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A novel, integrated, in vitro gastrointestinal (GI) system is presented to study oral bioavailability parameters of small molecules. Three compartments were combined into one hyphenated, flow-through set-up. In the first compartment, a compound was exposed dynamically to enzymatic digestion in three consecutive microreactors, mimicking the processes of the mouth, stomach, and intestine. The resulting solution (chyme) continued to the second compartment, a flow-through barrier model of the intestinal epithelium allowing absorption of the compound and metabolites thereof. The composition of the effluents from the barrier model were analysed either offline by electrospray-ionisation-mass spectrometry (ESI–MS), or online in the final compartment using chip-based ESI–MS. Two model drugs, omeprazole and verapamil, were used to test the integrated model. Omeprazole was shown to be broken down upon treatment with gastric acid, but reached the cell barrier unharmed when introduced to the system in a manner emulating an enteric-coated formulation. In contrast, verapamil was unaffected by digestion. Finally, a reduced uptake of verapamil was observed when verapamil was introduced to the system dissolved in apple juice, a simple food matrix. It is envisaged that this integrated, compartmentalised GI system has potential for enabling future research in the fields of pharmacology, toxicology, and nutrition.

In chapter 1 of this thesis, an overview of the main applications of NPs was provided and the main properties of NPs were briefly introduced. In addition, some of the key methods that are currently used in the toxicological safety assessment of NPs were presented. The aim of the thesis was introduced which was to investigate the potential of different in vitro methods combined with high-end analytical techniques as a testing strategy to study the toxicokinetic and toxicodynamic properties of silver (Ag) and polystyrene (PS) NPs and set priorities in their further safety testing. The current state of the art of the in vitro methods used in the studies in this thesis and the outline of the thesis were also presented.In chapter 2, the influences of the size and surface chemistry of pristine PSNPs on the protein corona formation and subsequent uptake/association and transport of these NPs through a Caco-2 intestinal cell model were studied. Four negatively charged PSNPs of different sizes (50 and 200 nm) and with different surface chemistries (sulfone or carboxyl groups) were studied. The protein coronas of these PSNPs were analysed by LC-MS/MS which showed subtle differences in the protein composition of especially the two PSNPs with different surface chemistries. In further experiments, the impact of surface chemistry on the cellular uptake/association and transport was characterised using high-content imaging analysis. These experiments demonstrated that the PSNPs with sulfone surface groups were associated with the cells to a significantly higher extent than the PSNPs with carboxyl surface groups. No clear effect of the size of the PSNPs on the cellular uptake/association was noted. Also, the transport of the PSNPs with sulfone surface groups through the monolayer of cells was significantly higher than that of PSNPs with carboxyl surface groups.The general conclusion was that the composition of the protein corona and the PSNPs surface chemistry influence the cellula

The small intestine is a crucial component of the digestive system allowing the digestion of food and absorption of nutrients to supply the body with energy to function properly. The complex anatomy and physiology of the human small intestine poses a challenge when defining alternative testing strategies to characterise intestinal absorption and toxicity. In the present thesis, the potential of an alternative in vitro gut-on-chip model with a continuous liquid flow to study intestinal absorption and toxicity was revealed. A glass-based chip was used to culture epithelial cells under the optimized dynamic conditions. Upon morphologic characterization a selected range of model compounds were used for transport studies including 17 dioxin congeners with different physicochemical properties that are well known food contaminants, and selected pharmaceuticals (i.e. antipyrine, ketoprofen, digoxin, amoxicillin) with well-known uptake profiles from traditional static in vitro models and from human in vivo studies. These studies were followed by the comparative gene expression study of cells cultured in the gut-on-chip and in Transwells and human intestinal tissues. Finally, a comparative gene expression study was performed evaluating the effects on gene expression profiles of a nanomaterial (TiO2 and ZnO) exposure in the gut-on-chip versus the Transwell model.

