study on the hydatid cyst membrane

8/2/2019 Study on the Hydatid Cyst Membrane

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224 T. Truong Cong et al. / International Journal of Pharmaceutics 353 (2008) 223232

Senyuz et al., 2001; Saimot, 2001 ). In the case of ABZ, inorder to prepare solutions, different approaches havebeen inves-tigated during the last decade. Since ABZ is basic in nature,its solubility could be notably increased by ionisation in anacid medium, although this rise is not enough for the prepa-ration of high ABZ concentration ( Garcia et al., 2003 ). Itis also possible to go up its water solubility by the use of surfactants, for instance, polysorbate, bile salts or cosolvents(Transcutol ) (Torrado et al., 1996; Redondo et al., 1998 ).Unfortunately, the absorption enhancer effects described withthe previous molecules are generally correlated to an irritationof the digestive mucosa. A similar approach was also used withhydroxypropyl- -cyclodextrin ( Garcia et al., 2003 ). Alterna-tively, the ABZ solubility could be improved by formulation of solid dispersion with polyvinylpyrrolidone ( Daniel-Mwambeteet al., 2004 ) or poly-lactic acid nanoparticles ( Rodrigues et al.,1995).

In reality, after oral administration,ABZis converted by rst-passhepatic metabolisminto its lessactivemetabolite, ABZ-SO,

that achieves variable concentrations in blood, bile, liver tissue,cystuid andwall,or crosses theblood-brainbarrier ( Smego andSebanego, 2005; Senyuz et al., 2001; Saimot, 2001; Lanusse etal., 1998).

The basic structure of the hydatid cyst was described as fol-lows. The cyst is lled with hydatid cyst uid which containsboth protoscoleces and host-derived proteins, and surroundedby a two-layered hydatid cyst wall. The innermost layer of this structure is the live parasite tissue, named germinal layer,which is a few cell bodies wide from which small packets of protoscoleces bud into the cyst lumen. This layer lays downaround it an extensive (12mm thick), acellular, carbohydrate-

rich, mechanically resistant structure named laminated layer(Klotz et al., 2000 ). That supportive and defense structure pro-tects the cyst from direct attack by host immune cells ( Diaz etal., 2000).

The success of the chemotherapeutic treatment of hydatiddisease is based upon the capacity of the drug to operate onthe germinal layer and the protoscolices of the hydatid cystinterior at adequate concentrations for sufcient periods. Toreach this goal, two of the most important parameters are thedrug diffusion trough the cyst membrane and the drug con-centration in the cyst environment. From this point of view,in the present work, we have confronted the cyst membranewith classical permeation method to clarify the drug physic-ochemical parameters required to allow good drug diffusiontowards the hydatid cyst interior. Because of the benzimida-zole low aqueous solubility, dyes have been used as modelmolecules. Then, we decided to formulate benzimidazole-loaded nanoparticles in order to increase the apparent solubilityof the drug and to avoid its hepatic metabolism. Furthermore,it is well known that after parenteral administration, a mas-sive accumulation of nanoparticles could be observed in theliver and lungs ( Soppimath et al., 2001; Moghimi and Szebeni,2003). These two tissues being the most frequent residentniches of the hydatid cyst in humans, benzimidazole-loadednanoparticles seem to be promising forms for the treatment of

hydatosis.

2. Materials and methods

2.1. Materials

Congo red, tartrazine and erythrosine were purchasedfrom Kuhlmann R.A.L. Carminic acid and gentian violetwere provided by Cooper. ABZ (ABZ) and poly( d ,l -lactide)(PLA, molecular weight 90,000120,000) were purchased fromSigmaAldrich. Methanol and acetonitrile were provided byProlabo. Acetic acid, sodium periodate (99%), acetone andpoly(vinyl alcohol) (PVA, molecular weight 13,00023,000,8789% hydrolysed) were obtained from Carlo Erba, Acrosorganics, Merck and Fluka, respectively.

All the solvents used without further purication were of analytical grade.

2.2. Albendazole sulphoxide synthesis

ABZ-SOwas preparedasreported ( DeLaurentiset al., 1996 ).

