Binding, Uptake, and Transport of Hypericin by Caco-2 Cell Monolayers

STEFFI SATTLER†‡, ULRICH SCHAEFER†, WERNER SCHNEIDER§, JOSEF HOELZL‡, AND CLAUS-MICHAEL LEHRX,†

Received January 6, 1997, from the †Department of Biopharmaceutics and Pharmaceutical Technology, University of the Saarland,Saarbruecken, Germany, ‡Department of Pharmaceutical Biology, Philipps-University, Marburg, Germany, and §SteigerwaldArzneimittelwerk GmbH, Darmstadt, Germany. Final revised manuscript received May 27, 1997. Accepted forpublication June 28, 1997X.

Abstract 0 The biological evaluation of hypericin in various test modelsis hampered by its very poor water solubility. In the present studycyclodextrin formulations and liposomal preparations were investigatedfor improved delivery and solubility of hypericin in aqueous buffer systems.Caco-2 cells, grown to tight monolayers on 96-well tissue culture platesas well as on Transwell polycarbonate filters, were used to study themembrane binding and the epithelial transport of hypericin. Cumulativetransport of hypericin, which could not be measured without the use ofcyclodextrins, in apical-to-basolateral direction from cyclodextrin−hypericinbuffer solutions was 3−5% at 37 °C and approximately 0.12% at 4 °Cafter 5 h. After an incubation time of 1 h at 37 and 4 °C, 12.7% ± 2.6%and 6.5% ± 0.8%, respectively, of hypericin were found to be bound toor taken up by Caco-2 cells. Liposomal formulations markedly increasedthe solubility of hypericin in Krebs−Ringer buffer, but there was no effectobserved on the binding and transport of hypericin delivered by liposomesin the Caco-2 cell model. Due to the fluorescence properties of hypericin,its interaction with the cells could be visualized by confocal laser scanningmicroscopy. The results indicate that a significant accumulation of thedrug in the cell membrane and the cell nucleus membrane takes place.We conclude that hypericin is absorbed through the intestinal epitheliumby passive transcellular diffusion and that increasing its solubility bycyclodextrin appears as a promising approach to increase its oralbioavailability for pharmaceutical formulations.

IntroductionHypericin is a natural drug belonging to the class of

naphthodianthrones that is isolated from plants of the genusHypericum, the most common of which is Hypericum perfo-ratum L., also known as St. John’s wort. The chemicalsynthesis of hypericin has been reported by Brockmann.1Crude Hypericum perforatum extract is approved in Germanyfor use as a mild-to-moderate antidepressant.2,3 Of all thecompounds isolated from the extract, the red-colored hyper-icins have been the center of interest. Recently, hypericin hasattracted scientific attention regarding its virucidal activityin vitro as well as in vivo against a broad range of envelopedviruses and retroviruses4,5,6,7 and its prevention of cell infec-tion by these viruses.8,9In a pilot study pharmacokinetics of hypericin were studied

in healthy male volunteers, following oral administration ofa standarized extract of H. perforatum L. in soft gelatinecapsules (Psychotonin forte, Steigerwald ArzneimittelwerkGmbH).10,11 In the dosage form used, the dried Hypericumperforatum extract was dispersed in a oily formulation,containing e.g. soy oil and soya lecithin. After a single oraldose, corresponding to 1.2 mg hypericin, median maximal

plasma levels of 9.5 ng/mL (cmax) were detected after 3-4 h(tmax) (Figure 1).Knowing about the bioavailability of hypericin in humans,

we wanted to investigate the mechanisms by which hypericinis able to overcome the epithelial barrier of the intestinal tract.For the pharmacological evaluation of hypericin in biologicaltest systems, as well as for its pharmaceutical formulation, itmust be considered that the compound is slightly soluble inwater, which causes problems.12 In water hypericin formsnonfluorescent high molecular weight aggregates which, inaddition, precipitate when salt solutions are added.To perform pharmacological studies in physiological media,

solubilizing agents, such as dimethyl sulfoxide,13 propyleneglycol, or ethanol,4 or detergents, like Tween 8014 or TritonX-100,15 have been added. It must be considered, however,that these agents may themselves have effects on biologicalsystems like cell cultures and moreover cannot be readily usedin oral pharmaceutical formulations.It is established that the bioavailability of many poorly

