Food Research International 51 (2013) 679–692

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Food Research International

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Intracellular fate of retinyl acetate-loaded submicron delivery systems by in vitrointestinal epithelial cells: A comparison between whey protein-stabilised submicrondroplets and micelles stabilised with polysorbate 80

Amal Benzaria a, Dominique Chevalier-Lucia a, Laëtitia Picart-Palmade a, Pauline Hue a,Tomás López-Pedemonte b, Eliane Dumay a,⁎a Université Montpellier 2, UMR 1208, Ingénierie des Agropolymères et Technologies Emergentes, Équipe de Biochimie et Technologie Alimentaires CC023, Place Eugène Bataillon,34095 Montpellier, cedex 5, Franceb Departamento de Ciencia y Tecnología de Alimentos, Facultad de Química, Universidad de la República, Montevideo, Uruguay

⁎ Corresponding author. Tel.: +33 467 143 351; fax:E-mail address: Eliane.Dumay@univ-montp2.fr (E. D

0963-9969/$ – see front matter © 2013 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.foodres.2012.12.059

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 October 2012Accepted 20 December 2012

Keywords:Submicron emulsionsUltra-high-pressure homogenisationIn vitro cell culturesWhey proteinsPolysorbate 80Retinyl acetate

Submicron peanut oil-in-water emulsion stabilised with whey proteins, was processed by ultra-high-pressurehomogenisation at 200 MPa (first-stage, 2-pass homogenisation) and an initial emulsion temperature (Tin) of24 °C. Retinyl acetate (RAC) was selected as a model of a lipophilic biomolecule to be carried by submicrondroplets. For comparison, micelles of retinyl acetate (RAC-micelles) were prepared at atmospheric pressureusing polysorbate 80 (Tween® 80) as emulsifier. Both types of bio-particles displayed submicron diameters.Their behaviour was investigated on TC7-cell monolayers to characterise their influence on monolayer integrity(TER measurements), cell metabolic activity (MTT-assay) and cell membrane integrity (LDH-leakage). TC7-cellexposure to submicron droplets UHPH-elaborated as shuttles for RAC, and stabilised with proteins, did notimpair TC7-cell viability compared with control cells (without deposit) or cells exposed to RAC-micelles. Suchresults demonstrated UHPH-processing innocuity in terms of Novel Food Regulation and implementation ofUHPH as a novel technology. TC7-cell internalisation of whey proteins coating submicron oil droplets wasobserved using confocal microscopy and fluorescent probes. Cellular uptake of RAC was determined for bothsystems, after extraction from TC7 cells and HPLC quantitation. Results revealed RAC incorporation into TC7cells and cellular turn-over of RAC into retinol for both systems without previous in vitro digestion. RACbioaccessibility appeared yet better in the form of RAC-micelles than after entrapment into oily submicrondroplets. Nevertheless, submicron droplets stabilised by proteins displayed a better physical stability than didRAC-micelles, which is the main feature for further developments of carrier systems.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Liposoluble bioactive compounds such as carotenoids and vitamins(A, D, E and K), polyphenols or bioactive lipids (omega-3, omega-6polyunsaturated fatty acids) recognised for their nutritional and healthyproperties (Krinsky & Johnson, 2005; Rice-Evans, Miller, & Paganga,1996), are not always synthesised in the human body and have to beprovided by food. The development of food or dietary supplements in-cluding lipophilic bioactive molecules is often limited due to the pooraqueous solubility of the latter compounds, their sensitivity to light,heat and/or oxygen, and their inherent poor bioavailability, which out-weighs the expected physiological benefits (Fernández-García et al.,2012). For such reasons, the interest in designing systems to protect,carry, deliver and control the release of lipophilic bioactive moleculesrecently greatly increased (Acosta, 2009; Gonnet, Lethuaut, & Boury,

+33 467 143 352.umay).

rights reserved.

2010). For food application, such delivery systems have to be composedof biocompatible, biodegradable, relatively inexpensive, food-grade orGRAS raw materials.

Different strategies have been developed to process oral food-gradenano/submicron vehicles (Cohen Benshitrit, Levi, Levi Tal, Shimoni, &Lesmes, 2012; Matalanis, Jones, & McClements, 2011) based on carbo-hydrates (Kosaraju, 2005), proteins (Chen, Remondetto, & Subirade,2006) or lipids (Fathi, Mozafari, & Mohebbi, 2012; Mozafari, Johnson,Hatziantoniou, & Demetzos, 2008). Among the lipid-based nanoscaledelivery systems, nano/submicron emulsions have been proposed withdroplet diameters well below the micron since small particle sizes aresupposed to improve cellular uptake (Acosta, 2009; Desai, Labhasetwar,Amidon, & Levy, 1996; Hussain, Jaitley, & Florence, 2001; McClean et al.,1998). Indeed, it will be easy to disperse lipophilic molecules into theoily phase as such, or in the form of an ethanolic solution. Furthermore,emulsion structuremay constitute physical and chemical barriers againstenvironment conditions, and particularly against pro-oxidant factors(free radicals, light, oxygen) during emulsion formulation and its further

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storage until consumption, and also during the digestion steps untilabsorption at the intestinal level (McClements, 2011; Talegaonkar,Mustafa, Akhter, & Iqbal, 2010).

Various processes categorised as either “high-energy or low-energyinput processes” are available to produce emulsions in the nano-/submicron range (Fathi et al., 2012; Tadros, Izquierdo, Esquena, &Solans, 2004). The required energy necessary to create the oil–water in-terface may come from (i) the mechanical devices used to generate in-tense disruptive forces, or (ii) the ingredient chemical potential leadingto almost spontaneous formation of oil droplets depending on the disper-sion composition and/or processing temperature. Among high-energyinput equipment, piston-gap type high-pressure homogenisers havebeen recently experimented to produce finely dispersed emulsions ina pulsating or continuous mode (Cortés-Muñoz, Chevalier-Lucia, &Dumay, 2009; Floury, Desrumaux, & Legrand, 2002; Keck & Müller,2006; Schubert, Ax, & Behrend, 2003). Ultra-high-pressure homogeni-sation (UHPH, also called dynamic high-pressure) consists in forcing afluid previously brought to high pressure (100–350 MPa) in a fewseconds in the pressure intensifier, through the valve-gap, a small ori-fice a few micrometres in width (Cortés-Muñoz et al., 2009; Dumay,Chevalier-Lucia, Picart-Palmade, & Benzaria, in press). The resultingpressure drop (ΔP) generates in theHP-valve intensemechanical forcesand elongational stress plus turbulence, cavitation phenomena and im-pacts with solid surfaces and between particles, all these mechanicalforces reducing particle size down to the submicron range. Due toshear effects, a temperature jump through the HP-valve is recorded,that can be controlled by efficient cooling devices located at the im-mediate outlet of the HP-valve (Chevalier-Lucia, Blayo, Gràcia-Julià,Palmade-Picart, & Dumay, 2011; Cortés-Muñoz et al., 2009; Gràcia-Juliàet al., 2008; Picart et al., 2006; Thiebaud, Dumay, Picart, Guiraud, &Cheftel, 2003). Submicron emulsions can be processed by UHPH upto 300 MPa without recycling, or by HPH at lower pressure levels(150–175 MPa) which necessitates multi-pass homogenisation (Dumayet al., in press). Lowmolecular weight (M.W.) surfactant such as polysor-bate or sucrose esters (Floury, Legrand, & Desrumaux, 2004; Sessa, Tsao,Liu, Ferrari, & Donsi, 2011), and macromolecules such as soy proteins(Floury et al., 2002) or whey proteins (Cortés-Muñoz et al., 2009;Floury, Desrumaux, & Lardières, 2000; He et al., 2011; Lee, Lefèvre,Subirade, & Paquin, 2009; Shukat & Relkin, 2011) have been used tostabilise UHPH-processed emulsion droplets. Comparing with low M.W.surfactants,milk proteins offer the advantage to be food-grade emulsifierswith excellent biocompatibility and biosafety, and to form a viscoelasticfilm at the oil–water interface able to protect oil droplets against coales-cence (Dickinson, 1997; McClements, 2004; Wilde, Mackie, Husband,Gunning, & Morris, 2004).

Medical and pharmaceutical areas, earlier than the food one, havedesigned nanoemulsions to improve the oral administration of drugsor natural chemo-preventive agents with very low aqueous solubilityand restricted permeation across the intestinal barrier (Brüsewitz,Schendler, Funke, Wagner, & Lipp, 2007; Gao et al., 2011; He et al.,2011; Qhattal, Wang, Salihima, Srivastava, & Liu, 2011; Ru, Yu, &Huang, 2010; Shen, Wang, & Zhang, 2011; Su, Zhang, & Ho, 2008).In combination or replacement of in vivo experiments, in vitro studiesbased on cellular uptake and transepithelial transport mainly throughCaco-2 intestinal adenocarcinoma cell-lines have been set to evaluatepermeation and transport of drugs included into nanoemulsions(Brüsewitz et al., 2007; He et al., 2011; Qhattal et al., 2011; Ru et al.,2010; Su et al., 2008). Embedding drugs into nanodroplets alloweda significant improvement of drug permeation through cell mono-layers, comparing with corresponding free drugs.