Three grams of NaIO 4 was added in 250 ml of a methanolwater (1:1) mixture in a two-necked ask. This mixture hasbeen stirred at room temperature until a complete dissolu-tion of the NaIO 4 . Then a solution made up of 3g of ABZand 60ml of acetic acid has been added from a droppingfunnel. After 24 h of stirring, the mixture was checked bythin layer chromatography and ltrate under vacuum. Then,a 50% solution of NaOH was slowly added to pH 7. Thesolution turned cloudy due to the formation of a precipitate.Three hundred millilitres of water has been incorporated toavoid the precipitation of AcONa and stirred weakly dur-ing 1 h. The suspension was ltered under vacuum and the

ltrate was evaporated in order to eliminate methanol. Anice bath was used to digest the ltrate and to obtain a sec-ond precipitate which was ltrate under vacuum. Similar bythin layer chromatography, the two precipitates were washedseveral times with water. The raw material was dried undervacuum until a constant weight was reached. Impurities (ABZsulphone and salt) were removed by silica gel column chro-matography eluting with a mixture dichloromethanemethanol(95:5).

The product was analyzed by the determination of themelt point, infra-red spectroscopy, high-performance liquidchromatography (HPLC) and nuclear magnetic resonance spec-troscopy ( 1H-NMR and 13C-NMR).

2.3. Nanoparticle preparation

The particles were obtained by an emulsion solvent evapo-ration method. d ,l -Polylactide (50 mg) and the drug (ABZ orABZ-SO, 5 mg) was dissolved in 5 ml of acetone. The solu-tion was added dropwise under magnetic stirring (900 rpm) for10 min with an aqueous phase (10ml) containing 100mg of poly(vinyl alcohol). The preparation was homogenized by a13000rpm stirring with an Ultra-turrax . After ltration (Iso-pore membrane, 0.5 m), acetone and water were evaporatedunder vacuum and the volume of the preparation was adjusted

to 5ml.
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T. Truong Cong et al. / International Journal of Pharmaceutics 353 (2008) 223232 225

ABZ aqueous suspension and unloaded nanospheres wereprepared by the same procedure, omitting polymer and ABZ,respectively.

RhodamineB-labellednanoparticle suspension waspreparedfollowing a similar procedureusingof 4.5mgRhodamineB basein place of ABZ in the acetone. 1ml of the obtained suspensionwas washed by water, ltered through an Ultrafree -CL lterunit (Filter Ultrafree -CL, pore size 0.1 m, Millipore, Bed-ford, USA) and subjected for 30 min centrifugation (1500 g) toremove free uorescent material.

2.4. Determination of the dye molecular descriptors

Thefollowing parameters havebeen used to characterized themolecular properties of the studied dyes: the molecular weight, M w, the octanolwater partition coefcient, log P , the numberof hydrogen bond acceptors, N acceptor , and donors, N donor , thenumber of rotatable bonds, N rotation , and the molecular polarsurface area, PSA.

All these parameters have been calculated using theMolinspiration property calculator software (MolinspirationCheminformatics, Bratislava, Slovak Republic). Log P is cal-culated by a methodology taking into account sum of fragment-based contributions and correction factors. The num-ber of rotatable bonds is a simple topological parameter whichdescribes the molecular exibility. Rotatable bond is denedas any single non-ring bond, bounded to non-terminal heavy(i.e., non-hydrogen) atom. Amine CN bonds are not con-sidered because of their high rotational energy barrier (ca.2022 kcal/mol). Themolecular polar surface area calculation isbased on a methodology published by Ertl et al. (2000) as a sum

of fragment contributions. O- and N-centered polar fragmentsare also considered.

2.5. Hydatid cyst removal

The membranesof hydatid cystswere obtained from infectedsheep. At autopsy, thehepatic cysts were macroscopically local-ized. A punction of hydatid uid was made and the cyst wasopen. The membranes of fertile cysts were taken off and keptat 80 C in a 10% DMSO (cryoprotectant) aqueous solutionuntil use.

2.6. Ex vivo permeation studies through hydatid cyst membranes

Before use, excised hydatid cysts were incubated three timesat room temperature in saline solution (0.9% NaCl) to removeimpurities. Hydatid cyst membrane samples (around 2 cm 2)were cut carefully with a scalpel and mounted onto static Franzglassdiffusioncellscontaining 9 ml of salinesolution(inner faceof thecyst membrane in contact with thereceptor compartment).Because of the low solubility of the drug, the saline solution hasbeen supplemented with 3% of ovalbumin during the experi-ment with ABZ. The diffusion area was 0.79cm 2 . The receptorcompartment was stirred with a magnetic bar at 37 0.2 C

throughout the study. Mounted membranes were kept onto dif-

fusion cells for 1 h before treatment. A 0.5ml sample of 1%dye aqueous solution, free-ABZ aqueous suspension or ABZ-loadednanoparticle suspension was then depositedonto hydatidcyst membrane. After 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6 and 24 h,0.5 ml sample of receptor uids were removed and replaced bythe same volume of fresh solution. The concentration of dyes orABZ in thereceptoruidswasassayedby theanalytical methodsbelow described.

