WO2020058329A1 - Stimulable biphilic polymer hydrogel particles for stabilising water-in-water emulsions - Google Patents

Stimulable biphilic polymer hydrogel particles for stabilising water-in-water emulsions Download PDF

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WO2020058329A1
WO2020058329A1 PCT/EP2019/074997 EP2019074997W WO2020058329A1 WO 2020058329 A1 WO2020058329 A1 WO 2020058329A1 EP 2019074997 W EP2019074997 W EP 2019074997W WO 2020058329 A1 WO2020058329 A1 WO 2020058329A1
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poly
water
polymer
heat
polysaccharide
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PCT/EP2019/074997
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French (fr)
Inventor
Valérie Ravaine
Lazhar BENYAHIA
Nicolai TACO
Théo MERLAND
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Université De Bordeaux
Institut Polytechnique De Bordeaux
Centre National De La Recherche Scientifique
Universite Du Mans
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Publication of WO2020058329A1 publication Critical patent/WO2020058329A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F290/00—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
    • C08F290/08—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups on to polymers modified by introduction of unsaturated side groups
    • C08F290/10—Polymers provided for in subclass C08B
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/24—Homopolymers or copolymers of amides or imides
    • C08L33/26—Homopolymers or copolymers of acrylamide or methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2333/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
    • C08J2333/24—Homopolymers or copolymers of amides or imides
    • C08J2333/26—Homopolymers or copolymers of acrylamide or methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2371/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
    • C08J2371/02—Polyalkylene oxides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/02—Dextran; Derivatives thereof

Definitions

  • the present invention relates to hydrogel particles of stimulable polymers and their use for the stabilization of water-in-water emulsions. It also relates to the emulsions thus obtained.
  • the present invention therefore aims to provide a system adapted to allow stabilization of water-in-water emulsions, and this for a satisfactory duration, in particular for at least 3 months.
  • the present invention also aims to provide water-in-water emulsions stable over time, but also having dilution stability for a satisfactory duration, in particular for at least 1 month.
  • the present invention also aims to provide water-in-water emulsions stable over time and which can be destabilized as a function of an external stimulus such as temperature.
  • Another object of the invention consists in providing aqueous systems, obtained by simple mixing, at low shear rate, based on capsules of controlled size, stable over time and at dilution, and which can be destroyed on demand, by application of an external stimulus such as temperature.
  • Another object of the invention is to provide a system for encapsulating water-soluble molecules, including fragile molecules such as proteins, enzymes or nucleic acids such as DNA.
  • the present invention relates to a hydrogel particle, in particular a nanogel, consisting of a copolymer of at least one polysaccharide and at least one heat-sensitive polymer.
  • the copolymer according to the invention contains from 10% to 50% by weight of polysaccharide and from 50% to 90% by weight of heat-sensitive polymer.
  • the copolymer according to the invention is a copolymer with two distinct units: a polysaccharide and a heat-sensitive polymer.
  • the two polymers are chemically crosslinked, at least partially, and form a swollen network of water.
  • the term “heat-sensitive polymer” designates a polymer which exhibits a drastic and discontinuous change in its physical properties with temperature.
  • this term designates polymers with LCST (critical lower solubility temperature) or LJGST (critical upper solubility temperature).
  • LCST critical lower solubility temperature
  • LJGST critical upper solubility temperature
  • These polymers are specific polymers whose solubility in water is modified above or below a certain temperature.
  • UCST Copper Critical Solution Temperature
  • the heat-sensitive polymer is an LCST polymer.
  • the heat-sensitive polymer is chosen from the group consisting of N-alkyl-substituted polyacrylamides such as poly (N-isopropylacrylamide), poly (N-isopropylmethacrylamide) or poly (N-ethylacrylamide), poly ((2- dimethylamino) ethyl methacrylate), oligoethylene glycol side chain poly (methacrylates), poly (N-vinylcaprolactam), poly (vinyl methyl ether), poly (2-alkyl-2-oxazoline), Poly (propylene oxide) ) and their mixtures.
  • N-alkyl-substituted polyacrylamides such as poly (N-isopropylacrylamide), poly (N-isopropylmethacrylamide) or poly (N-ethylacrylamide), poly ((2- dimethylamino) ethyl methacrylate), oligoethylene glycol side chain poly (methacrylates), poly (N-vinylcaprol
  • the heat-sensitive polymer can also be chosen from the group consisting of copolymers of acrylamide and acrylonitrile, homopolymers of methacrylamide, polymers with ureido derivative such as poly (allylamine) -co-poly (allylurea), zwittterionic polymers, such as poly (sulfobetaine) s or poly (phosphorylcholine) s, and mixtures thereof.
  • the heat-sensitive polymer is poly (N-isopropylacrylamide).
  • the polysaccharide according to the invention is at least difunctional, that is to say that it is substituted by at least two functional groups.
  • the term “functional group” designates a group carrying a chemical function, in particular chosen from methacrylate, acrylate, vinyl, acrylamide, methacrylamide, maleimide, vinyl sulfone, acrylonitrile, azide and amine functions.
  • the polysaccharide according to the invention has a degree of substitution (DS) of between 2% and 30% by moles.
  • the degree of substitution is defined as the molar ratio between the grafted functional groups, in particular the grafted methacrylate functions, and the repeat unit of the polysaccharide, here namely dextran. It can be measured by NMR (Nuclear Magnetic Resonance) of the proton, by integrating vinyl protons (5.8 and 6.25 ppm) and those of anomeric dextran (5 ppm).
  • the polysaccharide is chosen from the group consisting of hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate, pectin , dextran, dextrin, pullulan, glycogen and mixtures thereof.
  • the polysaccharide is dextran, in particular functionalized, in particular substituted by at least two methacrylate groups.
  • said particles are networks of polymer swollen with solvent.
  • the network is continuous through the particle, we can then speak of "full" particles.
  • the particles contain more solvent than polymer.
  • the particles according to the invention can also be designated as colloidal hydrogel particles.
  • the hydrogel or nanogel particles according to the invention preferably have a diameter of between 10 nm and 10 ⁇ m.
  • the present invention also relates to a series of hydrogel or nanogel particles as defined above.
  • the present invention is based on a new class of particles, consisting of copolymers, one of the entities having an affinity for the aqueous phase containing a polymer A and the other having an affinity for the aqueous phase containing a polymer B
  • copolymers are qualified as double hydrophilic or biphilic.
  • the present invention also relates to the use of at least one hydrogel or nanogel particle as defined above, for the stabilization of a water-in-water emulsion.
  • the particles of the invention consisting of copolymers, of which one of the entities has an affinity for the aqueous phase containing a polymer A (polA) and the other has an affinity for the aqueous phase containing a polymer B (polB)
  • polymers of which one of the entities has an affinity for the aqueous phase containing a polymer A (polA) and the other has an affinity for the aqueous phase containing a polymer B (polB)
  • the particles of the invention consisting of copolymers, of which one of the entities has an affinity for the aqueous phase containing a polymer A (polA) and the other has an affinity for the aqueous phase containing a polymer B (polB)
  • polymer A polymer A
  • polyB polymer B
  • the present invention also relates to a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B, and further comprising, at the interface of these two phases, particles of hydrogel or nanogel as defined above.
  • the concentration of polymers A and B in the continuous and dispersed aqueous phases is between 0.1% and 80% by mass relative to the mass of continuous or dispersed aqueous phase, respectively.
  • the concentration of the particles is between 0.005% and 30% by mass relative to the mass of emulsion.
  • the present invention also relates to a water-in-water emulsion as defined above, the dispersed aqueous phase of which is formed of at least one capsule comprising a core containing polymer A and a shell of hydrogel or nanogel particles. as defined above.
  • the polymer A is a water-soluble polymer which may have a phase separation with B.
  • a polymer can be chosen from the class of polysaccharides (dextran , chitosan, guar ...), proteins (casein, globular proteins ...), oligopeptides, nucleic acids (DNA, RNA) or synthetic polymers (POE, PPO, acrylates, ...) for example.
  • the polymer B is a water-soluble polymer which can have a phase separation with A.
  • Such a polymer can be chosen from the class of polysaccharides (dextran, chitosan, guar %), proteins (casein, globular proteins %), oligopeptides, nucleic acids (DNA, RNA) and synthetic polymers (POE, PPO, acrylates, %) by example.
  • the copolymer particles pass from the swollen state at a temperature below the LCST to contracted beyond the LCST.
  • the dispersibility of the particles in A or in B is also a function of the swelling state of the particle.
  • the emulsions of B-in-A will be stable at low temperature. A rise in temperature beyond the LCST causes the emulsion to become destabilized. If the emulsion is produced at high temperature, the emulsions from A to B will be stable. A decrease in temperature below the LCST causes destabilization of the emulsion.
  • one of the two polymers is a UCST polymer, that is to say that it is soluble at high temperature in water but precipitates for a temperature below the UCST, the particles of copolymers pass from the contracted state at a temperature lower than the UCST to swollen beyond the UCST.
  • the emulsions of B-in-A will be stable at high temperature. A decrease in temperature below the UCST causes destabilization of the emulsion. If the emulsion is made at low temperature, the emulsions from A to B will be stable. A decrease in temperature beyond the UCST causes destabilization of the emulsion.
  • the present invention is advantageous in that it is possible to modify the direction of the emulsion obtained in a simple manner.
  • the direction of the emulsion obtained differs depending on whether one is below the solubility temperature of the heat-sensitive polymer according to the invention or above.
  • the aqueous phase containing polysaccharides will be the dispersed phase whereas at high temperature it will become the continuous phase.
  • it is possible to choose the direction of the emulsion by adapting the phase transition temperature of the hydrogels or nanogels to the chosen use temperature.
  • the above-mentioned water-in-water emulsions, stabilized at low temperature can be destabilized on demand by increasing the temperature above the solubility temperature of the LCST polymer.
  • the emulsions according to the invention are stable at dilution, which is generally impossible for water-in-water emulsions because the dilution causes the reduction of the interfacial tension between the two phases rich in A and B and ends up at high dilution, to bring the polymers back to the monophasic field.
  • the present invention also relates to a method making it possible to change the direction of the abovementioned emulsions, by means of a temperature change, as described below.
  • a water-in-water emulsion comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B
  • a water-in-water emulsion comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A.
  • a water-in-water emulsion the continuous phase of which is the phase rich in polymer A and the dispersed phase, the phase rich in polymer B, A having an affinity for the LCST polymer.
  • Heating the emulsion at rest beyond the LCST leads to its destabilization, that is to say the return to the macroscopic phase separation. If the sample is kept under stirring, in particular using a vortex, during heating, heating beyond the LCST leads to a phase inversion of the emulsion, namely that the rich phase in polymer A becomes the dispersed phase while the phase rich in polymer B becomes the continuous phase.
  • the present invention therefore also relates to a process for the preparation of a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A, comprising a step of treating an emulsion such as defined above at a temperature below the LCST of the heat-sensitive polymer.