water soluble drugs is increased by coadministration withcyclodextrins16 or incorporation in liposomes.17 Given theabove-mentioned solubility problems of hypericin in buffersystems and in order to circumvent the addition of cosolventsand detergents, we wanted to use cyclodextrins and liposomesas a water/oily formulation to solubilize hypericin in aphysiological buffer medium having low ion concentration. Thelatter property being important because the solubility ofhypericin is strongly influenced by ions.12 The rationale forextending our studies to liposomes was motivated by the factthat some commercial preparations of Hypericum perforatumplant extracts, like the above mentioned soft gelatine capsules,are dispersed in an oily formulation.As a model to study cell interaction and epithelial transport

under controlled conditions in vitro, the human colon carci-noma cell line, Caco-2, which shows morphological andbiochemical similarity to normal intestinal enterocytes,18 wasused. Both binding and transport studies were carried outon Caco-2 cells grown on 96-well tissue culture plates orpolycarbonate membranes in Transwell cell culture chambers.To visualize the interaction between cells and drugs or drug-

carrier systems, confocal laser scanning microscopy (CLSM)

x Author to whom correspondence and reprint requests should be sentat Universitat des Saarlandes, FR 12.2, Department of Biopharmaceuticsand Pharmaceutical Technology, P.O. Box 15 11 50, 66041 Saarbruecken,Germany. Phone: +49-681-302-3039. Fax: +49-681-302-4677. E-mail:lehr@rz.uni-sb.de. http://www.uni-sb.de/matfak/fbiz/lehr/ .

X Abstract published in Advance ACS Abstracts, September 1, 1997.

Figure 1sPlasma levels of hypericin after oral administration of soft gelatinecapsules, corresponding to 1.2 mg hypericin, to two healthy volunteers.11

S0022-3549(97)00004-X CCC: $14.001120 / Journal of Pharmaceutical Sciences © 1997, American Chemical Society andVol. 86, No. 10, October 1997 American Pharmaceutical Association

has recently become available.19 Taking advantage of thefluorescence properties of hypericin, we used this techniqueto visualize drug uptake by living epithelial cells in culture.

Experimental Section

MaterialssCyclodextrins were donated by Wacker Chemie, GmbH,Munich, Germany, except for 2-hydroxypropyl â-cyclodextrin, whichwas provided by Roquette GmbH, Frankfurt, Germany, and â-cyclo-dextrin sulfobutyl ether 7-sodium salt, donated by Cydex. Phospho-lipon 80, a phospholipid fraction composed of 76% phosphatidylcholineand 8% phosphatic acid as negatively charged phospholipid, wasprovided by Rhone-Poulenc Rorer, Nattermann Phospholipid GmbH,Cologne, Germany. All high-performance liquid chromatography(HPLC) solvents were HPLC-grade, filtered, and degassed before use.All other chemicals were reagent grade or better and used withoutfurther purification.Hypericin: Extraction and PurificationsAerial parts of H. perfo-

ratum L. (St. John’s wort) were harvested at its flowering time, dried,cut, and milled. The crude hypericin-enriched extract was obtainedfollowing the Deutscher Arzneimittel Codex standard procedure.20After a 24 h light exposure, the extract was further chromatographi-cally purified by using a polyamide column (ICN Biomedicals,Eschwege, Germany) with methanol as solvent. When the eluent wasfairly pure, the solvent mixture was changed to methanol/ammoniasolution (99/1) to elute the hypericin fraction. Pure hypericin(C30H16O8,Mr 504.43) fractions were collected by using a preparativeRP18-HPLC column (2.5 × 25 cm, 7 µm, Merck, Darmstadt, Ger-many), with a mobile phase composed of methanol/acetonitrile/phosphoric acid (89.5/10/0.5), operated with a flow gradient (0-2 min,10 mL/min; 2-15 min, 12 mL/min; 15-30 min, 15 mL/min). Samplesfrom pure hypericin fractions were pooled, evaporated to remove thesolvent, transferred onto a polyamide column, and washed with a 50%methanolic solution to remove the phosphoric acid. Solutions yieldingpure hypericin, eluted from the column with methanol/ammoniasolution (99/1), were dried by slow evaporation. Hypericin, contentat least 93%, was characterized by 1H-NMR, mass, and UV spectro-scopic data, which were in agreement with data reported for synthe-sized hypericin.21 Hypericin was stored dry at 4 °C in the dark untiluse.Analytical HPLCMethodssThe content of hypericin in the samples

