WO2025005799A1

WO2025005799A1 - New method of producing an aqueous mesoparticle composition comprising a lipophilic compound

Info

Publication number: WO2025005799A1
Application number: PCT/NL2024/050344
Authority: WO
Inventors: Rients Pieter BEINTEMA; George Peter JOUBERT
Original assignee: Biogntx R&D B.V.
Priority date: 2023-06-30
Filing date: 2024-06-28
Publication date: 2025-01-02
Also published as: NL2035231B1

Abstract

Described is a method for the preparation of an aqueous mesoparticle composition comprising a lipophilic compound, comprising the steps of: a. Providing an emulsifier or a blend of emulsifiers in powder form; b. Mixing one or more oils at a temperature above 40°C where all oils have become liquid, wherein said oils differ in melting temperature and which mixture comprises at least a sufficient amount of medium chain triglycerides to enable the composition formed in step g have a partly liquid oil phase at temperatures around about 4°C; c. Adding the hydrophobic or amphiphilic compound in any hydrophobic solvent to the oil mixture; d. Optionally letting the mixture cool down to room temperature; e. Adding the emulsifier powder and water to the oil mixture and letting the mixture emulsify, under optional agitation and heating to 30-40°C; f. Subjecting the emulsified mixture to a sonication and optionally mixing or fluidisation treatment until the average particle size of the mixture remains stable; g. Cooling down the sonicated mixture allowing sufficient time for crystallisation; and h. Optionally, a second sonication treatment while keeping the mixture cold and compositions produced by the above method.

Description

NEW METHOD OF PRODUCING AN AQUEOUS MESOPARTICLE

COMPOSITION COMPRISING A LIPOPHILIC COMPOUND

BACKGROUND

Mesoparticles comprise particles having an average size of 80 - 300 nm and are sometimes indicated in the scientific literature as nanoparticles (NPs) and nanocarriers (NC). However, for the present invention nanoparticles are meant to be particles with an average size of 100 nm or less, while mesoparticles are particles having a size that is larger than this common nanoscale. Mesoparticle formulations of highly lipophilic drugs enable the delivery of compounds that previously could not be administered at therapeutic levels by conventional formulations. Complex NC constructs, such as liposomes, nanocapsules, polymeric NPs, micelles and polymersomes can improve the observed therapeutic effect of drug compounds by increasing solubility, improving pharmacokinetics or altering biodistribution. Metallic, organic, inorganic and polymeric structures, including dendrimers, micelles, and liposomes are frequently considered in designing the target-specific drug delivery systems. In particular, those drugs having poor solubility with less absorption ability are tagged with these mesoparticles. However, the efficacy of these mesostructures as drug delivery vehicles varies depending on the size, shape, and other inherent biophysical/chemical characteristics. For these reasons, there is still need for a new, stable mesoparticle composition comprising a lipophilic compound enabling longterm storage and easy application for producing a pharmaceutical composition comprising the lipophilic compound.

Many drugs are lipophilic and thus would require a different carrier system to reach their target than drugs that are hydrophilic. A non-exhaustive list of such compounds includes the well known drugs imipramine, verapamil, vortiotoxetine, lurasidone, posaconazole, diazepam (and various other benzodiazeptines like midazolam and oxazepam), propranolol, trazodone, phenytoin, various statins such as atorvastatin, simvastatin and lovastatin, bifonazole, ciprofloxacin, clarithromycin, tigecyclin and clindamycin. Outside the pharmaceutical compounds also many other compounds have a hydrophobic or amphiphilic character, such as the plant-derived cannabinoids and auxins. In practice, (oral) compositions with lipophilic substances, such as cannabinoid compositions are usually provided in the form of a solution in an oily solvent wherein, in the example of cannabinoids, compounds such as cannabidiol (CBD) and tetrahydrocannabinol (THC) dissolve, allowing a rather concentrated cannabinoid content. Most of the known compositions are oil-based, i.e. an oily solution wherein the cannabinoids are dissolved, or a water-in oil dispersion, wherein the cannabinoids are in the oily phase. For oral administration, the oily solvent needs to be food grade and acceptable for oral administration. The composition is defined as oily or oil-based when more than half of the volume of the composition is an oil, and in case of a dispersion, the oily phase should be the continuous phase. Ingestion of oil is however cumbersome and since the ingestion volume is limited, the cannabinoid compositions known in the art are highly concentrated, e.g. in concentrations of 5 w/w% to 60 w/w%. Such high concentrated composition are however difficult to dose properly and often, undesired side effects are observed. Furthermore, the bioavailability of lipophilic compounds from oily preparations is low, which means that much of the ingested active compound is not utilized.

In more recent times many approaches have been published to provide waterbased compositions comprising lipophilic substances, such as cannabinoids. The present invention, which provides an aqueous mesoparticle composition, is advantageously suited for comprising such a lipophilic substance, in particular a plant extract, such as a cannabinoid composition, and thereby overcomes many of the disadvantages of the presently available aqueous dosage forms of lipophilic substances, such as cannabinoids.

Mesoparticles have been a big step forward in getting lipophilic drugs, like some components of cannabis, into the body. They have helped drugs work better by making them dissolve more easily, getting them to the right places in the body, and changing how they spread out once they are there. However, there are still some problems to solve. One big issue is finding a way to create mesoparticles that are stable for a long time and are easy to apply, especially for drugs that do not like water. Therefore in the present invention water soluble mesocarriers have been developed in which the oil phase still is partly liquid. Further, such mesoparticles may not only be used in pharmaceutics, but they can also be used in cosmetics and even in uses not intended for humans (but e.g. for plant nutrition and protection). Right now, most methods for delivering lipophilic substances, such as cannabis components, use an oily solution. This is far from ideal. It is not easy to ingest, and the body does not absorb much of the drug. It also takes a long time (about 3.5 hours) for the drug to start working, and the dose (and thus the effects) can be unpredictable. Moreover, these methods usually have a high concentration of the drug, which can make dosing difficult and cause unwanted side effects. They also go through a process in the body called first pass metabolism, which can increase the drug's interaction with other substances in the body, complicating things further.

