Margot Stasse

Eric Laurichesse

Tiphaine Ribaut

Olivier Anthony

Valérie Héroguez
email: heroguez@enscbp.fr

Véronique Schmitt
email: veronique.schmitt@crpp.cnrs.fr

Formulation of concentrated oil-in-water-in-oil double emulsions for fragrance encapsulation

Keywords: emulsions, O/W/O emulsion, fragrance, encapsulation, formulation

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Introduction

Among multiple emulsions [START_REF] Leal Calderon | Emulsion science basic principles[END_REF][START_REF] Garti | Progress in Stabilization and Transport Phenomena of Double Emulsions in Food Applications[END_REF][START_REF] Seifriz | Studies in emulsions[END_REF][START_REF] Frenkel | Multiple emulsions: I. Stability: inversion, apparent and weighted HLB[END_REF][START_REF] Benna-Zayani | Stabilisation of W/O/W double emulsion by polysaccharides as weak gels[END_REF][START_REF] Dickinson | Double Emulsions Stabilized by Food Biopolymers[END_REF][START_REF] Liu | Protection of anthocyanin-rich extract from pH-induced color changes using water-in-oil-in-water emulsions[END_REF], double oil-in-water-in-oil emulsions are dispersions consisting of oil-in-water emulsions themselves dispersed as drops in an oil outer phase. Owing to their compartmentalized structure, they are interesting colloidal systems for encapsulating either aqueous or lipophilic or both kinds of species [START_REF] Goubault | Shear Rupturing of Complex Fluids: Application to the Preparation of Quasi-Monodisperse Water-in-Oil-in-Water Double Emulsions[END_REF][START_REF] Nollet | Water fluxes and encapsulation efficiency in double emulsions: impact of emulsification and osmotic pressure unbalance[END_REF][START_REF] Aserin | Recent Developments in OWO multiple emulsions[END_REF][START_REF] Sajjadi | Dynamic behaviour of drops in oil/water/oil dispersions[END_REF][START_REF] Nonomura | Multiple Pickering Emulsions Stabilized by Microbowls[END_REF][START_REF] Besnard | Breaking of the Bancroft rule for multiple emulsions stabilized by a single stimulable polymer[END_REF][START_REF] Besnard | Multiple Emulsions Controlled by Stimuli-Responsive Polymers[END_REF][START_REF] De Luca | Les émulsions multiples[END_REF][START_REF] Laugel | Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions[END_REF][START_REF] Chu | Monodisperse Multiple Emulsions[END_REF][START_REF] Nollet | Determination of Formulation Conditions Allowing Double Emulsions Stabilized by PGPR and Sodium Caseinate to Be Used as Capsules[END_REF][START_REF] O' Dwyer | Effects of Green Tea Extract and α-Tocopherol on the Lipid Oxidation Rate of Omega-3 Oils, Incorporated into Table Spreads, Prepared using Multiple Emulsion Technology[END_REF]. Such systems may be produced in a one-step process using one surfactant [START_REF] Aserin | Recent Developments in OWO multiple emulsions[END_REF][START_REF] Sajjadi | Dynamic behaviour of drops in oil/water/oil dispersions[END_REF][START_REF] Sajjadi | Phase Inversion in Abnormal O/W/O Emulsions. 2. Effect of Surfactant Hydrophilic-Lipophilic Balance[END_REF][START_REF] Sajjadi | Phase Inversion in Abnormal O/W/O Emulsions: I. Effect of Surfactant Concentration[END_REF][START_REF] Pradhan | A one-step process for oil-in-water-in-oil double emulsion formation using a single surfactant[END_REF][START_REF] Oh | O/W/O Multiple Emulsions via One-Step Emulsification Process[END_REF]. Although simple, this process does not offer sufficient versatility and does not allow the control of the globule size. Moreover, the inner and the outer oils are necessarily the same, which is not relevant for encapsulation. This is why we decided to use two-step process [START_REF] Goubault | Shear Rupturing of Complex Fluids: Application to the Preparation of Quasi-Monodisperse Water-in-Oil-in-Water Double Emulsions[END_REF][START_REF] Nollet | Water fluxes and encapsulation efficiency in double emulsions: impact of emulsification and osmotic pressure unbalance[END_REF][START_REF] De Luca | Les émulsions multiples[END_REF][START_REF] Laugel | Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions[END_REF][START_REF] Nollet | Determination of Formulation Conditions Allowing Double Emulsions Stabilized by PGPR and Sodium Caseinate to Be Used as Capsules[END_REF][START_REF] O' Dwyer | Effects of Green Tea Extract and α-Tocopherol on the Lipid Oxidation Rate of Omega-3 Oils, Incorporated into Table Spreads, Prepared using Multiple Emulsion Technology[END_REF][START_REF] Jiménez-Martín | Volatile compounds and physicochemical characteristics during storage of microcapsules from different fish oil emulsions[END_REF][START_REF] O' Dwyer | Formation, rheology and susceptibility to lipid oxidation of multiple emulsions (O/W/O) in table spreads containing omega-3 rich oils[END_REF][START_REF] Bernewitz | Structure of and diffusion in O/W/O double emulsions by CLSM and NMR-comparison with W/O/W[END_REF][START_REF] Mabille | Rheological and Shearing Conditions for the Preparation of Monodisperse Emulsions[END_REF] very common for W/O/W double emulsions. For W/O/W emulsions, first, water containing the hydrophilic drug (or the encapsulated hydrophilic species) is dispersed as small droplets in the oil containing a preferably lipophilic stabilizer, then, this reverse emulsion is dispersed in water containing a hydrophilic stabilizer [START_REF] Goubault | Shear Rupturing of Complex Fluids: Application to the Preparation of Quasi-Monodisperse Water-in-Oil-in-Water Double Emulsions[END_REF][START_REF] Nollet | Water fluxes and encapsulation efficiency in double emulsions: impact of emulsification and osmotic pressure unbalance[END_REF]. As a consequence of the simultaneous presence of a direct (oil-inwater) and a reverse (water-in-oil) emulsion, two kinds of stabilizers are required. In order to improve the stability of the water-in-oil emulsion, a water-soluble species like salt or glucose is added in the innermost aqueous phase. An osmotic regulator is also added in the outermost aqueous phase [START_REF] Wen | Effects of Osmotic Pressure on Water Transport in W1/O/W2 Emulsions[END_REF]. Due to the numerous constituents, these systems are often considered complex and their use as capsules are limited despite their large loading ability. Another difficulty comes from their preparation method: during the second emulsification step, fragmentation must be controlled in order to avoid destruction of the first emulsion or a connection of the two (innermost and outer) compartments that would lead to a dilution and immediate release of the encapsulated drug [START_REF] Nollet | Water fluxes and encapsulation efficiency in double emulsions: impact of emulsification and osmotic pressure unbalance[END_REF]. Also, their sensitivity towards the encapsulated species is often a brake to their use. For a recent review about double emulsions in the domain of food, the reader can refer to [START_REF] Muschiolik | Double Emulsions Relevant to Food Systems: Preparation, Stability, and Applications: Double emulsions in food…[END_REF]. Indeed, this paper is dedicated to a critical evaluation of the emulsification preparation methods, the range of encapsulated components and emulsion composition, the balancing of the osmotic pressure, the stabilization by increased viscosity or gelation…in the food domain considering both W/O/W and O/W/O double emulsions. Among the literature on double emulsions, the majority concerns W/O/W emulsions while O/W/O are more scarce [START_REF] O' Dwyer | Effects of Green Tea Extract and α-Tocopherol on the Lipid Oxidation Rate of Omega-3 Oils, Incorporated into Table Spreads, Prepared using Multiple Emulsion Technology[END_REF][START_REF] Sajjadi | Phase Inversion in Abnormal O/W/O Emulsions. 2. Effect of Surfactant Hydrophilic-Lipophilic Balance[END_REF][START_REF] Sajjadi | Phase Inversion in Abnormal O/W/O Emulsions: I. Effect of Surfactant Concentration[END_REF][START_REF] Jiménez-Martín | Volatile compounds and physicochemical characteristics during storage of microcapsules from different fish oil emulsions[END_REF][START_REF] O' Dwyer | Formation, rheology and susceptibility to lipid oxidation of multiple emulsions (O/W/O) in table spreads containing omega-3 rich oils[END_REF][START_REF] Schmidt | Investigation and application of measurement techniques for the determination of the encapsulation efficiency of O/W/O multiple emulsions stabilized by hydrocolloid gelation[END_REF][START_REF] Edris | Encapsulation of orange oil in a spray dried double emulsion[END_REF][START_REF] Liao | Preparation and characterization of succinic acid deamidated wheat gluten microspheres for encapsulation of fish oil[END_REF][START_REF] Okonogi | Method for producing low-fat spread and oil-in-water-in-oil type spread[END_REF], and are usually devoted to food [START_REF] O' Dwyer | Effects of Green Tea Extract and α-Tocopherol on the Lipid Oxidation Rate of Omega-3 Oils, Incorporated into Table Spreads, Prepared using Multiple Emulsion Technology[END_REF][START_REF] Jiménez-Martín | Volatile compounds and physicochemical characteristics during storage of microcapsules from different fish oil emulsions[END_REF][START_REF] O' Dwyer | Formation, rheology and susceptibility to lipid oxidation of multiple emulsions (O/W/O) in table spreads containing omega-3 rich oils[END_REF][START_REF] Edris | Encapsulation of orange oil in a spray dried double emulsion[END_REF][START_REF] Liao | Preparation and characterization of succinic acid deamidated wheat gluten microspheres for encapsulation of fish oil[END_REF][START_REF] Okonogi | Method for producing low-fat spread and oil-in-water-in-oil type spread[END_REF]. The most common HS in food are the Tween 20 surfactant [START_REF] Bernewitz | Structure of and diffusion in O/W/O double emulsions by CLSM and NMR-comparison with W/O/W[END_REF][START_REF] Schmidt | Investigation and application of measurement techniques for the determination of the encapsulation efficiency of O/W/O multiple emulsions stabilized by hydrocolloid gelation[END_REF] and the Sodium Caseinate protein [START_REF] O' Dwyer | Effects of Green Tea Extract and α-Tocopherol on the Lipid Oxidation Rate of Omega-3 Oils, Incorporated into Table Spreads, Prepared using Multiple Emulsion Technology[END_REF][START_REF] Jiménez-Martín | Volatile compounds and physicochemical characteristics during storage of microcapsules from different fish oil emulsions[END_REF][START_REF] O' Dwyer | Formation, rheology and susceptibility to lipid oxidation of multiple emulsions (O/W/O) in table spreads containing omega-3 rich oils[END_REF][START_REF] Okonogi | Method for producing low-fat spread and oil-in-water-in-oil type spread[END_REF] and the most common LS are Span 80 as for example in [START_REF] Liao | Preparation and characterization of succinic acid deamidated wheat gluten microspheres for encapsulation of fish oil[END_REF] and polyglycerol polyricinoleate (PGPR) [START_REF] O' Dwyer | Effects of Green Tea Extract and α-Tocopherol on the Lipid Oxidation Rate of Omega-3 Oils, Incorporated into Table Spreads, Prepared using Multiple Emulsion Technology[END_REF][START_REF] Jiménez-Martín | Volatile compounds and physicochemical characteristics during storage of microcapsules from different fish oil emulsions[END_REF][START_REF] O' Dwyer | Formation, rheology and susceptibility to lipid oxidation of multiple emulsions (O/W/O) in table spreads containing omega-3 rich oils[END_REF][START_REF] Bernewitz | Structure of and diffusion in O/W/O double emulsions by CLSM and NMR-comparison with W/O/W[END_REF][START_REF] Schmidt | Investigation and application of measurement techniques for the determination of the encapsulation efficiency of O/W/O multiple emulsions stabilized by hydrocolloid gelation[END_REF][START_REF] Okonogi | Method for producing low-fat spread and oil-in-water-in-oil type spread[END_REF]. Kinetic stability is often enhanced using gelation of the water phase as for example with pectin [START_REF] Bernewitz | Structure of and diffusion in O/W/O double emulsions by CLSM and NMR-comparison with W/O/W[END_REF][START_REF] Schmidt | Investigation and application of measurement techniques for the determination of the encapsulation efficiency of O/W/O multiple emulsions stabilized by hydrocolloid gelation[END_REF] or at the interface. In the present case, the application domains are cosmetics, laundry and detergency. In such domains, the use of animal proteins as sodium caseinate originating from milk should be avoided. Also, in the present study, double emulsions are only a first stage before polymerization of the intermediate aqueous phase [START_REF] Stasse | Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-inoil (O/W/O) double emulsion[END_REF] so that a kinetic stability of few hours is large enough.

