Measuring nanomaterials in the alimentary tract...

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1 Methods to Evaluate Uptake of Engineered Nanomaterials by the Alimentary Tract Heather Alger, Dragan Momcilovic, David Carlander, and Timothy V. Duncan Abstract: This article is one of a series of 4 that report on a task of the NanoRelease Food Additive project of the International Life Science Institute Center for Risk Science Innovation and Application to identify, evaluate, and develop methods that are needed to confidently detect, characterize, and quantify intentionally produced engineered nanomaterials (ENMs) released from food along the alimentary tract. This particular article focuses on the problem of detecting and characterizing ENMs in the various compartments of the alimentary tract after they have been ingested from dietary sources. An in depth analysis of the literature related to oral toxicity of ENMs is presented, paying particular attention to analytical methodology and sample preparation. The review includes a discussion of model systems that can be used to study oral uptake of ENMs in the absence of human toxicological data or other live-animal studies. The strengths and weaknesses of various analytical and sample preparation techniques are discussed. The article concludes with a summary of findings and a discussion of potential knowledge gaps and targets for method development in this area. Keywords: alimentary tract, characterization, detection, food safety, measurement methods, nanotechnology, nanotoxicology Project Background This article is the 4th in a series of 4 articles related to a task of the NanoRelease Food Additive project of the International Life Science Institute (ILSI). Center for Risk Science Innovation and Application to identify, evaluate, and develop methods that are needed to confidently detect, characterize, and quantify intentionally produced engineered nanomaterials (ENMs) released from food along the alimentary tract. A full description of the project s charge and scope, as well as an executive summary of the project s findings, is presented in the first article in this series (Szakal and others 2014). The focus area of the present article is measurement methods to identify, quantify, and characterize the ENMs in the alimentary tract, including postabsorption characteristics. The 2nd and 3rd articles in this series, respectively, describe methods to characterize and detect ENMs released into foods from food contact materials (Noonan and others 2014) and characterize and detect ENMs in foods (including sample preparation) (Singh and others 2014). While the present article is capable of stand- MS Submitted 28/2/2014, Accepted 13/3/2014. Author Alger s current affiliation is with The Pew Charitable Trusts, Food Additives Project, 901 E Street NW, Washington, DC, 20004, USA. Author Alger s current affiliation is with American Heart Assoc., Office of Science Operations, 7272 Greenville Ave, Dallas, TX 75231, USA. Author Momcilovic s current affiliation is with Center for Veterinary Medicine, United States Food and Drug Administration, 7519 Standish Place, Rockville, MD, 20855, USA. Author Carlander s current affiliation is with Nanotechnology Industries Assoc., 101 Ave. Louise, 1050, Brussels, Belgium. Author Duncan s current affiliation is with Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, 6502, South Archer Road, Bedford Park, IL, , USA. Direct inquiries to author Duncan ( timothy.duncan@fda.hhs.gov). ing alone, due to the fact that some experimental methods may have utility in multiple areas relevant to the project s overall scope, some methods discussed within this article may have additional descriptive detail offered in other articles in this series. Introduction and Scope Estimating consumer exposure to ENMs in food products and predicting their toxicological properties are necessary steps in the assessment and management of the risks of this technology. To this end, ENMs must be able to be characterized or measured as components of food packaging, in the final food product, and as they are absorbed, distributed, metabolized, and excreted (ADME) after being exposed to the alimentary tract. The previous 2 articles in this series report focused on the first 2 aspects (food packaging and food products), respectively (Noonan and others 2014; Singh and others 2014). The present article focuses on the 3rd aspect and offers a comprehensive analysis of methods that may be used to evaluate the manner or quantity ADME after ingestion. This article addresses the factors that may affect the behavior of ENMs in the alimentary tract, which therefore may be important for the performance of the methods. For the purposes of this article, the alimentary tract (or gut) includes the mouth, esophagus, stomach, and small and large intestines. To fully understand the impact of oral uptake of ENMs, we must have an understanding of what happens to ENMs as they transit through the alimentary tract, including information on whether they are absorbed and distributed to different tissues and organ systems. Our ability to predict the behavior of ENMs when ingested requires experimental data in 3 general areas. 1) Assuming that we know the characteristics of the ENMs at the point of ingestion, we must have data on how the properties of the ENMs Published This article is a U.S. Government work and is in the public domain in the USA. doi: / Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 705

2 change between their starting point and every possible point of absorption from the alimentary tract. 2) We must have data on the extent of absorption, including data that inform mechanistic details and elucidate factors related to ENM characteristics and gut physiology (including the impact of disease state, diet, age, and so forth) that are likely to impact absorption. 3) We must have data on where ENMs go after absorption (if they are absorbed) and what they do when they get there. Because of this diverse range of needed information, the range of techniques required to acquire these data is similarly broad. For point 1, we need techniques that can monitor compositional transformations of ENMs in a diverse range of gut environments. For point 2, we need analytical methods that can closely monitor how ENMs cross biological barriers, we need well-controlled model systems and experimental techniques that can be used to systematically study the impact of various biological conditions on these processes. For point 3, we need methods that can locate small concentrations of ENMs in complex living tissues, enumerate them and tell us about their properties, and educate us about what types of effects they have at the cellular, tissue, organ, and whole organism levels. This article considers available methods to model alimentary tract conditions and cellular boundaries, methods to assess how ENMs interact with cells, and methods to detect, quantify, and characterize ENMs in a diverse range of homogeneous and heterogeneousmatrices. Theterm methods includes, but is not limited to, gut models, in vitro and in vivo experiments to assess toxicity, analytical approaches (compositional and imaging) to measure the physical and chemical characteristics of ENMs, and biological sampling techniques that can assist in our understanding of the toxicological and pharmacological impact of exposure to ENMs in the food supply. We begin this undertaking with a summary of how ENMs are expected to behave in the alimentary tract and follow this with a discussion of the general challenges of detecting ENMs in living systems, after which we present the strengths and weaknesses of the many currently available techniques. Behavior of ENMs in the alimentary tract Prior to discussing available and emerging methods to assess what happens to ENMs after they are consumed from food sources (or produced de novo in the gut from non-nanoscale materials in foods), the task group did a brief survey of factors likely to affect the behavior of ENMs in various regions of the alimentary tract. While this information has been covered far more extensively by other NanoRelease Food Additive task groups, providing a brief review here is useful, particularly because many of these factors are likely to influence the value or applicability of specific experimental or theoretical approaches discussed later in this report. Therefore, this article begins with a basic accounting of materials and alimentary tract characteristics likely to impact ADME of ENMs, which is then followed by a focused discussion of some of the general challenges inherent to detection and characterization of ENMs in the alimentary tract. ENM characteristics that affect uptake by the alimentary tract. Task Group 1 of the NanoRelease Food Additive project analyzed much of the toxicological literature related to ingestion of ENMs potentially encountered in the food supply and found considerable evidence that the physical characteristics (size, shape, surface coating, agglomeration state, and so on) and composition of ENMs can have a significant impact on the bioavailability and toxicity of ENMs by oral routes of exposure (Yada and others 2014). For example, a study by Park and others (2010) found an inversely size-dependent distribution pattern in elemental silver content among various internal organs after AgNPs with diameters ranging from 22 to 323 nm were fed to mice at 1 mg/kg over a 14-d period, suggesting that smaller particles may more readily penetrate tissues and biological barriers. Surface charge also appears to be important for ENM absorption (Desai and others 1996; Cockburn and others 2012), as it has been reported that negatively charged gold nanoparticles crossed intestinal barriers and accumulated into internal organs more efficiently than positively charged particles of the same size (Schleh and others 2012). Particle shape has been less well studied in in vivo models by oral exposure routes, but some in vitro models have exhibited shape-dependent toxicological effects in cell culture (Yamamoto and others 2004; Chithrani and Chan 2007). While numerous studies have investigated the relationships between ENM characteristics and toxicological endpoints, at present most of these data are phenomenological and thus yield little insight into precise ADME mechanisms, which limits their ability to inform a broad predictive framework for ENM toxicity. For example, while most studies generally agree that smaller particles are more easily absorbed from the gut and become localized into peripheral organs, little is known about where and how these particles are absorbed. Likewise, while some in vivo and in vitro studies (Powers and others 2011; Yang and others 2012) have attributed variations in toxicity as a function of surface coating to differences in aggregation kinetics and dissolution rates, we need a better general understanding of how surface coating, aggregation/dissolution, and ADME are connected in the various alimentary tract environments, particularly because the surface coatings of ENMs are expected to be highly dependent on the presence of other substances in foods ingested at the same time. In this sense, the primary limitation is high-quality studies in relevant organismal models, which, in turn, may be limited by the availability of good analytical methods that can quantify ENMs and analyze their characteristics at various stages of digestion. Factors related to the alimentary tract that affect uptake. As will be elaborated upon in the upcoming report by NanoRelease Food Additive Task Group 2 (unpublished data, 2014), orally administered ENMs pass through many different environments on their journey through the alimentary tract. Chemically speaking, these environments are defined largely by ph variations inherent to different gastrointestinal organs. The luminal ph varies from moderately acidic in the mouth (ph about 5.5 to 6), to very acidic in the stomach (ph about 1.0 to 2.5), to neutral or slightly basic in the distal regions (ph about 6.6 in the proximal small intestine, ph about 7.5 in the terminal ileum, and ph about 6.4 in the caecum) (Evans and others 1988). Moreover, the ph values in each of these regions, and particularly in the mouth and stomach, can vary depending on the characteristics of recently consumed foods. These natural variations in the ph along the alimentary tract, as well as the presence of other proteins and biomolecules deriving either from codigested food substances or intrinsic to the gut environment, can affect the properties of ENMs, particularly those that are sensitive to ph-induced hydrolysis, deamination, or oxidation. They can also contribute to dissolution and aggregation of ENMs, or in some cases, de novo preparation of ENMs from non-nanoscale materials ingested with foods (Powell and others 2010). Dissolution is particularly relevant to the fate of inorganic particles, which can lead to compositional changes, alterations in physical characteristics like size and shape, and attenuation of 706 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