  • This process therefore consists in transforming a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B, into a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A.
  • the present invention also relates to a polymer capsule, comprising:
  • these capsules have a size of between 0.1 ⁇ m and 1000 ⁇ m, preferably between 500 nm and 100 ⁇ m.
  • the present invention also relates to the use of a capsule as defined above for the encapsulation of water-soluble molecules, in particular chosen from proteins, enzymes or nucleic acids such as DNA or RNA.
  • Such capsules thus allow the encapsulation of food, cosmetic or pharmaceutical assets.
  • these capsules can be used for the encapsulation of dyes, pigments, quantum dots, fluorescent markers, vitamins, peptides, oligonucleotides, antibiotics, hormones, anticancer agents or sun filters.
  • the heart of said capsules also comprises an active ingredient.
  • any water-soluble active ingredient with a partition coefficient different from 1.
  • said active ingredient will be in the dispersed phase.
  • the state of the hydrogel will be chosen so as to orient the direction of the emulsion so that the phase in which the active ingredient is dissolved becomes the dispersed phase.
  • the present invention provides a means of encapsulating fragile water-soluble molecules.
  • the latter in a process of separation of liquid-liquid phases in an aqueous medium, the latter have a greater affinity for one aqueous phase of polymers than for the other.
  • they can therefore be located selectively in the dispersed phase. If the dispersed phase drops are sufficiently stable, they can be concentrated without the risk of coalescing or diluted without dislocating. It is therefore possible to transport these fragile molecules, while protecting them from the outside environment via the capsule.
  • the hydrogel or nanogel capsule can then be destroyed by the application of a simple stimulus such as temperature.
  • a simple stimulus such as temperature.
  • the formation process used is not deleterious for fragile molecules since the mechanical agitation remains moderate and that the stability rests solely on physical processes (no reactivity, no change of medium such as pH or temperature).
  • Figure 2 Influence of the DS of dextran in nanogels on the evolution of their diameter as a function of temperature.
  • R is the reference without nanogel
  • 0 the emulsion stabilized by nanoparticles of pNIPAM without dextran
  • 1, 2 and 3 are stabilized by nanogels 1, 2 and 3 respectively (see Table 1).
  • Figure 4 Dextran-in-POE emulsion with 0.04% m of fluorescent T nanogels after 3 days at 25 ° C.
  • Figure 5 Inverse of the diameter of the drops as a function of the amount of nanogels for dextran-in-POE emulsions (1 ’nanogels) at 25 ° C.
  • Figure 8 Behavior of dextran-in-PEO emulsions stabilized by nanogels 1 'at 20 ° C following dilutions ranging from 1 to 5 times the initial volume of the emulsion. Temporal monitoring of the transmittance of emulsions under normal gravity. The emulsions contain 0.1% by weight of nanogels.
  • New nanoparticles made of copolymers of pNIPAM and dextran are synthesized.
  • dextran is functionalized by methacrylate patterns.
  • Dextran-methacrylate is obtained by reaction of methacrylic anhydride with the hydroxyls of dextran, in DMF / water medium (Pitarresi, G .; Pierro, P .; Palumbo, FS; Tripodo, G .; Giammona , G. Biomacromolecules 2006, 7, 1302-1310; Auzely, dx.doi.org/10.1021/bm300324m
  • dextran 40 kg / mol, Sigma-Aldrich
  • 175 mL of water / DMF 7/3 mixture to which 2 g of methacrylic anhydride is added .
  • the pH is maintained between 8 and 9 by additions of 0.5 M NaOH for 4 h at 4 ° C.
  • the medium is dialyzed via a dialysis membrane (cutoff threshold 3500 g / mol) for 6 days, changing the water at least once a day.
  • the solution is then lyophilized.
  • the substitution rate defined as the molar ratio between the grafted methacrylate functions and the repetition unit of dextran, is determined by proton NMR by integrating vinyl protons (5.8 and 6.25 ppm) and those of anomeric dextran ( 5 ppm). DS ranging from 5 to 40% of the dextran chain is obtained by modifying the concentration of methacrylic anhydride.
  • the pNIPAM-Dex particles are synthesized by precipitation polymerization.
  • NIPAM (796 mg) and dex-MA (between 10 and 37% by mass compared to NIPAM) are dissolved in 95 ml of deionized water.
  • the medium is heated at 70 ° C and degassed.
  • potassium persulfate (67 mg in 5 ml) is introduced to initiate the polymerization.
  • the medium becomes turbid, indicating the nucleation of the particles.
  • the reaction is carried out for 4 h.
  • fluorescein-O-methacrylate 0.1 mol% relative to NIPAM
  • fluorescein-O-methacrylate is introduced into the solution, at the same time as NIPAM.
  • the particles from the synthesis are filtered and then dialyzed for a minimum of 6 days using a dialysis membrane with a cut-off threshold of 100 kDa.
  • the particles are of uniform size, as evidenced by their analysis in the dry state by transmission electron microscopy (TEM Tecnai Biotwin (120 kV))
  • FIG. 1 The diameter of the particles d H is measured by dynamic light scattering (Zetasizer Nano S90, Malvern Instruments) equipped with a He-Ne laser at 90 ° incidence relative to the detector. It is reported in Table 1 for different particle compositions. The evolution of the diameter of the particles as a function of the temperature is given in Figure 2.
  • the nanogels are swollen at low temperature and contract for a temperature above 32 ° C, corresponding to the LCST of pNIPAM.
  • the emulsions were prepared with solutions containing, for one, a mass fraction of poly (ethylene oxide) POE (Sigma-Aldrich), of average molar mass by mass of 200,000 g / mol, and for the other a mass fraction of dextran (Sigma-Aldrich), of mass-average molar mass of between 450,000 and 650,000 g / mol. All solutions are obtained with milli-Q water with a resistivity of 18.2 MW-cm.
  • poly (ethylene oxide) POE Sigma-Aldrich
  • dextran Sigma-Aldrich
  • the POE solution has a concentration of 10% by mass and the dextran solution is 20% by mass.
  • the emulsions are produced by mixing a volume ratio of 75% / 25% of each phase.
  • a first system contains a volume fraction of 75% of the phase rich in POE and 25% of the phase rich in dextran.
  • the masses of dextran and POE are 4% and 6% respectively.
  • the concentration of the phase rich in POE is 15.8% and that in dextran is 8.2%.
  • the nanogels in solution are added to the mixture, so as to obtain a maximum of 0.12% by mass of nanogels in the total mixture.
  • the dilution ability was tested by adding up to 5 times the volume of the emulsion in water equivalent.
  • Nanogels containing the greatest amount of dextran better stabilize emulsions
  • Emulsions are produced with a batch of nanogels containing 37% Dex-MA of DS 10, into which a fluorescent comonomer (Fluorescein O-methacrylate, 0.1% mol relative to NIPAM) has been introduced.
  • a fluorescent comonomer Fluorescein O-methacrylate, 0.1% mol relative to NIPAM
  • the preparation of the emulsions with the addition of particles is carried out as described in Example 2.
  • the emulsions are observed under a confocal microscope. It is clear that nanogels are placed on the surface of the drops, thus forming capsules.
  • the size of the drops is a function of the quantity of particles ( Figure 5).
  • the linear evolution of the inverse of the diameter as a function of the quantity of particles is a signature characteristic of the limited coalescence of Pickering emulsions. From a practical point of view, this phenomenon constitutes a lever to control the size of the drops and therefore of the capsules.
  • the mechanical stability of dextran-in-PEO emulsions is evaluated by subjecting the emulsions to a centrifugal force.
  • the LUMIsizer ® gives the analysis simultaneous transmittance as a function of the altitude in a tube, it makes it possible to probe the phenomena of creaming / sedimentation, as well as force and centrifugal coalescence and controlled temperature.
  • a weak transmittance translates a turbid phase and therefore the existence of drops. They can be located in a small area after sedimentation / creaming.
  • a high transmittance over the entire height means that the sample has out of phase or that the interfaces have disappeared by coalescence.
  • Figure 6 shows the evolution of this signal for emulsions stabilized by nanogel 1 ’, used at different concentrations. For concentrations greater than 0.04% by mass, the persistence of turbidity at the bottom of the tube after a very long centrifugation (33 h) at 300 g testifies to a great resistance of the drops to coalescence under mechanical stress.
  • emulsions stabilized by nanogels having a composition of 37% m. in DS 10 dextran provide great emulsion stability.
  • a POE / dextran emulsion (75% / 25%) was prepared by adding 0.1% m of 1 ’particles.
  • this system has shown unprecedented stability at room temperature. After 1 day of stabilization of the system, the emulsions were diluted 1 to 5 times by adding water.
  • the emulsions are then observed over a period of 1 week

Abstract

The invention relates to hydrogel particles consisting of a copolymer of at least one polysaccharide and at least one heat-sensitive polymer. The invention also relates to the use thereof for stabilising a water-in-water emulsion and to the corresponding water-in-water emulsions.

Description

PARTICULES D’HYDROGELS DE POLYMÈRES BIPHILIQUES STIMULABLES POUR STABILISER DES ÉMULSIONS EAU-DANS-EAU  HYDROGEL PARTICLES OF STIMULABLE BIPHILIC POLYMERS FOR STABILIZING WATER-IN-WATER EMULSIONS
La présente invention a pour objet des particules d’hydrogels de polymères stimulables et leur utilisation pour la stabilisation d’émulsions eau-dans-eau. Elle a également pour objet les émulsions ainsi obtenues. The present invention relates to hydrogel particles of stimulable polymers and their use for the stabilization of water-in-water emulsions. It also relates to the emulsions thus obtained.
