Field of Invention

[0001] The invention relates to a composition comprising an emulsion, more specifically a double emulsion, for use in the delivery of active ingredients, a process for making said composition, a method of delivering active ingredients using the composition and methods of treatment using the composition.

Background

[0002] In order to achieve stable dispersions of one liquid in another, emulsions in the traditional sense require the addition of an interface-active substance (emulsifier). Emulsifiers have an amphiphilic molecular structure, consisting of a polar (hydrophilic) and a nonpolar (lipophilic) molecular moiety, which are spatially separate from one another. In simple emulsions, finely disperse droplets of one phase, surrounded by an emulsifier shell (water droplets in water-in-oil [W/O] emulsions or lipid vesicles in oil-in-water [O/W] emulsions) are present in the second phase. Emulsifiers lower the interfacial tension between the phases by positioning themselves at the interface between the two liquids. At the phase boundary, they form oil/water interfacial films, which prevent irreversible coalescence of the droplets. Emulsions are frequently stabilized using emulsifier mixtures.

[0003] Oil based formulations (oil as the continuous phase) are obtained by dissolving, emulsifying and/or suspending active materials in an oil phase. Such products are employed across a range of technology and business sectors that includes pharmaceutical agents, food additives, cleaning agents, complexing agents, personal care substances, lubricants, adhesives, heating/cooling agents, colourants, indicators and crop protection chemicals. Water-in-oil emulsions (oil continuous emulsions) contain water soluble active materials in the dispersed aqueous phase. Water-based formulations (water as the continuous phase) are obtained by dissolving, emulsifying and/or suspending active materials in water. The use of such products is widespread across many technology and business sectors but includes pharmaceutical agents, food additives, cleaning agents, complexing agents, personal care substances, lubricants, adhesives, heating/cooling agents, colourants, indicators and crop protection chemicals. Within crop protection, the efficient use of aqueous systems with certain crop protection agents, however, may be restricted due to their poor water-solubility. Therefore, active ingredients are often administered in the form of oil-in-water emulsions.

[0004] Hydrocolloids are compounds which swell when reacted with water to form gel like materials. Well known examples of hydrocolloids include gellatin and pectin which are readily obtainable from natural sources. These materials, when mixed with water and heated, form gel like structures and can be tailored in their properties by varying the ratio of water to hydrocolloid and treatment conditions. Such gel-like structures are very useful for supporting and/or entrapping active ingredients in order to preserve or protect said active ingredients from damage or to provide delayed or slow release in vivo.

[0005] The use of cross-linked gels containing cells has been known for a number of years. For example, using cells trapped within a gel matrix. For example, US2005/0003010A describes a cross-linked alginate obtained by cross-linking sodium alginate solution by the addition of calcium ions to form a gel. Such gels are used to induce cell proliferation, by, for example, injecting the solution into damaged tissue. The cross-linking is broken on shearing, for example by passing through a needle to a site to be treated.

[0006] US2007/0116680 describes embedding stem cells in a three dimensional hydrogel. Stem cells are suspended in a matrix solution, the matrix is gelled and the cells are contained within microbeads formed from the matrix. Hydrogel encapsulated stem cells are also disclosed in US2012/0027860A. Adipose-derived mesenchymal stem cells are mixed with a gel forming solution prior to causing the solution to gel.

[0007] Another method for introducing active ingredients into hydrocolloids is to heat the hydrocolloid above its melting point and then gradually allow this to cool in solution, encapsulating active ingredients as it does so. For instance, WO2011/077073 and WO2014/ 140549 each describe a processes wherein solutions containing hydrocolloids ("monoglyceride and triglyceride" and "alginate") are heated to temperatures of approximately 70°C and 90°C respectively in order to create the necessary hydrocolloid gels. US 5,698,188 also described how carrageenan gels can be made by heat treating carrageenan in an aqueous solution at 90°C.

[0008] However, many active ingredients are heat sensitive and so cannot be incorporated into hydrocolloid particles in this manner as the high temperatures required to melt the hydrocolloid greatly exceed the temperature limits over which the active ingredients are stable.

