Long-Term Accumulation, Biological Effects and Toxicity of BSA-Coated Gold Nanoparticles in the Mouse Liver, Spleen, and Kidneys.

Introduction: Gold nanoparticles are promising candidates as vehicles for drug delivery systems and could be developed into effective anticancer treatments. However, concerns about their safety need to be identified, addressed, and satisfactorily answered. Although gold nanoparticles are considered biocompatible and nontoxic, most of the toxicology evidence originates from in vitro studies, which may not reflect the responses in complex living organisms. Methods: We used an animal model to study the long-term effects of 20 nm spherical AuNPs coated with bovine serum albumin. Mice received a 1 mg/kg single intravenous dose of nanoparticles, and the biodistribution and accumulation, as well as the organ changes caused by the nanoparticles, were characterized in the liver, spleen, and kidneys during 120 days. Results: The amount of nanoparticles in the organs remained high at 120 days compared with day 1, showing a 39% reduction in the liver, a 53% increase in the spleen, and a 150% increase in the kidneys. The biological effects of chronic nanoparticle exposure were associated with early inflammatory and fibrotic responses in the organs and were more pronounced in the kidneys, despite a negligible amount of nanoparticles found in renal tissues. Conclusion: Our data suggest, that although AuNPs belong to the safest nanomaterial platforms nowadays, due to their slow tissue elimination leading to long-term accumulation in the biological systems, they may induce toxic responses in the vital organs, and so understanding of their long-term biological impact is important to consider their potential therapeutic applications.

The Effect of Chemical Structure of OEG Ligand Shells with Quaternary Ammonium Moiety on the Colloidal Stabilization, Cellular Uptake and Photothermal Stability of Gold Nanorods.

PURPOSE: Plasmonic photothermal cancer therapy by gold nanorods (GNRs) emerges as a promising tool for cancer treatment. The goal of this study was to design cationic oligoethylene glycol (OEG) compounds varying in hydrophobicity and molecular electrostatic potential as ligand shells of GNRs. Three series of ligands with different length of OEG chain (ethylene glycol units = 3, 4, 5) and variants of quaternary ammonium salts (QAS) as terminal functional group were synthesized and compared to a prototypical quaternary ammonium ligand with alkyl chain - (16-mercaptohexadecyl)trimethylammonium bromide (MTAB). METHODS: Step-by-step research approach starting with the preparation of compounds characterized by NMR and HRMS spectra, GNRs ligand exchange evaluation through characterization of cytotoxicity and GNRs cellular uptake was used. A method quantifying the reshaping of GNRs was applied to determine the effect of ligand structure on the heat transport from GNRs under fs-laser irradiation. RESULTS: Fourteen out of 18 synthesized OEG compounds successfully stabilized GNRs in the water. The colloidal stability of prepared GNRs in the cell culture medium decreased with the number of OEG units. In contrast, the cellular uptake of OEG+GNRs by HeLa cells increased with the length of OEG chain while the structure of the QAS group showed a minor role. Compared to MTAB, more hydrophilic OEG compounds exhibited nearly two order of magnitude lower cytotoxicity in free state and provided efficient cellular uptake of GNRs close to the level of MTAB. Regarding photothermal properties, OEG compounds evoked the photothermal reshaping of GNRs at lower peak fluence (14.8 mJ/cm2) of femtosecond laser irradiation than the alkanethiol MTAB. CONCLUSION: OEG+GNRs appear to be optimal for clinical applications with systemic administration of NPs not-requiring irradiation at high laser intensity such as drug delivery and photothermal therapy inducing apoptosis.

Gold Film over SiO2 Nanospheres-New Thermally Resistant Substrates for Surface-Enhanced Raman Scattering (SERS) Spectroscopy.

Surface-enhanced Raman scattering (SERS) sensors are constructed from metallic plasmonic nanostructures providing high sensitivity and spectral reproducibility. In many cases, irradiation of the SERS substrate by the laser beam leads to an increase of the local temperature and consequently to thermal degradation of metallic nanostructure itself and/or adsorbed analyte. We report here a "bottom-up" technique to fabricate new thermally resistant gold "film over nanosphere" (FON) substrates for SERS. We elaborated the simple and straightforward method of preparation of homogeneously and closely packed monolayer of SiO2 nanoparticles (50 nm in diameter) and covered it by a thin (20 nm) layer of magnetron-sputtered gold. The spectral testing using biologically important molecules (methylene blue, cationic porphyrin, and fungicide 1-methyl-1H-benzimidazole-2-thiol) proved a sensitivity and reproducibility of our AuSiO2 substrates. The main advantage of such SERS-active substrates is high thermal stability and low intensity of background and signal of graphitic carbon.

Biological safety and tissue distribution of (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTABGNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 µg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

Two-Step Mechanism of Cellular Uptake of Cationic Gold Nanoparticles Modified by (16-Mercaptohexadecyl)trimethylammonium Bromide.

