Neenu Singh

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.

Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION)

Superparamagnetic iron oxide nanoparticles (SPION) are being widely used for various biomedical applications, for example, magnetic resonance imaging, targeted delivery of drugs or genes, and in hyperthermia. Although, the potential benefits of SPION are considerable, there is a distinct need to identify any potential cellular damage associated with these nanoparticles. Besides focussing on cytotoxicity, the most commonly used determinant of toxicity as a result of exposure to SPION, this review also mentions the importance of studying the subtle cellular alterations in the form of DNA damage and oxidative stress. We review current studies and discuss how SPION, with or without different surface coating, may cause cellular perturbations including modulation of actin cytoskeleton, alteration in gene expression profiles, disturbance in iron homeostasis and altered cellular responses such as activation of signalling pathways and impairment of cell cycle regulation. The importance of protein–SPION interaction and various safety considerations relating to SPION exposure are also 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.

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.

The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles.

Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for revolutionising biomedical applications such as MRI, localised hyperthermia, and targeted drug delivery. Though evidence is increasing regarding the influence of nanoparticle physico-chemical features on toxicity, data however, is lacking that assesses a range of such characteristics in parallel. We show that iron redox state, a subtle though important physico-chemical feature of USPION, dramatically modifies the cellular uptake of these nanoparticles and influences their induction of DNA damage. Surface chemistry was also found to have an impact and evidence to support a potential mechanism of oxidative DNA damage behind the observed responses has been demonstrated. As human exposure to ferrofluids is predicted to increase through nanomedicine based therapeutics, these findings are important in guiding the fabrication of USPION to ensure they have characteristics that support biocompatibility.

Analysis of the Influence of Cell Heterogeneity on Nanoparticle Dose Response

Understanding the effect of variability in the interaction of individual cells with nanoparticles on the overall response of the cell population to a nanoagent is a fundamental challenge in bionanotechnology. Here, we show that the technique of time-resolved, high-throughput microscopy can be used in this endeavor. Mass measurement with single-cell resolution provides statistically robust assessments of cell heterogeneity, while the addition of a temporal element allows assessment of separate processes leading to deconvolution of the effects of particle supply and biological response. We provide a specific demonstration of the approach, in vitro, through time-resolved measurement of fibroblast cell (HFF-1) death caused by exposure to cationic nanoparticles. The results show that heterogeneity in cell area is the major source of variability with area-dependent nanoparticle capture rates determining the time of cell death and hence the form of the exposure–response characteristic. Moreover, due to the particulate nature of the nanoparticle suspension, there is a reduction in the particle concentration over the course of the experiment, eventually causing saturation in the level of measured biological outcome. A generalized mathematical description of the system is proposed, based on a simple model of particle depletion from a finite supply reservoir. This captures the essential aspects of the nanoparticle–cell interaction dynamics and accurately predicts the population exposure–response curves from individual cell heterogeneity distributions.

STEM mode in the SEM: A practical tool for nanotoxicology

Abstract The addition of a transmitted electron detector to a scanning electron microscope (SEM) allows the recording of bright and dark field scanning transmission electron microscope (STEM) images and the corresponding in-lens secondary electron images from the same region of a thin sample. These combined imaging techniques have been applied here to the analysis of ultrathin sections of cells exposed in vitro to nanomaterials for toxicology investigation. Electron microscopy in general permits the exact nature of the interaction of nanomaterials and cells to be elucidated, and in addition the use of STEM mode in the SEM enables the easy identification and exclusion of artefacts produced by ultramicrotome sectioning. The imaging and analysis obtained by using the STEM mode in the SEM configuration from three different nanomaterial systems of importance (iron oxide nanoparticles, single-walled carbon nanotubes and cadmium selenide quantum dots) indicate that it is a simple, practical and cost-effective tool for nanotoxicological research.

Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity

With the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo. This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity.

Quantum dot induced cellular perturbations involving varying toxicity pathways

The unique optical and electronic properties of quantum dots (QD) have led to rapid progress in their development and application, particularly in innovative therapeutic and diagnostic products. Along with the great pace at which QD are being developed, research is being focussed on fabricating less toxic QD with novel surface functionalities. The present study was therefore focused on assessing the impact of varying QD surface chemistry on cellular uptake and a range of indicators for cell perturbation following exposure. The study demonstrated that despite a low intrinsic cytotoxicity of three QD with different surface functional groups, they were all capable of inducing an acute inflammatory response and alterations in transcriptional gene activity, without affecting cell cycle regulation. Further, this investigation demonstrated that although the QD were capable of inducing an inflammatory and oxidative stress response, there was clearly variation in the degree of molecular change according to surface chemistry, which correlated with the degree of cellular uptake. These findings therefore highlight the potential for chronic inflammatory responses following exposure to QD, but in addition, they also demonstrate the importance of studying a wide range of toxicity pathways to generate a comprehensive picture of biological response to nanomaterials.

Electrosprayed mesoporous particles for improved aqueous solubility of a poorly water soluble anticancer agent: in vitro and ex vivo evaluation

ABSTRACT Encapsulation of poorly water‐soluble drugs into mesoporous materials (e.g. silica) has evolved as a favorable strategy to improve drug solubility and bioavailability. Several techniques (e.g. spray drying, solvent evaporation, microwave irradiation) have been utilized for the encapsulation of active pharmaceutical ingredients (APIs) into inorganic porous matrices. In the present work, a novel chalcone (KAZ3) with anticancer properties was successfully synthesized by Claisen‐Schmidt condensation. KAZ3 was loaded into mesoporous (SBA‐15 and MCM‐41) and non‐porous (fumed silica, FS) materials via two techniques; electrohydrodynamic atomization (EHDA) and solvent impregnation. The effect of both loading methods on the physicochemical properties of the particles (e.g. size, charge, entrapment efficiency, crystallinity, dissolution and permeability) was investigated. Results indicated that EHDA technique can load the active in a complete amorphous form within the pores of the silica particles. In contrast, reduced crystallinity (˜79%) was obtained for the solvent impregnated formulations. EHDA engineered formulations significantly improved drug dissolution up to 30‐fold, compared to the crystalline drug. Ex vivo studies showed EHDA formulations to exhibit higher permeability across rat intestine than their solvent impregnated counterparts. Cytocompatibility studies on Caco‐2 cells demonstrated moderate toxicity at high concentrations of the anticancer agent. The findings of the present study clearly show the immense potential of EHDA as a loading technique for mesoporous materials to produce poorly water‐soluble API carriers of high payload at ambient conditions. Furthermore, the scale up potential in EHDA technologies indicate a viable route to enhance drug encapsulation and dissolution rate of loaded porous inorganic materials.