WO2013045504A1

WO2013045504A1 - Perfluorinated nanoemulsions, method for preparing same, and use thereof as a contrast agent

Info

Publication number: WO2013045504A1
Application number: PCT/EP2012/068984
Authority: WO
Inventors: Caroline ROBIC; Samy CHADEL; Véronique SCHMITT; Fernando Leal Calderon; Marc Port; Olivier Lambert
Original assignee: Guerbet
Priority date: 2011-09-26
Filing date: 2012-09-26
Publication date: 2013-04-04
Also published as: FR2980365A1; FR2980365B1

Abstract

The present application relates to an oil-in-water emulsion, including: an aqueous phase; a fluorinated oil phase; and a surfactant at the interface between the aqueous phase and the fluorinated oil phase, the surfactant being an anionic surfactant, as well as to a method for preparing same, and to the use thereof as a contrast agent.

Description

PERFLUOROUS NANOEMULSIONS, PROCESS FOR THEIR PREPARATION, AND THEIR USE AS A CONTRAST AGENT.

The present invention relates to new nanoemulsions and methods for the preparation of nanoemulsions, and their use as contrast agents, especially in MRI. In the field of diagnostic imaging, a large number of studies have concerned lipid nanosystems of the emulsion type. Typically the emulsions used are in the form of vesicles prepared using lipid constituents (oil in particular) and surfactants (also called surfactants) serving as interface between the aqueous phase and the lipid core of the nanodroplet. The oil-in-water lipid emulsions incorporate a lipophilic oily phase, forming droplets in aqueous solution.

A particular class of emulsions described in particular in WO 03/062198 or US Pat. No. 6,676,963 is that of fluorinated nanoemulsions comprising, integrated within the lipid vesicles, fluorinated compounds comprising fluorine atoms F19 used for Magnetic Resonance Imaging MRI. . Indeed fluorine has particular interest compared to the MRI of the proton to be almost absent in the biological systems in the free state which allows it to be recognized as an excellent quantitative probe in the fluorine form F19 . The heart is formed of a fluorinated oil, and surrounded by a lipid layer formed by a surfactant (lecithin for example).

These fluorinated emulsions may furthermore comprise a very large number of paramagnetic metal complexes, in particular lanthanides, to associate the MRI of fluorine 19F and proton H. Thus, fluorine emulsions for MRI incorporating chelates capable of complexing are known. lanthanides, especially gadolinium. The chelates used are in particular derivatives of DTPA, DOTA, D03A, HPD03A and other chelates widely described in the prior art. These hydrophilic chelates are rendered lipophilic by grafting them with a lipophilic zone such as a phospholipid, which makes it possible to integrate them into the lipid membrane that forms the lipid surfactant of the composition. Several thousand (about 5000 to 100,000) of these complexes are integrated in the lipid membrane of these vesicles, which allows to obtain a high relaxivity (MRI signal) for a detection of the physiological zone studied and to modify the relaxation time of the 19F. The hydrophilic part (the hydrophilic part which represents the chelate to which a lipophilic group is attached so as to take the lipophilic chelate) is located on the outer surface of the nanodroplets, in contact with the aqueous phase of the nanodroplet solution.

Also known are oil-in-water and non-fluorinated emulsions, including lanthanide chelates for MRI only of the proton. In addition, in order to obtain a specific signal of pathological zones, for example associated with overexpression of a marker of these zones (for example receptors), targeting molecules (or biovectors, for example peptide having an affinity for the receptor ) were grafted onto the nanodroplets of these fluorinated emulsions. WO 03/062198 describes in particular the use of peptidomimetic compounds to target integrins overexpressed in tumor areas. For incorporation into the lipid membrane, biovectors are rendered lipophilic by combining them with lipophilic chains.

However, despite promising advances, fluorinated contrast agents and optionally vectorized described have not yet fully demonstrated their clinical effectiveness, and pose difficulties including stability over time. In particular a shelf life of more than 9 months is difficult to achieve, while the desired stability is at least one year and preferably of the order of 2 to 3 years.

A complex technical problem remains the optimization of constituents, including oil and surfactants. These are direct emulsions of oil-in-water (O / W) type, the dispersed phase of which is lipophilic and the continuous hydrophilic phase. The immiscibility of the two phases results in the existence of interface between the water and the oil, which costs energy to the system. This energy is quantified by the interfacial tension γ (energy per unit area). Thanks to their double affinity, the amphiphilic molecules used as surfactant (or surfactant) can be adsorbed at the interfaces and thus reduce the interfacial tension. The amphiphilic molecules particularly considered here are phospholipids.

