Eva Roblegg

Models for oral uptake of nanoparticles in consumer products

Presently, many consumer products contain nano-sized materials (NMs) to improve material properties, product quality and ease of use. NMs in food additives and in cosmetic articles (e.g., tooth paste) may be taken up by the oral route. As adverse effects of environmental nanoparticles, like ultrafine particles, have been reported, consumers worry about potential risks when using products containing NMs. The review focuses on metal and metal oxide NMs as common additives in tooth paste and in food industry and exposure by the oral route. Testing of NMs for oral exposure is very complex because differences in the diet, in mucus secretion and composition, in pH, in gastrointestinal transit time and in gastrointestinal flora influence NM uptake. Acellular (mucus, saliva) and epithelial layer of the orogastrointestinal barrier are described. Expected exposure doses, interaction of the NMs with mucus and permeation through the epithelium as well as in vivo data are mentioned. The role of in vitro models for the study of parameters relevant for ingested NMs is discussed.

Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes.

Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study. Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.

Development of sustained-release lipophilic calcium stearate pellets via hot melt extrusion

The objective of this study was the development of retarded release pellets using vegetable calcium stearate (CaSt) as a thermoplastic excipient. The matrix carrier was hot melt extruded and pelletized with a hot-strand cutter in a one step continuous process. Vegetable CaSt was extruded at temperatures between 100 and 130°C, since at these temperatures cutable extrudates with a suitable melt viscosity may be obtained. Pellets with a drug loading of 20% paracetamol released 11.54% of the drug after 8h due to the great densification of the pellets. As expected, the drug release was influenced by the pellet size and the drug loading. To increase the release rate, functional additives were necessary. Therefore, two plasticizers including glyceryl monostearate (GMS) and tributyl citrate (TBC) were investigated for plasticization efficiency and impact on the in vitro drug release. GMS increased the release rate due to the formation of pores at the surface (after dissolution) and showed no influence on the process parameters. The addition of TBC increased the drug release to a higher extent. After dissolving, the pellets exhibited pores at the surface and in the inner layer. Small- and Wide-Angle X-ray Scattering (SWAXS) revealed no major change in crystalline peaks. The results demonstrated that (nearly) spherical CaSt pellets could be successfully prepared by hot melt extrusion using a hot-strand cutter as downstreaming system. Paracetamol did not melt during the process indicating a solid suspension. Due to the addition of plasticizers, the in vitro release rate could be tailored as desired.

Evaluation of a physiological in vitro system to study the transport of nanoparticles through the buccal mucosa

Abstract A buccal physiological in vitro testing system for the evaluation of the permeability, the transport route and toxic effects of nanoparticles was developed. Carboxyl polystyrene (CP, 20 nm, 200 nm) and amine modified polystyrene (AP, 200 nm) particles were used as reference particles and characterized in biological media. The permeability through excised porcine buccal mucosa was investigated with Franz diffusion cells. To evaluate the transport route, particle uptake into oral H376 cells was recorded and the cell damage was measured. All particles immediately formed aggregates once dispersed in saliva. 20 nm CP particles permeated the mucus layer and penetrated into the stratum superficiale of the top third region of the epithelium by the transcellular route. The positively-charged 200 nm AP particles permeated the mucus-layer and penetrated into deeper regions of the tissue. By decreasing the temperature to 4°C, particle uptake was inhibited for 20 nm CP and 200 nm AP particles. 200 nm CP particles interacted with the mucus, formed agglomerates and did not penetrate into the epithelium. It can be concluded that the presented system serves as a valuable tool to evaluate the behavior of nanoparticles in the buccal mucosa.

Development of an Advanced Intestinal in Vitro Triple Culture Permeability Model To Study Transport of Nanoparticles

Intestinal epithelial cell culture models, such as Caco-2 cells, are commonly used to assess absorption of drug molecules and transcytosis of nanoparticles across the intestinal mucosa. However, it is known that mucus strongly impacts nanoparticle mobility and that specialized M cells are involved in particulate uptake. Thus, to get a clear understanding of how nanoparticles interact with the intestinal mucosa, in vitro models are necessary that integrate the main cell types. This work aimed at developing an alternative in vitro permeability model based on a triple culture: Caco-2 cells, mucus-secreting goblet cells and M cells. Therefore, Caco-2 cells and mucus-secreting goblet cells were cocultured on Transwells and Raji B cells were added to stimulate differentiation of M cells. The in vitro triple culture model was characterized regarding confluence, integrity, differentiation/expression of M cells and cell surface architecture. Permeability of model drugs and of 50 and 200 nm polystyrene nanoparticles was studied. Data from the in vitro model were compared with ex vivo permeability results (Ussing chambers and porcine intestine) and correlated well. Nanoparticle uptake was size-dependent and strongly impacted by the mucus layer. Moreover, nanoparticle permeability studies clearly demonstrated that particles were capable of penetrating the intestinal barrier mainly via specialized M cells. It can be concluded that goblet cells and M cells strongly impact nanoparticle uptake in the intestine and should thus be integrated in an in vitro permeability model. The presented model will be an efficient tool to study intestinal transcellular uptake of particulate systems.