The aim of the present study was to examine the behaviour of sub-micron droplets used as a shuttle for a model lipophilic biomolecule(retinyl acetate), on in vitro epithelial cell-monolayers and to correlatesuch behaviour with the particle physicochemical characteristics. In aprevious study, we have shown that whey protein isolate successfullystabilised peanut oil-in-water (O/W) emulsion processed by UHPH up

to 300 MPa, a high-energy input process (Cortés-Muñoz et al., 2009).In the present study, submicron O/W emulsion droplets with amonomodal distribution have been UHPH-processed by 2-pass homog-enisation at 200 MPa (initial temperature Tin of 24 °C) to embed retinylacetate. Such submicron oil droplets processed by UHPH are comparedto micelles of retinyl acetate prepared at atmospheric pressure usingpolysorbate 80 as emulsifier (a low-energy process), firstly in terms ofphysicochemical properties (size distribution curves, droplet size indi-ces, ζ-potential) then in terms of behaviour on TC7-cell monolayersused as a biological tool: TER measurements, MTT-assay and cellularLDH-leakage were performed to characterise monolayer integrity, cellmetabolic activity and cell membrane integrity, respectively, aftercell exposure to emulsion oil droplets or retinyl acetate micelles.Cellular uptake of retinyl acetate was determined for both systems,after extraction from TC7 cells and HPLC quantitation. Finally, TC7-cellinternalisation of whey proteins coating submicron oil droplets was in-vestigated using confocal microscopy and quantitation of transportedproteins.

2. Materials and methods

2.1. Materials

Whey protein isolate (WPI) has been industrially prepared byLactalis (Prolacta 90, lot 38, Retiers, France) in mild conditions usingmicrofiltration of milk followed by ultrafiltration then retentatespray drying. WPI powder contained 95.1 g dry solids per 100 g,and in dry basis (w/w), 1.04% non-protein nitrogen (NPN), 86.6%protein [(total N−NPN)×6.38], ~1.9% ash (including 0.34% calcium)and 1.5% lactose, as given by the producer. Protein solubility index(PSI) (g of soluble protein per 100 g of total protein) as determinedafter centrifugation of 1% (w/w) protein dispersion at 12,000 g and20 °C for 15 min, equalled 100.3±3% at pH 6.6, and 91.1±1.1% atpH 4.7, indicating the good native state of proteins. Protein constituentscorresponded mainly to β-lactoglobulin (β-Lg) and α-lactalbumin(α-La) (i.e., 68.5% β-Lg and 21.5% α-La per 100 g of soluble protein atpH 6.6) plus small amounts or traces of immunoglobulins, bovineserum albumin (BSA) and lactoferrin.

High-glucose Dulbecco'smodified Eaglemedium (Phenol red-DMEM,11960085), high-glucose Dulbecco's modified Eagle medium with-out L-glutamine and neither pyruvate (Phenol red-free DMEM,31053028), Dulbecco's phosphate-buffered saline containing MgCl2and CaCl2 (DPBS, pH 7.4, 14040174), penicillin–streptomycin solution(15140122), MEM non-essential amino acids (11140035), L-glutamine200 mmol L−1 (25030024), and foetal bovine serum (FBS, 10270106)for cell culture were obtained from Invitrogen (Villebon-sur-Yvette,France). β-Nicotinamide adenine dinucleotide hydrate (NAD, N7004),Trizma® base (Tris, T1503), L-(+)-lactic acid (L1750), bicinchoninicacid solution (B9643), copper (II) sulphate pentahydrate solution(C2284), bovine serum albumin (A9647), PIPES (P8203), EGTA (E4378),3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT,M2128), retinyl acetate (RAC, R7882), retinol (R7632), retinoic acid(R2625), 5-methoxyflavone (M8422), Tween® 80 (P1754), saponin(47036), dimethyl sulfoxide (DMSO, D8418), and formaldehyde so-lution (F1635) were purchased from Sigma-Aldrich (St-QuentinFallavier, France). Triton® X-100 (648466) was fromMerck (Darmstadt,Germany). NaCl (479687), KCl (471177), MgCl2, 6 H2O (459337),n-Hexane (446907), absolute ethanol (528131), acetone (400974) andacetonitrile (412412) came from Carlo Erba (Milano, Italy).

Molecular Probes® DAPI (4′,6-diamidino-2-phenylindole dihydro-chloride, D1306), Alexa Fluor® 680 Phalloidin Conjugate (A22286),Alexa Fluor® 488 goat anti-rabbit IgG antibody (A11034), Pro-long® Gold antifade reagent (P36934) were obtained fromInvitrogen (Villebon-sur-Yvette, France). Rabbit serum anti-bovineβ-lactoglobulin antibody (ASBBLG) was a generous gift from ID Biotech(Immuno-Diffusion Biotechnologies, Issoire, France).

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2.2. Processing of coarse O/W emulsions

WPI dispersion containing 6.1% (w/w) proteins was prepared atthe spontaneous pH (6.3±0.1) in deionised water (Millipore®) bygentle magnetic stirring at 22±2 °C for 2 h, avoiding foam formation.WPI dispersion with a density of 1020.1 kg m−3 (at 20 °C) wasstored overnight at 4 °C to allow protein hydration.

O/W emulsion was prepared at a final concentration (w/w) of 4.3%proteins and 30% peanut oil (Lesieur, Asnières-sur-Seine, France;density of 913 kg m−3; viscosity of 93±6 mPa s, at 20 °C) corre-sponding to an oil volume fraction Φ of 0.32 (i.e., volume of the oilydispersed phase/total volume of emulsion) and an emulsion density at20 °C of 988.0 kg m−3.

A coarse emulsion was firstly prepared using a Silverson emulsifier(model LR2, Silverson Machines LTD, Chesman, UK) equipped with itsemulsion perforated grid. Before emulsification, WPI dispersion andpeanut oil were equilibrated at 20±1 °C. When needed, an adequateamount of RAC previously dissolved in absolute ethanol awayfrom light, was added to peanut oil then dispersed using theSilverson emulsifier (5000 rpm, 10 s, 20±1 °C), so that the finalemulsion contained (w/w, final) 0.3% RAC and 1.6% ethanol. Peanutoil (±RAC) was added all at once to the WPI dispersion. Coarseemulsion was then prepared by stirring together the protein aque-ous phase and peanut oil (±RAC) using the Silverson emulsifier(5000 rpm, 10 min) avoiding air inclusion. The final temperatureof the coarse emulsion equalled 24±1 °C after stirring. A controlcoarse emulsion was also prepared without RAC but with ethanol(1.6%, w/w, final emulsion) to check the possible effect of ethanol onemulsion characteristics.

2.3. Processing of submicron O/W emulsions by ultra-high-pressurehomogenisation

The coarse emulsion at an initial temperature (Tin) of 24±0.5 °Cwas immediately run twice through the ultra-high-pressure (UHP)homogeniser (model FPG7400H, Stansted Fluid Power Ltd., Essex,UK), at 200 MPa (first-stage homogenisation). The UHP-homogeniserwas especially equipped with thermocouples, manometers, pressuregauges and a fast data acquisition system (Red Lion PAXP and PAXTcards, Controls BV, Amersfoort, The Netherlands; 10 Hz acquisition) tofollow temperature and pressure changes at different locations of thehomogeniser during the whole process. The high-pressure generatingsystem consisted in an intensifier driven by a hydraulic pump and oper-ating in a pulsating mode, as already described (Cortés-Muñoz et al.,2009; Picart et al., 2006). The pulsed flow rate was 13.8±0.1 L h−1 at200 MPa. Heat exchangers with circulating water at 8 °C were installedaround the HP-valve to avoid changes in the geometrical characteristicsof the high-pressure valve (HP-valve, first-stage) during processing,around the intensifier and at the immediate outlet of the HP-valve tolimit fluid overheating of the processed emulsion. In addition, a lastcooling device located before the homogeniser outlet allows thefinal temperature of the processed fluid to be adjusted at the desiredoutlet temperature for recycling. The low-pressure valve (LP-valve,second-stage) was not used in the present study. Pressure and temper-ature were recorded before (T1, P1) and after (T2, P2) the high-pressurevalve, and after the first rapid cooling of the processed fluid down-stream the HP-valve (T3). The initial temperature (Tin) of coarse emul-sion in the feeding tank, and the outlet temperature of the processedemulsion (T4)weremeasured using thermistors. Emulsion temperaturewas re-equilibrated at 24±0.5 °C (Tin) before the second pass throughthe homogeniser at 200 MPa (one recycling). Emulsion samples werecollected in brown glass tubes avoiding head-space, at the homogeniseroutlet after the first 500 mL, corresponding to about three-times themean residence time of a particle in the whole homogeniser (Picart etal., 2006).

For each UHPH experiment, samples of emulsion±RAC were col-lected as follows: non processed coarse emulsion (control A), coarseemulsion processed in the homogeniser without applied pressure(control B), and emulsion processed by UHPH at 200 MPa once(first-stage, single-pass homogenisation) or twice (first-stage, 2-passhomogenisation). After UHPH-processing, samples were stored imme-diately at 4 °C and protected from light until further analyses. Two orthree independent UHPH experiments carried out on different dayswere performed for emulsions with or without RAC, respectively.

2.4. Preparation of retinyl acetate micelles

Micelles of retinyl acetate (RAC-micelles) were prepared usingpolysorbate 80 (Tween® 80) as surfactant according to Anwar, Kayden,and Hussain (2006) with minor modifications. Tween® 80 was dis-persed in acetone at a concentration of 12 mg mL−1 at atmosphericpressure and 22±1 °C and RAC was dissolved in absolute ethanol at aconcentration of 0.15 mg mL−1. Twenty μL of the Tween® 80 dispersionwas then added to 11.56 mL of the RAC solution. The ethanol/acetonemixture was dried under a flush of gaseous pure nitrogen. After solventevaporation, the residue was re-suspended into 12 mL of phenolred-free DMEM, to obtain a final concentration of 15.3 μmol L−1

Tween® 80 and 440 μmol L−1 RAC.