Cationic colloidal gold nanorods (GNRs) have a great potential as a theranostic tool for diverse medical applications. GNRs' properties such as cellular internalization and stability are determined by physicochemical characteristics of their surface coating. GNRs modified by (16-mercaptohexadecyl)trimethylammonium bromide (MTAB), MTABGNRs, show excellent cellular uptake. Despite their promise for biomedicine, however, relatively little is known about the cellular pathways that facilitate the uptake of GNRs, their subcellular fate and intracellular persistence. Here we studied the mechanism of cellular internalization and long-term fate of GNRs coated with MTAB, for which the synthesis was optimized to give higher yield, in various human cell types including normal diploid versus cancerous, and dividing versus nondividing (senescent) cells. The process of MTABGNRs internalization into their final destination in lysosomes proceeds in two steps: (1) fast passive adhesion to cell membrane mediated by sulfated proteoglycans occurring within minutes and (2) slower active transmembrane and intracellular transport of individual nanorods via clathrin-mediated endocytosis and of aggregated nanorods via macropinocytosis. The expression of sulfated proteoglycans was the major factor determining the extent of uptake by the respective cell types. Upon uptake into proliferating cells, MTABGNRs were diluted equally and relatively rapidly into daughter cells; however, in nondividing/senescent cells the loss of MTABGNRs was gradual and very modest, attributable mainly to exocytosis. Exocytosed MTABGNRs can again be internalized. These findings broaden our knowledge about cellular uptake of gold nanorods, a crucial prerequisite for future successful engineering of nanoparticles for biomedical applications such as photothermal cancer therapy or elimination of senescent cells as part of the emerging rejuvenation approach.

Quantitative SERS analysis of azorubine (E 122) in sweet drinks.

Considering both the potential effects on human health and the need for knowledge of food composition, quantitative detection of synthetic dyes in foodstuffs and beverages is an important issue. For the first time, we report a fast quantitative analysis of the food and drink colorant azorubine (E 122) in different types of beverages using surface-enhanced Raman scattering (SERS) without any sample preparation. Seven commercially available sweet drinks (including two negative controls) with high levels of complexity (sugar/artificial sweetener, ethanol content, etc.) were tested. Highly uniform Au "film over nanospheres" (FON) substrates together with use of Raman signal from silicon support as internal intensity standard enabled us to quantitatively determine the concentration of azorubine in each drink. SERS spectral analysis provided sufficient sensitivity (0.5-500 mg L(-1)) and determined azorubine concentration closely correlated with those obtained by a standard HPLC technique. The analysis was direct without the need for any pretreatment of the drinks or Au surface. Our SERS approach is a simple and rapid (35 min) prescan method, which can be easily implemented for a field application and for preliminary testing of food samples.

In situ WetSTEM observation of gold nanorod self-assembly dynamics in a drying colloidal droplet.

Direct in situ visualization of nanoparticles in a liquid is an important challenge of modern electron microscopy. The increasing significance of bottom-up methods in nanotechnology requires a direct method to observe nanoparticle interactions in a liquid as the counterpart to the ex situ electron microscopy and indirect scattering and spectroscopy methods. Especially, the self-assembly of anisometric nanoparticles represents a difficult task, and the requirement to trace the route and orientation of an individual nanoparticle is of highest importance. In our approach we utilize scanning transmission electron microscopy under environmental conditions to visualize the mobility and self-assembly of cetyltrimethylammonium bromide (CTAB)-capped gold nanorods (AuNRs) in an aqueous colloidal solution. We directly observed the drying-mediated AuNR self-assembly in situ during rapid evaporation of a colloidal droplet at 4°C and pressure of about 900 Pa. Several types of final AuNR packing were documented including side-by-side oriented chains, tip-to-tip loosely arranged nanorods, and domains of vertically aligned AuNR arrays. The effect of local heating by electron beam is used to qualitatively asses the visco-elastic properties of the formed AuNR/CTAB/water membrane. Local heating induces the dehydration and contraction of a formed membrane indicated either by its rupture and/or by movement of the embedded AuNRs.

Methylacridinium and its cholinergic properties.

10-Methylacridinium iodide (methylacridinium; MA) is an inhibitor of cholinesterases. Inhibitors of acetylcholinesterase (AChE) are used in the treatment of myasthenia gravis, Alzheimer's disease, and in the prophylaxis of poisoning with organophosphates. Using spectrophotometric Ellman's method at 436 nm and commercial enzymes we found that MA inhibits AChE by binding with relatively high potency to the peripheral anionic site (IC(50) = 1.68 +/- 0.14 1M; human recombinant AChE) and equally to its inhibition of butyrylcholinesterase (BuChE; IC(50) = 3.54 +/- 0.27 1M; BuChE from human serum). MA also inhibits the binding of [(3)H]N-methylscopolamine to the muscarinic M2 receptor subtype, possibly in an allosteric manner (IC(50) = 1.90 1M). Functional effects on both the enzyme and the receptor could be observed in contractile studies on the isolated rat bladder. The ability of MA to cross the blood-brain barrier (log P = -0.32; polar surface area 3.88) provides prerequisites for a potential use of the drug in the treatment of neural disorders.