2.7. Quantication of dye amounts

The determination of the dye concentrations in thereceptor compartment was obtained by simple UVvisiblespectrophotometric measurements using a systronics model106A digital spectrophotometer. The concentration linearityranges were [1.239 g/ml], [2.4160 g/ml], [1.220 g/ml],[1.239 g/ml], and [0.69.8 g/ml] for tatrazine, carminicacid, gentianviolet, congored anderythrosine, respectively, withsquared correlation coefcient, r 2 , above 0.999 for all tested

molecules. The receptor solution samples could be diluted toreach the previous ranges.

2.8. Permeation result analysis

The permeation results have been treated with the Ficks rstlaw of diffusion:

J =dQdt

1A

=K s D

h C (1)

where J is the drug ux, d Q /dt is the amount of dye diffusedper unit of time, A is the treated membrane surface area, K s isthe partition coefcient, D is the diffusion coefcient, h is thediffusional path length and C is the concentration gradient of dye.

Then the permeability coefcient, P , was determined by thefollowing equation:

P =J

C 0(2)

where C 0 is the dye concentration in the donor compartment.

2.9. Intra- and inter-variability of the weight of different cyst membrane samples

The variability of sample weights was used to approach thevariability of the diffusional path length, h. After removal with a6 mm diameter punch, ve samples of three different cysts wereweighted. The intra- and inter-cyst variation coefcients werethen determined.

2.10. Quantication of drug loading

Because of their different water solubility, two procedureshave been used to determine the drug loading: ABZ was quan-tied inside particles and ABZ-SO outside.

The obtained ABZ nanoparticle suspension was ltered

through a 5 m Millex

-SV membrane (Milipore, Bedford,


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USA) to eliminateprecipitates formedduring preparation due toits poor aqueous solubility. Entrapment efciency is calculatedas follow:

2.10.1. ABZ loadingABZ entrapmentefciency (%) =

(ABZ total ABZ free )ABZ initial

100

in which, ABZ total is the concentration of drug in the nalnanoparticle suspension (loaded and soluble unloaded ABZ).ABZ free is the amount of soluble unloaded ABZ in the sus-pension. ABZ initial is the initial amount (5 mg) of ABZ used toprepare the ABZ particles. Owing to the very low water solubil-ity, the soluble unloaded ABZ is negligible. Then, the loadingefciency can be approximately calculated as follow:

ABZ entrapmentefciency (%) =ABZ totalABZ initial

100

2.10.2. ABZ-SO loadingAfterthepreparation, theABZ-SO loaded nanoparticleswere

puried by ultracentrifugation.The preparations weredivided inaliquots of 1.5 ml and centrifuged at 25000rpm during 30 minat 4 C. After re-suspension, the particles were re-centrifuged.Finally, three centrifugations were made on each aliquot. Theamount of drug entrapped into nanoparticles was calculated bydifference between the initial amount of drug and the amount of free ABZ-SO by the following relation:

ABZSOentrapmentefciency (%)

=(ABZSO initial ABZSO supernatant )

ABZSO initial 100

in which, ABZSO initial is the initial amount (5 mg) of ABZ-SO

for formulation and ABZSO supernatant is the sum of ABZ-SOamounts quantied in the three supernatants.

2.11. Particle shape determination

The morphological examination of nanoparticles was per-formedusing a transmissionelectron microscope JEM-1200 EXfrom JEOL (Japan). The negative staining was obtained witha 4% sodium phosphotungstate solution (pH 7.2) on carbon-parlodion grids.

