Margot Stasse

Tiphaine Ribaut

Véronique Schmitt

Valérie Héroguez

Encapsulation of lipophilic fragrance by polymerization of the intermediate aqueous phase of an oil-in-water-in-oil (O/W/O) double emulsion

Introduction

Encapsulation of lipophilic components exhibits several advantages. First of all, it allows the protection of the encapsulated species from the oxidation or deterioration, it also permits to spatially and kinetically control the release of the encapsulated compounds i.e. over a given time and/or on a specific area 1,2 . Encapsulation appeared in 1957 for carbonless paper [START_REF] Green | Oil-containing microscopic capsules and method of making them[END_REF] . Nowadays, encapsulation is present everywhere as in pharmaceutical [START_REF] Reis | [END_REF] , agro-food 5 , phyto-sanitary 6 industries or cosmetics and fragrance applications 7,8 . Two main categories of capsules are described in literature 2 : the core-shell and the matrix capsules. In the latter, the active ingredients are integrated within the matrix capsules. Various synthesizing pathways are described to elaborate all kinds of capsules: Layerby-Layer 9,10 , solvent evaporation 11 , interfacial polycondensation 12,13 or suspension polymerization [START_REF] Jahns | Microcapsule preparations and detergents and cleaning agents containing microcapsules[END_REF][START_REF] Berg | [END_REF] . However, for most of these techniques, monomer is located in the same phase as the encapsulated species. In the specific case of fragrance, the encapsulated species is a complex mixture of different molecules such as ketones, alcohols, aldehydes, amines, nitriles, aromatics, alkenes or conjugated alkenes. These compounds possess reactive functions enable to react during the polymerization step necessary to elaborate the capsules. Due to their reactive groups, the fragrance molecules can be altered, therefore modifying be the olfactory notes. To avoid these issues, we propose a strategy, which allows the compartmentalization of the polymerization and the fragrance storage. First, a double emulsion of fragrance-in-water-in-oil is formulated and then the intermediate aqueous phase is polymerized to create a polymeric barrier between the fragrance and the external oil phase. Likewise, we aim at obtaining capsules able to release the fragrance by diffusion through the polymeric barrier with a tunable kinetics of release that can be controlled by changing the monomers. In a previous study [START_REF] Stasse | Figure 10: Observation by optical microscopy of a) OEGMA/B b) OEGMA/A and c) MAM/B after polymerization and dispersion in water[END_REF] , we determined the best conditions to obtain a double emulsion of fragrance-in-water-in cosmetic oil without monomer in the aqueous phase. Several compositions, leading to stable enough double emulsions, have been identified. Herein, we focus on the formulation and polymerization of these double emulsions containing monomer inside the water phase. Double emulsions are complex systems containing two antagonistic stabilizers (a more hydrophilic surfactant (HS) to stabilize the oil-in-water emulsion and a more lipophilic one (LS) to stabilize the reverse water-in-oil emulsion). Consequently, each additional compound may interact with the established formula. Therefore, in the present paper we chose a progressive approach. In a first step, the reverse emulsion containing monomers inside the water phase will be studied in terms of feasibility and stability. This will allow selecting the most interesting monomers for the study in terms of stability and emulsions' size. In a second step, the obtained reverse emulsion will be polymerized. In the third step, double emulsions will be formulated with the previously selected monomers. In the last step, the double emulsion will be The relative solubility of the fragrance in water and in octanol, LogP, is defined as:

LogP = Log ( C octanol C water ) (1)
where P is the partition coefficient between water and octanol, Coctanol and Cwater are the concentration of the considered species in octanol and water respectively. A lipophilic species, more soluble in octanol (resp. hydrophilic species, more soluble in water) than in water (resp. than in octanol) leads to a high and positive (resp low and negative) value of LogP. If LogP is equal to zero, then the molecule exhibits the same affinity for both solvents. In the present case, as the fragrance is a mixture of molecules, the molar weighted average value of LogP is calculated 17 . For both fragrances, the values are positive, meaning a higher solubility in octanol than in water. Moreover, the LogP values of the two fragrances are very close to each other.

2-Emulsion size characterization

The emulsions drop size distributions were measured using static light scattering by means of a Malvern Mastersizer MS2000 granulometer. For simple emulsions, the collected scattered intensity as a function of the angle was transformed into the size distribution using the Mie theory. The emulsions were diluted prior to measurements to avoid multiple scattering effects. We checked by optical microscopy that dilution required for static light scattering did not alter the droplet size. The refractive indices were measured using an Abbé refractometer, they were equal to 1.333 for water or brine (no significant difference has been detected for 0.1M NaCl solution compared to water), 1.434 for IPM and to 1.454 and 1.475 for fragrances A and B respectively.

For the multiple emulsions, since oily globules contained water droplets, they were not optically homogeneous anymore. Therefore, Mie theory could not be used anymore. We chose the simplified Fraunhofer theory where drops are approximated by opaque discs and the principles of geometrical optics apply 18 .

For all types of emulsions, the granulometer gave the volume average diameter d and the polydispersity index P, defined by equation ( 2): d=

∑ 𝑁 𝑖 𝐷 𝑖 4 𝑖 ∑ 𝑁 𝑖 𝐷 𝑖 3 𝑖 P= 1 𝐷 𝑚 ∑ 𝑁 𝑖 𝐷 𝑖 3 |𝐷 𝑚 -𝐷 𝑖 | 𝑖 ∑ 𝑁 𝑖 𝐷 𝑖 3 𝑖 (2)
Where Di, Ni and Dm are the diameter of the drops, the number of the drops and the median diameter respectively.

3-Preparation of the reverse emulsion

In the following, all the inverted emulsions (with or without monomers in the aqueous phase), 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 PGPR with respect to the oil phase (for 100 g of reverse emulsion the composition was therefore as following: 75 g of solution containing 74.56 g of H2O in presence or not of monomer and 0.44 g of NaCl and 25 g of the oil phase composed of 22.5 g of IPM and 2.5 g of PGPR). This crude premixed emulsion was then sheared by a Couette cell composed of two concentric cylinders (commercialized by Ademtech). The inner cylinder of radius Ri=20 mm rotated with a speed of 150 rpm and was spaced out (gap) from the outer static cylinder by eCouette=200 µm. Therefore, the applied shear rate was equal to 1560 s -1 . The emulsion was injected in the device by a syringe pump. A Rayleigh instability developed leading to quasimonodisperse emulsions 19,20 .

The resulting reverse emulsion drop size distribution has been measured using a preparation cell dedicated to oil continuous system. It is characterized by a mean diameter of 15 µm (P = 47%) as can be seen in Figure 1.

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4-Preparation of the double oil-in-water-in-oil emulsion

Multiple oil-in-water-in-oil emulsions were prepared following a classical two-step procedure 21 as more precisely described earlier [START_REF] Stasse | Figure 10: Observation by optical microscopy of a) OEGMA/B b) OEGMA/A and c) MAM/B after polymerization and dispersion in water[END_REF] .

(a) Direct emulsion. First a polydisperse emulsion is obtained by manually incorporating the chosen A or B fragrance (90 wt%) into an aqueous phase composed of salted water (0.1 M of NaCl) and Tergitol 15-S-12 at 30 wt% with respect to the water phase. In other words, for 100 g of a direct emulsion, the composition was the following: 90 g of fragrance dispersed in an aqueous phase composed of 0.04 g of NaCl, 3 g of Tergitol 15-S-12 and 6.96 g of H2O. The premixed emulsion was then sheared at 14 280 s -1 thanks to the same Couette cell operating at 680 rpm with a 100 µm gap. The resulting direct emulsion is characterized by a mean diameter of 0.68 µm with a narrow size distribution (Pd=27%) as it can be seen Figure 2. (b) Double emulsion. In a second step, the just described direct emulsion composed of either fragrance A or fragrance B was diluted by a factor ranging from 1.5 to 18 (leading to a fragrance fraction comprised between 5 and 60 wt%), depending on the targeted fraction of inner droplets, with an aqueous phase containing salted water (0.1 M NaCl) hydrophilic surfactant, initiator and monomer. The direct emulsion whose fragrance volume fraction (d) has been set by the dilution served as the dispersed phase of the reverse emulsion. It was progressively and manually incorporated into an oil phase containing IPM and 10 wt% of PGPR. The advantage of this two-step procedure that allows dilution of an initially highly concentrated direct emulsion (90 wt%) is the possibility to tune both the mass fraction of inner droplets with respect to water drops (d) and the hydrophilic surfactant concentration in the aqueous phase %HS.

5-Interfacial tension measurement

The interfacial tensions were measured with the weighted drop method. It consisted in shaping a drop of the heaviest phase at the end of a straight needle, which was immersed in the lightest phase in a beaker. The volume of the drop was slowly increased. When the weight of the drop became larger than the capillary force which retained the drop at the end of the needle, the drop detached and felt in the beaker placed on a precision balance. The drop's precise mass was measured. The operation was repeated at least 10 times. Just before detachment, the capillary force and the drop weight were equal so that the following relation applied:

2πrfγ = ∆ρ m ρ g (3)
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 detaches (the expression of f can be found in supporting information Equations S1), m is the mass of the drop,  the density of the heaviest liquid,  the density difference between the two liquids and  the interfacial tension between them.

6-Protocol of polymerization

Polymerization of inverted and double emulsions were performed as described hereafter. KPS was used as an initiator. The total amount of salt (KPS and NaCl) was fixed to 0.1M with respect to the aqueous phase.

(a) Polymerization of the inverted emulsion. All the emulsions were polymerized in the same way. The inverted emulsion was prepared as described previously with an aqueous phase containing both the monomer and the initiator.

The polymerization was proceeded in a flask with a magnetic stirrer in olive shape at a speed of 100 rpm under nitrogen. The polymerization was initiated by increasing the temperature at 70°C. The temperature and agitation were maintained during 24 h and the polymerization was stopped with the exposure to air. The polymer was washed with pentane (in twice its volume, a good solvent of all the reactants to eliminate) then isolated by successive centrifugation steps during 20 minutes at 10000 g (g being the gravity constant equal to 9.81 m.s -2 ). Then the polymer was dried during 12 h at 50°C under vacuum. The conversion of the monomer was obtained by gravimetric measurement thanks to the following relation (

Synthesis of POEGMA

The reverse emulsion was prepared with this monomer with a concentration of 25 wt% of monomer in the aqueous phase. In other words, for 100 g of sample, the water phase was composed of 18.75 g of OEGMA, 0.04 g of KPS, 0.42 g of NaCl and 55.79 g of H2O. This water phase represented 75 wt% of the total sample (75 g). The 25 g of oil phase was composed of 22.5 g of IPM and 2.5 g (10 wt%) of PGPR.

Synthesis of PMAM

The reverse emulsion was prepared with this monomer with a concentration of 15 wt% of monomer in the aqueous phase.

