Journal of Controlled Release 172 (2013) 904913

Contents lists available at ScienceDirect

Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre l

NANOMEDICIN

E

An oral malaria therapy: Curcumin-loaded lipid-based drug deliverysystems combined with -arteether

Patrick B. Memvanga a,b, Rgis Coco a, Vronique Prat a,a Universit catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and drug delivery group, Avenue Mounier 73, B1.73.12, 1200 Brussels, Belgiumb University of Kinshasa, Faculty of Pharmaceutical Sciences, Laboratoire de Pharmacie galnique, BP 212 Kinshasa XI, Democratic Republic of Congo

Corresponding author. Tel.: +32 2 764 7320; fax: +3E-mail address: Veronique.preat@uclouvain.be (V. Pr

0168-3659/$ see front matter 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.jconrel.2013.09.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 June 2013Accepted 1 September 2013Available online 7 September 2013

Keywords:Curcumin-arteetherLipid-based formulationsOral deliveryCaco-2 cellsAntimalarial efficacy

Curcumin (CC), a potential antimalarial drug, has poor water solubility, stability and oral bioavailability. To cir-cumvent these pitfalls, lipid-based drug delivery systems (LBDDSs) with a high CC loading (30 mg/g) were for-mulated. In a biorelevant gastric medium, CC-LBDDSs formed particle sizes in the range of 3040 nm. Duringin vitro lipolysis, 9095% of the CC remained solubilized, whereas 510% of the CC precipitated as an amorphoussolid, with a high rate of re-dissolution in a biorelevant intestinal medium. The transport of the CC-LBDDS acrossCaco-2monolayers was enhanced compared with the transport of free drug because of the increased CC solubil-ity. In Plasmodium berghei-infected mice, modest antimalarial efficacy was observed following oral treatmentwith CC-LBDDSs. However, the combination therapy of CC-LBDDS with a subtherapeutic dose of -arteether-LBDDS provided an increase in protection and survival rate that was associated with a significant delay in recru-descence. These findings suggest that the combination of oral CC and -arteether lipid-based formulations mayconstitute a promising approach for the treatment of malaria.

2013 Elsevier B.V. All rights reserved.

1. Introduction

Malaria is the most prevalent parasitic disease and the foremostcause of morbidity and mortality in the world [1]. Currently, thetreatment of choice for malaria is based on the association of anartemisinin-type compound with another drug [1,2]. The combinationantimalarials with different mechanisms of action can ensure very highcure rates and prevent the onset of drug resistance or delay its emer-gence [1]. Indeed, these combination therapies may reduce the impactof artemisinin-induced dormancy (or quiescence) of a subpopulationof the ring stage parasites, a mechanism that allows the parasites tosurvive under the pressure of high doses of artemisinins [3,4]. How-ever, non-viable parasites can retain the ability to cytoadhere, whichcontributes to the disease [5]. Moreover, a variety of reasons, includ-ing cost considerations, pharmacokinetic mismatch, resistance, cross-resistance and side effects due to the partner drug, can also contributeto treatment failure [1,6].

Numerous investigations have demonstrated that curcumin, a hy-drophobic polyphenol extracted from the rhizomes of Curcuma longa,exhibits an antimalarial activity both in vitro and in vivo [7,8]. Therefore,curcumin (CC) has been proposed as an attractive partner drug for-arteether (an artemisinin derivative) due to its short-life (12 h),which closely matches the half-life of artemisinin derivatives [911].CC acts as an adjunctive therapeutic strategy in the artemisinin-combination therapies to protect against parasite recrudescence and

2 2 764 7398.at).

ghts reserved.

relapse [6,1014]. Indeed, CC synergizes with artemisinins as an anti-malarial to kill the parasites [6,15] and can also reverse cytoadherenceand subsequent sequestration of parasitized erythrocytes by the inhibi-tion of NF-B activation, which downregulates the proinflammatorycytokine responses and the expression of cytoadhesion molecules inendothelial cells [16,17]. By producing a transient induction of reactiveoxygen species in infected red blood cells, CC is able to damage theparasitic DNA [7] and to prime the immune system (the activation ofTLR2 and IL-10 and antiparasitic antibody production) [11]. CC alsoupregulates the cyto- and neuro-protective enzymes and the surfaceexpression of CD36 on monocytes/macrophages, which increases theirphagocytic activity [17]. Treatment with CC also inhibits the microtu-bule activity [18] and the formation of hemozoin, an intraerythrocyticpigment that is essential to the survival of the parasites [6,11,13].

Due to its safety, relative abundance and cost effectiveness, CC is aphytochemical of great interest in the treatment of several diseases,including malaria [16]. However, the major disadvantages of CC whenadministered orally are its low bioavailability due to its poor water sol-ubility, chemical instability at neutral and alkaline pH, poor absorptionassociated with its high rate of metabolism and its rapid elimination[16,19]. Encapsulations in nanoparticles [13,14,20], nanoemulsions[21,22], cyclodextrins [23], phospholipid complexes [24] or solid dis-persions [25] have been proposed to improve the oral bioavailabilityand the therapeutic efficacy of CC (see [26] for review). However, thesestrategies are characterized by several weaknesses, such as high costs,cytotoxicity, limited drug loading and entrapment efficiency, poor scaleup and the use of organic solvents. Self-emulsifying lipid-based systemsare a simple and effective approach to solubilize hydrophobic drugs in

http://crossmark.crossref.org/dialog/?doi=10.1016/j.jconrel.2013.09.001&domain=pdfhttp://dx.doi.org/10.1016/j.jconrel.2013.09.001mailto:Veronique.preat@uclouvain.behttp://dx.doi.org/10.1016/j.jconrel.2013.09.001http://www.sciencedirect.com/science/journal/01683659

Fig. 1. Pseudo-ternary phase diagram for Labrafil M2125CS-Labrasol-Cremophor ELmixturediluted in 0.1 N HCl, pH 1.2 (1:100, w/v). The gray area represents the range of existence ofa nanoemulsion. The red dot highlights the selected self-nanoemulsifying system: LabrafilM2125CS-Labrasol-Cremophor EL at weight ratio of 1:1:1. (For interpretation of the refer-ences to color in this figure legend, the reader is referred to the web version of this article.)

Table 1Composition and characteristics of selected CC-LBDDSs.

Formulation A Formulation B

Composition (mg)Labrafil M2125CS 100 100Labrasol 100 100Cremophor EL 100 100Transcutol HP 50CC (mg/g excipient) 30 30

Droplet size (nm), polydispersity index a

FaSSGF 38.0 3.1 (0.27) 42.0 2.7 (0.29)FaSSIF pH 6.8 49.2 1.2 (0.17) 56.4 0.5 (0.31)FaSSIF pH 7.5 (lipolysis medium) 50.2 2.8 (0.14) 61.8 2.3 (0.18)HBSS 32.1 0.1 (0.03) 29.5 0.3 (0.09)

a Polydispersity index (PDI) are given in the parentheses (n = 3).

905P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

the gastrointestinal tract and to protect these drugs from enzymaticand/or chemical hydrolysis, and these systems have been developed toimprove the oral bioavailability of CC [2729]. However, no antimalarialevaluation of CC in these systems has been reported.

The objective of this study was to develop novel oral lipid-baseddrug delivery systems (LBDDSs) with (i) a high CC loading, (ii) a highdegree of resistance to lipolysis, (iii) an increased intestinal absorptionand (iv) antimalarial efficacy either alone or in combination with-arteether loaded in lipid-based drug delivery systems.

2. Materials and methods

2.1. Materials

Curcumin (~80%), L--Phosphatidylcholine from egg yolk (~60%),pancreatin from porcine pancreas (activity 8 USP specifications),tributyrin and all the other chemicals were obtained from Sigma-Aldrich (Diegem, Belgium). Unless otherwise indicated, all cell cul-ture media and reagents were purchased from Invitrogen Gibco(Merelbeke, Belgium). Cremophor EL (polyoxyl 35 castor oil) waskindly provided by BASF (Burgbernheim, Germany). Labrafil M2125CS(linoleoyl polyoxyl glycerides), Labrafil M1944CS (oleoyl polyoxylglycerides), Labrasol (caprylocaproyl polyoxyl glycerides) and TranscutolHP (diethylene glycol monoethyl ether) were kind gifts from Gattefoss(Saint-Priest, France).

