The impact of gastrointestinal mucus on nanoparticle penetration – in vitro evaluation of mucus-penetrating nanoparticles for photodynamic therapy

European Journal of Pharmaceutical Sciences 133 (2019) 28–39

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The impact of gastrointestinal mucus on nanoparticle penetration – in vitro evaluation of mucus-penetrating nanoparticles for photodynamic therapy Laura Mahlert, Juliane Anderski, Dennis Mulac, Klaus Langer

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Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Corrensstraße 48, 48149 Muenster, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: HT-29-MTX Cell model Co-culture Mucus penetration Poly (ethylene glycol) PLGA Nanoparticles Photodynamic therapy Cellular uptake Intestinal cancer

Over the last years nanoparticles (NP) have become a promising vehicle as drug delivery systems for photodynamic therapy (PDT), combining the advantages of an effective drug transport to the target cells and the reduction of undesired side effects. The in vitro evaluation of new nanoparticulate formulations has become a rising problem since cell culture models differ from the in vivo situation of the human body to a large extent. Particularly, in case of gastrointestinal tumors, after peroral application nanoparticles are challenged by overcoming the mucus layer as a first physical barrier before reaching the target cells, an aspect often neglected in literature. However, the presence of mucus is crucial for in vitro models to evaluate mucus-penetrating potential of surface-modified nanoparticulate drug carrier systems. Biodegradable poly(DL-lactide-co-glycolide) (PLGA) NP loaded with the model photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (mTHPP) were surface modified with either poly(ethylene glycol) (PEG) or chitosan (CS) to gain mucus-penetrating or mucoadhesive particle properties. All NP systems were compared to each other and to free mTHPP regarding cytotoxicity and cellular uptake in HT-29 cells and mucus producing HT-29-MTX cells. For PEGylated mTHPP-PLGA-PEG-NP a significantly higher accumulation was obtained in HT-29-MTX cells compared to all other tested nanoparticles and the free drug. Additionally, a mucus-containing Transwell® model, consisting of HT-29-MTX cells, confirmed these results, representing a promising in vitro screening method for mucus-penetrating particle properties.

1. Introduction Photodynamic therapy (PDT) with its benefits of selectivity and minimal invasiveness bears great potential as an alternative treatment of cancer compared to conventional therapies. It has gained in importance since the last three decades and is used in oncological therapies for several forms of cancer nowadays (Dolmans et al., 2003; Schuller et al., 1985). In principle, PDT is based on the accumulation of a hydrophobic photosensitizer (PS) in malignant tumor cells and its subsequent illumination with light of a certain wavelength. This leads to an activation of the drug, generating reactive oxygen species (ROS) and causing cell death (Moreira, 2008). Due to the intravenous application of PS, therapies targeting tumors in deeper regions of the human body, like the gastrointestinal tract, are currently not covered by the drug approval and require new technological developments (Agostinis et al., 2011). Given the importance and applicability of nanoparticles as an innovative oral drug delivery system, a nanoparticle-based PDT therefore offers a promising alternative compared to conventional intravenous application handling undesired distribution in healthy tissue

and hydrophobicity of PS (Davis et al., 2008; Löw et al., 2011). For targeting gastrointestinal tumors by an oral application route, the major challenge for nanoparticles is represented in overcoming the gastrointestinal mucus layer in order to achieve a sufficient enrichment of PS in epithelial cells localized beneath the mucus (Chatterjee et al., 2008; Date et al., 2016; Ogawara, 2017). Knowing physicochemical characteristics of gastrointestinal mucus as an extremely complex network, the size, charge and surface modification of nanoparticles play a fundamental role for successfully overcoming the mucus barrier (Boegh and Nielsen, 2015; Ensign et al., 2012). Chitosan (CS) and poly(ethylene glycol) (PEG) have been described in literature extensively as promising surface modifications for efficiently crossing mucus barriers. This is obtained by either mucoadhesive properties of CS, resulting in a prolonged residence in the mucus, or faster mucus penetration due to the hydrophilic characteristics of PEG (Cu and Saltzman, 2009; Kawashima et al., 2000). Besides the development of innovative drug delivery systems, the requirement of suitable in vitro models, giving an adequate representation of the in vivo situation in the GI tract, has received

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Corresponding author. E-mail addresses: [email protected] (L. Mahlert), [email protected] (J. Anderski), [email protected] (D. Mulac), [email protected] (K. Langer). https://doi.org/10.1016/j.ejps.2019.03.010 Received 5 December 2018; Received in revised form 28 January 2019; Accepted 14 March 2019 Available online 15 March 2019 0928-0987/ © 2019 Elsevier B.V. All rights reserved.

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nanoparticles with the photosensitizer mTHPP embedded into the polymeric matrix. The preparation was performed by a previously described solvent displacement method to achieve a consistent particle diameter of 100 nm (Quintanar-Guerrero et al., 1998). A detailed nanoparticle preparation and characterization is described by Anderski et al. (2018). Briefly, for manufacturing of surface unmodified control NP, 30 mg PLGA was dissolved in 2 mL acetone. Subsequently, 3 mg of mTHPP was added and the organic solution was injected under stirring into 4 mL of an aqueous PVA solution (2% (w/v)). The nanoparticle suspension was stirred overnight, whereby evaporation of the organic solvent finally led to the particle formation. Afterwards, purification of the nanoparticle suspension was conducted three times via centrifugation (30,000g; 1 × 1 h, 2 × 0.5 h) and redispersion in water. The NP were denoted as mTHPP-PLGA-NP. For surface modification of NP, the previously described preparation protocol was varied. Chitosan modified mTHPP-CS-PLGA-NP were prepared by replacement of the aqueous stabilizer solution. An alternative stabilizer solution containing 1% (w/v) PVA and 0.3% (w/v) chitosan hydrochloride (pH 6–7) was used for the preparation. The manufacturing of PEGylated mTHPP-PLGAPEG-NP was performed using PLGA-PEG as functional matrix polymer. Therefore, 130 mg PLGA-PEG was dissolved in 2 mL acetone with the addition of 10 mg mTHPP. All other preparation steps for the surface modified NP were the same as previously described for the control NP.