When mechanical energy is supplied to the system, the amount of interface between the two immiscible fluids is increased and one of the two phases is fragmented into droplets. The presence of the amphiphilic molecules at the interface will slow down the return to phase separation (see Figure 1).

These surfactants can be of different natures: conventional surfactant, polymer, phospholipid, particle ... They induce steric or electrostatic repulsions between the drops.

Generally, the stabilizers are surfactants and are not anchored irreversibly at the interface. Above the critical micelle concentration (CMC) amphiphilic molecules self-assemble in the continuous phase and they adopt various forms depending on several factors. There is then a dynamic equilibrium with continuous exchange between the adsorbed molecules at the interface and in excess in the continuous phase. There are two mechanisms of destruction of emulsions (irreversible destabilization) that make the system evolve towards the macroscopic phase separation. These two instabilities are the coalescence and ripening of Ostwald.

The coalescence corresponds to the rupture of the film of surfactants separating two drops in contact, thus causing the fusion of these (See Figure 2).

The Ostwald ripening is due to the transfer of the dispersed phase through the continuous phase, from the small drops to the larger ones under the effect of their internal pressure difference: the pressure in the drops is indeed greater. that their radius is weak (Laplace's law: PLaplace = 2y / R). This transfer takes place in order to equalize the internal pressures (See Figure 3).

Ostwald ripening dominates for small drops while coalescence is the predominant mechanism for large drops. During the destabilization of the nanoemulsion, the increase in the size of the drops is accompanied by a decrease in the amount of water-oil interface S: (S = 6V / D), where V is the volume of oil and D the diameter of the droplets and desorption of the stablizer.

In an emulsion, the two mechanisms of destabilization take place, with Ostwald maturing and then coalescing over time.

The objective is to achieve direct nanoemulsions stable over time, whose interfaces are covered by a mixture of natural phospholipids (PL) and modified phospholipids (PLM).

The applicant has succeeded in obtaining nanoemulsions including droplets where appropriate vectorized:

- stable enough to be produced and kept for a long time (several months to several years), in particular by limiting the problems of coalescence of the lipid droplets between them

- sufficiently stable in vivo not to be degraded

- adapted to pharmacokinetics

sufficiently effective in terms of signal for clinical imaging (MRI in particular) in the patient.

For this purpose, the application provides oil-in-water nanoemulsions comprising an aqueous phase and a fluorinated oil phase.

For the purposes of the present application, the term "nanoemulsion" means that the droplet size is between 1 and 1000 nm. The size of the droplets is typically 50 to 400 nm, advantageously 100 to 350 nm, in particular 150 to 300 nm, in particular 200 to 250 nm. The droplets are also called "drops" below. The droplets are small enough to allow them to circulate in biological media without degradation of the product. The size of the droplets can be measured by photon correlation spectroscopy (PCS for "photon correlation spectroscopy" in English).

In the nanoemulsions according to the invention, the aqueous phase is advantageously water or a pharmaceutically acceptable aqueous solution such as a saline solution or a buffer solution. The aqueous phase may for example be water comprising 1 to 4%, preferably 2 to 3%, more preferably 2.5% by weight of glycerol relative to the weight of the aqueous phase.

In the nanoemulsions according to the invention, for the fluorinated oil phase, any suitable fluorinated oil may be used, in particular the fluorinated oils already used in medical imaging. The fluorinated oil is in particular an oil chosen from oils including linear or branched, cyclic or polycyclic, saturated or unsaturated perfluorocarbons, perfluorinated cyclic tertiary amines, perfluoro esters or thioesters, haloperfluorocarbons and analogous or derived analogous compounds. Advantageously, at least 60% of the hydrogen atoms of the corresponding hydrocarbon oil are replaced by a fluorine atom. Typically these fluorinated oils are chains of 2 to 16 atoms, perfluoroalkanes, bis (perfluoroalkyl) alkenes, perfluoroethers, perfluoroamines, perfluoroalkyl bromides, perfluoroalkyl chlorides.