obtained from the solubility studies as well as the samples from thetransport studies were analyzed by reversed phase HPLC. The assayswere performed on a Waters Chromatography pump 6000 A (Waters,Milford, MA), a Shimadzu RF-535 fluorescence HPLC detector(Shimadzu Europa, Duisburg, Germany), and a Hewlett-Packard 3393A integrator (Hewlett-Packard Company, Avondale, PA). The ana-lytical column was a LiChrospher 100 RP-18 (125 × 4 mm, particlesize 5 µm, Merck, Darmstadt, Germany) and the guard column aLiChrospher 100 RP-18 (4 × 4 mm, particle size 5 µm). Theseparation was operated with the mobile phase composed of methanol/acetonitrile/phosphoric acid (85%) (89.5/10/0.5), flow rate 1.5 mL/min,and injection volume 100 µL, resulting in retention times of 4.5 (0.2 min. The excitation wavelength was set to 580 nm and theemission wavelength to 600 nm.Preparation and Characterization of Hypericin-Cyclodextrin Solu-

tions and Solubility StudiessSolubility measurements were carriedout according to the methods of Higuchi et al.22 Excess amounts ofhypericin were added to phosphate-buffered saline (PBS, pH 7.4, madeisotonic with 230 mM mannitol instead of sodium chloride; referredto as M-PBS from here on) containing 0-5% of various cyclodextrins.After sonification for 3 min, the samples were stirred at 30 °C untilequilibration. The suspensions were centrifugated at 5000 rpm andpipetted through a 0.22 µm cellulose acetate filter (Schleicher &Schuell, Dassel, Germany). The first 200 µL of filtrate was discardedand a subsequent aliquot diluted with methanol (1:10 or 1:20) torender the hypericin fluorescent and permit it to be assayed by HPLC.UV spectra of the aqueous hypericin-cyclodextrin solutions wererecorded on an UV/vis Spectrometer Lamda 16 (Perkin-Elmer Corp.,Uberlingen, Germany) in the range of 250-700 nm. The phasesolubility diagram of hypericin in the presence of 0-30% 2-hydrox-ypropyl â-cyclodextrin in water was determined in the same way.Preparation and Characterization of Liposome Formulationss

Hypericin-containing liposomes were prepared at room temperature

essentially by the film method23 followed by extruding the liposomesthrough a microporous filter membrane. Briefly, weighed quantitiesof phospholipon 80 and hypericin were dissolved in a mixture ofchloroform and methanol (2/1). After removal of the organic solventunder reduced pressure, resulting in the formation of a homogeneousthin lipid film, the film was hydrated with water or Krebs-Ringerbuffer (KRB). Size reduction and reduced polydispersity wereachieved by extruding the liposome dispersion at room temperature25 times through a commercially available device (Liposofast, AvestinInc., Ottawa, Canada) with 0.2 µm polycarbonate filters (Nuclepore,Pleasanton, CA). Particle size analysis on liposomes was performedby using photon correlation spectroscopy (PCS) (ALV-Laser Goniom-eter, ALV-5000 Multiple Tau Digital Korrelator, ALV-Laser GmbH,Langen, Germany). The amount of encapsulated hypericin wasdetermined by HPLC after dilution with methanol (1:100). For UV/vis spectra liposomes were diluted in the corresponding aqueousbuffer.Freeze-Fracture Electron MicroscopysLiposome dispersions were

sandwiched between gold plates and quickly frozen in a cryo jet withliquid propane. Samples were prepared for electron microscopyfollowing standard procedures: freeze-fracture, shadowing withplatinum (1.5 nm) at 45°, carbon evaporation (8 nm) at 90° andwashing of the replicas in a tensid solution. The replicas wereexamined with a Zeiss EM 902 electron microscope (Carl Zeiss,Oberkochen, Germany) and a series of micrographs was taken of eachreplica.Cell CulturesCaco-2 cells were kindly donated by Prof. Dr. T. Kissel

(Department of Pharmaceutical Technology, Philipps University,Marburg, Germany). The cells were maintained at 37 °C in Dulbecco’sModified Eagle’s Medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% nonessential amino acids asdescribed elsewhere,24 and gentamycin (final concentration 50 µg/mL) in an atmosphere of 5% CO2 and 90% relative humidity. Alltissue culture media were obtained from Gibco through Life Technolo-gies (Eggenstein, Germany). The cells were seeded on flat bottom96-well tissue culture plates (Greiner Labortechnik, Frickenhausen,Germany), on polycarbonate filter inserts (0.45 µm, 12 mm diameter)of Transwell cell culture chambers (apical volume 0.5 mL, basolateralvolume 1.5 mL; Transwell Costar, Badhoevedorp, The Netherlands)or on glass cover slips (10 mm diameter, Assistent Hecht, Sondheim,Germany). Cells of passage number 46-60 and 90-107 were usedthroughout. The cells were allowed to grow and differentiate toconfluent monolayers for 14-21 days.Drug Transport across Filter-Grown Cell MonolayerssTransport

