Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Defining Structure in Electrospun Polymer Fibres

We use a combination of microscopy, x-ray scattering and neutron scattering to show how structure develops in micro and nano-size polymer fibres prepared by electrospinning. The technique has been applied to a range of different polymers, an amorphous system (polystyrene), a crystallisable polymer (poly-epsilon-caprolactone), a composite systems (polyethylene oxide or poly vinyl alcohol containing polypyrrole) and consider the possibility of self assembly (gelatin).

Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor κB (NF-κB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

Nano- and macroscale structural and mechanical properties of in situ synthesized bacterial cellulose/PEO-b-PPO-b-PEO biocomposites

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In situ sonochemical synthesis of ZnO particles embedded in a thermoplastic matrix for biomedical applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Formation and Characterization of Oriented Micro-and Nanofibers Containing Poly (ethylene oxide) and Pectin

Formation of oriented or aligned micro- and nanofibers using biocompatible materials opens the possibility to obtain engineered tissues that can be used in medicine, environmental engineering, security and defense, among other applications. Pectin, a heteropolysaccharide, is a promising material to be incorporated into the fibers because, besides being biocompatible, this material is also biodegradable and bioactive. In this work, the formation of oriented fibers using solutions containing pectin and polyethylene oxide (biocompatible polymers), and chloroform (as the solvent) is investigated. The injection of solution into an intense electric field defined between two parallel electrodes was used to obtain oriented fibers. This novel approach is a modification of the conventional electrospinning process. The presence of pectin in the fibers was confirmed by FTIR analysis. Fibers with diameters of hundreds of nanometers and several centimeters long can be collected. The incorporation of pectin leads to a higher variation of the diameter of the fibers, and a trend to larger fiber diameters. This behavior can be related to the presence of pectin clusters in the fibers. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.057203jes] All rights reserved.

Strukturbildung doppelthydrophiler Blockcopolymere mit mehrwertigen organischen Gegenionen

Sowohl die Komplexierung von Polyelektrolyten mit anorganischen Salzen, als auch die mit entgegengesetzt geladenen Polymeren wurde von vielen Autoren bereits intensiv untersucht. Doch gerade mit Molekülen die zwischen diesen beiden Extremen liegen, sollte es möglich sein, durch elektrostatische Wechselwirkungen gezielt nanometergroße Teilchen definierter Struktur herzustellen. Ziel dieser Arbeit war es deshalb, die Strukturbildung doppelthydrophiler Blockcopolymere mit mehrwertigen organischen Gegenionen zu untersuchen und insbesondere Parameter für die Bildung supramolekularer Strukturen in wässriger Lösung zu finden. Als Blockcopolymer wurde dabei Polyethylenoxid-b-methacrylsäure mittels anionischer Polymerisation hergestellt und mittels Gelpermeationschromatographie (GPC) und Kernresonanzspektroskopie (NMR)charakterisiert. Die Strukturbildung des Polyelektrolyten mit mehrwertigen organischen Gegenionen wurde in pH = 6- und pH = 7-Pufferlösung mit dynamischer und statischer Lichtstreuung, Kleinwinkelneutronenstreuung und Ultrazentrifugation untersucht. Mit Diaminobenzidin als Gegenion wurden dabei sphärische Komplexe mit einem hydrodynamischen Radius um 100 nm erhalten und mit Ultrazentrifugation der Anteil des Gegenions im Komplex quantifiziert. Die schlechte Löslichkeit des Diaminobenzidins in wässrigem Medium erschwerte allerdings die Interpretation der Ergebnisse. Trotzdem deuten diese darauf hin, dass keine Kolloidbildung des Diaminobenzidins, sondern eine Komplexierung der Einzelmoleküle mit dem Copolymer vorliegt. Um Probleme mit der Löslichkeit zu vermeiden, wurden schliesslich Polyamidoamin-Dendrimere als Gegenionen verwendet. Dabei wurde in pH = 6- und pH = 7-Pufferlösung für Dendrimere der Generation 4 mit steigender Gegenionenkonzentration ein kontinuierlicher Anstieg des hydrodynamischen Radius bis zu einer Größe von 70 nm gefunden. Mit Kleinwinkelneutronenstreuung konnte eine ellipsoidale Struktur dieser Komplexe beobachtet werden. Auch die Größe der Gegenionen spielt für die Bildung supramolekularer Aggregate eine Rolle. So zeigte sich, dass für Polyamidoamin-Dendrimere der Generation 2, analog zu denen der Generation 4, ein Anstieg des hydrodynamischen Radius mit steigender Gegenionenkonzentration zu beobachten ist. Für Generation 0-Dendrimere hingegen wurde ein umgekehrter Verlauf beobachtet, welcher dem für Diaminobenzidin gleicht. Somit kann man annehmen, dass die Aggregation mit kleinen Molekülen zu einer anderen Struktur der Komplexe führt, als die mit größeren Molekülen.