Currently, static in vitro cell culture assays are used in early phases of drug development, food research and hazard identification of chemicals. However, these in vitro models lack organ specific functionality, hampering mechanism-based research needed for novel drug development and next generation risk assessment. Recent advances in microchip- and bio-engineering enabled the development of organ-on-a-chip models. The main technical advantages of organ-on-a-chip technology are the ability to spatiotemporally control the microenvironment and the low reagent consumption. On the other hand, micro-engineered organ-on-a-chip models may still lack robustness for reproducible and bio-relevant studies. In addition, also the low flow rates present a major challenge of organ-on-a-chip technology: how to detect drug uptake and compound metabolism in real-time and at a high temporal resolution at the microscale.In this thesis a dynamic intestinal cell culture device is coupled to highly advanced mass spectrometry equipment, aiming for automated and online analysis of translocation of drugs, natural toxins and nanoparticles, while maintaining the bio-integrity of the cell system. Apart from technical robustness challenges, the fundamental chemical incompatibility between complex biological systems used in cell culturing and the clean sample requirements of sensitive advanced analytical instrumentation need to be resolved. Furthermore, the dynamic cell culture model is placed outside of an incubator for the integration with the mass spectrometer. However, physiological relevant temperatures and pH levels still need to be regulated for a reliable biological experiment. As well as the evaluation of the barrier integrity of the intestinal cell layer an aspect largely overlooked by current literature.In Chapter 2 an overview is provided of the current knowledge regarding analytical techniques integrated with organ-on-a-chip systems. In Chapter 3a a dynamic flow-through in vitro mod

Microplastics (MPs) are considered an emerging issue as environmental pollutants and a potential health threat. This review will focus on recently published data on concentrations in food, possible effects, and monitoring methods. Some data are available on concentrations in seafood (fish, bivalves, and shrimps), water, sugar, salt, and honey, but are lacking for other foods. Bottled water is a considerable source with numbers varying between 2600 and 6300 MPs per liter. Particle size distributions have revealed an abundance of particles smaller than 25 µm, which are considered to have the highest probability to pass the intestinal border and to enter the systemic circulation of mammals. Some studies with mice and zebrafish with short- or medium-term exposure (up to 42 days) have revealed diverse results with respect to both the type and extent of effects. Most notable modifications have been observed in gut microbiota, lipid metabolism, and oxidative stress. The principal elements of MP monitoring in food are sample preparation, detection, and identification. Identified data gaps include a lack of occurrence data in plant- and animal-derived food, a need for more data on possible effects of different types of microplastics, a lack of in silico models, a lack of harmonized monitoring methods, and a further development of quality assurance.

In oral bioavailability studies, evaluation of the absorption and transport of drugs and food components across the intestinal barrier is crucial. Advances in the field of organ-on-a-chip technology have resulted in a dynamic gut-on-a-chip model that better mimics the in vivo microenvironment of the intestine. Despite a few recent integration attempts, ensuring a biologically relevant microenvironment while coupling with a fully online detection system still represents a major challenge. Herein, we designed an online technique to measure drug permeability and analyse unknown product formation across an intestinal epithelial layer of Caco-2 and HT29-MTX cells cultured on a flow-through Transwell system, while ensuring the quality and relevance of the biological model. Chip-based ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was coupled to the dynamic Transwell system via a series of switching valves, thus allowing alternating measurements of the apical and basolateral sides of the in vitro model. Two trap columns were integrated for online sample pre-treatment and compatibility enhancement. Temporal analysis of the intestinal permeability was successfully demonstrated using verapamil as a model drug and ergotamine epimers as a model for natural toxins present in foods. Evidence was obtained that our newly developed dynamic system provided reliable results versus classical static in vitro models, and moreover, for the first time, epimer-specific transport is shown for ergotamine. Finally, initial experiments with the drug granisetron suggest that metabolic activity can be studied as well, thus highlighting the versatility of the bio-integrated online analysis system developed.

In this study, a Bayesian Network (BN) was developed for the prediction of the hazard potential and biological effects with the focus on metal- and metal-oxide nanomaterials to support human health risk assessment. The developed BN captures the (inter) relationships between the exposure route, the nanomaterials physicochemical properties and the ultimate biological effects in a holistic manner and was based on international expert consultation and the scientific literature (e.g., in vitro/in vivo data). The BN was validated with independent data extracted from published studies and the accuracy of the prediction of the nanomaterials hazard potential was 72% and for the biological effect 71%, respectively. The application of the BN is shown with scenario studies for TiO2, SiO2, Ag, CeO2, ZnO nanomaterials. It is demonstrated that the BN may be used by different stakeholders at several stages in the risk assessment to predict certain properties of a nanomaterials of which little information is available or to prioritize nanomaterials for further screening.

