Enhanced therapeutic action of Trastuzumab loaded ZnxMn1-xFe2O4 nanoparticles using a pre-treatment step for hyperthermia treatment of HER2+ breast cancer.

In this study, Ferrites (Fe3O4, MnFe2O4, ZnFe2O4) and different stoichiometric ratios of ZnxMn1-xFe2O4 (x = 0.2, 0.4, 0.6, and 0.8) nanoparticles (<15 nm) were synthesized by microwave-assisted method and optimised for hyperthermia studies. The selection of the optimised variant of ferrite i.e. Zn0.4Mn0.6Fe2O4 was found to be the best variant based on VSM (38.14 emu g-1) hyperthermia-based temperature rise (maximum ΔT of 38 °C), SAR and ILP values. Trastuzumab, which is known to bind with HER2 receptors of breast cancer was chemically tethered onto Zn0.4Mn0.6Fe2O4 nanoparticles through EDC/NHS coupling with a loading efficiency of 80%. The attached Trastuzumab aided during the pre-treatment step by aiding in the internalisation of Zn0.4Mn0.6Fe2O4 nanoparticles, with cellular uptake of 11% in SK-BR-3 (cancerous HER2+) cells compared to ∼5% for MDA-MB-231 (cancerous HER2-) and RPE-1 (non-cancerous) cells. In the presence of a hyperthermia trigger for 15 mins, ZnxMn1-xFe2O4 -Trastuzumab formulation had a maximum therapeutic effect by reducing the SK-BR-3 cell viability to 14% without adversely affecting the RPE-1 cells. The mechanism of ZnxMn1-xFe2O4-Trastuzumab combination was examined using an internalisation study, MTT-based viability, proliferation study, and ROS generation assay. By utilizing both Trastuzumab and hyperthermia, we achieve their synergistic anticancer properties while minimizing the drug requirement and reducing any effect on non-cancerous cells.

Toxicological Impact and in Vivo Tracing of Rhodamine Functionalised ZIF-8 Nanoparticles.

Metal Organic Frameworks (MOFs) are extensively used for a wide range of applications due to their exceptionally high surface area. MOF particles are conventionally in micron size, but the nanosized MOFs show good transportation/mobility due to their small size, and when combined with the high surface area of MOFs, it makes MOF nanoparticles an ideal candidate to study for environmental remediation. Therefore, it is important to study the ecotoxicological impact of these MOFs. In this study, we developed rhodamine labelled nanoparticles of zinc imidazolate metal organic framework (ZIF-8 MOFs) as a means of in vivo tracing the MOF translocation in C. elegans. Rhodamine B isothiocyanate functionalized ZIF-8 MOFs nanoparticles (RBITC@ZIF-8 MOF nanoparticles; size 44 ± 7 nm) were fed to the worms naturally within a concentration range of 0.16-16.4 µg mg-1. Fluorescence was detected in the pharyngeal and gut lumen regions of the worms after 4 h of treatment, for exposure concentrations >0.163 µg mg-1. A higher intensity of fluorescence was observed at the end of 24 h for all exposure concentrations. Worms treated with RBITC@ZIF-8 MOF concentrations of ≥1.63 µg mg-1 for 24 h showed a bright stable fluorescence signal at the tail region. The uptake of RBITC@ZIF-8 MOF for an exposure concentration of 0.163, 1.63, and 8.2 µg mg-1 was found to be 52.1, 11.4 and 28.6%, respectively. Through this study, we showed that RBITC@ZIF-8 MOFs can be exposed to C. elegans and imaged at low concentrations of ∼0.16 µg mg-1.

Microwave-assisted synthesis and upconversion luminescence of NaYF4:Yb, Gd, Er and NaYF4:Yb, Gd, Tm nanorods.

Anisotropic rare earth ion (RE3+) doped fluoride upconversion particles are emerging as potential candidate in diverse areas, ranging from biomedical imaging to photonics. Here, we develop a facile strategy to synthesize NaYF4: Yb, Gd, Er, and NaYF4: Yb, Gd, Tm upconversion nanorods via microwave synthesis route by controlling the synthesis time and compared the optical properties similar nanorods prepared via solvothermal technique. With the increase in synthesis time, the phase of the particle found to change from mixed phase to purely hexagonal and morphology of the particles change mixed phase of spherical and rod-shaped particles to completely nanorods for a synthesis time of 60 min. Further, the intrinsically hydrophobic particles changed to hydrophilic by removal of oleic capping via acid treatment and the amine functionalized silica coating. The upconversion luminescence as well as laser power dependent emission properties of the surface modified particles elucidate that surface modification route influence the upconversion luminescence as well as solvent dependent emission properties. Moreover, the laser power dependent studies elucidate that the upconversion process in a multi-photon process.

