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International Journal of Pharmaceutics 454 (2013) 775– 783

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

j o ur nal ho me page: www.elsev ier .com/ locate / i jpharm

udesonide-loaded nanostructured lipid carriers reducenflammation in murine DSS-induced colitis

na Beloquia,b,c,1, Régis Cococ,1, Mireille Alhouayekd,e, María Ángeles Solinísa,b,licia Rodríguez-Gascóna,b, Giulio G. Mucciolid, Véronique Préatc,∗

Pharmacokinetics, Nanotechnology and Gene Therapy Group, School of Pharmacy, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, SpainCentro de Investigación Lascaray Ikergunea, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, SpainUniversité catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, 1200 Brussels, BelgiumUniversité catholique de Louvain, Louvain Drug Research Institute, Bioanalysis and Pharmacology of Bioactive Lipids Research Group, 1200 Brussels,elgiumUniversité catholique de Louvain, Louvain Drug Research Institute, Medicinal Chemistry Research Group, 1200 Brussels, Belgium

a r t i c l e i n f o

rticle history:eceived 18 February 2013eceived in revised form 30 April 2013ccepted 3 May 2013vailable online 18 May 2013

a b s t r a c t

The challenge for the treatment of inflammatory bowel disease (IBD) is the delivery of the drug to the siteof inflammation. Because nanoparticles have the ability to accumulate in inflamed regions, the aim ofthe present study was to evaluate nanostructured lipid carriers (NLCs) as nanoparticulate drug deliverysystems for the treatment of IBD. Budesonide (BDS) was chosen as a candidate anti-inflammatory drug.BDS-loaded NLCs (BDS-NLC) produced by high-pressure homogenization had a size of 200 nm and a

eywords:anostructured lipid carriersacrophages

olonic deliverynflammatory bowel diseaseudesonide

negative zeta potential. BDS-NLCs reduced the TNF-� secretion by activated macrophages (J774 cells).BDS-NLCs were more active in a murine model of dextran sulfate-induced colitis when compared withBlank-NLCs or a BDS suspension: BDS-NLCs decreased neutrophil infiltration, decreased the levels of thepro-inflammatory cytokines IL-1� and TNF-� in the colon and improved the histological scores of thecolons. These data suggest that NLCs could be a promising alternative to polymeric nanoparticles as atargeted drug delivery system for IBD treatment.

. Introduction

Inflammatory bowel diseases (IBD) are chronic and relapsingnflammatory disorders of the gastrointestinal tract (GIT). Thewo major forms, ulcerative colitis and Crohn’s disease, present

colonic-specific or a widespread repartition of inflamed lesions,espectively (Baumgart and Carding, 2007; Khor et al., 2011; Sartor,006). Patients with IBD require life-long anti-inflammatory and

mmunosuppressive therapy. Although most of the drugs can bedministered by the oral route for IBD treatment, high daily dosesre required to obtain a therapeutic effect (Baumgart and Sandborn,007; Fatahzadeh, 2009; Mowat et al., 2011). Furthermore, sys-emic absorption is observed, leading to serious adverse effects

Chourasia and Jain, 2003; Friend, 2005).

Considering the colonic location of these diseases as well ashe systemic side effects of absorbed drugs, the specific targeted

∗ Corresponding author at: Université catholique de Louvain, Louvain Drugesearch Institute, Pharmaceutics and Drug Delivery Group, Avenue Mounier 73te B1 73.12, B-1200 Brussels, Belgium. Tel.: +32 2 7647320; fax: +32 2 7647398.

E-mail address: veronique.preat@uclouvain.be (V. Préat).1 These two authors contributed equally to the study.

378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.05.017

© 2013 Elsevier B.V. All rights reserved.

delivery of anti-inflammatory drugs to the inflamed colonic areasshould lead to higher local concentrations and maintain therapeuticefficacy while enhancing safety by decreasing absorption. Differentdelivery strategies to target the inflamed colon have been studied(Collnot et al., 2012). Sustained drug release devices, pH-sensitiveformulations, rectal formulations and pro-drugs are some of thestrategies that have been developed during the past several decadesto obtain efficient colonic drug delivery. However, the inefficientcontrol of drug release, patient sensitivity to IBD symptoms (e.g.,diarrhea, modified colonic environment) or the inability to reachthe inflamed areas are some of the observed drawbacks that causedecreased efficacy.

