ABSTRACT
Nanoparticles present special challenges to in vitro risk assessment, which relate to their small size and resulting properties, such as increased surface area and reactivity, their behavior in solution, the need for extensive physicochemical characterization, and their interaction with a number of assays. These features necessitate the implementation of additional controls in many assays and render some standard assays impracticable. This chapter comprises a set of standard operating protocols used by the partners of the FP7 project InLiveTox to give a brief overview of testing for possible adverse effects of nanoparticles in vitro, using methods applicable for most adherent cells. The protocols include culture of human endothelial cells, intestinal epithelial cells, and hepatocytes; preparation of nanoparticle suspensions; assessment of particle size distribution; cytotoxicity and viability assays; testing of specific cellular functionality in intestinal epithelial cells, endothelial cells, and hepatocytes; quantification of markers of inflammation on the RNA and protein level; and measurement of reduced glutathione (GSH) levels as a measure of oxidative stress. 31.1.1 Nanoparticles and Nanomaterials
Although there is no agreed-upon definition or nomenclature, materials with at least one dimension measuring 100 nm or less are often referred to as nanomaterials and include nanofilms (one dimension ≤100 nm), nanofibers and nanorods (two dimensions ≤100 nm), and nanoparticles (NPs; all dimensions ≤100 nm [1a, 1b, 1c]). Particles on the nanoscale can occur naturally, for example, soot particles after combustion processes or large organic molecules [2]. In the last few decades, however, manufacture and application of NPs have become a new and increasingly important branch of industry [3]. The small size of NPs has various implications in their properties when compared to larger-size particles. There is an increased surface area per mass unit of material, and therefore a larger percentage of the atoms are present on the surface (Fig. 31.1). In addition to the larger surface area, the decreased stability of bonds in the smaller-size particles increases the surface reactivity [4].NPs have become promising tools in many applications and are added to many consumer products: Nanosilver is used for
its antibacterial properties, for example, in medical products [5]; nanometal oxides are used in paints and cosmetics such as sunscreens [6, 7]; and various nanomaterials are candidates for medical applications such as imaging and drug delivery [8, 9]. Particle A B C Diameter d 0.1 d 0.01 d Particles per mass unit n 1,000 n 1,000,000 n
Surface area per mass unit A 10A 100 A
31.1.2 NanotoxicologyWith the increase in the manufacture of NPs and NP-containing products, there is an increased risk of occupational exposure and also an increased risk of intentional exposure, for example, to medical NPs and NPs in cosmetics and personal care products. The changed properties of NPs compared to larger, or “bulk,” particles of the same material mean that their effects on the human body and the environment can be more severe than the effects of an equal mass dose of larger particles [4]. It has been suggested that for particles generally associated with low toxicity, the surface
area dose is a better predictor of their adverse effects than their mass, meaning toxicity and inflammogenicity of the same mass of particles increase with decreasing particle radius [10]. Another concern is that NPs may cross barriers that larger particles are unable to cross, and indeed transition of NPs from the lungs and gastrointestinal (GI) tract into the bloodstream have been observed [11, 12].On a cellular level, many NPs are of a size where interaction with the deoxyribonucleic acid (DNA) is possible if transition into the nucleus takes place [13], and reactive surfaces as well as surface contamination by metals or organic molecules can cause oxidative damage to membranes and proteins and also potentially the DNA [14]. For these reasons, it is important that NPs be tested comprehensively for potential adverse effects. Regulators have acknowledged this problem in various publications and call for researchers to investigate NP toxicity [15]. InLiveTox (www.inlivetox.eu) is a 7th Framework Programme project, in which a Quasi-Vivo® system [16] will be used to investigate transport of particles through an in vitro model of the GI barrier [17], and effects of transported particles on endothelial cells and hepatocytes connected to the GI model by medium flow will be examined and compared to an in vivo study to evaluate the in vitro model.Initial experiments focused on establishing protocols for measuring endpoints associated with NP toxicity, such as cytotoxicity, pro-inflammatory gene expression cytokine release, cellular functionality, and antioxidant depletion. These protocols are summarized in this chapter to provide researchers with a set of procedures for the evaluation of NP toxicity, which can be used for most adherent human cells. 31.1.3 Importance of Appropriate Controls and
Physicochemical CharacterizationSome challenges in in vitro nanotoxicology are different from the ones faced when working with larger particles. For instance, it can be difficult to remove NPs from a cell culture medium by conventional methods such as centrifugation or filtering. Therefore, additional controls, for example, a cell-free medium containing NPs, need to be run to eliminate the possibility of NP interference with assays such as fluorescent or absorbance measurements [18].