particular attention for several years (Beloqui et al., 2016). The FDA approved and commonly used Caco-2 model, is limited by the absence of goblet cells to constitute a mucus layer. Further development has been made to co- and triple co-culture models by addition of HT-29MTX, HT-29-H or Raji-B cells, aiming at a closer imitation of the gastrointestinal barrier by its physiological cell composition (Antunes et al., 2013; Schimpel et al., 2014; Wikman-Larhed, 1995). Studies have already shown the importance of mucus in cell models for evaluation of substances, leading to divergent results compared to the simple Caco-2 model (Calatayud et al., 2012; Mahler et al., 2009; Yuan et al., 2013). The aim of this study was to evaluate the mucus-penetrating properties of surface-modified nanoparticles for a sufficient enrichment of PS in gastrointestinal target cells. Accordingly, by the examination of suitable in vitro cell models and experiments including the influence of the mucus layer a basis for an effective photodynamic therapy is provided. Addressing this issue, comparative experiments were performed using human colonic adenocarcinoma HT-29 cells and the mucus-producing sub clone HT-29-MTX E-12 (Lesuffleur, 1990) in a simplified monoculture model. Poly(DL-lactide-co-glycolide) (PLGA)-based nanoparticles in a size range of 100 nm with the model photosensitizer mTHPP embedded were modified with either chitosan CS or PEG and compared to unmodified mTHPP-PLGA-NP and the free drug regarding their cytotoxic potential and intracellular uptake at different degrees of mucus layer thickness. Afterwards, a transfer into a three-dimensional Transwell® setup with HT-29-MTX cells was performed in comparison to a conventional co-culture model with Caco-2/HT-29-MTX cells and visualization of the mucus-penetrating effect for PEG was conducted by live-cell imaging and confocal microscopy.

2.2.2. Nanoparticle characterization via PCS measurement To investigate the average hydrodynamic particle diameter and polydispersity index (PDI), PCS measurements were performed. Aqueous nanoparticle suspensions were diluted with ultrapure water and analyzed at a temperature of 22 °C and a backscattering angle of 173°. The surface charge of the formulations was determined by laser Doppler microelectrophoresis using the zeta potential mode. For both experiments a Malvern Zetasizer Nano ZS system (Malvern Instruments Ltd., Malvern, UK) was used.

2. Material & methods 2.1. Materials The two polymers poly(DL-lactide-co-glycolide) (PLGA) (Resomer® RG 502H; Mw 7–17 kDa; copolymer ratio 50:50) and poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PLGA-PEG) (Resomer® RGP d 50,155; Mw 48.3 kDa with 5 kDa PEG) were purchased from Evonik Industries (Darmstadt, Germany). Chitosan-HCl (CS) (degree of deacetylation 80–95%; Mw 30–400 kDa) was purchased from Heppe Medical Chitosan GmbH (Halle (Saale), Germany). Biolitec research GmbH (Jena, Germany) kindly provided the photosensitizer mTHPP. The HT-29 cells were a kindly gift from the Institute of Food Chemistry, University of Muenster (Germany), the mucus secreting subclone HT-29-MTX E-12 was purchased from “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH” (Braunschweig, Germany) and Caco-2 cells were purchased from “Institut für angewandte Zellkulturen Dr. Toni Lindl GmbH” (Munich, Germany). Dulbecco's Modified Eagle Medium (DMEM), gentamycin, non-essential amino acids (NEA), L-alanyl-L-glutamine, trypsin/EDTA, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were obtained from Biochrom AG (Berlin, Germany). Poly(vinyl alcohol) (PVA; 30–70 kDa), trifluoroacetic acid, and paraformaldehyde were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). Acetonitrile and dimethyl sulfoxide (DMSO) were purchased from Roth (Karlsruhe, Germany). Wheat germ agglutinin, Alexa Fluor® 350 conjugate was obtained from Thermo Fisher Scientific (Waltham, MS, USA). WST-1 reagent was purchased from Roche Diagnostics (Mannheim, Germany). Tissue-Tek® O.C.T.™ Compound was obtained from Sakura Finetek (Staufen, Germany).

2.2.3. Quantification of embedded photosensitizer mTHPP The amount of incorporated photosensitizer was determined using a quantification method previously described by Grünebaum et al. (2015). After dissolving the nanoparticles in acetone, the embedded mTHPP was quantified via high performance liquid chromatography (HPLC) with the aid of a calibration curve of pure mTHPP (concentration range from 10 to 100 μg/mL). A HPLC-DAD system (Agilent Technologies 1200 series) was used in combination with a reversed phase column (Gemini RP 18; 250 × 4.6 mm, particle diameter 5 μm (Phenomenex, Aschaffenburg, Germany)). A mobile phase consisting of 42.5% water with 0.1% (w/v) trifluoroacetic acid and 57.5% acetonitrile was utilized for isocratic elution at a flow rate of 1.0 mL/min. mTHPP was detected at a wavelength of 415 nm. 2.3. Cell culture conditions HT-29, HT-29-MTX, and Caco-2 cells were cultivated in 75 cm2 flasks with DMEM supplemented with 10% (v/v) FBS, 1% (v/v) NEA 200 mM, 1% (v/v) L-alanyl-L-glutamine 200 mM, and 1% (v/v) gentamicin. Cultivation was carried out in an incubator under constant conditions of 37 °C, 100% humidity, and 10% CO2. The cells were splitted twice a week at a ratio between 1:4 and 1:6. All cell culture experiments were carried out in triplicate and for each experiment a different passage of the cell line was used. All experiments were performed within the first 20 passages. 2.4. Cytotoxicity studies

2.2. Preparation and characterization of photosensitizer-loaded nanoparticles

Cytotoxicity was measured using WST-1 assay, based on a NADHdependent reduction of the water-soluble tetrazolium WST-1 reagent. For PS activation a self-constructed illumination system with six red light-emitting diodes placed in an incubator was used. All the other

2.2.1. Preparation of PLGA-based nanoparticles and modification with chitosan and PEG All in vitro experiments were carried out with PLGA-based 29

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2 confocal microscope (Leica TCS SP2, diode laser 405 nm, Argon laser 514 nm, Leica confocal software, Heidelberg, Germany).

steps of cell handling were carried out under light exclusion. For determination of dark toxicity, WST-1 assay was performed without light activation of the photosensitizer. Five days after seeding in 96 well plates at a density of 1 × 104 cells/well, the incubation of different nanoparticle formulations and free mTHPP (stock solution of 1 mg/mL, dissolved in DMSO) was conducted at mTHPP concentrations between 0.001 μM and 5 μM in DMEM without FBS. After 24 h incubation the cells were washed once with medium and 100 μL serum free medium was added. Subsequently, the cells were illuminated with a light dose of 5 J/cm2 followed by a recovery period of 1 h. Non-illuminated controls were stored in the incubator simultaneously. After addition of WST-1 reagent the absorbance was measured immediately at 460 nm with a Synergy MX multi-well spectrophotometer (BioTek Instruments GmbH, Bad Friedrichshall, Germany), blank measurement was done before application of WST-1 reagent. Medium (100% viability) and DMSO (0% viability) served as negative and positive control, respectively, and were simultaneously incubated on each well plate. For calculation, blank measurement was subtracted and all values were set in correlation to the positive and negative control.