2.2. Simulated media

Fasted state simulated gastric fluid (FaSSGF) contained 80 M sodi-um taurodeoxycholate, 20 M lecithin, 0.1 mg/ml pepsin and 34.2 mMsodium chloride (pH 1.6) [30]. Fasted state simulated intestinal fluid(FaSSIF) contained 50 mM Tris-maleate, 150 mM sodium chloride,5 mM calcium chloride dihydrate, 5 mM sodium taurodeoxycholateand 1.25 mM lecithin, pH 6.8 [31]. The medium in the receiver com-partment of transport studies contained Hank's balanced salt solu-tion (HBSS) + 10 mM Hydroxyethyl piperazine ethanesulfonic acid(HEPES) + 1% (w/v) bovine serum albumin (BSA) [32,33]. The lipolysismedia contained 50 mMTris-maleate, 150 mM sodium chloride, 5 mMcalcium chloride dihydrate, 5 mM sodium taurodeoxycholate and1.25 mM lecithin, pH 7.5 [31,34].

2.3. The development of CC lipid-based self-emulsifying systems

The solubility of CC in various excipientswas estimated bydissolvingincreasing quantities of CC in 10 g of each excipient at room tempera-ture (20 2 C). After 2 h of stirring, the solubilization of CC wasobserved visually. The absence of CC crystals was confirmed using ami-croscope [35]. The solubility of CC in modified biorelevant media(FaSSGF and FaSSIF) and in the receiver compartment of the transportexperiment was determined by dispersing a large amount of free CCin these solutions at 37 C [36]. Over a period of 2 h, aliquots from thebiorelevant media were centrifuged for 15 min at 5000 g and 37 C.The supernatants were then diluted with methanol: 3.6% glacial aceticacid (73:27, v/v) and assayed for CC content by HPLC analysis. All exper-iments were performed in triplicate.

Based on the solubility study, the pseudo-ternary phase diagram(Fig. 1) was constructed using the water titration method. Differentmixtures of the surfactants (Labrasol and Cremophor EL at differentweight ratios from 10:0 to 0:10) were prepared with stirring at roomtemperature. The oil phase (Labrafil M2125CS)was then added at ratios(w/w) of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10. To deter-mine the feasibility of the self-(nano)emulsification, 1 g of each solution(surfactants + oil) was slowly titrated with 0.1 N HCl, pH 1.2 (100 ml,37 C), gently stirred and visually examined for transparency. Anotherseries of self-emulsifying systems was assessed with varying concentra-tions of Labrafil M2125CS: Labrasol: Transcutol HPmixture (2:2:1, w/w)

from 70 to 100% (w/w) and of Cremophor EL from 0 to 30% (w/w). Thedroplet sizewas determined at 37 C by photon correlation spectroscopyusing a Ztasizer Nano ZS model ZEN 3600 (Malvern Instrument Ltd,UK). Finally, to determine the maximum loading content of the CC inthe lipid-based systems, its solubility in the selected formulations wasdetermined in triplicate.

2.4. Preparation and characterization of CC-LBDDS

2.4.1. Preparation of CC-LBDDSThe CC-LBDDSs were prepared by mixing under agitation (400 rpm,

30 min, 20 C) the appropriate quantities of excipients (see Table 1).Thirty milligrams of CC was then added to 1 g of each LBDDS pre-concentrate (drug loading = 30 mg/g) and mixed for dissolution underagitation (400 rpm, 120 min, 20 C), protected from light.

2.4.2. Self-emulsification of CC-LBDDS in gastrointestinal and transportmedia

The size of droplets, the solubility and stability of CC-loaded LBDDS inFaSSGF, FaSSIF, lipolysis and transport (HBSS) media were assessed aspreviously described [35]. Briefly, 1.03 g of CC-LBDDS was gently stirredwith 36 ml of lipolysis medium or 2501000 ml of gastrointestinal and

906 P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

transport media at 37 C. After 20 min, the samples were removed forparticle size analysis by photon correlation spectroscopy. To evaluatethe solubility and the stability of CC, the samples or supernatants fromcentrifugation (5000 g, 15 min, 37 C) were diluted twice in a mixtureof methanol: 3.6% glacial acetic acid (73:27, v/v). The quantity ofCC present in the solution of the various media with or without cen-trifugation was assayed by HPLC analysis. All the measurements wereperformed in triplicate.

2.4.3. Stability of CC-LBDDS over 180 daysThe stability of CC in the lipid formulations selected was studied for

180 days at room temperature. During storage, the self-emulsifyingsystems were kept in sealed glass vials and protected from light. Thepresence or absence of drug re-precipitationwas initially observed visu-ally and then by microscopy [35]. One milliliter of the lipid-based sys-tems was then centrifuged (5000 g, 30 min, 20 C). After dispersion ofthe supernatant in 0.1 N HCl and an appropriate dilution in a methanol:3.6% glacial acetic acid (73:27, v/v) mixture, the quantity of CC presentin the solution was determined in triplicate by HPLC. The efficiency ofself-emulsification and the droplet size in the gastric medium were alsoassessed.

2.5. CC analysis by HPLC

An Agilent 1100 Series HPLC systemwith a diode array andmultiplewavelength detector was used for CC analysis. The column was an EC250/2 Nucleodur 100-5 C18 ec (Macherey-Nagel, Dren, Germany).The sample (50 l) was eluted with the mobile phase composed ofmethanol: 3.6% glacial acetic acid (73:27, v/v) [27]. The different sam-ples were analyzed after an appropriate dilution in the mobile phase.The system was run isocratically at a flow rate of 0.2 ml/min, and sam-ple detectionwas achieved at 428 nm. The retention time of the CCwasapproximately 6 min under these conditions. The limits of detection(LOD) and of quantification (LOQ) of CCwere 5 ng/ml and 15 ng/ml, re-spectively. The coefficients of variation (CVs) for intra- and inter-assaywere all within 5%.

2.6. Lipolysis of CC-LBDDS

Dynamic in vitro lipolysiswas performed using a pH-stat titration sys-tem (Metrohm Titrando 842; Software Tiamo 1.3, Herisau, Switzerland)as previously described [34,36]. Briefly, 1.03 g of CC-LBDDS was dis-persed in 36 ml of a lipolysis medium and stirred for 15 min at 37 C.The enzymatic digestion was initiated by the addition of 4 ml of freshlyprepared pancreatin extract (lipase activity of 1000 tributyrin units perml of digest). The fatty acids released during in vitro lipolysis were auto-matically titrated with 0.2 M NaOH to maintain a pH of 7.5. At differenttimes (15, 30, 45 and 60 min), aliquots were sampled, and a lipolysisinhibitor (0.5 M 4-bromophenylboronic, 9 l/ml of digestion medium)was immediately added. Subsequently, the aliquots were centrifugedfor 60 min at 37 C and48,000 g to separate the sample into an aqueousand a pellet phase. The samples obtained from each separated phasewere assayed for the CC content by HPLC analysis. Control experimentswith the lipolysis medium alone were also performed.

2.7. Analysis of the pellet obtained after lipolysis

The dissolution of the pellet obtained from centrifugation fromall the media sampled at the end of lipolysis was performed at 37 Cusing the USP II Apparatus with the paddle rotating at 100 rpm. Afterdispersion of two pellets in 10 ml ofwater, 500 ml of intestinal medium(pH 6.8, 37 C) was added. At each sampling time point, the sampleswere filtered through 0.45-m filters (Acrodisc, Pall, France) and thenanalyzed by HPLC for drug content. These experiments were conductedin triplicate.

To elucidate the solid state of the CC present in the isolated pellet,X-ray powder diffraction was performed on a Siemens D5000 diffrac-tometer using the K radiation of Cu ( = 1.5418 ). The 2 rangebetween 2 and 60 was scanned at a rate of 0.02/s with an appliedvoltage and current of 40 kV and 40 mA, respectively. The isolated pel-lets (CC pellets) were spread on a zero-loss sample holder (Si singlecrystal) and then allowed to air dry. Control experimentswere conductedusing blank pellets obtained from the lipolysis of unloaded-LBDDS andblank pellets spiked with the corresponding amount of crystalline drugspresent in the CC pellets [36,37].

2.8. In vitro intestinal cell models used to study the transport of CC

2.8.1. Cell culturesCaco-2 cells (clone 1), obtained from the University of Milano-

Bicocca, Italy, were used [38]. The cells were maintained in Dulbecco'smodified Eagle's minimal essential medium (DMEM) supplementedwith 10% (v/v) heat inactivated fetal bovine serum (Hyclone, ThermoScientific, UK), 1% (v/v) L-glutamine and 1% (v/v) non-essential amino-acids at 37 C in an atmosphere of 10% CO2. Caco-2 cells were usedbetween passage numbers x + 17 and x + 30 [35,38]. The humanBurkitt's lymphoma Raji-B cell line (American Type Culture Collection,VA, USA) was maintained in RPMI 1640 supplemented with 10% (v/v)heat inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v)non-essential amino acids at 37 C under a 5% CO2 humidified atmo-sphere. Raji-B cells were used at passage numbers 106110 [38,39].