According to advantageous embodiments, the lipid nanodroplet includes perfluorocarbons as described in US Pat. No. 5,958,371, the liquid nanoemulsion containing nanodroplets comprising a perfluorocarbon with a relatively high boiling point (for example between 30 and 150 ° C., preferably between 50 and 150 ° C. ° C) surrounded by a coating composed of a lipid and / or a surfactant.

The perfluorinated oils for perfluorocarbon nanoemulsions for MRI imaging are recalled in particular in documents US 6,676,963, US 4,927,623, US 5,077,036, US 5,1 14,703, US 5,171,755, US 5,304,325, US 5,350,571, US 5,393,524, US 5,403,575; especially oils: perfluorooctylbromide PFOB, C8F17Br (PFOB or perfluorobron) perfluorooctyléthane (C8F17C2H5 PFOE) Perfluorodecalin FDC, perfluorooctane C8F18, perfluorodichlorooctane bromide, perfluoro-n-octyl, perfluoroheptane, perfluorodecane C10F22, perfluorododecyl bromide C10F22Br PFDB, perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine, perfluorotributylamine, perfluorodimethylcyclohexane, perfluorotrimethylcyclohexane, perfluorodicyclohexyl ester, perfluoro-n-butyltetrahydrofuran.

Included in the definition of fluorinated oils are oils of formula C _n F _{2n +} 1 X, XC _n F _2n X, where n is an integer ranging from 2 to 10, X = Br, Cl or I

in particular: 1-bromo-F-butane (nC ₄ F ₉ Br), 1-bromo-F-hexane (nC ₆ F ₃ Br), 1-bromo-F-heptane (nC ₇ F ₁₅ Br), 1, 4- dibromo-F-butane and 1,6-dibromo-F-hexane. Fluorinated compounds with chlorinated substituents are also included, for example: perfluorooctyl chloride (nC ₈ F ₇ Cl), 1,8-dichloro-F-octane (n-C ₈ F ₆ Cl), 1, 6-dichloro-F hexane (n-CIC ₆ F ₁₂ Cl), and 1,4-dichloro-F-butane (n-Cl C ₄ F ₈ Cl).

Fluorinated oils of formula C _n F _{2n + 1} OC _m F _{2m + 1} , C _n F _{2n +} i CH = CHC _m F _{2m + 1 are also included} , for example: C ₄ F ₉ CH = CHC ₄ F ₉ (F -44E), iC ₃ F ₉ CH = CHC ₆ F ₁₃ (F-36E), C ₆ F ₁₃ CH = CHC ₆ F ₁₃ (F-66E)) where n and m are identical or different, and are integers between 2 and 12.

Also includes polycyclic or cyclic compounds such as: d ₀ F ₁₈ (F- Decalin or Perfluorodecalin), and mixtures of perfluoroperhydrophenanthrene and perfluoro n-butyldecalin.

Perfluorinated amines, such as: F-tripropylamine ("FTPA"), F-tributylamine ("FTBA"), F-4-methyloctahydroquinolizine ("FMOQ"), FN-methyl-decahydroisoquinoline ("FMIQ"), are also included. FN-methyldecahydroquinoline ("FHQ"), FN-cyclohexylpyrrolidine ("FCHP"), F-2-butyltetrahydrofuran ("FC-75" or "FC-77").

The surfactant of the nanoemulsion according to the invention is an anionic surfactant. Thus, the invention relates to an oil-in-water nanoemulsion, especially for MRI, comprising:

an aqueous phase

a fluorinated oil phase

a surfactant at the interface between the aqueous phase and the fluorinated oil phase,

the surfactant being an anionic surfactant.

The applicant has indeed found that the use of at least one anionic surfactant (whose hydrophilic part is negatively charged, typically with a carboxylate or sulfonate group) makes it possible to increase the stability of the nanoemulsion.

The anionic surfactant is chosen from surfactants known and usable according to the Pharmacopoeia, in particular SDS (sodium dodecyl sulphate). In one embodiment, the anionic surfactant is a fatty acid.