2.12. Quantication of drug amounts

The drug amount (ABZ or ABZ-SO) was assayed by high-performance liquid chromatography (HPLC) with a HewlettPackard 1050 series HPLC system (Germany), an isocraticpump and an automatic sampler. For the two investigated drugs,the column was an Inerstil ODS-3 (5 m, 4.6mm 250mm,OAI6069) from GL Sciences Inc. (Japan). The other HPLCparameters depend on the drug of interest. For ABZ, the mobilephase was a methanolwater (75:25) mixture. Sample volumeinjected was 50 l. The ow rate was settled at 0.8 ml/minand the detection was performed at 291 nm. The retention timewas 10.6 min and for concentrations between 0 and 20 g/ml,peak area increases were linear with a squared correlation

factor, r 2, of 0.9999. Concerning the ABZ-SO, the mobile

phase was a mixture of acetonitrilewater85% phosphoric acid(200 ml:800ml:188 l).Samplevolume injected was 30 l. Theow rate was 1.0 ml/min and the detection was performed at290 nm. The retention time was 7.5 min and for concentrationsbetween 0 and 50 g/ml, peak area increases were linear with asquared correlation factor, r 2 , higher than 0.9999.

2.13. Particle size analysis

The mean particle size and the size distribution of nanopar-ticles were estimated by light scattering (dispersant refractiveindex= 1.33; detector angle = 90 ; wavelength = 670nm) witha Zetamaster II (Malvern Instruments, Orsay, France). Toestimate the stability at 4 C, the measurements were madeimmediately and during 60 days following preparation. Beforemeasurements, the nanoparticles suspensions were diluted indeionised water.

2.14. Particle zeta potential

Themeasurements of zeta potentialwere performedusingthetechnique of electrophoretic laser Doppler anemometry with aZetamaster II (Malvern Instruments, Orsay, France).Thedeter-minations were carried on in three different media: deionisedwater and HEPES buffer (pH 7.4, NaCl 150 mM) supplementedor not with 10% (v/v) of fetal bovine serum.

2.15. Distribution of uorescent probe across cyst germinalmembrane

Hydatid cyst germinal membrane was mounted onto Franz

static diffusion cell and 0.5 ml of rhodamine B base-labellednanoparticle suspension or ABZ aqueous suspension was addedto the donor compartment. The receptor compartment com-prised of 9 ml of ovalbumine saline solution was maintainedat 37 C and stirred with a magnetic bar. After 24 h the mem-brane was removed from the diffusion cell, residual formulationwas removed by washing with distilled water. The membranewas then examined by using a confocal laser scanning micro-scope (Leica-SP2, Mannheim, Germany; gain 1.000, excitationand emission wavelengths: 514 and 522 nm, pinhole: 121 m).All images were acquired with identical settings.

2.16. Permeation studies across whole cyst membrane

The entire hydatid cysts were incubated in either rhodamineB-loaded nanoparticle suspension or ABZ-loaded nanoparticlesuspension.After3 h, thecysts were takenoutand puncturedby aneedle to aspirate the intra-cystic media. These media were thensubjected for microscopic study and ABZ assay, respectively.

2.17. Statistical analysis

Statistical calculations wereexecutedusingthe Kaleidagraphtools (Synergy software). Comparisons were analyzed by usingone-way ANOVA with post hoc testing using Bonferronis cor-

rection. Except if noted, the chosen level of signicance was


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Table 1Molecular descriptors of the studied dyes

Dye M w (g/mol) Log P N acceptor N donor N violation PSA (A2) N rotation

Gentian violet 372.5 2.64 3 0 0 9.5 4Tartrazine 465.5 0.47 13 1 1 217.3 6Carminic acid 492.4 0.99 13 9 2 242.5 3Congo red 650.7 5.09 12 4 3 215.9 7Erythrosine 833.9 4.33 5 0 1 93.4 2

The underlined values are out of the rule of ve ranges (see text).

P < 0.05 or P < 0.01. As the group were small ( n = 3), normaldistribution was assumed and not further tested.

3. Results and discussion

3.1. Physicochemical properties of the dyes

The properties of the dyes are described in the Table 1 . It is

important to note that the obtained molecular descriptor values,especially the partition coefcient, are comparable with valuesexpressed in chemical databases ( http://chemdb.niaid.nih.gov ,http://hmdb.med.ualberta.ca and http://cdb.ics.uci.edu ). Foreach studied parameter, some dye describing values are higherthan the generally admitting limits between important and poorpermeation abilities. Thus, a set of rules imposing limitationson log P , molecular weight, and number of hydrogen bonddonors and acceptors introduced by Lipinski has become partic-ularly popular in the eld of membrane permeation prediction(Lipinski et al., 1997 ). These limitations are known as therule of ve because each border value deals with 5. Therule states that poor absorption and permeation are more likelywhen: there are more than 5 H-bond donors, the molecularweight is over 500, the log P is over 5 and there are morethan 10 H-bond acceptors. However, the compound classes thatare substrates for biological transporters are exceptions to therule.

