WO2010121307A1

WO2010121307A1 - Complex emulsions

Info

Publication number: WO2010121307A1
Application number: PCT/AU2010/000451
Authority: WO
Inventors: Chun-xia ZHAO
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 2009-04-21
Filing date: 2010-04-21
Publication date: 2010-10-28
Anticipated expiration: 2011-10-21

Abstract

A method of forming a complex emulsion including the steps of forming a dispersed phase fluid comprising two or more at least partially immiscible emulsion forming fluids maintained in an intimately mixed state by a co-solvent, introducing the dispersed phase fluid into a continuous phase fluid which is immiscible with at least one of the emulsion forming fluids and allowing the co-solvent to substantially separate from the at least two emulsion forming fluids to thereby form a complex emulsion.

Description

COMPLEX EMULSIONS

FIELD OF THE INVENTION

The present invention relates to the field of emulsions and in particular to a method of producing complex emulsions and an apparatus for working the method. More particularly, the invention relates to production of multiple emulsions.

BACKGROUND TO THE INVENTION

Multiple emulsions are emulsions having complex internal structure, where the droplets of the dispersed phase themselves contain even smaller dispersed droplets. They have significant potential in many applications including pharmaceuticals, foods, cosmetics, microspheres, microcapsules and for chemical separations. Double emulsions are typically formed through a two-step bulk emulsification process, by first emulsifying two immiscible liquids by high- shear mixing, and then undertaking a second emulsification step with a third immiscible liquid. Owing to hydrodynamic heterogeneity in large-scale equipment, it is difficult to prepare multiple emulsions having a narrow droplet size distribution and precise microstructure.

Microfluidic devices provide improved hydrodynamic definition and thus allow enhanced control of multiple-emulsion droplet size, structure, and composition. A variety of microchip formats have been investigated to provide hydrodynamic control over droplet formation, including T-junction and flow- focusing designs. For example, a W/O/W emulsion has been formed in microfluidic devices [T. Kawakatsu, G. Tragardh, C. Tragardh, Colloids Surf., A 2001 , 189, 257] by forcing a W/O emulsion, prepared by conventional homogenization, into a microchannel array. Double emulsions have also been produced by using this two-step emulsification process with straight-through microchannels [I. Kobayashi, X. F. Lou, S. Mukataka, M. Nakajima, J. Am. Oil Chem. Soc. 2005, 82, 65], although this method cannot precisely control encapsulation of the inner droplet. Cascading two T-junctions, two consecutive flow-focusing or one T-junction and one flow-focusing microchannel with locally modified surface chemistry has also produced double emulsions, with some control over the number and size of the inner droplets, although device fabrication is complicated by the need for precise and local surface wettability control. 3D microcapillaries, which relax the surface wetting constraints, have been used to prepare both W/O/W and O/W/O emulsions.

Utada et al. [A. S. Utada, E. Lorenceau, D. R. Link, P. D. Kaplan, H. A. Stone, D. A. Weitz, Science 2005, 308, 537] developed coaxial flow-focusing geometries consisting of cylindrical glass capillary tubes nested within a square glass tube to produce double emulsions. Chu et al. recently presented another microcapillary technique [L Y. Chu, A. S. Utada, R. K. Shah, J. W. Kim, D. A. Weitz, Angew. Chem., Int. Ed. 2007, 46, 8970], which was able to achieve precise control over the size and contents of each level of the emulsions. To generate tunable droplets with a wide range of dimensions, Lin et al. [Y. H. Lin, C. H. Lee, G. B. Lee, J. Microelectromech. Syst. 2008, 17, 573] developed a new microfluidic device able to make double emulsions by incorporating pneumatically controlled moving-wall structures.

However, all of these microfluidic methods seek to improve emulsion precision at the expense of more complicated device design. Invariably, hydrodynamic (in planar and 3D devices) and mechanical (in moving-wall microfluidic devices) control of emulsion formation trades greater device complexity for enhanced emulsion structure. Additionally, such devices lack robustness because of practical flow-control limits in confined flows dominated by viscous and interfacial factors. Despite the enormous research effort to date, the preparation of precise double and other multiple emulsions remains a significant research challenge, even on microfluidic platforms.

OBJECT OF THE INVENTION It is an object of the present invention to overcome or at least alleviate one or more of the problems identified above.

It is a further object to provide a method for the production of complex emulsions.

It is a yet further object of the invention to provide an apparatus to work the method.

Other objects will be evident from the detailed discussion.

