Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

High-Dose Exposure to Polymer-Coated Iron Oxide Nanoparticles Elicits Autophagy-Dependent Ferroptosis in Susceptible Cancer Cells.

Ferroptosis, a form of iron-dependent, lipid peroxidation-driven cell death, has been extensively investigated in recent years, and several studies have suggested that the ferroptosis-inducing properties of iron-containing nanomaterials could be harnessed for cancer treatment. Here we evaluated the potential cytotoxicity of iron oxide nanoparticles, with and without cobalt functionalization (Fe2O3 and Fe2O3@Co-PEG), using an established, ferroptosis-sensitive fibrosarcoma cell line (HT1080) and a normal fibroblast cell line (BJ). In addition, we evaluated poly (ethylene glycol) (PEG)-poly(lactic-co-glycolic acid) (PLGA)-coated iron oxide nanoparticles (Fe3O4-PEG-PLGA). Our results showed that all the nanoparticles tested were essentially non-cytotoxic at concentrations up to 100 µg/mL. However, when the cells were exposed to higher concentrations (200-400 µg/mL), cell death with features of ferroptosis was observed, and this was more pronounced for the Co-functionalized nanoparticles. Furthermore, evidence was provided that the cell death triggered by the nanoparticles was autophagy-dependent. Taken together, the exposure to high concentrations of polymer-coated iron oxide nanoparticles triggers ferroptosis in susceptible human cancer cells.

Design of TiO2-Surfactin Hybrid Systems with Multifunctional Properties.

In recent years, multifunctional inorganic-organic hybrid materials have been widely investigated in order to determine their potential synergetic, antagonist, or independent effects in terms of reactivity. The aim of this study was to design and characterize a new hybrid material by coupling well-known photocatalytic TiO2 nanoparticles with sodium surfactin (SS), a biosurfactant showing high binding affinity for metal cations as well as the ability to interact with and disrupt microorganisms' cell membranes. We used both chemical and colloidal synthesis methodologies and investigated how different TiO2:SS weight ratios affected colloidal, physicochemical, and functional properties. We discovered a clear breaking point between TiO2 and SS single-component trends and identified different ranges of applicability by considering different functional properties such as photocatalytic, heavy metal sorption capacity, and antibacterial properties. At low SS contents, the photocatalytic properties of TiO2 are preserved (conversion of organic dye = 99% after 40 min), and the hybrid system can be used in advanced oxidation processes, taking advantage of the additional antimicrobial SS properties. At high SS contents, the TiO2 photoactivity is inhibited, and the hybrid can be usefully exploited as a UV blocker in cosmetics, avoiding undesired oxidative effects (UV adsorption in the range between 300-400 nm). Around the breaking point (TiO2:SS 1:1), the hybrid material preserves the high surface area of TiO2 (specific surface area around 180 m2/g) and demonstrates NOx depletion of up to 100% in 80 min, together with improved adhesion of hybrid antibacterial coating. The last design demonstrated the best results for the concurrent removal of inorganic, organic, and biological pollutants in water/soil remediation applications.

Native Study of the Behaviour of Magnetite Nanoparticles for Hyperthermia Treatment during the Initial Moments of Intravenous Administration.

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve long-term stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle-proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

Smart Magnetic Nanocarriers for Multi-Stimuli On-Demand Drug Delivery.

In this study, we report the realization of drug-loaded smart magnetic nanocarriers constituted by superparamagnetic iron oxide nanoparticles encapsulated in a dual pH- and temperature-responsive poly (N-vinylcaprolactam-co-acrylic acid) copolymer to achieve highly controlled drug release and localized magnetic hyperthermia. The magnetic core was constituted by flower-like magnetite nanoparticles with a size of 16.4 nm prepared by the polyol approach, with good saturation magnetization and a high specific absorption rate. The core was encapsulated in poly (N-vinylcaprolactam-co-acrylic acid) obtaining magnetic nanocarriers that revealed reversible hydration/dehydration transition at the acidic condition and/or at temperatures above physiological body temperature, which can be triggered by magnetic hyperthermia. The efficacy of the system was proved by loading doxorubicin with very high encapsulation efficiency (>96.0%) at neutral pH. The double pH- and temperature-responsive nature of the magnetic nanocarriers facilitated a burst, almost complete release of the drug at acidic pH under hyperthermia conditions, while a negligible amount of doxorubicin was released at physiological body temperature at neutral pH, confirming that in addition to pH variation, drug release can be improved by hyperthermia treatment. These results suggest this multi-stimuli-sensitive nanoplatform is a promising candidate for remote-controlled drug release in combination with magnetic hyperthermia for cancer treatment.

