lecithin in mixed micelles attenuates the cytotoxicity of bile salts in caco-2 cells

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Lecithin in mixed micelles attenuates the cytotoxicity of bile salts in Caco-2 cells Ya’nan Tan a,b , Jianping Qi a,, Yi Lu a , Fuqiang Hu c , Zongning Yin b , Wei Wu a a Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai 201203, China b West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, China c School of Pharmacy, Zhejiang University, Hangzhou 310058, Zhejiang, China article info Article history: Received 3 August 2012 Accepted 28 November 2012 Available online 6 December 2012 Keywords: Mixed micelles Bile salt Lecithin Caco-2 cells Cytotoxicity abstract This study was designed to investigate the cytotoxicity of bile salt–lecithin mixed micelles on the Caco-2 cell model. Cell viability and proliferation after mixed micelles treatments were evaluated with the MTT assay, and the integrity of Caco-2 cell monolayer was determined by quantitating the transepithelial elec- trical resistance and the flux of tracer, FITC-dextran 4400. The apoptosis induced by mixed micelles treat- ments was investigated with the annexin V/PI protocol. The particle size of mixed micelles was all smaller than 100 nm. The mixed micelles with lower than 0.2 mM sodium deoxycholate (SDC) had no significant effects on cell viability and proliferation. When the level of SDC was higher than 0.4 mM and the lecithin/ SDC ratio was lower than 2:1, the mixed micelles caused significant changes in cell viability and prolif- eration. Furthermore, the mixed micelles affected tight junctions in a composition-dependent manner. Specifically, the tight junctions were transiently opened rather than damaged by the mixed micelles with SDC of between 0.2 and 0.6 mM. The mixed micelles with more lecithin also induced less apoptosis. These results demonstrate that relatively higher concentrations of mixed micelles are toxic to Caco-2 cells, while phospholipids can attenuate the toxicity of the bile salts. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. 1. Introduction Nanotechnology has been widely applied in medications and drug delivery systems. The demand for nanotechnology in medical products is expected to grow by more than 17% annually, with the largest share in pharmaceutical applications that may reach $18 billion in 2014 (Jones and Grainger, 2009). However, the effects of nanomaterials or nanodrug delivery systems on tissues or cells require further evaluations for their safe and effective applications. Indeed, the properties of particles, including pharmacology and toxicity, will change significantly from the normal states when their sizes are reduced to nanoscales. Different reports have reached distinct conclusions towards the toxicity of nanoparticles (Gelperina et al., 2002; Renwick et al., 2001). This controversy clearly needs new studies to be settled. The mixed micelles of bile salts (BSs) and phosphatidylcholine (PC) have been widely used in the oral delivery of insoluble drugs to increase their dissolution and bioavailability (Mrestani et al., 2010; van Hasselt et al., 2009; Wiedmann et al., 2002; Yu et al., 2010). The concentration of bile salts can reach 14 mM in human bile, and they can form micelles spontaneously when their concen- trations exceed 2–5 mM, the critical micellar concentrations (CMC) (Coleman et al., 1979; Dial et al., 2008). PC is another main ingre- dient of the bile (Balint et al., 1965), which binds to bile salts to form mixed micelles that can improve the dissolution of insoluble drugs (Alkanonyuksel and Son, 1992; Li et al., 1996; Magee et al., 2003). Although bile salts can dissolve the membrane lipids and dis- rupt the gastric mucosal barrier (Duane, 1980), the mucosal lining of the gastrointestinal (GI) tract is not damaged by the bile under physiological conditions (Martin et al., 1992). PC has been sug- gested to prevent the toxicity of bile salts on gastrointestinal epi- thelia and membrane (Dial et al., 2008; Narain et al., 1998). In this study, we evaluated the effects of lecithin on the bile salt, sodium deoxycholate, and the effects of mixed micelles formed by lecithin and bile salts on cell viability, proliferation, and the integ- rity of monolayer and apoptosis in Caco-2 cells. The Caco-2 cell model was chosen to evaluate the effects of mixed micelles on gas- trointestinal epithelia and membrane for the following two rea- sons: Caco-2 cells were derived from a human colon carcinoma thus serve as a cell culture model of intestinal epithelia cells; and the morphology and other properties of the Caco-2 monolayer mimic those of the intestinal epithelia (Shah et al., 2006; Turco et al., 2010). 0887-2333/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.11.018 Abbreviations: SDC, sodium deoxycholate; BS, bile salts; PC, phosphatidylcho- line; TEER, transepithelial electrical resistance; P app , apparent permeability coeffi- cient; FD-4, FITC-dextran 4400. Corresponding author. Address: Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China. Tel./ fax: +86 21 5198 0084. E-mail address: [email protected] (J. Qi). Toxicology in Vitro 27 (2013) 714–720 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit

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Page 1: Lecithin in mixed micelles attenuates the cytotoxicity of bile salts in Caco-2 cells

Toxicology in Vitro 27 (2013) 714–720

Contents lists available at SciVerse ScienceDirect

Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Lecithin in mixed micelles attenuates the cytotoxicity of bile salts in Caco-2 cells

Ya’nan Tan a,b, Jianping Qi a,⇑, Yi Lu a, Fuqiang Hu c, Zongning Yin b, Wei Wu a

a Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai 201203, Chinab West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, Chinac School of Pharmacy, Zhejiang University, Hangzhou 310058, Zhejiang, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 August 2012Accepted 28 November 2012Available online 6 December 2012

Keywords:Mixed micellesBile saltLecithinCaco-2 cellsCytotoxicity

0887-2333/$ - see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.tiv.2012.11.018

Abbreviations: SDC, sodium deoxycholate; BS, billine; TEER, transepithelial electrical resistance; Papp,cient; FD-4, FITC-dextran 4400.⇑ Corresponding author. Address: Department o

Pharmacy, Fudan University, 826 Zhangheng Road, Sfax: +86 21 5198 0084.

E-mail address: [email protected] (J. Qi).

This study was designed to investigate the cytotoxicity of bile salt–lecithin mixed micelles on the Caco-2cell model. Cell viability and proliferation after mixed micelles treatments were evaluated with the MTTassay, and the integrity of Caco-2 cell monolayer was determined by quantitating the transepithelial elec-trical resistance and the flux of tracer, FITC-dextran 4400. The apoptosis induced by mixed micelles treat-ments was investigated with the annexin V/PI protocol. The particle size of mixed micelles was all smallerthan 100 nm. The mixed micelles with lower than 0.2 mM sodium deoxycholate (SDC) had no significanteffects on cell viability and proliferation. When the level of SDC was higher than 0.4 mM and the lecithin/SDC ratio was lower than 2:1, the mixed micelles caused significant changes in cell viability and prolif-eration. Furthermore, the mixed micelles affected tight junctions in a composition-dependent manner.Specifically, the tight junctions were transiently opened rather than damaged by the mixed micelles withSDC of between 0.2 and 0.6 mM. The mixed micelles with more lecithin also induced less apoptosis. Theseresults demonstrate that relatively higher concentrations of mixed micelles are toxic to Caco-2 cells,while phospholipids can attenuate the toxicity of the bile salts.

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Nanotechnology has been widely applied in medications anddrug delivery systems. The demand for nanotechnology in medicalproducts is expected to grow by more than 17% annually, with thelargest share in pharmaceutical applications that may reach $18billion in 2014 (Jones and Grainger, 2009). However, the effectsof nanomaterials or nanodrug delivery systems on tissues or cellsrequire further evaluations for their safe and effective applications.Indeed, the properties of particles, including pharmacology andtoxicity, will change significantly from the normal states whentheir sizes are reduced to nanoscales. Different reports havereached distinct conclusions towards the toxicity of nanoparticles(Gelperina et al., 2002; Renwick et al., 2001). This controversyclearly needs new studies to be settled.

The mixed micelles of bile salts (BSs) and phosphatidylcholine(PC) have been widely used in the oral delivery of insoluble drugsto increase their dissolution and bioavailability (Mrestani et al.,

012 Published by Elsevier Ltd. All

e salts; PC, phosphatidylcho-apparent permeability coeffi-

f Pharmaceutics, School ofhanghai 201203, China. Tel./

2010; van Hasselt et al., 2009; Wiedmann et al., 2002; Yu et al.,2010). The concentration of bile salts can reach 14 mM in humanbile, and they can form micelles spontaneously when their concen-trations exceed 2–5 mM, the critical micellar concentrations (CMC)(Coleman et al., 1979; Dial et al., 2008). PC is another main ingre-dient of the bile (Balint et al., 1965), which binds to bile salts toform mixed micelles that can improve the dissolution of insolubledrugs (Alkanonyuksel and Son, 1992; Li et al., 1996; Magee et al.,2003).