2.5. Physicochemical characterisation of submicron emulsion dropletsand retinyl acetate micelles

The size distribution of oil droplets was evaluated at 25±1 °C bylaser light-scattering using a Mastersizer 2000 laser diffractometer(Malvern Instruments, Malvern, UK) at λ=633 nm. Emulsion sam-ples were diluted to 1/10 (v/v) in deionised water then introducedin the diffractometer cell under moderate stirring to reach a 4–5%laser obscuration value which corresponded to a further sampledilution of ~1/1000, v/v, in deionised water (Dumay, Lambert,Funtenberger, & Cheftel, 1996; Thiebaud et al., 2003). The refractiveindex was taken as 1.47 for peanut oil and 1.33 for deionised waterat 25 °C. Absorbance of peanut oil droplet covered by the whey pro-tein layer(s) was considered as 0.004 as previously evaluated formilk proteins (Dumay et al., 1996; Thiebaud et al., 2003). Dropletsizes were characterised by the size distribution curves in volume(%) and in number (%) as a function of droplet diameter, and by thed4.3 index, defined as ∑(nidi4) /∑(nidi3), where ni is the number ofoil droplets of diameter di. The d90 index and the dispersion indexexpressed as (d90−d10)/d50 (or span) were also indicated. The d10,d50 and d90 indices corresponded to the respective droplet diametersat 10, 50 and 90% of the cumulative droplet distribution or emulsionoil volume. For each emulsion sample, two dilutions were evaluatedand each diluted sample was measured five successive times to calculateameandroplet size distribution curve andmean size index values±stan-dard deviation (s.d.).

The size distribution of RAC-micelles was evaluated at 25±1 °Cby photon correlation spectroscopy (PCS) using a Nano-seriesZetasizer (Nano-ZS, Malvern Instruments, Malvern, UK) equippedwith a 4 mW He–Ne laser operating at 633 nm, and a photodiodedetector with a detection angle of 173°. Measurements were carriedout in polystyrene 4-sided polished cuvettes with a 40 μL workingvolume (ZEN0040, Sarstedt, Nümbrecht, Germany). Experimentaldata were assessed by NNLS algorithm taking the dispersant vis-cosity equal to 0.89 mPa s at 25 °C, and the dispersant refractiveindex to 1.33 as for water. Characteristics of RAC-micelles weretaken as 0.001 and 1.53 for the imaginary and the real refractiveindices, respectively. Size distributions were represented as inten-sity or number fraction (%) curves and called size distribution inlight intensity or size distribution in particle number frequency,respectively. For each independent experiment carried out on dif-ferent days, a mean distribution curve in intensity and in number

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was calculated from at least 5–6 measurements per sample. Thecorresponding mean diameters (i.e., arithmetical means calculatedfrom the size distribution curves in intensity), and the minimumand maximum diameters obtained from the distribution curves inintensity were indicated±s.d.

Zeta-potential (ζ) of emulsion droplets and RAC-micelles wasmeasured at 25 °C after 5 min equilibration, using the Nano-seriesZetasizer and clear disposable zeta-cells (DTS 1060C, Malvern Instru-ments, Malvern, UK) previously rinsed with deionized water thenethanol. Real and imaginary refractive indices at 25 °C were respec-tively taken as 1.47 and 0.004 for emulsion oil droplets, and 1.53 and0.001 for RAC-micelles. Water characteristics at 25 °C were taken as1.33 for the refractive index, 0.89 mPa s for the viscosity and 78.5 forthe dielectric constant. Emulsion and RAC-micelle sampleswere dilutedfrom 10- to 500-foldwithmilli-Qwater (18.2 mΩ cm) beforemeasure-ment to check eventual dilution effects on (ζ) measurements. Each (ζ)valuewas an average of 8–12 determinations carried out over the expe-rienced dilution range.

2.6. TC7-cell culture

The Caco-2 clone TC7 used in the present study was kindly provid-ed by Dr. Rousset (Centre de Recherche des Cordeliers, UMR S 872,Paris, France). TC7 cells (passage 36–40) were routinely cultured on75 cm2 cell culture flasks in culture medium composed of phenolred-DMEM containing 4.5 g L−1 glucose and supplemented with 1%(v/v) penicillin–streptomycin solution, 4 mmol L−1 L-glutamine, 1%(v/v) MEM non-essential amino acids and 20% (v/v) heat-inactivatedFBS. Cells were incubated in a humidified 8000DH incubator (ThermoFisher Scientific, Saint Herblain, France) at 37 °C, 8% CO2, 92% air, 100%relative humidity (RH).

For protein transport experiments and determination of cell met-abolic activity (MTT-assay) and membrane integrity (LDH-leakage),TC7 cells were seeded in sterile 12-well Transwell® plates withThinCert™ inserts (3 μm pore size; 1.13 cm2/well; PET, 392–0051,Greiner Bio-one, VWR International, Fontenay-sous-Bois, France)at a density of 4.5×105 cells/well. A 500 μL of cell suspension wasdeposed in the apical compartment and 1 mL of culture medium inthe basolateral one. Cells were cultured for 19 days at 37 °C in con-trolled atmosphere (8% CO2, 92% air, 100% RH; incubator 8000DH) toreach confluence and obtain differentiated cells. The culture mediumwas changed in both compartments every 2 days.

For experiments of retinyl acetate uptake, TC7 cells were seededin sterile 12-well plates (3.5 cm2/well; 734–2156, Nunc, VWR,Fontenay-sous-Bois, France) at a density of 2.5×105 cells/well. Theplates were then incubated for 19 days at 37 °C in controlled atmo-sphere (8% CO2, 92% air, 100% RH; incubator 8000DH) to reach con-fluence and obtain differentiated cells, the culture medium beingchanged every 2 days.

2.7. Determination of the transepithelial electrical resistance

The monolayer integrity was controlled by measurement oftransepithelial electrical resistance (TER) using a Millicell®-ERSvolt-ohm meter (Millipore, St-Quentin-en-Yvelines, France). TERmeasurements were performed at least in triplicate: (i) 48 h be-fore, (ii) just before and (iii) 3 h or 24 h after deposit of submicronemulsion droplet or RAC-micelle samples on TC7 cells followed bycell incubation (8% CO2, 92% air, 100% RH; incubator 8000DH). TER isan index of cell confluence achievement and cell monolayer homo-geneity (Huynh-Delerme, Huet, Noël, Frigieri, & Kolf-Clauw, 2005).The influence of submicron emulsion droplets (first-stage, 2-passhomogenisation at 200 MPa) elaborated±RAC was evaluated com-paring with RAC-micelles.

2.8. Determination of in vitro TC7-cell metabolic activity and cellmembrane integrity

TC7 monolayers grown on ThinCert™ inserts were washed twicewith FBS-free DMEM without Phenol red. Cells were then incubatedfor 3 h or 24 h after deposit of submicron emulsion droplets (first-stage,2-pass homogenisation at 200 MPa, Tin=24 °C) or RAC-micelles inthe apical compartment. Before deposit, submicron emulsionswere previously diluted 20-fold in phenol red-free DMEM whichcorresponded to a RAC concentration of 440 μmol L−1 in the culturemedium. RAC-micelles were deposed at the same final RAC concentra-tion of 440 μmol L−1 in phenol red-free DMEM. The same protocolwas also carried out for (i) submicron emulsions prepared withoutRAC, and (ii) submicron emulsions prepared without RAC but withethanol to check the possible effect of ethanol on cell viability. Theethanol concentration in the DMEM-diluted emulsion deposed ontothe cells was 0.08% (v/v).

MTT-assay was carried out according to Mosmann (1983) withminor modifications, to assess the influence of submicron emulsiondroplets (±RAC) or RAC-micelles on TC7 cell metabolic activity.Indeed, the ability of cells to reduce MTT into Formazan® providesan indication of mitochondrial integrity and activity which, in turn,may be interpreted as a measure of cell viability or metabolic activity.After 3 or 24 h of cell incubation with emulsion droplets orRAC-micelles at 37 °C in controlled atmosphere as described above,apical and basolateral culture media were removed. The 12-wellTranswell plates were washed twice with DPBS. Cells were incubatedfor a further 3 h with MTT (0.15 mg MTT mL−1 in FBS-free DMEMwithout Phenol red) added in the apical compartment of wells. Theapical medium was then removed, and 500 μL/well of DMSO wasadded to lyse cells. A hundred μL of each lysate was then transferredinto 96-well plates and diluted 2-fold with DMSO. Absorbance ofFormazan® liberated by cell lyse, was detected at 570 nm using amicroplate reader (Multiskan Spectrum, Thermo Electron, Vintaa,Finland). For each independent experiment, each submicron emul-sion or RAC-micelle samples were subjected to MTT-assay using 2–3ThinCert™ inserts. Results are the mean of 7–10 absorbance measure-ments. Three independent experiments (UHPH-processed submicronemulsions prepared on different days) were carried out to elaboratesubmicron emulsion without RAC and two for emulsions containingRAC. Results obtained with RAC-micelles were the means from twoindependent micelle preparations.

LDH-leakage outside TC7 cells was measured in the cellular apicalmedium according to Mahfoud, Maresca, Garmy, and Fantini (2002)with minor modifications. Indeed, an increase of LDH-release fromthe cytosol indicates a loss of cellular membrane integrity leading tofurther cell death. After TC7 cell incubation at 37 °C in controlledatmosphere (8% CO2, 92% air, 100% RH; incubator 8000DH), in thepresence of emulsion droplets (±RAC) or RAC-micelles, 25 μL of theapical medium was added to 250 μL of NAD–substrate mixture(1.65 mM NAD, 165 mM KCl, 54 mM L-lactic acid, 108 mM Tris,final pH 9.3) in 96-well microplates (Maxisorp™ microplates, VWRInternational, Fontenay-sous-Bois, France). LDH induced the lactateoxidation into pyruvate with the simultaneous reduction of NADto NADH. NADH absorbance in the plate-well content was recordedat 340 nm using the Multiskan plate-reader immediately, and after10 min incubation at 37 °C. A positive control corresponding to 100%LDH release by lysing cells completely was included in LDH-leakageassay using a 1% (v/v) Triton® X-100 solution in DMEM. LDH-releasewas expressed as the proportion (%) of the positive control value:

LDH−release %ð Þ ¼ ΔAbs of the sampleð Þ= ΔAbs of the positive controlð Þ�100 ð1Þ

where,ΔAbs is the difference between absorbancemeasured immediate-ly and 10 min after deposing the NAD–substrate mixture on plate-wells.

