On the Structure, Stability, and Cell Uptake of Nanostructured Lipid Carriers for Drug Delivery.

The efficacy of nanostructured lipid carriers (NLC) for drug delivery strongly depends on their stability and cell uptake. Both properties are governed by their compositions and internal structure. To test the effect of the lipid composition of NLC on cell uptake and stability, three kinds of liquid lipids with different degrees of unsaturation are employed. After ensuring homogeneous size distributions, the thermodynamic characteristics, stability, and mixing properties of NLC are characterized. Then the rates and predominant pathways of cell uptake are determined. Although the same surfactant is used in all cases, different uptake rates are observed. This finding contradicts the view that the surface properties of NLC are dominated by the surfactant. Instead, the uptake rates are explained by the structure of the nanocarrier. Depending on the mixing properties, some liquid lipids remain inside the nanocarrier, while other liquid lipids are present on the surface. Nanocarriers with liquid lipids on the surface are taken up more readily by the cells. This shows that the engineering of efficient lipid nanocarriers requires a delicate balance of interactions between all components of the nanocarrier on the molecular level.

Animals in Respiratory Research.

The respiratory barrier, a thin epithelial barrier that separates the interior of the human body from the environment, is easily damaged by toxicants, and chronic respiratory diseases are common. It also allows the permeation of drugs for topical treatment. Animal experimentation is used to train medical technicians, evaluate toxicants, and develop inhaled formulations. Species differences in the architecture of the respiratory tract explain why some species are better at predicting human toxicity than others. Some species are useful as disease models. This review describes the anatomical differences between the human and mammalian lungs and lists the characteristics of currently used mammalian models for the most relevant chronic respiratory diseases (asthma, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, and tuberculosis). The generation of animal models is not easy because they do not develop these diseases spontaneously. Mouse models are common, but other species are more appropriate for some diseases. Zebrafish and fruit flies can help study immunological aspects. It is expected that combinations of in silico, in vitro, and in vivo (mammalian and invertebrate) models will be used in the future for drug development.

Development and Characterization of an In Vitro Intestinal Model Including Extracellular Matrix and Macrovascular Endothelium.

In vitro intestinal models are used to study biological processes, drug and food absorption, or cytotoxicity, minimizing the use of animals in the laboratory. They usually consist of enterocytes and mucus-producing cells cultured for 3 weeks, e.g., on Transwells, to obtain a fully differentiated cell layer simulating the human epithelium. Other important components are the extracellular matrix (ECM) and strong vascularization. The former serves as structural support for cells and promotes cellular processes such as differentiation, migration, and growth. The latter includes endothelial cells, which coordinate vascularization and immune cell migration and facilitate the transport of ingested substances or drugs to the liver. In most cases, animal-derived hydrogels such as Matrigel or collagen are used as ECM in in vitro intestinal models, and endothelial cells are only partially considered, if at all. However, it is well-known that animal-derived products can lead to altered cell behavior and incorrect results. To circumvent these limitations, synthetic and modifiable hydrogels (Peptigel and Vitrogel) were studied here to mimic xenofree ECM, and the data were compared with Matrigel. Careful rheological characterization was performed, and the effect on cell proliferation was investigated. The results showed that Vitrogel exhibited shear-thinning behavior with an internal structure recovery of 78.9 ± 11.2%, providing the best properties among the gels investigated. Therefore, a coculture of Caco-2 and HT29-MTX cells (ratio 7:3) was grown on Vitrogel, while simultaneously endothelial cells were cultured on the basolateral side by inverse cultivation. The model was characterized in terms of cell proliferation, differentiation, and drug permeability. It was found that the cells cultured on Vitrogel induced a 1.7-fold increase in cell proliferation and facilitated the formation of microvilli and tight junctions after 2 weeks of cultivation. At the same time, the coculture showed full differentiation indicated by high alkaline phosphatase release of Caco-2 cells (95.0 ± 15.9%) and a mucus layer produced by HT29-MTX cells. Drug tests led to ex vivo comparable permeability coefficients (Papp) (i.e., Papp; antipyrine = (33.64 ± 5.13) × 10-6 cm/s, Papp; atenolol = (0.59 ± 0.16) × 10-6 cm/s). These results indicate that the newly developed intestinal model can be used for rapid and efficient assessment of drug permeability, excluding unexpected results due to animal-derived materials.

