In vitro cytotoxicity study of superparamagnetic iron oxide and silica nanoparticles on pneumocyte organelles.

Inhalation is the main route of nanoparticles (NP) exposure during manufacturing. Although many mechanisms of toxicity have been described, the interaction of NP with relevant pneumocytes organelles is not widely understood. Considering that the physicochemical properties of NP influence their toxicological responses, the objective of this study was to evaluate whether exposure to different NP, crystalline Fe3O4 NP and amorphous SiO2 NP could alter pneumocytes organelles in alveolar epithelial cells. To achieve this goal, cell viability, ultrastructural changes, lysosomal damage, mitochondrial membrane potential (MMP), lipid droplets (LD) formation and cytokines production were evaluated by MTT, electron microscopy, lysotracker red staining, JC-1, Oil Red staining and Milliplex® assay respectively. Both NP were observed within lamellar bodies (LB), lysosomes, and cytoplasm causing morphological changes. Exposure to SiO2 NP at 6 h induced lysosomal activation, but not Fe3O4 NP. MMP decreased and LD increased at the highest concentrations after both NP exposure. Pro-inflammatory cytokines were released only after SiO2 NP exposure at 48 h. These results indicate that SiO2 NP have a greater impact than Fe3O4 NP on organelles responsible for energy, secretion, degradation and metabolism in pneumocytes leading to the development of respiratory disorders or the exacerbation of preexisting conditions. Therefore, the established biocompatibility for amorphous NP has to be reconsidered.

Plasma protein adsorption on Fe3O4-PEG nanoparticles activates the complement system and induces an inflammatory response.

BACKGROUND: Understanding of iron oxide nanoparticles (IONP) interaction with the body milieu is crucial to guarantee their efficiency and biocompatibility in nanomedicine. Polymer coating to IONP, with polyethyleneglycol (PEG) and polyvinylpyrrolidone (PVP), is an accepted strategy to prevent toxicity and excessive protein binding. AIM: The aim of this study was to investigate the feature of IONP adsorption of complement proteins, their activation and consequent inflammatory response as a strategy to further elucidate their biocompatibility. METHODS: Three types of IONP with different surface characteristics were used: bare (IONP-bare), coated with PVP (IONP-PVP) and PEG-coated (IONP-PEG). IONPs were incubated with human plasma and adsorbed proteins were identified. BALB/c mice were intravenously exposed to IONP to evaluate complement activation and proinflammatory response. RESULTS: Protein corona fingerprinting showed that PEG surface around IONP promoted a selective adsorption of complement recognition molecules which would be responsible for the complement system activation. Furthermore, IONP-PEG activated in vitro, the complement system and induced a substantial increment of C3a and C4a anaphylatoxins while IONP-bare and IONP-PVP did not. In vivo IONP-PEG induced an increment in complement activation markers (C5a and C5b-9), and proinflammatory cytokines (IL-1ß, IL-6, TNF-α). CONCLUSION: The engineering of nanoparticles must incorporate the association between complement proteins and nanomedicines, which will regulate the immunostimulatory effects through a selective adsorption of plasma proteins and will enable a safer application of IONP in human therapy.

A comparison of the human and mouse protein corona profiles of functionalized SiO2 nanocarriers.

Nanoparticles are a promising cancer therapy for their use as drug carriers given their versatile functionalization with polyethylene glycol and proteins that can be recognized by overexpressed receptors in tumor cells. However, it has been suggested that in biological fluids, proteins cover nanoparticles, which gives the proteins a biological identity that could be responsible for unexpected biological responses: the so-called protein corona. A relevant biological event that is usually ignored in protein-corona formation is the interspecies differences in protein binding, which can be involved in the discrepancies observed in preclinical studies and the nanoparticle safety and efficiency. Hence, the aim of this study was to determine the differences between human and mouse plasma protein corona profiles in an active therapy model using silicon dioxide nanoparticles (SiO2 nanoparticles) functionalized with polyethylene glycol and transferrin. Functionalized SiO2 nanoparticles were made with a primary particle size of 25 nm and a transferrin content of 50 µg mg-1 of nanoparticles and were PEGylated with a cross-linker. The proteomic analysis by nanoliquid chromatography tandem-mass spectrometry (nanoLC-MS/MS) showed interspecies differences. The most abundant proteins found in the human protein corona profile were immunoglobulins, actin cytoplasmic 1, hemoglobin subunit beta, serotransferrin, ficolin-3, complement C3, and apolipoprotein A-1. Meanwhile, the mouse protein corona adsorbed the serine protease inhibitor A3K, serotransferrin, alpha-1-antitrypsin 1-2, hemoglobin subunit beta, and fibrinogen gamma and beta chains. These protein-corona profile differences in the functionalized SiO2 nanoparticles indicate that biological responses observed in in vivo models could not be translated to clinical use and must be considered in the interpretation of preclinical trials in order to design more efficient and safer nanomedicines.