Novel dual labelled nanoprobes for nanosafety studies: Quantification and imaging experiment of CuO nanoparticles in C. elegans.

Metal oxide nanoparticles have been extensively studied for their toxicological impacts. However, accurate tracing/quantification of the nanomaterials and their biological responses are difficult to measure at low concentrations. To overcome the challenge, we developed a dual-labelling technique of CuO nanoparticles with a stable isotope of 65Cu, and with rhodamine dye. In vivo experiments on C. elegans were performed using natural feeding of Rhodamine B isothiocyanate-(3 aminopropyl) triethoxysilane functionalized 65CuO nanoprobes (RBITC-APTES@65CuO) (size = 7.41 ± 1 nm) within the range of Predicted Environmental Concentration (PEC) of CuO nanoparticles in soil and sediments. Fluorescence emission (570 nm) was detected in the lumen of the intestine and the pharynx of C. elegans with no impact of nanoparticle exposure on the brood size and life span of worms. The ingested fluorescent labelled RBITC-APTES@65CuO nanoprobes did not enter the reproductive system and were distributed in the alimentary canal of C. elegans. Strong fluorescent signals from the ingested RBITC-APTES@65CuO nanoprobes were achieved even after 24 h of exposure demonstrating the high stability of these nanoprobes in vivo. The net accumulation measured of 65Cu in C. elegans after background subtraction was 0.001 µg mg-1 (3.52 %), 0.005 µg mg-1 (1.76 %) and 0.024 µg mg-1 (1.69 %) for an exposure concentration of 0.0284 µg mg-1, 0.284 µg mg-1, and 1.42 µg mg-1 of 65Cu, respectively. Using C. elegans as a model organism, we demonstrated that RBITC-APTES tagged 65CuO nanoparticles acted as novel nanoprobes for measuring the uptake, accumulation, and biodistribution through quantification and imaging the nanoprobes at a very low exposure concentration (65CuO concentration: 0.033 µg mg-1).

Transformation in band energetics of CuO nanoparticles as a function of solubility and its impact on cellular response.

Nanoparticles under a reactive microenvironment, have the propensity to undergo morphological and compositional changes, which can translate into band edge widening. Although cell membrane depolarization has been linked with the electronic band structure of nanomaterials in their native state, the change in band structure as a consequence of a soluble nanoparticle system is less studied. Therefore we studied the consequence of dissolution of CuO nanoparticles on the band structure and flat band potentials and correlated it with its ability to induce a intracellular oxidative stress. The temporal variation in bandgap, fermi energy level and valence band maxima were evaluated on the remnant CuO nanoparticles post dissolution. CuO nanoparticles showed a very high dissolution in simulated body fluid (51%) and cell culture media (75%). This dissolution resulted in an in situ physico-chemical transformation of CuO nanoparticles. A temporal increase in the bandgap energy as a result of media interaction was up to 107%. Temporal variation in the flat band potentials with the generation of intracellular ROS, cell viability, late and early apoptosis in addition to necrosis on RAW 264.7 cells was established due to biological redox potential overlap. The mRNA expression for TNF-α, IL-6, IL-1ß and IL-10 in response to the particle treatment was also evalulated for 6 hours. Through this study, we establish that the toxicological potential of CuO nanoparticles is a temporal function of band energies (its overlap with the intracellular redox potential) followed by release of ionic species in the cytotoxic regime.

Ion exchange based approach for rapid and selective Pb(II) removal using iron oxide decorated metal organic framework hybrid.