Although bile salts can dissolve the membrane lipids and dis-rupt the gastric mucosal barrier (Duane, 1980), the mucosal liningof the gastrointestinal (GI) tract is not damaged by the bile underphysiological conditions (Martin et al., 1992). PC has been sug-gested to prevent the toxicity of bile salts on gastrointestinal epi-thelia and membrane (Dial et al., 2008; Narain et al., 1998).

In this study, we evaluated the effects of lecithin on the bile salt,sodium deoxycholate, and the effects of mixed micelles formed bylecithin and bile salts on cell viability, proliferation, and the integ-rity of monolayer and apoptosis in Caco-2 cells. The Caco-2 cellmodel was chosen to evaluate the effects of mixed micelles on gas-trointestinal epithelia and membrane for the following two rea-sons: Caco-2 cells were derived from a human colon carcinomathus serve as a cell culture model of intestinal epithelia cells;and the morphology and other properties of the Caco-2 monolayermimic those of the intestinal epithelia (Shah et al., 2006; Turcoet al., 2010).

rights reserved.

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Y. Tan et al. / Toxicology in Vitro 27 (2013) 714–720 715

2. Materials and methods

2.1. Chemicals and regents

Lecithin (Lipoid S100, average Mw 770) and sodium deoxycho-late (SDC) were purchased from Lipoid (Germany) and Sionpharmchemical Co. Ltd (Shanghai, China), respectively. Methyl tetrazo-lium (MTT) and FITC-dextran (Mw 4400) were obtained from Sig-ma (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kitwas purchased from KeyGEN Company (Nanjing, China). The HPLCgrade acetonitrile and methanol were purchased from Merck(Darmstadt, Germany).

2.2. Preparation and characterization of mixed micelles

Different concentrations of lecithin (0.4, 0.2, 0.1, 0.05 mM) and0.1 mM sodium deoxycholate (SDC) were dissolved in 10 mLorganic solvent containing equal amounts of methanol anddichloromethane (v/v). Then, the organic solvent was removed byreduced-pressure evaporation for 12 h. At last, a series of mixedmicelles with different ratios of lecithin/SDC were obtained byadding 10 mL deionized water. The micelles were treated withultrasound (285 W, 60 s) (ultrasonic instrument, Scientz IID, China)to reduce the particle size and homogenize the micelles. The sizeand zeta potential were evaluated by Zeta-Sizer Nano ZS (Marlven,UK). Besides, control group (0/1 of lecithin to SDC) was prepared bydissolving SDC in deionized water and then diluted to differentconcentrations.

Each mixed micelle was diluted to various concentrations (0.1,0.2, 0.4, 0.6, 0.8, 1.0 mM, calculated by SDC) with culture mediumwithout fetal bovine serum for the cell experiments.

2.3. Cell culture

Human colonic carcinoma Caco-2 cells were purchased fromthe American Type Culture Collection (Rockville, MD). The cellswere incubated at 37 �C in a humidified atmosphere with 5% CO2

in Dulbecco’s modified minimum essential medium (DMEM) sup-plemented with 10% fetal bovine serum (FBS, Gibco), 4.5 mg/L glu-cose, 1% (v/v) nonessential amino acids, and antibiotics (penicillin200 U/mL and streptomycin 100 mg/mL). The cells were grown in25 cm2 culture flasks. The medium was changed every other day,and the cells were trypsinized every 5 days for passage. TheCaco-2 cells with passage numbers of 45–52 were used in theexperiments.

2.4. Assessment of cell viability

The cell viability of Caco-2 cells was measured by the MTT assayto evaluate the cytotoxicity of mixed micelles. 1 � 104 cells in200 lL culture medium per well were seeded into 96-well plate.Three days later, the medium was replaced with the same volumeof mixed micelles in culture medium. Blank culture medium wasused as negative control. After 2 h of micelle treatment, 20 lL of5 mg/mL MTT solution was added to each well, and the mediumwas removed after 4 h. Then, the formazan crystals were dissolvedin 150 lL of DMSO. The absorbance was measured at 490 nm.