The Variety of 3D Breast Cancer Models for the Study of Tumor Physiology and Drug Screening.

Breast cancer is the most common cancer in women and responsible for multiple deaths worldwide. 3D cancer models enable a better representation of tumor physiology than the conventional 2D cultures. This review summarizes the important components of physiologically relevant 3D models and describes the spectrum of 3D breast cancer models, e.g., spheroids, organoids, breast cancer on a chip and bioprinted tissues. The generation of spheroids is relatively standardized and easy to perform. Microfluidic systems allow control over the environment and the inclusion of sensors and can be combined with spheroids or bioprinted models. The strength of bioprinting relies on the spatial control of the cells and the modulation of the extracellular matrix. Except for the predominant use of breast cancer cell lines, the models differ in stromal cell composition, matrices and fluid flow. Organoids are most appropriate for personalized treatment, but all technologies can mimic most aspects of breast cancer physiology. Fetal bovine serum as a culture supplement and Matrigel as a scaffold limit the reproducibility and standardization of the listed 3D models. The integration of adipocytes is needed because they possess an important role in breast cancer.

Pars Distalis and Pars Tuberalis Thyroid-Stimulating Hormones and Their Roles in Macro-Thyroid-Stimulating Hormone Formation.

Thyroid-stimulating hormone (TSH) and thyroid hormone levels are standard parameters in blood analysis. However, the immunoassays employed may lead to false-positive or false-negative results when the sample contains certain materials that interfere with the assay. Macro-TSH, a complex of TSH with immunoglobulin or albumin, may cause apparently increased TSH concentrations. TSH is produced in the pars tuberalis (PT) of the pituitary gland and by thyrotrophs of the pars distalis (PD). It was found that variable glycosylation can render the molecule more strongly bound to antibodies or albumin in the blood, leading to the hypothesis that macro-TSH consists mainly of PT-TSH. Although less known than PD-TSH, PT-TSH plays an important role in the central regulation of thyroid metabolism. The present review summarizes the physiological function of human PT-TSH and its role in macro-TSH formation. The prevalence of macro-hyperthyrotropinemia, the structure of PT-TSH and macro-TSH, problems in the measurement of TSH, and the action of PT-TSH in animals with seasonal breeding are discussed. Despite the absence of a specific function of macro-TSH in the organism, the identification of macro-TSH is important for avoiding unnecessary treatment based on a falsified readout of increased TSH concentrations as numerous individual case reports describe.

Effect of differently coated silver nanoparticles on hemostasis.

With the emergence of nano-enabled medical devices (MDs) for the use in human medicine, ensuring their safety becomes of crucial importance. Hemocompatibility is one of the major criteria for approval of all MDs in contact with blood (e.g. vascular grafts, stents, or valves). Silver nanoparticles (AgNPs) are among the most used nanomaterials for MDs due to their biocidal activity; however, detailed knowledge on their hemostatic effects is still lacking.This study aimed to evaluate comprehensively AgNPs effects on hemostasis in human blood by exploiting combination of affordable and clinically relevant techniques.Differently stabilized AgNPs were prepared using sodium bis(2-ethylhexyl)sulphosuccinate (AOT), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL), and bovine serum albumin (BSA) as coating agents. They were tested for hemolytic activity, induction of platelet aggregation, plasmatic coagulation, thrombin generation, and hemostasis in whole blood.All AgNPs were found to cause dose-dependent hemolysis. The BSA-, AOT-, and PVP-coated AgNPs delayed plasmatic coagulation, while only PLL-AgNPs inhibited plasmatic coagulation, induced platelet activation, and interfered with hemostasis by delaying clotting time and decreasing clot firmness in whole blood.Obtained results demonstrate that a combination of different techniques should be used for reliable assessment of AgNPs hemostatic effects highlighting the need for a standardized approach in sampling and experimental protocols.

The forgotten effects of thyrotropin-releasing hormone: Metabolic functions and medical applications.