SUMMARY OF THE INVENTION

In one form, although it need not be the only or indeed the broadest form, the invention resides in a method of forming a complex emulsion including the steps of: forming a dispersed phase fluid comprising two or more at least partially immiscible emulsion forming fluids maintained in an intimately mixed state by a co-solvent; introducing the dispersed phase fluid into a continuous phase fluid which is immiscible with at least one of the emulsion forming fluids; and allowing the co-solvent to substantially separate from the at least two emulsion forming fluids to thereby form a complex emulsion.

If more than two immiscible emulsion forming fluids are present in the dispersed phase fluid then at least two will be immiscible with one another.

The two or more immiscible emulsion forming fluids in the dispersed phase fluid may be maintained in a state whereby they are physically indistinct, one from the other, by the co-solvent.

Preferably, the two or more immiscible emulsion forming fluids in the dispersed phase fluid form a substantially homogeneous phase with the co- solvent.

Suitably, the co-solvent solubilises the two or more immiscible emulsion forming fluids in the dispersed phase fluid. The co-solvent may be any solvent or combination of solvents which allows the two or more immiscible emulsion forming fluids in the dispersed phase fluid to remain intimately mixed.

Preferably, the co-solvent is a hydrophilic solvent. More preferably, the co-solvent is selected from the group consisting of an alcohol, a ketone, an ether, a formamide, a nitrile, a carboxylic acid and a sulfoxide.

Even more preferably, the co-solvent is selected from the group consisting of methanol, ethanol, acetone, terahydrofuran, N₁N- dimethylformamide, acetonitrile, acetic acid and dimethylsulfoxide.

More preferably still, the co-solvent is ethanol or acetone.

In one general embodiment, the method may further include the step of adding an emulsion stabilising agent to the dispersed phase fluid.

Suitably, the dispersed phase emulsion stabilising agent limits coalescence of one or more of the immiscible emulsion forming fluids and/or the co-solvent.

The dispersed phase emulsion stabilising agent may be a dispersed phase surfactant.

Preferably, the dispersed phase surfactant is an oil soluble dispersed phase surfactant such as a sorbitan-based surfactant or an oil soluble polymer.

More preferably, the dispersed phase surfactant is selected from the group consisting of Span 20, 60 and 80.

In one embodiment, the method may further include the step of adding an emulsion stabilising agent to the continuous phase fluid. The continuous phase emulsion stabilising agent may be a continuous phase surfactant.

Preferably, the continuous phase surfactant limits droplet coalescence of the dispersed phase fluid. The continuous phase surfactant may be a small molecular weight surfactant, a polysorbate surfactant, a peptide, a protein or a polymer surfactant.

Suitably, the continuous phase surfactant is sodium dodecyl sulphate (SDS), Tween 20 (Polyoxyethylene (20) sorbitan monolaurate), AM1 , AFD4 or lysozyme.

Preferably, the dispersed phase fluid comprises water droplets solubilised in oil by a co-solvent. The co-solvent is suitably an alcohol or a ketone, specifically ethanol or acetone.

Alternatively, the dispersed phase fluid comprises oil droplets solubilised in water by a co-solvent.

The dispersed phase fluid is suitably a ternary or quaternary system.

The continuous phase fluid is suitably water or oil and the complex emulsion is oil in water in oil or water in oil in water, respectively.

The dispersed phase fluid may be introduced into the continuous phase fluid by bulk mixing or by the use of a microfluidic channel.

The method is preferably worked in an apparatus comprising a microfluidic channel having an intersection of two channels, which may be T- junction, flow-focusing or co-flowing microfluidic devices. The microfluidic channel also suitably incorporates an expansion section. When a microfluidic apparatus is used, the method may further include the step of controlling the external size of a droplet in the complex emulsion by controlling the ratio of flow rate of the continuous phase fluid to the dispersed phase fluid.

The size of an internal droplet within the complex emulsion may be controlled by varying the relative amounts of one or more of the immiscible emulsion forming fluids and/or the co-solvent in the dispersed phase fluid.

In an alternative form the invention resides in a method of controlling the formation of a complex emulsion including the steps of: forming a dispersed phase fluid comprising two or more at least partially immiscible emulsion forming fluids maintained in an intimately mixed state by a co-solvent; introducing the dispersed phase fluid, via a first microfluidic channel, into a continuous phase fluid flowing through a second microfluidic channel, the continuous phase fluid being immiscible with at least one of the emulsion forming fluids; and allowing the co-solvent to substantially separate from the at least two emulsion forming fluids to thereby form a complex emulsion. In a further alternative form the invention resides in the use of a complex emulsion formed by the present method in the encapsulation and/or delivery of one or more agents.