Nanodispersions of TiO2 in Water for Removing Acrylic Films Used in Conservation.

The influence of a nanodispersion of TiO2 in water (nanoparticle size: 40 nm, polydispersity index: 0.25), brushed on a Paraloid film and subjected to UV-Vis irradiation was evaluated. The TiO2 nanodispersions showed a tendency to reduce the molecular weight of Paraloid due to its photocatalytic properties. FTIR and GPC analyses and SEM images suggested the degradation of the polymer, while chromatic variations of the films were scarcely detected. This study is very remarkable in the perspective of using this material for the removal of polymeric films used in conservation.

T Cell Delivery of Nanoparticles-Bound Anti-CD20 Monoclonal Antibody: Successful B Cell Depletion in the Spinal Cord during Experimental Autoimmune Encephalomyelitis.

We developed a nanotechnology based-cell mediated drug delivery system by loading myelin antigen-specific T cells with nanoparticles bound to anti-CD20 monoclonal antibody. Anti-CD20 antibody is a current treatment (ocrelizumab) for multiple sclerosis (MS), a chronic, inflammatory and autoimmune disease of the central nervous system (CNS). CD20-depletion has been associated with efficacy in active relapsing and progressive MS, but may not efficiently target inflammatory cells compartmentalized in the CNS. In our work, the intravenous transfer of T cells containing nanoparticle-anti-CD20 complex in mice causes B cell depletion in the spleen and in the brain, whereas the injection of anti-CD20 alone depletes B cells only in the spleen. Testing this system in Experimental Autoimmune Encephalomyelitis (EAE), animal model of MS, we found that spinal cord B cell depletion ameliorates the disease course and pathology. Graphical Abstract.

Microwave-Assisted Vacuum Synthesis of TiO2 Nanocrystalline Powders in One-Pot, One-Step Procedure.

A new method for fast and simple synthesis of crystalline TiO2 nanoparticles with photocatalytic activity was developed by carrying out a classic sol-gel reaction directly under vacuum. The use of microwaves for fast heating of the reaction medium further reduces synthesis times. When the solvent is completely removed by vacuum, the product is obtained in the form of a powder that can be easily redispersed in water to yield a stable nanoparticle suspension, exhibiting a comparable photocatalytic activity with respect to a commercial product. The present methodology can, therefore, be considered a process intensification procedure for the production of nanotitania.

Studies on Cell Compatibility, Antibacterial Behavior, and Zeta Potential of Ag-Containing Polydopamine-Coated Bioactive Glass-Ceramic.

Dopamine is a small molecule that mimics the adhesive component (L-DOPA) of marine mussels with a catecholamine structure. Dopamine can spontaneously polymerize to form polydopamine (PDA) in a mild basic environment. PDA binds, in principle, to all types of surfaces and offers a platform for post-modification of surfaces. In this work, a novel Ag-containing polydopamine coating has been developed for the functionalization of bioactive glass-ceramics. In order to study the interactions between the surface of uncoated and coated samples and the environment, we have measured the surface zeta potential. Results confirmed that PDA can interact with the substrate through different chemical groups. A strongly negative surface zeta potential was measured, which is desirable for biocompatibility. The dual function of the material, namely the capability to exhibit bioactive behavior while being antibacterial and not harmful to mammalian cells, was assessed. The biocompatibility of the samples with MG-63 (osteoblast-like) cells was determined, as well as the antibacterial behavior against Gram-positive Staphylococcus carnosus and Gram-negative Escherichia coli bacteria. During cell biology tests, uncoated and PDA-coated samples showed biocompatibility, while cell viability on Ag-containing PDA-coated samples was reduced. On the other hand, antibacterial tests confirmed the strong antimicrobial properties of Ag-containing PDA-coated samples, although tailoring of the silver release will be necessary to modulate the dual effect of PDA and silver.