The pH of the continuous aqueous phase is chosen so that the carboxylic acid function of the fatty acid is in the carboxylate form, taking into account the pKa of the carboxylic acid functions of the fatty acids (generally between 4 and 5). The skilled person is able to adjust the pH of the continuous aqueous phase in this sense. The continuous aqueous phase generally has a pH of about 7. The term "fatty acid" refers to aliphatic carboxylic acids having a carbon chain of at least 6 carbon atoms. Natural fatty acids have a carbon chain of 4 to 28 carbon atoms (usually an even number). It is called "long chain fatty acid" for a length of 14 to 22 carbons and "very long chain", if there are more than 22 carbons. On the contrary, we speak of "short-chain fatty acid" for a length of 6 to 10 carbons, in particular 8 or 10 carbon atoms. The skilled person knows the associated nomenclature and in particular uses:

- Cn-Cp to designate a range of fatty acids in On to Cp

- and Cn + Cp, the total of Cn fatty acids and Cp fatty acids

For example :

- the fatty acids between 14 and 18 carbon atoms are written C14-C18 fatty acids

the total of the C16 fatty acids and of the C18 fatty acids is written as C16 + C18

Preferably, the anionic surfactant may be an unsaturated fatty acid, preferably monounsaturated, especially C16-C24, advantageously C16-C18, more advantageously C18, in particular oleic acid (in the form of carboxylate).

The nanoemulsion typically comprises from 0.05 to 5% of anionic surfactant, especially from 0.2 to 3% of anionic surfactant, in particular 0.25, 0.5, 1, 1, 1 or 2% of anionic surfactant (for example: example of oleic acid), relative to the total weight of the fluorinated oil.

In one embodiment, said nanoemulsion further comprises a compatibilizer forming an additional layer interposed between the fluorinated oil phase and the surfactant layer.

According to preferred embodiments, the compatibilizing agent is a hydrocarbon-based (non-fluorinated) oil comprising at least 70%, advantageously at least 80%, advantageously at least 95% by weight, in particular at least 97%, of saturated fatty acids. C6-C18, advantageously C6-C14, more preferably C6-C10.

Very advantageously, the hydrocarbon oil comprises less than 10%, preferably less than 5% of unsaturated fatty acids, in particular less than 5%, and preferably less than 2%, less than 1% unsaturated fatty acids. C14-C18 or C14-C22.

For example the oil is MIGLYOL ^® ,

(R = C ₈ + C ₁₀ )> 95%

P03040

or one of its known derivatives, for example MIGLYOL ^® 810 or MIGLYOL ^® 812 (caprylic / capric triglyceride), MIGLYOL ^® 818 (caprylic / capric / linoleic triglyceride), MIGLYOL ^® 612 (glyceryl trihexanoate), others MIGLYOL ^® derivatives propylene glycol dicaprylate dicaprate.

For example Miglyol ^® 812 has the following composition:

Caproic acid (C ₆ -o): max 2%

Caprylic acid (C ₈ -o): 50 to 65%

Caprique acid (C _{1 0} -o): 30 to 45%

Lauric acid (C12-0): max 2%

Miretic acid (C-0): max 1%

Linoleic acid (C18-2):

According to variants, the saturated hydrocarbon oil is a mixture of saturated oils each comprising at least 70%, preferably at least 80, 90, 95% of saturated fatty acids of 6 to 10 carbon atoms.

According to embodiments, the saturated hydrocarbon oil is a saturated oil comprising at least 70%, preferably at least 80, 90, 95% of saturated fatty acids of 12 to 18 carbon atoms, or comprising a mixture of saturated oils. each comprising at least 70%, preferably at least 80, 90, 95% saturated fatty acids of 12 to 18 carbon atoms.

Preferably, the saturated fatty acids of the saturated oils used by the applicant are used in the form of mono-, di- or triglycerides, preferably triglycerides.

Preferably, the hydrocarbon oil of the applicant's nanoemulsions comprises saturated fatty acids in the following variants:

C6-C18> 70%, preferably C6-C18> 80%, preferably C6-C18> 95%, and more preferably C6-C18> 98%

C6-C14> 70%, preferably C6-C14> 80%, preferably C6-C14> 95%, and more preferably C6-C14> 98%

C8 + C10> 70%, preferably C8 + C10> 80%, preferably C8 + C10> 95%, and still preferably C8 + C10> 98%

- C8 between 40 and 70%, preferably 50 to 65% and / or C10 between 20 and 50%, preferably 30 to 45%, the total C8 + C10 being greater than 80%. For simplicity, it is stated that the nanoemulsion comprises a surfactant. It is clear to those skilled in the art that it is at least one surfactant (for example a surfactant mixture) forming a surfactant layer between the oily phase and the aqueous phase, and also referred to as "total surfactants". in the application. For example, the nanoemulsion may comprise an anionic surfactant and an amphiphilic lipid, and / or a pegylated lipid as defined hereinafter.