Polyacrylic acid capped Fe3O4 - Cu-MOF (i-MOF) hybrid was prepared for rapid and selective lead removal, with 93% removal efficiency, exceptional selectivity, and adsorption capacity of 610 mg/g and 91% of i-MOF hybrid could be easily separated from the contaminated water using magnetic separation. The adsorption process followed a pseudo-second-order model and the adsorption efficiency decreased from 93% to 83% on raising the temperature from 25 °C to 40 °C. The change in equilibrium adsorption capacity with respect to equilibrium adsorbate concentration followed the Langmuir isotherm model. i-MOF had a high selectivity coefficient and removal efficiency for lead ions even when exposed simultaneously with naturally abundant cations (Na(I), Ca(II), Mg(II)). Release of Cu(II) ions from the i-MOF after Pb(II) removal suggested suggested ion-exchange to be the dominant removal mechanism. This new finding for Pb(II) removal with excellent adsorption performance using i-MOF through ion exchange based approach is a viable option for treating lead contaminated water.

Montmorillonite clay and humic acid modulate the behavior of copper oxide nanoparticles in aqueous environment and induces developmental defects in zebrafish embryo.

Copper oxide nanoparticles (CuO NPs) is one of the most commonly used metal oxide nanoparticles for commercial and industrial products. An increase in the manufacturing and use of the CuO NPs based products has increased the likelihood of their release into the aquatic environment. This has attracted major attention among researchers to explore their impact in human as well as environmental systems. CuO NPs, once released into the environment interact with the biotic and abiotic constituents of the ecosystem. Hence the objective of the study was to provide a holistic understanding of the effect of abiotic factors on the stability and aggregation of CuO NPs and its correlation with their effect on the development of zebrafish embryo. It has been observed that the bioavailability of CuO NPs decrease in presence of humic acid (HA) and heteroagglomeration of CuO NPs occurs with clay minerals. CuO NPs, CuO NPs + HA and CuO NPs + Clay significantly altered the expression of genes involved in development of dorsoventral axis and neural network of zebrafish embryos. However, the presence of HA with clay showed protective effect on zebrafish embryo development. These findings provide new insights into the interaction of NPs with abiotic factors and combined effects of such complexes on developing zebrafish embryos genetic markers.

Tailoring of physicochemical properties of nanocarriers for effective anti-cancer applications.

Nanotechnology has emerged strongly as a viable option to overcome the challenge of early diagnosis and effective drug delivery, for cancer treatment. Emerging research articles have expounded the advantages of using a specific type of nanomaterial-based system called as "nanocarriers," for anti-cancer therapy. The nanocarrier system is used as a transport unit for targeted drug delivery of the therapeutic drug moiety. In order for the nanocarriers to be effective for anticancer therapy, their physicochemical parameter needs to be tuned so that bio-functionalisation can be achieved to (1) allow drugs being attached to the substrate and for their controlled release, (2) ensure the stability of the nanocarrier up to the point of delivery, and (3) clearance of the nanocarrier after the delivery. It is therefore envisaged that tailoring of the physicochemical properties of nanocarriers can greatly influence their reactivity and interaction in the biological milieu, and this is becoming an important parameter for increasing the efficacy of cancer therapy. This review emphasizes the importance of physicochemical properties of nanocarriers, and how they influence its usage as chemotherapeutic drug carriers. The goal of this review is to present a correlation between the physicochemical properties of the nanocarriers and its intended action, and how their design based on these properties can enhance their cancer combating abilities while minimizing damage to the healthy tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2906-2928, 2017.

Electrophoretic deposition of organic/inorganic composite coatings containing ZnO nanoparticles exhibiting antibacterial properties.

To address one of the serious problems associated with permanent implants, namely bacterial infections, novel organic/inorganic coatings containing zinc oxide nanoparticles (nZnO) are proposed. Coatings were obtained by electrophoretic deposition (EPD) on stainless steel 316L. Different deposition conditions namely: deposition times in the range 60-300s and applied voltage in the range 5-30V as well as developing a layered coating approach were studied. Antibacterial tests against gram-positive Staphylococcus aureus and gram-negative Salmonella enteric bacteria confirmed the activity of nZnO to prevent bacterial growth. Coatings composition and morphology were analyzed by thermogravimetric analysis, Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. Moreover, the corrosion resistance was analyzed by evaluation of the polarization curves in DMEM at 37°C, and it was found that coatings containing nZnO increased the corrosion resistance compared to the bare substrate. Considering all results, the newly developed coatings represent a suitable alternative for the surface modification of metallic implants.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

Inhibition of potential uptake pathways for silver nanoparticles in the estuarine snail Peringia ulvae.