Nanotechnology in the food industry I: applications

Nanotechnology is a multidisciplinary science that is having a boom today, providing new products with attractive physicochemical properties for many applications. In agri/feed/food sector, nanotechnology offers great opportunities for obtaining products and innovative applications for agriculture and livestock, water treatment and the production, processing, storage and packaging of food. To this end, a wide variety of nanomaterials, ranging from metals and inorganic metal oxides to organic nanomaterials carrying bioactive ingredients are applied. This review shows an overview of current and future applications of nanotechnology in the food industry. Food additives and materials in contact with food are now the main applications, while it is expected that in the future are in the field of nano-encapsulated and nanocomposites in applications as novel foods, additives, biocides, pesticides and materials food contact.

Atomic resolution studies of oxide superlattices and ultrathin films

The ability to grow ultrathin films layer-by-layer with well-defined epitaxial relationships has allowed research groups worldwide to grow a range of artificial films and superlattices, first for semiconductors, and now with oxides. In the oxides thin film research community, there have been concerted efforts recently to develop a number of epitaxial oxide systems grown on single crystal oxide substrates that display a wide variety of novel interfacial functionality, such as enhanced ferromagnetic ordering, increased charge carrier density, increased optical absorption, etc, at interfaces. The magnitude of these novel properties is dependent upon the structure of thin films, especially interface sharpness, intermixing, defects, and strain, layering sequence in the case of superlattices and the density of interfaces relative to the film thicknesses. To understand the relationship between the interfacial thin film oxide atomic structure and its properties, atomic scale characterization is required. Transmission electron microscopy (TEM) offers the ability to study interfaces of films at high resolution. Scanning transmission electron microscopy (STEM) allows for real space imaging of materials with directly interpretable atomic number contrast. Electron energy loss spectroscopy (EELS), together with STEM, can probe the local chemical composition as well as local electronic states of transition metals and oxygen. Both techniques have been significantly improved by aberration correctors, which reduce the probe size to 1 Å, or less. Aberration correctors have thus made it possible to resolve individual atomic columns, and possibly probe the electronic structure at atomic scales. Separately, using electron probe forming lenses, structural information such as the crystal structure, strain, lattice mismatches, and superlattice ordering can be measured by nanoarea electron diffraction (NED). The combination of STEM, EELS, and NED techniques allows us to gain a fundamental understanding of the properties of oxide superlattices and ultrathin films and their relationship with the corresponding atomic and electronic structure. In this dissertation, I use the aforementioned electron microscopy techniques to investigate several oxide superlattice and ultrathin film systems. The major findings are summarized below. These results were obtained with stringent specimen preparation methods that I developed for high resolution studies, which are described in Chapter 2. The essential materials background and description of electron microscopy techniques are given in Chapter 1 and 2. In a LaMnO3-SrMnO3 superlattice, we demonstrate the interface of LaMnO3-SrMnO3 is sharper than the SrMnO3-LaMnO3 interface. Extra spectral weights in EELS are confined to the sharp interface, whereas at the rougher interface, the extra states are either not present or are not confined to the interface. Both the structural and electronic asymmetries correspond to asymmetric magnetic ordering at low temperature. In a short period LaMnO3-SrTiO3 superlattice for optical applications, we discovered a modified band structure in SrTiO3 ultrathin films relative to thick films and a SrTiO3 substrate, due to charge leakage from LaMnO3 in SrTiO3. This was measured by chemical shifts of the Ti L and O K edges using atomic scale EELS. The interfacial sharpness of LaAlO3 films grown on SrTiO3 was investigated by the STEM/EELS technique together with electron diffraction. This interface, when prepared under specific conditions, is conductive with high carrier mobility. Several suggestions for the conductive interface have been proposed, including a polar catastrophe model, where a large built-in electric field in LaAlO3 films results in electron charge transfer into the SrTiO3 substrate. Other suggested possibilities include oxygen vacancies at the interface and/or oxygen vacancies in the substrate. The abruptness of the interface as well as extent of intermixing has not been thoroughly investigated at high resolution, even though this can strongly influence the electrical transport properties. We found clear evidence for cation intermixing through the LaAlO3-SrTiO3 interface with high spatial resolution EELS and STEM, which contributes to the conduction at the interface. We also found structural defects, such as misfit dislocations, which leads to increased intermixing over coherent interfaces.

