maria Cuffini,1,* Giuliana Banche1,* 1Department of Public Health and Pediatric Sciences, University of Torino, Turin, 10126, Italy; 2Department of Drug Science and Technology, University of Torino, Turin, 10125, Italy; 3Department of Life Sciences and Systems Biology, University of Torino, Turin, 10123, Italy; 4Department of Oncology, University of Torino, Turin, 10126, Italy; 5Istituto Nazionale di Ricerca Metrologica, Turin, 10135, Italy*These authors contributed equally to this workCorrespondence: Valeria Allizond, Department of Public Health and Pediatric Sciences, University of Torino, Via Santena 9, Turin, 10126, Italy, Tel +390116705644, Fax +390112365644, Email valeria.allizond@unito.itPurpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) – methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata – and for their physical interaction with cell walls/membranes.Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy.Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h.Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.Keywords: chitosan nanodroplets, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Candida spp, chronic wounds

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Additionally, CWs are often worsened by microbial infections. Oxygen-loaded nanobubbles (OLNBs), displaying a peculiar structure based on oxygen-solving perfluorocarbons such as perfluoropentane in the inner core and polysaccharydes including chitosan in the outer shell, have proven effective in delivering oxygen to hypoxic tissues. Antimicrobial properties have been largely reported for chitosan. In the present work chitosan/perfluoropentane OLNBs were challenged for biocompatibility with human skin cells and ability to promote wound healing processes, as well as for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans . After cellular internalization, OLNBs were not toxic to human keratinocytes (HaCaT), whereas oxygen-free NBs (OFNBs) slightly affected their viability. Hypoxia-dependent inhibition of keratinocyte migratory ability after scratch was fully reversed by OLNBs, but not OFNBs. Both OLNBs and OFNBs exerted chitosan-induced short-term bacteriostatic activity against MRSA (up to 6 h) and long-term fungistatic activity against C. albicans (up to 24 h). Short-term antibacterial activity associated with NB prolonged adhesion to MRSA cell wall (up to 24 h) while long-term antifungal activity followed NB early internalization by C. albicans (already after 3 h of incubation). Taken altogether, these data support chitosan-shelled and perfluoropentane-cored OLNB potential as innovative, promising, non-toxic, and cost-effective antimicrobial devices promoting repair processes to be used for treatment of MRSA- and C. albicans -infected CWs., Competing Interests: The authors report no declarations of interest., (© 2022 The Authors. Published by Elsevier B.V.)

Aim: To evaluate the performance of urinary fibrinogen β-chain (FBC) - either alone or associated with urinary tyrosine-phosphorylated proteins (UPY) - as bladder cancer (BCa) diagnostic biomarker., Materials & Methods: 164 subjects were tested., Results: Significantly different FBC and UPY levels were found between BCa patients and controls, as well as between low-grade and high-grade cancers. The diagnostic accuracy was 0.84 for FBC and 0.87 for UPY. The combination of FBC and UPY improved the accuracy to 0.91. The addition of clinical variables (age, gender, and smoking habit) to FBC and UPY into a model for BCa prediction significantly improved the accuracy to 0.99. The combination of FBC and UPY adjusted for clinical variables associates with the highest sensitivity and good specificity., Conclusion: Urinary FBC and UPY could be used as biomarkers for BCa diagnosis., Competing Interests: Financial & competing interests disclosure This study was supported by the CRT Foundation Grant (Italy) Progetti Ordinari 2015, Cod. ROL 46644 to G Giribaldi and P Gontero. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript., (© 2021 The Authors.)

Persistent hypoxia is a main clinical feature of chronic wounds. Intriguingly, oxygen-loaded nanodroplets (OLNDs), filled with oxygen-solving 2H,3H-decafluoropentane and shelled with polysaccharides, have been proposed as a promising tool to counteract hypoxia by releasing a clinically relevant oxygen amount in a time-sustained manner. Here, four different types of chitosan (low or medium weight (LW or MW), glycol-(G-), and methylglycol-(MG-) chitosan) were compared as candidate biopolymers for shell manufacturing. The aim of the work was to design OLND formulations with optimized physico-chemical characteristics, efficacy in oxygen release, and biocompatibility. All OLND formulations displayed spherical morphology, cationic surfaces, ≤500 nm diameters (with LW chitosan-shelled OLNDs being the smallest), high stability, good oxygen encapsulation efficiency, and prolonged oxygen release kinetics. Upon cellular internalization, LW, MW, and G-chitosan-shelled nanodroplets did not significantly affect the viability, health, or metabolic activity of human keratinocytes (HaCaT cell line). On the contrary, MG-chitosan-shelled nanodroplets showed very poor biocompatibility. Combining the physico-chemical and the biological results obtained, LW chitosan emerges as the best candidate biopolymer for future OLND application as a skin device to treat chronic wounds.

Vancomycin (Vm) currently represents the gold standard against methicillin-resistant Staphylococcus aureus (MRSA) infections. However, it is associated with low oral bioavailability, formulation stability issues, and severe side effects upon systemic administration. These drawbacks could be overcome by Vm topical administration if properly encapsulated in a nanocarrier. Intriguingly, nanobubbles (NBs) are responsive to physical external stimuli such as ultrasound (US), promoting drug delivery. In this work, perfluoropentane (PFP)-cored NBs were loaded with Vm by coupling to the outer dextran sulfate shell. Vm-loaded NBs (VmLNBs) displayed ∼300nm sizes, anionic surfaces and good drug encapsulation efficiency. In vitro, VmLNBs showed prolonged drug release kinetics, not accompanied by cytotoxicity on human keratinocytes. Interestingly, VmLNBs were generally more effective than Vm alone in MRSA killing, with VmLNB antibacterial activity being more sustained over time as a result of prolonged drug release profile. Besides, VmLNBs were not internalized by staphylococci, opposite to Vm solution. Further US association promoted drug delivery from VmLNBs through an in vitro model of porcine skin. Taken together, these results support the hypothesis that proper Vm encapsulation in US-responsive NBs might be a promising strategy for the topical treatment of MRSA wound infections., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Chronic wounds, characterized by hypoxia, inflammation and impaired tissue remodeling, are often worsened by bacterial/fungal infections. Intriguingly, chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLNs) have proven effective in delivering oxygen to hypoxic tissues., Aim: The present work aimed at investigating nanodroplet antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) or Candida albicans, toxicity on human keratinocytes (HaCaT) and ultrasound (US)-triggered transdermal delivery., Materials & Methods: Nanodroplet antibacterial/antifungal properties, human cytotoxicity, and US-triggered transdermal delivery were measured through microbiological, biochemical, and sonophoresis assays, respectively., Results: OLNs and oxygen-free nanodroplets (OFNs) displayed short- or long-term cytostatic activity against MRSA or Candida albicans, respectively. OLNs were not toxic to keratinocytes, whereas OFNs slightly affected cell viability. Complementary US treatment promoted OLN transdermal delivery., Conclusion: As such, US-activated chitosan-shelled OLNs appear as promising, nonconventional and innovative tools for adjuvant treatment of infected chronic wounds.