at 37 °C was determined first in the absence of cells, in order to assessthe barrier properties of the polycarbonate membrane alone, and thenacross Caco-2 cell monolayers in the apical-to-basolateral direction.For cell culture experiments, Transwell-grown cell monolayers at days14-21 after plating were used. Transport experiments were initiatedby washing the monolayers three times with buffer before the drugsolutions were added to the apical side of the cells. Hypericinsolutions were prepared on the one hand in M-PBS (pH 7.4, asdescribed above), supplemented with 5% of 2-hydroxypropyl â-cyclo-dextrin to yield a final concentration of hypericin in the donorcompartment of about 0.2 mM, and on the other hand from liposomescontaining hypericin in KRB to give a final hypericin concentrationof 0.396 and 0.799 mM. At the beginning of the experiment the initialdonor concentration was determined from a 50 µL sample. Nofluorescence was found in the basolateral chamber, in which hyperi-cin-free donor medium was used as receptor phase. Subsequently,samples (50 µL) were taken from the receiver side at specified timeintervals and replaced with an equal volume of transport medium.Before a sample was taken the integrity of the monolayer was checkedby measurement of the transepithelial electrical resistance (Evom,World Precision Instruments, Sarasota, FL). The transport experi-ments were carried out over a time period of 5 h. At the end of eachexperiment, the final concentration of the donor was determined froma 50 µL sample, the monolayer was washed three times with thetransport medium, and the hypericin bound to the cells was measuredby HPLC after solubilization with methanol.Binding and Uptake of Hypericin by Cells Grown on Well

PlatessCaco-2 cells were seeded in flat bottom 96-well plates andgrown to confluency within 14 days prior to use. Binding studies wereperformed in air (5% CO2) at 90% relative humidity and 37 °C or 4°C (refrigerator), depending on the experimental design. Hypericin

Journal of Pharmaceutical Sciences / 1121Vol. 86, No. 10, October 1997

dissolved in M-PBS containing 2.5% of 2-hydroxypropyl â-cyclodextrin(dilutions of hypericin from 0.0041 to 0.2646 mM) or hypericinincorporated in liposomes and dissolved in KRB (dilutions of hypericinfrom 0.012 to 0.77 mM) was used. After cell incubation for 1 or 2 hwith 100 µL/well of the drug solutions at the given concentrations,the cells were washed three times with buffer and solubilized withmethanol to terminate the uptake of hypericin. Samples wereimmediately analyzed in a CytoFluor II microplate fluorescence reader(PerSeptive Biosystems, Wiesbaden, Germany) at excitation andemission wavelengths of 530 and 590 nm, respectively. The hypericincontent of the stock solutions was measured in another scan afterdilution with methanol (1:50), which permitted calculation of the totalamount of cell-associated hypericin (Figure 2). Binding at eachhypericin concentration was determined 12 times and repeated withcells of different passages.Confocal Laser Scanning MicroscopysA laser scanning confocal

imaging system (MRC-1024, Bio-Rad Laboratories, Munchen, Ger-many), linked to a Zeiss Axiovert 100 microscope (Carl Zeiss,Oberkochen, Germany), was equipped with an argon ion laser(American Laser Corp., Salt Lake City, UT) set to the 514 nm line toexcite hypericin fluorescence. All images were taken with a ZeissNeofluor 40×, NA 1.3 oil objective, a scanning format of 1024 × 1024pixel, and a 585 LP emission filter setting. Unfixed cells, grown onglass cover slips, were treated with hypericin for 1 h in the sameconcentration and medium as that used in the transport and bindingexperiments (37 °C, humidified atmosphere, 5% CO2). Subsequently,the cells were washed three times with the corresponding buffersolution and mounted on glass microscope slides in a drop of buffer.The cell layer was thereby sandwiched between the glass cover slipand microscope slide to observe the hypericin fluorescence. Imageswere taken at room temperature within 5-15 min after positioningthe cells on the microscope slides.