2.5. Assessment of cell proliferation by MTT assay

The cells were seeded in 96-well plates at a density of 1 � 104

cells per well and cultured for 12 h before the mixed micelles sam-ples were added. Cells were thereafter cultured for 3 days andunderwent MTT assay (Yin et al., 2009). The cells without micelletreatment served as a control. The MTT assay was performed in

sextuplicates for each sample, and the proliferation ratio was de-fined as the proportion of treated cells to the control cells.

2.6. Examination of the integrity of Caco-2 monolayer

The Caco-2 cells were cultured on porous polycarbonate filtermembranes (Millicell�-PCF, 12 mm diameter, cell culture insert,Millipore) with a pore size of 0.4 lm and a diameter of 12 mm in24-well culture plate (Corning Costar, Cambridge, MA) for the mea-surement of cell monolayer integrity. The seeding density on thefilter was 1 � 104 cells/cm2. The cells were cultured for 21 days un-der normal conditions as in Section 2.3 before the measurement.The medium was changed every second day for the first weekand every day for the following two weeks.

The transepithelial electrical resistance (TEER) of Caco-2 cellmonolayer was measured once every three days during the cul-turing with a Millicell�-ERS (Millipore Corp., Bedford, MA) con-nected to a pair of chopstick electrodes. On the day ofmeasurement, the culture medium was replaced with equal vol-ume of HBSS (0.4 mL apically and 0.6 mL basolaterally), and thecells were allowed to equilibrate for 1 h. Then, the TEER was mea-sured to reveal the integrity of the monolayer formed on the fil-ters. TEER measurements were also performed during theexperiment to check the effects of mixed micelles on the integrityof Caco-2 cell monolayers at the intervals of 0, 30, 60 and120 min. After 120 min, the donor solutions were removed care-fully, and the monolayers were rinsed with HBSS and re-fed withculture medium for monolayer recovery. Twenty-four hours later,TEER was measured again.

The permeability of FITC-dextran MW 4400 (FD-4) was deter-mined after mixed micelle treatments to further characterize theintegrity of Caco-2 cell monolayers. Briefly, the HBSS solution ofFD-4 (5 mg/mL) containing different mixed micelles was addedto the apical side. After 15, 30, 45, 60, 90, and 120 min, 400 lL sam-ples in the basolateral side were collected and replaced with400 lL blank HBSS. The concentration of samples was determinedby HPLC-FLD (Agilent, USA). Apparent permeability (Papp) for eachsubstance was calculated according to the following formula:

Papp ¼dQdt� 1

AC0

where Papp is the apparent permeability (cm/s); dQ/dt is the perme-ability rate; A is the diffusion area of the monolayer (cm2); and C0 isthe initial concentration of the compound in the feed.

2.7. Determination of FD-4

FD-4 in the medium was isolated and characterized by usingAgilent 1100 HPLC system (Agilent, USA) consisting of a ternarypump, an automatic sampler, a fluorescence detector and a columnheater. The separation of FD-4 was carried out using an analyticalRP 18 column (150 � 4.6 mm, i.d. 5 lm) with a mobile phase of10% acetonitrile and 90% KH2PO4 (2 mM, pH 7.2). The flow rateof the mobile phase was 1 mL/min, and the detector was operatedat an excitation wavelength of 495 nm and an emission wave-length of 515 nm. The injection volume was 20 lL, and the detec-tion limit of FD-4 was 5 ng/mL.

2.8. Apoptosis assay

The Caco-2 cells were cultured in 6-well plates at a dendity of1 � 106 cells per well. After 5 days of incubation, the culture med-ium was removed, and the cells were washed 3 times with D-Hank’s buffer solution. Then the mixed micelle treatments started(0.2 mM mixed micelles) with blank culture medium as the nega-tive control. After being treated for 24 h, the cells were detached

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Table 1The size and zeta potential of mixed micelles with different lecithin/SDC (n = 3).

Ratios of lecithin to SDC

1/4 1/2 1/1 2/1 4/1

Size (nm) 92.17 ± 10.5 74.05 ± 15.3 57.18 ± 17.8 24.12 ± 9.2 11.98 ± 5.1PDI 0.317 ± 0.012 0.328 ± 0.018 0.467 ± 0.032 0.525 ± 0.056 0.598 ± 0.077Zeta potential (mv) �51.4 ± 7.6 �57.4 ± 11.2 �67.3 ± 5.5 �50.3 ± 7.6 �57.0 ± 7.1

Fig. 1. The viability of Caco-2 cells treated with mixed micelles with different lecithin/SDC ratios (n = 5). The concentrations of micelles were represented by theconcentrations of SDC, which were 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mM. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with control group (0/1).