Because nanoparticulate drug delivery systems have the abil-ity to accumulate in the inflamed regions of the gut, they offer anew targeting approach in IBD (Collnot et al., 2012; Ulbrich andLamprecht, 2010). Size is a critical factor in the design of drug deliv-ery systems for the inflamed colon. During active IBD, the luminalcontent undergoes a decrease in its transit time due to diarrhea, themost frequent symptom of IBD. In animal models of experimental

colitis and in human patients (Schmidt et al., 2013), a preferentialuptake of nano-sized particles was observed in the inflamed colonicmucosa. It is apparent that the pathophysiological changes associ-ated with mucosal inflammation, such as (i) a disrupted intestinal

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76 A. Beloqui et al. / International Journ

arrier due to the presence of mucosal surface alterations, cryptistortions and ulcers, (ii) increased mucus production and (iii) the

nfiltration of immune-related cells such as macrophages, lympho-ytes or dendritic cells, provide an environment favorable for thereferential accumulation of nanoparticles in the inflamed colonicegions (Lamprecht et al., 2001). Therefore, nano- and micropar-iculate drug carriers have been widely studied for targeting thenflamed intestinal mucosa. Liposomes (Jubeh et al., 2006), poly-

eric nanoparticles (Lamprecht et al., 2005; Schmidt et al., 2013;ong et al., 2012) and drug-loaded nanoparticles in combinationith polysaccharide hydrogels (Laroui et al., 2010), among others,ave been widely studied for effective drug targeting and release

n the inflamed colonic mucosa. Most of these strategies are basedn pH-, enzyme- or transit time-dependent systems (Laroui et al.,010; Makhlof et al., 2009; Ulbrich and Lamprecht, 2010). There-ore, these delivery systems encounter inter- and intra-individualariability in terms of the gut pH and microflora, which may evokearly release of the drug with subsequent decrease in therapeuticfficacy.

Among the wide variety of currently available nanocarriers,olid lipid nanoparticles (SLNs) are promising delivery vectors forhe treatment of inflammatory diseases due to the following rea-ons: (i) SLNs can be prepared on a large scale without organicolvent (Müller et al., 2000); (ii) SLNs are stable in the GIT comparedith other lipid-based nanoparticles such as liposomes; and (iii)

he ability of lipid-based nanoparticles to modulate the immuneystem and induce anti-inflammatory effects has been previouslyescribed (Bondi et al., 2011; Harel-Adar et al., 2011). SLNs thatre usually used for parental and topical formulations have beenecently proposed for colonic delivery. Sodium butyrate-loadedLNs displayed better anti-adhesive effects than butyrate alone onhe in vitro adhesion of polymorphonuclear cells to human umbil-cal vein endothelial cells, as this step is critical in the recruitmentnd infiltration of immune-related cells into inflamed tissues dur-ng active IBD (Dianzani et al., 2006). Furthermore, in a humanBD whole-blood model, after 24 h of lipopolysaccharide expo-ure, a significant decrease in pro-inflammatory cytokines (IL-1�nd TNF-�) secretion was observed following administration ofexamethasone- or butyrate-loaded SLNs, when compared withexamethasone or butyrate alone (Serpe et al., 2010). However,o date, no in vivo studies of SLNs targeting the inflamed colonic

ucosa have been published.Budesonide (BDS) is a corticosteroid with high topical efficacy

nd minimal systemic activity (Chopra et al., 2006; Makhlof et al.,009). This steroid exhibits low oral bioavailability and an exten-ive hepatic first pass effect, as 10–15% of the drug reaches systemicirculation (Lofberg et al., 1996). These characteristics give BDS aetter adverse effect profile than the conventional steroids and thusake it the first choice for the induction of remission in IBD. Ene-as and controlled-release formulations such as Budenofalk® or

ntocort® have been used in clinical practice for the topical deliveryf BDS. However, due to the long-term side effect profile inherent tohe corticosteroid class, BDS cannot be used for the maintenance ofemission (Klotz and Schwab, 2005; Yang and Lichtenstein, 2002).pecific mucosal targeting and delivery of BDS could reduce thenflammation in lesions while maintaining a good safety profile.

The aim of the present study was to evaluate nanostructuredipid carriers (NLCs), a second generation of SLNs with a higher sta-ility and drug loading capacity, as nanoparticulate drug deliveryystems for IBD treatment. BDS was chosen as a candidate anti-nflammatory drug for entrapment into NLCs. The evaluation ofDS-loaded NLCs (BDS-NLC) was performed (i) in vitro, by evaluat-

ng their anti-inflammatory potential in reducing TNF-� secretionrom activated macrophages (J774 cells) and (ii) in vivo, in a murineextran-sulfate (DSS)-induced colitis model, by quantitating neu-rophil infiltration and the concentrations of the pro-inflammatory

harmaceutics 454 (2013) 775– 783

cytokines IL-1� and TNF-� and by histological evaluation of inflam-mation severity in excised colons.