Further features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

To assist in understanding the invention and to enable a person skilled in the art to put the invention into practical effect, preferred embodiments of the invention will be described by way of example only with reference to the accompanying drawings, in which:

FIG 1 is a schematic of an apparatus for producing a complex emulsion; FIG 2 shows droplet formation in the apparatus of FIG 1 ;

FIG 3 shows a water-in-oil-in-water complex emulsion formed in the apparatus of FIG I ; FIG 4(a)displays the formation of a droplet of the dispersed phase in the continuous phase;

FIGs 4(b)-(d) show complex emulsions of water-in-ethanol/oil-in-oil-in-water formed using varying concentrations of surfactant; FIG 5 shows a complex emulsion of multiple droplets of water in a water-in-oil- in-water emulsion;

FIG 6 shows methylene blue dissolved in a water droplet (a); a water droplet without methylene blue (b); and the methylene blue encapsulation process (c) in a water-in-oil-in-water emulsion;

FIG 7 shows a double emulsion formed using the apparatus of FIG 1 with acetone as a co-solvent;

FIG 8 shows an apparatus for producing oil-in-water-in-oil double emulsions; FIG 9 is an expanded view of a portion of the apparatus of FIG 8; FIG 10 shows oil-in-water-in-oil complex emulsions formed in the apparatus of FIG 8;

FIG 11 shows a water-in-ethyl acetate-in-water complex emulsion formed in the apparatus of FIG 1 ;

FIG 12 shows a water-in-ethanol/oil-in-oil-in-water complex emulsion formed in the apparatus of FIG 1 ;

FIG 13 shows a water-in-ethyl acetate-in-water complex emulsion formed using a bulk mixing process;

FIG 14 shows an alternative apparatus for producing a complex emulsion; and FIG 15 shows another alternative apparatus for producing a complex emulsion.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention reside primarily in a method of forming complex emulsions and an apparatus for working the method. Many of the concepts associated with forming emulsions will be well known to persons skilled in the art. Accordingly, the devices and method steps described below have been illustrated in concise schematic form in the drawings, showing only those specific details that are necessary for understanding the embodiments of the present invention, but so as not to obscure the disclosure with excessive detail that will be readily apparent to those of ordinary skill in the art having the benefit of the present description.

In this specification, adjectives such as first and second, left and right, and the like may be used solely to distinguish one element or action from another element or action without necessarily requiring or implying any actual such relationship or order. Words such as "comprises" or "includes" are intended to define a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed, including elements that are inherent to such a process, method, article, or apparatus.

The terms "physically indistinct", "intimately mixed" and "a substantially homogeneous state" are employed herein to describe the physical state of the components of the dispersed phase in relation to one another prior to their introduction into the continuous phase. Their use herein is to clarify that, upon mixing of the immiscible liquids with the co-solvent, either a single phase solution of the dispersed phase is formed or the individual components are at least so intimately mixed (and remain so until a droplet has been released into the continuous phase) that they cannot be said any longer to reside in noticeably distinct phases. The terms "complex emulsion" and "multiple emulsions" are used herein to describe, as a minimum, emulsions in which an internal droplet is contained within an external droplet which is itself within a continuous phase i.e. a double emulsion. Further complex emulsions, such as a triple emulsion, have additional immiscible droplets within the emulsion. Referring to FIG 1 there is depicted a simple apparatus for forming a complex emulsion. The apparatus 1 comprises a T-junction 2 formed by a continuing microfluidic channel 3 and an intersecting microfluidic channel 4. The T-junction microfluidic apparatus is made of PMMA (Polymethylmethacrylate), although persons skilled in the art will appreciate that other materials will be suitable. The channel surface is made hydrophilic by coating with an aromatic epoxy, so that very regular oil droplets can be generated. Continuous phase fluid is urged through the continuing microfluidic channel 3 by pump 5 and dispersed phase fluid is urged through intersecting microfluidic channel 4 by pump 6. In the particular embodiment shown in FIG 1 the continuing microfluidic channel 3 has a width of 100 μm and the intersecting microfluidic channel 4 has a width of 50 μm. Flow rates of continuous phase fluid and dispersed phase fluid are controlled using, for example, motor-driven syringe pumps. When the continuous phase fluid and the dispersed phase fluid intersect at the T-junction 2, droplets are sheared off and continue downstream to a 500 μm expansion channel 7. The apparatus may connect to other devices after the expansion channel 7 depending on the proposed use of the complex emulsions formed.