In other words, for 100 g of sample, the aqueous phase was composed of 11.25 g of MAM, 0.1 g of KPS, 0.37 g of NaCl and 63.26 g of H2O. This aqueous phase represented 75 wt% of the total sample (75 g). The 25 g of oil phase was composed of 22.5 g of IPM and 2.5 g (10 wt%) of PGPR.

Synthesis of PHEMA

The reverse emulsion was prepared with this monomer with a concentration of 25 wt% and 50 wt% of monomer in the aqueous phase.

In other words, for 25 wt% of monomer, for 100 g of sample, the aqueous phase was composed of 18.75 g of HEMA, 0.09 g of KPS, 0.40 g of NaCl and 55.76 g of H2O. This aqueous phase represented 75 wt% of the total sample (75 g). The 25 g of oil phase was composed of 22.5 g of IPM and 2.5g (10 wt%) of PGPR.

For 50 wt% of monomer, if we considered 100 g of sample, the water phase was composed of 37.5 g of HEMA, 0.18 g of KPS, 0.36 g of NaCl and 36.96 g of H2O. This water phase represented 75 wt% of the total sample (75 g). The 25 g of oil phase was composed of 22.5 g of IPM and 2.5g (10 wt%) of PGPR.

(b) Polymerization of the oil-in-water-in-oil emulsion. The composition is the same as previously described apart that a portion of the water phase was replaced by fragrance. In the following the double emulsion was composed of d=20 wt% of fragrance in the water globules, themselves corresponding to 75 wt% of the total sample. The composition of surfactants was kept constant: %HS=7.5 wt% with respect to the aqueous phase and %LS=10 wt% with respect to the outer IPM (%HS, %LS, mass % of hydrophilic and lipophilic surfactants respectively). All the multiple emulsions were polymerized with the same protocol than the one described for inverted emulsion. The only difference was the purification. The washing was proceeded with ethanol instead of pentane because the residual reactants and fragrance to eliminate are very soluble in ethanol. After this step, the solid obtained was lyophilized. The conversion was also obtained by gravimetric measurement thanks to the following relation (5):

Conversion = m obtainedpolymer * % solidTGA % monomer * m collect (5)
mobtainedpolymer is the weight of polymer obtained after purification and drying subtracting the salt. mcollect represents the quantity of polymerized medium weighted before the purification. %monomer represents the initial weight percentage of monomer present initially in the sample and %solidTGA represents the quantity of polymer in the sample after the elimination of remaining water despite lyophilization. It was obtained by TGA.

Synthesis of PMAM

The monomer was added to the aqueous phase at a concentration of 15 wt% with respect to the globules (fragrance add aqueous phase) corresponding to 11.25 g of MAM and 0.1 g of KPS for a total sample of 100 g. The oil phase was unchanged.

Synthesis of POEGMA

The double emulsion was formulated with 25 wt% of monomer in the globules. In other words, if we considered 100 g of sample, it contained 18.75 g of OEGMA, 0.04 g of KPS. Again the oil phase was unchanged.

(c) Polymer characterization

The polymers were analyzed by Differential Scanning Calorimetry (DSC) with a DSC Q100 RCS apparatus. The protocol was the following: first cycle: heating from -80°C to 120°C with a temperature ramp of 10°C/min followed by cooling down to -80°C with a temperature ramp of 10°C/min. The second cycle started at -80°C up to 230°C with a ramp of 10°C/min followed by a cooling down to -50°C with the same temperature ramp.

The polymer was also characterized by TGA (Thermogravimetric Analysis) with an apparatus TGA Q500 commercialized by TA instruments. The sample was heated up to 600°C with a ramp of 10°C.mn -1 under air.

Results and Discussion

The double emulsion was prepared with a 2 step process as described previously [START_REF] Stasse | Figure 10: Observation by optical microscopy of a) OEGMA/B b) OEGMA/A and c) MAM/B after polymerization and dispersion in water[END_REF]20,21 in section 4b (Scheme 1). First of all, the direct emulsion was produced by emulsifying the inner lipophilic fragrance in a salted water phase in presence of the hydrophilic surfactant (Tergitol 15-S-12). After dilution of this direct emulsion, the second step consisted in dispersing it in the second oil phase (IPM) containing the lipophilic surfactant (PGPR). The obtained multiple emulsions were of the oil-in-water-in-oil type. Tergitol 15-S-12 and PGPR have been respectively selected in agreement with a previous study [START_REF] Stasse | Figure 10: Observation by optical microscopy of a) OEGMA/B b) OEGMA/A and c) MAM/B after polymerization and dispersion in water[END_REF] for their ability to produce a direct emulsion with a mean droplet size close to 1 µm and a reverse emulsion with a mean globule size of 15 µm respectively (see Figure 1 and Figure 2). IPM was chosen as the oil continuous phase because it is commonly used in cosmetics and perfumery. The sodium chloride was incorporated in the water phase to increase the reverse emulsion stability.

ARTICLE

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2) Double emulsion 1) Direct emulsion

A/B W + M +HS

Mixed

IPM + LS

Mixed Shear

To determine whether monomers may destabilize the double emulsion, we first prepared reverse emulsions in presence of monomer and initiator and studied the polymerization of this simple emulsion alone. Then, the formulation of the double emulsion with the monomers was tested to determine the best appropriate composition.

Scheme 1: Fabrication of double emulsion

Finally, the polymerization of the chosen double emulsion was performed for each monomer.

Preparation and polymerization of simple reverse emulsions

Three monomers chosen for their hydrophilicity and biocompatibility have been evaluated: HEMA, OEGMA, MAM.

The study of the reverse emulsions was divided in two parts. First of all, the formulation of the reverse emulsions with the different monomers was proceeded in order to identify the most interesting ones in terms of stability and emulsions drop size. The second part is the polymerization with the selected monomers.

(a) Formulation of the reverse emulsions. The different reverse emulsions were prepared by adding the monomer in the aqueous phase at different concentrations. The first studied monomer was HEMA. The preparation of the emulsion was performed for two concentrations (25 wt% and 50 wt% of HEMA). It turned out that the introduction of the monomer inside the aqueous phase induced a large decrease of the droplet size as it can be observed in Figures 3 and S1, 4.9 µm and 7.1 µm for 25 wt% and 50 wt% of HEMA in the water phase respectively to be compared with 15 µm for the reverse emulsion without HEMA).

Such a size decrease indicates that HEMA likely exhibits interfacial properties. To evidence it, interfacial measurements were performed. The interfacial tension between water and IPM was measured and compared to the one obtained between a water phase containing 25 wt% of HEMA and IPM. The addition of the monomer in the water phase induced a large decrease of the interfacial tension (38.1 mN.m -1 without monomer to be compared with 5.8 mN.m -1 with monomer) evidencing the interfacial affinity of HEMA and therefore its migration to the interface in agreement with the emulsion drop diameter decrease. As we targeted drops around 15 µm to be able to encapsulate the direct emulsion, work with this HEMA monomer was not continued.

As far as OEGMA and MAM are concerned, the preparation of the reverse emulsion at 25 wt% and 15 wt% (due to the relative solubility of MAM in water of 202 g.L -1 ) respectively, in the aqueous phase gave emulsions with appropriate droplet diameters as shown in Figure 4a (diameters of 8.2 µm and 9.2 µm for OEGMA and MAM respectively).

Moreover, as can be seen from Figure 4b, the emulsions were stable with no size evolution for more than 20 hours.

OEGMA and MAM were hence two appropriate candidates to study the reverse emulsion polymerization.

(b) Polymerization of the reverse emulsions. Free radical polymerization of the reverse emulsion prepared with MAM and with OEGMA will be considered in this part. These polymerizations were initiated by KPS. They were performed in flask with magnetic stirring for 24h and were quenched by exposure to air.

Synthesis of POEGMA

After the polymerization, the obtained suspension exhibited a particle diameter equal to 7.8 µm close to the initial emulsion drop size (8.2 µm). This means that no aggregation or As it can be seen on Figure 5, 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. Compared with those of the OEGMA macromonomer, a significant increase of glass temperature (Tg=-61°C) was observed (Supporting data, Figure S2). This behavior can be explained by the decrease of the mobility of the OEGMA grafted chains by comparison with free OEGMA chains attesting that the polymerization occurred.

The OEGMA conversion was close to 100 % (91%).

Synthesis of PMAM

After polymerization, the obtained suspension is analyzed by optical microscopy and size-distribution analysis. The presence of two main populations is evidenced (Figure 6). The population with a diameter of about 40 µm corresponds to aggregates of several polymerized drops that are also visible by optical microscopy. However, these aggregates are no coagulum since the particles can move with respect to each other when a shear is applied between the two glass slides. The population, with a diameter of about 10 µm corresponds to isolated polymerized drops (same diameter as drops before the polymerization). DSC analysis was proceeded. Three heating/cooling cycles were performed (see Figure 7). During the first cycle, a transition at 50-150°C was visible but this one disappeared during the second and third cycles. This endothermic change is probably caused by the evaporation of residual water. The endothermic change at a temperature above 150°C could be attributed to the thermal reaction of amide groups to convert to imide groups 22 . It can also be attributed to physical crosslinking due to hydrogen bonding formation 23 . In addition, the physical crosslinking of PMAM chains was validated by the non-solubility of the polymer except in strong acids known to be able to disrupt hydrogen bonds.

The MAM conversion obtained by gravimetric measurement was 64%.

Study of the double emulsion formulated with the monomer

The formulation of the double emulsion has been done in agreement with results published previously [START_REF] Stasse | Figure 10: Observation by optical microscopy of a) OEGMA/B b) OEGMA/A and c) MAM/B after polymerization and dispersion in water[END_REF] ; d wt% of droplets inside the water phase and %HS of hydrophilic surfactant. In other words, a part of the intermediate aqueous phase has been replaced by droplets fragrance and HS.

First, the effect of the monomer addition in the water phase, on the size and stability of double emulsion, has been investigated in the particular case of the fragrance B.

Then, the system with the best repartition of droplets inside the globules was fixed and polymerized with the two monomers and fragrances A and B.

(a) Formulation of the double emulsions.

Formulations with OEGMA

Six different compositions, for which only multiple drops could be obtained [START_REF] Stasse | Figure 10: Observation by optical microscopy of a) OEGMA/B b) OEGMA/A and c) MAM/B after polymerization and dispersion in water[END_REF] , have been selected. OEGMA (25 wt% and 50 wt%) was added in the water phase of these formulations (Table 2). Only three compositions allowed obtaining double emulsion (Figure 8). Moreover, even if we succeeded in formulating emulsions with 25 wt% of monomer, we failed with 50 wt%. This is probably because this monomer exhibits interfacial properties (8.4 mN.m -1 for interfacial tension of OEGMA in water and IPM) showing that it migrates to the interface as HEMA did but to a lower extent (5.8 mN.m -1 for the interfacial tension of HEMA in water against IPM). The lowered migration of OEGMA to the interface compared to HEMA could be explained by larger size of OEGMA. Interfacial properties of OEGMA have also been observed by Kasantsev 24 .