2.6. Evaluation of mucus permeation in Transwell® models 2.6.1. Permeation studies in co-culture (Caco-2/HT-29-MTX) and monoculture (HT-29, HT-29-MTX) models Caco-2 and HT-29-MTX cells were seeded in a ratio of 7:3 and 1:1 on 12 well polycarbonate filter (Transwell® Corning Incorporated, Tewksbury, USA) for permeation studies. A density of 1 × 105 cells was chosen for 1.12 cm2 growth area with a pore size of 3 μm. Full supplemented DMEM served as culture and transport medium for experiments and was changed every other day. Permeation experiments were conducted 21 days after seeding. The initial nanoparticle concentration for incubation in the apical compartment was adjusted to a concentration of 5 μM mTHPP. At different time points (1 h, 2 h, 4 h, 6 h, and 24 h) samples were taken from the apical (50 μL) and basolateral (100 μL) compartment and replaced by medium immediately. For quantification of mTHPP, the samples of each time point were diluted with DMSO in a ratio of 1:4. For mTHPP quantification in the cell and filter compartment the filters were cut out, washed once with PBS++ and lysed in 500 μL DMSO for at least 72 h in order to extract the PS. Centrifugation was used to purify diluted samples and filters (30,000 g; 30 min) prior to quantification via HPLC-FLD (Section 2.7). During the whole experiment, transepithelial electric resistance (TEER) was measured as a parameter for intact barrier using CellZScope® (nanoAnalytics GmbH, Muenster, Germany). As a second cell culture model for analysis of mucus permeation, either HT-29 or HT-29-MTX cells were seeded as a monoculture on previously described Transwell® filters, analogue to permeation studies in the co-culture model. The experiments were performed as described above except for the permeation studies which were conducted after 10 days of cultivation.

2.5. Determination of cellular uptake and interaction 2.5.1. Intracellular uptake in HT-29 and HT-29-MTX cells To determine the intracellular uptake of the photosensitizer in both cell lines, uptake studies were performed on the basis of literature described methods (Mulac et al., 2013). In brief, cells were cultivated for 5 or 10 days after seeding at a density of 1 × 105 cells/well in 24 well plates, respectively. After the growing period, medium was replaced by nanoparticle suspensions or free mTHPP (1 mg mTHPP/mL DMSO) diluted in DMEM corresponding to a concentration of 1 μM mTHPP. At specific time points (1 h, 2 h, 4 h, 6 h, and 24 h) incubation medium was removed in three wells and cells were washed twice with phosphate buffered saline (PBS++, containing Ca2+ and Mg2+). A fourth well was used to determine cell amount and cell volume for the calculation of cellular mTHPP concentration at each time point. Therefore, cells were detached with Trypsin/EDTA solution and measured with a CASY® TT Cell counter (OMNI Life Science GmbH, Bremen, Germany). After 24 h, cells of all time points were dissolved in 300 μL DMSO for a minimum of 72 h and were centrifuged (30,000 g; 15 min) to separate cell debris. The supernatant was used for HPLC-FLD analysis (see chapter 2.7).

2.6.2. Visualization of mucus on HT-29-MTX in a Transwell® model For visualization of HT-29 and HT-29-MTX cells and produced mucus in a Transwell® model, both cell lines were seeded on Transwell® filters and cultivated for 10 days with a half change of medium every other day. After growing period, filters were washed with PBS++, fixed with ethanol 70% (v/v), cut out and embedded in Tissue-Tek®. Samples were frozen at −80 °C and vertical filter slices with a thickness of 10 μm were prepared using a microtome cryostat (CryoStar NX70, Thermo Scientific, Walldorf, Germany). Afterwards, the mucus layer was stained with Alcian blue (Thermo Fisher, Karlsruhe, Germany), whereas Nuclear Fast red solution (Sigma Aldrich GmbH, Steinheim, Germany) was used for cell nuclei staining. All transmitted light images were taken with an Olympus IX81 microscope (Hamburg, Germany).

2.5.2. Determination of cellular interaction HT-29 and HT-29-MTX cells were seeded in 24 well plates at a density of 1 × 105 cells/well and treated as described above for uptake studies. Scanning of the whole plate was performed every hour for 24 h during incubation process using an IncuCyte® S3 Live-Cell Analysis Imaging System (Essen Bioscience, Inc., Michigan, USA). At all time points, nine images of each well were taken, covering nearly the whole well; each formulation was incubated in three wells. Image channel red (excitation: 565–605 nm / emission: 625–705 nm) was used to determine mTHPP. Calculation was carried out using the red object area detected by the system. Intensities of all taken images were averaged and background of control wells without photosensitizer (medium) was subtracted after analysis.

2.6.3. Visualization of mucus permeation in HT-29-MTX-model by fluorescence microscopy For visualization of nanoparticle permeation through the mucus layer of HT-29-MTX cells, after 10 days of cell growth Transwell® filters were incubated with the same sample concentration (referred to 1 μM mTHPP) as performed in permeation studies for a period of 4 h and 24 h, respectively. After removing the incubation medium, filter preparation was conducted using parameters as described before, except for the staining procedure of mucus. For fluorescence microscopy cells were stained with DAPI containing Vectashield mounting medium. The images were taken by an Olympus IX81 fluorescence microscope (Hamburg, Germany) with a filter system including visualization for DAPI (excitation: 360–370 nm, dichroic mirror: 400 nm, emission: 426–446 nm) and the photosensitizer mTHPP (excitation: 400–410 nm, dichroic mirror: 455nm, emission: 635–685 nm). All pictures were taken as multi-layer image stacks with a minimum of 20 images. To reduce out of focus fluorescence the stacks were processed by deconvolution (Wiener filter) using cellSens Dimension Software version

2.5.3. Visualization of cellular uptake by confocal microscopy For confocal microscopy, HT-29 and HT-29-MTX cells were seeded at a density of 5 × 104 cells/chamber on Millicell® EZ slides (Merck, Darmstadt, Germany) and incubated for 6 h after a growing period of 5 days. After incubation, cells were washed with PBS++ and incubated with wheat germ agglutinin (WGA) Alexa Fluor® 350 solution (10 μg/ mL) for staining of cell membranes (10 min). Following another washing step with PBS++, cells were fixed with 4% paraformaldehyde (m/v) for 15 min (room temperature). Finally, Vectashield mounting medium with DAPI (Vector Laboratories Inc., Burlingame, USA) was used to cover the cells and analysis was performed with a Leica DMIRE 30

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1.8.1 (Olympus, Hamburg, Germany).

higher than the CS content (p ≤ 0.05).

