Development of antileishmanial lipid nanocomplexes

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Research paper

Development of antileishmanial lipid nanocomplexes Q4

T.T.H. Pham a, d, C. Gueutin a, M. Cheron b, S. Abreu c, P. Chaminade c, P.M. Loiseau d, G. Barratt a, * a Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie, UMR CNRS 8612, Facult
e de Pharmacie, Universit
e Paris XI, ^tenay Malabry Cedex, France 92296 Cha b Laboratoire Acides Nucl
eïques et Biophotonique ANBIOPHY, FRE 3207 CNRS, Universit
e Pierre et Marie Curie, 75252 Paris Cedex, France c ^tenay Malabry Cedex, France Groupe de Chimie Analytique de Paris-Sud, EA4041, Facult
e de Pharmacie, Universit
e Paris XI, 92296 Cha d ^tenay Malabry Cedex, France Groupe Chimioth
erapie Antiparasitaire, UMR 8076 CNRS, Facult
e de Pharmacie, Universit
e Paris XI, 92296 Cha

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 February 2014 Accepted 3 June 2014 Available online xxx

Visceral leishmaniasis is a life-threatening disease that affects nearly a million people every year. The emergence of Leishmania strains resistant to existing drugs complicates its treatment. The purpose of this study was to develop a new lipid formulation based on nanocochleates combining two active drugs: Amphotericin B (AmB) and Miltefosine (HePC). Nanocochleates composed of dioleoylphosphatidylserine (DOPS) and Cholesterol (Cho) and Ca2þ, in which HePC and AmB were incorporated, were prepared. Properties such as particle size, zeta potential, drug payload, in-vitro drug release and storage stability were investigated. Moreover, in-vitro stability in gastrointestinal fluid was performed in view of an oral administration. AmBeHePC-loaded nanocochleates with a mean particle size of 250 ± 2 nm were obtained. The particles displayed a narrow size distribution and a drug payload of 29.9 ± 0.5 mg/g for AmB, and 14.0 ± 0.9 mg/g for HePC. Drug release occurred preferentially in intestinal medium containing bile salts. Therefore, AmBeHePC-loaded nanocochleates could be a promising oral delivery system for the treatment of visceral leishmaniasis. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Nanocochleates Amphotericin B Miltefosine Oral route Visceral leishmaniasis

1. Introduction Visceral leishmaniasis (VL), also known as kala-azar, caused by an intracellular protozoan of the Leishmania donovani complex, is a major public health problem throughout the world. There are an estimated 500,000 new cases and more than 50,000 deaths from the disease each year, mainly in developing countries [1e5]. Furthermore, VL is a frequent co-infection with human immunodeficiency virus-1 (HIV-1) [1e5]. Since control of the sandfly vector is difficult, chemotherapy is the main means of controlling the disease [6]. For the last 70 years, pentavalent antimonials have been the first-line treatment, but high resistance to this drug class, particularly in north-east India, and their severe side effects have led to a search for alternative treatments. The current second-line treatment is Amphotericin B (AmB), despite a number of drawbacks, including its renal toxicity, the emergence of resistance and its very low oral bioavailability which means that it must be given

* Corresponding author. UMR CNRS 8612, Centre d'Etudes Pharmaceutiques, 5
ment, 92296 Chatenay-Malabry Cedex, France. Tel.: þ33 (0)1 46 83 56 rue J.B. Cle 27; fax: þ33 (0)1 46 83 59 46. E-mail address: [email protected] (G. Barratt).

intravenously. Phospholipid-based systems such as AmBisome® reduce the acute toxicity of AmB and allow higher doses to be administered [7], but these formulations are prohibitively expensive for developing countries and still require intravenous administration. Recently, miltefosine (hexadecylphosphocholine or HePC) was approved as the first orally active drug against VL [8]. However, resistance to this molecule has already been observed in the field, as a result of itsnarrow therapeutic index and long half-life which has been estimated at around 7 days [9e10]. Therefore, an urgent solution is necessary to extend the life of this molecule as an effective drug, and to reduce its side effects. One such solution could be the development of a combined therapy with AmB, since the use of two drugs with different mechanisms of action would allow the dose of each to be reduced. The aim of this work was to prepare a lipid-based formulation containing HePC and AmB that could be administered by the oral route. A study of the literature indicated that nanocochleate cylinders would be a suitable drug delivery system. Nanocochleates are cigar-shaped nanostructures composed of rolled negatively charged lipid bilayers (phosphatidylserine e PS) bridged by calcium. They were first described by Papahadjopoulos

http://dx.doi.org/10.1016/j.biochi.2014.06.007 0300-9084/© 2014 Elsevier Masson SAS. All rights reserved.