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For each independent experiment, submicron emulsion or RAC-micellesamples were subjected to LDH-assay at least in duplicate. Data areexpressed as the mean of 4–6 measurements±s.d. obtained from 2ThinCert™ inserts.

2.9. Preparation of TC7-cell samples for confocal microscopy imaging

TC7 cells grown on ThinCert™ inserts (sterile 12-well Transwell®plates) were incubated for 5 min, 1 h, 3 h or 24 h (8% CO2, 92% air,100% RH; incubator 8000DH) in the presence of submicron emulsiondroplets prepared by 2-pass homogenisation at 200 MPa (Tin=24 °C).Submicron emulsion was 20-fold diluted in DMEMwhich correspondedto a final WPI protein concentration of 2.12 mg mL−1. Then 500 μL ofthe diluted submicron emulsion was deposed on the apical compart-ment of the insert which corresponded to a protein deposit of 1.06 mg.After incubation, cells were washed twice with DPBS warmed at 37 °Cthen fixed using a 4% (v/v) formaldehyde solution for 30 min. After 3washing-steps with DPBS to remove formaldehyde excess, the fixedcells were permeabilised using a PEM solution (100 mM PIPES, 5 mMEGTA, 2 mM MgCl2·6H2O, pH 6.9) containing 0.2% (w/v) saponin, for10 min. After washing twice with a PEM solution at 0.1% (v/v) saponinplus 0.1% (w/v) BSA, the fixed cell samples were let overnight at 4 °Cin a PEM solution containing 0.1% (v/v) saponin plus 2% (w/v) BSA toblock the free protein sites and reduce further non-specific backgroundstaining. After sucking the blocking solution, the fixed cell sampleswere successively treated for 1.5 h with rabbit serum anti-bovine β-Lgantibody (primary antibody; diluted 1:100), then for 1 h with AlexaFluor® 488-labelled goat anti-rabbit IgG antibody (secondary antibody;diluted 1:200). Antibodies were diluted with a PEM solution at 0.1%(v/v) saponin and 0.1% (w/v) BSA. Finally, F-actin (cytoskeleton) incells was labelled in purple with Alexa Fluor® 680 Phalloidin Conjugate(0.58 μg mL−1; 40 min of exposure time), and cell nuclei were labelledin blue with DAPI (1 μg mL−1; 1 min of exposure time), both fluores-cent probes being diluted at the desired concentration using the PEMsolution at 0.1% (v/v) saponin and 0.1% (w/v) BSA. After washing withthe latter PEM solution, cell samples were mounted on microscopyslides with Prolong® antifade reagent then covered with cover slips.All fixation/labelling treatments were conducted at 20±2 °C. Fixedand labelled cell samples were stored at 4 °C before imaging. Slideswere observed by confocal microscopy at the Montpellier RIO Imagingfacility of Human Genetics Institute (IGH) (CNRS, Montpellier) usingthe laser scanning microscope Axioplan2/LSM 510 Meta and LSM 510Software (version 2.8.) from Carl Zeiss (Jena, Germany).

2.10. Determination of protein amount transported through TC7-cellmonolayers

Protein concentration was determined in the basolateral com-partments of ThinCert™ inserts after 5 min, 3 h or 24 h incubationof TC7 cells grown on inserts in the presence of submicron emulsion(first-stage, 2-pass homogenisation at 200 MPa, Tin=24 °C), asexplained in Section 2.9. A 500 μL of previously 20-fold diluted submicronemulsion in phenol red-free DMEM (2.12 mg protein mL−1

final) wasdeposed onto the apical compartment of ThinCert™ inserts correspond-ing to 1.06 mg of protein deposit. After the desired exposure time, theprotein concentrationwas determined in aliquots taken in the basolateralcompartment. Protein content was measured by the bicinchoninic acid(BCA) method (Smith et al., 1985) and expressed as the means of 6–8determinations obtained from two ThinCert™ inserts per sample.

2.11. Determination of retinyl acetate cellular uptake

For cellular uptake experiments, differentiated TC7 cells grown onsterile 12-wells plates were washed twice with DPBS buffer then incu-bated at 37 °C in controlled atmosphere (8% CO2, 92% air, 100% RH;incubator 8000DH) for 5 min, 1 h, 3 h, 6 h or 24 h, in the presence of

emulsion droplets (±RAC) or RAC-micelles at a final RAC concentrationof 440 μmol L−1. After incubation, the culture medium was removedfrom the plate-wells then washed 3-times with ice-cold DPBS buffer.Cells were then scratched and collected using 1 mL of DPBS containing1% (v/v) of Triton® X-100 solution. Cell samples were stored at−40 °Cuntil retinyl acetate extraction and quantitation. The determination ofretinoid cellular uptake was carried out from three plate-wells persample and from at least two independent experiments (independentUHPH experiment followed by deposit of submicron or RAC-micelleson TC7 cells then retinoid extraction from the cells).

RAC was then extracted from cell samples using an ethanol–hexanemixture according to Chávez-Servín, Castellote, and López-Sabater(2006) with minor modifications. Briefly, 60 μL of an ethanolic solutioncontaining 5-methoxyflavone (final concentration 0.5 mmol L−1) wasadded as external standard to 1 mL of cell sample in a 12 mL centrifugetube (PPCO, Nalgene, Rochester, USA). Three mL of absolute ethanolwas added and the mixture was vigorously vortexed for 2 min. OnemL of hexane was then added, and the whole mixture was vortexedfor another 1 min. After 10 min standing at 20±1 °C away from light,3 mL of a NaCl-saturated aqueous solution was added, and the mixturewas manually shaken by inversion. After centrifugation at 1500 g and20 °C for 6 min (Sorvall RC5B Plus, rotor SS-34), the hexane phasewas recovered in brown vials and dried using a flush of gaseous purenitrogen. Cell-extracts from three wells were re-suspended into 200 μLhexane for high-performance liquid chromatography (HPLC) analysis.

HPLC was performed using a chromatography system fromWaters(St-Quentin-en-Yvelines, France) equipped with a 2695 separationmodule, a 2996 UV/visible photodiode array detector and a Waters-plusauto sampler injector. The chromatography separation was performedat 20±2 °C in 20 min with a reversed-phase Altima C18 column(150×4.6 mm; 5 μm particle size; Alltech, Epernon, France), using anisocratic acetonitrile–water (90%–10%) mobile phase at a flow rate of1 mL min−1. Detection was carried out at 327 nm. Injection volume ofcell extracts was 20 μL. Chromatography peaks were integrated usingthe Millenium 32 v4.0 Software from Waters. Retinyl acetate, retinoland retinoic acid were used for identification of chromatography peaks.For each cell-extract, retinoid quantitation was performed in triplicateand expressed as nanomoles per mg of cellular proteins present in thecorresponding plate-well. Protein concentration in the correspondingplate-wells was measured by the bicinchoninic acid (BCA) method(Smith et al., 1985) and expressed as the means of 3–4 determinations.

2.12. Statistical analysis

Statistical analyses of experimental data were carried out usingFischer's and Student's tests. Valueswere presented asmeans±standarddeviation (s.d.) for independent experiments carried out on differentdays.

3. Results and discussion

3.1. Physicochemical characteristics of submicron emulsion droplets andretinyl acetate micelles

Submicron droplets of O/W emulsion processed by UHPH (a high-energy process) and stabilised by whey proteins were firstly comparedto retinyl acetate micelles prepared at atmospheric pressure usingpolysorbate 80 as emulsifier (a low-energy process). Fig. 1 displaysthe particle size distribution curves for the 4–6 h-old emulsions:non-processed coarse emulsions (control A), coarse emulsion processedin the homogeniser without applied homogenisation pressure(control B), and emulsion processed by UHPH at 200 MPa once(first-stage, single-pass homogenisation) or twice (first-stage, 2-passhomogenisation).

The coarse O/W emulsion (control A) was characterised by a widebimodal distribution over 0.7–35 μm (distribution in volume; Fig. 1A)

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Fig. 1. Droplet size distribution curves in volume (A) or in number frequency (B) of O/Wcoarse emulsion (control A: ), coarse emulsion processed in the homogeniser withoutapplied pressure (control B: ), emulsion processed by UHPH at 200 MPa once(first-stage, single-pass homogenisation: ) or twice (first-stage, 2-pass homogenisation:

) at an initial emulsion temperature Tin of 24 °C for each homogenisation pass. Cumula-tive size distributions in volume (%, A) or in number (%, B) are shown for emulsionprocessed twice by UHPH at 200 MPa (first-stage, 2-pass homogenisation: ). Emul-sions contained (w/w) 4.3% whey proteins plus 30% peanut oil. Measurements were car-ried out using laser light-scattering after sample dilution (for details, see Section 2.5).Mean distribution curves were obtained from 5 successive measurements.

