Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant

European Journal of Pharmaceutical Sciences 91 (2016) 162–171

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant Pengli Bu a,b, Silpa Narayanan a, Damon Dalrymple c, Xingguo Cheng a, Abu T.M. Serajuddin a,⁎ a b c

Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, St. John's University, 8000 Utopia Parkway, Queens, NY 11439, United States Department of Biological Sciences, College of Liberal Arts and Sciences, St. John's University, 8000 Utopia Parkway, Queens, NY 11439, United States ABITEC Corporation, 501W 1st Avenue, Columbus, OH 43215, United States

a r t i c l e

i n f o

Article history: Received 2 March 2016 Received in revised form 24 May 2016 Accepted 15 June 2016 Available online 17 June 2016 Keywords: Lipid-based drug delivery system Medium-chain lipid Monoglyceride Triglyceride Surfactant Cremophor EL® Caco-2 cells Cytotoxicity assessment

a b s t r a c t Purpose: Caco-2 cells are used extensively for in vitro prediction of intestinal drug absorption. However, toxicity of excipients and formulations used can artificially increase drug permeation by damaging cell monolayers, thus providing misleading results. The present study aimed to investigate cytotoxicity of common lipid-based excipients and formulations on Caco-2 cells. Methods: Medium-chain monoglycerides alone or in mixture with the surfactant Cremophor EL, with and without a medium-chain triglyceride, were prepared and incubated with Caco-2 cells from a series of culture stages with varying maturity. Cell viability was evaluated and cell membrane integrity assessed. Results: Cytotoxicity of lipid-based formulations was influenced by the maturity of Caco-2 cells and formulation composition. One-day culture was most sensitive to lipids. When cultured for 5 days, viability of Caco-2 cells was significantly improved. The 21-day Caco-2 monolayers maintained the highest survival rate. Microemulsion formulations exhibited significantly less cytotoxicity than neat lipids or surfactant at all stages of cell maturity, and microemulsions containing 1:1 mixtures of monoglyceride and triglyceride appeared to be best tolerated among all the formulations tested. Mechanistically, the observed cytotoxicity was partially due to lipid-induced rupture of cell membrane. Conclusions: Microemulsions of lipid-surfactant mixtures have less cytotoxicity than lipid alone. Maturity of Caco-2 cells renders significant resistance to cytotoxicity, and monolayers with 21-day maturity are more relevant to in vivo conditions and appear to be a more accurate in vitro model for cytotoxicity assessment. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Oral drug administration is considered as the most convenient route and the preferred choice for patients. The absorption of orally administered drugs has thus been a subject of intense and continuous investigation in the pharmaceutical industry (Pang, 2003). Good oral bioavailability requires efficient drug absorption in the gastrointestinal (GI) tract, which highlights the importance of intestine in regulating the absorption of oral drugs. The single epithelial layer lining the intestinal lumen constitutes the rate-limiting barrier to absorption of orally Abbreviations: LBDDS, Lipid-based drug delivery system; LBSEDDS, Lipid-based selfemulsifying drug delivery system; SEDDS, Self-emulsifying drug delivery system; SMEDDS, Self-microemulsifying drug delivery system; MTT, (3-(4.5-Dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide); PG, Propylene glycol; HBSS, Hank's Balanced Salt Solution; DMEM, Dulbecco's Modification of Eagle's Medium; FBS, Fetal bovine serum; DMSO, Dimethyl sulfoxide; TEER, Transepithelial electrical resistance; P-gp, Pglycoprotein. ⁎ Corresponding author. E-mail address: [email protected] (A.T.M. Serajuddin).

http://dx.doi.org/10.1016/j.ejps.2016.06.011 0928-0987/© 2016 Elsevier B.V. All rights reserved.

administered drugs. A reliable and widely used in vitro model to assess intestinal drug absorption is the Caco-2 cell monolayer model (Shah et al., 2006; Artursson et al., 2001). Caco-2 cells are cells derived from human colon adenocarcinoma, which under experimental conditions, can differentiate into a monolayer with morphological and functional similarities to intestinal epithelium (Hubatsch et al., 2007; van Breemen and Li, 2005). Therefore, Caco-2 monolayer model has been accepted by pharmaceutical companies and regulatory authorities as a standard in vitro model for predicting intestinal drug permeability (Shah et al., 2006). It is also commonly used for screening dosage forms and drug delivery systems for their effects on drug permeation (Shah et al., 2006; Degim et al., 2004; Hugger et al., 2002). The extensive application of combinatorial chemistry and highthroughput screening in drug discovery has led to a myriad of new drug candidates, majority of which are extremely insoluble in water and require efficient drug delivery systems to increase solubility and promote absorption (Lipinski, 2000; Lipinski et al., 2001; Rahman et al., 2011; Williams et al., 2013). Lipid-based drug delivery systems (LBDDS) have been recognized as an attractive approach to solubilize

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

poorly water-soluble drugs and present them to the GI tract as solutions in lipids or mixtures of lipids, surfactants and/or co-surfactants. The LBDDS facilitate formation of solubilized phases that promote drug absorption (Porter et al., 2007, 2008; Gursoy and Benita, 2004; Singh et al., 2009; Serajuddin et al., 1985). Furthermore, components of LBDDS such as certain surfactants (Sha et al., 2005; Deli, 2009; Dimitrijevic et al., 2000) and lipids (Lindmark et al., 1998) have been shown to enhance drug permeation by modulating tight junction in Caco-2 monolayers. Such permeation enhancing effects of excipients have attracted considerable attention as a desirable addition to facilitating solubilization, which can boost bioavailability. However, it was noticed that increased permeation rendered by drug delivery systems in certain cases correlated with decreased viability of Caco-2 cells (Ujhelyi et al., 2012; El-Sayed et al., 2002). Damages in Caco-2 monolayers due to cytotoxicity of lipids and/or surfactants can yield artificial increase in permeation assessment. Therefore, it is of crucial importance to test excipients of interest for cytotoxicity prior to further biological evaluation. A prerequisite for any good lipid-based formulations is good tolerance at the sites of absorption. However, there was no generally accepted approach in the literature for the cytotoxicity assessment of excipients. It has been long established that initial seeding density and culture durations influence proliferation, differentiation and senescence of the Caco-2 cell population (Sambuy et al., 2005; Natoli et al., 2011, 2012). However, in the few available reports in the literature evaluating excipient cytotoxicity on Caco-2 cells, the culture conditions of Caco-2 cells employed appeared to be varied and cannot be compared with each other. For example, when evaluating cell viability using MTT assay in 96-well plates, Konsoula and Barile (2005) utilized a seeding density of 3.3 × 103 cells per well, cultured the cells for 7 days and then incubated these Caco-2 cells with various chemicals for 24 and 72 h. In other studies, Buyukozturk et al. (2010) seeded Caco-2 cells at a density of 5 × 104 cells/well, cultured the cells for 7 days and conducted the incubation with formulations for 3 h, while Sha et al. (2005) seeded Caco-2 cells at the same density (5 × 104 cells/well) but cultured them for 4 days prior to a 2-h incubation. Consequently, the inconsistency in crucial culture conditions inevitably leads to data variability and the lack of comparability among similar studies. One major objective of the present study was to develop a systematic approach that may be applied to assessing safety of common components of LBDDS as well as their mixtures by using the Caco-2 cell in vitro model. For comparison purposes, we established a series of Caco-2 cells with the same seeding density but varying culture durations and tested them for the most accurate cellular response to lipid-based formulations. We believe that the cytotoxicity assessment method established by the current study identified Caco-2 monolayers, which were fully differentiated on membrane supports, more relevant to in vivo situations and exhibited the best tolerance, as a suitable model for safety assessment. Another major objective of the present investigation was to study the effect of formulation variables on the cytotoxicity of LBDDS using the Caco-2 cell model. Lipid and surfactant are the two primary components of LBDDS. Among lipids, mono- and tri-glycerides of mediumchain fatty acids as well as mono- and di-esters of medium-chain fatty acids with propylene glycol (PG) are commonly used. Cremophor EL (PEG-35 castor oil ester) and Tween 80 (polysorbate 80) are two common surfactants. Prajapati et al. (2011, 2012) and Patel et al. (2012) reported that mixtures of Cremophor EL with either the monoglyceride or the propylene glycol (PG) monoester of medium-chain fatty acids lead to microemulsion formation upon dilution with aqueous media. In contrast, no microemulsion was formed when Cremophor EL was mixed with the triglyceride of medium-chain fatty acid. Although the triglyceride per se does not form microemulsion, the most optimal result with respect to the formation of microemulsion was obtained when mixtures of a triglyceride with a monoglyceride (or PG monoester) along with Cremophor EL was formulated instead of using the monoglyceride by