De nombreux systèmes ternaires aqueux contenant deux types de polymères présentent une séparation de phase liquide-liquide entre une phase riche en polymère A et une phase riche en polymère B (W. J. Frith, Advances in Colloid and Interface Science, 2010, 161 , 48-60). Parmi les exemples connus, on retiendra les systèmes eau-dextran-polyoxyde d’éthylène (POE) (D. Forciniti, C. K. Hall and M. R. Kula, Fluid Phase Equilibria, 1991 , 61 , 243-262) ou eau-dextran-gélatine (M. W. Edelman, E. van der Linden, E. de Hoog and R. H. Tromp, Biomacromolecules, 2001 , 2, 1 148-1 154). Ces systèmes sont intéressants du point de vue des applications car ils permettent de séquestrer certaines molécules d’intérêt dans l’une des phases, en particulier certaines protéines ou de l’ADN (J. P. Douliez, N. Martin, C. Gaillard, T. Beneyton, J.C. Baret, S. Mann, L. Beven, Angew. Chem. Int. Ed., 2017, 3689). Ces systèmes présentent une tension interfaciale très basse (G. Balakrishnan, T. Nicolai, L. Benyahia and D. Durand, Langmuir, 2012, 28, 5921 - 5926). Ils peuvent être mélangés de façon temporaire mais démixent avec le temps, à moins de gélifier l’une des deux phases (I. Capron, S. Costeux and M. Djabourov, Rheologica Acta, 2001 , 40, 441 -456). Une approche plus générale consisterait à préserver l’état dispersé d’une phase aqueuse dans l’autre à l’état liquide en ajoutant des agents de surface. Many aqueous ternary systems containing two types of polymers exhibit a liquid-liquid phase separation between a phase rich in polymer A and a phase rich in polymer B (WJ Frith, Advances in Colloid and Interface Science, 2010, 161, 48-60 ). Among the known examples, the water-dextran-polyethylene oxide (POE) systems (D. Forciniti, CK Hall and MR Kula, Fluid Phase Equilibria, 1991, 61, 243-262) or water-dextran-gelatin ( MW Edelman, E. van der Linden, E. de Hoog and RH Tromp, Biomacromolecules, 2001, 2, 1 148-1 154). These systems are interesting from the point of view of applications because they make it possible to sequester certain molecules of interest in one of the phases, in particular certain proteins or DNA (JP Douliez, N. Martin, C. Gaillard, T. Beneyton, JC Baret, S. Mann, L. Beven, Angew. Chem. Int. Ed., 2017, 3689). These systems have a very low interfacial tension (G. Balakrishnan, T. Nicolai, L. Benyahia and D. Durand, Langmuir, 2012, 28, 5921 - 5926). They can be mixed temporarily but demix over time, unless one of the two phases is gelled (I. Capron, S. Costeux and M. Djabourov, Rheologica Acta, 2001, 40, 441-456). A more general approach would be to preserve the dispersed state from one aqueous phase to the other in the liquid state by adding surfactants.
Contrairement aux émulsions eau-huile, ces émulsions eau-dans-eau sont difficiles à stabiliser. En effet, non seulement les tensioactifs de chimie adéquate n’existent pas - il faudrait concevoir des tensioactifs biphiliques - mais les petites molécules ne peuvent pas stabiliser des émulsions eau-dans-eau car la longueur de corrélation est inférieure à celle des solutions de polymères. Les protéines ou les particules aux interfaces peuvent constituer une alternative (T. Nicolai and B. Murray, Food Hydrocolloids, 2017, 68, 157-163 ; J. Esquena, Current Opinion in Colloid & Interface Science, 2016, 25, 109-1 19). Cependant, à ce jour, aucun composé n’a permis d’obtenir des émulsions eau-dans-eau stables à la dilution. De plus, aucun système n’a encore permis de contrôler le sens de l’émulsion et sa stabilité par la température. Unlike water-oil emulsions, these water-in-water emulsions are difficult to stabilize. In fact, not only do adequate chemistry surfactants not exist - biphilic surfactants should be designed - but small molecules cannot stabilize water-in-water emulsions because the correlation length is less than that of polymer solutions . Proteins or particles at interfaces can constitute an alternative (T. Nicolai and B. Murray, Food Hydrocolloids, 2017, 68, 157-163; J. Esquena, Current Opinion in Colloid & Interface Science, 2016, 25, 109-1 19). However, to date, no compound has made it possible to obtain water-in-water emulsions stable at dilution. In addition, no system has yet made it possible to control the direction of the emulsion and its stability by temperature.
La présente invention a donc pour but de fournir un système adapté pour permettre de stabiliser des émulsions eau-dans-eau, et ce pour une durée satisfaisante, notamment pendant au moins 3 mois. The present invention therefore aims to provide a system adapted to allow stabilization of water-in-water emulsions, and this for a satisfactory duration, in particular for at least 3 months.
La présente invention a également pour but de fournir des émulsions eau- dans-eau stables dans le temps, mais présentant également une stabilité à la dilution pour une durée satisfaisante, notamment pendant au moins 1 mois.  The present invention also aims to provide water-in-water emulsions stable over time, but also having dilution stability for a satisfactory duration, in particular for at least 1 month.
La présente invention a également pour but de fournir des émulsions eau- dans-eau stables dans le temps et pouvant être déstabilisées en fonction d’un stimulus externe tel que la température.  The present invention also aims to provide water-in-water emulsions stable over time and which can be destabilized as a function of an external stimulus such as temperature.
Un autre but de l’invention consiste à fournir des systèmes aqueux, obtenus par simple mélange, à faible taux de cisaillement, à base de capsules de taille contrôlée, stables dans le temps et à la dilution, et pouvant être détruites à la demande, par application d’un stimulus externe tel que la température.  Another object of the invention consists in providing aqueous systems, obtained by simple mixing, at low shear rate, based on capsules of controlled size, stable over time and at dilution, and which can be destroyed on demand, by application of an external stimulus such as temperature.
Un autre but de l’invention consiste à fournir un système permettant d’encapsuler des molécules hydrosolubles, y compris des molécules fragiles comme les protéines, les enzymes ou les acides nucléiques tels que l’ADN.  Another object of the invention is to provide a system for encapsulating water-soluble molecules, including fragile molecules such as proteins, enzymes or nucleic acids such as DNA.
Ainsi, la présente invention concerne une particule d’hydrogel, notamment de nanogel, constituée d’un copolymère d’au moins un polysaccharide et d’au moins un polymère thermosensible. Thus, the present invention relates to a hydrogel particle, in particular a nanogel, consisting of a copolymer of at least one polysaccharide and at least one heat-sensitive polymer.
Selon un mode de réalisation, le copolymère selon l’invention contient de 10% à 50% en poids de polysaccharide et de 50% à 90% en poids de polymère thermosensible.  According to one embodiment, the copolymer according to the invention contains from 10% to 50% by weight of polysaccharide and from 50% to 90% by weight of heat-sensitive polymer.
Selon un mode de réalisation, le copolymère selon l’invention est un copolymère avec deux unités distinctes : un polysaccharide et un polymère thermosensible.  According to one embodiment, the copolymer according to the invention is a copolymer with two distinct units: a polysaccharide and a heat-sensitive polymer.
De préférence, les deux polymères sont réticulés chimiquement, au moins partiellement, et forment un réseau gonflé d’eau.  Preferably, the two polymers are chemically crosslinked, at least partially, and form a swollen network of water.
Selon l’invention, le terme « polymère thermosensible » désigne un polymère qui présente un changement drastique et discontinu de ses propriétés physiques avec la température. En particulier, ce terme désigne des polymères à LCST (température inférieure critique de solubilité) ou LJGST (température supérieure critique de solubilité). Ces polymères sont des polymères particuliers dont la solubilité dans l’eau est modifiée au-delà ou en deçà d’une certaine température. Il s'agit des polymères présentant une température critique (ou point de trouble) définissant leur zone de solubilité dans l'eau. Cette température est appelée "LCST" (Lower Critical Solution Température) lorsqu'au-dessus de cette température, le polymère perd sa solubilité dans l’eau et devient soluble dans l'eau en dessous de cette température critique. Dans le cas où le polymère devient soluble au-delà d’une température critique, celle-ci est désignée par l’acronyme "UCST" (Upper Critical Solution Température). According to the invention, the term “heat-sensitive polymer” designates a polymer which exhibits a drastic and discontinuous change in its physical properties with temperature. In particular, this term designates polymers with LCST (critical lower solubility temperature) or LJGST (critical upper solubility temperature). These polymers are specific polymers whose solubility in water is modified above or below a certain temperature. These are polymers with a critical temperature (or cloud point) defining their zone of solubility in water. This temperature is called "LCST" (Lower Critical Solution Temperature) when above this temperature, the polymer loses its solubility in water and becomes soluble in water below this critical temperature. In the case where the polymer becomes soluble above a critical temperature, this is designated by the acronym "UCST" (Upper Critical Solution Temperature).
Selon un mode de réalisation, le polymère thermosensible est un polymère à LCST. According to one embodiment, the heat-sensitive polymer is an LCST polymer.
De préférence, le polymère thermosensible est choisi dans le groupe constitué des polyacrylamides N-alkyl-substitués comme le poly(N-isopropylacrylamide), le poly(N-isopropylméthacrylamide) ou le poly(N-éthylacrylamide), le poly((2- diméthylamino)éthyl méthacrylate), les poly(méthacrylates) à chaîne latérale oligoéthylène glycol, le poly(N-vinylcaprolactame), le poly(vinyl méthyl éther), le poly(2-alkyl-2-oxazoline), le Poly(propylène oxyde) et leurs mélanges. Preferably, the heat-sensitive polymer is chosen from the group consisting of N-alkyl-substituted polyacrylamides such as poly (N-isopropylacrylamide), poly (N-isopropylmethacrylamide) or poly (N-ethylacrylamide), poly ((2- dimethylamino) ethyl methacrylate), oligoethylene glycol side chain poly (methacrylates), poly (N-vinylcaprolactam), poly (vinyl methyl ether), poly (2-alkyl-2-oxazoline), Poly (propylene oxide) ) and their mixtures.
Le polymère thermosensible peut également être choisi dans le groupe constitué des copolymères d’acrylamide et d’acrylonitrile, des homopolymères de méthacrylamide, des polymères à dérivé ureido tels que le poly(allylamine)-co- poly(allylurée) et ses dérivés, des polymères zwittterioniques, tels que les poly(sulfobétaïne)s ou les poly(phosphorylcholine)s, et leurs mélanges.  The heat-sensitive polymer can also be chosen from the group consisting of copolymers of acrylamide and acrylonitrile, homopolymers of methacrylamide, polymers with ureido derivative such as poly (allylamine) -co-poly (allylurea), zwittterionic polymers, such as poly (sulfobetaine) s or poly (phosphorylcholine) s, and mixtures thereof.
Selon un mode de réalisation préféré, le polymère thermosensible est le poly(N-isopropylacrylamide). According to a preferred embodiment, the heat-sensitive polymer is poly (N-isopropylacrylamide).
Selon un mode de réalisation, le polysaccharide selon l’invention est au moins difonctionnel, c’est-à-dire qu’il est substitué par au moins deux groupes fonctionnels. Selon l’invention, le terme « groupe fonctionnel » désigne un groupe portant une fonction chimique, notamment choisie parmi les fonctions méthacrylates, acrylates, vinyliques, acrylamides, méthacrylamides, maléimides, vinylsulfones, acrylonitriles, azotures et amines. De préférence, le polysaccharide selon l’invention présente un degré de substitution (DS) compris entre 2% et 30% en moles. According to one embodiment, the polysaccharide according to the invention is at least difunctional, that is to say that it is substituted by at least two functional groups. According to the invention, the term “functional group” designates a group carrying a chemical function, in particular chosen from methacrylate, acrylate, vinyl, acrylamide, methacrylamide, maleimide, vinyl sulfone, acrylonitrile, azide and amine functions. Preferably, the polysaccharide according to the invention has a degree of substitution (DS) of between 2% and 30% by moles.
