The role of specific serum/plasma proteins in the modulation of the cellular response to amorphous silica nanoparticles

Nanoparticles are structures of different dimensions (1-100 nm) and composition (metal oxides, organic acid polymers, silica polymers) present in the environment as a consequence of natural processes (such as volcanic eruptions or dusts erosion) or anthropogenic activities (industrialization or pollution). In the last decades they have been intensely studied and engineered for industrial applications (as additive of food, cosmetics and building materials) and medical purposes, where they can be employed as drug carriers or imaging agents. Thanks to its abundance, cheapness and resistance to a variety of environmental perturbations, silicon dioxide (SiO2) is one of the most widely used materials in both industrial and biomedical fields in its amorphous form (contrary to crystalline silica that causes silicosis, a chronic pulmonary disease common in occupational categories largely exposed to silica crystals, such a miners and ceramic workers). Despite amorphous silica is considered much less dangerous than crystalline silica, recent evidences have shown cytotoxic and pro-inflammatory potential in in vitro and animal models. To shed more light on this topic, in the first part of my PhD work I have characterized amorphous silica toxicity and inflammatory effects in primary human monocytes and macrophages (myeloid professional phagocytic cells) and on primary human lymphocytes and HeLa cells (lymphoid and epithelial non-phagocytic cells), using as nanoparticles model the commercial non labeled Ludox TM40 (29 nm Ø) and the fluorescein labeled Stöber (35 nm Ø). In particular, the influence of serum and the possible mechanisms determining silica nanoparticles (SiO2-NPs) effects have been investigated. I have found that SiO2-NPs toxicity (evaluated as mitochondrial dysfunction and plasma membrane permeabilization) was stronger in phagocytes (LD50 after 18 h exposure 40 µg/ml) and, in these cells, associated to the production of the three main inflammatory cytokines (IL-1 beta, TNF- alpha and IL-6). On the contrary, non phagocytic cells were much more resistant (LD50 after 18 h exposure 300-500 µg/ml) and, curiously, HeLa cells were subjected apoptosis after SiO2-NPs treatment (while monocytes, macrophages and lymphocytes became necrotic). Cytofluorimetric and confocal microscopy analysis have shown that SiO2-NPs were engulfed in acidic compartments (phagolysosomes) in monocytes and macrophages, while they mostly localized onto plasma membrane in HeLa cells. This suggested that the higher sensitivity of the two phagocytic models could be due to the more efficient internalization of the particles, followed by lysosomal rupture (as indicated by experiments showing the decrease of the fluorescence associated to the lysosomotropic agent Lysotracker upon cellular exposure to high NPs doses) and the consequent liberation of lysosomal proteases (as reported in literature for crystalline silica). To investigate if acidic lysosomal pH could influence SiO2-NPs cytotoxicity in phagocytes, cells were treated with silica in the absence or in the presence of two pH neutralizing agents (NH4Cl and Bafilomycin AI), resulting in a significant protective effect in monocytes and in a negligible protection in macrophages. As mentioned above, we found that in myeloid cells SiO2-NPs induced an inflammatory response (more pronounced in monocytes), starting in the correspondence of the beginning of cytotoxicity, reaching a peak and decreasing at high NPs doses because of the strong and anticipated cellular death. In particular, IL-1b was the cytokine most represented in both cellular types, while TNF-alpha and IL-6 levels were lowers. Moreover, in monocytes (and, in less degree in macrophages) IL-1beta production was synergized by the co-stimulation with silica and lypopolisaccharide (LPS). Since crystalline silica is known to activate the NLRP3 inflammasome (a cytosolic multiprotein complex responsible of the production of some inflammatory cytokines, primarily IL-1beta) we investigated NLRP3 activation by amorphous SiO2-NPs in our myeloid cellular models. We found that monocytes and macrophages treatment with SiO2-NPs increased pro-IL1beta levels, and that its conversion into mature IL-1beta involved caspase 1 activation, intracellular ATP release and subsequent binding to ATP receptor P2X7. Interestingly, P2X7 blockage did not affect SiO2-NPs induced cellular death in both cells, and caspase 1 inhibition did not reduce SiO2-NPs toxicity in macrophages but showed a protective effect in monocytes, suggesting that amorphous silica might induce pyroptosis (a caspase 1 dependent cellular death) in these cells. Afterwards, I have studied how serum could modulate SiO2-NPs toxicity and pro inflammatory effects. In the presence of increasing FCS amount both SiO2-NPs cytotoxicity threshold and LD50 were shifted to higher NPs doses, indicating a serum protective action (more pronounced in non-phagocytic cells in comparison to phagocytes). In parallel, also inflammatory cytokines production in myeloid cells occurred at higher NPs concentrations with the increment of FCS percentage. To investigate if the protective serum effect reflected a modification in NPs cellular association I have performed experiments with the fluorescent Stöber NPs, finding that the presence of serum strongly decreased NPs cellular binding in lymphocytes and HeLa, with a moderate reduction in monocytes and macrophages. This result was in accord with the stronger serum protection in non phagocytic cells, and consolidated the hypothesis that SiO2-NPs toxicity depends on their interaction level with cells. Also SiO2-NPs cellular localization was affected by different serum concentrations. In particular, I have found that in the absence of serum SiO2-NPs mostly localized onto the plasma membrane in monocytes and HeLa, while in macrophages they were still internalized into phagolysosomes, even if with a lower efficiency. Considering this different NPs sub-cellular localization, I have investigated if the protective effect of NH4Cl, Bafilomycin AI and caspase 1 inhibitor observed in monocytes in standard culture conditions (10% FCS) was maintained also in the absence of serum. Interestingly, neither acidic compartments neutralization nor caspase 1 blockage were able to reduce SiO2-NPs toxicity in serum free conditions, suggesting that monocytes cellular death mechanism was different with or without serum. In the second part of my PhD work I have studied several aspects of nanoparticles interaction with plasma and serum proteins, since this is a very important point in nanomaterials biomedical applications. Indeed, when nanoparticles (functioning as drug-gene vectors or imaging agents) are introduced in the human body (primary, into the bloodstream), they are rapidly coated by a series of specific proteins, forming the so called “ nanoparticles corona” and mediating cellular response to nanomaterial. This problematic was approached by using Ludox TM40 as nanoparticles model (to maintain the continuity with the previous characterization work), and performing a comparative analysis between fetal calf serum and human plasma. I have found that the main proteins adsorbed to NPs surface were plasminogen, albumin, apolipoprotein AI (ApoAI), hemoglobin and apolipoprotein AII (ApoAII) from FCS, and immunoglobulins (IgG), histidine rich glycoprotein (HRG), albumin, apolipoprotein AI, apolipoprotein AII and apolipoprotein CIII (ApoCIII) from human plasma. In both situations, albumin (the most abundant plasma/serum protein) was the principal polypeptide bound to NPs at very low serum/plasma concentrations, while at greater serum/plasma doses it was displaced by less plentiful proteins (over all, apolipoproteins), indicating an higher affinity of these latter for SiO2-NPs surface. Congruent with this, NPs firstly absorbed plasminogen, apolipoproteins and hemoglobin, while once these proteins were depleted from serum albumin started to bind NPs surface proportionally to NPs dose. I have also found that the serum proteins pattern associated to NPs surface was not rearranged from a neutral environment (representative of cytosol) to an acidic environment (representative of endo-lysosomes). Finally, I have investigated how single plasma opsonines could influence SiO2-NPs biological activity, finding that IgG and HRG did not protect cells against NPs toxicity, while albumin and, over all, high density lipoproteins (the complexes containing ApoAI, ApoAII and ApoCIII) strongly reduced NPs adverse effects by inhibiting NPs cellular associatio

