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Journal of Colloid and Interface Science 293 (2006) 240–247www.elsevier.com/locate/jcis

Assembly of a model hydrophobic drug into cationic bilayer fragmen

Débora B. Vieira, Luis F. Pacheco, Ana M. Carmona-Ribeiro∗

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, Avenida Lineu Prestes 748, Butantã,CEP 05513-970, São Paulo, Brazil

Received 5 May 2005; accepted 10 June 2005

Available online 10 August 2005

Abstract

Our previous work has shown that dioctadecyldimethylammonium bromide (DODAB) bilayer fragments (BF) presented antimactivity, solubilized fungicides, e.g., amphotericin B and miconazole (MCZ), stabilized hydrophobic drug particles and were effevivo. Here, the interaction between MCZ and DODAB BF is evaluated from determination of BF loading capacity and effects oflipid molar proportion (MP) on particle size, zeta potential and gel-to-liquid-crystalline phase transitionTm. DODAB BF solubilized MCZover a range of MP. BF loading capacity was 0.5 mM MCZ at 5 mM DODAB. Above this limit, the drug aggregated in the disperspH 6.3, BF zeta potentials decreased with MP, suggesting insertion of deprotonated drug into the bilayer. MCZ optical spectra insimilar to those in best organic solvent, confirming drug solubilization. At MP� 1:10, BFTm remained unchanged, suggesting drug capat BF hydrophobic edges. At MP� 1:10,Tm decreased, showing MCZ insertion into DODAB bilayer. However, drug was expelled frobilayer core upon lowering temperature. Minimal fungicidal concentrations againstC. albicanswere synergically reduced by 10 times fdrug/BF. 2005 Elsevier Inc. All rights reserved.

Keywords:Miconazole; Dioctadecyldimethylammonium bromide; Sodium dihexadecylphosphate; Bilayer fragments; Fungicidal activity;Candida albicans

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1. Introduction

Over the past two decades membrane-derivatizedloidal systems have been introduced as potential apptions to a broad set of problems involving chemical eveon cell membrane surfaces[1–6]. Vesicles and other bilayer assemblies, such as bilayer fragments or disks, thephemeral, produced useful devices by depositing onto pmeric [7,8], mineral[9,10], or drug particles[3,11] or cellsurfaces[12–14] or by acting as templates for directioninorganic matter deposition, redox processes, or polymetion reactions[15–17]. In biology and medicine, they offerea suitable matrix to solubilize and/or carry drugs[18–20],either as entire entities, as complexes with the drug tocarried, or as bilayer capsules surrounding the hydropbic drug granule[21]. In medicinal chemistry, aggregatio

* Corresponding author. Fax: +55 11 3815 5579.E-mail address:mcribeir@iq.usp.br(A.M. Carmona-Ribeiro).

0021-9797/$ – see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2005.06.046

of hydrophobic drug molecules in aqueous solutionsshown to have an effect on drug bioavailability[22]. The up-take rate into systemic circulation would depend on havhydrophobic drug aggregates of appropriate size availfor absorption[22].

On the other hand, there are two clinically importaclasses of antifungal hydrophobic drugs: the polyenes (amphotericin B) and the azoles (e.g., miconazole);Ref. [23] for a review. The self-assembly of amphotericin(AB) or miconazole (MCZ) at hydrophobic sites of synthebilayer fragments has been reported[24,25], as has the invivo therapeutic activity of a novel AB formulation witcationic bilayer fragments[26] which exhibited low nephrotoxicity [27]. In fact, some double-chained synthetic lipisuch as dioctadecyldimethylammonium bromide (DODAor sodium dihexadecylphosphate (DHP) self-assemblaqueous solution yielding closed bilayers (vesicles) orrupted vesicles (bilayer fragments, BF, or disks or lipidlayer rafts), depending on the procedure used for disperthe lipid [3]. Furthermore, sterically (PEGylated) stabiliz

D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247 241

am-

-as-ing

Mnc-r abi-n a

il-nt

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orys e

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and

is-

oft 1,

f

eseri-on-d toid-

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e or

BF have recently been employed for rapid evaluation ofphiphatic drug partition[28].