2. Materials and methods

2.1. Materials

2.1.1. ChemicalsBDS, coumarin-6, O-dianisidine, HTAB (hexadecyltrimethyl-

ammonium bromide), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), hematoxylin, eosin and hydrogen per-oxide (30%) were purchased from Sigma–Aldrich (St. Louis, MO,USA). Precirol ATO®5 was kindly provided by Gattefossé (Madrid,ES). Polysorbate 80 (Tween 80) was purchased from Vencaser (Bil-bao, SP). Poloxamer 188 was a gift from BASF (Madrid, ES). Miglyol812 N/F was purchased from Sasol (Hamburg, GE). Potassiumdihydrogen phosphate (KH2PO4), disodium hydrogen phosphate(Na2HPO4) and dimethylsulfoxide (DMSO) were obtained fromMerck (Darmstadt, DE). Acetonitrile (gradient HPLC grade) andethanol were purchased from VWR (Leuven, BE). Dulbecco’sPhosphate-Buffered Saline (PBS) and Hank’s Balanced Salt Solu-tion (HBSS) were purchased from GibcoTM Invitrogen Corporation(Paisley, UK). Dextran sodium sulfate (DSS) was purchased fromTdB Consultancy (Uppsala, SE). Complete protease inhibitor cock-tail tablets were purchased from Roche Diagnostics (Vilvoorde, BE).Paraffin was provided by Sakura Finetek (Torrance, CA).

2.1.2. Cell cultureThe J774 murine macrophage cell line was kindly donated by Pr.

Marie-Paule Mingeot (Université catholique de Louvain, LDRI, BE).Roswell Park Memorial Institute (RPMI) 1640 medium, fetal

bovine serum (FBS), trypsin (0.05%) with EDTA was purchased fromGibcoTM Invitrogen Corporation (Paisley, UK). Lipopolysaccharide(LPS, Escherichia coli O111:B4) was purchased from Sigma–Aldrich(St. Louis, MO, USA).

2.2. Preparation of the formulations

2.2.1. NLC preparationBDS-NLCs were prepared using the high-pressure homogeniza-

tion technique (Beloqui et al., 2013; Müller et al., 2000). Briefly,Precirol ATO®5 (5 g) and Miglyol 812 (0.5 ml) were melted at 75 ◦Cuntil a uniform and clear oil phase was obtained and BDS (4 mg)(melting point above 200 ◦C) was homogenously dispersed in thementioned solution. The aqueous phase was prepared by dispers-ing Tween 80 (2%) (w/v) and poloxamer 188 (1%) (w/v) in water(50 ml) and heating to the same temperature as the lipid phase. Thehot aqueous phase was then added to the oil phase, and the mix-ture was sonicated for 15 s to form a hot pre-emulsion, which wassubsequently homogenized at 80 ◦C and 500 bar using a StanstednG12500 homogenizer (SFP, Essex, UK) for ten homogenizationcycles. A batch of blank nanoparticles was also prepared withoutBDS (Blank-NLCs).

For the in vivo localization study, the NLCs were labeled with thefluorescent dye coumarin-6. Briefly, 5 mg of coumarin-6 was incor-porated into the lipid phase of the formulation in place of BDS, andthe NLCs were prepared according to the aforementioned protocol(coumarin-6-NLCs).

2.2.2. BDS suspensionA BDS suspension (BDS sus) was prepared as a control. BDS

(4 mg) was dispersed in PBS (50 ml). The primary volume medianparticle diameter of the BDS suspension was measured by laserdiffraction (HELOS, Sympatec, Clausthal-Zellerfeld, DE). Before siz-ing, the suspension was homogenized in a 50 ml flask by sonication

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30–60 s) with the internal probe powered at 60 W while magnet-cally stirring at 1000 rpm.

.3. NLC characterization

.3.1. Size and zeta potential measurementsThe size of the NLCs was determined using photon correlation

pectroscopy (PCS), and the zeta potential was measured usingaser Doppler Velocimetry (LDV) with a Malvern Zetasizer NanoS (Malvern Instruments Ltd., Worcestershire, UK). Samples wereiluted in ultra-pure water (Arium 611 VF, Sartorius AG Gottingen,ermany) before measurement.

.3.2. Encapsulation efficiencyUntrapped BDS was quantified by ultrafiltration using Millipore

Madrid, ES) Amicon® ultracentrifugal filters (molecular weightutoff, MWCO, 100,000 Da) (1500 × g, 20 min). The free BDS presentn the filtrate was then measured by HPLC and was found to beelow the limit of quantification (LOQ < 1 �g/mL).

The drug content within BDS-NLCs was calculated by dissolvinghe samples (1 ml) in a solvent mixture of methanol/chloroform1:1 v/v) (1:10 dilution) and filtering through a 0.2 �m filter. Theesulting solution was analyzed by HPLC. The encapsulation effi-iency, determined in triplicate, was calculated as the percentagef the amount of BDS present in the NLCs (drug content – free drug)o the initial amount of BDS incorporated into the NLCs, as follows:

ncapsulation efficiency (%) = Amount of BDS in NLCsInitial amount of BDS

× 100

Coumarin-6 (�Ex = 460 nm, �Em = 505 nm) encapsulation wasssessed by ultracentrifuging the coumarin-6-loaded NLC suspen-ion as described for BDS. The free coumarin-6 present in the filtrateas then measured using fluorimetry (SFM 25 fluorometer, Kon-

ron Instruments, Basel, SW).