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as an intermediate in the preparation of large unilamellar vesicles [11,12]. Hydrophobic, amphiphilic, negatively charged or positively charged drugs can be easily inserted into the PS bilayers. Nanocochleates differ from liposomes in that they have a water-free interior, a rod-like shape and a rigid structure, imparting good stability in the gastrointestinal environment. Furthermore, because of their non aqueous structure nanocochleates are less susceptible to oxidation of the phospholipids, and can maintain their structure after lyophilization; giving them useful storage properties which would facilitate their use in developing countries. The drug delivery potential of nanocochleates for anti-fungal agents, DNA, and subunit vaccines were investigated by Zarif [13] and Mannino et al. [14]. In particular, it was observed that nanocochleates containing AmB were highly effective in treating a murine model of candidiasis [15,16] and murine model of systemic aspergillosis [17] by the oral route and compared well with AmBisome® and Fungizone®. Furthermore, cochleates containing AmB have been described as having minimal toxicity both in vitro and in vivo [13]. On the basis of these previous observations, we decided to develop a nanocochleate delivery system containing both AmB and HePC for administration by the oral route. 2. Materials and methods 2.1. Materials Dioleoylphosphatidylserine (DOPS) was purchased from Avanti Polar Lipids (Alabaster, Alabama). Solutions of DOPS were prepared in methanol/chloroform 1:2 (v/v) at an initial concentration of 5 mg/ml. AmB was purchased from Sigma (St. Louis, MO, USA). Stock solutions of AmB were prepared in methanol at an initial concentration of 0.1 mg/ml. HePC was kindly provided by Zentaris (Frankfurt, Germany. Cholesterol (99% pure) was purchased from Sigma, and a-tocopherol (a-toc, 99% pure) from Acros Organics (Geel, Belgium). The lipids were used without further purification. Stock solutions of HePC, Cho and a-tocopherol were prepared in methanol/chloroform 1:2 (v/v) at an initial concentration of 1 mg/ ml. All organic stock solutions were stored at 20  C until needed. Dextran (500,000 Da), poly(ethylene glycol) (PEG 8000), calcium chloride, ethylene diamine tetraacetic acid (EDTA), calcium chloride, acetic acid, sodium monobasic phosphate anhydrous, maleic acid, pancreatin (8  USP specification), pepsin (European Pharmacopeia), sodium taurocholate, lecithin, sodium hydroxide and all other chemical reagents (analytical grade) were obtained from Sigma. All solvents, methanol, chloroform and ethanol were purchased from Sigma, 99% pure and used without further purification. Water was purified by reverse osmosis (Milli-Q, Millipore).

any residual solvent was removed by a stream of nitrogen. The film was hydrated with 1 M TriseHCl buffer pH 7 by rotating the flask at room temperature and pressure for 4 h and then sonicated with a vibrating metallic tip (IBP7677, Ultrasons, Annemasse, France) at 180 V, for 10  1 min over ice with 1-min intervals. Liposome size was reduced and homogenized by sequential extrusion through polycarbonate membranes (Nucleopore®, Costar Corp., Cambridge, USA) ranging from 0.2 to 0.05 mm in pore size in a 10 ml extrusion barrel (Lipex Biomembranes, Canada). One passage was made through a 0.2-mm membrane, 2 passages through 0.1 mm and 15 passages through 0.05 mm. During this step, the insoluble fraction of the drugs was retained by the membranes. 2.2.2. Conversion of liposomes into nanocochleates by the hydrogel method The AmBeHePC loaded liposome suspension was mixed with 40% w/w dextran (500 kDa) at the ratio of 2/1 v/v dextran/liposome. This mixture was then injected using a syringe and a 23G needle into 15% w/w PEG-8000 under magnetic stirring (1000 rpm). An aqueouseaqueous emulsion of AmB liposomes/ dextran droplets dispersed in a PEG continuous phase was obtained. A CaCl2 solution (100 mM) was added to the emulsion to give a final calcium concentration of 1 mM. Stirring was continued for 1 h to allow the slow formation of small-sized AmBeHePCloaded nanocochleates sequestered within the dextran droplets. The PEG was removed by the addition of ten volumes of washing buffer containing 1 mM CaCl2 and 150 mM NaCl to one volume of the emulsion. The suspension was vortexed and centrifuged at 1000 g, 4  C, for 30 min. After removal of the supernatant, washing buffer was added at a ratio of 5:1 (v/v) and the centrifugation step was repeated. This purification procedure was carried out four times. Finally the preparation was rapidly frozen by immersing the vial in liquid nitrogen and freeze-dried using a Genesis 12EL freeze dryer (Virtis, USA) for 48 h in the dark. The final formulation in powder form was stored at 4  C until use. Unloaded cochleates were prepared in the same way without addition of AmB and HePC to the lipid mixture for liposome preparation. 2.3. Characterization of AmBeHePC-loaded nanocochleates

AmBeHePC-loaded nanocochleates were prepared by the hydrogel method, as described by Zarif et al. [18] and Jin et al. [19] with a number of modifications. The preparation comprised two steps: incorporation of AmB and HePC into small unilamellar liposomes and conversion of these liposomes into nanocochleates.

2.3.1. Size and zeta potential analysis The hydrodynamic diameter (dH) and polydispersity index (PDI) was measured before and after freeze-drying by quasi elastic light scattering, using a Zetasizer Nano ZS instrument (Malvern, France). Suspensions of nanocochleates before freeze-drying in the washing buffer were diluted in washing buffer that had been filtered over a 0.22 mm membrane. Nanocochleates in powder form were dispersed in the washing buffer filtered over a 0.22 mm membrane. Measurements were performed in triplicate at 25  C at an angle of 173 . Correlation data were acquired for 60 s. Zeta potential measurements were carried out with the same instrument, at 25  C after dilution in CaCl2 solution (1 mM) filtered over a 0.22 mm membrane. At least three different preparations of each formulation were analysed.