ResultsSolubilization of Hypericin by CyclodextrinssThe effect of

six different cyclodextrins in various concentrations on thesolubility of hypericin in M-PBS was measured (Figure 3).Comparing the chemically different cyclodextrins, we foundno significant difference in solubility behavior: the solubilityof hypericin increased as a function of the cyclodextrinconcentration, reaching a plateau at ca. 100-200 µg/mL,compared to ca. 40 µg/mL in cyclodextrin-free M-PBS and onlyca. 5 µg/mL in KRB. Solubilization of hypericin appeared tobe essentially independent of the number of interacting groups(hydrophobic side chains) in the cyclodextrin molecule. Insharp contrast, however, in the case of â-cyclodextrin sulfobu-tyl ether 7-sodium salt, the solubility decreased at higherconcentration, probably because of the evenly increased

sodium concentration. On the basis of phase solubilityanalysis, the interaction between 2-hydroxypropyl â-cyclo-dextrin and hypericin in water was investigated. Figure 4shows the solubility of hypericin as a function of the 2-hy-droxypropyl â-cyclodextrin concentration in water. Uponaddition of 30% cyclodextrin, the saturation solubility ofhypericin was clearly enhanced from 40 to 480 µg/mL, whichis approximately equal to the solubility of hypericin inmethanol. The initial rising segment of the solubility curveis followed by a plateau region. According to Higuchi et al.22this system is best fit by a solubility curve of type AN, whichexhibits negative curvature as indicated by the index. Furtherexamination of the interactions between substances andcomplexing agents can be performed by measuring UV spectraand calculating their differential spectra.25 The differentialUV spectra of 0.079 mM hypericin in the presence of 0.79-395 mM 2-hydroxypropyl â-cyclodextrin are presented inFigure 5. Under the influence of increasing amounts ofcyclodextrin, a shift of the aqueous hypericin spectrum towardthe methanolic spectrum, with an increase of the typical peakmaxima between 500 and 600 nm, can be recognized. Thesedata, based on the phase solubility technique and the spectraldisplacement technique26,27 (Scott plot not shown), are indica-tive of the formation of inclusion complexes between hypericinand cyclodextrins.

Figure 2sSchematic of the fluorescence binding assay in 96-well plates. Cellswere incubated with hypericin buffer solution and washed to remove the unbounddrug and to obtain the cell-associated hypericin. Bound hypericin was solubilizedin methanol and its fluorescence measured. In addition, stock hypericin buffersolutions were diluted and the total hypericin content determined.

Figure 3sEffects of different cyclodextrins (w/v) on dissolution of hypericin inM-PBS at 30 °C (mean ± SD; n ) 3) : b, R-cyclodextrin; 9, 2-hydroxypropylâ-cyclodextrin; +, â-1,8-dimethylcyclodextrin; 0, γ-cyclodextrin; [, â−1,0-acetyl-cyclodextrin; 1, â-cyclodextrinsulfobutyl ether 7-sodium salt.

Figure 4sPhase solubility diagram of hypericin under the influence of 2-hydrox-ypropyl â-cyclodextrin in water, pH 7.1, at 30 °C.

1122 / Journal of Pharmaceutical SciencesVol. 86, No. 10, October 1997

Incorporation of Hypericin in LiposomessPhospholipon 80liposomes, loaded with hypericin at different local concentra-tions (hypericin to phospholipon molar ratio), dispersed inwater or KRB, increased the aqueous solubility of hypericinmarkedly. Depending on the experimental conditions, lipo-some dispersions (average liposome diameter of 150 nm asmeasured by PCS) with a hypericin concentration of ap-proximately 400 and 900 µg/mL could be reproducibly achieved(for comparison, hypericin solubility in KRB is less than 5 µg/mL, and in water it is less than 50 µg/mL). Comparativeoptical measurements of hypericin embedded in liposomes,which were dispersed in water or KRB, and of hypericin inmethanol were performed. The two typical peak maxima ofthe methanolic UV spectrum between 500 and 600 nm arecentered at 545 and 588 nm. In contrast, those of hypericinin liposomes are centered at 552 and 595 nm, which meansthat a bathochromic shift of 7 nm, independent of the aqueoussolvent of the liposomes, could be identified when the chro-mophor was embedded in the phospholipon vesicles (Figure6). It should be pointed out that the spectra are qualitativelymore similar to the methanolic than to the aqueous hypericinspectra.Freeze-fracture electron micrographs of the liposome dis-

persions in water or KRB showed an average vesicle size of

70-100 nm (Figure 7), with occasional smaller particles andlarger ones up to 350 nm also present. The shell consistsmostly of one bilayer, though in larger vesicles multilamellarbilayers are found.Transport Studies of Hypericin across Caco-2 Monolayerss