[0009] Accordingly, it is desirable to have a method of making hydrocolloid particles containing one or more active ingredients without requiring high temperatures. The invention is intended to overcome or at least ameliorate this problem.

Summary of Invention

[0010] There is provided, in a first aspect of the invention, a process for making a double emulsion, comprising the steps of: a) providing a first aqueous solution comprising: a hydrocolloid and one or more active ingredients; b) shearing the solution of step a); c) adding the composition from step b) to a hydrophobic solution comprising a first emulsifier; d) shearing the solution of step c) to form a primary emulsion; e) adding the emulsion of step d) to a second aqueous solution comprising a second emulsifier; and f) shearing the composition of e) to form a double emulsion; wherein the first aqueous solution is charged.

[0011] The inventors have found that, despite the directions in the prior art to melt hydrocoUoids in order for active ingredients to be incorporated therein, this is not necessary providing that a series of shearing steps are conducted. This process therefore permits thermally unstable materials to be introduced into hydrocoUoids that would not be possible using conventional techniques.

[0012] The double emulsion that is produce by the process of the invention is typically for use in delivering active ingredients to a target site, typically this is in the body.

[0013] The term "primary emulsion" as used herein is intended to refer to a mixture of hydrocolloid particles which are substantially uniformly dispersed throughout a hydrophobic phase, the particles being prevented from agglomerating by virtue of an emulsifying agent associating with the hydrocolloid particles at the interface between the particles and the hydrophobic phase. This may be described as a, "gel-in-oil" emulsion.

[0014] The term "double emulsion" as used herein is intended to refer to the product of adding the primary emulsion to a hydrophilic phase with a further emulsifier. This results in particles having a hydrocolloid core and an outer "jacket" of hydrophobic solution. The particles are substantially uniformly dispersed throughout an aqueous phase (the second aqueous phase) and are prevented from prevented from rupturing due to emulsifiers at the core-jacket interface and the jacket-solution interface. This may be described as a, "gel-in- oil-in-water" emulsion.

[0015] The temperature of the process is typically less than the melting point of the hydrocolloid or hydrocolloids used in the process. Typically, the temperature is also lower than that required to denature or degrade the active ingredients for incorporation into the hydrocolloid particles. As such, although the absolute maximum temperature of the process is determine by the materials used in a given process, it is typically the case that the temperature is less than 80°C, more typically less than 70°C, more typically less than 60°C, more typically less than 50°C, more typically less than 40°C and most typically about 30°C. As the process occurs in aqueous media, it is also the temperature is typically controlled to be high enough to prevent freezing of the solvent or active ingredients (especially wherein the active ingredients are cells). Typically, the temperature is set to provide a balance between preventing degradation of active ingredients and providing adequate reaction kinetics.

[0016] The rate of shearing is determined by the robustness of the materials to be incorporated into the hydrocolloid particles. There is no particular restriction on the shear rate used provided that the components of the composition, particularly the active ingredients, will tolerate such shear rates without degrading. For example, where the active ingredients are cells, the shearing applied to the solution at each of the stages is typically low (around 500 s ^-1 ) in order to prevent damage to the cells. However, where the active ingredients are small molecules, not so easily damaged by shear forces, higher shear rates can be used.

[0017] Accordingly, it is typically the case that the shear rate at each of the stages are each independently less than 2000 s ^-1 , more typically less than 1800 s ^-1 , more typically still, less than 1500 s ^-1 , even more typically less than 1000 s ^-1 . Frequently, the shear rate will be less than 800 s ^-1 and more typically equal to or less than 500 s ^-1 . Although there is no lower limit on the shear rate, with very low shear rates, the process takes a long time. Accordingly, it is preferred that the shear rate is greater than 100 s ^-1 , typically greater than 200 s ^-1 and more typically greater than 400 s ^-1 .

[0018] The duration of the shearing steps is not particularly limited and varies depending on the temperature, choice of hydrocolloid and aqueous solutions used. Often, the shearing is conducted until no further change in the solution is observed. Typically this is less than one hour, more typically less than 30 minutes and even more typically less than 10 minutes.