Le degré de substitution est défini comme le rapport molaire entre les groupes fonctionnels greffés, notamment les fonctions méthacrylates greffées, et l’unité de répétition du polysaccharide, ici nommément le dextran. Il peut être mesuré par RMN (Résonance Magnétique Nucléaire) du proton, en intégrant les protons vinyliques (5.8 et 6.25 ppm) et ceux du dextran anomérique (5 ppm).  The degree of substitution is defined as the molar ratio between the grafted functional groups, in particular the grafted methacrylate functions, and the repeat unit of the polysaccharide, here namely dextran. It can be measured by NMR (Nuclear Magnetic Resonance) of the proton, by integrating vinyl protons (5.8 and 6.25 ppm) and those of anomeric dextran (5 ppm).
De préférence, le polysaccharide est choisi dans le groupe constitué de l’acide hyaluronique, de l’héparosane, de la chondroïtine, du sulfate de chondroïtine, de l’héparine, du sulfate d’héparane, de l’alginate, de la pectine, du dextran, de la dextrine, du pullulane, du glycogène et de leurs mélanges. Preferably, the polysaccharide is chosen from the group consisting of hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate, pectin , dextran, dextrin, pullulan, glycogen and mixtures thereof.
Préférentiellement, le polysaccharide est le dextran, notamment fonctionnalisé, en particulier substitué par au moins deux groupes méthacrylates.  Preferably, the polysaccharide is dextran, in particular functionalized, in particular substituted by at least two methacrylate groups.
Selon l’invention, lesdites particules sont des réseaux de polymère gonflé de solvant. Le réseau est continu à travers la particule, on peut alors parler de particules « pleines ». Généralement, les particules contiennent plus de solvant que de polymère. Les particules selon l’invention peuvent également être désignées comme des particules colloïdales d’hydrogel. According to the invention, said particles are networks of polymer swollen with solvent. The network is continuous through the particle, we can then speak of "full" particles. Generally, the particles contain more solvent than polymer. The particles according to the invention can also be designated as colloidal hydrogel particles.
Les particules d’hydrogel ou de nanogel selon l’invention présentent de préférence un diamètre compris entre 10 nm et 10 pm. The hydrogel or nanogel particles according to the invention preferably have a diameter of between 10 nm and 10 μm.
La présente invention concerne également une série de particules d’hydrogel ou de nanogel telles que définies ci-dessus. The present invention also relates to a series of hydrogel or nanogel particles as defined above.
Ainsi, la présente invention est basée sur une nouvelle classe de particules, constituées de copolymères, dont l’une des entités présente une affinité pour la phase aqueuse contenant un polymère A et l’autre a une affinité pour la phase aqueuse contenant un polymère B. Ces copolymères sont qualifiés de double- hydrophiles ou biphiliques. Thus, the present invention is based on a new class of particles, consisting of copolymers, one of the entities having an affinity for the aqueous phase containing a polymer A and the other having an affinity for the aqueous phase containing a polymer B These copolymers are qualified as double hydrophilic or biphilic.
La présente invention concerne également l’utilisation d’au moins une particule d’hydrogel ou de nanogel telle que définie ci-dessus, pour la stabilisation d’une émulsion eau-dans-eau. Lorsque les particules de l’invention, constituées de copolymères, dont l’une des entités présente une affinité pour la phase aqueuse contenant un polymère A (polA) et l’autre a une affinité pour la phase aqueuse contenant un polymère B (polB), sont ajoutées au mélange ternaire eau-polA-polB, elles se placent préférentiellement à l’interface entre les deux phases aqueuses incompatibles et stabilisent des gouttes de A-dans-B ou B-dans-A selon que les particules préfèrent être dispersées dans la phase riche en B ou dans la phase riche en A respectivement. Cette préférence découle de la composition du copolymère et de sa structure. The present invention also relates to the use of at least one hydrogel or nanogel particle as defined above, for the stabilization of a water-in-water emulsion. When the particles of the invention, consisting of copolymers, of which one of the entities has an affinity for the aqueous phase containing a polymer A (polA) and the other has an affinity for the aqueous phase containing a polymer B (polB) , are added to the ternary water-polA-polB mixture, they are preferably placed at the interface between the two incompatible aqueous phases and stabilize drops of A-in-B or B-in-A depending on whether the particles prefer to be dispersed in the phase rich in B or in the phase rich in A respectively. This preference results from the composition of the copolymer and from its structure.
La présente invention concerne également une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère A et une phase aqueuse continue contenant un polymère B, et comprenant en outre, à l’interface de ces deux phases, des particules d’hydrogel ou de nanogel telles que définies ci- dessus. The present invention also relates to a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B, and further comprising, at the interface of these two phases, particles of hydrogel or nanogel as defined above.
Selon un mode de réalisation, la concentration des polymères A et B dans les phases aqueuses continue et dispersée est comprise entre 0,1% et 80% en masse par rapport à la masse de phase aqueuse continue ou dispersée, respectivement. According to one embodiment, the concentration of polymers A and B in the continuous and dispersed aqueous phases is between 0.1% and 80% by mass relative to the mass of continuous or dispersed aqueous phase, respectively.
Selon un mode de réalisation, la concentration des particules est comprise entre 0,005% et 30% en masse par rapport à la masse d’émulsion.  According to one embodiment, the concentration of the particles is between 0.005% and 30% by mass relative to the mass of emulsion.
La présente invention concerne également une émulsion eau-dans-eau telle que définie ci-dessus, dont la phase aqueuse dispersée est formée d’au moins une capsule comprenant un cœur contenant le polymère A et une écorce de particules d’hydrogel ou de nanogel telles que définies ci-dessus. The present invention also relates to a water-in-water emulsion as defined above, the dispersed aqueous phase of which is formed of at least one capsule comprising a core containing polymer A and a shell of hydrogel or nanogel particles. as defined above.
Selon un mode de réalisation, dans les émulsions eau-dans-eau telles que définies ci-dessus, le polymère A est un polymère hydrosoluble pouvant présenter une séparation de phase avec B. Un tel polymère peut être choisi dans la classe des polysaccharides (dextran, chitosan, guar...), des protéines (caséine, protéines globulaires...), des oligopeptides, des acides nucléiques (ADN, ARN) ou des polymères synthétiques (POE, PPO, acrylates, ...) par exemple. Selon un mode de réalisation, dans les émulsions eau-dans-eau telles que définies ci-dessus, dans laquelle le polymère B est un polymère hydrosoluble pouvant présenter une séparation de phase avec A. Un tel polymère peut être choisi dans la classe des polysaccharides (dextran, chitosan, guar...), des protéines (caséine, protéines globulaires...), des oligopeptides, des acides nucléiques (ADN, ARN) et des polymères synthétiques (POE, PPO, acrylates, ...) par exemple. According to one embodiment, in water-in-water emulsions as defined above, the polymer A is a water-soluble polymer which may have a phase separation with B. Such a polymer can be chosen from the class of polysaccharides (dextran , chitosan, guar ...), proteins (casein, globular proteins ...), oligopeptides, nucleic acids (DNA, RNA) or synthetic polymers (POE, PPO, acrylates, ...) for example. According to one embodiment, in water-in-water emulsions as defined above, in which the polymer B is a water-soluble polymer which can have a phase separation with A. Such a polymer can be chosen from the class of polysaccharides (dextran, chitosan, guar ...), proteins (casein, globular proteins ...), oligopeptides, nucleic acids (DNA, RNA) and synthetic polymers (POE, PPO, acrylates, ...) by example.
Selon un mode de réalisation, lorsque l’un des deux polymères de la particule possède une température critique de solution, basse par exemple (LCST), c’est-à- dire qu’il est soluble à basse température dans l’eau mais précipite pour une température supérieure à la LCST, les particules de copolymères passent de l’état gonflé à une température inférieure à la LCST à contracté au-delà de la LCST. La dispersabilité des particules dans A ou dans B est également fonction de l’état de gonflement de la particule. According to one embodiment, when one of the two polymers of the particle has a critical temperature of solution, low for example (LCST), that is to say that it is soluble at low temperature in water but precipitates for a temperature above the LCST, the copolymer particles pass from the swollen state at a temperature below the LCST to contracted beyond the LCST. The dispersibility of the particles in A or in B is also a function of the swelling state of the particle.
Si les particules ont une affinité pour A à basse température, les émulsions de B-dans-A seront stables à basse température. Une élévation de température au- delà de la LCST provoque la déstabilisation de l’émulsion. Si l’émulsion est réalisée à haute température, les émulsions de A dans B seront stables. Une diminution de la température en-deçà de la LCST provoque la déstabilisation de l’émulsion.  If the particles have an affinity for A at low temperature, the emulsions of B-in-A will be stable at low temperature. A rise in temperature beyond the LCST causes the emulsion to become destabilized. If the emulsion is produced at high temperature, the emulsions from A to B will be stable. A decrease in temperature below the LCST causes destabilization of the emulsion.
Selon un mode de réalisation, lorsque l’un des deux polymères est un polymère à UCST, c’est-à-dire qu’il est soluble à haute température dans l’eau mais précipite pour une température inférieure à la UCST, les particules de copolymères passent de l’état contracté à une température inférieure à la UCST à gonflé au-delà de la UCST. According to one embodiment, when one of the two polymers is a UCST polymer, that is to say that it is soluble at high temperature in water but precipitates for a temperature below the UCST, the particles of copolymers pass from the contracted state at a temperature lower than the UCST to swollen beyond the UCST.
Si les particules ont une affinité pour A à haute température, les émulsions de B-dans-A seront stables à haute température. Une diminution de température en- deçà de la UCST provoque la déstabilisation de l’émulsion. Si l’émulsion est réalisée à basse température, les émulsions de A dans B seront stables. Une diminution de la température au-delà de la UCST provoque la déstabilisation de l’émulsion.  If the particles have an affinity for A at high temperature, the emulsions of B-in-A will be stable at high temperature. A decrease in temperature below the UCST causes destabilization of the emulsion. If the emulsion is made at low temperature, the emulsions from A to B will be stable. A decrease in temperature beyond the UCST causes destabilization of the emulsion.
Ainsi, la présente invention est avantageuse en ce qu’il est possible de modifier de façon simple le sens de l’émulsion obtenue. En effet, le sens de l’émulsion obtenue diffère selon que l’on est au-dessous de la température de solubilité du polymère thermosensible selon l’invention ou au-dessus. Par exemple, en utilisant un polymère à LCST, à basse température, la phase aqueuse contenant des polysaccharides sera la phase dispersée alors qu’à haute température elle deviendra la phase continue. Ainsi, il est possible de choisir le sens de l’émulsion en adaptant la température de transition de phase des hydrogels ou nanogels à la température d’utilisation choisie. Thus, the present invention is advantageous in that it is possible to modify the direction of the emulsion obtained in a simple manner. Indeed, the direction of the emulsion obtained differs depending on whether one is below the solubility temperature of the heat-sensitive polymer according to the invention or above. For example, using an LCST polymer, at low temperature, the aqueous phase containing polysaccharides will be the dispersed phase whereas at high temperature it will become the continuous phase. Thus, it is possible to choose the direction of the emulsion by adapting the phase transition temperature of the hydrogels or nanogels to the chosen use temperature.