Protein disulfide isomerase overexpression in vascular smooth muscle cells induces spontaneous preemptive NADPH oxidase activation and Nox1 mRNA expression: Effects of nitrosothiol exposure

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). Here, we further investigated PDI effects on vascular NADPH oxidase. VSMC transfected with wild-type PDI (wt-PDI) OF PDI mutated in all four redox cysteines (mut-PDI) enhanced (2.5-fold) basal cellular ROS production and membrane NADPH oxidase activity, with 3-fold increase in Nox1, but not Nox4 mRNA. However, further ROS production, NADPH oxidase activity and Nox1 mRNA increase triggered by angiotensin-II (AngII) were totally lost with PDI overexpression, suggesting preemptive Nox1 activation in such cells. PDI overexpression increased Nox4 mRNA after AngII stimulus, although without parallel ROS increase. We also show that Nox inhibition by the nitric oxide donor GSNO is independent of PDI. PDI silencing decreased specifically Nox1 mRNA and protein, confirming that PDI may regulate Nox1 at transcriptional level in VSMC. Such data further strengthen the role of PDI as novel NADPH oxidase regulator. (C) 2009 Elsevier Inc. All rights reserved.

Dual role of hydrogen sulfide in mechanical inflammatory hypernociception

Hydrogen Sulfide (H2S) is an endogenous gas involved in several biological functions, including modulation of nociception. However, the mechanisms involved in such modulation are not fully elucidated. The present Study demonstrated that the pretreatment of mice with PAG, a H2S synthesis inhibitor, reduced LPS-induced mechanical paw hypernociception. This inhibition of hypernociception was associated with the prevention of neutrophil recruitment to the plantar tissue. Conversely, PAG had no effect on LPS-induced production of the hypernociceptive cytokines, TNF-alpha, IL-1 beta and CXCL1/KC and on hypernociception induced by PGE(2), a directly acting hypernociceptive mediator. In contrast with the pro-nociceptive role of endogenous H2S. systemic administration of NaHS, a H2S donor, reduced LPS-induced mechanical hypernociception in mice. Moreover, this treatment inhibited mechanical hypernociception induced by PGE(2), suggesting a direct effect of H2S on nociceptive neurons. The antinociceptive mechanism of exogenous H2S depends on K-(ATP)(+) channels since the inhibition of PGE(2) hypernociception by NaHS was prevented by glibenclamide (K-(ATP)(+) channel blocker). Finally, NaHS did not alter the thermal nociceptive threshold in the hot-plate test, confirming that its effect is mainly peripheral. Taken together, these results suggest that H2S has a dual role in inflammatory hypernociception: 1. an endogenous pro-nociceptive effect due to up-regulation of neutrophil migration. and 2. an antinociceptive effect by direct blockade of nociceptor sensitization modulating K-(ATP)(+) channels. (c) 2008 Elsevier B.V. All rights reserved.

Macrophage Depletion Attenuates Chronic Cyclosporine A Nephrotoxicity

Background. Cyclosporine A (CsA)-induced chronic nephrotoxicity is characterized by renal dysfunction and interstitial fibrosis. Early and progressive renal macrophage influx, correlating with latter interstitial fibrotic areas, has been associated with CsA treatment. This study investigated the role of macrophages, the nitric oxide (NO) pathway, and the oxidative stress on chronic CsA nephrotoxicity. Methods. The macrophages were depleted by clodronate liposomes. Animals were distributed into four groups: vehicle (olive oil for 21 days), CsA 7.5 mg/kg per day (21 days), CsA plus clodronate (5 mg/mL intraperitoneally on days -4, 1, 4, 11, and 18 of CsA treatment), or vehicle plus clodronate. On day 22, glomerular filtration rate, renal blood flow, renal tubulointerstitial fibrosis, CsA blood levels, serum malondialdehyde and renal tissue immunohistochemistry for macrophages, inducible NO synthase, transforming growth factor-beta, nuclear factor-k beta, alpha-smooth muscle actin, vimentin, and nitrotyrosine were assessed. Results. CsA-induced increase in the macrophage was prevented by clodronate. Macrophage depletion attenuated the reductions in the glomerular filtration rate and renal blood flow, the development of tubulointerstitial fibrosis, malondialdehyde increase and increases in nuclear factor-k beta, transforming growth factor-beta, vimentin, inducible NO synthase, and nitrotyrosine expression provoked by CsA. Clodronate did not affect alpha-smooth muscle actin expression and CsA blood levels. Conclusions. Renal macrophage influx plays an important role in CsA-induced chronic nephrotoxicity. The NO pathway and oxidative stress are likely mechanisms involved in the genesis of this form of renal injury.

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint. (C) 2007 Elsevier Inc. All rights reserved.