SUMMARY OF THE INVENTION

The current invention provides a new method that uses an aqueous composition to deliver lipophilic, i.e. hydrophobic or amphiphilic components, such as lipophilic pharmaceuticals or cannabinoids. It is quick, customizable, and consistent. People taking the composition can often feel the effects within seconds, offering immediate and consistent efficacy. It gets around many of the issues with oil-based methods, like the unpredictable absorption of oils, leading to results that are more reliable. In addition, it reduces first pass metabolism, so it does not interact as much with other substances in the body.

Even better, the present method entails a (drug) delivery system that may be programmed to release the enclosed compound in a specific way, using triggers that are present inside the target of delivery or that can be applied externally. This means specific tissues can be targeted, which can make the drug more effective and which can reduce side effects. This invention addresses many of the problems with current drug delivery methods, and it could transform the way cannabinoids and other drugs that do not like water are administered.

The present invention relates to a method for the preparation of an aqueous mesoparticle composition (mesostructured lipid carrier) comprising a lipophilic compound, comprising the steps of: a. Providing an emulsifier or a blend of emulsifiers in powder form; b. Mixing one or more oils at a temperature above 40°C where all oils have become liquid, wherein said oils differ in melting temperature and which mixture comprises at least a sufficient amount of medium chain triglycerides to enable the composition formed in step g to have a partly liquid oil phase at temperatures around about 4°C; c. Adding the hydrophobic or amphiphilic compound in any hydrophobic solvent to the oil mixture; d. Optionally letting the mixture cool down to room temperature; e. Adding the emulsifier powder and water to the oil mixture and letting the mixture emulsify, under optional agitation and heating to 30-40°C; f. Subjecting the emulsified mixture to a sonication and optionally mixing or fluidisation treatment until the average particle size of the mixture remains stable; g. Cooling down the sonicated mixture allowing sufficient time for crystallization and h. Optionally, a second sonication treatment while keeping the mixture cold.

Preferably in such a method the emulsifier is a blend of emulsifiers, preferably wherein said emulsifiers are non-toxic emulsifiers, more preferably wherein said blend comprises sugar-based emulsifiers, such as sucrose ester and/or cyclodextrin. Especially preferred is an emulsifier blend comprising sucrose ester, cyclodextrin and lecithin, preferably sunflower lecithin, more preferably wherein the amount of lecithin is such, that in the final sonicated mixture from step g. the concentration of lecithin is less than 5%, preferably less than 2%, more preferably less than 1 %. In this embodiment it is further preferred that the amount of sugar-based emulsifiers is at least two times the amount of lecithin, preferably at least four times. It is also preferred that the ratio between sucrose ester, cyclodextrin and lecithin is 2 : 2 : 1.

In another embodiment, the method preferably comprises oils or fats that are non toxic. It is also preferred that the oil mixture comprises at least one oil with a melting point above 50°C, preferably above 60°C. In another preferred embodiment the oil mixture comprises an oil with a melting point in between room temperature and body temperature.

In order to achieve the above conditions, the oil mixture preferably comprises stearic acid, coconut oil and medium chain triglycerides. Then preferably the oil mixture, when mixed with the lipophilic compound, comprises the components in a ratio of stearic acid : coconut oil : medium chain triglycerides : solvent with lipophilic compound of 1 : 2 : 3 : 5. In a further preferred embodiment a non-toxic antioxidant is added to the oil mixture, preferably wherein said antioxidant is a blend of antioxidants, more preferably wherein said antioxidant or blend of antioxidants in total does not exceed the amount of 10% of the oil mixture, preferably not exceed the amount of 5% of the oil mixture, more specifically wherein said blend of antioxidants comprises linseed oil, hempseed oil, tocopherol and/or rosemary extract; preferably where it comprises linseed oil, hempseed oil, tocopherol and rosemary extract, preferably in a ratio of 2 : 2 : 2 : 1.

It is further preferred in the method of the invention that the water that is used to prepare the composition is food-grade water.

In a further preferred embodiment, the mesoparticles in the sonicated mixture will have a mean particle size of 10 - 600 nm, preferably of 50 - 150 nm and more preferably of 80 - 130 nm, most preferably about 110 nm.

Also preferred is a method as detailed herein wherein glycerol is added to the sonicated mixture, more preferably wherein the concentration of glycerol is more than 20%, preferably more than 25%.