Thyrotropin-releasing hormone (TRH) causes a variety of thyroidal and non-thyroidal effects, the best known being the feedback regulation of thyroid hormone levels. This was employed in the TRH stimulation test, which is currently little used. The role of TRH as a cancer biomarker is minor, but exaggerated responses to TSH and prolactin levels in breast cancer led to the hypothesis of a potential role for TRH in the pathogenesis of this disease. TRH is a rapidly degraded peptide with multiple targets, limiting its suitability as a biomarker and drug candidate. Although some studies reported efficacy in neural diseases (depression, spinal cord injury, amyotrophic lateral sclerosis, etc.), therapeutic use of TRH is presently restricted to spinocerebellar degenerative disease. Regulation of TRH production in the hypothalamus, patterns of expression of TRH and its receptor in the body, its role in energy metabolism and in prolactin secretion are addressed in this review.

In vitro toxicity screening of polyglycerol esters of fatty acids as excipients for pulmonary formulations.

One of the main problems for the development of pulmonary formulations is the low availability of approved excipients. Polyglycerol esters of fatty acids (PGFA) are promising molecules for acting as excipient for formulation development and drug delivery to the lung. However, their biocompatibility in the deep lung has not been studied so far. Main exposed cells include alveolar epithelial cells and alveolar macrophages. Due to the poor water-solubility of PGFAs, the exposure of alveolar macrophages is expected to be much higher than that of epithelial cells. In this study, two PGFAs and their mixture were tested regarding cytotoxicity to epithelial cells and cytotoxicity and functional impairment of macrophages. Cytotoxicity was assessed by dehydrogenase activity and lactate dehydrogenase release. Lysosome function, phospholipid accumulation, phagocytosis, nitric oxide production, and cytokine release were used to evaluate macrophage function. Cytotoxicity was increased with the increased polarity of PGFA molecules. At concentrations above 1 mg/ml accumulation in lysosomes, impairment of phagocytosis, secretion of nitric oxide, and increased release of cytokines were noted. The investigated PGFAs in concentrations up to 1 mg/ml can be considered as uncritical and are promising for advanced pulmonary delivery of high powder doses and drug targeting to alveolar macrophages.

Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs.

Phospholipidosis (PLD), the intracellular accumulation of phospholipids, is an adaptive response to toxic stimuli and serves as an important parameter in the biological assessment of compounds. Cationic amphiphilic drugs are the main inducers of PLD and may impair the function of alveolar macrophages. In vivo and in vitro models are used for PLD screening but the choice of the cellular model may be important because PLD develops in a cell- and species-specific manner. In this study, a panel of different staining (LysoSensor, Acridine Orange, Nile Red, HCS LipidTOX, LysoID) was evaluated in murine (DMBM-2, J774, RAW264.7) and human (THP-1, monocyte-derived macrophages from peripheral blood) cells to identify the most sensitive and easy to analyze staining method and to detect species-specific differences in the reaction pattern. Amiodarone and chloroquine served as inducers of PLD. High content screening was used to compare number, area, and intensity of the staining. Due to the fast staining protocol and the sensitivity of the detection, LysoID proved to be the most suitable dye of the testing. The lower induction of PLD by chloroquine reported in vivo was also seen in this study. THP-1 macrophages, followed by DMBM-2 cells, produced the most similar reaction pattern to human monocyte-derived macrophages.

Prazosin induced lysosomal tubulation interferes with cytokinesis and the endocytic sorting of the tumour antigen CD98hc.

The quinazoline based drug prazosin (PRZ) is a potent inducer of apoptosis in human cancer cells. We recently reported that PRZ enters cells via endocytosis and induces tubulation of the endolysosomal system. In a proteomics approach aimed at identifying potential membrane proteins with binding affinity to quinazolines, we detected the oncoprotein CD98hc. We confirmed shuttling of CD98hc towards lysosomes and upregulation of CD98hc expression in PRZ treated cells. Gene knockout (KO) experiments revealed that endocytosis of PRZ still occurs in the absence of CD98hc - suggesting that PRZ does not enter the cell via CD98hc but misroutes the protein towards tubular lysosomes. Lysosomal tubulation interfered with completion of cytokinesis and provoked endoreplication. CD98hc KO cells showed reduced endoreplication capacity and lower sensitivity towards PRZ induced apoptosis than wild type cells. Thus, loss of CD98hc does not affect endocytosis of PRZ and lysosomal tubulation, but the ability for endoreplication and survival of cells. Furthermore, we found that glutamine, lysomototropic agents - namely chloroquine and NH4Cl - as well as inhibition of v-ATPase, interfere with the intracellular transport of CD98hc. In summary, our study further emphasizes lysosomes as target organelles to inhibit proliferation and to induce cell death in cancer. Most importantly, we demonstrate for the first time that the intracellular trafficking of CD98hc can be modulated by small molecules. Since CD98hc is considered as a potential drug target in several types of human malignancies, our study possesses translational significance suggesting, that old drugs are able to act on a novel target.

