Utility of models of the gastrointestinal tract for assessment of the digestion and absorption of engineered nanomaterials released from food matrices

Figures & data

Table 1. In vivo techniques that have been commonly used to assess the digestion and absorption of conventional chemicals in the gastrointestinal tract, and could be relevant to the assessment of NMs.

Model	Species	Characteristics and applications for conventional chemicals	Limitations	References on conventional uses
Toxicokinetic feeding or gavage studies	Primarily rat, mouse, hamster or guinea pig, unless another species such as pigs or primates is required for regulatory purposes	Assesses systemic absorption, excretion, and persistence of the chemical	Gavage does not represent a realistic exposure scenario; recovery and quantification of administered compounds in animal tissues is a challenge; some species do not accurately predict chemical bioavailability in humans	OECD (2010)
In situ intestinal perfusion	Rats or other species	Cannulation of both ends of a segment of the gastrointestinal tract in a live, anesthetized subject allows infusion of a solution and collection of the perfusate at the distal end; determines permeability through the gastrointestinal tract wall	Requires surgery and anesthesia; can result in abnormal transit times or paralysis of the bowel; digestion is not modeled; not a high-throughput model	Lennernas et al. (1997), Kim et al. (2006)

Table 2. Ex vivo tissue models that have been commonly used to assess the absorption of conventional chemicals through the gastrointestinal tract, and that could be relevant to the assessment of NMs.

Model	Species and tissue of origin	Characteristics and applications for conventional chemicals	Limitations	References on conventional uses
Everted gut sac	Excised intestinal segments from rat or other species	The intestine is everted, ligated at both ends and cultured in an oxygenated nutrient solution; adhesion of an administered compound to the outer mucus layer and permeability through the tissue to the inside of the sac is measured; relatively simple and inexpensive methodology	Careful tissue handling is critical to avoid mechanical damage; appropriate culture conditions must be used to maintain tissue viability; the static fluid in the serosal compartment does not accurately model in vivo circulation	Alam et al. (2012)
Transmucosal perfusion  through isolated mucosa	Mucosal layer isolated from biopsies from the buccal cavity or intestine of rat, pig or other species	Perfusion device with a donor and receiver chamber, such as an Ussing chamber or Franz diffusion cell, determines permeability through the mucosal layer; results compare favorably to human data, particularly for passively absorbed substances	Tissue viability declines rapidly	Roblegg et al. (2012), Rozehnal et al. (2012)

Table 3. In vitro cell culture models that simulate the gastrointestinal tract epithelium, are commonly used to assess the absorption of conventional chemicals, and could be relevant for the assessment of NMs.

Model	Species and tissue of origin	Characteristics and applications for conventional chemicals	Limitations	References on conventional uses
TR146 cells	Human buccal carcinoma	Epithelial origin; forms a multi-layered squamous tissue in culture; a model of chemical permeability through the buccal epithelium	Transport mechanisms require further characterization	Teubl et al. (2013)
Caco-2 cells	Human colorectal carcinoma	Widely used model of small intestine enterocytes; develop tight junctions, polarization, brush border enzymes and membrane transporters that are responsible for the active uptake of chemicals	Cells vary in phenotype and function according to culture conditions; tight junction permeability is lower than in humans; expression level of some active transporters differs from the human intestine; lacks mucus	Sambuy et al. (2005), Prieto et al. (2010), Kim et al. (2012)
T84 cells	Human colon carcinoma cells that had metastasized to a pulmonary tumor	Less prone to differentiation than Caco-2 cells; an ideal model of the colon since the cells are capable of regulated chloride secretion and can secrete mucus	Few microvilli on the apical membrane; relatively low monolayer permeability	Donato et al. (2011)
2/4/A1 cells, IEC-6 cells, or  IEC-18 cells	Rat intestinal epithelial cells	Good predictors of paracellular permeability and passive transport in the human duodenum	Lack several active transporters and efflux systems; a poor model of active transport	Thomas & Oates (2002), Versantvoort et al. (2002), Tavelin et al. (2003)
Caco-2 with HT29 cells	Human colorectal carcinoma cell lines	Co-culture incorporates a mucus layer on the enterocytes and enhances paracellular permeability	The availability of different cell sub-clones results in interlaboratory variability	Walter et al. (1996), Chen et al. (2010)
Caco-2 with Raji B cells, or  Caco-2 with  Peyer’s patch lymphocytes	Human colorectal carcinoma cells in combination with human Burkitt’s lymphoma cells or murine Peyer’s patch lymphocytes	Co-culture induces a fraction of the Caco-2 cells to develop the morphology of Peyer’s patch epithelial microfold cells, including few microvilli and a soft mucus layer	Variations in primary lymphocyte preparations may influence co-culture characteristics; quantitative correlation to human uptake not fully understood	Tyrer et al. (2002), des Rieux et al. (2007)
Gut-on-a-chip	Human colorectal carcinoma	Two fluidic channels separated by a Caco-2 monolayer, with microcircuitry to detect absorption of substances	Cells vary in phenotype and function according to culture conditions; tight junction permeability is lower than in humans; expression level of some active transporters differs from the human intestine; lacks mucus	Kim et al. (2012); Vergeres et al. (2012)