To conclude, three different compositions were possible with the OEGMA. Among these compositions, we have chosen the one with 20 wt% of droplets inside the water phase and 7.5 wt% of Tergitol 15-S-12 (Figure 8a).

Formulation with MAM

We chose to keep the same emulsion composition as for OEGMA, e. g.20 wt% of droplets inside the water phase and 7.5 wt% of Tergitol 15-S-12 and to incorporate MAM (Figure 9).

As observed in Figure 9, the obtained emulsion was only composed of globules homogeneously filled with droplets (only a multiple emulsion). To conclude this part, addition of monomers in the double emulsion narrows the composition domains where double emulsions can be prepared.

(b) Polymerization of the chosen systems. To limit the number of polymerizations and to focus on the effect of the monomer, we selected one emulsion composition for the rest of the study: d=20 wt%, %HS=7.5 wt%, total salt concentration (NaCl and KPS) equal to 0.1M, G=75% and CPGPR=10 wt% with respect to external oil.

Three different systems were polymerized with the composition and the protocol described in the experimental part:

1) Emulsion with 25 wt% of OEGMA and the fragrance B (OEGMA/B)

2) Emulsion with 25 wt% of OEGMA and the fragrance A (OEGMA/A)

3) Emulsion with 15 wt% of MAM and the fragrance B (MAM/B) For the three systems, a gel was obtained because of the high concentration of globules (75 wt%). In order to characterize these globules, we attempted to disperse them in water (good solvent of the polymer) and in IPM (continuous phase of the initial double emulsion). Surprisingly, we noticed that none of the polymerized double emulsions was dispersible in IPM. IPM is thus not a good solvent of the resulting polymer and caused a polymer collapse as it can be observed in the Supporting Data Figure S3.

On the other hand, the three systems can be dispersed in water, the polymerized multiple drops can be then observed by optical microscopy. It is explained by the fact that water is a good solvent of POEGMA and PMAM, it likely swells the polymer particles (not enough to be observable) and separates them, which allows the observation of the better dispersed compartmentalized particles (Figure 10).

The DSC thermograms (Supporting data Figure S4) were very similar to those obtained for the polymerized reverse emulsions and confirmed the polymerization step.

The monomer conversions were: 35%, 6% and 18% for OEGMA/B, OEGMA/A and MAM/B respectively. Even if these values are relatively low, they show that polymerization of the intermediate phase of double emulsion was possible. One should keep in mind that none of these three polymerizations has been optimized. It is interesting to note the large difference between OEGMA/B and OEGMA/A in term of conversion whereas the only variance was the used fragrance. This is likely due to the difference of interfacial behavior between the two fragrances. Indeed, the interfacial tension between water and fragrance A was significantly smaller than with fragrance B (Table 1). The energy required to form an interface between the fragrance A and water is lower than the one required to form an interface between fragrance B and water even if the Log P (estimated with octanol) of both fragrances are close. In other words, the molecules of fragrance A are more likely to migrate towards the interface than the molecules of fragrance B. Please do not adjust margins

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This migration can provoke interaction between the fragrance molecules and the polymerization and can reduce the conversion of the polymerization.

Conclusions

The formulation of reverse emulsion with hydrophilic monomers allowed evidencing that not only the monomer should be hydrophilic but also it should not have interfacial properties to make it possible to prepare a double emulsion with a difference in size of droplet and globule large enough to allow the encapsulation of the droplets.

For example, we have shown that the presence of HEMA reduces too significantly the size of the globules because of its ability to migrate to the interface. This size reduction prevents the encapsulation of fragrance droplets. Two different monomers that do not alter the complex double emulsion structure were selected: OEGMA and MAM. When these two monomers are present in the dispersed phase of a reverse W/O emulsion, they can be polymerized with a conversion higher than 60%. The monomer incorporation leads to narrower accessible composition domains for the double emulsions. However, for a unique double emulsion composition: d=20 wt%, %HS=7.5 wt%, NaCl and KPS = 0.1M in the globules and G=75% and CPGPR=10 wt% with respect to external oil, compartmentalized capsules can be obtained for both fragrances with identical LogP values and despite their different interfacial behaviors. Indeed, fragrance A is composed of molecules that are more likely to migrate to the interface possibly hampering the polymerization. Consequently, the conversion is much lower for fragrance A than for fragrance B (for OEGMA: 6% of conversion for fragrance A versus 35% for fragrance B). For fragrance B, a lower conversion was obtained for MAM (18%) than OEGMA (35%). However, MAM remains an interesting monomer for future applications because of its physical crosslinking ability.

Figure 1 :

 1 Figure 1: Drop size distribution of the reverse emulsion.

Figure 2 :

 2 Figure 2: (a) Optical microscopy of direct emulsion and (b) corresponding droplet size distribution.

  weight of polymer obtained after purification and drying subtracting the salt. mcollect represents the quantity of polymerized medium weighted before the This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins purification and %monomer represents the weight percentage of monomer initially present in the sample.

Figure 3 :Please

 3 Figure 3: Observation by optical microscopy of the reverse emulsion prepared with (a) 25 wt% (b) 50 wt% and (c) 0 wt% of HEMA in the water phase.

Figure 5 :

 5 Figure 5: DSC thermogram of the polymerized reverse emulsion prepared with 25% of OEGMA

Figure 6 :

 6 Figure 6: Left: optical microscopy of the polymerized emulsion containing 15w% of MAM. The black circles enclose the aggregates. Right: particle size distribution before and after polymerization.

Figure 4 :

 4 Figure 4: (a) Observation by optical microscopy of reverse emulsion prepared with (a1) 25 wt% of OEGMA (a2) 15 wt% of MAM. (b) emulsion drop diameter vs time for the two previous emulsions. The error bars represent the diameter polydispersity of the drops in the emulsion.

Figure 7 :

 7 Figure 7: Thermogram obtained by DSC of the polymer resulting of the polymerization of the reverse emulsion formulated with 15 w% of MAM in the aqueous phase.

Figure 9 :

 9 Figure 9: Observation by optical microscopy of the double emulsion at d = 20 wt% and %HS = 7.5 wt% with 15 wt% of MAM inside the water phase.

Figure 8 :

 8 Figure 8: Observation by optical microscopy of the different formulations which allowed obtaining double emulsions containing 25 wt% of OEGMA a) d = 20 wt% and %HS = 7.5 wt% b) d = 20 wt% and %HS = 10 wt% c) d = 30 wt% and %HS = 7.5 wt%.

Table 1 :

 1 Physical properties of each fragrance

	Properties	LogP	Refractive	Density	
			Index		(mN/m)
	Fragrance A	3.38	1.454	0.869	21.8
	Fragrance B	3.52	1.475	0.887	34.6
	 Interfacial tension against H2O

Table 2 :

 2 Summary table of the different double emulsion formulations with the addition of OEGMA No double emulsion could be obtained Double emulsion, Not proceeded (NP)

	O/W/O composition	45/20	30/20	30/15	30/7.5	20/10	20/7.5
	d /%HS (wt%/ wt%)
	25 w% of OEGMA
	50 w% of OEGMA	NP	NP	NP		NP

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This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

This journal is © The Royal Society of Chemistry 20xx

Acknowledgements

The authors would like to thank T.E.P.L and ANRT (contract number N° 2014/1309) for financial support with contract number: 2014/1309.

Conflicts of interest

There is no conflict of interest to declare for any of the authors.

Supporting information

Table S1 and S2:

Equations S1: Formula of corrective factors.


Priscillia Laniesse

Céline Cau

Dit Coumes

Gwenn Le Saout

Adel Mesbah

Influence of the boron and aluminum concentrations in the mixing solution

Keywords:

come

Understanding the setting and hardening process of wollastonite-based brushite cement. Part 2: Influence of the boron and aluminum concentrations in the mixing solution

Introduction

Portland cement, possibly blended with fly ash and/or blastfurnace slag, is widely used for nuclear waste encapsulation for several reasons [START_REF] Atkins | Application of Portland cement-based materials to radioactive waste immobilization[END_REF][START_REF]Improved cement solidification of low-and intermediate-level radioactive waste[END_REF][START_REF] Glasser | Fundamental aspects of cement solidification and stabilisation[END_REF][START_REF] Ojovan | An Introduction to Nuclear Waste Immobilization[END_REF]. This binder, which has been investigated and used for more than 200 years, is inexpensive and easily available. Properly designed materials usually exhibit good mechanical strength. They are resistant to irradiation at doses of a few MGy representative of those integrated by cemented waste packages over their lifetime. In addition, cement wasteforms are prepared at ambient temperature, using rather simple and robust processes which do not require any special gas treatment. Finally, cement matrices are rather flexible since they can accommodate contaminated effluents, sludges, powders or massive solids. In the first two cases, water brought by the waste can be simply used for cement hydration. As a consequence, cementation is worldwide the reference process for the conditioning of low-level or intermediate-level radioactive waste.

However, Portland cement paste is a very alkaline medium, with a pore solution pH close to 13 [START_REF] Lothenbach | Effect of temperature on the pore solution, microstructure and hydration products of Portland cement pastes[END_REF], which is an issue for the direct stabilization of acidic wastes. Indeed, high temperature rise and flash setting of the grout can result from the fast and exothermic acid-base reaction occurring between protons from the waste and hydroxide ions released by the dissolution of cement phases. Neutralization or basification of such waste before its cementation is thus mandatory [START_REF] Coppens | On the immobilization of NaNO 3 -rich ILW-LL radioactive effluent[END_REF]. This additional step raises several issues such as (i) volume increase resulting from the addition of the base reagent, (ii) gelification of the waste due to precipitation of metallic cations (Al 3+ , Fe 3+ , Zn 2+ …) as hydroxides, which makes its transfer to the cementation unit difficult and can lead to poorly workable grout [START_REF] Collier | Immobilisation of Fe Floc: part 2, encapsulation of floc in composite cement[END_REF], and (iii) enhanced risk of alkali-silica reaction in the long term if the pre-treatment solution is a concentrated solution of sodium or potassium hydroxide and if the aggregates used in the concrete formulation are not carefully selected [START_REF] Fournier | Alkali-aggregate reaction in concrete: a review of basic concepts and engineering implications[END_REF][START_REF] Courtois | Rheological and swelling properties of alkali-silica gels produced in cemented waste packages[END_REF].