In this work, effects of the MCZ:DODAB molar proportion on physicochemical properties of the drug/BFsemblies were investigated. At room temperature, loadcapacity of 5 mM DODAB bilayer fragments was 0.5 mMCZ. Although the loading capacity increased as a fution of temperature, returning to room temperature afteheating–cooling cycle caused drug expulsion from thelayer and appearance of drug aggregates in dispersiodrug-to-lipid molar proportionsM higher than 1:8. Belowthis limit, there was drug solubilization with excellent stabity of the drug/bilayer fragment complex plus improvemeof drug antifungal activity againstCandida albicans.

2. Materials and methods

2.1. Drugs, lipids, and microorganisms

Dioctadecyldimethylammonium bromide (DODAB) ansodium dihexadecylphosphate (DHP) were purchasedSigma at the highest purity available. Miconazole nitr(MCZ) (batch BEA53, Jansen Research Foundation, BeeBelgium) was used as such from a 10 mg/ml (ca. 20 mM)stock solution in ethanol 43% v/v. This solution was incbated for 1 h at 50◦C to obtain complete dissolution of drucrystals.Candida albicansATCC 90028 was obtained fromthe American Type Culture Collection. While methanolethanol were used as cosolvents percentages were alwapressed as volume ratios.

2.2. Preparation of lipid dispersions and analyticaldeterminations of lipid concentration

Lipids were dispersed in water, using proceduresyielded either large and closed vesicles (LV) or bilayer frments (BF), depending on the dispersion method. Sonicawith tip above the bilayerTm [30] was the method employefor producing cationic DODAB or anionic DHP bilayer framents[31]. At room temperature, BF were in the gel staThis procedure dispersed the amphiphile powder in wusing a high-energy input, which not only produced bilavesicles but also disrupted these vesicles, thereby generopen bilayer fragments. Thus, lipids were dispersed in wusing procedures that yielded either large and closed ves(LV) or bilayer fragments (BF). Sonication with tip at 60◦Cwas the method employed for producing cationic DODor anionic DHP bilayer fragments in the rigid gel stateroom temperature[29–31]. DODAB was alternatively dispersed by vortexing at 60◦C to obtain large vesicles (LV)Analytical concentration was determined either by micrtration for DODAB[32] or by inorganic phosphorus analysfor DHP[33].

t

,

x-

g

2.3. Determination of surface tension

Surface tension at the air–water interface was measur25◦C as a function of MCZ concentration at 5 mM DODABF in pure water with a Du Nouy ring. Controls were pformed for methanol, which was the drug solvent, andDODAB, which is soluble in this solvent.

2.4. Determination of miconazole optical absorptionspectra in different media

UV–visible optical spectra for MCZ were determined uing a Hitachi 2000 spectrophotometer in the double-bemode against a blank of lipid dispersions and solvent (wout drug). Light scattered by the lipid dispersions was stracted from light absorption spectra for the drug. The MCBF mixtures were obtained by adding 20–100 µl of MCstock solution in 43% ethanol (10 mg/ml MCZ) to 1 ml lipiddispersion (10 mg/ml, 15 mM DODAB). All optical spectrawere obtained at room temperature (25◦C) just after mixing.

2.5. Determination of mean average diameter and zetapotential of dispersions

The size distributions for DODAB, MCZ, and DODABMCZ (mean zeta-average diameter,Dz) were determined bymeans of a ZetaPlus zeta potential analyzer (BrookhaInstruments Corporation, Holtsville, NY) equipped with570-nm laser and dynamic light scattering at 90◦ for parti-cle sizing[34]. The mean diameters referred to in this wofrom now on should be understood as the mean hydronamic diametersDz. Zeta potentials (ζ ) were determinedfrom the electrophoretic mobilityµ and Smoluchowski‘sequation,ζ = µη/ε, whereη and ε are medium viscosityand dielectric constant, respectively. For zeta potentialsize distribution measurements, 0.3 ml MCZ (10 mg/ml) in43% ethanol was added to 2.7 ml pure water or lipid dpersion. All size distributions were obtained at 25 and 50◦Cjust after MCZ and DODAB BF were mixed. As a controlformulation stability, mean sizes were determined also a24, 72, and 288 h after mixing.