Recent Advances in Biomedical, Therapeutic and Pharmaceutical Applications of Microbial Surfactants

The spread of antimicrobial-resistant pathogens typically existing in biofilm formation and the recent COVID-19 pandemic, although unrelated phenomena, have demonstrated the urgent need for methods to combat such increasing threats. New avenues of research for natural molecules with desirable properties to alleviate this situation have, therefore, been expanding. Biosurfactants comprise a group of unique and varied amphiphilic molecules of microbial origin capable of inter-acting with lipidic membranes/components of microorganisms and altering their physicochemical properties. These features have encouraged closer investigations of these microbial metabolites as new pharmaceutics with potential applications in clinical, hygiene and therapeutic fields. Mounting evidence has indicated that biosurfactants have antimicrobial, antibiofilm, antiviral, immunomodu-latory and antiproliferative activities that are exploitable in new anticancer treatments and wound healing applications. Some biosurfactants have already been approved for use in clinical, food and environmental fields, while others are currently under investigation and development as antimicrobials or adjuvants to antibiotics for microbial suppression and biofilm eradication strategies. Moreover, due to the COVID-19 pandemic, biosurfactants are now being explored as an alternative to current products or procedures for effective cleaning and handwash formulations, antiviral plastic and fabric surface coating agents for shields and masks. In addition, biosurfactants have shown promise as drug delivery systems and in the medicinal relief of symptoms associated with SARS-CoV-2 acute respiratory distress syndrome