3 surface charge. For organic particles, enzymatic activity may be a more important driver of ENM modification in the alimentary tract, as enzymes are capable of degrading and digesting orally administered nanoscale organic matter such as particles composed of or coated with proteins, carbohydrates, and lipids. Aside from chemical considerations, differences in biological structures and functions of the various gut organs play a role in determining the characteristics of ENMs, as well as their ability to be absorbed into an organism s tissues. In most cases, the mucus layer functions as the first chemical and enzymatic barrier (McGuckin and others 2011) and it serves as a semipermeable gateway that allows nutrients, water, and other small molecules to pass through, while stopping bacteria and pathogens (Ensign and others 2012). The mucus is negatively charged and any orally administered ENM has to be able to pass through the mucus in order to interact with the epithelial cells on its way to being absorbed, suggesting that any chemical changes to the ENM surface due to the local environment will potentially impact this process. Once materials pass the mucus layer, they must be absorbed by epithelial cells in order to pass into the bloodstream and become distributed throughout the organism. Mechanisms of intestinal absorption can be broadly assigned to 2 categories: active endocytosis and passive diffusion of molecules across the intestinal wall. Active transport is a mechanism for the movement of large substances, including proteins and ENMs, across the cell membrane against a concentration gradient and is usually energetically driven via the use of membrane-spanning proteins that open channels through which these materials can pass (Frohlich and Roblegg 2012). Passive translocation of ENMs and other material through the intestinal wall is a multistep process that involves diffusion through the mucus lining of the gut wall, contact with enterocytes or M- cells, cellular or paracellular transport, and posttranslocation events (Hoet and others 2004), all without expenditure of cellular energy. Uptake of ENMs via either mechanism is likely to depend on local gut biology (each segment of the alimentary tract), general organism health (including age and disease status), and other environmental factors (especially diet) (Frohlich and Roblegg 2012). A better understanding of these relationships is critical toward a broad assessment of ENM risk from food, which, of course, requires readily available analytical methods as a prerequisite. Challenges of analyzing ENM characteristics in the alimentary tract For test methods to be capable of detecting and quantifying ENMs in the alimentary tract, it is essential that they overcome the substantial challenges imposed on their performance by the complexities of the alimentary tract and/or the significant variation in properties of ENMs introduced into the tract by food or synthesized within the tract de novo from non-enm food components. For example, ENMs in food could be altered when exposed to the stomach contents so that they may agglomerate or aggregate, lose their coatings, or experience other changes, all of which could affect the performance of analytical methods. Owing to these challenges, researchers have questioned whether the standard toxicological toolset is sufficient to determine the safety of ENMs in food. Methods may need to be developed or adapted to specifically measure ADME of ENMs introduced into the gut. Importantly, many existing toxicological methods require sample preparation (for example, isolation or purification) to reduce signal interference from the surrounding tissue matrix, which may alter the physicochemical properties of the ENMs being analyzed (Peters and others 2011). Before describing currently available specific techniques to assess ENM behavior and characteristics in the alimentary tract, or those that are under development, we first describe some of the general difficulties associated with detection and characterization of ENMs in this challenging environment. Some of these difficulties will be similar to those described in previous articles in this series dealing with detection of ENMs in foods (Singh and others 2014) and detection of ENM migration from food contact materials (Noonan and others 2014), and thus will not be elaborated here. However, as described in the previous section, the natural variability in chemical and biological properties experienced by ENMs during passage through the alimentary tract creates new problems related to the complexity and shifting nature of the gut environment, difficulty of analysis in living tissue, and the crucial need for good baseline characterization data for ENMs before they are introduced to the gastrointestinal tract. Some of the outstanding issues related to detection and characterization of ENMs in the alimentary tract are discussed below and were recently reviewed (Powell and others 2010). Compositional analysis and distinguishing between conventional and nanoscale materials. Information regarding the chemical composition of an ENM is an essential parameter for characterization and identification of the material (European Food Safety Authority 2011). Therefore, it seems reasonable to expect that a chemical test should be included in the list of test methods for characterization of the ENM in the alimentary tract. However, a substance of a given chemical identity (for example, titanium oxide) may be introduced to the alimentary tract in a conventional/bulk form or as a nanoscale material, and these various forms can potentially have very different pharmacological or toxicological endpoints. Distinguishing between macroscale and nanoscale versions of a substance requires the availability of an analytical approach that goes beyond simple compositional analysis. Thus, while ascertaining the chemical composition of an ENM is important for identification, this alone is not sufficient to fully describe uptake or exposure. A pertinent example would be chemical composition analysis of ingested silver particles. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of gut contents and internal organs after ingestion can confirm that the material ingested or absorbed into the organism contained silver, but this straightforward chemical analysis provides no information about whether the absorbed silver was nanoscale, macroscale (or agglomerate/aggregate), or dissolved ions at the terminal location of the material. It also provides no information about what transformations this material may have undergone as it made its way from the food matrix, through the alimentary tract, during absorption, and during transit to the target organ. Moving beyond straightforward chemical analysis is particularly important in the case of nanoscale materials that may not be deliberately introduced into foods. For instance, digestion has a capacity for reducing the conventional material to a nanosize particle either through dissolution, enzymatic degradation, or mechanical action. In such a case, distinguishing between a conventional material reduced by digestion to nanosize and an ENM originally ingested as nanomaterial could be very challenging because their basic compositions might be similar. Similar complications would arise in trying to distinguish between nanoparticles that are humanmade and nanoparticles that are naturally present in food stuffs (Dudkiewicz and others 2011). Some clever methods have been used to work around these challenging situations, such as the use of isotopic tracers to distinguish between exogenous and endogenous materials (Moller and others 2009; He and others 2013). C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 707

4 However, such approaches may not be generally applicable (available isotopes are required) and may be too expensive or cumbersome to be used. They also may only provide chemical composition information without necessary details about the form of the materials as they pass through the digestive tract. It is important to stress that chemical compositional analysis can provide useful information, and can even provide hints as to the changes a material may undergo after it is consumed orally. For instance, compositional analysis of AgNPs (scanning electron microscopy [SEM] with energy dispersive X-ray spectroscopy [EDS]) after exposure to simulated human gastric fluid revealed significant portions of AgCl bound to particle surfaces (Mwilu and others 2013; Walczak and others 2013). This has been used as supportive evidence that AgNPs are able to partially dissolve in the highly acidic stomach environment, and then the dissolved silver cations coprecipitate with chlorine anions as insoluble AgCl layers on the AgNP surfaces; these AgCl layers then can mediate the formation of large AgNP aggregates. While imaging methods or light scattering could show the aggregation aspect, only compositional analysis sheds light on the potential mechanism, which highlights the fact that multiple analytical approaches are usually necessary to fully characterize ENM behavior in a complex system. Methodological limitations related to behavior of ENMs in the alimentary tract. Some of the transformative effects of the various gut environments on ingested ENMs can complicate detection and characterization of these ENMs as well as analyses of their uptake. One of the most significant and problematic transformative effects in this regard is aggregation and agglomeration. Because of their high surface areas, small particles tend to stick together in order to minimize the energy of interaction between their surfaces and the external environment. In most deliberately engineered applications, surfactants or other surface modifications are employed to keep the particles well dispersed and thus preserve their unique nanoscale properties. When such ENMs are introduced into the caustic and high-dielectric environment of the stomach, however, surface molecules or the neutral surface charge can be disrupted, which can interfere with these protective measures and cause clumping of the particles. Both agglomeration and aggregation effects are anticipated to impact the fate of ENMs introduced into the alimentary tract, and such phenomena are already known to affect the toxicity of ENMs in cell cultures (Bae and others 2010; Zook and others 2011). However, agglomeration and aggregation can also adversely impact analytical approaches to studying the behavior of ENMs as they pass through the gut. For example, dynamic light scattering (DLS), which is used to analyze particle size distribution and monitor aggregation or agglomeration events, can be impacted by particle sedimentation or, more severely, by large-particle bias, in which just a few large (micron size) aggregates can wash out the scattering signal created by, in some cases, orders of magnitude more dispersed, nanosized particles or clusters of particles. The presence of organic aggregates of partially digested material and other biomolecules that may be present in the intestinal environment can also impact such measurements. Interactions between ENMs and other materials present in the gut can also impact analysis methods. While aggregation and agglomeration likely occur in the stomach, readsorption of surface substrates in higher ph intestinal regions where enzymes and other biomolecules that are well digested by the stomach adhere to particles to form a protein corona, which has been shown to be able to redisperse ENMs lower in the gut (Walczak and others 2013) in synthetic human gut models. The redispersion of gut-aggregated ENMs during sample preparation for imaging or other analytical methods could be similarly facile, suggesting that care needs to be taken in order to acquire information about the aggregation state of ingested ENMs in their native environment. Inorganic salts and dissolved ions can also impact agglomeration/aggregation of ENMs in the gut as well as mediate binding of surface ligands to ENM surfaces; this may impact absorption and uptake, of course, but may also be problematic in certain analysis methods that utilize salts for their operation or for sample preparation. For example, Powell and others (2010) use an example of calcium ions, plentiful in the gut as well as a critical component of some cell culture media, which can precipitate as calcium phosphate on the surfaces of titanium dioxide particles and significantly impact measured particle sizes and other ENM attributes. Other analytical limitations or complications related to ENM behavior either in the alimentary tract or elsewhere include the following: interference from endogenous ENMs with similar compositions formed de novo in the gut, the role of intestinal cell sloughing in ENM uptake and distinguishing between freshly absorbed and reabsorbed ENMs, adsorption of surface-stabilizerdenuded ENMs on sample introduction hardware for analytical instrumentation prior to measurement, as well as equipment used in animal feeding studies (syringes, vials, gavage tubes, and so on), and cherry-picking or failing to survey an adequate quantity of tissue samples in microscopic analysis of gut organs, particularly since some regions of the gut (such as Peyer s patches) seem more likely to accumulate absorbed ENMs than others. These and other limitations of current methodologies were recently reviewed (Powers and others 2011). Analysis of organic particles and coatings. The relative difficulty of evaluating organic ENMs or organic coatings on inorganic ENMs has been a running theme in this series of articles, and this problem is perhaps no more difficult or important to solve than in the case of detection of ENMs in the alimentary tract. Organic ENMs may be presented in the form of carbon-based structures like liposomes or micelles aimed at encapsulating organic or inorganic flavors, colors, or other additives, or as constitutive structural elements of food based on biodegradable polymers (such as carbohydrates, lipids, and proteins) (Peters and others 2011). Such materials are perhaps more likely to undergo deleterious transformations in the gut because of their susceptibility to both enzymatic and chemical (ph effects) degradation. Moreover, the overabundance of proteins and other biological molecules capable of interacting with ENMs severely complicates the analysis of ENM coatings, both for materials with inorganic and organic core compositions. Nevertheless, finding suitable techniques that can tease out this information seems of utmost importance because the nature of the organic surface coating plays such a dominant role in moderating both aggregation effects and, possibly, transport of ENMs from the gut through the intestinal or mucosal wall. The primary difficulty in this area appears to be related to high backgrounds involved in analyzing an organic substrate in a matrix that has fundamentally the same composition. This has the effect of rendering elemental and other compositional analysis techniques practically useless and imaging techniques challenging or even impossible because of a lack of visible contrast. An additional issue is the sheer quantity and variety of organic matter than can comprise the protein corona of an ENM dispersed in the gut. Quantifying and identifying this nonhomogeneous collection of organic matter bound to the surface of an ENM, and especially acquiring data that can inform our understanding of how these bound substrates change the aggregative and absorptive behavior 708 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