2.6. Determination of temperature effects on turbidity ofDODAB or DODAB/MCZ dispersions and assessment obilayer phase transition

Incubation of DODAB LV and MCZ was done at 50◦C/

18 h because the usualTm for DODAB LV is ca. 45–47◦C[40] and drug insertion in the bilayer core of LV requirthese relatively high temperatures and long incubation pods. For samples containing DODAB BF, such drastic cditions were not required, since the drug readily attacheavailable BF sites, as shown in Results. The gel-to-liqucrystalline phase transition of DODAB BF or LV was ivestigated by means of turbidimetry at 310 and 440 nma function of temperature, respectively, in the presenc

242 D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247

de-yer,ine

hanbe-

ereper

lder.eans-ctedrn-310dedtem-t-s

-

Bted

ort

ud

ed

on-

)U

lled

ornotesi-ainive-esi-s

g

icinr

d

asnts-itsa flatsi-ec-hase

mi-

onas averten-

absence of MCZ. This procedure maker it possible totermine major changes in the physical state of the bilasuch as its transition from the gel to the liquid-crystallstate, expected to occur around 45–47◦C [40,41]. Althoughturbidity is generally considered to be less sensitive tdifferential scanning calorimetry, excellent agreementtweenTm for DODAB LV, obtained by turbidimetry[40]or DSC[41], was previously reported. The dispersions wplaced inside a thermostatted quartz cuvette and the temature was changed at a constant rate of 0.3◦C per minute bymeans of a circulating bath connected to the cuvette hoTemperature inside the cuvette was determined by mof a Ni/Cr (+) and Ni/Al (−) thermocouple in direct contact with the dispersion. The thermocouple was conneto a Thermolyne digital pyrometer, Model PM 20700 (Bastead/Thermolyne Corporation, Singapore). Turbidity atand 440 nm, for BF and LV, respectively, was then recoras a function of temperature with mean phase-transitionperatures (Tm) determined from inflection points in the heaing/cooling curves. The MCZ/DODAB LV dispersion waincubated above LVTm (50◦C) for 18 h in an attempt to improve the poor drug solubilization in LV[24,25]. Thereafter,heating–cooling cycles were obtained. For MCZ/DODABF, due to good drug solubilization, incubation of preheasamples was not needed.

2.7. Determination of minimal fungicidal concentration fMCZ, MCZ/DODAB, and MCZ/DHP formulations againsC. albicans

C. albicansATCC 90028 was subcultured on Sabouradextrose agar (SDA) plates and grown at 35◦C for 24 hprior to testing. The macrodilution method was performaccording to the recommendations of the NCCLS[35]. Themedium used for diluting cells was 0.264 mMD-glucose,isotonic solution. Counts for cell suspension were cfirmed by plating on SDA plates after adjusting to 2.5 ×103 CFU/ml. Minimum fungicidal concentrations (MFCfor DODAB/MCZ formulations were determined by CF

-

counts after plating 0.1 ml and incubating for 48 h at 35◦C.The endpoint was the lowest drug concentration that ki�99.9% of the inoculum[36].

