The invention relates to a method of producing nanocrystals or microcrystals or amorphous particles, by heating the compound dissolved in a mixture of a solvent and an anti-solvent with microwave radiation to form nanocrystals or microcrystals or amorphous particles of the compound.

A problem associated with a number of hydrophobic, less hydrophilic, or lipophobic drugs and other bioactive compounds including nutraceuticals, polymers and lipids, are low bioavailability, bioaccessibility, stability, and solubility. This can be improved by producing nano-sized or micro-sized particles of drugs to address or alleviate these problems. Using these nano-sized/micro-sized particles increases their stability, distribution and absorption index (bioavailability, bioaccessibility). This may also be used, for example, for bioactives, polymers, lipids etc.

In the past attempts have been made to increase the pharmaceutical properties (bioavailability, stability, solubility, drug release) of organic molecules using nanocrystalization technology by top-down or bottom-up techniques. The main aim of nanonization technology is to increase the surface area of the formed crystals and thereby enhance their dissolution kinetics that in turn helps to overcome the problem of low bioavailability, stability, solubility, uncontrolled drug release etc.

Top down processes involve a breaking down of larger particles by milling or homogenization (HPH, Microfiuidizer, media milling etc.)

Following the Noyes-Whitney equation:

Q

Vk

Here, dc/dt is the change of concentration over time (dissolution rate), D is the diffusion coefficient, S is the surface area, V is the volume of the dissolution medium, h is the thickness of the diffusion layer, CS is the saturation solubility and C is the instantaneous concentration at time. Some examples of top-down technique are High Pressure Homogenizer (HPH), microfiuidizer, bear ball milling etc. In the bottom-up technique organic molecules are precipitated from the solvent in which they are dissolved to form crystals with less size compared to their unaltered counterparts. A number of different generally known methods are used, including precipitation using antisolvent; precipitation using supercritical fluids; precipitation by solvent removal; precipitation combined with high energy processes.

Precipitation by liquid solvent-antisolvent addition is a generally known technique for fabrication of nanocrystals, due to its simplicity and cost effectiveness. In this technique two solvents are used. One in which the desired compound is highly soluble in, and the second in which the compound is sparingly soluble. Precipitation is typically conducted under conditions such as sonication, centrifugation etc. It allows the production of nanocrystals with smaller and narrower size distribution. Evaporative precipitation can be used in which solute, which is dissolved in solvent, is precipitated by evaporating solvent by heating to its boiling point. This result in suspension of solute in antisolvent (solvent in which solute is less soluble or insoluble).

Supercritical fluids, such as ethane, ammonia, carbon dioxide and fluroform, may be used to dissolve the compound. The solution may then be passed around a narrow nozzle with low pressure. This immediately reduces the pressure of the supercritical fluid and results in the supersaturation of the compound in the solvent which initiates nucleation and causes precipitation. One major advantage of this is the requirement of using a supercritical fluid, such as ammonia, many of which are not suitable to be used for food and pharmaceutical products. The precipitation by solvent removal is also generally known. A solvent is added to an aqueous solution (antisolvent) with or without a cryoprotectant and lyophilized. The problem with this is that solutes with low glass transition temperature are preferred for fabrication of nanocrystals. Low glass transition temperature assists in obtaining the freezing of the compounds at temperatures that are higher than the glass transition temperature of the compound.

Precipitation combined with high energy processes, carried out by removing solvent from the anti-solvent and solvent mixture and in addition the formed nanosuspension, is subjected to high energy such as high pressure homogenisation, micro fluidisation, sonication etc. The major disadvantage of this process is the requirement of long processing procedures. Radasci and co-workers (Crystal Growth and Design (2013) 13(10) 4186-4189 and J.

Pharma. Biomed. Analysis (2014) 98, 16-21, describe solvent evaporation using microwaves. Niflumic acid was dissolved in a solvent (ethanol) and the solvent evaporated using microwave heating. This resulted in crystal powders being formed. This also created an increase in viscosity during the end point of the evaporation which resulted in higher than ideal nuclei formation and polydispersed, bimodular niflumic acid crystals. Caffeine/nucleic acid co-crystals have been similarly produced (Pagires et al Cryst. Eng. Comm. (2013), 15, 3705-3710).

The inventors have realised that the use of antisolvents allows the improved production and control of nano or micro sized particles (both amorphous and crystaline). The system allows the antisolvent ratio to be maintained and to obtain uniform and rapid supersaturation diffusion distance, collision distance and viscosity to be controlled.

This is readily automated. Moreover, the safety of the process is improved by reducing the need to heat the solvent to around or above the flashpoint of the solvent.

The invention provides a method for nanonization or micronization of a compound comprising heating the mixture of a solvent, in which compound of interest is solubilized and antisolvent with microwave radiation for nanonization or micronization of the compound.