En outre, les émulsions eau-dans-eau susmentionnées, stabilisées à basse température, peuvent être déstabilisées à la demande en augmentant la température au-delà de la température de solubilité du polymère à LCST.  In addition, the above-mentioned water-in-water emulsions, stabilized at low temperature, can be destabilized on demand by increasing the temperature above the solubility temperature of the LCST polymer.
Enfin, les émulsions selon l’invention sont stables à la dilution, ce qui est généralement impossible pour des émulsions eau-dans-eau car la dilution provoque la diminution de la tension interfaciale entre les deux phases riches en A et B et finit, à forte dilution, de ramener les polymères dans le domaine monophasique.  Finally, the emulsions according to the invention are stable at dilution, which is generally impossible for water-in-water emulsions because the dilution causes the reduction of the interfacial tension between the two phases rich in A and B and ends up at high dilution, to bring the polymers back to the monophasic field.
La présente invention concerne également un procédé permettant de changer le sens des émulsions susmentionnées, par l’intermédiaire d’un changement de température, comme décrit ci-dessous. The present invention also relates to a method making it possible to change the direction of the abovementioned emulsions, by means of a temperature change, as described below.
Par exemple, il est possible de transformer une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère A et une phase aqueuse continue contenant un polymère B, en une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère B et une phase aqueuse continue contenant un polymère A.  For example, it is possible to transform a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B, into a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A.
Ainsi, pour une température inférieure à la LCST du polymère, on obtiendra préférentiellement une émulsion eau-dans-eau dont la phase continue est la phase riche en polymère A et la phase dispersée, la phase riche en polymère B, A ayant une affinité pour le polymère à LCST. Le chauffage de l’émulsion au repos au-delà de la LCST conduit à sa déstabilisation, c’est-à-dire le retour à la séparation de phases macroscopique. Si l’on maintient l’échantillon sous agitation, notamment à l’aide d’un vortex, pendant le chauffage, le chauffage au-delà de la LCST conduit à une inversion de phase de l’émulsion, à savoir que la phase riche en polymère A devient la phase dispersée alors que la phase riche en polymère B devient la phase continue.  Thus, for a temperature below the LCST of the polymer, there will preferably be obtained a water-in-water emulsion the continuous phase of which is the phase rich in polymer A and the dispersed phase, the phase rich in polymer B, A having an affinity for the LCST polymer. Heating the emulsion at rest beyond the LCST leads to its destabilization, that is to say the return to the macroscopic phase separation. If the sample is kept under stirring, in particular using a vortex, during heating, heating beyond the LCST leads to a phase inversion of the emulsion, namely that the rich phase in polymer A becomes the dispersed phase while the phase rich in polymer B becomes the continuous phase.
La présente invention concerne donc également un procédé de préparation d’une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère B et une phase aqueuse continue contenant un polymère A, comprenant une étape de traitement d’une émulsion telle que définie ci-dessus à une température inférieure à la LCST du polymère thermosensible. Ce procédé consiste donc à transformer une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère A et une phase aqueuse continue contenant un polymère B, en une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère B et une phase aqueuse continue contenant un polymère A. The present invention therefore also relates to a process for the preparation of a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A, comprising a step of treating an emulsion such as defined above at a temperature below the LCST of the heat-sensitive polymer. This process therefore consists in transforming a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B, into a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A.
La présente invention concerne également une capsule de polymère, comprenant : The present invention also relates to a polymer capsule, comprising:
- un cœur contenant une phase aqueuse et au moins un polymère A ; et - a core containing an aqueous phase and at least one polymer A; and
- une écorce de particules d’hydrogel ou de nanogel telles que définies ci- dessus, ladite écorce encapsulant totalement ledit cœur à sa périphérie. - a bark of hydrogel or nanogel particles as defined above, said bark completely encapsulating said heart at its periphery.
De préférence, ces capsules présentent une taille comprise 0,1 pm et 1 000 pm, de préférence comprise entre 500 nm et 100 pm.  Preferably, these capsules have a size of between 0.1 μm and 1000 μm, preferably between 500 nm and 100 μm.
La présente invention concerne également l’utilisation d’une capsule telle que définie ci-dessus pour l’encapsulation de molécules hydrosolubles, notamment choisies parmi les protéines, les enzymes ou les acides nucléiques tels que l’ADN ou l’ARN. The present invention also relates to the use of a capsule as defined above for the encapsulation of water-soluble molecules, in particular chosen from proteins, enzymes or nucleic acids such as DNA or RNA.
De telles capsules permettent ainsi l’encapsulation d’actifs alimentaires, cosmétiques ou pharmaceutiques.  Such capsules thus allow the encapsulation of food, cosmetic or pharmaceutical assets.
En particulier, ces capsules peuvent être utilisées pour l’encapsulation de colorants, de pigments, de quantum-dots, de marqueurs fluorescents, de vitamines, de peptides, d’oligonucléotides, d’antibiotiques, d’hormones, d’anticancéreux ou de filtres solaires.  In particular, these capsules can be used for the encapsulation of dyes, pigments, quantum dots, fluorescent markers, vitamins, peptides, oligonucleotides, antibiotics, hormones, anticancer agents or sun filters.
Selon un mode de réalisation, le cœur desdites capsules comprend en outre un actif.  According to one embodiment, the heart of said capsules also comprises an active ingredient.
A titre d’actif, on peut par exemple citer tout actif hydrosoluble avec un coefficient de partage différent de 1. Selon l’invention, ledit actif sera dans la phase dispersée. L’état de l’hydrogel sera choisi de façon à orienter le sens de l’émulsion pour que la phase dans lequel l’actif est dissous devienne la phase dispersée.  As an active ingredient, one can for example quote any water-soluble active ingredient with a partition coefficient different from 1. According to the invention, said active ingredient will be in the dispersed phase. The state of the hydrogel will be chosen so as to orient the direction of the emulsion so that the phase in which the active ingredient is dissolved becomes the dispersed phase.
Ainsi, comme expliqué ci-dessus, la présente invention propose un moyen d’encapsuler des molécules hydrosolubles fragiles. En particulier, dans un processus de séparation de phases liquide-liquide en milieu aqueux, ces dernières présentent une affinité plus importante pour une phase aqueuse de polymères que pour l’autre. En adaptant les conditions, elles peuvent donc se localiser sélectivement dans la phase dispersée. Si les gouttes de phase dispersée sont suffisamment stables, elles peuvent être concentrées sans risquer de coalescer ou diluées sans de disloquer. Il est donc possible de transporter ces molécules fragiles, tout en les protégeant du milieu extérieur via la capsule. Thus, as explained above, the present invention provides a means of encapsulating fragile water-soluble molecules. In particular, in a process of separation of liquid-liquid phases in an aqueous medium, the latter have a greater affinity for one aqueous phase of polymers than for the other. By adapting the conditions, they can therefore be located selectively in the dispersed phase. If the dispersed phase drops are sufficiently stable, they can be concentrated without the risk of coalescing or diluted without dislocating. It is therefore possible to transport these fragile molecules, while protecting them from the outside environment via the capsule.
La capsule d’hydrogel ou de nanogel peut ensuite être détruite à façon par l’application d’un simple stimulus tel que la température. Le processus de formation utilisé est non délétère pour des molécules fragiles puisque l’agitation mécanique reste modérée et que la stabilité repose uniquement sur des processus physiques (pas de réactivité, pas de changement de milieu tel que le pH ou la température). The hydrogel or nanogel capsule can then be destroyed by the application of a simple stimulus such as temperature. The formation process used is not deleterious for fragile molecules since the mechanical agitation remains moderate and that the stability rests solely on physical processes (no reactivity, no change of medium such as pH or temperature).
FIGURES FIGURES
Figure 1 : Image de TEM (microscopie électronique en transmission) des nanogels 1’ (cf. Tableau 1 ci-après) Figure 1: TEM image (transmission electron microscopy) of 1 ’nanogels (see Table 1 below)
Figure 2 : Influence du DS du dextran dans les nanogels sur l’évolution de leur diamètre en fonction de la température. Exemple pour les nanogels de composition contenant 37% massique de dextran-MA (dextran-méthacrylate), avec différents taux de substitution du dextran. Figure 2: Influence of the DS of dextran in nanogels on the evolution of their diameter as a function of temperature. Example for nanogels of composition containing 37% by mass of dextran-MA (dextran-methacrylate), with different rates of substitution of dextran.
Figure 3 : Stabilité des émulsions produites à partir de différents nanogels pour des compositions contenant 75% de phase aqueuse de dextran et 25% de phase PEO (gauche) et 25% de phase aqueuse de dextran et 75% de phase PEO - comparaison entre t=0 (haut) et t=1 jour (bas), à température ambiante. Pour chaque série, R est la référence sans nanogel, 0 l’émulsion stabilisée par des nanoparticules de pNIPAM sans dextran, 1 , 2 et 3 sont stabilisées par les nanogels 1 , 2 et 3 respectivement (voir tableau 1 ). Figure 3: Stability of emulsions produced from different nanogels for compositions containing 75% dextran aqueous phase and 25% PEO phase (left) and 25% dextran aqueous phase and 75% PEO phase - comparison between t = 0 (high) and t = 1 day (low), at room temperature. For each series, R is the reference without nanogel, 0 the emulsion stabilized by nanoparticles of pNIPAM without dextran, 1, 2 and 3 are stabilized by nanogels 1, 2 and 3 respectively (see Table 1).
Figure 4 : Emulsion dextran-dans-POE avec 0,04% m de nanogels T fluorescents après 3 jours à 25°C. Figure 4: Dextran-in-POE emulsion with 0.04% m of fluorescent T nanogels after 3 days at 25 ° C.
Figure 5 : Inverse du diamètre des gouttes en fonction de la quantité de nanogels pour des émulsions de dextran-dans-POE (nanogels 1’) à 25°C. Figure 5: Inverse of the diameter of the drops as a function of the amount of nanogels for dextran-in-POE emulsions (1 ’nanogels) at 25 ° C.
Figure 6 : Influence de la concentration en nanogels sur la stabilité des émulsions dextran-dans-PEO stabilisés par les nanogels T (37% massique de dextran DS 10), à 25°C. Suivi temporel de la transmittance des émulsions soumises à une force centrifuge de 300g (t=2 min, 6 min, 33h). Les émulsions contiennent 0, 0,01 %, 0,04% et 0,08% en poids en nanogels. Figure 6: Influence of the nanogel concentration on the stability of dextran-in-PEO emulsions stabilized by T nanogels (37% by mass of dextran DS 10), at 25 ° C. Temporal monitoring of the transmittance of emulsions subjected to a centrifugal force of 300g (t = 2 min, 6 min, 33 h). The emulsions contain 0, 0.01%, 0.04% and 0.08% by weight in nanogels.
