Immunology of biodegradable nanoparticles: a brief overview on a wide growing field

Immunity is continuously evolving by evolutionary mechanisms shaped by pathogenic stimuli of different kinds. Man-made nanomaterials (NMs) have been developed in the last decades and represent a novel challenge for our immune system, especially when applied to medical science. Toxicological studies of such nanoparticles (NPs) revealed that size, shape, and surface chemistry are key parameters to understand their noxious effects on cellular mechanisms. Less is known on the immune reactions to NMs since prolonged exposure data are not so detailed as the results for acute administration. The importance of immunity to biocompatible NPs is underlined by their increasing use as drug or gene delivery carriers in common pharmaceutical preparations and vaccines. In the latter case, the immunomodulatory properties of NMs allow their use also as efficient adjuvants to enhance the innate immune response. In the current manuscript, the authors discuss the main concepts in this fast-growing field by restricting our view to NMs with consolidated application in biomedicine

Studio dell'effetto di metaboliti secondari sul potenziale di transattivazione di p53 in S.cerevisiae

p53, codificato dal gene oncosoppressore TP53, è un fattore di trascrizione cruciale per la vita della cellula. La sua funzione principale è, infatti, quella di mantenere l’integrità del genoma in risposta a vari tipi di stress come, per esempio, danno al DNA o attivazione di oncogeni. La risposta allo stress si esplica tramite l’avvio di vari processi come arresto del ciclo cellulare, senescenza ed apoptosi, grazie alla regolazione, da parte di p53, di diversi geni coinvolti nei medesimi processi. A sua volta l’attività di p53 può essere influenzata da numerosi fattori come proteine regolatrici e piccole molecole. Lo scopo della tesi è quello di valutare l’effetto di metaboliti secondari vegetali, saggiati singolarmente e/o in combinazione con farmaci antitumorali, sul potenziale di transattivazione di p53 in Saccharomyces cerevisiae, utilizzando un saggio funzionale di luciferasi precedentemente sviluppato. Il saggio è stato messo a punto con lo scopo di valutare l’effetto delle sostanze sul potenziale di transattivazione di un TF potendo usufruire di numerosi vantaggi, quali: - Espressione variabile del TF sotto il controllo del promotore inducibile GAL1,10 - Presenza in singola copia di un Responsive Element del TF e di geni reporter di controllo - Miglior assorbimento e accumulo delle molecole in esame grazie alla presenza di trasportatori ABC modificati - Utilizzo di piccoli volumi per i trattamenti e il saggio funzionale - Possibilità di co-esprimere il TF con altri cofattori - Adattabilità a diversi TF così come p53 - Applicabilità a diversi sistemi cellulari I metaboliti secondari testati, comunemente noti come saponine, sono stati estratti della pianta Astragalus verrucosus, in particolare in questo studio sono state saggiate frazioni contenenti diverse combinazioni di saponine e saponine singole quali Astraverrucina I, Astraverrucina III, Astraverrucina VI e Cicloalaroside D. Queste sostanze sono note per le loro proprietà farmaco-terapiche tra cui riduzione del colesterolo, riduzione del rischio di cancro, attività antiossidante ed immuno-stimolante. Come farmaci antitumorali sono stati utilizzati due agenti chemioterapici, quali Mitomicina C e Doxorubicina, sia singolarmente sia in combinazione con i suddetti metaboliti al fine di valutare un eventuale effetto delle saponine sull’azione del farmaco. Dai risultati ottenuti si evince che le frazioni di saponine mostrano effetti inibitori significativi sulla transattivazione di p53, mentre se testate in combinazione con Mitomicina C e Doxorubicina esercitano un effetto di ripristino dell’attività di p53 quando paragonate all’azione singolare del farmaco. Le saponine singole allo stesso modo mostrano un significativo effetto inibitoro sul potenziale di transattivazione di p53 quando testate singolarmente mentre incrementano l’azione inibitoria dei farmaci quando usate in combinazione. L'effetto delle frazioni di saponine è stato saggiato anche sul potenziale di transattivazione di p53 co-espressa con la proteina regolatrice MDM2, in grado di inibirne l'azione, al fine di verificare un comportamento simile a Nutlin, da parte delle medesime sostanze. Nutlin è, infatti, una molecola in grado di rompere il legame tra p53 e MDM2. Dai risultati è emerso che la maggior parte delle frazioni si comporta in modo simile a Nutlin, talvolta producendo un effetto maggiore. Infine si è verificato un eventuale effetto sull’espressione di p53 da parte delle sostanze utilizzando la tecnica del Western Blot. Nessuna sostanza ha mostrato di esercitare effetti significativi sulla sintesi di p53