The Work Agentic Capabilities (WAC) questionnaire: validation of a new measure

Agentic capabilities refer to the basic capabilities of mind that, according to social cognitive theory, allow people to proactively influence their functioning and external context. This study presents a new scale, namely the Work Agentic Capabilities (WAC) questionnaire, that consists of 28 items and measures forethought capability, self-regulation capability, self-reflection capability and vicarious capability in the organizational context. Accordingly, an exploratory (N = 290) and a confirmatory factor analysis (N = 300) demonstrated a four-factor structure. Agentic capabilities were positively correlated with psychological capital and its dimensions (i.e., self-efficacy, hope, optimism and resiliency), positive job attitudes (work engagement and job satisfaction), proactive organizational behaviours (job crafting and organizational citizenship behaviours), perceived job performance, and promotion prospects. Finally, we discuss meaningful differences in the mean values of agentic capabilities associated with sociodemographic and organizational variables. Results suggest that the WAC questionnaire can be reliably used to measure agentic capabilities.Las capacidades agénticas aluden a aquellas capacidades de la mente que según la teoría social cognitiva permiten a las personas influir de un modo proactivo en su funcionamiento y en el contexto externo. El presente estudio presenta una nueva escala, el cuestionario de Capacidades Agénticas en el Trabajo (WAC, por sus siglas en inglés), compuesto de 28 ítems que mide la capacidad de previsión, autorregulación, autorreflexión y vicaria en el contexto organizativo. Un estudio con análisis factorial exploratorio (N = 290) y confirmatorio (N = 300) descubrió una estructura de cuatro factores. Las capacidades agénticas correlacionaban positivamente con el capital psicológico y sus dimensiones (es decir, autoeficacia, esperanza, optimismo y resiliencia), las actitudes laborales positivas (engagement y satisfacción en el trabajo), la percepción del desempeño laboral y las perspectivas de promoción. Por último abordamos las diferencias significativas de los valores medios de las capacidades agénticas asociadas a las variables sociodemográficas y organizativas. Los resultados indican que el cuestionario WAC puede utilizarse de modo fiable para medir las capacidades agénticas

Formyl-peptide Receptor Agonists and Amorphous SiO2-NPs Synergistically and Selectively Increase the Inflammatory Responses of Human Monocytes and PMNs

We tested whether amorphous SiO2-NPs and formyl-peptide receptor (FPRs) agonists synergistically activate human monocytes and neutrophil polymorphonuclear granulocytes (PMNs). Peptide ligands specifically binding to FPR1 (f-MLP) and to FPR2 (MMK-1, WKYMVM and WKYMVm) human isoforms did not modify the association of SiO2-NPs to both cell types or their cytotoxic effects. Similarly, the extent of CD80, CD86, CD83, ICAM-1 and MHCII expression in monocytes treated with SiO2-NPs was not significantly altered by any FPRs agonist. However, FPR1 stimulation with f-MLP strongly increased the secretion of IL-1β, IL-6 and IL-8 by human monocytes, and of IL-8 by PMNs in the presence of SiO2-NPs, due to the synergic stimulation of gene transcription. FPR2 agonists also up-modulated the production of IL-1β induced by monocytes treated with SiO2-NPs. In turn, SiO2-NPs increased the chemotaxis of PMNs toward FPR1-specific ligands, but not toward FPR2-specific ones. Conversely, the chemotaxis of monocytes toward FPR2-specific peptides was inhibited by SiO2-NPs. NADPH-oxidase activation triggered by FPR1- and FPR2-specific ligands in both cell types was not altered by SiO2-NPs. Microbial and tissue danger signals sensed by FPRs selectively amplified the functional responses of monocytes and PMNS to SiO2-NPs, and should be carefully considered in the assessment of the risk associated with nanoparticle exposure