Encapsulation may be achieved through very diverse processes among which one can cite the following: i) layer-by-layer [START_REF] Johnston | Layer-by-layer engineered capsules and their applications[END_REF] deposition of alternate layers of polyelectrolytes of opposite charges onto a sacrificial particle that has to be removed. The fragrance filling occurs after the capsule preparation, ii) spray drying [START_REF] Gharsallaoui | Applications of spray-drying in microencapsulation of food ingredients: An overview[END_REF] of an emulsion that solidifies the drops during drying iii) Spray-coating [START_REF] Dewettinck | Fluidized bed coating in food technology[END_REF] allowing encapsulating continuously solid particles. Particles are fluidized and a coater is pulverized onto the particles. The coater is then solidified by decreasing the temperature or by solvent evaporation, iv) solvent evaporation [START_REF] Gürsel | Properties and drug release behaviour of poly(3hydroxybutyric acid) and various poly(3-hydroxybutyrate-hydroxyvalerate) copolymer microcapsules[END_REF]. Usually the organic phase containing a volatile solvent, a polymer and the species to be encapsulated is emulsified in a water phase in which the polymer is not soluble. Stirring is maintained until the volatile solvent has diffused through the aqueous phase and has evaporated while the polymer has precipitated. The obtained capsules are usually very porous, v) coacervation [START_REF] Yow | Formation of liquid core polymer shell microcapsules[END_REF] is based on the separation between a polymer-rich and a polymer-poor phase provoked by the polymer desolvation induced by temperature, pH… A cross-linker may be added to rigidify the shell, vi) colloïdosomes [START_REF] Hsu | Self-assembled Shells Composed of Colloidal Particles: Fabrication and Characterization[END_REF] are obtained through the sintering of particles adsorbed at the interface of a so called Pickering emulsion, that is to say a particle stabilized emulsion, vii) interfacial polycondensation, widely used, is based on the formation of an emulsion containing a hydrophobic monomer in its dispersed lipophilic phase and a hydrophilic monomer in its continuous aqueous phase. The two monomers can react at the interface forming a primary membrane that thickens during polymerization [START_REF] Teixeira | A case study of product engineering: Performance of microencapsulated perfumes on textile applications[END_REF][START_REF] Jacquemond | Perfume-containing polyurea microcapsules with undetectable levels of free isocyanates[END_REF], viii) suspension polymerization [START_REF] Jahns | Microcapsules, the production and use thereof[END_REF][START_REF] Berg | Microencapsulation of emulsified oil droplets by <i>in-situ</i> vinyl polymerization[END_REF].

The monomer, the initiator and the species to be encapsulated are solubilized in the oily dispersed phase of a direct emulsion containing the stabilizer. Polymerization is initiated and occurs in the oily phase. As the chain length increases, the polymer becomes less soluble in the oily phase and precipitates at the interface forming a shell, ix) emulsion polymerization is one of the most used encapsulation technique. A lipophilic monomer is progressively added in an aqueous solution containing surfactant, at a concentration that can be chosen below its critical micellar concentration and an initiator. This leads to the formation, under stirring, of monomer drops stabilized by the surfactant. After polymerization initiation, the oligomers in formation become water insoluble and migrate into the monomer drops in which nucleation occurred. The species to be encapsulated may be directly added with the monomer or added after so that it diffuses into the capsules.

In the present work, we wish to elaborate fragrance capsules in which the fragrance is not soluble in the polymerization locus. This is the reason why we chose in a first step to formulate fragrance-in-water-in-perfumery oil emulsions in order to, later, polymerize the aqueous intermediate phase. In this context, the aim of the present paper is the formulation of a fragrance-in-water-in-oil double emulsion able to be used for different fragrances.

Encapsulation of fragrance is a widely spread first step in many applications like softeners, deodorants and so on. The studied fragrances were model systems composed of a variety of molecules with different functional groups that are commonly employed in perfumery. Rather than just diluting the lipophilic fragrance into an oily perfumery solvent, the proposed strategy is the encapsulation of the fragrance in an emulsion. The choice of an O/W/O is imposed by the polarities of the fragrance and the solvent. The advantage of these multiple emulsions is the presence of the intermediate water phase to slow down the leakage of the fragrance. In order to improve the stability of the water-in-oil emulsion, a water soluble species like salt or glucose is added in the intermediate aqueous phase [START_REF] Bernewitz | Structure of and diffusion in O/W/O double emulsions by CLSM and NMR-comparison with W/O/W[END_REF]. O/W/O systems are very rare in the literature compared to W/O/W emulsions. As described above, the two-step process has been chosen and the use of a couple of antagonist surfactants is required. Therefore, a hydrophilic one has been selected to stabilize the direct emulsion and a lipophilic one to stabilize the inverted emulsion containing the direct emulsion. To determine a possible composition, we adopted a progressive strategy by first seeking surfactants able to stabilize concentrated simple emulsions (direct and reverse), authorized in cosmetics and not dependent on pH to avoid restriction of possible uses.

Usually, the choice of the surfactant to stabilize an emulsion with almost equal volumes of both phases is based on its Hydrophilic Lipophilic Balance (HLB) [START_REF] Bancroft | The Theory of Emulsification, V[END_REF] given by the provider.

However, in the present case, this is only an indication, since we targeted concentrated emulsions with a much larger amount of dispersed phase compared to the continuous one (i.e. 90 wt% for the direct emulsion and 75 wt% for the reverse emulsion). Such high dispersed phase volume fractions were chosen to reach larger loadings of the capsules and a better control over the drop sizes [START_REF] Mabille | Monodisperse fragmentation in emulsions: Mechanisms and kinetics[END_REF]. Therefore, we swept different available surfactants and tested their ability to stabilize the simple concentrated emulsions. In a second part of the present paper, we report the formulation of the double emulsions where we varied the compositions in dispersed phase and hydrophilic surfactant (in the intermediate phase) concentrations for the two model fragrances. This led us to plot stability or feasibility diagrams, based on macroscopic and optical microscopy observations of the systems. From these diagrams, we selected a composition common to the two fragrances and tested its robustness against a third fragrance. Finally, the encapsulation rate of the selected system of double emulsion was assessed using gas chromatography.

Materials and Methods

Chemicals

All the reagents were used without further purification. The tested lipophilic surfactants (LS) were polyglycerol polyricinoleate (PGPR) with a molar mass of 3000 g.mol -1 (HLB = 1.5) graciously provided by Paalsgard and Span80 (molar mass around 420 g.mol -1 , HLB = 4.3) purchased from Sigma-Aldrich. The selected hydrophilic surfactants (HS) were Tween 60 (molar mass around 1310 g.mol -1 , HLB = 14.9), Tween 20 (molar mass around 1220 g.mol -1 , HLB = 16%) and Tergitol 15-S-12 (molar mass around 760 g.mol -1 and HLB = 14.5). All these HS were purchased from Sigma-Aldrich. The external oil was isopropyl myristate (IPM, density equal to 0.850 g.mL -1 ) chosen because it is widely used in perfumery. It was provided by Takasago Europe Perfumery Laboratory (T.E.P.L). Two different fragrances, hereafter named A and B and composed of 10 and 13 molecules respectively, have been specially assembled by T.E.P.L. Their composition is detailed in Tables 1 and2 and their main global characteristics are summed up in Table 3. Sodium Chloride (NaCl, molar mass 58.44 g.mol -1 ), incorporated in the water phase to increase the reverse emulsion stability was purchased from Sigma-Aldrich.

Milli-Q water was used in all emulsions preparation. LogP, widely used in perfumery and in pharmacy to describe the molecules' polarity, is defined as:

LogP = Log( C C ) ( 1
)
where P is the partition coefficient, Coctanol and Cwater the respective concentrations of the species in octanol and water. Log P is an indication of the relative solubility of the molecule in water and in octanol. Indeed, a positive and high value of LogP means that the considered species is more soluble in octanol than in water or, in other words, it means that the species is lipophilic.

Inversely, a negative LogP value is obtained for a hydrophilic species while LogP equals to zero means that the molecule is equally soluble in water and in octanol. Perfumes are mixtures of a large number of molecules, it is assumed that LogP of a mixture is given, with a good approximation [START_REF] Laane | Rules for optimization of biocatalysis in organic solvents[END_REF], by the molar fraction weighted average value. For both fragrances A and B, the values are positive meaning they are more soluble in octanol than in water. It is also worth noticing that the fragrances have close LogP values (see Table 3).

Emulsion size characterization

The emulsions size distributions were measured using static light scattering by means of a Malvern Mastersizer MS2000 granulometer.

For the simple emulsion, the measured scattered intensity as a function of the angle was transformed into the size distribution using the Mie theory. Reverse emulsions were diluted with IPM prior to measurement. The refractive index of the dispersed water phase was taken equal to 1.333 as no significant difference has been detected for the brine (0.1 M NaCl) compared to pure water. The refractive index of the continuous IPM oil was taken equal to 1.434. For the direct emulsion, the refractive indices of fragrances A and B were 1.454 and 1.475 respectively. All the refractive indices have been measured using an Abbé refractometer.

For the multiple emulsions, as oily globules were themselves composed of water droplets, they were not optically homogeneous; we then considered that the Mie theory was not adapted. To avoid this difficulty, we chose to use the simplified Fraunhofer theory where the principles of geometrical optics apply and the drops are approximated by opaque discs. This approximation is usually valid for drops larger than, or of the order of, 10 µm. However Nollet et al. [START_REF] Nollet | Water fluxes and encapsulation efficiency in double emulsions: impact of emulsification and osmotic pressure unbalance[END_REF] showed that it can also be applied down to 5 µm with a correct determination of the mean drop size even if the drops size distribution is misestimated.