163

itself. For example, combinations of Cremophor EL with a 1:1 w/w mixture of Capmul MCM EP (medium-chain monoglyceride) and Captex 355 EP/NF (medium-chain triglyceride) formed microemulsions spontaneously upon dilution with aqueous media without going through any gel phase and provided the largest microemulsion region in the lipid-surfactant-water phase diagram (Prajapati et al., 2011). Prajapati et al. (2012) also described the possible mechanism for the effect of monoglyceride-triglyceride mixture on the formation of microemulsion. For these reasons, it was of interest to study the relative effects of different excipients, such as surfactant alone (Cremophor EL), binary mixtures of surfactants with monoglyceride or PG monoester, and ternary mixtures of surfactants with monoglyceride (or PG monoester) and triglyceride, on the viability of Caco-2 cell monolayers. In this way, the optimal LBDDS formulation with the best tolerance on the GI membrane may possibly be identified. 2. Materials and Methods 2.1. Lipids and Surfactant Surfactant (Cremophor EL, PEG-35 castor oil) and medium-chain lipids used (mono- and triglycerides; PG monoesters), along with their trade names, manufacturers, chemical structures and compositions, are listed in Table 1. It should be noted that the lipids investigated in the present study do not exist as pure species, but are mixtures of glycerides (or PG esters) with differing degrees of esterification and different fatty acid compositions (Prajapati et al., 2012). Therefore, only the chemical structure of the predominant component of each lipid is provided in the table. Aqueous buffer Hank's Balanced Salt Solution (HBSS) (Hyclone, cat. # SH30588.02) was used to prepare preconcentrate of the surfactant alone formulation (30% surfactant and 70% buffer) and used as the diluent for all formulations in particle size measurement. 2.2. Preparation of Lipid-based Formulations Various formulations tested are listed in Table 2. Each test lipid was mixed with the surfactant Cremophor EL at the following ratios (v/v): (1) 30% Cremophor EL and 70% monoglyceride; (2) 30% Cremophor EL and 70% PG monoester; (3) 30% Cremophor EL, 52.5% monoglyceride and 17.5% triglyceride; (4) 30% Cremophor EL, 52.5% PG monoester and 17.5% triglyceride; (5) 30% Cremophor EL, 35% monoglyceride and 35% triglyceride; (6) 30% Cremophor EL, 35% PG monoester and 35% triglyceride. The triglyceride could not be mixed with the monoglyceride at higher than the 1:1 ratio as it led to phase separation of lipid upon dilution with water (Prajapati et al., 2011). As listed in Table 1, Capmul PG-8 NF was used as the PG monoester and Captex 355 was used as the triglyceride, while two monoglycerides with differences in their chemical compositions were used. All formulations as per Table 2 were prepared in 50 mL polycarbonate tubes, mixed by vortex at the highest setting followed by further mixing in a rotating shaker at room temperature for 3 h, and then equilibrated at room temperature for at least 24 h prior to use. 2.3. Particle Size Analysis A DelsaNano C particle size analyzer (Beckman Coulter Inc., Brea, CA) was used to measure the mean particle size of lipid-surfactant mixtures at 0.1%, 0.2%, and 0.5% (v/v) in aqueous HBSS solution. To ensure reproducibility of particle size determination, three replicate solutions of each concentration were prepared. Approximately 2 mL of sample was added to a disposable plastic cuvette (Beckman Coulter disposable cell, Beckman Coulter Inc., Brea, CA) for particle size determination using the dynamic light scattering technique at 25 °C. To determine the effect of equilibration time on particle size, the particle size

164

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

Table 1 Chemical name, trade name, structure and composition of surfactant and lipids. Generic name

Trade name/Manufacturer

Surfactant PEG-35 castor oil

Lipid

Primary component structure

Composition The main component (83%) is polyethylene glycol ester of ricinoleic acid, HLB 13

Cremophor EL BASF Tarrytown, NY, USA

Glyceryl Capmul MCM EP ABITEC monocaprylocaprate Corp. Columbus, OH, USA

Medium chain length mono (60%) and diglyceride (35%) consisting of 83% w/w caprylic acid (C8) and 17% w/w capric acid (C10), HLB 4.7

Glyceryl mono- and dicaprylate

Capmul 708G ABITEC Corp. Columbus, OH, USA

Mono- and diglyceride of caprylic acid (C8), N99% Wt w/w

Propylene glycol monocaprylate

Capmul PG-8 NF ABITEC Corp. Columbus, OH, USA

Propylene glycol ester of mono- (90%) and dicaprylate (10%), HLB 5–6

Caprylic/capric triglyceride

Captex 355 ABITEC Corp. Columbus, OH, USA

Medium chain triglyceride consisting of mixture of caprylic acid (C8) and capric acid (C10) at 55:45 ratio, HLB 0

measurement of each sample was performed at 0, 60 and 120 min in triplicates. Table 2 Mean particle size of lipid-surfactant mixtures at different concentrations (v/v% in HBSS buffer) and time intervals. Particle size (nm)a Formulation

Dilution

0 min

60 min

120 min

Cremophor EL 30% HBSS 70%

0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5% 0.1% 0.2% 0.5%

38 ± 14 18 ± 1 17 ± 2 64 ± 9 157 ± 24 849 ± 87 121 ± 3 161 ± 11 1471 ± 359 76 ± 3 122 ± 11 92 ± 9 52 ± 1 45 ± 2 107 ± 19 78 ± 5 35 ± 2 30 ± 2 50 ± 1 41 ± 2 43 ± 2 56 ± 3 47 ± 3 38 ± 2 56 ± 3 47 ± 3 38 ± 2 68 ± 2 71 ± 4 73 ± 1