Fig. 2. MTT assay for proliferation of Caco-2 cells within 3 days in the presence of mixed micelles with different lecithin/SDC ratios (n = 5). The proliferation was representedas cell viability compared with no treatment group. The concentrations of micelles were represented by the concentrations of SDC, which were 0.1, 0.2, 0.4 and 0.6 mM.⁄p < 0.05 and ⁄⁄p < 0.01 compared with group 1/4 (lecithin/SDC).

716 Y. Tan et al. / Toxicology in Vitro 27 (2013) 714–720

from the plates by trypsinization and collected by centrifugation at100g. Pellets were washed in phosphate buffered saline (PBS) andstained with both propidium iodide (PI) and annexin V-FITC forflow cytometry with FL1 (annexin V) and FL2 (PI) bivariate analy-ses. The cells were separated into four fractions, i.e., PI-negativeand annexin V-negative, PI-positive and annexin V-negative, PI-negative and annexin V-positive, and PI-positive and annexin V-positive, and the percentage of cells in each fraction wascalculated.

2.9. Statistical analysis

One-way analysis of variance (ANOVA) was performed on allexperimental data and the means were compared using Student’st-test at the 5% level with SPSS 13.0 software (SPSS, Inc).

3. Results

3.1. In vitro properties of mixed micelles

The mixed micelles with different lecithin/SDC ratios wereformed spontaneously. Their sizes were all below 100 nm and in-creased with more lecithin (Table 1). The zeta potentials of allmixed micelles were negative and without any significant regular-ity (Table 1).

3.2. Effects of lecithin/SDC mixed micelles on cell viability

The effects of SDC and mixed micelles on the viability of Caco-2cells were evaluated with MTT assay. As shown in Fig. 1, SDCseriously damaged the Caco-2 cells at 0.2 mM or higher, while

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Y. Tan et al. / Toxicology in Vitro 27 (2013) 714–720 717

significantly less severe damages were caused by mixed micelles.The toxicity was reduced with increased lecithin/SDC ratios, andthe cell viability was significantly increased when the lecithin/SDC ratio was higher than 2/1.

3.3. Effects of lecithin/SDC mixed micelles on cell proliferation

Higher SDC concentrations led to higher growth inhibition ofCaco-2 cells. As shown in Fig. 2, the cell proliferation was increased

A

B

C

Fig. 3. TEER changes after the administration of 0.2 mM (A), 0.4 mM (B), and 0.6 mM (C) m(n = 3). ⁄p < 0.05 and ⁄⁄p < 0.01 compared with initial value.

significantly by the micelles with higher than 1/1 of lecithin/SDCratios, consistent with the change patterns of cell viability.

3.4. Effects of lecithin/SDC mixed micelles on the integrity of Caco-2monolayer

Caco-2 cells were incubated with 0.2 mM, 0.4 mM and 0.6 mMmixed micelles with different lecithin/SDC ratios at 1/2, 1/1, 2/1and 4/1. The mixed micelles with 0.4 mM or higher SDC affected

ixed micelles with different lecithin/SDC ratios of 1/2, 1/1, 2/1, and 4/1, respectively

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718 Y. Tan et al. / Toxicology in Vitro 27 (2013) 714–720

the integrity of Caco-2 monolayers. However, the decline of TEERwas correlated with not only the SDC concentration but also thelecithin/SDC ratio. Specifically, when the SDC concentration ofmixed micelles was 0.6 mM, the rate of TEER declines was in-creased with higher lecithin contents. However, when the mixed

A

B

C

Fig. 4. Cumulative transport of FD-4 after the co-culture with 0.2 mM (A), 0.4 mM (B), and2, 1/1, 2/1, and 4/1, respectively (n = 3).

micelles were removed, the degree of recovery of the TEER was dif-ferent with lecithin/SDC (Fig. 3). The changes in the permeability ofFD-4 also demonstrated the effects of the mixed micelles on theintegrity of Caco-2 monolayers (Fig. 4). However, when the SDCconcentration was 0.2 mM, the mixed micelles failed to increase

0.6 mM (C) mixed micelles that were formed with different lecithin/SDC ratios of 1/

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Table 2Papp of FD-4 after the co-culture with mixed micelles (n = 3).