2.7. Quantification of mTHPP by high performance liquid chromatography (HPLC)

3.2. Cytotoxicity studies The examination of acute light-induced cytotoxicity is a first necessary precondition for evaluation of new photosensitizer formulations and was performed by WST-1 assay after 24 h incubation with mTHPP concentrations ranging from 0.001 μM to 5 μM. Evaluation of dark toxicity was investigated using the same assay without illumination step after incubation of the cells. No influence on cell viability was observed which confirms the absence of dark toxicity (data not shown). The results of the cytotoxicity studies are shown in Fig. 1A and B for HT-29 and HT-29-MTX cells, respectively. By comparing the concentration-dependent cell viability course of both cell lines, in case of HT-29 cells a slightly sharper decline was observed for all formulations, indicating a lower effective concentration for cells without a mucus layer. This effect was underlined by comparison of EC50 values for both cell lines. HT-29 cells exhibited a higher sensitivity for all tested nanoparticle formulations, with EC50 values ranging from 0.47 ± 0.28 μM for free mTHPP to 0.79 ± 0.19 μM for mTHPP-CSPLGA-NP while unmodified mTHPP-PLGA-NP (0.48 ± 0.08 μM) and PEG-modified mTHPP-PLGA-NP (0.53 ± 0.20 μM) showed comparable values to free mTHPP. In contrast HT-29-MTX cells displayed higher EC50 values between 0.88 ± 0.47 μM (mTHPP-PLGA-PEG-NP) and 1.73 ± 0.74 μM (mTHPP-CS-PLGA-NP). Therefore, an effective cytotoxicity for mTHPP-PLGA-NP, mTHPP-PLGA-PEG-NP, mTHPP-CSPLGA-NP, and mTHPP was observed in HT-29 and HT-29-MTX cells with slightly lower effective concentrations for all formulations in cells without mucus. Although no statistically significant differences between the EC50 values in HT-29 and HT-29-MTX cells were observed the by trend increased EC50 values in HT-29-MTX cells provide an indication that the mucus layer impedes the drug transport capability of NPs.

For uptake and permeation studies, the photosensitizer mTHPP was determined using a HPLC-system (Agilent Technologies 1200 series) according to a previously described quantification method (Grünebaum et al., 2015). Because of lower PS concentrations in cell culture experiments, a more sensitive FLD system (excitation 421 nm, emission 653 nm) was chosen for mTHPP detection with a calibration curve ranging from 0.005 μg/mL to 5 μg/mL and the injection volume was increased to 20.0 μL. All samples were dissolved in DMSO and purified from cell debris via centrifugation (30,000 g; 30 min). 2.8. Statistical methods The data shown in the graphics represents the mean value and standard deviation of experiments performed in triplicate. The comparison of independent groups was conducted by one-way ANOVA and Holm-Sidak post test using Sigma Plot 12.5 (Systat Software GmbH, Erkrath, Germany). Significance levels are denoted with * p ≤ 0.05. 3. Results 3.1. Preparation and characterization of surface modified PLGA nanoparticles For the preparation of photosensitizer-loaded NP, the model photosensitizer mTHPP was embedded into polymeric nanoparticles with varied surface modifications. As an unmodified control formulation, mTHPP-PLGA-NP were manufactured by a literature described solvent displacement method (Quintanar-Guerrero et al., 1998). The surfacemodified mTHPP-PLGA-PEG-NP and mTHPP-CS-PLGA-NP were prepared using a varied preparation technique. All formulations were physicochemically characterized in detail (Table 1). The formulations showed average hydrodynamic diameters in a range of about 90 to 120 nm. mTHPP-PLGA-PEG-NP were significantly smaller and mTHPPCS-PLGA-NP were significantly larger than unmodified mTHPP-PLGANP (p ≤ 0.05). All particle systems were obtained with PDI values below 0.2, reflecting a narrow size distribution. PEGylation of the unmodified NP led to a significant decrease of the zeta potential of about 10 mV from −29.7 to −19.8 mV (p ≤ 0.05). A positive surface charge of about +10 mV was observed for CS-modified NP (p ≤ 0.05). Incorporated mTHPP was determined via HPLC-DAD analysis. The quantified drug load is presented in Table 1. The highest drug load of 66.5 μg mTHPP/mg NP was detected for mTHPP-PLGA-PEG-NP and was thereby significantly higher (p ≤ 0.05) in comparison to the unmodified standard system. In vitro release studies demonstrated the stable entrapment of mTHPP in all formulations (data not shown). The extent of surface modification via CS or PEG was quantified via two spectrophotometric assays already described by Anderski et al. (2018). The CS content in mTHPP-CS-PLGA-NP was detected as 0.7 ± 0.1% (m/m) referred to the particle bulk. In comparison, the PEG content of mTHPP-PLGA-PEG-NP was 13.3 ± 0.3% (m/m) and was significantly

3.3. Determination of cellular uptake and interaction A high intracellular photosensitizer accumulation is a crucial parameter for a successful PDT and was evaluated by various methods. The used nanoparticle concentrations corresponding to 1 μM mTHPP had no negative influence on cell viability, since the formulations showed no dark toxicity. All experiments regarding cellular uptake and interaction were performed under light exclusion. First, uptake studies over 24 h were performed with all formulations in HT-29 and HT-29-MTX cells followed by mTHPP quantification after cell lysis. Prior to the experiments, in our previous study the NP stability including a stable entrapment of mTHPP in all tested NP was verified (Anderski et al., 2018). Therefore, in the present study the recovery of mTHPP in the samples can be ascribed to drug transport by NP. Prior to NP incubation the cells were cultivated for either 5 or 10 days in order to achieve a different mucus thickness for HT-29-MTX cells. The intracellular uptake after 5 days of cell cultivation and mucus formation (Fig. 2A-B) showed no significant differences in the accumulation for all tested formulations in both cell lines. The highest intracellular concentration of mTHPP was achieved after 24 h by mTHPP-PLGA-PEG-NP, representing a 23-fold cellular accumulation compared to the initial concentration, independent of the cells with and without mucus (HT-29:

Table 1 Physicochemical characteristics of mTHPP-loaded PLGA-based nanoparticles (mean ± SD; n ≥ 3). Significance values versus the standard formulation (mTHPPPLGA-NP) are marked with * p ≤ 0.05. Nanoparticle system

Hydrodynamic diameter [nm]

Polydispersity index

Zeta potential [mV]

Drug load [μg mTHPP/mg NP]

mTHPP-PLGA-NP mTHPP-PLGA-PEG-NP mTHPP-CS-PLGA-NP

107.6 ± 6.8 93.4 ± 2.3* 120.1 ± 4.2*

0.07 ± 0.01 0.04 ± 0.01 0.20 ± 0.01

−29.7 ± 2.6 −19.8 ± 1.7* +10.3 ± 0.6*

51.8 ± 1.9 66.5 ± 2.5* 43.8 ± 7.0

31

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Fig. 1. Cell viability of (A) HT-29 and (B) HT-29-MTX cells after 24 h incubation with different formulations and illumination with 5 J/cm2 measured by WST-1 assay. A recovery period of 1 h after illumination was included before measurement (mean ± SD, n = 3).

Fig. 2. Cellular mTHPP uptake after incubation with 1 μM mTHPP in different formulations: (A) HT-29 and (B) HT-29-MTX cells after 5 days of growth and (C) HT-29 cells and (D) HT-29-MTX cells after 10 days of growth (mean ± SD, n = 3). Significant differences after 24 h incubation are marked with * p ≤ 0.05. 32

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incubation with an equivalent concentration of 1 μM mTHPP, revealing differences in intracellular accumulation between HT-29 (Fig. 4A-D) and HT-29-MTX cells (Fig. 4E-H). Control images of cells without incubation of nanoparticles were performed and exhibited no red fluorescence (data not shown). Comparing both cell lines, the punctuated distribution of nanoparticles for HT-29 cells differed from a slightly diffuse allocation in HT-29-MTX cells, suggesting a general influence of the mucus on the nanoparticle distribution around the cells. The intracellular accumulation of mTHPP-PLGA-PEG-NP in HT-29-MTX cells (Fig. 4F) led to the highest red intensity compared to unmodified, CSmodified PLGA-NP, and free mTHPP, confirming the tendency of an PEG-mediated increased accumulation in mucus-producing cells, which is consistent with the previous results of uptake studies after 10 days of mucus production.

Table 2 Intracellular mTHPP concentrations of different formulations after a growth period of 10 days in HT-29 and HT-29-MTX cells. Incubation was performed over 24 h followed by quantification of mTHPP via HPLC analysis. Significant differences between formulations are marked with * p ≤ 0.05.

mTHPP-PLGA-NP mTHPP-PLGA-PEG-NP mTHPP-CS-PLGA-NP mTHPP

HT-29 [μM/cell]

HT-29-MTX [μM/cell]

9.72 ± 0.89 12.31 ± 0.71 9.40 ± 0.81 11.58 ± 3.11

11.24 17.43 10.00 12.68

± ± ± ±

0.28 1.38* 0.84 2.36

23.3 ± 7.5 μM/cell vs. HT-29-MTX: 23.3 ± 7.2 μM/cell). The other formulations mTHPP-PLGA-NP, mTHPP-CS-PLGA-NP, and mTHPP reached comparable accumulation after 24 h in both cell lines between 17.0 ± 7.8 μM (HT-29-MTX: mTHPP-PLGA-NP) and 19.1 ± 9.4 μM (HT-29-MTX: mTHPP). However, no significant differences between both cell types as well as between the different formulations were observed. In comparison, the results of the uptake studies after 10 days of mucus production revealed a significantly higher accumulation for mTHPP-PLGA-PEG-NP in HT-29-MTX cells (24 h: 17.4 ± 1.4 μM/cell; p ≤ 0.05), indicating a beneficial effect of PEG-modification on mucus penetration (Fig. 2C-D). In comparison the mTHPP accumulation of the other NP formulations and free mTHPP remained at a decreased level (9- to 12-fold) with a certain similarity for HT-29 and HT-29-MTX cells (Table 2). The effect of a higher intracellular enrichment of PEGylated PLGANP in HT-29-MTX cells was evaluated by further visualization methods. The cellular interaction with HT-29-MTX cells over a period of 24 h was conducted by live-cell imaging (Fig. 3). The total red object area was quantified, representing the mTHPP autofluorescence. An increased cellular interaction was observed for mTHPP-PLGA-PEG-NP with 95.8 ± 8.2 mm2/well total red object area compared to both other NP formulations. Merely, free mTHPP exhibited higher values (130.1 ± 4.6 mm2/well total red object area). However, control experiments with free mTHPP as well as either mTHPP or mTHPP-PLGANP in combination with a HT-29-MTX cell lysate were performed and revealed a drastic increase in fluorescence intensity over time for the free drug in combination with the lysate whereas no increase was observed for the nanoencapsulated drug (data not shown). Additionally, confocal microscopy images were taken after 6 h of