The surfactant generally comprises one or more amphiphilic lipids, which comprise a hydrophilic part and a lipophilic part. They are generally chosen from compounds whose lipophilic part comprises a saturated or unsaturated, linear or branched chain having from 8 to 30 carbon atoms.

Advantageously, the amphiphilic lipid is a phospholipid, preferably chosen from: phosphatidylcholine dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol. Lecithin is a preferred amphiphilic lipid. Lipoid, for example E80 is also a preferred lipid. The amphiphilic lipid may also be chosen also from cholesterols, lysolipids, sphingomyelins, tocopherols, glucolipids, stearylamines, cardiolipins of natural or synthetic origin; molecules composed of a fatty acid coupled to a hydrophilic group by an ether or ester function such as sorbitan esters such as, for example, sorbitan monooleate and monolaurate; polymerized lipids; sugar esters such as mono- and di-laurate, mono- and di-palmitate, sucrose mono- and distearate; said amphiphilic lipids may be used alone or in mixtures.

Advantageously, the amphiphilic lipid is a lipoid, in particular lipoid E80. According to a particular embodiment, all or part of the amphiphilic lipid may have a reactive function, such as a maleimide, thiol, amine, ester, oxyamine or aldehyde group. The presence of reactive functions allows the grafting of functional compounds at the interface between the nanodroplets and the continuous aqueous phase.

It will be possible to use the surfactant layer, in addition to the amphiphilic lipid, in a non-mandatory manner, and in particular to act on the stealthiness of the product in the body, pegylated lipids that is to say carriers of polyethylene oxide (PEG) groups, such as polyethylene glycol / phosphatidyl ethanolamine (PEG-PE). For the purposes of the present application, the term "polyethylene glycol" PEG generally denotes compounds comprising a chain -CH ₂ - (CH ₂ -O-CH ₂ ) _k -CH ₂ OR ₃ in which k varies from 2 to 100 (for example 2, 4, 6, 10, 50), and R 3 is chosen from H, alkyl or - (CO) Alk, the term "alkyl" or "alk" denoting a linear or branched hydrocarbon aliphatic group having about 1 to 6 carbon atoms in the chain. The term "polyethylene glycol" as used herein includes aminopolyethylene glycol compounds, such as DSPE-PEG (DSPE: Distearoyl phosphatidyl ethanolamine). PEGs 350, 750, 2000, 3000, 5000, modified by the addition of amphiphilic groups to be inserted into the surfactant layer of the nanodroplet, include:

-1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy (Polyethylene glycol) - 350]

-1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy (Polyethylene glycol) -550],

- 1, 2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy (Polyethylene glycol) - 750]

In particular, the pegylated lipid will be used:

In a preferred embodiment, the surfactant comprises a mixture of lipoid (amphiphilic lipid) and DSPE-PEG (pegylated lipid), in particular Lipoid E80 and DSPE-PEG 2000.

Advantageously, the surfactant layer includes at least one amphiphilic targeting ligand of a physiological zone (also called a biovector), the amphiphilic targeting ligand representing 0.1 to 10 mol% of the total of the surfactant layer, advantageously 0.05 to 5%. %, especially 1 to 2%.

The droplets of the nanoemulsions typically each comprise a number of amphiphilic targeting ligands of the order of 100 to 5000, in particular 500 to 2000, which allows the targeting according to the affinity and the multivalence of the targeting ligand to be effective. According to very advantageous embodiments, the oil-in-water nanoemulsion comprises:

an aqueous phase, preferably representing 29.3 to 80% by weight of the composition, advantageously 55 to 65%, more advantageously 58 to 62%, a fluorinated phase comprising at least one fluorinated oil, representing 19.3 to 70% by weight of the composition, advantageously 35 to 45%, more preferably 37 to 42%,

a surfactant (forming the surfactant layer) at the interface between the aqueous and fluorinated phases,

a compatibilizing agent forming a layer between the fluorinated oil phase and the surfactant layer,

the total content of surfactant by weight relative to the oil being between 3 and 15%, advantageously between 6 and 12%;

the total content of surfactant by weight relative to the composition being between 0.6 and 10%, advantageously between 1 and 3%;

the total content of compatibilizing agent being between 0.1% and 5% by weight relative to the aqueous phase.

