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Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation
European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation Marie Boegh, María García-Díaz, Anette Müllertz, Hanne Mørck Nielsen ⇑ Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen O, Denmark
a r t i c l e
i n f o
Article history: Received 18 October 2014 Accepted in revised form 14 January 2015 Available online xxxx Keywords: Biosimilar mucus Mucosal drug delivery Oral Caco-2 cell culture model Diffusion Peptides Nanoparticles Diffusive wave spectroscopy
a b s t r a c t The mucus lining of the gastrointestinal tract epithelium is recognized as a barrier to efficient oral drug delivery. Recently, a new in vitro model for assessment of drug permeation across intestinal mucosa was established by applying a biosimilar mucus matrix to the surface of Caco-2 cell monolayers. The aim of the present study was to gain more insight into the steric and interactive barrier properties of intestinal mucus by studying the permeation of peptides and model compounds across the biosimilar mucus as well as across porcine intestinal mucus (PIM). As PIM disrupted the Caco-2 cell monolayers, a cell-free mucus barrier model was implemented in the studies. Both the biosimilar mucus and the PIM reduced the permeation of the selected peptide drugs to varying degrees illustrating the interactive properties of both mucus matrices. The reduction in peptide permeation was decreased depending on the cationicity and H-bonding capacity of the permeant clearly demonstrated by using the biosimilar mucus, whereas the larger inter sample variation of the PIM matrix obstructed similarly clear conclusions. Thus, for mechanistic studies of permeation across mucus and mucosa the biosimilar mucus offers a relevant and reproducible alternative to native mucus. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Mucus is a viscoelastic hydrophilic gel mainly structured by a network of high molecular weight extensively glycosylated mucin fibers. It lines the mucosal surfaces of the body including the gastrointestinal (GI) tract, where it lubricates and protects the underlying epithelium from toxic substances and pathogens, while enabling the exchange of nutrients [1]. Mucus is composed mainly of water (90–95%) in addition to 2–5% of mucins and smaller quantities of lipids, proteins and minor amounts of electrolytes and DNA [2]. Mucin molecules form a crosslinked and entangled network and are key responsible for the hydrogel-like properties of mucus [3]. The brush-like structure and the mesh space of the network can act as a size exclusion filter reducing the mobility of large molecules [2,4,5]. In addition to this steric barrier, mucus also comprises an important interactive component as the formation of multiple non-covalent bonds may immobilize a drug or particle in the mucus network [2,5,6]. The non-glycosylated regions of the mucin protein backbone and the lipids associated to the mucins allow for hydrophobic interactions with drugs leading to ⇑ Corresponding author. Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O, Denmark. Tel.: +45 3533 6346; fax: +45 3533 6001. E-mail address: [email protected] (H.M. Nielsen).
decreased diffusion rates in the mucus [1,7]. The oligosaccharides O-linked to the mucin backbone constitute 40–80% by weight of the mucins [8]. Many of these oligosaccharides carry a terminal carboxyl group or ester sulfate groups, which gives mucus a net negative charge and the capacity of forming electrostatic interactions in addition to hydrogen bonds [1]. Furthermore, the dynamic barrier also play a role for the overall mucus barrier properties [9]. Therefore, the availability of an in vitro model incorporating a mucus barrier is of key importance when evaluating the mucosal drug permeation of oral drug candidates or delivery systems. A biosimilar mucus matrix with in vivo relevant composition and rheological properties has recently been described [10,11]. The biosimilar mucus is composed of mucin, bovine serum albumin (BSA), polyacrylic acid (PAA), cholesterol, phosphatidylcholine and linoleic acid. The mixture is biocompatible; thus it can be applied to a monolayer of cultured cells, which then constitute a suitable in vitro model for the intestinal mucosa for studies of mucus penetration and transmucosal drug permeation. It was demonstrated that this biosimilar mucus forms a diffusion barrier to small molecules such as testosterone and mannitol and hydrophilic macromolecules as dextrans (4 and 10 kDa) and BSA [11]. It is also known that the properties of mucus vary depending on whether they are investigated on a micro- or macroscale [3,12]; purified porcine gastric mucin was e.g. found to have a 100-fold
http://dx.doi.org/10.1016/j.ejpb.2015.01.014 0939-6411/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Boegh et al., Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.014
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lower microscopic viscosity as compared to the macroscopic viscosity [13]. It is therefore of importance to characterize mucus at the microscale as well as the macroscale. The overall purpose of the present study was to increase the knowledge about the steric and interactive barriers of mucus to nanoparticle and peptide delivery by further investigations using the biosimilar mucus and porcine intestinal mucus (PIM) applied onto agar-coated filters in a novel cell-free model, or onto Caco-2 monolayers as the combined model developed previously [11] (Fig. 1). Increased evidence of the importance of implementing these barriers in in vitro models is crucial in order to improve the design of future drug delivery systems for successful oral administration of biomacromolecules. Thus, in the present study we aimed to gain further insight into the functional importance of the different components of biosimilar mucus and the resemblance to PIM in terms of mucus microstructure. This is supplemented by investigations of the barrier properties in relation to the permeation of a range of drugs across biosimilar mucus or PIM and the combined in vitro model, respectively. 2. Materials and methods 2.1. Materials Fluorescein-isothiocyanate (FITC) dextran of 4000 (FD4) and 10,000 Da (FD10) and FITC-labeled BSA (FITC-BSA) were purchased from Sigma–Aldrich (Broendby, Denmark). 3H-metoprolol (40–80 Ci/mmol) was purchased from American Radiolabeled Chemicals (Saint Louis, MO, USA), 3H-testosterone (85–105 Ci/mmol) and 14C-mannitol (45–60 mCi/mmol) from Perkin Elmer (Skovlunde, Denmark). Desmopressin acetate was purchased from Zhejiang Medicines & Health Products I/E (Chengguan Town, China), octreotide acetate from PolyPeptide Group (Hilleroed, Denmark), vancomycin hydrochloride hydrate from Sigma– Aldrich, plectasin and novicidin were kindly supplied by Novozymes A/S (Copenhagen, Denmark). Mucin from porcine stomach type II, bovine serum albumin (BSA) (P98%), linoleic acid (>99%), cholesterol (>99%), polysorbate 80 (Tween 80), agar, Hank’s Balanced Salt Solution (HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin, L-glutamine, non-essential amino acids, phenazine methosulfate (PMS) and trifluoroacetic acid (TFA), Alcian Blue 8GX (AB) and DPX Mountant were purchased from Sigma Aldrich. Polyacrylic acid (PAA) (CarbopolÒ 974P NF) was purchased from Lubrizol (Brussels, Belgium), phosphatidylcholine (PC, purity 98%) from Lipoid (Ludwigshafen, Germany), hydroxyethyl piperazineethanesulfonic acid (HEPES) from AppliChem (Darmstadt, Germany), fetal bovine serum (FBS) from Fisher Scientific (Slangerup, Denmark), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) from Promega (Madison, WI, USA), Ultima Gold Scintillation fluid from Perkin Elmer and Optimal Cutting Temperature (OCT) medium from VWR Chemicals (Herlev,
Denmark). All other chemicals were obtained commercially at analytical grade and the chromatographic solvents at HPLC grade. 2.2. Mucus sample preparation The small intestine was obtained from three pigs immediately after euthanization. The pigs were around three months of age and 30 kg, and fasted for 24 h prior to surgery. The intestine was cut into shorter pieces and rinsed with 10 mM HEPES isotonic buffer containing 1.3 mM CaCl2, 1.0 mM MgSO4 and 137 mM NaCl in order to remove undigested matter. Using a spoon, the mucus was gently scraped off the luminal side, collected, aliquoted and stored at 20 °C until further use. Mucus was obtained from three different pigs and named PIM1, PIM2 and PIM3, respectively. The animals were handled as approved by the Danish Animal Experiments Inspectorate (license number 2012-15-2934-00077). The isolated PIM was analyzed on a cone and plate rheometer as described previously [11] and normalized by dilution to display comparable rheological profiles. The PIM used for DWS was undiluted. Biosimilar mucus was prepared in 10 mM HEPES buffer with 1.3 mM CaCl2 and 1.0 mM MgSO4 as previously described [11]. Briefly, 0.9% (w/v) PAA was dissolved under magnetic stirring and 5% (w/v) mucin was added. The pH of the PAA and mucin solution was increased toward neutral by addition of NaOH prior to the addition of the lipid mixture. The lipid mixture was prepared separately with polysorbate 80 and added to the mucus to final concentrations of 0.36% (w/v) cholesterol, 0.18% (w/v) PC, 0.11% (w/v) linoleic acid and 0.16% (w/v) polysorbate 80. Finally, 3.1% (w/v) BSA was added to the mucus mixture and the pH was adjusted to 7.4 with NaOH. For some experiments, the linoleic acid or the whole lipid mixture was omitted. The biosimilar mucus was prepared one day prior to the experiment and stored overnight at 4 °C. 2.3. Diffusive wave spectroscopy (DWS) Diffusive wave spectroscopy (DWS) measurements were performed on biosimilar mucus and PIM with the transmission mode setting using a DWS Research Lab (LS Instruments, Fribourg, Switzerland) equipped with a laser beam of wavelength 685 nm. Samples were prepared and placed in disposable cuvettes with 5 mm optical path length. PolybeadÒ polystyrene tracer particles (Polysciences, Warrington, PA, USA) with a diameter of 456 nm were added to the mucus samples to a concentration of 0.65% (w/w). Samples were measured at 37 °C, using an integrated Peltier temperature controller and equilibrated for 10 min at the required temperature prior to the measurement. The scattered light was collected and cross-correlated with a single-photoncounting avalanche photodiode correlator (APD). The Rheo Lab software (LS instruments) was used for data collection and analysis.
