US20100144899A1

US20100144899A1 - Method for preparing nano-emulsions

Info

Publication number: US20100144899A1
Application number: US12/527,371
Authority: US
Inventors: Mathieu Goutayer; Isabelle Texier-Nogues; Jerome Bibette
Original assignee: Commissariat a lEnergie Atomique CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2007-02-14
Filing date: 2008-02-14
Publication date: 2010-06-10
Also published as: WO2008102065A1; IL200115A0; EP2129455A2; WO2008104717A2; WO2008104717A3; JP2010518152A; JP2010517768A; WO2008125747A3; ES2482090T3; US9072775B2; IL200351A0; JP5620111B2; WO2008125747A2; EP2129455B1; JP5837279B2; EP2131869A2; IL200351A; EP2131869B1; IL200115A; US20100284932A1

Abstract

The present invention relates to a method for preparing nano-emulsions comprising at least one aqueous continuous phase and at least one oily dispersed phase, comprising the steps of:

(i) preparing the oily phase comprising at least one amphiphilic lipid and at least one solubilising lipid;
(ii) dispersing the oily phase in an aqueous phase under the effect of sufficient shear force to form a nano-emulsion; and
(iii) recovering the nano-emulsion thus formed.

Description

The present invention relates to a method for preparing nano-emulsions.
Nano-emulsions, also known as mini-emulsions, ultrafine emulsions or even submicron emulsions, are emulsions in which the dispersed phase has an average diameter generally of between 10 and 200 nm.
It will be remembered that an emulsion is a mixture of two immiscible liquid substances consisting of a continuous phase and a dispersed phase. One substance is dispersed in the second substance (continuous phase) in the form of small droplets (dispersed phase). The mixture remains stable due to the action of amphiphilic molecules, termed “emulsifying” or “surfactant”, which position themselves at the interface between the two phases. Emulsions are metastable supramolecular structures. These structures are shown in FIG. 3B and are distinct from polymersomes and micelles.
Polymersomes (the family containing liposomes) are vesicles having an average diameter varying from tens to thousands of nanometres. These vesicles are composed of one or more bilayers of surfactants which allow the intravesicular medium to be separated from the external medium, the two media being of the same type, most often aqueous (FIG. 3A).
Micelles consist of aggregates of self-assembled surfactants measuring a few nanometres in diameter. Surfactants organise themselves in such a way as to orient the hydrophilic part thereof towards the outside (the solvent) and the hydrophobic chains thereof towards the centre of the micelle (see FIG. 3C).
A number of methods for preparing oil-in-water nano-emulsions are known.
A method has thus been proposed in which the surfactants are solubilised in the continuous phase, i.e. the water (Jafari, S. M.; He, Y.; Bhandari, B., Production of sub-micron emulsions by ultrasound and microfluidization techniques. Journal of Food engineering 2007, 82, (4), 478-488 and Jafari, S. M.; He, Y.; Bhandari, B., Nano-emulsion production by sonication and microfluidization—A comparison. International Journal of Food Properties 2006, 9, (3), 475-485 and Mason, T. G.; Wilking, J. N.; Meleson, K.; Chang, C. B.; Graves, S. M., Nanoemulsions: formation, structure, and physical properties. Journal of Physics: Condensed Matter 2006, 18, (41), R635-R666).
However, a large number of surfactants, in particular phospholipids, do not dissolve easily in the aqueous phase and in the oily phase. It is therefore hard to incorporate into one of the phases the amount of surfactant necessary to obtain nano-emulsions with a low-diameter dispersed phase.
In order to be able to solubilise a larger amount of surfactant, it has been proposed to solubilise the surfactants in a solvent, which is subsequently evaporated so as to form a film of surfactants. The water or aqueous solution is subsequently added and the mixture is sonicated in order to disperse the film of surfactants. The oil is subsequently dispersed in the solution of liposomes obtained. The diphasic solution obtained is sonicated once again or treated with a microfluidiser to produce the nano-emulsion (Vyas, T. K.; Shahiwala, A.; Amiji, M. M., Improved oral bioavailability and brain transport of Saquinavir upon administration in novel nanoemulsion formulations. International Journal of Pharmaceutics 2008, 347 (1-2), 93-101) and US 2005/0079131. However, this method is long and requires a plurality of sonication steps. Moreover, the presence of residual liposomes in the nano-emulsion cannot be excluded.
The main object of the present invention is to propose a method for preparing nano-emulsions which overcomes one or more of the above drawbacks.
In particular, the object of the present invention is to propose a simple method which gives access to nano-emulsions with a low-diameter dispersed phase.
Another object of the invention is to propose a method for preparing nano-emulsions which makes it possible to control the size of the dispersed phase and/or the physicochemical properties of the interface.
The object is achieved by a method of preparing a nano-emulsion in which at least one low-solubility surfactant, in particular a phospholipid, is solubilised in the oily phase due to the presence of a solubilising lipid.
The present invention therefore relates to a method for preparing nano-emulsions comprising at least one aqueous continuous phase and at least one oily dispersed phase, comprising the steps of:
(i) preparing the oily phase comprising at least one amphiphilic lipid and at least one solubilising lipid;
(ii) dispersing the oily phase in an aqueous phase under the effect of sufficient shear force to form a nano-emulsion; and
(iii) recovering the nano-emulsion thus formed.
The method according to the present invention has the following advantages in particular:

- it includes only a single step of sonication;
- it allows significant amounts of surfactants to be dissolved and thus allows the preparation of nano-emulsions with a low-diameter dispersed phase;
- it is quick, because the aqueous phase has a low proportion of surfactant and is therefore less viscous and can be emulsified quickly; and
- it avoids the formation of liposomes and thus allows access to a homogeneous nano-emulsion.

Within the meaning of the present invention:

- the term “droplet” encompasses droplets of liquid oil as such, as well as the solid particles from oil-in-water emulsions in which the oil used is a crystallisable oil. In the latter case, the term “solid emulsion” is also often used.
- the term “lipid” refers to small molecules consisting primarily of carbon, hydrogen and oxygen and having a density lower than that of the water. The lipids can be in a solid state, as in waxes, or liquid, as in oils.
- the term “amphiphilic” refers to species having a hydrophilic group and a hydrophobic group. Amphiphilic compounds have surfactant properties, i.e. they alter the surface tension between two surfaces.
- the term “phospholipid” refers to lipids having a phosphate group, in particular phosphoglycerides. Most often, phospholipids comprise a hydrophilic end formed by the optionally substituted phosphate group and two hydrophobic ends formed by fatty acid chains. Particular phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin.
- the term “lecithin” refers to phosphatidylcholine, i.e. a lipid formed from a choline, a phosphate, a glycerol and two fatty acids. More broadly, it includes phospholipids extracted from living sources, of plant or animal origin, as long as they primarily consist of phosphatidylcholine. These lecithins generally consist of mixtures of lecithins carrying different fatty acids.
- within the meaning of the invention, the term “nano-emulsion” refers to an emulsion in which the dispersed phase has an average diameter of between 10 and 200 nm, preferably between 10 and 150 nm and in particular between 10 and 80 nm.

[Oily Phase]