Particularly advantageously, the nanoemulsions of the applicant oil in water, especially for MRI, include:

an aqueous phase

a fluorinated oil phase

a surfactant at the interface between the aqueous phase and the fluorinated oil phase, the fluorinated phase comprising a first fluorinated oil and at least a second fluorinated oil.

The applicant has indeed found that a mixture of fluorinated oils increases the stability of the nanoemulsion. Advantageously, the first fluorinated oil represents between 70 and 95% by weight of the fluorinated oil phase and the second fluorinated oil represents 5 to 30% by weight of the fluorinated oil phase.

Advantageously, the fluorinated oils are chosen from the fluorinated oils described in the present application.

Preferably, the second fluorinated oil is an oil with a longer molecular chain than the first fluorinated oil (that is to say comprising more carbon atoms, and whose molecular mass is generally higher).

Very advantageously, the first fluorinated oil is PFOB, the second fluorinated oil is an oil with a longer molecular chain, preferably the perfluorohexadecane PFHD or perfluorodecylbromide PFDB. For example, the ratio is 10% by weight in PFHD or PFDB and 90% by weight in PFOB.

This embodiment is particularly advantageous in the case of the nanoemulsions described in the application which comprise a compatibilizing agent forming a layer between the fluorinated oil phase and the surfactant layer. The invention thus relates, in one embodiment, to an oil-in-water nanoemulsion, in particular for MRI, comprising:

an aqueous phase

a fluorinated oil phase

a surfactant at the interface between the aqueous phase and the fluorinated oil phase, the surfactant forming a surfactant layer between the aqueous phase and the fluorinated oil phase,

said nanoemulsion further comprising a compatibilizing agent forming an additional layer interposed between the fluorinated oil phase and the surfactant layer,

the fluorinated phase comprising a first fluorinated oil and at least one second fluorinated oil.

In another aspect, the invention relates to an oil-in-water nanoemulsion for MRI comprising:

an aqueous phase

a fluorinated oil phase

said nanoemulsion further comprising at least one stabilizing agent selected from proteins and polysaccharides, preferably proteins.

The applicant has indeed found that the use of at least one protein or polysaccharide makes it possible to increase the stability of the nanoemulsion. The protein is chosen from the proteins known and usable according to the Pharmacopoeia, in particular casein or lactoglobulin and preferentially lactoglobulin. The polysaccharide is preferably starch. Preferably, the protein or the polysaccharide is used in a proportion of between 0.1 and 5% by weight relative to the aqueous phase.

In one embodiment, the nanoemulsion comprises a compatibilizing agent as defined above.

In one embodiment, the oily phase of the nanoemulsion comprises a first fluorinated oil and at least one second fluorinated oil. In one embodiment, the nanoemulsion comprises a compatibilizing agent as defined above and the oily phase of the nanoemulsion comprises a first fluorinated oil and at least one second fluorinated oil. According to another aspect, the invention relates to a process for preparing oil-in-water emulsions, in particular for MRI, comprising an aqueous phase, a fluorinated oil phase, a surfactant at the interface between the aqueous and lipid phases, the process comprising Steps :

a) mixing the constituents of the aqueous phase, the fluorinated phase, and the surfactant, so as to obtain a nanoemulsion

b) at least one washing step of the nanoemulsion prepared in a).

In embodiments, during the washing step, the nanoemulsion is centrifuged in order to separate the droplets from the continuous phase. Since the oil is heavier than water, the droplets are the opaque and viscous sub-oil, while the continuous phase containing water and phospholipids form the supernatant. This supernatant is replaced by a continuous phase (water or salt water) without phospholipids.

According to another aspect, the invention relates to the use of a nanoemulsion as defined above or of a nanoemulsion obtainable by the method as defined above as a contrast agent, in particular for detection. magnetic resonance imaging (MRI).

The invention also relates to a contrast agent comprising a nanoemulsion as defined above or a nanoemulsion obtainable by the process as defined above.