Mortality from infectious diseases in diabetes

Background and Aims: to investigate the risk of mortality from infections by comparing the 33 underlying causes of death versus the multiple causes of death in known diabetic subjects living in 34 the Veneto Region, Northern Italy. 35 Methods and Results: 185,341 diabetic subjects aged 30-89 years were identified in the year 2010 36 and causes of death were assessed from 2010 to 2015. Standardized Mortality Ratios (SMR) with 37 95% confidence intervals were computed with regional mortality rates as reference. The underlying 38 causes of death and all the diseases reported in the death certificates were scrutinized. At the end of 39 the follow-up, 36,382 subjects had deceased. We observed an increased risk of death from 40 infection-related causes in subjects affected by diabetes with a SMR of 1.83 (95 % CI, 1.71-1.94). 41 The SMR for death from septicemia was 1.91 (95 % CI, 1.76-2.06) and from pneumonia 1.47 (95 % 42 CI, 1.36-1.59). The use of the multiple causes of death approach emphasized the contribution of 43 infectious diseases to mortality. 44 CONCLUSION: the results of the present study demonstrate an excess mortality from infection45 related diseases in patients affected by diabetes and, more interestingly, show a possible 46 underestimation of the impact of these conditions by routine mortality analyses

C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity (Kd = 11 \ub1 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps

A straightforward multiparametric quality control protocol for proton magnetic resonance spectroscopy: Validation and comparison of various 1.5 T and 3 T clinical scanner systems

Purpose: The aim of this study was to propose and validate across various clinical scanner systems a straightforward multiparametric quality assurance procedure for proton magnetic resonance spectroscopy (MRS). Methods: Eighteen clinical 1.5 T and 3 T scanner systems for MRS, from 16 centres and 3 different manufacturers, were enrolled in the study. A standard spherical water phantom was employed by all centres. The acquisition protocol included 3 sets of single (isotropic) voxel (size 20 mm) PRESS acquisitions with unsuppressed water signal and acquisition voxel position at isocenter as well as off-center, repeated 4/5 times within approximately 2 months. Water peak linewidth (LW) and area under the water peak (AP) were estimated. Results: LW values [mean (standard deviation)] were 1.4 (1.0) Hz and 0.8 (0.3) Hz for 3 T and 1.5 T scanners, respectively. The mean (standard deviation) (across all scanners) coefficient of variation of LW and AP for different spatial positions of acquisition voxel were 43% (20%) and 11% (11%), respectively. The mean (standard deviation) phantom T2 values were 1145 (50) ms and 1010 (95) ms for 1.5 T and 3 T scanners, respectively. The mean (standard deviation) (across all scanners) coefficients of variation for repeated measurements of LW, AP and T2 were 25% (20%), 10% (14%) and 5% (2%), respectively. Conclusions: We proposed a straightforward multiparametric and not time consuming quality control protocol for MRS, which can be included in routine and periodic quality assurance procedures. The protocol has been validated and proven to be feasible in a multicentre comparison study of a fairly large number of clinical 1.5 T and 3 T scanner systems

Prolonged higher dose methylprednisolone vs. conventional dexamethasone in COVID-19 pneumonia: a randomised controlled trial (MEDEAS)

Dysregulated systemic inflammation is the primary driver of mortality in severe COVID-19 pneumonia. Current guidelines favor a 7-10-day course of any glucocorticoid equivalent to dexamethasone 6 mg·day-1. A comparative RCT with a higher dose and a longer duration of intervention was lacking

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

The role of specific serum/plasma proteins in the modulation of the cellular response to amorphous silica nanoparticles

