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Nanotoxikologie (Fach) / Teil 5 (Lektion)

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• a) What are the parts of the upper GI-tract and what is the environmental pH there? b) What are the parts of the lower GI-tract and what is the environmental pH there? a) •esophagus•stomach (pH 1,5-3,5) b) •Small Intestine => Duodenum, Jejunum, Ileum (pH 6-8) •Large Intestine => Cecum, Colon, Rectum    (pH 5,5-6,8)
• a) What kind of uptake methods or animals do exist? b) Why is the one with the more stress for animals preferred? c) How long is an oral uptake test usually performed? a) •Oral Gavage => typically a substance is dispersed in a liquid dispersion (Volumes up to 10 ml/kg in mice and 20 ml/kg rats) and syringed in the animals mouth •Uptake ad libitum => Substance can be added to food or water •Pill formulation b) •Uptake ad libitum => dosing and treatment time more difficult to control c) •28 days
• a) What are the cell types which are the structural basis of barrier in stomach, esophagus/oral cavity and small and large intestine? b) Which of them is the thickest and what's the inference we can draw from this fact? a) •Oral cavity and the esophagus: Keratinocytes•Stomach: Gastric epithelial cells •Small & large intestine: Enterocytes b) •Oral Cavity: 550 and 800 µm •Esophagus: 300–500 µm •GI Tract: 20–25 µm=> the barrier of the GI-T is the thinnest, so it can be passed via diffusion very easily
• a) Which kind of in vitro models for modeling of the oral uptake do you already know? b) The transport through the GI-T is dependent on the Size, surface-coating and pretreatment of the NP. You read a paper, where a increased transport of NP is observed. How is this reasonable? a) •CaCo2-cells => in vitro barrier model (is been transformed into a barrier after 21 days) b) •maybe trough the digestion process the amount of protein corona (or proteins are low weight-proteins) decreased
• a) Can you state in vivo/in vitro models for skin penetration? b) Can you state in vivo/in vitro models for skin sensitization (contact allergy)? c) Can you state in vivo/in vitro models for skin irritation/corrosion? d) Can you state in vivo/in vitro models for photo toxicity? a) •in vivo: Skin Absorption•in vitro: Franz Cells b) •in vivo: Local Lymph Node Assay (mice) •in vitro: Direct Peptide Reactivity Assay (DPRA), ARE-Nrf2 Luciferase Assay, H-CLAT Assay c) •in vitro: Reconstructed Human Epidermis d) •in vitro: 3T3 NRU phototoxicity test
• a) Do you expect that NP can be taken up from the gut and transported to secondary organs? Is this possible? b) On which NP characteristics will this depend (please mention two)? c) Why are mostly co-cultured cell models used for modeling the GI-Tract uptake? a) •amounts are very low, but this can occur b) •mainly NM type & size (smaller, the higher the penetration)•solubility (the more soluble, the higher the penetration)•surface charge => cationic are penetrating well (preventing mucoadhesion) c) •In vitro models have limitations (often fail to accurately reflect in vivo responses) => cells in isolation suffer from reduced survival, disrupted metabolic competence, reduced cell-cell interaction, disrupted organ topology, and absence of tissue communication•To overcome these limitations and improve physiological relevance with respect to the GI-T system, multicell cultures that incorporate mucosa-resident cells in addition to barrier epithelia (e.g. mucus secreting goblet cells) Microfold-cells and even immunocompetent macrophages and dendritic cells are combined
• How can one test via uptake via GI tract is possible in vitro? Please briefly explain the experimental set-up (mention the cell type that can be used and explain some details on the study design)! •co-cultured In vitro model with mucosa-resident cells in addition to barrier epithelia (e.g. mucus secreting goblet cells) Microfold-cells and even immunocompetent macrophages and dendritic cells•the GI-T ingestion model should respect the different pH-values, enzymes, proteins and the ionic strength of the juices (saliva 6,8; gastric juice 1,3; duodenal juice 8,1; bile juice 8,2) in a realistic composition•NP should be tested in different sizes and coatings•Impact of digestion measuring via DLS, Single NP-ICP-MS (inductively coupled plasma) => for metals and their oxides•to characterize particles (elemental composition/morphology) during digestion (SEM-EDX = scanning electron microscopy-energy dispersive x-ray spectroscopy)
• The skin is a very effective barrier to the entry of particulate material. Which skin components have the most important barrier functions? •sebum: high lipophilic composition (triglycerides, fatty acids…) •stratum corneum: matrix of lipids and corneocytes with 20-200 keratinized cell layers
• a) Which properties of nanoparticles are of advantage when you want to use them as nanocarriers for delivery of substances or drugs to skin? b) Where does the delivery ends in the skin and how does the drug delivery continue? a) •particle size (should be between 50-100nm => uptake ↑)•shouldn’t be hygroscopic (take to much water)surface properties: •protection against GI degradation (coating)•mucoadhesive properties (positive charge, coating e.g. PLGA)•through skin, they should be lipophilic b) •in the stratum corneum (strong barrier) => then the drug is penetrating into deeper skin layer

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