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with a main population at 11–13 μm and a minor one close to 1.5 μm(peak maxima) as shown by the distribution curves in volume(Fig. 1A), or a main peak at 0.8–1 μm and a second one at 5–6 μm,as shown by the distribution curves in number frequency (Fig. 1B).The differences observed between both types of distribution areexplained by the fact that the size distribution in volume reflectsthe influence of the largest particles and, conversely, the size distribu-tion in number frequency is sensitive to particles of the smallest sizes.Processing the coarse emulsion through the homogeniser withoutapplied pressure (control B) slightly decreased the number of largestdroplets (Fig. 1B) and slightly but significantly (Pb0.001) decreasedthe d4.3 index (Table 1) due to the size reduction of pipe diametersin the equipment and HP-valve gap. As expected, processing thecoarse emulsion once by UHPH at 200 MPa decreased the droplet sizedown to the submicron range, with a main population at ~120–138 nmplus a secondary one at 550 nm (peak maxima) (Fig. 1A) in accordancewith previous results (Cortés-Muñoz et al., 2009). A second homogenisa-tion pass (recycling once) at 200 MPa allowed amonomodal and narrowsize distribution to be obtained, with a peak maximum at 138 nm(distribution in volume, %, Fig. 1A) or 60 nm (distribution in numberfrequency, Fig. 1B), and significantly (Pb0.001) lower span, d4.3 and d90values compared to a sole homogenisation pass (Table 1). Cumulate

distribution indicated that 90% of the emulsion oil volume correspondedto droplet diameters below 200 nm, and 98% below 300 nm (Fig. 1A).Processing twice at 200 MPawas therefore selected as optimised condi-tions to get a homogeneous emulsion sample with droplets in thesubmicron/nano-size range to be later studied on cell-monolayers. Fur-thermore, processing at 200 MPa avoided UHPH-induced aggregationof whey proteins as already observed (Gràcia-Julià et al., 2008).

Despite a slight decrease in d4.3 and d90 values observed for bothcontrol samples (A and B) after addition of 1.6% ethanol (w/w, final;data not shown) or the ethanolic RAC solution (0.3% RAC and 1.6%ethanol, w/w, final; Table 1), similar results were obtained after pro-cessing at 200 MPa (one- or two-pass homogenisation) for emulsionsloaded or not with RAC (Table 1), which indicated that addition ofretinyl acetate in absolute ethanol at the indicated concentrationdid not really impair the surface-active properties of whey proteins.

ζ-potential values (Table 1) confirmed that droplets covered bywhey proteins at pH 6.3±0.1 were negatively charged, as expected.The absolute value of ζ-potential was slightly but significantly(P=0.01) higher for UHPH-processed (200 MPa, 1–2 homogenisationpasses) than non-processed emulsion droplets (control A), which willimprove droplet stability by favouring electrostatic repulsions betweendroplets.

The highest temperature reached by an emulsion sample through-out UHPH-processing was 62.9±1.0 °C as measured at the immedi-ate HP-valve outlet (T2). The residence time of the processed fluidat temperature T2 was b0.25 s (Picart et al., 2006). Emulsion temper-ature rapidly decreased to 44.9±0.8 °C after the first cooling device(T3) then to 30.0±2.0 °C (T4) at the homogeniser outlet after thesecond cooling device. Controlling the fluid temperature throughoutthe process thus avoided or limited sample-overheating that couldbe detrimental for heat-sensitive biomolecules such as RAC embed-ded in oil droplets.

The oil–water interfacial area calculated from the size distributioncurves of emulsion sample processed twice by UHPH at 200 MPa was16.7 m2 per mL of emulsion, which corresponded to 55.8 m2 per g oil.Assuming that all whey proteins present in emulsion (4.3% protein,w/w) are available to cover droplet interface, a 2.55 mg proteins perm2 of interface was calculated, which corresponded to 1.75 mg β-Lgand 0.55 mg α-La from the WPI (Section 2.1.). Amounts of 1–4 mgof food proteins per m2 interface are usually determined after separa-tion of the O/W emulsion creamy phase from the bulk emulsion,results depending on the methodology (emulsion composition, ageand pH; protein interfacial properties and the presence of otherproteins or surfactants; processing pressure-level and temperature;centrifugation conditions to separate oil droplets from the aqueousphase, and number of emulsified-phasewashing-steps; protein determi-nation in the creamy phase or in the aqueous phase) (Dalgleish, 1996;Robin, Kalab, Britten, & Paquin, 1996). For emulsions prepared byhomogenisation at 30 MPa and pH 7.0 with (w/w) ~0.5% purified β-Lgor α-La and 10–20% n-tetradecane, amounts of 1.7–3 mg of β-Lg and1.5–2.7 mg of α-La have been found per m2 of interface (Courthaudon,Dickinson, Matsumura, & Williams, 1991; Dickinson, Rolfe, & Dalgleish,1989). Using 10% soy oil and 0.45% WPI proteins (%, w/w, final emul-sion), Lee, Subirade, and Paquin (2008) prepared by homogenisation(50–200 MPa) O/W emulsion with d3.2 size index of 500–320 nm, andprotein load of 1.70–1.58 mg m−2. Using 20% soy oil and 0.4–1.8% WPIproteins (%, w/w, final emulsion), Hunt and Dalgleish (1994a, 1994b)found that β-Lg and α-La adsorbed to the interface in proportion totheir concentration. A minimum limiting surface concentration of1.5 mg m−2 of whey proteins was required to stabilise emulsions(droplet apparent average-diameter of 0.4 μm), and a maximal sur-face concentration of ~3.2 mg m−2 was found for protein concentra-tions>1.8% (w/w, final emulsions). Considering the latter figures andthe large interface area created in UHPH-processed emulsion samplesin the present study, it looks like that the major part of WPI proteinscould stabilise the oil–water interface, and that a very small amount

Table 1Droplet size indices (d4.3, d90 and span) and zeta-potential (ζ) of O/W emulsions processed by UHPH (one- or two-pass homogenisation at 200 MPa and Tin=24 °C) with (w/w,final) 4.3% whey proteins plus 30% peanut oil, and in the presence or absence of retinyl acetate (0.3%).

Retinyl acetate (%, w/w) Pressure(MPa)

d4.3a

(μm)d90

a

(μm)Spana ζ-valueb

(mV)

0 Control A 13.47±0.01 23.04±0.03 1.383±0.002 −19.6±1.7Control B 13.08±0.05 22.39±0.06 1.395±0.006 n.d.200—1 pass 0.177±0.001 0.307±0.001 1.743±0.001 −22.0±2.1200—2 passes 0.147±0.003 0.227±0.001 1.064±0.054 −22.8±2.0

0.3 Control A 12.52±0.01⁎⁎⁎ 21.43±0.02⁎⁎⁎ 1.392±0.001⁎⁎⁎ −21.0±2.2Control B 12.20±0.01⁎⁎⁎ 20.81±0.04⁎⁎⁎ 1.395±0.002 n.d.200—1 pass 0.176±0.004 0.309±0.012 1.769±0.048 −24.1±2.0⁎

200—2 passes 0.149±0.002 0.226±0.002 1.021±0.010 −24.3±1.7

The emulsion content in absolute ethanol used to solubilise retinyl acetate was 1.6% (w/w, final).a Means values±standard deviation from 5 measurements. Values were calculated from the size distribution in volume (%) as determined by laser light scattering. In the same

series (0% or 0.3% retinyl acetate), values of d4.3, d90 and span were all significantly different for at least P=0.05.b Means values±standard deviation from 8 to 12 measurements. In the same series (0% or 0.3% retinyl acetate), ζ-values (zeta-potential) of submicron emulsion droplets

(one- or two-pass homogenisation at 200 MPa) were significantly different from ζ-value of non-processed emulsion droplets (control A) for P=0.01.⁎⁎⁎ When comparing both series, significant differences between the corresponding emulsion samples prepared without (0%) or with (0.3%) retinyl acetate for P=0.001.

⁎ When comparing both series, significant differences between the corresponding emulsion samples prepared without (0%) or with (0.3%) retinyl acetate for P=0.05.

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of β-Lg could remain free in emulsion aqueous phase. Following the fateof β-Lg through cell monolayers, could therefore bring some informa-tion on internalisation of droplet materials in TC7 cells (Section 3.4.).

Fig. 2 shows the size distribution curves of RAC-micelles in lightintensity (Fig. 2A) or number frequency (Fig. 2B) for fresh samplesanalysed within 1 h of preparation at 22±1 °C, and after storage at20 °C for 6 h or 24 h. Fresh RAC-micelles displayed a broad andbimodal size distribution over 44–615 nm (Fig. 2A and B), with

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Fig. 2. Particle size distribution curves of retinyl acetatemicelles as determined by photoncorrelation spectroscopy (PCS) in light intensity (A) or in particle number frequency (B).Micelles of retinyl acetate were analysed within 1 h of preparation at 22±1 °C (♦) orafter storage at 20 °C for 6 h ( ) or 24 h ( ). PCS measurements were carried out at25 °C (for details, see Section 2.5). Mean curves were calculated from 6 to 8 PCSmeasurements.

peak maxima at 164 nm (Fig. 2A) or 80 nm (Fig. 2B) and mean micellediameters (arithmetic means) of 204±30 nm or 100±30 nm, respec-tively. After 6 h storage at 20 °C, large micelles appeared up to 5–6 μm(Fig. 2B) indicating some micelle coalescence which was emphasisedafter 24 h of storage at 20 °C leading to a mean diameter of 279±89 nm (distribution in light intensity; Fig. 2A). From the size distribu-tion in number frequency (Fig. 2B), the disappearance of large micelles(>600 nm)was accompanied by an apparent increase in the number ofthe remaining droplets of smallest sizes (40–60 nm). The markedchanges observed in the size distribution curves of RAC-micelles preparedwith Tween® 80 resulted from a time-limited physical stability followedby coalescence, despite the initial submicron RAC-micelle sizes. Higherconcentration of Tween® 80 was not chosen in the present study, toavoid further detrimental effects on cell viability (He et al., 2011).

In opposite, UHPH-processed submicron emulsion droplets stabilisedwith whey proteins displayed an excellent time-stability vs. creamingand coalescence over 9 days of storage at 4 °C, as already studied(Cortés-Muñoz et al., 2009). The better stability against aggregation andcoalescence of oil droplets stabilised with proteins could partly resultfrom higher repulsive forces between submicron droplets as suggestedby ζ-potential values (ζ=−24.3±1.7 mV) (Table 1), comparing toRAC-micelles stabilisedwith Tween® 80 (ζ=−12.7±2.7 mV). Further-more,milk proteins forma viscoelasticfilm at the oil–water interface ableto protect oil droplets against coalescence (Dalgleish, 1996, 1997; Lee etal., 2009). However, it would be interesting to determine the film interfa-cial thickness and viscoelasticity of nanodroplets stabilised with proteinscompared to micelles stabilised with polysorbate as it has been carriedout for other systems (Torcello-Gómez, Maldonado-Valderrama, deVicente, et al., 2011).