24 ± 19 17 ± 2 15 ± 1 65 ± 6 162 ± 35 1301 ± 52 118 ± 3 160 ± 9 1570 ± 62 80 ± 5 128 ± 3 100 ± 7 50 ± 1 46 ± 3 105 ± 22 79 ± 6 35 ± 2 30 ± 2 54 ± 2 63 ± 2 82 ± 2 50 ± 5 41 ± 2 42 ± 3 57 ± 4 47 ± 3 37 ± 2 69 ± 4 70 ± 4 74 ± 2

28 ± 5 18 ± 3 16 ± 2 63 ± 5 161 ± 40 1804 ± 321 115 ± 2 150 ± 13 2061 ± 701 79 ± 4 138 ± 5 102 ± 10 46 ± 6 46 ± 3 105 ± 20 77 ± 5 35 ± 3 31 ± 2 55 ± 5 59 ± 1 84 ± 2 52 ± 4 41 ± 1 41 ± 3 57 ± 5 49 ± 4 38 ± 2 70 ± 3 71 ± 5 75 ± 2

Cremophor EL 30% Capmul MCM EP 70% Cremophor EL 30% Capmul 708G 70% Cremophor EL 30% Capmul PG-8 NF 70% Cremophor EL 30% Capmul MCM EP 52.5% Captex 17.5% Cremophor EL 30% Capmul 708G 52.5% Captex 17.5% Cremophor EL 30% Capmul PG-8 NF 52.5% Captex 17.5% Cremophor EL 30% Capmul MCM EP 35% Captex 35% Cremophor EL 30% Capmul 708G 35% Captex 35% Cremophor EL 30% Capmul PG-8 NF 35% Captex 35% a

Values are mean ± SD (n = 3).

2.4. Cell Culture Caco-2 cells (ATCC, cat. # HTB-37) were cultured in Dulbecco's Modification of Eagle's Medium (DMEM/High Glucose, Hyclone, cat. # SH30243.01) supplemented with 10% fetal bovine serum (FBS) at 37 ° C in a humidified atmosphere of 5% CO2, and 95% air. For all experiments, Caco-2 cells were maintained in T-75 flasks in growth medium and used between passages 3 and 8 after recovering from cryogenic stocks. For the images presented in Fig. 1, Caco-2 cells were grown in tissue culture grade 24-well plates with a seeding density of 1.0 × 105 in a final volume of 0.5 mL culture medium per well and cultured for indicated duration. For 1-day and 5-day cultures, Caco-2 cells were seeded into regular tissue culture 96-well plates at 2 × 104 cells per well in a volume of 200 μL growth medium and cultured for designated duration. For 21day monolayer culture, Caco-2 cells were seeded at 2 × 104 cells per well into 96-well transwell plates (Falcon, 1.0 μm pore size PET membrane-based 96-well insert system, cat. # 351131) with a feeder tray and cultured for 21 days, where the medium was changed every other day. 2.5. Cytotoxicity Assessment After being cultured for designated durations, Caco-2 cells were treated with individual lipids or formulations in a final volume of 200 μL per well in 96-well plates at indicated concentrations diluted with culture medium for two hours. Cells incubated with only culture medium (no lipid) and medium containing digitonin (30 μg/mL) served as negative and positive controls, respectively. Immediately after treatment, cells were evaluated for cytotoxicity with either trypan blue exclusion test or MTT cell viability assay. For trypan blue exclusion test,

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

165

Fig. 1. Caco-2 cells in culture: forming monolayer. Cultured Caco-2 cells were in the process of forming monolayer, images captured at day 1 (A), day 3 (B), day 5 (C) and day 10 (D) after seeding, the day of seeding was designates as day 0. Scale bar: 500 μm.

cells were incubated with 0.05% trypan blue for 3 min followed by microscopic examination. For MTT assay, cells were incubated with 0.45 mg/mL 3-(4.5-dimethylthiazol-2-yl)-2.5- diphenyltetrazolium bromide (MTT, Alfa Aesar, cat. # L11939) for 3.5 h at 37 °C in a tissue culture incubator. Upon completion of incubation, the medium was replaced with dimethyl sulfoxide (DMSO, Fisher Scientific, cat. # BP 2311) (100 μL/well) and the plate was covered with foil to minimize exposure to light and agitated on an orbital shaker at low speed for 15 min. Absorbance was then measured using a microplate reader at a wavelength of 570 nm. For MTT assay with 21-day Caco-2 monolayer culture, monolayers grown in each insert were incubated with individual formulations (in a volume of 100 μL in the apical compartment) on day 22 for two hours, with a volume of 300 μL medium in the basal compartment (96-square angled bottom well plate, Falcon, cat. # 353925). Upon completion of treatment, cells were incubated with 1.67 mg/mL MTT solution for another 3.5 h. Then the apical solution was removed and membrane insert (with cells) was transferred to a new 96-square angled bottom well plate followed by addition of 100 μL DMSO into the apical compartment. The plate was covered from light and incubated at 37 °C with orbital shaking (150 rpm) for 45 min. An aliquot of 25 μL DMSO solution from each apical compartment of the transwell plate was transferred to a microplate reader compatible 96-well pate, mixed with 75 μL fresh DMSO solution and was proceeded to absorbance measurement.

to manufacturer's instructions. Briefly, cells were incubated with a fluorogenic and cell-impermeant peptide substrate specific for an intracellular protease. Only when cell membrane is damaged this protease leaks into the culture medium and converts the substrate into a fluorescent product. The amount of fluorescent products, which can be determined by a fluorescence microplate reader, correlates to the level of membrane damage within a well. In the present study, upon completion of treatment with 5-day Caco-2 culture, cells were incubated with bisAAF-R110 Substrate (fluorogenic and cell-impermeant substrate) and buffer in a volume of 200 μL for 1 h at 37 °C. Fluorescence intensity was determined using a fluorescence microplate reader (BioTek, Synergy H1 Multi-Mode Microplate Reader) with an excitation wavelength at 485 nm and an emission wavelength at 520 nm. 2.7. Statistical Analysis Representative data of two or three independent experiments are presented as mean ± S.E.M., n = 5–6 replicates for each treatment groups. Data were analyzed by one-way ANOVA, followed by Duncan's post hoc test. Statistical significance was set at p b 0.05. 3. Results

2.6. Membrane Integrity Assessment

3.1. Caco-2 Cells Grown in Culture: A Dynamic Population with Distinct Morphologies

Caco-2 cells were seeded into 96-well tissue culture grade black plates with clear bottom (Corning, COSTAR, cat. # 3603) at 2 × 104 cells per well in a volume of 200 μL growth medium and cultured for 5 days with medium changed on day 2, 4 and 5. Treatment was conducted on day 5. Immediately after treatment, cells were processed for MultiTox-Fluor Cytotoxicity Assay (Promega, cat. # G9200) according

Images of Caco-2 cells grown in culture were captured at days 1, 3, 5 and 10 after seeding (Day 0) (Fig. 1). After overnight growth following seeding, most cells were present as single cells (Fig. 1A). As cell divided, small colonies were formed after 3 days of culture (Fig. 1B). These small colonies continued to expand to form larger colonies by joining with nearby colonies on Day 5 (Fig. 1C). Eventually all the available space

166

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

was filled with cells and a continuous monolayer was formed in 10 days (Fig. 1D). These images demonstrate that the Caco-2 cells grown in culture were exhibiting dynamic features. Cells from different confluences showed distinct morphologies (i.e. single cells, small colonies, larger colonies or monolayer) and were very likely associated with distinct cellcell contact and communications. Such differences of the Caco-2 cell populations may possibly cause distinct cellular responses to the same treatment. Therefore, this finding provides a rationale for the later selection of Caco-2 cells with varying culture durations for the cytotoxicity assessment.