2.2.1. Incorporation of AmB and HePC into liposomes Liposomes containing AmB and HePC in the lipid bilayer were prepared by thin film hydration. Firstly, AmB, HePC and DOPS were mixed in the ratio 100DOPS/5AmB/5HePC (mol/mol) from their stock solutions as described in “Materials”. Different proportions of Cho and were added and the mixture was dried to a film at 50  C under reduced pressure using a Büchi rotary evaporator RE-111 (Büchi, Switzerland). When a dry homogeneous film was formed,

2.3.2. Transmission electron microscopy (TEM) Transmission electron microscopy was performed at CCME (Orsay, France) using a Philips EM208 instrument operating at 80 kV. Suspensions of nanocochleates before and after freezedrying (0.1 mg/mL) and at various stages during the preparation process were deposited on copper grids covered with a formvar film (400 mesh) for 2 min. The excess solution was blotted off using filter paper and grids were air-dried before observation. Images

2.2. Preparation of AmBeHePC loaded cochleates

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were acquired using a high-resolution camera, Advantage HR3/ 12GO4 (AMT-Hamamatsu). TEM was used also to examine the reconversion of nanocochleates into liposomes. An equal volume of EDTA solution (100 mM, pH 9) is added to the suspension of nanocochleates and the mixture was examined immediately by TEM. 2.3.3. Determination of encapsulation efficiency of AmB and HePC within nanocochleates The encapsulation efficiency of AmB and HePC within the nanocochleates, the drug loading as a percentage of the total mass of the preparation and the product yield were calculated as described below.

Encapsulation efficiency ¼

Measured drug loading  100 Theoretical drug loading

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[20] using a Hypersil ODS C18 column (150 mm  4.6 mm ID, 5 mm particle size) (Thermo Fisher Scientific, Villebon, France) thermostated at 30  C (7956R, Grace, Deerfield, United States). The mobile phase was a 1% aqueous solution of triethylamine adjusted to pH 5.2 with 1% formic acid, acetonitrile and tetrahydrofuran (1000/ 385/154 v/v). The flow rate (600 Controller, Waters, Guyancourt, France) was fixed at 0.3 mL min1. 100 mL of sample were injected by an automatic injector (717 Autosampler, Waters, Guyancourt, France). AmB was detected by its absorbance at 409 nm (2996 Photodiode Array Detector, Waters, Guyancourt, France) and the peaks were automatically integrated (Empower software, Waters, Guyancourt, France). Under these conditions, the retention time for AmB was approximately 6 min. This method showed a good linearity between 20 ng and 10,000 ng mL1 with a mean correlation coefficient of 0.9985. The limit of quantification was 0.01 mg mL1. 2.6. HePC quantification by LCeMS/MS

Mass of drug encapsulated  100 Drug payload ¼ Mass of cochleates Product yield ð%Þ ¼

Recovered mass ðcochleatesÞ  100 Theoretical mass ðdrugs þ lipidsÞ

AmB and HePC were extracted from nanocochleates as follows. Freeze-dried nanocochleates were weighed precisely (5 mg) and 10 ml of methanol/chloroform 1:2 (v/v) was added to completely dissolve all the components, followed by vigorous vortexing for 15 min. The mixture was then divided into two aliquots for extraction. Each aliquot was dried to a film at 50  C under reduced pressure using a Büchi rotary evaporator RE-111 (Büchi, Switzerland) to extract the two drugs. 1 ml of dimethyl sulfoxide (DMSO) was added to one aliquot to solubilize AmB from the dried film under vigorous vortexing, followed by an equal 5 ml of methanol (5 ml). In order to extract HePC, 5 ml of methanol was added with vigorous vortexing to the second aliquot. The drug content of the resulting solutions was quantified by HPLC for AmB or by LCeMS/MS for HePC, as described in the analysis section. Each experiment was repeated three times. 2.4. Circular dichroism and UVevisible spectroscopy Circular dichroism and UVevisible spectroscopy were used to evaluate the aggregation state of AmB in AmBeHePC-loaded nanocochleates. Absorbance measurements were made by using a Cary 1E UVevisible spectrometer (Varian, France). CD spectra were recorded with a JobineYvon Mark V dichrograph, and expressed as Dε (M1 cm1); that is, the differential molar absorption dichroic coefficient. All measurements were made at 20  C. The organization of AmB was investigated in the drug-loaded liposomes as described in Section 2.2.1 before their conversion into nanocochleates by the hydrogel method and also after reconversion of nanocochleates into liposomes by EDTA solution (100 mM, pH 9). The molar proportions of the constituents were 9DOPS/1Cho/0.5AmB or 9DOPS/1Cho/ 0.5AmB/0.5HePC. The samples were diluted in water to final AmB concentrations of 105 M and 2$105 M for the liposome samples and the liposome samples derived from the nanocochleates respectively. As a control, spectroscopic measurements were also on a solution of AmB after dilution in water to a final AmB concentration of 3  105 M. Quartz cuvettes with pathlengths of 1 cm were used. The spectra are presented as molar extinction coefficient (ε) to take into account the different AmB concentrations of the samples. 2.5. AmB quantification by HPLC AmB was quantified by reversed phase high-performance liquid chromatography in accordance with a previously validated method

