Annika Leifert

Gold Nanoparticles of Diameter 1.4 nm Trigger Necrosis by Oxidative Stress and Mitochondrial Damage

Gold nanoparticles (AuNPs) are generally considered nontoxic, similar to bulk gold, which is inert and biocompatible. AuNPs of diameter 1.4 nm capped with triphenylphosphine monosulfonate (TPPMS), Au1.4MS, are much more cytotoxic than 15-nm nanoparticles (Au15MS) of similar chemical composition. Here, major cell-death pathways are studied and it is determined that the cytotoxicity is caused by oxidative stress. Indicators of oxidative stress, reactive oxygen species (ROS), mitochondrial potential and integrity, and mitochondrial substrate reduction are all compromised. Genome-wide expression profiling using DNA gene arrays indicates robust upregulation of stress-related genes after 6 and 12 h of incubation with a 2 x IC50 concentration of Au1.4MS but not with Au15MS nanoparticles. The caspase inhibitor Z-VAD-fmk does not rescue the cells, which suggests that necrosis, not apoptosis, is the predominant pathway at this concentration. Pretreatment of the nanoparticles with reducing agents/antioxidants N-acetylcysteine, glutathione, and TPPMS reduces the toxicity of Au1.4MS. AuNPs of similar size but capped with glutathione (Au1.1GSH) likewise do not induce oxidative stress. Besides the size dependency of AuNP toxicity, ligand chemistry is a critical parameter determining the degree of cytotoxicity. AuNP exposure most likely causes oxidative stress that is amplified by mitochondrial damage. Au1.4MS nanoparticle cytotoxicity is associated with oxidative stress, endogenous ROS production, and depletion of the intracellular antioxidant pool.

Differential hERG ion channel activity of ultrasmall gold nanoparticles

Understanding the mechanism of toxicity of nanomaterials remains a challenge with respect to both mechanisms involved and product regulation. Here we show toxicity of ultrasmall gold nanoparticles (AuNPs). Depending on the ligand chemistry, 1.4-nm-diameter AuNPs failed electrophysiology-based safety testing using human embryonic kidney cell line 293 cells expressing human ether-á-go-go-Related gene (hERG), a Food and Drug Administration-established drug safety test. In patch-clamp experiments, phosphine-stabilized AuNPs irreversibly blocked hERG channels, whereas thiol-stabilized AuNPs of similar size had no effect in vitro, and neither particle blocked the channel in vivo. We conclude that safety regulations may need to be reevaluated and adapted to reflect the fact that the binding modality of surface functional groups becomes a relevant parameter for the design of nanoscale bioactive compounds.

High-sensitivity real-time analysis of nanoparticle toxicity in green fluorescent protein-expressing zebrafish.

Gold nanoparticles (AuNP) show great potential for diagnostic and therapeutic application in humans. A great number of studies have tested the cytotoxicity of AuNP using cell culture. There is, however, an urgent need to test AuNP in vertebrate animal models that interrogate biodistribution and complex biological traits like organ development, whole body metabolism, and cognitive function. The sheer number of different compounds precludes the use of small rodent model for initial screening. The extended fish embryo test (FET) is used here to bridge the gap between cell culture and small animal models. A study on the toxicity of ultrasmall AuNP in wild type and transgenic zebrafish is presented. FET faithfully reproduce all important findings of a previous study in HeLa cells and add new important information on teratogenicity and hepatotoxicity that could not be gained from studying cultured cells.