Chemokine functionalized nanoparticles interactions with acute monocytic leukemia cells.

Biodegradable nanoparticles (NPs) aimed at drug/gene delivery can be functionalized with different molecules to have specific characteristics, like short bio-persistence, protection of the NP cargo and controlled release, specific cell/tissue targeting, as well as modulation of cellular signaling. To specifically improve NP selective targeting and modulation of immune responses, I focused my PhD project on the interactions between biocompatible NPs and immune cells. This goal was pursued by PLGA/Pluronic NP-surface decoration with two different chemokines, namely CXCL12 and CCL5. The first is a key mediator of homeostatic cell trafficking and it is involved in several pathologies, including cancers\u2019 metastasis. On the contrary, CCL5 is an induced chemokine attracting leukocytes during the inflammatory leukocyte effector phase. The rationale of the project relies on these molecules\u2019 roles to drive NPs towards specific cells exclusively expressing their cognate receptors, CXCR4 and CCR5. During the first two years I functionalized, characterized and tested the biological activity of chemokine-decorated PLGA-NPs in CXCR4 and CCR5 expressing human monocytic cells in vitro. I demonstrated CXCL12/CCL5-NPs stability in cell culture media and I proved that CXCL12-NPs enhanced binding/internalization abrogating CXCR4-mediated THP-1 leukemia cell chemotaxis. This result contributes to the search of biocompatible nanotools to quench tumor cell dysregulated migration. In the last year I optimized CCL5-NPs, further enhancing their cell targeting and internalization. I proved the dependence of CCL5-NP binding/internalization on the amount of surface-linked CCL5 and the unspecific adhesion of \u201cnaked\u201d PLGA/Pluronic-NPs

Epidemiology, ventilation management and outcomes of COVID–19 ARDS patients versus patients with ARDS due to pneumonia in the Pre–COVID era

Background: Ventilation management may differ between COVID–19 ARDS (COVID–ARDS) patients and patients with pre–COVID ARDS (CLASSIC–ARDS); it is uncertain whether associations of ventilation management with outcomes for CLASSIC–ARDS also exist in COVID–ARDS. Methods: Individual patient data analysis of COVID–ARDS and CLASSIC–ARDS patients in six observational studies of ventilation, four in the COVID–19 pandemic and two pre–pandemic. Descriptive statistics were used to compare epidemiology and ventilation characteristics. The primary endpoint were key ventilation parameters; other outcomes included mortality and ventilator–free days and alive (VFD–60) at day 60. Results: This analysis included 6702 COVID–ARDS patients and 1415 CLASSIC–ARDS patients. COVID–ARDS patients received lower median VT (6.6 [6.0 to 7.4] vs 7.3 [6.4 to 8.5] ml/kg PBW; p < 0.001) and higher median PEEP (12.0 [10.0 to 14.0] vs 8.0 [6.0 to 10.0] cm H2O; p < 0.001), at lower median ΔP (13.0 [10.0 to 15.0] vs 16.0 [IQR 12.0 to 20.0] cm H2O; p < 0.001) and higher median Crs (33.5 [26.6 to 42.1] vs 28.1 [21.6 to 38.4] mL/cm H2O; p < 0.001). Following multivariable adjustment, higher ΔP had an independent association with higher 60–day mortality and less VFD–60 in both groups. Higher PEEP had an association with less VFD–60, but only in COVID–ARDS patients. Conclusions: Our findings show important differences in key ventilation parameters and associations thereof with outcomes between COVID–ARDS and CLASSIC–ARDS. Trial registration: Clinicaltrials.gov (identifier NCT05650957), December 14, 2022

