Systematic determination of the relationship between nanoparticle core diameter and toxicity for a series of structurally analogous gold nanoparticles in zebrafish.

Predictive models for the impact of nanomaterials on biological systems remain elusive. Although there is agreement that physicochemical properties (particle diameter, shape, surface chemistry, and core material) influence toxicity, there are limited and often contradictory, data relating structure to toxicity, even for core diameter. Given the importance of size in determining nanoscale properties, we aimed to address this data gap by examining the biological effects of a defined series of gold nanoparticles (AuNPs) on zebrafish embryos. Five AuNPs samples with narrowly spaced core diameters (0.8-5.8 nm) were synthesized and functionalized with positively charged N,N,N-trimethylammonium ethanethiol (TMAT) ligands. We assessed the bioactivity of these NPs in a high-throughput developmental zebrafish assay at eight concentrations (0.5-50 µg/mL) and observed core diameter-dependent bioactivity. The smaller diameter AuNPs were the most toxic when expressing exposures based on an equal mass. However, when expressing exposures based on total surface area, toxicity was independent of the core diameter. When holding the number of nanoparticles per volume constant (at 6.71 × 1013/mL) in the exposure medium across AuNPs diameters, only the 5.8 nm AuNPs exhibited toxic effects. Under these exposure conditions, the uptake of AuNPs in zebrafish was only weakly associated with core diameter, suggesting that differential uptake of TMAT-AuNPs was not responsible for toxicity associated with the 5.8 nm core diameter. Our results indicate that larger NPs may be the most toxic on a per particle basis and highlight the importance of using particle number and surface area, in addition to mass, when evaluating the size-dependent bioactivity of NPs.

Potential Environmental Impacts and Antimicrobial Efficacy of Silver- and Nanosilver-Containing Textiles.

For textiles containing nanosilver, we assessed benefit (antimicrobial efficacy) in parallel with potential to release nanosilver (impact) during multiple life cycle stages. The silver loading and method of silver attachment to the textile highly influenced the silver release during washing. Multiple sequential simulated household washing experiments for fabric swatches in deionized water with or without detergent showed a range of silver release. The toxicity of washing experiment supernatants to zebrafish (Danio rerio) embryos was negligible, with the exception of the very highest Ag releases (∼1 mg/L Ag). In fact, toxicity tests indicated that residual detergent exhibited greater adverse response than the released silver. Although washing the fabrics did release silver, it did not affect their antimicrobial efficacy, as demonstrated by >99.9% inhibition of E. coli growth on the textiles, even for textiles that retained as little as 2 µg/g Ag after washing. This suggests that very little nanosilver is required to control bacterial growth in textiles. Visible light irradiation of the fabrics reduced the extent of Ag release for textiles during subsequent washings. End-of-life experiments using simulated landfill conditions showed that silver remaining on the textile is likely to continue leaching from textiles after disposal in a landfill.

Structurally similar triphenylphosphine-stabilized undecagolds, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, exhibit distinct ligand exchange pathways with glutathione.

Ligand exchange is frequently used to introduce new functional groups on the surface of inorganic nanoparticles or clusters while preserving the core size. For one of the smallest clusters, triphenylphosphine (TPP)-stabilized undecagold, there are conflicting reports in the literature regarding whether core size is retained or significant growth occurs during exchange with thiol ligands. During an investigation of these differences in reactivity, two distinct forms of undecagold were isolated. The X-ray structures of the two forms, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, differ only in the number of TPP ligands bound to the core. Syntheses were developed to produce each of the two forms, and their spectroscopic features correlated with the structures. Ligand exchange on [Au11(PPh3)8Cl2]Cl yields only small clusters, whereas exchange on Au11(PPh3)7Cl3 (or mixtures of the two forms) yields the larger Au25 cluster. The distinctive features in the optical spectra of the two forms made it possible to evaluate which of the cluster forms were used in the previously published papers and clarify the origin of the differences in reactivity that had been reported. The results confirm that reactions of clusters and nanoparticles may be influenced by small variations in the arrangement of ligands and suggest that the role of the ligand shell in stabilizing intermediates during ligand exchange may be essential to preventing particle growth or coalescence.

Synthesis of ligand-stabilized metal oxide nanocrystals and epitaxial core/shell nanocrystals via a lower-temperature esterification process.

The properties of metal oxide nanocrystals can be tuned by incorporating mixtures of matrix metal elements, adding metal ion dopants, or constructing core/shell structures. However, high-temperature conditions required to synthesize these nanocrystals make it difficult to achieve the desired compositions, doping levels, and structural control. We present a lower temperature synthesis of ligand-stabilized metal oxide nanocrystals that produces crystalline, monodisperse nanocrystals at temperatures well below the thermal decomposition point of the precursors. Slow injection (0.2 mL/min) of an oleic acid solution of the metal oleate complex into an oleyl alcohol solvent at 230 °C results in a rapid esterification reaction and the production of metal oxide nanocrystals. The approach produces high yields of crystalline, monodisperse metal oxide nanoparticles containing manganese, iron, cobalt, zinc, and indium within 20 min. Synthesis of tin-doped indium oxide (ITO) can be accomplished with good control of the tin doping levels. Finally, the method makes it possible to perform epitaxial growth of shells onto nanocrystal cores to produce core/shell nanocrystals.

