Development of an Advanced Intestinal in Vitro Triple Culture

Article pubs.acs.org/molecularpharmaceutics

Development of an Advanced Intestinal in Vitro Triple Culture Permeability Model To Study Transport of Nanoparticles Christa Schimpel,† Birgit Teubl,† Markus Absenger,‡ Claudia Meindl,‡ Eleonore Fröhlich,‡ Gerd Leitinger,§ Andreas Zimmer,† and Eva Roblegg*,† †

Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria Center for Medical Research and §Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria

‡

S Supporting Information *

ABSTRACT: 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. KEYWORDS: intestinal mucosa, in vitro triple culture, Caco-2 cells, goblet cells, M cells, nanoparticles

1. INTRODUCTION Oral administration with intestinal absorption is the most common method of drug delivery. However, it faces serious obstacles, including enzymatic degradation of the drug in the stomach and the “first pass” metabolism, making this route a considerable challenge for biopharmaceuticals, degradationprone molecules and molecules with poor solubility and/or permeability. Over recent years the number of peptide and protein drugs has increased significantly. Thus, carriers that protect the drug during gastrointestinal transit and impact their permeability behavior in the intestine are of enormous interest.1−4 Nanoparticles are expected to play an important role in this field. However, in order to design such systems in a rational way, physiologically relevant biological models are necessary to study the impact of nanoparticle properties on the © 2014 American Chemical Society

permeability, the transport mechanisms and various interactions. Currently, the best known in vitro model used for permeability studies via the gastrointestinal barrier is the Caco-2 cell line.5−7 Caco-2 cells are human intestinal carcinoma cells (enterocytes) that form intact confluent monolayers on cell culture bottles/dishes or filter materials. Moreover, they offer a high morphological and physiological similarity to the human intestinal epithelium, including characteristic features such as the formation of a brush border, expression of typical Received: Revised: Accepted: Published: 808

August 23, 2013 December 18, 2013 January 28, 2014 February 6, 2014 dx.doi.org/10.1021/mp400507g | Mol. Pharmaceutics 2014, 11, 808−818

Molecular Pharmaceutics

Article

nanoparticles across the in vitro model were comparable with data gathered from studies using an ex vivo rat ileum model. They showed that the chitosan-modified nanoparticles were mucoadhesive and enhanced paracellular permeation of insulin. The goal of our work was to develop and carefully characterize an in vitro triple culture model intended to study transcellular mechanisms and cellular interactions of nanoparticles taking into account particle properties (i.e., size, surface charge). We correlated permeability data from in vitro investigations with a commonly used ex vivo method (Ussing chambers, porcine intestine) and compared the results with in vivo and ex vivo data from the literature. Permeability studies of the in vitro model were performed with model drugs and, in addition, with reference nanoparticles since the model is intended to investigate nanoparticle mechanisms. Furthermore, we compared in vitro double culture models, comprising enterocytes and M cells as well as enterocytes and goblet cells, with the triple culture model, to evaluate to which extent mucus and M cells impact nanoparticle uptake.