The solvent and antisolvent differ by their ability to dissolve the compound. Typically the compound is able to dissolve more readily in the solvent, compared to the antisolvent. The solvent or antisolvent is typically susceptible to dielectric heating on exposure to microwaves.

Mixtures of two or more solvents and/or two or more antisolvents may be used to more readily control the production of the nanocrystals.

By nanonization or micronization, we mean particles having an average size of typically between 1 and 5000 nm, more typically between 1 and 500 nm.

Typically the volumetric ratio of solvent to antisolvent is 1:0.5 to 1:50, especially 1: 1 to 1:40 or 1:2 to 1:4. Solvents and antisolvents are typically those that are acceptable in food, pharmaceutical and cosmetic products and which can be used in the production of nano and micro sized particles. These include (but are not limited to), for example, ethanol, water hexane, glycerol, t-butanol, isopropanol, ethyl acetate,

The nanocrystals may be organic compounds. They may include, for example, pharmaceuticals or drugs, bioactives, nutraceuticals, polymers or lipids.

The mixture may additionally comprise one or more surfactants or co- surfactants.

Surfactants and co- surfactants include, for example, Tween, a non-ionic detergent widely used in biochemical applications. It is also known as PEG(20) Sorbitan monolaurate. Other emulsifiers include poloxomer, a hydrophilic non-ionic surfactant which is a non-ionic triblock copolymer, Tween 80, and lecithins.

Further examples of drugs and nutritional compounds include curcumin, quercetin, rutin, artemether, sirolimus, aprepitant and fenofibrate.

The ratios and amounts of those compounds may be adjusted according to the compound, solvents, antisolvents and emulsifier physicochemical properties, such as solubility, melting point, purpose of use, stability, polymorphism, etc.

Subjecting the compound to microwaves in the mixture under vacuum improves the precipitation of the compound. Typically the vacuum may be between ambient to -98 mbar (absolute pressure).

The microwave radiation used is dependent on the scale and economic power requirements, but frequencies are typically 2500-200 MHz and but will also include 2400-2500, 900-915 and 433 MHz.

Typically the microwave power used is 0.1-100 kW, or 10 to 1000W or 50 to 800W. The method may be used as a continuous process in which the mixture is passed through a region of an apparatus in which the mixture and compound is subjected to microwaves, or alternatively as a batch or semi batch process. Typically the compound is subjected to microwaves for 0.1-10 minutes, or more typically 10 to 360 seconds or 10 to 240 seconds or

10-30 seconds, although this will vary depending on the solvent, compound, co-solvent and microwave power source.

Preferably the amount of microwave energy supplied in the mixture of solvent, antisolvent and compound is 0.1 to 5000 j/g mixture, typically 10 to 1000, 100 to 800, 200 to 500 or 400 j/g-

Once the nanocrystals are formed, they may be removed from the mixture by, for example, filtration or centrifugation, spray drying, freeze drying etc.

The invention also provides an apparatus for use in the method according to the invention, comprising a sample container containing a compound dissolved in a mixture of a solvent and an antisolvent, and a microwave generator. This may additionally comprise a vacuum source arranged to allow the compound in the mixture to be subjected to a vacuum when under microwave radiation. The sample container may be a closed container or alternatively, for example, a pipe through which the mixture is passed, or allowed to fall past the microwave source (coupling aperture).

The apparatus may be automated, for example, computer operated, and may contain one or more sensors measuring one or more of microwave power, solvent or antisolvent concentration, solute concentration, mixture temperature or pressure.

The invention will now be described by way of example only, with reference to the following figure:

Figure 1A shows a close up of the arrangement of a microwave generator with a sample chamber.

Figure IB shows an expanded view showing the arrangement of the sample container with a vacuum pump.

Figure 2 is a graph showing the differential scanning calorimetry (DSC) results for curcumin having different particle polymorphism. Solid line native curcumin; dotted line (F2) nanonised curcumin precipitated by evaporation in ethanol at 400 j/g microwave power at a solvent : antisolvent ratio of 1:30; dashed line (F7) curcumin precipitated by evaporating ethanol at 400 j/g microwave power at solvent to antisolvent ratio of 1: 10.

Figure 3 shows Scanning electron Micrograph (SEM) images of curcumin nanosuspension made using microwaves under vacuum as described below.