Nanoparticles are structures of different dimensions (1-100 nm) and composition (metal oxides, organic acid polymers, silica polymers) present in the environment as a consequence of natural processes (such as volcanic eruptions or dusts erosion) or anthropogenic activities (industrialization or pollution). In the last decades they have been intensely studied and engineered for industrial applications (as additive of food, cosmetics and building materials) and medical purposes, where they can be employed as drug carriers or imaging agents. Thanks to its abundance, cheapness and resistance to a variety of environmental perturbations, silicon dioxide (SiO2) is one of the most widely used materials in both industrial and biomedical fields in its amorphous form (contrary to crystalline silica that causes silicosis, a chronic pulmonary disease common in occupational categories largely exposed to silica crystals, such a miners and ceramic workers). Despite amorphous silica is considered much less dangerous than crystalline silica, recent evidences have shown cytotoxic and pro-inflammatory potential in in vitro and animal models. To shed more light on this topic, in the first part of my PhD work I have characterized amorphous silica toxicity and inflammatory effects in primary human monocytes and macrophages (myeloid professional phagocytic cells) and on primary human lymphocytes and HeLa cells (lymphoid and epithelial non-phagocytic cells), using as nanoparticles model the commercial non labeled Ludox TM40 (29 nm Ø) and the fluorescein labeled Stöber (35 nm Ø). In particular, the influence of serum and the possible mechanisms determining silica nanoparticles (SiO2-NPs) effects have been investigated. I have found that SiO2-NPs toxicity (evaluated as mitochondrial dysfunction and plasma membrane permeabilization) was stronger in phagocytes (LD50 after 18 h exposure 40 µg/ml) and, in these cells, associated to the production of the three main inflammatory cytokines (IL-1 beta, TNF- alpha and IL-6). On the contrary, non phagocytic cells were much more resistant (LD50 after 18 h exposure 300-500 µg/ml) and, curiously, HeLa cells were subjected apoptosis after SiO2-NPs treatment (while monocytes, macrophages and lymphocytes became necrotic). Cytofluorimetric and confocal microscopy analysis have shown that SiO2-NPs were engulfed in acidic compartments (phagolysosomes) in monocytes and macrophages, while they mostly localized onto plasma membrane in HeLa cells. This suggested that the higher sensitivity of the two phagocytic models could be due to the more efficient internalization of the particles, followed by lysosomal rupture (as indicated by experiments showing the decrease of the fluorescence associated to the lysosomotropic agent Lysotracker upon cellular exposure to high NPs doses) and the consequent liberation of lysosomal proteases (as reported in literature for crystalline silica). To investigate if acidic lysosomal pH could influence SiO2-NPs cytotoxicity in phagocytes, cells were treated with silica in the absence or in the presence of two pH neutralizing agents (NH4Cl and Bafilomycin AI), resulting in a significant protective effect in monocytes and in a negligible protection in macrophages. As mentioned above, we found that in myeloid cells SiO2-NPs induced an inflammatory response (more pronounced in monocytes), starting in the correspondence of the beginning of cytotoxicity, reaching a peak and decreasing at high NPs doses because of the strong and anticipated cellular death. In particular, IL-1b was the cytokine most represented in both cellular types, while TNF-alpha and IL-6 levels were lowers. Moreover, in monocytes (and, in less degree in macrophages) IL-1beta production was synergized by the co-stimulation with silica and lypopolisaccharide (LPS). Since crystalline silica is known to activate the NLRP3 inflammasome (a cytosolic multiprotein complex responsible of the production of some inflammatory cytokines, primarily IL-1beta) we investigated NLRP3 activation by amorphous SiO2-NPs in our myeloid cellular models. We found that monocytes and macrophages treatment with SiO2-NPs increased pro-IL1beta levels, and that its conversion into mature IL-1beta involved caspase 1 activation, intracellular ATP release and subsequent binding to ATP receptor P2X7. Interestingly, P2X7 blockage did not affect SiO2-NPs induced cellular death in both cells, and caspase 1 inhibition did not reduce SiO2-NPs toxicity in macrophages but showed a protective effect in monocytes, suggesting that amorphous silica might induce pyroptosis (a caspase 1 dependent cellular death) in