5 of ENMs, remains a considerable challenge that is nevertheless an urgent one to solve from a health and environmental safety perspective. Analytical metrics and relevance of experimental parameters (dosage, detection limits, and so on). While certain research methods may be aimed at acquiring primarily qualitative data (for example, some aspects of imaging techniques), any analytical technique that intends to generate quantitative results (which is to say, any technique which attempts to answer the How much is there? question) requires reliable, reproducible, and standardized metrics by which results can be calibrated and effectively compared to results acquired by other individuals using identical or different quantitative methods. Analytical metrics in conventional quantitative chemical analysis are well known and have unambiguous meaning: molar concentrations, detection limits, and dosage can easily be compared across studies and detection platforms with little trouble. As previously discussed (Szakal and others 2014), quantification of ENM concentrations and characteristics introduces new challenges, because these simple concepts take on ambiguous meaning. What is the concentration of an element we should report by ICP-MS analysis? Is it particle density, mass concentration, or something else? When we speak of dosage, should we refer to the number of particles per unit volume administered, or the amount of mass or surface area? Such issues are not trivial, because if 2 research studies report different effects and the measurement metrics are not identical, it is hard to know whether the anomaly stems from a difference in the way the system behaves or in the way in which the system was measured. The challenge of identifying appropriate metrics for ENM analysis, which is difficult even for pristine particle analysis, is particularly troublesome for analysis of ENMs in the alimentary tract. The prevalent process of aggregation, for example, may result in changes in reactivity and functionality, of course (Dudkiewicz and others 2012), but also can yield differing conclusions about particle concentration depending on what the metric is. In measuring concentration present in the stomach or when it is absorbed by intestinal cells, does an aggregate count as a single particle or many particles? We might use a mass-based concentration metric to get around this problem; however, in this case, we would then lose potentially valuable information about the nature of particle particle interactions that could be vital to understanding uptake mechanism. Without some form of supplementary information, it is virtually impossible to compare results from one study that presents ENM concentration in a liver in micrograms per kilogram of organ weight to another study that presents the same experiment in particles per milliliter. Clearly, some kind of standardization is a crucial step in achieving a broad understanding of what the current and future body of literature pertaining to ENM uptake by the alimentary tract is telling us. Failing that, a conscious effort on the part of research scientists to clearly define what is being measured in each case and to present results in as many forms as possible (for example, particle concentration and mass concentration) will ensure maximum usefulness and relevance of data. A final remark worth making here concerns the challenge of detection limits. Based on expected levels of ENMs used in test samples, quantitative techniques with detection limits in a partsper-billion or subparts-per-billion area will likely be required (He and others 2013). This is typically not an issue for ICP-MS analysis of inorganic particles, although characteristics (particularly aggregation/agglomeration) of the particles may become altered from those of their native environments in cases in which dilution or concentration of samples is required to meet this target range. If ICP-MS in single-particle mode is desired, there is a mass concentration limit as well as a size-based detection limit, which could become problematic if ingested particles are smaller than approximately 20 nm. Organic particles are more challenging to analyze (see above) and the limit of detection or quantification typically becomes higher due to elevated background levels. In cases in which interferences from exogenous food components or other substances endogenous to the gut are likely to raise limits of quantitation beyond the useful range, extraction methodologies may be necessary (Dudkiewicz and others 2012), although these involve disadvantages of their own. These are discussed in more detail in another article in this series (Singh and others 2014). Reference materials, method validation, and pristine particle analysis. Beyond choosing reliable and useful measurement metrics, ensuring that experimental design parameters are realistic and have a good chance of providing relevant information is another challenge for researchers. The in vivo nanotoxicology literature primarily consists of acute or subacute studies in which massive amounts of ENMs (sometimes as much as 5 g/kg body wt per dose) are fed to animals on a single occasion or multiple occasions spread over a short time frame. We currently have a poor understanding of whether such scenarios are realistic models of likely exposure. In a sense, the challenges inherent to detection of ENMs in foods and in food contact materials are indirectly responsible for many of the challenges facing our understanding of ENM uptake from the alimentary tract. Because of the analytical method shortcomings discussed in previous articles in this series (Noonan and others 2014; Singh and others 2014; Szakal and others 2014), the quantity and form of ENMs likely to be ingested are still a matter of considerable uncertainty. This makes it difficult to pick a suitable frame of reference from which to design in vivo and in vitro toxicological studies that we can be confident are realistic reflections of exposure. As a result, while the development of standardized reference materials validated analytical methods for ENM quantification and characterization, and pristine particle analysis will have direct benefits to the study of ENM toxicology by oral exposure routes, particularly when systematic model systems are being developed, indirect benefits will also be manifested. A full understanding of the starting materials before ENMs were introduced to foods or food contact materials will have a trickle-down effect that will aid in experimental design at the toxicology level. Available Methods to Evaluate ENM Characteristics in and Uptake by the Alimentary Tract Robust safety evaluations and ultimately public confidence in the use of food nanotechnology require extensive knowledge about how the body absorbs, distributes, accumulates, metabolizes, and excretes ENMs. There are many techniques, both in vivo and in vitro, that can inform our understanding of the behavior and fate of ENMs introduced to the alimentary tract, intentionally or otherwise, via food sources. It is unlikely that any single all-performing test method will be sufficient to provide all of the necessary information; therefore, multiple techniques may be best used in concert (Tiede and others 2008; He and others 2013). In effect, understanding the behavior of ENMs in the alimentary tract requires 2 methodological components: a suitable test environment and an analytical technique (or combination of techniques) that can qualify or quantify ENM behavior in that environment. The test environment can be a living organism s alimentary tract or an artificial model that can adequately simulate the C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 709

6 conditions in the former, or a portion thereof. In either case, a necessary counterpart to the test environment is a suitable method to extract or isolate the ENMs from the organism (or model) so that they can be studied. This can include study (such as imaging) of ENMs directly in gut organs or monitoring the state of ENMs after they are excreted (such as in feces) or absorbed by the intestinal lumen (such as blood or peripheral organ analysis postdissection). Below, we describe some of the test environments that have been used for ENM toxicological research in the recent past, as well as their advantages and disadvantages. We also discuss a few available extraction or isolation methods as well as analytical techniques that ultimately collect data on quantity and character of ENMs that are excreted or absorbed. Some of the analytical techniques presented below were detailed in the companion articles in this series. Thus, this section will focus specifically on the information that these techniques can provide on ADME of ENMs and their limitations in this regard, as well as any specific sample preparation steps that may be required. Model systems for simulating the ENM uptake The most direct and realistic approach to measuring the toxicity of an unknown substance after it is eaten by a human is to feed the substance to an appropriate living model organism and observe what happens. Observation in this case usually means looking for changes in behavior or other gross pathological signs after the dose is administered, which is then often followed by humanely euthanizing the animal, performing dissection, and then investigating the various internal tissues and organs for damage and traces of the substance or its known metabolites. The downside to this approach is the need to use live animals. Aside from ethical concerns, animals are expensive to handle and keep, are prone to variability, and may require significant time before some types of effects can be manifested. It has also been questioned as to whether mice and rats are good model organisms in the case of toxicity by oral routes of exposure, particularly for ENMs, because the chemical environment in their digestive tracts differs substantially from that of humans (McConnell and others 2008). Model systems that mimic the conditions of the human alimentary tract can be especially useful alternatives to live animals in situations in which information about the process of ENM digestion is desired. In vivo experiments typically are most useful for studying the outcome of digestion, because the state of the ENM at each stage of digestion in the animal (mouth, stomach, and so on) is usually not possible to be sampled only the gross anatomic effects and postabsorption state of ENMs are typically observed. Model systems also offer the potential to study the process of ENM digestion under tightly controlled conditions, without much of the inherent variability (due to diet or disease state) that can complicate interpretation of in vivo experiments. The primary disadvantage of in vitro alimentary tract models is that they yield little direct information about ENM toxicity, because there is no greater organism to manifest toxicological effects. However, owing to the fact that in vitro models can offer rapid information about key structure-function relationships without the concern of sampling effects from living organisms makes them a valuable addition to the methodological toolset available for understanding ENM behavior in the gut. Three distinct in vitro model systems are described below. Basic benefits and limitations of these model systems are summarized in Table 1. Artificial/in vitro gastrointestinal systems. Artificial gastrointestinal tract systems simulate digestion from the mouth to the intestine, or can be devised to investigate specific steps, such as chemical changes in the mouth or digestion effects in the mouth and stomach. One such model is depicted in Figure 1. For digestion of ENMs from food sources, this 3-step technique is useful to model the physical transformations that ENMs undergo as they pass from 1 organ to the next in the gastrointestinal tract. When validated against in vivo digestion experiments, this model can reduce the need for experimental animals because the method uses in vitro compartments to model each of the steps in digestion in a systematic way. This approach can give useful information that is difficult to obtain from in vivo methods. For instance, Peters and others (2012) used this model to analyze the transformative effects of silica nanoparticles (and macroscale silica) in the alimentary tract environment after the nanoparticles were consumed as an additive in hot water, coffee with creamer, instant soup, and pancakes. Introduction of the test material sequentially into each compartment of the model system, followed by separation of the digestate with a size-exclusion based chromatographic technique (in this case, hydrodynamic chromatography [HDC], supported by DLS and SEM) and elemental analysis (ICP-MS) yielded information on how the silica materials dissolved, agglomerated, or changed their physical state after each stage in digestion compared to undigested control samples. Using this approach, the researchers determined that although both the silica engineered to be in the nanoscale range as well as the macroscale silica powder contained nanoscale particles in the mouth portion of the model, these nanoparticles predominantly disappeared (formed aggregates) in the lower ph stomach chamber, and then reappeared again in the intestinal region in amounts that exceeded those observed in the earlier digestive stages. Data on what the intestinal lumen is likely to be exposed to, as described by Peters and others (2012), would be difficult to obtain in a live organism because only the final outcome of silica digestion (excretion, transport to peripheral organs, and pathological effects) can be observed in live organisms. In vitro digestion models have also been employed to obtain similar information related to AgNP digestion (Rogers and others 2012; Mwilu and others 2013; Walczak and others 2013). Nevertheless, there are some limitations to this approach. It is vital, for instance, to verify that the ENMs do not adhere to the reaction vials and that the detection method distinguishes between nanosized and conventionally sized materials. These models may not account for the significant variations in real digestive tracts (due to disease state, age of the organism, diet, genetic variation, and so forth) unless they are specifically designed to do so. Finally, a limitation of this protocol is that it does not show whether, or how much, absorption occurs from the digestive tract into the body; therefore, traditional in vivo approaches will not be replaced by these in vitro based methods. Intestinal epithelial monolayer assays. Intestinal epithelial (such as Caco-2) monolayer assays allow researchers to simulate in vitro intestinal absorption (U.S. Food and Drug Administration 2000) without the use of live animals. Caco-2 cells originate from a collection of cell lines derived from gastrointestinal (colorectal) tumors generated in the late 1970s, and although they are derived from the large intestine, they express numerous similarities to endothelium cells of the small intestine (Sambuy and others 2005). In particular, they express numerous proteins associated with active transport on their cell membranes, similar to those of intestinal enterocytes, which makes them ideal candidates as intestinal absorption models. In a typical experiment, researchers grow a monolayer of polarized Caco-2 cells on a permeable membrane that divides 2 chambers (Figure 2). If the substance of interest is an ENM, one 710 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