The lipophilic compound used in the method of the invention preferably is a plantbased extract in oil, more preferably the plant-based extract is an extract of Cannabis sativa, preferably, said extract comprises a cannabinoid, more preferably, said extract comprises a cannabinoid chosen from the group consisting of A9-tetrahydrocannabinol (THC), A9-tetrahydrocannabinolic acid (A9-THCA or THCA), A9-tetrahydrocannabio- rolic acid (A9-THCA-C1 or THCA-C1), A9-tetra-hydrocannabiorcol (A9-THCO-C1 or THCO-C1), A9-tetrahydrocanna-biorcolic acid (A9-THCOA or THCOA), A9-tetra- hydrocannabivarin (A9-THCV or THCV), A9-tetrahydrocannabivarinic acid (A9- THCVA or THCVA), trihydroxy-A9-tetrahydro-cannabinol (TRIOH-THC), A10- tetrahydro-cannabinol (A10-THC), tetrahydro-cannabiphorol (THCP), THC-0 acetate (THCO), hexa-hydrocannabinol (HHC), 10-oxo- A6a-tetrahydrocannabinol (OTHC), A8-tetra-hydrocannabinol (A8-THC), A8-tetrahydrocannabinolic acid (A8-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiorcol (CBDC1), cannabidiol- C4 (CBDC4), cannabidiol dimethyl ether (CBDD), cannabidiol monomethyl ether (CBDM), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), nabilone, nabiximol, anandamide, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerolic acid A monomethykether (CBGAM), canna-bigerovarin (CBGV), cannabigerovarinic acid (CBGVA), cannabigerol mono-methylether (CBGM), cannabinol (CBN), cannabinolic acid (CBNA), cannabdiorcol (CBN-C1), cannabinol-C2 (CBN-C2), cannabivarin (CBN- C3), cannabinol-C4 (CBN-C4), cannabinodivarin (CBNDC3), cannabinol methylether (CBNM-C5), cannabichromene (CBC), cannabichromenc acid (CBCA), cannabichromanon (CBCN-C5), cannabicoumaronone (CBCON-C5), cannabi- chromanone-C3 (CBCN-C3), cannabichromevarin (CBCV), cannabichromevarinic acid (CBCVA), cannabielsoin (CBE-C5), cannabigelndol-C3 (OH-iso-HHCVC3), C3- canna-bielsoicacid B (CBEA-C3 B), cannabifuran (CBF), dehydrocannabifuran (DCBF-C5), cannabifuran (CBF-C5), dehydrocannabifuran (DCBF or CBFD), cannabicyclol (CBL-C5), cannabicyclovarin (CBLV-C3), cannabitriol (CBT), cannabitriolvarin (CBTV), cannabiripsol (CBR), cannabinodivarin (CBV or CBVD), 2- arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE), isotetrahydrocannabinol, isotetrahydrocannabivarin, palmitoylethanolamide, epigallo- catechin (EGG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG), most preferably said extract comprises THC or a blend with THC.

In another embodiment the present invention relates to a method as described above , wherein an additional step of diluting the sonicated mixture obtained in step g. is performed to obtain a diluted composition, preferably, wherein the mixture is diluted with water, more preferably wherein the mixture is diluted in such a way that the dilution comprises between 0.001 % and 5% of the lipophilic compound, preferably between 0.005% and 1 %, more preferably between 0.01 % and 0.5%, more preferably between 0,02 % and 0.1 %. Preferably the water used for the dilution is food grade. Further it is preferred to add a stabiliser to the composition, preferably wherein said stabiliser is a food grade stabiliser, more preferably wherein said stabiliser is a gum, more preferably wherein said stabiliser comprises guar gum and/or xanthan gum, more preferably wherein the concentration of guar gum and/or xanthan gum in the diluted composition is between 0.01 and 0.05%, more preferably about 0.02%. In another preferred embodiment a preservative is added to the composition, preferably wherein said preservative is a food grade preservative, more preferably, wherein said preservative is chosen from the group consisting of ascorbic acid, sodium ascorbate, isoascorbic acid, sodium isoascorbicate, citric acid, sorbic acid, calcium sorbate, benzoic acid, potassium benzoate, acetic acid, erythorbic acid, sodium erythorbate, ethyl lauroyl arginate, long-chain glycolipids from Dacryopinax spathularia MLICL 53181 , methyl-p-hydroxybenzoate, nisin, sulphurous acid, dimethyl dicarbonate, ascorbyl palmitate and blends thereof, more preferably .wherein said preservative comprises ascorbic acid, citric acid or sorbic acid or a blend thereof, preferably wherein the ascorbic acid, if present, is present at a concentration between 0.01 % and 0,1 %, preferably about 0.05%, and wherein the citric acid, if present, is present at a concentration between 0.005% and 0.05%, preferably at about 0.01 %, and wherein the sorbic acid, if present, is present at a concentration between 0.05% and 0.5%, preferably about 0, 1 %. Further, it is preferred to add a flavouring compound, preferably a food grade flavouring compound.

In another preferred embodiment panthenol is added to the oil mixture, preferably wherein panthenol is added to an amount between 0.5 and 5% of the oil mixture, more preferably an amount between 1 and 3% of the oil mixture.

The present invention also relates to an aqueous mesoparticle composition provided by a method as described above. Further, the invention also relates to a pharmaceutical composition comprising such an aqueous mesoparticle composition.

FIGURES

Fig. 1 shows a comparison a solid mesoparticle and a mesoparticle according to the invention that comprises an oil phase where part of the oil phase is liquid.

Fig. 2 shows the corrected peak area for the HPLC measurements of the fast and the slow mesoparticle compositions prepared according to Example 4. This data shows the increased release of auxin when samples are treated at 37°C compared to lower temperatures. Additionally, the release at auxin from slow mesoparticles is less than fast mesoparticles after incubation for both 12 and 45 minutes. It is observed that probably a peak is reached after incubation for 12 minutes at 37°C by the fast release mesoparticle composition, since a further increase was not visible.

Figure 3: Auxin release results in degradation of GFP in BOX632 nematodes

Quantification of (a) mean and corrected total fluorescence intensity levels of eGFP::AID::bb/n-7 in the intestine of BOX632 (mib111) nematodes, and (b) the ratio between intestinal and pharyngeal intensity of GFP after 120 minutes of meso-auxin exposure. MQ denotes non-auxin controls. Mann-Whitney U tests were performed with multiple comparison correction using false discovery rate (FDR), where FDR-corrected p-values of <0.05 were considered significant, n.s. = not significant with an FDR- corrected p-value >0.05; + = a trend with an FDR-corrected p-value of <0.1 ; * = FDR- corrected p-value <0.05; ** = FDR-corrected p-value <0.01.

