Water-in-oil emulsions and preparation thereof

Abstract

The invention relates to a water-in-oil emulsion, a method of preparing the same, a method of manipulating the composition of template droplets in the water-in-oil emulsion, and methods of assembling microparticles or providing cross-linked polymer microparticles using said water-in-oil emulsion. The water-in-oil emulsion comprises (a) a first aqueous phase forming template droplets, (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute, (c) a continuous oil phase, and (d) one or more surfactants.

Description

[0001] WATER-IN-OIL EMULSIONS AND PREPARATION THEREOF

[0002] Field of the invention

[0003] This invention relates to water-in-oil emulsions wherein the compositions of the droplets may be manipulated.

[0004] Background to the invention

[0005] Processing materials on the nano- to micro-scale into controlled structures is challenging and presents a significant obstacle to many promising applications. Emulsion droplets with dimensions on this length scale can be effectively used as templates or containers to manipulate and assemble materials, for instance to form functional microparticles. However, the process of droplet manipulation or microparticle formation often relies on controlled solvent or solute transport which is difficult to achieve in emulsions without destabilisation processes or loss of microparticle structure / internal assembly control.

[0006] Thus, there is a need to develop new methods of manipulating emulsion droplet compositions which enable processing control and the creation of microscale structures. The present invention addresses this need by providing a water-in-oil emulsion having a complex structure.

[0007] Summary of the invention

[0008] In a first aspect, there is provided a water-in-oil emulsion, comprising:

[0009] (a) a first aqueous phase forming template droplets,

[0010] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0011] (c) a continuous oil phase, and

[0012] (d) one or more surfactants.

[0013] In a second aspect, there is provided a method of preparing a water-in-oil emulsion, the emulsion comprising:

[0014] (a) a first aqueous phase forming template droplets,

[0015] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0016] (c) a continuous oil phase, and

[0017] (d) one or more surfactants; wherein the method comprises:

[0018] (i) preparing a secondary pre-emulsion by emulsifying the second aqueous phase and the solute in a continuous oil phase with the one or more surfactants to provide the plurality of secondary droplets; and

[0019] (ii) forming the template droplets in the secondary pre-emulsion thereby to provide the water-in- oil emulsion. In another aspect, there is provided the water-in-oil emulsion obtainable by the methods according to the second aspect.

[0020] In another aspect, there is provided a method of manipulating the composition of template droplets in a water-in-oil emulsion, comprising: (A) providing a water-in-oil emulsion comprising:

[0021] (a) a first aqueous phase forming template droplets,

[0022] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0023] (c) a continuous oil phase, and

[0024] (d) one or more surfactants, wherein (B) water and / or solute transported between the template droplets and secondary droplets, thereby manipulating the composition of the template droplets in the water-in-oil emulsion.

[0025] In another aspect, there is provided a method of assembling microparticles from nanomaterials or polymers, comprising: a) preparing a water-in-oil emulsion according to the methods of the second aspect described herein, wherein the template droplets comprise nanomaterials or polymers and the concentration of the solute of the secondary phase is such that there is an osmotic pressure gradient between the template droplets and the secondary droplets; b) assembling the microparticles by removing water by transporting water from the template droplets to the secondary droplets; and c) optionally washing and filtering the assembled microparticles.

[0026] In another aspect, there is provided a method of providing crosslinked polymer microparticles from a polymer, comprising: a) preparing a water-in-oil emulsion according to the methods of the second aspect described herein, wherein the template droplets comprise a polymer and the solute of the secondary droplets is a crosslinker, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing an osmotic pressure gradient and a solute concentration gradient between the template droplets and the secondary droplets; b) crosslinking the polymer by addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, and removing water by transporting water from the template droplets to the secondary droplets, thereby to provide crosslinked polymer microparticles.

[0027] In another aspect, there is provided a kit for preparing a water-in-oil emulsion according to the first aspect, comprising:

[0028] (a) a first aqueous phase comprising a template component selected from: organic molecules or their derivatives, polymers, monomers, inorganic salts, or nanomaterials, and (b) a secondary water-in-oil pre-emulsion comprising a second aqueous phase forming a plurality of secondary droplets, the secondary droplets comprising a solute, dispersed in a continuous oil phase.

[0029] In another aspect, there is provided a kit for preparing a water-in-oil emulsion according to the first aspect, comprising:

[0030] (a) a first water-in-oil pre-emulsion comprising a first aqueous phase forming template droplets, dispersed in a continuous oil phase, the template droplets comprising a template component selected from: organic molecules or their derivatives, polymers, monomers, inorganic salts, or nanomaterials, and

[0031] (b) a secondary water-in-oil pre-emulsion comprising a second aqueous phase forming a plurality of secondary droplets, the secondary droplets comprising a solute, dispersed in a continuous oil phase.

[0032] Brief Description of the Drawings

[0033] Figure 1 shows a schematic summary of factors related to the continuous phase influencing the rate of water loss from template droplets containing 0.05 wt.% methyl blue dye in Example 1 .

[0034] Figure 2 shows a schematic summary of factors related to the dispersed phase(s) influencing the rate of water loss from template droplets containing 0.05 wt.% methyl blue dye in the emulsions of Example 2.

[0035] Figure 3 demonstrates the effect of solidification rate on SiO2 nanoparticle assembly into microparticles including (c) optical images and (d) reflectance spectra of polydisperse crystalline microparticles with structural colour generated through processing monodisperse SiO2 nanoparticles with sizes from 200-250 nm inside template droplets.

[0036] Figure 4 shows the results of Method 2.2 of Example 3 where microparticles were produced with a single channel microfluidic droplet generator and solidified in-line, showing optical microscopy of (a) emulsified droplet templates and (b) microparticles produced by Method 2.2; (c) size distributions of emulsified droplets and solid microparticles with the conditions in (a) and (b); and (d)-(f) scanning electron microscopy of colloidal crystalline microparticles.

[0037] Figure 5 shows microparticles generated from carbon nanotube dispersions, where (a) shows the influence of secondary emulsion droplet size on microparticle morphology, (b) compares water removal rate from template droplets with differing secondary emulsion droplet sizes, (c) compares the initial measured water transport rate for different template droplet sizes, (d) shows polydisperse CNT microparticles produced by Method 3.3 of Example 4; and (e) shows monodisperse CNT microparticles produced by Method 3.5 of Example 4.

[0038] Figure 6 shows the resulting polydisperse microparticles generated from nanoparticle dispersions using Method 3.3 of Example 4.

[0039] Figure 7 shows solidification rates of template droplets of different sizes containing sodium alginate with simultaneous water removal and cross-linking in Example 5. Figure 8 shows an example of how to determine coalescence stability for water droplets of -350 pm in diameter over a period of 1 hour. Figure 8(a) shows images of droplets at 0 minutes (top left), and after 1 hour static at room temperature (bottom left), after 1 hour stirred at 150 rpm at room temperature (top right), and 1 hour static with heating at 60 °C (scale bar is 500 pm);

[0040] Figure 8(b) shows mean droplet size for each of the emulsions at different conditions.

[0041] Detailed Description

[0042] In a first aspect there is provided a water-in-oil emulsion, comprising:

[0043] (a) a first aqueous phase forming template droplets,

[0044] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0045] (c) a continuous oil phase, and

[0046] (d) one or more surfactants.

[0047] The emulsions according to this disclosure demonstrate enhanced stabilisation against coalescence and demonstrate a tuneable rate of selective solvent or solute transport. Furthermore, as demonstrated herein, these effects are observed for a variety of oil phases and surfactants, indicating these principles are more generally applicable to other systems.

[0048] The presence of the secondary droplets surrounding the template droplets imparts improved resistance to coalescence compared with an emulsion lacking the secondary droplets. Without wishing to be bound by theory, it is believed the smaller secondary droplets form a protective layer surrounding the template droplets, which thereby reduces template droplet coalescence.

[0049] It will be appreciated that the oil phase should be immiscible with water and compatible with the one or more surfactant(s). A particular advantage of the emulsions described herein is that the viscosity, and chemical nature of the oil phase can also be tuned to adjust control over mass transport between the template and secondary droplets.

[0050] The first and second aqueous phases are preferably distinct in composition.

[0051] Preferably, the volume-to-volume ratio of the continuous oil phase to the second aqueous phase may be of from about 1 :1 to 100:1 , preferably of from about 2:1 to about 80:1 , more preferably of from about 4:1 to about 60:1 , even more preferably of from about 5:1 to about 40:1.

[0052] The template droplets may comprise a template component, or more than one template component, which may be any component(s) capable of being solubilised or dispersed within the droplets. The selected template component thus may preferably be selected to be soluble or dispersible within the first aqueous phase. The template component may be dried, precipitated, co-precipitated, crystallised, phase separated, or reacted with other components added by the secondary droplets, according to the methods described herein.

[0053] The template component may be selected from: an organic molecule, a biomolecule, a polymer, a monomer, an inorganic salt, or a nanomaterial, or a combination thereof.

[0054] Preferably, the template droplets may comprise an organic molecule or its derivative. The organic molecule may be selected from, but is not limited to: a carbohydrate, an organic acid or base, a dye, a pharmaceutical, a peptide, a cross-linker, a monomer, or a functional additive.

[0055] Preferably, the template droplets may comprise an inorganic salt, including but not limited to sodium chloride (NaCI), sodium hydroxide (NaOH), calcium chloride (CaCI2), potassium permanganate (KMnO4), potassium iodide (KI), or mixtures thereof.

[0056] The template droplets may comprise a polymer. It will be appreciated that the polymer should be readily soluble in aqueous solution. The polymer may include but is not limited to: biodegradable hydrogels, sodium alginate, poly(acrylic acid) (PAA), chitosan, hyaluronic acid, carboxymethyl cellulose, hydroxypropyl cellulose, collagen, gelatine. If the template droplets comprise a polymer, then in some instances it may be preferred that the secondary droplets may comprise a functional agent that reacts with or modifies the polymer, such as a crosslinker or monomer. Alternatively, a polymer microparticle can also be made without a functional agent, e.g. through solvent removal.

[0057] The template droplets may comprise a nanomaterial. A nanomaterial, as defined herein, is a material wherein at least one external dimension lies in the size range of 0.1 nm to 1000 nm. This definition includes, but is not limited to, carbon nanoparticles (in particular, carbon nanotubes), metal oxide nanoparticles, and other inorganic nanoparticles (e.g., silica or titania). This definition is inclusive of nanomaterials that are natural, incidental, or manufactured. Preferably, the inorganic oxide may be silicon dioxide. The metal oxide nanoparticle may preferably be selected from titania or lithium titanate. Preferably, the template droplets comprise a functionalised carbon nanoparticle, and titania and / or lithium titanate.

[0058] Droplet diameter

[0059] The initial sizes of the template and secondary droplets may be selected depending on the desired application. The initial size of the droplets also impacts on the rate of transport of water and / or solute between the droplets, allowing the size of the droplets to be tuned according to the needs of a particular application.