2.8.2. CytotoxicityThe viability of the Caco-2 cells was assessed after the incubation of

2 104 Caco-2 cells/well with 100 l of unloaded-LBDDS dispersed inthe culturemedium in a 96-well tissue culture plate (Corning Costar).After 2 h of incubation, the supernatants of eachwellwere removed andkept at 4 C until the LDH assay. The cells were then incubated for 3 hwith 100 l of 0.5 mg/mlMTT [35]. The IC50 values of the lipid formula-tions were calculated using the GraphPad Prism5. The LDH activityreleased from the cytosol of damaged cells was measured according tothe manufacturer's instructions. Each experiment was performed intriplicate.

2.8.3. Caco-2 and FAE monolayersCaco-2 cells (5 105 cells/well) were seeded on 12-well cell culture

inserts with a 1 m pore diameter and 0.9 cm2 area (Corning Costar,NY, USA) and were grown in supplemented DMEM + 1% PEST for21 days [35,36,38]. The culture medium was changed every 2 days. Fortransport studies, only Caco-2 cell monolayerswith initial transepithelialelectrical resistance (TEER) values higher than 400 cm2 were used.

The inverted follicle-associated epithelium (FAE) model (includingM-like cells) was obtained by co-culturing Caco-2 and Raji-B cells[36,38,39]. Briefly, after 5 days of Caco-2 seeding at a density of5 105 cells/well onto 12-well cell culture inserts with a 3 m porediameter and 0.9 cm2 area (Corning Costar), the insertswere invertedand a piece of silicon tube was plated on the basolateral side of eachinsert. The inverted inserts were transferred into pre-filled Petri disheswith supplemented DMEM + 1% PEST and maintained for 11 days.The basolateral medium was refreshed every 2 days. Raji-B cells(2.5 105 cells/well) were added to the basolateral compartmentof the inserts. The co-cultures were maintained inverted for 5 days.Monocultures of Caco-2 cells, cultivated as described above but withoutthe addition of Raji-B cells, were used as controls. After removing thesilicon tube, the insertswere placed in 12-well multiwell plates (BectonDickinson, France) in their original orientation for the transport exper-iment. Permeation studies were conducted on cell monolayers withinitial TEER values greater than 200 cm2 for the monocultures andgreater than 100 cm2 for the co-cultures.

907P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

2.8.4. Permeation studies of free CC and CC-LBDDS across intestinal in vitromodels

The apical (A) to basolateral (B) transport experiments acrossCaco-2 monolayers were conducted by adding 0.5 ml of the CC solution(0.015 mg/ml in HBSS + 3 mMMES + 4% [v/v] methanol) or 0.5 ml ofdispersed formulations inHBSS (1.03 mg/ml of CC-LBDDS i.e. 0.03 mg/mlof CC, unless otherwise stated) on the apical compartment of the insertsand 1.2 ml of HBSS + 10 mM HEPES + 1% BSA in the basolateral com-partment. For the basolateral to apical transport experiments (B to A),1.2 ml of CC solution (0.015 mg/ml) was added at the basolateralside, while the apical side was filled with 0.5 ml of HBSS + 10 mMHEPES + 1% BSA. After 2 h, sampling of the acceptor compartment(basolateral for A to B transport or apical for B to A transport) wasperformed to determine the permeation of CC in solution or loadedin LBDDS. The amount of CC that had crossed the Caco-2 monolayerswas determined by HPLC.

The CC apparent permeability coefficient (Papp) was calculatedaccording to the following equation:

Papp dQdt

1CoA

;

where dQ/dt (transport rate) is the amount of CC (g) appearing pertime unit (s) in the receiver compartment, Co is the initial concentrationin the donor compartment (g/ml) and A is the surface area of themonolayer (A = 0.9 cm2).

The integrity of cell monolayers after 2 h of exposure to the CC-LBDDSs was assessed by TEER measurements using an electrodeconnected to an EVOM volt ohmmeter (World Precision Instruments,USA).

During the transport studies in the FAEmodels, A to B transport wasinvestigated as described above.

2.8.5. Intracellular CC in the cell monolayerAfter the transport studies, the intracellular amount of CC in the

Caco-2 cells was evaluated quantitatively by HPLC and qualitatively byconfocal laser scanning microscopy. For the HPLC analysis, the cellswere washed three times with HBSS + 3 mM MES, detached fromthe plates by trypsinization and then centrifuged (1500 g, 10 min).The supernatant was discarded, and the cells in the pellet were lysedin HBSS + 3 mM MES + 1% Triton (w/v) for 120 min at 37 C. Theamount of CC in the Caco-2 monolayers was then quantified.

For the confocal microscopy study, inserts fixed in 2% (v/v) bufferedparaformaldehyde (pH 7.4, 30 min) were washed twice in PBS. Tovisualize the cell boundaries, actin was immunostained with 250 lof rhodaminephalloidin (4 U/ml) (Molecular Probes, OR, USA) in PBSfor 30 min protected from light. After washing in PBS, the inserts werecut and mounted on glass slides with 4,6-diamidino-2-phenylindole(DAPI) (1:20) to visualize cell nuclei [40]. Images were captured usinga Carl Zeiss confocal microscope equipped with a multi-Argon laser.The fluorescence of CC was detected at 488 nm [41]. The data wereanalyzed by the Axio Vision software (version 4.8) to obtain yz, xzand xy views of the cell monolayers.

2.9. In vitro hemolytic study

To estimate the damage caused by the formulations on the eryth-rocyte membrane, a hemolysis test was performed as previously de-scribed [18,42]. Briefly, the blood from human healthy volunteers wascentrifuged for 10 min at 2000 g and the plasma discarded. The eryth-rocytes werewashed three times and then diluted 12-fold with isotonicphosphate buffer saline (PBS). Two milliliters of this erythrocyte sus-pensionwere incubatedwith 2 ml of the unloaded-LBDDS in dispersionin PBS (010 mg/ml). Incubation was carried out at 37 C for 30 minunder agitation. PBS was used as a negative control and Triton X-100(1%, w/v) as a positive control. After centrifugation (2000 g, 5 min),

the absorbance of the supernatants was measured on HumanLyzer2000 (Human, Germany) at 540 nm. The percentage of hemolysis wasdetermined by the following formula:

%Hemolysis astancapcanc

100;

where ast = absorbance of sample-test, apc = absorbance of positivecontrol, anc = absorbance of negative control. All the experimentswere performed in triplicate.

2.10. In vivo antimalarial efficacy of CC-LBDDS

The in vivo evaluation of the antimalarial activity of CC-LBDDS inPlasmodium berghei ANKA infection was performed by the curativetest as described previously [35,43]. Briefly, on day 0, mice were inocu-lated intraperitoneally with 1 107 P. berghei parasitized erythrocytes.Three days after infection (48% parasitemia), the mice were orallyadministered 0.1 ml of CC-LBDDS (at 0, 60 and 100 mg/kg/day) or aCC solution in dimethyl sulfoxide (DMSO) (at 0 and 100 mg/kg/day),once daily, for 4 consecutive days. Four others groups of mice receivedCC-LBDDS (at 0, 60, 80 and 100 mg/kg/day) and -arteether-LBDDS(at 0 and 12 mg/kg/day) in combination. -arteether (AE)-loadedLBDDSs were composed of groundnut oil (300 mg), Maisine 35-1(300 mg), Cremophor EL (300 mg), ethanol (100 mg), AE (200 mg)(Formulation C) or of sesame oil (300 mg), Maisine 35-1 (300 mg),Tween 80 (300 mg), ethanol (100 mg), AE (200 mg) (Formulation D)[35,36]. The final group of mice was infected but not treated. Parasitecounts were made on days 3, 7, 12 and 28 post-infection, and survivaltimes were also recorded. All animal experiments were approved byethical committee of animal use of the University of Kinshasa.

Antimalarial efficacy was assessed by the parasitemia level, theactivity, the mean survival time and the survival rate of the mice forup to 46 weeks following inoculation. Parasitemia was monitored bylight microscopy (oil immersion, 1000 magnification) by examiningthin smears of blood from the tail veins of themice, fixedwithmethanoland stained with Giemsa. The parasitemia level was determined bycounting, in random fields of themicroscope, the number of parasitizederythrocytes per 1000 erythrocytes. The average percent antimalarialactivity (equal to the percent suppression) was determined accordingto the following formula [44]:

Activity 1Meanparasitemiaof treatedgroupMeanparasitemiaof controlgroup

:

2.11. Statistical analyses

Significant differences between the droplet sizes, permeabilitycoefficients and percent distribution of CC into the various digestionphases were compared by one-way ANOVA with Tukey's post-hoc test(significance set at of p b 0.05). The cumulative survival rates from eachtreatment groupwere compared by the KaplanMeier survival analysis.