3.2. Influence of submicron emulsion droplet or retinyl acetate micelledeposit on the characteristics of in vitro TC7-cell monolayers

The behaviour of both systems (RAC-loaded emulsion droplets orRAC-micelles) was investigated after deposit on TC7-cells then incu-bation for 3 h or 24 h at 37 °C in controlled atmosphere, both beingdeposed at a final RAC concentration of 440 μmol L−1 in the culturemedium. Effects of particle deposit on TC7 cells were evaluated throughTERmeasurements (Table 2), determination of cellularmetabolic activ-ity (MTT-assay) (Fig. 3) and cell membrane integrity (LDH-leakage)(Fig. 4).

The integrity of epithelial cell monolayers is used as a marker ofcell viability, since the loss of monolayer integrity caused by toxi-cants often occurs much earlier than the cell death or dysfunction(Biganzoli, Cavenaghi, Rossi, Brunati, & Nolli, 1999; des Rieux et al.,

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Fig. 4. Cell membrane integrity as assessed by LDH leakage (for details, see Section 2.8)measured after 3 h (■) or 24 h ( ) incubation of TC7 cells in the presence of submicronemulsion droplets loaded without or with retinyl acetate (RAC), or in the presence ofRAC-micelles stabilised with Tween 80®. Control cell-samples were incubated withDMEM (cell culture medium) alone. Submicron O/W emulsion was processed twice byUHPH at 200 MPa (first-stage, 2-pass homogenisation, Tin=24 °C). Submicron emulsiondroplets or RAC-micelle deposits corresponded to 440 μmol L−1 of retinyl acetate in thecell culture medium. Results were expressed as the proportion (%) of the positive controlvalue obtained with Triton® X-100 solution to lyse the cells completely (for details, seeSection 2.8). For each sample, data are expressed as themeanof 4–6measurements±stan-dard deviation obtained from 2 ThinCert™ inserts. Means with different letters (a, b, c)were significantly (P=0.05) different between themselves.

Table 2TER valuesa determined before and after 3 h or 24 h of TC7-cell exposure to emulsiondroplets processed by UHPH±retinyl acetate, or to retinyl acetate micelles.

TER measurement 48 h beforeTC7 cellexposure

Just beforeTC7 cellexposure

After TC7 cellexposure for3 h or 24 h

Series “3 h” of exposure timeEmulsion droplets without retinylacetate

298±11 347±11 335±09

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297±23 358±06 342±24

Micelles of retinyl acetate(440 μmol L−1 in DMEM)

307±08 347±11 478±25b

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293±12 357±06 410±20c

Emulsion droplets loaded with retinylacetate (440 μmol L−1 in DMEM)

303±10 369±01 416±15c

Micelles of retinyl acetate(440 μmol L−1 in DMEM)

319±06 377±16 469±97

Emulsions processed by UHPH at 200 MPa (two-pass homogenisation) and Tin=24 °C.a Samples of submicron emulsion and retinyl acetate micelles were diluted with

DMEM before being deposed onto TC7-cell monolayers for a 3 h or 24 h of exposuretime. Means±standard deviation obtained from 3 to 4 TER determinations.

b Significant difference (P=0.01) between retinyl acetate micelles and submicronemulsion samples within the same series of exposure time.

c Significant difference (P=0.05) between TER values determined before and afterexposure of TC7 cells to submicron emulsion droplets or retinyl acetate micelles withinthe same series of exposure time.

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2005; Narai, Arai, & Shimizu, 1997; Tyrer et al., 2002). In the presentstudy, TER values were close to 302±14 Ω cm−2 as measured48 h before TC7 cell incubation with emulsion droplets (±RAC)or RAC-micelles, and close to 359±13 Ω cm−2 just before cell expo-sure (means±s.d. obtained fromboth series 3 h and 24 h, Table 2) indi-cating cell layer confluence and integrity of the tight junctions.

After 3 h of TC7-cell exposure to emulsion droplets without RAC,no significant change (P=0.05) in TER values was noticed comparedto TER values measured just before exposure (Table 2). After 3 h ofTC7-cell exposure to droplets loaded with RAC, TER values did notsignificantly change comparing with droplets without RAC. The latterresults indicate that submicron droplets with or without RAC, did notimpair TC7 cells.

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In opposite, TER value significantly (P=0.01) increased after 3 hof TC7-cell exposure to RAC-micelles comparing with TER valuesdetermined (i) just before exposure to RAC-micelles, or (ii) afterexposure to submicron emulsion droplets loaded with RAC, whichsuggested that RAC as a metabolic factor improved TC7-cell develop-ment, and was more available in the form of RAC-micelles than afterincorporation into submicron oily droplets. A similar trendwas observedafter 24 h of TC7-cell exposure to RAC-micelles (Table 2) although theincrease in TER value (469±97, Table 2) was no more significant(P=0.05) due to the large standard deviation which suggested someinhomogeneity in the cell culture probably due to the formation ofcell multilayer areas. To conclude, results of Table 2 indicate that(i) submicron emulsion droplets or RAC-micelles as formulated inthe present study did not induce any detrimental effect on TC7-cellviability, and (ii) RAC favoured cell development rather in the form ofRAC-micelles than after incorporation into submicron oily droplets.

Fig. 3 displays TC7-cell metabolic activity as assessed by theMTT-assay. Results indicated that TC7-cell mitochondrial activity (orcell viability) was not impaired by exposure to submicron emulsiondroplets (±RAC) or to RAC-micelles, suggesting again that all formu-lations were biocompatible with TC7 cells. Furthermore, TC7-cellmetabolic activity slightly but significantly (P=0.05) increased after3 h exposure for both submicron emulsion droplets loaded with RAC,and for RAC-micelles, comparing with control cells (DMEM alone)(Fig. 3), suggesting a positive effect of RAC on cell metabolic activity.The latter positive effect no more appeared after 24 h of cell exposure,comparing with control cells (DMEM alone) (Fig. 3).

The percent of LDH-release relative to that induced by Triton®X-100 remained low after 3 h or 24 h of TC7-cell incubation in thepresence of emulsion droplets (±RAC) or after 3 h of cell incubationwith RAC-micelles, and most of the values were not significantly dif-ferent from that obtained for control cells (DMEM alone) (Fig. 4). Cellincubation with RAC-micelles for 24 h, displayed yet a significant(P=0.05) increase in LDH-release (~44%; Fig. 4) indicating somedamageof cellular membrane probably induced by Tween® 80 although in littleconcentration in RAC-micelles. In opposite, emulsion droplets±RAC didnot cause cellular membrane damage at the experimental RAC and pro-tein concentrations in the culture medium, even after 24 h incubation.

Few studies concern the behaviour of food emulsions on model cellmonolayers. He et al. (2011) prepared submicron droplets 200–250 nm

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in diameter by 10-pass homogenisation at 80 MPa, and stabilised bypreviously heat-denatured food proteins (soybean protein, wheyprotein isolate or β-lactoglobulin). In accordance with our results, aCaco-2-cell viability (as evaluated by the MTT-assay)≥85% was foundafter 4 h of cell exposure for all emulsion samples stabilised with foodproteins. In opposite, emulsions stabilised with Tween® 80 caused amarked decrease in cell viability when deposed in the culture mediumat a concentration≥2 mg mL−1 (He et al., 2011). For comparison, theamount of Tween® 80 deposed with RAC-micelles on TC7-cells in ourstudy, was much lower (~0.02 mg mL−1 of culture medium) and didnot impair cell viability as assessed byMTT-assay (Fig. 3). Nevertheless,the more sensitive LDH-leakage test (Fig. 4) revealed some membranedamage after 24 h of cell exposure to RAC-micelles stabilised withTween® 80, in line with the results of He et al. (2011) that highlightsome detrimental effect of polysorbate.

The pooled results indicated that submicron droplets elaboratedby UHPH as shuttles for RAC, and stabilised with whey proteins, didnot impair TC7-cell metabolic activity or membrane integrity whichmay not always be the case of a lowM.W. surfactant such as polysorbate.Such results are of importance to ascertain the UHPH-process innocuityin terms of Novel Food Regulation, and implementation of UHPH as anovel technology.

3.3. In vitro cellular uptake of retinyl acetate

Retinyl acetate uptake by TC7 cells was determined after TC7-cellexposure for 5 min and 1, 3, 6 or 24 h, to submicron emulsion droplets(±RAC) or RAC-micelles at a final concentration of 440 μmol L−1 in theculture medium. After RAC extraction from cell-samples then HPLCanalysis, retinyl acetate (RAC) plus retinol (ROH)were found in cellularextracts for both systems (Fig. 5A, B), indicating that some retinylacetate has been converted into retinol by a cellular hydrolase. RACwas eluted at 12.8±0.2 min with a spectrum maximum (not shown)

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

0.24

0.20

0.16

0.12

0.08

0.04

0.00

0.40

0.3

0.20

0.10

0.00

A

B

(3’)

(3)

(2)

(1)

(3)

(2)

(1)

Ab

sorb

ance

at

327

nm

(A

.U.)