metabolic enzymes and efflux transporters (p-glycoprotein transporter). Despite these advantages, one cell type cannot reflect the complete physiology of the intestine. The difference in permeability values could be attributed to the variable expression level and substrate specifity of the carrier and efflux systems found in Caco-2 cells. Thus, in vivo/in vitro correlations are often inappropriate.6,8−11 The second major phenotype in the small intestine is represented by goblet cells (interspersed between enterocytes). These cells continuously secrete mucus that covers the epithelium and prevents particles or pathogens to penetrate into the epithelium and/or wraps them up due to its tenacious property.11,12 Thus, the uptake into the underlying epithelia is restricted, and particles are eliminated via clearance mechanisms. In addition, it enables the exchange of water, gases or nutrients, just to mention some examples.10 These data indicate that a permeability model for studying the transport of nanoparticles requires the integration of this barrier. Wikman-Larhed and Artursson11 first characterized an in vitro coculture model comprising Caco-2 cells and goblet-like HT29-H cells. Later Walter and co-workers12 established an in vitro model using HT29-MTX and absorptive Caco-2 cells in coculture. Mahler et al.13 used the same coculture components but varied the ratio, so that a mucus layer, which completely covers the cell monolayer, was achieved. The model was used to study iron bioavailability, and the resulting data correlated well with human in vivo data. Apart from enterocytes and mucus, epithelial M cells located in the epithelium overlaying the Peyer’s patches also play a dominant role. Although they represent a minor population in the follicle-associated epithelium, M cells are responsible for the uptake of microorganisms and foreign substances and deliver them via transepithelial transport from the external environment to the lymphoid follicles.14 Several reports suggest that nanoparticles are capable to enter intestinal epithelia via M cells and that uptake by absorptive enterocytes only plays a minor role.15−17 Kernéis et al.18 constructed the first intestinal in vitro coculture model based on Caco-2 cells (on inverted inserts) and mouse Peyer’s patch lymphocytes, which settled into the epithelial monolayer and triggered M cell development. They did not alter the polarity and the integrity of Caco-2 cells. However, villin and sucrose isomaltase, two proteins that exhibit brush-border organization, disappeared from the apical side of the Caco-2 cells and enzymes were downregulated. Additionally, it was demonstrated that, compared to Caco-2 cells, translocation of microorganisms and latex particles occurred only via M cells. Gullberg et al.19 developed a similar model based on a coculture of Caco-2 cells (on normally oriented inserts) and human Raji B lymphocytes instead of mouse cells, since Raji cells exhibit B cell markers, which are major inductive partners in reconstruction of M cells.20 Using this model, des Rieux et al.17,20 investigated the impact of nanoparticle properties on the intestinal in vitro permeability. They showed that particle transport through the M cell model was 50-fold higher compared to a pure Caco-2 monolayer. Recent studies by Mahler et al. and Antunes et al.21,22 combined enterocytes, goblet cells and M cells in their in vitro models. Mahler et al. tested the impact of polystyrene nanoparticles on iron absorption and showed that data obtained by the in vitro model correlated well with in vivo data gained from intestinal chicken loop studies. Antunes et al. conducted insulin transport studies, both in solution and associated with mucoadhesive dextran/sulfate chitosan nanoparticles, and demonstrated that Papp values for insulin-loaded

2. EXPERIMENTAL SECTION 2.1. Cell Culture and in Vitro Permeability Models. Caco-2 cells (ACC169, HTB-37 clone from the German Collection of Microorganismen and Cell Cultures), provided by E. Fröhlich (Medical University of Graz, Austria), were cultivated at 37 °C under 10% CO2 water saturated atmosphere in complete medium consisting of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine and 1% penicillin and streptomycin (PEST) according to the protocol of de Rieux et al.20 HT29-MTX cells were kindly provided by T. Lesuffleur (INSERM UMR S 938, Paris, France) and were grown in the same medium under a 5% CO2 water saturated atmosphere.23 Raji B cells were a kind gift from R. Fuchs (Medical University of Graz, Austria). Raji B cells (nonadherent cells) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% L -glutamine and 1% penicillin and streptomycin (PEST), at 37 °C in a 5% CO2 water saturated atmosphere. Routinely, Caco-2 cells and HT29-MTX cells were subcultured once a week with trypsin-EDTA (0.25%, 0.53 mM) and seeded at a density of 4 × 105 per 75 cm2 flask. Equally, Raji B cells were subcultured once a week seeding an appropriate volume of the cell suspension into fresh medium, restoring the cell concentration to 1 × 106 per 75 cm2 flask. Medium was changed every other day. The double culture (Caco-2/Raji B coculture) was cocultivated following previously described protocols.17,19 Initially 5 × 105 Caco-2 cells (passage 8−20) suspended in 0.5 mL of supplemented DMEM were seeded onto polycarbonate 12-well Transwell filters (Corning Incorporated, USA; 3 μm mean pore size, 1.12 cm2 surface area). Caco-2 cells were maintained under standard incubation conditions for 14−16 days, and medium on both the apical (0.5 mL) and basolateral sides (1.5 mL) was changed every other day. Subsequently, 5 × 105 Raji B cells (passage 8− 20), resuspended in supplemented DMEM, were added to the basolateral compartment of inserts to trigger M cell differentiation. The cocultures were maintained for 4−5 days. Corresponding monocultures of Caco-2 cells on matched inserts served as controls. Medium in the apical side was changed every second day. For the establishment of the triple culture model (Caco-2/HT29-MTX/Raji B triple culture) predetermined cell numbers of Caco-2 and HT29-MTX cells 809