these cells. Afterwards, I have studied how serum could modulate SiO2-NPs toxicity and pro inflammatory effects. In the presence of increasing FCS amount both SiO2-NPs cytotoxicity threshold and LD50 were shifted to higher NPs doses, indicating a serum protective action (more pronounced in non-phagocytic cells in comparison to phagocytes). In parallel, also inflammatory cytokines production in myeloid cells occurred at higher NPs concentrations with the increment of FCS percentage. To investigate if the protective serum effect reflected a modification in NPs cellular association I have performed experiments with the fluorescent Stöber NPs, finding that the presence of serum strongly decreased NPs cellular binding in lymphocytes and HeLa, with a moderate reduction in monocytes and macrophages. This result was in accord with the stronger serum protection in non phagocytic cells, and consolidated the hypothesis that SiO2-NPs toxicity depends on their interaction level with cells. Also SiO2-NPs cellular localization was affected by different serum concentrations. In particular, I have found that in the absence of serum SiO2-NPs mostly localized onto the plasma membrane in monocytes and HeLa, while in macrophages they were still internalized into phagolysosomes, even if with a lower efficiency. Considering this different NPs sub-cellular localization, I have investigated if the protective effect of NH4Cl, Bafilomycin AI and caspase 1 inhibitor observed in monocytes in standard culture conditions (10% FCS) was maintained also in the absence of serum. Interestingly, neither acidic compartments neutralization nor caspase 1 blockage were able to reduce SiO2-NPs toxicity in serum free conditions, suggesting that monocytes cellular death mechanism was different with or without serum. In the second part of my PhD work I have studied several aspects of nanoparticles interaction with plasma and serum proteins, since this is a very important point in nanomaterials biomedical applications. Indeed, when nanoparticles (functioning as drug-gene vectors or imaging agents) are introduced in the human body (primary, into the bloodstream), they are rapidly coated by a series of specific proteins, forming the so called “ nanoparticles corona” and mediating cellular response to nanomaterial. This problematic was approached by using Ludox TM40 as nanoparticles model (to maintain the continuity with the previous characterization work), and performing a comparative analysis between fetal calf serum and human plasma. I have found that the main proteins adsorbed to NPs surface were plasminogen, albumin, apolipoprotein AI (ApoAI), hemoglobin and apolipoprotein AII (ApoAII) from FCS, and immunoglobulins (IgG), histidine rich glycoprotein (HRG), albumin, apolipoprotein AI, apolipoprotein AII and apolipoprotein CIII (ApoCIII) from human plasma. In both situations, albumin (the most abundant plasma/serum protein) was the principal polypeptide bound to NPs at very low serum/plasma concentrations, while at greater serum/plasma doses it was displaced by less plentiful proteins (over all, apolipoproteins), indicating an higher affinity of these latter for SiO2-NPs surface. Congruent with this, NPs firstly absorbed plasminogen, apolipoproteins and hemoglobin, while once these proteins were depleted from serum albumin started to bind NPs surface proportionally to NPs dose. I have also found that the serum proteins pattern associated to NPs surface was not rearranged from a neutral environment (representative of cytosol) to an acidic environment (representative of endo-lysosomes). Finally, I have investigated how single plasma opsonines could influence SiO2-NPs biological activity, finding that IgG and HRG did not protect cells against NPs toxicity, while albumin and, over all, high density lipoproteins (the complexes containing ApoAI, ApoAII and ApoCIII) strongly reduced NPs adverse effects by inhibiting NPs cellular associationLe nanoparticelle sono strutture di diverse dimensioni (1-100 nm) e varia composizione (ossidi metallici, polimeri di silice, polimeri di acidi organici), presenti nell’ambiente come conseguenza di processi naturali (eruzioni vulcaniche, erosione di rocce) o antropogenici (inquinamento, attività industriale). Negli ultimi decenni esse sono state intensamente studiate e ingegnerizzate a scopo industriale (come additivi di cibi, cosmetici e materiali utilizzati nell’edilizia) e nell’ambito biomedico, in cui possono essere impiegate come vettori per farmaci o agenti di imaging. Grazie alla sua abbondanza, economicità e resistenza, il biossido di silicio (SiO2) è, nella sua forma