Fig. 5. Typical chromatogram of ethanol–hexane extracts from TC7 cells, analysed by HPLC.retinyl acetate. (B) Cell extract in the case of TC7 cell incubation with RAC-micelles. For details o(1) void volume. (2) 5-methoxyflavone (internal standard). (3) Retinol (ROH). (3′) peak non-

at 327 nm and ROH at 7.4±0.2 min with a spectrum maximum(not shown) at 325.9 nm. Indeed, it is known that dietary retinylesters are either hydrolysed into retinol in the lumen of intestineor undergo hydrolysis at the intestinal brush-border by a retinyl esterhydrolase (Borel et al., 2001; D'Ambrosio, Clugston, & Blaner, 2011;Harrison, 2012; Sauvant, Cansell, Sassi, & Atgié, 2012; Schreiber et al.,2012). Retinol in the enterocyte binds to the cellular retinol-bindingprotein II (CRBP-II) and is esterified into retinyl ester. The resultingretinyl ester is then packed with dietary fat and cholesterol into chylo-microns to be secreted into the lymphatic system (D'Ambrosio et al.,2011). In the present study, RAC amounts found in cellular extractsafter TC7-cell exposure to submicron emulsion droplets varied between1 and2 nmol permg of cellular proteins, the RACuptakemeasured after3 h of incubation being the only one significantly (P=0.05) higherthan the other values. In opposite, ROH amounts increased signifi-cantly (P=0.05) in a time-dependent manner over 24 h of exposure,from 0.1 (5 min) to 1.4 (24 h) nmol per mg proteins (i.e., a 14-foldincrease) indicating a progressive turnover of RAC into ROH (Fig. 6A).

RAC uptake determined after exposure of TC7-cells to RAC-micelles(1–3.2 nmol per mg of cellular proteins; Fig. 6B) remained ≥ to thatfound for submicron droplets but much higher ROH amounts weredetected after exposure toRAC-micelles than submicrondroplets. Indeed,ROH amounts increased significantly (P=0.05) in a time-dependentmanner from 1.3 (5 min) to a maximal value of 15.5 nmol per mg pro-teins after 6 h of exposure time (which was ~10-fold higher than forsubmicron droplets after 24 h exposure), then decreased down to 9.9after 24 h (Fig. 6B). Although both systems initially displayed similarparticle sizes in the nanosize range (peakmaxima at 60–80 nm in num-ber frequency), uptake experiments clearly indicated a faster cellularincorporation of RAC then turnover into ROH, for RAC-micelles com-pared to oily droplets, suggesting therefore a better bioaccessibility ofRAC in the former case. Looking at the chromatograms (Fig. 5B), anon-identified peak (3′) eluted at 7.1±0.1 min just before ROH peak

10 11 12 13 14 15 18 19

10 11 12 13 14 15

16 17

16 17 18 19

Min

Min

(4)

(4)

(A) Cell extract in the case of TC7 cell incubation with emulsion droplets loaded withf cellular extraction and chromatography conditions see Section 2.11. Peak identification:identified. (4) Retinyl acetate (RAC).

0.0

1.0

2.0

3.0

4.0

5.0

5 min 1 h 3 h 6 h 24 h

Exposure time

Nan

om

ole

s / m

g o

f ce

llula

r p

rote

ins

0

5

10

15

20

5 min 1 h 3 h 6 h 24 h

Exposure time

Nan

om

ole

s / m

g o

f ce

llula

r p

rote

ins

A

B

Fig. 6. Effect of incubation time on the cellular uptake of retinyl acetate by TC7 cellsafter deposit of submicron emulsion droplets loaded with retinyl acetate (A), or depos-it of RAC-micelles stabilised with Tween 80® (B). Retinyl acetate (RAC) ( ) and retinol(ROH) ( ) were quantitated by HPLC after extraction from TC7 cells at the indicatedtimes of incubation. Submicron O/W emulsion was processed twice by UHPH at200 MPa (first-stage, 2-pass homogenisation, Tin=24 °C). Submicron emulsion drop-lets and RAC-micelle deposits on TC7 cells corresponded to 440 μmol L−1 of retinylacetate in the cell culture medium. Data are the mean of 3 HPLC determinations obtainedfrom three different plate-wells and extracts per independent experiment. Means fromtwo independent experiments±standard deviation are shown. For details of cellular ex-traction and chromatography conditions see Section 2.11.

688 A. Benzaria et al. / Food Research International 51 (2013) 679–692

(3) that appeared in slightly greater amount relative to ROH especiallyafter 3 h (Fig. 6B) and 6 h (not shown) of incubation could correspondto cis-retinol. Retinoic acidwhichwas eluted at 6.9 minwith a spectrummaximum at ~356 nm (Sigma standard, not shown) in the presentHPLC conditions was not observed in cellular extracts.

The present results show a significant RAC incorporation in TC7cells without previous digestion of the delivery systems, althoughRAC bioaccessibility appeared lower when entrapped into oily drop-lets than in the form of polysorbate-stabilised micelles. Such a resultcould be explained by the weak physical stability of RAC-micelles(Section 3.1) facilitating RAC-accessibility to cellular enzymes. Fur-thermore, the significantly smaller absolute value of ζ-potential(−12.7±2.8 mV) measured for RAC-micelles compared to that ofsubmicron droplets (−24.3±1.7 mV) could facilitate RAC-micelleadsorption to the negatively charged cellular brush-border. Indeedit is known that the particle charge affects cellular adsorption (He, Hu,Yin, Tang, & Yin, 2010; Yue et al., 2011). In opposite, the slower cellularincorporation and turn-over of RAC embedded into the oily submicrondroplets (which droplets are time-stable and protected by the interfa-cial protein film), may correspond to an expected protection plus de-layed release of biomolecules for food formulated products.

In view of nutraceutical or enteral nutrition applications, it remainsto study how such submicron droplets obtained in the nanoscale range

(b300 nm) by UHPH-processing will undergo the travelling throughthe digestive tract as such (liquid emulsion; Singh & Sarkar, 2011) orin a gelled state (gelled emulsion; Liang, Leung Sok Line, Remondetto,& Subirade, 2010), or after inclusion in food matrix in a bottom upapproach. Armand et al. (1999) and Borel et al. (2001) observed thatO/W emulsion droplets prepared for enteral nutrition (10.8% oil and6.8% milk proteins (w/v) plus vitamin A and E) and characterised by amedian diameter of 0.7±0.2 μm (i.e., fine emulsion), increased in sizeup to 2.7–6.2 μm during the gastric digestion, and that no further in-crease in size was noticed in the duodenum (as evaluated in vivo, aftersampling through nasogastric/duodenal tubing in human volunteers).An increase in droplet diameters accompanied by a gradual change inζ-potential, was also observed by Sarkar, Goh, Singh, and Singh (2009)using this time, in vitro digestion of an O/W emulsion (20%, w/w, soyoil; 1.0% purified β-Lg; pH 7.0) with synthetic gastric fluid at pH 1.2(37 °C). It must be noticed yet, that gastric pH does not remain as lowthan 1.2 during in vivo digestion. In their in vivo study, Armand et al.(1999) and Borel et al. (2001) observed that despite its increase insize, the median diameter of the fine emulsion stabilised with milkproteins remained significantly lower than that of the correspondingcoarse emulsion (median diameter of 10.1±0.0 μm), and that dropletgastro-duodenal lipolysis was greater for the fine than the coarseemulsion. In addition, the triacylglycerol and retinyl-ester peaks(from oily droplets) recorded in blood serum and chylomicron fractionwere significantly delayed for the fine emulsion compared to the coarseone, which may be an expected delivery feature. Studying in vitrosimulated duodenum digestion of olive O/W emulsion prepared in themicron range with a non-ionic synthetic surfactant (Pluronic F68)or a zwitterionic biological surfactant (soy lecithin), Torcello-Gómez,Maldonado-Valderrama, Martín-Rodríguea, and McClements (2011b),observed a ~10-fold increase in particle size induced by bile-extractwhateverwas the surfactant, due to a partial displacement of the surfac-tant by bile-extract at the oil–water interface, but the rate and extent oflipid digestionwith amixture of lipase/bile-extract vs. the initial dropletsizes appeared to be surfactant-dependent. Comparing emulsion drop-lets (~9 μm in diameter) stabilised by either sodium caseinate or soylecithin, Vors et al. (2012) also observed that the gastric and duodenallipolysis levels depended on the surfactant type, as well as did the totaltriacylglycerols secreted by Caco-2/TC7 cells after exposure to lipolysisproducts.

The mechanism of retinoid internalisation from emulsion dropletswithout a previous in vitro gastrointestinal lipolysis as proposed byVors et al. (2012) remained to be elucidated. Ribeiro et al. (2006)studied cellular uptake of β-carotene and astaxanthine from O/Wemulsions prepared by homogenisation at 120 MPa with palm oiland Tween® 20 as the dispersed phase. HT-29 and Caco-2 cell-lineswere used as models for human intestinal epithelial cells. Results in-dicated that the cellular uptake of carotenoids depended on the emul-sifier used (Tween® 20 or sucrose laurate) and its combination withWPI proteins. It looks like WPI increased significantly the carotenoiduptake into cell monolayers. Authors suggested that β-Lg could ac-commodate β-carotene and astaxanthine in its hydrophobic pocketas already known for retinol, increasing their bioavailability. Indeed,it is possible that the presence of proteins at the O/W interface allowsthe diffusion of the lipophilic compound towards the droplet outside.Such a more efficient uptake in the case of WPI proteins used as sur-factant compared to polysorbate was not observed for RAC uptake inthe present study.

3.4. Cellular fate of emulsion proteins through TC7-cell monolayers

To follow the fate of emulsion droplets through TC7-cellmonolayers,a protocol for confocal laser-scanning microscopy (CLSM) examinationwas firstly set up using fluorochromes to delimit cell architecture(nuclei and cytoskeleton) plus aβ-Lg antibody–IgG antibody complex la-belled with Alexa® 488 to locate β-Lg inside the cells according to x, y, z

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three-dimensional axes. Indeed, β-Lg, the major protein in WPI, plays amain role to stabilise the oil–water droplet interface (Dalgleish, 1996,1997). Fluorochromes have been chosen avoiding/limiting the overlapof their excitation/emission spectra. Cell nuclei were stained in bluewith DAPI (λex=405 nm;λem=420–480 nm) (Fig. 7A-1), the cytoskel-eton was stained in purple with Alexa Fluor® 680 Phalloidin Conjugate

A-1

B

C

A-3

A-2

A-4

(λex=633 nm; λem=650 nm) (Fig. 7A-2), and the β-Lg antibody–IgGantibody complex labelled with Alexa Fluor® 488 (λex=488 nm;λem=505–530 nm) appeared as green spots (Fig. 7A-3). Fig. 7A-4 dis-plays the corresponding merged images.