Conc. of SDC Ratio of lecithin to SDC Papp of FD-4 (�10�8)

0.2 mM 1/2 2.32 ± 0.351/1 2.14 ± 0.062/1 2.19 ± 0.274/1 2.28 ± 0.63

0.4 mM 1/2 3.78 ± 0.34⁄

1/1 3.47 ± 0.62⁄

2/1 2.42 ± 0.434/1 2.34 ± 0.79

0.6 mM 1/2 4.34 ± 0.44⁄

1/1 3.91 ± 0.34⁄

2/1 3.12 ± 0.27⁄

4/1 2.31 ± 0.38Control 1.95 ± 0.19

Note: ⁄p < 0.05 compared with control group. Papp is the abbreviation of apparentpermeability coefficient.

Fig. 5. Effects of 0.2 mM mixed micelles with different lecithin/SDC ratios on theapoptosis of Caco-2 cells (n = 5). ⁄p < 0.05 and ⁄⁄p < 0.01 compared with controlgroup.

Y. Tan et al. / Toxicology in Vitro 27 (2013) 714–720 719

significantly the permeability of FD-4. Furthermore, higher leci-thin/SCD ratios attenuated the effects of mixed micelles withincreasing concentrations (Table 2).

3.5. Apoptosis of Caco-2 cells induced by mixed micelles

As shown in Fig. 5, apoptosis was induced in Caco-2 cells bymixed micelles with 0.2 mM SDC and different lecithin/SDC ratios.The mixed micelles of 0.2 mM SDC with 1/2–2/1 of lecithin/SDC ra-tios induced significant apoptosis compared with the controlgroup. However, the highest apoptosis was induced by the mixedmicelles with 1/2 of lecithin/SDC ratio (p < 0.05) and the cells trea-ted with micelles with 4/1 of lecithin/SDC had not evident apopto-sis. These results indicate that more lecithin in mixed micellesleads to less apoptosis.

4. Discussion

Bile salts bind to phosphatidylcholine to form mixed micellesthat can facilitate the solubilization and absorption of dietary fatsand drug molecules. The mixed micelles formed by bile salts andlecithin have been widely used as carriers for insoluble oral drugs.The increase in the bioavailability of drugs by mixed micelles maybe caused mainly by the increased solubility and permeabilityacross gastrointestinal epithelia (Pithavala et al., 1995). However,detailed studies on the effects of mixed micelles on the gastroin-

testinal epithelia have been lacking to reveal their potentialtoxicity.

The phosphatidylcholine or phospholipid in bile may preventthe bile salt-induced injury either by promoting the formation ofless toxic mixed micelles or by enhancing or restoring the mucosa’shydrophobic properties (Barrios and Lichtenberger, 2000; Dialet al., 2008; Narain et al., 1998). In this study, we performed de-tailed analyses using various assays to evaluate the cytotoxicityof mixed micelles as drug delivery systems. Specifically, the ‘‘cellviability’’ assay revealed that the mixed micelles with lower than0.2 mM SDC caused no apparent cellular damages within 4 h. Thecellular damages were increased significantly when the SDC con-centration was more than 0.4 mM. However, the damages causedby higher concentrations of SDC were attenuated or even pre-vented by adding lecithin. This is different from a previous reportthat that no toxicity to gastrointestinal tract was observed formuch higher levels of bile salts (Coleman et al., 1979). The longterm effects on cell proliferation were evaluated with longerexposure (3 days) of mixed micelles using the MTT assy. The cellproliferation was inhibited significantly by mixed micelles in aSDC-concentration-dependent manner, consistent to what hadbeen observed in the ‘‘cell viability’’ assay. It also implies that thatincreased lecithin contents in mixed micelles can attenuate theinhibition of cell proliferation.

