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International Journal of Pharmaceutics 446 (2013) 130– 135

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

jo ur n al homep age: www.elsev ier .com/ locate / i jpharm

hamnolipids enhance epithelial permeability in Caco-2 monolayers

ifang Jiang, Xuwei Long, Qin Meng ∗

epartment of Chemical Engineering and Biochemical Engineering, Zhejiang University, Zhejiang 310027, PR China

r t i c l e i n f o

rticle history:eceived 10 October 2012eceived in revised form 28 January 2013ccepted 3 February 2013vailable online 10 February 2013

eywords:hamnolipids

a b s t r a c t

This work aimed to evaluate the applicability of rhamnolipids as permeation enhancers for oral drugs. Inthis study, rhamnolipids were found to effectively increase the paracellular and transcellular transportof Transwell®-cultured Caco-2 cells, an in vitro model of the human small intestinal epithelium, in aconcentration-dependent manner. Rhamnolipids at 150 mg/L increased the paracellular apparent per-meability (Papp) of phenol red almost 7- to 8-fold, the largest enhancement ever reported, while Tween-80exhibited no such effect. Regarding the transcellular pathway, rhamnolipids at 150 mg/L enhanced thePapp of propranolol 2-fold, similar to the performance of Tween-80 at 400 mg/L. Moreover, rhamnolipids

ween-80bsorption enhancerransporter-glycoprotein

like Tween-80, significantly inhibited P-glycoprotein (P-gp) activity reflected by the reduced efflux ratio(basolateral-to-apical/apical-to-basolateral) of rhodamine 123 (R123), a P-gp substrate, on Caco-2 cells.Inhibition of P-gp activity was confirmed on plate cultured Caco-2 monolayers by assaying accumula-tion/efflux of R123 and R110, a non-P-gp substrate. Finally, rhamnolipids were demonstrated to be safeby cell viability and hemolysis assays. In conclusion, rhamnolipids were highly effective regulators of allthree transport pathways, suggesting their use as a safe absorption enhancer for oral drugs.

. Introduction

Oral drug administration is the most convenient and preferredhoice for patients, but most hydrophilic drugs and some higholecular weight hydrophobic drugs are poorly absorbed due to

he intestinal barrier function (Lipinski et al., 2001; Miyoshi andakai, 2005).

The intestinal barrier function of the mucosal epitheliums dominated by two passive barriers: transcellular and para-ellular barriers. The transcellular barrier is largely due toestricted permeability across cell membranes, while the para-ellular barrier is mainly controlled by tight junctions betweendjacent cells (Turner, 2009). Another important barrier in thentestinal epithelium consists of specialized transport pathways,

here active efflux transporters such as P-glycoprotein (P-gp)educe drug absorption by pumping drugs out of cells (Hanket al., 2010). To overcome these three epithelial barriers, vari-us absorption enhancers are used to improve drug absorption,

articularly for drugs taken orally, by enhancing the transcellu-

ar/paracellular pathways or inhibiting P-gp activity (Maher et al.,008, 2009).

∗ Corresponding author at: Department of Chemical Engineering and Biochem-cal Engineering, College of Materials Science and Chemical Engineering, Zhejiangniversity, 38 Zheda Road, Hangzhou, Zhejiang, 310027, PR China.el.: +86 571 87953193; fax: +86 571 87951227.

E-mail address: mengq@zju.edu.cn (Q. Meng).

378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.02.003

© 2013 Elsevier B.V. All rights reserved.

Currently, at least one drug-transport pathway across theintestinal epithelium barrier has been enhanced by chemicals thatcould be potentially developed into drug-absorption enhancers. Afew surfactants such as sodium lauryl sulfate and Tween-80, whichhave been traditionally used as drug excipients, were shown to havegreat potential as absorption enhancers (Takahashi et al., 2002).For example, Tween-80 increased passive transcellular transport(Takahashi et al., 2002) and inhibited P-gp activity (Rege et al.,2001), while sodium lauryl sulfate has an extra function in enhanc-ing paracellular transport (Takahashi et al., 2002). Sodium caprate(C10), a medium-chain fatty acid promoter, enhanced drug per-meability by mediating all three drug-transport pathways, amongwhich paracellular transport was most significantly improved(Maher et al., 2009). Some peptides, represented by Zonula occludentoxin (Fasano and Uzzau, 1997), Clostridium perfringens entero-toxin (Kondoh et al., 2012) and melittin (Maher et al., 2007), couldenhance both paracellular and transcellular transport, but couldnot inhibit P-gp activity.