Figure 4: Auxin release results in degradation of GFP in BOX817 nematodes

Quantification of (a) mean and corrected total fluorescence intensity levels of eGFP::AID::bbln-1 in the intestine of BOX817 (mib171) nematodes, and (b) the ratio between intestinal and pharyngeal intensity of GFP after 60 minutes of meso-auxin exposure. MQ denotes non-auxin controls. Two Sample f-tests were performed with multiple comparison correction using false discovery rate (FDR), where FDR-corrected p-values of <0.05 were considered significant, n.s. = not significant with an FDR- corrected p-value >0.05; + = a trend with an FDR-corrected p-value of <0.1 ; * = FDR- corrected p-value <0.05; ** = FDR-corrected p-value <0.01.

DETAILED DESCRIPTION

In the present invention a mesoparticle composition is made by emulsifying an oil composition comprising a lipophilic compound of interest with water by adding an emulsifier, after which the emulsion is sonicated to produce an aqueous mesoparticle solution.

A first step in the present invention for preparing the aqueous mesoparticle composition of the present invention is to provide an emulsifier or, preferably, a blend of emulsifiers. The goal of these emulsifiers is to provide a system from which mesoparticles may be produced and for this purpose, the emulsifier should be able to provide a sufficient stability. Further, since it is highly likely that eventually the composition is taken orally, it should also have a sufficient safety profile and it should also provide for an acceptable taste. One preferred emulsifier is lecithin, this in itself already being a blend of glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid. The lecithin may be derived from various sources, such as soybean, rapeseed, cotton seed or sunflower. Lecithin is a food-grade product, has GRAS status and is also admitted in Europe as food additive E322. Use of lecithin is advantageous since it can regulate the release of the active compound on basis of pH. At high pH values (pH >9) no degradation of the lecithin takes place and thus no release of the active compound will occur, while at lower pH values (such as the pH in living organisms) release may take place (see also Haidar, I. et al., Int. J. Pharm. 528 (1 -2) 524-535, 2017). However, due to its off-flavour, it is desirable that the amount of lecithin in the final composition is relatively low. So, care should be taken that the concentration of lecithin is less than 5%, preferably less than 2%, more preferably less than 1 % of the final sonified mixture as obtained in the process according to the present claims. Next to this low amount, it is also deemed advantageous to include further emulsifiers that would be able to mask the bad taste of lecithin. For this purpose sugar-based emulsifiers are preferred, since they provide a sweet taste. Any sugar-based emulsifier that is non-toxic and which has a sweet taste can be used, such as sucrose esters, cyclodextrin, sucralose esters, sophorolipids, and the like. For an optimal taste masking effect the concentration of these sugar-based emulsifiers is at least two times the concentration of lecithin and preferably at least four times. The most preferable combination of emulsifiers is a blend of sunflower lecithin, sucrose ester (e.g. Ryoto™ sugar ester P-1670 obtainable from Mitsubishi Chemical Corporation) and p- cyclodextrin, most preferably in a ratio of 1 :2:2. This blend also shows a lower toxicity profile than traditional emulsifiers or emulsifier blends with a lower formation of harmful free radicals and degradation products. Also, the presence of cyclodextrin means that the size of the mesoparticles will be approximately 110 nm, which size is determined by the internal bond angles of cyclodextrin encouraging particle sizes of this diameter. Particles below 110nm that incorporate beta-cyclodextrin do so with torsion energy supplied by sonication (provided in a later step) which is a thermodynamically unstable arrangement. It is expected that these particles will spontaneously reform themselves over time to a larger, more stable and energetically favourable conformation of about 110 nm.

Particle size is also determined by the ratio between oil and emulsifier. As can be seen from Figure 1 , the size of the mesoparticle is also defined by the surrounding emulsifier which acts as a surfactant. This means that the balance between the oil components and the emulsifier also influences the size that can be obtained by the meso-particles. It has been observed that a weight ratio oil vs. emulsifiers between 3.0 and 5.0 gives good results, but preferably the ratio is between 3.2 and 4.0. More preferred is a ratio of about 3.5 which causes an average particle size of about 110 nm.

One further advantage of the possibility to be able to vary the average particle size of the composition by choosing different emulsifier blends and/or a different oil/emulsifier ratio, is the fact that the mesoparticles may be sensitive to breakdown by UV radiation at a wavelength that is identical to the their average size. Thus, to enhance the degradation of mesoparticles with an average particle size of 110 nm radiation with UV light having a wavelength of 110 nm could be used. This is especially useful when the compositions are used outside the human or animal body, e.g. in or on plants.