Results of the transport of hypericin from hypericin-2-hydroxypropyl â-cyclodextrin solutions from the apical-to-basolateral side of the cells are presented in Figure 8, wherethe cumulative transport is plotted against the experimentaltime. The clear temperature-dependence of the transportrates of hypericin was observed. At 37 °C, about 3% ofhypericin is transported after 5 h, whereas at 4 °C only 0.1%of hypericin is transported. These results may be partlyexplained by the fact that the TEER was increased at 4 °Ccompared to 37 °C (see Figure 8 inset). Separately performedcontrol experiments with hypericin and 2-hydroxypropylâ-cyclodextrin-free buffer, however, demonstrated that cyclo-dextrin alone had no effect on this parameter (see Figure 8inset).

Figure 5sUV/vis differential spectra of solutions of hypericin (0.079 mM) and2-hydroxypropyl â-cyclodextrin (0.79−395 mM) in water.

Figure 6sUV/vis spectra of hypericin in methanol and in liposomes: s, methanol;− ‚ −, water; − −, liposomes hydrated in KRB; ‚‚‚, liposomes hydrated in water.

Figure 7sExample of electron micrographs of liposomes in KRB after freeze−fracture preparation (M 12 000, bar ) 300 nm).

Figure 8sCumulative transport of hypericin−cyclodextrin solutions in M-PBS at37 °C and l 4 °C. Inset: TEER-measurements at (+) 37 and (b) 4 °C. (1)Control experiment at 37 °C without cyclodextrin and hypericin (mean ± SD; n )3).

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The apparent permeability coefficient (Papp) was determinedaccording to the following equation:24

where V is the volume of the receiver compartment (mL), Ais the membrane surface area (cm2), c0 is the initial donorconcentration of hypericin (µg/mL), and dc/dt the permeabilityrate (µg/s) which is the slope of a plot of the cumulativereceiver concentration with time. From the data from thelinear portion of the transport curve (2-5 h) at 37 °C, Pappwas calculated to have a value of 1.274 ( 0.231 × 10-6 and of0.048 ( 0.031 × 10-6 cm/s (n ) 3, (SD) at 4 °C. Thecalculated apparent permeability coefficients show that thetransport of hypericin across Caco-2 cell monolayers isstrongly temperature dependent, at 4 °C the Papp is signifi-cantly reduced compared to 37 °C.Comparing these transport rates with the results obtained

from a study across the blank polycarbonate filters withoutcells, in which equilibrium between the donor and acceptorcompartment is reached after 5 h, we recognize clearly thebarrier function of the Caco-2 cells. Additionally, this studyproves that there was no binding of hypericin to the blankpolycarbonate membrane (Table 1). From the amount ofhypericin determined in the donor and acceptor compartmentsat the end of the transport experiment across the cellmonolayer at 37 °C, only about 60-70% of the initial dosecould be recovered. As there was no binding of hypericin-2-hydroxypropyl â-cyclodextrin solutions to the polycarbonatemembrane, hypericin was obviously bound to or internalizedby the cells. In agreement with these considerations, at 37°C more than 20% and even at 4 °C still more than 5% ofhypericin was found to be bound or taken up by the Caco-2cells.From liposomes containing 200 or even 400 µg/mL hyperi-

cin, no epithelial transport from apical-to-basolateral at 37as well as 4 °C could be measured. The recovery of hypericinafter the 5 h experiment in the donor compartment wasapproximately 100%, corresponding to binding rates to theCaco-2 cells of less than 0.4% (at 37 °C) and 0.2% (at 4 °C).Under these experimental conditions, hypericin present inliposomes seems not to interact with, nor to be transportedacross, the Caco-2 monolayer.Hypericin Binding to Caco-2 Cell MonolayerssBinding of

hypericin from hypericin-cyclodextrin solutions in M-PBS toCaco-2 cells grown on well plates was found to be saturable.Equilibrium of the binding was reached after 1 h and did notchange when incubation was prolonged up to 2 h (data notshown). Therefore an incubation period of 1 h was chosenfor all quantitative binding studies. Total binding wascalculated as the mean of 12 measurements at each hypericinconcentration. The results of the studies show a stronglytemperature-dependent binding of hypericin. At an experi-mental temperature of 4 °C, only about half of the amount ofbinding measured at 37 °C was observed. The results werefound to be independent of the cell passage number used: no

significant difference in binding in cells for early passage ofCaco-2 cells (passage 46) or for later passage of cells (passage107) was obtained (Figure 9).Total binding was calculated as the mean of 12 measure-