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trogen) according to the manufacturer’s specifications. In our case, ELF 97 staining appeared blue against a black background when visualized with a fluorescence microscope (Axio Observer, Zeiss; camera: Axio Cam) at 405 excitation wavelength in conjunction with BP 420/80 for the blue spectral region. 2.3. Morphological Characterization of the in Vitro Model. To distinguish between different cell types, scanning electron microscopy (SEM) was used to evaluate morphological changes of cell surface architectures. After cultivation in Transwell systems cells were washed twice with PBS. Fixation was performed in Schaffer’s fixative (37% formol/100% ethanol) for 2 h to avoid changes in mucin structure.26 Subsequently, dehydration was carried out through a graded series of ethanol (80%−100%), following a standard procedure. Subsequently, samples were dried with hexamethyldisilazane and the removed filter membranes were sputtered with gold palladium (Bal-Tec SCD 500). The samples were coated at 25 mA for 60 s under argon atmosphere, and images were acquired using a scanning electron microscope (Zeiss DSM 950). 2.4. Permeability Studies of the in Vitro/ex Vivo Model Using Antipyrine. For permeability studies of the in vitro model, antipyrine (Sigma Aldrich) was used as model drug. The data were compared with ex vivo results obtained from experiments performed with porcine mucosa and Ussing chambers as well as data from literature. The buffer in the apical compartment of the in vitro triple culture model was replaced by antipyrine (10 mM) suspended in Krebs-Ringer bicarbonate buffer (KRBB). After 4 h samples of 100 μL were withdrawn from the basolateral compartment and determined by high performance liquid chromatography (HPLC). The apparent permeability coefficient (Papp; cm/s) representing the apical-to-basolateral permeability of the test compound was calculated from the following equation:27