Molecular polar surface area (PSA), i.e., surface belongingto polar atoms, is a descriptor that was shown to correlate wellwith passive molecular transport through membranes as Caco-2layer or blood-brain barrier and to allow prediction of transportproperties of drug ( Veber et al., 2002 ). With the chosen dyes,a large range of PSA was investigated, from 9.5 to 242.5 A2 .Navia and Chaturvedi (1996) suggested the importance of the

molecular exibility, quantied by the N rotation parameter, forexample in the case of the digestive absorption.

3.2. Permeation coefcient

The permeation coefcient values are summarized in theTable 2 . In a previous work, Garcia-Llamazares et al. stud-ied the membrane permeability of secondary hydatid cyst toABZ-SO by incubation of whole cysts in a drug-containingmedium ( Garcia-Llamazares et al., 1998 ). In spite of the dif-ferent methodology, the authors obtained ABZ-SO permeationcoefcients between0.56 10 5 and0.81 10 5 cm/sdepend-

ing on the treated or untreated state of the cysts. In the present

Table 2Values of the dye permeation coefcient

Dye Permeation coefcient (cm/s) 10 5

Gentian violet 1.14 0.50Tartrazine 0.62 0.46 a

Carminic acid 0.27 0.25 b ,c

Congo red 1.31 0.47 c

Erythrosine 1.52 0.08 a ,b

The curve tting was conducted for each individual diffusion experiment, anddata represent the mean standard deviation of three or four determinations.a p < 0.01.b p < 0.05.c p < 0.05.

study, the permeation coefcients of the dyes are in a similarrange, from 0.27 10 5 to 1.52 10 5 cm/s.

Except for erythrosine, the permeation standard deviationsare relatively important (variation coefcients from 5 to 93%depending on the dye). Several parameters dealing with theexperimental method or with the membrane state could explainsuch important variation coefcients. First, the membrane of thehydatid cyst is irregular in thickness. The weight of cyst mem-brane samples having similar surface area is summarized in theTable 3 . The inter-cyst variation coefcient is around 30% con-rming morphological differences among cysts. In addition, asimilar intra-cyst variation coefcient should be noted, suggest-ing membrane thickness variations even in a same cyst. Withthe Franz cells we used in the present experiments, an approachto overcome this problem was to normalize the results by theweight of each membrane sample as classically made with skinfor example. Actually, thecyst membrane was thin andvery eas-ily damaged, it was not possible to manipulate the same samplefor the weighting and the permeation experiments. It is evident

from the Eqs. (1) and (2) that a change of sample weight, cor-responding to the change of the membrane thickness, modiesthe diffusional path length, h, as well as the permeation coef-cient. However, GarciaLlamazes and co-authors, who worked

Table 3Weight intra- and inter-variations of hydatid cyst membrane samples

Cyst 1 5.6 1.2mg (21.6%)Cyst 2 9.8 2.6mg (26.5%)Cyst 3 10.0 3.3mg (33.0%)Inter-cyst variation coefcient 30%

Data represent the mean standard deviation of three (inter-cyst variation) or

ve (intra-cyst variation) determinations.
http://chemdb.niaid.nih.gov/http://hmdb.med.ualberta.ca/http://cdb.ics.uci.edu/http://cdb.ics.uci.edu/http://hmdb.med.ualberta.ca/http://chemdb.niaid.nih.gov/


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with whole cysts and avoided the intra-cyst variation, obtainedvariation coefcient from 29 to 46% ( Garcia-Llamazares et al.,1998). Besides the inter-cysts membrane thickness uctuations,these authors observed permeation coefcient differences dueto the past of the cysts. Indeed, they noted a loss of cyst per-meability to ABZ-SO following its treatment with Netobimin.Furthermore, Xiao et al.(1992) observeda signicantly decreaseof the glucose content in the hydatid uid of cysts from micetreated with mebendazole after intravenous glucose administra-tion, suggesting that mebendazole had an inhibitory effect onthe transport of exogenous glucose to the cyst wall. Similarly,the stage of development, the maturation and the localization of the cysts would also alter their permeation properties ( Holcmanand Heath, 1997 ).