3. Results and discussion

3.1. Solubilization of CC in various excipients and in biorelevant media

To select the excipients for the self-emulsifying systems, the solubil-ity of CC in different excipients was determined (n = 3). CC exhibitedpoor solubility (b25 mg/g) in most of the lipid vehicles and surfactants(ethyl oleate, sesameoil, groundnut oil,Maisine 35-1, LabrafilM2125CS,Labrafil M1944CS, Tween 80 and Cremophor EL). This solubility reachedapproximately 85 mg/g in Labrasol. In the tested co-solvents, PEG 400yielded the highest solubility for CC (~140 mg/g), followed by TranscutolHP (~100 mg/g) and ethanol (~10 mg/g).

Fig. 2. Solubilization of CC-loaded LBDDS in FaSSGF (A) and in FaSSIF (B) after 60 min. Thetotal CC () and the solubilized CC () are expressed as the percentage of CC recoveredbefore and after centrifugation. Data are presented as the mean SD, n = 3.

908 P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

Due to their emulsion forming capability, Cremophor EL and Labrasolwere selected as surfactants. Labrafil M2125CS was also chosen becauseof its miscibility with Cremophor EL and Labrasol. After lipolysis, LabrafilM2125CS can also release oleic and linolenic acid that could contrib-ute to the antimalarial activity [4547]. Transcutol HP was selected asa solubilizer and absorption promoter; PEG-400 was not chosen dueto its high aqueous partition, which caused the precipitation of a largeamount of CC upon dilution (data not shown).

The solubility of CC in the simulated fluids containing bile saltsand phospholipids or albumin dramatically increased compared withwater (solubility = 11 ng/ml). Indeed, up to 0.5 g/ml, 5.4 g/ml and4.2 g/ml of CC were dissolved in FaSSGF, FaSSIF (pH 6.8 and pH 7.5)and HBSS + 10 mM HEPES + 1% BSA, respectively, after 2 h.

3.2. The development of oral CC self-emulsifying systems

To obtain self-emulsifying systems, pseudo-ternary phase diagramswere constructed using a water titration method with three differentmixtures: Labrafil M2125CS-Labrasol-Transcutol HP, Labrafil M2125CS-Cremophor EL-Transcutol HP and Labrafil M2125CS-Labrasol-CremophorEL. Labrafil M2125CS-Labrasol-Cremophor EL provided the highest self-nanoemulsion area (particle size b 100 nm), in agreement with previousresults (data not shown, [48]); hence, it was selected as the desirablemix-ture for the self-nanoemulsifying formulation development. When theaforementioned selected mixture contained relatively high quantities ofCremophor EL, it formed a viscous gel that slowly dissolved over time. Inaddition, its cytotoxicity was enhanced as the concentrations of Labrasolincreased (data not shown). Based on self-emulsification efficiency andcytotoxicity, a first self-nanoemulsifying system (Formulation A) wasretained and is highlighted by a dot in the phase diagram (Fig. 1). Thedroplet size of Formulation A in gastric media (1%, w/v) was approx-imately 30 nm (Polydispersity index, PDI b 0.25).

The mixture of Labrafil M2125CS, Labrasol and Transcutol HP(2:2:1, v/v) was previously shown to be well-tolerated in rats [49]but was not able to self-nanoemulsify and yielded an unstable emulsionin 0.1 N HCl (a droplet size of 3 m on average). The incorporation ofincreasing concentrations of Cremophor EL (0 to 30%, w/w) facilitatedits self-nanoemulsification and decreased the size of the resulting oildroplets from 3 m to 35 nm. Hence, a second self-nanoemulsifyingsystem (Formulation B) containing LabrafilM2125CS, Labrasol, TranscutolHP and Cremophor EL (2:2:1:2, w/w) was selected. The composition ofthe two selected LBDDSs is detailed in Table 1.

The solubility of CC in the selected self-emulsifying systems wasthen evaluated. Up to 42 mg/g of CC were solubilized in both Formula-tion A and Formulation B. However, to avoid the rapid precipitation ofCC after dilution in the aqueous medium, the CC loading was reducedto 30 mg/g (70% of saturation solubility).

3.3. Dilution of CC-LBDDS in gastrointestinal and transport media

To verify whether the CC-loaded LBDDSs could self-emulsify in thegastrointestinal tract, 1.03 g of the formulation was diluted in 2501000 ml of gastro-intestinal or in 36 ml of the lipolysis medium. Thesize of the droplets, the solubility and the stability of CC were thenassessed. In the gastrointestinal medium, Formulation A and Formu-lation B dispersed spontaneously to form nanoemulsions with sizesof 3040 nm (PDI b 0.25). Dilution did not induce changes in thesize of droplets, confirming the kinetic stability that characterizesself-nanoemulsifying drug delivery systems (SNEDDSs). Additionally,no phase separation was observed after centrifugation for 15 min at5000 g. The incorporation of CC in the self-emulsifying systems didnot change significantly the size and the polydispersity index of drop-lets compared to unloaded-LBDDS (p N 0.05, Table 1). In the lipolysismedium, CC-LBDDSs formed particles with a size of approximately60 nm and a polydispersity index of 0.15 (Table 1).

In 250 ml of FaSSGF, 8095% of the CC (~96114 g/ml) containedin LBDDS was dissolved after 1 h, whereas more than 96% of CC(~115 g/ml) was dissolved in the FaSSIF (pH 6.8) (Fig. 2). The dilutionof eachmedium up to 1000 ml did not significantly change the quantityof solubilized CC (p N 0.05, Fig. 2). After 1 h, the total CC content wasmore than 95% in gastric and intestinal media, indicating an improve-ment in CC stability (Fig. 2). Over periods of 1 h and 3 h, CC-LBDDS(1.03 to 4.12 mg/ml) showed a similar solubility and stability in thetransport medium (HBSS buffer) and in the intestinal medium (datanot shown).

The stability of free CC in the transport media is also crucial to avoidan erroneous estimation of its permeability. Consistent with resultspreviously reported [50], approximately 5060%of the CCwasdegradedin the HBSS buffer (pH 7.4) after 2 h. The impact of this degradation inthe donor compartment was suppressed to less than 5% by the adjust-ment of the pH of HBSS to 6.5 with 3 mM MES. Additionally, the useof HBSS + 10 mM HEPES + 1% BSA (pH 7.4) in the receiver chamberwas highly effective at stabilizingmore than 85%of the CC, which agreeswith previously reported results [51].

A preliminary study of stability was conducted at room temperature(20 2 C) and protected from light. Over 180 days, the percentageof CC remaining in the formulations was greater than 98.5% (data notshown). None of the LBDDS samples showed any change in color or ap-pearance under the above-described storage conditions. Moreover, nochange in the droplet size and in the self-nanoemulsification efficiencywas observed (data not shown).

3.4. In vitro dynamic lipolysis

3.4.1. In vitro lipolysisTo determine whether CC precipitates due to intestinal digestion of

the lipid vehicles present in LBDDS, a dynamic lipolysis procedure was

909P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

performed, as previously described [34,36]. The course of NaOH addi-tion as a function of time during the lipolysis of CC-LBDDS is shown inFig. 3A. The quantity of NaOH used to titrate the fatty acids releasedby the lipid formulations ranged from 1.15 to 1.35 mmol for both theformulations.

After centrifugation of the digested formulations, a pellet and anaqueous phase were isolated. The amount of the non-precipitated CCin the aqueous phase and the amount of the precipitated CC in the pelletas a function of time are shown in Fig. 3B. For all the formulations, 9095% (2728.5 mg) of the CC was present in the aqueous phase at eachtime point of the lipolysis, and 510% (1.53 mg) was present in thepellet phase. The dispersion of CC-LBDDS in the lipolysis medium

Fig. 3. In vitro dynamic lipolysis of Formulation A (, ) and Formulation B (, ).(A) Quantity of 0.2 M NaOH added to titrate the fatty acids that were released duringlipid digestion. (B)Distribution profile of CC in the aqueousphase (open shapes anddottedlines) and in the pellet phase (filled shapes and lines) as a function of lipolysis time.(C) Dissolution rate of CC in pellets at the end of lipolysis (60 min). (D and E) The X-raypowder diffraction pattern of (a) crystalline, (b) CC pellet, (c) blank pellet spiked withCC and (d) blank pellet from the lipolysis of Formulation A (D) and Formulation B (E).The numbers over the peaks indicate d-spacings.

