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.

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.

In-vitro permeability of neutral polystyrene particles via buccal mucosa.

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.

Rational Design and Characterization of a Nanosuspension for Intraoral Administration Considering Physiological Conditions.

The oral cavity displays an attractive route in drug administration that is not associated with gastric transit and hepatic first-pass metabolism. However, limiting factors for an efficient transit of drugs through the oral mucosa are poor water solubility and permeability. Hence, various strategies exist to enhance solubility. Specifically, nanotechnology has attracted much research interest in the past decade. This study aimed at developing a stable nanosuspension of the model compound phenytoin via wet media milling. The nanosuspensions were carefully characterized regarding hydrodynamic particle sizes, crystallinity, and dissolution characteristics under nonphysiological or physiological (salivary) conditions. The permeability of bulk phenytoin and nanophenytoin through a buccal in vitro and ex vivo model was investigated, and the apparent permeability coefficients were determined. Moreover, cytotoxicity studies were conducted. The addition of Tween 80 as stabilizer resulted in a stable crystalline nanosuspension (330 nm). The solubility characteristics significantly increased under salivary conditions, which further impacted the permeability, as the steady state appearance rate of nanosized phenytoin was 1.4-fold higher. Cytotoxicity studies demonstrated that bulk-/nano-phenytoin exhibited no harmful effects. It can be concluded that the salivary environment (i.e., ionic strength, pH) strongly impacts the solubility and consequently the permeability of crystalline nanosuspensions across the buccal mucosa.

Interactions between nano-TiO2 and the oral cavity: impact of nanomaterial surface hydrophilicity/hydrophobicity.

Titanium dioxide (TiO2) nanoparticles are available in a variety of oral applications, such as food additives and cosmetic products. Thus, questions about their potential impact on the oro-gastrointestinal route rise. The oral cavity represents the first portal of entry and is known to rapidly interact with nanoparticles. Surface charge and size contribute actively to the particle-cell interactions, but the influence of surface hydrophilicity/hydrophobicity has never been shown before. This study addresses the biological impact of hydrophilic (NM 103, rutile, 20 nm) and hydrophobic (NM 104, rutile, 20 nm) TiO2 particles within the buccal mucosa. Particle characterization was addressed with dynamic light scattering and laser diffraction. Despite a high agglomeration tendency, 10% of the particles/agglomerates were present in the nanosized range and penetrated into the mucosa, independent of the surface properties. However, significant differences were observed in intracellular particle localization. NM 104 particles were found freely distributed in the cytoplasm, whereas their hydrophobic counterparts were engulfed in vesicular structures. Although cell viability/membrane integrity was not affected negatively, screening assays demonstrated that NM 104 particles showed a higher potential to decrease the physiological mitochondrial membrane potential than NM 103, resulting in a pronounced generation of reactive oxygen species.

The buccal mucosa as a route for TiO2 nanoparticle uptake.

The oral cavity, although part of the aero-digestive tract, is still neglected in terms of risk assessment with respect to nanoparticle uptake. If nanoparticles enter the oral cavity, either via oral products or inhaled materials, it is not clear whether they rapidly interact with the mucosae or are swallowed. In this study, interactions of three distinct titanium dioxide (TiO2) particles (i.e. NM 100, NM 101 and NM 105) with oral tissues are presented. Physicochemical properties were addressed in relevant media, and particle penetration was investigated with an ex vivo model using porcine mucosa. To avoid modification of the particle surfaces via labeling, multiphoton microscopy was introduced as an accurate method to detect TiO2 particles within the tissue. The spatiotemporal aspects of nanoparticle uptake, as well as the intracellular localization in human epithelial cells, were studied and potential toxic effects were evaluated. Although TiO2 particles formed large aggregates once dispersed in media, 10-50% remained in the nanoscale range, rapidly interacting with the mucus layer and infecting the epithelium. However, differences in the penetration depth were observed depending on the particle characteristics. NM 100 and NM 105 were found in both the upper part and the lower part of the buccal mucosa, while NM 101 (smallest particle sizes) only penetrated the upper parts. Transport studies revealed that TiO2 nanoparticles were found in vesicles, as well as freely distributed in the cytoplasm. Cell viability/integrity was not affected negatively; however, NM 105 triggered the production of reactive oxygen species. These data clearly suggest that the oral cavity should be considered in further risk assessment studies.

Atomic force microscopy as analytical tool to study physico-mechanical properties of intestinal cells.

The small intestine is a complex system that carries out various functions. The main function of enterocytes is absorption of nutrients, whereas membranous cells (M cells) are responsible for delivering antigens/foreign substances to the mucosal lymphoid tissues. However, to get a fundamental understanding of how cellular structures contribute to physiological processes, precise knowledge about surface morphologies, cytoskeleton organizations and biomechanical properties is necessary. Atomic force microscopy (AFM) was used here as a powerful tool to study surface topographies of Caco-2 cells and M cells. Furthermore, cell elasticity (i.e., the mechanical response of a cell on a tip indentation), was elucidated by force curve measurements. Besides elasticity, adhesion was evaluated by recording the attraction and repulsion forces between the tip and the cell surface. Organization of F-actin networks were investigated via phalloidin labeling and visualization was performed with confocal laser scanning fluorescence microscopy (CLSM) and scanning electron microscopy (SEM). The results of these various experimental techniques revealed significant differences in the cytoskeleton/microvilli arrangements and F-actin organization. Caco-2 cells displayed densely packed F-actin bundles covering the entire cell surface, indicating the formation of a well-differentiated brush border. In contrast, in M cells actins were arranged as short and/or truncated thin villi, only available at the cell edge. The elasticity of M cells was 1.7-fold higher compared to Caco-2 cells and increased significantly from the cell periphery to the nuclear region. Since elasticity can be directly linked to cell adhesion, M cells showed higher adhesion forces than Caco-2 cells. The combination of distinct experimental techniques shows that morphological differences between Caco-2 cells and M cells correlate with mechanical cell properties and provide useful information to understand physiological processes/mechanisms in the small intestine.

The oral cavity as a biological barrier system: design of an advanced buccal in vitro permeability model.

An important area for future research lies in finding a drug delivery system across or into the oral mucosa. However, to design such systems, simplified biological models are necessary so that the mechanisms and/or interactions of interest can readily be studied. The oral epithelium is covered by a complex mucus layer, which enables exchange of nutrients and provides lubrication. However, it has been demonstrated that mucus has an impact on the mobility of nanoparticles and drug molecules. Thus, we aimed to develop an advanced buccal in vitro model for studying transport of nanoparticles, taking the mucus layer into account. First, animal mucins (porcine gastric, bovine submaxillary) were compared with natural human mucin regarding chemical and morphological structure. Second, an "external" mucus layer was prepared by a film method and deposited onto an oral cell line (TR 146), cultured on transwells®. Adherence of the mucin fibers was evaluated and the viability of the model was assessed. Nanoparticle transport studies were performed with this advanced in vitro model and an ex vivo diffusion system. The results revealed that porcine mucin is most similar to human natural mucin in chemical structure and morphology. Both the bovine and porcine mucin fibers adhered onto the oral cells: Due to the different morphology of bovine mucin, the viability of the oral cells decreased, whereas porcine mucin maintained the viability of the model for more than 48 h. Comparison of in vitro data with ex vivo data suggested reliability of the advanced buccal in vitro model. Additionally, it was demonstrated that the mucus layer in the oral cavity also acts as a strong barrier for the mobility of nanoparticles.

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

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.