Table 4. In vitro non-cellular fluid models that simulate the gastrointestinal tract, are commonly used to assess the digestion and absorption of conventional chemicals, and could be relevant to the assessment of NMs.

Model	Characteristics and applications for conventional chemicals	Limitations	References on conventional uses
Static digestion models	Synthetic fluids simulate the enzymatic and chemical digestive conditions in the various compartments of the gastrointestinal tract	Lack the mechanical forces that contribute to digestion in vivo; metabolites accumulate and can interfere with digestion	Versantvoort et al. (2005), Guhmann et al. (2013)
Dynamic digestion models	Synthetic digestive fluids are mixed and propelled by mechanical forces similar to those encountered in vivo; bioaccessibility and digestion are modeled for the various compartments of the gastrointestinal tract	Specialized equipment required to generate the physical forces	Minekus et al. (1999), McAllister (2010)
Natural and synthetic mucus	Isolated gastrointestinal mucus incubated in vitro; models mucoadhesion and permeation through the mucus	Variations in mucus source and preparation can influence characteristics	Crater & Carrier (2010)
Artificial membrane  permeability assays	Non-cellular synthesized membrane on a filter support models the cell membrane; lipid composition can be specified; passive transcellular permeability is modeled; both hydrophilic and hydrophobic compounds can be applied	Membrane lacks pores and transporter proteins; does not model active transport or paracellular permeability	Reis et al. (2010)

Table 5. In silico computational models that simulate the gastrointestinal tract, are increasingly used to assess the dissolution and absorption of conventional chemicals, and could be relevant to the assessment of NMs.

Model	Capabilities for conventional chemicals	Limitations	References on conventional uses
Compartmental absorption and transit	The dissolution and bioavailability of chemicals in different compartments of the human gastrointestinal tract is modeled based on inputted solubility and permeability data on that chemical from in vivo, ex vivo or in vitro studies; can incorporate food effects and physiological inter-individual variability	Uses generalized mathematical models; prediction of intrinsic solubility is a challenge	Yu & Amidon (1999), Bolger et al. (2009), Jamei et al. (2009), Sugano (2009), Sinha et al. (2012)
Statistical machine learning and Quantitative Structure Property Relationship	Prediction of human intestinal chemical permeability or bioavailability, based on the observed or calculated physico-chemical properties of the molecule; trained with existing permeability data from similar molecules in the human intestine or in Caco-2 cells	Molecular descriptor selection is algorithm- and descriptor software package- dependent, leading to variability in prediction results among methods; training data sets limit the applicability domain; relies on correlations with property values only, not on structural features	Linnankoski et al. (2008), Ahmed & Ramakrishnan (2012)
Quantitative Structure-Activity Relationship	Toxicokinetic database that predicts human intestinal absorption based on chemical descriptors or fingerprints in 1-, 2-, and 3-dimensions; computes from patterns of chemical fragments or substructural features; uses multiple algorithmic techniques and decision-tree analysis	Training data sets limit the applicability domain; quality of training sets vary; different validation methods lead to different predictive performance characteristics	Suenderhauf et al. (2011), Moda & Andricopulo (2012), Winkler et al. (2012)