Fig. 1. Schematic representation of the mucus containing in vitro models: PIM or biosimilar mucus applied onto agar-coated Transwell filters (A) and biosimilar mucus applied onto Caco-2 cell monolayers (B).
Please cite this article in press as: M. Boegh et al., Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.014
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2.4. Cell culturing Caco-2 cells (from DSMZ Braunschweig, Germany) were cultured in DMEM supplemented with 10% (v/v) FBS, penicillin/streptomycin (100 U/ml and 100 lg/ml), 1% (v/v) L-glutamine and 1% (v/ v) non-essential amino acids at 37 °C in an atmosphere of 5% CO2. The T75 culture flasks were passaged weekly. The cells were seeded at a density of 1 105 cells/insert onto 12-well polycarbonate TranswellÒ filter inserts (1.12 cm2 growth area, 0.4 lm pore size) (Corning Costar, Sigma–Aldrich). The medium at the apical and basolateral side of the cell layer was replaced every other day and cells of passages 3–9 after thawing were seeded and used after 18 or 20 days of culturing. 2.5. Mucus and mucosa permeability Four hour long permeability experiments were carried out at 37 °C on a horizontal shaker at 90 rpm exploring the mucus alone or in combination with an epithelium. Agar-coated filter inserts were used for mucus permeability studies without the epithelium. A total amount of 2% (w/v) agar was dissolved in ultrapure water from Barnstead NanoPure Systems (Thermo Scientific, Waltham, MA, USA) by microwave heating. A volume of 200 ll was added to each 12-well polycarbonate TranswellÒ filter insert (Corning Costar, Sigma–Aldrich), allowed to dry at room temperature for 30 min and stored overnight at 4 °C. For permeability experiments using the combined in vitro model incorporating the Caco-2 cell epithelium, the transepithelial electrical resistance (TEER) was measured prior to and following the experiments after an equilibration period of 20 min at room temperature with the assay buffer (10 mM HEPES HBSS, pH 7.4). After the permeation experiments, the TEER values of the cell monolayers were, unless stated otherwise, comparable with the starting value, which in all cases were between 430 and 550 O cm2. For comparison the resistance of the agar-coated filter inserts was approximately 10 O cm2. The inserts with agar coating or a Caco-2 cell monolayer were covered with 250 ll of buffer, biosimilar mucus or PIM (PIM2 and PIM3). Then, a volume of 100 ll test solution was added to the apical compartment with the following concentrations: 0.13, 0.66 and 3.32 mg/ml novicidin, 10.6 mg/ml plectasin, 14.9 mg/ml vancomycin, 10.7 mg/ml desmopressin and 10.2 mg/ml octreotide. The concentration of 14C-mannitol was 0.2 lCi/insert, whereas 0.1 lCi/insert was used for 3H-testosterone and 3H-metoprolol. Samples of 100 ll were taken from the basolateral compartment at fixed time points during the four hours and replaced with prewarmed assay buffer. A volume of 5 ll 1% (v/v) formic acid in methanol was added to each HPLC sample to prevent further enzymatic degradation and the samples were stored at 20 °C until analysis. 2.6. Analytical methods The samples from the peptide permeability experiments were analyzed by reverse-phase high-pressure liquid chromatography (HPLC) (Shimadzu, Prominence system, Kyoto, Japan) using a Kinetex (Phenomenex, Vaerloese, Denmark) C18 (50 4.6 mm) column and water/acetonitrile-based solvents containing 0.1% (v/ v) trifluoroacetic acid (TFA). The test compounds were eluted using a linear gradient of solvent B (95:5:0.1 (v/v/v) acetonitrile/water/ TFA) in solvent A (95:5:0.1 (v/v/v) water/acetonitrile/TFA) from 10% to 40% over 3.5 min at a flow rate of 1.85 ml/min. A PDA detector (SPD-M20A, Shimadzu) was set to record the 200–400 nm range. In the case of desmopressin the gradient of solvent B in solvent A was from 0% to 30% over 3.5 min. Vancomycin was quantified using a Kinetex C18 (50 2.1 mm) column and a gradient
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from 2% to 8% over 12 min at a flow rate of 0.5 ml/min. The injection volume was 10 ll for all samples. The compounds were quantified using automatic integration of the peak at 218 nm. The limits of detection (LOD) for novicidin, plectasin, octreotide, desmopressin and vancomycin were 1.4 lg/ml, 2.4 lg/ml, 1.6 lg/ml, 1.3 lg/ml and 3.7 lg/ml (n = 3), respectively. The samples from the permeability experiments with radiolabelled isotopes were analyzed on a Tri-Carb 2100 TR liquid scintillation analyzer (Canberra Packard, Dreieich, Germany). Each sample was mixed with 2 ml of Ultima Gold scintillation fluid prior to the analysis. The apparent permeability coefficient (Papp) was calculated according to Eq. (1):
Papp ¼ ðdQ =dtÞ 1=ðA C 0 Þ
ð1Þ
where dQ/dt is the steady state rate of permeation, A is the diffusion area and C0 the initial donor concentration. 2.7. Cellular viability The cellular viability after the permeability experiments was determined by utilizing the MTS/PMS assay. Cell monolayers were gently washed twice with the assay buffer and 320 ll of freshly prepared MTS/PMS solution was added. After 1.25 h of incubation on a horizontal shaker at 90 rpm and 37 °C, 100 ll of apical reagent mixture was transferred to a 96-well plate and the absorbance was measured at 492 nm on a FLUOstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany). 2.8. Microscopy Agar-coated filter inserts were covered with 250 ll of PIM or biosimilar mucus and frozen at 20 °C. The filter inserts were then removed from their plastic supports, cut it into a strip of few millimeters width and embedded between two thin slices of chicken liver in order to preserve the mucus during the processing. The sandwich was snap frozen in liquid nitrogen and mounted in OCT medium and cut into 10 lm sections using a Leica cryostat CM3050 S (Leica Microsystems, Ballerup, Denmark) and collected onto SuperFrostÒ plus microscope slides. Sections on slides were allowed to air dry and were then used immediately or stored at 20 °C until use. The slides were defrosted and stained with 1% (w/v) Alcian Blue in 3% (v/v) acetic acid (pH 2.5) for 10 min. Sections were gently rinsed with tap water, dehydrated in toluene and mounted in DPX. Imaging was performed on a Zeiss Axio Scan Z1 brightfield slide scanner (Carl Zeiss Microscopy, Jena, Germany). Images were processed using ZEN 2012 lite software from Zeiss. The mucus thickness was measured for approximately every 250 lm and averaged. 2.9. Statistics GraphPadPrism (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The significance between two findings was determined by the student’s t test or by the one-way ANOVA for multiple comparisons, using in both cases a 95% confidence interval. 3. Results and discussion 3.1. The steric barrier property of biosimilar mucus is independent of lipid content In order to investigate the effect of the different components of the biosimilar mucus on the micro-rheological properties, diffusive wave spectroscopy (DWS) measurements were carried out using
Please cite this article in press as: M. Boegh et al., Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.014
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polystyrene tracer particles. The DWS technique offers much more spatial and temporal resolution, which cannot be proved in a mechanical rheometer, yet as compared to microscope-based methodologies applied for particle tracking the actual diffusion distance may be considered low. The analysis of the normalized intensity correlation function provided the mean squared displacement (MSD) of the particles in the different variants of biosimilar mucus. Assuming that there are no differences in the chemical adhesive interactions between the polystyrene particles and the different mucus matrices, the differences in the diffusion of the particles would mainly be governed by the steric barrier. The diffusion of the particles is reflected in the slope a of the logarithmic MSD vs. logarithmic time scale plot, where a is known as the anomalous diffusion coefficient (a = 1 for pure Brownian diffusion). The tracer particles present subdiffusive motion in all mucus samples (Fig. 2A). No significant differences in the a value of the particles were observed when measured in biosimilar mucus devoid of lipids as compared to a value in the biosimilar mucus composed as described previously (Fig. 2B) [11]. Thus, the lipid components are not causing a rearrangement of the matrix of the biosimilar mucus to a degree that influences the diffusion of particles with a diameter around 450 nm. Indications of faster mobility of the particles in the biosimilar mucus without lipids can be perceived, though not statistically different. This tendency would correlate with the previous findings describing an effect of the lipids in the macrostructure of the biosimilar mucus [11]. The particle diffusion was significantly increased when the synthetic polymer, polyacrylic acid (PAA), in the biosimilar mucus was omitted from the mixture. As previously described, the use of this non-natural mucus component was necessary to counteract the low viscosity of commercial mucins when used at relevant concentrations in order to obtain a macrorheological profile similar to PIM [11]. Interestingly, particles in the PAA gel diffused even more freely
Fig. 2. Mean squared displacement of 456 nm polystyrene tracer particles at 37 °C in water (black), 0.9% (w/v) of polyacrylic acid (PAA, blue), biosimilar mucus without PAA (red), without lipids (green), without linoleic acid (LA, purple) or complete biosimilar mucus (orange) (A). Solid lines represent mean values and dotted lines represent SD (n = 3). Anomalous diffusion coefficients (a) of the tracer particles in the different mucus samples (B). Data represent mean ± SD (n = 3). n.s. not significant, ⁄⁄⁄p < 0.001. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
as compared to the biosimilar mucus without PAA, indicating a larger mesh spacing of the matrix formed by the polymer alone. In order to elaborate on the previous comparison of PIM to biosimilar mucus [11], undiluted mucus samples from two animals were included in the study. It was, however, clear that the contribution of self-scattering from the PIM was too high to extract the MSD from the normalized intensity correlation function. Further, the correlation function revealed an enormous difference between the PIM isolations (Fig. 3), which was also observed visually during the sample handling and in accordance with measurements on the macrorheology (Fig. S1A) and also from previous studies on intestinal mucus [11,14].