Example 1

The apparatus of FIG 1 was used to produce water-in-oil-in-water (W/OΛ/V) double emulsions, in which each external oil droplet within the water continuous phase fluid contains a single internal water droplet. Miglyol 812 oil, which is a commercially available material and is generally regarded as a pharmaceutically safe product, is chosen as the oil component of the dispersed oil phase, and water is used as the continuous phase. A ternary system comprising Miglyol 812 oil, ethanol and water formed one homogeneous phase at a volume ratio of 1 :1 :0.04 (v/v/v), and this was used as the dispersed phase fluid. Peptide surfactant AFD4, which forms a cohesive mechanically strong interfacial film in the presence of Zn" at neutral pH, was added to the water continuous phase to prevent droplet coalescence. AFD4 is discussed in a number of journal articles including A.F. Dexter, A.P.J. Middleberg, Journal of physical chemistry C, 2007, 111 , 10484 and A.F. Dexter, A.S. Malcolm, A.P.J. Middleberg, Nature Materials, 2006, 5, 502 and was synthesised for present use by GenScript Corporation, Piscataway, NL, USA. This experiment employed 100 mM AFD4 with 200 mM ZnSO₄ at pH 7.0 in the water continuous phase.

The ternary dispersed oil phase was introduced from the intersecting channel 4 while the continuous water phase flowed through the continuing channel 3. When the dispersed phase fluid penetrated into the continuing channel, a droplet began to grow until the ternary oil droplet containing Miglyol 812 oil, ethanol, and water was sheared off at the T-junction 2. This generated a droplet having a blurred interface, (shown in FIG 2), mainly due to rapid diffusion of ethanol from the oil phase to the continuous water phase. The diffusion time t_f of ethanol from the droplet to the continuous phase can be estimated by:

t_f * 50 - t_D - Pe-²" (1)

where t_D (=h²/D, h is the height of the channel, and D the diffusion constant) is the characteristic time of diffusion, Pe is Peclect number (defined as

Pe=ud/D, where u is the typical velocity, d is the size of the droplet). For D=10 ^"9 m²/s, and droplet size d =100 μm, Equation (1) predicts a diffusion time of 2.5 s at the flow rate of 1.0 ml/hr. As it takes about 2 s for the droplet to travel to the expansion channel, and 200~500 ms for droplet formation, ethanol transfer from the oil to the continuous water phase will be nearly complete when droplets reach the downstream expansion channel, giving an emulsion with small internal water droplets inside the external oil droplet which are held within the aqueous continuous phase (FIG 3).

As can be seen in FIG 3, very regular water-in-oil-in-water double emulsions with narrow size distribution of both the internal and external droplets were generated. The inventors speculate that the peptide surfactant stabilized the double emulsion against coalescence. The external organic droplet size can be adjusted by the flow rate ratio of the dispersed phase fluid to the continuous phase fluid. Other factors including the dimensions of the microfluidic channel and solvent properties such as viscosity, density and interfacial tension have a role to play in the final external droplet size achieved. The size of the internal water droplets depends largely on the relative content of water in the dispersed phase.

In the example, emulsion stabilisation was provided by the peptide surfactant. Emulsion stabilisation could also be provided by placing a charge on the oil drops so that they repel and thus resist coalescence.

Example 2

In a second example the apparatus of FIG 1 was used to generate multiple emulsions. The same ternary Miglyol 812-ethanol-water dispersed phase with a water continuous phase was used but two small non-peptide surfactants were used as emulsion stabilising agents, one for the dispersed oil phase and another for the continuous water phase. The common surfactant sodium dodecyl sulphate (SDS) was used for the water phase at a concentration of 1 mM, and Span 80 (sorbitan oleate) was used for the ternary Miglyol 812- ethanol-water dispersed phase. Water-in-ethanol/oil-in-oil-in-water triple emulsions are generated as shown in FIG 4, with only a single emulsification step. The second-level inner droplet within this emulsion (surrounding the internal water droplet) is a mixture of ethanol and Miglyol 812 which, because of its particular solubility and the effect of the surfactant, remains as a discrete layer rather than combining with either of the oil or water layers between which it is located.

FIG 4(a) shows the droplet formation in a similar manner to that shown in FIG 2. In FIG 4(a), however, the newly formed droplet has a relatively clear interface compared to that seen in FIG 2. This is believed to be due to the comparatively smaller quantities of ethanol which would have been able to escape the dispersed phase and diffuse into the continuous phase. In this example the ethanol is substantially retained within the dispersed phase although it is no longer intimately mixed with the water and oil i.e. the ethanol co- solvent has substantially separated from the water and oil components (some oil remains in the ethanol layer) of the dispersed phase and is maintained that way due to the presence of the Span 80 surfactant and the solubility of the ethanol/oil mixture.