3. Results and discussion

3.1. Hydrophobic drugs at the air–water and bilayerfragments interfaces

The sonication with tip procedure dispersed DODABDHP powders in water using a high-energy input, whichonly produced bilayer vesicles but also disrupted these vcles, thereby generating open bilayer fragments. The mevidence for their existence was (1) osmotic nonresponsness of the dispersion, indicative of absence of inner vcle compartment[31]; (2) imaging of dispersions by meanof transmission electron microscopy (TEM)[30]; (3) cryo-TEM micrographies[37]; (4) quasi-elastic light scatterinand electron paramagnetic resonance spectroscopy[29,38];(5) solubilization of hydrophobic drugs such as amphoterB in DODAB bilayer fragments, which did not occur foclosed bilayer vesicles[24,25]; (6) coexistence of fluid ansolid bilayer phases for DODAB sonicated dispersions[39].The possibilities for the interaction between miconazolea model hydrophobic drug and DODAB bilayer fragmeis schematically represented inScheme 1. The packing geometric parameter for DODAB is close to 1, suggestingtendency to self-assemble as a large vesicle or even asbilayer [31]. Regarding the hydrophobic sides, it is posble that after bilayer disruption by sonication some molular rearrangement takes place to form a less packed pat the BF edges. In fact, Cocquyt and co-workers[39] re-ported such a phase in their H NMR experiments with silar DODAB dispersions.

Fig. 1A shows the effect of miconazole concentrationthe surface tension at the air–water interface. There wslight decay in surface tension as a function of [MCZ] othe 0.05–1 mM concentration range. The lowest surface

onome

Scheme 1. Chemical structures of DODAB and MCZ, MCZ half-dissociation at pH 6.5, and schematic representation of solubilization sites for MCZ mrsin the MCZ/DODAB BF dispersions.

D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247 243

Fig. 1. Effect of MCZ (A) or methanol (B) concentration on surface tension at the air-water interface in pure water (1) or in 5 mM DODAB ("). In (A) MCZwas added from a methanolic stock solution to yield the same methanol final concentrations shown in (B).

n atre-thisen-ten-

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ZtingtheBFged.nally, de-larCZ

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sion achieved was 53 mN/m at 1 mM MCZ. Thus, the drugpresented a limited capacity of lowering surface tensiothe air–water interface. The stock MCZ solution was ppared in methanol, which is the best organic solvent fordrug. The control for the effect of methanol on surface tsion at the air–water interface showed also a very limitedsioactive effect for this solvent, with ca. 65 mN/m attainedat the highest methanol concentration of 5% v/v (Fig. 1B).The composite effect of methanol and drug yielded a tdecay in surface tension of ca. 19 mN/m. On the other handmethanol solubilized a sufficient amount of DODAB to dcrease surface tension from 72 to 37 mN/m (Fig. 1B). Thisdid not occur for DODAB BF in pure water: surface tesion remained unchanged and equal to 72 mN/m even at arelatively large concentration of 5 mM DODAB (Fig. 1B),in agreement with previously reported data on effectsDODAB on surface tension at the air–water interface[40].

It was interesting to notice the stabilizing effect of MCon DODAB BF: whereas DODAB molecules from DODABF went to the air–water interface upon methanol addi(Fig. 1B), in the presence of MCZ, DODAB BF were so sble that DODAB barely went to the air–water interface inpresence of methanol (Fig. 1A).

These simple experiments have shown that the drugnot assemble as a packed monolayer at the air–water iface but preferred to assemble either at the DODAB BFat other hydrophobic surfaces, such as drug crystallitethe surface of the Teflon vessel where dispersions weretained. Dynamic light-scattering experiments allowed fther insights on aggregates present in MCZ/DODABdispersions.

At 5 mM DODAB, saturation of DODAB BF with MCZat room temperature took place between 0.2 and 0.5MCZ (Fig. 2). Comparison between size distributionsDODAB BF (Fig. 2A), MCZ (Fig. 2B), and MCZ/DODABBF (Figs. 2C, 2D, 2E) showed that it was possible to detemine the MCZ concentration where MCZ aggregates stato reappear in the MCZ/DODAB BF mixtures. MCZ aggrgates were ca. 329 nm mean diameter, in contrast to

-

-

92 nm mean diameter, of the DODAB BF with or withodrug. After saturating DODAB BF with MCZ, presenceMCZ aggregates in dispersion was observed, so that a0.5 mM MCZ, aggregates started to be visualized by naeye.