[0019] There is no particular restriction on how the shear force is applied to the composition and the skilled person would be able to use a range of different process to impart the necessary shear to the composition. For instance, a rheometer may be used to impart the shear force. This could be a dynamic shear rheometer, pipe or capillary rheometer, rotational cylinder rheometer or cone and plater rheometer, linear rheometer or combination thereof.

[0020] The term "hydrocoUoid" as used herein is intended to take its typical meaning. Namely, a material which is hydrophilic and which, in the presence of water, swells to form particles. Typically, the hydrocoUoid forms gels and most typically the hydrocoUoid may be selected from one or more polymers, which may in some embodiments be cross -linked or cross-linkable.

[0021] There is no particular restriction on the choice of hydrocoUoid that is used in the invention. The hydrocoUoid may be a naturally occurring hydrocoUoid such as polysaccharides or may be synthetic hydrocoUoid such as, for example, polyacrylates, and polyethylene glycol. Typically, where the hydrocoUoid is a naturally occurring hydrocoUoid it is typically selected from: agar, agarose, arabinoxylan, carrageenan, gelatin, gellan gum, glucan, curdlan, pectin, xanthan gum, gum arabic, guar gum, locust bean gum, cellulose and derivatives thereof, alginate, fibrin and starch or combinations thereof. Other suitable hydrocolloids include chitosan, dextran, collagen and hyaluronic acid. Typical examples of cellulose derivatives are those compounds wherein one or more of the hydroxyl groups have been functioned. Typically, these groups are reacted to form alloy groups, alkoxycarboxylic acid groups, alkyoxyesters, alkoxyethers, or combinations thereof. Typical cellulose derivatives include, carboxymethyl cellulose, methyl cellulose and ethyl cellulose.

[0022] Typically, the hydrocoUoid is selected from carrageenan, gelatin, agar, alginate, cellulose, cellulose derivatives and combinations thereof. Carrageenan is most typically used.

[0023] The melting point of the hydrocolloids used in the present invention is typically greater than 30°C, more typically greater than 40°C, even more typically greater than 50°C, more typically still greater than 60°C, even more typically still greater than 70°C, and most typically greater than 80°C.

[0024] The size of the hydrocolloid particles are typically in the range Ιμιη to ΙΟΟΟμιη, more typically Ιμιη to 500μιη, more typically still Ιμιη to ΙΟΟμιη, and even more typically still

[0025] The term "active ingredients" used herein is intended to refer the compound or compounds that are incorporated into the hydrocolloid particles at the centre of the particles of the emulsion produced by the method of the first aspect of the invention.

[0026] The composition obtainable by the process of the first aspect of the invention can be used in a wide range of different applications, some of which are described below in more detail. However, one typical application is as a delivery system for active ingredients for use in vivo. Accordingly, in one typical embodiment, the active ingredients are therapeutic agents. There is no particular restriction on the type of therapeutic agent used and one or more different therapeutic agents may be combined within the emulsion particles. However, the therapeutic agents may be selected from the group consisting of, proteins, antibodies, DNA, RNA, siRNA, hormones, nutrients, growth factors, inflammatory compounds, stimulating factors, cells, small molecule drugs, or combinations thereof. Other compounds useful in maintaining cells with the hydrocolloid may be added and/or compounds to help appropriate differentiation of cells or direct the active ingredients to appropriate cell targets.

[0027] In some embodiments, the active ingredients are cells. The cells may be prokaryotic or eukaryotic. Typically the cells are plant or animal cells, for example, bird, inspect, reptile, more typically mammal cells.