3.2. Mucus covered agar-coated inserts constitute a relevant in vitro mucus model It was previously reported that exposing well-differentiated Caco-2 cell monolayers to porcine intestinal mucus (PIM) was detrimental to the epithelium, as measured by a decreased transepithelial electrical resistance as well as a reduced cellular viability [11]. Therefore, a mucus barrier model that would allow for comparison between different PIM samples, as well as comparison of biosimilar mucus to PIM, was necessarily a cell-free model. This simplified model was established utilizing agar-coated filter inserts supplemented with the relevant mucus sample. The mucus layer thickness was measured in order to extrapolate information from results obtained using this model. Sections of the minimal applicable volume of biosimilar mucus and PIM applied to agarcoated filter inserts that would ensure full coverage of the filter were imaged after Alcian Blue staining (Fig. 4). In both cases the mucus was evenly distributed across the filter area and the thickness of biosimilar mucus and PIM was found to be 800 ± 190 lm and 1050 ± 210 lm (n > 15), respectively. These values correspond well with the mean mucus thickness of 830 ± 110 lm found in the colon of rats, and is about 4-fold thicker than the value measured in the rat duodenum [15]. The Alcian Blue staining of biosimilar mucus was more diffuse than for the PIM, which structure was more granular, probably due to the degradation of the commercially available mucin used for the biosimilar mucus preparation. In order to validate the use of this mucus model for permeability studies, the effect of mucus on the permeability of model compounds was studied. Since the agar-coated filter insert does not constitute a biological barrier, the absolute values of the apparent permeability coefficient (Papp) should not be compared. Thus, the fold-reduction in the permeability of biosimilar mucus applied to the cell-free model was compared to those previously found with the biosimilar mucus applied to Caco-2 cell monolayers
Fig. 3. The correlation function of 456 nm polystyrene tracer particles in water (black), porcine intestinal sample 1 (PIM1, dark gray), and PIM2 (light gray). Data represent mean values (n = 3).
Please cite this article in press as: M. Boegh et al., Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.014
M. Boegh et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx
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Fig. 4. Alcian Blue staining of cryosections of biosimilar mucus (A) or porcine intestinal mucus (B) applied to TranswellÒ inserts (T) coated with agar (Ag) and embedded between slices of chicken liver tissue (L). Size bars represent 500 lm. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
[11]. Overall, comparable results were obtained with the cell-free model with regard to reduction in the apparent permeability coefficient (Papp) of the hydrophilic and lipophilic model compounds investigated (Fig. 5). It should be mentioned that for FD4, a higher level of reduction by the presence of mucus was observed in the cell-free model as compared to when the mucus was applied to the Caco-2 model. This is believed to be due to slightly higher variations in the raw data from the cell-free model, which was used to calculate the reduction. Thus, it appears that agar coating of the filter inserts could be a useful alternative to using cultured cell monolayers, especially since this would also allow studies on the barrier properties of PIM. As the PIM2 and PIM3 samples used in the present study displayed a higher viscosity as compared to PIM1 previously used, they were difficult to handle and dilution was necessary in order to apply a mucus layer on to the agar coatings. Thus, PIM2 was diluted 40% (v/w) and PIM3 was diluted 25% (v/w) with an isotonic version of the buffer used for preparation of the biosimilar mucus in order to obtain a macrorheological profile
corresponding to PIM1 (Fig. S1). The reduction in the Papp value of the hydrophilic macromolecules by the presence of PIM tended to be higher as compared to the reduction caused by biosimilar mucus. For one of the PIM samples the permeability reduction increased with increasing molecular weight of the macromolecules as observed previously, while no clear pattern was observed for the other PIM sample (Fig. 5). Generally, larger differences in the reduction caused by the two PIM samples were observed in the permeability studies with macromolecules compared to those with small molecules. This finding is possibly due to a greater impact of the heterogeneity of the mesh spacing of native mucus on the diffusion of high molecular weight compounds as compared to small molecules. As previously mentioned, the agar displays different barrier properties as compared to the epithelium, thus small hydrophilic compounds have a higher Papp value than lipophilic compounds when using the agar model (Fig. S2), whereas the opposite is the case for epithelium monolayers. Still, the agar model seems to be a good alternative to the mucus-covered cell culture monolayers for the evaluation of the relative effect of mucus as a barrier to drug permeation. Also, the time and costs for culturing of cells can be avoided and the biological variation related to using cell culture models is also avoided. 3.3. The interactive barrier property of biosimilar mucus is not affected by the linoleic acid content
Fig. 5. Reduction in apparent permeability coefficient (Papp) of the test compounds by biosimilar mucus applied to Caco-2 cells monolayers (black bars) or agar coated filter inserts (gray bars), or two different PIM secretions applied to agar-coated filter inserts (striped bars). With the exception of estradiol results, the data from biosimilar mucus applied to Caco-2 cells monolayers (black bars) are previously reported [11] and have been included for comparative reasons. FD4: Fluorescein isothiocyanate (FITC)-dextran 4,000, FD10: FITC-dextran 10,000, FITC-BSA: FITCbovine serum albumin. Data represent means ± SD (n = 3–6).
As the presence of linoleic acid and other lipids in the biosimilar mucus had no significant effect on the diffusion of tracer particles (Fig. 2) it was relevant to investigate whether the presence of lipid was important for the interactive barrier properties of biosimilar mucus. This would increase the general applicability of the mucus, as the addition of a fatty acid-containing test solution was shown to render the otherwise biocompatible mucus toxic to the underlying Caco-2 cell monolayers (Fig. S3). Linoleic acid has earlier been shown to be the most critical component for biocompatibility of the mucus mixture [10,11] and it was hypothesized that the addition of e.g. capric acid as a permeability enhancer increased the total fatty acid content above the threshold for cellular compatibility. The relative reduction in the Papp value for permeation of mannitol, metoprolol and testosterone was unaffected by the presence or absence of linoleic acid in the biosimilar mucus applied to
Please cite this article in press as: M. Boegh et al., Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.014
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phosphatidylcholine in the general biosimilar mucus barrier properties. Thus, the omission of linoleic acid from the biosimilar mucus broadens the applicability of the model by ensuring compatibility with Caco-2 cell monolayers also when lipid-based formulations or lipid excipients are tested. Previously, differences in mucus microstructure between biosimilar mucus with and without all the lipids have been observed by cryo-SEM [11] and a tendency toward an increase particle mobility was observed by DWS when the lipids were not included in the mucus. Thus, cholesterol and phosphatidylcholine seem to influence the microstructure to a degree, and these lipids should be included to resemble the properties of native mucus. 3.4. The permeation of peptide drugs across biosimilar mucus varies with molecular properties Fig. 6. The reduction in apparent permeability of mannitol, metoprolol and testosterone across agar-coated filter insert caused by biosimilar mucus (black columns) or biosimilar mucus without linoleic acid (gray columns). Data represent mean values ± SD (n = 3).
agar-coated inserts (Fig. 6). In line with this, two separate experiments demonstrated that the Papp value for the permeation of mannitol and metoprolol across biosimilar mucus or biosimilar mucus without any lipids applied to Caco-2 cell monolayers did not differ significantly (Fig. S4). Furthermore, it was shown that the permeation of three peptide drugs across biosimilar mucus without linoleic acid or without any lipids applied to Caco-2 cell monolayers was also comparable (Fig. S5). Overall, the data from studies of the biosimilar mucus in terms of macrorheology, microstructure and permeation of small molecules and peptide drugs imply that there is no effect of the presence of linoleic acid or the other two lipids, cholesterol and
The barrier property of biosimilar mucus without linoleic acid was further studied by assessing the permeation of five different peptides (Table 1) across Caco-2 cell monolayers in the absence and presence of biosimilar mucus. The Papp values were all obtained at steady state (Fig. S6) and the permeation of octreotide and desmopressin across Caco-2 cell monolayers was comparable, which corresponds well with their structural similarities (Table 2), and in the range of previously published data for desmopressin [21,22]. However, the reduction of the Papp value by the presence of biosimilar mucus was for octreotide more than 2-fold higher than the reduction for desmopressin (Table 2). Octreotide has one hydrophobic residue more than desmopressin and thereby more potential for hydrophobic interactions with mucus components, and also a more pronounced cyclic structure. For vancomycin the reduction in permeation caused by mucus was around 7-fold probably due to the more bulky structure and the fact that vancomycin has a significantly higher number of
Table 1 The amino acid sequence, molecular weight (Mw) and the isoelectric point (pI) of the five peptides. Single letter denotation of amino acid residues is used. Cationic residues are marked with red and hydrophobic residues with blue.