To date, few of these potential drug-absorption enhancers havebeen licensed for clinical use in oral formulations due to limitationssuch as toxicity and high cost. A major limitation of ionic surfac-tants is their toxicity. For example, sodium lauryl sulfate enhancesall three transportation pathways, but its application is limitedby significant disruption of the intestinal mucosal epithelium

(Uchiyama et al., 1999). Although Tween-80 and other polyethoxy-lated pharmaceutical surfactants (Ardavanis et al., 2004; Price andHamilton, 2007) are much safer than ionic surfactants, they couldalter pharmacological properties of active ingredients and/or cause

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L. Jiang et al. / International Journa

athophysiological symptoms such as acute hypersensitivity reac-ions in susceptible patients (ten Tije et al., 2003; Coors et al., 2005).n the other hand, sodium caprate generally works under higheroncentrations in vivo and was confronted with its high cost in useLindmark et al., 1997), while peptide-based enhancers had poortability when subjected to enzymes in the gastrointestinal tractChung et al., 1979).

Thus, a new drug-absorption enhancer is desirable in view ofafety, efficacy, and cost effectiveness. A good candidate for suchn enhancer would be the class of glycolipids called rhamnolipids,hich are composed of mono- and disaccharides joined to hydroxy-

ated carboxylic acids by glycosidic bonds (Soberon-Chavez et al.,005). These bacteria-derived biosurfactants are commerciallyvailable and, more importantly, have been well accepted for theirood safety and strong surface activity. However, rhamnolipidsave never been reported as drug-transport enhancers. There-

ore, this study examined the effects of rhamnolipids on oral drugbsorption. The impact of rhamnolipids on each absorption path-ay was compared to that of Tween-80, a commonly accepted drug

xcipient and a potential absorption enhancer.

. Materials and methods

.1. Materials

Collagens (type IV), Dulbecco’s modified Eagle’s mediumDMEM) and non-essential amino acid were purchased fromibco (Gaithersburg, USA). Methyl thiazolyl tetrazolium (MTT), l-lutamine were purchased from Amresco Inc. (Solon, OH, USA).etal bovine serum (FBS) was obtained from Hangzhou Sijiqing Bio-ogical Eng Material Co., Ltd. (Hangzhou, China), propranolol wasenerously donated by Mr. Wang Yanming (College of Pharma-eutical Sciences, Zhejiang University, Hangzhou, China), HEPES,hodamine 123 (R123), Rhodamine 110 (R110), and Tween-80ere purchased from Sigma (Deisenhofer, Germany). Rhamnoli-ids were purchased from Huzhou Gemking Biotech Ltd. (Huzhou,hina) as a mixture of mono-(30%) and di-rhamnolipids (70%). Theemaining chemicals were obtained from local chemical suppliersnd were all of reagent grade.

.2. Cell culture

Caco-2 cells were grown in Dulbecco’s modified Eagle’s mediumDMEM) with 2 mM l-glutamine, 100 U/ml penicillin, 100 �g/mltreptomycin, 1% (v/v) non-essential amino acids and 10% (v/v) fetalovine serum. Caco-2 cell were cultivated in tissue culture flaskst 37 ◦C in a humidified atmosphere with 5% CO2. For the transportxperiments, the cells reaching 70–80% confluence were detachedrom the flasks and seeded onto polycarbonate Transwell® inserts3 �m pore size, 10 mm membrane diameter) at a density of

× 104 cells/insert. The cells were cultured for 19–22 days depend-ng on the integrity of the cell monolayer on DMEM medium as

entioned above which was changed bilaterally every other day.or accumulation and efflux study, the detached cells were seededn 24-well plate at a density of 3 × 105 cells/well and used untilonfluence. The passage numbers of the cells were between 27 and0. The integrity of each cell monolayer was examined by the per-eability of phenol red (Cano-Cebrian et al., 2005) before and after

he experiments.

