Determination of the LD50 with the chick embryo chorioallantoic membrane (CAM) assay as a promising alternative in nanotoxicological evaluation.

Toxicity tests in rodents are still considered a controversial topic concerning their ethical justifiability. The chick embryo chorioallantoic membrane (CAM) assay may offer a simple and inexpensive alternative. The CAM assay is easy to perform and has low bureaucratic hurdles. At the same time, the CAM assay allows the application of a broad variety of analytical methods in the field of nanotoxicological research. We evaluated the CAM assay as a methodology for the determination of nanotoxicity. Therefore we calculated the median lethal dose (LD50), performed in vivo microscopy and immunohistochemistry to identify organ-specific accumulation profiles, potential organ damage, and the kinetics of the in vivo circulation of the nanoparticles. Zinc oxide nanoparticles were intravascularly injected on day 10 of the egg development and showed an LD50 of 17.5 µM (1.4 µg/mLeggcontent). In comparison, the LD50 of equivalent amounts of Zn2+ was 4.6 µM (0.6 µg/mLeggcontent). Silica encapsulated ZnO@SiO2 nanoparticles conjugated with fluorescein circulated in the bloodstream for at least 24 h. Particles accumulated mostly in the liver and kidney. In immunohistochemical staining, organ damage was detected only in liver tissue after intravascular injection of zinc oxide nanoparticles in very high concentrations. Zinc oxide nanoparticles showed a different pharmacokinetic profile compared to Zn2+ ions. In conclusion, the CAM assay has proven to be a promising methodology for evaluating nanotoxicity and for the assessment of the in vivo accumulation profiles of nanoparticles. These findings may qualify the methodology for risk assessment of innovative nanotherapeutics in the future.

Zinc overload mediated by zinc oxide nanoparticles as innovative anti-tumor agent.

The predicted global cancer burden is expected to surpass 20 million new cancer cases by 2025. Despite recent advancement in tumor therapy, a successful cancer treatment remains challenging. The emerging field of nanotechnology offers great opportunities for diagnosis, imaging, as well as treatment of cancer. Zinc oxide nanoparticles (ZnO NP) were shown to exert selective cytotoxicity against tumor cells via a yet unknown mechanism, most likely involving the generation of reactive oxygen species (ROS). These nanoparticles are a promising therapeutic opportunity as zinc is a nontoxic trace element and its application in medically-related products is considered to be safe. We could show that ZnO NP can exert cytotoxic effects on several human tumor cell lines. There can be found ZnO NP concentrations which selectively damage tumor cells while human fibroblasts do not sustain lasting damage. Cytotoxicity is attributable to the release of zinc ions from the nanoparticles outside the cells as well as to a direct cell-nanoparticle interaction. This involves uptake of the particles into the tumor cells. With a silica shell the cytotoxicity can be delayed which can help in the future for a safe transport in the blood stream. Cellular damage finally cumulates in apoptotic cell death via zinc overload within 48 h after treatment with ZnO NP. A therapeutical perspective could be the targeted accumulation of ZnO NP at the tumor side to induce local zinc overload that substantially damages the tumor cells with no or low side effects. We suggest further studies to explore the potential of ZnO NP as an innovative anti-tumor agent.

Controlling the Morphology of Au-Pd Heterodimer Nanoparticles by Surface Ligands.