[0060] References herein to the average diameter of the template or secondary droplets is intended to refer to initial droplet diameter. Or in other words, the droplet diameter upon or shortly after preparation of the emulsion. The average diameter of the template droplets may be of from about 0.5 pm to about 1500 pm, preferably of from 1 to about 1000 pm. The average diameter of the secondary droplets may be of from about 0.01 to about 500 pm, preferably from about 0.1 to about 100 pm. The ratio of the average template droplet diameter to average secondary droplet diameter may be about 1 or less, preferably about 0.8 or less. Advantageously, the diameter of the secondary droplets for a given template droplet can be tuned to control the texture of a microparticle surface or the rate of mass transport.

[0061] Continuous oil phase

[0062] It will be appreciated that the water-in-oil emulsions according to this disclosure are stable against coalescence to the extent that the emulsions substantially maintain a droplet structure enabling use with the methods described herein. Coalescence stability is determined according to methods described herein.

[0063] Preferably, the continuous oil phase and the one or more surfactants are selected to provide an emulsion coalescence stability such that an average template droplet diameter increase of less than about 15%, less than 10%, or less than 5% is observed during 60 minutes when static and at room temperature.

[0064] Preferably, the continuous oil phase and the one or more surfactants are selected to provide an emulsion coalescence stability such that around 90% of the droplets do not coalesce i.e. to form larger droplets, when observed over 60 minutes when static at room temperature, and / or at 60 °C, and / or with agitation in an orbital shaker at 150 rpm at room temperature.

[0065] Preferably, the emulsion has a coalescence stability such that an average template droplet diameter increase of about 20% or less, more preferably about 10% or less when observed during 60 minutes when static and at room temperature.

[0066] Preferably, the emulsion has a coalescence stability such that an average template droplet diameter increase is about 20% or less, more preferably about 10% or less when observed during 60 minutes when static and at a temperature of 60°C.

[0067] Preferably, the emulsion has a coalescence stability such that an average template droplet diameter increase is about 20% or less, more preferably about 10% or less when observed during 60 minutes with agitation in an orbital shaker at 150 rpm and at room temperature.

[0068] The dynamic viscosity of the oil phase may be from about 1 .5 to about 15 mPa.s at room temperature, preferably from about 2 to about 3 mPa.s at room temperature. Dynamic viscosity is measured according to the methods herein. It will be appreciated that the oil phase should be immiscible with water. The selection of an appropriate oil phase may depend on the specific application for which the emulsion is to be used for and depending on the desired viscosity. The identity of the oil phase is therefore not critical, so long as there is formation of a stable water-in-oil emulsion with the selected surfactants.

[0069] However, it is preferred that the continuous oil phase comprises an oil selected from saturated and unsaturated hydrocarbon oils, silicone-based oils, vegetable oils or their derivatives, fatty acids, or mixtures thereof.

[0070] More preferably, the continuous oil phase may comprise an oil selected from: isoparaffinic oil; oleic acid; oleyl alcohol, sunflower oil, medium-chain triglyceride oil, mineral oil; silicone oil; 1 octadecene; or a mixture thereof. Most preferably, the continuous oil phase may comprise isoparaffinic oil and one or more of oleic acid, mineral oil, or silicone oil.

[0071] The identity of the components in the continuous oil phase and the volume ratios of those components may be selected to provide a desired dynamic viscosity in the range of from about 1.5 to about 15 mPa.s at room temperature.

[0072] For example, the continuous oil phase may comprise: a mixture of isoparaffinic oil and oleic acid in a volume to volume ratio of from about 1 :1 to 5:1 ; a mixture of isoparaffinic oil and mineral oil in a volume- to-volume ratio of from about 5:2 to 1 :3 ; a mixture of isoparaffinic oil, oleic acid, and mineral oil in a volume-to-volume ratio of from about 5:2:1 to 3:2:2; a mixture of isoparaffinic oil and silicone oil in a volume-to-volume ratio of from about 5:1 to 5:2; or 1-octadecene. Each ofthese continuous oil phases provided a dynamic viscosity from about 1 .8 mPa to about 3.7 mPa (see Table 1)

[0073] Surfactants

[0074] The one or more surfactants may be matched, as necessary, to the identity of the continuous oil phase according to known principles. The surfactant(s) should be selected to be able to provide stabilisation of interfacial films between droplets against coalescence.

[0075] The selection of appropriate surfactants is a common process and typically requires small scale tests comparing stability of oil / surfactant(s) mixtures, which may generally be evaluated based on sedimentation / creaming time, and optical inspection of droplets. The precise chemical identity of the surfactants is therefore not essential to the emulsions described herein and their use in the methods.

[0076] The concentration of the surfactant may be from about 0.2 to about 17 wt.% based on the total weight of the composition. The total Hydrophilic-Lipophilic Balance (HLB) of the one or more surfactants may be from about 2 to about 7, preferably from about 2 to about 6. The total HLB can be selected depending on the continuous oil phase, and adjusted through using a mixture of surfactants if needed. It will be appreciated that the actual desired HLB may depend on the oil phase (to a greater extent) and the aqueous phase composition (to a lesser extent). HLB may be determined according to Griffin’s and Davies’ methods, which are well-known and standard in the field of surfactant science for calculating the Hydrophilic-Lipophilic Balance (HLB) of a surfactant(s).

[0077] The one or more surfactants may be ionic or non-ionic surfactants, preferably non-ionic surfactants. Non-ionic surfactants are preferred as they do not have counter-ions, the presence of which may remain in the material at the end of processing.

[0078] The one or more surfactants may be selected from: sorbitan esters and polyoxyethylene sorbitan esters, mono- and di-glycerides of fatty acids, polyglycerol esters, polyoxyethylene alcohols, natural emulsifiers such as lecithin and mixtures thereof. Preferably, the one or more surfactants may be selected from: sorbitan monooleate, polyglycerol polyricinoleate and ethoxylated sorbitan trioleate, or mixtures thereof. Preferably, the surfactants are sorbitan monooleate and ethoxylated sorbitan trioleate.

[0079] For instance, the continuous oil phase may comprise a saturated or unsaturated hydrocarbon oil and a fatty acid, and the one or more surfactants may be selected from sorbitan esters, polyoxyethylene sorbitan esters, or mixtures thereof. In particular, the continuous oil phase may comprise isoparaffinic oil and oleic acid, and the one or more surfactants may be selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0080] Alternatively, the continuous oil phase may comprise isoparaffinic oil and silicone oil, and the one or more surfactants may be selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0081] Alternatively, the continuous oil phase may comprise isoparaffinic oil and mineral oil, and the one or more surfactants may be selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0082] Alternatively, the continuous oil phase may comprise isoparaffinic oil, oleic acid, and mineral oil, and the one or more surfactants may be selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0083] Alternatively, the continuous oil phase may comprise 1 -octadecene, and the one or more surfactants may be selected from sorbitan monooleate and sorbitan trioleate.

[0084] Mass transport of solute and / or water

[0085] The emulsions according to this disclosure advantageously provide a method by which mass transport of solute (or solutes, as the case may be) and / or water may occur in an emulsion, between the template droplets and secondary droplets. This is primarily driven by differences in chemical potential e.g., concentration gradients across the template and secondary droplets. For instance, for water transport between the droplets an osmotic pressure gradient can be provided between the template and secondary droplets, by way of a concentrated solute in either the template or secondary droplet. Depending on the solute and oil phase selected, the solute may or may not (or may only at a gradual rate) be able to transport across the droplets itself, and may be used to induce an osmotic pressure gradient.

[0086] Substances are generally transported down their concentration gradient (in the case of water this can be thought of in terms of osmotic pressure difference), which can be adjusted to achieve directionality to or from the template droplet.

[0087] The rate of substance transport may also depend on other factors, including but not limited to the properties of each aqueous phase, viscosity, the size of the substance to be transported, the solubility and partitioning of each substance in the selected oil phase, and intermolecular or inter-particle interactions.

[0088] The emulsions of this disclosure therefore provide droplet compositions and arrangements that can be tailored to ensure the required concentration gradient; and to ensure selected substance(s) are able to leave the aqueous phase it is being supplied from at the desired rate, and the substance is able to cross the oil phase at the desired rate; and finally, to ensure the substance is able to enter the aqueous phase it is being supplied to at the desired rate.

[0089] It is also shown in the examples herein that by using the emulsions of this disclosure it is possible to transport more than one material at the same time. For instance, in the example of simultaneous water removal and cross-linker addition (see Example 5), one osmotically active material is required to generate a gradient for water transport; to achieve the desired rates of water and cross-linker transport this material may be the same as the cross-linking material, or may be different to the cross-linking material. In the latter case this allows for more independent control over each substance being transported.

[0090] Without wishing to be bound by any theory, it is believed the osmotic or concentration gradient thus provides a non-equilibrium situation of different concentrations within the droplets, with mass transport rates which may change over time as the system moves towards equilibrium. The oil layers may be considered as selective ‘membranes’ with tuneable and substance specific permeability.

[0091] Preferably, the composition of the emulsion is such that the solute(s) of the secondary droplets provides an osmotic pressure gradient and / or solute concentration gradient between the template and secondary droplets such that water and / or solute is transported between the template droplets and the secondary droplets. The water and / or solute may be transported from the template droplets to the secondary droplets, or alternatively, the water and / or solute is transported from the secondary droplets to the template droplets. The water and solute(s) may be transported in the same direction, or in opposite directions.

[0092] Solutes

[0093] The choice of solute in the template and / or secondary droplets may depend on the specific application, and the intended manipulation of the template droplet.

[0094] It will be appreciated that the emulsions described herein comprise at least one solute, but in some cases may comprise multiple solutes in the secondary droplets and / or template droplets, each having the ability to drive transport of solvents or solutes between the template droplets and the secondary droplets. By way of non-limiting example, an emulsion may comprise a first solute that is a crosslinker in low amounts, and a second solute that provides the desired osmotic pressure gradient for water removal.

[0095] An emulsion where there are multiple solutes in the template and / or secondary droplets advantageously provides a system whereby the control of the rate of water transport can be controlled independently of the one or more rates of solute transport for the solutes, e.g. by varying the starting concentrations of the solutes.

[0096] The template droplets may also comprise a solute and in some cases may comprise multiple solutes. The solute(s) of the template droplets may be the same as the solute of the secondary droplets, or, the template droplets may be different to the solute of the secondary droplets.

[0097] The solute of the template droplets may be selected from: an organic molecule, a polymer, inorganic salts, a crosslinker, a monomer, or mixtures thereof.

[0098] The solute of the template droplets may be an inorganic salt including but not limited to sodium chloride (NaCI), calcium chloride (CaCh), potassium permanganate (KMnO4), potassium iodide (KI), or mixtures thereof.

[0099] The concentration of a solute in the secondary droplets therefore can be selected to tune the direction and rate of transport of the solute or water as required.

[0100] The concentration of a solute in the secondary droplets may be higher than the concentration of a solute in the template droplets, or vice versa, providing a gradient such that water and / or a solute is transported between the template droplets and the secondary droplets.

