Copper oxide nanoparticle toxicity profiling using untargeted metabolomics.

BACKGROUND: The rapidly increasing number of engineered nanoparticles (NPs), and products containing NPs, raises concerns for human exposure and safety. With this increasing, and ever changing, catalogue of NPs it is becoming more difficult to adequately assess the toxic potential of new materials in a timely fashion. It is therefore important to develop methods which can provide high-throughput screening of biological responses. The use of omics technologies, including metabolomics, can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. These techniques thus provide the opportunity to identify specific toxicity pathways and to generate hypotheses on how to reduce or abolish toxicity. RESULTS: We have used untargeted metabolome analysis to determine differentially expressed metabolites in human lung epithelial cells (A549) exposed to copper oxide nanoparticles (CuO NPs). Toxicity hypotheses were then generated based on the affected pathways, and critically tested using more conventional biochemical and cellular assays. CuO NPs induced regulation of metabolites involved in oxidative stress, hypertonic stress, and apoptosis. The involvement of oxidative stress was clarified more easily than apoptosis, which involved control experiments to confirm specific metabolites that could be used as standard markers for apoptosis; based on this we tentatively propose methylnicotinamide as a generic metabolic marker for apoptosis. CONCLUSIONS: Our findings are well aligned with the current literature on CuO NP toxicity. We thus believe that untargeted metabolomics profiling is a suitable tool for NP toxicity screening and hypothesis generation.

Poly(N-vinylimidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids.

In this study we report the novel polymeric resin poly(N-vinyl imidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids. The monomer to crosslinker ratio and the porogen composition were optimized for isolating phenolic acids diluted in acetonitrile at normal phase chromatography conditions, first. Acetonitrile serves as polar, aprotic solvent, dissolving phenolic acids but not interrupting interactions with the stationary phase due to the approved Hansen solubility parameters. The optimized resin demonstrated high loading capacities and adsorption abilities particularly for phenolic acids in both, acetonitrile and aqueous solutions. The adsorption behavior of aqueous standards can be attributed to ion exchange effects due to electrostatic interactions between protonated imidazole residues and deprotonated phenolic acids. Furthermore, adsorption experiments and subsequent curve fittings provide information of maximum loading capacities of single standards according to the Langmuir adsorption model. Recovery studies of the optimized polymer in the normal-phase and ion-exchange mode illustrate the powerful isolation properties for phenolic acids and are comparable or even better than typical, commercially available solid phase extraction materials. In order to prove the applicability, a highly complex extract of rosemary leaves was purified by poly(N-vinyl imidazole/ethylene glycol dimethacrylate) and the isolated compounds were identified using UHPLC-qTOF-MS.

Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides.

In this study, a novel method for the highly selective enrichment of phosphopeptides using erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns is presented. Erbium phosphate was synthesized by precipitation from boiling phosphoric acid and incubated overnight in erbium chloride solutions. The resulting powder was embedded in a monolithic poly(glycidyl methacrylate/ethylene dimethacrylate) polymer. The monolith was synthesized in a spin column by radical polymerization. Erbium phosphate demonstrated a high affinity and selectivity for phosphopeptides due to the strong interaction of trivalent erbium ions with the phosphate groups of phosphopeptides. The high selectivity and performance of the designed spin columns were demonstrated by successfully enriching phosphopeptides from tryptically digested protein mixtures containing the model phosphoproteins α- and ß-casein, bovine milk, and human saliva. By the implementation of several washing steps, unspecific components were removed and the enriched phosphopeptides were effectively eluted from the spin columns under alkaline conditions. The selective performance of the presented method was further demonstrated by the enrichment of two synthetic phosphopeptides, which were spiked in tryptically digested and dephosphorylated HeLa cell lysates at low ratios. Finally, the presented approach was compared to conventional phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the erbium phosphate doped monoliths.

Nucleophilic cross-linked, dextran coated iron oxide nanoparticles as basis for molecular imaging: synthesis, characterization, visualization and comparison with previous product.