[0028] Stem cells may be used. Stem cells are typically human or non-human, pluripotent or totipotent, typically not human totipotent stem cells. The stem cells may be obtained from cell banks or, for example, embryonic, non-embryonic (typically non-embryonic human stem cells), cord blood stem cells or adult mesenchymal stem cells. They may be obtained from single blastomere biopsy, a non-destructive method of producing embryonic stem cells, or from adult cells such as iPS (induced pluripotent stem) cells. Other cells such as differentiated cell lines, or cells isolated from the blood or tissue may be used. The cells may be a patient's own cells. They may be autologous cells. [0029] They may be osteoblasts/MC 3T3 osteoblast like cells, chondrocytes, keratinocytes, fibroblasts, dermal fibroblasts, tenocytes, neurons, osteocytes, osteoclasts, adipocytes or any other cell type with therapeutic activity.

[0030] In addition, additives may be used in tandem with the one or more therapeutic agents. For example, flavourings, co-drugs (i.e. drugs which work synergistically with another therapeutic agent), vitamins, pre-biotics, pro-biotics and other additives may be introduced. Typical additives include, for example, flavourings in order to mask unpleasant taste, co- drugs to counteract side-effects and/or other compounds to promote other synergistic effects.

[0031] Whilst there is no particular restriction on the choice of emulsifiers used in the present invention, the first and second emulsifiers are typically selected from: anionic surfactants, cationic surfactants, non-ionic surfactants, or combinations thereof. The person skilled in the art would be familiar with typical anionic surfactants. However, typical anionic surfactants include: carboxylates (such as fatty acids), sulfonates (such as alkyl sulfonates and aryl sulfonates), sulfates (such as alkyl sulfates, alkylether sulfates, sulfated alkanolamides and/or phosphonated sulfates) and combinations thereof.

[0032] Although there is no particular restriction on the choice of cationic surfactants, typical cationic surfactants include: alkyl amines, and alkyl ammonium salts, aromatic or saturated heterocylic compounds (such as alkyl pyridines and alkyl imidazoles), or combinations thereof.

[0033] It is also the case that non-ionic surfactants may be used. There is no particular limitation on the choice of non-ionic surfactant. Typical examples of non-ionic surfactants are selected from: ethoxylated linear alcohols, ethoxylated alkyl phenols, alkyl esters (such as fatty acid esters), polyalcohols, poly ethers copolymers, thiols and combinations thereof.

[0034] It is typically the case that the one or more first and second emulsifiers used in the process of the invention are polymeric. The inventors have found that emulsifiers with a high molecular weight, comparable with polyglycerol polyricinoleate (PGPR) tend to provide improved stabilisation of double emulsions. Typical emulsifiers include, but are not limited to, polyglycerols, typically functionalised with one or more alkyl chains such as fatty acids. In particular, wherein the alkyl chains are greater than 10 carbons in length, typically greater than 30, or more typically greater than 50, or even more typically greater than 100 carbons in length. High molecular weight emulsifiers with large structures have been found to offer improved stability to double emulsion particles, especially where such emulsifiers are used as the first emulsifier.

[0035] The first aqueous solution typically comprises cations, anions, zwitterions or a combination thereof. The inventors have found that having a charged aqueous solution improves the dissolution and hydrocoUoid particle forming process of step a) however there is no particular restriction on the charged particles that provide charge. For the avoidance of doubt, the term "charged aqueous solution" is intended to refer to a solution comprising some water and at least some charged particles or species, typically ions. It is often the case that the first aqueous solution is anionic, however the first aqueous solution may also be cationic. The first aqueous solution will often be a buffer solution, preferably a biological buffer suitable for medical applications.

[0036] It is typically the case that, after the first aqueous solution has been made (typically by adding the hydrocoUoid and active ingredients to a charged aqueous solvent) it is then immediately sheared according to step b). The inventors have found that if the first aqueous solution of step a) is left to stand for a prolonged period of time, the hydrocoUoid solution may aggregate and make micronisation of the hydrocoUoid difficult. Accordingly, the delay between making the first aqueous solution and shearing the solution in step b) is typically less than 5 minutes, more typically less than 1 minute, and more typically still less than 30 seconds.