FIG 4(b-d) show triple emulsions formed with Span 80 concentrations of 14 mM, 16 mM and 18 mM respectively. The respective flow rates for the continuous phase fluid and the dispersed phase fluid were 1.0 ml/hr and 0.01 ml/hr. It can be generally seen that increasing the concentration of the Span 80 surfactant in the dispersed phase results in an increase in the size of the internal water droplets within the complex emulsions, which is mainly due to the decrease of mass transfer of ethanol from the disperse phase to the continuous phase in the presence of high concentration surfactant. The morphologies described herein are prepared reproducibly. The ability to produce triple emulsions with three different fluids inside offers new opportunities to engineer novel materials and broadens their potential application, without necessitating complex device redesign. Preferably, the dispersed phase surfactant is an oil soluble dispersed phase surfactant such as a sorbate or sorbitan-based surfactant or an oil soluble polymer.

More preferably, the dispersed phase surfactant is selected from the group consisting of Span 20 (sorbitan monolaurate), Span 60 (sorbitan monostearate) and Span 80 (sorbitan monooleate).

The continuous phase surfactant may be a small molecular weight surfactant, a polysorbate surfactant, a peptide, a protein or a polymer surfactant.

Example 3

In a third example the apparatus of FIG 1 was used to produce double emulsions with multiple fine internal droplets. A ternary sunflower oil, ethanol and water mixture was used as the dispersed phase to prove the adaptability of this method, since sunflower oil is commonly used in food and in cosmetic formulations. A 1 mM aqueous SDS solution was employed as the continuous phase fluid. To generate a homogenous dispersed phase the sunflower oil was mixed with ethanol and water vigorously at a volume ratio of 1 :2:0.04 (v/v/v) in the presence of 20 mM Span 80, and stood until partition equilibrium had been attained. The homogeneous heavy phase, which contains a high concentration of sunflower oil with a certain amount of ethanol and water, was collected for use as the dispersed phase fluid. The complex double emulsion formed is shown in FIG 5, with many small droplets inside due to the high concentration of Span 80 in the dispersed oil phase. As Span 80 is not soluble in ethanol and water, it will primarily partition into the heavy phase. As a result, the concentration of Span 80 is very high in the dispersed oil phase, resulting in double emulsions having many fine inner droplets.

From the results above, it was found that the concentration of surfactant in the dispersed phase influences the type of emulsions prepared by the simple apparatus and method. Without any surfactants in the dispersed phase, double emulsions with one single inner droplet are formed (FIG 3). For very high concentrations of surfactants in the dispersed phase, double emulsions with many fine internal droplets are likely to be generated (FIG 5). In some cases, with an intermediate concentration of surfactant, monodispersed multiple emulsions are produced, such as the triple emulsion shown in FIG 4.

Example 4

A fourth example visually demonstrates the ability of the complex emulsions formed according to this invention to encapsulate active ingredients that can be released from the inner phase to the outer phase by a controlled and sustained mechanism. To demonstrate this encapsulating ability the water- soluble dye, methylene blue, was loaded into a double emulsion, and a blue inner droplet was observed inside the double emulsions (FIG 6(a)), in contrast to a white core (indicating water only) in the double emulsions lacking methylene blue (FIG 6(b)). The oil droplets are generated as previously described in Example 1 but with the additional presence of 0.072% methylene blue in the dispersed phase. Ethanol diffuses from the droplet to the continuous water phase and Miglyol 812 adds an additional barrier that separates the innermost fluid from the continuous phase, preventing methylene blue and water from escaping the oil droplet. The encapsulation process is shown in FIG 6(c). Initially a very small internal water droplet is observed, with some methylene blue dots scattered inside the oil droplet due to low solubility in the oil phase. Then methylene blue begins to accumulate in the internal water droplet as it grows. At about 2 seconds, a single larger water droplet containing methylene blue is formed in the centre of the oil droplet, demonstrating that the water-soluble cargo remains encapsulated within the oil droplets. This result opens a simple method for the encapsulation of active chemicals. Example 5

The examples described above have used ethanol as a co-solvent in each case but the invention is not limited to the use of any particular co-solvent or co-solvents, or indeed any particular combination of components. Persons skilled in the art of emulsions will envisage other emulsion systems that can be formed by the method of the invention using the apparatus of the invention. The inventors speculate that water-in-oil-in-water systems can be formed with various oils and various co-solvents and surfactants. Similarly, oil-in-water-in-oil emulsions can also be formed, as can further multiple emulsions of this kind.