The appended figures represent respectively:

Figure 1: Section of a drop of oil stabilized by surfactants

Figure 2: diagram representing the mechanism of coalescence

Figure 3: Schematic diagram of Ostwald ripening

Figure 4: size in nm of the droplets in the poor diet (3% mass.) In pure water as a function of time in days (example II.1)

Figure 5: size in nm of the droplets in the rich diet (12% mass.) In pure water as a function of time in days (example II.1)

Figure 6: Size in nm of the droplets comprising anionic (diamond), cationic (triangles) and nonionic (square) surfactants of carbon chains C12, in pure water as a function of time in days (Example II.2) The following examples illustrate the embodiments of the invention.

PART I: Manufacture and method of characterization of nanoemulsions 1. Compounds used

The products used to formulate nanoemulsions are:

- Dissimilated phases: (Fluorinated oils): perfluorooctylbromide (PFOB).

- Continuous phases: Distilled water or salt water (NaCl = 0.154M) or water containing 2.5% by weight of glycerol (pH 7).

- Stabilizers: The natural lipoid phospholipid E80 (PL) from egg yolk, composed mainly of phosphatidylcholine and the modified pegylated phospholipid DSPE-PEG 2000 (PLM). This modification of the phospholipid brings stealth to the nanoemulsion, so that the nanoemulsion is not immediately evacuated by the immune system.

- Anionic surfactant: Oleic acid or SDS

2. Manufacture and characterization of nanoemulsions

The phospholipids (PL) and modified phospholipids (PLM) and the anionic surfactant are dissolved with magnetic stirring and heating (45%) in the continuous phase containing pure water or salt water. In a first step, a so-called coarse nanoemulsion is formed by gradually incorporating the oil into the aqueous phase with vigorous stirring using Ultra-Turrax. This pre-emulsion is then passed to the microfluidizer to reduce the size of the drops. The average droplet size d _h as well as an indicator of the droplet size distribution width (PDI) are measured by the dynamic scattering of light.

The following nanoemulsions have been prepared

References Oil Tensioactive Phase Agent Ratio of Anionic Fluoride Anionic Compatibilizer Mixture (PFOB) ^* (Pure Water (Mygliol) ^** Molar of Except for Oleic) ^** Lipid The Last Amphiphile Nanoemuls (Lipoide E80) SC ^* and Lipid

PEGylé (DSPE-PEG)

97: 3 95: 5

SC166-F1 20% Up to 80% 0.425% 3%

(Comp.)

SC161-F1 20% Up to 80% 0.25% - 3% -

SC67-F1 20% Up to 80% 3%

(Comp.)

SC167-F1 20% Up to 80% 0.85% 3%

(Comp.)

SC162-F1 20% Up to 80% 0.5% - 3% -

SC164-F1 20% Up to 80% 1% - 12% -

SC165-F1 20% up to 80% 2% - 12% -

SC31-F2 20% to 80% 12%

(Comp.)

se 20% Water 1, 1% 3% added

2.5% in

weight of

glycerol

^* % by mass with respect to the weight of the composition

^** % by mass with respect to the weight of the fluorinated oil (PFOB in this case) Table 1: Composition of the nanoemulsions of FIGS. 4 and 5 The following nanoemulsions were prepared (the percentages being expressed by weight relative to the CMC (critical micelle concentration):

Table 2: Composition of the nanoemulsions of Figure 6

"Comp. »Means that the example is comparative

The size of the nanoemulsions was measured by photon correlation spectroscopy (PCS for "photon correlation spectroscopy" in English) using a particle size analyzer Zetasizer NanoS (Malvern company).

The particles of the SC nanoemulsion (last row of Table 1) had an average size of 190 nm. At 14 days after their manufacture, this size was 223 nm. At 191 days, it was 175 nm. These results therefore demonstrate that a nanoemulsion according to the invention is stable over time.

PART II: Improving the stability of nanoemulsions

11.1) Addition of a compatibilizer

During this study, the improvement of the compatibility between the PL and PLM system and the fluorinated dispersed interface was sought. From FIGS. 4 and 5, it can be seen that the addition of a compatibilizer, in particular Miglyol, improves the stability of the nanoemulsion when compared with the reference system (SC67-F1 or SC31-F2). II .2) Interface load: use of surfactants

In order to orient the studies towards the most appropriate type of stabilizer, the phospholipids PL and PLM were replaced by anionic (diamond), cationic (triangles) and nonionic (square) surfactants of carbon chains C12 (FIG. . The results show that the anionic surfactants are the ones that best stabilize the nanoemulsion. The nonionic and cationic surfactants do not remain at the interface and the nanoemulsion destabilizes very quickly.