Natural Polysaccharide Nanomaterials: An Overview of Their Immunological Properties

Natural occurring polymers, or biopolymers, represent a huge part of our planet biomass. They are formed by long chains of monomers of the same type or a combination of different ones. Polysaccharides are biopolymers characterized by complex secondary structures performing several roles in plants, animals, and microorganisms. Because of their versatility and biodegradability, some of them are extensively used for packaging, food, pharmaceutical, and biomedical industries as sustainable and renewable materials. In the recent years, their manipulation at the nanometric scale enormously increased the range of potential applications, boosting an interdisciplinary research attempt to exploit all the potential advantages of nanostructured polysaccharides. Biomedical investigation mainly focused on nano-objects aimed at drug delivery, tissue repair, and vaccine adjuvants. The achievement of all these applications requires the deep knowledge of polysaccharide nanomaterials&rsquo; interactions with the immune system, which orchestrates the biological response to any foreign substance entering the body. In the present manuscript we focused on natural polysaccharides of high commercial importance, namely, starch, cellulose, chitin, and its deacetylated form chitosan, as well as the seaweed-derived carrageenan and alginate. We reviewed the available information on their biocompatibility, highlighting the importance of their physicochemical feature at the nanoscale for the modulation of the immune system

Chemokine-Decorated Nanoparticles Target Specific Subpopulations of Primary Blood Mononuclear Leukocytes

Specific cell targeting to deliver nanoparticles can be achieved by tailored modifications of the material surface with chemical moieties. The selection of the cell targets can be optimized by covering the nanoparticle with molecules, the receptor expression of which is restricted to particular cell subsets. Chemokines perform their biological action through 7-TM Gi-protein-coupled receptors differently expressed in all tissues. We decorated the surface of biocompatible polymer nanoparticles with full-length CCL5, an inflammatory chemokine that attracts leukocytes by binding CCR5, which is highly expressed in blood-circulating monocytes. Our observations showed that CCL5 functionalization does not affect the nanoparticle biocompatibility. Notably, CCL5 NPs delivered to PBMCs are selectively internalized by CCR5+ monocytes but not by CCR5- lymphocytes. The efficacy of PBMC subpopulation targeting by chemokine-decorated nanoparticles establishes an easy-to-use functionalization for specific leukocyte delivery

CXCL12-PLGA/Pluronic Nanoparticle Internalization Abrogates CXCR4-Mediated Cell Migration

Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as well as several processes involving cell migration. Among them, the role of CXCL12/CXCR4 signaling in cancer and metastasis is well known, and CXCR4 has been often targeted with small molecule-antagonists or short CXCL12-derived peptides to limit the pathological processes of cell migration and invasion. To reduce CXCR4-mediated chemotaxis, we adopted a different approach. We manufactured poly(lactic acid-co-glycolic acid) (PLGA)/Pluronic F127 nanoparticles through microfluidics-assisted nanoprecipitation and functionalized them with streptavidin to docking a biotinylated CXCL12 to be exposed on the nanoparticle surface. Our results show that CXCL12-decorated nanoparticles are non-toxic and do not induce inflammatory cytokine release in THP-1 monocytes cultured in fetal bovine and human serum-supplemented media. The cell internalization of our chemokine receptor-targeting particles increases in accordance with CXCR4 expression in FBS/medium. We demonstrated that CXCL12-decorated nanoparticles do not induce cell migration on their own, but their pre-incubation with THP-1 significantly decreases CXCR4+-cell migration, thereby antagonizing the chemotactic action of CXCL12. The use of biodegradable and immune-compatible chemokine-mimetic nanoparticles to reduce cell migration opens the way to novel antagonists with potential application in cancer treatments and inflammation