The nano-emulsion prepared using the method according to the invention comprises an oily phase and an aqueous phase.
The oily phase comprises at least one amphiphilic lipid and at least one solubilising lipid.
To form a stable nano-emulsion, it is generally necessary to include at least one amphiphilic lipid in the composition as a surfactant.
The amphiphilic nature of the surfactant or surfactants causes the droplets of oil to stabilise within the aqueous continuous phase. Usually, at least two surfactants are used to prepare a nano-emulsion. The surfactant may also have other effects in the intended application of the nano-emulsion.
According to the invention, the oily phase comprises at least one amphiphilic lipid.
These amphiphilic lipids may be of natural or synthetic origin. They are preferably selected from phospholipids; cholesterol; lysolipids; sphingomyelins; tocopherols; glucolipids; stearylamines and cardiolipins.
Lecithin is the preferred amphiphilic lipid.
In one specific embodiment, all or part of the amphiphilic lipid may have a reactive function, such as a maleimide, thiol, amine, ester, oxyamine or aldehyde group. This variant allows functional compounds to graft at the interface. The reactive amphiphilic lipid is incorporated into the layer formed at the interface stabilising the dispersed phase, where it is liable to couple to a reactive compound present in the aqueous phase.
Generally, the oily phase will comprise 1 to 99% by weight, preferably 5 to 75% by weight and in particular 20 to 60% by weight amphiphilic lipid.
The solubilising lipid is a lipid having a sufficient affinity for the amphiphilic liquid to allow it to be solubilised. In the case where the amphiphilic lipid is a phospholipid, possible solubilising lipids are, in particular, glycerol derivatives, especially glycerides obtained by esterifying glycerol with fatty acids.
The solubilising lipid used is advantageously selected in dependence on the amphiphilic lipid used. It will generally have a close chemical structure so as to bring about the desired solubilisation. It may be an oil or a wax.
The preferred solubilising lipids, in particular for phospholipids, are glycerides of fatty acids, in particular of saturated fatty acids, and in particular of saturated fatty acids comprising 8 to 18 carbon atoms, even more preferably 12 to 18 carbon atoms.
Preferably, saturated fatty acid glycerides comprising 0% to 20% by weight C8 fatty acids, 0% to 20% by weight C10 fatty acids, 10% to 70% by weight C12 fatty acids, 5% to 30% by weight C14 fatty acids, 5% to 30% by weight C16 fatty acids and 5% to 30% by weight C18 fatty acids are involved.
The semi-synthetic glyceride mixtures sold by Gattefossé under the trade name Suppocire® NC, which are solid at room temperature, are particularly preferred. The type N Suppocire® glycerides are obtained by direct esterification of fatty acids and glycerol. These are semi-synthetic glycerides of C8 to C18 saturated fatty acids, of which the quali-quantitative composition is shown in the table below.
The amount of solubilising lipid may vary widely as a function of the type and amount of amphiphilic lipid present in the oily phase.
Generally, the oily phase will comprise 1 to 99% by weight, preferably 5 to 75% by weight and in particular 10 to 50% by weight solubilising lipid.

TABLE

Fatty acid composition of Suppocire NC ® from Gattefossé

	Chain length	[% by weight]

	C8	0.1 to 0.9
	C10	0.1 to 0.9
	C12	25 to 50
	C14	10 to 24.9
	C16	10 to 24.9
	C18	10 to 24.9

Preferably, the oily phase further comprises one or more other oils.
The oils used in the method according to the invention preferably have a hydrophilic-lipophilic balance (HLB) of less than 8 and even more preferably of between 3 and 6.
Advantageously, the oils are used without any chemical or physical modification in advance of the formation of the emulsion.
In the proposed applications, the oils may be selected from biocompatible oils, in particular from oils of natural (plant or animal) or synthetic origin.
Oils of this type include, in particular, oils of natural plant origin, including in particular soybean, linseed, palm, peanut, olive, grape seed and sunflower oils; and synthetic oils, including in particular triglycerides, diglycerides and monoglycerides. These oils may be in their natural form, refined or interesterified.
The preferred oils are soybean oil and linseed oil.
Generally, the oily phase comprises 1 to 70% by weight, preferably 2 to 50% by weight and in particular 5 to 30% by weight of oil.
Naturally, the oily phase may also contain other additives, such as colourings, stabilisers, preservatives, fluorophores or pharmacological active ingredients in an appropriate amount.
In the method according to the invention, the different oily constituents are initially mixed to prepare an oily premix for the dispersed phase of the emulsion. The mixing may optionally be facilitated by placing one of the constituents or the complete mixture in solution in an appropriate organic solvent, followed by evaporating the solvent, leaving a homogeneous oily premix for the dispersed phase.
Furthermore, it is preferred to produce the premix at a temperature at which all of the ingredients are liquid.

[Aqueous Phase]