Comparison of two in vitro systems to assess cellular effects of nanoparticles-containing aerosols

Highlights ► A new VITROCELL – Pariboy system was evaluated for testing of aerosolized NPs. ► Deposition rates differed between marker compounds and NPs. ► The manual aerosolizer MicroSprayer was suitable for cytotoxicity testing of NPs. ► Polystyrene nanoparticles acted more cytotoxic as aerosols than as suspensions.

Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity.

BackgroundData from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded.ResultsCPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced.ConclusionsDespite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.

Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats

The aim of the present study was the in vivo evaluation of thiomer-coated liposomes for an oral application of peptides. For this purpose, salmon calcitonin was chosen as a model drug and encapsulated within liposomes. Subsequently, the drug loaded liposomes were coated with either chitosan–thioglycolic acid (CS–TGA) or an S-protected version of the same polymer (CS–TGA–MNA), leading to an increase in the particle size of about 500 nm and an increase in the zeta potential from approximately − 40 mV to a maximum value of about + 44 mV, depending on the polymer. Coated liposomes were demonstrated to effectively penetrate the intestinal mucus layer where they came in close contact with the underlying epithelium. To investigate the permeation enhancing properties of the coated liposomes ex vivo, we monitored the transport of fluoresceinisothiocyanate-labeled salmon calcitonin (FITC-sCT) through rat small intestine. Liposomes coated with CS–TGA–MNA showed the highest effect, leading to a 3.8-fold increase in the uptake of FITC-sCT versus the buffer control. In vivo evaluation of the different formulations was carried out by the oral application of 40 μg of sCT per rat, either encapsulated within uncoated liposomes, CS–TGA-coated liposomes or CS–TGA–MNA-coated liposomes, or given as a solution serving as negative control. The blood calcium level was monitored over a time period of 24 h. The highest reduction in the blood calcium level, to a minimum of 65% of the initial value after 6 h, was achieved for CS–TGA–MNA-coated liposomes. Comparing the areas above curves (AAC) of the blood calcium levels, CS–TGA–MNA-coated liposomes led to an 8.2-fold increase compared to the free sCT solution if applied orally in the same concentration. According to these results, liposomes coated with S-protected thiomers have demonstrated to be highly valuable carriers for enhancing the oral bioavailability of salmon calcitonin.

Inline monitoring and a PAT strategy for pharmaceutical hot melt extrusion.

Implementation of continuous manufacturing in the pharmaceutical industry requires tight process control. This study focuses on a PAT strategy for hot melt extrusion of vegetable calcium stearate (CaSt) as matrix carrier and paracetamol as active pharmaceutical ingredient (API). The extrusion was monitored using in-line near-infrared (NIR) spectroscopy. A NIR probe was located in the section between the extrusion screws and the die, using a novel design of the die channel. A chemometric model was developed based on premixes at defined concentrations and was implemented in SIPAT for real time API concentration monitoring. Subsequently, step experiments were performed for different API concentrations, screw speeds and screw designs. The predicted API concentration was in good agreement with the pre-set concentrations. The transition from one API plateau to another was a smooth curve due to the mixing behaviour of the extruder. The accuracy of the model was confirmed via offline HPLC analysis. The screw design was determined as the main influential factor on content uniformity (CU). Additionally the influence of multiple feeders had a significant impact on CU. The results demonstrate that in-line NIR measurements is a powerful tool for process development (e.g., mixing characterization), monitoring and further control strategies.

Cytotoxity of nanoparticles is influenced by size, proliferation and embryonic origin of the cells used for testing

Abstract Cytotoxicity screening is a common technique in drug compound screening for the identification of adverse cellular effects. Nanoparticles may cause interference in these assays. For the interpretation of cytotoxicity data it is important to study also the influence of other factors like pre-treatment of the nanoparticles, the choice of the cell culture medium and type of cell used for testing. Carboxyl polystyrene particles (CPS, 20–1000 nm) were physicochemically characterized and cytotoxicity assessed with seven screening assays in 20 cell lines, which differed in species, growth pattern, cell size, doubling time, embryonic origin and capacity for phagocytosis. Small CPS acted more cytotoxic in all cell lines, larger CPS only in phagocytic cells. Small differences in cytotoxicity were noted between the screening assays. Growth pattern and cell size determined cytotoxicity more than proliferation rate and embryonic origin of cells. Non-adherent cells, cells of mesenchymal origin and with high proliferation rate may be more susceptible to damage by nanoparticles.