In a second step of the method a mixture of oils is provided. Similar to the blend of emulsifiers, also for the oil mixture only oils should be included which are non-toxic and food-grade. Further, in order to be able to be able to regulate the viscosity and stability of the mesoparticle solution, oils of different melting temperatures should be used. For obtaining a good stability of the mesoparticles at least one oil with a melting temperature of more than 50°C, preferably more than 60°C should be used, such as myristic acid, palmitic acid, stearic acid or arachidic acid. Preferably stearic acid (E570), that has a melting point of nearly 70°C, is used. Stearic acid is one of the most common saturated fatty acids found in nature and in the food supply and it is often used in (nonalcoholic) beverages. Stearic acid is preferably used since it advantageously stabilises the mesoparticles that will be formed in the process. Further preferred in the oil mix is an oil that has a melting point that lies between room temperature and body temperature. Such an oil may for example be chosen from coconut oil, cocoa butter, palm kernel oil, peanut oil and babassu oil. Preferable is coconut oil since this is cheap and easily commercially obtainable. Lastly, the oil mixture should also contain a component that would provide for a low melting point, such that the oil phase in the final mesocarrier emulsion still comprises oils that are liquid at about 4°C. The advantage of having an oil phase in the mesoparticles that is at least partly liquid is that the lipophilic compound that is contained in these mesoparticles is more readily available for absorption and uptake into the body. The characteristics of such a mesoparticle is shown in Fig. 1 , where it can be seen that the oil phase of the particle comprises both liquid and solid oil. For this ingredient an oily substance with a very low melting point should be taken, such as olive oil, rapeseed oil, sunflower oil, soybean oil, castor oil, tung oil, cotton seed oil, or medium chain triglycerides (MCT). Medium-chain triglycerides (MCTs) are triglycerides with two or three fatty acids having an aliphatic tail of 6 - 12 carbon atoms, i.e. medium-chain fatty acids (MCFAs). Preferably, medium chain triglycerides are used since these are completely saturated, which means that they are unlikely to react during sonication or mixing. Further MCTs are stable over a wide temperature range through all processing conditions. Further, it is a cheap source of oil and safely, rapidly metabolized by the body into known, safe metabolites with an extremely favourable safety profile. Also, they produce a small particle size that easily sonicates and MCTs can easily be obtained in high purity. In a further preferred embodiment, C8 MCT (caprylic acid MCT) is used. The oil mixture is prepared by adding all components at a temperature at which all the oils/fats are liquid (and which is below the boiling temperature of any of the present components).

The use of food-grade oil components is preferable, since this also means that release out of the mesoparticles is triggered inside living organisms that have enzymes (lipases) that can degrade the oil components of the mesoparticles.

To this oil mixture the lipophilic compound of interest is added which may or may not be present in a hydrophobic solvent. If such a hydrophobic solvent is used, care should be taken that the solvent is nontoxic and acceptable in food applications, at least at a concentration at which it will be available in the final product. Preferably the lipophilic compound in a solvent is a plant extract in oil, preferably an extract from hemp (Cannabis sativa) comprising one or more cannabinoids, more preferably comprising at least THC (A9-tetrahydrocannabinol). However, any cannabinoid compound may be included, such as selected from the group of A9- tetrahydrocannabinol (THC), A9-tetrahydrocannabinolic acid (A9-THCA or THCA), A9- tetrahydrocannabiorolic acid (A9-THCA-C1 or THCA-C1), A9-tetra-hydrocannabiorcol (A9-THCO-C1 or THCO-C1), A9-tetrahydrocanna-biorcolic acid (A9-THCOA or THCOA), A9-tetra-hydrocannabivarin (A9-THCV or THCV), A9- tetrahydrocannabivarinic acid (A9-THCVA or THCVA), trihydroxy-A9-tetrahydro- cannabinol (TRIOH-THC), A10-tetrahydro-cannabinol (A10-THC), tetrahydro- cannabiphorol (THCP), THC-0 acetate (THCO), hexa-hydrocannabinol (HHC), 10- oxo- A6a-tetrahydrocannabinol (OTHC), A8-tetra-hydrocannabinol (A8-THC), A8- tetrahydrocannabinolic acid (A8-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiorcol (CBDC1), cannabidiol-C4 (CBDC4), cannabidiol dimethyl ether (CBDD), cannabidiol monomethyl ether (CBDM), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), nabilone, nabiximol, anandamide, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerolic acid A monomethykether (CBGAM), canna-bigerovarin (CBGV), cannabigerovarinic acid (CBGVA), cannabigerol mono-methylether (CBGM), cannabinol (CBN), cannabinolic acid (CBNA), cannabdiorcol (CBN-C1), cannabinol-C2 (CBN-C2), cannabivarin (CBN-C3), cannabinol-C4 (CBN-C4), cannabinodivarin (CBNDC3), cannabinol methylether (CBNM-C5), cannabichromene (CBC), cannabichromenc acid (CBCA), cannabichromanon (CBCN-C5), cannabicoumaronone (CBCON-C5), cannabi- chromanone-C3 (CBCN-C3), cannabichromevarin (CBCV), cannabichromevarinic acid (CBCVA), cannabielsoin (CBE-C5), cannabigelndol-C3 (OH-iso-HHCVC3), C3- canna-bielsoicacid B (CBEA-C3 B), cannabifuran (CBF), dehydrocannabifuran (DCBF-C5), cannabifuran (CBF-C5), dehydrocannabifuran (DCBF or CBFD), cannabicyclol (CBL-C5), cannabicyclovarin (CBLV-C3), cannabitriol (CBT), cannabitriolvarin (CBTV), cannabiripsol (CBR), cannabinodivarin (CBV or CBVD), 2- arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE), isotetrahydrocannabinol, isotetrahydrocannabivarin, palmitoylethanolamide, epigallo- catechin (EGG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG). The lipophilic compound may be added to the oil mixture in the form as is, i.e. in the hydrophobic solvent, but, if available, it may also be added in dry or semidry form.

It has been found that a mixture with stearic acid, coconut oil and medium chain triglycerides is yielding excellent results with respect to the production of stable mesoparticles in which the oil phase in the mesoparticles is at least partly liquid at a temperature of 4°C. The ratio between the oils/fats with different melting points enables control of the melting behaviour of the mesoparticles and thereby release of the lipophilic compound associated with these mesoparticles after entering the human body. An increase in the amount of oils with a low melting point causes a larger part of the oil phase in the mesoparticle to be in liquid form, which provides a more rapid release, while an increase in the amount of oils with a high melting point causes a larger part of the oil phase in the mesoparticle to be solid, which provides for a retarded release of the lipophilic compound. This is illustrated by the experimental evidence provided in Example 4 and 5 below. The ratio of components that provides a very stable mesoparticle composition with the desired (fast) release properties may be achieved by mixing stearic acid, coconut oil, medium chain triglycerides and solvent with lipophilic compound in a ratio of approximately 1 : 2 : 3 : 5. However, depending on the nature and melting points of the individual components other ratios may be equally applicable. The skilled person will know how to vary the parameters involved in preparing the oil mixture to obtain the desired release characteristics. It is especially advantageous to ensure that the (solid) mesoparticles that will be formed after the emulsifying and sonication steps will melt at body temperature so that the mesoparticle becomes unstable and will disintegrate, which is aided by the enzymatic digestion of the oil and emulsifier components. Also the use of lecithin in the meso-emulsion may lead to instability of the mesoparticle in situations with a low pH. If thus an aqueous mesoparticle composition is desired that is able to safely pass the stomach after ingestion care should be taken not to use too much lecithin in the emulsifier mixture.