Figure 1. Control treatments for in vivo, ex vivo and in vitro nanomaterial digestion and absorption assays. (A) Background levels of the nanomaterial in the model should be measured in a control well receiving the vehicle components. (B) The controls are compared with the nanomaterial treatment group. (C) If a fraction of the nanomaterial surface dissolved in simulated gastrointestinal fluids, that concentration of ions can be administered alone, to determine if the ions are responsible for any absorption. (D) If the label dissociated from the nanomaterials in simulated gastrointestinal fluids, that concentration of label can be administered alone, to control for false positives from its absorption.

Figure 2. Categories of gastrointestinal tract models that demonstrate potential utility for the assessment of the digestion and absorption of engineered nanomaterials released from food. Specific examples are provided of models that have been used, or could be further developed, to quantify digestion and absorption parameters relevant to nanomaterials. The general parameters that the models could measure are listed.

Table 6. Gaps and proposed research strategies for models of the digestion and absorption of engineered nanomaterials in the gastrointestinal tract.

Identified gap	Approaches to address gaps
Overarching strategy required to assess uptake of NMs via the gastrointestinal tract	• A tiered strategy: (i) thorough physical and chemical characterization in the matrix as dosed, taking into account NM changes over time, (ii) high-throughput toxicokinetic approaches such as human-relevant in vitro or in silico systems, (iii) further testing in vivo.
Require validated and standardized models and analytical tools for the detection of NMs in food, complex media, chyme and feces, as well as within cells and tissues of the GI tract	• Assess reliability of analytical digestion and detection for routine use (e.g. Szakal et al., 2014b). • Radiolabeled materials have been used more reluctantly used in recent years, but these may need to be applied to answer scientific uptake questions.
Require validation and standardization of GI models - general comments	• Adapt test models used for conventional drugs.
Require assessment of the appropriateness of in vivo models	• Assess and modify if necessary the current OECD 2010 guidelines for toxicokinetics studies. • Assess relevance of administration via gavage versus introduction into food or water. • Either confirm relevance of existing techniques (e.g. neutron activation, radioactivity or fluorescent tags) or develop improved (or simpler) approaches to better quantify uptake of NM from the gastrointestinal tract into blood over time, and their subsequent biodistribution. • Extend existing and newly improved techniques to a wider array of NMs with different physicochemical properties. • Determine differences between humans and rodent models in terms of uptake and how parameters (e.g. food intake) modify uptake. • Utilize in situ perfusion models in live, anesthetized subjects to assess uptake.
Need to understand factors affecting mucus penetration by NMs	• Assess physicochemical characteristics of NM that influence their interaction with mucus.
Require improved alternative models – cell based systems	• Develop and utilize models to better represent the diversity of cell types in the gastrointestinal mucosa. • Incorporate permeable synthetic membranes into 3D human tissue models. • Determine the relationship between NM concentration in the media, internal dose and toxicity in vitro and relate this to the in vivo uptake. • Evaluate in vitro models compared to ex vivo tissue and animal models, followed by standardization of these novel models to clarify their applicability and accuracy for the prediction of particle uptake rate in humans. • Combine the data from more than one in vitro assay to address limitations of simple in vitro systems. • Simultaneously validate analytical tools with human-relevant in vitro or in silico methods. • Assess the effects of NM agglomeration and sedimentation on ex vivo models such as the gut sac. • NMs administered to test models can be either pristine particles, useful for mechanistic studies, or manipulated to more realistically reflecting the range of particles that might be found in food products.
Simple cell culture systems using established cell lines do not incorporate factors such as age and sex.	• Cell lines with different genotypes could be developed, e.g. via induced pluripotent stem cells.