The effect of saliva on the fate of nanoparticles.

OBJECTIVES: The design of nanocarriers for local drug administration to the lining mucosa requires a sound knowledge of how nanoparticles (NPs) interact with saliva. This contact determines whether NPs agglomerate and become immobile due to size- and interaction-filtering effects or adsorb on the cell surface and are internalized by epithelial cells. The aim of this study was to examine the behavior of NPs in saliva considering physicochemical NP properties. MATERIALS AND METHODS: The salivary pore-size distribution was determined, and the viscosity of the fluid inside of the pores was studied with optical tweezers. Distinct functionalized NPs (20 and 200 nm) were dispersed in saliva and salivary buffers and characterized, and surface-bound MUC5B and MUC7 were analyzed by 1D electrophoresis and immunoblotting. NP mobility was recorded, and cellular uptake studies were performed with TR146 cells. RESULTS: The mode diameter of the salivary mesh pores is 0.7 µm with a peak width of 1.9 µm, and pores are filled with a low-viscosity fluid. The physicochemical properties of the NPs affected the colloidal stability and mobility: compared with non-functionalized particles, which did not agglomerate and showed a cellular uptake rate of 2.8%, functionalized particles were immobilized, which was correlated with agglomeration and increased binding to mucins. CONCLUSION: The present study showed that the salivary microstructure facilitates NP adsorption. However, NP size and surface functionalization determine the colloidal stability and cellular interactions. CLINICAL RELEVANCE: The sound knowledge of NP interactions with saliva enables the improvement of current treatment strategies for inflammatory oral diseases.

Diverse action of lipoteichoic acid and lipopolysaccharide on neuroinflammation, blood-brain barrier disruption, and anxiety in mice.

Microbial metabolites are known to affect immune system, brain, and behavior via activation of pattern recognition receptors such as Toll-like receptor 4 (TLR4). Unlike the effect of the TLR4 agonist lipopolysaccharide (LPS), the role of other TLR agonists in immune-brain communication is insufficiently understood. We therefore hypothesized that the TLR2 agonist lipoteichoic acid (LTA) causes immune activation in the periphery and brain, stimulates the hypothalamic-pituitary-adrenal (HPA) axis and has an adverse effect on blood-brain barrier (BBB) and emotional behavior. Since LTA preparations may be contaminated by LPS, an extract of LTA (LTAextract), purified LTA (LTApure), and pure LPS (LPSultrapure) were compared with each other in their effects on molecular and behavioral parameters 3h after intraperitoneal (i.p.) injection to male C57BL/6N mice. The LTAextract (20mg/kg) induced anxiety-related behavior in the open field test, enhanced the circulating levels of particular cytokines and the cerebral expression of cytokine mRNA, and blunted the cerebral expression of tight junction protein mRNA. A dose of LPSultrapure matching the amount of endotoxin/LPS contaminating the LTAextract reproduced several of the molecular and behavioral effects of LTAextract. LTApure (20mg/kg) increased plasma levels of tumor necrosis factor-α (TNF-α), interleukin-6 and interferon-γ, and enhanced the transcription of TNF-α, interleukin-1ß and other cytokines in the amygdala and prefrontal cortex. These neuroinflammatory effects of LTApure were associated with transcriptional down-regulation of tight junction-associated proteins (claudin 5, occludin) in the brain. LTApure also enhanced circulating corticosterone, but failed to alter locomotor and anxiety-related behavior in the open field test. These data disclose that TLR2 agonism by LTA causes peripheral immune activation and initiates neuroinflammatory processes in the brain that are associated with down-regulation of BBB components and activation of the HPA axis, although emotional behavior (anxiety) is not affected. The results obtained with an LTA preparation contaminated with LPS hint at a facilitatory interaction between TLR2 and TLR4, the adverse impact of which on long-term neuroinflammation, disruption of the BBB and mental health warrants further analysis.

Multilayered Polysaccharide Nanofilms for Controlled Delivery of Pentoxifylline and Possible Treatment of Chronic Venous Ulceration.