3.4. Permeation studies in co-culture (Caco-2/HT-29-MTX) and monoculture (HT-29, HT-29-MTX) models For a detailed analysis of the mucus-permeating behavior of surfacemodified nanoparticles, different in vitro models were used, focusing either on the imitation of the gastrointestinal barrier with Caco-2 cells or the mucus layer itself in a monoculture model with mucus-producing cells. All cell models were incubated for 24 h with nanoparticles and free drug in a concentration equivalent to 5 μM mTHPP. Experiments in the co-culture model were performed with Caco-2 cells representing epithelial cells and additionally HT-29-MTX cells in order to produce a mucus layer on the formed barrier. The recovery of mTHPP in the basolateral and apical compartment as well as in the cells´ containing filter compartment is depicted in Fig. 5. Both cell ratios (Caco-2/HT-29MTX 7:3 (A) and 1:1 (B)) exhibited < 0.4% mTHPP recovery in the basolateral compartment for all tested NP formulations and free drug. TEER measurements showed constant values above 180 Ω × cm2, indicating an intact cell barrier during the whole experiment. Regarding the NP accumulation in the filter and mucus compartment, no significant differences were obtained in the co-culture model at both cell ratios between mTHPP-PLGA-NP, mTHPP-PLGA-PEG-NP, mTHPP-CSPLGA-NP, and free mTHPP. The results of the permeation studies in a monoculture model, consisting of either HT-29 or HT-29-MTX cells, are depicted in Fig. 6. The mucus layer on HT-29-MTX cells after a growth period of 10 days was observed by staining with Alcian blue for transmission light microscopy, whereas images of HT-29 cells solely showed cells stained with Nuclear Fast Red (Fig. 7). The permeation studies in the monoculture model were performed similarly to the co-culture model. Regarding the recovery of mTHPP in the basolateral compartment, the HT29-MTX model delivered comparable results to the co-culture model (< 0.4%), whereas HT-29 showed values between 2.6 and 5.4% (Fig. 6). With regard to the enrichment in the filter compartment containing cells and mucus, a significantly higher recovery was observed for mTHPP-PLGA-PEG-NP in combination with the HT-29-MTX model (30.4 ± 10.2%; p ≤ 0.05) whereas no significant increase was detected for the HT-29 cells. TEER measurements demonstrated values of approximately 50 Ω × cm2 for HT-29-MTX cells, while decreased values were observed for HT-29 cells (< 30 Ω x cm2). The effects of the permeation studies for the HT-29-MTX model were confirmed by fluorescence microscopy with vertical slices of the incubated Transwell® filters after 4 h and 24 h incubation (Fig. 8). Whereas slight differences in intensity of mTHPP were already obtained after 4 h of incubation (Fig. 8B), the higher accumulation of mTHPP-PLGA-PEG-NP in the mucus layer on the cells and already in the pores below the cell monolayer becomes apparent after 24 h (Fig. 8F), confirming a higher drug transport across the monolayer by PEGylated nanoparticles compared to all other tested nanoparticles and the free photosensitizer.

Fig. 3. Cellular interaction of mTHPP-PLGA-NP, mTHPP-PLGA-PEG-NP, mTHPP-CS-PLGA-NP, and free mTHPP with HT-29-MTX cells over a period of 24 h analyzed by IncuCyte® live-cell imaging system. mTHPP was visualized by red autofluorescence and calculated as total red object area. 33

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Fig. 4. Visualization of cellular uptake after 6 h incubation with 1 μM mTHPP in different formulations: (A-D) HT-29 and (E-H) HT-29-MTX cells. mTHPP was visualized by red autofluorescence, cell nuclei were stained with DAPI (blue) and membranes with WGA Alexa Fluor® 350 (blue). A/E: mTHPP-PLGA-NP; B/F: mTHPP-PLGA-PEG-NP; C/G: mTHPP-CS-PLGA-NP; D/H: mTHPP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

factor for formulation of photosensitizers such as mTHPP for future peroral application.

4.1. Cytotoxicity studies 4.2. Cellular uptake and interaction

The acute cytotoxicity plays an essential role for the general efficacy of new drug delivery systems, especially regarding promising surface modifications leading to a higher accumulation in desired target regions. An effective tumor cell cytotoxicity for PLGA-NP loaded with mTHPC, a structurally related photosensitizer to mTHPP, was already demonstrated in HT-29 cells by Löw et al. (2011) and these earlier results are in accordance with the results of the WST-1 assay of the present study. Besides the general efficiency of PS-loaded nanoparticles for PDT, the comparison between both chosen cell lines was performed to investigate if the mucus produced by HT-29-MTX cells already has an impact on cytotoxicity. The comparison of EC50 values revealed, that the effective concentration for all formulations is 2-fold higher in case of HT-29-MTX cells, indicating that after 5 days of cell cultivation small amounts of mucus formed a barrier for nanoparticle and drug transport. An expected higher cytotoxic effect resulting from a more effective transport of mTHPP across the mucus layer by chitosan- or PEG-modified PLGA-NP compared to unmodified NP was not obtained in a statistically significant manner. The formation of an insufficient mucus layer on HT-29-MTX cells after the chosen cell cultivation over 5 days might be an explanation as some groups described the onset of mucus production only after cell confluence (Lesuffleur, 1990; Pontier et al., 2001). In the present study WST-1 assay was selected for cytotoxicity studies due to its higher sensitivity compared to other colorimetric assays. A more precise assessment of cytotoxicity caused by surfacemodified NP might be feasible after a longer period of cell growth to attain a thicker mucus layer, but the colorimetric assay was interfered by an increasing number of cells and mucus, respectively. Therefore, the investigation of cytotoxicity after longer periods of cell growth could potentially be determined by a suitable non-colorimetric cell viability assay. Nevertheless, EC50 values for all NP formulations ranging from 0.5 to 1.7 μM in HT-29 and HT-29-MTX cells were achieved which were comparable to free mTHPP in solution (HT-29: 0.5 μM; HT29-MTX: 1.0 μM). Therefore, nanoencapsulation is not a detrimental

Cytotoxicity studies were followed by investigation of the cellular uptake as another crucial precondition for a sufficient nanoparticlemediated accumulation of mTHPP in gastrointestinal carcinoma cells. By the comparison of the cellular uptake after 5 and 10 days of cell growth (Fig. 2) a different mucus thickness could be simulated. Under these conditions a lower accumulation of mTHPP-PLGA-PEG-NP in HT29-MTX cells became clear if the cells were grown for a longer period of time (10 days; 17.4 ± 1.4 μM/cell) compared to the 5 day protocol (23.3 ± 7.2 μM/cell). This is caused by the increased thickness of the mucus layer after 10 days of cell growth. Additionally, it has to be considered that the lower accumulation values after 10 days, calculated as concentration per cell, are furthermore a consequence of an increased cell number due to a prolonged period of cell cultivation. Nevertheless, the significant 17-fold increased accumulation of mTHPPPLGA-PEG-NP in mucus-secreting HT-29-MTX cells after 10 days of growth was significantly higher compared to mTHPP-PLGA-NP, mTHPP-CS-PLGA-NP, and free mTHPP. The results verified the hypothesized mucus-penetrating effect of PEG due to its hydrophilic characteristics and underlines the importance of mucus to be included in in vitro experiments (Huckaby and Lai, 2017; Yuan et al., 2013). Referring to Maisel et al. (2015), a successful mucus diffusion is dependent on the molecular weight of PEG (5 kDa) and the surface density of PEG coating (minimum of 5%). Both conditions were fulfilled with the manufactured mTHPP-PLGA-PEG-NP (Anderski et al., 2018). Furthermore, with regard to the results of intracellular uptake after 5 days, it supports the assumption, that a shorter growing period is insufficient for an evaluation of the mucus influence. This agrees with the results of the cytotoxicity studies mentioned above. The effects for mTHPP-PLGAPEG-NP were confirmed by confocal microscopy, where the highest intensity of red photosensitizer fluorescence was observed after 6 h in HT-29-MTX cells. Likewise, the findings of live-cell imaging were in accordance with the previous results with mTHPP-PLGA-PEG-NP