[0101] That is, the concentration of the solute in the secondary droplets may be higher than the concentration of the solute in the template droplets providing an osmotic pressure gradient that causes water to be transported from the template droplets to the secondary droplets. Alternatively, or in addition, the concentration of the solute in the secondary droplets may be higher than the concentration of the solute in the template droplets providing a concentration gradient that causes solute to be transported from the secondary droplets to the template droplets.

[0102] Similarly, the concentration of a solute in the template droplets therefore can be selected to tune the direction and rate of transport of the solute or water as required.

[0103] For instance, the concentration of the solute in the template droplets may be higher than the concentration of the solute in the secondary droplets providing an osmotic pressure gradient that causes water to be transported from the secondary droplets to the template droplets.

[0104] Alternatively, or in addition, the concentration of the solute in the template droplets may be higher than the concentration of the solute in the secondary droplets providing a concentration gradient that causes solute to be transported from the template droplets to the secondary droplets.

[0105] The solute of the secondary droplets may be selected from: an organic molecule, inorganic salts, a crosslinker, a monomer, a solvent, or mixtures thereof. The solute of the secondary droplets may be a carbohydrate selected from glucose, sucrose, or fructose, preferably glucose.

[0106] The solute of the secondary droplets may be an inorganic salt selected from sodium chloride (NaCI), sodium hydroxide (NaOH), calcium chloride (CaCI2), potassium permanganate (KmnO4), potassium iodide (KI), or mixtures thereof.

[0107] The concentration of the solute in the second aqueous phase may be from about 0.05 mol / L to about 10 mol / L.

[0108] In some instances, substantially no solute of the secondary droplets may be transported from the secondary droplets to the template droplets.

[0109] Similarly, substantially no solute of the template droplets may, alternatively or in addition, be transported from the template droplets to the secondary droplets.

[0110] The concentration of the solute in the first aqueous phase may be from about 0.05 mol / L to about 10 mol / L, preferably from about 0.9 mol / L to about 5 mol / L.

[0111] For an emulsion with the aim of removing water from the template droplets, then

[0112] (i) the template droplets may comprise an organic molecule, a biomolecule, a polymer, or a nanomaterial, and (ii) the concentration of the solute of the secondary droplets may be selected such that it provides an osmotic pressure gradient between the template and secondary droplets such that water is transported from the template droplets to the secondary droplets.

[0113] The solute may be such that substantially no amount is transported from the secondary droplets to the template droplets. This allows for the osmotic pressure gradient to be maintained for longer, as little, or no reverse transport of the solute occurs. The template component may preferably be present in the first aqueous phase in an initial amount of up to about 50 wt.%.

[0114] The solute of the secondary droplets may preferably be selected from: an organic molecule such as a carbohydrate, an inorganic salt, a polymer. If a carbohydrate, then it may be selected from glucose, sucrose, or fructose. The concentration of the solute in the second aqueous phase may be from about 0.05 mol / L to about 10 mol / L.

[0115] The organic molecule may preferably be a pharmaceutical or therapeutic ingredient.

[0116] In an emulsion where transport of solute and water is desired, then:

[0117] (i) the template droplets may comprise a polymer, and

[0118] (ii) the concentration of the solute of the secondary droplets may be higher than the concentration of the solute in the template droplets providing an osmotic pressure gradient and a solute concentration gradient between the template and secondary droplets such that water and solute is transported between the template droplets and the secondary droplets.

[0119] The osmotic pressure gradient may be such that water is transported from the template droplets to the secondary droplets. The solute concentration gradient may be such that solute is transported from the secondary droplets to the template droplets. The polymer is preferably present in the first aqueous phase in an amount of up to about 50 wt% based on the weight of the first aqueous phase. The solute of the secondary droplets or template droplets may be a crosslinker, a monomer, or a mixture thereof. The concentration of the solute in the second aqueous phase may be from about 0.05 mol / L to about 10 mol / L. The first aqueous phase and / or second aqueous phase may comprise one or more polymerisation initiators.

[0120] Kit

[0121] There is also provided a first kit for preparing a water-in-oil emulsion according to the first aspect, comprising:

[0122] (a) a first aqueous phase comprising a template component selected from: organic molecules or their derivatives, polymers, monomers, inorganic salts, or nanomaterials, and

[0123] (b) a secondary water-in-oil pre-emulsion comprising a second aqueous phase forming a plurality of secondary droplets, the secondary droplets comprising a solute, dispersed in a continuous oil phase. There is also provided a second kit for preparing a water-in-oil emulsion according to the first aspect, comprising:

[0124] (a) a first water-in-oil pre-emulsion comprising a first aqueous phase forming template droplets, dispersed in a continuous oil phase, the template droplets comprising a template component selected from: organic molecules or their derivatives, polymers, monomers, inorganic salts, or nanomaterials, and

[0125] (b) a secondary water-in-oil pre-emulsion comprising a second aqueous phase forming a plurality of secondary droplets, the secondary droplets comprising a solute, dispersed in a continuous oil phase.

[0126] It will be appreciated that the first aqueous phase (either itself or as a pre-emulsion) can be added to the secondary water-in-oil pre-emulsion in various ways, depending on the desired system.

[0127] Method of preparation

[0128] In another aspect there is provided a method of preparing a water-in-oil emulsion, the emulsion comprising:

[0129] (a) a first aqueous phase forming template droplets,

[0130] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0131] (c) a continuous oil phase, and

[0132] (d) one or more surfactants; wherein the method comprises:

[0133] (i) preparing a secondary pre-emulsion by emulsifying the second aqueous phase and the solute in a continuous oil phase with the one or more surfactants to provide the plurality of secondary droplets; and

[0134] (ii) forming the template droplets in the secondary pre-emulsion thereby to provide the water-in- oil emulsion.

[0135] The water-in-oil emulsion prepared may be according to any embodiments or examples provided as part of the first aspect.

[0136] There are multiple ways of preparing the water in oil emulsions described herein.

[0137] For instance, step (ii) may comprise:

[0138] (ii-a) preparing a template pre-emulsion by emulsifying the first aqueous phase in a continuous oil phase with one or more surfactants to provide the template droplets, and

[0139] (ii-b) combining the secondary and template pre-emulsions, thereby to provide the water-in-oil emulsion.

[0140] It will be appreciated that steps (i) and (ii-a) may comprise emulsifying in the same continuous oil phase. Alternatively, steps (i) and (ii-a) involve emulsifying in different continuous oil phases that when combined provide the continuous oil phase of the water-in-oil emulsion. For instance, the template droplets could be emulsified in a lower viscosity oil, the secondary droplets could be emulsified in a higher viscosity oil, and these two emulsions could then be mixed together to achieve the correct viscosity range and droplet arrangement. The choice of whether to use the same or different oil phases will also be affected by the chosen method of preparing each emulsion, the chosen method by which the two emulsions are combined, and the dispersed phase volume fraction of the emulsion(s).

[0141] Any suitable method for emulsifying may be used, depending on for example the desired droplet diameters to be obtained. Thus, steps (i) and / or (ii-a) may comprise emulsifying using one or more of ultrasonication, high shear mixing, stirring, homogenisation, high pressure homogenisation, phase inversion, rotor-stator mixing, impingement mixing, controlled emulsification methods, membrane emulsification, or microfluidics.

[0142] Another method of preparing may be wherein step (ii) may comprise:

[0143] (ii-a) introducing the first aqueous phase into the continuous oil phase of the secondary preemulsion (e.g., by injection) such that the template droplets are formed, thereby to provide the water- in-oil emulsion.

[0144] Steps (i) and / or (ii) comprises emulsification using ultrasonication, high shear mixing, stirring, homogenisation, controlled emulsification methods, membrane emulsification, or microfluidics.

[0145] There is also provided the water-in-oil emulsion obtainable by the methods according to the second aspect.

[0146] Method of manipulating composition

[0147] There is also provided a method of manipulating the composition of template droplets in a water-in-oil emulsion, comprising: (A) providing a water-in-oil emulsion comprising:

[0148] (a) a first aqueous phase forming template droplets,

[0149] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0150] (c) a continuous oil phase, and

[0151] (d) one or more surfactants, wherein (B) water and / or solute is transported between the template droplets and secondary droplets, thereby manipulating the composition of the template droplets in the water-in-oil emulsion.

[0152] It will be appreciated that the water-in-oil emulsion may be according to any embodiments or examples described in relation to the first aspect, or obtainable by the second aspect.

[0153] Methods of assembling particles There is also provided a method of assembling microparticles from nanomaterials or polymers, comprising: a) preparing a water-in-oil emulsion according to the method of any embodiments in relation to the second aspect, wherein the template droplets comprise the nanomaterials or polymers and the concentration of the solute of the secondary phase is such that there is an osmotic pressure gradient between the template droplets and the secondary droplets; causing the removal of water by transporting water from the template droplets to the secondary droplets and assembling of the microparticles; and b) optionally washing and filtering the assembled microparticles.

[0154] There is also provided a method of providing crosslinked polymer microparticles from a polymer, comprising: a) preparing a water-in-oil emulsion according to the method of any embodiments in relation to the second aspect, wherein the template droplets comprise a polymer and the solute of the secondary droplets is a crosslinker, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing an osmotic pressure gradient and a solute concentration gradient between the template droplets and the secondary droplets; thereby causing addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, and removing water by transporting water from the template droplets to the secondary droplets, thereby to provide crosslinked polymer microparticles, b) optionally washing and filtering the crosslinked polymer microparticles.

[0155] There is also provided a method of providing crosslinked polymer microparticles from a polymer, comprising: a) preparing a water-in-oil emulsion to the method of any embodiments in relation to the second aspect, wherein the template droplets comprise a polymer and the secondary droplets comprise a first solute that is a crosslinker and a second solute, wherein the concentration of the second solute in the secondary droplets is higher than the concentration of second solute in the template droplets providing an osmotic pressure between the template droplets and the secondary droplets; thereby removing water by transporting water from the template droplets to the secondary droplets, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing a crosslinker concentration gradient between the template droplets and the secondary droplets; thereby causing addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, thereby to provide crosslinked polymer microparticles, b) optionally washing and filtering the crosslinked polymer microparticles. Preferably, the cross-linker is present in an amount which is lower than the amount of the second solute. This allows for a system where the control of the rate of water removal from the template droplets can be controlled independently of the rate of crosslinker addition e.g. by varying the starting concentration of the second solute. Similarly, the starting concentration of the crosslinker could be varied to control independently the rate of crosslinker addition to the template droplets.

[0156] The diameter of the obtained microparticles may be measured according to the methods here, e.g., optical microscopy. The obtained microparticle may undergo further processing, e.g., cross-linking, washing, drying, or extraction, to obtain a final product.

[0157] The microparticle diameter may be from about 1 pm to about 1000 pm, preferably, from about 1 to about 300 pm, for example.

[0158] Definitions and Measurement methods

[0159] An organic molecule as defined herein is a molecule that contains carbon atoms covalently bonded to atoms of other elements, most commonly hydrogen, oxygen, or nitrogen. This definition includes, but is not limited to, carbohydrates, dyes, pharmaceuticals, organic cross-linkers, monomers, and functional additives.