The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are epithelial breast cancer cells with extensive well-characterized toxicogenomics profiles. In the present study we aimed to gain a deeper understanding of the cellular molecular responses in Caco-2 and MCF-7 cells after AgNP exposure in order to evaluate whether epithelial cells derived from different tissues demonstrated similar responses. These insights could possibly reduce the size of cell panels for NP hazard identification screening purposes. AgNPs of 20, 30, 60, and 110 nm, and AgNO3 were exposed for 6h and 24h. AgNPs were shown to be taken up and dissolve intracellularly. Compared with MCF-7 cells, Caco-2 cells showed a higher sensitivity to AgNPs, slower gene expression kinetics, and absence of NP size-dependent responses. However, on a molecular level, no significant differences were observed between the two cell types. Transcriptomic analysis showed that Ag(NP) exposure caused (oxidative) stress responses, possibly leading to cell death in both cell lines. There was no indication for effects specifically induced by AgNPs. Responses to AgNPs appeared to be induced by silver ions released from the AgNPs. In conclusion, differences in mRNA responses to AgNPs between Caco-2 and MCF-7 cells were mainly related to timing and magnitude, but not to a different underlying mechanism.

The increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from singlecell- type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using cocultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testing.

The likelihood of oral exposure to nanoparticles (NPs) is increasing, and it is necessary to evaluate the oral bioavailability of NPs. In vitro approaches could help reducing animal studies, but validation against in vivo studies is essential. Previously, we assessed the translocation of 50 nm polystyrene NPs of different charges (neutral, positive and negative) using a Caco-2/HT29-MTX in vitro intestinal translocation model. The NPs translocated in a surface charge-dependent manner. The present study aimed to validate this in vitro intestinal model by an in vivo study. For this, rats were orally exposed to a single dose of these polystyrene NPs and the uptake in organs was determined. A negatively charged NP was taken up more than other NPs, with the highest amounts in kidney (37.4 µg/g tissue), heart (52.8 µg/g tissue), stomach wall (98.3 µg/g tissue) and small intestinal wall (94.4 µg/g tissue). This partly confirms our in vitro findings, where the same NPs translocated to the highest extent. The estimated bioavailability of different types of NPs ranged from 0.2 to 1.7 % in vivo, which was much lower than in vitro (1.6–12.3 %). Therefore, the integrated in vitro model cannot be used for a direct prediction of the bioavailability of orally administered NPs. However, the model can be used for prioritizing NPs before further in vivo testing for risk assessment.

Synthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range. Food products containing SAS have been shown to contain silica in the nanometer size range (i.e. 5 – 200 nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica.

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Item does not contain fulltext, Amongst the engineered nanomaterials, especially carbon nanotubes (CNTs) have received considerable attention for application in tissue engineering scaffolds. CNTs are considered promising on behalf of their physicochemical properties, yet such nanomaterials also have been associated with potentially hazardous effects on human health. To gain insight into the toxicity aspects of CNTs in vivo, the present study presents a systematic review of literature. After screening of literature through defined inclusion and exclusion criteria, and subsequent data extraction, it can be concluded that pulmonary administered CNTs have the capacity to induce toxicity in the lung area. However, conclusions for other organs, or on systemic toxicity, are yet premature. In addition, the carcinogenic potential of CNTs is also still ambiguous, because contradictive results are presented. Intrinsic factors, such as material characteristics, and associated distribution and agglomeration patterns influence the toxic potential of CNTs. Similarly, environmental factors such as the exposure route, preexisting allergies, pathological infections, or air pollutant exposure are significant. Despite the many reports published currently, more studies will be required to gain full understanding of the toxic potential of CNTs and especially the underlying mechanisms. For this end, development of standardized protocols and reliable nanodetection techniques will form prerequisites.

Radboud Universiteit Nijmegen, 31 maart 2011, Promotor : Jansen, J.A. Co-promotor : Walboomers, X.F., Contains fulltext : 85860.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, There is an evident clinical need for artificial bone restorative materials. In this respect, novel composites based on poly(L-lactic acid) (PLLA) have been described. The bone response of such polymer-based composites is usually improved by the addition of bone morphogenetic protein-2 (BMP-2). However, released BMP-2 is cleared almost immediately from the site of implantation by diffusion, whereas a prolonged retention of BMP-2 onto the scaffold has been suggested to be more favourable. Besides the ability to improve the mechanical strength and osteoconductivity of polymeric scaffolds, both carbon nanotubes (CNTs) and microhydroxyapatite (microHA) have been described to facilitate such retention of BMP-2 when incorporated into a composite scaffold. Therefore, in the current study, radiolabelled BMP-2 was loaded onto plain PLLA and composite PLLA-CNT-microHA scaffolds. Subsequently, the scaffolds were implanted subcutaneously for 5 weeks in rats and BMP-2 release was measured. Release started with an initial phase of quick release, followed by a gradual release of BMP-2. Both scaffold types comprised the same in vivo release properties for BMP-2. The bioactivity of the BMP-2 remained unaltered. It can be concluded that incorporated CNTs and microHA did not affect BMP-2 release from composite scaffold materials.