Optionally one or more antioxidants may be added to the oil mixture. However, in order to maintain the antioxidant activity of such compounds, these should only be added when the oil mixture is cooled down to about RT. Antioxidants prevent free radical induced cell and biological targets damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition . Moreover, antioxidants prevent the oxidative reaction which is responsible for rancid odors and flavors within fats and oils which reduces nutritional quality of foods. Thus, antioxidants play an important role to enable a long-term storage of compositions comprising oils and fats and also act advantageously in the body. Luckily, there are sufficient lipophilic compounds that may be added to the oil mixture that can function as antioxidant (see e.g. Papas AM. Oil-Soluble Antioxidants in Foods. Toxicology and Industrial Health. 1993;9(1-2):123-149; Fan L and Micheal Eskim NA, The Use of Antioxidants in the Preservation of Edible oils, In: Handbook of Antioxidants for Food Preservation, Woodhead Publishing Series in Food Science, Technology and Nutrition, 2015, 373- 388). Many plant oils, such as olive oil, rapeseed oil, linseed oil, peanut oil, sunflower oil, carrot seed oil, palm oil, corn oil, hempseed oil or cottonseed oil can be used, but also other plant derived components, such as vitamin E (tocopherol), extracts from rosemary, sage, thyme, and the like. The main purpose of adding these antioxidants to the oil mixture is to protect the active lipophilic ingredient during sonication without presenting a toxicity threat to the user of the composition. Although all of the mentioned antioxidants as a single component or as a blend may be added in such an amount to achieve the desired protection, we found that a mixture of linseed oil, hempseed oil, tocopherol and rosemary extract provided sufficient antioxidant protection in the process of the invention. Tocopherol also enhances tissue absorption of the lipophilic compound.

A further component that may be added to the oil mixture is panthenol, which is a provitamin of vitamin B5. It is a moisturizer and humectant that is often found in shampoos and skin care products. In the present invention it enhances the binding of the mesoparticles to water, i.e. it increases the Zeta potential of the mesoparticle solution. Because of this, it enhances tissue absorption rates of the lipophilic compound of interest. Other additions may be pyridoxal 5’-phosphate or pyridoxine hydrochloride (vitamin B6) or melatonin.

The oil mixture is mixed with the emulsifier (blend) and water at a slightly elevated temperature (about 30 - 40°C). On 1 litre of the oil mixture a total of 25 grams of the emulsifier (blend) and 500 ml water may be used. The water preferably is food-grade water.

The addition of these three components results in an emulsion with discontinuous oil droplets containing a load of the lipophilic component dispersed in the continuous aqueous medium. The mixture is preferably homogenised to obtain an emulsion in which the oil droplets are uniformly dispersed in the continuous phase. Such homogenisation can be performed with any type of mixing apparatus, such as a highspeed blender, a homogenizer, an immersion blender, an overhead stirrer, a magnetic stirrer or even a kitchen mixer or whisk (for small batches). It is also possible to obtain homogenisation through fluidisation. After this optional step of homogenisation the process of sonication is started. By this process, the oil droplets in the emulsion will fall apart into smaller droplets, finally resulting into mesoparticles. The result is an aqueous medium in which meso-size oil droplets loaded with the lipophilic component are available, i.e. the droplets are a mixed solid and liquid composition. As indicated above, the nature and the amounts and ratios of the oils determine largely the distribution of the solid and liquid oil in the mesoparticles and with this the release characteristics of the mesoparticles. The sonication process may be performed with any commercially available sonicator and should be continued until the moment that the average particle size of the mesoparticles no longer decreases, i.e. until the mean particle size of the mesoparticles is stable. Care should be taken not to overheat the sonicated mixture. The sonication process itself produces heat which may jeopardize the formation and stability of the mesodroplets formed. Cooling can be performed by external cooling of the container in which the sonication process takes place, but a better way is to immediately cool down the formed mesoparticle solution at the moment that the sonication process is (nearly) completed. This can be achieved by putting the solution on ice, which can be done already during the sonication process, but this can also be achieved by adding glycerol in an amount up to 25% of the mesoparticle solution. Cooling down has the additional advantage that it crystallizes the particles, thereby increasing the shelf life of the product. If no crystallisation is allowed for, a so- called amorphous type mesoparticle is formed (type 3 mesoparticle, see Sharma, A. and Baldi, A. J. Develop. Drugs, 2018, (7): 1) and Khan, S. et al., Adv. Pharm. Bull. 2023, 13(3): 446-460). However, since crystallisation may cause drug extrusion, which can eventually lead to clumping of the mesoparticles, it may be advantageous to perform the sonication process a second time to recapture the imperfection formed by the extruded compound of interest. This sonication then should take place while cooling the mixture.