Fig. 3 (continued)

(36 ml, no lipase)maintained the CC in a solubilized state; no precipita-tion was observed after 30 min.

3.4.2. X-ray powder diffraction and CC dissolution in pellets after lipolysisTo determine the solid state of theprecipitated CC, X-raypowder dif-

fraction was performed on the CC pellets. These results show similarcharacteristic reflections between the typical X-ray powder diffractionof crystalline CC and of the blank pellets spiked with CC, but not withCC present in the pellets, suggesting that the CC precipitated in an amor-phous form and/or in molecular dispersion during in vitro lipolysis(Fig. 3D and E). This phenomenon was also reported with cinnarizine[37], halofantrine [52] and simvastatine [53], but to our knowledge,this result is the first report of the phenomenon with CC. The transfor-mation of crystalline CC to amorphous solids had already been reportedafter encapsulation in PLGA nanoparticles [20,56] and in solid disper-sions [25]. The difference observed in the organization of the fattyacids phase between the CC pellets and the blank pellets spiked withCC suggests a possible intermolecular interaction between the drugand the colloidal structure during lipolysis (Fig. 3D and E).

Potential re-dissolution of the precipitated CC in the intestinalmedium prior to absorption was assessed by performing a dissolu-tion test of the pellet obtained from the lipolysis of CC-LBDDS. Inter-estingly, 0.81 mg (~3070%) of the CC present in these pellets wasre-dissolved in the intestinal medium after 60 min (n = 3, Fig. 3C).The re-dissolution of CC could occur as lipid digestion progresses. Thedrug micellization in various colloidal structures containing bile salts,phospholipids and lipid digestion products and the amorphization ofcrystalline CC may have a strong influence on the re-dissolution of CCpellets.

Fig. 4. (A)Apparent permeability coefficient of the free CC across Caco-2monolayers in theapical to basolateral (A to B) and the basolateral to apical (B to A) directions. (B) Apparentpermeability and transport rate of CC-loaded LBDDS across Caco-2 monolayers with twodifferent drug concentrations (0.03 and 0.05 mg/ml). (C) The influence of M cells on thetransport of CC-LBDDS in Caco-2 monolayers and in the follicle-associated epitheliummodel (co-cultures). Each value represents the mean SD (n = 36).

910 P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

3.5. Caco-2 cell in vitro studies

3.5.1. Cytotoxic assessment of the LBDDSTo select the range of concentrations used for the permeation stud-

ies, the cytotoxicity of the LBDDS was investigated using the MTT test.The IC50 ranged between 2.0 and 3.0 mg/ml (data not shown). The pres-ence of co-solvent (Transcutol HP) in Formulation B did not result in asignificant difference in the metabolic activity of Caco-2 cells comparedto Formulation A. After exposure of cells with 3 mg/ml of LBDDSs, lessthan 20% of the LDH was released (data not shown).

3.5.2. Free CC transport across Caco-2 cell monolayersTo evaluate the permeability of free CC (0.015 mg/ml in HBSS +

3 mM MES + 4% methanol), absorptive (A to B) and secretive (B to A)transports across Caco-2 cell monolayers were performed for 120 min.To improve the recovery of CC by the enhancement of its solubility and

its stability, a solution of 1% BSA in HBSS + 10 mM HEPES was used asa receiver medium [32,51]. In these conditions, the Papp (A to B) of CCwas 3.85 0.45 106 cm/s at 37 C (Fig. 4A). A similar permeabilityof 2.93 106 cm/s has been observed by Wahlang et al. [50]. Yu andHuang also reported a CC Papp value in the same rank order(7.1 106 cm/s and 8.4 106 cm/s) [22,54]. Each report indi-cated a high level of permeability of CC (Papp N 106 cm/s).

The existence of potential active efflux was also investigated duringthe transport. The Papp (B to A) of CC was 3.68 106 cm/s (Fig. 4A).The ratio Papp (B to A)/Papp (A to B) corresponds to 0.96, indicatingthat CC is not a substrate of the Pg-P efflux transporter [50,54,55].

3.5.3. The transport of CC-LBDDS across Caco-2 and FAE monolayersTo determine the intestinal permeability of CC formulated in LBDDS,

transport studies were carried out in Caco-2 cells. Based on cytotoxicitystudies, Caco-2 monolayers were incubated for 120 min at 37 C with1.03 mg/ml of CC-LBDDS, which corresponded to 0.03 mg/ml of CC.The TEER values before and after incubation with the CC-LBDDSs didnot change (p N 0.05). As shown in Fig. 4B, the Papp of CC was 1.10 0.07 106 cm/s (Formulation A) and 1.20 0.06 106 cm/s(Formulation B). The quantity of CC transported across the Caco-2monolayers ranged between 1.4 and 1.6% (0.21 and 0.24 g) of thedonor CC-LBDDS. Transcutol HP, the absorption enhancer employedin Formulation B, did not significantly increase the transport of CCacross Caco-2 monolayers compared to Formulation A (p N 0.05).The transport of CC in suspension (0.15 mg/ml in HBSS) was belowthe limit of detection (data not shown).

CC is known to be retained in enterocytes [50]. To determine theamount of CC within Caco-2 cells, CC was quantified by HPLC aftercell lysis. Approximately 1.6% of the CC from LBDDS (i.e., 0.24 g)was present in Caco-2 monolayers after a 2 h incubation, suggestingthat the amount of CC transported across Caco-2 cells may increasebeyond this period of time. Fig. 5 shows the confocal microscopyimages corresponding to the cellular uptake of CC in Caco-2 cells.CC-loaded LBDDS was taken up more by enterocytes than the CC insuspension. An intracellular staining was observed, which confirmsan increased uptake of CC from LBDDS.

The effect of the concentration of CC-LBDDS onCCpermeation acrossthe Caco-2 cell monolayer was investigated. At 0.03 and 0.05 mg/mlof CC loaded in LBDDS, the transport rates were 3.40 102 ng/s and4.01 102 ng/s for Formulation A and 3.62 102 ng/s and 4.54 102 ng/s for Formulation B. These results indicate that the transportrates were similar at the same concentration of CC (p N 0.05) but werenot linearly dependent on drug concentration (Fig. 4B).

Using the inverted M cell model, no significant difference in thePapp of CC between the co-culture and monoculture (Papp 1.3 to1.5 106 cm/s) (p N 0.05, Fig. 4C) was detected, confirming that theuptake of the encapsulated drug in LBDDS was not enhanced by Mcells [36]. Before and after the experiment, the average TEER was notmodified for the monocultures and for the co-cultures (p N 0.05).

A series of complex processes or factors (e.g., bile salts, phospholipidmicelles, lipid and surfactant digestion) occurring in vivo may largelyinfluence the predictive permeability of CC-LBDDS observed in thein vitro Caco-2 cell system. Therefore, we attempted to assess the per-meability of CC across intestinal cells after a lipid digestion of CC-LBDDS. However, the cytotoxic effects of the lipolysis buffer (bile saltsand lipase) prevented the realization of this experiment. Appropriateexperimental conditions could not be obtained even after the additionof lipase inhibitors, thermal inactivation of enzymes or a dilution ofdigested CC-LBDDS to non-cytotoxic concentrations of enzymes andbile salts to maintain cell integrity (data not shown, [57,58]).

3.6. Hemolysis test

To check if emulsions transported to systemic blood circulationcould cause damage on the membrane of erythrocytes which is the

Fig. 5. Confocal laser scanning microscopy images of the inserts after 2 h of incubationwith Formulation A (Panel A), Formulation B (Panel B) and CC suspension (Panel C).Green fluorescence indicates the localization of CC in cell monolayers (excitation wave-length = 488 nm). Cell membranes are immunostained (red) with rhodaminephalloidin and cell nuclei (blue) with DAPI. (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Survival of mice treated with CC-DMSO (at 100 mg/kg), CC-LBDDS (at 0, 60 and100 mg/kg) (Panel A and B) or with CC-LBDDS (at 0, 60, 80 and 100 mg/kg) in com-bination with AE-LBDDS (at 0 and 12 mg/kg) (Panel C and D).

911P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

main target in malaria therapy, hemolytic activity of unloaded-LBDDSwas assessed as previously described [18,42]. All the formulations(010 mg/ml in PBS) showed negligible hemolytic effect (1.23.7%).Nevertheless, this in vitro hemolytic study does not exactly predictin vivo observations due to lipid digestion in gastrointestinal tract.Therefore, the estimated in vitro erythrocyte toxicity may be largely re-duced in vivo.