7 Table 1 Summary of benefits and limitations of model systems available for simulating ENM behavior in or uptake by the alimentary tract. Method Description Benefits Limitations References Artificial in vitro gastrointestinal systems In lieu of research animals, the artificial gastrointestinal system allows researchers to model physicochemical transitions of ENM after each stage of digestion by simulating conditions in artificial containers Does not require animals and can be used with food matrices Properties of ENMs at each stage of digestion can be closely monitored Can be used to make conclusions about transformative behavior of ENMs in the gut Rapid technique This technique does not inform questions about mechanisms or extent of ADME May not account for natural variations in gut environments, including diet and organism health, unless specifically designed to do so (Peters and others 2012; Rogers and others 2012; Mwilu and others 2013; Walczak and others 2013) Intestinal epithelial monolayer assay Polarized intestinal cells mimic the anatomy and physiology of the intestinal wall to simulate ENM absorption in the gut FDA has guidance for testing pharmaceuticals in cultured monolayers Does not require research animals Cells can be collected and imaged after uptake assays Can distinguish between active or passive ENM transportation mechanisms Monolayer assays do not provide information on ENM absorption in a complex food matrix May not account for natural variations in gut environments, including diet and organism health, unless specifically designed to do so (Jia 2005; Lin and others 2005; Jia and others 2009; Zhang and others 2010b; Brun and others 2011; Lin and others 2011b) Everted gut sac Everted (turned inside-out) sections of small intestine from animals are turned into pouches and used to predict translocation of ENMs Well-established technique (used since the 1950s) If appropriate animal models exist, can be used to differentiate across the intestinal wall between normal and diseased absorption Can systematically determine the influence of other substances on uptake of the primary analyte, and uptake in different intestinal regions May be useful for study of organic ENMs Microsurgery requires skill to perform Viability of living tissue can be a challenge The loss of enzymatic activity may lead to unrealistic conclusions about uptake for ENMs that are vulnerable to enzymatic actions Cannot determine organ distribution, metabolism, or excretion of ENMs after absorption (Carreno-Gomez and others 1999; Sandri and others 2007; Lafforgue and others 2008; Alam and others 2012; Frohlich and Roblegg 2012) C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 711

8 Figure 1 Three-step in vitro model of ENM digestion in the human mouth, stomach, and intestine. Reprinted with permission from Peters and others (2012). Copyright C 2012 American Chemical Society. Figure 2 Depiction of the intestinal epithelial monolayer experiment (Caco-2 assay), which measures translocation of an analyte across a monolayer of cells with characteristics similar to gut epithelium. Green arrows show possible unidirectional transit of the analyte across intestinal barriers. chamber contains the ENM and the other chamber is free of ENMs. The cells are polarized to quantify the directional uptake (absorption) of chemicals from the apical face of intestine cells (the lumen) to the basolateral face (circulation), and transport is typically measured in both directions to establish vectorality of the transport mechanism (so as to distinguish between active versus passive transport mechanisms). ENMs have unique physical properties and may cross epithelial cells differently than their conventionally sized counterparts. In 2005, researchers at the Natl. Cancer Inst. used cultured monolayer assays to show that nanosized pharmaceuticals have higher bioavailability than larger-sized pharmaceuticals (Jia 2005). Using Caco-2 cell monolayers to investigate TiO 2 nanoparticle translocation across epithelial cells, Brun and others (2011) coupled in vitro translocation studies with transmission electron microscopy (TEM) to show that the TiO 2 nanoparticles accumulated in the cells. Other studies that have used Caco-2 transport models to investigate ENM uptake or interactions with intestinal cells have also been published (Jia 2005; Lin and others 2005, 2011a; Jia and others 2009; Zhang and others 2010a). In most of these studies, ENM locations within the cells could be conveniently imaged using a technique like TEM and their sizes and other morphological characteristics before and after translocation could be ascertained by light scattering (DLS) methods. In addition, monolayer Caco-2 assays can elucidate key mechanistic details of translocation via the use of measurements such as trans-epithelial electrical resistance (TEER), a measurement of electrical resistance across the monolayer. The decrease of TEER values can signal when the ENM weakens the junctions between adjacent cells and increases rates of paracellular transport, an effect that has been shown via this technique to be dependent on ENM surface coating (Lin and others 2011b). Everted gut sac. Everted gut sac experiments are a hybrid of in vivo and in vitro experiments to simulate and measure uptake of ENMs from the intestine (Carreno-Gomez and others 1999; Frohlich and Roblegg 2012). In this approach, portions of the small intestine are removed from a living organism (usually a rat), washed with physiological solution, everted on a glass rod, filled with physiological solution, tied closed at both ends, and placed in a suitable medium that contains the ENM of interest (Alam and others 2012). The interior of the everted gut sac can then be sampled at regular intervals to assess the kinetics of ENM absorption and also probe the characteristics of ENMs (or residuals) that passed through the intestinal barrier. For instance, researchers may estimate absorption by the intestinal lumen with detection methods such as DLS to determine whether and how much of the ENM crossed the intestine wall, whether the ENM is agglomerated or in a dispersed state, or whether the ENM was actively transported across cell membranes and packaged onto nascent lipoproteins or passively diffused. The everted gut sac technique has been used for several decades to support toxicological research and has numerous advantages for ENM uptake analysis: 1) it is reasonably inexpensive and quick compared to in vivo methods; 2) it can be used to compare differences between normal and diseased organs where animal models are available; 3) it can be used to systematically determine 712 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

9 the influence of other substances on uptake of the primary analyte; 4) the gut sac tissue can be recovered after the assay to determine the organelles in which ENM accumulate (if any); 5) because it offers an idealized analysis matrix with few interferences, it could be valuable for investigating uptake of organic ENMs (Carreno- Gomez and others 1999); 6) relative rates of uptake in different regions of the intestine can be specifically determined; and 7) it may also be a better model for paracellular transit than other in vitro techniques such as intestinal Caco2 cell monolayers (Barthe and others 1998). On the other hand, the everted gut sac method has some disadvantages as well, including the following: 1) it may not reliably model absorption rates for passively diffused ENMs if they reach equilibrium across the intestinal wall or if the ENM is absorbed earlier in digestion (oral absorption); 2) it still requires the use of laboratory animals; 3) the viability of living intestinal tissue can be problematic, particularly in unskilled hands; 4) the loss of enzymatic activity may lead to unrealistic conclusions about uptake, particularly for ENMs that are vulnerable to enzymatic actions; and 5) while it provides information on absorption and intracellular distribution, it is not possible to determine organ distribution, metabolism, or excretion of ENMs. The last point above implies that like the artificial gastrointestinal system, it is unlikely everted gut sac methods will ever fully replace in vivo experiments because the type of information resulting from these approaches is quite different. Nevertheless, all of the in vitro approaches discussed here offer unique ways to probe specific structure function relationships that would be difficult if not impossible to investigate using whole, live organisms. Moreover, the epithelial monolayer assay and inverted gut sac approaches yield similar types of information, and therefore, offer a convenient means of methodological validation, a convenience that was recently employed in an in vitro study related to transport and uptake of chitosan nanoparticles (Sandri and others 2007). Thus, although these in vitro models are fairly well established for drug interaction and oral toxicity studies of conventional molecular substances, more work needs to be done to evaluate their usefulness for nanoscale materials. In vivo techniques and sampling methods Despite the utility of in vitro models of the alimentary tract to investigate the behavior of ENMs in various gut organs as well as mechanisms of ENM uptake by the intestinal lumen, in vivo methods particularly those utilizing rodents are and likely will continue to be indispensable to study the downstream effects of intestinal absorption of ENM introduced via ingestion. Aside from the traditional difficulties of in vivo experiments, detection and characterization of ENMs after ingestion by an organism presents a unique methodological challenge because the biological effects, target organs, and physical characteristics of absorbed ENMs are relatively unknown. To determine whether an ENM is absorbed and where it accumulates in the body after absorption, researchers must design protocols to feed the ENM to the animal according to a specific dosing schedule and collect tissue samples to assay for the ENM or its residuals. This section is primarily concerned with methods that can be used to assay for ENM location and physical characteristics after ingestion by organisms in in vivo experiments. In some cases, particularly where background interferences may complicate detection (as in organic particles), these methods can be supported by modifications to the ENMs that make them easier to find. For example, radiolabels and fluorescent tags may make detection of ENM easier when the ADME is unknown. Radiolabels, such as 14 C-labeled poly(methyl methacrylate) (PMMA) nanoparticles (Araujo and others 1999), do not change the chemical structure of the ENM and they are detectable at relatively low concentrations with high tissue penetration. However, they typically have poor spatial resolution, and can incur significant infrastructural challenges for laboratories (Liu and others 2010; Quek and Leong 2012). Fluorescently tagged ENMs (He and others 2008; Huang and others 2011), on the other hand, allow for repeated measurements on live animals, but unless the tags are incorporated into the interior of the particle, they may add bulk and alter the ADME of the ENM. In the unusual case that the ENM itself is fluorescent, as in the case of semiconductor nanocrystals (QDs), the ENM itself can be the fluorescent probe. This property, intrinsic to some classes of ENMs, has been used specifically in in vivo biodistribution experiments (Michalet and others 2005) in which accumulation of fluorescent particles in various internal organs is easy to monitor as a function of time. However, these particles are unlikely to be utilized in food applications and their fluorescent properties may fail to persist after exposure to the harsh chemical environment of the stomach; therefore, this approach may be of little use to study ADME of particles by oral routes of exposure at this time. Whether or not labeled ENMs are used, biological tissues usually have to be prepared in some way before examination by analytical instruments to determine ENM (or residual) content. Researchers also need to consider advantages and limitations for different dosing methods. Gavage, for example, is an established method to deliver a known amount of material directly into the stomach of an animal, but this step bypasses the first important digestive steps in the mouth. Alternatively, researchers may also provide experimental animals with the ENM in their food, which would include mouth digestion, but this approach requires careful monitoring of ingestion of food to determine the dose of ENM that animals actually consume during the experiment. Regardless of how an animal is dosed or ENMs detected in biological tissues, ADME of ENMs can be monitored in several ways. The following are brief explanations of some of the most popular methods currently available to acquire useful information about ENM uptake and postuptake behavior. The basic advantages and disadvantages of these techniques are also summarized in Table 2. Necropsy and direct organ analysis. The most direct method of ascertaining where ENMs go in a live animal after they are absorbed from the alimentary tract is to humanely euthanize the animal and analyze each of its organs for traces of the ENM. If the ENM is then found to persist in the organs at sacrifice, the researcher can then do routine histology to determine whether the ENMs are associated with cellular damage and structural changes in the organs. As a representative example of this approach, Kim and others (2010) fed 56-nm AgNPs to rats at several doses over a 90-d period and observed the rats for changes in their feeding behavior and body weight, as well as their blood chemistry. At the study conclusion, Kim and others (2010) sacrificed the animals and harvested their organs, including the adrenal glands, bladder, testes/ovaries, uterus, epididymis, seminal vesicle, heart, thymus gland, thyroid gland, trachea, esophagus, tongue, prostate gland, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain. These organs were weighed and portions were fixed for histological analysis. Additional portions of each organ were digested in nitric acid under microwave conditions and analyzed by graphite furnace atomic absorption spectroscopy (AAS) for silver content. Via this approach, C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 713