HePC was quantified by liquid chromatographyetandem mass spectrometry by a previously validated method [21]. Chromatographic separation of miltefosine was carried out using an HP1050 € blingen, liquid chromatograph system (HewlettePackard GmbH, Bo Germany) consisting of a quaternary pump, degasser and autosampler. The analytical column was an C18 Uptispher 3 mm ODB, 50 mm  2.0 mm I.D., 5 mm particle size; Interchrom. The HPLC system was connected to an API-3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source (Abi Sciex, Les Ulis, France). The quadrupoles were operated with 0.3 unit resolution in the positive ion mode. Data were processed using AnalystTM software (version 1.4; Sciex). An isocratic eluent (ratio 10 mM ammonia in water: 10 mM ammonia in methanol, 5:95, v/v) was used for chromatographic separation, with a flow-rate of 0.3 mL/min and an injection volume of 10 mL. The autosampler temperature was set at 10  C. The mass transition of m/z 408.5 to 124.8 was optimized for HePC with a dwell-time of 200 ms. Nebulizer and turbo gas (zero air) were set at 350 kPa and 700 kPa, respectively. Curtain gas (nitrogen grade 5.0) was set at 300 kPa and the collision gas (nitrogen grade 5.0) at a pressure setting of 600 kPa. The ion spray voltage was set at 3500 V, while the source temperature was 400  C. The range of linear response was 4e2000 ng/mL. The lower limit of detection was 0.05 ng/mL and the lower limit of quantification was 0.2 ng/mL. 2.7. In-vitro release study 10 mg of AmBeHePC-loaded nanocochleates containing 300 mg of AmB and 140 mg of HePC were dispersed in 100 ml phosphatebuffered saline (PBS, pH ¼ 7.4) and stirred at 150 rpm at 37  C. The pH of the incubation medium was checked frequently and readjusted if necessary with 0.01 M NaOH. Sample of 0.5 ml were removed and replaced by PBS at various times intervals. These samples were centrifuged in a JOUAN MR1812 centrifuge at 5000 g for 5 min in order to remove intact nanocochleates and fragments. The released drug in the supernatant was determined by HPLC for AmB and by LCeMS/MS for HePC and was expressed as a percentage of the theoretical drug payload. After each sampling, the theoretical drug payload was adjusted to take into account the removal of nanocochleates by sampling. 2.8. Gastrointestinal stability of AmBeHePC nanocochleates 2.8.1. Composition of simulated intestinal media Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to USP XXIV [22]. The SGF medium contained 0.32% (w/v) of pepsin; the pH was 1.2. The SIF medium was

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2.8.2. Stability study The conditions of the stability study were designed to mimic the passage of the formulations through the gastrointestinal tract. 10 mg of AmBeHePC-loaded nanocochleates were added to 200 ml of simulated gastric fluid (SGF). After 55 min, 200 mL of either SIF, FaSSIF or FeSSIF at twice the desired final concentration were added. The stirring speed was set at 100 rpm and the temperature of the medium was maintained at 37 ± 0.5  C. 0.5 ml samples was removed and replaced by fresh medium at different time intervals: 5, 15, 30 and 55 min in SGF and 1 h, 1 h 30, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h and 9 h of incubation in SIF, FaSSIF or FeSSIF. The size distribution of the particles in suspension was measured by dynamic light scattering on a Zetasizer Nano ZS instrument (Malvern, France). TEM was used in order to observe the changes in morphology over time in each media. The samples were then centrifuged with a JOUAN MR1812 centrifuge (5000 g, 5 min) in order to remove intact nanocochleates and aggregates of pancreatin, which were returned into the medium to maintain constant conditions. The amount of released AmB and HePC in the supernatants was determined by HPLC for AmB or LSeMS/MS for HePC (Section 2.5 and 2.6). All experiments were run in triplicate. In the results section, we use the terms SGF-SIF, SGF-FaSSIF and SGF-FeSSIF for the SGF/double concentration SIF, SGF/double concentration FaSSIF and SGF/double concentration FeSSIF mixtures (50/50; v/v), respectively.

a regulator of membrane fluidity by promoting tight lipid packing and thereby to stabilize liposomes against disruption by surfactants [24e27]. Preliminary experiments were performed to determine the optimal proportion of cholesterol which would be able to stabilize the membranes in the presence of HePC without diluting the DOPS content and thus the negative charge of the bilayers so that the interaction with Ca2þ could still occur. Nanocochleates prepared with different ratios of DOPS/Cho (7/3, 8/2, 8.5/1.5, 9/1) were examined by TEM. It was observed that when the proportion of cholesterol was 20% or higher, the liposomes did not fuse to form cylindrical objects but rather formed large sheets. When the proportion of cholesterol was lower and the proportion of DOPS higher, the addition of Ca2þ ions allowed nanocochleates to be formed (results not shown). Based on these observations, the ratio of DOPS/Cho of 9/1 was chosen for the formulation. These results were complementary to a previous study performed using lipid monolayers on a Langmuir balance [28]. This allowed us to understand the interaction between the different components of the nanocochleate formulation and yielded much interesting information about the interaction and the stability, and the orientation of AmB and HePC in the lipid monolayer. It was observed that, despite its high solubility in water, HePC was retained at the interface in the presence of lipid, particularly when cholesterol was present and therefore could be expected to be located within the lipid bilayers in the nanocochleates, like AmB, rather than in the water phase between the bilayers. This work highlighted a second role of cholesterol in the formulation, since this sterol also improved the retention of both AmB and HePC in the monolayer. Based on these results, the optimal drugs/lipids ratio for the development of the formulation was determined to be 9DOPS/ 1Cho/0.5AmB/0.5HePC. A number of factors intervening in the preparation: hydration time, time and frequency of sonication and the number of passages by extrusion were also optimized in preliminary experiments.

2.9. Stability of nanocochleates over time

3.2. Preparation and characterization of nanocochleates

The stability of AmBeHePC-loaded nanocochleates was evaluated after storage of the lyophilized powder at 2e8  C for 6 months. The particle size distribution, zeta potential and drug payload of samples after 1, 3 and 6 months were determined as previously described after resuspension in water.