.3. Transport studies

The transport of drugs was examined in the presence orbsence of Tween-80 and rhamnolipids. The compounds evalu-ted were phenol red (100 �M), a paracellular transport marker

armaceutics 446 (2013) 130– 135 131

(Cano-Cebrian et al., 2005), and propranolol (10 �M), a transcellu-lar transport marker (Karyekar et al., 2003). Cells were rinsed withpre-warmed Hank’s balanced salt solution (HBSS) supplementedwith glucose (11 mM) and HEPES (25 mM, pH 7.4) and equilibratedin this HBSS for 30 min. Phenol red or propranolol in the presenceof Tween-80 or rhamnolipids over a range of concentrations wereadded to the apical side. Apical sample was carried out at time 0and 180 min. Basolateral samples were taken every 20 min over3 h, replenishing with fresh pre-warmed HBSS at each sample timepoint (Maher et al., 2007). The samples were analyzed by spec-trophotometer, 479 nm for phenol red (Lewis, 2002) and 290 nmfor propranolol (Jagdale et al., 2009).

Apparent permeability coefficients (Papp) of test agents in thepresence or absence of rhamnolipids or Tween-80 were determinedfollowing triplicate experiments (n = 3). Permeability coefficientswere determined at sink conditions from the following equation:

Papp = dc

dt· V

AC0

where dc/dt is equal to the rate of linear appearance of mass in thereceiver solution, A is the cross-sectional area of the membrane incm2, and C0 is the initial drug concentration in the donor compart-ment. All values are represented as mean and standard deviationof the values from three monolayer Transwell® inserts.

2.4. Transport of R123 across the Transwell® monolayer cells inboth directions

In the apical-to-basolateral (AP-BL) transport studies, 10 �MR123 solution containing various concentrations of Tween-80 (0,200, 400 mg/L) or rhamnolipids (0, 20, 50, 100, 150 mg/L) was addedto the apical side, whereas blank HBSS solution was added to thebasolateral side (Zhu et al., 2009). In the basolateral-to-apical (BL-AP) direction, the R123 solution as mentioned above was added tothe basolateral side. The samples were taken from the added sideand the receiver side as 2.3 carried out. R123 levels in the transportstudy were determined with a fluorescence spectroscope (Perkin-Elmer LS50B; Perkin-Elmer Life and Analytical Sciences, Boston,MA, USA), at an excitation wavelength of 485 nm and an emissionwavelength of 535 nm. The efflux ratio (B/A) was taken as the BP-ALversus AL-BP (Rege et al., 2002).

2.5. Cellular accumulation and efflux of R123 and R110

The cellular accumulation and efflux of the fluorescent dyes(R123 and R110) were used to examine the effects of Tween-80 andrhamnolipids on the cell monolayers cultured on 24-well platesas previously reported (Batrakova et al., 1998). Cell monolayerswere preincubated for 30 min at 37 ◦C with HBSS. Then, HBSS wasremoved and the cells were exposed to 3.2 �M R123 (or R110) ineither blank HBSS or HBSS with Tween-80 (200 mg/L), or rhamnoli-pids (10, 20, 50 mg/L). The cells were incubated with dye solutionsfor up to 30 min at 37 ◦C. Then, the dye solutions were removed andcell monolayers were washed three times with ice-cold HBSS.

For the accumulation study, the cells were then solubilized in1.0% Triton X-100 and aliquots were removed for determinationof cellular dyes using a fluorescence spectrophotometer (Perkin-Elmer LS50B; Perkin-Elmer Life and Analytical Sciences, Boston,MA, USA), at an excitation wavelength of 485 nm and an emissionwavelength of 535 nm. For the efflux study, the remaining mono-layers were incubated at 37 ◦C in blank HBSS solutions. The effluxof R123 and R110 in each treatment was determined by sampling

buffer solutions at 30 min, and the measurement of fluorescencein each sample using fluorescent spectroscopy as described above.Samples were taken for protein assay using the BCA method. Theefflux was expressed for each treatment as a percentage of R123

1 l of Pharmaceutics 446 (2013) 130– 135

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Fig. 1. Effects of Tween-80 (T80) and rhamnolipids (Rha) on the apparent perme-ability coefficients (Papp) of (A) phenol red and (B) propranolol transport acrossCaco-2 cell monolayers. The permeability experiments were performed in the apical

To exclude the paracellular transport of R123, we conductedexperiments to detect the accumulation and efflux of R123, whichreflects P-gp activity, in plates cultured Caco-2 cell monolayers.