Depending on the sonication equipment used and the components that were used in the oil mixture and emulsifier blend the mesoparticles will have a mean particle size of 10 - 600 nm, preferably of 50 - 150 nm and more preferably of 80 - 130 nm, most preferably about 110 nm. Calculation of the mean particle size may be expressed as D50 determined in accordance with ISO 9276-2 (14th Edition, September 4, 2019) or with tuneable resistive pulse sensing (TRPS) such as obtainable by using an Izon Exoid™ apparatus. Other methods of measuring the droplet size in a meso-emulsion may be used, such as dynamic light scattering, mesoparticle tracking analysis, transmission electron microscopy, scanning electron microscopy or laser diffraction. Monitoring the particle size during the sonication is preferably achieved by TRPS on samples taken during sonication.

After sonication, but before glycerol is added, the mixture is preferably filtered to remove larger particles and microorganisms, such as bacteria. For such a filtration, a filter with a cut-off at e.g 200 nm is used. Several filter types may be used, such as polyetherculfone (PES) filter, polyvinylidene fluoride (PVDF) filters, polytetrafluoroethylene (PTFE) filters, mixed cellulose ester (MCE) filters, polypropylene (PP) filters or nylon filters. All such filters may be pre-sterilized or can be sterilized by the user and these are readily commercially available. .

The presence of the meso-sized oil droplets (nanostructured lipid carriers, NLC) enables a controlled release of the lipophilic component: there will be a fraction of solid lipid and a fraction of liquid lipid dependent on the temperature to which the aqueous solution is exposed. This enables the function of a controlled release as when the mesoparticles crystallize they displace the lipophilic component from the core into the surrounding medium. This is also what differentiates the presently claimed system from earlier drug delivery mesoparticle systems (see e.g. Khan et al. supra). These either use a solid base (solid lipid nanoparticles, SLN) which suffers from low encapsulation efficiency and poor drug release kinetics, or a liquid base (mesoemulsion) which suffers from poor shelf stability as the mesoparticles just coalesce with each other and fuse to stop being mesoparticles. The partial crystallisation, which is dependent on the melting temperature of the oils used in the oil mixture, gives stability to the mesoparticles that resist flocculation without sacrificing the ability to load the mesoparticles with high amounts of hydrophilic or amphiphilic compounds and maintain encapsulation efficiency.

For shelfing the product obtained according to the above-described process, the product should be packaged in a sterile packaging, which can be of any inert material, such as glass or vacuum packaging materials, which are normally used for airtight packaging of food products. When packaged in such a way the shelf life of the product is extremely long.

The dispersion that is obtained according to the claimed method as described above is a highly stable solution/dispersion of mesostructured lipid particles, loaded with the lipophilic compound of interest, herein also called mesoparticles. It has a very high shelf life of many years without any noticeable change in the composition. Further, it is a highly concentrated source of the lipophilic compound of interest.

If needed this product can be diluted to decrease the amount of active ingredient in order to obtain a suitable dose form. Dilution normally will take place by adding (food-grade) water. When diluting additionally stabilizers, colourants, preservatives and/or flavourants may be added. For stabilizers preferably a food grade stabiliser is chosen. Preferably the stabiliser is a gum, such as guar gum (E412), arabic gum (E414), xanthan gum (E415), alginic acid (E400), carrageenan (E407), ghatti gum, tragacanth gum (E413), karaya gum (E416), locust bean gum (E410), dammar gum, glucomannan (E425), tara gum (E417), gellan gum or beta-glucan. We have found that addition of a combination of guar gum and xanthan gum works well as a stabilizer when it is applied in a concentration in the diluted composition of between 0.01 and 0.05%, more preferably about 0.02%.

Preservatives may be chosen from the group consisting of ascorbic acid, sodium ascorbate, isoascorbic acid, sodium isoascorbicate, citric acid, sorbic acid, calcium sorbate, benzoic acid, potassium benzoate, acetic acid, erythorbic acid, sodium erythorbate, ethyl lauroyl arginate, long-chain glycolipids from Dacryopinax spathularia MLICL 53181 , methyl-p-hydroxybenzoate, nisin, sulphurous acid, dimethyl dicarbonate, ascorbyl palmitate and blends thereof. In the present invention it was found that a combination of ascorbic acid, citric acid and sorbic acid provides the desired result when the ascorbic acid, if present, is present at a concentration between 0.01 % and 0,1 %, preferably about 0.05%, and when the citric acid, if present, is present at a concentration between 0.005% and 0.05%, preferably at about 0.01 %, and when the sorbic acid, if present, is present at a concentration between 0.05% and 0.5%, preferably about 0,1 %

Although the composition as produced according to the above described method already has an acceptable, if not pleasant taste, a further flavouring compound may be added, if desired. Any flavouring compound that can be used in food, including drinks, may be added, such as flavouring essences. These are readily available in any taste and the skilled person will know how to apply these and which concentration is needed for the flavouring to provide a pleasant taste.

In the products that were obtained in which the lipophilic compound of interest was a cannabinoid a dilution of 1 :8 was made, which resulted in a product that comprises approximately 8 to 16 mg of the cannabinoid in 25 ml of product (used as a dosage form), which is a concentration of about 0.04 - 0.08% (meaning that the undiluted product had a concentration of about 0.3 - 0.6%). However, it has appeared that in the current system concentrations up to 5% of lipophilic compound of interest may be reached, of course depending on the nature of the lipophilic compound, the concentration of the lipophilic compound in the solvent in which it is added to the oil mixture, the amount of oil mixture and the amount of water that is used in the emulsifying reaction, etc.