[0037] There is no particular restriction on the concentration of hydrocoUoid used in the first aqueous solution. Typically, the amount of hydrocoUoid is less than 25% by weight of the first aqueous solution, more typically less than 20%, more typically still less than 10%. It is often the case that the amount of hydrocoUoid used is in the range of 1% to 10% by weight of the first aqueous solution, more typically, in the range of 5% to 10% and even more typically about 8% hydrocoUoid by weight of the first aqueous solution. Some applications favour higher concentrations of hydrocoUoid than others and, using the process of the first aspect of the invention, it is possible to create compositions with higher levels of hydrocoUoid than with conventional methods.

[0038] The hydrophobic solution used in the process of the invention is not particularly limited, however it is typically the case that the hydrophobic solution is biocompatible and is often a lipid. Typical hydrophobic solutions include any non-polar solution typical examples of which include: naturally occurring and synthetic oils, waxes, fats and combinations thereof. These may be derived from animals or plants, such as sunflower oil, or mineral oils and/or fractions thereof.

[0039] The ratio of the first aqueous solution to the hydrophobic solution is typically in the range of 50: 1 to 1:50 respectively, more typically 20: 1 to 1:20, more typically still 10: 1 to 1: 10 and even more typically still in the range 5: 1 to 1:5. It is often the case that the ratio will be in the range 2: 1 to 1:2 and furthermore, the ratio may be about 1: 1.

[0040] The second aqueous solution is less limited on its composition as there is no need for this solution to be charged, although the solution may be charged and/or as described for the first aqueous solution. The ratio of the primary emulsion to the second aqueous solution is typically in the range of 50: 1 to 1:50 respectively, more typically 20: 1 to 1:20, more typically still 10: 1 to 1: 10 and even more typically still in the range 5: 1 to 1:5. It is often the case that the ratio will be in the range 2: 1 to 1:2 and furthermore, the ratio may be about 1: 1.

[0041] The process of the first aspect of the invention may further comprising a sterilisation step which may be via irradiation or chemical means. Irradiation may utilise, for example, ultraviolet, x-ray or gamma ray radiation to sterilise the medium, for example, to remove unwanted bacterial contamination. The choice of sterilisation depends on the particular active ingredients being incorporated into the hydrocolloid particles.

[0042] There is also provided, in a second aspect of the invention, an aqueous composition comprising an aqueous solvent and one or more particles, the particles comprising: a hydrocolloid core comprising one or more active ingredients; an hydrophobic layer adjacent the hydrocolloid core; a first emulsifier positioned between the hydrocolloid core and the hydrophobic layer; and a second emulsifier positioned between the hydrophobic layer and the aqueous solvent; wherein the one or more active ingredients are thermally sensitive.

[0043] The term "thermally sensitive" is intended to encompass those compounds, substances and/or organic matter that cannot be incorporated into hydrocolloids using traditional methods that involve melting of the hydrocolloids. Therefore, it is usually the case that the active ingredient used in the composition of the invention are only thermally stable below the melting point of the hydrocolloid. There is no particular restriction on the temperature as this depends largely on the particular choice of hydrocolloid. However, the term "thermally sensitive" is typically intended to cover compounds, substances and/or organic matter which degrades (or at least begins to degrade) above 80°C, more typically above 70°C, even more typically above 60°C, more typically still above 50°C, even more typically still above 40°C and most typically above 30°C.

[0044] The composition is typically a delivery medium. The term "delivery medium" is intended to mean, a system for delivery active ingredients to a particular target site. The emulsion described herein can be used to entrap active ingredients that would otherwise breakdown in certain environments. This is particularly true when used in vivo and the composition may be used to transport active ingredients to regions where they would not otherwise reach, for instance due to incompatible solubility, poor pH stability or premature reaction with other materials in vivo. Typically, where the active ingredient comprises one or more cells, the delivery medium is a cell delivery medium.

[0045] It is typically the cases that the active ingredients are substantially confined to the hydrocolloid core. The active ingredient may be dispersed through the hydrocolloid core and/or distributed across the surface of the core. Typically, the active ingredients are distributed throughout the hydrocolloid core.

[0046] Typically, the composition is a double emulsion.