Fig. 5. Permeation studies in a co-culture model of Caco-2 and HT-29-MTX cells in a ratio of (A) 7:3 and (B) 1:1 after 21 days of growth. All nanoparticle formulations and mTHPP were incubated for 24 h (mean ± SD, n = 3). 35

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Fig. 6. Permeation studies in a monoculture model of (A) HT-29 and (B) HT-29-MTX cells after 10 days of growth (mean ± SD, n = 3). All nanoparticle formulations and mTHPP were incubated for 24 h. Significant differences are marked with * p ≤ 0.05.

(Walter et al., 1996), which is not of relevance for our evaluation of mucus permeating NP. The optimization of such an intestinal model with regard to i.e. cell ratio is frequently reported in literature (Chen et al., 2010; Pan et al., 2015). According to Mahler et al. (2009), who showed that a cell ratio of 9:1 already led to an continuous mucus layer after 16 days of growth, experiments were conducted after 21 days at a cell ratio of 7:3 in order to increase the mucus formation compared to the literature described conditions. Additional experiments with an increased ratio for mucus-producing HT-29-MTX cells up to 1:1 were performed with the purpose of obtaining a thicker mucus layer. Contrary to our expectations, even with a higher ratio of HT-29-MTX cells, no differences in mucus permeation resulting in a higher enrichment of PS in cells and filter could be observed. This is contradictory to earlier reports which show a positive correlation in cell uptake with an increasing ratio of HT-29-MTX cells in the co-culture model (Calatayud et al., 2012). Furthermore, Beduneau (2014) described the seeding procedure of HT-29-MTX cells between day 0 and 3 in the co-culture model as an elementary factor for the permeation characteristics of the co-culture. In the present study the seeding day was taken into account and was fixed on day 0. Under the chosen conditions no drug enrichment in the basolateral compartment and therefore no cell permeation was observed for all of the NP formulations. However, with regard to a local PDT of gastrointestinal tumors only a local enrichment but not a systemic availability of the drug is required. Nonetheless, due to the indistinct results of the co-culture model in relation to the mucus-penetrating behavior of the NP, a second focus was set on an in vitro model, consisting solely of HT-29-MTX cells, aiming a thicker continuous mucus layer. Even with the drawback of reflecting only on cell type, the HT-29-MTX model is known in literature as a permeation setup for examination of mucus impact on NP (Behrens et al., 2001). The HT-29-MTX model was shown to be superior to Caco-2 and HT-29 model, representing a simple experimental setup for specific adhesion studies (Gagnon et al., 2013). At a first sight, the TEER values of the HT-29-MTX model (50 Ω × cm2) were significantly lower compared to the Caco-2 model and therefore an impaired barrier has to be expected. But on the other hand the mucus of the HT-29-MTX contributes to the barrier characteristics of the cell model and is the crucial aspect for the NP evaluation in the present study. In order to evaluate the impact of the mucus layer on NP permeation, the experiments were conducted in comparison to a simple HT-29 model, where no mucus layer was formed on the cell surface.

leading to the highest amount of red object area compared to the other NP formulations. Solely mTHPP in solution exhibits higher values even after a short period of 2 h. However, this effect has to be explained by an increase in mTHPP fluorescence in the presence of cell components as could be observed in control experiments in which different formulations were incubated with a HT-29-MTX cell lysate (data not shown). This phenomenon is explainable by the aggregation behavior of porphyrin molecules in solution leading to a reduced fluorescence quantum yield (Ricchelli, 1995). An increased fluorescence is caused by a subsequent disaggregation of mTHPP after contact with cell fragments leading to drug solubilisation. On the other hand the stable entrapment of mTHPP in nanoparticles showed no influence on fluorescence intensity. Therefore, the live-cell imaging and confocal laser microscopy results of the free drug were biased by an overestimation of the drugs´ fluorescence and could not directly be compared to the nanoparticle formulations. Moreover, the calculation of the red object area is based on images taken from the bottom of the well plates, which allows no differentiation of cell associated and intracellular photosensitizer. As a consequence, the results from this analytical method have rather to be discussed as cellular interaction, giving a tendency for NP accumulation, whereas the uptake experiments of the present study with HPLC quantification of mTHPP provide a clear measure of cell entrapped drug.

4.3. Evaluation of mucus permeation in different cell models The mucus layer in the gastrointestinal tract plays an important protective role for the human body and simultaneously represents the major obstacle for potent nanocarriers to enter epithelial cells underneath the layer (Cone, 2009). For a differentiated investigation of mucus permeation, we transferred our results of intracellular uptake to a three-dimensional setup consisting of cells grown on a semipermeable Transwell® filter, a widely used in vitro method for examination of mucus interaction (Grießinger et al., 2015). The pore size 3 μm of the filter was chosen to enable a sufficient permeation of the NP from the apical to basolateral compartment. At first, the literature known coculture model consisting of Caco-2 and HT-29-MTX cells was selected, representing epithelial and goblet cells. It mimicks a cellular barrier with additional mucus, allowing the evaluation of efficacy and safety of potential drug carriers (Ciappellano et al., 2016). Recent studies have shown that an active drug transport is underestimated in this model 36

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Fig. 7. HT-29 (A) and HT-29-MTX (B) cells visualized by transmission light microscopy after 10 days of growth on Transwell® filter. Cell nuclei were stained with Nuclear Fast red (red) and mucus was stained with Alcian blue (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

PLGA-NP previously observed in the uptake studies, resulting in a significantly higher enrichment in the mucus and cell compartment. Furthermore, this was observed in the fluorescence microscopy images after 4 h. In this context, the basolateral recovery for mTHPP in the mucus-free HT-29 model compared to the mucus-containing HT-29MTX cells confirms the theory of the mucus layer as a crucial barrier. Based on the results of both in vitro models, the conclusion can be drawn, that the produced mucus layer in the monoculture is a suitable setup for the evaluation of mucus-penetrating NP.