We present a new synthesis protocol for a multivalent, multimodality, nucleophilic nanoparticle ideal for in vivo imaging. Stability requirements necessitated covalent cross-linking of the carbohydrate cage, easy functionalization the introduction of sterically accessible amine groups. The new protocol aimed at more uniform particle size, less clustering and superior magnetic properties compared with commercial nanoparticles. Particles were precipitated from Fe(2+) and Fe(3+) in the presence of 10 kDa dextran monodispersed from the aerosol phase. Cross-linking was achieved with epichlorhydrin, nuclophilication with NH3, purification with ultrafiltration and dialysis. Particles and a commercial product (Rienso®, Takeda Pharma) underwent physicochemical characterizations. Biocompatibility was assessed by Resazurin on LLC-PK1 cells; the internalization rate was measured for three cell lines (HAEC, HASMC, HT29). Core size was 5.61 ± 1.25 nm; hydrodynamic size was 49.56 ± 11.73 nm. The number of sterically accessible amine groups averaged 9.9. The cores showed cubic magnetite structure. Values of r1 and r2 were 10.9 and 148.17 mM(-1) s(-1). Cellular viability was unchanged after incubation. Introduction of aerosol phase dextran resulted in a reduction of the overall hydrodynamic diameter and a narrower size distribution of the synthesized particles. Electron tomography visualized for the first time the postulated 'hairy layer' of the dextran coating and enabled the measurement of the overall diameter of 100.2 ± 7.92 nm. The resulting nanoparticle is biocompatible, functionalizable and detectable at nanomolar concentrations with MRI and optical imaging. It can potentially serve as a platform for multimodal molecular imaging and targeted therapy approaches.

Separation of small molecules on novel monolithic poly(vinylphosphonic acid/ethylene dimethacrylate) columns.

In HPLC, monolithic organic stationary phases are usually restricted to the separation of high-molecular-weight compounds such as proteins or oligonucleotides. The aim of this study was to enlarge the applicability of monolithic stationary phases to the micro-liquid chromatography separation of smaller molecules. For this, a new monolithic stationary phase was synthesized by radical polymerization of vinylphosphonic acid (VPA) and ethylene dimethacrylate (EDMA) using azobisisobutyronitrile as radical initiator. In situ reactions at two different temperatures and reaction times resulted in poly(VPA/EDMA) capillaries and allowed fast separations for small molecules, especially parabens and alkylbenzenes. The capillaries showed high mechanical stability, low-swelling properties, high permeability and lower surface area as expected. Polymerization at 75°C for 20 min exhibited efficient separation of parabens within 1.5 min with short half-peak widths and satisfactory resolutions. Apart from attenuated total reflectance Fourier transform infrared (ATR-IR) measurements, the pH-dependent separation of alkylbenzenes confirmed the incorporation of phosphonate groups into the polymeric network, resulting into deprotonation of the stationary phase at pH >4. Moreover, methylparaben and propylparaben were quantitatively determined in human saliva after treatment with paraben-containing tooth paste.

Non-formaldehyde, crease resistant agent for cotton fabrics based on an organic-inorganic hybrid material.

1,2,3,4-Butanetetracarboxylic acid (BTCA) was reacted with (3-aminopropyl)triethoxysilane (APTES) to a poly(amic)acid (PAA). The molar ratios of BTCA and APTES were 1/1 (B/A-1/1), 1/2 (B/A-1/2), 1/3 (B/A-1/3), and 1/4 (B/A-1/4). The as-prepared precursor solution was applied to cotton substrates. After thermal treatment (180°C) the physical-mechanical properties of the cotton samples were tested by means of dry crease recovery angle and tensile strength. For B/A-1/1 treated fabrics a significant improvement of the crease resistance was observed. FT-IR spectra revealed the formation of an imide group and an ester linkage, indicating the cross-linking of the cellulosic material. SEM images showed a smooth surface. As evidenced by TGA data the thermal stability of the cotton samples was not increased. No hydrophobicity could be observed. For B/A-1/3 and (B/A-1/4) modified cotton samples no crease resistant properties were detected. However, enhanced contact angle values were measured. A reaction mechanism for the formation of the ladder-like polysilsesquioxane and the cross-linking reaction is proposed.

Monolithic poly(N-vinylcarbazole-co-1,4-divinylbenzene) capillary columns for the separation of biomolecules.