The aqueous phase used in the method according to the invention preferably consists of water and/or a buffer, such as a phosphate buffer, for example PBS (“phosphate buffer saline”) or another saline solution, in particular sodium chloride.
Moreover, it optionally comprises other ingredients, preferably including a cosurfactant.
The cosurfactants which may be used in emulsions according to the present invention are preferably water-soluble surfactants.
The water-soluble surfactants preferably comprise at least one chain composed of ethylene oxide units (PEO or PEG) or ethylene oxide and propylene oxide units. Preferably, the number of units in the chain varies between 2 and 500.
Examples of co-surfactants include, in particular, the conjugated compounds polyethyleneglycol/phosphatidylethanolamine (PEG-PE), fatty acid and polyethylene glycol ethers such as the products sold under the Brij® trade names (for example Brij® 35, 58, 78 or 98) by ICI Americas Inc., fatty acid and polyethylene glycol esters such as the products sold under the Myrj® trade names by ICI Americas Inc. (for example Myrj® 45, 52, 53 or 59) and block copolymers of ethylene oxide and propylene oxide such as the products sold under the Pluronic® trade names by BASF AG (for example Pluronic® F68, F127, L64, L61, 10R4, 17R2, 17R4, 25R2 or 25R4) or the products sold under the Synperonic® trade name by Unichema Chemie BV (for example Synperonic® PE/F68, PE/L61 or PE/L64).
The aqueous phase preferably comprises 0 to 50% by weight, preferably 1 to 30% by weight and in particular 5 to 20% by weight of a water-soluble cosurfactant.
In a preferred embodiment, the continuous phase also comprises a thickening agent such as a glycerol, a saccharide, oligosaccharide or polysaccharide, a gum or even a protein, preferably glycerol. In fact, the use of a continuous phase of a higher viscosity facilitates emulsification and thus allows the sonication time to be reduced.
The aqueous phase advantageously comprises 0 to 50% by weight, preferably 1 to 30% by weight and in particular 5 to 20% by weight of a thickening agent.
Naturally, the aqueous phase may further comprise other additives such as colourings, stabilisers and preservatives in appropriate amounts.
The aqueous premix for the continuous phase of the emulsion may be prepared by simply mixing the different constituents with the selected aqueous medium.

[Emulsification]

In the method according to the invention, the nano-emulsion is prepared by dispersing appropriate amounts of oily phase and aqueous phase under the effect of a shear force.
The proportion of oily phase and aqueous phase is highly variable. However, usually, the nano-emulsions will be prepared with 1 to 50%, preferably 5 to 40%, and in particular 10 to 30% by weight oily phase and 50 to 99%, preferably 60 to 95% and in particular 70 to 90% by weight aqueous phase.
Advantageously, the oily phase is subsequently dispersed in the aqueous phase in a liquid state. If one of the phases solidifies at room temperature, it is preferable to make the mixture with the two phases heated to a temperature greater than or equal to the fusion temperature.
The emulsification under shear force effect is preferably produced using a sonicator or a microfluidiser. Preferably, the aqueous phase and then the oily phase are introduced into an appropriate cylindrical receptacle in the desired proportions and the sonicator is dipped into the medium and switched on for long enough to obtain a nano-emulsion, usually a few minutes.
This produces a homogeneous nano-emulsion in which the average diameter of the oil droplets is greater than 10 nm and less than 200 nm.
Before conditioning, the emulsion may be diluted and/or sterilised, for example by filtration. The filtration step also makes it possible to eliminate any aggregates which might have formed during preparation of the emulsion.

The invention will be explained in greater detail by means of the examples, given by way of illustration, and the appended figures, in which:

FIG. 1 is a schematic drawing of the production of nano-emulsions by ultrasound according to the prior art;

FIG. 2 is a comparison drawing of the production of nano-emulsions by ultrasound according to a preferred embodiment of the invention;

FIG. 3 is a schematic drawing of the structure of a nano-emulsion (b) in terms of the micellar systems (c) and the polymersomes (a), the structures all having water as the continuous phase;

FIG. 4 shows the size distribution, weighted by volume, of a nano-emulsion prepared according to example 1, the percentage by volume being shown as a function of the size in nm;

FIG. 5 shows the average diameter of the dispersed phase of the nano-emulsions according to example 1 (▪) and example 2 as a function of the total amount of surfactants at an even surfactant/cosurfactant molar ratio;

FIG. 6 shows the average diameter of the dispersed phase of the nano-emulsions according to example 1 (▪) and example 3 as a function of the amount of surfactants and cosurfactants; and

FIG. 7 shows the progression over time of the average diameter of nano-emulsions prepared according to example 1.