Multifunctional graphene-based magnetic nanocarriers for combined hyperthermia and dual stimuli-responsive drug delivery.

The synthesis of hydrophilic graphene-based yolk-shell magnetic nanoparticles functionalized with copolymer pluronic F-127 (GYSMNP@PF127) is herein reported to achieve an efficient multifunctional biomedical system for mild hyperthermia and stimuli-responsive drug delivery. In vitro tests revealed the extraordinary ability of GYSMNP@PF127 to act as smart stimuli-responsive multifunctional nanomedicine platform for cancer therapy, exhibiting (i) an outstanding loading capacity of 91% (w/w, representing 910 µg mg-1) of the chemotherapeutic drug doxorubicin, (ii) a high heating efficiency under an alternating (AC) magnetic field (intrinsic power loss ranging from 2.1-2.7 nHm2 kg-1), and (iii) a dual pH and thermal stimuli-responsive drug controlled release (46% at acidic tumour pH vs 7% at physiological pH) under AC magnetic field, in just 30 min. Additionally, GYSMNP@PF127 presents optimal hydrodynamic diameter (DH = 180 nm) with negative surface charge, high haemocompatibility for blood stream applications and tumour cellular uptake of drug nanocarriers. Due to its physicochemical, magnetic and biocompatibility properties, the developed graphene-based magnetic nanocarrier shows high promise as dual exogenous (AC field)/endogenous (pH) stimuli-responsive actuators for targeted thermo-chemotherapy, combining magnetic hyperthermia and controlled drug release triggered by the abnormal tumour environment. The presented strategy and findings can represent a new way to design and develop highly stable added-value graphene-based nanostructures for the combined treatment of cancer.

Spectroscopic and photoacoustic characterization of encapsulated iron oxide super-paramagnetic nanoparticles as a new multiplatform contrast agent.

Recently, a number of photoacoustic (PA) agents with increased tissue penetration and fine spatial resolution have been developed for molecular imaging and mapping of pathophysiological features at the molecular level. Here, we present bio-conjugated near-infrared light-absorbing magnetic nanoparticles as a new agent for PA imaging. These nanoparticles exhibit suitable absorption in the near-infrared region, with good photoacoustic signal generation efficiency and high photo-stability. Furthermore, these encapsulated iron oxide nanoparticles exhibit strong super-paramagnetic behavior and nuclear relaxivities that make them useful as magnetic resonance imaging (MRI) contrast media as well. Their simple bio-conjugation strategy, optical and chemical stability, and straightforward manipulation could enable the development of a PA probe with magnetic and spectroscopic properties suitable for in vitro and in vivo real-time imaging of relevant biological targets.

Design, Synthesis, and Structure-Property Relationships of Er3+-Doped TiO2 Luminescent Particles Synthesized by Sol-Gel.

Titania particles doped with various concentrations of Erbium were synthesized by the sol-gel method followed by different heat treatments. The shape and the grain growth of the particles were noticeably affected by the concentration of Erbium and the heat treatment conditions. An infrared emission at 1530 nm, as well as green and red up-conversion emissions at 550 and 670 nm, were observed under excitation at 976 nm from all of the synthesized particles. The emission spectra and lifetime values appeared to be strongly influenced by the presence of the different crystalline phases. This work presents important guidelines for the synthesis of functional Er3+-doped titania particles with controlled and tailored spectroscopic properties for photonic applications.

EGFR-Targeted Magnetic Nanovectors Recognize, in Vivo, Head and Neck Squamous Cells Carcinoma-Derived Tumors.

Head and neck squamous cell carcinomas (HNSCC) are a diverse group of tumors with high morbidity and mortality that have remained mostly unchanged over the past decades. The epidermal growth factor receptor (EGFR) is often overexpressed and activated in these tumors and strongly contributes to their pathogenesis. Still, EGFR-targeted therapies such as monoclonal antibodies and kinase inhibitors have demonstrated only limited improvements in the clinical outcome of this disease. Here, we take advantage of the extraordinary affinity of EGF for its cognate receptor to specifically target magnetite-containing nanoparticles to HNSCC cells and mediate, in vitro, their cellular upload. On the basis of this, we show efficient accumulation, in vivo, of such nanoparticles in subcutaneous xenograft tumor tissues in sufficient amounts to be able to mediate visualization by magnetic resonance imaging. Overall, our EGF-coated nanosystem may warrant, in the near future, novel and very efficient theranostic approaches to HNSCC.

Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells.

Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe3O4-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 µg/mL of Fe3O4-NPs. Significant dose-response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death.

Bioactive Glasses with Low Ca/P Ratio and Enhanced Bioactivity.

Three new silica-based glass formulations with low molar Ca/P ratio (2-3) have been synthesized. The thermal properties, the crystalline phases induced by thermal treatments and the sintering ability of each glass formulation have been investigated by simultaneous differential scanning calorimetry-thermogravimetric analysis (DSC-TG), X-ray diffraction (XRD) and hot stage microscopy (HSM). The glasses exhibited a good sintering behavior, with two samples achieving shrinkage of 85%-95% prior to crystallization. The bioactivity of the glasses in simulated body fluid (SBF) has been investigated by performing XRD and Fourier transform infrared spectroscopy (FTIR) on the samples prior and after immersion. The glasses with lower MgO contents were able to form a fully crystallized apatite layer after three days of immersion in simulated body fluid (SBF), while for the glass exhibiting a higher MgO content in its composition, the crystallization of the Ca-P layer was achieved after seven days. The conjugation of these properties opens new insights on the synthesis of highly bioactive and mechanically strong prosthetic materials.

Nanomaterial applications in multiple sclerosis inflamed brain.

In the last years scientific progress in nanomaterials, where size and shape make the difference, has increased their utilization in medicine with the development of a promising new translational science: nanomedicine. Due to their surface and core biophysical properties, nanomaterials hold the promise for medical applications in central nervous system (CNS) diseases: inflammatory, degenerative and tumors. The present review is focused on nanomaterials at the neuro-immune interface, evaluating two aspects: the possible CNS inflammatory response induced by nanomaterials and the developments of nanomaterials to improve treatment and diagnosis of neuroinflammatory diseases, with a focus on multiple sclerosis (MS). Indeed, nanomedicine allows projecting new ways of drug delivery and novel techniques for CNS imaging. Despite the wide field of application in neurological diseases of nanomaterials, our topic here is to review the more recent development of nanomaterials that cross blood brain barrier (BBB) and reach specific target during CNS inflammatory diseases, a crucial strategy for CNS early diagnosis and drug delivery, indeed the main challenges of nanomedicine.

In vivo anticancer evaluation of the hyperthermic efficacy of anti-human epidermal growth factor receptor-targeted PEG-based nanocarrier containing magnetic nanoparticles.

Polymeric nanoparticles with targeting moieties containing magnetic nanoparticles as theranostic agents have considerable potential for the treatment of cancer. Here we report the chemical synthesis and characterization of a poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-based nanocarrier containing iron oxide nanoparticles and human epithelial growth factor receptor on the outer shell. The nanocarrier was also radiolabeled with (99m)Tc and tested as a theranostic nanomedicine, ie, it was investigated for both its diagnostic ability in vivo and its therapeutic hyperthermic effects in a standard A431 human tumor cell line. Following radiolabeling with (99m)Tc, the biodistribution and therapeutic hyperthermic effects of the nanosystem were studied noninvasively in vivo in tumor-bearing mice. A substantial decrease in tumor size correlated with an increase in both nanoparticle concentration and local temperature was achieved, confirming the possibility of using this multifunctional nanosystem as a therapeutic tool for epidermoid carcinoma.

Magnetic Glass Ceramics by Sintering of Borosilicate Glass and Inorganic Waste.

Ceramics and glass ceramics based on industrial waste have been widely recognized as competitive products for building applications; however, there is a great potential for such materials with novel functionalities. In this paper, we discuss the development of magnetic sintered glass ceramics based on two iron-rich slags, coming from non-ferrous metallurgy and recycled borosilicate glass. The substantial viscous flow of the glass led to dense products for rapid treatments at relatively low temperatures (900-1000 °C), whereas glass/slag interactions resulted in the formation of magnetite crystals, providing ferrimagnetism. Such behavior could be exploited for applying the obtained glass ceramics as induction heating plates, according to preliminary tests (showing the rapid heating of selected samples, even above 200 °C). The chemical durability and safety of the obtained glass ceramics were assessed by both leaching tests and cytotoxicity tests.