Table 1Effects of rhamnolipids and Tween-80 on P-glycoprotein-mediated permeability ofrhodamine 123 across Caco-2 cells.

Concentration (mg/L) Papp (×10−6 cm/s) Ratio B/A

AP-BL a BL-AP b

0 1.58 ± 0.02 10.34 ± 0.14 8.40 ± 0.02Tween-80

200 5.15 ± 0.08 8.75 ± 0.12 1.70 ± 0.03**

400 16.80 ± 0.24 6.97 ± 0.09 0.41 ± 0.01**

Rhamnolipids20 2.31 ± 0.19 9.98 ± 0.12 4.32 ± 0.07*

50 6.06 ± 0.09 24.10 ± 0.33 3.90 ± 0.05*

100 20.32 ± 0.30 37.04 ± 0.50 1.82 ± 0.02**

150 24.30 ± 0.35 45.80 ± 0.62 1.88 ± 0.03**

32 L. Jiang et al. / International Journa

nd R110 in the media to R123 and R110 accumulation in cellonolayers during the loading period.

.6. Cytotoxicity and hemolysis assays

Viability of the Caco-2 cells upon treatment with rhamnolipidsr Tween-80 was examined by MTT assay as previously describedWang et al., 1996). Briefly, cells were immerged in 0.65 ml of.15 mg/ml MTT-phosphate buffer solution in 24-well plates before

ncubated in the incubator at 5% CO2 (37 ◦C) for 3 h. At the endf incubation, MTT solution was discarded, and then 1.5 ml iso-ropanol (containing 10 mM HCl) was added to each well. Thelates were then shaken for 1 h to extract the blue products andhe absorbance of the solution was read at a wavelength of 570 nmy a spectrophotometer.

Rat blood was chosen to evaluate the hemolytic activity ofween-80 or rhamnolipids. Briefly, fresh defibrinated rat erythro-ytes were rinsed three times with normal saline (0.9%, w/v),esuspended in saline to obtain a suspension with A540 = 1 (Sanchezt al., 2010) and incubated with an equal volume of test agent for

h at 37 ◦C. The cell suspension was then centrifuged at 1000 × g for5 min after which an aliquot of the supernatant was transferred to

new well with hemoglobin release measured spectrophotomet-ically at 540 nm. Percent hemolysis was calculated from equation:

ate of hemolysis % = ODtest agent − IDnegative

ODpositive − ODnegative× 100

.7. Data analysis

All values reported in the text are presented as mean ± standardrror. Comparisons between multiple groups were performed withne-way ANOVA or results for two different treatments were com-ared using t-test for statistical comparisons. A p-value < 0.05 wasetermined to be significant.

. Results

.1. Effect of rhamnolipids on drug transport in Caco-2 cells onranswell®

Rhamnolipids and Tween-80 were compared for their effectPapp) on the transport of two drugs (phenol red and propra-olol) across Caco-2 monolayers cultured on Transwell® plates. Ashown in Fig. 1A, rhamnolipids at concentrations above 20 mg/Lncreased Papp of phenol red, e.g., 150 mg/L of rhamnolipidsnhanced the Papp of phenol red from 4.37 × 10−6 cm/s (control)o 32.43 × 10−6 cm/s (p < 0.01). In contrast, Tween-80 at all concen-rations tested (200–400 mg/L) did not affect the Papp of phenoled.

To determine if rhamnolipids altered transcellular perme-bility, transport studies were conducted with propranolol. Theapp of propranolol was significantly increased by rhamnolipidst concentrations above 50 mg/L and by Tween-80 at 400 mg/LFig. 1B). Rhamnolipids failed to improve transcellular permeabilityt 20 mg/L (Fig. 1B) or at lower concentrations (data not shown).