amorfa, uno dei materiali maggiormente usati sia in ambito industriale che biomedico (contrariamente alla forma cristallina che provoca l’insorgenza della silicosi, una malattia polmonare cronica molto diffusa nei minatori, lavoratori di ceramica e altre categorie occupazionali quotidianamente esposte a cristalli/fibre di silicio). Nonostante la silice amorfa sia considerata molto meno pericolosa di quella cristallina, studi recenti condotti in vitro e su modelli animali hanno messo in luce un potenziale citotossico e pro infiammatorio. Per chiarificare questo aspetto, nella prima parte del mio dottorato ho caratterizzato la citotossicità e l’induzione di una risposta pro infiammatoria di nanoparticelle di silice amorfa (la variante commerciale non fluorescente Ludox TM40 e la variante fluorescente Stöber) su due modelli di cellula fagocitica (monociti e macrofagi primari umani) e in due modelli di cellula non fagocitica (linfociti primari umani e la linea stabile epiteliale HeLa). In particolare, mi sono concentrata sui possibili meccanismi coinvolti nell’azione delle nanoparticelle e su come il siero possa influenzarli. Un primo risultato ha indicato una maggior tossicità delle nanoparticelle di silice (valutata in termini di disfunzione mitocondriale e permeabilizzazione della membrana) nelle cellule fagocitiche, associata anche alla produzione delle tre principali citochine pro infiammatorie (IL-1beta, TNF-alfa and IL-6). Più in dettaglio, la dose di nanoparticelle in grado di uccidere il 50% delle cellule (LD50) dopo un trattamento di 18 h è risultata essere di 40 µg/ml, mentre le cellule non fagocitiche hanno mostrato una maggior resistenza alle nanoparticelle (LD50 300-500 µg/ml). Analizzando il tipo di morte indotta dalle nanoparticelle di silice, le cellule HeLa hanno mostrato un fenotipo apoptotico, mentre i monociti, macrofagi e linfociti sono risultati andare incontro a necrosi. Da una valutazione al citofluorimetro e al microscopio confocale dell’associazione e della localizzazione cellulare delle nanoparticelle è emerso che queste venivano internalizzate in compartimenti acidi nelle due cellule fagocitiche, mentre nelle cellule HeLa rimanevano legate alla membrana plasmatica. Questo risultato ha suggerito che la maggior sensibilità dei fagociti fosse dovuta a una maggior captazione delle nanoparticelle che, una volta accumulate all’interno di endo-lisosomi, potessero provocarne la rottura con la conseguente liberazione di enzimi litici (proteasi, idrolasi, lipasi) in grado di danneggiare la cellula. A questo proposito, è stato valutato il contributo dell’ambiente acido degli endo-lisosomi alla tossicità delle nanoparticelle di silice nei fagociti trattando le cellule in presenza o in assenza di due agenti neutralizzanti (NH4Cl o Bafilomicina AI), ottenendo una diminuzione della citotossicità della silice nei monociti e solo un lieve effetto nei macrofagi. Come anticipato precedentemente, le nanoparticelle di silice si sono mostrate in grado di indurre una risposta infiammatoria (più evidente nei monociti) caratterizzata da un’iniziale fase di latenza fino al raggiungimento della soglia di tossicità, un picco centrale e una fase finale decrescente (in corrispondenza delle dosi più alte di nanoparticelle) a causa della forte e anticipata morte cellulare. In particolare, l’ IL-1beta è risultata essere la citochina prodotta più abbondantemente, seguita dal TNF-alfa e dall’IL-6; inoltre, in copresenza di nanoparticelle e LPS essa veniva secreta in modo sinergico. Dal momento che la silice cristallina è in grado di attivare l’inflammasoma NLRP3 (un complesso multi proteico citosolico responsabile della produzione di alcune citochine pro infiammatorie, prima fra tutte l’ IL-1beta una parte del lavoro di tesi è stata dedicata allo studio dell’attivazione di NLRP3 da parte di nanoparticelle di silice amorfa nei nostri due modelli di cellula fagocitica. Inizialmente è stata evidenziata sia in monociti che in macrofagi la capacità delle nanoparticelle di silice amorfa di aumentare i livelli di pro IL1-beta e stimolarne la conversione nella forma matura IL-1beta tramite un processo dipendente dall’attivazione della caspasi 1, della secrezione di ATP ed dal successivo legame di ATP al suo recettore P2X7. Inoltre, a seguito del blocco di P2X7 con uno specifico inibitore la mortalità indotta dalle nanoparticelle di silice non ha subito variazioni sia nei monociti che nei macrofagi, mentre i monociti hanno mostrato una maggior resistenza alle nanoparticelle in presenza di un inibitore della caspasi 1, segno di una possibile morte per piroptosi causata dalle