Local drug delivery systems made from nontoxic polysaccharide nanofilms have an enormous potential in wound care. A detailed understanding of the structural, surface, physicochemical, and cytotoxic properties of such systems is crucial to design clinically efficacious materials. Herein, we fabricated polysaccharide-based nanofilms onto either a 2D model (SiO2 and Au sensors) or on nonwoven alginate 3D substrates using an alternating assembly of N,N,N-trimethylchitosan (TMC) and alginic acid (ALG) by a spin-assisted layer-by-layer (LbL) technique. These TMC/ALG multilayered nanofilms are used for a uniform encapsulation and controlled release of pentoxifylline (PTX), a potent anti-inflammatory drug for treatment of the chronic venous ulceration. We show a tailorable film growth and mass, morphology, as well as surface properties (charge, hydrophilicity, porosity) of the assembled nanofilms through control of the coating during the spin-assisted assembly. The uniform distribution of the encapsulated PTX in the TMC/ALG nanofilms is preserved even with when the amount of the incorporated PTX increases. The PTX release mechanism from the model and real systems is studied in detail and is very comparable for both systems. Finally, different cell-based assays illustrated the potential of the TMC/ALG multilayer system in wound care (e.g., treatment chronic venous ulceration) applications, including a decrease of TNF-α secretion, a common indicator of inflammation.

Role of omics techniques in the toxicity testing of nanoparticles.

Nanotechnology is regarded as a key technology of the twenty-first century. Despite the many advantages of nanotechnology it is also known that engineered nanoparticles (NPs) may cause adverse health effects in humans. Reports on toxic effects of NPs relay mainly on conventional (phenotypic) testing but studies of changes in epigenome, transcriptome, proteome, and metabolome induced by NPs have also been performed. NPs most relevant for human exposure in consumer, health and food products are metal, metal oxide and carbon-based NPs. They were also studied quite frequently with omics technologies and an overview of the study results can serve to answer the question if screening for established targets of nanotoxicity (e.g. cell death, proliferation, oxidative stress, and inflammation) is sufficient or if omics techniques are needed to reveal new targets. Regulated pathways identified by omics techniques were confirmed by phenotypic assays performed in the same study and comparison of particle types and cells by the same group indicated a more cell/organ-specific than particle specific regulation pattern. Between different studies moderate overlap of the regulated pathways was observed and cell-specific regulation is less obvious. The lack of standardization in particle exposure, in omics technologies, difficulties to translate mechanistic data to phenotypes and comparison with human in vivo data currently limit the use of these technologies in the prediction of toxic effects by NPs.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

The Development of Indicator Cotton Swabs for the Detection of pH in Wounds.

Indicator cotton swabs have been developed in order to enable faster, less expensive, and simpler information gathering of a wound status. Swabs are normally used for cleaning the wound, but here, they were covalently functionalized with indicator chemistry. Thus, they in principle enable simultaneous wound cleaning and wound pH detection. Using an indicator dye with a color change from yellow to red, combined with an inert dye of blue color, a traffic light color change from green to red is induced when pH increases. The indicator cotton swabs (ICSs) show a color change from green (appropriate wound pH) to red (elevated wound pH). This color change can be interpreted by the naked eye as well as by an optical color measurement device in order to obtain quantitative data based on the CIE L*a*b* color space. Two types of swabs have been developed-indicator cotton swabs ICS1 with a sensitive range from pH 5 to 7 and swabs ICS2 with a sensitive range from 6.5 to 8.5. The swabs are gamma-sterilized and the effect of sterilization on performance was found to be negligible. Furthermore, cytotoxicity testing shows cell viability and endotoxin levels to be within the allowable range.

Cognitive impairment by antibiotic-induced gut dysbiosis: Analysis of gut microbiota-brain communication.