In all cases, we checked by optical microscopy that the drop size was not altered by the dilution required for static light scattering measurements.

For all types of emulsions, the volume-average diameter D [START_REF] Frenkel | Multiple emulsions: I. Stability: inversion, apparent and weighted HLB[END_REF][START_REF] Seifriz | Studies in emulsions[END_REF] and the polydispersity index U were determined, they are defined as following:

D[4,3]= ∑ 𝐍 𝐢 𝐃 𝐢 𝟒 𝐢 ∑ 𝐍 𝐢 𝐃 𝐢 𝟑 𝐢 U= 𝟏 𝐃 𝐦 ∑ 𝐍 𝐢 𝐃 𝐢 𝟑 |𝐃 𝐦 𝐃 𝐢 | 𝐢 ∑ 𝐍 𝐢 𝐃 𝐢 𝟑 𝐢 (2)
where Ni is the total number of droplets with diameter Di. Dm is the median diameter, i.e., the diameter for which the cumulative undersized volume fraction is equal to 50%. The polydispersity index U is therefore the volume average difference with the median diameter normalized by the median diameter. It is an indicator of the drops size distribution width.

Both D [START_REF] Frenkel | Multiple emulsions: I. Stability: inversion, apparent and weighted HLB[END_REF][START_REF] Seifriz | Studies in emulsions[END_REF] and U were directly given by the Malvern Mastersizer software.

Emulsion preparation 2.3.1. Simple direct emulsion

All the direct emulsions were prepared following the same procedure. First, a concentrated polydisperse emulsion was obtained by manually incorporating the chosen A or B fragrance (90 wt% with respect to total emulsion weight) into an aqueous phase composed of salted water (0.1 M of NaCl) and HS at 30 wt% with respect to the water phase (in other words, for 100 g of a direct emulsion, the composition is the following: 90 g of fragrance dispersed in an aqueous phase composed of 0.04 g of NaCl, 3 g of HS and 6.96 g of H2O). This crude premixed emulsion was then sheared in a Couette cell (commercialized by Ademtech) composed of two concentric cylinders spaced by a 100 µm gap.

The inner cylinder of radius Ri = 20 mm rotated with a speed of 680 rpm while the outer cylinder was static, leading to an applied shear rate of 14 280 s -1 . If the conditions are fulfilled for a Rayleigh instability to develop, quasi-monodisperse emulsions can be obtained [START_REF] Pradhan | A one-step process for oil-in-water-in-oil double emulsion formation using a single surfactant[END_REF]. An average droplets size close to 1µm was aimed at, in order to be encapsulated in a second step. In the following, the drops of the direct emulsion will be named droplets.

Simple reverse emulsions

All the inverted emulsions were prepared following the same procedure. First, a polydisperse emulsion was obtained by manually incorporating 75 wt% of 0.1 M NaCl brine in IPM containing 10 wt% of LS with respect to the oil phase (for 100 g of reverse emulsion the composition was therefore as following: 75 g of aqueous solution containing 74.56 g of H2O and 0.44 g of NaCl and 25 g of the oily phase composed of 22.5 g of IPM and 2.5 g of LS). The crude emulsion was then sheared with the same Couette cell using stators with a gap of 100 or 200 µm. The speed has been varied to determine the most appropriate one as a function of the surfactant. The drops of inverted emulsions will be named globules in the following. In order to be able to encapsulate the direct emulsion droplets, the targeted globule size was around 15 µm.

double emulsions

Multiple oil-in-water-in-oil emulsions were prepared following a classical two-step procedure [START_REF] Seifriz | Studies in emulsions[END_REF][START_REF] Frenkel | Multiple emulsions: I. Stability: inversion, apparent and weighted HLB[END_REF]. First, a direct emulsion was prepared with the protocol described in the 2.3.1 section. However, in order to expand the concentration range of HS in the intermediate aqueous phase, the concentration of HS used for the stabilization of the direct emulsion was varied from 10 wt% to 30 wt% with respect to the direct emulsion aqueous phase (corresponding to 1 wt% to 3 wt% with respect to the total fragrance-in-water emulsion). This direct emulsion was sheared and the highest achievable rate (~14 000 s -1 ). In a second step, the direct emulsion described just above and composed of either fragrance A or fragrance B was diluted with salted water (0.1 M NaCl) and served as the dispersed phase of the reverse emulsion. The direct emulsion was diluted by a factor ranging from 1.5 to 18 according to the targeted fraction of inner droplets d (corresponding to a droplets fraction varying from 5 to 60% with respect to globule volume). Moreover, the aqueous solution used for dilution could contain HS. This dilution procedure allowed controlling both the fragrance droplet fraction d, defined as the ratio of the encapsulated droplets' volume to the globule volume, and the final hydrophilic surfactant concentration with respect to water. This diluted direct emulsion was then progressively and manually incorporated into the oil phase containing IPM and 10 wt% of LS using a spatula. The amount of incorporated direct emulsion determines the globule volume fraction G defined as the ratio of globules' volume to total sample volume.

To sum-up the emulsions compositions are listed in Table 4 for 100 g of emulsion. The encapsulation rate, defined as the amount of encapsulated species to total species ratio, was deduced from the measurement of the non-encapsulated fragrance (fragrance in IPM):

ε = = (3)
For this aim, the emulsion was centrifuged at 1500 rpm during 25 min to induce separation of the continuous phase from the emulsion without double emulsion rupture. The continuous phase was recovered, filtrated and diluted up to 5 wt% in ethanol. This phase was analyzed with a gas chromatograph (Agilent GC 7890B) equipped with flame ionization detector (FID) coupled with Mass spectrometer 5977A (MS), a split/splitless injector (1:40 split ratio) and a data system (Enhanced ChemStation MSD Chemstation). The detector and injector temperatures were set at 250°C. A 30 m×250 µm internal diameter capillary column coated with 5%-phenyl poly(methylsiloxane) (0.250 µm film thickness) (HP-5) was used. The oven temperature was programmed from 50 °C (2 min) to 280 °C (5 min) at 10 °C/min. Two microliters of the sample were injected. Fragrance molecules are identified with MS and quantified according to an external calibration with different solutions of fragrance in ethanol on FID.

Interfacial tension measurement

The interfacial tensions were measured with the weighing drop method. This technique consisted in shaping a drop of the heaviest phase at the end of a straight needle immersed in the lighter phase in a beaker. The volume of the drop was slowly increased. When the weight of the drop became larger than the capillary forces that maintained the drop at the end of the needle, the drop detached. The beaker was placed on a precision balance. When the drop detached, its precise weight was measured, and its volume could be deduced. The operation was repeated 10 times. Just before detachment, the capillary force and the drop force are equal so that the following equation applied:

2πrfγ = ∆ρ g (4)
where r is the radius of the needle (r = 0.3mm), f is a corrective factor reflecting the complexity of the shape of the drop when it detached, m its mass,  the drop density, ∆ρ the density difference between the two liquids and g the gravity acceleration taken equal to 9.81 m.s -2 . The corrective f factor is given in Supporting Information S1).

Results and Discussion

The first step to formulate oil-in-water-in-oil double emulsions was the selection of the two antagonistic, the hydrophilic and the lipophilic, stabilizers. A first criterion was their theoretical ability to stabilize either direct emulsions (for the hydrophilic one) or reverse emulsions (for the lipophilic one) so we focused on the Hydrophilic-Lipophilic Balance (HLB)

that should be lower than 7 for the lipophilic one and larger than 8 for the hydrophilic one. To avoid sensitivity toward pH, we ruled out all surfactants bearing amine, acid and other pHsensitive groups. We also chose to privilege stabilizers inducing steric repulsions rather than electrostatic ones. A very important criterion was their acceptance in cosmetic applications (meaning that they were not harmful, carcinogenic, mutagenic or reprotoxic CMR). After this first selection, formulations of simple, direct and reverse emulsions were studied. The second step of the work consisted in formulating double emulsions. Due to the presence of the two antagonist surfactants, the formulation could be something tricky and different for each couple of hydrophilic surfactant (HS)/lipophilic surfactant (LS). Thus, we varied the quantity of the droplets inside the globules and the amount of HS (by controlled dilution of the direct emulsion previously to incorporation in the oily continuous phase) and defined the droplet volume fraction d as the ratio of the encapsulated droplets' volume to the globule volume. The study of the direct and double emulsions was carried out for both fragrances. The resistance of the double emulsion formulation to a change in the fragrance has also been tested by using a third fragrance named C made of 12 molecules (see Supporting Information S2 for its composition).

By this progressive strategy, we think that we determined the best composition in terms of homogeneity of the globules and droplet sizes and in term of droplets encapsulation in the globules. The homogeneity of the globules and droplet sizes were appraised by optical microscopy. Finally, the encapsulation rate of the double emulsion was measured for the "optimized" system by gas chromatography.

Formulation of the direct emulsion

Among all the hydrophilic surfactants initially selected on the basis of their HLB, only three were maintained for their ability to stabilize concentrated direct emulsions with at least one of the two fragrances: Tween 60, Tween 20 and Tergitol 15-S-12. For each surfactant, a crude concentrated direct (90 wt% of dispersed phase) emulsion was obtained. Then this emulsion was sheared in the Couette cell at the maximum accessible shearing rate (14 280 s -1 ) in order to get the smallest droplet sizes. The size distribution was determined by static light scattering using Mie theory. Their characteristics, defined by Eq. 2, are reported in Table 5. The droplet size distributions measured by static light scattering are reported in Fig. 1 for The impossibility to formulate the direct emulsion for fragrance A with Tween 60 can be noticed. Indeed, as soon as 10 wt% of fragrance were incorporated, large and unstable drops were visible by naked eye. With the same surfactant, Tween 60, emulsions are obtained with fragrance B. As can be seen in Table 3, the interfacial tension between fragrance A and water is much lower than between fragrance B and water. As both fragrances exhibit similar LogP values, we think that this could possibly indicate the ability of some fragrance molecules to adsorb at the interface during the emulsification of fragrance A, interfering with the adsorption of Tween 60. This phenomenon was not observed with Tween 20 that differs only by about one unit in HLB (14.9 and 16 for Tween 60 and Tween 20 respectively). As the targeted globule size is 15 µm, the required size for the direct emulsion to be encapsulated is of the order of 1 µm. As the same formula should be feasible with both fragrances, Tween 60 was not adapted to prepare double fragrance/W/O emulsions.