3.3. Permeation coefcient versus dye descriptors

With our results, no linear relationship between permeationcoefcient and the N rotation , polar surface area, and N violationsparameters could be observed (gures not shown, correlationcoefcients, r , lower than 0.68), suggesting that these molecu-lar descriptors could not be used as simple diffusion predictivefactor. Contrary to the previous ones, the permeation coefcientcorrelates closely with the partition coefcient log P (r = 0.951,P < 0.05) as described in Fig. 1. From the present results, it ispossible to propose a simple model in order to determine the dif-fusionof molecule through thehydatidcyst membrane.Thus,thepermeation coefcient, P , could be estimated from the partitioncoefcient, log P , by using the following equation:

P = 5.98 10 6 + 1.77 10 6 log P (3)

In their study, Garcia-Llamazares et al. (1998) measured a per-meationcoefcient equalto 8.06 10 6 2.30 10 6 cm/sforthe ABZ-SO diffusion through untreated hydatid cyst mem-brane (Garcia-Llamazares et al., 1998 ). With the Eq. (3), and

Fig.1. Permeation coefcients vs.partitioncoefcient, log P , ofthe studied dyes

with linearization established by the Kaleidagraph tools (Synergy software).

using the partition coefcient calculated with the molinspirationtools (log P = 1.60), the calculated permeation coefcient of theABZ-SO is equal to 8.81 10 6 cm/s. These two permeationcoefcients are remarkably similar.

Our results conrm the preponderant role played by thelipophilicity of the anthelmintic drugs and the barrier functionof the cyst germinal layer. By considering the general diffu-sion routes, the laminated and germinal layer model provides aninteresting structural base for the understanding of the relevantmolecule transport process across the cyst wall. Compounds areable to movedue to their thermic energy in the direction ofa con-centrationgradient (passive diffusion). Passivediffusion througha biomembrane may occur via its lipid structures (transcellularpathway) or in its water-lled pores (inter-, para-cellular poreor tight junction pathway) ( Camenisch et al., 1996 ). The log Pdependence of thedyediffusion strongly suggests a transcellularpathway through the lipid structures instead of along aqueouspores. The laminated layer of the cyst wall is made up of anumber of acellular laminations and besides giving support to

the cyst, it is presumed to protect the parasite from the hostsimmune responses. It consists of a protein-polysaccharide com-plex with a carbohydratecomponent that builds up with glucose,galactose, glucosamine and galactosamine. If these carbohy-drates could protect the parasite from the immune response of the host, probably inhibiting complement activation, they couldnot be a barrier for the diffusion of hydrophilic molecules due totheir important polarities. Contrary to the previous one, the ger-minal layer is madeof undifferentiated proliferative totipotentialcells, which produce brood capsules projecting into the lumenof the mother cysts. This lipid-rich syncytium would certainlyfavour the diffusion of lipophilic molecules (important log P )

anddrastically reduce thediffusionof themore hydrophilic ones(negative log P ).Mottier et al. (2003) reported a high correlation between

molecular lipid solubility (expressed as log P ) and the avail-ability (expressed as Area Under Curve) of benzimidazolecompounds within the helminth parasite used as a model( Moniezia benedeni ). In their study, the total drug amountrecovered in Moniezia benedeni increases linearly with theoctanolwater partition coefcient with a correlation coef-cient, r , equal to 0.87. With a similar approach Alvarez et al.(2001) described the more important total ABZ recovered in Ascaris suum and Fasciola hepatica compared with ABZ-SO.Theauthorsexplained such difference bythefact that a lipophilicdrug as ABZ may havea greater capability to across the externalsurface of theparasite than itsmore polar sulphoxide metabolite.Traditionally, thenematodes cuticle, thecestodes tegumentandthe hydatid cyst membrane have been considered to be barrierslimiting entry of molecules into the parasite. However, amongothers (Geary et al., 1995; Reisin et al., 1977 ), the two last citedworks and the present report conrm that the passive diffusioncould be a major route of entryfor substances in parasite tissues.