[0160] A functional additive as defined herein refers to a substance incorporated into a product to enhance or modify its properties. This can include, but is not limited to, dyes, imaging agents, contrast agents, preservatives or viscosity modifiers, pharmaceutical ingredients contained within a microparticle scaffold for drug delivery applications, conductive additives present in low concentrations to improve a product’s conductivity, pore-forming agents to allow for tuneable porosity, a stimuli-responsive material which can impart properties such as temperature or humidity sensitivity, substances to enhance solubility or stability of a given material.

[0161] Average droplet diameter, as defined herein and unless otherwise specified, is measured by optical microscopy, or using a camera. For measurements of changing template droplet size due to mass transport this is conducted by measuring the sizes of at least three droplets and taking the mean of the result. For measurements of emulsion coalescence stability for a given set of conditions, the mean of at least 10 droplets should be taken, preferably at least 50.

[0162] It will be appreciated that the appropriate method of droplet diameter measurement may depend on the droplet diameter range to be measured. For instance, whilst microscale droplets and milliscale droplets can be measured by optical microscopy, or by using a camera, other methods may be used for measuring the diameter of droplets of other sizes, including but not limited to, techniques such as dynamic light scattering, laser diffraction, other scattering techniques, also cryo-SEM measurement. Viscosity, unless specified otherwise herein, is intended to refer to dynamic (or absolute) viscosity. Viscosity was measured with a very high shear rate vibrational viscometer (Hydramotion Viscolite 700) at room temperature (~20°C).

[0163] Coalescence stability as defined here refers to the ability of a water-in-oil emulsion to resist coalescence, wherein coalescence is where emulsion droplets in close proximity merge together as the interfacial film between them breaks. For a given emulsion system here consisting of dissolved or dispersed substances in template and secondary droplets, the emulsion stability must be such that coalescence does not substantially interfere with mass transport processes over the desired time duration, which may be, for example, from 1 minute to 10 days. In systems undergoing mass transport between droplets, coalescence can be observed directly through microscopy to allow it to be distinguished from mass transport without coalescence. It will be appreciated that both of these processes affect droplet size and size distribution. However, coalescence generally results in more rapid changes in a population of droplets’ size compared to controlled mass transport, so these processes can be qualitatively distinguished e.g. by microscopy alongside monitoring of template droplet shrinkage / swelling.

[0164] The coalescence stability of suitable continuous phases can be assessed by measuring the sizes and size distribution of a population of droplets over time, where minimal mass transport is taking place. A continuous phase is considered to have good stability if around 90% of the droplets do not coalesce i.e. to form larger droplets.

[0165] Coalescence stability is determined herein according to the following method. Immediately after emulsification, an image of the droplets was taken with a camera attached to an optical microscope. After leaving the emulsion in a controlled environment for a period of time, another image was taken. The droplet sizes were then measured for both states, taking at least 10, preferably at least 50 droplets to give a representative size distribution. Comparison of the two size distributions, and the mean and standard deviation of the droplet population reveals the proportion of droplets which have coalesced.

[0166] Figure 8 shows an example of how to determine coalescence stability for water droplets of -350 pm in diameter over a period of 1 hour. Figure 8(a) shows images of droplets at 0 minutes (top left), and after 1 hour static at room temperature (bottom left), after 1 hour stirred at 150 rpm at room temperature (top right), and 1 hour static with heating at 60 °C (scale bar is 500 pm). These droplets were emulsified in a continuous phase of 5:2 v:v Isopar: oleic acid containing 5 wt.% sorbitan monooleate and ethoxylated sorbitan trioleate (4:1 w:w) surfactants. It can be seen from Figure 8(b) that droplet diameter did not increase after 1 hour, thus indicating the emulsions were stable against coalescence.

[0167] As used herein, singular forms ”a“ ”an" and "the" also include plural forms unless the context clearly dictates otherwise. Use of the singular includes the plural unless specifically stated otherwise. The terms “comprising”, “containing”, "including" and “having” as well as other forms (e.g., "include," "comprise", "contain" and “has”) do not exclude the presence of other features. As used herein, wherever “comprising” is referenced, this may also be interpreted to mean “consisting essentially of’ and “consisting of’.

[0168] The aspects provided herein are also described in the following clauses:

[0169] 1 . A water-in-oil emulsion, comprising:

[0170] (a) a first aqueous phase forming template droplets,

[0171] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0172] (c) a continuous oil phase, and

[0173] (d) one or more surfactants.

[0174] 2. A water-in-oil emulsion according to clause 1 , wherein the first and second aqueous phases are distinct in composition.

[0175] 3. A water-in-oil emulsion according to clause 1 or clause 2, wherein the volume-to-volume ratio of the continuous oil phase to the second aqueous phase is from about 1 :1 to 100:1 .

[0176] 4. A water-in-oil emulsion according to any one of the preceding clauses, wherein the volume-to- volume ratio of the continuous oil phase to the second aqueous phase of from about 2:1 to about 80:1 , more preferably of from about 4:1 to about 60:1 , even more preferably of from about 5:1 to about 40:1 .

[0177] 5. A water-in-oil emulsion according to any one of the preceding clauses, wherein the template droplets comprise a template component selected from: an organic molecule, a biomolecule, a polymer, a monomer, an inorganic salt, or a nanomaterial, or a combination thereof.

[0178] 6. A water-in-oil emulsion according to clause 5, wherein the template droplets comprise an organic molecule or its derivative.

[0179] 7. A water-in-oil emulsion according to clause 5 or 6, wherein the organic molecule is selected from a carbohydrate, a dye, a pharmaceutical, a cross-linker, a monomer, or a functional additive.

[0180] 8. A water-in-oil emulsion according to any of clauses 5 to 7, wherein the organic molecule is a pharmaceutical.

[0181] 9. A water-in-oil emulsion according to any of clauses 5 to 8, wherein the carbohydrate is glucose, fructose, or sucrose. 10. A water-in-oil emulsion according to any of clauses 5 to 9, wherein the template droplets comprise an inorganic salt.

[0182] 11. A water-in-oil emulsion according to clause 10, wherein the inorganic salt is selected from sodium chloride (NaCI), sodium hydroxide (NaOH), calcium chloride (CaCI2), potassium permanganate (KMnO4), potassium iodide (KI), or mixtures thereof.

[0183] 12. A water-in-oil emulsion according to any of clauses 5-11 , wherein the template droplets comprise a polymer.

[0184] 13. A water-in-oil emulsion according to clause 12, wherein the polymer is sodium alginate.

[0185] 14. A water-in-oil emulsion according to any of clauses 5-13, wherein the template droplets comprise a nanomaterial.

[0186] 15. A water-in-oil emulsion according to clause 14, wherein the nanomaterial is selected from a functionalised carbon nanoparticle, a metal oxide nanoparticle, an inorganic nanomaterial.

[0187] 16. A water-in-oil emulsion according to clause 15, wherein the inorganic nanomaterial is silicon dioxide.

[0188] 17. A water-in-oil emulsion according to clause 15, wherein the metal oxide nanoparticle is selected from titania or lithium titanate.

[0189] 18. A water-in-oil emulsion according to clause 15, wherein the template droplets comprise a functionalised carbon nanoparticle, and titania and / or lithium titanate.

[0190] 19. A water-in-oil emulsion according to any one of the preceding clauses, wherein the average diameter of the template droplets is from about 0.5 pm to about 1500 pm.

[0191] 20. A water-in-oil emulsion according to any one of the preceding clauses, wherein the average diameter of the secondary droplets is from about 0.01 to about 500 pm.

[0192] 21 . A water-in-oil emulsion according to clause 20, wherein the average diameter of the secondary droplets is from about 0.01 to about 100 pm.

[0193] 22. A water-in-oil emulsion according to any one of the preceding clauses, wherein the ratio of the average template droplet diameter to average secondary droplet diameter is about 1 or less. 23. A water-in-oil emulsion according to clause 22, wherein the ratio of the average template droplet diameter to average secondary droplet diameter is less than about 0.5.

[0194] 23a. A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase and the one or more surfactants are selected to provide an emulsion coalescence stability such that around 90% of the droplets do not coalesce to form larger droplets, when observed over 60 minutes when static at room temperature, and / or at 60 °C, and / or with agitation in an orbital shaker at 150 rpm at room temperature.

[0195] 24. A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase and the one or more surfactants are selected to provide an emulsion coalescence stability such that an average template droplet diameter increase of less than about 15%, less than 10%, or less than 5% is observed during 60 minutes when static and at room temperature.

[0196] 25. A water-in-oil emulsion according to any one of the preceding clauses, wherein the emulsion has a coalescence stability such that an average template droplet diameter increase of about 20% or less, more preferably about 10% or less when observed during 60 minutes when static and at room temperature.

[0197] 26. A water-in-oil emulsion according to any one of the preceding clauses, wherein the emulsion has a coalescence stability such that an average template droplet diameter increase is about 20% or less, more preferably about 10% or less when observed during 60 minutes when static and at a temperature of 60°C.

[0198] 27. A water-in-oil emulsion according to any one of the preceding clauses, wherein the emulsion has a coalescence stability such that an average template droplet diameter increase is about 20% or less, more preferably about 10% or less when observed during 60 minutes with agitation in an orbital shaker at 150 rpm and at room temperature.

[0199] 28. A water-in-oil emulsion according to any one of the preceding clauses, wherein the dynamic viscosity of the oil phase is from about 1 .5 to about 15 mPa.s at room temperature.

[0200] 29. A water-in-oil emulsion according to clause 28, wherein the dynamic viscosity of the oil phase is from about 2 to about 3 mPa.s at room temperature.

[0201] 29. A water-in-oil emulsion according to any one of the preceding clauses, wherein the oil phase is immiscible with water. 30. A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase comprises an oil selected from saturated and unsaturated hydrocarbon oils, silicone-based oils, vegetable oils or their derivatives, fatty acids, or mixtures thereof.

[0202] 31 . A water-in-oil emulsion according to clause 30, wherein the fatty acid is oleic acid.

[0203] 32. A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase comprises an oil selected from: isoparaffinic oil; oleic acid; oleyl alcohol; sunflower oil; medium chain triglyceride oil ; mineral oil; silicone oil; 1 -octadecene; or a mixture thereof.

[0204] 33. A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase comprises isoparaffinic oil and one or more of oleic acid, mineral oil, or silicone oil.

[0205] 34. A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase comprises: a mixture of isoparaffinic oil and mineral oil in a volume-to-volume ratio of from about 5:2 to 1 :3 ; a mixture of isoparaffinic oil, oleic acid, and mineral oil in a volume-to- volume ratio of from about 5:2:1 to 3:2:2; a mixture of isoparaffinic oil and silicone oil in a volume-to- volume ratio of from about 5:1 to 5:2; or 1-octadecene.

[0206] 35. A water-in-oil emulsion according to any one of the preceding clauses, wherein the concentration of the surfactant is from about 0.2 to about 17 wt.% based on the total weight of the composition.