3.2. Miconazole solubilization in DODAB bilayers andstability of miconazole/DODAB BF assemblies

Miconazole optical spectra in different media are shoin Fig. 3. MCZ optical spectra in ethanol 4.3% indicatsuperposition of turbidity by insoluble MCZ granules in wter solution plus light absorption by the drug (Fig. 3A). Incontrast, spectra very similar to those of MCZ in its bestganic solvent (methanol)[39] were obtained in 43% ethan(Fig. 3B) or in 10 mg/ml DODAB/BF in 4.3% ethano(Fig. 3C). A 43% ethanol solvent could advantageouslyused to solubilize MCZ in the stock solution, instead oftoxic methanol.

Mean diameters (D) and zeta potentials (ζ ) for drug,lipid, and drug plus lipid dispersions are given inTable 1.In 2% methanol, 0.4 mM MCZ formed relatively large, poitively charged granules with mean diameter 334 nmmean zeta potential 32 mV. In 4.3% ethanol, 2 mM MCalso formed large and positively charged particles exhibislightly larger sizes and zeta potentials, possibly due todecrease in dielectric constant of the medium. DODABmean diameters were 90–107 nm, also positively charUpon mixture of MCZ granules with DODAB BF, meadiameters were 73–102 nm: large MCZ granules basicdisappeared. Furthermore, BF zeta potential, 39–41 mVcreased to 20 or 32 mV at 1:3 and 1:8 MCZ:DODAB moratios, respectively, revealing insertion of deprotonated Min the DODAB bilayer.

Fig. 4 shows the large colloid stability of MCZ/DODABBF assembly at 25 or 50◦C from determination of size distribution as a function of time. Mean sizes were constana function of time up to 12 days of observation time (nshown).

244 D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247

me-

n

Z/-geli-mithlay-nat-

%

0.6;

lus

er-

oomi-CZtial

d/orag-Bn--Z

Fig. 2. Size distributions for DODAB BF in water (A), MCZ in 4.3%ethanol (B), and mixtures of both in 4.3% ethanol (C, D, E). Mean diaters are indicated in each subfigure.

3.3. Effect of drug-to-lipid molar proportion on fluidity ofDODAB bilayers and stability of MCZ/DODAB formulatio

The temperature effect on turbidity at 310 nm for MCDODAB BF is shown inFig. 5. Upon heating and cooling of samples, the path for the transition from thestate to the liquid-crystalline state of the DODAB BF blayer was not identical to the one for the transition frothe liquid-crystalline to the gel state, in accordance wpreviously described phase transitions for charged biers[40]. At MCZ:DODAB � 1:10, the main phase transitioof the bilayer (Tm) was barely affected by the drug and he

Fig. 3. MCZ optical absorption spectra in different media: (A) MCZ in 4.3ethanol; (B) MCZ in 43% ethanol; (C) MCZ in 15 mM (10 mg/ml) DODABBF and 4.3% ethanol. Final MCZ concentrations are (a) 0.2; (b) 0.4; (c)(d) 0.8; and (e) 1 mg/ml.

Table 1Mean diameters (D) and zeta potentials (ζ ) for drug (MCZ), lipid(DODAB) as BF, or drug plus lipid dispersions in water or in water porganic solvent, pH 6.3, at 25◦C

[MCZ] [DODAB] [MCZ]:[DODAB] D ± δ ζ ± δ

(mM) (mM) (nm) (mV)

0.4a – – 334±3 32±2– 1.3 – 79±1 41±20.4a 1.3 1:3 102±1 20±22.0b – – 380±8 48±2– 15.0 – 90±1 39±22b 15.0 1:8 73±1 32±1

a Final methanol concentration= 2%.b Final ethanol concentration= 4.3%.

ing/cooling cycles did not change turbidity at room tempature (b and c inFig. 5A). At MCZ:DODAB � 1:10,Tm de-creased and heating/cooling cycles reduced turbidity at rtemperature, indicating MCZ solubilization in DODAB blayer, increased bilayer fluidity, and disappearance of Mgranules in dispersion that were contributing to the initurbidity value (d inFig. 5A; b in Fig. 5B).