Figure 7 : Influence de la concentration en nanogels sur la stabilité des émulsions PEO-dans-dextran stabilisés par les nanogels T (37% m de dextran DS 10), à 50°C. Suivi temporel de la transmittance des émulsions soumises à une force centrifuge de 300g (t=6 min, 33h). Les émulsions contiennent 0, 0,01%, 0,04%, 0,08% et 0,12% en poids de nanogels. Figure 8 : Comportement des émulsions dextran-dans-PEO stabilisées par les nanogels 1’ à 20°C suite à des dilutions allant de 1 à 5 fois le volume initial de l’émulsion. Suivi temporel de la transmittance des émulsions sous gravité normale. Les émulsions contiennent 0,1 % en poids de nanogels. Figure 7: Influence of the nanogel concentration on the stability of PEO-in-dextran emulsions stabilized by T nanogels (37% m of dextran DS 10), at 50 ° C. Temporal monitoring of the transmittance of emulsions subjected to a centrifugal force of 300g (t = 6 min, 33h). The emulsions contain 0, 0.01%, 0.04%, 0.08% and 0.12% by weight of nanogels. Figure 8: Behavior of dextran-in-PEO emulsions stabilized by nanogels 1 'at 20 ° C following dilutions ranging from 1 to 5 times the initial volume of the emulsion. Temporal monitoring of the transmittance of emulsions under normal gravity. The emulsions contain 0.1% by weight of nanogels.
EXEMPLES EXAMPLES
Exemple 1 : Préparation de particules selon l’invention Example 1: Preparation of particles according to the invention
De nouvelles nanoparticules constituées de copolymères de pNIPAM et de dextran sont synthétisées. Pour cela, le dextran est fonctionnalisé par des motifs méthacrylates. New nanoparticles made of copolymers of pNIPAM and dextran are synthesized. For this, dextran is functionalized by methacrylate patterns.
Le dextran-méthacrylate (Dex-MA) est obtenu par réaction de l’anhydride méthacrylique avec les hydroxyles du dextran, en milieu DMF/eau (Pitarresi, G.; Pierro, P.; Palumbo, F. S.; Tripodo, G.; Giammona,G. Biomacromolecules 2006, 7, 1302-1310 ; Auzely, dx.doi.org/10.1021/bm300324m | Biomacromolecules) selon le schéma suivant :  Dextran-methacrylate (Dex-MA) is obtained by reaction of methacrylic anhydride with the hydroxyls of dextran, in DMF / water medium (Pitarresi, G .; Pierro, P .; Palumbo, FS; Tripodo, G .; Giammona , G. Biomacromolecules 2006, 7, 1302-1310; Auzely, dx.doi.org/10.1021/bm300324m | Biomacromolecules) according to the following diagram:
Figure imgf000013_0001
Figure imgf000013_0001
anhydride anhydride
Figure imgf000013_0002
méthacrylique
Figure imgf000013_0002
methacrylic
Figure imgf000013_0003
Figure imgf000013_0003
A titre d’exemple, pour obtenir un taux de substitution de 10%, le dextran (40 kg/mol, Sigma-Aldrich) est introduit dans 175 mL de mélange eau/DMF 7/3 auquel est ajouté 2 g d’anhydride méthacrylique. Le pH est maintenu entre 8 et 9 par des ajouts de NaOH 0,5 M pendant 4 h à 4°C. A l’issue de la réaction, le milieu est dialysé via une membrane de dialyse (seuil de coupure 3500 g/mol) pendant 6 jours, en changeant l’eau au moins une fois par jour. La solution est ensuite lyophilisée. Le taux de substitution (DS), défini comme le rapport molaire entre les fonctions méthacrylates greffées et l’unité de répétition du dextran, est déterminé par RMN du proton en intégrant les protons vinyliques (5.8 et 6.25 ppm) et ceux du dextran anomérique (5 ppm). Des DS allant de 5 à 40% de la chaîne de dextran sont obtenus en modifiant la concentration de l’anhydride méthacrylique. For example, to obtain a substitution rate of 10%, dextran (40 kg / mol, Sigma-Aldrich) is introduced into 175 mL of water / DMF 7/3 mixture to which 2 g of methacrylic anhydride is added . The pH is maintained between 8 and 9 by additions of 0.5 M NaOH for 4 h at 4 ° C. At the end of the reaction, the medium is dialyzed via a dialysis membrane (cutoff threshold 3500 g / mol) for 6 days, changing the water at least once a day. The solution is then lyophilized. The substitution rate (DS), defined as the molar ratio between the grafted methacrylate functions and the repetition unit of dextran, is determined by proton NMR by integrating vinyl protons (5.8 and 6.25 ppm) and those of anomeric dextran ( 5 ppm). DS ranging from 5 to 40% of the dextran chain is obtained by modifying the concentration of methacrylic anhydride.
Les particules de pNIPAM-Dex sont synthétisées par polymérisation par précipitation. Le NIPAM (796 mg) et le dex-MA (entre 10 et 37% massiques par rapport au NIPAM) sont dissous dans 95 mL d’eau désionisée. Le milieu est chauffé à 70°C et dégazé. Puis le persulfate de potassium (67 mg dans 5 ml_) est introduit pour amorcer la polymérisation. Au bout d’une dizaine de minutes, le milieu devient turbide, indiquant la nucléation des particules. La réaction est conduite pendant 4 h. Dans le cas de la synthèse de particules fluorescentes, la fluorescéine-O- méthacrylate (0,1% mol. par rapport au NIPAM) est introduite dans la solution, en même temps que le NIPAM. The pNIPAM-Dex particles are synthesized by precipitation polymerization. NIPAM (796 mg) and dex-MA (between 10 and 37% by mass compared to NIPAM) are dissolved in 95 ml of deionized water. The medium is heated at 70 ° C and degassed. Then potassium persulfate (67 mg in 5 ml) is introduced to initiate the polymerization. After about ten minutes, the medium becomes turbid, indicating the nucleation of the particles. The reaction is carried out for 4 h. In the case of the synthesis of fluorescent particles, fluorescein-O-methacrylate (0.1 mol% relative to NIPAM) is introduced into the solution, at the same time as NIPAM.
Les particules issues de la synthèse sont filtrées, puis dialysées pendant 6 jours au minimum à l’aide d’une membrane de dialyse de seuil de coupure 100 kDa. Les particules sont de taille uniforme, comme en témoigne leur analyse à l’état sec par microscopie électronique en transmission (TEM Tecnai Biotwin (120 kV)) The particles from the synthesis are filtered and then dialyzed for a minimum of 6 days using a dialysis membrane with a cut-off threshold of 100 kDa. The particles are of uniform size, as evidenced by their analysis in the dry state by transmission electron microscopy (TEM Tecnai Biotwin (120 kV))
(Figure 1 ). Le diamètre des particules dH est mesuré par diffusion dynamique de la lumière (Zetasizer Nano S90, Malvern Instruments) équipée d’un laser He-Ne sous incidence de 90° par rapport au détecteur. Il est reporté dans le tableau 1 pour différentes compositions de particules. L’évolution du diamètre des particules en fonction de la température est donnée en Figure 2. Les nanogels sont gonflés à basse température et se contractent pour une température supérieure à 32°C, correspondant à la LCST du pNIPAM. (Figure 1 ). The diameter of the particles d H is measured by dynamic light scattering (Zetasizer Nano S90, Malvern Instruments) equipped with a He-Ne laser at 90 ° incidence relative to the detector. It is reported in Table 1 for different particle compositions. The evolution of the diameter of the particles as a function of the temperature is given in Figure 2. The nanogels are swollen at low temperature and contract for a temperature above 32 ° C, corresponding to the LCST of pNIPAM.
Tableau 1 : Exemples de nanogels obtenus pour différentes compositions. Chaque synthèse utilise une quantité définie de NIPAM, de Dex-MA dont le degré de substitution (DS) varie. Toutes les teneurs sont données pour 100 mL d’eau. La quantité d’amorceur, le persulfate de potassium, est de 2,5 mM. Le dextran de masse molaire Mn=40 kDa est utilisé. Les compositions des nanogels résultants sont déterminées par RMN du 1H. Après purification par dialyse, le taux de solide (extrait sec, exprimé en % m.) est mesuré par pesée après évaporation de la phase aqueuse. Table 1: Examples of nanogels obtained for different compositions. Each synthesis uses a defined quantity of NIPAM, of Dex-MA whose degree of substitution (DS) varies. All contents are given for 100 mL of water. The amount of initiator, potassium persulfate, is 2.5 mM. The dextran of molar mass Mn = 40 kDa is used. The compositions of the resulting nanogels are determined by 1 H NMR. After purification by dialysis, the level of solid (dry extract, expressed in% m.) Is measured by weighing after evaporation of the aqueous phase.
Figure imgf000014_0001
Exemple 2 : Préparation de systèmes ternaires
Figure imgf000014_0001
Example 2: Preparation of ternary systems
Les émulsions ont été préparées avec des solutions contenant pour l’une une fraction massique de poly(oxyde d’éthylène) POE (Sigma-Aldrich), de masse molaire moyenne en masse de 200 000 g/mol, et pour l’autre une fraction massique de dextran (Sigma-Aldrich), de masse molaire moyenne en masse comprise entre 450 000 et 650 000 g/mol. Toutes les solutions sont obtenues avec de l’eau milli-Q dont la résistivité est de 18,2 MW-cm. The emulsions were prepared with solutions containing, for one, a mass fraction of poly (ethylene oxide) POE (Sigma-Aldrich), of average molar mass by mass of 200,000 g / mol, and for the other a mass fraction of dextran (Sigma-Aldrich), of mass-average molar mass of between 450,000 and 650,000 g / mol. All solutions are obtained with milli-Q water with a resistivity of 18.2 MW-cm.
Deux solutions mères sont préparées à l’avance. La solution de POE a une concentration de 10% en masse et la solution de dextran est à 20% massique. La poudre de POE contenant des particules de silice, celles-ci ont été retirées en passant les solutions de POE à la centrifugeuse pendant 4h à 50 000 g. Two stock solutions are prepared in advance. The POE solution has a concentration of 10% by mass and the dextran solution is 20% by mass. The POE powder containing silica particles, these were removed by passing the POE solutions to the centrifuge for 4 h at 50,000 g.
Ces solutions ont été préparées par pesée de chaque solution, puis agitées pendant 24h avant utilisation afin d’assurer leur homogénéité. La solution mère de dextran a été obtenue par exemple en pesant 20g de polymère puis en complétant à 100 g total par ajout d’eau milli-Q.  These solutions were prepared by weighing each solution, then stirred for 24 hours before use to ensure their homogeneity. The stock solution of dextran was obtained for example by weighing 20 g of polymer and then making up to 100 g total by adding milli-Q water.
Les émulsions sont réalisées en mélangeant un rapport volumique 75%/25% de chaque phase. Un premier système contient une fraction volumique de 75% de la phase riche en POE et 25% de la phase riche en dextran. Pour le système à 75% volumique de POE, les masses du dextran et du POE sont de de 4% et 6% respectivement. Après séparation de phase, la concentration de la phase riche en POE est de 15,8% et celle en dextran est de 8,2%. The emulsions are produced by mixing a volume ratio of 75% / 25% of each phase. A first system contains a volume fraction of 75% of the phase rich in POE and 25% of the phase rich in dextran. For the 75% by volume POE system, the masses of dextran and POE are 4% and 6% respectively. After phase separation, the concentration of the phase rich in POE is 15.8% and that in dextran is 8.2%.