To overcome these issues and suppress the pre-treatment step, an alternative strategy may be to use a binder showing better chemical compatibility with acidic wastes than Portland cement [START_REF] Cau Dit Coumes | Alternative binders to ordinary Portland cement for radwaste solidification and stabilization[END_REF]. In this way, wollastonite-based brushite cement is a potential candidate as its setting and hardening process occurs in acidic medium (between 0 and 6) [START_REF] Semler | Lime Silico Phosphate Ceramics[END_REF][START_REF] Semler | A quick-setting wollastonite phosphate cement[END_REF][START_REF] Mosselmans | Thermal hardening and structure of a phosphorus containing cementitious model material[END_REF]. This binder is prepared by mixing wollastonite, a natural metasilicate (CaSiO 3 ), with a solution containing phosphoric acid, borax and metallic cations [START_REF] Wu | Inorganic Resins Composition, Their Preparation and Use Thereof[END_REF][START_REF] Alshaaer | Evaluation of a low temperature hardening inorganic phosphate cement for high-temperature applications[END_REF]. In a previous paper [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF], the authors have showed that the hydration of wollastonite with a commercial mixing solution comprising broax, zinc and aluminum cations is a multi-step process which involves the formation of a transient crystalline phase (Monocalcium phosphate monohydrate Ca(H 2 PO 4 ) 2 ⋅H 2 O (MCPM)), and produces brushite (CaHPO 4 ⋅2H 2 O) as well as an amorphous aluminophosphate phase containing zinc and calcium. However, data about the influence of the mixing solution composition on the properties of the resulting material are scarce in the literature. A previous work [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF] was focussed on the impact of the phosphoric acid concentration, Ca/P molar ratio and liquid-to-solid (l/s) ratio. Increasing the Ca/P ratio (at constant H 3 PO 4 concentration) and decreasing the l/s ratio were shown to accelerate the early stages of hydration but limited its progress at 7 d. Moreover, basic calcium orthophosphates formed in addition to brushite. At constant l/s ratio, increasing the H 3 PO 4 concentration between 7 and 10 mol⋅L -1 delayed cement hydration and inhibited the setting at concentrations above 12 mol⋅L -1 . A good compromise was obtained for H 3 PO 4 concentrations within the range 9-10 mol⋅L -1 : setting occurred between 10 and 48 h and the amount of precipitated brushite was at its maximum. However, the sole presence of phosphoric acid in the mixing solution produced materials with poor mechanical strength. Since commercial binders prepared with mixing solutions comprising metallic cations and borax usually have compressive strengths of several tens of MPa [START_REF] Semler | A quick-setting wollastonite phosphate cement[END_REF][START_REF] Mosselmans | Thermal hardening and structure of a phosphorus containing cementitious model material[END_REF][START_REF] Alshaaer | Evaluation of a low temperature hardening inorganic phosphate cement for high-temperature applications[END_REF], these additions must play a key role in the hardening process of the material. This work investigates their influence into more details.

Experimental

Materials

Wollastonite was provided by the French company Sulitec. The powder comprised wollastonite and small contents of calcite and quartz that were evidenced by XRD and TGA (diagrams are given in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]). The CaCO 3 content was calculated from the weight loss recorded at 654 • C by TGA to be 1.6 ± 0.1 wt%. The particle size distribution of the powder (laser granulometry) was comprised between 0.5 μm and 135 μm (d 10 = 2.8 μm, d 50 = 15.2 μm, d 90 = 48.3 μm). The wollastonite particles had a needle shape with a mean aspect ratio (ratio of the length-to-diameter) close to 4:1 (determined manually using SEM images of c.a. 100 particles).

The mixing solution was a 9 mol⋅L -1 phosphoric acid solution, prepared from 85%wt. H 3 PO 4 (14.6 mol⋅L -1 ) analytical grade commercial solution provided by VWR.

First, boron and aluminum species were introduced to the phosphoric acid solution separately from each other to investigate their influence on the cement hydration process. Boron was added either as borax (Na 2 B 4 O 7 ⋅10H 2 O) or boric acid (B(OH) 3 ). These two borated species were investigated for the following reasons:

-borax was a cheaper additive, -boric acid had less influence on the initial acidity of the solution and did not bring any additional cations (Na + ) to the system.

Aluminum was added as metal grade aluminum powder (purity > 99.9%) to the mixing solution. Under such acidic conditions, metallic aluminum is unstable and oxidized into Al 3+ cations according to Eq. [START_REF] Atkins | Application of Portland cement-based materials to radioactive waste immobilization[END_REF].

Al + 3H + →Al 3+ + 3 / 2H 2 (1)
Al 3+ cations could thus be produced without any addition of supplementary anions. The temperature rise due to the exothermic nature of reaction (Eq. ( 1)) and the gas evacuation led to partial evaporation of water which was compensated after complete oxidation of the Al powder by an addition of demineralized water.

Then, the joint effect of aluminum and boron was studied using design of experiments (D.O.E) (cf. Section 2.2).

The addition of each species may have an impact on the acidity of the mixing solution. As the solution was initially very acidic, its pH could not be measured directly with a pH electrode. It was then necessary to determine its Hammett acidity function H 0 [START_REF] Hammett | A series of simple indicators[END_REF] Measurement of such function value has been detailed in a previous work [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF].

Specimen preparation

Paste preparation was performed by mixing the powder and solution for 5 min using a laboratory mixer equipped with an anchor stirrer rotating at 250 rpm. Paste samples were then cast into airtight polypropylene boxes (20 mL of paste per box) and cured at 25 ± 1 • C.

At first, the influences of boron and aluminum on the hydration and mechanical properties of the cement paste were investigated separately from each other. Then, to look for possible interactions, boron and aluminum were simultaneously added to the mixing solution.

A first series of cement pastes was thus prepared by varying the concentration (from 0.05 to 0.2 mol⋅L -1 ) and the type of boron species in the mixing solution (Table 1). In a second set of experiments, the concentration of aluminum varied from 0.5 to 2.5 mol⋅L -1 . A third series of pastes was formulated with a mixing solution containing various amount of aluminum and borax as described in Section 2.2. In all experiments, the l/s ratio, phosphate concentration and Ca/P ratio were kept constant and respectively equal to 0.76 mL⋅g -1 , 9 mol⋅L -1 and 1.25.

Experimental design

The combined influence of B and Al on the paste properties was studied using D.O.E [START_REF] Montgomery | Design and Analysis of Experiments[END_REF] to evidence any synergistic or antagonist effects between these two species. The objective was to fit and analyse response surfaces in order to optimize the concentrations of these two additives. The factors (or variables) were the Al (introduced at Al powder) and B (introduced as borax) concentrations in the mixing solution. Their range of variation defined the experimental domain.

-Al: The maximum Al concentration (2.5 mol⋅L -1 ) was fixed by the solubility limit of Al 3+ in the H 3 PO 4 solution. Since preliminary experiments with Al 3+ concentrations of 1 mol⋅L -1 or less led to materials with poor mechanical strength (<1 MPa), the minimum Al concentration was set to 1.5 mol⋅L -1 . -B: Borax was preferred to boric acid since it had similar retarding effect and was less expensive. The B concentration varied between 0.2 mol⋅L -1 (retardation being too weak for lower concentrations) and 0.6 mol⋅L -1 (the solubility limit in the H 3 PO 4 solution).

Coded dimensionless variables varying within the range [-1; 1] were defined according to Eq. (2) (Table 2).

( x j -x 0 j Δx j ) = ( U j -U 0 j ΔU j ) (2)
with x j = value of coded variable j.

x j 0 = value of coded variable j at the center of the domain (in this work, x j 0 = 0) U j = value of natural variable j U j • = value of natural variable j at the center of the domain

ΔU j = U max j -U min j 2 Δx j = x max j -x min j 2
The P concentration, Ca/P and l/s ratios were maintained constant at 9 mol⋅L -1 , 1.25 (mol/mol) and 0.76 mL⋅g -1 in all runs. The acidity varied however since it depended on the B and Al concentrations.

Eight responses were measured in order to characterize the cement hydration rate, the mineralogical assemblage and compressive strength of pastes after 28 d of curing in sealed bag at 25 • C. More precisely, the cement hydration, investigated by isothermal calorimetry, was characterized by the maximum heat flow, the corresponding time and the maximum cumulative heat. The phase assemblage was characterized by XRD and calculated quantitatively by Rietveld refinement in order to quantify the amount of brushite, wollastonite and amorphous aluminophosphate at a 28 days. Finally, the compressive strength and the mean diameter of pores were measured in order to characterize and explain mechanical performances of the binders. More details are given in Section 2.4.

Since the objective of the study was to predict response values within the experimental domain, the postulated empirical model was a quadratic polynomial (Eq. ( 3)).

y = β 0 + β 1 x 1 + β 2 x 2 + β 11 x 1 2 + β 22 x 2 2 + β 12 x 1 ⋅x 2 + ε (3)
with: y = predicted response, x i = level of factor X i , β i = model coefficient to be estimated, and ε = error.

The positioning of experimental points within the experimental domain is of great importance to obtain a good precision on the estimates of the model parameters and on the model-predicted response values. The selected design was a face-centered central composite design (α = 1) [START_REF] Box | On the experimental attainment of optimum conditions[END_REF] comprising 9 experiments located at the corners, center and edge centroids of the experimental domain (Fig. 1). This design exhibits interesting properties such as limited number of trials and good quality of predicted response values (maximum prediction variance d max of 0.81 over the domain of interest, meaning that, if the model is validated, the variance of predicted response is ≤0.81 σ 2 , where σ 2 is the experimental variance). The experiment at the center of the domain was repeated 3 times to assess the variance of the experimental error. Table 3 summarizes the experimental design and the measured response values (see Section 2.3 for the characterization methods used).

Table 1

Composition of cement pastes comprising boron (series 1) or aluminum (series 2) or both (series 3) and responses as described in the following section (H 0 stands for the acidity function, the pastes are labelled P BAorB/Y/Z where BA stands for boric acid, B for Borax, Y and Z for the boron and aluminum concentrations respectively).

Table 2

Correspondence between natural and coded variables.

Characterization techniques

The hydration process of the cement pastes was characterized using a TAM AIR conduction microcalorimeter under isothermal conditions at 25 • C. The pastes samples were prepared outside the calorimeter. About 2 g were poured into a glass ampoule and introduced in the calorimeter (more details about the technique are given in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]).

A typical curve obtained by this technique is given in Fig. 2. Three parameters were extracted for further analysis:

-the maximum heat flow (HF max ): the higher the heat flow, the faster the dissolution of wollastonite which was the main exothermic reaction in the hydration process [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF], -the time corresponding to the maximum heat flow (t max ), which gave information on the setting time of the material (previous experiments showed that it approximately corresponded to the end of setting [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]) and on the duration of the low thermal activity period that preceded the peak, -the cumulative heat (H max ), which provided insight into the selfheating behaviour of the material during hydration and strongly depended on the progress of exothermic reactions. Note that the first 30 min were omitted for H max calculation due to extra undesirable heat resulting from the introduction of the ampoule in the calorimetric chamber.

The hydration process of paste samples was stopped by successively immersing the crushed paste into isopropanol for few minutes, filtrating the suspension and drying it in an oven at 38 • C for 24 h. Preliminary trials showed that the drying at 38 • C for 24 h had no effect on the samples mineralogy.