10 Table 2 Summary of benefits and limitations of in vivo techniques and sampling methods available for measuring ENM uptake by the alimentary tract. Method Description Benefits Limitations References Necropsy and direct organ analysis After ENM ingestion by a live animal, the animal is humanely euthanized Established guidelines exist for short-term and long-term feeding Expense and ethics of using live animals Used by most in vivo studies of ENM oral toxicity. For examples, see Kim and its organs and other tissues are studies Concepts like dosage and and others (2010); Baek and harvested and analyzed for ENM Compatibility with radiolabels and concentration have ambiguous others (2012); Fu and others content and characteristics or fluorescent markers for convenient meaning for ENMs (2013) other cellular or macroscopic signs tracking of ENM distribution and Natural variability in gut biology of toxicity accumulation can obscure relationships between Offers the most direct and realistic ENM characteristics and portrayal of the effects of ENM toxicological endpoints ingestion Analyzing ENMs in tissues is Compatible with histological analysis to determine cellular effects of ENM exposure challenging, particularly sample preparation Blood collection and monitoring After ENM ingestion by a live animal, blood samples are removed from the tail vein or ophthalmic vein at timed intervals and analyzed for ENM content to measure kinetics of absorption by the alimentary tract Samples will inform ADME questions by determining the rates of appearance and disappearance from the blood ENMs may be measured directly or biochemical markers may be detected to inform toxicological impact Expense and ethics of using live animals Delayed absorption or biliary excretion can impact kinetics Gives no information about ENM distribution in organ tissues Biliary metabolism may impact ENM properties Procedure description: (Diehl and others 2001); literature examples: (Rohner and others 2007; Wang and others 2008) Fecal and urinary excretion After ENM ingestion by a live animal, feces and/or urine is collected from experimental animals and assayed for ENM content or residuals Offers an indirect estimate of absorption Offers insight into transformative effects of ENM between first and last stages of digestion Amenable to fluorescent or the diet radionucleotide labeling experiments Complimentary to blood collection excretion Expense and ethics of using live animals Does not work well for ENMs that are present in the cage materials or composed of materials naturally in Does not consider other mechanisms of metabolism or and necropsymethods Biliary or other excretion mechanisms can complicate results interpretation Feces example: (Loeschner and others 2011); Urine example: (He and others 2008) Lymph duct cannulation After dosing animals with a known amount of ENM, lymph is collected Most direct means of measuring ENM absorption by the alimentary Expense and ethics of using live animals from experimental animals to tract in live animals No information on distribution, others 2010) directly determine absorption of Tight experimental control over metabolism, or excretion of ENMs ENMs from the intestine analyte concentration and perfusion rate Contrasted with blood analysis, details about the absorbed ENMs prior to metabolism by the liver can be acquired No information on the mechanism of translocation across the gut wall Invasive and technically challenging, particularly in small animals Procedure description (Ionac 2003); Literature example (Peng and 714 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

11 the authors determined lowest observed and no observed adverse effect levels (LOAEL/NOAEL), sex- and dose-dependent distribution differences in organ silver levels, and evidence of slight liver damage from light microscopy of histological slides. It is worth mentioning that the study was based on the Organisation for Economic Co-operation and Development (OECD) test guideline 408 for measuring subchronic toxicity of chemicals, a procedure, last revised in 1998, which has not been validated for measurement of subchronic toxicological effects of ENMs (Organisation for Economic Co-operation and Development 1998). It would be fair to say that a good portion of the oral toxicology literature related to ENMs involves studies that employ some variation of necropsy and direct organ analysis, and an exhaustive account of the experimental details of such studies is beyond the scope of this report. A brief discussion of oral toxicity of ENMs, as measured by feeding studies and subsequent gross organ and histological analysis, is provided in the NanoRelease Food Additive Task Group 1 report (Yada and others 2014), and other reviews are available in the literature (Bouwmeester and others 2009). The clear advantage of this experimental tool is the ability to track exactly where ENMs go after they are absorbed by the gut and what (if any) damage to tissues they may cause. The value of the information obtained is dependent, of course, on the analytical toolset used for tissue analysis. In the study by Kim and others (2010), the AAS used for determining silver content provided no information about the form of silver distributed in the various assayed organs (that is, nanoparticulate, ionic, and so on). This particle/ion question can be answered usually by combining elemental analysis with some form of imaging. TEM analysis of tissue samples, for example, was used in a study published by Fu and others (2013) who investigated tissue distribution of mesoporous silica nanoparticles in mice after ingestion. These authors also utilized fluorescein isothiocyanate (FITC) doped silica particles and localized them in tissues with fluorescence microscopy; this latter approach could find utility in localization studies of carbon-based ENMs postingestion, where ICP-MS and TEM would be less useful. Additional chemical imaging techniques that can be helpful in these areas include MALDI, SIMS, and CARS, as described in an earlier article in this series (Szakal and others 2014). Blood collection and monitoring. Blood collection (such as from the tail or ophthalmic veins) is an in vivo technique to monitor how much of an ENM is absorbed from the gastrointestinal tract into circulation. Timed collection of blood samples allows researchers to estimate the rate and peak of absorption of the ENM, either directly by quantifying particles themselves or indirectly by monitoring biological effects of absorption. For example, Rohner and others (2007) fed ferric phosphate nanoparticles (30.5 and 10.7 nm, amorphous) to rats and collected timed blood samples from the tail to monitor for signs of oxidative stress in the form of thiobarbituric acid-reactive substances to determine whether the nanoform of the material incorporated into blood hemoglobin more efficiently than larger particles. Wang and others (2008) dosed animals with zinc nanoparticles and detected blood zinc levels from ophthalmic veins by ICP-MS. Similar to the artificial gastrointestinal digestion method, researchers must couple this sample collection technique with a measurement method such as HDC-ICP-MS or Raman spectroscopy. If the sample appears in the blood below the analytical method s limit of quantification, then the analytical method may not detect the presence of the ENM. Researchers also should keep in mind that any ENMs or residuals detected in the blood after absorption by the gut have been processed or metabolized by the liver, and thus, the characteristics of the ENMs measured in blood samples may not reflect their characteristics immediately after absorption. If information related to the latter is crucial, lymph duct cannulation may be a more appropriate, albeit more technically challenging, alternative (see above). Fecal or urinary excretion. Excretion is an important factor in determining whether an ingested ENM may be absorbed by the alimentary tract. If a research animal consumes a specific amount of ENM, it is important to know whether the animal excretes the entire amount of the ENM or if the animal retains a portion of the material in its tissues. A basic method to determine excretion starts with giving experimental animals a known dose of ENMs by gavage or in food and collecting and analyzing feces for the ENM. For example, Loeschner and others (2011) studied uptake of AgNPs and silver acetate control in rats over a 28-d period. In addition to typical organ analysis as described above, the authors also collected feces for each animal, dried it in an oven for 60 h at 80 C, and then homogenized the samples, after which the samples were analyzed by ICP-MS and TEM. While the authors detected that >60% of the administered AgNPs was excreted in the feces, there was no significant difference observed between the AgNPs and the silver acetate control, and the authors found no evidence of whole Ag- NPs in the feces by TEM or SEM. The authors noted the need for optimization of sample preparation methods, as well as (in their case) the collection of a larger number of samples to reduce error rates in the analysis. While fecal excretion analysis can give important information about mass balance, as well as generate key insight into transformation of ENMs between the first and last stages of digestion, this approach may not work well for ENMs that are already present in the diet or the caging material (for example, certain proteins or lipids) or for ENMs that can be similar in composition to nanomaterials that are also synthesized de novo in the gut from nonnanoscale materials. Radiolabeled or tagged chemicals are often easier to track than unlabeled chemicals, and therefore may help in some of these situations. Some caution must also be used in interpreting fecal excretion results, as ENMs that are absorbed by the intestine may end up in the feces via biliary excretion, and delayed excretion is also possible if particles bind to the intestinal walls or other partially digested food materials in the gut (Loeschner and others 2011). Fecal excretion studies are also challenging to perform because of the high-potential contamination from food sources elsewhere in animal cages. Finally, the method does not answer questions about metabolism, distribution, or accumulation and does not consider ENMs that are excreted in the urine or exhaled. Urinary excretion is also possible to study, and this approach gives very different information from fecal excretion because any ENMs that end up in the urine have usually been absorbed by the alimentary tract and processed by the kidneys. Some of the challenges of urinary excretion experiments are similar to those of feces analysis (notably, the potential for contamination). Nevertheless, they can be quite informative, particularly if the ENMs are tagged with fluorophores that make tracking by fluorescence imaging convenient (He and others 2008). Lymph duct cannulation. After absorption from the intestine, the lymphatic system is one of the first places that digested materials go, particularly fat-soluble substances. Thoracic lymph duct cannulation is a method to determine uptake of materials from the intestine in rodents by surgically accessing the lymph duct of a living animal and removing lymphatic material for analysis C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 715