3.2. Validation of Methods for Assessing Lipid-Associated Cytotoxicity and Impact of Neat Lipids on Viability of Caco-2 Cells We chose two commonly used methods, namely, trypan blue exclusion test (Fig. 2A) and MTT cell viability assay (Fig. 2B), for assessing cytotoxicity. The principle of trypan blue exclusion test is that healthy cells are not permeable to certain dyes such as trypan blue, whereas dead cells are (Strober, 2001). Therefore, viable cells will maintain a clear cytoplasm whereas nonviable cells will have a blue cytoplasm when incubated with trypan blue and examined under a microscope. The MTT assay developed by Mossman (Mosmann, 1983) is one of the most used assays for evaluating cytotoxicity. The MTT assay involves the conversion of the water-soluble tetrazolium dye MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) by cellular NADH to insoluble purple-colored formazan. The absorbance of solubilized formazan solution is then quantified by a spectrophotometer at a certain wavelength (usually at 570 nm) (Liu et al., 1997). The MTT assay is a sensitive assay with excellent linearity (up to 106 cells per well),

with optical density reading directly reflecting the metabolic activities and thus the number of viable cells present (Berridge et al., 2005). Caco-2 cells after 1-day culture were treated with neat lipids at indicated concentrations, and results from the trypan blue exclusion test and the MTT assay are shown in Fig. 2A and B, respectively. Although the principles of the two tests are different, the results were similar, where severe cytotoxicity was observed at concentrations equal and higher than 0.1% (v/v) of all three lipids tested. Particularly, Caco-2 cells appeared to be somewhat more sensitive to one lipid, Capmul PG-8, with a significantly reduced survival rate at concentration of 0.03% (Fig. 2). These findings suggest that neat lipids can cause prominent cytotoxicity in Caco-2 cells with a short culture period (1 day). However, the Caco-2 monolayers used in permeation studies are usually cultured for 21 days and, therefore, these initial studies indicated that cells from longer culture periods may be more suitable for cytotoxicity assessments. It was also observed in this study that the neat lipids were not miscible with aqueous culture medium, which necessitated the use of lipid-surfactant mixtures in subsequent studies to achieve homogeneous mixtures. Further, since the initial results of neat lipid cytotoxicity obtained from the trypan blue exclusion test (Fig. 2A) and the MTT assay (Fig. 2B) were very similar and consistent with each other, the MTT assay was chosen for all the future cytotoxicity assessment. 3.3. Preparation of Lipid-based Formulations and Particle Size Analysis Chemical name, structure and composition of surfactant and lipids used in this study are listed in Table 1. Lipid-based formulations containing surfactant Cremophor EL were prepared as described in Materials and Methods. The particle size analysis was performed on all the formulations that were later evaluated for cytotoxicity in Caco-2 cells. As shown in Table 2, the addition of aqueous buffer Hank's

Fig. 2. Cytotoxicity assessment of neat lipids by trypan blue exclusion test and MTT cell viability assay. Caco-2 cells (1-day culture) were incubated with medium-chain monoglyceryl lipids (Capmul MCM EP or Capmul 708G) or propylene glycol monoester (Capmul PG-8 NF) at the indicated concentrations for 2 h followed by cytotoxicity assessment with trypan blue exclusion test (A) or MTT cell viability assay (B). Negative control: cells incubated with culture medium only; positive control: cells incubated with culture medium containing digitonin (30 μg/mL). Data are presented as mean ± S.E.M., n = 5–6 replicates per each treatment. Asterisks (*) indicate statistically significant differences, p b 0.05, compared to control. MCM, Capmul MCM EP; 708G, Capmul 708G; PG8, Capmul PG-8 NF.

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

167

Balanced Salt Solution (HBSS) to liquid preconcentrates (liquid-surfactant mixtures) produced microemulsions with particle sizes b200 nm, except for cases of Cremophor EL-Capmul MCM EP and Cremophor EL-Capmul 708G mixtures at the concentration of 0.5% (v/v). The results with Capmul MCM and Capmul 708G are in agreement with previous studies (Prajapati et al., 2011, 2012; Patel et al., 2012), where it was observed that binary mixtures of a surfactant with the monoester of fatty acids do not always lead to microemulsion formation. Indeed, the results of the present study confirm that when ternary mixtures were prepared by adding a triglyceride Captex to the preconcentrate, very fine microemulsions (nanoemulsions) with particle size around 100 nm or less were formed upon dilution with aqueous buffer and slightly coarse emulsions (particle sizes larger than 1000 nm) in the case of Cremophor EL 30% Capmul 708G 70% at 0.5% (v/v) concentration. Table 2 also summarizes the impact of time on the particle size of the lipid-surfactant mixtures during the dispersion test. Once microemulsion forms in the aqueous solution (at 0 min), the mean particle size remained almost unchanged with minimal fluctuations throughout the 2-h test period (see value of S.D. for each mean value in Table 2). Our results are in agreement with previous findings (Shah, 1998), demonstrating that microemulsions are thermodynamically equilibrium systems and the particle size of microemulsion systems remains stable over the test period. Unlike microemulsions, the particle size grew in case of coarse emulsions (Cremophor EL-Capmul MCM EP and Cremophor EL-Capmul 708G mixtures at 0.5% (v/v)). 3.4. Cytotoxicity Assessment of Lipid-Surfactant Mixtures on Caco-2 Cells from Various Culture Stages Lipid-based formulations containing surfactant Cremophor EL, as described in Table 1, were tested for their impact on viability of Caco-2 cells. Caco-2 cells cultured for various periods of time (1, 5 and 21 days) were used in the study. Two treatment groups were included as controls, the surfactant alone group and the buffer (70%)-surfactant (30%) mixture (the buffer occupies the same percent as the test lipid does in the lipidsurfactant mixtures). Within every treatment group for each formulation, the control group received no treatment but growth medium, and was considered as 100% viable. On 1-day old Caco-2 cells, which were young and fragile, most of the formulations caused a significant reduction on cell survival (Fig. 3). However, surprisingly, one particular composition, which contained 30% surfactant, 35% monoglyceride (Capmul MCM or Capmul 708G) or PG monoester (Capmul PG8) and 35% triglyceride (Captex), exhibited the least toxicity (Fig. 3B–D, red-patterned bars). With this composition, a concentration-dependent increase in cytotoxicity was also obvious for all three lipids tested (Fig. 3B–D, 0.1% vs. 0.2%). Especially, the formulation containing 30% Cremophor EL, 35% Capmul MCM and 35% Captex appeared to be most tolerant by cells as no decrease in the cell viability at 0.1% concentration with respect to the control (cells receiving no treatment) was observed (Fig. 3B, control vs. 0.1% red-patterned bar), and, even at 0.2% concentration (Fig. 3B, 0.2% red-patterned bar), the cell viability is much higher than those with Capmul 708G (Fig. 3C, 0.2% red-patterned bar) and Capmul PG8 (Fig. 3D, 0.2% red-patterned bar). As shown in Fig. 4, the overall tolerance of the 5-day old Caco-2 cells to lipid-surfactant mixtures improved greatly as compared to that of the 1-day old cells (Fig. 3). Although cytotoxicity was still obvious in the surfactant alone control group, the buffer-surfactant mixture control group showed no toxicity at concentrations of 0.1% and 0.2%, and only mild effect with the highest concentration (0.5%) (Fig. 4A). It should be noted that in the buffer-surfactant formulation only 30% of the formulation was occupied by surfactant, while the remaining 70% was occupied by the buffer and later replaced by the lipid in the lipidsurfactant formulations. Cell viability was markedly improved on almost all 0.1% concentration treatments (Fig. 4B–D) and on 0.2% concentration treatments with the test lipid Capmul 708G (Fig. 4C). Although