By way of example a water-in-oil-in-water emulsion was formed using acetone as the co-solvent. This example was carried out in a similar manner to Example 1 described above. The dispersed phase fluid was formed from Miglycol 812 oil with acetone and water in the volume ratio of 1.5:1.5:0.04, (v/v/v). The continuous phase fluid was a 10 mM aqueous SDS solution. The respective flow rates were 1.0 ml/hr for the continuous phase fluid and 0.01 ml/hr for the dispersed phase fluid. The resultant complex emulsions are shown in FIG 7.

The Examples demonstrate the use of ethanol or acetone as co-solvents. However, any solvent with intermediate hydrophilicity may be suitable for use as a co-solvent in the present inventive method. The co-solvent has the effect of breaking down the phase barrier between the immiscible fluids of the dispersed phase, e.g. oil and water, to allow them to become intimately mixed or to form a homogeneous phase.

The co-solvent may be any solvent or combination of solvents which allows the two or more immiscible emulsion forming fluids in the dispersed phase fluid to remain intimately mixed.

Preferably, the co-solvent is a hydrophilic solvent.

More preferably, the co-solvent is selected from the group consisting of an alcohol, a ketone, an ether, a formamide, a nitrile, a carboxylic acid and a sulfoxide. Even more preferably, the co-solvent is selected from the group consisting of methanol, ethanol, acetone, terahydrofuran, N₁N- dimethylformamide, acetonitrile, acetic acid and dimethylsulfoxide.

More preferably still, the co-solvent is ethanol or acetone. Persons skilled in the art will be aware of other suitable co-solvents for any particular emulsion system.

Example 6

The microfluidic device of FIG 8 was used to produce oil-in-water-in-oil double emulsions by the method described above. The microfluidic device is made of PDMS (polydimethylsiloxane) with a hydrophobic channel surface and has the structure shown in FIG 8. The microfluidic channel apparatus 11 comprises a flow-focusing junction 12 formed by two side channels 13 carrying the continuous phase fluid and a central channel 14 supplying the dispersed phase fluid. Miglyol 812 is used as the continuous phase fluid and is urged through the two side channels 13 by pump 15. The dispersed phase fluid is water-ethanol-Miglyol 812 (1 :8:0.1 , v/v/v) and is urged through the central channel 14 by pump 16. The respective flow rates are 0.2 ml/hr for the continuous phase fluid and 0.02 ml/hr for the dispersed phase fluid.

FIG 9 shows an expanded view of the channel intersection 12 in the area surrounded by dotted lines in FIG 8. When the continuous phase fluid and the dispersed phase fluid intersect at the junction 12 droplets are sheared off and continue downstream through continuing channel 17 to expansion channel 18.

The resultant oil-in-water-in-oil double emulsions are shown in FIG 10. The internal droplet size of FIG 10 is smaller than the internal droplets produced in example 1. This is because the residence time in the continuing channel 17 used was not sufficient for all ethanol in the droplet to transfer to the oil phase. A longer residence time would result in a larger internal droplet.

It is postulated that the reason for this delay is that the ethanol takes a longer time to diffuse out into the oil continuous phase due to the viscosity of Miglyol 812 (30 mPa.s) being much greater than that of water (1 mPa.s) thus presenting a greater barrier to the transfer. However, this result shows that, in some cases, the residence time in the continuing channel can be used as a control factor for the size of the internal droplet. For example, in the above example a longer residence time in the channel would have resulted in a larger internal droplet as all of the ethanol eventually escapes and the entrapped oil coalesces. Thus, for double emulsions such as this it is necessary to use a co- solvent which can diffuse through the oil to escape into the water continuous phase.

Example 7

The apparatus of FIG 1 was used in essentially the manner described for example 1 except the dispersed phase was a ternary phase comprising ethyl acetate and ethanol and water in the volume ratio of 1.0:1.0:1.0, (v/v/v). The continuous phase was a 10 mM aqueous solution of SDS with a flow rate ratio of 1.0 to 0.01 mL/h continuous to dispersed phase. This produced a water-in-ethyl acetate-in-water double emulsion as shown in FIG 11. The internal water droplets are considerably larger than those obtained using the method of example 1. This indicates that employing a greater relative amount of water in the dispersed phase will result in a larger internal water droplet in the final emulsion thereby providing a further mechanism for morphology control.

Example 8

The apparatus of FIG 1 was used with a dispersed phase of Miglyol 812 and ethanol in a volume ratio of 1.0:1.0 (v/v) with 18 mM of Span 80 and a continuous phase of 10 mM SDS solution. A flow rate ratio of 1.0 to 0.01 ml_/h continuous to dispersed phase was employed. This produced a water-in- ethanol(with some oil)-in-oil-in-water triple emulsion, much as was seen in example 2, and which can be seen in FIG 12. The water droplet in this emulsion comes from a small amount of water which is inherent within or is absorbed into the ethanol during the experiment.