12 before it has been distributed to the various bodily endpoints (Ionac 2003). While this technique has the potential to provide detailed absorption rate information in different intestinal segments by offering tight experimental control over the analyte concentration and perfusion rate and can also provide details about the absorbed materials prior to metabolism by the liver (contrasted to blood analysis), the method is a technically challenging and invasive microsurgery in rodents. This method does not distinguish between active and passive translocation of ENMs and needs to be coupled with a characterization technique (such as Raman spectroscopy) to determine whether the physical properties of the absorbed ENM change during translocation. Lymph duct cannulation has found acceptance, especially among researchers interested in pharmaceutical bioavailability studies and, likewise, oral bioavailability of pharmaceutical nanoencapsulates has also been studied in this way. For instance, Peng and others (2010) used this method to analyze uptake and bioavailability of an orally administered pharmaceutical in a phospholipid nanoencapsulate, an analysis that was supported by both fluorescence and conventional visible microscopy. Given the technical difficulty involved, it seems unlikely this will become a widespread technique to study uptake of ENMs from the alimentary tract, at least in the near future, but prospective researchers should be aware that it offers some of the advantages of the in vitro gut models, while still being an in vivo system. AnalyticalmethodsforquantifyingandcharacterizingENMs in vivo There are 2 chief challenges facing scientists who want to study absorption of ENMs from the alimentary tract after they have been ingested from food sources. First, the absorbed ENMs have to be removed from the test organism. Second, the removed samples have to be analyzed. The previous several sections were primarily concerned with the 1st problem. This section addresses the 2nd issue. It is obvious that ENMs absorbed by the alimentary tract cannot be magically removed from an organism in their pristine state: they are removed along with biological tissue, which can range from something liquid and reasonably homogeneous like urine or blood to something solid and/or heterogeneous like a liver or fecal sample. Previous articles in this series discussed, in general terms, some of the many analytical methods available for analyzing the presence, composition, concentration, and/or location of ENMs in complex matrices. Here, we focus on some of the methods specifically useful for analysis of ENMs in biological tissues to support studies on the ADME of ENMs following oral ingestion. Other methods presented in the other articles in this series (Noonan and others 2014; Singh and others 2014; Szakal and others 2014) may also be useful toward compositional or imaging analysis of ENMs in the alimentary tract or other biological tissues. Some analytical methods that may be useful in this area could be applied toward the analysis of ENMs in their native environment with little secondary sample preparation, whereas other methods require that the ENM be relatively pure. In the latter cases, the surrounding tissue or material that is obtained along with the ENMs during the sampling process must either be separated from or otherwise homogenized/digested before analysis. A previous article in this series presented some of the challenges and techniques available to prepare ENM-containing food samples for analysis as well as detection methods that may be applied after the samples have been prepared (Singh and others 2014). Many of the sample preparation methods (for example, filtration, centrifugation, digestion, and so on) described in the context of food analysis in the previous article will likewise find utility in preparing biological tissue samples for analysis methods described below; thus, further elaboration will not be provided on these sampling techniques in this article. What follows is a detailed description of the general classes of analytical techniques most likely to be useful for analysis of ENMs in the alimentary tract or other living tissues. This information is also summarized in Table 3. Light scattering techniques. When photons interact with phase interfaces or grain boundaries, they become reflected and refracted in a process known as light scattering. The characteristics of the scattering phenomenon depend upon the properties of the material doing the scattering. Thus, measurements of how the light properties change in a light scattering experiment can provide information about, among other things, the refractive index of the material as well as the nature of the material s surface. With respect to ENM characterization, the primary value of light scattering experiments is to provide information on particle size. DLS, also referred to as quasi-elastic light scattering (QELS) or photon-correlation spectroscopy (PCS), is a technique that is primarily used to measure the hydrodynamic radius of particles in a solution as they move due to Brownian motion, which causes fluctuations in the intensity of scattered light. DLS instruments (with appropriate measurement cuvettes) are also often equipped with the ability to apply an electrical field to the sample, which can be used to measure the surface charge (zeta potential) of ENMs. This is a rather unique advantage of DLS, as zeta potential is difficult to measure with any other technique. Because DLS equipment is relatively inexpensive, and because it is quick and easy to use, DLS has become a fairly standard tool in the analyst s toolbox and has been used to characterize ENMs in numerous studies (Peters and others 2011, 2012; Raz and others 2012). In a relatively pure suspension of particles, DLS can be used to measure a distribution of particle sizes but does not provide information about composition of the materials doing the scattering. A primary drawback of DLS is that it is not the most appropriate method for characterizing ENMs with large polydispersity (Stamm and others 2012), as the method returns a disproportionately large signal for larger particles or agglomerates (Linsinger and others 2012). Sample purification therefore is quite important, because a small number of micron-sized contaminants can completely wash out the signal from a significantly larger number of nanosized particles. In this regard, while the technique is useful for identifying the presence of particle aggregates, samples that have a combination of isolated particles and large-particle agglomerates (such as might be the case in samples extracted from the stomach or gastric models) can be difficult to accurately measure. Moreover, it is worth bearing in mind that the method is highly sensitive to sample quality and can return significantly different values for particle size than those obtained by microscopic methods like TEM. This is because DLS measures hydrodynamic radius, which can be affected by the protein corona or other bound biomolecules that would be transparent in TEM. Multiangle light scattering (MALS) is another light scattering technique that relies on similar physics as DLS to acquire particle size information, except that the method measures static light scattering intensity at multiple angles rather than DLS intensity at a single angle. The particle size determined from MALS, properly called a radius of gyration, is often similar to that measured by DLS (Kato and others 2012). The primary advantage of MALS is that it is faster than DLS and can be slightly more sensitive (Alvarez and others 2011). As such, it is most often encountered in combination 716 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

13 Table 3 Summary of benefits and limitations of analytical methods available for measuring ENM characteristics in biological tissues or uptake by the alimentary tract. Method Description Sampling Benefits Limitations Light scattering techniques General attributes Benchtop techniques primarily used for determining particle size Generally requires a pure, transparent sample, such as Relatively inexpensive and does not require significant expertise Sample preparation can alter properties of the ENM Cell culture media Data easyto analyze Reproducibility can be low No compositional information Artificial gastrointestinal fluid Pristine solution Widely available in analytical laboratories Dynamic light scattering (DLS) Records time-dependent intensity fluctuations of scattered light to measure size-dependent diffusion speeds of ENMs Cuvette-based method. Zeta potential requires cuvette equipped with electrodes Can reveal ENM size distribution and agglomeration under various conditions Hydrodynamic radius can differ substantially from other particle sizing methods (TEMs) Properly equipped instrument can measure surface charge (zeta potential) Large particles can skew the measurement or obscure the presence of smaller particles Highly sensitive to impurities Multiangle light scattering (MALS) Records light scattered off of particle fluid interfaces as a function of incidence angle to estimate size Most often encountered as a detector which accompanies a chromatographic method like asymmetric field flow fractionation (afff) Real-time analysis and complexation to separation technologies Slightly more sensitive than DLS Coupled with ICP-MS, simultaneous size, and composition data are possible Chromatographic separation entails additional sample preparation complexities Large particles can interfere with afff separations Long analysis times make it unsuitable as a rapid screening method Elemental analysis methods General attributes Methods designed for compositional analysis of ENMs in either pristine or complex matrices Most modes require a low-viscosity, homogeneous fluid, which requires extraction of ENMs or digestion of the matrix. Direct measurement: Cell culture media Artificial gastrointestinal fluid Pristine solution Measurement after digestion or extraction: Solids and tissue samples Provide compositional information to verify ENM identity and locate ENMs in tissues and organs after ingestion Provide mechanistic detail about transformations in the gut Most are widely available in analytical laboratories High throughput for convenient screening of numerous samples (useful for kinetics) Difficult or impossible to quantify organic ENMs due to compositional similarities with the background matrix In general provide little information on ENM characteristics (size, shape, surface) (Continued) C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 717

14 Table 3 Continued. Method Description Sampling Benefits Limitations Inductively coupled plasma mass spectrometry (ICP-MS) Highly sensitive and flexible analytical technique for trace metal analysis based on atomic mass Same as above. Laser ablation method can be used for direct sampling of solids, but finding suitable concentration standards can be a challenge Simultaneous detection of multiple elements (good screening tool) <0.01 µg/l sensitivity for many elements Single particle mode can distinguish single particles from ionic background as well as determine ENM sizes Can be interfaced with chromatographic separation techniques (for example, HPLC, afff) for fractional analysis High level of method development expertise Sample digestion can destroy information about ENM characteristics In basic modes cannot distinguish between whole ENMs and ions Generally intolerant of high concentrations of dissolved solids Inductively coupled plasma atomic emission spectroscopy (ICP-AES) Highly sensitive and flexible analytical technique for trace metal analysis based on atomic spectral signatures Sometimes referred to as ICP-OES Same as above Simultaneous detection of multiple elements (good screening tool) Tolerant of high concentrations of dissolved solids Less expensive alternative to ICP-MS Less method development skill needed Fewer chemical interferences than ICP-MS Can easily process organic matrices, useful for biological samples Generally limited to simple analysis mode (compositional analysis only) 0.1 to 100 µg/llodsmaynotbe good enough for tissue analysis if extent of absorption is small Typically cannot be interfaced with chromatographic techniques Atomic absorption spectroscopy (AAS) Highly sensitive and flexible analytical technique for trace metal analysis based on atomic spectral signatures; comes in graphite furnace (GFAAS) and basic flame (FAAS) versions Same as above Easy to use and inexpensive GFAAS has detection limits approaching those of ICP-MS Fewer spectral interferences than ICP-AES Only can measure a few elements at a time (no screening ability) >1 µg/l LODs for FAAS may not be sufficient if ENM concentrations in tissues are too small Does not perform well for some elements due to low excitation temperatures (Continued) 718 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

15 Table 3 Continued. Method Description Sampling Benefits Limitations Imaging analysis and microscopy General attributes Appropriate for visualizing ENMs in their native environments and/or the impacts of ENMs living cells For studies related to absorption of ENMs from the alimentary tract, relevant categories include light-based microscopy and electron microscopy For imaging biological tissues: Most methods require some form of fixing, staining, and/or sectioning of the sample prior to analysis For pristine particle analysis: ENMs can usually be deposited on an appropriate substrate and imaged directly Reveal how the ENMs are integrated in the surrounding matrix Can specify where ENMs are located at both the organ and cellular level Offer visual clues about toxicological effects of ENM absorption A variety of available techniques can fulfill many different needs Most methods provide no compositional information Usually less useful for organic ENMs due to poor contrast with the background matrix Laborious sample preparation Very low throughput; not a good general screening tool Representative sampling is a challenge Transmission electron microscopy (TEM) Electron microscopy technique that measures passage of a high energy beam of electrons through the sample Images contrast generated by electron density differences in the substrate: electron dense areas (for example, metals) appear dark Requires a uniformly thin sample Tissue samples need to be desiccated, fixed and thinly sectioned, usually with an ultramicrotome Newer techniques like focused ion beam (FIB) improve sample quality but are expensive Very high (<1 nm) resolution possible, single ENMs resolved Most inorganic ENMs show high contrast ENM size and/or shape revealed Distinguish between whole ENMs and ions in tissues Scanning TEM with EDS can do compositional analysis Counting numerous particles can be expensive and slow High level of expertise required Sample preparation may change ENM or matrix characteristics May not be reliable for low (ENM concentrations in tissues) No information on ENM surface features (Continued) C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 719