were mixed in different ratios (i.e., 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9) for Caco-2 to HT29-MTX cells) prior to seeding. Cocultures (5 × 105 per insert) were seeded on the upper side of the Transwell filter inserts. Subsequently, 5 × 105 Raji B cells were added to the basolateral insert compartment, whereas medium in the upper compartment was changed every other day. Cocultures of Caco-2 and HT29-MTX cells (cultivated as described above) served as controls. To ensure intactness and confluence of fully differentiated monolayers after 19−21 days in culture, the integrity of cell monolayers was monitored by TEER measurements with an Endohm culture cup connected to an epithelial volt ohm meter (World Precision Instruments, Sarasota, FL, USA). The integrities of cell monolayers were calculated by the transepithelial electrical resistance (R; Ω) according to Ranaldi et al.24 Only monolayers representing TEER values between 300 and 450 Ω cm2 were used for further experiments. 2.2. Histochemical Characterization of the in Vitro Model. Identification of the appropriate enterocytes/goblet cell ratio was performed with Alcian Blue (Sigma Aldrich). Briefly, cells were placed in 3% acetic acid for 3 min and stained with Alcian Blue solution (1 g of Alcian Blue 8GX in 100 mL of 3% acetic acid) for 30 min. Afterward, cells were rinsed twice with distilled water before cell nuclei were counterstained using Nuclear Fast Red Solution (Sigma Aldrich). The reaction was stopped by washing once with water, until red staining became obvious. Finally, cells were visualized by light microscopy (Olympus BX-51, camera: DP-71). Two methods were used in order to first monitor and second verify the differentiation of Caco-2 cells into M cells in the presence of Raji B cells. M cell development takes advantage of the increased binding affinity of lectins on the apical membrane of M cells.19 We performed Alexa Fluor 488 fluorescencelabeled wheat germ agglutinin/Hoechst double staining to monitor glycosidic moieties that might be differentially expressed by M cells. For this purpose cells were cultivated as previously described and were washed once with prewarmed HBSS. Subsequently, cells were costained with 0.5 mL of a WGA/Hoechst staining solution (consisting of 1 μg/mL Hoechst and 5 μg/mL WGA in HBSS) and were incubated for 10 min at 37 °C/5% CO2 in the dark. Afterward, cells were washed two times with HBSS before samples were observed using fluorescence microscopy (Axio Observer, Zeiss; camera: Axio Cam) at 488 nm excitation wavelength using a BP 505/ 550 nm band-pass detection for the green channel and 405 excitation wavelength in conjunction with BP 420/80 for the blue spectral region. Next, M cells were identified. M cells show a downregulation of the brush border enzyme alkaline phosphatase due to the absence of a brush border membrane.25 Thus alkaline phosphatase activity was determined by using SIGMAFAST p-nitrophenyl phosphate tablets (Sigma Aldrich) according to the manufacturer’s instructions. 200 μL/well of the substrate solution was added, and after an incubation of 30 min absorbance was measured at 405 nm with a VIS-plate reader (FLUOstar Optima, BMG, Labortechnik). Alkaline phosphatase is capable of hydrolyzing p-nitrophenyl phosphate (pNPP) substrates releasing p-nitrophenol. The rate of change in absorbance at 405 nm is directly proportional to the alkaline phosphatase enzyme activity. To verify the alteration regarding alkaline phosphatase activity, a novel fluorescence-based method for detecting phosphatases was performed using ELF 97 Endogenous Phosphatase Detection Kit (E6601) (Invi-

Papp = (dC /dt )/(C0A)

where dC/dt is the appearance rate of the test compound in the receiver compartment over time, C0 is the initial concentration in the donor compartment and A is the exposed area of the tissue. Ex vivo experiments were performed with porcine intestinal mucosa due to the morphological similarities with human mucosa.28 The tissue was immediately isolated after the pigs’ slaughter (age triple culture > Caco-2 and > Caco-2/HT29-MTX coculture (Table 4).

coculture nanoparticles were completely entrapped and immobilized due to adhesive/hydrophobic interactions with mucins (Figure 5c). In the triple culture model nanoparticle uptake was significantly reduced for enterocytes and mainly occurred via M cells (Figure 5d). Experiments with 200 nm PP particles revealed similar results. In the Caco-2 monolayer (Figure 6a) particles were found to be attached to the extracellular cell surface. Highest particle uptake was observed in the double culture model (Figure 6b). Again, in the Caco-2/HT29-MTX monolayer (Figure 6c), 200 nm PP particles were immobilized. In contrast, in the triple culture model, particles were also found inside of the cells (Figure 6d). Since no mucus is secreted in the microvilli-free region of M cells, we assume that again M cells provide portals through which some of the particles are able to cross the cell membrane. The ex vivo data revealed that 50 and 200 nm PP particles permeated the mucus layer and penetrated the epithelial tissue of the porcine intestinal mucosa (Figure 7). However, 50 nm particles were able to overcome the barriers more efficiently than 200 nm particles. Radial sections of the porcine intestinal mucosa showed that 50 nm PP were able to penetrate deeper regions of the porcine epithelium (Figure 7a), whereas the 200 nm PP particles permeated only the upper part of the tissue and penetrated into superficial regions of the epithelium (Figure 7b). Since individual intestinal tissue samples show autofluorescence intensities, the mean autofluorescence intensity of unexposed tissue samples served as control (Figure 7c). The amount of particles that permeated the mucosa increased with increasing time (Table 4). The calculated rate for 50 nm PP was 0.42 μg/mL and for 200 nm PP particles 0.24 μg/mL. Considering the thickness of the mucosa it can be concluded that the results of the in vitro triple culture model correlated well with the data from the ex vivo experiments, indicating a high reliability of the model.