[0207] 36. A water-in-oil emulsion according to any one of the preceding clauses, wherein the total Hydrophilic-Lipophilic Balance (HLB) of the one or more surfactant is from about 2 to about 7, preferably from about 2 to about 6.

[0208] 37. A water-in-oil emulsion according to any one of the preceding clauses, wherein the one or more surfactants are non-ionic surfactants.

[0209] 38. A water-in-oil emulsion according to any one of the preceding clauses, wherein the one or more surfactants are selected from: sorbitan esters and polyoxyethylene sorbitan esters, mono- and di-glycerides of fatty acids, polyglycerol esters, polyoxyethylene alcohols, natural emulsifiers such as lecithin and mixtures thereof.

[0210] 39. A water-in-oil emulsion according to any one of the preceding clauses, wherein the one or more surfactants are selected from: sorbitan monooleate, sorbitan trioleate, polyglycerol polyricinoleate and ethoxylated sorbitan trioleate, or mixtures thereof. 40. A water-in-oil emulsion according to any one of the preceding clauses, wherein the surfactants are sorbitan monooleate and ethoxylated sorbitan trioleate in a weight-to-weight ratio of from about 1 :1 to 7:1 .

[0211] 41 . A water-in-oil emulsion according to any one of the preceding clauses, wherein the continuous oil phase comprises a saturated or unsaturated hydrocarbon oil and a fatty acid, and the one or more surfactants are selected from sorbitan esters, polyoxyethylene sorbitan esters, mono- and di-glycerides of fatty acids, polyglycerol esters, polyoxyethylene alcohols, natural emulsifiers such as lecithin, or mixtures thereof.

[0212] 42. A water-in-oil emulsion according to any one of clauses 1-40, wherein the continuous oil phase comprises isoparaffinic oil and oleic acid, and the one or more surfactants are selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0213] 43. A water-in-oil emulsion according to any one of clauses 1-40, wherein the continuous oil phase comprises isoparaffinic oil and silicone oil, and the one or more surfactants are selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0214] 44. A water-in-oil emulsion according to any one of clauses 1-40, wherein the continuous oil phase comprises isoparaffinic oil and mineral oil, and the one or more surfactants are selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0215] 45. A water-in-oil emulsion according to any one of clauses 1-40, wherein the continuous oil phase comprises isoparaffinic oil, oleic acid, and mineral oil, and the one or more surfactants are selected from sorbitan monooleate and ethoxylated sorbitan trioleate.

[0216] 46. A water-in-oil emulsion according to any one of the preceding clauses, wherein the composition of the emulsion is such that the solute of the secondary droplets provides an osmotic pressure gradient and / or solute concentration gradient between the template and secondary droplets such that water and / or solute is transported between the template droplets and the secondary droplets.

[0217] 47. A water-in-oil emulsion according to clause 46, wherein the water and / or solute is transported from the template droplets to the secondary droplets.

[0218] 48. A water-in-oil emulsion according to clause 46, wherein the water and / or solute is transported from the secondary droplets to the template droplets. 49. A water-in-oil emulsion according to any one of the preceding clauses, wherein the template droplets comprise a solute.

[0219] 50. A water-in-oil emulsion according to clause 49, wherein the solute of the template droplets is the same as the solute of the secondary droplets.

[0220] 51 . A water-in-oil emulsion according to clause 49, wherein the solute of the template droplets is different to the solute of the secondary droplets.

[0221] 52. A water-in-oil emulsion according to any one of the preceding clauses, wherein the solute of the template droplets is selected from: an organic molecule, a polymer, a nanoparticle, inorganic salts, a crosslinker, a monomer, a solvent.

[0222] 53. A water-in-oil emulsion according to clause 52, wherein the carbohydrate is selected from glucose, sucrose, or fructose.

[0223] 54. A water-in-oil emulsion according to clause 52, wherein the solute of the template droplets is an inorganic salt is selected from sodium chloride (NaCI), sodium hydroxide (NaOH), calcium chloride (CaCh), potassium permanganate (kMnC ), potassium iodide (kl), or mixtures thereof.

[0224] 55. A water-in-oil emulsion according to any one of the preceding clauses, wherein the concentration of a solute in the secondary droplets is higher than the concentration of a solute in the template droplets, or vice versa, providing a gradient such that water and / or a solute is transported between the template droplets and the secondary droplets.

[0225] 56. A water-in-oil emulsion according to any one of the preceding clauses, wherein the concentration of the solute in the secondary droplets is higher than the concentration of the solute in the template droplets providing an osmotic pressure gradient that causes water to be transported from the template droplets to the secondary droplets

[0226] 57. A water-in-oil emulsion according to any one of the preceding clauses, wherein the concentration of the solute in the secondary droplets is higher than the concentration of the solute in the template droplets providing a concentration gradient that causes solute to be transported from the secondary droplets to the template droplets.

[0227] 58. A water-in-oil emulsion according to any of clauses 49-57, wherein the concentration of the solute in the template droplets is higher than the concentration of the solute in the secondary droplets providing an osmotic pressure gradient that causes water to be transported from the secondary droplets to the template droplets. 59. A water-in-oil emulsion according to any of clauses 49-58, wherein the concentration of the solute in the template droplets is higher than the concentration of the solute in the secondary droplets providing a concentration gradient that causes solute to be transported from the template droplets to the secondary droplets.

[0228] 60. A water-in-oil emulsion according to any one of the preceding clauses, wherein the solute of the secondary droplets is selected from: an organic molecule, a carbohydrate, a polymer, inorganic salts, a crosslinker, a monomer.

[0229] 61 . A water-in-oil emulsion according to clause 60, wherein the solute of the secondary droplets is a carbohydrate selected from glucose, sucrose, or fructose.

[0230] 62. A water-in-oil emulsion according to clause 60, wherein the solute of the secondary droplets is an inorganic salt is selected from sodium chloride (NaCI), sodium hydroxide (NaOH), calcium chloride (CaCL), potassium permanganate (KMnC ), potassium iodide (KI), or mixtures thereof.

[0231] 63. A water-in-oil emulsion according to any preceding clause, wherein the concentration of the solute in the second aqueous phase is from about 0.05 mol / L to about 10 mol / L.

[0232] 64. A water-in-oil emulsion according to clause 63, wherein the concentration of the solute in the second aqueous phase is from about 0.9 mol / L to about 5 mol / L.

[0233] 65. A water-in-oil emulsion according to any one of clauses 1-56, wherein substantially none of the solute of the secondary droplets is transported from the secondary droplets to the template droplets.

[0234] 66. A water-in-oil emulsion according to clause 49-58, wherein substantially none of the solute of the template droplets is transported from the template droplets to the secondary droplets.

[0235] 67. A water-in-oil emulsion according to any one of the preceding clauses, wherein the concentration of the solute in the first aqueous phase is from about 0.05 mol / L to about 10 mol / L.

[0236] 68. A water-in-oil emulsion according to clause 67, wherein the concentration of the solute in the first aqueous phase is from about 0.9 mol / L to about 5 mol / L.

[0237] 69. A water-in-oil emulsion according to any one of the preceding clauses, wherein:

[0238] (i) the template droplets comprise an organic molecule, a biomolecule, a polymer, or an inorganic nanomaterial, and (ii) the concentration of the solute of the secondary droplets is selected such that it provides an osmotic pressure gradient between the template and secondary droplets such that water is transported from the template droplets to the secondary droplets.

[0239] 70. A water-in-oil emulsion according to clause 69, wherein the solute is not transported from the secondary droplets to the template droplets.

[0240] 71 . A water-in-oil emulsion according to clause 69 or 70, wherein the template component is present in the first aqueous phase in an amount of up to about 50 wt.%.

[0241] 72. A water-in-oil emulsion according to any of clauses 69-72, wherein the solute of the secondary droplets is selected from: an organic molecule, a biomolecule, a carbohydrate, an inorganic salt, a solvent.

[0242] 73. A water-in-oil emulsion according to clause 72, wherein the carbohydrate is selected from glucose, sucrose, or fructose.

[0243] 74. A water-in-oil emulsion according to any of clauses 69-73, wherein the concentration of the solute in the second aqueous phase is from about 0.01 mol / L to about 15 mol / L.

[0244] 75. A water-in-oil emulsion according to clause 74, wherein the concentration of the solute in the second aqueous phase is from about 0.05 mol / L to about 10 mol / L.

[0245] 75a. A water-in-oil emulsion according to any of clauses 69-73, wherein the solute in the second aqueous phase is glucose and the concentration of the solute in the second aqueous phase is from about 0.05 mol / L to about 10 mol / L, preferably from about 0.05 mol / L to about 3.3 mol / L.

[0246] 76. A water-in-oil emulsion according to any of clauses 69-75, or 75a, wherein the organic molecule is a pharmaceutical.

[0247] 77. A water-in-oil emulsion according to any one of the preceding clauses, wherein:

[0248] (i) the template droplets comprise a polymer, and

[0249] (ii) the concentration of the solute of the secondary droplets is higher than the concentration of the solute in the template droplets providing an osmotic pressure gradient and a solute concentration gradient between the template and secondary droplets such that water and solute is transported between the template droplets and the secondary droplets.

[0250] 78. A water-in-oil emulsion according to clause 77, wherein the osmotic pressure gradient is such that water is transported from the template droplets to the secondary droplets. 79. A water-in-oil emulsion according to clause 77 or 78, wherein the solute concentration gradient is such that solute is transported from the secondary droplets to the template droplets.

[0251] 80. A water-in-oil emulsion according to any of clauses 77-79, wherein the polymer is present in the first aqueous phase in an amount of up to about 50 wt.%.

[0252] 81. A water-in-oil emulsion according to any of clauses 77-80, wherein the solute of the secondary droplets is a crosslinker, a monomer, or a mixture thereof.

[0253] 82. A water-in-oil emulsion according to any of clauses 77-81 , wherein the solute of the template droplets is a crosslinker, a monomer, or a mixture thereof.

[0254] 83. A water-in-oil emulsion according to any of clauses 77-82, wherein the concentration of the solute in the second aqueous phase is from about 0.01 mol / L to about 15 mol / L.

[0255] 84. A water-in-oil emulsion according to clause 83, wherein the concentration of the solute in the second aqueous phase is from about 0.05 mol / L to about 10 mol / L.

[0256] 84a. A water-in-oil emulsion according to clause 84, wherein the solute in the second aqueous phase is glucose and the concentration of the solute in the second aqueous phase is from about 0.05 mol / L to about 10 mol / L, preferably from about 0.05 mol / L to about 3.3 mol / L.

[0257] 85. A water-in-oil emulsion according to any of clauses 77-84, or 84a, wherein the first aqueous phase comprises one or more initiators.

[0258] 86. A kit for preparing a water-in-oil emulsion according to any one of the preceding clauses, comprising:

[0259] (a) a first aqueous phase comprising a template component selected from: organic molecules or their derivatives, polymers, monomers, inorganic salts, or nanomaterials, and

[0260] (b) a secondary water-in-oil pre-emulsion comprising a second aqueous phase forming a plurality of secondary droplets, the secondary droplets comprising a solute, dispersed in a continuous oil phase.