Fig. 3. Cytotoxicity assessment of lipid-based self-emulsifying drug delivery systems on 1day Caco-2 culture. Caco-2 cells (1-day culture) were incubated with medium-chain monoglyceryl lipids (Capmul MCM EP or Capmul 708G) or propylene glycol monoester (Capmul PG-8 NF) in mixture with surfactant (Cremophor EL) with and without a medium-chain triglyceride (Captex 355) at indicated ratio for 2 h followed by cytotoxicity assessment with MTT viability assay. Data are presented as mean ± S.E.M., n = 5–6 replicates per each treatment. Asterisks (*) indicate statistically significant differences, p b 0.05, compared to control. Cre. EL, Cremophor EL; MCM, Capmul MCM EP; Cap, Captex 355; 708G, Capmul 708G; PG8, Capmul PG-8 NF. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

the toxicity was still obvious at 0.5% concentration treatments, a consistent trend was that the optimal composition of surfactant, monoglyceride or PG monoester and triglyceride mentioned earlier (30% surfactant, 35% monoglyceride or PG monoester and 35% triglyceride) showed the least toxicity (Fig. 4B–D, red-patterned bars). On 21-day Caco-2 monolayers, which were cultured on filter membrane support in transwell plates following the same procedure used in permeation studies reported in the literature (van Breemen and Li, 2005; Sha et al., 2005; Ujhelyi et al., 2012), excellent tolerance was seen in the control treatment group containing surfactant alone and in the control treatment group containing a mixture of 70% buffer and

168

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

Fig. 4. Cytotoxicity assessment of lipid-based self-emulsifying drug delivery systems on 5day old Caco-2 culture. Caco-2 cells (5-day culture) were incubated with medium-chain monoglyceryl lipids (Capmul MCM EP or Capmul 708G) or propylene glycol monoester (Capmul PG-8 NF) in mixture with surfactant (Cremophor EL) with and without a medium-chain triglyceride (Captex 355) at indicated ratio for 2 h followed by cytotoxicity assessment with MTT viability assay. Data are presented as mean ± S.E.M., n = 5–6 replicates per each treatment. Asterisks (*) indicate statistically significant differences, p b 0.05, compared to control. Cre. EL, Cremophor EL; MCM, Capmul MCM EP; Cap, Captex 355; 708G, Capmul 708G; PG8, Capmul PG-8 NF. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

30% surfactant (Fig. 5A). Consistent with what was observed in the 5day cell culture, the particular formulation containing 30% surfactant, 35% monoglyceride or PG monoester and 35% triglyceride showed the best tolerance (Fig. 5B–D, red-patterned bars). Additionally, the survival rates of cells were higher with the 21-day monolayer cells than the 5-day old Caco-2 cells. More than 75% cell survival was maintained with the treatment of formulation containing 30% surfactant, 35% Capmul MCM and 35% Captex, at the highest concentration (0.5%) (Fig. 5B, red-patterned bars), and there was no significant toxicity with the same formulation containing Capmul 708G at the same concentration (Fig. 5C, red-patterned bars). 3.5. Assessment of Membrane Integrity in Caco-2 Cells Treated with LipidSurfactant Mixtures We next investigated the mechanism(s) responsible for toxicity induced by lipid-surfactant mixtures. Our working hypothesis was that

Fig. 5. Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system on 21day Caco-2 culture. Caco-2 cells were maintained under the same condition as in permeation studies (growing in transwell plates for 21 days with medium changed every other day) prior to incubation with lipid-surfactant mixtures and cytotoxicity assessment. Data are presented as mean ± S.E.M., n = 5–6 replicates per each treatment. Asterisks (*) indicate statistically significant differences, p b 0.05, compared to control. Cre. EL, Cremophor EL; MCM, Capmul MCM EP; Cap, Captex 355; 708G, Capmul 708G; PG8, Capmul PG-8 NF.

lipids and lipid-surfactant mixtures disrupt cell membrane integrity by interacting with the phospholipid bilayer of plasma membrane (McCall, 2010; Moquin and Chan, 2010). Consequently, loss of membrane integrity leads to cellular injury and eventually cell death, which is reflected by reduced metabolic activities at the cell population level. Because 5-day culture of Caco-2 cells appeared to tolerate lipid/surfactant mixtures much better than 1-day culture and close to 21-day culture, we decided to use the 5-day culture for this assessment. Cell membrane integrity was evaluated using MultiTox-Fluor Cytotoxicity assay, which measures the activity of a marker protease specific to cells with compromised plasma membrane. A fluorogenic and cellimpermeant peptide substrate was converted to fluorescent products only by the marker protease that leaks out through damaged cell

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

Fig. 6. Assessment of membrane integrity of 5-day Caco-2 culture treated with lipid-based self-emulsifying drug delivery system. Caco-2 cells (5-day culture) were incubated with various lipid-surfactant mixtures for 2 h followed by assessment of cell membrane integrity with MultiTox-Fluor Assay. RFU, relative fluorescence unit; Neg cont: cells received no treatment; Pos cont: cells were treated with 30 μg/mL digitonin for 15 min prior to membrane integrity assessment. Data are presented as mean ± S.E.M., n = 5–6 replicates per each treatment. Asterisks (*) indicate statistically significant differences, p b 0.05, compared to Neg cont.