Les nanogels en solution sont ajoutés au mélange, de façon à obtenir au maximum 0,12% massique de nanogels dans le mélange total. L’aptitude à la dilution a été testée en rajoutant jusqu’à 5 fois le volume de l’émulsion en équivalent d’eau.  The nanogels in solution are added to the mixture, so as to obtain a maximum of 0.12% by mass of nanogels in the total mixture. The dilution ability was tested by adding up to 5 times the volume of the emulsion in water equivalent.
Les émulsions ont ensuite été mélangées à l’agitateur Fisherbrand pendant 10 s. Il a été vérifié que la force et le temps d’agitation n’avaient pas d’influence sur le comportement des émulsions. Exemple 3 : Emulsions réalisées à température ambiante The emulsions were then mixed in the Fisherbrand shaker for 10 s. It was verified that the strength and the agitation time had no influence on the behavior of the emulsions. Example 3 Emulsions Made at Room Temperature
Les essais réalisés avec les différents nanogels sont reportés sur la Figure 3.The tests carried out with the various nanogels are shown in Figure 3.
Seule l’émulsion dont la phase continue est le PEO avec le nanogel 1 est stable sur une journée. La phase PEO étant moins dense que la phase dextran, elle crème lors de la séparation de phases. Only the emulsion whose continuous phase is PEO with nanogel 1 is stable over one day. The PEO phase being less dense than the dextran phase, it creams during the phase separation.
Les essais de la Figure 3 montrent que : The tests in Figure 3 show that:
1 ) Les nanogels contenant la plus grande quantité de dextran stabilisent mieux les émulsions ;  1) Nanogels containing the greatest amount of dextran better stabilize emulsions;
2) Le système dextran-dans-PEO est stable alors que le système PEO-dans- dextran est instable ;  2) The dextran-in-PEO system is stable while the PEO-in-dextran system is unstable;
3) Quelle que soit leur composition, les particules ont tendance à migrer dans la phase PEO après déstabilisation.  3) Whatever their composition, the particles tend to migrate in the PEO phase after destabilization.
Des émulsions sont réalisées avec un lot de nanogels contenant 37% en Dex- MA de DS 10, dans lequel a été introduit un comonomère fluorescent (Fluorescein O-methacrylate, 0.1% mol par rapport au NIPAM). La préparation des émulions avec l’ajout de particules se fait comme décrit dans l’exemple 2. Les émulsions sont observées au microscope confocal. Il apparaît clairement que les nanogels se mettent à la surface des gouttes, formant ainsi des capsules. Ces capsules renfermant le dextran, plus lourd que le POE, sédimentent sous l’effet de la gravité, se concentrent mais elles restent intègres tout en étant au contact (Figure 4). Leur taille évolue peu au cours du temps. Elle varie d’environ 30% sur les 3 premiers jours puis se stabilise. Les gouttes sont donc très résistantes au stockage. Emulsions are produced with a batch of nanogels containing 37% Dex-MA of DS 10, into which a fluorescent comonomer (Fluorescein O-methacrylate, 0.1% mol relative to NIPAM) has been introduced. The preparation of the emulsions with the addition of particles is carried out as described in Example 2. The emulsions are observed under a confocal microscope. It is clear that nanogels are placed on the surface of the drops, thus forming capsules. These capsules containing dextran, heavier than POE, sediment under the effect of gravity, concentrate but they remain intact while being in contact (Figure 4). Their size changes little over time. It varies by around 30% over the first 3 days and then stabilizes. The drops are therefore very resistant to storage.
Il convient également de noter que la taille des gouttes est une fonction de la quantité de particules (Figure 5). L’évolution linéaire de l’inverse du diamètre en fonction de la quantité de particules est une signature caractéristique de la coalescence limitée des émulsions de Pickering. D’un point de vue pratique, ce phénomène constitue un levier pour contrôler la taille des gouttes et donc des capsules. It should also be noted that the size of the drops is a function of the quantity of particles (Figure 5). The linear evolution of the inverse of the diameter as a function of the quantity of particles is a signature characteristic of the limited coalescence of Pickering emulsions. From a practical point of view, this phenomenon constitutes a lever to control the size of the drops and therefore of the capsules.
La stabilité mécanique des émulsions dextran-dans-PEO est évaluée en soumettant les émulsions à une force centrifuge. Le LUMIsizer® donne l’analyse simultanée de la transmittance en fonction de l’altitude dans un tube, il permet de sonder les phénomènes de crémage/sédimentation, ainsi que la coalescence à force et centrifuge et température contrôlées. Ainsi une transmittance faible traduit une phase turbide et donc l’existence de gouttes. Elles peuvent être localisées sur une petite zone après sédimentation/crémage. Une transmittance élevée sur l’ensemble de la hauteur signifie que l’échantillon a déphasé ou que les interfaces ont disparues par coalescence. The mechanical stability of dextran-in-PEO emulsions is evaluated by subjecting the emulsions to a centrifugal force. The LUMIsizer ® gives the analysis simultaneous transmittance as a function of the altitude in a tube, it makes it possible to probe the phenomena of creaming / sedimentation, as well as force and centrifugal coalescence and controlled temperature. Thus a weak transmittance translates a turbid phase and therefore the existence of drops. They can be located in a small area after sedimentation / creaming. A high transmittance over the entire height means that the sample has out of phase or that the interfaces have disappeared by coalescence.
La figure 6 montre l’évolution de ce signal pour des émulsions stabilisées par le nanogel 1’, utilisé à différentes concentrations. Pour des concentrations supérieures à 0,04% massique, la persistance d’une turbidité dans le bas du tube après une très longue centrifugation (33h) à 300g témoigne d’une grande résistance des gouttes à la coalescence sous contrainte mécanique.  Figure 6 shows the evolution of this signal for emulsions stabilized by nanogel 1 ’, used at different concentrations. For concentrations greater than 0.04% by mass, the persistence of turbidity at the bottom of the tube after a very long centrifugation (33 h) at 300 g testifies to a great resistance of the drops to coalescence under mechanical stress.
Ainsi, les émulsions stabilisées par les nanogels ayant une composition de 37% m. en dextran de DS 10, assurent une grande stabilité à l’émulsion. La comparaison des lots 1 et 1’, de même composition mais de taille différente, montrent que cette stabilité est d’autant plus grande que la taille des nanogels est élevée. Thus, emulsions stabilized by nanogels having a composition of 37% m. in DS 10 dextran, provide great emulsion stability. The comparison of lots 1 and 1 ′, of the same composition but of different size, show that this stability is all the greater the higher the size of the nanogels.
Exemple 4 : Inversion de phase des émulsions EXAMPLE 4 Phase Reversal of the Emulsions
Les mêmes émulsions que dans la partie précédente ont subi le même traitement au LUMiSizer® mais à 50°C. Il s’en dégage une inversion des comportements par rapport à la température ambiante, comme avec les particules 1’. The same emulsions as in the previous section underwent the same treatment with LUMiSizer ® but at 50 ° C. This results in an inversion of behavior with respect to ambient temperature, as with particles 1 '.
Ainsi, les émulsions dextran-dans-POE sont peu stables. Les émulsions POE- dans-dextran sont, en revanche, très stables (Figure 7).  Thus, dextran-in-POE emulsions are not very stable. POE-in-dextran emulsions, on the other hand, are very stable (Figure 7).
Les résultats obtenus montrent donc que la température est un levier pour moduler l’affinité du nanogel pour l’une des phases aqueuses. Le sens de l’émulsion eau-dans-eau stabilisée par des nanogels est dicté par la préférence des particules pour l’une ou l’autre des phases. Les émulsions produites sont extrêmement stables à la température considérée. A fortiori, les émulsions peuvent être déstabilisées par l’application d’un stimulus qui réduit l’affinité des particules pour la phase continue et provoque leur dispersion dans la phase dispersée. Exemple 5 : Stabilité à la dilution The results obtained therefore show that temperature is a lever for modulating the affinity of the nanogel for one of the aqueous phases. The direction of the water-in-water emulsion stabilized by nanogels is dictated by the preference of the particles for one or the other of the phases. The emulsions produced are extremely stable at the temperature considered. A fortiori, the emulsions can be destabilized by the application of a stimulus which reduces the affinity of the particles for the continuous phase and causes their dispersion in the dispersed phase. Example 5: Stability at dilution
Une émulsion POE/dextran (75%/25%) a été préparée en rajoutant 0.1% m de particules 1’. A POE / dextran emulsion (75% / 25%) was prepared by adding 0.1% m of 1 ’particles.
Comme énoncé plus haut, ce système a montré une stabilité inédite à température ambiante. Après 1 journée de stabilisation du système, les émulsions ont été diluées de 1 à 5 fois en rajoutant de l’eau.  As stated above, this system has shown unprecedented stability at room temperature. After 1 day of stabilization of the system, the emulsions were diluted 1 to 5 times by adding water.
Les émulsions sont ensuite observées sur une période de 1 semaine  The emulsions are then observed over a period of 1 week
La quantification de cette stabilité à la dilution des émulsions a été réalisée sur le LUMiReader®, l’équivalent du LUMiSizer® mais travaillant à 1g, ainsi aucune contrainte mécanique supplémentaire ou aucune perturbation ne sont infligées aux émulsions (Figure 8). Il en ressort qu’une dilution jusqu’à 5 fois n’altère pas la stabilité des émulsions même après une semaine après la dilution. C’est le premier système eau-dans-eau qui à ce jour montre une telle stabilité à la dilution. The quantification of this dilution stability of the emulsions was carried out on the LUMiReader ® , the equivalent of the LUMiSizer ® but working at 1g, so no additional mechanical stress or any disturbance is inflicted on the emulsions (Figure 8). It appears that a dilution up to 5 times does not alter the stability of the emulsions even after a week after the dilution. It is the first water-in-water system which to date has shown such dilution stability.

Claims

REVENDICATIONS
1. Utilisation d’au moins une particule d’hydrogel constituée d’un copolymère d’au moins un polysaccharide et d’au moins un polymère thermosensible, pour la stabilisation d’une émulsion eau-dans-eau. 1. Use of at least one hydrogel particle consisting of a copolymer of at least one polysaccharide and at least one heat-sensitive polymer, for the stabilization of a water-in-water emulsion.
2. Utilisation selon la revendication 1 , dans laquelle le copolymère contient de 10% à 50% en poids de polysaccharide et de 50% à 90% en poids de polymère thermosensible. 2. Use according to claim 1, in which the copolymer contains from 10% to 50% by weight of polysaccharide and from 50% to 90% by weight of heat-sensitive polymer.