3.7. In vivo antimalarial efficacy of CC-LBDDS

To determine whether treatment of CC-LBDDSs could delay para-sitemia in an established infection, their antimalarial efficacy was in-vestigated in a P. berghei mouse model of malaria. The results aresummarized in Fig. 6 and Table 2. At a dosage of 100 mg/kg for four

consecutive days, parasitemia suppression by CC-loaded LBDDSs rangedbetween 40 and 50% (Table 2). The mean survival time was approxi-mately 14 days, whereas the survival rate was 43% and 0% at day-14and day-21, respectively. No significant differences in the antimalarialefficacy of Formulation A and Formulation B were observed (p N 0.05).Oral delivery of CC-LBDDS at 60 mg/kg for 4 days decreased the survivalrate to less than 25% at day-14 with a mean survival time of approxi-mately 9 days.

Table 2Antimalarial activity of formulations and mean survival time of mice.

Formulation Drug Dose(mg/kg/day)

% Activity a Mean survivaltime (days) b

AE CC

A CC 0 11.5 7.3 1.6 60 20.8 9.6 4.1 100 46.0 14.0 4.9

B CC 0 10.6 8.3 1.4 60 24.4 10.3 4.4 100 43.0 13.9 4.2

C + A AE + CC 0 0 8.7 8.1 2.312 0 99.8 18.7 5.312 60 99.8 20.7 4.812 80 99.8 22.9 5.212 100 99.8 25.1 3.7

D + B AE + CC 0 0 9.6 7.7 1.112 0 99.5 18.9 6.012 60 99.5 21.7 4.712 80 99.7 22.9 4.912 100 99.8 24.9 3.7

DMSO CC 100 32 9.3 4.5Untreated 6.7 0.8

a Average activity of formulations was determined on day 4 of treatment.b Mean survival time of mice determined during an observation period of 28 days.

912 P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

Administered in DMSO at 100 mg/kg for 4 days, CC decreasedparasitemia by 32% with a mean survival time of 9 days (Table 2),which is in agreement with previous studies [10,11]. The survival rateof mice was 30% at day-14 and 0% at day-21. These results contrastwith a survival rate of 29% at day-21 reported byReddy et al. [8]. Somedif-ferences in the experimental conditions (administration protocols andparasitemia levels) may account for these observations. At 100 mg/kg,the efficacy of CC-DMSO was lower than the efficacy of CC-LBDDS inboth parasite suppression and survival time (p b 0.05). The possibilityof re-precipitation and degradation of CC dissolved in DMSO occurringin the gastrointestinal tract may reduce the intestinal absorption andtherefore the activity of CC.

The LBDDS without CC prolonged survival time up to 11 days post-infection for 15% of the mice compared with 67 days post-infectionin untreated mice. This finding suggests that lipid vehicles may havesome inherent antimalarial activity and immunomodulatory proper-ties [35,45,47,59]. Indeed, C18 fatty acids (linolenic, linoleic and oleicacid) inhibit parasite growth both in vitro and in vivo [46,47]. Fattyacids may also stimulate a protective immune response by the activa-tion of neutrophils, Th2 type CD4+ T cells and other effector cells toincrease the clearing of parasitemia [47,59].

These results demonstrate that, at the dose of 100 mg/kg, LBDDS(and DMSO) containing CC have a moderate effect on the reductionof parasitemia and are not able to delay the course of an establishedinfection on mice. Hence, to overcome an incomplete cure and recru-descence occurring during this monotherapy, a combinatorial approachusing a subtherapeutic dose of AE-LBDDS (12 mg/kg) [35] and CC-LBDDS was investigated. When administered, combinations of AE andCC-loaded lipid-based systems drastically decreased blood parasitemia(~0%) (data not shown) but were not fully effective in suppressingrecrudescence (Fig. 6 C andD). Nevertheless, they significantly enhancedthe survival rate of mice compared with their respective monother-apy groups. Indeed, the combination of Formulation C (12 mg/kg) +Formulation A (100 mg/kg) resulted in a survival rate of 60% in compar-ison with a survival rate of 15% for Formulation C (12 mg/kg) +Formulation A (0 mg/kg) (Fig. 6C). The co-administration of Formula-tion D with Formulation B at 12 mg/kg + 0 mg/kg and 12 mg/kg +100 mg/kg led to a 15% and 45% 28-day survival, respectively (Fig. 6D).Similar results were previously obtained after oral administrationof Formulation C and Formulation D (alone) at the subtherapeuticdose of 12 mg/kg [35]. The moderate antimalarial activity and the

immunomodulatory effect of CC (and fatty acids to a lesser extent)may explain the increasing rate of survival (4565%), regardless ofthe high recrudescence of parasites on day-12 (~2% parasitemia onaverage). No significant differences in survival time were observedwhen Formulation C +Formulation B or Formulation D + Formula-tion A were used at the different doses (p N 0.05 and data notshown).

The potential of AE-CC combination therapy in experimentalmalariahave been demonstrated previously [1012]. In these reports, CC wasorally administered in DMSO or intravenously administered in lipo-somes and AE intramuscularly administered. The present study showsthe utility of LBDDS in the oral delivery of CC: enhanced drug's solubili-zation and transport across Caco-2 monolayers as well as increasedantimalarial efficacy. The suitability of oral AE-LBDDS in the malariatherapy has been also demonstrated previously [35]. Oral AE-LBDDSconstitute a promising alternative to the marketed oil-based intramus-cular injectable formulations that are characterized by slow and erraticabsorption [2]. In order to be able to appreciate the potential effectof oral AE-LBDDS and CC-LBDDS when administered in combination,a subtherapeutic dose of each drug was tested in P. berghei-infectedmice. The results clearly demonstrate for the first time that the oraladministration of both CC-LBDDS and AE-LBDDS display a significantanti-malarial efficacy against P.berghei-infected mice. The resultsare supportive but not conclusive of a synergistic effect of CC in anartemisinin-combination therapy. In this combination, an increaseddose of AE (to 1824 mg/kg) and/or CC (to 150200 mg/kg) maylead to complete cure and long-term protection. Additionally, thestart of treatment at a very early stage of the disease may also favora quick resolution of symptoms and prevention of hyperparisitemia,which would reduce mortality. Additional in vivo studies are neededto confirm these hypotheses and to assess the oral bioavailability ofCC loaded in LBDDS.

4. Conclusion

In this study, two LBDDSs containing a high CC loading (30 mg/g)were formulated. In a gastrointestinal medium, CC-LBDDSs spontane-ously self-emulsify to form nanosized particles. During in vitro lipolysis,510% of CC precipitated from LBDDS as an amorphous solidwith a highrate of re-dissolution in FaSSIF. Due to the increased CC solubility,LBDDS enhanced the transport of CC across Caco-2 cell monolayersin comparison with the transport of free drug. In mice, a modest an-timalarial efficacy was observed following treatment with CC-LBDDS(100 mg/kg). However, the combination therapy of CC-LBDDS (80100 mg/kg) with a subtherapeutic dose of AE-LBDDS (12 mg/kg)showed an increased protection and survival rate associated with asignificant delay in recrudescence. The combination of oral CC and AElipid-based formulations may constitute a promising approach for thetreatment of malaria.

Acknowledgments

The authors thank the Universit Catholique de Louvain (Boursede la coopration au dveloppement) for financing and supporting theproject and the scholarship of Patrick Bondo Memvanga. We are alsoindebted to Dr Pascal Somville (UCB Pharma, Belgium) for access tothe pH-stat titration system. The scientific and technical support ren-dered by Pierre Eloy (Institute of Condensed Matter and Nanosciences,Universit Catholique de Louvain) during the realization of X-ray pow-der diffraction is acknowledged.We are grateful toDr StomyKarhemere(Institut Nationale de Recherches Biomdicales, Democratic Republicof Congo) for providing the facilities for the in vivo study. The technicalassistance rendered by Guy Midingi is acknowledged. We extend ourthanks to Gattefoss and BASF for kind gifts of excipients used in thisstudy.

913P.B. Memvanga et al. / Journal of Controlled Release 172 (2013) 904913

NANOMEDICIN

E

References

[1] Who Press, World Malaria Report, 2012.[2] World Health Organization, Guidelines for the Treatment of Malaria, 2010.[3] A. Codd, F. Teuscher, D. Kyle, Q. Cheng, M. Gatton, Artemisinin-induced parasite

dormancy: a plausible mechanism for treatment failure, Malar. J. 10 (2011) 56.[4] B. Witkowski, J. Lelievre, M.J. Lopez Barragan, V. Laurent, X.Z. Su, A. Berry, F. Benoit-

Vical, Increased tolerance to artemisinin in Plasmodium falciparum is mediated by aquiescence mechanism, Antimicrob. Agents Chemother. 54 (2010) 18721877.