Early kidney damage induced by subchronic exposure to PM2.5 in rats.

BACKGROUND: Particulate matter exposure is associated with respiratory and cardiovascular system dysfunction. Recently, we demonstrated that fine particles, also named PM2.5, modify the expression of some components of the angiotensin and bradykinin systems, which are involved in lung, cardiac and renal regulation. The endocrine kidney function is associated with the regulation of angiotensin and bradykinin, and it can suffer damage even as a consequence of minor alterations of these systems. We hypothesized that exposure to PM2.5 can contribute to early kidney damage as a consequence of an angiotensin/bradykinin system imbalance, oxidative stress and/or inflammation. RESULTS: After acute and subchronic exposure to PM2.5, lung damage was confirmed by increased bronchoalveolar lavage fluid (BALF) differential cell counts and a decrease of surfactant protein-A levels. We observed a statistically significant increment in median blood pressure, urine volume and water consumption after PM2.5 exposure. Moreover, increases in the levels of early kidney damage markers were observed after subchronic PM2.5 exposure: the most sensitive markers, ß-2-microglobulin and cystatin-C, increased during the first, second, sixth and eighth weeks of exposure. In addition, a reduction in the levels of specific cytokines (IL-1ß, IL-6, TNF-α, IL-4, IL-10, INF-γ, IL-17a, MIP-2 and RANTES), and up-regulated angiotensin and bradykinin system markers and indicators of a depleted antioxidant response, were also observed. All of these effects are in concurrence with the presence of renal histological lesions and an early pro-fibrotic state. CONCLUSION: Subchronic exposure to PM2.5 induced an early kidney damage response that involved the angiotensin/bradykinin systems as well as antioxidant and immune imbalance. Our study demonstrates that PM2.5 can induce a systemic imbalance that not only affects the cardiovascular system, but also affects the kidney, which may also overall contribute to PM-related diseases.

Protein corona acts as a protective shield against Fe3O4-PEG inflammation and ROS-induced toxicity in human macrophages.

Protein corona (PC) is the main biological entity of initial cell interaction and can define the toxicological response to Fe3O4 nanoparticles (IONP). Polymer coating to IONP, polyethilenglycol (PEG) and polyvinylpyrrolidone (PVP), is a widely accepted strategy to prevent toxicity and avoid excessive protein binding. The aim of this study was to assess the role of PC as a potential protector for ROS-induced cytotoxicity and pro-inflammatory response in THP-1 macrophages (exposed to three different IONP: bare, PVP or PEG coated). Cells were exposed to either IONP in RPMI-1640 media or IONP with a preformed human PC. All three IONP showed cytotoxic effects, which in the presence of PC was abolished. IONP-PEG exposure significantly increased ROS, mitochondrial dysfunction and pro-inflammatory cytokines release (IL-1ß and TNF-α). PC presence on IONP-PEG promoted a decrease in ROS and prevented cytokine secretion. Also, presence of PC reduced cell uptake for IONP-bare, but had no influence on IONP-PVP or IONP-PEG. Hence, the reduction in IONP-PEG cytotoxicity can be attributed to PC shielding against ROS generation and pro-inflammatory response and not a differential uptake in THP-1 macrophages. The presence of the PC as a structural element of NP biological entity provides in vivo-relevant conditions for nanosafety testing.

Exposure to inhaled particulate matter activates early markers of oxidative stress, inflammation and unfolded protein response in rat striatum.

To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32 µg/m³), fine (178 µg/m³) or ultrafine (107 µg/m³) concentrated PM or filtered air. Different brain regions (olfactory bulb, frontal cortex, striatum and hippocampus), were harvested from the rats following exposure to airborne PM. Subsequently, prooxidant (HO-1 and SOD-2), and inflammatory markers (IL-1ß and TNFα), apoptotic (caspase 3), and unfolded protein response (UPR) markers (XBP-1S and BiP), were also measured using real-time PCR. Activation of nuclear transcription factors Nrf-2 and NF-κB, associated with antioxidant and inflammation processes, respectively, were also analyzed by GSMA. Ultrafine PM increased HO-1 and SOD-2 mRNA levels in the striatum and hippocampus, in the presence of Nrf-2 activation. Also, ultrafine PM activated NF-κB and increased IL-1ß and TNFα in the striatum. Activation of UPR was observed after exposure to coarse PM through the increment of XBP-1S and BiP in the striatum, accompanied by an increase in antioxidant response markers HO-1 and SOD-2. Our results indicate that exposure to different size fractions of PM may induce physiological changes (in a neuroanatomical manner) in the central nervous system (CNS), specifically within the striatum, where inflammation, oxidative stress and UPR signals were effectively activated.

Temporal variation of nitro-polycyclic aromatic hydrocarbons in PM10 and PM2.5 collected in Northern Mexico City.