[0047] The aqueous composition may be obtainable by way of the process of the first aspect of the invention. The process of the first aspect of the invention has made possible the fabrication of new compositions and emulsions wherein heat sensitive materials can be introduced into hydrocolloid particles.

[0048] The components and dimensions of the one or more particles are as described above.

[0049] In one embodiment the composition is used as an active ingredient delivery system. Typically, the composition is used as a delivery system for a therapeutic agent such as those described above. More typically, the composition is a cell delivery system. The inventors have found that the present invention is particular suitable to encapsulating therapeutic agents, especially cells. [0050] In one embodiment, the composition may comprise two or more therapeutics. This is particularly desirable wherein the disease to be treated is very resistant to a single therapeutic and/or wherein one therapeutic induces side-effects which can be counteracted by the second therapeutic.

[0051] Additives may also be used together with the one or more active ingredients. Typical additives include: flavourings (especially useful for paediatric applications, in order to mask the taste of unpleasant tasting drugs), co-drugs (compounds that work synergistically with other active ingredients, for instance to inhibit side-effects or improve efficacy), vitamins, minerals or nutrients (for instance to supply a patient with nutrition or to keep any cells to be delivered healthy for a desired duration in transits through the body or for storage purposes), colourings or combinations thereof. There is no particular restriction on the choice of additive, providing that it does not negatively interfere with the operation of the composition.

[0052] In an alternative embodiment, the composition may be used as an additive in food products. The emulsion of the invention allows a range of nutrients (such as amino acids, proteins, polyunsaturated fats and oil), nutraceuticals (such as vitamins and/or minerals) and additives (e.g. flavourings, drugs, pH stabilisers, etc.) to be introduced into many common food products that would not otherwise be soluble or stable therein. This reduces the amount of fats needed in conventional products to "carry" such materials. Typical examples of food products include: mayonnaise, sauces, water or lipid continuous spreads, dips, confectionary, and drinks.

[0053] There is also provided in a third aspect of the invention, a composition according to the second aspect of the invention for use in the treatment of disease. Typical diseases include, but are not limited to: cancer, bacterial or viral infection and nutrient deficiency. The composition may also be used treat diseased or damaged tissue.

[0054] There is also provided a fourth aspect of the invention, a method of treating a disease comprising the steps of administering the composition, containing one or more therapeutic agents, to a patient.

[0055] The composition of the invention can be used to deliver therapeutic agents to target sites within the body and may be used to repair or augment the repair of damaged or diseased tissue. This is particularly the case when the active ingredient comprises cells, such as stem cells.

[0056] There is also provided in a fifth aspect of the invention, the use of the composition of according to the second aspect of the invention in the delivery of active ingredients. Typically this is intended to cover specific regions within the body but could also include the delivery of active ingredients to cells, or materials outside the body for instance in industrial processes or research applications.

[0057] The invention will now be described by way of example only, with reference to the accompanying figures.

Brief Description of Figures

[0058] FIGURE 1 shows Kappa carrageenan in DMEM (Dulbecco's Modified Eagle's Medium) at a range of concentrations (2% (a), 4% (b), 5% (c), 6% (d), 7% (e) and 8% (f)). Samples were formed at 30°C, and sheared at 500 s ^-1 for 60 minutes, using a rheometer.

[0059] FIGURE 2 shows inverted vials of 6% (a), 7% (b) and 8% (c) kappa carrageenan in DMEM. Photographs were taken after vials had been inverted for 3 hours.

[0060] FIGURE 3 shows differential scanning calorimetry (DSC) heating (a) and cooling (b) profiles for different concentrations of KC (0% (□), 2% (·), 4% (Δ), 6% ( T ), 7% (0), and 8% (►)) dispersed in DMEM buffer.

[0061] FIGURE 4 shows Kappa carrageenan in HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) at a range of concentrations (2% (a), 4% (b), and 6% (c)). Samples were formed at 30°C, and sheared at 500 s-1 for 60 minutes, using a rheometer.

[0062] FIGURE 5 shows differential scanning calorimetry (DSC) heating (a) and cooling (b) profiles for different concentrations of KC (0% (□), 2% (·), 4% (Δ), 5% ( T ) and 6% (0)) dispersed in HEPES buffer.