In order to get better insight into the understanding of the observed phenomenon, and check if our hypothesis might be correct, we carried out interfacial measurements, by the weighing drop method, between air and water in presence of the surfactant (7.5wt% well above the critical micellar concentration where it is independent of surfactant concentration). It is equal to 39.1, 43.8 and 32.6 for Tween 20, Tween 60 and Tergitol 15-S-12 respectively. Note that for emulsification one should consider the interfacial tension between fragrances and the aqueous phases so that the same experiments were carried out between fragrance B and water in presence of the same amount of surfactant. The interfacial tension is equal to 5.5, 11.3 and 2.5 for Tween 20, Tween 60 and Tergitol 15-S-12 respectively. These values can be faced to the droplets size (Fig. 2). As put into evidence by Figure 2, the resulting droplet size depends linearly on the interfacial tension. Such a plot seems indicating that, as expected, the drop size depends linearly on the interfacial tension in agreement with the definition of the critical capillary number Ca = where  is the applied stress, d the obtained drop size and  the interfacial tension [START_REF] Mabille | Monodisperse fragmentation in emulsions: Mechanisms and kinetics[END_REF]. This helps understanding the obtained sizes for the three surfactants but it does not explain the impossibility to emulsify fragrance A with Tween 60. Indeed the interfacial tension between fragrance A and an aqueous phase containing Tween 60 is equal to 8.9 mN/m to be compared with 11.3 for fragrance B. The interfacial tension for fragrance A is lower than for fragrance B as it was already the case for the fragrance/water interface (see Table 3). Nevertheless, they remain of the same order of magnitude, so that interfacial tension alone is not a determining parameter.

As a conclusion, the two surfactants Tween20 and Tergitol 15-S-12 lead to satisfactory emulsions for both fragrances so that these two surfactants have been selected for the rest of the study.

Formulation of reverse emulsion

A similar work has been done for lipophilic surfactants to stabilize the reverse brine-in-IPM emulsions. However, lipophilic surfactants (LS) are less numerous and after a first selection, we only considered two: Span 80 and PGPR. Crude emulsions were first prepared by manually incorporating the aqueous phase in the IPM oil containing 10 wt% of LS. After that, the premixed emulsion was sheared with a Couette Cell at various shear rates in order to determine the process conditions to reach 15 µm sized globules. As can be observed from Figure 3, where the globule size is plotted as a function of the shear rate for each LS, the size of the globules decreases steeply when the shear rate is increased. The distributions become also narrower as deduced from the polydispersity index decrease. This phenomenon is similar to the one observed in direct emulsions and has been explained by the development of a Rayleigh instability under shear [START_REF] Mabille | Monodisperse fragmentation in emulsions: Mechanisms and kinetics[END_REF]. Example of two emulsions are given in Fig. 4. The high polydispersity at low shear rates is evidenced by the presence of smaller droplets as can be seen on both the micrograph and the drop size distribution on the left of Figures 4a and4b. It can also be noticed that small droplets surround the larger ones as the van der Waals interaction is larger between a small drop and a large drop than between two large drops. This is commonly observed in polydisperse emulsions. It is worth noticing that from the micrograph the small drops seem very numerous while from the drop size distribution it can be concluded than they represent about 5% of the volume (Figure 4b less sensitive to stirring during use than an equivalent emulsion obtained with an initially lower shear rate. For this reason, we chose the 15 µm-sized reverse emulsion obtained at 1560 s -1 with PGPR for the following.

Formulation of the double emulsion

After this first step, that allowed determining adequate surfactants, two possible couples for the formulation of the double emulsion were selected: PGPR with Tween 20 and PGPR with Tergitol 15-S-12 and two fragrances. For clarity reasons, the systems will be referred as HS/LS/fragrance.

Study of the Tween 20/PGPR/fragrance A or fragrance B system

To vary the HS surfactant concentrations in the intermediate aqueous phase as well as the amount of fragrance, beginning with a direct emulsion composed of 90 wt% of fragrance and 10 wt% to 30 wt% of HS with respect to the aqueous phase, dilutions have been applied.

The dilution can be done either with pure water or with an aqueous phase containing the same HS. This direct emulsion was then dispersed in the outer IPM phase containing the LS. Due to the numerous experiments to carry out and time consumed by using the Couette cell, the second emulsification, that is to say the incorporation of the direct emulsion into the IPM, was manually operated (the second shearing with the Couette cell has been skipped). Moreover, the obtained sizes were adequate and a better monodispersity was not targeted. In addition, for application reducing the number of stages is always beneficial. Once obtained, the double emulsions were observed by means of an optical microscope. Several morphologies could be seen. Either the obtained emulsion was indeed double or it turned out to be simple. In many cases, observation was not sharply defined, a coexistence between double or simple drops was seen. Then, the dominant behavior was reported. In some other cases, no emulsion at all could be obtained. All the results are reported in Figure 5 in the form of diagrams. Despite the fact that these observations are qualitative, they are reproducible. The limit of dilution of the direct emulsion ( ) represents the limit of accessible HS concentration that depends on the minimal concentration possible for the stabilization of the direct concentrated emulsion. Indeed, in the case of Tween 20/PGPR/Fragrance B, the direct emulsion could be prepared with HS concentration from 10 wt% to 30 wt%. As an example, to obtain a system at d = 30 wt% and %HS = 7.5 wt%, a direct emulsion at 90 wt% of droplets stabilized by 20 wt% of Tween20 was diluted three times with water containing 0.83 wt% of HS.

The line at 0 wt% of droplets fraction (x-axis) corresponds to emulsions prepared with an aqueous phase (0.1 M NaCl) containing different concentrations of Tween 20 dispersed and an oil phase composed of IPM and 10% of PGPR. This line without fragrance (d=0) allows evidencing interaction between the two surfactants. Indeed, two different behaviors can be observed. Until 5 wt% of Tween 20, the obtained emulsions are simple reverse emulsions,

showing that PGPR dominates the emulsion type. Above 5 wt% of Tween 20, multiple emulsions can be observed (see the micrographs in Supporting information S5 for 1 and 10 wt% of Tween 20), showing the competition of the two surfactants.

Several behaviors are visible on the "diagrams". It is worth noticing that we could not find any composition where only double globules could be observed, the samples were always "polluted" by the coexistence with empty globules. Moreover, only small areas of compositions allowed obtaining multiple emulsions (Figure 6) and the domains even almost vanished for fragrance B. The main area of the diagrams corresponds to compositions where multiple globules coexist with a high number of simple globules or where only simple globules are visible. The fact that there exist no common zone where only double emulsions could be observed, that is to say globules filled with fragrance droplets, indicate that the mixture of surfactant PGPR/Tween 20 is not efficient in stabilizing direct and reverse emulsions. Indeed, coexistence has already been

C B A )
observed in zones of formulation-composition maps where there is a conflict on the type of emulsions [START_REF] Salager | Phase transformation and emulsion inversion on the basis of catastrophe theory[END_REF][START_REF] Salager | Physicochemical parameters influencing the emulsion drop size[END_REF][START_REF] Izquierdo | Formation and stability of nano-emulsions prepared using the phase inversion method[END_REF].

In order to get better insight into the reason of such a difficulty to get double emulsions, we measured the interfacial tension between the aqueous phase containing various concentrations of Tween 20 and the oil phase: IPM containing PGPR (Figure 7) without fragrance (corresponding to the x-axis of Figure 5). A sharp decrease of interfacial tension can be observed in presence of Tween 20. Such a high interaction between the two surfactants likely explains the difficulty to stabilize multiple emulsions. Moreover it can be noticed that the resulting interfacial tensions are very low in presence of Tween 20 (<1 mN/m). This very low interfacial tension favors the formation of an IPM-in water-in IPM emulsion (inner and outer oils are identical) in a one-step process.

However, we think that a low interfacial tension is not appropriate when the two oils are different, that is to say when d>0. Indeed, it is our understanding that the direct fragrance-inwater emulsion should be resistant during its incorporation into the IPM oil. It is known, in Compared to the system with Tween 20, less compositions are accessible by dilution because the minimal concentration needed to stabilize the direct emulsion was higher. Thus, the dilution maximum leads to higher HS surfactant. However, more compositions allow obtaining only or a majority of multiple globules (dark and light green symbols respectively in Figure 8). It is also noticeable that the diagrams with both fragrances look very similar. It seems that Tergitol 15-S-12 is less sensitive to the fragrance. From the diagrams, it can also be concluded that theoretical encapsulations from 15 wt% (g =20%) to about 34 wt% (g=45wt%) of fragrance are accessible. Some rules can be deduced from these diagrams. The higher the droplet fraction is, the more multiple globules are observed up to a limiting concentration of 50 wt%. Also, the lower the amount of hydrophilic surfactant in the aqueous phase is, the larger proportion of multiple globules (globules containing small droplets) are observed. Thus, a balance has to be found in terms of homogeneous filling and globule size, and the best composition we have identified is 20 wt% of fragrance droplets and 7.5 wt% of Tergitol 15-S-2. In order to test the resistance of this formula with respect to the fragrance variability, a third fragrance C has been tested, and identically a double emulsion could be obtained (see supporting Information S6). The further goal was to polymerize the intermediate aqueous phase. This requires the introduction of monomers into the aqueous phase. Again, the formulation was very resistant to addition of various monomers, cross-linkers and initiators, showing that the proposed formulation is not specific and therefore is of special interest [START_REF] Stasse | Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-inoil (O/W/O) double emulsion[END_REF].

As polymerization of the aqueous intermediate phase is targeted, the time stability of the double emulsions is not an issue, only a few hours stability was required. Nevertheless, the kinetic stability of some emulsions containing fragrance B was studied over a period of several days.

The emulsions were observed by optical microscopy after several days and micrographs are reported in Figure 10 and compared with the initial emulsion (Figure 9) to determine whether destabilization occurred. From the comparison between Figure 9 and Figure 10, different observations can be made.

First, all the emulsions remained double and are still composed of multiple globules only, so that no catastrophic and massive destabilization occurred over this period. It is worth noticing that we did not observe any macroscopic phase separation over a period larger than two weeks.

Secondly, the emulsion with d = 20 wt% 7.5wt% has evolved in 3 days while the other emulsions are more identical to the initial ones. Globules contained less droplets but they are larger (Figure 10 a). This means that coalescence between inner droplets occurred at this time scale, a phenomenon already described by Pays et al. [START_REF] Pays | Coalescence in Surfactant-Stabilized Double Emulsions[END_REF] and coalescence between globules also occurred. Note that these phenomena do not change the encapsulation rate it only modifies the size of the compartments. It can also be seen that massive coalescence between droplets and globule, a mechanism described earlier [START_REF] Nollet | Determination of Formulation Conditions Allowing Double Emulsions Stabilized by PGPR and Sodium Caseinate to Be Used as Capsules[END_REF][START_REF] Pays | Coalescence in Surfactant-Stabilized Double Emulsions[END_REF][START_REF] Ficheux | Some Stability Criteria for Double Emulsions[END_REF][START_REF] Chávez-Páez | Coalescence in Double Emulsions[END_REF] and detrimental for fragrance encapsulation, neither occurred. As a conclusion, despite some coalescence events, the multiple emulsions remained stable for at least 3 days. This delay was sufficient in the present case, as our goal was to formulate double emulsions, a first step before incorporating monomers and polymerizing the intermediate phase [START_REF] Stasse | Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-inoil (O/W/O) double emulsion[END_REF]. Note that the stability of double emulsion against encapsulation may vary over a very large range of time depending on the used stabilizers. Kinetic stability is increased by replacing at least one of the surfactant by a protein, a polysaccharide, fat crystals, particles… that do not desorb and exchange as easily as surfactants.