The Caco-2 cells grown on permeable filters display importantcharacteristics of normal intestinal epithelia, including theexpression of enzymes involved in tight junctions, microvilliand brush border (Misra et al., 2006). The tight junctions (TJs)are a barrier controlling the paracellular transport of ions andmolecules by size and charges. The compactness of TJs indicatesthe integrity of gastrointestinal epithelia and can be quantitatedby the trans-epithelial electrical resistance (TEER) and the fluxof tracers (Gonzalez-Mariscal et al., 2008). TJs can be reversiblyopened by chitosan (Wood et al., 2004), surfactants (Anderberget al., 1993) and Ca2+ chelating agents (Tomita et al., 1996).Although transient opening of TJs cannot induce permanent tox-icity of gastrointestinal tract, it may cause other toxicity for tem-porary deprivation of barriers. In this study, all mixed micelleswith different SDC concentrations (0.2, 0.4, 0.6 mM) did not leadto permanent damages in the TJs of Caco-2 cell monolayers. How-ever, the Papp of FD-4 was increased significantly and the TEERwas reduced significantly when the SDC concentration of mixedmicelles was higher than 0.4 mM. The SDC/lecithin ratio was an-other important factor that affected the integrity of TJs, furthersuggesting the attenuating effects of lecithin on SDC-induced cel-lular damages.

Apoptosis is a critical cell death mechanism in many physiolog-ical processes, including embryogenesis and tissue/organ involu-tion. Many exogenous substances can induce apoptosis, whichmay affect significantly the cell growth (Wang et al., 2000). In thisstudy, we observed that the 0.2 mM mixed micelles with lowerthan 4/1 lecithin/SDC induced significant apoptosis, although thecell viability was not changed at this concentration, which maybe caused by different exposure time of micelles to Caco-2 cells.The ‘‘cell viability’’ experiment was carried by treating with mixedmicelles for only 4 h, but the cells were exposed in mixed micellesfor 24 h during ‘‘apoptosis’’ experiment.

5. Conclusions

The mixed micelles formed by lecithin and SDC affected thegrowth of Caco-2 cells. SDC treatments led to inhibited prolifera-tion, disrupted tight junctions, and apoptosis, all of which wereattenuated by the lecithin in mixed micelles. More lecithin wasneeded for higher concentrations of mixed micelles to preventthe toxicity of SDC. Therefore, the content of lecithin in the

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720 Y. Tan et al. / Toxicology in Vitro 27 (2013) 714–720

lecithin/SDC mixed micelles should be optimized to reduce thetoxicity in their versatile applications.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the Shanghai Commission of Sci-ence and Technology (1052nm03600), the Shanghai Commissionof Education (10SG05), the Ministry of Education (NCET-11-0114), and the National Key Basic Research Program of China(2009CB930300).

References

Alkanonyuksel, H., Son, K., 1992. Mixed micelles as proliposomes for thesolubilization of teniposide. Pharm. Res. 9, 1556–1562.

Anderberg, E.K., Lindmark, T., Artursson, P., 1993. Sodium caprate elicits dilatationsin human intestinal tight junctions and enhances drug absorption by theparacellular route. Pharm. Res. 10, 857–864.

Balint, J.A., Kyriakid, E.C., Spitzer, H.L., Morrison, E.S., 1965. Lecithin fatty acidcomposition in bile and plasma of man dogs rats and oxen. J. Lipid. Res. 6, 96–99.

Barrios, J.M., Lichtenberger, L.M., 2000. Role of biliary phosphatidylcholine in bileacid protection and NSAID injury of the ileal mucosa in rats. Gastroenterology118, 1179–1186.

Coleman, R., Iqbal, S., Godfrey, P.P., Billington, D., 1979. Membranes and bileformation – composition of several mammalian biles and their membrane-damaging properties. Biochem. J. 178, 201–208.

Dial, E.J., Rooijakkers, S.H.M., Darling, R.L., Romero, J.J., Lichtenberger, L.M., 2008.Role of phosphatidylcholine saturation in preventing bile salt toxicity togastrointestinal epithelia and membranes. J. Gastroen. Hepatol. 23, 430–436.

Duane, W.C., 1980. Mechanism by which bile-salts disrupt the gastric-mucosalbarrier. Gastroenterology 78, 1159-1159.

Gelperina, S.E., Khalansky, A.S., Skidan, I.N., Smirnova, Z.S., Bobruskin, A.I., Severin,S.E., Turowski, B., Zanella, F.E., Kreuter, J., 2002. Toxicological studies ofdoxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate)nanoparticles in healthy rats and rats with intracranial glioblastoma. Toxicol.Lett. 126, 131–141.

Gonzalez-Mariscal, L., Tapia, R., Chamorro, D., 2008. Crosstalk of tight junctioncomponents with signaling pathways. BBA-Biomembranes 1778, 729–756.

