The in vitro gastrointestinal digestion-associated protein corona of polystyrene nano- and microplastics increases their uptake by human THP-1-derived macrophages.

BACKGROUND: Micro- and nanoplastics (MNPs) represent one of the most widespread environmental pollutants of the twenty-first century to which all humans are orally exposed. Upon ingestion, MNPs pass harsh biochemical conditions within the gastrointestinal tract, causing a unique protein corona on the MNP surface. Little is known about the digestion-associated protein corona and its impact on the cellular uptake of MNPs. Here, we systematically studied the influence of gastrointestinal digestion on the cellular uptake of neutral and charged polystyrene MNPs using THP-1-derived macrophages. RESULTS: The protein corona composition was quantified using LC‒MS-MS-based proteomics, and the cellular uptake of MNPs was determined using flow cytometry and confocal microscopy. Gastrointestinal digestion resulted in a distinct protein corona on MNPs that was retained in serum-containing cell culture medium. Digestion increased the uptake of uncharged MNPs below 500 nm by 4.0-6.1-fold but did not affect the uptake of larger sized or charged MNPs. Forty proteins showed a good correlation between protein abundance and MNP uptake, including coagulation factors, apolipoproteins and vitronectin. CONCLUSION: This study provides quantitative data on the presence of gastrointestinal proteins on MNPs and relates this to cellular uptake, underpinning the need to include the protein corona in hazard assessment of MNPs.

Impact of nanoparticle surface functionalization on the protein corona and cellular adhesion, uptake and transport.

BACKGROUND: Upon ingestion, nanoparticles can interact with the intestinal epithelial barrier potentially resulting in systemic uptake of nanoparticles. Nanoparticle properties have been described to influence the protein corona formation and subsequent cellular adhesion, uptake and transport. Here, we aimed to study the effects of nanoparticle size and surface chemistry on the protein corona formation and subsequent cellular adhesion, uptake and transport. Caco-2 intestinal cells, were exposed to negatively charged polystyrene nanoparticles (PSNPs) (50 and 200 nm), functionalized with sulfone or carboxyl groups, at nine nominal concentrations (15-250 µg/ml) for 10 up to 120 min. The protein coronas were analysed by LC-MS/MS. RESULTS: Subtle differences in the protein composition of the two PSNPs with different surface chemistry were noted. High-content imaging analysis demonstrated that sulfone PSNPs were associated with the cells to a significantly higher extent than the other PSNPs. The apparent cellular adhesion and uptake of 200 nm PSNPs was not significantly increased compared to 50 nm PSNPs with the same surface charge and chemistry. Surface chemistry outweighs the impact of size on the observed PSNP cellular associations. Also transport of the sulfone PSNPs through the monolayer of cells was significantly higher than that of carboxyl PSNPs. CONCLUSIONS: The results suggest that the composition of the protein corona and the PSNP surface chemistry influences cellular adhesion, uptake and monolayer transport, which might be predictive of the intestinal transport potency of NPs.

Potential Health Impact of Environmentally Released Micro- and Nanoplastics in the Human Food Production Chain: Experiences from Nanotoxicology.

High concentrations of plastic debris have been observed in the oceans. Much of the recent concern has focused on microplastics in the marine environment. Recent studies of the size distribution of the plastic debris suggested that continued fragmenting of microplastics into nanosized particles may occur. In this review we assess the current literature on the occurrence of environmentally released micro- and nanoplastics in the human food production chain and their potential health impact. The currently used analytical techniques introduce a great bias in the knowledge, since they are only able to detect plastic particles well above the nanorange. We discuss the potential use of the very sensitive analytical techniques that have been developed for the detection and quantification of engineered nanoparticles. We recognize three possible toxic effects of plastic particles: first due to the plastic particles themselves, second to the release of persistent organic pollutant adsorbed to the plastics, and third to the leaching of additives of the plastics. The limited data on microplastics in foods do not predict adverse effect of these pollutants or additives. Potential toxic effects of microplastic particles will be confined to the gut. The potential human toxicity of nanoplastics is poorly studied. Based on our experiences in nanotoxicology we prioritized future research questions.