nanoparticelle in queste cellule. Un altro aspetto importante presentato in questa tesi di dottorato riguarda l’influenza del siero (FCS) sugli effetti citotossici e pro infiammatori indotti dalle nanoparticelle di silice amorfa. Innanzitutto, all’aumentare della concentrazione del siero sia la soglia di tossicità sia l’LD50 delle nanoparticelle di silice sono risultate spostate verso valori più alti, indicando un effetto protettivo dell’ FCS (più evidente nei non fagociti rispetto ai fagociti), cosi come la produzione di citochine pro infiammatorie nei monociti e nei macrofagi. Per capire se questo spostamento fosse dovuto a una diversa associazione delle nanoparticelle alle cellule sono stati fatti esperimenti con le nanoparticelle fluorescenti Stöber, che hanno evidenziato come la presenza di siero fosse in grado di diminuire il legame fra la nanoparticelle e le cellule, in particolare nel caso dei linfociti e delle HeLa. Questo risultato è in accordo con la precedente osservazione di un maggior effetto protettivo del siero nei non fagociti, e rafforza l’ipotesi che la tossicità delle nanoparticelle sia in qualche modo legata al loro livello di interazione con le cellule. Inoltre, anche la localizzazione intracellulare delle nanoparticelle è risultata essere influenzata dalla concentrazione di siero. In particolare, in assenza di siero le nanoparticelle erano prevalentemente distribuite sulla membrana cellulare nei monociti e nelle HeLa, mentre nei macrofagi venivano internalizzate in fago-lisosomi, anche se meno efficientemente che con 10% FCS. Vista la diversa localizzazione subcellulare nelle due diverse condizioni di siero, ci si è chiesti se l’effetto protettivo dell’ NH4CL, Bafilomicina AI e dell’inibitore della caspasi 1 osservato nei monociti nelle condizioni di coltura standard (10% FCS) venisse mantenuto anche in assenza di siero. Né la neutralizzazione dei compartimenti acidi né l’inibizione della caspasi 1 si sono dimostrati efficaci nel prevenire la tossicità, indicando che nei monociti il meccanismo di morte cellulare fosse diverso in assenza o in presenza di siero. Nella seconda parte della mia tesi di dottorato ho analizzato diversi aspetti dell’interazione delle nanoparticelle con le proteine del plasma e del siero, essendo questo un aspetto cruciale nell’applicazione dei nanomateriali in campo biomedico. Infatti, nanoparticelle introdotte nell’organismo (in particolare del circolo sanguigno come vettori per farmaci o agenti d’immagine) vengono rapidamente rivestite da una serie di proteine (costituenti la cosiddetta “corona di proteine”) in grado di mediare l’interazione cellula-nanoparticella. Questa problematica è stata affrontata utilizzando come nanoparticelle modello le Ludox TM40 (per mantenere la continuità con la caratterizzazione fatta in precedenza) ed eseguendo esperimenti in parallelo con il siero fetale bovino ed il plasma umano. Le principali proteine del siero bovino legate alle nanoparticelle di silice sono risultate essere il plasminogeno, l’albumina, le apolipoproteine AI e AII e l’emoglobina, mentre quelle del plasma umano le immunoglobuline, l’histidine rich glycoprotein, l’albumina e le apolipoproteine AI, AII e CIII. Il pattern di proteine adsorbite alle nanoparticelle di silice ha evidenziato che a basse concentrazioni di siero/plasma la principale proteina legata era l’albumina (la proteina più abbondante del siero/plasma), mentre all’aumentare della concentrazione di siero/plasma essa veniva “spiazzata” da proteine meno abbondanti (in primis dalle apolipoproteine), suggerendo una maggiore affinità di queste ultime per la superficie di nanoparticelle di silice amorfa. In accordo con questa ipotesi, le nanoparticelle inizialmente legavano il plasminogeno, l’emoglobina e le apolipoproteine, mentre solo quando queste proteine venivano esaurite dal siero (in presenza di alte dosi di nanoparticelle) iniziava a legarsi l’albumina, proporzionalmente alla quantità di nanoparticelle presenti. Dall’analisi del pattern di proteine legate alla silice in presenza di differenti pH è emerso che esso non subiva variazioni rilevanti in presenza di un ambiente neutro (rappresentativo del citoplasma) o di un ambiente acido (rappresentativo degli endo-lisosomi). Infine, esperimenti preliminari su come le singole proteine della corona potessero influenzare l’attività biologica delle nanoparticelle hanno indicato né le immunoglobuline né l’HRG erano in grado di diminuire gli effetti citotossici delle nanoparticelle, mentre l’albumina e, in particolare, le HDL erano fortemente protettive, riducendo l’associazione delle nanoparticelle alle cellul