Marine Collagen-Based Bioink for 3D Bioprinting of a Bilayered Skin Model

Marine organisms (i.e., fish, jellyfish, sponges or seaweeds) represent an abundant and eco-friendly source of collagen. Marine collagen, compared to mammalian collagen, can be easily extracted, is water-soluble, avoids transmissible diseases and owns anti-microbial activities. Recent studies have reported marine collagen as a suitable biomaterial for skin tissue regeneration. The aim of this work was to investigate, for the first time, marine collagen from basa fish skin for the development of a bioink for extrusion 3D bioprinting of a bilayered skin model. The bioinks were obtained by mixing semi-crosslinked alginate with 10 and 20 mg/mL of collagen. The bioinks were characterised by evaluating the printability in terms of homogeneity, spreading ratio, shape fidelity and rheological properties. Morphology, degradation rate, swelling properties and antibacterial activity were also evaluated. The alginate-based bioink containing 20 mg/mL of marine collagen was selected for 3D bioprinting of skin-like constructs with human fibroblasts and keratinocytes. The bioprinted constructs showed a homogeneous distribution of viable and proliferating cells at days 1, 7 and 14 of culture evaluated by qualitative (live/dead) and qualitative (XTT) assays, and histological (H &amp; E) and gene expression analysis. In conclusion, marine collagen can be successfully used to formulate a bioink for 3D bioprinting. In particular, the obtained bioink can be printed in 3D structures and is able to support fibroblasts and keratinocytes viability and proliferation

Incorporation of Copper Nanoparticles on Electrospun Polyurethane Membrane Fibers by a Spray Method

Electrospinning is an easy and versatile technique to obtain nanofibrous membranes with nanosized fibers, high porosity, and pore interconnectivity. Metal nanoparticles (e.g., Ag, Cu, ZnO) exhibit excellent biocide properties due to their size, shape, release of metal ions, or reactive oxygen species production, and thus are often used as antimicrobial agents. In this study, a combined electrospinning/spray technique was employed to fabricate electrospun polyurethane membranes loaded with copper nanoparticles at different surface densities (10, 20, 25, or 30 mu g/cm(2)). This method allows particle deposition onto the surface of the membranes without the use of chemical agents. SEM images showed that polyurethane fibers own homogeneous thickness (around 650 nm), and that spray-deposited copper nanoparticles are evenly distributed. STEM-EDX demonstrated that copper nanoparticles are deposited onto the surface of the fibers and are not covered by polyurethane. Moreover, a uniaxial rupture test showed that particles are firmly anchored to the electrospun fibers. Antibacterial tests against model microorganisms Escherichia coli indicated that the prepared electrospun membranes possess good bactericidal effect. Finally, the antiviral activity against SARS-CoV-2 was about 90% after 1 h of direct contact. The obtained results suggested that the electrospun membranes possess antimicrobial activities and can be used in medical and industrial applications

One-step silver coating of polypropylene surgical mask with antibacterial and antiviral properties

Face masks can filter droplets containing viruses and bacteria minimizing the transmission and spread of respiratory pathogens but are also an indirect source of microbes transmission.A novel antibacterial and antiviral Ag-coated polypropylene surgical mask obtained through the in situ and one-step deposition of metallic silver nanoparticles, synthesized by silver mirror reaction combined with sonication or agitation methods, is proposed in this study.SEM analysis shows Ag nanoparticles fused together in a continuous and dense layer for the coating obtained by sonication, whereas individual Ag nanoparticles around 150 nm were obtained combining the silver mirror reaction with agitation. EDX, XRD and XPS confirm the presence of metallic Ag in both coatings and also oxidized Ag in samples by agitation. A higher amount of Ag nanoparticles is deposited on samples by sonication, as calculated by TGA. Further, both coatings are biocompatible and show antibacterial properties: coating by sonication caused 24 % and 40 % of bacterial reduction while coating by agitation 48 % and 96 % against S. aureus and E. coli, respectively. At 1 min of contact with SARS-CoV-2, the coating by agitation has an antiviral capacity of 75 % against 24 % of the one by sonication. At 1 h, both coatings achieve 100 % of viral inhibition. Nonetheless, larger samples could be produced only through the silver mirror reaction combined with agitation, preserving the integrity of the mask.In conclusion, the silver-coated mask produced by silver mirror reaction combined with agitation is scalable, has excellent physico-chemical characteristics as well as significant biological properties, with higher antimicrobial activities, providing additional protection and preventing the indirect transmission of pathogens