.3.3. Determination of BDS by HPLCHPLC was performed to determine the BDS concentration using

Waters 1525 HPLC Binary Pump (Waters Corp., Milford, USA).he UV-detector was a Waters 2487. The system was controlled byhe Breeze software (Waters, UK). A Nucleodur 100-5 C18 5 �m4 mm × 125 mm) column was used at room temperature. The

obile phase contained 55% (v/v) acetonitrile and 45% (v/v) of.025 M KH2PO4 adjusted to pH 3.2 with phosphoric acid, as pre-iously reported by Gupta and Bhargava (2006). BDS was detectedt 244 nm using an ultraviolet detector. The flow rate was set to.1 ml/min for isocratic elution, and the injected sample volumeas 50 �l. The assay was linear over the BDS concentration range of

–30 �g/ml. The intra- and inter-day coefficients of variation wereoth within ±5%. The limits of detection (LOD) and of quantita-ion (LOQ) of BDS were 0.05 �g/ml and 1 �g/ml, respectively. Nonterfering peaks were detected in the assay.

.4. In vitro studies

.4.1. Cell cultureThe J774 macrophage cell line was maintained in RPMI 1640

upplemented with 10% fetal bovine serum. Cells were grown in5 cm2 flasks (Corning, Lowell, MA, USA) in an atmosphere of 5%O2/95% air (v/v) at 37 ◦C.

.4.2. Effect of NLCs on cell viability

The cytotoxicity of the NLCs on J774 cells was evaluated using

he MTT method. Briefly, J774 cells (20,000/well) were seeded in6-well plates (Nunc, Roskilde, DK) and maintained for 24 h at 37 ◦Cnd 5% CO2. After 4 h of exposure at different NLC concentrations,

harmaceutics 454 (2013) 775– 783 777

the cells were washed 3 times with HBSS and incubated for 3 h with0.5 mg/ml MTT in RPMI. The medium was then removed, and thepurple formazan crystals were dissolved in 100 �l of DMSO. Theabsorbance was measured at 570 nm using a MultiSkan Ex platereader (Thermo Fisher Scientific, Waltham, MA, USA). The IC50sfor the different formulations were calculated using the Graph-Pad Prism 5 program (CA, USA). All MTT assays were repeated intriplicate.

2.4.3. Inhibition of TNF- ̨ production from J774 macrophages byBDS-NLCs

The J774 cells were seeded in 12-well plates and allowed toadhere for 24 h. The cells were exposed for 4 h to the following for-mulations: (i) BDS-NLCs (0.4 mg/ml containing 0.29 �g/ml BDS, (ii)Blank-NLCs (0.4 mg/ml) or (iii) BDS suspension (0.29 �g/ml). Theincubation medium was then collected for the lactate dehydroge-nase release assay and replaced with fresh medium supplementedwith 0.1 �g/ml of LPS to activate macrophages (Fernandes et al.,2012; Kitamura et al., 2008). Cell-free supernatants were harvested24 h later and stored at −20 ◦C for cytokine quantitation. TNF-� wasassayed using a DuoSet® ELISA kit (R&D Systems, Minneapolis, MN,USA).

The absence of cytotoxicity of the different formulations onmacrophages was also assessed in the cell culture media after 4 hof incubation by measuring the activity of lactate dehydrogenase.A cytotoxicity detection kit (Roche Diagnostics GmbH, Mannheim,DE) was used according to the manufacturer’s instructions. Theresults were expressed as a percentage of the positive control,which consisted of cells exposed to 1% (v/v) Triton X-100.

2.5. In vivo efficacy of BDS-loaded formulations

An in vivo study was performed in C57BL/6 female mice(18–20 g, 6 weeks; Janvier Laboratories, FR). Animals were housedunder standard conditions and supplied with food and tap waterad libitum. Protocols were approved by the Université catholiquede Louvain animal committee (UCL/MD/2009-001). Colitis wasinduced by administration of drinking water containing 3% (w/v)DSS for 5 consecutive days, and colonic inflammation was assessed7 days after the beginning of DSS treatment (Coco et al., 2013;Laroui et al., 2010). The animals were divided into the followingfive groups (8 mice per group): untreated control group (healthymice), untreated DSS group, DSS+BDS-NLCs group, DSS+Blank-NLCs group and DSS+BDS suspension group (0.4 mg of BDS/kg;550 mg/kg NLCs). Food (but not water) was withdrawn 12 h priorto the administration of the treatment. One hundred microliters ofeach formulation were administered by gavage at days 1, 3 and 5;the untreated control group and the DSS control group received PBSinstead of the formulation. At day 7, the animals were sacrificed,and colon samples were collected for evaluation of the severity ofcolitis.

2.5.1. Clinical activity scoringThe clinical activity score for weight loss, stool consistency and

rectal bleeding was calculated as described by Melgar et al. (2005).Inflammation was scored on a scale of 0–4.