The internal water droplet is considerably smaller in this emulsion as becomes clear when FIGs 4 (b)-(d) are compared with FIG 12. This is due to the lower relative amount of water contained within the dispersed phase in this example and once again demonstrates that emulsion morphology is highly tunable.

Example 9

The invention described herein is not limited to the use of an apparatus comprising a microfluidic channel and to demonstrate this a bulk double emulsion was generated using a ternary solvent system comprising ethyl acetate and ethanol and water in the volume ratio of 1.0:1.0:1.0, (v/v/v) in the presence of 10 mM Span 80. This solvent system was added to a 50 mM SDS solution and emulsification achieved by sonication for 30 seconds (x3). This one step solvent shift process produced a double emulsion of water-in-ethyl acetate-in- water as shown in FIG 13.

This is an extremely simple way of achieving a bulk double emulsion and will have many applications where a narrow dispersion of droplet size is not crucial. It is clear, however, that when a reproducible and uniform external droplet size is required then a device employing a microfluidic channel is preferred.

Other Embodiments

As discussed in relation to example 9, T-junction and flow-focusing microfluidic devices have been used for examples one to eight, described above, but the invention is not limited to these particular apparatus or to any particular apparatus geometry. An apparatus may merely comprise a container in which to mix the two phases and a device to achieve the mixing such as a sonicator or shear generating device. The requirement for the apparatus suited for use with examples one to eight is that it is a microfluidic channel device that produces droplets at the intersection of immiscible continuous phase and dispersed phase fluids such that discrete droplets of the dispersed phase fluid are formed within the continuous phase fluid. Two other embodiments of a suitable apparatus are shown in FIG 14 and FIG 15. Persons skilled in the art will be able to envisage other microfluidic channel apparatuses which, while different in appearance, do not depart from the spirit of the invention.

FIG 14 shows a microchannel apparatus 21 comprising a Y-junction 22 formed by a continuing microfluidic channel 23 and an intersecting microfluidic channel 24. As with the embodiment of FIG 1 and FIG 8, the microchannel apparatus may be formed from PMMA, PDMS or any other suitable material. The continuing microfluidic channel 23 has a width of 100 μm and the intersecting microfluidic channel 24 has a width of 50 μm. Continuous phase fluid is urged through the continuing microfluidic channel 23 by pump 25 and dispersed phase fluid is urged through intersecting microfluidic channel 24 by pump 26. When the continuous phase fluid and the dispersed phase fluid intersect at the Y-junction 22 droplets are sheared off and continue downstream to an expansion channel 27.

FIG 15 shows a microfluidic apparatus 31 comprising a concentric junction 32 formed by a continuing microfluidic channel 33 and an intersecting microfluidic channel 34. As with the embodiment of FIG 1 and FtG 8, the microfluidic apparatus may be formed from PMMA, PDMS or any other suitable material. Continuous phase fluid is urged through the continuing microfluidic channel 33 by pump 35 and dispersed phase fluid is urged through intersecting microfluidic channel 34 by pump 36. When the continuous phase fluid and the dispersed phase fluid intersect at the concentric junction 32, droplets are sheared off and continue downstream to an expansion channel 37. In this case the intersecting microfluidic channel 34 has a width of 50 μm and the continuing microfluidic channel 33 has a width of 150 μm.

In conclusion, when emulsion droplet size distribution control is critical then a number of simple microfluidic device types have been described that can generate complex emulsions, including double emulsions having either a single internal droplet or many fine droplets, or multiple emulsions. The emulsions are formed without the need for complex and difficult to manufacture microfluidic device geometries. The invention enables the ability to encapsulate substances in the inner droplets, which is of great importance for potential delivery applications.

The method relies on mass-transfer control of emulsion formation instead of more complex hydrodynamic or mechanical control as found in the prior art. It relies specifically on initial solubilisation of a droplet component in another component (such as water in oil or oil in water) using a co-solvent to form a substantially single phase or homogeneous solution, formation of a single emulsion (if required, at a channel intersection), and then autocatalytic formation of a complex emulsion from the single emulsion through co-solvent movement into either the continuous phase fluid or into a separate layer within the emulsion.