Simple cell culture systems using established cell lines lack systemic circulation pathways such as the enterohepatic secretion.	• Develop and exploit multi-tissue fluidic model that allows flow.
Standard culture media does not contain GI lumen factors	• More physiologically relevant culture fluids can be used to reflect the fasted and fed states.
Agglomeration and settling vary according to the physicochemical properties of NMs and this then influences the dose rate to cells.	• Mathematical models such as the In Vitro Sedimentation, Diffusion and Dosimetry (ISDD) model can calculate the apparent density of the NMs in the medium and the dose to the cells (Hinderliter et al., 2010; Teeguarden et al., 2007; Watson et al., 2014). • Further research is required to demonstrate whether modifying dose according to the ISDD model alters the conclusions made about the relative toxicity of different NMs. • Research is required to compare the ISDD model to data from animal or human studies. • Research is required to assess whether mass is the best metric for dose, or whether other options (e.g. surface area or particle number) might be more useful.
Require improved alternative models – non-cellular fluid models	• Use fluid models that simulate the human GI tract to evaluate certain aspects of NM digestion including bioaccessibility, stability, dissolution and aggregation. • Static in vitro digestion assays using simulated gastric fluids could be used to distinguish a variety of the possible outcomes for NMs. • Dynamic gastric simulators which recreate physical mixing and emptying conditions at work in the human can be applied to determine their influence on the retention, release, and breakdown of food particles and NM, but requires validation for evaluation of the behavior of NMs in food. • NMs administered to test models can be either pristine particles, useful for mechanistic studies, or manipulated to more realistically reflecting the range of particles that might be found in food products.
Many non-cellular fluid models do not accurately mimic the complex physical forces that are an important factor in the disintegration of food particles in vivo.	• Chewing and swallowing forces encountered in vivo can be modeled using devices with mobile parts.
In vitro fluid systems that mimic the lumen of the buccal cavity	• Use several fluids with different osmotic, amylase and pH values. • Research is required to standardize in vitro fluid system models that mimic the lumen of the buccal cavity for the application of NMs.
Impacts of NM on microflora	• In vivo assess diversity of microorganisms in faeces over time after NM exposure. • In vitro assess impact on commensal and pathogenic microorganisms. • Develop and validate in vitro models of epithelial and microorganism co-culture to investigate how such interactions influence NM uptake.
Modelling of uptake	• Mathematical approaches can be used to approximately equate uptake values to human in situ transcellular permeability. • Characterize not only permeability, but also digestion in GI fluids.
There are a lack of disease models relevant for studying inter-individual variability in NM uptake from the gastrointestinal tract	• Development of models of specific disease states (e.g. inflammatory bowel disease, Crohn’s disease and cancer). Examples of such models include: ° In vivo rodent models induced to develop premalignant lesions and tumors of the buccal cavity, esophagus, stomach, small intestine and colon. ° The rat model of dinitrobenzene sulphonic acid-induced colitis. • Development of models that reflect differences in human digestion and permeability associated with factors such as age and pregnancy. • Include alternative approaches to represent different physiological and diseases states Examples of such models include: ° The in vitro culture of neoplastic buccal cavity keratinocytes (Costea et al., 2005). ° Caco-2 or T84 cells treated with tumor necrosis factor-alpha and interferon-gamma, or infected with enteropathogenic Escherichia coli, to induce barrier dysfunction (Zolotarevsky et al., 2002). ° Caco-2 cells co-cultured with primary macrophages and dendritic cells derived from human blood, and then treated with interleukin 1-beta to induce inflammation (Leonard et al., 2010). • Use of ex vivo tissue biopsies from human volunteers with ulcerative colitis. • Such models would not only identify how disease influences uptake, but could provide beneficial targeting of ingested pharmaceutical or imaging NMs to gastrointestinal disease sites.

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