membrane. Consistent with cytotoxicity results obtained from Caco-2 cells with the same maturity (5-day culture), membrane damage was seen in a dose-dependent manner, i.e., the higher the concentrations of lipid-surfactant mixtures, the more intense were the fluorescent signals (Fig. 6). For example, the highest cytotoxicity was seen in treatments with 0.5% lipid-surfactant mixtures (Fig. 4B–D) and the same treatment regimen gave the highest fluorescent reading (Fig. 6B–D), which indicates a positive correlation between cell membrane impairment assessed by the MultiTox-Fluor Cytotoxicity assay and the cytotoxicity measured by the MTT cell viability assay. 4. Discussion Oral dosage form is the most popular way of drug administration for several reasons, one major reason being patient compliance, which is important for long-term therapy (Sastry et al., 2000). However, the development of a large majority of newly discovered drug molecules into

169

orally bioavailable and clinically effective dosage forms is not possible due to their low aqueous solubility (Yewale et al., 2015). Lipid-based drug delivery systems (LBDDS) present an effective means to improve absorption and bioavailability of such drugs. LBDDS are a diverse group of formulations, range from simple oil solutions to complex selfemulsifying drug delivery systems (SEDDS) or self-microemulsifying drug delivery systems (SMEDDS) (Patel et al., 2012; Shah and Serajuddin, 2012; Prajapati et al., 2013). SEDDS and SMEDDS are mixtures of lipids and surfactants, into which the drug is dissolved (Patel et al., 2012; Prajapati et al., 2013). Co-surfactants can be added to improve emulsification. For example, cyclosporine A, a poorly water-soluble drug, was solubilized in a formulation consisting of lipid, surfactant, co-surfactant and organic solvent (Beauchesne et al., 2007). It has been demonstrated that the lipid and surfactant components within the formulation are crucial to the success of cyclosporine oral capsule product (Rahman et al., 2011; Beauchesne et al., 2007). Despite the importance of LBDDS in oral dosage development, studies on the performance of LBDDS and the components within with respect to their interaction with GI tract after oral ingestion and the mechanism of their effect on drug transport across the GI membrane are rather limited. Among all the important effects of LBDDS components, an essential one is the toxicity towards sites of absorption, i.e., intestinal epithelium in vivo or an appropriate in vitro model, such as Caco-2 cells. The current investigation focused on cytotoxicity assessment of formulations containing selected lipids and one common surfactant at varying ratios. As indicated by Prajapati et al. (2012, 2013), until recently there were no systematic approaches of developing lipid-based formulations for poorly water-soluble drugs. Often large amounts of surfactants and only limited amounts of lipids are used in a formulation to enable emulsification, and high concentrations of organic solvents are added to increase drug solubility and prevent gel formation. Porter et al. (Porter et al., 2007) also mentioned that a rational basis for the selection of excipients for LBDDS was rather ‘elusive’. For this reason, we have undertaken an extensive investigation in our laboratory to identify ‘a rational basis’ for the selection of different components of a LBDDS by systematically constructing phase diagrams and conducting dispersion testing (Prajapati et al., 2011, 2012, 2013; Patel et al., 2012). It was observed that mixing monoglyceride and triglyceride (or PG monoester) at 1:1 (v/v) ratio as the lipid component and combining them with the surfactant Cremophor EL provided most optimal results by greatly reducing the gel phase and promoting microemulsion formation. Furthermore, in the present study, the lipid portion of the formulation may be increased as high as 70% without compromising the formation of microemulsions. It is also observed in the present study that such a mixture formed very fine microemulsions with particle size b100 nm at all dilutions studied. An unexpected but very desirable finding of the present study is that a specific LBDDS formulation containing 30% surfactant, 35% monoglyceride (or PG monoester) and 35% triglyceride exhibited the safest profile to Caco-2 cells under all concentrations tested. In a study evaluating cytotoxicity of lipid-based formulations, Buyukozturk et al. (Buyukozturk et al., 2010) also reported that the surfactant-to-oil ratio played a role in determining toxicity in Caco-2 cells. As mentioned earlier, two different medium-chain monoglycerides, Capmul MCM EP and Capmul 708G, were used in the present study. Since both of them are commonly used in the development of LBDDS, it was of interest to determine their safety and possible cytotoxic effects towards Caco-2 cells. Between them, Capmul MCM appeared to perform better on the 1-day old cell culture as its 1:1 (v/v) mixture with Captex 355 showed less cytotoxic effects at 0.1 and 0.2% concentration levels (Fig. 3). However, the difference disappeared in the 5-day old cultures, where Capmul 708G rather appeared to have performed better than Capmul MCM (Fig. 4). As the cells matured for 21 days, there was practically no difference between the effects of Capmul MCM and Capmul 708G, and both of them were well tolerated (Fig. 5). From the perspective of biological models used, we employed Caco2 cells from a series of culture stages as well as fully differentiated Caco-

170

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171

2 monolayers as recipients for LBDDS. Caco-2 monolayers have been widely accepted as a standard in vitro model of the intestinal barrier (Shah et al., 2006). Caco-2 cells in culture undergo spontaneous differentiation forming a monolayer, which exhibits morphological and functional characteristics of in vivo enterocytes (Hubatsch et al., 2007; Sambuy et al., 2005). Furthermore, improved culture conditions such as the usage of transwell plates led to better differentiation of Caco-2 cells. The transwell plates were shown to effectively promote Caco-2 cell differentiation by providing a permeable filter membrane support for the cells to grow upon and allowing free access of growth medium to both sides of the monolayer (Sambuy et al., 2005). Therefore, fully differentiated Caco-2 cell monolayers grown on filter membrane for a period of 21–27 days are considered as a more relevant model to in vivo conditions and have been used extensively (Hubatsch et al., 2007). Although the usage of Caco-2 cell model is extensive, considerable variability exists in culture protocols of Caco-2 cells utilized by different laboratories, which causes reproducibility issues and difficulties in data comparison (Sambuy et al., 2005; Natoli et al., 2011, 2012). Among the several culture-related factors, two were particularly important, namely the initial seeding density and culture duration (Sambuy et al., 2005). For example, in published studies, the initial seeding density of Caco-2 cells varied considerably, ranging from 3.5 × 103, 3 × 104, 1.5 × 105, to 5 × 105 cells/cm2 (Ujhelyi et al., 2012; Natoli et al., 2011; Buyukozturk et al., 2010; Anderle et al., 2003; Ranaldi et al., 2003). The intermediate seeding density (6 × 104 cells/cm2), as used in the current study, was shown to achieve good differentiation after three weeks of culture (Sambuy et al., 2005). Higher or lower seeding density, however, was demonstrated to affect the morphology of Caco-2 monolayers and carrier-mediated transcellular transport although no significant impact on transepithelial electrical resistance (TEER) value or paracellular permeability (Behrens and Kissel, 2003). High seeding density causes multilayer formation (Tavelin et al., 2002), which was by nature different from the structure and functions of the desired monolayer. Moreover, suboptimal seeding densities were shown to down-regulate transporter expression including HPT1 and P-gp in Caco-2 monolayers (Behrens and Kissel, 2003). Caco-2 cells started from different seeding density are likely to possess distinct properties with respect to maturity and/ or differentiation status and likely result in incomparable data, even after being cultured in the same type of tissue culture vessel for the same duration. Another important cell culture-related factor is the culture duration. Caco-2 cells in culture go through a process from homogeneously undifferentiated to heterogeneously polarized and differentiated to homogeneously polarized and differentiated (Vachon and Beaulieu, 1992). The morphological feature and functional differentiation status of Caco-2 cells is, therefore, a function of culture duration. Upon reaching confluence, Caco-2 cells continue to differentiate and gradually acquire expression of membrane transporters and brush border enzymes, polarized nuclei, cell height comparable to that of human intestinal epithelium, and tight junction formation (Sambuy et al., 2005). Thus, Caco-2 populations from distinct culture stage would possess different maturity. As observed in the current report, Caco-2 cells grown in culture were exhibiting dynamic morphological features when they were in the process of forming monolayer (Fig. 1). In addition to microscopic observation, we demonstrated that Caco-2 cells with shorter culture time were more sensitive to LBDDS exposure, while as Caco-2 cells were cultured for a longer period and became more mature their tolerance towards LBDDS improved markedly (Figs. 3–5). Based on the present study, it is recommended that Caco-2 cells with the maturity of about 3 weeks should preferably be used for safety assessment of surfactants, lipids and lipid-surfactant mixtures. The compositions that do not show toxicity on such cells should be used in drug permeation studies to further investigate the effect of lipid-based formulations, such as Pgp inhibition, modulation of tight junctions, etc. Otherwise, the cells may not mimic the in vivo condition existing in the GI membranes in humans.