ments at each hypericin concentration. In order to find outwhether there are any specific receptors for hypericin on theapical membrane of the Caco-2 cells, the data were fitted tothe following model equation by nonlinear regression (Slide-Write 2.0, Advanced Graphics Software, Carlsbad, CA):

where C is the concentration of free hypericin (nmol/well) andB is the amount of bound hypericin (nmol/well). Kd (M), theapparent affinity constant, indicates the hypericin concentra-tion at which 50% of the binding places are occupied. Fromthe data obtained from passage 46, as a representativeexperiment, Bmax was calculated to have a value of 5.86 ( 0.76nmol/well (37 °C) and 2.65 ( 0.11 nmol/well (4 °C). Kd wasdetermined as 1.81 ( 0.41 × 10-4 M (37 °C) and 1.36 ( 0.11

Table 1sComparison of Cumulativ e 5 h Transport ofHypericin −2-Hydroxypropyl â-Cyclodextrin Solutions across the Caco-2Monolayer with Experiments across the Polycarbonate Membrane withoutCells (mean ± SD; n ) 3)

CumulativeTransport (%)

MembraneBinding (%)

Caco-2 monolayer, 37 °C 3.22 ± 0.13 23.81 ± 2.67Caco-2 monolayer, 4 °C 0.12 ± 0.08 5.39 ± 1.96Polycarbonate membrane 45.1 ± 5.2 0

Papp ) dcdt

VAc0

(cm/sec)

Figure 9sBinding of hypericin from hypericin−cyclodextrin solutions to Caco-2cells at 37 °C (closed symbols) and at 4 °C (open symbols) after an incubationtime of 1 h. Data from passage numbers 46 (full line with squares) and 107(dotted line with circles) are shown (mean ± SD; n ) 12).

Figure 10sBinding to Caco-2 cells of hypericin from hypericin−cyclodextrinsolutions at 37 °C (9), and at 4 °C (0), in comparison to the binding results ofhypericin from phospholipon 80 liposomes at 37 °C (b), and at 4 °C (O), afteran incubation time of 1 h, also shown in the insert graph (mean ± SD; n ) 6).

B )BmaxCKd + C

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× 10-4 M (n ) 12, ( SD). The calculated affinity constantsshow that there is only a low affinity of hypericin to theassumed receptor, suggesting that there is no specific interac-tion between the drug and Caco-2 cells.Again, in contrast to the results of our studies of hypericin-

cyclodextrin solutions, the binding of hypericin from hyperi-cin-containing liposomes was very low (Figure 10). At 37 °Conly about 5% of the binding capacity of that of cyclodextrinsolutions is achieved, at 4 °C it is less than 3%.Imaging of Hypericin Uptake by Confocal Laser Scanning

MicroscopysIn Caco-2 monolayers grown on glass cover slips,the incubation with hypericin-2-hydroxypropyl â-cyclodextrinsolutions resulted primarily in the appearance of hypericinin the cell membrane. CLSM images revealed the presenceof hypericin mainly in the cell surface membrane of Caco-2cells after a 1 h incubation time, but hypericin seems toassociate also with the cell nucleus membrane (Figure 11).The observed effects in the CLSM measurements wereuniform over the whole monolayer. Images from hypericin-loaded liposomes comparable to those taken of the hypericin-cyclodextrin solutions could be obtained only at higher laserintensities, indicative of a reduced transport of hypericin intothe cells (data not shown). These results are in good agree-ment with the performed cell binding studies. Control cells,which were treated with cyclodextrin solutions or liposomedispersions without hypericin, showed no fluorescence.

DiscussionSince the low aqueous solubility of drugs represents a

potential barrier and limiting factor in the bioavailability andthe process of drug uptake as well as in the evaluation of thepharmacological properties of drug compounds in biologicaltest systems, ways must be found to overcome these problems.In the present work, instead of using cosolvents and deter-gents, we studied the influence of cyclodextrins and liposomeson the solubility behavior of hypericin in the hope of gaininga better insight into the interaction of hypericin with cellsusing the Caco-2 cell culture model. Our experiments showedthat the solubility of hypericin can be enhanced by the use ofcyclodextrins. To elucidate the mechanism of the solubility-enhancing effect of cyclodextrins, the physicochemical proper-ties of hypericin in this system were studied. Both, the phasesolubility diagram (Figure 4) and the UV/vis spectra of thesolutions are indicative of the formation of inclusion com-plexes.With the thereby increased solubility of hypericin, it was