Crystallization process involves two key phenomenon i.e. nucleation and crystal growth. This phenomenon mainly depends on supersaturation (S), Diffusivity (DA _B ) and interfacial energy (γ). It is highly difficult to control these key parameters with required precision using existing fabrication techniques (sonoprecipitation, high gravity centrifugation, super critical fluid precipitation etc.). However, using MVP technique, these parameters can be precisely controlled as follows,

Diffusivity and super-saturation: Most of the solvents and anti-solvents that are used in the evaporative crystallization e.g. water, ethanol, ethyl ether etc., have low thermal conductivity (diffusivity). This slower diffusivity results in the longer evaporation time that has the propensity to delay supersaturation. This condition leads to the formation of larger crystals with wider particle size distribution and un-controlled shape. However, microwave heating depends on dielectric properties (dielectric loss and dielectric constant) of the solution rather than thermal conductivity. Since the solvents and anti-solvents like water, ethanol, ethyl ether etc. are very susceptible to the dielectic heating; there are two major advantages. First it will lead to the uniform heating throughout the reaction mixture that will bring uniformity in diffusivity that will result in homogeneous supersaturation. Secondly, since most of the polar solvents have excellent dielectric loss, mixture will heat up rapidly leading to rapid supersaturation. Obtaining homogeneous supersaturation and rapid supersaturation is important to obtain shape-controlled nanocrystals. Further, according to Werling and Debenedetti model, diffusivity and supersaturation of solvent and anti-solvent depends on the reaction ambience (temperature, pressure, gas composition etc.). Due to the possibility for maneuverability in the highly automated microwave instrumentation, we can easily control the reaction ambience and in turn diffusivity and supersaturation.

In addition, as each individual solvent has a distinct dielectric constant and dielectric loss in a reaction mixture of polar reactants (solvent and anti- solvent), volumetric heating is specific and distinct for individual reactants. By utilizing this difference in dielectric property between solvent and anti- solvent, solvent can be efficiently evaporated rapidly creating supersaturation leading to the short burst of nucleation.

Figure 1A shows a close up of the arrangement of the microwave generator with the sample container. This shows a microwave generator (1), isolator (2) and manual or automatic stub tuning device (3). The tuning device is used to maximise power transference into the sample by reducing reflected power. Choke structure (4) allows the insertion of a sample into the wave guide of the microwave whilst preventing microwaves being transmitted into the surrounding. This may be used, for example, to prevent leakage over 5mW/cm ² A sliding short circuit (5) is typically used to allow the highest energy region (maxima) of the wave to be set over the sample to ensure efficient energy transfer. This also acts as a reflecting device that allows for a standing wave to form. Sample chamber (6) is provide to contain the mixture of compound, solvent and antisolvent. This may be, for example, a closed container however, it may also be, for example, a tube or a tunnel through which the mixture is passed. This allows a continuous process to be used rather than a batch or semi batch process.

Figure IB shows an expanded part of the apparatus. It shows a transfer line (7) attached to the sample container (6). A condenser (8), vacuum/pressure gauge (9), isolation valve (10) and pump (11) is used to reduce the pressure and condense evaporated solvent.

Typically the mixture contains solvent, antisolvent and one or more compounds to be precipitated (solute). Surfactants and co- surfactants may be used to reduce or avoid crystal growth, cryoprotectant etc.

In the example below, operating parameters were -98 kPa - 800 kPa, 0.01 kW - 2 kW, 2.45 GHz, 915 MHz, 433 MHz, under batch or flow conditions. Curcumin was used with ethanol as a solvent and water as an antisolvent. The results are shown in the attached table.

Table. 1. Effect of processing parameters on nanoparticle size

This shows that particle size and polydispersity can be varied by adjusting microwave solvent: antisolvent ratio and solute concentration.

Figure 2 shows the possibility of obtaining nanonized particles both in amorphous form and crystalline form (nanocrystals or microcrystals) by varying the processing parameters and composition. Curcumin as received is the native curcumin without any modification. F2 is the nanonized curcumin obtained by precipitating curcumin by evaporating ethanol at ca. 400j/g (e.g. 50 W for 240 s) microwave power at solvent to antisolvent ratio of 1:30. It is crystalline in nature. Whereas in F7, curcumin nanonization was obtained by precipitating curcumin by evaporating ethanol at ca. 400j/g (e.g. 50 W for 240 s) microwave power at solvent to antisolvent ratio of 1: 10. It is amorphous in nature.

Figure 3 shows the possibility of obtaining curcumin nanoparticles by varying the processing parameters and composition. Curcumin as received is the native curcumin without any modification.

F2 is the nanonized curcumin obtained by precipitating curcumin by evaporating ethanol at ca. 400j/g (e.g. 50 W for 240 s) microwave power at solvent to antisolvent ratio of 1:30 and drug concentration in the solvent is 2mg/mL.

F6, curcumin nanonization was obtained by precipitating curcumin by evaporating ethanol at ca. 400j/g (e.g. 800 W for 15 s) microwave power at solvent to antisolvent ratio of 1:30 and drug concentration in the solvent is 2mg/mL. F7 is the nanonized curcumin obtained by precipitating curcumin by evaporating ethanol at ca. 400j/g (e.g. 50 W for 240 s) microwave power at solvent to antisolvent ratio of 1: 10 and drug concentration in the solvent is 2mg/mL.

F13 is the nanonized curcumin obtained by precipitating curcumin by evaporating ethanol at ca. 400j/g (e.g. 50 W for 240 s) microwave power at solvent to antisolvent ratio of 1:30 and drug concentration in the solvent is 4mg/mL.