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 O) and the fluorescein labeled Stober (35 nm O). 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 Stober 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 association