Gold nanoparticles disrupt zebrafish eye development and pigmentation.

Systematic toxicological study is still required to fully understand the hazard potentials of gold nanoparticles (AuNPs). Because their biomedical applications are rapidly evolving, we investigated developmental toxicity of AuNPs in an in vivo embryonic zebrafish model at exposure concentration ranges from 0.08 to 50mg/l. Exposure of zebrafish embryos to 1.3 nm AuNPs functionalized with a cationic ligand, N,N,N-trimethylammoniumethanethiol (TMAT-AuNPs), resulted in smaller malpigmented eyes. We determined that TMAT-AuNPs caused a significant increase of cell death in the eye, which was correlated with an increase in gene expression of p53 and bax. Expression patterns of key transcription factors regulating eye development (pax6a, pax6b, otx2, and rx1) and pigmentation (sox10) were both repressed in a concentration-dependent manner in embryos exposed to TMAT-AuNPs. Reduced spatial localization of pax6a, rx1, sox10, and mitfa was observed in embryos by whole-mount in situ hybridization. The swimming behavior of embryos exposed to sublethal concentrations of TMAT-AuNPs showed hypoactivity, and embryos exhibited axonal growth inhibition. Overall, these results demonstrated that TMAT-AuNPs disrupt the progression of eye development and pigmentation that continues to behavioral and neuronal damage in the developing zebrafish.

Surface functionalities of gold nanoparticles impact embryonic gene expression responses.

Incorporation of gold nanoparticles (AuNPs) into consumer products is increasing; however, there is a gap in available toxicological data to determine the safety of AuNPs. In this study, we utilised the embryonic zebrafish to investigate how surface functionalisation and charge influence molecular responses. Precisely engineered AuNPs with 1.5 nm cores were synthesised and functionalized with three ligands: 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol. Developmental assessments revealed differential biological responses when embryos were exposed to the functionalised AuNPs at the same concentration. Using inductively coupled plasma-mass spectrometry, AuNP uptake was confirmed in exposed embryos. Following exposure to MES- and TMAT-AuNPs from 6 to 24 or 6 to 48 h post fertilisation, pathways involved in inflammation and immune response were perturbed. Additionally, transport mechanisms were misregulated after exposure to TMAT and MES-AuNPs, demonstrating that surface functionalisation influences many molecular pathways.

Media ionic strength impacts embryonic responses to engineered nanoparticle exposure.

Embryonic zebrafish were used to assess the impact of solution ion concentrations on agglomeration and resulting in vivo biological responses of gold nanoparticles (AuNPs). The minimum ion concentration necessary to support embryonic development was determined. Surprisingly, zebrafish exhibit no adverse outcomes when raised in nearly ion-free media. During a rapid throughput screening of AuNPs, 1.2-nm 3-mercaptopropionic acid-functionalized AuNPs (1.2-nm 3-MPA-AuNPs) rapidly agglomerate in exposure solutions. When embryos were exposed to 1.2-nm 3-MPA-AuNPs dispersed in low ionic media, both morbidity and mortality were induced, but when suspended in high ionic media, there was little to no biological response. We demonstrated that the media ionic strength greatly affects agglomeration rates and biological responses. Most importantly, the insensitivity of the zebrafish embryo to external ions indicates that it is possible, and necessary, to adjust the exposure media conditions to optimize NP dispersion prior to assessment.

Single-molecule atomic force microscopy force spectroscopy study of Aß-40 interactions.

Misfolding and aggregation of amyloid ß-40 (Aß-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aß-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aß-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.

Nanomedicine and protein misfolding diseases.

Misfolding and self assembly of proteins in nano-aggregates of different sizes and morphologies (nano-ensembles, primarily nanofilaments and nano-rings) is a complex phenomenon that can be facilitated, impeded, or prevented, by interactions with various intracellular metabolites, intracellular nanomachines controlling protein folding and interactions with other proteins. A fundamental understanding of molecular processes leading to misfolding and self-aggregation of proteins involved in various neurodegenerative diseases will provide critical information to help identify appropriate therapeutic routes to control these processes. An elevated propensity of misfolded protein conformation in solution to aggregate with the formation of various morphologies impedes the use of traditional physical chemical approaches for studies of misfolded conformations of proteins. In our recent alternative approach, the protein molecules were tethered to surfaces to prevent aggregation and AFM force spectroscopy was used to probe the interaction between protein molecules depending on their conformations. It was shown that formation of filamentous aggregates is facilitated at pH values corresponding to the maximum of rupture forces. In this paper, a novel surface chemistry was developed for anchoring of amyloid beta (Abeta) peptides at their N-terminal moieties. The use of the site specific immobilization procedure allowed to measure the rupture of Abeta-Abeta contacts at single molecule level. The rupture of these contacts is accompanied by the extension of the peptide chain detected by a characteristic elasto-mechanical component of the force-distance curves. Potential applications of the nanomechanical studies to understanding the mechanisms of development of protein misfolding diseases are discussed.