One should notice that increasing temperature andrug concentration promoted drug insertion in bilayer frments. MCZ solubilization by entire and closed DODAvesicles (DODAB LV) only took place under drastic coditions such as 18 h incubation at 50◦C for complete dissolution of MCZ granules. In contrast to DODAB BF, MCsolubilization was poor in DODAB LV at 25◦C. A heating–

D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247 245

ize

he, in

rinVABingyerin

al-

.dad-f

hy-in

erghe

ed

le-ing-

nsB

chntcialion

atea-

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theth

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-ve

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Fig. 4. Size distributions and meanDz for DODAB (A) and DODAB/MCZ(B–D) formulation as a function of time at two different temperatures. Sdistributions were obtained at 25◦C (A, B, C) or 50◦C (D) for two differentinteraction times between drug and DODAB BF: 1 (B) or 24 h (C, D). Tfinal DODAB and MCZ concentrations were 15 and 2 mM, respectively4.3% ethanol.

cooling cycle for MCZ/DODAB LV at 1:1 molar ratio aftethe incubation period showed that there was a decreaseTmof about 10◦C compared to the one typical of DODAB L(not shown). This was consistent with increase in DODbilayer fluidity caused by drug insertion. However, returnto room temperature caused drug expulsion from the bilawith a large increase in turbidity and visible flocculationa few minutes after returning temperature to 20–25◦C (notshown).

The effect of drug concentration on gel-to-liquid-crystline phase transition temperature (Tm) for the DODAB BFwas shown inTable 2 over a range of drug:lipid ratiosDODAB BF alone presentedTm values equal to 43.4 an39.8◦C for heating and cooling runs, respectively. Upondition of MCZ to DODAB BF, over a drug:lipid range o1:25–1:5,Tm decreased slightly from 43.4 to 42.5–43.0◦C,as expected for predominance of drug capture at thedrophobic edges of the DODAB BF and drug insertion

the bilayer barely affecting bilayer fluidity. On the othhand, upon addition of MCZ to DODAB LV at a 1:1 druto lipid ratio, there was a remarkable fluidification of tDODAB LV bilayer shown by ca. 10◦C decrease inTmboth for heating and cooling procedures (Table 2). At 1:1MCZ:DODAB ratio, there was insertion of deprotonatMCZ in the DODAB LV bilayer, which reduced bothTm(Table 2) and zeta potential (Table 1). At pH 6.5, in pure wa-ter, MCZ is at half-dissociation so that neutral MCZ mocules would be available to insert in the bilayer, displacthe equilibrium onScheme 1to the left up to bilayer saturation.

3.4. In vitro activity against C. albicans formiconazole/bilayer fragment assemblies

The fungicidal activities againstC. albicansATCC 90028were obtained from minimal fungicidal concentratio(MFC) for Zoltec (as a control), MCZ alone, MCZ/DODABF, and MCZ/DHP BF as 2, 4, 1, and 2 mg/ml, respectively(Table 3). Therefore, when compared to MCZ MFC, whiwas 4 mg/ml, both BF formulations were more efficiethan MCZ alone, requiring lower MCZ doses. In the specase of DODAB BF, there was possibly synergistic actof DODAB and MCZ against the fungus (Table 3). ForMCZ alone or MCZ/anionic sodium dihexadecylphosph(DHP) BF in 4.3% ethanol, minimal fungicidal concentrtions (MFC) againstCandida albicansATCC 90028 were4 and 2 mg/ml (Table 3), respectively, indicating improvedrug efficiency also in anionic BF assemblies. The improperformance obtained also for MCZ/DHP BF could posbly be understood from the monomeric state of the druthe BF formulation.