[0261] 87. A kit for preparing a water-in-oil emulsion according to any of clauses 1-85, comprising:

[0262] (a) a first water-in-oil pre-emulsion comprising a first aqueous phase forming template droplets, dispersed in a continuous oil phase, the template droplets comprising a template component selected from: organic molecules or their derivatives, polymers, monomers, inorganic salts, or nanomaterials, and (b) a secondary water-in-oil pre-emulsion comprising a second aqueous phase forming a plurality of secondary droplets, the secondary droplets comprising a solute, dispersed in a continuous oil phase.

[0263] 88. A method of preparing a water-in-oil emulsion, the emulsion comprising:

[0264] (a) a first aqueous phase forming template droplets,

[0265] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0266] (c) a continuous oil phase, and

[0267] (d) one or more surfactants; wherein the method comprises:

[0268] (i) preparing a secondary pre-emulsion by emulsifying the second aqueous phase and the solute in a continuous oil phase with the one or more surfactants to provide the plurality of secondary droplets; and

[0269] (ii) forming the template droplets in the secondary pre-emulsion thereby to provide the water-in- oil emulsion.

[0270] 89. A method according to clause 88, wherein the water-in-oil emulsion is according to any of clauses 1-85.

[0271] 90. A method according to clause 88 or clause 89, wherein step (ii) comprises

[0272] (ii-a) preparing a template pre-emulsion by emulsifying the first aqueous phase in a continuous oil phase with one or more surfactants to provide the template droplets, and

[0273] (ii-b) combining the secondary and template pre-emulsions, thereby to provide the water-in-oil emulsion.

[0274] 91 . A method according to clause 90, wherein steps (i) and (ii-a) comprise emulsifying in the same continuous oil phase.

[0275] 92. A method according to clause 90, wherein steps (i) and (ii-a) involve emulsifying in different continuous oil phases that when combined provide the continuous oil phase of the water-in-oil emulsion.

[0276] 93. A method according to any of clauses 90-92, wherein steps (i) and / or (ii-a) comprise emulsifying using one or more of ultrasonication, high shear mixing, stirring, homogenisation, controlled emulsification methods, membrane emulsification, or microfluidics.

[0277] 94. A method according to clause 88, wherein step (ii) comprises: (ii-a) introducing the first aqueous phase into the continuous oil phase of the secondary pre-emulsion (e.g., by injection) such that the template droplets are formed, thereby to provide the water-in-oil emulsion.

[0278] 95. A method according to clause 94, wherein steps (i) and / or (ii) comprises emulsification using ultrasonication, high shear mixing, stirring, homogenisation, controlled emulsification methods, membrane emulsification, or microfluidics.

[0279] 96. The water-in-oil emulsion obtainable by the method of any of clauses 88-95.

[0280] 97. A method of manipulating the composition of template droplets in a water-in-oil emulsion, comprising: (A) providing a water-in-oil emulsion comprising:

[0281] (a) a first aqueous phase forming template droplets,

[0282] (b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,

[0283] (c) a continuous oil phase, and

[0284] (d) one or more surfactants, wherein (B) water and / or solute is transported between the template droplets and secondary droplets, thereby manipulating the composition of the template droplets in the water-in-oil emulsion.

[0285] 97a. A method according to clause 97, wherein the concentration of the solute is higher in the secondary droplets than the concentration of the solute in the template droplets, providing an osmotic pressure gradient and / or solute concentration gradient between the template and secondary droplets such that water and / or solute is transported between the template droplets and the secondary droplets, thereby manipulating the composition of the template droplets in the water-in-oil emulsion.

[0286] 98. A method according to clause 97 or 97a, wherein the water-in-oil emulsion is according to any one of clauses 1-85 and 96.

[0287] 99. A method of assembling microparticles from nanomaterials or polymers, comprising: a) preparing a water-in-oil emulsion according to the method of any one of clauses 88-95, wherein the template droplets comprise the nanomaterials or polymers and the concentration of the solute of the secondary phase is such that there is an osmotic pressure gradient between the template droplets and the secondary droplets; causing the removal of water by transporting water from the template droplets to the secondary droplets and assembling of the microparticles; and b) optionally washing and filtering the assembled microparticles.

[0288] 100. A method of providing crosslinked polymer microparticles from a polymer, comprising: a) preparing a water-in-oil emulsion according to the method of any one of clauses 88-95, wherein the template droplets comprise a polymer and the solute of the secondary droplets is a crosslinker, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing an osmotic pressure gradient and a solute concentration gradient between the template droplets and the secondary droplets; thereby causing addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, and removing water by transporting water from the template droplets to the secondary droplets, thereby to provide crosslinked polymer microparticles, b) optionally washing and filtering the crosslinked polymer microparticles.

[0289] 101. A method of providing crosslinked polymer microparticles from a polymer, comprising: a) preparing a water-in-oil emulsion according to the method of any one of clauses 88-95, wherein the template droplets comprise a polymer and the secondary droplets comprise a first solute that is a crosslinker and a second solute, wherein the concentration of the second solute in the secondary droplets is higher than the concentration of second solute in the template droplets providing an osmotic pressure between the template droplets and the secondary droplets; thereby removing water by transporting water from the template droplets to the secondary droplets, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing a crosslinker concentration gradient between the template droplets and the secondary droplets; thereby causing addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, thereby to provide crosslinked polymer microparticles, b) optionally washing and filtering the crosslinked polymer microparticles.

[0290] 102. A method according to clause 101 , wherein the cross-linker is present in an amount which is lower than the amount of the second solute.

[0291] The present invention will now be described by way of reference to the following examples. These examples are not to be construed as being limiting on the invention.

[0292] General methods:

[0293] Oil phase preparation: The selected oil phase was mixed with the selected surfactant(s) in the desired concentration and vigorously mixed. The oil / surfactant mixture was then left to equilibrate for at least 1 day. Oil phases consisting of mixtures were prepared by mixing all phases together in the desired quantities prior to surfactant dissolution. The dynamic viscosity of the oil phase was measured before use as a quality check. Dispersed phase preparation (for both the template droplet phase and the secondary phase): The desired material(s) were added to de-ionised (DI) water in the required concentration and mixed by one of the following methods (according to material): vigorous stirring, ultrasonication, planetary centrifugal mixer. In cases where more than one material was involved, materials may be mixed together or sequentially according to their specific requirements. A homogeneous solution should be obtained prior to emulsification. the rate of water extraction of oil continuous

[0294] This example demonstrates how the properties of the continuous oil phase can be altered to provide selective water removal, with extremely fine control over the rate of water removal. As a model substance, methyl blue (an organic hydrophilic dye) was used in the template droplet. The rate of water removal from template droplets under different conditions was quantified by measuring template droplet shrinkage from optical microscope images (see Figure 1) over 1 hour. The influence of different parameters affecting the rate of water transport between template and secondary droplets is shown in Figure 1 , which focuses on factors related to the oil phase properties.

[0295] System composition 0.05 wt.% methyl blue in water, individual pipetted droplets -700 pm in diameter, emulsified in secondary emulsion. 5 mol / kg glucose (3.2 mol / L) in water, 1 :10 dispersed phase:continuous phase, emulsified by stirring 30 s at 2500 rpm to produce < 150 pm droplets. ases mixtures of isoparaffin mineral oil, oleic acid, silicone oil. Also 1-octadecene. mixtures of sorbitan ester and ethoxylated sorbitan ester surfactants dissolved in the oil phase at 5 wt.%.

[0296] Emulsification: The secondary droplet phase was produced by adding the oil phase to the secondary dispersed phase at a selected volume ratio. The secondary phase comprised a solute in water. This mixture underwent vigorous stirring with a vortex mixer for 30 seconds at 2500 rpm to produce < 150 pm secondary droplets. Shortly after this, the required volume was transferred to a dish. Static template droplets containing a solute e.g. methyl blue dye in water (0.05 wt.%) were generated directly in this emulsion by injection of 0.125 pl of solution at a time. Individual pipetted droplets were -700 pm in diameter, emulsified in the secondary emulsion.

[0297] Characterisation of mass transport and stability: Immediately after the two emulsions were brought into contact, an image of the template droplets was taken with a camera attached to an optical microscope. Further images were taken over time to reveal the change in size of the template droplet, and enable qualitative observation of coalescence (either between the template droplet and secondary droplets, or between secondary droplets by themselves). Experiments were conducted at room temperature, imaging at least three template droplets for each oil phase composition. Table 1 describes conditions in the continuous phase used to fine tune the rate of water extraction from a droplet containing at least one solute. Table 1

[0298] Results:

[0299] Each of the above emulsions possesses coalescence stability as determined herein, measured by optical microscopy, and evaluated over 60 minutes. Without wishing to be bound by theory, it is believed the smaller secondary droplets form a protective layer surrounding the template droplets, which thereby reduces template droplet coalescence.

[0300] Figure 1 demonstrates factors related to the continuous phase that influence the rate of water loss from template droplets containing 0.05 wt.% methyl blue dye. In particular, it can be seen that surfactant concentration and HLB, and the oil phase viscosity and polarity all can be altered to finely tune the rate of water removal from template droplets (indicated by the change in diameter of the droplets). This advantageously provides a system wherein the rate of water removal can be finely tuned depending on the desired application, e.g. precipitation or microparticle assembly or formation. In Figure 1 , shown is (a) the influence of surfactant concentration in an oil phase consisting of 5:2 isoparaffin oil: oleic acid with the surfactants sorbitan monooleate and ethoxylated sorbitan trioleate at a 4:1 w:w ratio (HLB 5.6). (b) The effect of surfactant HLB value and composition in the oil phase 1- octadecene (ODE) where S80 is sorbitan monooleate, and S85 is sorbitan trioleate, (c) The effect of increasing oil phase viscosity using mineral oil (MO) while maintaining a similar polarity with oleic acid (OA) using 5 wt.% of the same surfactants as in (a), (d) The effect of changing oil phase composition and polarity with mixtures of mineral oil, silicone oil, oleic acid, and isoparaffin while maintaining a viscosity -f around 2 - 3 mPa.s. The same surfactant concentration and composition as in (d) was used for each phase. Method 1.1 was used to prepare all tested emulsions.

[0301] It will be appreciated that the chemical identity of the components of the emulsion are not critical, and other oil phase(s) and surfactant(s) could be used so long as a water-in-oil emulsion is obtained which is stable against coalescence during the manipulation of the emulsion composition, such as for the duration of material assembly into microparticles. the rate of water extraction

[0302] This example demonstrates how substance(s) dissolved or dispersed in the template droplet can be concentrated to a desired droplet size or substance concentration through selective water removal, with extremely fine control over the rate of water removal. As a model substance, methyl blue (an organic hydrophilic dye) was used in the template droplet. The rate of water removal from template droplets under different conditions was quantified by measuring template droplet shrinkage from optical microscope images (see Figure 2) over 1 hour. The influence of different parameters affecting the rate of water transport between template and secondary droplets is shown in Figure 2. . Figure 2 focuses on factors related to the two dispersed phase compositions and emulsion structure.