Jones, C.F., Grainger, D.W., 2009. In vitro assessments of nanomaterial toxicity. Adv.Drug Deliver. Rev. 61, 438–456.

Li, C.Y., Zimmerman, C.L., Wiedmann, T.S., 1996. Solubilization of retinoids by bilesalt/phospholipid aggregates. Pharm. Res. 13, 907–913.

Magee, G.A., French, J., Gibbon, B., Luscombe, C., 2003. Bile salt/lecithin mixedmicelles optimized for the solubilization of a poorly soluble steroid moleculeusing statistical experimental design. Drug Dev. Ind. Pharm. 29, 441–450.

Martin, G.P., Elhariri, L.M., Marriott, C., 1992. Bile salt-induced andlysophosphatidylcholine-induced membrane damage in human erythrocytes.J. Pharm. Pharmacol. 44, 646–650.

Misra, A., Shah, P., Jogani, V., Bagchi, T., 2006. Role of Caco-2 cell monolayers inprediction of intestinal drug absorption. Biotechnol. Progr. 22, 186–198.

Mrestani, Y., Behbood, L., Hartl, A., Neubert, R.H.H., 2010. Microemulsion and mixedmicelle for oral administration as new drug formulations for highly hydrophilicdrugs. Eur. J. Pharm. Biopharm. 74, 219–222.

Narain, P.K., DeMaria, E.J., Heuman, D.M., 1998. Lecithin protects against plasmamembrane disruption by bile salts. J. Surg. Res. 78, 131–136.

Pithavala, Y.K., Odishaw, J.L., Han, S.M., Wiedmann, T.S., Zimmerman, C.L., 1995.Retinoid absorption from simple and mixed micelles in the rat intestine. J.Pharm. Sci. 84, 1360–1365.

Renwick, L.C., Donaldson, K., Clouter, A., 2001. Impairment of alveolar macrophagephagocytosis by ultrafine particles. Toxicol. Appl. Pharm. 172, 119–127.

Shah, P., Jogani, V., Bagchi, T., Misra, A., 2006. Role of Caco-2 cell monolayers inprediction of intestinal drug absorption. Biotechnol. Prog. 22, 186–198.

Tomita, M., Hayashi, M., Awazu, S., 1996. Absorption-enhancing mechanism ofEDTA, caprate, and decanoylcarnitine in Caco-2 cells. J. Pharm. Sci. 85, 608–611.

Turco, L., Caloni, F., Consiglio, E.D., Testai, E., Stammati, A., 2010. Caco-2/TC7 cell linecharacterization for intestinal absorption: how reliable is this in vitro model forthe prediction of the oral dose fraction absorbed in human? Toxicol. In Vitro.

van Hasselt, P.M., Janssens, G.E.P.J., Slot, T.K., van der Ham, M., Minderhoud, T.C.,Talelli, M., Akkermans, L.M., Rijcken, C.J.F., van Nostrum, C.F., 2009. Theinfluence of bile acids on the oral bioavailability of vitamin K encapsulated inpolymeric micelles. J. Control. Release 133, 161–168.

Wang, T.G., Gotoh, Y., Jennings, M.H., Rhoads, C.A., Aw, T.Y., 2000. Lipidhydroperoxide-induced apoptosis in human colonic CaCo-2 cells is associatedwith an early loss of cellular redox balance. Faseb J. 14, 1567–1576.

Wiedmann, T.S., Liang, W., Kamel, L., 2002. Solubilization of drugs by physiologicalmixtures of bile salts. Pharm. Res. 19, 1203–1208.

Wood, E., Smith, J., Dornish, M., 2004. Effect of chitosan on epithelial cell tightjunctions. Pharm. Res. 21, 43–49.

Yin, L.C., Zhao, X., Cui, L.M., Ding, J.Y., He, M., Tang, C., Yin, C.H., 2009. Cytotoxicityand genotoxicity of superporous hydrogel containing interpenetrating polymernetworks. Food Chem. Toxicol. 47, 1139–1145.

Yu, J.N., Zhu, Y.A., Wang, L., Peng, M., Tong, S.S., Cao, X., Qiu, H., Xu, X.M., 2010.Enhancement of oral bioavailability of the poorly water-soluble drug silybin bysodium cholate/phospholipid-mixed micelles. Acta Pharmacol. Sin. 31, 759–764.