5. Conclusions Lipid-based drug delivery systems (LBDDS) are effective dosage forms for improving bioavailability of poorly water-soluble drugs. Toxicity assessment of lipid-based formulations and their individual components is a prerequisite for selecting dosage forms and for testing such dosage forms for in vitro and in vivo drug permeation and absorption. In this study, we presented a systematic method for cytotoxicity evaluation of common components of LBDDS using the Caco-2 cell model. We have identified optimal microemulsion formulations with markedly reduced toxicity and determined 21-day Caco-2 monolayers as a more accurate in vitro model for cytotoxicity assessment. Based on the findings from the present study, well-tolerated lipid-based formulations will be selected for further assessment in our laboratory as potential permeability enhancers in Caco-2 monolayer models.

Acknowledgements & Disclosures This study was supported, in part, by a generous research grant from ABITEC Corporation, 501 W. 1st Avenue, Columbus, OH 43215. The authors also thank Ms. Yue Ji and Ms. Maria Ines Uemura for assistance with particle size measurement.

References Anderle, P., Rakhmanova, V., Woodford, K., Zerangue, N., Sadee, W., 2003. Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines. Pharm. Res. 20 (1), 3–15 (PubMed PMID: 12608530). Artursson, P., Palm, K., Luthman, K., 2001. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46 (1–3), 27–43 (PubMed PMID: 11259831). Beauchesne, P.R., Chung, N.S., Wasan, K.M., 2007. Cyclosporine A: a review of current oral and intravenous delivery systems. Drug Dev. Ind. Pharm. 33 (3), 211–220. http://dx. doi.org/10.1080/03639040601155665 (PubMed PMID: 17454054). Behrens, I., Kissel, T., 2003. Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers? Eur. J. Pharm. Sci. 19 (5), 433–442 (PubMed PMID: 12907294). Berridge, M.V., Herst, P.M., Tan, A.S., 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127–152. http://dx. doi.org/10.1016/S1387-2656(05)11004-7 (PubMed PMID: 16216776). Buyukozturk, F., Benneyan, J.C., Carrier, R.L., 2010. Impact of emulsion-based drug delivery systems on intestinal permeability and drug release kinetics. J. Control. Release 142 (1), 22–30. http://dx.doi.org/10.1016/j.jconrel.2009.10.005 (PubMed PMID: 19850092). Degim, Z., Unal, N., Essiz, D., Abbasoglu, U., 2004. The effect of various liposome formulations on insulin penetration across Caco-2 cell monolayer. Life Sci. 75 (23), 2819–2827. http://dx.doi.org/10.1016/j.lfs.2004.05.027 (PubMed PMID: 15464833). Deli, M.A., 2009. Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery. Biochim. Biophys. Acta 1788 (4), 892–910. http://dx.doi.org/10.1016/j.bbamem.2008.09.016 (PubMed PMID: 18983815). Dimitrijevic, D., Shaw, A.J., Florence, A.T., 2000. Effects of some non-ionic surfactants on transepithelial permeability in Caco-2 cells. J. Pharm. Pharmacol. 52 (2), 157–162 (PubMed PMID: 10714945). El-Sayed, M., Ginski, M., Rhodes, C., Ghandehari, H., 2002. Transepithelial transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers. J. Control. Release 81 (3), 355–365 (PubMed PMID: 12044574). Gursoy, R.N., Benita, S., 2004. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs. Biomed. Pharmacother. 58 (3), 173–182. http://dx.doi.org/10.1016/j.biopha.2004.02.001 (PubMed PMID: 15082340). Hubatsch, I., Ragnarsson, E.G., Artursson, P., 2007. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2 (9), 2111–2119. http://dx.doi.org/10.1038/nprot.2007.303 (PubMed PMID: 17853866). Hugger, E.D., Novak, B.L., Burton, P.S., Audus, K.L., Borchardt, R.T., 2002. A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro. J. Pharm. Sci. 91 (9), 1991–2002. http://dx.doi.org/10. 1002/jps.10176 (PubMed PMID: 12210046). Konsoula, R., Barile, F.A., 2005. Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells. Toxicol. in Vitro 19 (5), 675–684. http://dx.doi.org/10. 1016/j.tiv.2005.03.006 (PubMed PMID: 15896555). Lindmark, T., Kimura, Y., Artursson, P., 1998. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J. Pharmacol. Exp. Ther. 284 (1), 362–369 (PubMed PMID: 9435199). Lipinski, C.A., 2000. Drug-like properties and the causes of poor solubility and poor permeability. J. Pharmacol. Toxicol. Methods 44 (1), 235–249 (PubMed PMID: 11274893).