2.5.2. Colon weight/colon length ratioAt the time of sacrifice, 7 days after colitis induction, the excised

colons were measured and weighed (after rinsing the luminal

contents), and the colon weight/colon length ratio was deter-mined. This ratio is considered a reliable and sensitive indicatorof the severity and extent of the inflammatory response in colitis(Alhouayek et al., 2011; Galvez et al., 2000).

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.5.3. Histological assessment of colonic inflammationFor histological scoring, small segments of the colon were fixed

n 4% buffered formalin overnight, washed with 70% ethanol andubsequently embedded in paraffin. Two sets of 3 serial sections10 �m) were cut 100 �m apart, and 6 sections were evaluated forach mouse. Sections were stained with hematoxylin–eosin, andistological scores were determined in a blind manner accordingo a scoring procedure for colitis reported by Geboes et al. (2000),ith slight modifications. Briefly, a five-grade classification system

or inflammation was employed. Inflammation was graded semi-uantitatively from 0 to 4 as follows: 0, no structural change; 1,

nflammation visible; 2, leukocytes present in the lamina propria;, leukocytes present in the epithelium (3.1 < 5% crypts involved,.2 < 50% crypts involved, 3.3 > 50% crypts involved); and 4, cryptestruction visible. Mice were scored individually. Each score rep-esented the mean of six sections.

.5.4. Myeloperoxidase activity (MPO)The tissue-associated MPO assay was performed to quanti-

ate the degree of inflammatory infiltration, which correlates withhe severity of colitis (Krawisz et al., 1984). Briefly, colon speci-

ens from both the proximal and the distal portion of the colonogether (∼25 mg) were snap frozen in liquid N2 at the timef sacrifice and stored for later assessment. For determinationf MPO activity, tissue was placed in HTAB buffer (0.5% HTABn 50 mM potassium phosphate buffer, pH 6) on ice and gentlyomogenized. The homogenate was centrifuged (Allegra X-15R,eckman Coulter, CA) at 2000 × g for 10 min and subsequentlyltracentrifuged (Biofuge 15 R, Heraeus Sepatech, Chandler, Ari-ona, USA) at 18,393 × g for 20 min at 4 ◦C. The supernatant7 �l) was added to 96-well plates (Nunc, Roskilde, DK) togetherith 200 �l of a 50 mM potassium phosphate buffer containing

.167 mg/ml O-dianisidine and 500 ppm hydrogen peroxide. Sam-les were analyzed in duplicate. MPO activity in the supernatantas measured spectrophotometrically (Spectophotometer Spec-

ramax M2e & program SoftMax Pro, Molecular Devices, LLC, USA)t 460 nm for 30 min. The results were expressed as MPO units perram of tissue, and one unit of MPO activity was defined as themount that degrades 1 mmol/min of hydrogen peroxide at 25 ◦CAlhouayek et al., 2011).

.5.5. Tissue cytokine quantitation by ELISAThe concentrations of pro-inflammatory cytokines (IL-1� and

NF-�) in the colon were determined by a sandwich-type ELISAechnique using the Ready-Set-Go! Kit (eBioscience, Vienna, AU)ollowing the manufacturer’s instructions (Alhouayek et al., 2011).olon homogenates were directly prepared from frozen tissue sam-les. Briefly, colonic tissue samples were homogenized in 1 mlf extraction buffer (PBS with 1% SDS and 1 tablet of completerotease inhibitor cocktail (Roche Diagnostics, Vilvoorde, BE), per0 ml of solution) with an UltraTurrax (IKA T18 Basic, Staufen,ermany) and centrifuged first at 900 × g for 10 min at 4 ◦C and

hen for 15 min at 7000 × g at 4 ◦C. The supernatants were col-ected and stored at −80 ◦C for later assessment. The total proteinoncentration was determined using the Lowry method proteinssay.

able 1ummary of formulation particle size, zeta potential, polydispersity index (PdI) and entra

NLC characterization

Size (nm) Zeta potential (m

Blank-NLCs 243.8 ± 12.3 −40.1 ± 7.0

BDS-NLCs 204.1 ± 8.7 −42.2 ± 7.2

Coumarin-6-NLCs 196.8 ± 12.0 −43.4 ± 7.1

harmaceutics 454 (2013) 775– 783

2.6. In vivo localization of coumarin-6-NLCs in thegastrointestinal tract

The localization of coumarin-6-NLCs in the GIT in vivo wasstudied quantitatively by fluorophotometry and qualitatively byfluorescence microscopy. Inflammatory colitis was induced in miceas previously described in Section 2.4. After 7 days, mice receivedby gavage 100 �l of coumarin-6-NLCs. Food (but not water) waswithdrawn 12 h prior to the administration of the treatment. Micewere sacrificed 3, 8 or 12 h after administration, and the wholeGIT (including the luminal content) was then collected. The sam-ples were divided into four sections: stomach, small intestine,colon and cecum. The different samples were homogenized in1 ml of PBS, and the homogenate was subsequently extracted witha methanol/chloroform mixture as described by Makhlof et al.(2009). Coumarin-6 content was quantitated by fluorophotometry.Specimens excised from the non-treated mice were homogenizedas mentioned above and used as blanks. Samples emitting less thandouble the blank’s signal were discarded and not included for thequantitation.