The phase assemblage was analysed using XRD in a Debbye Scherrer configuration (transmission mode) according to the procedure described in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. To reduce the preferred orientation effect, the samples, ground to a particle size less than 80 μm and containing 10 wt% Si used as an internal standard, were introduced in Lindeman tubes (0.7 mm by diameter) and mounted on a spinning goniometric head. Measurement was performed using the Debye Scherrer configuration (transmission mode) with copper radiation (λ = 1.5418 Å) at room temperature in the 2θ range 5-120 • (step size of 0.017 • , total counting time of 6 h). Refinement of the X-ray diagrams was then performed using the Rietveld method. The internal standard method made it possible to calculate the amount of amorphous phases as explained in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. Calculations were carried out using the Fullproof_suite software [START_REF] Frontera | FullProf as a new tool for flipping ratio analysis[END_REF]. ICDS files used for the assignment of crystalline phases are given in Table 3.

The mechanical strength of 4 × 4 × 4 cm paste specimens was characterized after 28 d of curing at 25 • C in a sealed bag (endogenous conditions). The compressive strength was measured following EN 196-1 European standard (loading rate: 2.4 kN⋅s -1 ) with the 3R RP 40/ 400FC testing machine.

Polished fractions of paste samples were observed by Scanning Electron Microscopy (FEI Inspect S50, high vacuum mode, acceleration voltage of 15 kV, current intensity of 50 nA, working distance of 9.7 mm). More details about the polishing protocol are given in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. Elemental mapping was performed using X-ray microanalysis (high vacuum mode, Bruker X-flash SDD detector (10 mm 2 )). The Ca/P, Al/P and Zn/P ratios in the hydrates were determined with statistics on 20 to 30 measurement points, after calibration of the detector using jadeite for Na, aluminum metal for Al, zinc metal for Zn, GaP for P and wollastonite for Si and Ca.

The pore structure (in the range of mesopores) was characterized using the nitrogen adsorption technique. Samples were first freezedried, and then stored for 12 h under vacuum at 75 • C to remove the water and gas impurities adsorbed on the pores surface. Preliminary characterizations by XRD showed that this drying procedure did not change the mineralogy of the samples. Gas sorption experiments were performed using Micromeritics ASAP 2020 device. Analyses were carried out at the liquid nitrogen temperature (77 K) with a relative pressure ranging from 5.10 -6 to 0.99. The pore size distribution was determined using the Barret-Joyner-Halenda (BJH) method.

Results and discussion

Influence of the type and concentration of boron-containing species (first series of experiments)

Hydration rate

At a given boron concentration, boric acid and borax had almost the same effect on the cement hydration rate (Fig. 3). The higher the boron concentration, the higher the time corresponding to the maximum heat flow, and the longer the duration of the low thermal activity, meaning that boron retarded the hydration process. Moreover, whereas the heat flow curve of the reference paste sample exhibited one main peak, three thermal events were evidenced in the presence of boron, as previously observed for commercial cement pastes having more complex mixing solution (additional presence of Zn and Al cations) [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. The maximum heat flow was reduced from 72 to 7 mW⋅g -1 when the boron concentration was increased from 0 to 0.2 mol⋅L -1 . This heat flow value was close to that of commercial products [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF].

Pastes prepared with 0 and 0.05 mol⋅L -1 of boron displayed the same cumulative heat at the end of the experiment, suggesting similar progress of hydration. For higher boron concentrations, the plateau was not reached at the end of experiment, making the cumulative heat flows difficult to compare.

Phase assemblage

The phase evolution of pastes P B/0.05/0 and P BA/0.05/0 was characterized by XRD. Regardless of the boron additive, MCPM precipitated rapidly after mixing and was destabilized to form brushite after the heat flow peak. The precipitation sequence was thus similar to that evidenced for the commercial binder [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF] (Fig. 4):

Even if the hydration process was slowed down, wollastonite was almost totally consumed at 48 h. Brushite (CaHPO 4 .2H 2 O) precipitated instead of monetite (CaHPO 4 ), the product observed for the boron-free reference [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF]. This result could be explained by the lower selfheating of the boron-containing material which tended to promote the precipitation of metastable brushite instead of thermodynamically stable monetite [START_REF] Alshaaer | Production of monetite-based Inorganic Phosphate Cement (M-IPC) using hydrothermal post curing (HTPC)[END_REF].

The main role of boron was to delay the hydration and reduce the maximum heat flow. The X-Ray diffraction results showed that boron mainly influenced the conversion of MCPM to brushite which occurred Fig. 2. Typical curves obtained (experiment P 0/2.0 ) by isothermal microcalorimetry and parameters used for their analysis. later as compared to the MCPM-monetite transition evidenced in the boron-free reference paste [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF]. The retarding effect of boron could thus result from a delay in MCPM dissolution and/or brushite precipitation.

Mechanical properties

All the paste samples containing boron (P B/0.05/0 , P BA/0.05/0 , P B/0.2/0 , P BA/0.2/0 ) had a very poor compressive strength (< 1 MPa, the lower limit of the measurement range of the testing machine) at 28 d. Moreover, their free surface exhibited a dome, indicating a strong volume instability.

Influence of the aluminum concentration (second series of experiments)

Hydration rate

Increasing the aluminum concentration in the mixing solution from 0 to 2.5 mol⋅L -1 decreased the length of the low thermal activity period, the maximal heat flow and cumulative heat (Fig. 5). However, the heat flow reduction was much less important than in the presence of boron. It must be kept in mind that increasing the Al concentration also decreased the acidity of the mixing solution (see Eq. ( 1)). In a previous work, it was shown that decreasing the acidity (or, equivalently, increasing the acidity function H 0 within the range [-2.13, -1.28]) decreased the period of low thermal activity and cumulative heat but increased the maximal heat flow [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF]. Consequently, the reduction in the maximal heat flow observed with increasing [Al 3+ ] could not be attributed to an acidity effect, but to the presence of metallic cations. As for the variation of the cumulative heat and duration of the period of low thermal activity, both parameters (acidity and [Al 3+ ]) could be involved. The evolution of the cumulative heat was plotted versus the acidity function and compared to the curve obtained for solutions containing phosphoric acid only [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF] (Fig. 6). For low aluminum concentrations, the effect of the acidity seemed to be predominant, whereas, for Al concentrations higher than 1 mol⋅L -1 , the decrease in the acidity resulting from the introduction of aluminum to the mixing solution could not explain by itself the decrease in the cumulative heat, meaning that Al 3+ cations were also involved. Paste P 0/2.5 seemed to show a distinct behaviour, with a period of low thermal activity intermediate between those of pastes P 0/0 and P 0/2. Complementary experiments would be needed to clarify this point.

Phase assemblage

The mineralogy of paste P 0/2 prepared with 2 mol⋅L -1 of aluminum was characterized by XRD at relevant hydration times: 1 h (period of low thermal activity), 5 h (maximum of the heat flow), 8 h (end of the second peak) and 48 h (Fig. 7).

The presence of MCPM was evidenced shortly after mixing. Its amount seemed to increase until the first heat flow peak. Then, this phase was destabilized and brushite precipitated. Wollastonite was not totally depleted at 48 h. Residual wollastonite (in orange) even persisted after 28 d, as shown by the elemental mapping performed on a polished section of paste P0/2.5 (Fig. 8). Silica (in green) was detected at the initial location of wollastonite grains, suggesting an incongruent dissolution of wollastonite as observed previously [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF][START_REF] Weisshart | Wollastonite: incongruent dissolution and leached layer formation[END_REF][START_REF] Green | Incongruent dissolution of wollastonite measured with vertical scanning interferometry[END_REF]. The presence of brushite was also clearly evidenced (zones in purple with intense colour). In addition, EDS mapping and EDS spectra (Fig. 9) showed the precipitation of a phase containing Al, P and Ca (in light purple). This phase, which was not detected by XRD, was likely amorphous or poorly crystalline (Fig. 8). Similarly, precipitation of an amorphous aluminophosphate phase containing calcium and zinc was reported for a commercial binder prepared with a mixing solution comprising aluminum and zinc cations [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF].

Mechanical properties

The compressive strength of 28 d-old paste samples increased from less than 1 MPa (reference paste P 0/0 -[Al 3+ ] = 0 mol⋅L -1 ) to 14 MPa (Paste P0/2 -[Al 3+ ] = 2 mol⋅L -1 ) and 52 MPa (Paste P0/2.5 -[Al 3+ ] = 2.5 mol⋅L -1 ). The aluminum concentration thus seemed to have an outstanding effect on the mechanical properties of the hardened material. Note that the strength increase with the aluminum concentration could not be explained by the decrease in acidity since all the pastes prepared with Al-free mixing solutions of variable acidity exhibited a compressive strength less than 1 MPa [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF]. In addition, visual observation showed that unlike the hardened pastes with boric acid or borax, those with aluminum did not show any expansion.

Discussion about the influence of aluminum on the rate of hydration

Increasing the Al concentration reduced the period of low thermal activity, but also the cumulative heat flow. This effect could be partly explained by the decreased acidity of mixing solutions with high Al concentrations, but the contribution of aluminum was also evidenced.

The presence of aluminum changed the mineralogical assemblage formed by hydration since an amorphous aluminophosphate phase precipitated in addition to MCPM and brushite or monetite. It was evidenced from the first characterization time, in good agreement with previous observations [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. The rapid precipitation of this amorphous phase consumed ions from the solution, which in turn could promote the dissolution of wollastonite and decrease the period of low thermal activity.

The cumulative heat decreased however in the presence of aluminum, which indicated a smaller extent of dissolution of wollastonite since this process was shown to govern the heat production [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF]. Increasing the Al concentration likely favoured the precipitation of the aluminophosphate phase (this will be shown in Section 3.3 for pastes prepared with mixing solutions containing B ad Al), which resulted in a denser microstructure, evidenced by the SEM observations and higher mechanical strength. This denser microstructure might contribute to slowing down the subsequent progress of hydration because of a more difficult access to wollastonite or water, or a lack of space for the precipitation of hydrates.

Joint influence of aluminum and boron concentrations (third series of experiments)

The joint influence of the Al and B concentration in the mixing solution on the paste properties was investigated using the experimental design shown in Table 1. For each response, the model coefficients were estimated from the results by standard least squares regression using the Nemrod software [START_REF] Draper | Applied Regression Analysis[END_REF] (Table 4). Possible model deficiencies were looked for by analysis of variance [START_REF] Mathieu | NEMROD-W Version[END_REF] (Table 5). The variance of the experimental error was first estimated using the replicated runs. Since there were degrees of freedom to calculate the residuals, model adequacy was then checked by verifying that the lack of fit of the model was statistically not significant. Finally, a test for significance of regression was carried out by assuming that all coefficients β i were zero. Rejection of this hypothesis implied that at least one of the regressor variables x i contributed significantly to the model, and that this model was thus meaningful. Validated models could be built for all the responses (Table 5), with predictive power ranging from good (H max , Amorphous, Brushite, R c ) to excellent (HF max , t max , d p ), as indicated by the R 2 pred values (Table 4). Note that, for response "%wt Amorphous", one replicate run (P B/0.4/2.0 -3), characterized by a residual almost twice that of the experimental error, was considered as erratic and discarded for the calculation of the model.