EXAMPLES

Example 1

Formulation of a Water-in-Oil Nano-Emulsion

In an appropriate receptacle, a premix was prepared consisting of 0.125 g of soybean oil (Sigma-Aldrich), 0.375 g of semi-synthetic glycerides sold under the trade name Suppocire® NC (Gattefossé) and 0.350 g of soy lecithin (enriched with 75% phosphatidylcholine) sold by Lipoïd under the trade name Lipoïd® S75.
These compounds were dissolved in chloroform, then the solution was evaporated at low pressure and dried at 50° C. so as to obtain a premix in the form of a viscous oil which solidifies as it cools. The mixture obtained was heated to 50-60° C. so as to keep it liquid for emulsification.
The continuous phase was prepared by mixing 0.125 g of glycerol, 0.55 g of polyoxyethylene stearate with 50 moles ethylene oxide sold under the trade name Myrj® 53 by ICI Americas Inc. and phosphate buffer solution, PBS, to make the mixture up to 4.150 g. This solution was kept hot (50-60° C.) before emulsification.
The aqueous solution was subsequently added to the oil/lecithin mixture. Then, the diphasic solution was brought into contact with an AV505® sonicator provided with a conical probe measuring 3 mm in diameter (Sonics, Newtown) which was dipped about 1 cm into the solution. The solution was sonicated for 5 minutes with the sonicator set to 30% of the maximum power, with the following pulse sequence: 10 seconds sonication/30 seconds rest. During sonication, the solution was kept at 40° C.
The average diameter of the dispersed phase of the prepared emulsion was determined by dynamic light diffusion (Zetasizer nano ZS, Malvern Instrument)
The dispersed phase of the nano-emulsions thus obtained had an average diameter of 35 nm (FIG. 4). Table 1 summarises the composition of the formulation of the nano-emulsion obtained. FIG. 7 shows the stability over time of nano-emulsions prepared according to example 1.

TABLE 1

formulation of the nano-emulsion of example 1

Example 1		Mass [mg]	% by weight	[mmol]

Dispersed phase	Soybean oil	125	2.5
	Suppocire	375	7.5
Surfactants	Lecithin		350	7	0.467
	Myrj 53	550	11	0.233
Aqueous phase	Glycerol	125	2.5
	Buffer solution	3475	69.5
Total		5000	100

Examples 2A to 2K

Effect of the Total Amount of Surfactant and Cosurfactant

In order to study the effect of surfactants on the formulation of example 1, said example was reproduced while varying the amount of lecithin and Myrj 53 but keeping the molar ratio between them [lecithin/(lecithin+Myrj® 53)] constant at a value of 0.67. The amount and the composition of the dispersed phase were unchanged as compared with the formulation of example 1. The amount of buffer solution was altered as a result to obtain 5 g of solution.
In examples 2A and 2B, where there is a large amount of cosurfactant, glycerol was replaced with buffer solution because the solution was sufficiently viscous. Table 2 summarises the concentrations used for the different formulations, prepared as stated in example 1.

TABLE 2

formulation of the emulsions of examples 2A to 2K

	Surfactant	Cosurfactant	Total
Ex-	(lecithin)	(Myrj ® 53)	amount of	Average

am-		% by		% by	surfactant	diameter
ple	Mass [mg]	weight	Mass [mg]	weight	[mmol]	[mm]

1	350	7	550	11	0.7	36.6
2A	420	8.4	660	13.2	0.84	35.1
2B	385	7.7	605	12.1	0.77	34.2
2C	315	6.3	495	9.9	0.63	37.4
2D	280	5.6	440	8.8	0.56	40.3
2E	245	4.9	385	7.7	0.49	45.7
2F	210	4.2	330	6.6	0.42	72.7
2G	175	3.5	275	5.5	0.35	92.9
2H	140	2.8	220	4.4	0.28	127
2I	105	2.1	165	3.3	0.21	170
2J	70	1.4	110	2.2	0.14	177
2K	35	0.7	55	1.1	0.07	272

It is found that the average diameter of the dispersed phase of the nano-emulsions obtained decreases as the total amount of surfactants increases (see FIG. 5). The formulation of example 1 is shown by a square in FIG. 5.
It is therefore possible to control the size of the dispersed phase of the nano-emulsion by adjusting the amount of surfactants. However, two different patterns can be distinguished: below 0.5 mmol of surfactants, the average diameter varies rapidly with the amount of surfactants, whereas above this concentration, the average diameter does not change much. Formulations comprising more than 0.5 mmol of surfactants therefore seem more stable, given that a slight variation in the amount of surfactant only has a small effect on the eventual size of the dispersed phase of the nano-emulsions.

Examples 3A to 3F

Effect of the Surfactant/Cosurfactant Molar Ratio

Example 1 was then reproduced but the surfactants/cosurfactants ratio was varied, whilst the molar amount of surfactants was kept constant however.
In examples 3A to 3F, where there is a large amount of cosurfactant, glycerol was replaced with buffer solution because the solution was sufficiently viscous. Table 3 summarises the concentrations used for the different formulations, prepared as stated in example 1.