For fluorescence microscopy, colon specimens were embeddedin Tissue-Tek® O.C.T. compound (Sakura Finetek, Torrance, CA)and frozen at −80 ◦C for subsequent experiments. Sections werethen cut at a thickness of 10 �m using a cryostat (Leica Microsys-tems, Wetzlar, GE), and images were captured using a fluorescencemicroscope (Carl Zeiss® Axio Observer) equipped with a structuredillumination module (ApoTome).

2.7. Statistical analyses

Statistical analyses were performed using the GraphPad Prism5.0 program for Windows (GraphPad Software Inc., San Diego, CA,USA). The normal distribution was assessed by the Shapiro–Wilknormality test. The one-way ANOVA test of multiple comparisonsfollowed by Bonferroni’s post hoc test was applied according to theresult of Bartlett’s test of homogeneity of variances for the pro-tein concentration comparisons. All other analyses were performedusing Student’s t-test. Differences were considered statistically sig-nificant at *p < 0.05. The results are expressed as the means ± SD(SD: standard deviation).

3. Results

3.1. NLC characterization

The particle characterization of Blank-NLCs, BDS-NLCs andcoumarin-6-NLCs is summarized in Table 1. Their sizes wereapproximately 200 nm and all presented a negative zeta potential.The mean particle size of the BDS suspension was 41.2 ± 3.41 �m.BDS was completely encapsulated in the NLCs.

3.2. In vitro studies with J774 cells

3.2.1. Assessment of the cytotoxicity of the nanoparticles on J774cells

To select a non-toxic concentration, the cell viability of the BDS-NLCs was measured using the MTT assay on proliferating J774 cells.

pment efficiency (E.E.) per formulation (n = 3; data are expressed as mean ± SD).

V) PdI Encapsulation efficiency (%)

0.2 ± 0.0 –0.2 ± 0.0 95.53 ± 2.410.2 ± 0.0 ∼100

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A. Beloqui et al. / International Journ

he IC50s of Blank-NLCs and BDS-NLCs were 1.74 and 1.59 mg/ml,espectively. Blank-NLCs and BDS-NLCs did not affect the metabolicctivity of J774 cells (∼100% of cell viability) at concentrationselow 0.4 mg/ml. The absence of toxicity of these three formula-ions on J774 cells was corroborated by the lactate dehydrogenaseelease assay (lactate dehydrogenase release <10% after 4 h incuba-ion at 0.4 mg/ml, data not shown).

.2.2. Inhibition of TNF- ̨ production from J774 cellsThe inhibition of TNF-� secretion by J774 macrophages was

valuated by comparing BDS-NLCs with blank-NLCs and free BDS.hile the BDS suspension did not exhibit any significant differ-

nce in the TNF-� levels (measured by ELISA) when comparedith the control medium, no TNF-� secretion was observed from

774 macrophages treated with blank NLCs nor BDS-NLCs (Fig. 1)***p < 0.001).

.3. In vivo effects of BDS-NLCs on murine colitis

.3.1. Inflammatory clinical activity and histological scoringAt day 7, mice were sacrificed, and the severity of inflamma-

ion was characterized by clinical activity scoring (Fig. 2A) andolon structure and histology (Figs. 2B and 3, respectively). Ashown in Fig. 2B, the colon weight/length ratio was increased in thelank-NLCs-treated and BDS-sus-treated DSS groups when com-ared with both the control and the PBS-treated DSS group, but noignificant differences were observed for the BDS-NLCs group.

Histological scoring (Fig. 3) demonstrated a decrease in inflam-ation for the BDS-sus group (**p < 0.01), the BDS-NLCs group

***p < 0.001) and the Blank-NLCs-treated group (*p < 0.05) whenompared with the PBS group. Moreover, the decrease in inflamma-ion was significantly higher in the BDS-NLCs group (*p < 0.05) thann the Blank-NLCs-treated and the BDS-sus groups. As shown Fig. 3Bnd E, the inflammation observed in the colon was strongly reducedn the BDS-NLCs-treated group, and the mucosal architecture was

aintained.

.3.2. MPO activity and pro-inflammatory cytokines (IL-1 ̌ andNF-˛) in the colon

MPO activity, which is a measure of neutrophil infiltration, andhe concentrations of the pro-inflammatory cytokines IL-1� and

NF-� in the colon are shown in Fig. 4. It is noteworthy thatPO activity and cytokine concentrations for the BDS-NLCs-treatedSS group were decreased in all cases when compared with theBS-treated DSS group (*p < 0.05). No significant differences were

ig. 2. (A) Clinical activity scoring after 7 days (B) colon weight/length ratio for controlDS-NLCs-treated DSS group (n = 7) and BDS-sus-treated DSS group (n = 8). Values are me

Blank NLCs (0.4 mg/ml); BDS-NLCs (BDS 0.29 �g/ml, NLC 0.4 mg/ml). The resultswere expressed as the absolute (mean ± SD; n = 4) TNF-� levels (pg/ml) and werecompared to medium-treated cells (***p < 0.001 vs. medium).

observed when mice were treated with control, Blank-NLCs, or freeBDS sus.