Hydration rate

The contour plots for responses HF max , t max and H max (Fig. 10) showed that both boron and aluminum affected the cement hydration rate. Indeed, the higher the boron concentration, the lower the maximum heat flow and the higher the corresponding hydration time t max . In addition, increasing the boron concentration slightly decreased the maximum cumulative heat. Therefore, boron kept its retarding effect in the presence of aluminum. As for aluminum, raising its concentration strongly decreased t max , and thus the duration of the low thermal activity period, as well as the maximum cumulative heat, and increased to a minor extent the maximum heat flow. Thus, in the presence of boron, aluminum still accelerated the first stages of hydration and limited its progress afterwards. Its influence on the maximum heat flow changed however, since this parameter was shown to decrease when the Al concentration increased in the absence of boron (cf Section 3.2.1). From a methodological point of view, this change of behaviour indicated the existence of an interaction between the two factors [Al] and [B]. Complementary investigations would be needed to explain its origin from a phenomenological point of view.

Mineralogy

The mass fractions of wollastonite, brushite and amorphous phase in 28 d-old cement pastes were estimated using Rietveld refinement.

The mass fraction of residual wollastonite did not evolve significantly as a function of the mixing solution composition. The observed variations were of the same magnitude order as the experimental error estimated from the replicates.

In contrast, the fractions of brushite and amorphous phase strongly depended on the aluminum concentration in the mixing solution (Fig. 11). The higher this concentration, the higher the content of amorphous phase and the lower that of brushite. The boron concentration had comparatively very little influence. It is interesting to note that, within all the investigated experimental domain, the fraction of brushite remained much higherand that of the amorphous phase much lowerthan those of a binder prepared with the same wollastonite powder, but with a mixing solution additionally containing zinc at a concentration of 1.3 mol⋅L -1 [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. This latter comprised indeed 21 wt% brushite and 48 wt% amorphous phase at the same age. Therefore, the presence of zinc in the mixing solution seemed to inhibit the formation of brushite and promote the formation of amorphous phase. This hypothesis is supported by previous work showing the inhibiting effect of zinc onto brushite crystallization [START_REF] Lundager Madsen | Influence of foreign metal ions on crystal growth and morphology of brushite (CaHPO 4 .2H 2 O) and its transformation to octacalcium phosphate and apatite[END_REF].

The amorphous phase present in samples P B/0.6/1.5 and P B/0.6/2.5 (mixing solution containing 0.6 mol⋅L -1 of boron and 1.5 or 2.5 mol⋅L -1 of aluminum) was studied by SEM-EDS after 28 d of curing in sealed bag. Elemental mapping showed that the amorphous phase (in light purple on Fig. 12) was more dispersed in the aluminum-rich sample. Moreover, its composition varied as a function of the initial amount of aluminum: Al concentration of 1.5 mol⋅L -1 and 2.5 mol⋅L -1 yielded Ca/P ratios of 0,53 ± 0,03 and 0,86 ± 0,08 respectively, and Al/P ratios of 0,54 ± 0,02 and 0,4 ± 0,1 respectively. The higher Ca/P ratio of the amorphous phase in sample P B/0.6/2.5 was consistent with its lower brushite content (the amount of depleted wollastonite being approximatively the same in both pastes, calcium ions precipitated in the amorphous phase preferentially to brushite). Nevertheless, it cannot be excluded that this higher Ca/P ratio also resulted from a submicronic mix of brushite with the better dispersed amorphous phase. On the contrary, the Al/P ratio was slightly lower, which could arise from the higher content of amorphous phase formed during the hydration process.

Compressive strength

The aluminum concentration in the mixing solution not only influenced the phase assemblage of the hardened cement paste, but also its mechanical strength (Fig. 13). Increasing this concentration from 1.5 to 2.5 mol⋅L -1 almost doubled the compressive strength. With Al concentrations above 2.2 mol⋅L -1 , the paste samples exhibited higher strength than the commercial binder investigated in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. The boron concentration was much less influent. However, raising its value within the range 0.4-0.6 mol⋅L -1 also tended to improve slightly the compressive strength. It was shown in Section 3.3.2 that increasing the Al concentration promoted the precipitation of the amorphous alumino-phosphate phase at the expense of brushite. This amorphous phase thus seemed to play a key role in the strength development of the material because it had better binding properties than brushite and/or led to denser microstructure.

Observation by SEM of polished cross sections of P B0.2/1.5 , P B0.2/2.5 , P B0.6/1.5 and P B0.6/2.5 samples prepared with mixing solutions containing 0.2 or 0.6 mol⋅L -1 of boron and 1.5 or 2.5 mol⋅L -1 of aluminum showed that these two species had not the same effect on porosity (Fig. 14). Pastes prepared with the highest Al concentration showed a denser cement matrix, but also a higher macroporosity. Note that this latter mainly resulted from CO 2 release within the paste due the destabilization in acidic medium of calcite present as traces in the wollastonite powder. Evacuation of the gas bubbles from the fresh paste was likely more difficult at high Al concentration since setting occurred faster. Increasing the boron concentration from 0.2 to 0.6 mol⋅L -1 decreased the macroporosity since the gas bubbles were filled with brushite crystals. This observation suggests that, in the presence of boron, calcium ions released by the dissolution of wollastonite could diffuse within the matrix before precipitating. This filling of macropores may explain the beneficial effect on the compressive strength evidenced at high boron concentrations.

Finally, aluminum and boron were shown to have opposite effects on the mean mesopore diameter assessed using the BJH method (Fig. 13): the pore size tended to decrease mainly by increasing the Al concentration -and thus the content of amorphous phaseand, to a minor extent, by decreasing the B concentration. It is worth mentioning however that, whatever the Al and B concentrations, the mean pore diameter of the pastes investigated in this work was always much smaller than that (138 Å) of the paste prepared in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF] with similar formulation parameters except for the mixing solution which also contained zinc cations.

Multicriteria optimization

To be used for waste conditioning, cement-based materials have to meet a number of quality demands depending on their elaboration process, but also on their subsequent storage in a repository. General requirements may include in particular a setting time ideally comprised between 5 h and 24 h (to avoid any risk of setting in the mixer in case of technical hitch, while keeping a good output of the conditioning unit), a rise in temperature resulting from cement hydration that does not affect the package integrity and a compressive strength higher than 20 MPa.

The time corresponding to the maximum heat flow giving a good estimation of the setting time, the results showed that to get setting in less than 24 h, mixing solutions with a small concentration of aluminum and a large concentration of boron should be avoided. However, such conditions would be interesting to get a low heat flow and a high cumulative heat (corresponding to a significant progress of hydration). Finding a compromise will then be necessary to meet the demands of not too long setting, limited self-heating and high progress of reaction within the first days after mixing.

A multi-criteria optimization [START_REF] Harrington | The desirability function[END_REF][START_REF] Derringer | Simultaneous optimization of several response variables[END_REF][START_REF] Khuri | Simultaneous optimization of multiple responses represented by polynomial regression functions[END_REF] was performed to point out the compositions of the mixing solution making it possible to produce brushite cement-based materials checking these requirements. The fitted regression models were used to define individual desirability functions for responses t max , HF max and R c . Every calculated response y i was transformed into a d i value, comprised between 0 and 100%, that represented the satisfaction level associated to the response value depending on the requirements. d i was set to 100% if the calculated response value was equal to the target, and to 0 if it was outside the tolerance interval. Fig. 15 shows the individual desirability functions defined for the 3 investigated responses. Overall desirability function D was calculated as the geometric mean of the 3 individual desirability functions (Eq. ( 4)).

D = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ d 1 ⋅d 2 ⋅d 3 3 √ (4)
D was plotted as a function of the Al and B concentrations in the mixing solution (Fig. 16). The maximum value, corresponding to a satisfaction level of 98%, was obtained for Al and B concentrations of 2.1 mol⋅L -1 and 0.56 mol⋅L -1 respectively (R c = 46.8 MPa, t max = 25.7 h and HF max = 4,97 mW⋅g -1 ). The time for maximum heat flow slightly exceeded 24 h. More generally, function D took interesting values (above 90%) when the Al and B concentrations were within the ranges [1.8; 2.4 mol⋅L -1 ] and [0.45; 0.6 mol⋅L -1 ] respectively. P-value: probability that a random variable having a F-distribution is greater than the F-test; ***: P < 0.1%.

Conclusion

The mixing solution of wollastonite-based brushite cement usually contains orthophosphoric acid, borax or boric acid, and metallic cations (such as Zn 2+ or Al 3+ ). However, data about the optimal composition and the role of each component are very limited. After a first study dedicated to the influence of the Ca/P molar ratio, H 3 PO 4 initial concentration and l/s weight ratio [START_REF] Laniesse | Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H 3 PO 4 concentration of the mixing solution[END_REF], the focus was placed in this work on the separate and joint effects of boron and aluminum. The main conclusions can be summarized as follows. 1. Whatever the type of addition (borax or boric acid), boron retards the cement hydration process and makes it possible to strongly reduce the self-heating of the material. As a consequence, brushite precipitates instead of monetite. 2. Adding Al 3+ cations to the mixing solution is necessary to get significant mechanical strength of hardened cement pastes. Precipitation of an amorphous alumino-phosphate phase seems to play a key role in the hardening process. Increasing the Al concentration also reduces the maximum heat flow and promotes the precipitation of brushite instead of monetite. However, it strongly reduces the duration of the low thermal activity period and setting times can become too short for field application. 3. The joint addition of boron and aluminum in the mixing solution is thus beneficial. High compressive strengths can be achieved thanks to the presence of aluminum while boron makes it possible to control the hydration rate, and thus the setting time. Empirical polynomial models have been built to predict several paste properties (maximal heat flow and corresponding hydration time, maximum cumulative heat, phase content and compressive strength at 28 d) as a function of the Al and B concentrations in the mixing solution. These models have then been used for multicriteria optimization: to design a conditioning matrix for waste stabilization / solidification, the B and Al concentrations should advantageously lie within the ranges [0.45; 0.6 mol⋅L -1 ] and [1.8; 2.4 mol⋅L -1 ] respectively. 4. As compared to previous work [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF], it is possible to prepare a wollastonite-based brushite cement paste using a simplified mixing solution (without zinc) and obtain good properties both in the fresh and hardened states. Future work should focus on the long-term evolution of such material under the environment of a repository.

Declaration of competing 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.