Synthesis of well-defined tower-shaped 1,3,5-trisubstituted adamantanes incorporating a macrocyclic trilactam ring system.

We describe the synthesis of two novel well-defined tower-shaped 1,3,5-trisubstituted adamantanes 30 and 33 that incorporate a macrocyclic trilactam ring system. Each nanoscale molecule has a broad tripodal base consisting of three identical sulfur-containing termini as the tripod feet, 4-acetylsulfanylmethylphenyl units in the case of 30 and 3,5-bis(acetylsulfanylmethyl)phenyl units in the case of 33. The sulfur atoms are designed to bind the molecules trivalently to the apex of a gold-coated commercial AFM tip through formation of three S-Au bonds. The rigid adamantane-derived head unit with a single hydrogen atom at the apex is designed to scan the sample. Molecules 30 and 33 are synthesized from 1,3,5-triethynyladamantane by a series of Sonogashira coupling reactions involving terminal alkynes and aryl iodides. A macrocyclic trilactam unit is included for added rigidity. We demonstrate that molecule 30 is sufficiently large and rigid to be visualized by a conventional AFM tip. These nanoscale molecules may also find application as chemically well-defined nanoscale objects for calibration of AFM tips.

Nanoscale tripodal 1,3,5,7-tetrasubstituted adamantanes for AFM applications.

The synthesis of four novel nanoscale 1,3,5,7-tetrasubstituted adamantanes 22 and 25-27 designed for atomic force microscopy (AFM) applications is described. Each tetrahedrally shaped molecule incorporates a broad tripodal base made up of three identical legs that terminate with a sulfur-containing moiety, which is either a 4-acetylsulfanylmethylphenyl unit or else a (1,2,5-dithiazepan-1-yl)phenyl unit. The sulfur atoms are intended for eventual binding of the molecule multivalently to the apex of a gold-coated commercial AFM tip through formation of multiple S-Au bonds. In each molecule, the fourth terminus is a para-substituted benzoic acid methyl ester that is designed to scan the sample. We demonstrate that 27 is sufficiently large and rigid to be imaged by a conventional AFM tip. Adamantanes 22 and 25-27 may also find application as chemically well-defined nanoscale objects for calibration of AFM tips.

Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C.

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.

Listeria monocytogenes phosphatidylinositol-specific phospholipase C: activation and allostery.

The animal and human pathogen Listeria monocytogenes secretes several virulence factors, including a phosphatidylinositol-specific phospholipase C (PI-PLC). Sufficient quantities of L. monocytogenes PI-PLC for biophysical studies were obtained by overexpression of the enzyme in Escherichia coli. The purified PI-PLC was examined in enzyme kinetics experiments using a new fluorogenic substrate, methyl-FLIP. Methyl-FLIP is a water-soluble monomeric substrate cleaved in a manner similar to the natural aggregate substrate, phosphatidylinositol (PI). Michaelis-Menten kinetics were observed with K(M) = 61 +/- 7 microM and V(max) = 120 +/- 5 micromol min(-1) mg(-1), corresponding to k(cat) = 66+/-3 s(-1). The catalysis is activated by the addition of a short-chain phospholipid, dihexanoyl phosphatidylcholine (diC(6)PC). The kinetics were fitted to a two-site model in which the substrate binds to the active site and diC(6)PC binds to a second site, with an interaction between the two sites. The result is a decrease in K(M) and an increase in V(max), producing an overall four to five-fold increase in catalytic efficiency (k(cat)/K(M)). The interaction is not a regulatory mechanism, as is the case for multimeric enzymes; rather, it suggests interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site.

Nanoscale 1,3,5,7-tetrasubstituted adamantanes and p-substituted tetraphenyl-methanes for AFM applications.

[structure: see text] Tetrahedrally shaped nanoscale molecules 18-20 were synthesized from the corresponding tetraiodide by a series of Sonogashira coupling reactions. Three of the sulfur-containing termini are intended for eventual binding to a gold-coated conventional AFM tip, while the fourth terminus scans the sample. AFM images of 19 demonstrate that the molecule is sufficiently large and rigid to be imaged by a conventional AFM tip.