[0303] System composition: ase 0.05 wt.% methyl blue in de-ionised water without or with glucose at various concentrations. ase up to 5 mol / kg (3.2 M) glucose in de-ionised water.

[0304] Oil ases mixtures of isoparaffin mineral oil, oleic acid, silicone oil. Also 1-octadecene. mixtures of sorbitan ester and ethoxylated sorbitan ester surfactants dissolved in the oil phase at 5 wt.%.

[0305] System preparation:

[0306] Method 1.1 : the secondary droplet phase was produced by mixing the oil phase with the secondary dispersed phase at a 10:1 v:v ratio. This mixture was emulsified by vigorous stirring for 30 seconds at 2500 rpm. Template droplets were generated directly in the resulting emulsion by injection of 0.125 pl of solution. The total volume of the secondary droplet phase exceeded the total volume of the template droplet phase by at least two orders of magnitude. The shrinkage rate of these droplets was measured by optical microscope image analysis of droplet diameter over 1 hour; an average (mean) of at least 3 droplet sizes was taken for each system condition.

[0307] Method 1.2: As for Method 1.1 but where the secondary emulsion was generated by homogenisation (IKA Ultra-turrax T25 equipped with S25N-8G-ST dispersing tool) for 30s at 24000 rpm. Method 1.3: As for Method 1.1 but where the secondary emulsion was generated by tip sonication for 30 s at 50% amplitude (Misonix S-4000, output frequency 20 KHz, equipped with a microtip probe 1 .6 mm in diameter).

[0308] Table 2 describes conditions in the dispersed phases used to fine tune the rate of water extraction from a droplet containing at least one solute.

[0309] Method 1.1 was used to prepare all tested emulsions. In all experiments in Table 2 the continuous phase was composed of 5:2 v:v isoparaffimoleic acid, containing 5 wt.% total surfactants (4:1 w:w sorbitan monooleate: ethoxylated sorbitan trioleate (HLB 5.6)).

[0310] Table 2

[0311] Results:

[0312] Figure 2 demonstrates factors related to the dispersed phase components that influence the rate of water loss from template droplets containing 0.05 wt.% methyl blue dye. In particular, it can be seen that adjusting the solute concentration inside the template droplets and inside the secondary droplets (i.e. changing the osmotic pressure gradient) allowed fine tuning of the rate of water removal. In addition, it is shown that changing the initial droplet diameter of the template and secondary droplets advantageously provides a system wherein the rate of water removal can be finely tuned. Such fine tuning is especially advantageous as it allows for an optimised system depending on the desired application, e.g. precipitation, or microparticle assembly or formation. Figure 2 shows (a) The influence of surfactant concentration in an oil phase consisting of 5:2 isoparaffin oil: oleic acid with the surfactants sorbitan monooleate and ethoxylated sorbitan trioleate at a 4:1 w:w ratio (HLB 5.6). (b) The effect of surfactant HLB value and composition in the oil phase 1- octadecene (ODE) where S80 is sorbitan monooleate, and S85 is sorbitan trioleate, (c) The effect of increasing oil phase viscosity using mineral oil (MO) while maintaining a similar polarity with oleic acid (OA) using 5 wt.% of the same surfactants as in (a), (d) The effect of changing oil phase composition and polarity with mixtures of mineral oil, silicone oil, oleic acid, and isoparaffin while maintaining a viscosity of around 2 - 3 mPa.s. The same surfactant concentration and composition as in (d) was used for each phase. Method 1.1 was used to prepare all tested emulsions.

[0313] Example 3: Assembly of monodisperse silica (SiO2) colloids within a template droplet with control over porosity and structure.

[0314] This example demonstrates selective concentration and assembly of nanoparticles within a droplet. Monodisperse spherical silica nanoparticles were used as an exemplar colloidal system which can adopt crystalline arrangements depending on the assembly conditions. By controlling the rate of water extraction, it was possible to control colloidal assembly and create microparticles which were hollow or solid, with crystalline or amorphous primary nanoparticle arrangements. 200-250 nm nanoparticles were used as a periodic arrangement composed of these nanoparticles selectively reflects visible wavelengths of light. Microparticles with a well-controlled porosity have applications in many fields.

[0315] System composition: up to 20 wt.% silica nanoparticles dispersed in de-ionised water by up to 5 mol / kg (3.2 M) glucose in de-ionised water.

[0316] Oil ase isoparaffin, and oleic acid at a 5:2 v:v ratio. sorbitan monooleate and ethoxylated sorbitan trioleate at a 4:1 w:w ratio (HLB 5.6), dissolved in the oil phase at 5 wt.% total.

[0317] System preparation:

[0318] Method 2.1 : The secondary droplet phase was produced by ultrasonication for 30 s at 50% amplitude (Misonix S-4000, output frequency 20 KHz, equipped with a microtip probe 1.6 mm in diameter) after adding the oil phase to the secondary dispersed phase at a 5:1 v:v ratio. Template droplets were produced by vigorous stirring (5s at 300-2500 rpm) in the oil phase at a volume fraction of 0.13-0.33 prior to mixing with the secondary emulsion, to yield a final secondary phase volume ratio of 10:1 . After the allocated time for assembly, solid particles were extracted, filtered, and washed with ethyl acetate and isopropyl alcohol before drying.

[0319] Method 2.2: The secondary emulsion was produced as in Method 2, using a 5:1 v:v ratio. This emulsion was then pumped through a flow-focusing microfluidic droplet generator alongside the template droplet dispersed phase to produce monodisperse droplets. A flow rate of 3 pl / min was used for the SiO2 dispersion, 40 pl / min was used for the continuous phase, and 100 pl / min was used for the secondary emulsion absorbing component (143 pl / min total). This produced template droplets with an average diameter of 44 ± 2 pm. Template droplets were solidified in tubing, then extracted, filtered, and washed before drying.

[0320] Results:

[0321] This enabled the production of solid crystalline microparticles produced according to the above methods wherein the rate of water removal was tuneable. The solid crystalline microparticles prepared had a particle size from -5-50 pm, and depending on the rate of water removal from the template droplet, some particles provided a hollow or broken structure (see Figure 3). This suggest the size and porosity of the microparticles can be finely tuned using the methods described herein.

[0322] Some exemplary embodiments are shown in the table below, wherein solidification occurred at room temperature, and the secondary droplet phase was emulsified by tip Bonification providing a secondary droplet diameter of <1 pm. Figure 3 shows (a) (b): demonstration of the effect of solidification rate on SiO2 assembly into microparticles using glucose solutions with 2-5 mol / kg concentration in the secondary emulsion droplets to selectively remove water; (c), (d): optical images (c) and reflectance spectra (d) of polydisperse crystalline microparticles with structural colour generated through processing monodisperse SiO2 nanoparticles with sizes from 200-250 nm inside template droplets. Particles in (c) and (d) were generated using Method 2.1 with 4 mol / kg glucose in the secondary emulsion.

[0323] Figure 4 shows the results of Method 2.2 where microparticles were produced with a single channel microfluidic droplet generator and solidified in-line, showing optical microscopy of (a) emulsified droplet templates and (b) microparticles produced where the flow rates were: 3 pl / min for SiO2 / H2O, 40 pl / min for the continuous phase, and 100 pl / min for the solvent absorbing component (143 pl / min total); (c) size distributions of emulsified droplets and solid microparticles with the conditions in (a) and (b); and (d)-(f) scanning electron microscopy of colloidal crystalline microparticles. carbon nanotubes (CNTs) into

[0324] This example demonstrates CNT assembly into microparticles from emulsion droplets using the processing method of this disclosure. Firstly, the optimization of secondary droplet solutes is described for CNT assembly based on microparticle solidification speed. Next, described is the effect of secondary droplet size on CNT particle shape and solidification rate, and the formation of monodisperse and polydisperse template droplets of different sizes. Finally, described is the co-assembly of CNTs with lithium titanate nanoparticles to form composite microparticles.

[0325] System composition:

[0326] Template droplet phase: 0.05-1 wt.% oxidized carbon nanotubes (CNTs) dispersed in de-ionised water by ultrasonication. Also 0.25 wt.% oxidized CNTs and 2 wt.% lithium titanate nanoparticles (LTO).

[0327] Secondary droplet phase: Various solutes and concentrations (see Table 2) in de-ionised water.

[0328] Oil phase: isoparaffin, and oleic acid at a 5:2 v:v ratio.

[0329] Surfactant(s): sorbitan monooleate and ethoxylated sorbitan trioleate at a 4:1 w:w ratio (HLB 5.6), dissolved in the oil phase at 5 wt.% total.

[0330] System preparation:

[0331] Method 3.1 : the secondary droplet phase was produced by mixing the oil phase with the secondary dispersed phase at a 10:1 v:v ratio. This mixture underwent vigorous stirring for 30 seconds at 2500 rpm. Shortly after this, the emulsion was transferred to a dish. Static template droplets were generated directly in this emulsion by injection of 0.5 pl of solution at a time. Afterthe allocated time for incubation, solid particles were filtered and washed before drying.

[0332] Method 3.2: as for Method 3.1 but where the secondary droplet phase was produced by tip sonication to yield a droplet size of < 1 um. Method 3.3: as for Method 3.2 but where the template droplets were produced by vigorous stirring in isoparaffin oil prior to mixing with the secondary emulsion.

[0333] Method 3.4: as for Method 3.2 but where the template droplets were produced by vigorous stirring in the secondary emulsion after it had been emulsified by ultrasonication.

[0334] Method 3.5: as for method 3.2 but where the template droplets were produced by microfluidic emulsification in a flow of the secondary emulsion as the continuous phase.

[0335] Results:

[0336] Solute testing: A variety of different solutes were tested in the secondary emulsion for compatibility, stability, and rate of forming CNT microparticles from aqueous droplets. All systems were tested by Method 3.1 and some exemplary results are summarised in the table below.

[0337] Investigating droplet size and microparticle formation rate: The solidification rate and microparticle morphology of CNT microparticles produced from the same size of template droplet by Method 3.1 and Method 3.2 are compared in Figure 5a and 5b. Larger secondary droplets give a faster solidification rate but imprinted on the final microparticle whereas smaller secondary droplets give a smoother microparticle but are slower to extract water. Figure 5c shows that the rate of solidification also depends on CNT-containing template droplet size where draw emulsion size and composition remain constant (tip sonicated draw emulsion). This method was tested with polydisperse template droplets created by Methods 3.3 and 3.4 to generate polydisperse CNT microparticles (example in Figure 5d). The same secondary emulsion was also used in conjunction with microfluidic template droplet emulsification in Method 5 which created CNT microparticles shown in Figure 5e. Emulsions were also heated to 60 and 80°C to investigate solidification rate.