The process of nanocochleate formulation is schematized in Fig. 1. TEM observations were used to follow this process and confirm the mechanism (Fig. 2). The first step was the preparation of multi-lamellar vesicles (MLV). As shown in Fig. 2A, these were very polydisperse with a mean diameter of around 1 mM. It was necessary to reduce the size of the liposomes before forming the nanocochleates. Our

composed of 1% (w/v) pancreatin at a pH of 7.5. The FaSSIF/FeSSIF fluids represent a simplification of the proximal small intestine composition in the fasted state (FaSSIF) or in the fed state (FeSSIF). While FeSSIF contains low concentrations of lecithin and sodium taurocholate, (0.8 mM and 3 mM respectively) and has a pH of 6.5, FaSSIF contains 3.8 mM lecithin and 15 mM taurocholate and is at pH 5. SIF, FeSSIF and FaSSIF were prepared at twice the final concentration.

2.10. Statistical analysis Results were expressed as mean values ± S.D. A Student's t-test was used for statistical comparison. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Formulation AmBeHePC-loaded nanocochleates were prepared according to a process described by Zarif et al. [18] and Jin et al. [19]. However, the methodology was adapted to allow two drugs to be incorporated into the same formulation. The incorporation of HePC into the formulation was a critical step because of the surfactant properties of this molecule. HePC has a critical micellar concentration (CMC) of approximately 2.5 mM in distilled water [23] and therefore forms micelles in aqueous media that may be able to solubilize other amphiphilic molecules such as phospholipids. This behaviour could have hindered the formation of liposomes from DOPS. In order to counteract this effect, cholesterol was included in the formulation. This sterol is known to act as

Fig. 1. General procedure for preparing the AmBeHePC-loaded cochleates.

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preliminary observations and those of Zarif et al. [18] indicated that the smaller the liposomes, the smaller the final nanocochleate particles were. Small particle size is a prerequisite for the formulation of poorly water-soluble drugs because the increased surface area and the decreased diffusion layer thickness leads to an enhanced dissolution rate [29,30]. We chose to use a combination of sonication and extrusion to produce small unilamellar liposomes (SUV) as illustrated in Fig. 2B. This procedure was found to be the most efficient at obtaining the required liposome size while avoiding prolonged sonication or a very larger number of passages through the extruder which might provoke degradation of drugs and lipids. The SUV had a mean diameter of 80 nm as measured by quasi-elastic light scattering. This was slightly larger than

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liposomes prepared by the same procedure without cholesterol (62 nm), which can be explained by the increased rigidity of the bilayers containing cholesterol. The zeta potential of the SUV, measured in washing buffer (1 mM CaCl2 and 150 mM NaCl) was about 40 mV whatever the cholesterol content, consistent with the use of DOPS as the main component. The addition of the divalent cation Ca2þ is able to promote aggregation of these negatively charged SUV as a result of electrostatic interactions, so that these SUV fuse and form large sheets. This phenomenon can be seen in the TEM images in Fig. 2C and D. These sheets then roll up into the cigar-like structure of nanocochleates, excluding water (Fig. 2E). The nanocochleate suspensions obtained were homogeneous in size and shape, with an

Fig. 2. TEM images of AmBeHePC-loaded MLV (A) or SUV (B), large sheets formed from the SUV (C, D), AmBeHePC-loaded nanocochleates (E), AmBeHePC-loaded nanocochleates converted to liposomes after addition of EDTA (F).

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Table 1 Encapsulation efficiency of AmB and HePC in MLV and SUV; encapsulation efficiency, payload and product yield of AmBeHePC-loaded into nanocochleates (mean of 3 preparations). AmB encapsulation yield (%) in MLV HePC encapsulation yield (%) in MLV AmB encapsulation yield (%) in SUV HePC encapsulation yield (%) in SUV AmB encapsulation yield (%) in nanocochleates HePC encapsulation yield (%) in nanocochleates AmB payload (mg/g) in nanocochleates HePC payload (mg/g) in nanocochleates Product yield (%) in nanocochleates

79 81 55 60 54 59 29.9 14.6 75

± ± ± ± ± ± ± ± ±

1 3 2 2 1 4 0.5 0.5 4

average diameter of 252 ± 2 nm and a slightly negative potential zeta 2.3 ± 0.6 mV. This change in zeta potential compared with liposomes measured under the same conditions reflects the neutralization of the charge on DOPS by Ca2þ ions. No modification in the morphology and size of these nanocochleates was observed after lyophilisation and resuspension. This result is in agreement with the publication of Zarif et al. [31] and is in contrast to the behaviour of liposomes, which have a tendency to aggregate and fuse during lyophilisation unless large quantities of cryoprotectors are added [32]. This difference is probably due to the fact that the core of the nanocochleates excludes water. The amenability of nanocochleates to lyophilization is a very important advantage for their use as a drug delivery system for antiparasitic drugs, because it allows long-term storage and transport without the need for refrigeration. Moreover, the powder form opens up a number of possibilities for the final formulation: capsule, tablet or suspension. Fig. 2F shows that the formation of nanocochleates can be reversed by the removal of Ca2þ. When EDTA, a chelating agent, was added, the nanocochleates open up to form very large liposomes. This is further proof of the mechanism of formation illustrated in Fig. 1. 3.3. Determination of encapsulation efficiency of AmB and HePC within nanocochleates The encapsulation yields in the original MLV were 79% and 81% for AmB and HePC respectively (Table 1). However, after the transformation into SUV, these values fell to 55% and 60% for AmB and HePC respectively. After nanocochleate formation, an encapsulation efficiency of 54 ± 1% for AmB and 59 ± 4% for HePC was found (Table 1). A drug payload of about 29.9 ± 0.5 mg/g for AmB and 14.6 ± 0.5 mg/g for HePC was obtained (Table 1). Thus, when SUV were converted into nanocochleates, there was no significant difference in the encapsulation yield of the drugs. On the other hand, when nanocochleates were formulated directly from MLV without any size reduction, the payloads obtained were