With the aim to determine the presence of individual nitro-PAH contained in particles in the atmosphere of Mexico City, a monitoring campaign for particulate matter (PM(10) and PM(2.5)) was carried out in Northern Mexico City, from April 2006 to February 2007. The PM(10) annual median concentration was 65.2µgm(-3) associated to 7.6µgm(-3) of solvent-extractable organic matter (SEOM) corresponding to 11.4% of the PM(10) concentration and 38.6µgm(-3) with 5.9µgm(-3) SEOM corresponding to 15.2% for PM(2.5). PM concentration and SEOM varied with the season and the particle size. The quantification of nitro-polycyclic aromatic hydrocarbons (nitro-PAH) was developed through the standards addition method under two schemes: reference standard with and without matrix, the former giving the best results. The recovery percentages varied with the extraction method within the 52 to 97% range depending on each nitro-PAH. The determination of the latter was effected with and without sample purification, also termed fractioning, giving similar results. 8 nitro-PAH were quantified, and their sum ranged from 111 to 819pgm(-3) for PM(10) and from 58 to 383pgm(-3) for PM(2.5), depending on the season. The greatest concentration was for 9-Nitroanthracene in PM(10) and PM(2.5), detected during the cold-dry season, with a median (10th-90th percentiles) concentration in 235pgm(-3) (66-449pgm(-3)) for PM(10) and 73pgm(-3) (18-117pgm(-3)) for PM(2.5). The correlation among mass concentrations of the nitro-PAH and criteria pollutants was statistically significant for some nitro-PAH with PM(10), SEOM in PM(10), SEOM in PM(2.5), NO(X), NO(2) and CO, suggesting either sources, primary or secondary origin. The measured concentrations of nitro-PAH were higher than those reported in other countries, but lower than those from Chinese cities. Knowledge of nitro-PAH atmospheric concentrations can aid during the surveillance of diseases (cardiovascular and cancer risk) associated with these exposures.

Hematotoxicity response in rats by the novel copper-based anticancer agent: casiopeina II.

The in vivo toxicity of the novel copper-based anticancer agent, casiopeina II (Cu(4,7-dimethyl-1,10-phenanthroline)(glycine)NO3) (CII), was investigated. Casiopeinas are a family of copper-coordinated complexes that have shown promising anticancer activity. The major toxic effect attributed to a single i.v. administration of CII (5 mg/kg dose) in the rat was an hemolytic anemia (reduced hemoglobin concentration (HB), red blood cell (RBC) count and packed cell volume (PCV) accompanied by a marked neutrophilic leukocytosis) 12 h and 5 days after administration, attributed to a direct erythrocyte damage. Increased reticulocyte levels and presence of normoblasts in peripheral blood 5 days post-administration indicated an effective erythropoietic response with recovery at 15 days. Increase in spleen weight and the morphological evidence of congestion of the red pulp (RP) with erythrocytes (E) resulting in a higher ratio of red to white pulp (WP) was consistent with increased uptake of damaged erythrocytes by the reticuloendothelial system observed by histopathology and electron microscopy. Extramedullary hemopoiesis was markedly increased at 5 days giving further evidence of a regenerative erythropoietic response that had an effective recovery by 15 days. Morphological changes in spleen cellularity were consistent with hematotoxicity, mainly a reduction of the red pulp/white pulp ratio, increase in erythrocyte content at 12 h, and an infiltration of nucleated cells in the red pulp at 5 days, with a tendency towards recovery 15 days after administration. The erythrocyte damage is attributed to generation of free radicals and oxidative damage on the membrane and within cells resulting from the reduction of Cu(II) and the probable dissociation of the CII complex.

Induction of apoptosis by a novel copper-based anticancer compound, casiopeina II, in L1210 murine leukaemia and CH1 human ovarian carcinoma cells.

The activity of casiopeina II [Cu(1,4-dimethyl-1, 10-phenanthroline)(glycine)NO(3)], a novel anticancer agent, was tested in two cell lines, L1210 murine leukaemia, CH1 human ovarian carcinoma, cisplatin-resistant and sensitive. Exposure of the cells to a range of concentrations of casiopeina II indicates that this copper complex kills cells by apoptosis and necrosis. Condensed chromatin and nuclear fragmentation were observed after exposure to casiopeina II. The caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) almost completely inhibited apoptosis induced by cisplatin; however, casiopeina II-induced apoptosis was inhibited only by 50-70%. These data are consistent with caspase activation (measured by Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; Z-DEVD-AFC) by casiopeina II and cisplatin and confirm that caspases are activated in the apoptotic cell death induced by casiopeina II. DNA fragmentation was observed in L1210 cells, but not in CH1 cells. No difference in susceptibility to induction of apoptosis by casiopeina II was found between sensitive and cisplatin resistant cells. In this work we show that the novel copper-based antineoplastic agent casiopeina II is highly active against murine and human cancer cell lines, including cell lines resistant to cisplatin.