16 Table 3 Continued. Method Description Sampling Benefits Limitations Scanning electron microscopy (SEM) Electron microscopy technique that scanssamplewithhighenergy beam of electrons Image generated by measuring concentration of secondary electrons ejected from the sample surface Provides information on surface topography Same as above (TEM) Organic surfaces must be coated (for example, gold) or stained (for example, O-osmium) with a conductive material Newer techniques like environmental SEM allow measurement of ligand samples without complicated sample preparation High resolution (2 to 5 nm) possible, single ENMs resolved ENM size and/or shape revealed EDS-equipped SEM for compositional mapping: ENM identification and compositional transformations 3D detail might show localization of ENM within the cellular structures May not be reliable for low doses Counting numerous particles can be expensive and slow High level of expertise required Topological information may not be useful for tissue specimens No information on ENM surface features Sample preparation may change ENM or matrix characteristics Confocal laser scanning microscopy (CLSM) (also: confocal fluorescence microscopy) Light-based microscopy technique which provides 3D images of cellular structure via optical sectioning to localize fluorescent-tagged ENMs or cellular structures Because samples are optically sectioned by a confocal pinhole, thin physical sectioning is not required Some sample fixation may be necessary and fluorescent staining is usually needed to image cellular structures If ENMs are not fluorescent, tagging with a fluorescent marker is necessary Shows localization of ENMs within the cells and tissues Real-time scanning capability Optical sectioning provides rapid 3D distribution information Widely available fluorescent ENMs (QDs, dye-incorporated silica) can be convenient models for gastrointestinal uptake Organic ENMs can be tagged and imaged Well established for study of tissues Laser penetration, photobleaching, and spherical aberrations can limit resolution and signal intensity Optical resolution is too poor to image single ENMs (no structural information) No compositional information Tagging ENMs with fluorophores can alter their chemical and/or pharmacologic properties Staining can change biological properties of tissues (Continued) 720 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

17 Table 3 Continued. Method Description Sampling Benefits Limitations Light microscopy Established technique that images samples by transmission of light through the sample Most useful for analysis of cellular abnormalities in various tissues Some sectioning and staining required Inexpensive Well established for study of tissues Widely available Indispensible for histological evaluations Can reveal impacts of ENM absorption and distribution Optical resolution too poor to image ENMs Little information on ENM presence, composition, or characteristics Suitable controls are necessary to establish that tissue abnormalities are due to ENMs Examples of methods in development Surface plasmon resonance (SPR) SPR measures the presence of an ENM based on interaction with antibodies or other binding agents attached to a metal substrate Purification usually required to minimize interference (nonspecific binding), but sample preparation not well-understood for ENM detection Sensitive in the µg/l range Can measure directly in complex liquids Chip-based technology is reusable Multiplexing Requires targets that bind specifically with the ENM Chips targeting each ENM type need to be individually designed Enzyme-linked immunosorbent assay (ELISA) Assays that rely on the binding of antibodies to a specific antigen (for example, ENM), typically for optical (fluorescence or colorimetric) based detection Purification usually required to minimize interference (nonspecific binding), but sample preparation not well-understood for ENM detection Multiplexing Rapid No sensitive equipment required Concentration or localization of targets (for example, ENMs) within cells is possible Well-established for conventional biological analytes Requires targets that bind specifically with the ENM No compositional information Potential for matrix interferences is not well studied for ENM antibodies Qualitative or semiquantitative (Continued) C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 721

18 Table 3 Continued. Method Description Sampling Benefits Limitations Asymmetric field flow fractionation afff Separates ENMs based on their size, which impacts ENM motility Generally requires a purified, homogeneous sample Usually coupled with an analytical technique like ICP-MS to provide simultaneous composition and structural information Very broad size range Very large particles can interfere with afff fractionation There are no standardized methods for ENM separation Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) Identifies chemical composition based on mass fragmentation patterns Samples need to be reasonably pure and dilute to simplify the analysis and limit interferences Broad molecular size range that is useful for lipids, proteins, and other biomolecules Widely available Rapid technique Suitable for compositional analysis of organic ENMs Useless for inorganic ENM analysis Must be coupled with a separation technique which can dilute and purify the sample Better adapted to answer is it there? than how much is there? Flow cytometry and fluorometry These methods count or sort cells with fluorescent markers by passing them one at a time through a detector Can be used to determine whether cells contain luminescent ENMs Atmospheric scanning electron microscopy (ASEM) A version of SEM that allows samples to be scanned without fixation (for example, not under vacuum) Also known as environmental SEM Raman spectroscopy and microscopy Light scattering technique that provides qualitative and quantitative information about some ENMs Chemical imaging and mapping capability in microscope format Can be used to monitor changes in chemical or physical properties (for example, agglomeration) and for basic identification of ENMs in complex matrices Samples need to be reasonably pure and dilute to ensure that single cells pass through the detector. Background matrix should be free of fluorescent signatures Detect whether a population of cells took up ENMs Widely available Rapid technique Wet samples can be imaged directly Mostly the same as standard SEM (see above) Typically laborious preparation of EM samples not required Samples need to be reasonably transparent to the irradiation wavelength. Since Raman excitations are in the nearinfrared, this is often not a problem for tissue samples No need for radiolabels or fluorescent tags High tissue penetration Very good spatial resolution in microscopy configuration Tagging metallic ENMs with reporter molecules can take advantage of surface enhanced Raman scattering (SERS) effects for high contrast No compositional or characteristics analysis: identification and localization only Midrange sensitivity Tagging ENMs with fluorophores can alter their physical or toxicological properties Gravity bias can skew measured particle size distributions Most commercial versions are currently not compatible with EDS Usefulness for inorganic materials may be limited without tagging In most cases, signal strengths are low, so sensitivity is poor, particularly in inhomogeneous samples where Raleigh scattering is pronounced Slow due to long data acquisition times LOD, limit of detection. 722 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

19 with a chromatographic separation technique such as asymmetric field flow fractionation (afff), which allows one to separate a sample of ENMs by size and measure in real time from the size of each fraction before directly aspirating into an ICP-MS for elemental analysis, as a study by Schmidt and others (2009) related to detection of clays migrated from food contact materials. On the other hand, chromatographic separation of ENMs entails its own set of experimental difficulties; thus, when a quick particle size measurement is desired and concomitant compositional analysis is not desired, DLS may be the more appropriate method to choose. Both DLS and afff-mals require reasonably pure samples, because interferences in more complex matrices can interfere with the ENM scattering signal. Therefore, ENMs need to be extracted from the tissue matrix prior to acquisition of sizing data via these methods. Insofar as such sample preparation methods can destroy information about the ENMs in their native environment, light scattering techniques may not be the best methods to use if information such as aggregation state in the native environment is desired. Elemental analysis. Elemental analysis methods, which include AAS and ICP methods (MS and atomic emission spectroscopy), are highly sensitive analytical techniques that identify chemical composition based on unique properties of individual elements. In the case of AAS and ICP-AES, the unique property is spectroscopic, whereas in ICP-MS, elements are uniquely identified by their atomic weights. All of these techniques provide the same basic information and involve the same sample preparation, but differ in their relative sensitivity, analysis time, and adaptability. Flame AAS, for example, has the least sensitivity and typically only allows for analysis of a few elements at a time, but is also the least expensive technique. ICP-AES has better sensitivity than AAS (typically limits of detection in the parts-per-billion range) and can measure virtually every element on the periodic table simultaneously. ICP-MS has the best sensitivity (parts per trillion or even parts per quadrillion, in some cases), but is by far the most expensive, may be more vulnerable to interferences, and can be troublesome for some elements that have many naturally occurring isotopes. On the other hand, ICP-MS also offers the potential for speciation experiments via interface on the front end with chromatographic separation and, more recently, can be run in single-particle mode for distinguishing between ions and particulates in solution. Graphite furnace AAS actually can have detection limits that rival ICP-MS in some cases but is not as flexible in terms of measuring many elements simultaneously. Still, this technique may find utility as a more cost-effective alternative to ICP-MS in cases in which only a few elements need to be detected. Currently, ICP-MS is in the ascendancy among elemental analysis techniques and is virtually indispensable in any laboratory involved in research related to inorganic ENMs. ICP-MS instruments are highly flexible and can measure liquids, gases, and even solids (via laser ablation). If liquid aspiration is used to introduce the sample into the instrument, the sample needs to be homogeneous and relatively dilute, so as to facilitate nebulization without clogging the narrow pump tubing, nebulizer, or torch nozzle: ICP-MS is far more challenging than ICP-AES in this regard because of its high sensitivity and comparatively low tolerance to high concentrations of dissolved solids. This typically necessitates sample digestion prior to measurement, which implies that physical properties of ENMs fed into the plasma if they even survive the digestion are not retained at the time of measurement. This is discussed in more detail in the previous article in this series by Singh and others (2014). ICP-MS and associated elemental analysis techniques provide information on the composition of ENMs but do not provide any detail on their characteristics, or even whether they are ENMs at all. Research protocols often couple ICP-MS with other methods such as afff or HDC to determine size and concentration of particles (Dekkers and others 2011; Peters and others 2012). ICP- MS can also be run in a time-dependent single-particle mode as discussed previously (Singh and others 2014), which, if the solution is sufficiently dilute, registers single particles passing into the plasma as spikes on an intensity versus time plot. In principle, particle size and possibly even shape can be determined via this technique, although this is still an active area of research. In addition, these modifications to traditional ICP-MS do not bypass the need for a completely homogeneous, low-viscosity liquid sample, which means that, even if measures are taken to extract information about ENM characteristics in addition to basic compositional data, information about the ENMs as they exist in their native environment is still lost during sample preparation. As such, ICP-MS almost always needs to be accompanied by a supplemental imaging technique like TEM. Most of the studies presented earlier in this section related to detecting ENMs in the alimentary tract or peripheral tissues employ some version of ICP-MS, ICP-AES, or AAS for compositional analysis. This is usually related to the assay of peripheral organs after necropsy for ENM concentration. Elemental analysis techniques can be used for analysis of virtually any ENM in living animal tissue in support of oral toxicity studies, including ENMs composed of silver (Fondevila and others 2009; Kim and others 2010; Park and others 2010, 2011; Loeschner and others 2011; Chiao and others 2012), titanium oxide (Wang and others 2007, 2013; Novak and others 2012), zinc oxide (Wang and others 2008; Baek and others 2012; Li and others 2012; Sharma and others 2012), alumina (Balasubramanyam and others 2009; Park and others 2011; Yang and others 2012), and silica (Fu and others 2013). It must be said that some of these elements are not without their unique challenges to analyze (silicon, for example, is a component of glass and, therefore, glass-free sample introduction hardware is required for analysis of silica nanoparticles), and other elements (like carbon) are practically impossible to measure because of their ubiquity. Nevertheless, and regardless of the other aforementioned limitations related to sample preparation, ICP-MS and related technologies still remain the primary means of ENM detection in biological tissues. Imaging analysis and microscopy. As was pointed out in the 1st article in this series (Szakal and others 2014), elemental analysis and other characterization techniques can provide compositional information about ENMs in tissue samples effectively answering questions such as Is it there?, How much is there?, and, in some cases, What are its characteristics? However, because of the realities of sample preparation, much of the information about how the ENMs behave or what the ENMs look like in their native environment is lost. Imaging analysis is therefore a critical component of ENM characterization because it reveals details about how the ENM is integrated in a particular matrix. While imaging samples does often require quite a bit of preparation before the samples can be placed in a microscope, the external matrix is typically still retained in some fashion, so questions related to whether ENMs absorbed by the alimentary tract are located in certain types of cells, or whether they are ENMs at all, can be answered. With the exception of scanning probe techniques like atomic force microscopy (AFM), most microscopy techniques involve C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 723