[5] K.R.Hughes, G.A. Biagini, A.G. Craig, Continued cytoadherence of Plasmodium falciparuminfected red blood cells after antimalarial treatment, Mol. Biochem. Parasitol. 169(2010) 7178.

[6] G. Padmanaban, V.A. Nagaraj, P.N. Rangarajan, Artemisinin-based combinationwith curcumin adds a new dimension to malaria therapy, Curr. Sci. 102 (2012)704711.

[7] L. Cui, J. Miao, L. Cui, Cytotoxic effect of curcumin on malaria parasite Plasmodiumfalciparum: inhibition of histone acetylation and generation of reactive oxygen species,Antimicrob. Agents Chemother. 51 (2007) 488494.

[8] R.C. Reddy, P.G. Vatsala, V.G. Keshamouni, G. Padmanaban, P.N. Rangarajan,Curcumin for malaria therapy, Biochem. Biophys. Res. Commun. 326 (2005)472474.

[9] A. Martinelli, L.A. Rodrigues, P. Cravo, Plasmodium chabaudi: efficacy of artemisinin +curcumin combination treatment on a clone selected for artemisinin resistance inmice, Exp. Parasitol. 119 (2008) 304307.

[10] D.N. Nandakumar, V.A. Nagaraj, P.G. Vathsala, P. Rangarajan, G. Padmanaban,Curcumin-artemisinin combination therapy formalaria, Antimicrob.Agents Chemother.50 (2006) 18591860.

[11] P.G. Vathsala, C. Dende, V.A. Nagaraj, D. Bhattacharya, G. Das, P.N. Rangarajan, G.Padmanaban, Curcumin-arteether combination therapy of Plasmodium berghei-infected mice prevents recrudescence through immunomodulation, PLoS One 7(2012) e29442.

[12] N.P. Aditya, G. Chimote, K. Gunalan, R. Banerjee, S. Patankar, B. Madhusudhan,Curcuminoids-loaded liposomes in combination with arteether protects againstPlasmodium berghei infection in mice, Exp. Parasitol. 131 (2012) 292299.

[13] F. Akhtar, M.M. Rizvi, S.K. Kar, Oral delivery of curcumin bound to chitosannanoparticles cured Plasmodium yoelii infected mice, Biotechnol. Adv. 30 (2012)310320.

[14] A.P. Nayak,W. Tiyaboonchai, S. Patankar, B.Madhusudhan, E.B. Souto, Curcuminoids-loaded lipid nanoparticles: novel approach towards malaria treatment, Colloids Surf.B: Biointerfaces 81 (2010) 263273.

[15] J.G. Bilmen, S.Z. Khan, M.H. Javed, F. Michelangeli, Inhibition of the SERCA Ca2+pumps by curcumin. Curcumin putatively stabilizes the interaction between thenucleotide-binding and phosphorylation domains in the absence of ATP, Eur. J.Biochem. 268 (2001) 63186327.

[16] B.B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Curcumin: the Indian solid gold,Adv. Exp. Med. Biol. 595 (2007) 175.

[17] P.N. Mimche, D. Taramelli, L. Vivas, The plant-based immunomodulator curcuminas a potential candidate for the development of an adjunctive therapy for cerebralmalaria, Malar. J. 10 (Suppl. 1) (2011) S10.

[18] R. Chakrabarti, P.S. Rawat, B.M. Cooke, R.L. Coppel, S. Patankar, Cellular effects ofcurcumin on Plasmodium falciparum include disruption of microtubules, PLoS One8 (2013) e57302.

[19] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability ofcurcumin: problems and promises, Mol. Pharm. 4 (2007) 807818.

[20] J. Shaikh, D.D. Ankola, V. Beniwal, D. Singh, M.N. Kumar, Nanoparticle encapsulationimproves oral bioavailability of curcumin by at least 9-fold when compared tocurcumin administered with piperine as absorption enhancer, Eur. J. Pharm. Sci.37 (2009) 223230.

[21] K. Ahmed, Y. Li, D.J.McClements,H. Xiao, Nanoemulsion-and emulsion-baseddeliverysystems for curcumin: encapsulation and release properties, Food Chem. 132 (2012)799807.

[22] H. Yu, Q. Huang, Improving the oral bioavailability of curcumin using novelorganogel-based nanoemulsions, J. Agric. Food Chem. 60 (2012) 53735379.

[23] K. Desai, Curcumin cyclodextrin combination for preventing or treating variousdiseases. Patent US 2010/0779103.

[24] K. Maiti, K. Mukherjee, A. Gantait, B.P. Saha, P.K. Mukherjee, Curcumin phospholipidcomplex: preparation, therapeutic evaluation and pharmacokinetic study in rats,Int. J. Pharm. 330 (2007) 155163.

[25] S.W. Seo, H.K. Han, M.K. Chun, H.K. Choi, Preparation and pharmacokinetic evalua-tion of curcumin solid dispersion using Solutol HS15 as a carrier, Int. J. Pharm. 424(2012) 1825.

[26] M.M. Yallapu, M. Jaggi, S.C. Chauhan, Curcumin nanoformulations: a futurenanomedicine for cancer, Drug Discov. Today 17 (2012) 7180.

[27] J. Cui, B. Yu, Y. Zhao, W. Zhu, H. Li, H. Lou, G. Zhai, Enhancement of oral absorption ofcurcumin by self-microemulsifying drug delivery systems, Int. J. Pharm. 371 (2009)148155.

[28] S. Setthacheewakul, S. Mahattanadul, N. Phadoongsombut, W. Pichayakorn, R.Wiwattanapatapee, Development and evaluation of self-microemulsifying liquidand pellet formulations of curcumin, and absorption studies in rats, Eur. J. Pharm.Biopharm. 76 (2010) 475485.

[29] Y.D. Yan, J.A. Kim, M.K. Kwak, B.K. Yoo, C.S. Yong, H.G. Choi, Enhanced oral bioavail-ability of curcumin via a solid lipid-based self-emulsifying drug delivery system usinga spray-drying technique, Biol. Pharm. Bull. 34 (2011) 11791186.

[30] E. Jantratid, N. Janssen, C. Reppas, J.B. Dressman, Dissolution media simulating con-ditions in the proximal human gastrointestinal tract: an update, Pharm. Res. 25(2008) 16631676.

[31] C.J. Porter, W.N. Charman, In vitro assessment of oral lipid based formulations, Adv.Drug Deliv. Rev. 50 (Suppl. 1) (2001) S127S147.

[32] G. Krishna, K. Chen, C. Lin, A.A. Nomeir, Permeability of lipophilic compounds indrug discovery using in-vitro human absorption model, Caco-2, Int. J. Pharm. 222(2001) 7789.

[33] S. Yamashita, T. Furubayashi, M. Kataoka, T. Sakane, H. Sezaki, H. Tokuda, Optimizedconditions for prediction of intestinal drug permeability using Caco-2 cells, Eur. J.Pharm. Sci. 10 (2000) 195204.

[34] J.F. Cuine, C.L. McEvoy, W.N. Charman, C.W. Pouton, G.A. Edwards, H. Benameur, C.J.Porter, Evaluation of the impact of surfactant digestion on the bioavailability ofdanazol after oral administration of lipidic self-emulsifying formulations to dogs,J. Pharm. Sci. 97 (2008) 9951012.

[35] P.B. Memvanga, V. Prat, Formulation design and in vivo antimalarial evaluation oflipid-based drug delivery systems for oral delivery of beta-arteether, Eur. J. Pharm.Biopharm. 82 (2012) 112119.

[36] P.B. Memvanga, P. Eloy, E.M. Gaigneaux, V. Prat, In vitro lipolysis and intestinaltransport of beta-arteether-loaded lipid-based drug delivery systems, Pharm. Res.30 (2013) 26942705.

[37] P.J. Sassene, M.M. Knopp, J.Z. Hesselkilde, V. Koradia, A. Larsen, T. Rades, A. Mullertz,Precipitation of a poorly solublemodel drugduring in vitro lipolysis: characterizationand dissolution of the precipitate, J. Pharm. Sci. 99 (2010) 49824991.

[38] A. des Rieux, E.G. Ragnarsson, E. Gullberg, V. Prat, Y.J. Schneider, P. Artursson,Transport of nanoparticles across an in vitro model of the human intestinal follicleassociated epithelium, Eur. J. Pharm. Sci. 25 (2005) 455465.

[39] A. des Rieux, V. Fievez, I. Theate, J. Mast, V. Prat, Y.J. Schneider, An improved in vitromodel of human intestinal follicle-associated epithelium to study nanoparticletransport by M cells, Eur. J. Pharm. Sci. 30 (2007) 380391.