A versatile, compartmentalised gut-on-a-chip system for pharmacological and toxicological analyses.

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.

Digestion-on-a-chip: a continuous-flow modular microsystem recreating enzymatic digestion in the gastrointestinal tract.

In vitro digestions are essential for determining the bioavailability of compounds, such as nutrients. We have developed a cell-free, miniaturized enzymatic digestive system, employing three micromixers connected in series to mimic the digestive functions of the mouth, stomach and small intestine. This system continuously processes samples, e.g. containing nutrients, to provide a constant flow of digested materials which may be presented to a subsequent gut-on-a-chip absorption module, containing living human intestinal cells. Our system incorporates three-compartment enzymatic digestion, one of the key functions of the gastrointestinal tract. In each of these compartments, we modify the chemical environment, including pH, buffer, and mineral composition, to closely mimic the local physiological environment and create optimal conditions for digestive processes to take place. It will therefore provide an excellent addition to existing gut-on-a-chip systems, providing the next step in determining the bio-availability of orally administered compounds in a fast and continuous-flow ex vivo system. In this paper, we demonstrate enzymatic digestion in each separate compartment using compounds, starch and casein, as model nutrients. The use of transparent, microfluidic micromixers based on chaotic advection, which can be probed directly with a microscope, enabled enzyme kinetics to be monitored from the very start of a reaction. Furthermore, we have digested lactoferrin in our system, demonstrating complete digestion of this milk protein in much shorter times than achievable with standard in vitro digestions using batch reactors.

Effects of food-borne nanomaterials on gastrointestinal tissues and microbiota.

Ingestion of engineered nanomaterials is inevitable due to their addition to food and prevalence in food packaging and domestic products such as toothpaste and sun cream. In the absence of robust dosimetry and particokinetic data, it is currently challenging to accurately assess the potential toxicity of food-borne nanomaterials. Herein, we review current understanding of gastrointestinal uptake mechanisms, consider some data on the potential for toxicity of the most commonly encountered classes of food-borne nanomaterials (including TiO2 , SiO2, ZnO, and Ag nanoparticles), and discuss the potential impact of the luminal environment on nanoparticle properties and toxicity. Much of our current understanding of gastrointestinal nanotoxicology is derived from increasingly sophisticated epithelial models that augment in vivo studies. In addition to considering the direct effects of food-borne nanomaterials on gastrointestinal tissues, including the potential role of chronic nanoparticle exposure in development of inflammatory diseases, we also discuss the potential for food-borne nanomaterials to disturb the normal balance of microbiota within the gastrointestinal tract. The latter possibility warrants close attention given the increasing awareness of the critical role of microbiota in human health and the known impact of some food-borne nanomaterials on bacterial viability. WIREs Nanomed Nanobiotechnol 2018, 10:e1481. doi: 10.1002/wnan.1481 This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Surface PEG Grafting Density Determines Magnetic Relaxation Properties of Gd-Loaded Porous Nanoparticles for MR Imaging Applications.

Surface PEGylation of nanoparticles designed for biomedical applications is a common and straightforward way to stabilize the materials for in vivo administration and to increase their circulation time. This strategy becomes less trivial when MRI active porous nanomaterials are concerned as their function relies on water/proton-exchange between the pores and bulk water. Here we present a comprehensive study on the effects of PEGylation on the relaxometric properties of nanozeolite LTL (dimensions of 20 × 40 nm) ion-exchanged with paramagnetic GdIII ions. We evidence that as long as the surface grafting density of the PEG chains does not exceed the "mushroom" regime (conjugation of up to 6.2 wt % of PEG), Gd-LTL retains a remarkable longitudinal relaxivity (38 s-1 mM-1 at 7 T and 25 °C) as well as the pH-dependence of the longitudinal and transverse relaxation times. At higher PEG content, the more compact PEG layer (brush regime) limits proton/water diffusion and exchange between the interior of LTL and the bulk, with detrimental consequences on relaxivity. Furthermore, PEGylation of Gd-LTL dramatically decreases the leakage of toxic GdIII ions in biological media and in the presence of competing anions, which together with minimal cytotoxicity renders these materials promising probes for MRI applications.