Mechanisms involved in the uptake of Ag NPs, and NPs in general, have been long debated within nano-ecotoxicology. In vitro studies provide evidence of the different available uptake pathways, but in vivo demonstrations are lacking. In this study, pharmacological inhibitors were employed to block specific uptake pathways that have been implicated in the transport of metal NPs and aqueous metal forms; phenamil (inhibits Na(+) channel), bafilomycin A1 (H(+) proton pump), amantadine (clathrin-mediated endocytosis), nystatin (caveolae-mediated endocytosis) and phenylarsine oxide (PAO, macropinocytosis). Peringia ulvae (snails) were exposed to 150 µg Ag L(-1) added as citrate capped Ag NPs or aqueous Ag (AgNO3) in combination with inhibitor treatment (determined by preliminary studies). Reductions in accumulated tissue burdens caused by the inhibitors were compared to control exposures (i.e. no inhibition) after 6 and 24 h. No inhibitor treatment completely eliminated the uptake of Ag in either aqueous or NP form, but all inhibitor treatments, except phenamil, significantly reduced the uptake of Ag presented as Ag NPs. Clathrin- and caveolae-mediated endocytosis appear to be mechanisms exploited by Ag NPs, with the latter pathway only active at 24 h. Inhibition of the H(+) proton pump showed that a portion of Ag NP uptake is achieved as aqueous Ag and is explained by the dissolution of the particles (∼25% in 24 h). This in vivo study demonstrates that uptake of Ag from Ag NPs is achieved by multiple pathways and that these pathways are simultaneously active.

Bioaccumulation and toxicity of CuO nanoparticles by a freshwater invertebrate after waterborne and dietborne exposures.

The incidental ingestion of engineered nanoparticles (NPs) can be an important route of uptake for aquatic organisms. Yet, knowledge of dietary bioavailability and toxicity of NPs is scarce. Here we used isotopically modified copper oxide ((65)CuO) NPs to characterize the processes governing their bioaccumulation in a freshwater snail after waterborne and dietborne exposures. Lymnaea stagnalis efficiently accumulated (65)Cu after aqueous and dietary exposures to (65)CuO NPs. Cu assimilation efficiency and feeding rates averaged 83% and 0.61 g g(-1) d(-1) at low exposure concentrations (<100 nmol g(-1)), and declined by nearly 50% above this concentration. We estimated that 80-90% of the bioaccumulated (65)Cu concentration in L. stagnalis originated from the (65)CuO NPs, suggesting that dissolution had a negligible influence on Cu uptake from the NPs under our experimental conditions. The physiological loss of (65)Cu incorporated into tissues after exposures to (65)CuO NPs was rapid over the first days of depuration and not detectable thereafter. As a result, large Cu body concentrations are expected in L. stagnalis after exposure to CuO NPs. To the degree that there is a link between bioaccumulation and toxicity, dietborne exposures to CuO NPs are likely to elicit adverse effects more readily than waterborne exposures.

Bioaccumulation and effects of different-shaped copper oxide nanoparticles in the deposit-feeding snail Potamopyrgus antipodarum.

Copper oxide (CuO) nanoparticles (NPs) are among the most widely used engineered NPs and are thus likely to end up in the environment, predominantly in sediments. Copper oxide NPs have been found to be toxic to a variety of (mainly pelagic) organisms, but to differing degrees. In the present study, the influence of CuO NP shape on bioavailability and toxicity in the sediment-dwelling freshwater gastropod Potamopyrgus antipodarum was examined. In 2 separate studies, snails were exposed to either clean sediment or sediment spiked with either aqueous Cu or CuO NPs of different shapes (rods, spheres, or platelets) at 240 µg Cu/g dry weight of sediment (nominal). In neither of the studies was survival found to be related to Cu form (i.e., free ion vs particle) or shape, whereas snail growth was severely influenced by both form and shape. Reproduction was affected (by CuO NP spheres and aqueous Cu) only when estimated as the total number (live plus dead) of juveniles produced per snail per week. Both the aqueous and particulate forms of Cu were available for uptake by snails when mixed into sediment. However, Cu body burden was not directly related to observed effects. The present study stresses the need for both a better understanding of uptake mechanisms and internal distribution pathways of NPs and an assessment of long-term consequences of NP exposure.

Toxicity and accumulation of silver nanoparticles during development of the marine polychaete Platynereis dumerilii.

