Bella Manshian

NanoGenotoxicology : The DNA damaging potential of engineered nanomaterials

With the rapid expansion in the nanotechnology industry, it is essential that the safety of engineered nanomaterials and the factors that influence their associated hazards are understood. A vital area governing regulatory health risk assessment is genotoxicology (the study of genetic aberrations following exposure to test agents), as DNA damage may initiate and promote carcinogenesis, or impact fertility. Of late, considerable attention has been given to the toxicity of engineered nanomaterials, but the importance of their genotoxic potential on human health has been largely overlooked. This comprehensive review focuses on the reported abilities of metal nanoparticles, metal-oxide nanoparticles, quantum dots, fullerenes, and fibrous nanomaterials, to damage or interact with DNA, and their ecogenotoxicity is also considered. Many of the engineered nanomaterials assessed were found to cause genotoxic responses, such as chromosomal fragmentation, DNA strand breakages, point mutations, oxidative DNA adducts and alterations in gene expression profiles. However, there are clear inconsistencies in the literature and it is difficult to draw conclusions on the physico-chemical features of nanomaterials that promote genotoxicity, largely due to study design. Hence, areas that require that further attention are highlighted and recommendations to improve our understanding of the genotoxic potential of engineered nanomaterials are addressed.

Confounding experimental considerations in nanogenotoxicology

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Cytotoxic effects of gold nanoparticles: a multiparametric study.

The in vitro labeling of therapeutic cells with nanoparticles (NPs) is becoming more and more common, but concerns about the possible effects of the NPs on the cultured cells are also increasing. In the present work, we evaluate the effects of poly(methacrylic acid)-coated 4 nm diameter Au NPs on a variety of sensitive and therapeutically interesting cell types (C17.2 neural progenitor cells, human umbilical vein endothelial cells, and PC12 rat pheochromocytoma cells) using a multiparametric approach. Using various NP concentrations and incubation times, we performed a stepwise analysis of the NP effects on cell viability, reactive oxygen species, cell morphology, cytoskeleton architecture, and cell functionality. The data show that higher NP concentrations (200 nM) reduce cell viability mostly through induction of reactive oxygen species, which was significantly induced at concentrations of 50 nM Au NPs or higher. At these concentrations, both actin and tubulin cytoskeleton were deformed and resulted in reduced cell proliferation and cellular differentiation. In terms of cell functionality, the NPs significantly impeded neurite outgrowth of PC12 cells up to 20 nM concentrations. At 10 nM, no significant effects on any cellular parameter could be observed. These data highlight the importance of using multiple assays to cover the broad spectrum of cell-NP interactions and to determine safe NP concentrations and put forward the described protocol as a possible template for future cell-NP interaction studies under comparable and standardized conditions.

In vitro genotoxicity testing strategy for nanomaterials and the adaptation of current OECD guidelines

Highlights ► We consider current in vitro OECD genotoxicity tests for nanomaterials. ► Ames test does not appear to be suitable for nanomaterial assessment. ► In vitro HPRT and micronucleus assays require nanomaterial specific protocols. ► We recommend a strategic in vitro genotoxicity testing strategy for nanomaterials.

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

The number of newly engineered nanomaterials is vastly increasing along with their applications. Despite the fact that there is a lot of interest and effort is being put into the development of nano-based biomedical applications, the level of translational clinical output remains limited due to uncertainty in the toxicological profiles of the nanoparticles (NPs). As NPs used in biomedicines are likely to directly interact with cells and biomolecules, it is imperative to rule out any adverse effect before they can be safely applied. The initial screening for nanotoxicity is preferably performed in vitro, but extrapolation to the in vivo outcome remains very challenging. In addition, generated in vitro and in vivo data are often conflicting, which consolidates the in vitro-in vivo gap and impedes the formulation of unambiguous conclusions on NP toxicity. Consequently, more consistent and relevant in vitro and in vivo data need to be acquired in order to bridge this gap. This is in turn in conflict with the efforts to reduce the number of animals used for in vivo toxicity testing. Therefore the need for more reliable in vitro models with a higher predictive power, mimicking the in vivo environment more closely, becomes more prominent. In this review we will discuss the current paradigm and routine methods for nanotoxicity evaluation, and give an overview of adjustments that can be made to the cultivation systems in order to optimise current in vitro models. We will also describe various novel model systems and highlight future prospects.

Exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticle-induced autophagy in biomedical research.

Nanoparticle-Induced Autophagy in Biomedical Research Karen Peynshaert,†,‡ Bella B. Manshian, Freya Joris,†,‡ Kevin Braeckmans,†,‡ Stefaan C. De Smedt,*,†,∥ Jo Demeester,† and Stefaan J. Soenen*,†,§ †Lab of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, ‡Centre for Nanoand Biophotonics, and Ghent Research Group on Nanomedicine, Ghent University, B9000 Ghent, Belgium Biomedical MRI Unit/MoSAIC, Department of Imaging and Pathology, Faculty of Medicine, Catholic University of Leuven, B3000 Leuven, Belgium

Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles that Determine Their Interaction with Cells

A homologous nanoparticle library was synthesized in which gold nanoparticles were coated with polyethylene glycol, whereby the diameter of the gold cores, as well as the thickness of the shell of polyethylene glycol, was varied. Basic physicochemical parameters of this two-dimensional nanoparticle library, such as size, ζ-potential, hydrophilicity, elasticity, and catalytic activity ,were determined. Cell uptake of selected nanoparticles with equal size yet varying thickness of the polymer shell and their effect on basic structural and functional cell parameters was determined. Data indicates that thinner, more hydrophilic coatings, combined with the partial functionalization with quaternary ammonium cations, result in a more efficient uptake, which relates to significant effects on structural and functional cell parameters.

Cytotoxicity of cadmium-free quantum dots and their use in cell bioimaging.

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.

Single-walled carbon nanotubes: differential genotoxic potential associated with physico-chemical properties

Abstract Single-walled carbon nanotubes (SWCNTs) have recently attracted great attention because of their fibrous structure and high aspect ratio. Here the genotoxic potential of 400–800 nm, 1–3 μm and 5–30 μm SWCNT with respect to their geometry and surface characteristics was studied. Following thorough physico-chemical characterisation, human bronchial epithelial (BEAS−2B) and lymphoblastoid (MCL-5) cells were treated with SWCNT for 24 or 48 h. This showed significant increases in micronucleus frequency in a time- and dose-dependent manner in both cell types in the absence of cytotoxicity. Over the same dose range, only 1–3 μm SWCNT gave rise to significant increases in hprt point mutations at doses ≥25 μg/ml. Cellular 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA) fluorescence assay and RT-PCR for oxidative pathway gene profiling revealed a possible oxidative mechanism for the genotoxicity observed in the 1–3 μm SWCNT. Consequently, this study has demonstrated that SWCNT genotoxicity is dependent on its secondary structure under experimental conditions and oxidative stress alone cannot account for the observed damage.

Fluorescent non-porous silica nanoparticles for long-term cell monitoring: Cytotoxicity and particle functionality

Inorganic nanoparticles such as silica particles offer many exciting possibilities for biomedical applications. However, the possible toxicity of these particles remains an issue of debate that seriously impedes their full exploitation. In the present work, commercially available fluorescent silica nanoparticles 25, 45 and 75 nm in diameter optimized for cell labelling (C-Spec® particles) are evaluated with regard to their effects on cultured cells using a novel multiparametric setup. The particles show clear concentration and size-dependent effects, where toxicity is caused by the number and total surface area of cell-associated particles. Cell-associated particles generate a short burst of oxidative stress that, next to inducing cell death, affects cell signalling and impedes cell functionality. For cell labelling purposes, 45 nm diameter silica particles were found to be optimally suited and no adverse effects were noticeable at concentrations of 50 μg ml(-1) or below. At this safe concentration, the particles were found to still allow fluorescence tracking of cultured cells over longer time periods. In conclusion, the data shown here provide a suitable concentration of silica particles for fluorescent cell labelling and demonstrate that at safe levels, silica particles remain perfectly suitable for fluorescent cell studies.