32.6 ± 0.3 mg/g for AmB and 16.4 ± 0.8 mg/g for HePC, respectively 8% and 10% higher than those obtained from SUV converted into nanocochleates. This suggests that the size reduction procedure leads to a loss of drugs and/or lipids. The total product yield, taking into account all the components, for the procedure from SUV was 75% (Table 1); about 20% higher than the encapsulation yields for the drugs. Therefore, the loss of drugs was greater than that of the lipids. This may be due to breaking and structural rearrangement of the lipid bilayer during the sonication and extrusion process. It would be interesting to follow the yield of each individual component at each stage of the preparation to obtain more detailed information about the process of nanocochleate formation. Despite the higher encapsulation yields in nanocochleates formed from MLV, these formulations had a higher final particle size than those obtained from SUV. The formulation of drugs as nanosized particles would greatly increase their surface area and thereby facilitate dissolution in the GI tract [29,30]. In this present study, we chose to optimize the size of the nanocochleates, and accept the small reduction in encapsulation efficiency. In future work, a study of the compromise between the size and the drug payload will be carried out with a view to improving oral bioavailability and minimizing the adverse effects associated with AmB and HePC. 3.4. Stability of nanocochleates over time Unloaded nanocochleates and AmBeHePC-loaded nanocochleates were physically stable when stored in the lyophilized form at 2e8  C for at 6 months. No significant changes in size, pH and zeta potential in the reconstituted particles were observed (Table 2). This result is in agreement with those of Zarif et al. [31]. 3.5. CD and UVevisible spectroscopy The AmB molecule has a particular cyclic structure with seven conjugated double bonds which exhibit intense peaks of absorption between 250 and 450 nm. This means that electronic absorption and CD spectroscopy are useful tools for studying the organization of this molecule, since its spectral properties depend on whether it is monomeric, aggregated or complexed with other molecules [33e37]. Since the toxicity of AmB is known to be related to its aggregation state [38,39], we were interested in the molecular organization of AmB within the nanocochleates. It is important that the samples for UV and CD spectroscopy are homogeneous and free of highly light-scattering particles (more than 0.2 mm) in order to prevent interference [40]. In fact, the UV and CD spectra of the suspension of AmB-loaded-nanocochleates showed too much non-specific light scattering to allow interpretation (data not shown). Therefore, the organization of AmB was investigated in the drug-loaded liposomes before the conversion of liposomes into nanocochleates and also after the reconversion of

Table 2 Stability of nanocochleates stored at 2e8  C (n ¼ 6). Times (months) (A) AmBeHePC-loaded nanocochleates Mean hydrodynamic diameter (nm) Polydispersity index Zeta potential (mV) AmB payload (mg/g of powder) HePC payload (mg/g of powder) (B) Unloaded nanocochleates Mean hydrodynamic diameter (nm) Polydispersity index Zeta potential (mV)

Q1 At preparation 252 0.155 2.3 29.9 14.1

± ± ± ± ±

2 0.026 0.6 0.2 1.0

254 ± 2 0.167 ± 0.016 2.5 ± 0.6

1 253 0.186 2.0 29.9 14.2

3 ± ± ± ± ±

1 0.013 1.4 0.5 1.4

252 ± 1 0.152 ± 0.055 2.0 ± 1.0

253 0.175 2.6 29.9 14.7

6 ± ± ± ± ±

1 0.020 1.1 0.5 0.9

253 ± 2 0.175 ± 0.025 3.6 ± 0. 5

254 0.164 2.3 30.0 14.8

± ± ± ± ±

2 0.019 0.2 0.6 1.4

254 ± 2 0.182 ± 0.032 2.6 ± 0.5

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cochleates into liposomes by the addition of EDTA followed by extrusion to reduce particle size. The absorption spectrum of AmB prepared from a stock solution in DMSO diluted in water to a sub-micellar concentration (107 M) has been described in literature as showing a small band at 344 nm and three bands at 365, 385 and 410 nm and, characteristic of the monomeric form [41]. As the AmB concentration is increased up to 105 M, the small band at 344 nm increases in intensity and in width; the other three bands undergo red shifts to respectively, 368, 390 and 420 nm, together with a reduction in intensity, all these changes being considered as indicating AmB self-association. The UVevisible absorption spectrum of 3  105 M AmB incorporated into liposomes presented a scattering background and four bands at 327, 368, 391, and 415 nm (Fig. 3A). The shift of the peak from 340 nm to 327 nm was accompanied by a decrease of the intensity (once the scattering has been removed). It is well established that the monomeric form of AmB yields a CD spectrum with three weak positive bands at 365, 385 and 410 nm. On the other hand, the CD spectrum of the self-associated form of AmB displays a very intense dichroic doublet centred at