Emerging evidence indicates that disruption of the gut microbial community (dysbiosis) impairs mental health. Germ-free mice and antibiotic-induced gut dysbiosis are two approaches to establish causality in gut microbiota-brain relationships. However, both models have limitations, as germ-free mice display alterations in blood-brain barrier and brain ultrastructure and antibiotics may act directly on the brain. We hypothesized that the concerns related to antibiotic-induced gut dysbiosis can only adequately be addressed if the effect of intragastric treatment of adult mice with multiple antibiotics on (i) gut microbial community, (ii) metabolite profile in the colon, (iii) circulating metabolites, (iv) expression of neuronal signaling molecules in distinct brain areas and (v) cognitive behavior is systematically investigated. Of the antibiotics used (ampicillin, bacitracin, meropenem, neomycin, vancomycin), ampicillin had some oral bioavailability but did not enter the brain. 16S rDNA sequencing confirmed antibiotic-induced microbial community disruption, and metabolomics revealed that gut dysbiosis was associated with depletion of bacteria-derived metabolites in the colon and alterations of lipid species and converted microbe-derived molecules in the plasma. Importantly, novel object recognition, but not spatial, memory was impaired in antibiotic-treated mice. This cognitive deficit was associated with brain region-specific changes in the expression of cognition-relevant signaling molecules, notably brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, serotonin transporter and neuropeptide Y system. We conclude that circulating metabolites and the cerebral neuropeptide Y system play an important role in the cognitive impairment and dysregulation of cerebral signaling molecules due to antibiotic-induced gut dysbiosis.

Peptides at the Interface: Self-Assembly of Amphiphilic Designer Peptides and Their Membrane Interaction Propensity.

Self-assembling amphiphilic designer peptides have been successfully applied as nanomaterials in biomedical applications. Understanding molecular interactions at the peptide-membrane interface is crucial, since interactions at this site often determine (in)compatibility. The present study aims to elucidate how model membrane systems of different complexity (in particular single-component phospholipid bilayers and lipoproteins) respond to the presence of amphiphilic designer peptides. We focused on two short anionic peptides, V4WD2 and A6YD, which are structurally similar but showed a different self-assembly behavior. A6YD self-assembled into high aspect ratio nanofibers at low peptide concentrations, as evidenced by synchrotron small-angle X-ray scattering and electron microscopy. These supramolecular assemblies coexisted with membranes without remarkable interference. In contrast, V4WD2 formed only loosely associated assemblies over a large concentration regime, and the peptide promoted concentration-dependent disorder on the membrane arrangement. Perturbation effects were observed on both membrane systems although most likely induced by different modes of action. These results suggest that membrane activity critically depends on the peptide's inherent ability to form highly cohesive supramolecular structures.

Oral uptake of nanoparticles: human relevance and the role of in vitro systems.

Nanoparticles (NPs) present in environment, consumer and health products, food and medical applications lead to a high degree of human exposure and concerns about potential adverse effects on human health. For the general population, the exposure through contact with the skin, inhalation and oral uptake are most relevant. Since in vivo testing is only partly able to study the effects of human oral exposure, physiologically relevant in vitro systems are being developed. This review compared the three routes taking into account the estimated concentration, size of the exposed area, morphology of the involved barrier and translocation rate. The high amounts of NPs in food, the large absorption area and the relatively high translocation rate identified oral uptake as most important portal of entry for NPs into the body. Changes of NP properties in the physiological fluids, mechanisms to cross mucus and epithelial barrier, and important issues in the use of laboratory animals for oral exposure are mentioned. The ability of in vitro models to address the varying conditions along the oro-gastrointestinal tract is discussed, and requirements for physiologically relevant in vitro testing of orally ingested NPs are listed.

Cytotoxicity of Nanoparticles Contained in Food on Intestinal Cells and the Gut Microbiota.

Toxicity of nanoparticles (NPs) upon oral exposure has been studied in animals using physiological changes, behavior, histology, and blood analysis for evaluation. The effects recorded include the combination of the action on cells of the exposed animal and the reaction of the microorganisms that populate the external and internal surfaces of the body. The importance of these microorganisms, collectively termed as microbiota, for the health of the host has been widely recognized. They may also influence toxicity of NPs but these effects are difficult to differentiate from toxicity on cells of the gastrointestinal tract. To estimate the likelihood of preferential damage of the microbiota by NPs the relative sensitivity of enterocytes and bacteria was compared. For this comparison NPs with antimicrobial action present in consumer products were chosen. The comparison of cytotoxicity with Escherichia coli as representative for intestinal bacteria and on gastrointestinal cells revealed that silver NPs damaged bacteria at lower concentrations than enterocytes, while the opposite was true for zinc oxide NPs. These results indicate that silver NPs may cause adverse effects by selectively affecting the gut microbiota. Fecal transplantation from NP-exposed animals to unexposed ones offers the possibility to verify this hypothesis.