20 focusing a beam of particles (photons or electrons) to a small spot on the sample and measuring how the substances in the sample at that spot reflect or scatter the particles to various degrees, thus forming an image. Light-based microscopy (such as confocal fluorescence microscopy) has some utility in ENM research, but the fundamental limitation of most of these techniques is the diffraction limit of light, which for even the best current optics is generally around 200 nm using a standard argon ion laser excitation wavelength of 514 nm. This typically means that particles or structures with dimensions<200 nm cannot be resolved, which is a significant problem for locating ENMs that can be over an order of magnitude smaller. Light microscopy techniques based on UV or X-ray photon sources, as well as certain near-field techniques, can extend this diffraction limit to smaller size regimes, but these techniques are expensive and have not been fully developed for biological tissue analysis as of yet. Owing to their larger masses/momenta, electrons have significantly shorter de Broglie wavelengths than photons and thus have theoretical resolving power several orders of magnitude smaller than light. As a result, EM has become the go-to class of imaging technology in ENM research. Other articles in this series have described in detail the usefulness of EM-based methods toward ENM analysis (Szakal and others 2014). In the context of the alimentary tract, EM is a valuable technique to quantify/locate ENMs within cells or organ tissue and characterize the physical properties of the materials (including size, shape, and agglomeration state) at a very high resolution (Dudkiewicz and others 2011; Linsinger and others 2012). Generally speaking, the volume of sample that can be analyzed in any one experiment is very small, and samples that contain water, such as a tissue section, must be fixed or frozen before analysis due to the need to perform measurements under vacuum. Because of the time, cost, and skill associated with EM analysis, it is not currently a good candidate for quick screening of tissue samples for the presence and location of ENMs. EM techniques also suffer from the same general drawbacks of all microscopic analysis: namely, the difficulty of ensuring representative sampling, generation of artifacts, and limited chemical identification. Below are brief descriptions of both EM and light microscopy techniques useful for ENM analysis in support of alimentary tract uptake studies. TEM is an EM technique in which a beam of electrons is focused onto a uniformly thin section (optimally no more than a few 100 nm thick) of sample. Images are usually generated by the differential absorption or interaction of electrons with materials in the sample. Because different materials interact with electrons to different degrees, TEM images in principle contain compositional information, although, in practice, this information is hard to express with any precision. In conventional bright field mode, contrast is typically generated as a function of the electron density of the material. Therefore, structures and materials composed of heavy, electron-dense elements (such as gold nanoparticles) appear darker than those composed of lighter elements (organic tissue). As a consequence, TEM, at least in conventional modes of operation, tends to be more useful for detection and characterization of metallic, semiconductor, or metal oxide nanostructures and less useful for analysis of organic ENMs. TEM is used ubiquitously for analysis of ENMs in complex matrices and is almost always used in tandem with some kind of elemental analysis technique. Numerous nanotoxicology studies published in the last few years have used TEM to determine the presence, identity, and/or atomic structure of ENMs, either in the pristine state prior to administration, in a tissue matrix after administration, or both. Figure 3 shows a representative example of the application of TEM to locate ENMs (in this case, composed of silver, after tail vein injection) in various organ tissues. Dziendzikowska and others (2012) could locate ENMs, determine the aggregation state, and localize with specificity at both the organ and cell-type levels. The most significant drawback of TEM analysis is the challenging sample preparation. Tissue samples must be desiccated, fixed, stained (if applicable), and sectioned; sections must be thin and of uniform thickness, and require great skill to prepare, usually via microtomy. Nagashima and others (2011) recently reviewed this process. As a result of these considerations, TEM is not an efficient method to determine the quantity of an ENM that has been absorbed from the alimentary canal and distributed to various sites in the organism. Nevertheless, since the information it provides often cannot be obtained in any other way, it continues to be an indispensable tool. SEM is an alternative EM technique that scans a sample in an xy plane and (usually) operates in a mode that detects secondary electrons ejected from the sample surface. The signal intensity at any point on the sample is dependent on the angle between the plane tangent to surface at that point and the incoming electron beam. Therefore, SEM primarily is used to analyze the topography of a sample, and (by itself) provides no compositional information. Electrons utilized in SEM microscopes are usually of lower energy than those used in TEM, which means that SEM is a lower resolution technique. However, the resolution of SEM is still high enough to image most nanostructures larger than a few nanometers in diameter and, in any case, the advantage of a larger image area compensates for this deficiency. Moreover, absorption of the incident electrons by surface atoms also stimulates the release of X-ray radiation, and the wavelengths of released X-rays are dependent on the atomic identity. As a result, SEM instruments are often equipped with accessories that can measure the energies of emitted X-ray photons, and thereby do compositional mapping of the surface topography. This supplementary technique is called EDS (sometimes also abbreviated as EDX), and it is of enormous value in ENM research. EDS is also sometimes an available option for TEM and scanning TEM equipment as well, although pointby-point compositional mapping is generally not possible in the former case. To study nanomaterials in the alimentary tract, researchers can use thin tissue sections from ENM-fed animals or ENM-treated cell culture experiments (chemically fixed and dried or frozen) for SEM analysis. Since SEM provides shape and topology information of 3D structures, the primary use of SEM in in vivo and in vitro toxicity studies is compositional analysis with EDS. Specifically, SEM/EDS is used to confirm that spots in a microscopic analysis that are suspected to be ENMs are indeed ENMs composed of the target material. However, in some cases, SEM/EDS can also give mechanistic information, as in an in vitro gastrointestinal model study in which SEM/EDS was used to show that chlorine in the gastric environment mediates AgNP aggregation (Walczak and others 2013). A sample of the data from this study is provided in Figure 4. As with TEM, the primary challenges of SEM analysis are complicated sample preparation as well as expense and expertise required to use the equipment. SEM equipment requires highvacuum conditions, requiring the usual desiccation and fixing of tissue samples. In addition, SEM requires a conductive surface for analysis, and because tissue samples from the alimentary tract are 724 Comprehensive Reviews in Food Science and Food Safety Vol. 13, 2014 C 2014 Institute of Food Technologists

21 Figure 3 Representative transmission electron microscope (TEM) images of silver nanoparticles in rat tissues extracted from the spleen (A), liver (B), and lung (C) after tail vein injection. TEM analysis can provide information on ENM location and quantity in various organ types and cell types, and it provides information on aggregation state. Adapted from Dziendzikowska and others (2012), with permission from John Wiley and Sons, Ltd. Copyright C 2012 John Wiley and Sons, Ltd. Figure 4 Sample of an SEM image (A) and an EDS spectrum (B) of AgNPs in an in vitro gastrointestinal model. The EDS spectrum was acquired for an aggregate and showed the presence of chlorine, which was used to conclude that hydrochloric acid in the gastric medium precipitated as AgCl on the AgNP surfaces, leading to aggregation. Chlorine was not observed in the EDS spectra of isolated AgNPs (data not shown). Adapted from Walczak and others (2013) with permission of Informa Healthcare. Copyright C 2012 Informa Healthcare. predominantly organic (nonconductive), they need to be treated with an electrically conductive coating, which may hinder analysis of some ENMs. In the case of tissue analysis, the high-energy electron beam may also damage the sample (Dudkiewicz and others 2011). Light microscopy, based on the irradiation of samples with light rather than electrons, has some utility in research related to ENM behavior in tissues, particularly when it is not necessary to resolve single particles. An example would be where researchers want to examine the effects of ENMs on tissues and cells rather than the particles themselves, as in histopathological analysis. For example, Sharma and others (2012) used light microscopy to identify pathological changes to kidney and liver cells in mice after oral ingestion of zinc oxide ENMs at 300 mg/kg and Hadrup and others (2012) did the same light microscopy histological analysis of AgNPs in mouse liver and kidney cells. The advantage of these techniques is that they are readily available and well established in the biological community. The disadvantage is that, in general, they can offer no information about the ENMs themselves; therefore, experimental design and the presence of suitable controls is important to establish that observed abnormalities are due to the presence of the ENM (or its derivatives) and not some artifact. CLSM, also called confocal fluorescence microscopy, is another light-based technique that does not have the optical resolution capable of imaging individual ENMs, but if the ENMs are fluorescent, or if fluorescent tags are used, the general location of ENMs (in a particular cell type or organ tissue) in 3 dimensions can be ascertained. The 3D information is easy to obtain by CLSM because unlike in EM techniques in which samples must usually be physically and individually sectioned, the use of a confocal pinhole in CLSM permits optical sectioning of biological samples in much the same way as a radiologist uses computed tomography to quickly scan sections of a patient s tissue for disease. As a result, confocal fluorescence microscopy can be used, in some specific situations, to conveniently answer the Is it there and, if so, where is it? question. Silica, for example, is fairly easy to tag with fluorescent molecules, and so fluorescence microscopy can be used to localize silica nanoparticles in cells after ingestion, as in a recent study by Fu and others (2013). As this study shows, it is typically necessary to also stain the background tissue with a fluorescent dye to provide positional reference points for locating the particles within cells. Other particles, like semiconductor quantum dots, are inherently fluorescent and have been used for some time as fluorescent model ENMs to determine how ENMs are processed by living organisms (Michalet and others 2005). On the other hand, metallic nanoparticles (such as gold nanoparticles), although not fluorescent, often possess their own unique optical properties like surface plasmon resonance (SPR) and/or efficient C 2014 Institute of Food Technologists Vol. 13, 2014 Comprehensive Reviews in Food Science and Food Safety 725