Octreotide Desmopressin
Amino acid sequence or structure
Peptide Mw (Da)
H-D-F-C-F-W-K-T-C-T(ol) SCH2CH2CO- Y-F-Q-N-C-P-R-G -NH2
1019
Vancomycin
Novicidin Plectasin
K-N-L-R-R-I-I-R-K-G-I-H-I-I-K-K-Y-F [16] G-F-G-C-N-G-P-W-D-E-D-D-M-Q-C-H-N-H-CK-S-I-K-G-Y-K-G-G-Y-C-A-K-G-G-F-V-C-K-C-Y
pI 8.06
1069
8.22
1485
7.20
2297
11.75
4408
8.85
[17]
The Mw and pI were calculated from the sequences using the ProtParam tool [18], except for vancomycin [19] and plectasin [20]. The structure of vancomycin is shown instead of the sequence, as it is a glycosylated peptide with non-proteinogenic amino acids.
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Table 2 The apparent permeability coefficients (Papp) for the permeation of five peptides across Caco-2 cell monolayers and across the mucosa model; i.e. biosimilar mucus covered epithelium and the reduction in Papp by two samples of porcine intestinal mucus (PIM).
Octreotide Desmopressin Vancomycin Novicidin Plectasin
Papp epithelium (108 cm/s ± SD)
Papp mucosa (108 cm/s ± SD)
Fold reduction by biosimilar mucus
Fold reduction by PIM2/PIM3
15.5 ± 2.5 14.4 ± 3.5 19.2 ± 5.7 861 ± 55a 6.2 ± 2.3
2.8 ± 0.4 7.4 ± 0.7 2.6 ± 0.7
5.5 1.9 7.2 – –
6/40 33/46 36/75 ND/7 145/151
LOD: limit of detection. a The integrity of the cell monolayer was compromised. The Papp values are means ± SD, (n = 6–9), conducted on 2–3 different cell passages. ND: not detected. The reduction caused by PIM is calculated from mean of triplicates. Biosimilar mucus without linoleic acid was used.
hydrogen bond acceptors and donors and a greater potential for electrostatic interactions [23], as compared to desmopressin and octreotide. Thus, extensive interaction between vancomycin and mucus is likely the reason for the reduced permeation of this compound. The diffusion of macromolecules in porcine mucus was previously found to decrease with increasing molecular weight, when peptide and proteins in the range 14.4–186 kDa and 3.4–66 kDa were tested in gastric and intestinal mucus, respectively [24,25]. Although, smaller peptides are tested here, the same tendency may apply. Further, a great reduction in the diffusion of anionic and especially cationic synthetic peptides in reconstituted mucin gels as compared to in water has been demonstrated, whereas it was not the case for a peptide composed of a cationic as well as anionic block and hence with a lower net charge [26]. No permeation of plectasin and novicidin across the mucosa model could be detected. Thus, it is likely that these larger, more cationic and structured peptides interact strongly with the negative mucin fibers in addition to forming hydrophobic interactions, which limits their diffusion significantly. Permeated novicidin could only be detected when using the highest concentration. However, the viability of the Caco-2 cell monolayers was reduced to approximately 20% at this condition (Fig. 7A) and this disruption of the cell monolayer resulted in a high flux and Papp value (Table 2). Novicidin is an unstable peptide [18] with known eukaryotic cellular toxicity at low concentrations as confirmed by the concentration-dependent decrease in viability of the epithelial cells (Fig. 7A). There was a similar trend for the effect on the epithelium covered by biosimilar mucus, but interestingly the biosimilar mucus to a large degree protected the underlying epithelium (Fig. 7A). In comparison, exposure to plectasin reduced the cellular viability of the epithelium only to 85%, whereas plectasin was fully tolerated in the presence of biosimilar mucus (Fig. 7B) and neither desmopressin, octreotide nor vancomycin had any negative effect on the cellular viability under the tested conditions. 3.5. Porcine intestinal mucus reduces peptide permeation with large variations When assessing the permeation of peptide drugs across porcine intestinal mucus the reduction caused by PIM was in most cases found to be significantly higher than the reduction caused by biosimilar mucus (Table 2). The reduction in permeation of octreotide and vancomycin varied greatly between the two PIM samples, while the reduction in permeation of desmopressin and plectasin was in the same range for both samples. As the molecular weight of desmopressin and octreotide is comparable, these findings suggest that it is the interactive barrier more than the steric barrier, which differs between the two PIM samples. Overall, in most cases the PIM on agar seemed to reduce the peptide permeation to a slightly greater extent than the biosimilar mucus on Caco-2 cells, while similar results were obtained for the reduction of octreotide
Fig. 7. The viability of Caco-2 cells in monolayers after four hours of incubation with novicidin (A) or desmopressin, octreotide, vancomycin and plectasin (B) in the absence or presence of biosimilar mucus without linoleic acid (BM) covering the Caco-2 epithelium. Different concentrations of novicidin were tested and for the other peptides, the concentrations were: 10.6 mg/ml plectasin, 14.6 mg/ml vancomycin, 10.7 mg/ml desmopressin and 10.2 mg/ml octreotide. Data represent means ± SD (n = 3) and B is representative of two individual experiments. ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.
permeation by PIM2 and biosimilar mucus. This observation supports that the specific physicochemical properties of the permeant as well as the complexity of the mucus determines the overall permeation. However, the large variation between the results from the two PIM samples precluded to rank the peptides with respect to the reduction and therefore also the comparison to the interactive barrier properties of biosimilar mucus. Thus, the great variability of
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porcine intestinal mucus reconfirms one of the main drawbacks of this model, whereas biosimilar mucus provides a reproducible and robust alternative to in vitro testing of mucus barrier properties.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2015.01.014.
4. Conclusion References The steric barrier properties of the biosimilar mucus are to some extent maintained by the non-natural polymer component in the biosimilar mucus, PAA, but PAA is in itself less of a steric barrier as compared to the mixture of the other biosimilar mucus components. At the microscale, the polymer itself constitutes a less significant steric barrier as compared to the mixture with the natural components in the biosimilar mucus. The presence of linoleic acid, was found not to be crucial for the steric or interactive barrier properties of the biosimilar mucus, and is suggested to be omitted from the mucus matrix to broaden the applicability of the mucosa model e.g. to allow for testing of lipid-based formulations or excipients. Mucus permeation studies utilizing a cell-free alternative to the mucosal in vitro model composed of the biosimilar mucus and Caco-2 cell monolayers demonstrated to be advantageous by allowing studies with PIM, which is not compatible with cells, but merely to investigate a relative influence of the mucus barrier. The transmucosal permeation of five peptide drugs with varying physicochemical properties illustrated the barrier properties of the biosimilar mucus combined with epithelium. The corresponding study using PIM revealed large variations between the two PIM samples, which impeded conclusions regarding the barrier effect. In general, the reduction on peptide permeability was significantly higher for PIM compared to the biosimilar mucus, probably due to differences in the mucin network integrity. In conclusion, the steric and interactive barrier properties of biosimilar mucus have been further elucidated. Although the differences in peptide permeation and mucin network integrity, the biosimilar mucus appears to be a useful and more reproducible alternative to native mucus for mechanistic studies on mucus diffusion and mucosal drug permeation. Acknowledgments Novozymes A/S is acknowledged for kindly supplying the novicidin and plectasin. Special thanks to Dr. Marc Obiols Rabasa at Lund University and the European Commision under the Seventh Framework Program for technical and financial support for conducting DWS studies as part of the European Soft Matter Infrastructure (ESMI) Project (Grant No. 262348). Thanks to Laboratory Technician Karina Vissing and PhD Fellow Søren Steffensen for great assistance with the HPLC analysis. DVM Camilla Schumacher-Petersen and Animal Technician Mette Olesen from Department of Experimental Medicine, University of Copenhagen, are acknowledged for providing the porcine small intestine. This work was funded by the Drug Research Academy, The Danish Agency for Science, Technology (mobility stipend and the innovation consortium Predicting Drug Absorption). Furthermore, the research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115363 resources, which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution.