[0063] FIGURE 6 shows oil continuous emulsions with DMEM as the aqueous phase (a) and with warm (30°C) water hydrated kappa Carrageenan at 2% (b), 4% (c), 5% (d), 6% (e), 7% (f) and 8% (g) kappa carrageenan in DMEM, emulsified with 1% PGPR. [0064] FIGURE 7 shows micrographs of duplex emulsions with the inclusion of 6% (a, d), 7% (b, e) and 8% (c, f) KC dispersed in DMEM buffer. Inner droplets were stabilised with PGPR, and outer droplets stabilised with Tween 20 (a, b, c) and Tween 80 (d, e, f). Micrographs were taken immediately after forming the duplex emulsion.

[0065] FIGURE 8 shows micrographs of duplex emulsions with the inclusion of 5% (a, c) and 6% (b, d) KC dispersed in HEPES buffer. Inner droplets were stabilised with PGPR, and outer droplets stabilised with Tween 20 (a, b) and Tween 80 (c, d). Micrographs were taken immediately after forming the duplex emulsion.

[0066] FIGURE 9 shows micrographs of duplex emulsions with the inclusion of 6% (a, d), 7% (b, e) and 8% (c, f) KC dispersed in DMEM buffer. Inner droplets were stabilised with Span 80, and outer droplets stabilised with Tween 20 (a, b, c) and Tween 80 (d, e, f). Micrographs were taken immediately after forming the duplex emulsion.

[0067] FIGURE 10 shows micrographs of duplex emulsions with the inclusion of 5% (a, c) and 6% (b, d) KC dispersed in HEPES buffer. Inner droplets were stabilised with Span 80, and outer droplets stabilised with Tween 20 (a, b) and Tween 80 (c, d). Micrographs were taken immediately after forming the duplex emulsion.

Examples

[0068] Example 1 - Method of Making Hydrocolloid Double Emulsion

[0069] Hydrocolloid particulate systems are formed through shearing the dry hydrocolloid powder (up to 10%) with an anion/buffer solution, at 30°C (using a Malvern Instruments rheometer - cup and vane geometry). The shear used is a low shear (500 s ^-1 ). As the temperature is not increased above the temperature of ordering, solubilisation/hydration of the hydrocolloid occurs. At higher concentrations, the particulate systems are self-supporting, but flow under low strains. This behaviour is similar to a fluid gel system.

[0070] The particulate system is mixed with oil (paraffin oil) and an emulsifier (PGPR, Span 80) to form oil continuous primary emulsions, whereby the hydrocolloid particulate structure is contained within the droplet. The particulate to oil phase was in a 1 : 1 ratio. [0071] The formation of the primary emulsions was carried out using low shear and a controlled low temperature (using a Malvern Instruments rheometer - cup and vane geometry). However, the formation of primary emulsions can be carried out using high shear rates.

[0072] Stable primary emulsion formulations were then emulsified with a polysorbate (Tween 20, Tween 80) to form water continuous double (or duplex) emulsions (gel/oil/water). The formation of the duplex emulsions was carried out using a Malvern Instruments rheometer, at low shear rates. Typically the formation of duplex emulsions is carried out at lower shear rates than for primary emulsions to avoid droplet breakup. However, the formation of these emulsions is not typically as low as 500 s ^-1 (the shear rate used here).

[0073] The primary emulsions were stable irrespective of emulsifier, double emulsions were less stable when Span 80 was used to stabilise the first interface between the hydrocolloid and oil layer. Figures 9 and 10 show that one or less primary emulsion droplets were seen inside secondary droplets. The use of PGPR (a larger emulsifier that can aggregate and bind more strongly to the interface than Span 80) formed duplex emulsions whereby multiple internal droplets are present within every secondary droplet. Double emulsions using PGPR were found to be stable for multiple weeks.

Table 3 - Droplet sizes for the stable emulsions, determined using a mastersizer. Values quoted are the peak maximum, or modal number.