In the present paper kinetic stability was not a goal as double emulsion were thought as an intermediate towards capsules.

Encapsulation efficiency

The encapsulation rate  of the double emulsion composed of 75 wt% of globules in IPM containing 10 wt% of PGPR with respect to the oil phase and 20 wt% of fragrance B in the globules and 7.5 wt% of Tergitol 15-S-2 with respect to the intermediate aqueous phase has been measured by GC-FID chromatography, immediately after preparation. Possibly the capsules can contain up to 15 wt% of fragrance with respect to the total sample. The GC results

showed that the IPM phase diluted at 5% by weight in ethanol contains 0.0315% of fragrance (see Table 6). It means 0.63% of fragrance in IPM phase. 0.63*0,25/0,15=1.05% of the fragrance was not encapsulated in the double emulsion but was solubilized in the outer IPM phase. In other words, the initial encapsulation rate is as high as 98.95%. GC experiments allow a deeper analysis of the fragrance composition (Table 6). The proportion of each molecule composing the fragrance contained in the IPM phase is given in the eighth column and can be compared to the proportion of the initial fragrance, reminded in the seventh column. Molecules with the largest deviation are noted in red. No clear correlation can be evidenced between these molecules and their hydrophilicity/lipophilicity through its Log P value. Only 1.05% of the initial fragrance amount has not been encapsulated and is solubilized in the outer IPM.

Assuming the amount preservation of each molecule the composition of the encapsulated fragrance can be deduced (last column) and compared to the initial fragrance. It can be observed that there is no detectable difference between the initial fragrance and the encapsulated fragrance (comparison between the seventh and ninth column). a A coelution is observed between one peak corresponding to (2-tert-butylcyclohexyl) acetate (isomer cis) and the peak corresponding to 10undecenal

Conclusion

The aim of this paper was to determine a unique formulation of fragrance-in-water-inoil double emulsion able to encapsulate large amounts of fragrance and that resists to fragrance modifications so that all the formulation work has not to be started again when considering another fragrance. The considered model fragrances were composed of a large number of molecules representative of the ones usually employed in industry. As the fragrance is usually lipophilic and the emulsion has to be dispersible in a perfumery solvent, the strategy is to formulate O/W/O emulsions that are not much described in literature (especially in domains others than food). The role of the intermediate aqueous phase is to create a barrier for the diffusion of fragrance. To obtain such double emulsions, two antagonist surfactants have to be chosen: the hydrophilic one for the formulation of the direct (fragrance-in-water) emulsion and the lipophilic one for the stabilization of the globules of inverted emulsion. After sweeping various non ionic stabilizers, PGPR has been selected for the reverse emulsion and Tergitol 15-S-2 has been selected for the direct emulsions. By varying the composition, we could draw "feasibility diagrams" useful to select the optimal composition: 75wt% of globules in IPM containing 10wt% of PGPR with respect to the oil phase, 20wt% of fragrance in the globules and 7.5wt% of Tergitol 15-S-2 in water. This optimal composition is summed up in Table 7 for an easier overview.

Figure 1 :

 1 Figure 1: Drop size distributions measured by static light scattering of the direct emulsions formulated with Tween20 (top) or Tergitol 15-S-12 (bottom). The dispersed phase is fragrance A (left) and fragrance B (right). Micrographs of the corresponding emulsions are reported in Supporting Information S3.

  tension (mN/m)

Figure 2 :

 2 Figure 2: Obtained droplet size D[START_REF] Frenkel | Multiple emulsions: I. Stability: inversion, apparent and weighted HLB[END_REF][START_REF] Seifriz | Studies in emulsions[END_REF] with fragrance B as a function of the air/water containing surfactant interfacial tension (full circles) and as a function of the fragrance B/water containing surfactant interfacial tension (full squares).

  left).

Figure 3 :FromFig. 3 ,Figure 4

 334 Figure 3: Evolution of the globule volume-average diameter as function of the applied shear rate for both LS: full squares PGPR and empty triangles Span 80. The error bars represent the polydispersity U of the emulsion and the targeted 15 µm size is made visible by the dashed line. Examples of drop size distributions with PGPR and Span 80 are reported in Supporting Information S4.

Figure 5 :

 5 Figure 5: Feasibility diagrams of double emulsions stabilized by 10 wt% of PGPR and various amounts of Tween 20 (x-axis). The inner fragrance droplet volume fraction in the globules d has also been varied. Top for fragrance A and bottom for fragrance B. Very few multiple droplets, Few multiple droplets in coexistence with simple ones, Multiple droplets with few simple ones, Multiple emulsions, Multiple droplets (without fragrance), Reverse emulsion Limit of dilution for the direct emulsion Area not accessible for this system of double emulsion.

Figure 6 :

 6 Figure 6: Multiple emulsions for A) fragrance A with d=20 wt% and 10 wt% of Tween 20, B) fragrance B with d=45 wt% and 5 wt% of Tween 20 and C) aspect of the double emulsion corresponding to the micrograph B.

Figure 7 :

 7 Figure 7: Evolution of the interfacial tension between the aqueous phase containing Tween 20 or not and the IMP oil phase containing PGPR.

  Interfacial tension (mN/m)enhanced oil recovery, that low interfacial values are detrimental for the stability of emulsions and systems leading to very low interfacial tension were sought after for this reason[START_REF] Förster | Calculation of optimum emulsifier mixtures for phase inversion emulsification[END_REF][START_REF] Shinoda | The Correlation between the Dissolution State of Nonionic Surfactant and the Type of Dispersion Stabilized with the Surfactant[END_REF][START_REF] Groeneweg | On the mechanism of the inversion of emulsions[END_REF][START_REF] Brooks | Phase inversion in non-ionic surfactant-oil-water systems, II. Drop size studies in catastrophic inversion with turbulent mixing[END_REF][START_REF] Brooks | Phase inversion in non-ionic surfactant-oil-water systems, I. The effect of transitional inversion on emulsion drop size[END_REF].3.3.2. Study of the PGPR/Tergitol 15-S-12/Fragrance A or B systemThe other hydrophilic surfactant still under consideration was Tergitol 15-S-12, with the same lipophilic surfactant (PGPR at 10 wt% with respect to IPM oil). Again, "feasibility diagrams" have been established. They are reported on Figure8.

Figure 8 :

 8 Figure 8: Feasibility diagrams of doubles emulsions stabilized by 10wt% of PGPR and various amount of both Tergitol 15-S-12 and inner fragrance droplet volume fraction in the globules d. Top for fragrance A and bottom for fragrance B. Very few multiple droplets Few multiple

Figure 9 :

 9 Figure 9: Optical microscopy pictures of the different double emulsions with fragrance A (top a to c) and B (bottom d to f). In all cases, PGPR=10 wt%, globule fraction 75 wt% various amounts of fragrance d and Tergitol 15-S-12 (a) d = 20 wt% and HS=15 wt%, (b) d = 30

Figure 10 :

 10 Figure 10: Observation by optical microscopy of the different systems at different times (a) d = 20 wt% and %HS = 7.5 wt% after 3 days, (b) d = 30 wt% and HS = 15 wt% after 4 days and (c) d = 45 wt% and HS = 20 wt% after 10 days.

Table 1 :

 1 Composition of fragrance A in wt%.* LogP corresponds to the relative solubility in water and octanol and is defined by Eq. 1 ** Tb is the boiling temperature taken at 760 mmHg, otherwise specified.

	Perfumery name	Chemical name	wt % Molecular	LogP* Tb **
				Weight		(°C)
				(g/mol)
	ALDEHYDE C12	Dodecanal	5	184.32	5.07	257
	LAURIC
	TH LINALOOL	3,7-dimethyloctan-3-ol	15	158.28	3.52	155
	HEDIONE	methyl 2-(3-oxo-2-	20	226.32	2.91	110 (0.2
		pentylcyclopentyl)acetate				mmHg)
	METHYL HEXYL	2-Octanone	10	128.22	2.44	173
	KETONE
	BOISAMBRENE	Ethoxymethyl-cyclododecyl ether	5	242.40	5.48	94	(1
						mmHg)
	DIMETHYL	Methyl N-methylanthranilate	5	165,19	2.66	256
	ANTHRANILATE
	DECALACTONE	5-Hexyloxolan-2-one	10	170,25	2.36	270
	CLONAL	Dodecane nitrile	5	181.32	4.90	277
	EUCALYPTOL	oxabicyclo[2,2,2]octane 1,3,3-triméthyl-2-	5	154.25	2.91	176
	ISO BORNYL	1,7,7-Trimethylbicyclo[2.2.1]hept-2-	20	196.29	4.04	225
	ACETATE	yl acetate

Table 2 :

 2 Composition of fragrance B in wt%.

Table 3 :

 3 Properties of both fragrances.

	Fragrance A	Fragrance B

Table 4 :

 4 Composition of the various emulsions. The double emulsion was obtained by incorporation into the continuous phase of the simple direct emulsion after dilution at various rates.

	Simple direct	Dispersed phase:	Continuous phase:
	O/W emulsion,
		Fragrance A or B	0.04 g NaCl
		90 g	3 g HS
			6.96 g H2O
	Simple reverse	Dispersed phase:	Continuous phase:
	W/O emulsion
		0.44 g NaCl	22.5 g IPM
		74.56 g H2O	2.5 g LS
	Double O/W/O	Innermost phase:	Intermediate phase:	Continuous phase:
	emulsion
		Fragrance A or B	NaCl brine 0.1M with HS	22.5 g IPM
		3.5 to 45 g	at various concentrations	2.5 g LS
			30 g to 71.5 g
	2.4. Double emulsion encapsulation rate determination

Table 5

 5

	: Droplet size distribution characteristics D[4,3] and U (defined by Eq 2) as a function
	of surfactant and fragrance
		Fragrance A		Fragrance B
		D [4,3] (µm)	U (%)	D [4,3] (µm)	U (%)
	Tween 20	1.8	64.4	1.1	30.9
	Tween 60	unstable		1.4	28.1
	Tergitol 15-S-12	0.7	28.0	0.8	28.0

Table 6 :

 6 Dosage of the non-encapsulated fragrance molecules from fragrance B in the continuous IPM phase (dilution at 5%w/w in ethanol): main peaks detected by GC-MS

	Retention time (min)	Attribution	Isomer	Proportion of each peak area in the case of the isomers in the fragrance (%)	Proportion of each peak area in the case of the isomers in the sample (%)	Concentration of the fragrance molecule(s) in the diluted sample (10 -3 %)	Proportion of the molecule in the original fragrance (wt%)	Proportion of the molecule in the fragrance in IPM (wt%)	Proportion of the molecule in the encapsulated fragrance (wt%)
	7.23	cis-3-Hexenyl acetate	/	/	/	1.1	5	3.5	5.0
	8.44 8.88	2,4-dimethylcyclohex-3-ene-1-carbaldehyde	(E)-isomer (Z)-isomer	51 49	51 49	2.3	5	7.3	5.0
	9.98	prop-2-enyl heptanoate	/	/	/	1.2	5	3.8	5.0
	10.71	3,7-dimethyloct-6-en-1-ol	/	/	/	2.9	5	9.3	5.0
	10.91		(Z)-isomer: neral	34	68
		3,7-dimethyl-2,6-Octadienal				2.1	5	6.7	5.0
	11.33		(E)-isomer: geranial	66	32
	11.71	(2-tert-butylcyclohexyl) acetate + 10-undecenal a	Cis-isomer	88	49	11.2	43	35.6	43.1
	11.99	(2-tert-butylcyclohexyl) acetate	Trans-isomer	12	51
	12.58	2-methoxy-4-(2-propenyl)-phenol	/	/	/	2	5	6.3	5.0

Table 7 :

 7 Composition of the double emulsion valid for three different fragrances. The amounts are given for 100 g of double emulsion

	Innermost phase	Intermediate phase	Outermost (continuous) phase
	15 g of fragrance	55.5 g of brine 0.1M NaCl	22.5 g IPM
		60 g	25 g
		4.5 g Tergitol 15-S-2	2.5 g PGPR

Acknowledgements

The authors would like to thank Magali Vandroux for developing the dosage method. M.S. would like to thank ANRT and T.E.P.L. for their financial support (contract number N° 2014/1309).