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Research paper
Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation Marie Boegh, María García-Díaz, Anette Müllertz, Hanne Mørck Nielsen ⇑ Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen O, Denmark
a r t i c l e
i n f o
Article history: Received 18 October 2014 Accepted in revised form 14 January 2015 Available online xxxx Keywords: Biosimilar mucus Mucosal drug delivery Oral Caco-2 cell culture model Diffusion Peptides Nanoparticles Diffusive wave spectroscopy
a b s t r a c t The mucus lining of the gastrointestinal tract epithelium is recognized as a barrier to efficient oral drug delivery. Recently, a new in vitro model for assessment of drug permeation across intestinal mucosa was established by applying a biosimilar mucus matrix to the surface of Caco-2 cell monolayers. The aim of the present study was to gain more insight into the steric and interactive barrier properties of intestinal mucus by studying the permeation of peptides and model compounds across the biosimilar mucus as well as across porcine intestinal mucus (PIM). As PIM disrupted the Caco-2 cell monolayers, a cell-free mucus barrier model was implemented in the studies. Both the biosimilar mucus and the PIM reduced the permeation of the selected peptide drugs to varying degrees illustrating the interactive properties of both mucus matrices. The reduction in peptide permeation was decreased depending on the cationicity and H-bonding capacity of the permeant clearly demonstrated by using the biosimilar mucus, whereas the larger inter sample variation of the PIM matrix obstructed similarly clear conclusions. Thus, for mechanistic studies of permeation across mucus and mucosa the biosimilar mucus offers a relevant and reproducible alternative to native mucus. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Mucus is a viscoelastic hydrophilic gel mainly structured by a network of high molecular weight extensively glycosylated mucin fibers. It lines the mucosal surfaces of the body including the gastrointestinal (GI) tract, where it lubricates and protects the underlying epithelium from toxic substances and pathogens, while enabling the exchange of nutrients [1]. Mucus is composed mainly of water (90–95%) in addition to 2–5% of mucins and smaller quantities of lipids, proteins and minor amounts of electrolytes and DNA [2]. Mucin molecules form a crosslinked and entangled network and are key responsible for the hydrogel-like properties of mucus [3]. The brush-like structure and the mesh space of the network can act as a size exclusion filter reducing the mobility of large molecules [2,4,5]. In addition to this steric barrier, mucus also comprises an important interactive component as the formation of multiple non-covalent bonds may immobilize a drug or particle in the mucus network [2,5,6]. The non-glycosylated regions of the mucin protein backbone and the lipids associated to the mucins allow for hydrophobic interactions with drugs leading to ⇑ Corresponding author. Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O, Denmark. Tel.: +45 3533 6346; fax: +45 3533 6001. E-mail address: [email protected] (H.M. Nielsen).
decreased diffusion rates in the mucus [1,7]. The oligosaccharides O-linked to the mucin backbone constitute 40–80% by weight of the mucins [8]. Many of these oligosaccharides carry a terminal carboxyl group or ester sulfate groups, which gives mucus a net negative charge and the capacity of forming electrostatic interactions in addition to hydrogen bonds [1]. Furthermore, the dynamic barrier also play a role for the overall mucus barrier properties [9]. Therefore, the availability of an in vitro model incorporating a mucus barrier is of key importance when evaluating the mucosal drug permeation of oral drug candidates or delivery systems. A biosimilar mucus matrix with in vivo relevant composition and rheological properties has recently been described [10,11]. The biosimilar mucus is composed of mucin, bovine serum albumin (BSA), polyacrylic acid (PAA), cholesterol, phosphatidylcholine and linoleic acid. The mixture is biocompatible; thus it can be applied to a monolayer of cultured cells, which then constitute a suitable in vitro model for the intestinal mucosa for studies of mucus penetration and transmucosal drug permeation. It was demonstrated that this biosimilar mucus forms a diffusion barrier to small molecules such as testosterone and mannitol and hydrophilic macromolecules as dextrans (4 and 10 kDa) and BSA [11]. It is also known that the properties of mucus vary depending on whether they are investigated on a micro- or macroscale [3,12]; purified porcine gastric mucin was e.g. found to have a 100-fold
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lower microscopic viscosity as compared to the macroscopic viscosity [13]. It is therefore of importance to characterize mucus at the microscale as well as the macroscale. The overall purpose of the present study was to increase the knowledge about the steric and interactive barriers of mucus to nanoparticle and peptide delivery by further investigations using the biosimilar mucus and porcine intestinal mucus (PIM) applied onto agar-coated filters in a novel cell-free model, or onto Caco-2 monolayers as the combined model developed previously [11] (Fig. 1). Increased evidence of the importance of implementing these barriers in in vitro models is crucial in order to improve the design of future drug delivery systems for successful oral administration of biomacromolecules. Thus, in the present study we aimed to gain further insight into the functional importance of the different components of biosimilar mucus and the resemblance to PIM in terms of mucus microstructure. This is supplemented by investigations of the barrier properties in relation to the permeation of a range of drugs across biosimilar mucus or PIM and the combined in vitro model, respectively. 2. Materials and methods 2.1. Materials Fluorescein-isothiocyanate (FITC) dextran of 4000 (FD4) and 10,000 Da (FD10) and FITC-labeled BSA (FITC-BSA) were purchased from Sigma–Aldrich (Broendby, Denmark). 3H-metoprolol (40–80 Ci/mmol) was purchased from American Radiolabeled Chemicals (Saint Louis, MO, USA), 3H-testosterone (85–105 Ci/mmol) and 14C-mannitol (45–60 mCi/mmol) from Perkin Elmer (Skovlunde, Denmark). Desmopressin acetate was purchased from Zhejiang Medicines & Health Products I/E (Chengguan Town, China), octreotide acetate from PolyPeptide Group (Hilleroed, Denmark), vancomycin hydrochloride hydrate from Sigma– Aldrich, plectasin and novicidin were kindly supplied by Novozymes A/S (Copenhagen, Denmark). Mucin from porcine stomach type II, bovine serum albumin (BSA) (P98%), linoleic acid (>99%), cholesterol (>99%), polysorbate 80 (Tween 80), agar, Hank’s Balanced Salt Solution (HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin, L-glutamine, non-essential amino acids, phenazine methosulfate (PMS) and trifluoroacetic acid (TFA), Alcian Blue 8GX (AB) and DPX Mountant were purchased from Sigma Aldrich. Polyacrylic acid (PAA) (CarbopolÒ 974P NF) was purchased from Lubrizol (Brussels, Belgium), phosphatidylcholine (PC, purity 98%) from Lipoid (Ludwigshafen, Germany), hydroxyethyl piperazineethanesulfonic acid (HEPES) from AppliChem (Darmstadt, Germany), fetal bovine serum (FBS) from Fisher Scientific (Slangerup, Denmark), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) from Promega (Madison, WI, USA), Ultima Gold Scintillation fluid from Perkin Elmer and Optimal Cutting Temperature (OCT) medium from VWR Chemicals (Herlev,
Denmark). All other chemicals were obtained commercially at analytical grade and the chromatographic solvents at HPLC grade. 2.2. Mucus sample preparation The small intestine was obtained from three pigs immediately after euthanization. The pigs were around three months of age and 30 kg, and fasted for 24 h prior to surgery. The intestine was cut into shorter pieces and rinsed with 10 mM HEPES isotonic buffer containing 1.3 mM CaCl2, 1.0 mM MgSO4 and 137 mM NaCl in order to remove undigested matter. Using a spoon, the mucus was gently scraped off the luminal side, collected, aliquoted and stored at 20 °C until further use. Mucus was obtained from three different pigs and named PIM1, PIM2 and PIM3, respectively. The animals were handled as approved by the Danish Animal Experiments Inspectorate (license number 2012-15-2934-00077). The isolated PIM was analyzed on a cone and plate rheometer as described previously [11] and normalized by dilution to display comparable rheological profiles. The PIM used for DWS was undiluted. Biosimilar mucus was prepared in 10 mM HEPES buffer with 1.3 mM CaCl2 and 1.0 mM MgSO4 as previously described [11]. Briefly, 0.9% (w/v) PAA was dissolved under magnetic stirring and 5% (w/v) mucin was added. The pH of the PAA and mucin solution was increased toward neutral by addition of NaOH prior to the addition of the lipid mixture. The lipid mixture was prepared separately with polysorbate 80 and added to the mucus to final concentrations of 0.36% (w/v) cholesterol, 0.18% (w/v) PC, 0.11% (w/v) linoleic acid and 0.16% (w/v) polysorbate 80. Finally, 3.1% (w/v) BSA was added to the mucus mixture and the pH was adjusted to 7.4 with NaOH. For some experiments, the linoleic acid or the whole lipid mixture was omitted. The biosimilar mucus was prepared one day prior to the experiment and stored overnight at 4 °C. 2.3. Diffusive wave spectroscopy (DWS) Diffusive wave spectroscopy (DWS) measurements were performed on biosimilar mucus and PIM with the transmission mode setting using a DWS Research Lab (LS Instruments, Fribourg, Switzerland) equipped with a laser beam of wavelength 685 nm. Samples were prepared and placed in disposable cuvettes with 5 mm optical path length. PolybeadÒ polystyrene tracer particles (Polysciences, Warrington, PA, USA) with a diameter of 456 nm were added to the mucus samples to a concentration of 0.65% (w/w). Samples were measured at 37 °C, using an integrated Peltier temperature controller and equilibrated for 10 min at the required temperature prior to the measurement. The scattered light was collected and cross-correlated with a single-photoncounting avalanche photodiode correlator (APD). The Rheo Lab software (LS instruments) was used for data collection and analysis.