This setup differs from other publications, where mucus was removed from HT-29-MTX cells by N-acetyl-cystein or buffer agitation, but it minimizes the risk of damaging the cell layer by such techniques (Hilgendorf et al., 2000; Meaney and O'Driscoll, 1999). To confirm a mucus layer on HT-29-MTX cells after cultivation on Transwell® filters, the staining of acidic mucus substances was performed with Alcian blue, a commonly used procedure for the evidence of a mucus layer (Antunes et al., 2013). A growth period of 10 days was selected for the monoculture model to ensure a better comparability to the uptake results after the same time interval. Permeation studies with HT-29-MTX monolayers between 9 and 23 days of growth were already reported by Hagesaether et al. (2013), who showed that drug permeation was not significantly influenced by the period of cell growth. Visualization of mucus after 9 days additionally supports the assumption, that a mucus layer is already existing (Fig. 7). The selected time period of 10 days for experiments is additionally strengthened by results of permeation studies in the HT-29-MTX model after 20 days of growth, where no differences in NP accumulation were observed (data not shown). Overall, the results of the permeation studies in the monoculture model confirmed the assumption of the mucus-penetrating potential of PEGylated

5. Conclusion The present study shows, that PEGylation of PLGA-based nanoparticles is a suitable modification for a better mucus penetration and consequently higher intracellular uptake of the transported photosensitizer in targeted cells. Therefore, PEGylated nanoparticles are a promising drug delivery system for a local PDT treatment of gastrointestinal cancer. The ability of PEGylated particles to permeate faster through the mucus layer and subsequently achieve a higher intracellular accumulation of mTHPP makes such systems superior to 37

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Fig. 8. Vertical slices of Transwell® filters with HT29-MTX cells after 10 days of mucus-production period and (A-D) 4 h and (E-H) 24 h incubation with different formulations: mTHPP-PLGA-NP (A/E), mTHPP-PLGA-PEGNP (B/F), mTHPP-CS-PLGA-NP (C/G), and free mTHPP (D/H) were incubated with a concentration of 5 μM mTHPP. Filters and cell nuclei were stained with DAPI (blue), mTHPP was visualized by red autofluorescence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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unmodified NP, CS-modified NP, and free photosensitizer. On the other hand experiments without mucus revealed no significant differences in cellular uptake supporting the importance of mucus for in vitro evaluation. Furthermore, HT-29-MTX cells in a Transwell® setup present a simple and efficient alternative besides the commonly used co-culture model for evaluation of mucus-penetrating nanoparticles. However, further parameters like transition time, pH conditions and mucus turnover need to be taken into account for an assessment of the complex physiology in the gastrointestinal tract and, therefore, for an optimized prediction and closer correlation to the in vivo situation. In conclusion, the HT-29-MTX model represents a suitable in vitro model for examination of new potent pharmaceutical formulations targeting not only intestinal cells, but also cells under mucosal tissues in the human body.

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Funding The authors want to thank the German Federal Ministry of Education and Research (BMBF; project 13N13423) for the financial support and biolitec research GmbH Jena for kindly providing the photosensitizer mTHPP. The authors state, that both contributors were not involved in this study. References Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti, A.W., Gollnick, S.O., Hahn, S.M., Hamblin, M.R., Juzeniene, A., Kessel, D., Korbelik, M., Moan, J., Mroz, P., Nowis, D., Piette, J., Wilson, B.C., Golab, J., 2011. Photodynamic therapy of cancer: an update. CA Cancer J. Clin. 61, 250–281. Anderski, J., Mahlert, L., Mulac, D., Langer, K., 2018. Mucus-penetrating nanoparticles: promising drug delivery systems for the photodynamic therapy of intestinal cancer. Eur. J. Pharm. Biopharm. 129, 1–9. Antunes, F., Andrade, F., Araújo, F., Ferreira, D., Sarmento, B., 2013. Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs. Eur. J. Pharm. Biopharm. 83, 427–435. Beduneau, A., 2014. A tunable Caco-2/HT-29-MTX co-culture model mimicking variable permeabilities of the human intestine obtained by an original seeding procedure. Eur. J. Pharm. Biopharm. 87, 290–298. Behrens, I., Stenberg, P., Artursson, P., Kissel, T., 2001. Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells. Pharm. Res. 18, 1138–1145. Beloqui, A., des Rieux, A., réat, V., 2016. Mechanisms of transport of polymeric and lipidic nanoparticles across the intestinal barrier. Adv. Drug Deliv. Rev. 106, 242–255. Boegh, M., Nielsen, H.M., 2015. Mucus as a barrier to drug delivery - understanding and mimicking the barrier properties. Basic Clin. Pharmacol. Toxicol. 116, 179–186. Calatayud, M., Vazquez, M., Devesa, V., Velez, D., 2012. In vitro study of intestinal transport of inorganic and methylated arsenic species by Caco-2/HT29-MTX cocultures. Chem. Res. Toxicol. 25, 2654–2662. Chatterjee, D.K., Fong, L.S., Zhang, Y., 2008. Nanoparticles in photodynamic therapy: an emerging paradigm. Adv. Drug Deliv. Rev. 60, 1627–1637. Chen, X.-M., Elisia, I., Kitts, D.D., 2010. Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design. J. Pharmacol. Toxicol. Methods 61, 334–342. Ciappellano, S.G., Tedesco, E., Venturini, M., Benetti, F., 2016. In vitro toxicity assessment of oral nanocarriers. Adv. Drug Deliv. Rev. 106, 381–401. Cone, R.A., 2009. Barrier properties of mucus. Adv. Drug Deliv. Rev. 61, 75–85. Cu, Y., Saltzman, W.M., 2009. Controlled surface modification with poly(ethylene)glycol enhances diffusion of PLGA nanoparticles in human cervical mucus. Mol. Pharm. 6, 173–181. Date, A.A., Hanes, J., Ensign, L.M., 2016. Nanoparticles for oral delivery: design, evaluation and state-of-the-art. J. Control. Release 240, 504–526. Davis, M.E., Chen, Z.G., Shin, D.M., 2008. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discov. 7, 771–782. Dolmans, J., Fukumura, D., Jain, R.K., 2003. Photodynamic therapy for cancer. Nat. Rev. Cancer 3, 380–387. Ensign, L.M., Cone, R., Hanes, J., 2012. Oral drug delivery with polymeric nanoparticles:

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