Fig. 1 .

 1 Fig. 1. Positioning of the experiments within the experimental design.

Fig. 3 .

 3 Fig. 3. Normalized heat flow and cumulative heat of cement pastes prepared with various concentrations of boron (A: 0.05 mol⋅L -1 ; B: 0.2 mol⋅L -1 ) introduced as borax or boric acid.

Fig. 4 .

 4 Fig. 4. X-Ray diffraction patterns (background subtracted) of paste samples prepared with 0.05 mol⋅L -1 of boron, added as borax (left) or boric acid (right).

Fig. 5 .

 5 Fig. 5. Heat flow (solid lines) and cumulative heat (dotted lines) of pastes prepared with different concentrations of aluminum in the mixing solution.

Fig. 6 .

 6 Fig. 6. Evolution of the cumulative heat in function of the solution aciditycomparison with values obtains for solutions containing only phosphoric acid from [17].

Fig. 7 .

 7 Fig. 7. X-Ray diffraction patterns (background subtracted) of paste P 0/2 prepared with 2 mol⋅L -1 of aluminum after 1 h to 48 h of hydration.

Fig. 8 .

 8 Fig. 8. SEM image (BSE, WD 9.7 mm, 15 kV) and elemental mapping of a polished section of paste P 0/2.5 prepared with 2.5 mol⋅L -1 of aluminum and cured for 28 days in sealed bag at 25 • C (1: wollastonite; 2: amorphous silica; 3: brushite; 4: amorphous aluminum phosphate).

Fig. 9 .

 9 Fig. 9. X-ray microanalysis in spots 3 and 4 of Fig. 8: brushite (left) and calcium aluminophosphate (right).

Fig. 11 .

 11 Fig. 11. Response contour plots for the mass fractions of brushite (left) and amorphous phase (right) in 28 d-old cement pastes (curing in sealed bag).

Fig. 12 .Fig. 13 .

 1213 Fig. 12. Microstructure of 28 d-old paste samples P B/0.6/1.5 and P B/0.6/2.5 (mixing solution comprising 0.6 mol⋅L -1 B and 1.5 or 2.5 mol⋅L -1 Al): SEM images (backscattered electronspolished cross section) and elemental mapping. 1: amorphous alumino-phosphate phase; 2: brushite; 3: wollastonite and 4: amorphous silica.

Fig. 14 .Fig. 15 .Fig. 16 .

 141516 Fig. 14. Microstructure of 28 d-old paste samples P B/0.2/1.5 , P B/0.2/2.5 P B/0.6/1.5 and P B/0.6/2.5 (mixing solution comprising 0.2 or 0.6 mol⋅L -1 B and 1.5 or 2.5 mol⋅L -1 Al): SEM images (backscattered electronspolished cross section) and EDS elemental mapping.

Table 3

 3 ICDS files associated to crystalline phase.

	Crystalline phase	ICDS file
	Brushite	72-0713
	Wollastonite	84-0654
	Quartz	70-7344
	Silicon	27-1402
	MCPM	09-0347

Table 4

 4 Estimated coefficients of polynomial models for the different investigated responses.

	Response	HF max	t max	H max	brushite	Amorphous	R c	d p
	Unit	mW/g	h	J/g	wt%	wt%	MPa	Å
	b 0	7.250	20.814	466.057	46.022	16.784	41.750	48.981
	b 1	2.032	-7.978	-40.225	-9.171	9.392	10.667	-8.953
	b 2	-4.275	8.529	-18.342	-0.157	-1.337	1.400	3.587
	b 11	0.327	1.169	-	-1.851	2.601	-	1.988
	b 22	1.226	0.477	5.086	-	-	2.517	-
	b 12 R 2 R 2 A R 2 pred	-0.460 0.998 0.997 0.988	-4.230 0.998 0.996 0.985	5.646 0.960 0.937 0.862	-0.890 0.848 0.800	-0.917 0.881 0.806	-0.864 0.813 0.725	-2.178 0.973 0.958 0.912

Table 5

 5 Checking for accuracy and significance of models using ANOVA.

	Response	Source	Sum of squares	df	Mean squares	F-test	P-value
	HF max	Regression	140.9352	5	28.1870	701.2372	<0.01***
		Residuals	0.2412	6	0.0402
		Lack of fit	0.1651	3	0.0550	2.1696	27.1
		Replication error	0.0761	3	0.0254
		Total	141.1764	11
	t max	Regression	895.9243	5	179.1849	540.8123	<0.01***
		Residuals	1.9880	6	0.3313
		Lack of fit	1.4230	3	0.4743	2.5190	23.4
		Replication error	0.5649	3	0.1883
		Total	897.9122	11
	H max	Regression	1.193E + 04	4	2.983E+003	41.57	<0.01***
		Residuals	5.023E + 02	7	7.176E+01
		Lack of fit	2.011E + 02	4	5.029E+01	0.501	74.3
		Replication error	3.012E + 02	3	1.004E+02
		Total	1.243E + 04	11
	wt% brushite	Regression	515.0152	3	171.6717	21.5293	0.0347***
		Residuals	63.7908	8	7.9739
		Lack of fit	17.0748	5	3.4150	0.2193	93.2
		Replication error	46.7160	3	15.5720
		Total	578.8060	11
	wt% amorphous	Regression	558.4234	3	186.1411	25.7816	0.0371***
		Residuals	50.5394	7	7.2199
		Lack of fit	30.9680	5	6.1936	0.6329	70.6
		Replication error	19.5714	2	9.7857
		Total	608.9628	10
	R c	Regression	713.4275	3	237.8092	16.9801	0.0789***
		Residuals	112.0417	8	14.0052
		Lack of fit	53.1217	5	10.6243	0.5410	74.4
		Replication error	58.9200	3	19.6400
		Total	825.4692	11
	d p	Regression	588.9969	4	147.2492	63.9925	<0.01***
		Residuals	16.1073	7	2.3010
		Lack of fit	5.7032	4	1.4258	0.4111	79.5
		Replication error	10.4041	3	3.4680
		Total	605.1041	11

  HF max , t max and H max contour plots in {[Al], [B]} plane. The red line corresponds to values obtained for a commercial binder investigated in[START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

											HFmax				t (HFmax)
		0.55 0.60		4	4.7				6	14 16 (mW/g)			0.55 0.60	40	(h)	39 43
		0.50											12			0.50	35	35
	) L / l o m ( ] B [	0.35 0.40 0.45								10 8			7.6 9.0 11	) L / l o m ( ] [ B	0.35 0.40 0.45	30 25	31 27 23
		0.30								12			6.0		0.30	18
		0.20 0.25	1.6	1.8		2.0	2.2	2.4 14			3.0 4.5		0.25 0.20	1.6 20 17	1.8 15	2.0	2.2	2.4	14 10
					[Al] (mol/L)										[Al] (mol/L)
																	Hmax
									0.60								(J/g)	536
									0.55								520
									0.50								506
							)	0.45								491
							L
							/
							l o m	0.40								476
							( ] [ B	0.35								460
									0.30								446
									0.25	520	500	482	480	460 440	431
									0.20								417
										1.6	1.8		2.0	2.2		2.4
											[Al] (mol/L)
											Brushite					Amorphous
		0.55 0.60	52.5	50.0	47.5	45.0	42.5	40.0 37.5	(wt.%)	51.2 53.5				0.55 0.60	10	14	18	22 26	(wt.%)	28 30
		0.50										48.8				0.50	25
	) L	0.45										46.4			) L	0.45	22
	/ l o m ( ] B	0.35 0.40										41.7 44.0			/ l o ] B ( m	0.35 0.40	17 19
	[															[
		0.30										39.3				0.30	14
		0.25										36.9				0.25	11
		0.20										34.6				0.20	12	16	20	24	28	8.7
			1.6	1.8	2.0	2.2	2.4							1.6	1.8	2.0	2.2	2.4
				[Al] (mol/L)										[Al] (mol/L)

Fig. 10.

Acknowledgements

Acknowledgements are due to Pascal Antonucci for his help in the laboratory.


Fabienne Rayne

Solène Debaisieux

Annie Tu

Christophe Chopard

Petra Tryoen-Toth

Bruno Beaumelle
email: bruno.beaumelle@irim.cnrs.fr

Petra Tryoen

Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot

Keywords: HIV-1, Tat, secretion, ELISA, immunoblots

come

Introduction

HIV-1 Tat is a small (86 to 101 residues) and basic protein that enables efficient viral transcription and is required for HIV-1 multiplication [1]. The capacity of HIV-1 infected cells to secrete Tat in the absence of cell lysis was demonstrated in the 1990s [2]. We recently showed that this secretion process, although unconventional, is very efficient since most Tat is exported by infected cells [3]. Circulating Tat can be taken up by various cell types such as T-cells, macrophages, endothelial cells and neurons. Tat will then cross the endosome membrane to reach the target cell cytosol and elicit a number of cell responses [4]. We first developed a sandwich ELISA assay, so that minute concentrations of Tat can be reliably assayed in the media of suspension cells such as T-cells that are the main HIV-1 target [3]. We used Jurkat cells for most experiments. During the development of this assay, we noticed that Tat in media was extremely sensitive to oxidation. We observed that reliable quantitation of Tat in the extracellular medium required prior degassing of the medium to remove oxygen as much as possible, the addition of freshly diluted -mercaptoethanol and the presence of immobilized anti-Tat antibodies in wells. This is why we called this assay a "cellular ELISA" because Tat-producing cells are added to wells pre-coated with the anti-Tat antibody. This enables antibodies to capture secreted Tat as soon as it is secreted by cells, before Tat would be oxidated that would render it unreactive toward antibodies. Another assay was developed for the use of adherent cells. In this case, we used Tat-FLAG transfected PC12 cells. Anti-FLAG antibodies covalently linked to agarose beads were directly added to the medium before immunoprecipitation and Western blot. For both techniques, it is necessary to assay both intracellular Tat and secreted Tat in order to calculate the secretion efficiency. 7. PBS/Tween (PBS containing 0.05 % Tween-20). 8. PBS/BME (PBS containing 20 µM -mercaptoethanol). Degas before use. 9. Citrate buffer (150 mM NaCl, 30 mM citrate, 20 mM phosphate, pH 7.2). 10. Lysis buffer (150 mM NaCl, 10% glycerol, 1% Triton X-100, 20 mM Tris-HCl pH 7.5).

Aliquots in 1.5 ml Eppendorf tubes and store them at -20°C. Just before use, add 1 mM PMSF (from a 100 mM stock solution in isopropanol) and a protease inhibitor cocktail. 11. 3,3',5,5' Tetramethylbenzidine (TMB) 12. 0.5M H2SO4 13. A 96-well plate OD450 reader is needed.

Luciferase assays.