3.3.3. In vivo localization of coumarin-6-NLCsTo evaluate the localization of the NLCs in the gastrointesti-

nal tract, coumarin-6-NLCs were administered, and the amountswere quantitated in the stomach, the small intestine, the cecumand the colon. Fig. 5 shows the amounts of coumarin-6 per organ(% dose/g organ) in control and DSS-treated mice 3, 8 and 12 hafter the oral administration of coumarin-6-NLCs. Healthy mice

exhibited a higher amount of coumarin-6 in the cecum and thecolon at 3 h after NLC administration than after 12 h. In DSS-treatedmice, the amounts of coumarin-6 were lower at 3 h but more sus-tained than in the control group in the cecum and colon. In control

(PBS) (n = 8), PBS-treated DSS group (n = 8), Blank-NLCs-treated DSS group (n = 8),ans ± SD. **p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. PBS-treated DSS group.

780 A. Beloqui et al. / International Journal of Pharmaceutics 454 (2013) 775– 783

Fig. 3. Histological scoring of colonic inflammation in DSS-induced colitis (top left). Values are the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. PBS-treated DSS group.Histological sections of the colon stained with hematoxylin–eosin. (A) PBS-treated control, (B) PBS-treated DSS group, (C) BDS sus-treated DSS group, (D) Blank-NLCs-treatedDSS group and (E) BDS-NLCs-treated DSS group.

Fig. 4. Reduction of inflammation in DSS-induced colitis. The BDS-NLCs-treated group showed a significant reduction in MPO activity (A) and in the expression of IL-1� (B)and TNF-� (C) in the colon as measured by an ELISA assay when compared with the PBS-treated DSS group. *p < 0.05 vs. PBS-treated DSS group.

A. Beloqui et al. / International Journal of Pharmaceutics 454 (2013) 775– 783 781

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Fig. 5. Amount of coumarin-NLCs in the stomach, small intestine, cec

ice, the NLCs appeared to accumulate at the surface of the villi.n DSS-treated rats, the NLCs penetrated the mucosae (data nothown).

. Discussion

Currently, strategies for targeting the inflamed mucosa inBD using polymeric nano- and microparticulate drug carriersre particularly promising (Collnot et al., 2012; Laroui et al.,012, 2011; Schmidt et al., 2013). In this study, a nanoparticu-

ate system was developed based on NLCs loaded with BDS andested it in vitro and in vivo to evaluate its potential for IBDreatment.

The BDS-NLC formulation obtained using the high-pressureomogenization technique exhibited physico-chemical character-

stics suitable for delivery to the inflamed colonic mucosa, as theyxhibited a mean size distribution in the range of 200 nm and

fairly monodispersed (PdI < 0.30) population (Table 1). With anncapsulation efficiency close to 95%, BDS was efficiently incor-orated into NLCs, most likely due to their solid lipid matrixhich was mixed with liquid oil (Precirol ATO®5 and Miglyol 812).egatively charged BDS-NLCs (zeta potential −40 mV) may alsoxhibit a strong attraction to the positively charged proteins inlcerated tissues. Moreover, high absolute potential zeta values

ndicate that BDS-NLC formulations are stable (Freitas and Müller,998; Heurtault et al., 2003). BDS is a poorly water-soluble drug.hen suspended in PBS, 41 �m-sized crystals were found in the

uspension. In this conformation, BDS displays poor absorptionharacteristics and poor targeting to the inflamed mucosa, as theres a preferential uptake of nano-sized particles (Lamprecht et al.,001).

The idea of the central role of innate immunity in IBD hasrogressively emerged from studies of IBD pathogenesis. The accu-ulation of experimental evidence from in vitro and animal models,

he genetic knowledge of IBD genetic and the emergence of bio-ogical therapy support this concept (Kaser et al., 2010). Thus, thistudy was focused on the contribution of the innate immune sys-

em in IBD pathogenesis. As nano-sized particles are taken up

ore easily by increased immune-related cells in active inflam-ation (Lamprecht et al., 2001), the anti-inflammatory potential

f NLCs was evaluated in vitro. A reduction in TNF-� secretion by

d colon 3 h, 8 h and 12 h post administration in control and DSS mice.

activated macrophages (J774 cells) was observed (Fig. 1). It is a well-accepted fact that BDS and steroids have an inhibitory effect onthe production of most cytokines by macrophages (Friend, 2005).Interestingly, the pretreatment of J774 cells with Blank-NLCs orBDS-NLCs for 4 h was found to reduce LPS-induced TNF-� levels, butno difference was observed when cells were pretreated with BDSsuspension. These results are in line with Serpe et al. (2010), whoalso observed a significantly reduced production of IL-1� and TNF-�in vitro after 4 h and 24 h of co-incubation of solid lipid nanoparti-cles (SLNs) in a human IBD whole-blood model. Furthermore, theimmunomodulatory ability of lipid-based nanoparticles has beendiscussed elsewhere (Landesman-Milo and Peer, 2012; Peer, 2012).The nature of the NLCs could contribute to the ability of both BDS-NLCs and Blank-NLCs to reduce TNF-� secretion by LPS-activatedmacrophages.