Fig. 1. Schematic representation of the mucus containing in vitro models: PIM or biosimilar mucus applied onto agar-coated Transwell filters (A) and biosimilar mucus applied onto Caco-2 cell monolayers (B).
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2.4. Cell culturing Caco-2 cells (from DSMZ Braunschweig, Germany) were cultured in DMEM supplemented with 10% (v/v) FBS, penicillin/streptomycin (100 U/ml and 100 lg/ml), 1% (v/v) L-glutamine and 1% (v/ v) non-essential amino acids at 37 °C in an atmosphere of 5% CO2. The T75 culture flasks were passaged weekly. The cells were seeded at a density of 1 105 cells/insert onto 12-well polycarbonate TranswellÒ filter inserts (1.12 cm2 growth area, 0.4 lm pore size) (Corning Costar, Sigma–Aldrich). The medium at the apical and basolateral side of the cell layer was replaced every other day and cells of passages 3–9 after thawing were seeded and used after 18 or 20 days of culturing. 2.5. Mucus and mucosa permeability Four hour long permeability experiments were carried out at 37 °C on a horizontal shaker at 90 rpm exploring the mucus alone or in combination with an epithelium. Agar-coated filter inserts were used for mucus permeability studies without the epithelium. A total amount of 2% (w/v) agar was dissolved in ultrapure water from Barnstead NanoPure Systems (Thermo Scientific, Waltham, MA, USA) by microwave heating. A volume of 200 ll was added to each 12-well polycarbonate TranswellÒ filter insert (Corning Costar, Sigma–Aldrich), allowed to dry at room temperature for 30 min and stored overnight at 4 °C. For permeability experiments using the combined in vitro model incorporating the Caco-2 cell epithelium, the transepithelial electrical resistance (TEER) was measured prior to and following the experiments after an equilibration period of 20 min at room temperature with the assay buffer (10 mM HEPES HBSS, pH 7.4). After the permeation experiments, the TEER values of the cell monolayers were, unless stated otherwise, comparable with the starting value, which in all cases were between 430 and 550 O cm2. For comparison the resistance of the agar-coated filter inserts was approximately 10 O cm2. The inserts with agar coating or a Caco-2 cell monolayer were covered with 250 ll of buffer, biosimilar mucus or PIM (PIM2 and PIM3). Then, a volume of 100 ll test solution was added to the apical compartment with the following concentrations: 0.13, 0.66 and 3.32 mg/ml novicidin, 10.6 mg/ml plectasin, 14.9 mg/ml vancomycin, 10.7 mg/ml desmopressin and 10.2 mg/ml octreotide. The concentration of 14C-mannitol was 0.2 lCi/insert, whereas 0.1 lCi/insert was used for 3H-testosterone and 3H-metoprolol. Samples of 100 ll were taken from the basolateral compartment at fixed time points during the four hours and replaced with prewarmed assay buffer. A volume of 5 ll 1% (v/v) formic acid in methanol was added to each HPLC sample to prevent further enzymatic degradation and the samples were stored at 20 °C until analysis. 2.6. Analytical methods The samples from the peptide permeability experiments were analyzed by reverse-phase high-pressure liquid chromatography (HPLC) (Shimadzu, Prominence system, Kyoto, Japan) using a Kinetex (Phenomenex, Vaerloese, Denmark) C18 (50 4.6 mm) column and water/acetonitrile-based solvents containing 0.1% (v/ v) trifluoroacetic acid (TFA). The test compounds were eluted using a linear gradient of solvent B (95:5:0.1 (v/v/v) acetonitrile/water/ TFA) in solvent A (95:5:0.1 (v/v/v) water/acetonitrile/TFA) from 10% to 40% over 3.5 min at a flow rate of 1.85 ml/min. A PDA detector (SPD-M20A, Shimadzu) was set to record the 200–400 nm range. In the case of desmopressin the gradient of solvent B in solvent A was from 0% to 30% over 3.5 min. Vancomycin was quantified using a Kinetex C18 (50 2.1 mm) column and a gradient
3
from 2% to 8% over 12 min at a flow rate of 0.5 ml/min. The injection volume was 10 ll for all samples. The compounds were quantified using automatic integration of the peak at 218 nm. The limits of detection (LOD) for novicidin, plectasin, octreotide, desmopressin and vancomycin were 1.4 lg/ml, 2.4 lg/ml, 1.6 lg/ml, 1.3 lg/ml and 3.7 lg/ml (n = 3), respectively. The samples from the permeability experiments with radiolabelled isotopes were analyzed on a Tri-Carb 2100 TR liquid scintillation analyzer (Canberra Packard, Dreieich, Germany). Each sample was mixed with 2 ml of Ultima Gold scintillation fluid prior to the analysis. The apparent permeability coefficient (Papp) was calculated according to Eq. (1):
Papp ¼ ðdQ =dtÞ 1=ðA C 0 Þ
ð1Þ
where dQ/dt is the steady state rate of permeation, A is the diffusion area and C0 the initial donor concentration. 2.7. Cellular viability The cellular viability after the permeability experiments was determined by utilizing the MTS/PMS assay. Cell monolayers were gently washed twice with the assay buffer and 320 ll of freshly prepared MTS/PMS solution was added. After 1.25 h of incubation on a horizontal shaker at 90 rpm and 37 °C, 100 ll of apical reagent mixture was transferred to a 96-well plate and the absorbance was measured at 492 nm on a FLUOstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany). 2.8. Microscopy Agar-coated filter inserts were covered with 250 ll of PIM or biosimilar mucus and frozen at 20 °C. The filter inserts were then removed from their plastic supports, cut it into a strip of few millimeters width and embedded between two thin slices of chicken liver in order to preserve the mucus during the processing. The sandwich was snap frozen in liquid nitrogen and mounted in OCT medium and cut into 10 lm sections using a Leica cryostat CM3050 S (Leica Microsystems, Ballerup, Denmark) and collected onto SuperFrostÒ plus microscope slides. Sections on slides were allowed to air dry and were then used immediately or stored at 20 °C until use. The slides were defrosted and stained with 1% (w/v) Alcian Blue in 3% (v/v) acetic acid (pH 2.5) for 10 min. Sections were gently rinsed with tap water, dehydrated in toluene and mounted in DPX. Imaging was performed on a Zeiss Axio Scan Z1 brightfield slide scanner (Carl Zeiss Microscopy, Jena, Germany). Images were processed using ZEN 2012 lite software from Zeiss. The mucus thickness was measured for approximately every 250 lm and averaged. 2.9. Statistics GraphPadPrism (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The significance between two findings was determined by the student’s t test or by the one-way ANOVA for multiple comparisons, using in both cases a 95% confidence interval. 3. Results and discussion 3.1. The steric barrier property of biosimilar mucus is independent of lipid content In order to investigate the effect of the different components of the biosimilar mucus on the micro-rheological properties, diffusive wave spectroscopy (DWS) measurements were carried out using
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polystyrene tracer particles. The DWS technique offers much more spatial and temporal resolution, which cannot be proved in a mechanical rheometer, yet as compared to microscope-based methodologies applied for particle tracking the actual diffusion distance may be considered low. The analysis of the normalized intensity correlation function provided the mean squared displacement (MSD) of the particles in the different variants of biosimilar mucus. Assuming that there are no differences in the chemical adhesive interactions between the polystyrene particles and the different mucus matrices, the differences in the diffusion of the particles would mainly be governed by the steric barrier. The diffusion of the particles is reflected in the slope a of the logarithmic MSD vs. logarithmic time scale plot, where a is known as the anomalous diffusion coefficient (a = 1 for pure Brownian diffusion). The tracer particles present subdiffusive motion in all mucus samples (Fig. 2A). No significant differences in the a value of the particles were observed when measured in biosimilar mucus devoid of lipids as compared to a value in the biosimilar mucus composed as described previously (Fig. 2B) [11]. Thus, the lipid components are not causing a rearrangement of the matrix of the biosimilar mucus to a degree that influences the diffusion of particles with a diameter around 450 nm. Indications of faster mobility of the particles in the biosimilar mucus without lipids can be perceived, though not statistically different. This tendency would correlate with the previous findings describing an effect of the lipids in the macrostructure of the biosimilar mucus [11]. The particle diffusion was significantly increased when the synthetic polymer, polyacrylic acid (PAA), in the biosimilar mucus was omitted from the mixture. As previously described, the use of this non-natural mucus component was necessary to counteract the low viscosity of commercial mucins when used at relevant concentrations in order to obtain a macrorheological profile similar to PIM [11]. Interestingly, particles in the PAA gel diffused even more freely
Fig. 2. Mean squared displacement of 456 nm polystyrene tracer particles at 37 °C in water (black), 0.9% (w/v) of polyacrylic acid (PAA, blue), biosimilar mucus without PAA (red), without lipids (green), without linoleic acid (LA, purple) or complete biosimilar mucus (orange) (A). Solid lines represent mean values and dotted lines represent SD (n = 3). Anomalous diffusion coefficients (a) of the tracer particles in the different mucus samples (B). Data represent mean ± SD (n = 3). n.s. not significant, ⁄⁄⁄p < 0.001. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
as compared to the biosimilar mucus without PAA, indicating a larger mesh spacing of the matrix formed by the polymer alone. In order to elaborate on the previous comparison of PIM to biosimilar mucus [11], undiluted mucus samples from two animals were included in the study. It was, however, clear that the contribution of self-scattering from the PIM was too high to extract the MSD from the normalized intensity correlation function. Further, the correlation function revealed an enormous difference between the PIM isolations (Fig. 3), which was also observed visually during the sample handling and in accordance with measurements on the macrorheology (Fig. S1A) and also from previous studies on intestinal mucus [11,14].