P. Bu et al. / European Journal of Pharmaceutical Sciences 91 (2016) 162–171 Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 2001. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46 (1–3), 3–26 (PubMed PMID: 11259830). Liu, Y., Peterson, D.A., Kimura, H., Schubert, D., 1997. Mechanism of cellular 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) reduction. J. Neurochem. 69 (2), 581–593 (PubMed PMID: 9231715). McCall, K., 2010. Genetic control of necrosis - another type of programmed cell death. Curr. Opin. Cell Biol. 22 (6), 882–888. http://dx.doi.org/10.1016/j.ceb.2010.09.002 (PubMed PMID: 20889324; PMCID: PMC2993806). Moquin, D., Chan, F.K., 2010. The molecular regulation of programmed necrotic cell injury. Trends Biochem. Sci. 35 (8), 434–441. http://dx.doi.org/10.1016/j.tibs.2010.03.001 (PubMed PMID: 20346680; PMCID: PMC2904865). Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1–2), 55–63 (PubMed PMID: 6606682). Natoli, M., Leoni, B.D., D'Agnano, I., D'Onofrio, M., Brandi, R., Arisi, I., Zucco, F., Felsani, A., 2011. Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. J. Cell. Physiol. 226 (6), 1531–1543. http://dx.doi.org/10. 1002/jcp.22487 (PubMed PMID: 20945374). Natoli, M., Leoni, B.D., D'Agnano, I., Zucco, F., Felsani, A., 2012. Good Caco-2 cell culture practices. Toxicol. in Vitro 26 (8), 1243–1246. http://dx.doi.org/10.1016/j.tiv.2012. 03.009 (PubMed PMID: 22465559). Pang, K.S., 2003. Modeling of intestinal drug absorption: roles of transporters and metabolic enzymes (for the Gillette Review Series). Drug Metab. Dispos. 31 (12), 1507–1519. http://dx.doi.org/10.1124/dmd.31.12.1507 (PubMed PMID: 14625347). Patel, D.P., Li, P., Serajuddin, A.T.M., 2012. Enhanced microemulsion formation in lipidbased drug delivery systems by combining mono-esters of medium chain fatty acids with di- or tri-esters. J. Excip. Food Chem. 3 (2), 29–44. Porter, C.J., Trevaskis, N.L., Charman, W.N., 2007. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat. Rev. Drug Discov. 6 (3), 231–248. http://dx.doi.org/10.1038/nrd2197 (PubMed PMID: 17330072). Porter, C.J., Pouton, C.W., Cuine, J.F., Charman, W.N., 2008. Enhancing intestinal drug solubilisation using lipid-based delivery systems. Adv. Drug Deliv. Rev. 60 (6), 673–691. http://dx.doi.org/10.1016/j.addr.2007.10.014 (PubMed PMID: 18155801). Prajapati, H.N., Patel, D.P., Patel, N.G., Dalrymple, D.M., Serajuddin, A.T., 2011. Effect of difference in fatty acid chain lengths of medium-chain lipids on lipid-surfactant-water phase diagrams and drug solubility. J. Excip. Food Chem. 2 (3), 73–88. Prajapati, H.N., Dalrymple, D.M., Serajuddin, A.T., 2012. A comparative evaluation of mono-, di- and triglyceride of medium chain fatty acids by lipid/surfactant/water phase diagram, solubility determination and dispersion testing for application in pharmaceutical dosage form development. Pharm. Res. 29 (1), 285–305. http://dx. doi.org/10.1007/s11095-011-0541-3 (PubMed PMID: 21861203; PMCID: PMC3246583). Prajapati, H., Dalrymple, D.M., Serajuddin, A.T.M., 2013. In vitro dispersion test can serve as a predictive method for assessing performance of lipid-based drug delivery systems. J. Excip. Food Chem. 4 (4), 111–125. Rahman, M.A., Harwansh, R., Mirza, M.A., Hussain, S., Hussain, A., 2011. Oral lipid based drug delivery system (LBDDS): formulation, characterization and application: a review. Curr. Drug Deliv. 8 (4), 330–345 (PubMed PMID: 21453264). Ranaldi, G., Consalvo, R., Sambuy, Y., Scarino, M.L., 2003. Permeability characteristics of parental and clonal human intestinal Caco-2 cell lines differentiated in serum-supplemented and serum-free media. Toxicol. in Vitro 17 (5–6), 761–767 (PubMed PMID: 14599474).

171

Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M.L., Stammati, A., Zucco, F., 2005. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol. Toxicol. 21 (1), 1–26. http://dx.doi.org/10.1007/s10565-005-0085-6 (PubMed PMID: 15868485). Sastry, S.V., Nyshadham, J.R., Fix, J.A., 2000. Recent technological advances in oral drug delivery — a review. Pharm. Sci. Technol. Today 3 (4), 138–145 (PubMed PMID: 10754543). Serajuddin, A.T., Rosoff, M., Goldberg, A.H., 1985. In situ intestinal absorption of a poorly water-soluble drug from mixed micellar solutions of bile salt and lipolysis products in rats. Pharm. Res. 2 (5), 221–224. http://dx.doi.org/10.1023/A:1016312827680 (PubMed PMID: 24272840). Sha, X., Yan, G., Wu, Y., Li, J., Fang, X., 2005. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. Eur. J. Pharm. Sci. 24 (5), 477–486. http://dx.doi.org/10.1016/j.ejps.2005.01.001 (PubMed PMID: 15784337). Shah, D.O., 1998. Micelles, Microemulsions, and Monolayers: Science and Technology. M. Dekker, New York, p. xvii (610 pp.). Shah, A.V., Serajuddin, A.T., 2012. Development of solid self-emulsifying drug delivery system (SEDDS) I: use of poloxamer 188 as both solidifying and emulsifying agent for lipids. Pharm. Res. 29 (10), 2817–2832. http://dx.doi.org/10.1007/s11095-0120704-x (PubMed PMID: 22371051). Shah, P., Jogani, V., Bagchi, T., Misra, A., 2006. Role of Caco-2 cell monolayers in prediction of intestinal drug absorption. Biotechnol. Prog. 22 (1), 186–198. http://dx.doi.org/10. 1021/bp050208u (PubMed PMID: 16454510). Singh, B., Bandopadhyay, S., Kapil, R., Singh, R., Katare, O., 2009. Self-emulsifying drug delivery systems (SEDDS): formulation development, characterization, and applications. Crit. Rev. Ther. Drug Carrier Syst. 26 (5), 427–521 (PubMed PMID: 20136631). Strober, W., 2001. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. http://dx.doi.org/10.1002/0471142735.ima03bs21 (PubMed PMID: 18432654. Appendix 3:Appendix 3B). Tavelin, S., Grasjo, J., Taipalensuu, J., Ocklind, G., Artursson, P., 2002. Applications of epithelial cell culture in studies of drug transport. Methods Mol. Biol. 188, 233–272. http://dx.doi.org/10.1385/1-59259-185-X:233 (PubMed PMID: 11987548). Ujhelyi, Z., Fenyvesi, F., Varadi, J., Feher, P., Kiss, T., Veszelka, S., Deli, M., Vecsernyes, M., Bacskay, I., 2012. Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in Caco-2 cell monolayer. Eur. J. Pharm. Sci. 47 (3), 564–573. http://dx.doi.org/10.1016/j.ejps. 2012.07.005 (PubMed PMID: 22841998). Vachon, P.H., Beaulieu, J.F., 1992. Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line. Gastroenterology 103 (2), 414–423 (PubMed PMID: 1634060). van Breemen, R.B., Li, Y., 2005. Caco-2 cell permeability assays to measure drug absorption. Expert Opin. Drug Metab. Toxicol. 1 (2), 175–185. http://dx.doi.org/10.1517/ 17425255.1.2.175 (PubMed PMID: 16922635). Williams, H.D., Trevaskis, N.L., Charman, S.A., Shanker, R.M., Charman, W.N., Pouton, C.W., Porter, C.J., 2013. Strategies to address low drug solubility in discovery and development. Pharmacol. Rev. 65 (1), 315–499 (PubMed PMID: 23383426). Yewale, C., Patil, S., Kolate, A., Kore, G., Misra, A., 2015. Oral absorption promoters: opportunities, issues, and challenges. Crit. Rev. Ther. Drug Carrier Syst. 32 (5), 363–387 (PubMed PMID: 26559432).