The acute DSS colitis model has been shown to be a good mousemodel for the study of the innate immune aspect of colitis, as itdoes not involve the entire adaptive immune system (Wirtz andNeurath, 2007). The infiltration of leukocytes is one of the earlysteps of the innate response occurring during the active intestinalinflammation. The increased number of leukocytes in the activeinflamed mucosa leads to the recrudescence of reactive oxygenspecies (Holma et al., 2001). MPO, the most abundant protein inneutrophils, is used as a standard test in animal models of IBD andalso as a biological marker in clinical practice, as it can be detectedin the feces of IBD patients (Mendoza and Abreu, 2009). In themurine DSS-induced colitis model, the MPO level is significantlyhigher at all stages of DSS treatment and even after DSS withdrawal(Yan et al., 2009). At day 7, after a 3-day treatment, only BDS-NLCDSS-treated mice exhibited a reduction in MPO activity when com-pared with the non-treated-DSS group (Fig. 4). Interestingly, nodifference was observed from the control group when mice weretreated with either Blank-NLCs or the BDS-suspension, suggestingthat the encapsulation of BDS in NLCs induces a reduction in theinflammation of DSS-induced colitis in mice. In the active inflamedarea, the activation of macrophages leads to the secretion of thepro-inflammatory cytokines TNF-� and IL-11�. The quantitation ofpro-inflammatory cytokines in the colon by ELISA showed a sig-

nificant reduction in the expression of both TNF-� and IL-1� inthe colon for the BDS-NLCs DSS-treated group compared with thePBS-treated DSS group. Although BDS is known to suppress TNF-�production by macrophages, only BDS-NLCs were effective in vivo,

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82 A. Beloqui et al. / International Journ

ighlighting the contribution of the NLCs to the effect of BDS. A his-ological evaluation of the severity of inflammation in the treatedroups was performed. DSS-induced colitis changed the structuref the colon, with a loss in the protective epithelial layer and exten-ive infiltration of immune cells. For the BDS-NLC treatment group,

colonic architecture similar to the control group was observed.he in vivo data demonstrate that in a DSS-colitis model, 3 admin-strations of BDS-NLC reduce the cellular infiltration, the levelsf inflammatory cytokines and maintain the mucosal architectureimilar to the control group.

By reducing the diameter of the particles, it is possible to avoidapid carrier elimination by diarrhea, which is a common symptomn IBD and to allow the preferential uptake of the nano-sized parti-les by immune-related cells (Lamprecht et al., 2001). The decreasen size is believed to increase the residence time of the formulationn the large intestine. The in vivo localization study of coumarin--NLCs allowed us to confirm that even after DSS treatment inice and in mice subjected to serious diarrhea, higher amounts

f NLCs were observed in the colon 12 h after the administration ofoumarin-6-loaded NLCs (Fig. 5). These results suggest an increasedesidence time of the formulation in the colons of DSS-induced col-tis mice. Recently, an in vivo study in human patients based onano- and microscaled particles for drug targeting to the inflameducosa has been published, highlighting the importance of parti-

le size on colon targeting and indicating promising results for theotential application of these formulations (Schmidt et al., 2013).

. Conclusion

To our knowledge, this is the first study evaluating the localelivery of NLCs to the inflamed mucosa in a murine model of

nduced colitis. In vitro, NLCs and BDS-NLCs reduced TNF-� secre-ion by activated macrophages by ∼100%. In vivo, we demonstratedhe sustained presence of NLCs in the colon over 12 h and moremportantly their efficacy in reducing inflammation in DSS-inducedolitis after a 3-day dosage, as observed by a reduction in MPO activ-ty, a reduction in the pro-inflammatory cytokines IL-1� and TNF-�n the colon and by an improved histological score of the colon.

cknowledgements

A. Beloqui wishes to thank the University of the Basque Coun-ry UPV/EHU for the fellowship grants (Personal Investigador enormación 2008 and Ayudas para la movilidad y divulgación deesultados de investigación en la Universidad del País Vasco, movil-dad de investigadores en estancias 2011). This work was partiallyupported by the Basque Government’s Department of Education,niversities and Investigation (IT-341-10) and the Fonds pour laormation à la Recherche dans l’Industrie et dans l’AgricultureF.R.I.A.) for the financial support of R. Coco.

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