Recently, the coexistence of fluid and solid states forDODAB bilayer fragment was reported by H NMR, wi50% of all DODAB molecules remaining fluid at room temperature[39]. These findings corroborated the excellent subilization capability of these structures at room temperafor hydrophobic drugs. MCZ/DODAB BF with mean dameter 49–50 nm were remarkably stable and adequaprinciple to drug delivery beyond the circulatory barrier,that in vivo the complex will possibly reach sites of infectiin the tissues and drug bioavailability will be improved[42].In fact, a previous evaluation of in vivo activity for another DODAB BF formulation carrying amphotericin B ga100% mouse survival at low amphotericin B doses[26].

Several cationic antimicrobial peptides have emergea promising new class of antibiotics[43]; among them sompresent synergistic activity with miconazole against sevyeast species[44], but their synthesis is often very expesive, as is their isolation when they are biologically pduced. Furthermore, peptides usually require carriers tdelivered and the cost of drugs delivered by carriers isgeneral, higher than the cost of free drugs. The improtherapeutic index of carrier-delivered drugs does not alwjustify the expensive treatment. In this respect, DODAB

246 D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247

) or 5

Fig. 5. Effect of drug-to-lipid molar ratio on turbidity of MCZ/DODAB dispersions as a function of temperature. In (A), 5 mM DODAB BF only (amM DODAB BF plus MCZ at 0.1 (b), 0.25 (c), or 0.5 mg/ml (d). In (B), 15 mM DODAB BF only (a) or 15 mM DODAB BF plus MCZ at 1 mg/ml (b).MCZ-to-DODAB molar ratios are specified inTable 2. Heating and cooling are represented by full and empty circles, respectively.

,

car-as

Table 2Effect of drug-to-lipid molar ratio on mean phase transition temperature(Tm) of cationic DODAB bilayers in the absence or presence of MCZ inethanol, 4.3%

[DODAB] (mM) [MCZ] [MCZ]:[DODAB] Tm (◦C)

BF LV (mM) Heating Cooling

5 – 43.4 39.8

5 0.2 1:25 43.0 39.6

5 0.5 1:10 42.5 38.3

5 1.0 1:5 – 38.3

15 – 43.4 40.1

15 2.0 1:8 – 38.6

– 2 – – 44.3 40.1

– 2 2.0 1:1 36.5 31.4

Table 3Minimal fungicidal concentration (MFC) for MCZ in ethanol 4.3%MCZ/DODAB or DHP, and Zoltec (trade name for fluconazol) forCan-dida albicansATCC 90028

Drug or drug/lipiddispersion

MFC

(µM) (mg/ml)

Zoltec (FCZ) 6.5 2DODAB 4000 250MCZ 8 4MCZ and DODAB 2 1MCZ and DHP 4 2

seems to represent an efficient and inexpensive novelrier for azoles, possibly acting synergistically with themcationic peptides do[44].

D.B. Vieira et al. / Journal of Colloid and Interface Science 293 (2006) 240–247 247

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4. Conclusions

At low drug-to-lipid molar ratios (smaller than 1:10MCZ/DODAB BF zeta potentials and bilayer phase trasitions were very similar to those of DODAB BF. At higmolar ratios (above 1:10), MCZ/DODAB BF zeta potetials decreased and bilayer fluidity increased, suggestincreased participation of the bilayer core in drug solubilition. Therefore, two major types of interaction sites for MCwith DODAB bilayers were identified: (1) sites at the hdrophobic edges readily available to hydrophobic drugroom temperature; (2) sites in the bilayer core, whichcome increasingly occupied by the drug with increasing dconcentration and/or temperature and bilayer fluidity.proved drug performance against the fungus was achifor drug/BF assemblies.

Acknowledgments

This work was supported by FAPESP, CAPES, aCNPq.

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