Pollutants affecting species at the population level generate ecological instability in natural systems. The success of early life stages, such as those of aquatic invertebrates, is highly affected by adverse environmental conditions. Silver released into the environment from emerging nanotechnology represents such a threat. Sediments are sinks for numerous pollutants, which aggregate and/or associate with depositing suspended particles. Deposit feeder such as the annelid Platynereis dumerilii, which has a large associated literature on its development, is an excellent model organism for exposure studies in coastal environments. We exposed eggs, larvae, juveniles and adults of P. dumerilii to various concentrations of citrate (cit-Ag NPs) or humic acid (HA-Ag NPs) capped silver nanoparticles (Ag NPs) as well to dissolved Ag (added as AgNO3). We showed that mortality and abnormal development rate increased with younger life stages. While adults and juvenile were the most tolerant life stages, fertilized eggs were highly sensitive to AgNO3, cit-Ag NPs and HA-Ag NPs. Exposures to HA-Ag NPs triggered the highest cute toxicity responses in P. dumerilii and in most cases both Ag NPs were more toxic than AgNO3. Uptake rate of HA-Ag NPs in adult worms was also higher than from other Ag forms, consistent with toxicity to other life stages. The early stages of the life cycle of marine coastal organisms are more affected by Ag NPs than the juvenile or adult life stages, indicating that exposure experiments at the larval level contribute to realistic eco-toxicological studies in aquatic environments.

Comparative study using spheres, rods and spindle-shaped nanoplatelets on dispersion stability, dissolution and toxicity of CuO nanomaterials.

Copper oxide nanoparticles with different shapes were used to examine the effect of shape on the various physicochemical properties (reactivity, aggregation, suspension stability) and to examine the behaviour by which CuO nanoparticles exhibit their biological response towards alveolar type-I cells. The different shapes examined in this study include spherical-, rod- and spindle-shaped platelet particles. In vitro dissolution studies (7 days) in 1 mM NaNO3 matrix showed a marked difference in dissolved Cu release between the nanoparticles. However, in serum-free cell-culture media (exposure media to cells), the particles' dissolution was found to be significantly enhanced with close to complete dissolution reported for all particle types. Biological studies showed both shape and size of the CuO nanoparticles tested to have a significant effect on TT-1 cell viability and release of pro-inflammatory cytokines IL-6 and IL-8. This study shows a complex interplay between particulate and dissolved species triggering the biological response. Upon immediate exposure of CuO nanoparticles of different shapes, the particulate form contributes towards the toxicity. However, for any biological response observed over and beyond a period of 24 h, the dissolved fraction becomes significant.

Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells.

Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT.

Bioaccumulation, toxicokinetics, and effects of copper from sediment spiked with aqueous Cu, nano-CuO, or micro-CuO in the deposit-feeding snail, Potamopyrgus antipodarum.

The present study examined the relative importance of copper (aqueous Cu and CuO particles of different sizes) added to sediment to determine the bioaccumulation, toxicokinetics, and effects in the deposit feeder Potamopyrgus antipodarum. In experiment 1, the bioaccumulation of Cu (240 µg Cu/g dry wt of sediment) added as aqueous Cu (CuCl2 ), nano- (6 nm, 100 nm), or micro- (<5 µm) CuO particles in adult snails was measured. In experiment 2, a more comprehensive analysis of the toxicokinetics of Cu (aqueous Cu, 6 nm, or 100 nm) was conducted. In experiment 3, the effects of Cu form (aqueous Cu and 6 nm CuO) on juvenile growth and survival at 0, 30, 60, 120, and 240 µg Cu/g dry weight sediment were assessed. Snails took up less of the 5-µm CuO particles than nano-CuO or aqueous Cu. A substantial fraction of Cu taken up was associated with shell, and this was rapidly lost when snails were transferred to clean sediment. Net uptake rates from sediment amended with 6 nm CuO and aqueous Cu were significantly higher (∼40-50%) than from sediment amended with 100 nm CuO. During 2 wk of depuration, there were no significant differences in depuration rates (kd ) among forms (aqueous Cu: kd = -0.12 wk(-1) ; 6 nm CuO: kd = -0.22 wk(-1) ; 100 nm CuO: kd = -0.2 wk(-1) ). Average juvenile growth was reduced by 0.11 mm (41%) at measured exposure concentrations of 127.2 µg Cu/g dry weight sediment for aqueous Cu and 71.9 µg Cu/g dry weight sediment for 6 nm CuO compared with control; however, differences between forms were not statistically significant. Juvenile snails in the highest exposure concentrations (aqueous Cu and 6-nm CuO groups pooled) reduced their growth by 0.18 mm on average (67%) compared with the control group. Although we observed minor differences in toxicity among Cu forms, effects on juvenile snail growth occurred at bulk sediment concentrations lower than those in the Canadian interim sediment quality guidelines. Characterization of the CuO particles showed that particle size distributions of commercially prepared particles deviated substantially from the manufacturers' specifications and highlighted the importance of fully characterizing particles when using them in toxicity tests.