	Surfactant (lecithin)	Cosurfactant	Average

% by

(Myrj ® 53)

diameter

Example	Mass [mg]	weight	Mass [mg]	% by weight	[mm]

1	350	7.0	550	11	34.3
3A	516	10.3	0	0	215
3B	387	7.7	428	9	48.8
3C	258	5.2	856	17	30.6
3D	172	3.4	1142	23	27.4
3E	129	2.6	1284	26	27.5
3F	0	0.0	1712	34	26.1

The size of the dispersed phase of the emulsions obtained is given as a function of the concentration of lecithin and Myrj® 53, the nominal formulation being represented by a square. The data are shown in the diagram of FIG. 6.
It is found that the size of the droplets decreases with the amount of surfactant, but also with the amount of cosurfactant. It may further be noted that the effect of the cosurfactant seems to be considerably more significant in this respect.
The surfactant/cosurfactant ratio thus also makes it possible to control the diameter of the dispersed phase by simultaneously altering the composition of the interface of the emulsion.

Example 4

Effect of the Length of the Hydrophilic Chains of the Cosurfactant

In order to study the effect of the length of the hydrophilic chains of the cosurfactant, the formulation of example 1 was reproduced, but Myrj® 53 was replaced with cosurfactants carrying polyoxyethylene chains of a different length.
The following cosurfactants were tested: Tween® 80, Myrj® 45, Myrj® 49 and Myrj® 59. Table 4 below summarises the properties of these cosurfactants.
The cosurfactant in the formulation of example 1 was replaced with the same amount in moles, i.e. 0.233 mmol.

TABLE 4

properties of the polyethoxylated cosurfactants

Polyethoxylated	Number of polyoxyethylene
cosurfactant	(POE) units	Molecular weight (g/mol)

Tween ® 80	20	1310
Myrj ® 45	8	636
Myrj ® 49	20	1164
Myrj ® 53	50	2484
Myrj ® 59	100	4684

Example 5

Effect of the Length of the Hydrophilic Chains of the Cosurfactant in a Formulation Comprising Surfactant and Cosurfactant in an Equimolar Amount

The study of the effect of the length of the hydrophilic chains of the cosurfactants was corroborated with a second series of experiments performed as in example 4, but based on a formulation in which the surfactant and the cosurfactant are present in an equimolar amount (0.344 mmol).
The results in terms of the average diameter of the dispersed phase are summarised for examples 4 and 5 in table 5 below.

TABLE 5

average diameter of the dispersed phase of the emulsions according
to examples 4 and 5

	Cosurfactant	Example 4 [nm]	Example 5 [nm]

Tween ® 80	65.5	28.2
Myrj ® 45	182	—
Myrj ® 49	132	31.4
Myrj ® 53	34.7	29.5
Myrj ® 59	15.3	37.7

It was found that the length of the hydrophilic chains in the cosurfactant has some influence on the average diameter of the dispersed phase of the emulsion obtained for the formulations of example 4. Specifically, the average diameter of the dispersed phase of the emulsion decreases considerably as the length of the chains increases.
However, it should be noted that the nano-emulsion of example 4 with a cosurfactant having very long chains (Myrj® 59) proved to be unstable. The very low diameter of the dispersed phase (15.3 nm) also suggests the presence of a micellar solution rather than a nano-emulsion.
On the other hand, the formulation of example 5, which is equimolar in surfactants, results in nano-emulsions in which the diameter of the dispersed phase remains close to 30 nm. It is therefore possible in this case to alter the length of the hydrophilic chains without significantly affecting the diameter of the particles.

Examples 6A to 6G

Effect of the Composition of the Dispersed Phase

Finally, the effect of the composition of the oily phase on the average diameter of the dispersed phase was studied by varying the type and proportion of oil and solubilising lipid.
In the formulation of example 1, the dispersed phase is composed of a mixture of 75% by weight solubilising lipid (Suppocire® NC) and 25% by weight soybean oil. The solubilising lipid is rich in saturated medium-chain fatty acids, whilst the soybean oil is rich in ω6-type unsaturated acids (C18:2) and the linseed oil is rich in ω3-type unsaturated acids (C18:3).
Nano-emulsions were therefore prepared of which the dispersed phase was composed of pure solubilising lipid or of a mixture of solubilising lipid and soybean oil or linseed oil. The respective composition of the nano-emulsions in solubilising lipid and oil is shown in percent by weight in table 8 below.
Otherwise, the formulation of example 5 is maintained, this formulation having the advantage of reducing the amount of lecithin by comparison with the formulation of example 1, thus allowing the amount of solubilising lipid to be reduced whilst allowing good solubilisation of the lecithin. The formulation of the nano-emulsions excluding the oily phase is summarised in table 7 below.