The effects of rhamnolipids and Tween-80 on the bidirectionalermeability of P-gp activity were compared using rhodamine23 (R123) as a P-gp substrate in Caco-2 monolayers cultured onranswell® inserts. Bi-directional permeability coefficients (apical-o-basolateral [AP-BL] and basolateral-to-apical [BL-AP]) were

ssessed as well as the B/A ratio (BL-AP versus AP-BL) of R123ith or without surfactants (Table 1). Under the two selected

ween-80 concentrations (200 and 400 mg/L), AP-BL permeabilityncreased, BL-AP permeability decreased, and the B/A ratio of R123

to basal direction. Asterisks (*) indicate a statistically significant difference com-pared to the control group (no addition) (*p < 0.05, **p < 0.01). Data are expressed asthe mean ± SD of three independent experiments.

was thereby reduced. Rhamnolipids, like Tween-80, significantlyreduced the B/A ratio, but the BL-AP permeability unexpectedlyincreased when rhamnolipids concentrations were over 20 mg/L.This unexpected enhancement of BL-AP R123 permeability byrhamnolipid treatment might have been caused by facilitated pas-sive (especially paracellular) transport.

3.2. Effect of rhamnolipids on accumulation and efflux of the P-gpsubstrate R123 in Caco-2 monolayers

Results are expressed as the mean ± SEM (n = 3).* Statistically different from control with no addition (p < 0.05).

** Statistically different from control with no addition (p < 0.01).a Twenn-80 and rhamnolipids were added at the apical side.b Tween-80 and rhamnolipids were added at the basal side.

L. Jiang et al. / International Journal of Pharmaceutics 446 (2013) 130– 135 133

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Concentration (mg/L)

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Control T80 200 Rha10 Rha20 Rha50

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Fig. 2. (A) The accumulation and (B) efflux of rhodamine 123 (R123) or rhodamine110 (R110) in Caco-2 cell monolayers in the presence or absence of Tween-80 (T80)and rhamnolipids (Rha) Asterisks (*) indicate a statistically significant differencebetween R123 values and the control group (no addition) (*p < 0.05, **p < 0.01).Pound signs (#) indicate a statistically significant difference between R110 valuesao

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Fig. 3. (A) Evaluation of the cytotoxic potential of rhamnolipids or Tween-80 onCaco-2 cell monolayers following 3 h treatment. Cell viability was assessed usingthe MTT assay. (B) The hemolytic rate of rhamnolipids at different concentrations.

rhamnolipids increased the paracellular transport of phenol red by

nd the control group (no addition) (#p < 0.05). Data are expressed as the mean ± SDf three independent experiments.

o reduce transcellular transport, lower concentrations of Tween-0 (200 mg/L) or rhamnolipids (10, 20 and 50 mg/L) were used.nother strategy to reflect possible transcellular transport was toompare the effects of surfactants on transport of R110, a non-P-p-dependent analog of R123.

Compared to the control without any surfactants, the presencef Tween-80 at 200 mg/L or rhamnolipids at two relatively highoncentrations (20 mg/L and 50 mg/L) increased the intracellularccumulation of R123 by up to 4-fold (Fig. 2A), while significantlyeducing its efflux (Fig. 2B). These effects were specific for R123,xcept at 50 mg/L of rhamnolipids, which enhanced both the intra-ellular accumulation and efflux of R110 (Fig. 2). Rhamnolipids at0 mg/L did not influence accumulation and efflux of R123 or R110.

.3. Effects of rhamnolipids on cytotoxicity and hemolytic activity

The cytotoxicity of Caco-2 cells treated with rhamnolipids orween-80 was evaluated by cell viability and impermeability tohenol red. Exposure to surfactants at the test concentrations (50,00 and 150 mg/L for rhamnolipids; 200 and 400 mg/L for Tween-

0) showed no significant influence on cell viability. In addition,he impermeability of phenol red was all recovered at 8 h after eachurfactant treatment for 3 h (data not shown).

Asterisks (*) indicate a statistically significant difference compared to the controlgroup (no addition) (*p < 0.05, **p < 0.01). Data are expressed as the mean ± SD ofthree independent experiments.

The hemolytic activity of Tween-80 and rhamnolipids wasdetermined by analyzing hemolysis of defibrinated rat erythro-cytes. As shown in Fig. 3B, rhamnolipids shows low hemolysiswithin 0–150 mg/L. Tween-80 shows no hemolysis even at400 mg/L (data not shown).

4. Discussion

This study shows that biosurfactant rhamnolipids can safely andeffectively enhance absorption of drugs in Caco-2 monolayers. Thisresult offers the possibility of a new absorption enhancer for oraldrugs, which have been studied for many years, but their use hasbeen limited due to safety problems or cost-ineffectiveness.