In the specic case of the hydatid cyst wall, as the log P of the most important benzimidazole drugs is comprised between2 and 4 (Mottier et al., 2003 ), corresponding to a permeationcoefcient around 1 10 5 cm/s (Fig. 1), the penetration of the

drugs into the cyst will not constitute a limiting factor for the


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Table 4Characterization results of the unloaded and loaded nanoparticles

Unloaded particles ABZ-loaded particles ABZSO-loaded particles

Mean size (nm)After preparation 194.6 14.4 288.2 10.8 197.5 13.9After 15 days 185.6 6.4 278.2 11.6 212.8 13.2After 60 days 225.2 29.1 322.6 42.9 205.4 29.1

Zeta potential (mV)In water 5.7 1.5 9.7 1.6 14.8 4.7In HEPES buffer 3.0 0.3 2.7 1.2 2.3 1.2In FBS medium 6.8 0.3 3.6 1.4 4.5 1.3

Entrapment efciency (%) 36.4 6.4 6.4 5.1

Data represent the mean standard deviation of three determinations.

their effectiveness. By way of contrast, the passive diffusion isgoverned by the concentration gradient across the membrane of interest (Eqs. (1) and (2) ). Because of their poor water solubility(Dayan, 2003 ), leading to a low concentration gradient, the ben-zimidazole uxshould be reduced. To optimize theanthelmintictherapy, the challenge is now to increase this concentration gra-dient with appropriate formulations.

3.4. Nanoparticle characterization

The characterization results have been summarized in theTable4 . The size of the particles is around 200 nm, from 194.6 to288.2 nm, depending on the loaded-drug. The weak increase of size(andstandard deviation) observedfor eachformulationafter2 months is not statistically signicant ( P < 0.05). This stabilityof size suggests the absence of matrix surface erosion and thegood physicochemical stability of our nanoparticles in storage

conditions despite a quite low zeta potential. Thespherical shapeof the nanoparticles has been clearly observed with microscopyas described in the Fig. 2. Furthermore, the micrographs seemto conrm the mean size increase obtained with ABZ-loadedparticles in comparison with ABZ-SO-loaded one.

Highly negative zeta potential values, around 20 to 30 mV, are expected for pure polyester nanoparticles thanksto the presence of the carboxyl groups on the polymeric chainextremities ( Quintanar-Guerreroet al.,1996;Konanet al.,2003 ).In the present work, the zeta potential values tend to zero inrapport with the residual PVA chains on the nanoparticle sur-face. Leading to a shield between the nanoparticle surface andthe surrounding medium, PVA would mask the carboxyl groupsexisting on the particle surface ( Konan et al., 2003 ). Duringthe process of preparation, it has been frequently describedthat drug could be adsorb on nanoparticles. This adsorptionhas often explained zeta potential uctuations. ABZ and ABZ-

Fig. 2. Transmission electron microscopy pictures of the nanoparticles.


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SO are amphoteric molecule where the protonation steps arewell separated from each other ( log K > 4). Thus, the pK a val-ues of ABZ are 3.37 and 9.93 and then, only the neutral formwill be present from pH 5.4 to 7.9. Furthermore, Botsoglou etal. (1997) described that ABZ-SO is a free base form for pHbetween 4.7 and a superior limit higher than 7.4. More recently,Wu et al. (2005) , proposed a more accurate model concerningthe ABZ-SO ionisation steps: the molecule will be protonatedon the amine group in position1 for pH values inferior to 3.5 andthe deprotonation occurs on the amine group in position 3 at pHhigher than 9.8. Then, at the two studied pH, ABZ and ABZ-SOmostly remain in non-ionised states. Moreover, a pH decreasewould lead to protonation of the position 1 amine group and thenthe surface charge would increase.

It is interesting to note the absence of quantitative maskingeffect due to our drugs in their neutral forms, suggesting theabsence of drug adsorption on the particle surface.

The entrapment efciencies of the two drugs are completelydifferent: it is six times more important with ABZ in relation to

its sulphoxide metabolite ( Table 4 ). As ABZ-SO is more solublein a 1% PVA solution than ABZ (640 g/ml versus 1.3 g/ml),we could assume that an important amount of ABZ-SO has beensolubilized in the aqueous phase during the emulsication stepof the nanoparticle preparation process.

For the drug release studies, only ABZ-loaded nanoparti-cles have been investigated for essentially three reasons, (i) themore important entrapment efciency obtained with this drug,(ii) the more important log P of ABZ, leading to a better diffu-sion through the cyst membrane (see above) and (iii) the highertherapeutic efcacydescribedwith ABZin theliterature ( Smegoand Sebanego, 2005; Senyuz et al., 2001; Saimot, 2001 ).