possible to study the epithelial transport of hypericin-cyclodextrin solutions in a buffer medium adopted to therequirements of cell cultures. Under controlled conditions in

the Caco-2 cell culture model, both transport rates and cellularbinding, which were strongly temperature dependent, at 4 °Cwere significantly reduced compared to 37 °C. Together withthe relatively weak affinity constant (about 10-4 M), thesedata suggest that hypericin does not interact with Caco-2 cellsvia a specific receptor, but rather nonspecifically interacts withthe membrane lipids due to its pronounced lipophilicity.Miskovsky et al.28 performed confocal laser microspectrof-

luorometric measurements and observed, after a short-termincubation (20 min) of 1 µM hypericin-containing culturemedium (0.1% DMSO), that in human T47Dmammary tumorcells hypericin is located in the cell membrane and cytoplasma,whereas after long-term incubation (3.5 h) it is located insideof the cell nucleus. This is not in agreement with previousresults from Thomas et al.,29 who showed by fluorescencephotomicrographs at the EMT6 mouse mammary carcinomacell line an association of hypericin in the plasma and cytosolicmembranes. With this in mind and to further establishqualitatively the nature of hypericin interaction with Caco-2monolayers, we report in the present paper the resultsobtained by CLSM. This technique allows horizontal andvertical optical sectioning through living, nonfixed specimenswith excellent resolution. The binding of the fluorescentcompound hypericin is imaged after incubating the Caco-2cells with the hypericin-2-hydroxypropl â-cyclodextrin con-taining buffer solution. Hypericin is mainly accumulated inthe cell membrane and the cell nucleus membrane, which isin principle in agreement with the results of Miskovsky.28Naturally, the visualization of the fluorescent compoundhypericin is influenced by its surrounding solvent. Forinstance, hypericin is strongly fluorescent in ethanol butnonfluorescent in water.30 Therefore, it cannot be entirelyexcluded that the fluorescence signal intensity of hypericinassociated with different cellular structures may be changeddue to local quenching by other components or due toconformational changes of the drug molecule resulting in areduced quantum yield. Nevertheless, both the bindingstudies and the results obtained by CLSM at least suggestthat hypericin is not taken up or bound by specific receptors.Hypericin seems to be transported through and accumulatedin lipophilic cell structures31,32 because of its hydrophobiccharacter. The strongly temperature-dependent binding ofhypericin can be explained by the different fluidity of the cellmembrane. The reduced fluidity at low temperature impedesthe binding and uptake of hypericin.A second approach to increase the water solubility of

hypericin was the use of liposomes. A strongly enhancedsolubility of hypericin in aqueous buffers was achieved byincorporation in phospholipon 80 liposomes. Transport andbinding studies were undertaken to determine whether or not

Figure 11sHorizontal optical sections of Caco-2 cells after incubation with hypericin−cyclodextrin solutions for 1 h at 37 °C performed with CLSM; parts a−c are aseries of confocal images taken at successively deeper focal levels: (a) near apical membrane, (b) at height of the nucleus, (c) near basolateral membrane.

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the enhanced solubility of hypericin incorporated in liposomeswould provide further information regarding the interactionsbetween hypericin and cells. Our findings showed that, incontrast to the cyclodextrin solutions, the liposome approachdid not yeld any measurable transport rates across Caco-2cell monolayers, although a small amount of hypericin uptakeby the cells could be measured in binding studies and bevisualized by CLSM. Under the experimental conditionsliposomes enhanced the solubility of hypericin, but as hyperi-cin was not released from the liposomes, this approach wasnot suitable to enhance the cellular uptake or transport ofthis compound.In summary, cyclodextrins were found to be useful to

enhance the solubility of hypericin in biological buffers, whichsubsequently allowed us to perform drug transport studiesacross tight epithelial cell monolayers under controlled condi-tions in vitro. When present in the donor medium as acyclodextrin complex, transport of hypericin across Caco-2monolayers was measurable. The compound rapidly binds tocell surface membranes and is also present in the cell nucleusmembrane, suggesting that hypericin is transported acrossthe intestinal epithelium by transcellular diffusion via lipo-philic domains.

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AcknowledgmentsWe wish to thank the Rhone-Poulenc Rorer/Nattermann Phospho-

lipid Corp. for the electron micrographs and the Institut fur NeueMaterialien (INM, Saarbrucken, Germany) for allowing us to use theirPCS equipment. The Steigerwald Arzneimittelwerk (Darmstadt,Germany) and the Fonds der Chemischen Industrie are thanked forsupport.

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