In our study, rhamnolipids increased the paracellular trans-port of drugs in a dose-dependent manner. We also showed thatTween-80 did not affect paracellular transport, consistent with pre-vious findings (Rege et al., 2001; Takahashi et al., 2002). In contrastto Tween-80, rhamnolipids increased the permeability of phenolred in a dose-dependent manner in Caco-2 cells, in agreementwith a previous report that rhamnolipids enhanced the perme-ability of inulin, a paracellular transport marker, in respiratoryepithelial cells (Zulianello et al., 2006). The mechanism by whichrhamnolipids regulate the paracellular pathway of Caco-2 cellsmay be similar to that of respiratory epithelial cells, which reg-ulate tight-junction proteins (ZO-1, occludin, claudin-1) instead ofdegrading them (Halldorsson et al., 2010). Notably, we found that

7.4-fold at 150 mg/L, while sodium caprate (C10) only increased thistransport by 4-fold at a 25-times high concentration (3940 mg/L)(Sugiyama et al., 1997) Thus, rhamnolipids showed much superior

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34 L. Jiang et al. / International Journa

p-regulation of paracellular drug transport than C10, which is cur-ently in advanced clinical trials for its distinguished enhancementf paracellular transport (Sugiyama et al., 1997; Badawi et al., 2008;aher et al., 2009). Rhamnolipids appear to be one of the most

ffective paracellular transport enhancers ever reported.Like most surfactants, rhamnolipids effectively enhanced

ranscellular transport. Rhamnolipids, like Tween-80 and otherbsorption promoters (Whitehead et al., 2008), demonstratedoncentration-dependent transcellular enhancement in Caco-2ells. The rhamnolipid-enhanced transcellular permeability wasonfirmed by the significantly improved accumulation and efflux of110, a non-P-gp-dependent analog of R123. Moreover, rhamnoli-ids are more effective than Tween-80 in promoting transcellularransport since rhamnolipids elicited enhancement at the criti-al micelle concentration (CMC) of 50 mg/L (data not shown), anmportant index of surface activity in surfactants, while Tween-80id not show this effect at 200 mg/L which is much higher than

ts CMC value of 14 mg/L (Haque et al., 1999). We would like toention that the CMC value (50 mg/L) of the rhamnolipid mixture

sed in this paper close to the previously reported CMC of purifiedirhamnolipid at 71 mg/L (Aranda et al., 2007) and well within theange of 50–230 mg/L for the CMC of other rhamnolipid mixturesBenincasa et al., 2004; Whang et al., 2008). As previously stated, theranscellular enhancement of surfactants was caused by perturb-ng the enterocyte plasma membrane as a result of surface activityMaher et al., 2009); naturally, such transcellular enhancement ofurfactants has been closely associated with their surface activitiesXia and Onyuksel, 2000). Nevertheless, membrane-permeabilitynhancement lacks a connection to CMC (Walters et al., 1981;wenson et al., 1994a,b). This could explain the much lower activ-ty in enhancing transcellular transport by Tween-80, which has aower CMC than rhamnolipids. Alternatively, a close predictor coulde the surface pressure � determined by the difference betweenhe surface tension of buffer in the absence and presence of sur-actants above the CMC (Swenson et al., 1994b; Xia and Onyuksel,000). In this respect, rhamnolipids, which decreased water sur-ace tension from 72.3 dyne/cm to a low of 30 dyne/cm (Parrat al., 1989), should have superior permeability-enhancing activ-ty over Tween-80 which only reduced water surface tension to0 dyne/cm (Haque et al., 1999). We would like to mention thathe unique molecular shape of rhamnolipids, showing comple-

entarity to phosphatidylethanolamine, should contribute to itsermeability-enhancing activity by facilitating membrane inser-ion when rhamnolipids present in a bilayer (Ortiz et al., 2010).