Translocation of positively and negatively charged polystyrene nanoparticles in an in vitro placental model.

To obtain insight in translocation of nanoparticles across the placental barrier, translocation was studied for one positively and two negatively charged polystyrene nanoparticles (PS-NPs) of similar size in an in vitro model. The model consisted of BeWo b30 cells, derived from a human choriocarcinoma grown on a transwell insert forming a cell layer that separates an apical from a basolateral compartment. PS-NPs were characterized with respect to size, surface charge, morphology and protein corona. Translocation of PS-NPs was not related to PS-NP charge. Two PS-NPs were translocated across the BeWo transwell model to a lower extent than amoxicillin, a model compound known to be translocated over the placental barrier to only a limited extent, whereas one PS-NP showed a slightly higher translocation. Studies on the effect of transporter inhibitors on the translocation of the PS-NPs indicated that their translocation was not mediated by known transporters and mainly dependent on passive diffusion. It is concluded that the BeWo b30 model can be used as an efficient method to get an initial qualitative impression about the capacity of NPs to translocate across the placental barrier and set priorities in further in vivo studies on translocation of NPs to the fetus.

In vitro gastrointestinal digestion increases the translocation of polystyrene nanoparticles in an in vitro intestinal co-culture model.

The conditions of the gastrointestinal tract may change the physicochemical properties of nanoparticles (NPs) and therewith the bioavailability of orally taken NPs. Therefore, we assessed the impact of in vitro gastrointestinal digestion on the protein corona of polystyrene NPs (PS-NPs) and their subsequent translocation across an in vitro intestinal barrier. A co-culture of intestinal Caco-2 and HT29-MTX cells was exposed to 50 nm PS-NPs of different charges (positive and negative) in two forms: pristine and digested in an in vitro gastrointestinal digestion model. In vitro digestion significantly increased the translocation of all, except the "neutral", PS-NPs. Upon in vitro digestion, translocation was 4-fold higher for positively charged NPs and 80- and 1.7-fold higher for two types of negatively charged NPs. Digestion significantly reduced the amount of protein in the corona of three out of four types of NPs. This reduction of proteins was 4.8-fold for "neutral", 3.5-fold for positively charged and 1.8-fold for one type of negatively charged PS-NPs. In vitro digestion also affected the composition of the protein corona of PS-NPs by decreasing the presence of higher molecular weight proteins and shifting the protein content of the corona to low molecular weight proteins. These findings are the first to report that in vitro gastrointestinal digestion significantly affects the protein corona and significantly increases the in vitro translocation of differently charged PS-NPs. These findings stress the importance of including the in vitro digestion in future in vitro intestinal translocation screening studies for risk assessment of orally taken NPs.

Translocation of differently sized and charged polystyrene nanoparticles in in vitro intestinal cell models of increasing complexity.

Intestinal translocation is a key factor for determining bioavailability of nanoparticles (NPs) after oral uptake. Therefore, we evaluated three in vitro intestinal cell models of increasing complexity which might affect the translocation of NPs: a mono-culture (Caco-2 cells), a co-culture with mucus secreting HT29-MTX cells and a tri-culture with M-cells. Cell models were exposed to well characterized differently sized (50 and 100 nm) and charged (neutral, positively and negatively) polystyrene NPs. In addition, two types of negatively charged NPs with different surface chemistries were used. Size strongly affected the translocation of NPs, ranging up to 7.8% for the 50 nm NPs and 0.8% for the 100 nm NPs. Surface charge of NPs affected the translocation, however, surface chemistry seems more important, as the two types of negatively charged 50 nm NPs had an over 30-fold difference in translocation. Compared with the Caco-2 mono-culture, presence of mucus significantly reduced the translocation of neutral 50 nm NPs, but significantly increased the translocation of one type of negatively charged NPs. Incorporation of M-cells shifted the translocation rates for both NPs closer to those in the mono-culture model. The relative pattern of NP translocation in all three models was similar, but the absolute amounts of translocated NPs differed per model. We conclude that for comparing the relative translocation of different NPs, using one intestinal model is sufficient. To choose the most representative model for risk assessment, in vivo experiments are now needed to determine the in vivo translocation rates of the used NPs.