Size-dependent multispectral photoacoustic response of solid and hollow gold nanoparticles

Photoacoustic (PA) imaging attracts a great deal of attention as an innovative modality for longitudinal, non-invasive, functional and molecular imaging in oncology. Gold nanoparticles (AuNPs) are identified as superior, NIR-absorbing PA contrast agents for biomedical applications. Until now, no systematic comparison of the optical extinction and PA efficiency of water-soluble AuNPs of various geometries and small sizes has been performed. Here spherical AuNPs with core diameters of 1.0, 1.4 and 11.2 nm, nanorods with longitudinal/transversal elongation of 38/9 and 44/12 nm and hollow nanospheres with outer/inner diameters of 33/19, 57/30, 68/45 and 85/56 nm were synthesized. The diode laser set-up with excitations at 650, 808, 850 and 905 nm allowed us to correlate the molar PA signal intensity with the molar extinction of the respective AuNPs. Deviations were explained by differences in heat transfer from the particle to the medium and, for larger particles, by the scattering of light. The molar PA intensity of 1.0 nm AuNPs was comparable to the commonly used organic dye methylene blue, and rapidly increased with the lateral size of AuNPs.

Probing Structural Dynamics of an Artificial Protein Cage Using High-Speed Atomic Force Microscopy

A cysteine-substituted mutant of the ring-shaped protein TRAP (trp-RNA binding attenuation protein) can be induced to self-assemble into large, monodisperse hollow spherical cages in the presence of 1.4 nm diameter gold nanoparticles. In this study we use high-speed atomic force microscopy (HS-AFM) to probe the dynamics of the structural changes related to TRAP interactions with the gold nanoparticle as well as the disassembly of the cage structure. The dynamic aggregation of TRAP protein in the presence of gold nanoparticles was observed, including oligomeric rearrangements, consistent with a role for gold in mediating intermolecular disulfide bond formation. We were also able to observe that the TRAP-cage is composed of multiple, closely packed TRAP rings in an apparently regular arrangement. A potential role for inter-ring disulfide bonds in forming the TRAP-cage was shown by the fact that ring-ring interactions were reversed upon the addition of reducing agent dithiothreitol. A dramatic disassembly of TRAP-cages was observed using HS-AFM after the addition of dithiothreitol. To the best of our knowledge, this is the first report to show direct high-resolution imaging of the disassembly process of a large protein complex in real time.

Size dependent photoacoustic signal response of gold nanoparticles using a multispectral laser diode system

Photoacoustic imaging is based on the generation of ultrasound using laser irradiation. Recently, gold nanoparticles received a lot of attention due to their excellent suitability for use as contrast agents. For contrast agent analysis, Nd:YAG laser systems are commonly employed, but pulsed laser diodes, which are much smaller and cheaper, can be an attractive alternative. They emit significantly lower pulse energies, but fast averaging is feasible due to high repetition rates. Analysis of contrast agents using laser diodes suffers from vastly different properties of different laser diodes. Here, we present a calibration procedure to correct for some of these differences and enable comparison of results across multiple laser diodes. The calibration procedure is performed for analysis of nanoparticles ranging from diameters of 1 nm to 85 nm. The method is analyzed using two reference materials, Copper Chloride (CuCl2) and Copper Sulfate (CuSO4).

Joint Experimental and Theoretical Investigation of the Interaction Between Antimicrobial Peptides, Gold Nanoparticles and Membranes

Antimicrobial peptides and gold nanoparticles (AuNPs) are interesting novel classes of pharmaceutically active compounds. To understand and optimize their efficacy, interactions of these systems with biological membranes need to be characterized. Given the wide range of synthetic possibilities, either by sequence design (peptides) or size, composition and ligand shell (nanoparticles), computational methods may help designing active compounds with predictable membrane permeability. Here we investigate a range of implicit membrane models, as extensions of our implicit solvent force field PFF02, to understand details of experimentally observed membrane association properties of naturally occurring antimicrobial peptides, in particular Gramicidin A/S and ligand stabilized gold nanoparticles of different size. Membrane association and penetration were studied in black lipid membrane (BLM) experiments using DOPC or DiphPC/DiphPG and DiphPE, model membranes. For Gram A we observed the transient formation of individual pores in the experiments, which are rationalized by simulations showing the dimerization of the helical peptides in the membrane. For Gram S, in agreement with the modeling results, we observe only small, fluctuating currents. We also observe size selective membrane association of the gold nanoparticles, where membrane integration of nanoparticles of 15 nm diameter generated ion-selective currents, while smaller 1.4 nm particles did not show such effects.