Multiple cytotoxic and genotoxic effects induced in vitro by differently shaped copper oxide nanomaterials.

In nanotoxicology, the capacity of nanoparticles of the same composition but different shape to induce cytotoxicity and genotoxicity is largely unknown. A series of cytotoxic and genotoxic responses following in vitro exposure to differently shaped CuO nanoparticles (CuO NPs, mass concentrations from 0.1 to 100 µg/ml) were assessed in murine macrophages RAW 264.7 and in peripheral whole blood from healthy volunteers. Cytotoxicity, cytostasis and genotoxicity were evaluated by the colorimetric assay of formazan reduction [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)] and by the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. The comet assay was applied for detecting DNA strand breaks and information on oxidative damage to DNA (oxidised purines and pyrimidines). The MTT assay revealed a decrease in cell viability in RAW 264.7 cells and peripheral blood lymphocytes (PBL) with significant dose-effect relationships for the different CuO NP shapes. The comet assay revealed a dose-dependent increase in primary DNA damage, and a significant increase in oxidative damage to DNA was also detectable, as well as increased frequency of micronuclei in binucleated cells, often in a dose-related manner. Proliferative activity, cytotoxicity and apoptotic markers showed a significant trend in the two cell types. Finally, we have differentiated clastogenic events from aneugenic events by fluorescence in situ hybridisation with human and murine pancentromeric probes, revealing for the first time characteristic aneugenic responses related to the shape of CuO NPs and cell type. Independently of size and shape, all CuO NPs revealed a clear-cut cytotoxic and genotoxic potential; this suggests that CuO NPs are good candidates for positive controls in nanotoxicology.

The complexity of nanoparticle dissolution and its importance in nanotoxicological studies.

Dissolution of nanoparticles (NPs) is an important property that alters their abundance and is often a critical step in determining safety of nanoparticles. The dissolution status of the NPs in exposure media (i.e. whether they remain in particulate form or dissolve - and to what extent), strongly affects the uptake pathway, toxicity mechanisms and the environmental compartment in which NPs will have the highest potential impact. A review of available dissolution data on NPs demonstrates there is a range of potential outcomes depending on the NPs and the exposure media. For example two nominally identical nanoparticles, in terms of size and composition, could have totally different dissolution behaviours, subject to different surface modifications. Therefore, it is imperative that toxicological studies are conducted in conjunction with dissolution of NPs to establish the true biological effect of NPs and hence, assist in their regulation.

Bioaccumulation dynamics and modeling in an estuarine invertebrate following aqueous exposure to nanosized and dissolved silver.

Predicting the environmental impact of engineered nanomaterials (ENMs) is increasingly important owing to the prevalence of emerging nanotechnologies. We derived waterborne uptake and efflux rate constants for the estuarine snail, Peringia ulvae, exposed to dissolved Ag (AgNO(3)) and silver nanoparticles (Ag NPs), using biodynamic modeling. Uptake rates demonstrated that dissolved Ag is twice as bioavailable as Ag in nanoparticle form. Biphasic loss dynamics revealed the faster elimination of Ag from Ag NPs at the start of depuration, but similar slow efflux rate constants. The integration of biodynamic parameters into our model accurately predicted Ag tissue burdens during chronic exposure with 85% of predicted values within a factor of 2 of observed values. Zeta potentials for the Ag NPs were lower in estuarine waters than in waters of less salinity; and uptake rates in P. ulvae were slower than reported for the freshwater snail Lymnaea stagnalis in similar experiments. This suggests aggregation of Ag NPs occurs in estuarine waters and reduces, but does not eliminate, bioavailability of Ag from the Ag NPs. Biodynamic modeling provides an effective methodology to determine bioavailable metal concentrations (originating from metal and metal-oxide nanoparticles) in the environment and may aid future ENM risk assessment.