Controlling the morphology of noble-metal nanoparticles is mandatory to tune specific properties such as catalytic and optical behavior. Heterodimers consisting of two noble metals have been synthesized, so far mostly in aqueous media using selective surfactants or chemical etching strategies. We report a facile synthesis for Au@Pd and Pd@Au heterodimer nanoparticles (NPs) with morphologies ranging from segregated domains (heteroparticles) to core-shell structures by applying a seed-mediated growth process with Au and Pd seed nanoparticles in 1-octadecene (ODE), which is a high-boiling organic solvent. The as-synthesized oleylamine (OAm) functionalized Au NPs led to the formation of OAm-Au@Pd heteroparticles with a "windmill" morphology, having an Au core and Pd "blades". The multiply twinned structure of the Au seed particles (⌀ ≈ 9-11 nm) is associated with a reduced barrier for heterogeneous nucleation. This leads to island growth of bimetallic Au@Pd heteroparticles with less-regular morphologies. The reaction process can be controlled by tuning the surface chemistry with organic ligands. Functionalization of Au NPs (Ø ≈ 9-11 nm) with 1-octadecanethiol (ODT) led to the formation of ODT-Au@Pd NPs with a closed Pd shell through a strong ligand-metal binding, which is accompanied by a redistribution of the electron density. Experiments with varied Pd content revealed surface epitaxial growth of Pd on Au. For OAm-Pd and ODT-Pd seed particles, faceted, Au-rich domain NPs and impeded core-shell NPs were obtained, respectively. This is related to the high surface energy of the small Pd seed particles (⌀ ≈ 5-7 nm). The metal distribution of all bimetallic NPs was analyzed by extended (aberration-corrected) transmission electron microscopy (HR-TEM, HAADF-STEM, EDX mapping, ED). The Au and Pd NPs, as well as the ODT-Au@Pd and OAm-Pd@Au heteroparticles, catalyze the reduction of 4-nitrophenol to 4-aminophenol with borohydride. The catalytic activity is dictated by the particle structure. OAm-Au@Pd heteroparticles with faceted Au domains had the highest activity because of a mixed Au-Pd surface structure, while ODT-Au@Pd NPs, where the active Au core is covered by a Pd shell, had the lowest activity.

Monitoring Thiol-Ligand Exchange on Au Nanoparticle Surfaces.

Surface functionalization of nanoparticles (NPs) plays a crucial role in particle solubility and reactivity. It is vital for particle nucleation and growth as well as for catalysis. This raises the quest for functionalization efficiency and new approaches to probe the degree of surface coverage. We present an (in situ) proton nuclear magnetic resonance (1H NMR) study on the ligand exchange of oleylamine by 1-octadecanethiol as a function of the particle size and repeated functionalization on Au NPs. Ligand exchange is an equilibrium reaction associated with Nernst distribution, which often leads to incomplete surface functionalization following "standard" literature protocols. Here, we show that the surface coverage with the ligand depends on the (i) repeated exchange reactions with large ligand excess, (ii) size of NPs, that is, the surface curvature and reactivity, and (iii) molecular size of the ligand. As resonance shifts and extensive line broadening during and after the ligand exchange impede the evaluation of 1H NMR spectra, one- and two-dimensional 19F NMR techniques (correlation spectroscopy and diffusion ordered spectroscopy) with 1H,1H,2H,2H-perfluorodecanthiol as the fluorinated thiol ligand were employed to study the reactions. The enhanced resolution associated with the spectral range of the 19F nucleus allowed carrying out a site-specific study of thiol chemisorption. The widths and shifts of the resonance signals of the different fluorinated carbon moieties were correlated with the distance to the thiol anchor group. In addition, the diffusion analysis revealed that moieties closer to the NP surface are characterized by a broader diffusion coefficient distribution as well as slower diffusion.

Glycine-functionalized copper(ii) hydroxide nanoparticles with high intrinsic superoxide dismutase activity.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O2-) radicals into molecular oxygen (O2) and H2O2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(ii) hydroxide nanoparticles (Gly-Cu(OH)2 NPs) are functional SOD mimics, whereas bulk Cu(OH)2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH)2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH)2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concentrations of toxic reactive oxygen species (ROS, >1016 molecules per puff) including superoxide and reactive nitrogen species which lead to the development of chronic and degenerative diseases via oxidative damage and subsequent cell death. Embedded in cigarette filters Gly-Cu(OH)2 NPs efficiently removed ROS from smoke, thereby protecting lung cancer cell lines from cytotoxic effects. Their stability, ease of production and versatility make them a powerful tool for a wide range of applications in environmental chemistry, biotechnology and medicine.