This composition is adequate for three different fragrances made of numerous molecules representative in perfumery industry, showing its promising use. Such a double emulsion allows encapsulating in theory 15 wt% of fragrance with respect to the total sample. The encapsulation yield is as high as 98.95% of the fragrance that is not altered by the emulsification process.

These double emulsions are kinetically stable for at least 3 days, making these systems very interesting by themselves or for more advanced capsules as for example capsules that could be obtained by polymerizing the intermediate aqueous phase.

Declaration of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Margot Stasse

Tiphaine Ribaut

Valérie Héroguez
email: valerie.heroguez@enscbp.fr

Véronique Schmitt
email: veronique.schmitt@crpp.cnrs.fr

Elaboration of double emulsion-based polymeric capsules for fragrance

Keywords: fragrance, capsules, encapsulation, double emulsions, free radical polymerization, monomers, cross-linkers, release, diffusion, network

Introduction

Encapsulation of fragrances concern various applications such as detergency, cosmetics, pharmaceutics, foods, textiles, agriculture, building and so on [START_REF] Doane | Encapsulation of pesticides in starch by extrusion[END_REF][START_REF] Tsuiji | Microencapsulation of pesticides and their improved handling safety[END_REF][START_REF] Zuidam | Encapsulation technologies for active food ingredients and food processing[END_REF][START_REF] Nelson | Application of microencapsulation in textiles[END_REF][START_REF] Singh | Microencapsulation: A promising technique for controlled drug delivery[END_REF][START_REF] Konuklu | Review on using microencapsulated phase change materials (PCM) in building applications[END_REF]. Once deposited on a substrate, they act as a fragrance reservoir that can deliver their content. In the present work, the targeted application is encapsulation for softeners. For such an application, the specifications are the following: i) the same formulation should be valid for various fragrances, ii) the capsules should resist pH variations, iii) they should be 15 µm-sized to deposit on the textile, iv) they should be dispersible in water and v) fragrance is expected to be released during drying. Of course, all the compounds should be compatible with the application meaning that they should be non-toxic, non-irritant, non-mutagenic… Today, capsules containing fragrance are already widely spread in detergents, cleaning and softeners [START_REF] Warr | New microcapsules[END_REF][8][9][10][START_REF] Jahns | Microcapsule preparations and detergents and cleaning agents containing microcapsules[END_REF][START_REF] Jahns | Microcapsules, the production and use thereof[END_REF]. Such capsules are usually synthesized by a suspension polymerization process [START_REF] Warr | New microcapsules[END_REF][8][9][10][START_REF] Jahns | Microcapsules, the production and use thereof[END_REF][START_REF] Jacquemond | Perfume-containing polyurea microcapsules with undetectable levels of free isocyanates[END_REF] meaning that the fragrance, the monomer and the initiator are miscible, and this blend is dispersed in water. Polymerization occurs during stirring. The obtained capsules are core-shell capsules made of a fragrance core and a polymer shell. They also often contain lattices in the fragrance. So the two main drawbacks are firstly the fact that, due to the miscibility with the fragrance, polymerization occurs in the fragrance phase while fragrance contains usually reactive molecules and secondly the presence of latex (Figure 1).

In the present work, we propose an alternate strategy based on a double O/W/O emulsion that is to say a fragrance-in-water-in-solvent double emulsions (Figure 1). This means that the aqueous globules contain fragrance droplets. The idea is to separate the place where the polymerization takes place from the fragrance phase. Therefore, double emulsions exhibit the advantage of being composed of two kinds of compartments with different affinities: more lipophilic compartments containing the fragrance and aqueous compartments where we aim at inducing polymerization. Once obtained these capsules likely need to be transferred into an aqueous phase. In the following, we will describe the progressive steps required to elaborate such capsules.

Materials

Fragrances

Two model fragrances noted A and B composed of 10 and 13 molecules respectively have been assembled by Takasago Europe Perfumery Laboratory to mimick real sytems. Their exact composition is given in supporting information S1. They include the classical class of molecules composing fragrances that is to say molecules with alkene, aldehyde, hydroxyl, ketone and ester moieties.

As a test to check the formulation resistance, we also used a third fragrance noted C. Its exact composition is also reported in S1.

Hydrophilic Surfactants

The selected hydrophilic non-ionic surfactants are: Tergitol 15-S-12 (molar mass = 760 g.mol -1 and HLB = 14.5), Tween 60 (molar mass around 1310 g.mol -1 , HLB = 14.9), Tween 20 (molar mass around 1220 g.mol -1 , HLB = 16) all purchased from Sigma-Aldrich.

Lipophilic Surfactant

The selected lipophilic surfactants (LS) is polyglycerol polyricinoleate (PGPR) with a molar mass of 3000 g.mol-1 (HLB = 1.5) graciously provided by Paalsgard.

Perfumery solvents

The solvents are water possibly containing NaCl and isopropyl myristate (noted IPM in the following, density equal to 0.850 g.mL -1 ) chosen because it is widely used in perfumery and graciously provided by Takasago Europe Perfumery Laboratory.

Monomers

Four monofunctionnal monomers have been selected for their hydrophilicity and the agreement to use them in cosmetics: Hydroxyethyl-methacrylate (98%) noted HEMA, Oligo(ethyleneglycol) methacrylate noted OEGMA with Mn=360 g.mol -1 (containing 500-800 ppm of monomethyl ether hydroquinone), Methacrylamide noted MAM (98%), and Methyl vinyl ketone noted MKV.

Cross-linkers

Two different cross-linkers, namely difunctional monomers, have been considered: Tetra(ethyleneglycol) diacrylate noted TEGDA and N,N-Methylene-bis-Acrylamide noted MbA. The 4 units of ethylene glycol for TEGDA between the two acrylate functions is expected to confer flexibility to the tridimensional sought after network whereas the MbA can add physical cross-linking due to the possible hydrogen bonding [START_REF] Kuo | Thermal behavior and specific interaction in high glass transition temperature PMMA copolymer[END_REF].

Methods

3.1) Optical and electronic microscopy observations

Emulsions and capsules were observed with a Leica Leitz inverted optical microscope and the images were recorded with an IDS video camera and the Scion image program. The globule or the capsule size was estimated through a statistic analysis over about 50 particles.

For the capsules, scanning electron microscopy (SEM) pictures were taken with a Hitachi TM-1000 apparatus. To avoid deformation of the capsules possibly provoked by vacuum, Cryo-SEM was performed with a JEOL 6700FEG electron microscope equipped with liquid nitrogen cooled sample preparation and transfer units. A small sample of the dispersion of capsules was first deposited on the aluminum specimen holder. The sample was then frozen in the slushing station with boiling liquid nitrogen and transferred under vacuum from the slushing station to the preparation chamber. The latter was held at T = -150°C and P = 10 -5 Pa and was equipped with a blade used to fracture the sample. Once fractured, the sample was coated by a layer of Au-Pd and then inserted into the observation chamber equipped with a SEM stage cold module held at -150°C.

3.2) Static light scattering

The emulsions size distributions were measured using static light scattering by means of a Malvern Mastersizer MS2000 granulometer. The software gives access to the volumeaveraged drops size D [START_REF] Nelson | Application of microencapsulation in textiles[END_REF][START_REF] Zuidam | Encapsulation technologies for active food ingredients and food processing[END_REF] and to the polydispersity index called uniformity and noted U defined as the volume-average difference between the diameter and the median diameter normalized by the median diameter. Let us remind that the median diameter corresponds to the midpoint of the size distribution meaning the diameter for which half of the dispersed phase is distributed in drops smaller than the median diameter and the other half is distributed in drops larger than the median diameter.

3.3) Gas chromatography

Gas chromatography (GC) has been used to analyze and quantify the amount of fragrance inside or outside the emulsion and capsules. The gas chromatograph (Agilent GC 7890B) is equipped with flame ionization detector (FID) coupled with Mass spectrometer 5977A (MS), a split/splitless injector (1:40 split ratio) and a data system (Enhanced ChemStation MSD Chemstation). The detector and injector temperatures were set at 250°C. A 30 m×250 µm internal diameter capillary column coated with 5%-phenyl poly(methylsiloxane) (0.250 µm film thickness) (HP-5) was used. The oven temperature was programmed from 50 °C (2 min) to 280 °C (5 min) at 10 °C/min. Two microliters of the sample were injected.

Fragrance molecules are identified with MS and quantified according to an external calibration with different solutions of fragrance in ethanol on FID.

3.4) DSC

The polymeric capsules were analyzed by Scanning Differential Calorimetric (DSC) with a DSC Q100 RCS apparatus. The protocol was the following: first an increase of temperature from -80°C to 120°C with a speed at 10°C/mn was applied followed by a decrease down to -80°C. The second cycle started at -80°C up to 230°C with a speed of 10°C/mn followed by a last decrease down to -50°C at the same speed.

3.5) Mechanical compression

The resistance of capsules against pressure was estimated using a homemade device. It is composed of three parts. The first part is an empty cup where the capsules are deposited. The second is a hollow piston equipped with a circle-shaped stainless-steel filter of 5 µm. The filter only allows the transfer of IPM containing the non-encapsulated fragrance while the capsules are maintained at the bottom below the filter. The piston is hollow to allow the external phase recovery under pressure. The third part is the load of different weights to access different pressures: 100 and 150 g corresponding to 780 Pa and to 1170 Pa respectively.

The protocol for the measurement is the following: the capsules are put in the cup and covered by IPM (1:1). The piston is placed on the sample and the weight is deposited on it. The pressure is applied for at least one hour to reach a stationary state. Then the IPM with the possibly released fragrance is recovered thanks to the hollow piston while maintaining the pressure. This recovered phase is then analyzed by GC to quantify the possibly leaked fragrance in IPM.