Table 4. Cellular Uptake of 50 and 200 nm Plain Particles in the in Vitro/ex Vivo Models polystyrene nanoparticles [μg/mL] 50 nm Caco-2 monolayer Caco-2/ HT29MTX Caco-2/M cells triple culture ex vivo (Ussing chamber)

200 nm

60 min

120 min

240 min

60 min 120 min

240 min

0.10

0.22

0.39

0.06

0.11

0.28

0.01

0.02

0.05

0.01

0.02

0.02

0.18

0.34

0.59

0.12

0.24

0.42

0.15 0.11

0.29 0.26

0.52 0.42

0.08 0.07

0.16 0.13

0.33 0.24

To discriminate between cell association and actual internalization of the particles on/into the cell, z-stack scans (x−z and y−z projections) were performed. Optical cross sections illustrate that 50 nm PP particles were located in the Caco-2 monolayer (Figure 5a). However, the uptake amount was higher in the Caco-2/M cell double culture model (Figure 5b). These results suggest that both cell types are involved in nanoparticle uptake. In contrast, in the Caco-2/HT29-MTX

4. CONCLUSIONS In the current study an advanced intestinal in vitro triple culture permeability model including enterocytes, mucussecreting HT29-MTX cells and M cells was developed. All cells within the model were characterized according to their confluence, integrity, M cell expression and surface architecture. The model was used to investigate the permeability of active

Figure 5. Fluorescence microscopic z-scans of a Caco-2 monolayer (a), the Caco-2/M cell double culture model (b), the Caco-2/HT29-MTX coculture (c) and the triple culture model (d) treated with 50 nm green PP particles. Cell nuclei were stained with Hoechst (blue), and actin was labeled with Phalloidin Rhodamine (red). The circles indicate nanoparticle uptake/penetration, whereas arrows depict entrapped/immobilized particles on the cell surface. 815

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Figure 6. Fluorescence microscopic z-scans of a Caco-2 monolayer (a), the Caco-2/M cell double culture model (b), the Caco-2/HT29-MTX coculture (c) and the triple culture model (d) treated with 200 nm red PP particles. Cell nuclei were stained with Hoechst (blue), and cell borders were stained with wheat germ agglutinin Alexa Fluor 488 (green). The circles indicate nanoparticle uptake/penetration, whereas arrows depict entrapped/immobilized particles on the cell surface.

Figure 7. Radial sections of the intestinal mucosa. The permeation of the PP particles was recorded with fluorescence microscopy after 4 h. Panels are overlays of the phase contrast and green fluorescence channel for PP 50 or red fluorescence channel for PP 200; panel c depicts the mean autofluorescence intensity of the nonlabeled control. (a) 50 nm PP particles permeated the cell layer and penetrated deeper regions of the epithelium; (b) 200 nm PP particles permeated the mucus layer and penetrated into superficial regions of the epithelium.

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pharmaceutical ingredients and of nanoparticles. The obtained data correlate well with data from ex vivo permeability studies through porcine intestinal mucosa indicating that the model is reliable. Furthermore we demonstrated that nanoparticle uptake in the small intestine is strongly impacted by the mucus layer and that smaller particles show a greater uptake compared to larger particles. However, the higher uptake amounts compared to single Caco-2 monolayers are attributed to the presence of M cells, which are hardly covered with mucus and, thus, play a major role in nanoparticle entry of the intestinal epithelium. This alternative in vitro permeability model closely recapitulates the physiology of the small intestine and provides a useful tool to study transcytosis of nanoparticles.

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AUTHOR INFORMATION

Corresponding Author

*Karl Franzens University of Graz, Institute of Pharmaceutical Sciences/Pharmaceutical Technology, Humboldtstrasse 46, 8010 Graz, Austria. E-mail: [email protected]. Phone: +43 316 380-8888. Fax: +43 316 380-9100. Notes

The authors declare no competing financial interest.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Histochemical characterization and permeability studies. This material is available free of charge via the Internet at http:// pubs.acs.org. 816

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