[0338] Figure 5 shows microparticles generated from carbon nanotube dispersions. (a),(b) comparison of rate, (c) comparison of initial measured shrinkage rate with water droplet transport, (d) Polydisperse CNT microparticles produced by Method 3.3; (e) Monodisperse CNT microparticles produced by

[0339] Method 3.5

[0340] Co-assembly of CNTs with lithium titanate nanoparticles (LTO): Lithium titanate is a commercially used active material for Li-ion battery anodes, but has poor electronic conductivity and a low Li diffusion coefficient. The combination of nanosized LTO particles and the incorporation of a conductive carbon network is therefore desirable and can be achieved through the formation of composite microparticles. Using Method 3.3, polydisperse microparticles ranging from 1-20 pm in diameter were formed from either LTO nanoparticles alone (2 wt.% LTO dispersed in DI water by ultrasonication), or LTO and CNT nanoparticles (2 wt.% LTO and 0.25 wt.% ox-CNT dispersed by ultrasonication) to form a composite particle.

[0341] Figure 6 shows the resulting polydisperse microparticles generated from nanoparticle dispersions using Method 3.3: (a) microparticles generated from a 2 wt.% LTO aqueous dispersion; (b) microparticles generated from 2 wt. LTO and 0.25 wt.% oxidized CNTs in water. and solvent removal from a

[0342] In this example processing of a polymer by simultaneous solvent removal from the template droplet, and cross-linking to the template droplet is demonstrated. Sodium alginate and ionic cross-linking is shown but it should be appreciated that this method could be applied to other systems of polymer and chemical cross-linking or solidification. Two different contrast agents are used here as model cargo: one small molecule dye and one nanoparticle dispersion.

[0343] System composition:

[0344] Template droplet phase: 0-1 wt.% sodium alginate dissolved in de-ionised water, with 0.05 wt.% methyl blue dye, or 0.02 wt.% Prussian blue nanoparticles as contrast agent.

[0345] Secondary droplet phase: 5 mol / kg or 6 mol / kg calcium chloride dissolved in de-ionised water.

[0346] Oil phase: isoparaffin and oleic acid mixed in a 5:2 v:v ratio.

[0347] Surfactant(s): sorbitan monooleate and ethoxylated sorbitan trioleate (4:1 w:w ratio, HLB 5.6) dissolved in the oil phase at 5 wt.% total.

[0348] System preparation:

[0349] Method 4.1 : the secondary droplet phase was produced by mixing the oil phase with the secondary dispersed phase, which underwent vigorous stirring for 30 seconds at 2500 rpm. Shortly after this, the emulsion was transferred to a dish. Static template droplets were generated directly in this emulsion by injection of up to 0.125 pl of aqueous solution. After the allocated time for incubation, particles were extracted and transferred to pure water to confirm solidification.

[0350] Method 4.2: the secondary droplet phase was produced by ultrasonication. Polydisperse template droplets containing alginate were emulsified in the oil phase by vigorous mixing. The two emulsions were then combined by mixing. After the allocated time for incubation, particles were extracted and transferred to pure water to confirm solidification. Method 4.3: the secondary droplet phase was produced by ultrasonication with a 20:1 v:v ratio of the continuous phase to secondary dispersed phase. Template droplets with a narrow size distribution were emulsified in the secondary emulsion phase by membrane emulsification (Micropore Technologies AXF-mini).

[0351] Figure 7 shows solidification rates of template droplets containing sodium alginate with simultaneous water removal and cross-linking from a secondary emulsion containing CaCh. (a) Average water removal rate (left) and optical microscopy (right) of template droplets containing 0, 0.25 and 0.5 wt.% sodium alginate and 0.05 wt.% methyl blue dye, produced with Method 4.1 , where the secondary droplet phase contains 5 mol / kg CaCh in water; (b) average droplet shrinkage rate (left) and optical microscopy (right) of droplets in a polydisperse emulsion containing 1 wt.% sodium alginate, produced with Method 4.2 where the secondary droplet phase contains 5 mol / kg CaCh in water; (c) average water removal rate (left) and optical microscopy (right) of template droplets containing 0, 0.5, 1 , and 3 wt.% sodium alginate and 0.2 wt.% Prussian blue nanoparticles, produced with Method 4.2, where the secondary droplet phase contains 6 mol / kg CaCh in water; (d) template droplets containing 1 wt.% alginate and 0.05 wt.% methyl blue produced by membrane emulsification with Method 4.2 (i) after emulsification; (ii) during solidification and (iii) after solidification and shrinkage: the secondary droplet phase contained 5 mol / kg CaCh in water.

[0352] In all experiments the continuous phase was composed of 5:2 v:v isoparaffimoleic acid, containing 5 wt.% total surfactants (4:1 w:w sorbitan monooleate: ethoxylated sorbitan trioleate (HLB 5.6))

[0353] Table 3 shows the tested conditions for the above example.

[0354] Table 3

Claims

Claims1 . A water-in-oil emulsion, comprising:(a) a first aqueous phase forming template droplets,(b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,(c) a continuous oil phase, and(d) one or more surfactants.

2. A water-in-oil emulsion according to claim 1 , wherein the first and second aqueous phases are distinct in composition.

3. A water-in-oil emulsion according to claim 1 or claim 2, wherein the volume-to-volume ratio of the continuous oil phase to the second aqueous phase is from about 2:1 to about 80:1 .

4. A water-in-oil emulsion according to any one of the preceding claims, wherein the template droplets comprise a template component selected from: an organic molecule, a biomolecule, a polymer, a monomer, an inorganic salt, or a nanomaterial, or a combination thereof.

5. A water-in-oil emulsion according to any one of the preceding claims, wherein the average diameter of the template droplets is from about 1 to about 1000 pm.

6. A water-in-oil emulsion according to any one of the preceding claims, wherein the average diameter of the secondary droplets is from about 0.01 to about 500 pm.

7. A water-in-oil emulsion according to any one of the preceding claims, wherein the dynamic viscosity of the oil phase is from about 1 .5 to about 15 mPa.s at room temperature.

8. A water-in-oil emulsion according to any one of the preceding claims, wherein emulsion has a coalescence stability such that about 90% of the droplets do not coalesce to form larger droplets.

9. A water-in-oil emulsion according to any one of the preceding claims, wherein the continuous oil phase comprises an oil selected from saturated and unsaturated hydrocarbon oils, silicone-based oils, vegetable oils or their derivatives, fatty acids and fatty alcohols, or mixtures thereof.

10. A water-in-oil emulsion according to any one of the preceding claims, wherein the concentration of the surfactant is from about 0.2 to about 17 wt.% based on the total weight of the composition.

11. A water-in-oil emulsion according to any one of the preceding claims, wherein the total Hydrophilic-Lipophilic Balance of the one or more surfactants is from about 2 to about 7, preferably from about 2 to about 6.

12. A water-in-oil emulsion according to any one of the preceding claims, wherein the composition of the emulsion is such that the solute of the secondary droplets provides a chemical potential gradient e.g. an osmotic pressure gradient and / or solute concentration gradient between the template and secondary droplets such that water and / or solute is transported between the template droplets and the secondary droplets.

13. A water-in-oil emulsion according to any one of the preceding claims, wherein the concentration of the solute in the secondary droplets is higher than the concentration of the solute in the template droplets providing:(i) an osmotic pressure gradient that causes water to be transported from the template droplets to the secondary droplets; and / or(ii) providing a concentration gradient that causes solute to be transported from the secondary droplets to the template droplets.

14. A water-in-oil emulsion according to any one of the preceding claims, wherein the solute of the secondary droplets is selected from: an organic molecule, a biomolecule, a polymer, a nanomaterial, inorganic salts, a crosslinker, a monomer.

15. A water-in-oil emulsion according to any preceding claim, wherein the concentration of the solute in the second aqueous phase is from about 0.05 mol / L to about 10 mol / L.

16. A method of preparing a water-in-oil emulsion, the emulsion comprising:(a) a first aqueous phase forming template droplets,(b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,(c) a continuous oil phase, and(d) one or more surfactants; wherein the method comprises:(i) preparing a secondary pre-emulsion by emulsifying the second aqueous phase and the solute in a continuous oil phase with the one or more surfactants to provide the plurality of secondary droplets; and(ii) forming the template droplets in the secondary pre-emulsion thereby to provide the water-in- oil emulsion.

17. A method according to claim 16, wherein step (ii) comprises(ii-a) preparing a template pre-emulsion by emulsifying the first aqueous phase in a continuous oil phase with one or more surfactants to provide the template droplets, and(ii-b) combining the secondary and template pre-emulsions, thereby to provide the water-in-oil emulsion.

18. A method according to claim 16, wherein step (ii) comprises:(ii-a) introducing the first aqueous phase into the continuous oil phase of the secondary pre-emulsion such that the template droplets are formed, thereby to provide the water-in-oil emulsion.

19. A method of manipulating the composition of template droplets in a water-in-oil emulsion, comprising: (A) providing a water-in-oil emulsion comprising:(a) a first aqueous phase forming template droplets,(b) a second aqueous phase forming a plurality of secondary droplets surrounding the template droplets, the secondary droplets comprising a solute,(c) a continuous oil phase, and(d) one or more surfactants, wherein (B) water and / or solute is transported between the template droplets and secondary droplets, thereby manipulating the composition of the template droplets in the water-in-oil emulsion.

20. A method of assembling microparticles from nanomaterials or polymers, comprising: a) preparing a water-in-oil emulsion according to the method of any one of claims 16-18, wherein the template droplets comprise nanomaterials or polymers and the concentration of the solute of the secondary phase is such that there is an osmotic pressure gradient between the template droplets and the secondary droplets; causing the removal of water by transporting water from the template droplets to the secondary droplets and assembling of the microparticles; and b) optionally washing and filtering the assembled microparticles.21 . A method of providing crosslinked polymer microparticles from a polymer, comprising: a) preparing a water-in-oil emulsion according to the method of any one of claims 16-18, wherein the template droplets comprise a polymer and the solute of the secondary droplets is a crosslinker, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing an osmotic pressure gradient and a solute concentration gradient between the template droplets and the secondary droplets; thereby causing addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, and removing water by transporting water from the template droplets to the secondary droplets, thereby to provide crosslinked polymer microparticles, b) optionally washing and filtering the crosslinked polymer microparticles.

22. A method of providing crosslinked polymer microparticles from a polymer, comprising:a) preparing a water-in-oil emulsion according to the method of any one of claims 16-18, wherein the template droplets comprise a polymer and the secondary droplets comprise a first solute that is a crosslinker and a second solute, wherein the concentration of the second solute in the secondary droplets is higher than the concentration of second solute in the template droplets providing an osmotic pressure between the template droplets and the secondary droplets; thereby removing water by transporting water from the template droplets to the secondary droplets, wherein the concentration of the crosslinker in the secondary droplets is higher than the concentration of crosslinker in the template droplets providing a crosslinker concentration gradient between the template droplets and the secondary droplets; thereby causing addition of the crosslinker by transporting the crosslinker from the secondary droplets to the template droplets, thereby to provide crosslinked polymer microparticles, b) optionally washing and filtering the crosslinked polymer microparticles.

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