3. Utilisation selon la revendication 1 ou 2, dans laquelle le polysaccharide est choisi dans le groupe constitué de l’acide hyaluronique, de l’héparosane, de la chondroïtine, du sulfate de chondroïtine, de l’héparine, du sulfate d’héparane, de l’alginate, de la pectine, du dextrane, de la dextrine, du pullulane, du glycogène et de leurs mélanges. 3. Use according to claim 1 or 2, wherein the polysaccharide is selected from the group consisting of hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate , alginate, pectin, dextran, dextrin, pullulan, glycogen and mixtures thereof.
4. Utilisation selon l’une quelconque des revendications 1 à 3, dans laquelle le polymère thermosensible est choisi dans le groupe constitué des polyacrylamides N-alkyl-substitués comme le poly(N-isopropylacrylamide), le poly(N-isopropylméthacrylamide) ou le poly(N-éthylacrylamide), du poly((2- diméthylamino)éthyl méthacrylate), des poly(méthacrylates) à chaîne latérale oligoéthylène glycol, du poly(N-vinylcaprolactame), du poly(vinyl méthyl éther), du poly(2-alkyl-2-oxazoline), du poly(propylène oxyde), des copolymères d’acrylamide et d’acrylonitrile, des homopolymères de méthacrylamide, des polymères à dérivé ureido tels que le poly(allylamine)-co-poly(allylurée) et ses dérivés, des polymères zwittterioniques, tels que les poly(sulfobétaïne)s ou les poly(phosphorylcholine)s, et leurs mélanges. 4. Use according to any one of claims 1 to 3, in which the heat-sensitive polymer is chosen from the group consisting of N-alkyl-substituted polyacrylamides such as poly (N-isopropylacrylamide), poly (N-isopropylmethacrylamide) or poly (N-ethylacrylamide), poly ((2-dimethylamino) ethyl methacrylate), oligoethylene glycol side chain poly (methacrylates), poly (N-vinylcaprolactam), poly (vinyl methyl ether), poly (2 -alkyl-2-oxazoline), poly (propylene oxide), copolymers of acrylamide and acrylonitrile, homopolymers of methacrylamide, polymers with ureido derivative such as poly (allylamine) -co-poly (allylurea) and its derivatives, zwitterionic polymers, such as poly (sulfobetaine) s or poly (phosphorylcholine) s, and mixtures thereof.
5. Utilisation selon l’une quelconque des revendications 1 à 4, dans laquelle le diamètre de la particule d’hydrogel est compris entre 10 nm et 10 pm. 5. Use according to any one of claims 1 to 4, in which the diameter of the hydrogel particle is between 10 nm and 10 pm.
6. Emulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère A et une phase aqueuse continue contenant un polymère B, et comprenant en outre, à l’interface de ces deux phases, des particules d’hydrogel, lesdites particules d’hydrogel étant constituées d’un copolymère d’au moins un polysaccharide et d’au moins un polymère thermosensible. 6. Water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer A and a continuous aqueous phase containing a polymer B, and further comprising, at the interface of these two phases, hydrogel particles, said hydrogel particles consisting of a copolymer of at least one polysaccharide and at least one heat-sensitive polymer.
7. Emulsion eau-dans-eau selon la revendication 6, dans laquelle le copolymère contient de 10% à 50% en poids de polysaccharide et de 50% à 90% en poids de polymère thermosensible. 7. A water-in-water emulsion according to claim 6, in which the copolymer contains from 10% to 50% by weight of polysaccharide and from 50% to 90% by weight of heat-sensitive polymer.
8. Emulsion eau-dans-eau selon la revendication 6 ou 7, dans laquelle le polysaccharide est choisi dans le groupe constitué de l’acide hyaluronique, de l’héparosane, de la chondroïtine, du sulfate de chondroïtine, de l’héparine, du sulfate d’héparane, de l’alginate, de la pectine, du dextrane, de la dextrine, du pullulane, du glycogène et de leurs mélanges. 8. A water-in-water emulsion according to claim 6 or 7, in which the polysaccharide is chosen from the group consisting of hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate, pectin, dextran, dextrin, pullulan, glycogen and mixtures thereof.
9. Emulsion eau-dans-eau selon l’une quelconque des revendications 6 à9. Water-in-water emulsion according to any one of claims 6 to
8, dans laquelle le polymère thermosensible est choisi dans le groupe constitué des polyacrylamides N-alkyl-substitués comme le poly(N-isopropylacrylamide), le poly(N-isopropylméthacrylamide) ou le poly(N-éthylacrylamide), du poly((2- diméthylamino)éthyl méthacrylate), des poly(méthacrylates) à chaîne latérale oligoéthylène glycol, du poly(N-vinylcaprolactame), du poly(vinyl méthyl éther), du poly(2-alkyl-2-oxazoline), du poly(propylène oxyde), des copolymères d’acrylamide et d’acrylonitrile, des homopolymères de méthacrylamide, des polymères à dérivé ureido tels que le poly(allylamine)-co-poly(allylurée) et ses dérivés, des polymères zwittterioniques, tels que les poly(sulfobétaïne)s ou les poly(phosphorylcholine)s, et leurs mélanges. 8, in which the heat-sensitive polymer is chosen from the group consisting of N-alkyl-substituted polyacrylamides such as poly (N-isopropylacrylamide), poly (N-isopropylmethacrylamide) or poly (N-ethylacrylamide), poly ((2 - dimethylamino) ethyl methacrylate), poly (methacrylates) with oligoethylene glycol side chain, poly (N-vinylcaprolactam), poly (vinyl methyl ether), poly (2-alkyl-2-oxazoline), poly (propylene oxide), copolymers of acrylamide and acrylonitrile, homopolymers of methacrylamide, polymers with ureido derivative such as poly (allylamine) -co-poly (allylurea) and its derivatives, zwitterionic polymers, such as poly ( sulfobetaine) or poly (phosphorylcholine) s, and mixtures thereof.
10. Emulsion eau-dans-eau selon l’une quelconque des revendications 6 à10. Water-in-water emulsion according to any one of claims 6 to
9, dans laquelle le diamètre des particules d’hydrogel est compris entre 10 nm et 10 pm. 9, in which the diameter of the hydrogel particles is between 10 nm and 10 pm.
11. Emulsion eau-dans-eau selon l’une quelconque des revendications 6 à11. Water-in-water emulsion according to any one of claims 6 to
10, dans laquelle le polymère thermosensible est un polymère à LCST. 10, wherein the heat-sensitive polymer is an LCST polymer.
12. Procédé de préparation d’une émulsion eau-dans-eau, comprenant une phase aqueuse dispersée contenant un polymère B et une phase aqueuse continue contenant un polymère A, comprenant une étape de traitement d’une émulsion selon la revendication 1 1 à une température inférieure à la LCST du polymère thermosensible. 12. A method of preparing a water-in-water emulsion, comprising a dispersed aqueous phase containing a polymer B and a continuous aqueous phase containing a polymer A, comprising a step of treating an emulsion according to claim 1 1 to a temperature below the LCST of the heat-sensitive polymer.
13. Capsule de polymère, comprenant : 13. Polymer capsule, comprising:
- un cœur contenant une phase aqueuse et au moins un polymère A ; et - a core containing an aqueous phase and at least one polymer A; and
- une écorce de particules d’hydrogel, ladite écorce encapsulant totalement ledit cœur à sa périphérie, lesdites particules d’hydrogel étant constituées d’un copolymère d’au moins un polysaccharide et d’au moins un polymère thermosensible. - A bark of hydrogel particles, said bark completely encapsulating said core at its periphery, said hydrogel particles consisting of a copolymer of at least one polysaccharide and at least one heat-sensitive polymer.
14. Capsule de polymère selon la revendication 13, dans laquelle le copolymère contient de 10% à 50% en poids de polysaccharide et de 50% à 90% en poids de polymère thermosensible. 14. The polymer capsule according to claim 13, in which the copolymer contains from 10% to 50% by weight of polysaccharide and from 50% to 90% by weight of heat-sensitive polymer.
15. Capsule de polymère selon la revendication 13 ou 14, dans laquelle le polysaccharide est choisi dans le groupe constitué de l’acide hyaluronique, de l’héparosane, de la chondroïtine, du sulfate de chondroïtine, de l’héparine, du sulfate d’héparane, de l’alginate, de la pectine, du dextrane, de la dextrine, du pullulane, du glycogène et de leurs mélanges. 15. Polymer capsule according to claim 13 or 14, in which the polysaccharide is chosen from the group consisting of hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, sulfate of heparan, alginate, pectin, dextran, dextrin, pullulan, glycogen and mixtures thereof.
16. Capsule de polymère selon l’une quelconque des revendications 13 à16. Polymer capsule according to any one of claims 13 to
15, dans laquelle le polymère thermosensible est choisi dans le groupe constitué des polyacrylamides N-alkyl-substitués comme le poly(N-isopropylacrylamide), le poly(N-isopropylméthacrylamide) ou le poly(N-éthylacrylamide), du poly((2- diméthylamino)éthyl méthacrylate), des poly(méthacrylates) à chaîne latérale oligoéthylène glycol, du poly(N-vinylcaprolactame), du poly(vinyl méthyl éther), du poly(2-alkyl-2-oxazoline), du poly(propylène oxyde), des copolymères d’acrylamide et d’acrylonitrile, des homopolymères de méthacrylamide, des polymères à dérivé ureido tels que le poly(allylamine)-co-poly(allylurée) et ses dérivés, des polymères zwittterioniques, tels que les poly(sulfobétaïne)s ou les poly(phosphorylcholine)s, et leurs mélanges. 15, in which the heat-sensitive polymer is chosen from the group consisting of N-alkyl-substituted polyacrylamides such as poly (N-isopropylacrylamide), poly (N-isopropylmethacrylamide) or poly (N-ethylacrylamide), poly ((2 - dimethylamino) ethyl methacrylate), poly (methacrylates) with oligoethylene glycol side chain, poly (N-vinylcaprolactam), poly (vinyl methyl ether), poly (2-alkyl-2-oxazoline), poly (propylene oxide), copolymers of acrylamide and acrylonitrile, homopolymers of methacrylamide, polymers with ureido derivative such as poly (allylamine) -co-poly (allylurea) and its derivatives, zwitterionic polymers, such as poly ( sulfobetaine) or poly (phosphorylcholine) s, and mixtures thereof.
17. Capsule de polymère selon l’une quelconque des revendications 13 à17. Polymer capsule according to any one of claims 13 to
16, dans laquelle le diamètre des particules d’hydrogel est compris entre 10 nm et 10 pm. 16, in which the diameter of the hydrogel particles is between 10 nm and 10 μm.
18. Utilisation d’une capsule selon la revendication 13 à 17, pour l’encapsulation de molécules hydrosolubles, notamment choisies parmi les protéines, les enzymes ou les acides nucléiques tels que l’ADN ou l’ARN. 18. Use of a capsule according to claim 13 to 17, for the encapsulation of water-soluble molecules, in particular chosen from proteins, enzymes or nucleic acids such as DNA or RNA.
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