[40] R. Coco, L. Plapied, V. Pourcelle, C. Jerome, D.J. Brayden, Y.J. Schneider, V. Preat, Drugdelivery to inflamed colon by nanoparticles: comparison of different strategies, Int. J.Pharm. 440 (2013) 312.

[41] C.F. Chignell, P. Bilski, K.J. Reszka, A.G. Motten, R.H. Sik, T.A. Dahl, Spectral and pho-tochemical properties of curcumin, Photochem. Photobiol. 59 (1994) 295302.

[42] G. Vandermeulen, L. Rouxhet, A. Arien, M.E. Brewster, V. Prat, Encapsula-tion of amphotericin B in poly(ethyleneglycol)-block-poly(-caprolactone-co-trimethylenecarbonate) polymeric micelles, Int. J. Pharm. 309 (2006) 234240.

[43] J.F. Ryley, W. Peters, The antimalarial activity of some quinolone esters, Ann. Trop.Med. Parasitol. 64 (1970) 209222.

[44] D.A. Fidock, P.J. Rosenthal, S.L. Croft, R. Brun, S. Nwaka, Antimalarial drug dis-covery: efficacy models for compound screening, Nat. Rev. Drug Discov. 3(2004) 509520.

[45] N.M. Carballeira, New advances in fatty acids as antimalarial, antimycobacterial andantifungal agents, Prog. Lipid Res. 47 (2008) 5061.

[46] M. Krugliak, E. Deharo, G. Shalmiev, M. Sauvain, C. Moretti, H. Ginsburg, Antimalarialeffects of C18 fatty acids on Plasmodium falciparum in culture andon Plasmodiumvinckeipetteri and Plasmodium yoelii nigeriensis in vivo, Exp. Parasitol. 81 (1995) 97105.

[47] L.M. Kumaratilake, B.S. Robinson, A. Ferrante, A. Poulos, Antimalarial properties ofn-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparumand in vivo effects on P. berghei, J. Clin. Invest. 89 (1992) 961967.

[48] W. Li, S. Yi, Z. Wang, S. Chen, S. Xin, J. Xie, C. Zhao, Self-nanoemulsifying drug deliv-ery system of persimmon leaf extract: optimization and bioavailability studies, Int. J.Pharm. 420 (2011) 161171.

[49] J.L. Delongeas, G.V. de Conchard, A. Beamonte, H. Bertheux, C. Spire, C.Maisonneuve, N.Becourt-Lhote, F. Goldfain-Blanc, N. Claude, Assessment of Labrasol/Labrafil/Transcutol(4/4/2, v/v/v) as a non-clinical vehicle for poorly water-soluble compounds after4-week oral toxicity study in Wistar rats, Regul. Toxicol. Pharmacol. 57 (2010)284290.

[50] B. Wahlang, Y.B. Pawar, A.K. Bansal, Identification of permeability-related hurdles inoral delivery of curcumin using the Caco-2 cell model, Eur. J. Pharm. Biopharm. 77(2011) 275282.

[51] M.H. Leung, T.W. Kee, Effective stabilization of curcumin by association to plasmaproteins: human serum albumin and fibrinogen, Langmuir 25 (2009) 57735777.

[52] N. Thomas, R. Holm, A. Mullertz, T. Rades, In vitro and in vivo performance of novelsupersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS), J. Control.Release 160 (2012) 2532.

[53] N. Thomas, R. Holm, M. Garmer, J.J. Karlsson, A. Mullertz, T. Rades, Supersaturatedself-nanoemulsifying drug delivery systems (Super-SNEDDS) enhance the bioavail-ability of the poorly water-soluble drug simvastatin in dogs, AAPS J. 15 (2013)219227.

[54] H. Yu, Q. Huang, Investigation of the absorption mechanism of solubilized curcuminusing Caco-2 cell monolayers, J. Agric. Food Chem. 59 (2011) 91209126.

[55] X.L. Hou, K. Takahashi, K. Tanaka, K. Tougou, F. Qiu, K. Komatsu, K. Takahashi, J.Azuma, Curcuma drugs and curcumin regulate the expression and function ofP-gp in Caco-2 cells in completely opposite ways, Int. J. Pharm. 358 (2008) 224229.

[56] K.K. Chereddy, R. Coco, P.B. Memvanga, B. Ucakar, A. des Rieux, G. Vandermeulen, V.Prat, Combined effect of PLGA and curcumin on wound healing activity, J. Control.Release 171 (2013) 208215.

[57] M.B. Gangloff, R.P. Glahn, D.D. Miller, D.R. Van Campen, Assessment of iron avail-ability using combined in vitro digestion and Caco-2 cell culture, Nutr. Res. 16 (1996)479487.

[58] D.A. Garrett, M.L. Failla, R.J. Sarama, Development of an in vitro digestionmethodto assess carotenoid bioavailability from meals, J. Agric. Food Chem. 47 (1999)43014309.

[59] C. Carrillo, C.M. Del Mar, H. Roelofs, G. Wanten, S.R. Alonso-Torre, Activation ofhuman neutrophils by oleic acid involves the production of reactive oxygen speciesand a rise in cytosolic calcium concentration: a comparisonwith N-6 polyunsaturatedfatty acids, Cell. Physiol. Biochem. 28 (2011) 329338.

http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0270http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0275http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0005http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0005http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0010http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0010http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0010http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0015http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0015http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0015http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0020http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0020http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0020http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0025http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0025http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0025http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0030http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0030http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0030http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0035http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0035http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0035http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0040http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0040http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0040http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0045http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0045http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0045http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0045http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0050http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0050http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0050http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0055http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0055http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0055http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0060http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0060http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0060http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0065http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0065http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0065http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0065http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0065http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0070http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0070http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0075http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0075http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0075http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0080http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0080http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0080http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0085http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0085http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0090http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0090http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0090http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0090http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0095http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0095http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0095http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0100http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0100http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0105http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0105http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0105http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0110http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0110http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0110http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0115http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0115http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0120http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0120http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0120http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0125http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0125http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0125http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0125http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0130http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0130http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0130http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0135http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0135http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0135http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0140http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0140http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0145http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0145http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0145http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0150http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0150http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0150http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0155http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0155http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0155http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0155http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0160http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0160http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0160http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0280http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0280http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0280http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0165http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0165http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0165http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0285http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0285http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0285http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0290http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0290http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0290http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0170http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0170http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0170http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0175http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0175http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0180http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0180http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0180http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0185http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0185http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0190http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0190http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0190http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0195http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0195http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0200http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0200http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0200http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0205http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0205http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0205http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0210http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0210http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0210http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0215http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0215http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0215http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0215http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0215http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0220http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0220http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0220http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0225http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0225http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0230http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0230http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0230http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0235http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0235http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0235http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0235http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0240http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0240http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0245http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0245http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0245http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0295http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0295http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0295http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0255http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0255http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0255http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0260http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0260http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0260http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0265http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0265http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0265http://refhub.elsevier.com/S0168-3659(13)00763-3/rf0265

An oral malaria therapy: Curcumin-loaded lipid-based drug delivery systems combined with -arteether1. Introduction2. Materials and methods2.1. Materials2.2. Simulated media2.3. The development of CC lipid-based self-emulsifying systems2.4. Preparation and characterization of CC-LBDDS2.4.1. Preparation of CC-LBDDS2.4.2. Self-emulsification of CC-LBDDS in gastrointestinal and transport media2.4.3. Stability of CC-LBDDS over 180days

2.5. CC analysis by HPLC2.6. Lipolysis of CC-LBDDS2.7. Analysis of the pellet obtained after lipolysis2.8. In vitro intestinal cell models used to study the transport of CC2.8.1. Cell cultures2.8.2. Cytotoxicity2.8.3. Caco-2 and FAE monolayers2.8.4. Permeation studies of free CC and CC-LBDDS across intestinal in vitro models2.8.5. Intracellular CC in the cell monolayer

2.9. In vitro hemolytic study2.10. In vivo antimalarial efficacy of CC-LBDDS2.11. Statistical analyses

3. Results and discussion3.1. Solubilization of CC in various excipients and in biorelevant media3.2. The development of oral CC self-emulsifying systems3.3. Dilution of CC-LBDDS in gastrointestinal and transport media3.4. In vitro dynamic lipolysis3.4.1. In vitro lipolysis3.4.2. X-ray powder diffraction and CC dissolution in pellets after lipolysis

3.5. Caco-2 cell in vitro studies3.5.1. Cytotoxic assessment of the LBDDS3.5.2. Free CC transport across Caco-2 cell monolayers3.5.3. The transport of CC-LBDDS across Caco-2 and FAE monolayers

3.6. Hemolysis test3.7. In vivo antimalarial efficacy of CC-LBDDS

4. ConclusionAcknowledgmentsReferences