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.

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.

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Drugs can be absorbed well in the oral cavity, which eliminates problems related to intestinal and hepatic first-pass metabolism. Although it is well-established that nanoparticles are small enough to penetrate/permeate epithelial barriers, there is no clear understanding of how they interact with the buccal mucosa. This work provides useful information regarding particle properties with regard to mucosal uptake and can be used for the rational design of nanocarriers. In the buccal mucosa, the uptake of neutral polystyrene nanoparticles (PP) is size-dependent. Compared to 25 and 50 nm particles, 200 nm PP particles penetrate into deeper regions of the mucosa. This is attributed to the structure of the buccal mucosa, i.e., mucus layer and microplicae. The particles permeate the mucus layer and deposit in ridge-like folds of superficial buccal cells. Thus, the effects of thermodynamic driving forces and/or interparticle electrostatic repulsion are enhanced and cellular uptake might be reduced for smaller particle sizes.

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.