Behaviour of silver nanoparticles and silver ions in an in vitro human gastrointestinal digestion model.

Oral ingestion is an important exposure route for silver nanoparticles (AgNPs), but their fate during gastrointestinal digestion is unknown. This was studied for 60 nm AgNPs and silver ions (AgNO3) using in vitro human digestion model. Samples after saliva, gastric and intestinal digestion were analysed with SP-ICPMS, DLS and SEM-EDX. In presence of proteins, after gastric digestion the number of particles dropped significantly, to rise back to original values after the intestinal digestion. SEM-EDX revealed that reduction in number of particles was caused by their clustering. These clusters were composed of AgNPs and chlorine. During intestinal digestion, these clusters disintegrated back into single 60 nm AgNPs. The authors conclude that these AgNPs under physiological conditions can reach the intestinal wall in their initial size and composition. Importantly, intestinal digestion of AgNO3 in presence of proteins resulted in particle formation. These nanoparticles (of 20-30 nm) were composed of silver, sulphur and chlorine.

Presence of nano-sized silica during in vitro digestion of foods containing silica as a food additive.

The presence, dissolution, agglomeration state, and release of materials in the nano-size range from food containing engineered nanoparticles during human digestion is a key question for the safety assessment of these materials. We used an in vitro model to mimic the human digestion. Food products subjected to in vitro digestion included (i) hot water, (ii) coffee with powdered creamer, (iii) instant soup, and (iv) pancake which either contained silica as the food additive E551, or to which a form of synthetic amorphous silica or 32 nm SiO(2) particles were added. The results showed that, in the mouth stage of the digestion, nano-sized silica particles with a size range of 5-50 and 50-500 nm were present in food products containing E551 or added synthetic amorphous silica. However, during the successive gastric digestion stage, this nano-sized silica was no longer present for the food matrices coffee and instant soup, while low amounts were found for pancakes. Additional experiments showed that the absence of nano-sized silica in the gastric stage can be contributed to an effect of low pH combined with high electrolyte concentrations in the gastric digestion stage. Large silica agglomerates are formed under these conditions as determined by DLS and SEM experiments and explained theoretically by the extended DLVO theory. Importantly, in the subsequent intestinal digestion stage, the nano-sized silica particles reappeared again, even in amounts higher than in the saliva (mouth) digestion stage. These findings suggest that, upon consumption of foods containing E551, the gut epithelium is most likely exposed to nano-sized silica.

A review of analytical methods for the identification and characterization of nano delivery systems in food.

Detection and characterization of nano delivery systems is an essential part of understanding the benefits as well as the potential toxicity of these systems in food. This review gives a detailed description of food nano delivery systems based on lipids, proteins, and/or polysaccharides and investigates the current analytical techniques that can be used for the identification and characterization of these delivery systems in food products. The analytical approaches have been subdivided into three groups; separation techniques, imaging techniques, and characterization techniques. The principles of the techniques together with their advantages and drawbacks, and reported applications concerning nano delivery systems, or otherwise related compounds are discussed. The review shows that for a sufficient characterization, the nano delivery systems need to be separated from the food matrix, for which high-performance liquid chromatography or field flow fractionation are the most promising techniques. Subsequently, online photon correlation spectroscopy and mass spectrometry seem to be a convenient combination of techniques to characterize a wide variety of nano delivery systems.