1

3.Methods

1. Jurkat cells are grown at 37 °C in the presence of 5 % of CO2 and 95% humidity (i.e. in a cell culture incubator). It is cultured in RPMI/FBS. Cell density should not exceed 1.5 x 10 6 /ml and dilutions are obtained by adding fresh medium. 2. PC12 cells are cultured in complete medium. Cells are maintained in 10-cm diameter Petri dishes containing 10 ml of culture medium. PC12 cells are adherent cells and have no contact inhibition. Confluent cells should be passed by trypsinization at least once a week and be diluted 40-fold. To this end the cell monolayer is washed with PBS before adding 2 ml of trypsin/EDTA solution (see Note 2) and incubating at 37°C until the cells detach (see Note 3).

After incubation, stop the enzymatic reaction by adding 8 ml of culture medium. Flush 3-4 times back and forth the cell suspension using a plastic pipette to obtain a homogenous cell suspension. Centrifuge at 1000 rpm (~300 g) for 5 min. The cell pellet is resuspended in complete medium by pipetting several times before counting (see Note 4). Cells are then transferred to a new tissue culture flask or plate (see Note 5). Use 1.5 million cells in 5 ml of culture medium / 6-cm diameter Petri dish. Incubate cells overnight before transfection. 3. To prepare CD4+ human primary T-cells, human blood from healthy volunteers should be obtained from the local blood bank following the local laws and regulations. Isolate peripheral blood mononuclear cells (PBMCs) using Ficoll-Hypaque gradients following the manufacturer instructions. Resuspend PBMCs in RPMI/FBS at 1-4 x 10 6 cells /ml. Leave them for 2-4 h in tissue culture flasks to allow monocyte adherence to the flask. Non-adherent cells are peripheral blood lymphocytes (PBLs). Centrifuge PBLs (5 min x 1000 rpm) and isolate CD4 + T-cells using the protocol provided by the kit manufacturer. Resuspend CD4 + Tcells at 1-2 x 10 6 cells /ml and activate them using phytohemagglutinin (1 µg/ml) for 24 h. Centrifuge CD4 + T-cells, resuspend them at 1-2.10 6 cells /ml then add interleukin-2 (50 U/ml) for 5-6 days. 3. Transfer 0.7 ml (9 x 10 6 cells) to a standard 4 mm electroporation cuvette. 4. Dilute endotoxin-free plasmids in a maximum of 30 µl of water. We routinely used 20 µg / transfection with 18 and 2 µg of Tat and luciferase vector, respectively. An empty vector without Tat should be used for control cells. Mix gently without making bubbles. 5. Place the cuvette in the cuvette holder and electroporate (using an exponential decay pulse) at 270 V, 1000 µF, infinite resistance value. The pulse length should be < 25 msec. Above this value, cell viability drops dramatically. 6. Transfer cells to a 25 cm 2 tissue culture flask containing 10 ml of RPMI/FBS/BME at 37°C. 7. Cultivate cells overnight at 37°C, 5% CO2.

Transfection of primary T-cells

1. Transfect cells using a kit for activated T-cells and following the manufacturer's instructions. 2. Cultivate cells overnight at 37°C, 5% CO2 in RPMI/FBS/BME.

T-cell infection

Infect purified CD4+ T-cells overnight with HIV-1 (NL4.3) using an MOI >0.5. Jurkat cells can also be used.

Day 2

1. Wash T-cells 3x with RPMI/FBS/, count and dilute cells to 0.5 x 10 6 /ml (Jurkats) or 1 x 10 6 / ml (primary CD4 + T-cells). 2. Prepare the ELISA plates: two plates are needed. The first one will be used for the secretion assay that will capture secreted Tat and the second one to assay intracellular Tat to normalize secretion data. 3. Dilute anti-Tat monoclonal antibody (1/2000 is a good starting point) in sodium carbonate buffer. 4. Add 100 µl / well of the diluted antibody. 5. Leave overnight at 4°C. 6. Wash wells 2 x with 200 µl PBS. 7. Saturate wells for 1.5 h with PBS/milk 8. Wash wells 3 x with 200 µl PBS, leaving wells in the last wash to prevent drying.

Place the plates back on the orbital shaker under strong agitation until a blue colour appears in

the key wells of the plates. It takes from 10 min (most often) to 1 h (very weak signal). 7. Stop the reaction by adding 50 µl of 0.5 M H2SO4 / well. 8. Read the optical density at 450 nm using a plate reader.

Luciferase Assay

1. Use a white 96-well plate. 2. To assay luciferase activity in cell lysates, add 6 µl of lysate to 30 µl of LAR/well. 3. To assay luciferase activity in media, add 20 µl of medium to 100 µl of LAR /well. 4. Typical settings for the luminometer are delay 2 s and measure 10 s.

Calculations of secretion efficiency (Jurkat cells)

S being the amount of secreted Tat (in ng), and I the amount of intracellular Tat (in ng), the secretion efficiency (%) is S x 100 /(S+I) the error on this ratio being 100 x (IS + SI)/ (S+I) 2 with S and I being the SEM of S and I, respectively (see Note 7). Luciferase secretion efficiency is calculated using the same procedure except that relative light units (RLU) are used instead of ng (see Note 8). For ELISA troubleshooting, see Note 9. The experimental flow chart (Fig. 1) summarizes the cellular ELISA procedure used to assay Tat secretion by T-cells.

Western blot procedure for adherent cells 3.2.1. Transfection of PC12 cells.

1. Because most anti-Tat antibodies are poorly effective for immunoprecipitation, we use Tat-FLAG and thus transfect cells with Tat-FLAG instead of Tat. Control experiments performed in Jurkat cells showed that this carboxyl-terminal tag does not affect Tat secretion efficiency. This tag also enables one to use commercially available anti-FLAG gel beads that allow one to capture Tat-FLAG in solution before it becomes oxidized and thus unreactive to antibodies. Tat is quantified using anti-FLAG Western blotting. 2. One hour before cell transfection, change the PC12 culture medium to 5 ml of Opti-MEM.

Return cells to the cell culture incubator for 1 h. 3. During the incubation period prepare the mix of plasmids with Lipofectamine 2000: mix 9 µg of Tat-FLAG (or empty vector) in a volume of 500 µl of Opti-MEM. Vortex briefly. In a separate tube, dilute 18 µl of Lipofectamine 2000 in 500 µl of Opti-MEM. Vortex briefly, then add the diluted DNA to the diluted Lipofectamine 2000. Incubate the DNA-Lipofectamine mix for 20 min at room temperature (RT) to allow complex formation. 4. Add the mix drop by drop to the cell culture medium. Gently agitate the culture dish back and forth to homogenously distribute the complexes. Incubate for 4 h in the cell culture incubator. After 4 h, replace the transfection medium with 5 ml of culture medium and incubate the cells overnight.

Tat-FLAG immunocapture

1. Gently rinse the cell cultures with warm DMEM and add 2 ml of immunocapture medium / 6cm diameter Petri dish. 2. Prepare the anti-FLAG affinity agarose beads for immunocapture: add a 20 µl aliquot of Anti-FLAG affinity gel / dish to 1 ml of DMEM in an Eppendorf tube. 3. Centrifuge the tube at 200 g for 1 min. Discard the supernatant. Repeat this step 3 x to remove all traces of glycerol from the bead-suspension. 4. Resuspend the gel in 500 µl of immunocapture medium.

Quantify relative band intensities using a quantification software such as ImageJ. This will enable one to evaluate the amount of Tat released into the cell culture medium (S) and present intracellularly (I). Calculations are then performed as described above for T-cells (3.1.12). It is also possible to use cotransfected luciferase to monitor transfected cell lysis, essentially as detailed for T-cells.

4.Notes.

1. Important: use a milk package opened less than 2 months before.

2.

It is also possible to use EDTA without trypsin to detach PC12 Cells. The procedure is the same.

3. To favour cell detachment, gently hit the side of the Petri dish. When all cells are detached they float in the medium and this can be easily seen with the naked eyes, or using an inverted microscope if necessary. Usually 2 min are sufficient to detach PC12 cells.

4. PC12 cells tend to form small clusters in cell suspension, hence complicating counting. A confluent 10-cm diameter Petri dish contains ~10 millions cells.

5. If needed, it is possible to increase the adherence of PC12 cells by pretreating the Petri dish surface with a PLL solution before cell plating. To end, add 2 ml of sterile PLL solution to a 6 cm diameter Petri dish and incubate 1 h at 37 °C. After incubation, remove the PLL solution and use the dish directly for cell plating.

6. Incubation can be longer if the medium does not become too much acidic, i.e. yellow. 7. ELISA Calculations. It should be noted that for most anti-Tat antibody pairs (monoclonal / polyclonal), standard curves will be different for the secretion and intracellular plate (Fig. 2). This is presumably due to the presence of detergent (from the lysis buffer) in the expression plate. Both standard curves are thus needed to first calculate the amount of Tat in the medium and intracellular, before calculating the secretion efficiency. Calculating directly from OD values can lead to erroneous secretion values. The extent of the error will depend on the difference between the standard curves and thus on the antibody pair.

8. Sensitivity and specificity of the ELISA assay. Fig. 3 shows that amount of secreted Tat is linear with the number of Jurkat cells present in the well, while Fig. 4 shows that luciferase release is negligible compared to Tat secretion. 9. ELISA Troubleshooting. If no signal is detected, the affinity of the antibodies should be checked by Western blotting. If high background is observed, it is possible to use a biotinylated rabbit anti-Tat detected using streptavidin-peroxidase [5].

10. Alternatively, Tat can also be efficiently separated on 15 % PAGE/ Tricine gels [6].

11.Tat is a small protein (~ 11 kDa) that, depending on the gel system, can easily run out of the gel.

Figure 1 :

 1 Figure 1 : Experimental flow chart for the cellular ELISA secretion assay (T-cells)

Figure 2

 2 Figure 2 Standard curves. Purified recombinant Tat was diluted in the indicated medium and assayed by ELISA. Means + SEM (n=3). Results from a typical experiment.

Figure 3 Secretion

 3 Figure 3Secretion efficiency. Jurkat T cells were transfected with Tat and luciferase (Tat) or with an empty vector and luciferase (Mock). The indicated number of cells was then plated in wells of a 96-well plate (n=4). After 6 h at 37°C secreted and intracellular Tat were assayed by ELISA. Tat secretion is linearly dependent on cell number.

Figure 4 Specificity

 4 Figure 4 Specificity of Tat secretion. Jurkat T cells were transfected with Tat and luciferase (Tat), before essaying their extracellular and intracellular levels by ELISA (n=4) to calculate Tat secretion efficiency as described in the text. Results from a typical experiment. Luciferase release is negligible compared to Tat secretion.

Acknowledgement

This work was funded by the CNRS, the ANRS and Sidaction.


Sylvain Bouley

James Tuttle Keane

David Baratoux

Benoit Langlais

Isamu Matsuyama

F Costard

Roger Hewins

Valérie Payré

Violaine Sautter

Antoine Séjourné

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