3.2. Mucus covered agar-coated inserts constitute a relevant in vitro mucus model It was previously reported that exposing well-differentiated Caco-2 cell monolayers to porcine intestinal mucus (PIM) was detrimental to the epithelium, as measured by a decreased transepithelial electrical resistance as well as a reduced cellular viability [11]. Therefore, a mucus barrier model that would allow for comparison between different PIM samples, as well as comparison of biosimilar mucus to PIM, was necessarily a cell-free model. This simplified model was established utilizing agar-coated filter inserts supplemented with the relevant mucus sample. The mucus layer thickness was measured in order to extrapolate information from results obtained using this model. Sections of the minimal applicable volume of biosimilar mucus and PIM applied to agarcoated filter inserts that would ensure full coverage of the filter were imaged after Alcian Blue staining (Fig. 4). In both cases the mucus was evenly distributed across the filter area and the thickness of biosimilar mucus and PIM was found to be 800 ± 190 lm and 1050 ± 210 lm (n > 15), respectively. These values correspond well with the mean mucus thickness of 830 ± 110 lm found in the colon of rats, and is about 4-fold thicker than the value measured in the rat duodenum [15]. The Alcian Blue staining of biosimilar mucus was more diffuse than for the PIM, which structure was more granular, probably due to the degradation of the commercially available mucin used for the biosimilar mucus preparation. In order to validate the use of this mucus model for permeability studies, the effect of mucus on the permeability of model compounds was studied. Since the agar-coated filter insert does not constitute a biological barrier, the absolute values of the apparent permeability coefficient (Papp) should not be compared. Thus, the fold-reduction in the permeability of biosimilar mucus applied to the cell-free model was compared to those previously found with the biosimilar mucus applied to Caco-2 cell monolayers
Fig. 3. The correlation function of 456 nm polystyrene tracer particles in water (black), porcine intestinal sample 1 (PIM1, dark gray), and PIM2 (light gray). Data represent mean values (n = 3).
Please cite this article in press as: M. Boegh et al., Steric and interactive barrier properties of intestinal mucus elucidated by particle diffusion and peptide permeation, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.014
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Fig. 4. Alcian Blue staining of cryosections of biosimilar mucus (A) or porcine intestinal mucus (B) applied to TranswellÒ inserts (T) coated with agar (Ag) and embedded between slices of chicken liver tissue (L). Size bars represent 500 lm. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
[11]. Overall, comparable results were obtained with the cell-free model with regard to reduction in the apparent permeability coefficient (Papp) of the hydrophilic and lipophilic model compounds investigated (Fig. 5). It should be mentioned that for FD4, a higher level of reduction by the presence of mucus was observed in the cell-free model as compared to when the mucus was applied to the Caco-2 model. This is believed to be due to slightly higher variations in the raw data from the cell-free model, which was used to calculate the reduction. Thus, it appears that agar coating of the filter inserts could be a useful alternative to using cultured cell monolayers, especially since this would also allow studies on the barrier properties of PIM. As the PIM2 and PIM3 samples used in the present study displayed a higher viscosity as compared to PIM1 previously used, they were difficult to handle and dilution was necessary in order to apply a mucus layer on to the agar coatings. Thus, PIM2 was diluted 40% (v/w) and PIM3 was diluted 25% (v/w) with an isotonic version of the buffer used for preparation of the biosimilar mucus in order to obtain a macrorheological profile
corresponding to PIM1 (Fig. S1). The reduction in the Papp value of the hydrophilic macromolecules by the presence of PIM tended to be higher as compared to the reduction caused by biosimilar mucus. For one of the PIM samples the permeability reduction increased with increasing molecular weight of the macromolecules as observed previously, while no clear pattern was observed for the other PIM sample (Fig. 5). Generally, larger differences in the reduction caused by the two PIM samples were observed in the permeability studies with macromolecules compared to those with small molecules. This finding is possibly due to a greater impact of the heterogeneity of the mesh spacing of native mucus on the diffusion of high molecular weight compounds as compared to small molecules. As previously mentioned, the agar displays different barrier properties as compared to the epithelium, thus small hydrophilic compounds have a higher Papp value than lipophilic compounds when using the agar model (Fig. S2), whereas the opposite is the case for epithelium monolayers. Still, the agar model seems to be a good alternative to the mucus-covered cell culture monolayers for the evaluation of the relative effect of mucus as a barrier to drug permeation. Also, the time and costs for culturing of cells can be avoided and the biological variation related to using cell culture models is also avoided. 3.3. The interactive barrier property of biosimilar mucus is not affected by the linoleic acid content
Fig. 5. Reduction in apparent permeability coefficient (Papp) of the test compounds by biosimilar mucus applied to Caco-2 cells monolayers (black bars) or agar coated filter inserts (gray bars), or two different PIM secretions applied to agar-coated filter inserts (striped bars). With the exception of estradiol results, the data from biosimilar mucus applied to Caco-2 cells monolayers (black bars) are previously reported [11] and have been included for comparative reasons. FD4: Fluorescein isothiocyanate (FITC)-dextran 4,000, FD10: FITC-dextran 10,000, FITC-BSA: FITCbovine serum albumin. Data represent means ± SD (n = 3–6).
As the presence of linoleic acid and other lipids in the biosimilar mucus had no significant effect on the diffusion of tracer particles (Fig. 2) it was relevant to investigate whether the presence of lipid was important for the interactive barrier properties of biosimilar mucus. This would increase the general applicability of the mucus, as the addition of a fatty acid-containing test solution was shown to render the otherwise biocompatible mucus toxic to the underlying Caco-2 cell monolayers (Fig. S3). Linoleic acid has earlier been shown to be the most critical component for biocompatibility of the mucus mixture [10,11] and it was hypothesized that the addition of e.g. capric acid as a permeability enhancer increased the total fatty acid content above the threshold for cellular compatibility. The relative reduction in the Papp value for permeation of mannitol, metoprolol and testosterone was unaffected by the presence or absence of linoleic acid in the biosimilar mucus applied to
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phosphatidylcholine in the general biosimilar mucus barrier properties. Thus, the omission of linoleic acid from the biosimilar mucus broadens the applicability of the model by ensuring compatibility with Caco-2 cell monolayers also when lipid-based formulations or lipid excipients are tested. Previously, differences in mucus microstructure between biosimilar mucus with and without all the lipids have been observed by cryo-SEM [11] and a tendency toward an increase particle mobility was observed by DWS when the lipids were not included in the mucus. Thus, cholesterol and phosphatidylcholine seem to influence the microstructure to a degree, and these lipids should be included to resemble the properties of native mucus. 3.4. The permeation of peptide drugs across biosimilar mucus varies with molecular properties Fig. 6. The reduction in apparent permeability of mannitol, metoprolol and testosterone across agar-coated filter insert caused by biosimilar mucus (black columns) or biosimilar mucus without linoleic acid (gray columns). Data represent mean values ± SD (n = 3).
agar-coated inserts (Fig. 6). In line with this, two separate experiments demonstrated that the Papp value for the permeation of mannitol and metoprolol across biosimilar mucus or biosimilar mucus without any lipids applied to Caco-2 cell monolayers did not differ significantly (Fig. S4). Furthermore, it was shown that the permeation of three peptide drugs across biosimilar mucus without linoleic acid or without any lipids applied to Caco-2 cell monolayers was also comparable (Fig. S5). Overall, the data from studies of the biosimilar mucus in terms of macrorheology, microstructure and permeation of small molecules and peptide drugs imply that there is no effect of the presence of linoleic acid or the other two lipids, cholesterol and
The barrier property of biosimilar mucus without linoleic acid was further studied by assessing the permeation of five different peptides (Table 1) across Caco-2 cell monolayers in the absence and presence of biosimilar mucus. The Papp values were all obtained at steady state (Fig. S6) and the permeation of octreotide and desmopressin across Caco-2 cell monolayers was comparable, which corresponds well with their structural similarities (Table 2), and in the range of previously published data for desmopressin [21,22]. However, the reduction of the Papp value by the presence of biosimilar mucus was for octreotide more than 2-fold higher than the reduction for desmopressin (Table 2). Octreotide has one hydrophobic residue more than desmopressin and thereby more potential for hydrophobic interactions with mucus components, and also a more pronounced cyclic structure. For vancomycin the reduction in permeation caused by mucus was around 7-fold probably due to the more bulky structure and the fact that vancomycin has a significantly higher number of
Table 1 The amino acid sequence, molecular weight (Mw) and the isoelectric point (pI) of the five peptides. Single letter denotation of amino acid residues is used. Cationic residues are marked with red and hydrophobic residues with blue.