Results and discussion

Elaboration of emulsions

Firstly, we need to formulate emulsions since we aim at polymerizing the intermediate aqueous phase of an established double emulsion. This is very different from the suspension polymerization where polymerization may be initiated under stirring, without need of stabilizing an emulsion that forms and destroys continuously. As double emulsions are complex morphologies composed of the two types of direct oil-in-water and reverse water-in-oil simple emulsions, these two types of emulsions need to be formulated before combining them.

Simple direct emulsion

Following the Brancroft rule [START_REF] Bancroft | The theory of emulsification[END_REF], the direct fragrance-in-water emulsion requires the use of a more hydrophilic surfactant. A first selection led to the choice of three non-ionic surfactants namely Tween 20®, Tween 60® and Tergitol 15-S-12® allowed in cosmetics. Since fragrance droplets have to be themselves dispersed in aqueous globules, the targeted droplet size is of the order of 1 µm.

The fragrance, corresponding to the dispersed phase of the direct emulsion, is progressively incorporated in the aqueous phase containing sodium chloride and the hydrophilic surfactant, up to a droplet mass fraction d=90%. This crude emulsion is then sheared using a Couette cell applying a shear rate of 14 280 s -1 during about 10 s. We chose preparing highly concentrated emulsions in order to better reduce the average drop size and to narrow the drop size distribution [START_REF] Mabille | Monodisperse fragmentation in emulsions: Mechanisms and kinetics[END_REF][START_REF] Mabille | Rheological and Shearing Conditions for the Preparation of Monodisperse Emulsions[END_REF]. Indeed, it has been demonstrated that shearing a concentrated emulsion is highly efficient. Also for dilute emulsions, the resulting drop size depends upon the interfacial tension: the lower the interfacial tension, the smaller the drop size [START_REF] Mabille | Monodisperse fragmentation in emulsions: Mechanisms and kinetics[END_REF]. More precisely, the size is proportional to the interfacial tension all the other parameters being kept identical, as a result of a constant critical capillary number defined as Ca = where σ is the applied stress, ddroplet the obtained droplet size and γ the interfacial tension. Obviously, this result remains valid in the present case of highly sheared concentrated emulsions (see Figure 2). As micrometric droplets are targeted to be incorporated in the double emulsion, we chose the Tergitol 15-S-12® surfactant for the following. The obtained emulsions exhibit a uniform drop size distribution as can be quickly visualized by the large field optical microscopy pictures and as can be quantified by the drops size distributions reported in Figure 2. In both cases, the volume-average droplet size is 0.7 µm and the polydispersity index U is 28%. Adapted from [START_REF] Stasse | Formulation of concentrated oil-in-water-in-oil double emulsions for fragrance encapsulation[END_REF].

Simple reverse emulsion

For the reverse brine-in-IPM emulsion, the widely spread PGPR surfactant has been selected [START_REF] Nollet | Determination of Formulation Conditions Allowing Double Emulsions Stabilized by PGPR and Sodium Caseinate to Be Used as Capsules[END_REF]. Brine (0.1 M NaCl) is preferred to pure water as it is known that adding salt in water is very efficient to prevent Ostwald ripening [START_REF] Taylor | Ostwald ripening in emulsions[END_REF] in reverse emulsions. Ostwald ripening is one of the two mechanisms responsible for the drop size evolution with time in emulsions. It proceeds by molecular diffusion of the dispersed phase through the continuous phase due to its solubility. Indeed salted water is less soluble in oil than pure water. The emulsion is prepared by progressively adding brine into IPM containing 10 wt% of PGPR up to a mass fraction of globule G=75%. This crude emulsion is then sheared at various shear rates. The targeted size should be of the order of 10-15 µm as this range of size is thought to be efficient for deposition on textiles [START_REF] Rodrigues | Scentfashion®: Microencapsulated perfumes for textile application[END_REF] (Figure 3)

Preparation of the O/W/O double emulsion

The double fragrance A or B-in-brine-in isopropyl myristate emulsions are prepared by a two-step procedure. First, the direct fragrance in brine emulsion is prepared as described previously in the Simple direct emulsion section. This concentrated emulsion is then diluted with an aqueous solution containing or not additional Tergitol 15-S-12®. This dilution allows fixing both the mass fraction of droplets droplet as well as the composition of the intermediate aqueous phase (salt and surfactant concentrations). Secondly, this diluted direct emulsion is progressively incorporated in IPM containing 10 wt% of PGPR. The emulsions are then observed by optical microscopy to determine whether the emulsion is indeed double or if the aimed morphology is not obtained. All the results may be reported on (droplet -Tergitol 15-S-12®) diagrams (Figure 4). The green squares correspond to formulations where double emulsion morphology is obtained. By comparing the diagrams from both fragrances, it can be noticed that the domains are the same for both fragrances showing the solidity of the formulation toward a fragrance variation. In order to even better test this resistance we prepared a double emulsion with a formulation in the green domain with the third fragrance C. Again a double emulsion morphology is observed. This shows that the proposed composition of double emulsion may be used with all the various molecules composing these three fragrances, none of them is deleterious for preparation of the double emulsion.

In the following, we will consider the emulsion composed of fragrance B, with droplet = 20 wt%, 7.5 wt% of Tergitol 15-S-12® in brine, G = 75 wt%, 10 wt% of PGPR in IPM. The details are given in Table 1.

Table 1: Composition of the selected double emulsion for the rest of the paper, the same composition is expressed differently. In the first row, the composition is given with respect to the considered phase. The last row gives the amounts for 100 g of total sample. Encapsulation efficiency and composition of the encapsulated fragrance A major point is the determination of the encapsulation efficiency  defined as the proportion of fragrance that is inside the double emulsion.

Inner phase

ε = (1)
It is deduced from the determination of the non-encapsulated fragrance that is to say the fragrance that could be solubilized in the outer IPM. To assess the amount of fragrance in IPM, the emulsion is centrifuged at 1500 rpm during 25 min to induce separation of the continuous phase from the emulsion without double emulsion rupture. The continuous phase is then recovered, filtrated and diluted in ethanol up to 5 wt%. This phase is then analyzed by gas chromatography. The amount of fragrance in IPM is 1.05 wt% of initial amount leading to an encapsulation rate of =98.5 wt%. Usually, the encapsulated species is a unique molecule of interest but as fragrance B is a blend of 13 molecules, it is also of great interest to determine the composition of the encapsulated fragrance and to compare it with the initial fragrance. The results obtained by gas chromatography are reported on Figure 5. This means that the double emulsion allows a high encapsulation rate and does not alter the fragrance.

Polymerization of the emulsions

Again, we chose a progressive strategy to select the monomer for the aqueous phase polymerization beginning with the simple reverse emulsion. The composition is the same as previously that is to say 10% of PGPR in IPM for the continuous phase and 75 wt% of brine, the only difference is the use, in the aqueous phase, of KPS that will be used as initiator of the free radical polymerization.

Note that, at this point, neither the study of the kinetics of polymerization, nor the optimization of the conversion are sought after. However, even if not optimized, the conversion C is assessed by gravimetric measurements using:

C = (2)
where the mass of obtained polymer is determined by weighting the purified and dried polymer, subtracting the non-volatile substances (surfactants and salt), the solid content by ATG takes

Polymerization of the reverse simple emulsion

A first selection of non-CMR hydrophilic monomers leads to the choice of four monomers: 2-Hydroxyethyl methacrylate (HEMA), Methyl vinyl ketone (MVK), Methacrylamide (MAM) and Oligo(ethyleneglycol) methacrylate (OEGMA 360). In all cases, KPS has been chosen as initiator. Note that when KPS is used, the amount of NaCl is reduced as KPS releases ions. A first criterion to be tested is the possibility to incorporate the monomers in the reverse emulsion.

HEMA:

The introduction of HEMA at 25 wt% and 50 wt% with respect to the aqueous phase leads to a strong decrease of the globule size to 7.1 µm and 4.9 µm respectively. This size decrease can be understood by a strong decrease of the interfacial tension between water and IPM (38.1 mN.m -1 ) and between water containing 25 wt% of HEMA and IPM (5.8 mN.m - 1 ) showing the strong affinity of HEMA towards the water/oil interface. As we are looking for polymerization to occur in the bulk water phase, HEMA is not a good candidate.

MVK: Addition of MVK in the aqueous phase does not modify the globule size of the reverse emulsion. However, it can be observed that the reverse emulsion sediment and coexist with a yellowish liquid at the top of the emulsion. This liquid is composed of IPM that is colorless and MVK that gives its yellow color. This means that MKV migrates into IPM and therefore is in fact too hydrophobic.

OEGMA 360: The introduction of 25 wt% of OEGMA in the aqueous phase does almost not alter the globule size (8.4 µm). In addition, the size does not evolve over a period of 18 hours large enough to induce polymerization by heating the sample 24 hours (the time optimization was not the goal) at 70°C. Polymerization is performed in a flask with a magnetic stirring and is quenched by exposure to air. The determined conversion is equal to 91%. The obtained capsule size is very similar to the initial double emulsion globule size showing that no emulsion destabilization occurred during polymerization and no coagulum was formed.

Due to the very low solubility of polyOEGMA in usual solvents available for NMR or SEC, the polymer was analyzed by DSC. The DSC curve of the synthesized polymer exhibits a glass temperature (Tg=-53°C), a crystallization temperature (Tc=-8°C) and a melting point (Tm=7°C) characteristic of polyethylene glycol (PEG) chains. Comparison with the OEGMA macromonomer [START_REF] Stasse | Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-inoil (O/W/O) double emulsion[END_REF] shows a significant increase of glass temperature (Tg=-61°C) that can be understood as a decrease of the mobility of the OEGMA grafted chains by comparison with free chains, therefore attesting that the polymerization occurred.

Figure 6: Optical microscopy of the polymerized simple reverse emulsion containing OEGMA.

Adapted from [START_REF] Stasse | Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-inoil (O/W/O) double emulsion[END_REF].

MAM:

The introduction of 15 wt% of MAM in the aqueous phase does not modify the globule size (9.5 µm). In addition, the size does not evolve over a period of 18 hours large enough to induce polymerization by heating the sample at 70°C. Polymerization is performed in a flask with a magnetic stirring for 24 hours (again the time optimization was not targeted) and is quenched by exposure to air. The determined conversion is equal to 64%.

The obtained capsule size is very similar to the initial double emulsion globule size showing that no emulsion destabilization occurred during polymerization and no coagulum was formed. Three heating/cooling cycles are performed (Figure 7). During the first cycle, a transition at 50-150°C is visible that disappears during the second and third cycles.

This endothermic change is likely due to the evaporation of residual water. The endothermic change above 150°C can be attributed to physical cross-linking due to hydrogen bonding. This polyMAM chains physical crosslinking is validated by the non-solubility of the polymer except in strong acids known to be able to disrupt hydrogen bonds.

Figure 7: DSC analysis of the polymerized simple reverse emulsion containing MAM. Adapted from [START_REF] Stasse | Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-inoil (O/W/O) double emulsion[END_REF] These polymerization tests in the reverse simple emulsion lead to the selection of the two MAM and OEGMA monomers for further use in the double emulsions.