Monolithic capillary columns were prepared by thermally initiated free radical copolymerization of N-vinylcarbazole (NVC) and 1,4-divinylbenzene (DVB) within the confines of 200 and 100 µm i.d. fused silica capillaries. The reaction was carried out under the influence of inert micro-(toluene) and macroporogen (1-decanol) and α,α'-azoisobutyronitrile (AIBN) as a free radical initiator. The material proved high mechanical stability applying water and acetonitrile as mobile phases. The morphological and porous properties were studied by scanning electron microscopy (SEM), nitrogen sorption (BET) and mercury intrusion porosimetry (MIP). The homogeneity of the copolymerization process was confirmed by elemental analysis and monomer conversion measurements. The newly developed NVC/DVB monolithic supports showed high separation efficiency towards biomolecules, applying reversed-phase (RP) and ion-pair reversed-phase (IP-RP) separation modes, which is exemplified by the separations of peptides, proteins and oligonucleotides. Furthermore the maximum loading capacity was evaluated. The chromatographic performance under isocratic elution was determined in terms of theoretical plate number and plate height, where up to 41,000 plates per column and a minimum plate height value of 1.7 µm were achieved, applying oligonucleotide separations. In gradient elution mode, peak capacities of 96 and 127 were achieved within a gradient time window of 60 min for protein and oligonucleotide separations, respectively. The material proved to have high permeability, good repeatability of the fabrication process and high surface areas in the range of 120-160 m(2) g(-1).

Efficient MRI labeling of endothelial progenitor cells: design of thiolated surface stabilized superparamagnetic iron oxide nanoparticles.

The aim of this study was to design thiolated surface stabilized superparamagnetic iron oxide nanoparticles (TSS-SPIONs) for efficient internalization with high MRI sensitivity. TSS-SPIONs were developed by chelation between thiolated chitosan-thioglycolic acid (chitosan-TGA) hydrogel and iron ions (Fe(2+)/Fe(3+)). Likely, unmodified chitosan hydrogel SPIONs (UC-SPIONs) and uncoated SPIONs were used as control. Moreover, TSS-SPIONs were investigated regarding to their iron core size, hydrodynamic diameter, zeta potential, iron contents, molar relaxivities (r1 and r2), and cellular internalization. TSS-SPIONs demonstrated an iron oxide core diameter (crystallite size by XRD) of 3.1 ± 0.02 nm, a hydrodynamic diameter of 94 ± 20 nm, a zeta potential of +21 ± 5 mV, and an iron content of 3.6 ± 0.9 mg/mL. In addition, internalization of TSS-SPIONs into human endothelial progenitor cells (EPC) from umbilical cord blood was more than threefold and 17-fold higher in contrast to UC-SPIONs and SPIONs, respectively. With twofold lower incubation iron concentration of TSS-SPIONs, more than threefold higher internalization was achieved as compared to Resovist®. Also, cell viability of more than 90% was observed in the presence of TSS-SPIONs after 24h. The molar MR relaxivities (r2) value at 1.5 T was threefold higher than that of Resovist® and demonstrated that TSS-SPIONs have the potential as very effective T2 contrast-enhancement agent. According to these findings, TSS-SPIONs with efficient internalization, lower cytotoxicity, and high MRI sensitivity seem to be promising for cell tracking.

In vitro evaluation of the toxicity induced by nickel soluble and particulate forms in human airway epithelial cells.

Epidemiological studies show that exposure to nickel (Ni) compounds is associated with a variety of pulmonary adverse health effects, such as lung inflammation, fibrosis, emphysema and tumours. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. In the current study we used Calu-3, a well differentiated human bronchial cell line, to investigate in vitro the effect of Ni in soluble form (NiCl(2)) and in the form of micro-sized Ni particles on the airway epithelium. For this purpose, we evaluated the effect of Ni compounds on the epithelial barrier integrity by monitoring the transepithelial electrical resistance (TEER) and on oxidative stress pathways by measuring reactive oxygen species (ROS) formation and induction of stress-inducible genes. Our results showed that exposure to NiCl(2) and Ni particles resulted in a disruption of the epithelial barrier function observed by alterations in TEER, which occurred prior to the decrease in cell viability. Moreover, Ni compounds induced oxidative stress associated with ROS formation and up-regulation of the stress-inducible genes, Metallothionein 1X (MT1X), Heat shock protein 70 (HSP70), Heme oxygenase-1 (HMOX-1), and gamma-glutamylcysteine synthetase (γGCS). Furthermore, we have demonstrated that the induced effects by Ni compounds can be partially attributed to the increase in Ni ions (Ni(2+)) intracellular levels.