Octreotide Desmopressin
Amino acid sequence or structure
Peptide Mw (Da)
H-D-F-C-F-W-K-T-C-T(ol) SCH2CH2CO- Y-F-Q-N-C-P-R-G -NH2
1019
Vancomycin
Novicidin Plectasin
K-N-L-R-R-I-I-R-K-G-I-H-I-I-K-K-Y-F [16] G-F-G-C-N-G-P-W-D-E-D-D-M-Q-C-H-N-H-CK-S-I-K-G-Y-K-G-G-Y-C-A-K-G-G-F-V-C-K-C-Y
pI 8.06
1069
8.22
1485
7.20
2297
11.75
4408
8.85
[17]
The Mw and pI were calculated from the sequences using the ProtParam tool [18], except for vancomycin [19] and plectasin [20]. The structure of vancomycin is shown instead of the sequence, as it is a glycosylated peptide with non-proteinogenic amino acids.
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Table 2 The apparent permeability coefficients (Papp) for the permeation of five peptides across Caco-2 cell monolayers and across the mucosa model; i.e. biosimilar mucus covered epithelium and the reduction in Papp by two samples of porcine intestinal mucus (PIM).
Octreotide Desmopressin Vancomycin Novicidin Plectasin
Papp epithelium (108 cm/s ± SD)
Papp mucosa (108 cm/s ± SD)
Fold reduction by biosimilar mucus
Fold reduction by PIM2/PIM3
15.5 ± 2.5 14.4 ± 3.5 19.2 ± 5.7 861 ± 55a 6.2 ± 2.3
2.8 ± 0.4 7.4 ± 0.7 2.6 ± 0.7
5.5 1.9 7.2 – –
6/40 33/46 36/75 ND/7 145/151
LOD: limit of detection. a The integrity of the cell monolayer was compromised. The Papp values are means ± SD, (n = 6–9), conducted on 2–3 different cell passages. ND: not detected. The reduction caused by PIM is calculated from mean of triplicates. Biosimilar mucus without linoleic acid was used.
hydrogen bond acceptors and donors and a greater potential for electrostatic interactions [23], as compared to desmopressin and octreotide. Thus, extensive interaction between vancomycin and mucus is likely the reason for the reduced permeation of this compound. The diffusion of macromolecules in porcine mucus was previously found to decrease with increasing molecular weight, when peptide and proteins in the range 14.4–186 kDa and 3.4–66 kDa were tested in gastric and intestinal mucus, respectively [24,25]. Although, smaller peptides are tested here, the same tendency may apply. Further, a great reduction in the diffusion of anionic and especially cationic synthetic peptides in reconstituted mucin gels as compared to in water has been demonstrated, whereas it was not the case for a peptide composed of a cationic as well as anionic block and hence with a lower net charge [26]. No permeation of plectasin and novicidin across the mucosa model could be detected. Thus, it is likely that these larger, more cationic and structured peptides interact strongly with the negative mucin fibers in addition to forming hydrophobic interactions, which limits their diffusion significantly. Permeated novicidin could only be detected when using the highest concentration. However, the viability of the Caco-2 cell monolayers was reduced to approximately 20% at this condition (Fig. 7A) and this disruption of the cell monolayer resulted in a high flux and Papp value (Table 2). Novicidin is an unstable peptide [18] with known eukaryotic cellular toxicity at low concentrations as confirmed by the concentration-dependent decrease in viability of the epithelial cells (Fig. 7A). There was a similar trend for the effect on the epithelium covered by biosimilar mucus, but interestingly the biosimilar mucus to a large degree protected the underlying epithelium (Fig. 7A). In comparison, exposure to plectasin reduced the cellular viability of the epithelium only to 85%, whereas plectasin was fully tolerated in the presence of biosimilar mucus (Fig. 7B) and neither desmopressin, octreotide nor vancomycin had any negative effect on the cellular viability under the tested conditions. 3.5. Porcine intestinal mucus reduces peptide permeation with large variations When assessing the permeation of peptide drugs across porcine intestinal mucus the reduction caused by PIM was in most cases found to be significantly higher than the reduction caused by biosimilar mucus (Table 2). The reduction in permeation of octreotide and vancomycin varied greatly between the two PIM samples, while the reduction in permeation of desmopressin and plectasin was in the same range for both samples. As the molecular weight of desmopressin and octreotide is comparable, these findings suggest that it is the interactive barrier more than the steric barrier, which differs between the two PIM samples. Overall, in most cases the PIM on agar seemed to reduce the peptide permeation to a slightly greater extent than the biosimilar mucus on Caco-2 cells, while similar results were obtained for the reduction of octreotide
Fig. 7. The viability of Caco-2 cells in monolayers after four hours of incubation with novicidin (A) or desmopressin, octreotide, vancomycin and plectasin (B) in the absence or presence of biosimilar mucus without linoleic acid (BM) covering the Caco-2 epithelium. Different concentrations of novicidin were tested and for the other peptides, the concentrations were: 10.6 mg/ml plectasin, 14.6 mg/ml vancomycin, 10.7 mg/ml desmopressin and 10.2 mg/ml octreotide. Data represent means ± SD (n = 3) and B is representative of two individual experiments. ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.
permeation by PIM2 and biosimilar mucus. This observation supports that the specific physicochemical properties of the permeant as well as the complexity of the mucus determines the overall permeation. However, the large variation between the results from the two PIM samples precluded to rank the peptides with respect to the reduction and therefore also the comparison to the interactive barrier properties of biosimilar mucus. Thus, the great variability of
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porcine intestinal mucus reconfirms one of the main drawbacks of this model, whereas biosimilar mucus provides a reproducible and robust alternative to in vitro testing of mucus barrier properties.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2015.01.014.
4. Conclusion References The steric barrier properties of the biosimilar mucus are to some extent maintained by the non-natural polymer component in the biosimilar mucus, PAA, but PAA is in itself less of a steric barrier as compared to the mixture of the other biosimilar mucus components. At the microscale, the polymer itself constitutes a less significant steric barrier as compared to the mixture with the natural components in the biosimilar mucus. The presence of linoleic acid, was found not to be crucial for the steric or interactive barrier properties of the biosimilar mucus, and is suggested to be omitted from the mucus matrix to broaden the applicability of the mucosa model e.g. to allow for testing of lipid-based formulations or excipients. Mucus permeation studies utilizing a cell-free alternative to the mucosal in vitro model composed of the biosimilar mucus and Caco-2 cell monolayers demonstrated to be advantageous by allowing studies with PIM, which is not compatible with cells, but merely to investigate a relative influence of the mucus barrier. The transmucosal permeation of five peptide drugs with varying physicochemical properties illustrated the barrier properties of the biosimilar mucus combined with epithelium. The corresponding study using PIM revealed large variations between the two PIM samples, which impeded conclusions regarding the barrier effect. In general, the reduction on peptide permeability was significantly higher for PIM compared to the biosimilar mucus, probably due to differences in the mucin network integrity. In conclusion, the steric and interactive barrier properties of biosimilar mucus have been further elucidated. Although the differences in peptide permeation and mucin network integrity, the biosimilar mucus appears to be a useful and more reproducible alternative to native mucus for mechanistic studies on mucus diffusion and mucosal drug permeation. Acknowledgments Novozymes A/S is acknowledged for kindly supplying the novicidin and plectasin. Special thanks to Dr. Marc Obiols Rabasa at Lund University and the European Commision under the Seventh Framework Program for technical and financial support for conducting DWS studies as part of the European Soft Matter Infrastructure (ESMI) Project (Grant No. 262348). Thanks to Laboratory Technician Karina Vissing and PhD Fellow Søren Steffensen for great assistance with the HPLC analysis. DVM Camilla Schumacher-Petersen and Animal Technician Mette Olesen from Department of Experimental Medicine, University of Copenhagen, are acknowledged for providing the porcine small intestine. This work was funded by the Drug Research Academy, The Danish Agency for Science, Technology (mobility stipend and the innovation consortium Predicting Drug Absorption). Furthermore, the research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115363 resources, which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution.
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