A confocal (x,y)-merged image of TC7 cells that have not beenexposed to emulsion droplets, accompanied with the (z-axis) of theoptical cell sections is proposed in Fig. 7B, and displays the organisa-tion of the TC7 cell monolayer. As expected, no β-Lg was detected asfluorescent green spots at the TC7-cell brush-border or inside thecells. It must have been noticed that the ThinCert™ insert permeablefilter was stained in green by Alexa Fluor® 488-labelled goatanti-rabbit IgG antibody at the bottom of the z-axis analysis of the op-tical sections (Fig. 7B).

After 3 h of exposure to submicron emulsion droplets (Fig. 7C),numerous areas stained in green were clearly observed at thebrush-border and inside the cells around nuclei, indicating the up-take of emulsion material and permeation of β-Lg into the cell mono-layer. β-Lg detected in cells could come from the thin protein layer atthe submicron droplet interface, since the major part of β-Lg wasprobably adsorbed at the oil–water interface (Section 3.1). It is notyet excluded that remaining free β-Lg could also be incorporated intoTC7 cells. The uptake of β-Lg by TC7 cells was followed vs. exposuretime (5 min, 1 h, 3 h and 24 h) after apical deposit of submicron drop-lets, by the way of CLSM z-axis merged projections (called x,z-plan) asshown in Fig. 8. The presence of labelled β-Lg gradually increased inthe brush-border area from 5 min to 3 h of exposure time to submicrondroplets, accompanied by the progression of labelled β-Lg from theapical towards the basolateral membrane (Fig. 8B–D). After 24 h of ex-posure time, very few labelled β-Lg remained (Fig. 8E), suggesting thatthe protein material has been degraded.

The passage of food proteins included β-Lg, through epithelial bar-rier proceeds by a major degradative pathway together with a minorintact route (Caillard & Tomé, 1995; Heyman & Desjeux, 1992;Isolauri, Gotteland, Heyman, Pochard, & Desjeux, 1990). The passageof labelled β-Lg is mostly transcellular as suggested by Bernasconi,Fritsché, and Corthésy (2006) and Rytkönen et al. (2006). Particularly,Bernasconi et al. (2006) observed the presence of β-Lg predominantlyin the apical cell region after 5 min exposure of Caco-2 cell to 20 μg ofbiotinylated β-Lg, then from the apical zone to the basal one after 1 hof exposure time. Results of Fig. 8 are in agreement with the latterstudy. The uptake of β-Lg at the apical cell membrane was believedmostly as a non-specific endocytosis mechanism since endocytosisappeared as a non-saturable phenomenon (Caillard & Tomé, 1995).Then after, two pathways were proposed concerning the fate of β-Lg:(i) a very small percentage of intact β-Lgwas recycled to the incubationside or was directly transported by transcytosis without alteration tothe basolateral side; and (ii) the main fraction of β-Lg was degradedduring the passage through the monolayer (Caillard & Tomé, 1995;Isolauri et al., 1990; Puyol, Perez, Sanchez, Ena, & Calvo, 1995) thenthe degraded products were largely excreted outside the cells (bothapical and basolateral sides), a small amount only being retained insidethe cells (Caillard & Tomé, 1995). In the present study, a significant

Fig. 7. Confocal laser scanning microscopy of TC7 cells grown on ThinCert™ insertsafter apical exposure to submicron emulsions (UHPH at 200 MPa, 2-pass homogenisa-tion, Tin=24 °C) for 3 h. Fig. 7A-1 to A-3 show the different channels separately and Fig.7A-4, the merged image, by the way of z-axis projection (x,z-plan). Cell nuclei werestained in blue with DAPI (λex=405 nm; λem=420–480 nm) (Fig. 7A-1), the cytoskel-eton was stained in purple with Alexa Fluor® 680 Phalloidin Conjugate (λex=633 nm;λem=650 nm) (Fig. 7A-2), and the β-Lg antibody–IgG antibody complex labelled withAlexa Fluor 488 (λex=488 nm; λem=505–530 nm) appeared as green spots (Fig. 7A-3).Fig. 7B displays themerged (x,y) image of control TC7 cells without exposure to submicronemulsion. Fig. 7C displays the merged (x,y) image of TC7 cells after apical exposure to sub-micron emulsions for 3 h. The (x,y) images (Fig. 7B and C) are completed with thethird-dimensional analysis (z-axis) of the cell optical sections. Submicron emulsioncontained (w/w) 4.3% whey proteins plus 30% peanut oil. Microscopy imaging wasperformed using the Laser scanning microscope Axioplan2/LSM 510 Meta and LSM 510Software (version 2.8.) from Carl Zeiss (Jena, Germany).

A

B

C

D

E

Fig. 8. Confocal laser scanning microscopy imaging of control TC7 cells without exposureto submicron emulsion (A), and TC7 cells after apical exposure to submicron emulsion for5 min (B), 1 h (C), 3 h (D) and 24 h (E). z-axis merged projections (x,z-plans). Cell nucleiwere stained in blue with DAPI, the cytoskeleton in purple with Alexa Fluor® 680Phalloidin Conjugate, and the β-Lg antibody–IgG antibody complex labelled with AlexaFluor 488 appeared as green spots, as for Fig. 7. Each image is a projection of 12 optical sec-tions collected at 2 μm intervals in the z-axis. Submicron emulsion contained (w/w) 4.3%whey proteins plus 30% peanut oil. Microscopy imaging was performed using the Laserscanning microscope Axioplan2/LSM 510 Meta and LSM 510 Software (version 2.8.)from Carl Zeiss (Jena, Germany).

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(P=0.01) increase of protein amount was found in the basolateralcompartment after 3 h and 24 h of TC7 exposure time to the submicrondroplets (Fig. 9). Although such results obtained by BCAmeasurementsmay reflect the presence of proteins as well as peptides in the basal in-sert compartment, and therefore the whole cell metabolic activity afterdeposit of emulsion materials, they are consistent with the results of

0.00

0.05

0.10

0.15

0.20

0.25

0.30

5 min 3 h 24 h

Pro

tein

co

nce

ntr

atio

n (

mg

/mL

)

Exposure time

a

b

c

Fig. 9. Net protein concentration determined in the basolateral compartment ofThinCert™ inserts after apical exposure of TC7 cells grown on the inserts, to submicronemulsions for 5 min, 3 h or 24 h. For each exposure time, the net protein concentrationwas calculated as the difference, between the protein concentrations determined inthe basolateral compartment of inserts after exposure to submicron emulsion andwithout exposure (corresponding control cells). Submicron emulsion was processedtwice by UHPH at 200 MPa (first-stage, 2-pass homogenisation, Tin=24 °C). Emulsioncontained (w/w) 4.3% whey proteins plus 30% peanut oil. Protein content was mea-sured by the bicinchoninic acid (BCA) method. For each indicated exposure time,data are the means values±standard deviation obtained from 6 to 9 determinationsfrom two ThinCert™ inserts. Means with different letters (a, b, c) were significantly(P=0.01) different between themselves.

RAC uptake into TC7 cells, and with confocal microscopy observations,showing evidence of internalisation of submicron emulsion materials.

4. Conclusions

In the present study, we have successfully elaborated submicronO/W emulsion droplets (b300 nm) stabilised by proteins, using amechanical process (high-pressure homogenisation) and controllingprocessing thermal effects, to embed retinyl acetate selected as abiomolecule to be transported. Indeed, operating at 200 MPa andusing an appropriate homogenisation-valve design allowed a limitednumber of homogenisation passes (two) for obtaining droplets witha monomodal distribution in the nanoscale range (which is a goodstability criterion for emulsion droplets).

It remains to evaluate howmuch themain advantage acquired foremulsion time-stability (i.e., small particle size and monomodaldistribution) will be conserved through the gastro-duodenal digestivetract. Indeed, preservation of emulsion droplet integrity is expected toprotect the embedded molecule and deliver it at the targeted locationin the digestive tract in view of tailoring delivery systems for food andpharmaceutical applications.

In vitro TC7-cell viability assays (TER, MTT-assay, LDH-leakage)indicated a good biocompatibility of submicron emulsion dropletsstabilised by whey proteins, compared to retinyl acetate-micellesprepared at atmospheric pressure with polysorbate 80 as emulsifier.Such result is important in terms of Novel Food Regulation for productdevelopment using high-pressure homogenisation and European im-plementation of UHPH as a novel technology in the food area.

In vitro uptake experiments allowed the internalisation of emul-sion materials and particularly of the embedded retinyl acetate to bedemonstrated, without previous emulsion digestion steps. Since, thecellular turn-over of retinyl acetate into retinol was about 5 to 7-foldgreater in the case of retinyl acetate-micelles compared to the oilyemulsion droplets some delayed delivery of retinyl acetate/retinolcan be expected in the latter case. Such results remain to be validatedby in vivo experiments, taking care to avoid risk of overdoses in theformulation (Sauvant et al., 2012).

Acknowledgements

The authors thank the French “Agence Nationale de la Recherche”(ANR, Paris) who funded in part the team research in the frameworkof the NANOLIA project (ANR, Program ALIA 2009–2011), as well asthe Comité Ecos-Sud (Action ECOS n°U08B01, Paris) who grantedthe research stay of Dr. López-Pedemonte at Montpellier 2 University.

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