3.5. Permeation studies across cyst membrane

The Fig. 3 describes the ABZ permeation through hydatidcyst membrane from ABZ-loaded nanoparticles in comparison

Fig. 3. In vitro delivery of albendazole from albendazole-loaded nanoparticlesand raw albendazole suspension through hydatid cyst membranes.

with a free ABZ suspension byusingverticalFranzcells. Despiteof an important variability (probably due membrane thicknessdifferences as suggested in the previous parts of this paper),an increase of the drug in the receptor compartment could beobserved in thecase of thenanoparticles. This could be probablyexplained by the better dispersion of the drug obtained withnanoparticles compared with raw suspensions.

To complete this experiment, whole cysts were incubatedin ABZ-loaded nanoparticle suspension during 3 h. After thattime, the cysts were washed, punctured by a needle to aspiratethe intra-cystic media and the drug quantied in it. After threehours of incubation, the intra-cystic ABZ concentration was

Fig. 4. Confocal microscopy pictures describing Echinococcus granulosus scolex penetrated by rhodamine B base (a) and rhodamine B base heterogeneous

accumulation in the outer (b) and inner (c) face of the hydatid cyst membrane.


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1 0.5 g/ml. A similar incubation was used with rhodamine-loaded nanoparticles. Such uorescent dye was used as modelmolecule to follow the drug accumulation in the cyst. The cystliquid ( Fig. 4a) and the cyst membrane ( Fig. 4b and c) were thenobserved with confocal microscopy. After the 3 h incubationwith rhodamine B base-loaded nanoparticles, the intra-cysticmedia was coloured in red, due to rhodamine B. Moreover,there were many protoscoleces labelled with the uorescentdye. Reconstructed confocal microscopy ( Fig. 4b and c) sug-gests important knowledge concerning our system. First thereis an important accumulation of uorescent probe in the mem-brane. As a matter of fact, this retention seems to take placeextensively in the inner layer of the membrane as shown the redcolour distribution in the Fig. 4b and c. This accumulation inthe innermost layer conrms the role of diffusion controllingbarrier played by the germinal layer of the cyst wall as con-cluded after the dye permeation experiments. In addition, in asame plan, the co-existence of coloured and uncoloured regionsshould benoted. Thisheterogeneousdistribution evokesan accu-

mulation of thenanoparticles in particular regionof thecyst wallrather than a simple diffusion of the dye which generally leadsto continuous layer of uorescence. In reality, we have shownthat during the incubation time (3 h), approximately 35% of theencapsulated rhodamine B is able to leave the nanoparticle (datanot shown) and from the confocal microscopy picture, it is notpossible to discriminate the loaded nanoparticles and the freerhodamine B.

It is interesting to note that the success of the chemothera-peutic treatment of hydatid disease requires the ability of thedrug to operate on the germinal layer and on the protoscolecesof the hydatid cyst interior at adequate concentrations ( Morris,

1987). By considering rhodamine B base as a model drug of benzimidazole (similar log P range) (Mottier et al., 2003 ), thesetwo goals are clearly reached. In vitro , effective concentra-tion of intra-cystic ABZ is 0.1 g/ml (Morris et al., 1987 ).This concentration is largely obtained in vitro with ABZ-loadednanoparticles (1 g/ml after a 3 h incubation).

4. Conclusion

Thediffusionof severalmodel molecules through thehydatidcystmembrane hasbeendescribedin this report. Thepermeationcoefcients correlate closely with the octanolwater partitioncoefcient of the studied compounds: the more lipophilic theyare, the more important is their diffusion. This penetrationbehaviour conrms the role of diffusion barrier played by theinnermost layer of the cyst wall (germinal layer). The prepa-ration of benzimidazole-loaded nanoparticles seems interestingfor the treatment of hydatid cyst, especially in the case of ABZ.Indeed, this drug has a good encapsulation rate and an inter-esting partition coefcient. In comparison with a simple drugsuspension, we observed a signicant increase of the drug diffu-sion from the formulation to the interior of the cyst. This passivediffusion through the hydatid cyst membrane was favoured bythe important log P of ABZ as concluded in the rst part of thispaper and by the increase of the ABZ apparent solubility with

regard to the nanoparticle form.

Thanks to the liver accumulation afternanoparticles injectionand the good candidate of ABZ for a passive diffusion throughthe cyst wall, ABZ-loaded nanoparticles seem to be promisingforms for the treatment of the hydatidosis. Therefore, the invivo behaviour of these nanoparticles should be investigated tocomplete the present work.

Acknowledgments

The authors would like to thank S. Peyrol and Y. Tourneurfor their help in transmission electron microscopy and confocalmicroscopy, respectively, and C. Morel.

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