The above description of various embodiments of the present invention is provided for purposes of description to one of ordinary skill in the related art. It is not intended to be exhaustive or to limit the invention to a single disclosed embodiment. As mentioned above, numerous alternatives and variations to the present invention will be apparent to those skilled in the art of the above teaching. Accordingly, while some alternative embodiments have been discussed specifically, other embodiments will be apparent or relatively easily developed by those of ordinary skill in the art. Accordingly, this invention is intended to embrace all alternatives, modifications and variations of the present invention that have been discussed herein, and other embodiments that fall within the spirit and scope of the above described invention.

Claims

CLAIMS 1. A method of forming a complex emulsion including the steps of: forming a dispersed phase fluid comprising two or more at least partially immiscible emulsion forming fluids maintained in an intimately mixed state by a co-solvent; introducing the dispersed phase fluid into a continuous phase fluid which is immiscible with at least one of the emulsion forming fluids; and allowing the co-solvent to substantially separate from the at least two emulsion forming fluids to thereby form a complex emulsion.

2. The method of claim 1 wherein the two or more immiscible emulsion forming fluids in the dispersed phase fluid are maintained in a physically indistinct state by the co-solvent.

3. The method of claim 1 wherein the two or more immiscible emulsion forming fluids in the dispersed phase fluid form a substantially homogeneous phase with the co-solvent.

4. The method of claim 1 wherein the co-solvent solubilises the two or more immiscible emulsion forming fluids in the dispersed phase fluid.

5. The method of any one of the preceding claims wherein the co-solvent is a hydrophilic solvent.

6. The method of claim 5 wherein the co-solvent is selected from the group consisting of an alcohol, a ketone, an ether, a formamide, a nitrile, a carboxylic acid and a sulfoxide.

7. The method of claim 5 wherein the co-solvent is selected from the group consisting of methanol, ethanol, acetone, terahydrofuran, N₁N- dimethylformamide, acetonitrile, acetic acid and dimethylsulfoxide.

8. The method of claim 7 wherein the co-solvent is ethanol or acetone.

9. The method of any one of the preceding claims further including the step of adding an emulsion stabilising agent to the dispersed phase fluid prior to its introduction to the continuous phase fluid.

10. The method of claim 9 wherein the emulsion stabilising agent limits coalescence of one or more of the immiscible emulsion forming fluids and/or the co-solvent.

11. The method of claim 9 wherein the emulsion stabilising agent is a surfactant.

12. The method of claim 11 wherein the surfactant is an oil soluble surfactant and/or a polymer surfactant.

13. The method of claim 11 wherein the surfactant is selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate and sorbitan monooleate.

14. The method of claim 9 wherein the concentration of the emulsion stabilising agent in the dispersed phase fluid influences the morphology of the resulting emulsion.

15. The method of any one of the preceding claims further including the step of adding an emulsion stabilising agent to the continuous phase fluid.

16. The method of claim 15 wherein the emulsion stabilising agent is a surfactant.

17. The method of claim 16 wherein the surfactant is a small molecular weight surfactant, a polysorbate surfactant, a peptide, a protein or a polymer surfactant.

18. The method of claim 16 wherein the surfactant is selected from the group consisting of sodium dodecyl sulfate, Tween 20, AM1 , AFD4, and lysozyme.

19. The method of any one of the preceding claims wherein the dispersed phase fluid is introduced into the continuous phase fluid through a first microfluidic channel to form a droplet.

20. The method of claim 19 wherein the first microfluidic channel intersects with a second microfluidic channel containing the continuous phase fluid.

21. The method of claim 20 further including the step of controlling the external size of the droplet by controlling the ratio of flow rate of the continuous phase fluid to the dispersed phase fluid.

22. The method of any one of the preceding claims further including the step of controlling the size of an internal droplet to be formed within the complex emulsion by varying the relative amounts of one or more of the immiscible emulsion forming fluids and/or the co-solvent in the dispersed phase fluid.

23. The method of any one of the preceding claims further including the step of controlling the formation of the complex emulsion by introducing the dispersed phase fluid, via a first microfluidic channel, into a continuous phase fluid flowing through a second microfluidic channel.

24. The method of claim 23 wherein the first and second microfluidic channels intersect at a junction which is a T-junction, Y-junction or concentric junction.

25. The method of claim 23 wherein the first and second microfluidic channels are continuous with a continuing channel and an expansion section.

26. The method of claim 25 wherein the residence time of the complex emulsion in the continuing channel influences the size of an internal droplet within the complex emulsion.

27. The method of any one of the preceding claims wherein the complex emulsion is a double emulsion containing a plurality of internal droplets.

28. Use of a complex emulsion formed by the method of any one of the preceding claims in the encapsulation and/or delivery of one or more agents.