Polymerization of the double emulsions

The following step consists in combining the previous results obtained in formulating double emulsions and polymerizing in simple reverse emulsion. In other words, double emulsions are prepared as previously described in presence either of 15 wt% of MAM or of 25 wt% of OEGMA. Polymerization is induced with the same protocol as applied to the reverse simple emulsion. Again, no coagulum is observed and capsules have sizes similar to the globule sizes of the double emulsions. More surprising, in opposition to the double emulsion globules, the obtained capsules are not dispersible in IPM but in water. This is likely linked to the nature of the obtained polymers, IPM is not a good solvent for the polyMAM or polyOEGMA. This means that no transfer of the capsules is required before use.

Polymerization in the double emulsions in presence of cross-linkers.

In order to elaborate a 3D network in the intermediate aqueous phase a cross-linker is added before polymerization. Two cross-linkers have been chosen again for their water affinity and non-toxicity: Methylene-bis-acrylamide noted MbA in the following and Tetra(ethylene glycol) diacrylate referred to as TEGDA in the following. A first formulation work consists in determining the maximum amount of cross-linker that can be incorporated in the double emulsion without altering the formula. The results are summarized in Figure 8a where the amount of cross-linker is given in mol% with respect to total amount of monomer and cross- linker. The polymerization is initiated as at 70°C and lasts for 24 hours in a double envelope reactor with a mechanical stirrer operating at 100 rpm

The result of polymerization is summarized in Figure 8b where CryoSEM picture are reported.

The obtained conversions are also noted. Total amount of fragrance after polymerization and encapsulation efficiency

The capsules are diluted with ethanol that is thought to remove all the encapsulated and non-encapsulated fragrance. After 30 minutes of contact, ethanol containing fragrance is removed and analyzed by gas chromatography. This protocol is the same as the one classically used for core-shell capsules. Indeed ethanol and fragrance are miscible and ethanol is a good solvent for the polymer shell that swells allowing fragrance recovery. The proportion of recovered fragrance in ethanol with respect to the initial amount of fragrance incorporated in the double emulsion is reported in Table 2. Among the fragrance extracted by ethanol, the proportion that is encapsulated can be determined using Equation 1, for that the non encapsulated fragrance is extracted using a dilution in IPM and a soft centrifugation. The results are reported in Table 2b. High encapsulation rates are achieved.

Despite the high rates of recovery, a small fraction of fragrance is not present in ethanol. This may due to an incomplete recovery by ethanol linked to the matrix nature of the capsules making the polymer swelling more difficult. An alternate explanation could be evaporation during heating required to induce polymerization, interactions between monomers and fragrance molecules or degradation of some fragrance molecules during polymerization. To get better insight about the possible phenomenon, the recovered fragrance is analyzed by gas chromatography (figure 9 top) as well a pure fragrance heated 24 hours at 80°C and fragrance in contact with water containing KPS (demixed phases) also heated 24 hours at 80°C (Figure 9 bottom). From Figure 9 (bottom), it can be seen that some molecules are very sensitive to the presence of KPS when heated: Triplal, Citronellol, Citral, Aldehyde C11 undecylenic and hexyl cinnamic acid in a much smaller extend. Nevertheless, these molecules are present in the capsules in a larger proportion (Figure 9 top) showing that the proposed strategy has a protective effect on them.

From the previous results, the fragrance composition after polymerization can also be plotted and compared to the initial one (Figure 10)

Evolution of encapsulated fragrance with time in closed vials

A small amount (1 g) of capsules is deposited on the bottom of 20 ml vials with a lid.

This means that a large volume of air occupies the vials. At regular time intervals, a small amount of the capsules is taken and diluted in IPM in order to measure the amount of fragrance still encapsulated. The results are normalized by the initial amount of fragrance (in the double emulsion formulation before polymerization). Note that possible escaped fragrance in the air of the vials is not considered so that the measure is not biased. The results are reported on Figure 11. No visible evolution over 5 weeks could be detected showing that fragrance is not released in close vials and that the capsules exhibit a high kinetic stability for storage.

In opposition for use, these capsules are required to release the encapsulated fragrance.

Evolution of encapsulated fragrance with time in open vials at 20 and 40°C

The mass loss of capsules deposited on small cups without lid in a thermally controlled oven is measured as well as each of the constituting phase. The results are reported on Figure

Figure 12: Evolution of the normalized weight at 20°C (top) and 40°C (bottom) for the isolated phases (left) and the capsules (right): MAM/MbA, MAM/TEGDA, OEGMA/MbA and X OEGMA/TEGDA Adapted from [START_REF] Stasse | Cross-linking of double oil-in-water-in-oil emulsions: a powerful way to obtain microcapsules for with attractive mechanical properties[END_REF].

At 20°C, a first fast decrease of the capsule weight is observed that can be attributed to the water loss, the second slowly loss is due to fragrance and it can be noted that the mass of IPM does not evolve at all over 150 days. At 40°C, the same accelerated tendency is observed but IPM also evaporates. Taking into account the composition of the capsules and the water and when required the IPM losses, the fragrance release can be estimated for the various capsules.

The results are reported as examples after 150 days at 20°C and after 40 days at 40°C on Figure 13.

Mechanical resistance

It is worth noticing that all capsules resisted to a 10 -3 Pa vacuum during 12 hours without any rupture or morphology evolution. Then a home-made set-up [START_REF] Stasse | Cross-linking of double oil-in-water-in-oil emulsions: a powerful way to obtain microcapsules for with attractive mechanical properties[END_REF] allowed simultaneously applying a compression (using a load of 100 g corresponding to 780 Pa and 150 g corresponding to 1170 Pa) and taking a part of the recovered fragrance in an excess of IPM. Fragrance is release by compression as plotted in Figure 14 Figure 14: Amount of released fragrance under compression normalized by the initial amount of fragrance. Adapted from [START_REF] Stasse | Cross-linking of double oil-in-water-in-oil emulsions: a powerful way to obtain microcapsules for with attractive mechanical properties[END_REF].

When the load is removed and the capsules can relax, no fracture and no morphology evolution can be observed, showing the elastic nature of the capsules that behave as small sponges.

Conclusion

Herein we presented a novel strategy to elaborate polymer capsules containing multicomponent fragrance from the formulation of double fragrance-in-aqueous phase-in-perfumery solvent (isopropyl myristate) emulsions. This strategy aims at compartmentalizing the fragrance and the polymerization locus. The selected double emulsion formulation resists changes in fragrance composition in the innermost droplets and addition of monomers, cross-linkers and initiator in the aqueous intermediate phase. This allows polymerizing and cross-linking this intermediate phase to elaboration a tri-dimensional network achieving matrix capsules. This strategy is also versatile as various monomers, cross-linkers may be used, and the obtained properties are tunable. Indeed, a judicious choice of monomers and cross-linkers allows adjusting the fragrance passive release. In addition to the fragrance diffusion across the capsules, the release may also be triggered by applying a compression leading to an additional boost effect. All the proposed capsules exhibit morphologies that are resistant to compression, no fracture has been observed and the polymer capsules act as small sponges. Moreover, unexpectedly, beginning with a double emulsion dispersed in a perfumery oil, no transfer of the

Figure 1 :

 1 Figure 1: Schematic representation of a) and b) the nowadays most widely spread strategy to elaborate fragrance core-shell capsules and c) the alternate proposed strategy based on the polymerization of double emulsions for producing matrix capsules.

Figure 2 :

 2 Figure 2: Result of the direct emulsion preparation, top) obtained droplet size as a function of the interfacial tension between fragrance B and brine or fragrance B and air in presence of the selected surfactant, middle) large view of the emulsions prepared with Tergitol 15-S-12® and fragrance A (left) and B (right) and bottom corresponding droplet size distribution measured by

Figure 3 :

 3 Figure 3: Result of the reverse emulsion preparation, top) obtained globule size as a function of the applied shear rate. The error bars represent the polydispersity index U, middle) Optical microscopy picture of the emulsion after shearing at 3150 s -1 and bottom corresponding globule size distribution measured by static light scattering. The volume-average globule size is 9.5 µm and the polydispersity index U is 35.5%. Adapted from [18].

Figure 4 :

 4 Figure 4: Feasibility of doubles emulsions: effect of the droplet fraction and the amount of hydrophilic surfactant keeping the fraction of PGPR (10 wt%) and the globule fraction (G=75 wt%) constant. Top) (droplet -Tergitol 15-S-12®) diagrams for fragrance A (left) and B (right). Caption: ( ) very few multiple droplets ( ) few multiple droplets in coexistence with simple ones ( ) only multiple droplets ( ) Multiple droplets with few simple ones ( ) multiple droplets (without fragrance)Limit of dilution of the direct emulsion. In grey, inaccessible area by the proposed two-steps process including a dilution of the direct emulsion. Bottom) Double emulsions with (droplet = 30 wt% and 15 wt% of Tergitol 15-S-12® for fragrance A, B and C from left to right. Adapted from[START_REF] Stasse | Formulation of concentrated oil-in-water-in-oil double emulsions for fragrance encapsulation[END_REF].

Figure 5 :

 5 Figure 5: composition of bulk (initial) and encapsulated (in the double emulsion) fragrance B.

  into account the possibility of remaining water despite the lyophilization, the proportion of monomers represents the initial weight percentage of monomer initially present in the sample and the collected mass is the considered sample mass after polymerization.

Figure 8 :

 8 Figure 8: a) Addition of cross-linker in the double emulsion and b) obtained capsules.Adapted from[START_REF] Stasse | Cross-linking of double oil-in-water-in-oil emulsions: a powerful way to obtain microcapsules for with attractive mechanical properties[END_REF]

Figure 9 :

 9 Figure 9: Fragrance analysis contained in the capsules (top) and control sample without capsules. Adapted from [23].

Figure 10 :

 10 Figure 10: Composition of the fragrance in the capsules, derived from Figure 9 top and from the initial fragrance composition.

Figure 11 :

 11 Figure 11: Evolution with time of the fragrance encapsulation rate (with respect to initial amount of fragrance). Adapted from [23].

Figure 13 :

 13 Figure 13: Proportion of the initial fragrance still encapsulated after 150 days at 20°C and after 40 days at 40°C.

  capsules into water was necessary after polymerization as the capsules were already dispersible in water owing to the affinity of the polymer towards water.

Table 2

 2

	: a) proportion of fragrance recovered in ethanol after polymerization and b)
	encapsulation efficiency as defined by (1).
	a)		Cross-linker
		MbA	TEGDA
	MAM	97.5 %	96.3 %
	monomer
	OEGMA	96.5 %	99.7 %
	b)		Cross-linker
		MbA	TEGDA
	MAM	84.7 %	70.8 %
	monomer
	OEGMA	80.6 %	80.9 %

Acknowledgment

The authors would like to thank Olivier Anthony for fruitfull discussions, Eric Laurichesse for his technical support, Maud Bassereau for her support in interpreting some GC chromatograms and Takasago Europe Perfumery Laboratory for assembling the fragrances. The ANRT, Takasago Europe Perfumery Laboratory, CNRS, Université de Bordeaux and INP are acknowledged for financial support (contract number 2014/1309).

Conflict of Interest

The authors declare no conflict of interest that could influence or impart bias on the work