Rhamnolipids, for the first time, were shown to inhibit P-gpctivity. The presence of rhamnolipids significantly reduced the B/Aatio, representing a similar inhibition of P-gp activity as Tween-0 (Neuhoff et al., 2003). In contrast to regular P-gp inhibitorsRege et al., 2002) and Tween-80, rhamnolipids increased BL-APermeability following the same trend as SDS (Gundogdu et al.,012). The enhanced BL-AP permeability was due to the influencef passive transport, as indicated by comparison of R123 and R 110ccumulation/efflux in plated Caco-2 monolayers. Specifically, rha-nolipids at 20 mg/L, which lower than half its CMC, had no impact

n transcellular transport, but inhibited P-gp activity as shown bynhanced accumulation versus decreased efflux of R123. As ouresults clearly presented, rhamnolipids impacted each transportathway at concentrations higher than 10 mg/L or had no functionst a lower concentrations including 10 mg/L. Taken together, ouresults show that rhamnolipids reduced P-gp activity at concentra-ions as low as half CMC, and this inhibition was highly impacted byhe two passive transport pathways at concentrations over 20 mg/L.

uch impact at higher concentrations was not common for otherurfactants.

The safety of rhamnolipids as potential absorption enhancersas well supported by our cytotoxicity and hemolysis studies.

armaceutics 446 (2013) 130– 135

Both surfactants were considered to be safe for Caco-2 cells withinthe effective concentration range (20–150 mg/L for rhamnolipidsand 200–400 mg/L for Tween-80) by the MTT assay (Fig. 3A). Thesafety of rhamnolipids on Caco-2 cells was further confirmed bythe recovered paracellular barrier to phenol red permeability aftertreatment with rhamnolipids (data not shown). As rhamnolipidsshowed slight hemolysis of about 16% at the highest effective con-centration of 150 mg/L at RBC concentration of 1% and showedno hemolysis at RBC concentration of 8% (data not shown), wepresumed that rhamnolipids are safe within the effective con-centration range in vivo where RBC concentration is 100%. Ourpresumption could be supported by the fact that C10, though of100% hemolysis at 10 mM under RBC concentration of 2%, has neverreported of hemolysis in vivo even under a much higher concentra-tion compared with that of in vitro (Maher et al., 2009). We wouldlike to mention that the large discrepancy of the reported rhamno-lipid hemolytic concentration ranging from 30 mg/L (Sanchez et al.,2010) to 125 mg/L (Goran PILJAC et al., 1995); could be due to thedifferentially used rhamnolipids in purity and compositions as wellas the tested RBC concentration. Taken together, the performanceof rhamnolipids on safety did not follow the trend of other anionicsurfactants like sodium dodecyl sulfate, which possess inherent celltoxicity (Uchiyama et al., 1999). Actually, naturally produced rha-mnolipids have long been enthusiastically proposed as safe agentsin cosmetics and pharmaceutics (Ishigami, 1997; Ortiz et al., 2006)due to their low toxicity and biodegradability. Recently, rhamno-lipids were approved by the FDA for use in fruit, vegetable, andlegume crops for their low acute mammalian toxicity and non-mutagenicity (Nitschkea and Costa, 2007).

To the best of our knowledge, the majority of reported perme-ability enhancers, like peptides (Kondoh et al., 2012) and Tween-80(Rege et al., 2001; Takahashi et al., 2002), were shown to enhanceone or two pathways of drug transport across the epithelial bar-rier. Although C10 (Maher et al., 2009) and sodium lauryl sulfate(Takahashi et al., 2002) could affect all three drug-transport path-ways, they were limited either by high dosage or severe safetyconcerns. In comparison, rhamnolipids exhibited high effectivenessnot only at low concentrations, but also acted in a multi-regulatorymanner for each transport pathway. Moreover, rhamnolipids arecommercially produced from microbial fermentation, making themcost-effective as well as safe. Hence, rhamnolipids could be supe-rior to reported permeability enhancers for potential application inthe future.

5. Conclusions

Rhamnolipids at low concentrations not only enhanced paracel-lular and transcellular transport pathways in Caco-2 monolayers,an in vitro model of the human small intestinal epithelium, but alsoinhibited P-gp activity. Moreover, rhamnolipids showed low toxic-ity to Caco-2 monolayers and erythrocytes. Overall, rhamnolipidssignificantly enhanced permeability via three transport pathwaysand have a large safety window, suggesting their future use as asafe absorption enhancer for oral drugs.

Acknowledgement

This work was supported by Grants Nos. 21176216 and21076186 from NSFC (National Natural Science Foundation ofChina).

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