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340 nm, and negative bands at 368, 390 and 420 nm [41]. In water at 105 M, the CD spectrum of AmB shown in Fig. 3B exhibited a strong dichroic doublet centred on 339 nm accompanied by three negative bands, characteristic of self-associated AMB [41]. Fig. 3B shows that the association with liposomes led to modifications in the CD spectra of AmB: (i) appearance of a strong positive peak at the wavelength of absorption, i.e. 327 nm, resulting in the shift of the intense dichroic doublet from 340 to 332 nm, (ii) decrease of the intensity of the negative bands and (iii) appearance of a positive band at 428 nm. All these characteristics are largely documented in former studies about the interaction of cholesterol-containing egg yolk phosphatidylcholine vesicles [41e44]. They can be interpreted as an indication of AmB interaction with the membrane lipids. The UV and CD spectrum of AmBeHePC-loaded nanocochleates reconverted into liposomes were similar to those obtained with the original liposomes (Fig. 3A, B), suggesting that the interactions between AmB and the lipids are established during liposome preparation and are not changed by the subsequent conversion into nanocochleates. In fact, the transformation from liposomes to nanocochleates and their reconversion back to liposomes when calcium is chelated by EDTA do not involve modifications in the bilayer structure, the only important change is that the lipid sheets made from the fusion of liposomes due to the addition of the divalent cations roll up into a dehydrated cigar-like structure. The UV and CD results were therefore in agreement with our results obtained with monolayer study: in the nanocochleate structures, AmB can integrate into the lipid bilayers and form complexes with the components [28]. The fact that the UV and CD spectra of the formulations also containing HePC were identical to those with AmB alone indicates that there is no significant interaction between the two drugs. This would be expected given that each was present at only 5% molar proportions in the formulation. The observation that AmB within the nanocochleates is probably not self-associated but complexed with lipids has important consequences for the biological properties of the formulation. It is likely that AmB will be released in its monomeric form in the gastro-intestinal tract after oral administration. This will protect intestinal cells from AmB-mediated toxicity and improve drug absorption, leading to higher bioavailability. 3.6. Stability of AmBeHePC nanocochleates in simulated gastrointestinal media

Fig. 3. UV spectra (ε M1 cm1) (A) and CD spectra (Dε M1 cm1) (B) of AmB obtained from different preparations.

After oral administration of a drug delivery system, the first barrier to overcome is the physicochemical environment of the gastrointestinal tract. Therefore we investigated the stability of AmBeHePC-loaded nanocochleates in different media mimicking the gastrointestinal environment. Fig. 4 shows the evolution of mean particle hydrodynamic diameter with time when the preparations were incubated in stimulated gastric medium for 55 min followed by the addition of concentrated medium to convert the conditions to those of the intestinal environment in different circumstances. Samples were taken at intervals for a total period of 9 h. As well as analysis by quasi-elastic light scattering, observations by TEM were made. The hydrodynamic diameters of the particles showed a high degree of variability but a clear tendency emerged (Fig. 4): an initial increase in size followed by a decrease. By considering these results together with the TEM images shown in Fig. 6, we can put forward a hypothesis about the behaviour of the nanocochleates. The size increase over the first 55 min, in the SGF medium, can be interpreted as the unrolling of the cochleate cylinders into large sheets, as illustrated in Fig. 5A, B and C. This could be the result of the dilution factor, displacing Ca2þ from the interaction with DOPS

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2000 1500 Size (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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observations (Fig. 5D, E, F). SIF contains a pancreatin extract rich in enzymes, including lipases, which could disrupt the nanocochleate structure by converting DOPS to lysoDOPS [45]. FaSSIF and FeSSIF both contain a bile salt, sodium taurocholate, which is much more concentrated in FeSSIF than in FaSSIF. It is well known that bile salts can form mixed micelles with phospholipids and disrupt vesicular structures [47]. As a phospholipid with unsaturated acyl chains, DOPS would be particularly susceptible to forming mixed micelles with bile salts, although the presence of cholesterol can protect to a certain extent [48].

0 0

3

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3.7. In-vitro release study

Time (h) Fig. 4. AmBeHePC-loaded nanocochleate particle size during incubation in simulated gastric fluidesimulated intestinal fluid (SGFeSIF), simulated gastric fluidesimulated intestinal fluid in a fasted state (SGFeFaSSIF) or simulated gastric fluidesimulated intestinal fluid in a fed state (SGFeFeSSIF). (n ¼ 3; data are shown as mean ± S.D.).

according to the law of mass action. Binder et al. [45] investigated the specificity of ion binding to phospholipids in terms of headgroup structure, hydration and lyotropic phase behaviour by means of infrared spectroscopy. Their results divided the investigated ions into three groups: Ba2þ, Sr2þ, Naþ, Kþ, Liþ only weakly affected hydration and structure of the polar interphase; Mg2þ and Ca2þ caused partial dehydration, a conformational change and immobilization of the phosphodiester groups; Be2þ, Cu2þ and Zn2þ which ions cause considerable dehydration of the phosphate and carbonyl groups by direct, strong interactions and/or by a conformational change in this region. According to this classification, the Ca[DOPS]2 complex would only be partially stable and susceptible to disruption on dilution. Furthermore, the SGF is extremely acidic and it is possible that divalent calcium cations are replaced by monovalent protons, leading to decomplexation and unrolling of the nanocochleates. On addition of the simulated intestinal fluid, a large reduction in the mean hydrodynamic diameter of the AmBeHePC-loaded nanocochleates was observed, regardless of the type of medium (SIF, FaSSIF or FeSSIF). This decrease was particularly rapid in the case of the FeSSIF medium, and was corroborated by the TEM

The release of AmB and HePC from the nanocochleates was first studied in a simple medium (PBS buffer 0.05 M pH 7.4). Release occurred in two phases (results not shown). During the first hour, only about 1.5% of AmB and 3% of HePC were released. This is consistent with a strong interaction in the form of a complex between the drugs and lipids used to prepare the nanocochleates, as previously observed in monolayer experiments [28] and in the CD spectra as described above. The second stage was a relatively slow release phase which lasted for almost 24 h. The release mechanism at this stage could be the diffusion of drug localized at the surface of large sheets. In fact, when the nanocochleates begin to unfurl into large sheets as observed with TEM images (data not shown) the surface area would increase and facilitate drug release. Although the curves were very similar for the two drugs, the percentage of HePC release was slightly higher than that of AmB, probably as a result of the amphiphilic properties of the former. 3.8. Release in simulated gastro-intestinal medium Drug release from the nanocochleates was studied at the same time as the evolution of nanocochleate size and shape, in the simulated gastric and intestinal media described in Section 3.6. As shown in Fig. 6, only approximately 13% of the initial amount of encapsulated AmB and 15% of the initial amount of encapsulated HePC were released over the first 55 min, corresponding to the

Fig. 5. TEM images of AmBeHePC-loaded nanocochleates in SGF-FeSSIF during time: 10 min (A), 30 min (B), 55 min (C), 2 h (D), 4 h (E), 9 h (F).