Claims

1. Nanoemulsion oil in water comprising:

an aqueous phase,

a fluorinated oil phase,

a surfactant at the interface between the aqueous phase and the fluorinated oil phase, the surfactant being an anionic surfactant.

The nanoemulsion of claim 1, wherein the anionic surfactant comprises a carboxylate or sulfonate group.

3. Nanoemulsion according to claim 1 or 2, wherein the anionic surfactant is a fatty acid.

4. Nanoemulsion according to any one of claims 1 to 3, wherein the anionic surfactant is an unsaturated fatty acid, preferably monounsaturated, especially C16-C24, advantageously C16-C18, more preferably C18.

A nanoemulsion according to any one of claims 1 to 4, wherein the anionic surfactant is oleic acid.

6. Nanoemulsion according to any one of claims 1 to 5, comprising an amphiphilic lipid, in particular a phospholipid, such as lipoid E80.

7. Nanoemulsion according to any one of claims 1 to 6, comprising a pegylated lipid, especially an aminopolyethylene glycol lipid, such as distearoyl phosphatidyl ethanolamine.

8. Nanoemulsion according to any one of claims 1 to 7, comprising a compatibilizer forming an additional layer interposed between the fluorinated oil phase and the surfactant layer, the compatibilizing agent being a non-fluorinated hydrocarbon oil comprising at least 70 %, advantageously at least 80%, advantageously at least 95% by weight, especially at least 97%, of saturated C6-C18 fatty acids.

9. Nanoemulsion according to claim 8, characterized in that the compatibilizing agent is a non-fluorinated hydrocarbon oil comprising at least 70% saturated fatty acids C6-C14, more preferably C6-C10.

10. Nanoemulsion according to claim 8, characterized in that the saturated hydrocarbon oil is a saturated oil comprising at least 70%, preferably at least 80, 90, 95% of saturated fatty acids of 12 to 18 carbon atoms, or comprising a mixture of saturated oils each comprising at least 70%, preferably at least 80, 90, 95% saturated fatty acids of 12 to 18 carbon atoms.

1 1. Nanoemulsion according to any one of Claims 1 to 10, characterized in that the fluorinated oil is an oil chosen from oils including linear or branched perfluorocarbons, or cyclic or polycyclic saturated or unsaturated perfluorocarbons, cyclic perfluorinated tertiary amines, perfluoro esters or thioesters, haloperfluorocarbons; advantageously perfluorooctylbromide PFOB, C ₈ F ₇ Br (PFOB or perfluorobron), perfluorooctylethane (C ₈ F ₁₇ C ₂ H ₅ PFOE), perfluorodecalin FDC, perfluorooctane C ₈ Fi ₈ , perfluorodichlorooctane, perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane Ci ₀ F ₂₂ , perfluorododecyl bromide Ci ₀ F ₂₂ Br PFDB, perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine, perfluorotributylamine, perfluorodimethylcyclohexane, perfluorotrimethylcyclohexane, perfluorodicyclohexyl ester, perfluoro-n-butyltetrahydrofuran.

12. Nanoemulsion according to any one of claims 1 to 1 1, characterized in that the fluorinated phase comprises a first fluorinated oil and at least a second fluorinated oil; advantageously, the first fluorinated oil represents between 70 and 95% by weight of the fluorinated oil phase and the second fluorinated oil represents 5 to 30% by weight of the fluorinated oil phase.

13. Nanoemulsion according to claim 12, characterized in that the first fluorinated oil is PFOB, the second fluorinated oil is an oil of longer molecular chain, preferably perfluorohexadecane PFHD or perfluorodécylbromide PFDB.

14. Process for the preparation of oil-in-water nanoemulsions according to any one of claims 1 to 13, comprising an aqueous phase, a fluorinated oil phase, a surfactant at the interface between the aqueous and lipid phases, the process comprising the steps of: a) mixing the constituents of the aqueous phase, the fluorinated phase, and the surfactant, so as to obtain a nanoemulsion

b) at least one washing step of the nanoemulsion prepared in a).

15. Contrast agent comprising a nanoemulsion according to any one of claims 1 to 13 or a nanoemulsion obtainable by the process according to claim 14.