TABLE 7

formulation of the nano-emulsions of example 6

	Mass in mg	%	[mmol]

Dispersed phase	Total oil	500	10
Surfactants	Lecithin	258	5	0.344
	Myrj ® 53	827.5	16.55	0.344
Aqueous phase	Glycerol	125	2.5
	Buffer solution	3289.5	65.95

Table 8 below summarises the average diameter of the dispersed phase in the nano-emulsions obtained with the composition shown

TABLE 8

change in the average diameter as a function of the composition
of the oily phase

	Suppocire ®
	NC	Soybean oil	Linseed oil	Average
Example	% by weight	% by weight	% by weight	diameter (d, nm)

6A	75	25	0	29
6B	50	50	0	30.4
6C	25	75	0	30.9
6D	100	0	0	29.2
6E	75	0	25	29
6F	50	0	50	28.3
6G	25	0	75	30

These results show that the composition of the dispersed phase of the emulsion can be altered without changing the diameter thereof, thus making it possible to alter the solubilisation characteristics without affecting the diameter of the dispersed phase of the nano-emulsions.
The method described above therefore makes it possible to obtain stable, homogeneous nano-emulsions quickly and easily. The method further makes it possible to control the average diameter of the dispersed phase, the interface and/or the dispersed phase of the nano-emulsions via the concentration and the type of surfactants used, as described above.

Claims

1. Method for preparing nano-emulsions comprising at least one aqueous continuous phase and at least one oily dispersed phase, comprising the steps of:

(i) preparing the oily phase comprising at least one amphiphilic lipid and at least one solubilising lipid;

(ii) dispersing the oily phase in an aqueous phase under the effect of sufficient shear force to form a nano-emulsion; and

(iii) recovering the nano-emulsion thus formed,

wherein the solubilising lipid consists of a mixture of saturated fatty acid glycerides comprising at least 10% by weight of C12 fatty acids, at least 5% by weight of C14 fatty acids, at least 5% by weight of C16 fatty acids and at least 5% by weight of C18 fatty acids.

2. Method according to claim 1, wherein the solubilising lipid consists of a mixture of saturated fatty acid glycerides comprising 0% to 20% by weight of C8 fatty acids, 0% to 20% by weight of C10 fatty acids, 10% to 70% by weight of C12 fatty acids, 5% to 30% by weight of C14 fatty acids, 5% to 30% by weight of C16 fatty acids and 5% to 30% by weight of C18 fatty acids.

3. Method according to claim 1, wherein the amphiphilic lipid is selected from phospholipids, cholesterol, lysolipids, sphingomyelins, tocopherols, glucolipids, stearylamines or cardiolipins of natural or synthetic origin.

4. Method according to claim 1, wherein the amphiphilic lipid is a phospholipid.

5. (canceled)

6. Method according to claim 1, wherein the oily phase further comprises at least one oil.

7. Method according to claim 6, wherein the oil has a hydrophilic-lipophilic balance (HLB) of between 3 and 6.

8. Method according to claim 1, wherein the oily phase comprises at least one oil selected from soybean oil and linseed oil.

9. Method according to claim 1, wherein the aqueous phase further comprises a cosurfactant.

10. Method according to claim 9, wherein the cosurfactant comprises at least one chain composed of ethylene oxide units or ethylene oxide and propylene oxide units.

11. Method according to claim 9, wherein the cosurfactant is selected from the conjugated compounds polyethyleneglycol/phosphatidylethanolamine (PEG-PE), fatty acid and polyethyleneglycol ethers, fatty acid and polyethyleneglycol esters, and block copolymers of ethylene oxide and propylene oxide.

12. Method according to claim 1, wherein the continuous phase of the emulsion comprises a physiologically acceptable buffer.

13. Method according to claim 1, wherein the shear force effect is produced by sonication.

14. Method according to claim 1, wherein the oily phase is prepared by placing all or some of the constituents in solution in an appropriate solvent and subsequently evaporating the solvent.