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(A)

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Percentage of AmB released (%)

80 70 60 50 40 30 20

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Time (h) Fig. 6. Percentage of AmB (A) and HePC (B) released in a simulated gastric fluidesimulated intestinal fluid (SGFeSIF), in a simulated gastric fluidesimulated intestinal fluid in a fasted state (SGFeFASSIF) and in a simulated gastric fluidesimulated intestinal fluid in a fed state (SGFeFESSIF) (n ¼ 3; data are shown as mean ± S.D.).

period spent in the simulated gastric medium. This medium has a pH of 1.2 and contains pepsin, a protease present in gastric secretions. Zarif et al. [13] demonstrated the AmB-cochleate formulations were able to resist the degradation in the gastrointestinal tract due to their stable phospholipid-cation crystalline structures consisting of a spiral lipid bilayer sheet with no internal aqueous space. However, our observations of particle size and morphology by TEM suggest that there is an unrolling of the phospholipid sheets during this phase. However, the release results indicate that even under these conditions, the interactions between the lipids and the drugs are strong enough to retain AmB and HePC within the formulation. After the addition of the simulated intestinal medium, the release rate increased and was dependent on the type of medium added. SIF medium contains digestive enzymes in the pancreatin extract, whereas FaSSIF and FeSSIF contain lecithin and bile salts, with higher concentrations in the latter. For both drugs, the most rapid release was obtained in FeSSIF, followed by FaSSIF and then SIF (Fig. 6). The percentages of drug released after 2 h were in the range of 40e46% and 51e58% for AmB

and HePC respectively in SIF compared with 52e59% and 60%e69% in FaSSIF and 63e72% and 66%e78% in FeSSIF. These results demonstrate that destabilization of the nanocochleates by bile salts could be a more important release mechanism that lipolytic destruction and correlate well with the evolution of nanocochleate size and morphology shown in Figs. 4 and 5. The interaction of the nanocochleates with bile salts could have important consequences for the oral bioavailability of the two drugs, especially AmB. Firstly, mixed micelles and small fragments of bilayer would present a very large surface area for drug dissolution. Secondly, bile salts are known to promote intestinal absorption of drugs [49]. Thirdly, Dangi et al. [50] showed that mixed micelles formed with bile salts lipids and AmB increased the permeability of the drug across the intestinal barrier in a gut perfusion model by a factor of 20. 4. Conclusion The nanocochleate formulation described in this work would appear to have suitable properties for an orally active formulation

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containing both AmB and HePC. The formulation is able to retain and protect the drugs in the gastric environment and provide gradual release in the intestine. Furthermore, the formulation is extremely stable in a lyophilized form. In-vivo studies are now in progress to evaluate the bioavailability of AmBeHePC-loaded nanocochleates in catheterized rats. Uncited reference [46]. Acknowledgements This work was supported by a Ph.D. scholarship to T.T.H. Pham from the Region Ile-de-France. We are grateful to Zentaris (Frankfurt, Germany) for providing the HePC used in this study. This work benefited from the facilities and expertise of the Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr)
ne
ral de l'Essonne. The auwhich is supported by the Conseil Ge thors thank Valentina Facciolo and Serena Lombardi from University of Ferrara (Italy) for their technical help and Dr Claire Boulogne (Imagif Cell Biology) for electron microscopy observations. References [1] P. Desjeux, Leishmaniasis: public health aspects and control, Clin. Dermatol. 14 (1996) 417e423. [2] B.L. Herwaldt, Leishmaniasis, Lancet 354 (1999) 1191e1199. [3] P. Desjeux, Leishmaniasis: current situation and new perspectives, comparative immunology, Microbiol. Infect. Dis. 27 (2004) 305e318. [4] P. Desjeux, The increase in risk factors for leishmaniasis worldwide, Trans. R. Soc. Trop. Med. Hyg. 95 (2001) 239e243. [5] S. Sundar, M. Rai, Laboratory diagnosis of visceral leishmaniasis, Clin. Diagn. Lab. Immunol. 9 (2002) 951e958. [6] J. Alvar, S. Croft, P. Olliaro, H.M. David, Chemotherapy in the treatment and control of leishmaniasis, in: Advances in Parasitology, Academic Press, 2006, pp. 223e274. [7] J. Berman, Recent developments in leishmaniasis: epidemiology, diagnosis, and treatment, Curr. Infect. Dis. Rep. 7 (2005) 33e38. [8] S. Sundar, F. Rosenkaimer, M.K. Makharia, A.K. Goyal, A.K. Mandal, A. Voss, P. Hilgard, H.W. Murray, Trial of oral miltefosine for visceral leishmaniasis, Lancet 352 (1998) 1821e1823. [9] P.J. Guerin, P. Olliaro, S. Sundar, M. Boelaert, S.L. Croft, P. Desjeux, M.K. Wasunna, A.D.M. Bryceson, Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda, Lancet Infect. Dis. 2 (2002) 494e501.
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