With the specific compositions as exemplified herein it was found that a superior system was made that surpasses the stability, flavor, and safety profiles of existing counterparts currently in the market. The chosen co-emulsifiers (sunflower lecithin, beta-cyclodextrin, and sucrose ester) interact synergistically to enhance mesoparticle stability, offering a robust formulation capable of maintaining product quality under various storage conditions. The combination also enhances the flavor profile of the beverage, ensuring an enjoyable consumption experience. Further, the blend exhibits a lower toxicity profile compared to traditional emulsifiers, thus preventing the formation of harmful free radicals and degradation products during high-intensity processing. The thermal process, as exemplified herein, involving a hot emulsion phase followed by cooling before homogenization, results in a more efficient procedure with minimized energy requirements and side reactions, especially since a natural antioxidant blend is employed to shield the active ingredients, mitigating potential oxidative damage to consumers. Further, some molecules (tocopherol, vitamin B5 precursor) have been incorporated to enhance tissue absorption rates and facilitate efficient drug release kinetics.

By adjusting the solid-to-liquid lipid ratios, the formulation allows precise manipulation of drug release kinetics, offering a customizable delivery experience , which is unknown for at least cannabinoids. The unique temperature-responsive release mechanism in the mesoparticles ensures a stable product at room temperature that allows controlled release upon ingestion. The formulation, however, is versatile and allows for ultra-stable mesoparticles capable of being loaded with a variety of hydrophobic drugs, expanding potential applications beyond cannabinoid delivery. The optimized mesoparticle size in the formulation supports efficient tissue penetration and helps in overcoming drug resistance mechanisms. The unique combination of emulsifiers, lipid vehicles, and natural antioxidants in the formulation not only provides a safe and stable cannabinoid delivery system but also enhances bioavailability and release kinetics. The tested formulation offered enhanced bioavailability of cannabinoids, being up to 10 times more bioavailable than normal cannabinoid oil and showing effects in minutes rather than hours. Nevertheless, the release kinetics of compounds from the mesoparticles can be programmed to suit the needs of different consumers, ranging from rapid to delayed release. The technology also supports the targeted delivery of cannabinoids or other therapeutic agents specifically inside tumors, providing a valuable tool for personalized medicine . The mesoparticle design supports potential sequential release of multiple therapeutic agents, facilitating a coordinated treatment approach. The technology can be extended to controlled-release drug delivery systems, improving patient compliance and therapeutic outcomes.

Further, the mesoparticles are transdermally bioavailable in areas where the skin is relatively thin and where there is a sufficient blood flow, due to their semi-solid partially-crystallised nature which allows the particles to maintain their structure while diffusing through the various layers of tissues in the skin, thereby expanding the delivery routes for therapeutic agents. The technology can be applied to improve the efficacy of cosmetic formulations, potentially enabling better skin penetration and longer-lasting effects. Also, the mesoparticle design allows for encapsulation and preservation of volatile or sensitive substances, extending shelf-life and maintaining compound efficacy. The formulation enables the loading and delivery of lipoophilic, amphiphilic, or charged bioactive compounds, expanding potential applications . For instance, the technology can be used to improve oral delivery of drugs with low bioavailability due to first-pass metabolism. Incorporation of bioessential compounds can potentially improve the stability and bioavailability of probiotics or other beneficial gut microflora and thus co-administered with these. The formulation can also potentially protect and enhance the delivery of probiotics, supporting their survival during transit through the harsh stomach environment.

The present technology may also be used to produce stable solutions of nanoparticles, i.e. particles having an average diameter of 10-100 nm. Such small particles may be achieved by omitting the use of beta-cyclodextrin from the current recipe and adding larger amounts of lecithin and sucrose ester. Depending on the total amount of emulsifier being added, nanoparticles of between 10 nm and 100 nm can be produced (more emulsifier will produce smaller particles). However, the smaller the nanoparticle, the less loading capacity for hydrophilic compounds will be, due to a greater (hydrophilic) surface area and the smaller internal volume. The particles will, however, be able to load more amphiphilic and hydrophilic compounds because of the increased surface area. The smaller size of the particles would allow for a reduced metabolic clearance (less renal clearance and less prone to endocytosis) which translates into an increased and quicker bioavailability in the body. Also, because of the smaller size, the nanoparticles are able to move faster within the body, thereby increasing their ability to penetrate into deep tissues, such as solid cancers , where chemotherapies historically have failed to be of use. Leaky tumor vessels also are important contributors to the enhanced permeability and retention (EPR) effect, which can be used to promote passive accumulation of nanoparticles in tumor tissue (Zhu, D. et al., J. Nanobiotechnol. 19, 435, 2021). Further, the deeper penetration also may be effective for antibiotic treatment of biofilms.

Nanoparticles may also be applicable to overcome drug resistance, which is especially helpful in resistance to chemotherapy and antibiotic resistance, which greatly impede the efficacy of pharmacological treatment (see Wang, H. et al., Nature Commun. 12 312, 2021). Many different mechanisms may enable or promote multidrug resistance, but drug efflux pumps have been considered as one of the key mechanisms, which are located on the cell membrane to e.g. efflux anticancer drugs from cancer cells. Because the drug efflux pumps cannot efflux nanoparticles from (cancer) cells, nanoparticles may be used to deliver anticancer drugs into multidrugresistant cancer cells thereby overcoming the resistance to chemotherapy. Similarly, nanoparticles may be used to deliver antibiotic to the target bacterial cells. Advantageously, together with the effective compound, e.g. chemotherapeutic agent or antibiotic, and efflux pump inhibitor (such as ritonavir, verapamil, erythromycin, cyclosporine, ketoconazole, tamoxifen, quinine or HM30181A,) is concomitantly encompassed in the nanoparticle.

Nanoparticles may also be readily pass the blood-brain barrier, whereas mesoparticles are blocked because of tight junctions that effectively filter particles that are larger than 100 nm.

Although the mesoparticles of the present invention are able to penetrate through the skin, such a use is even better achievable by nanoparticles that will meet less resistance in passing through the skin. This means that for smaller particles the site of application is less critical.

Both the he nanoparticle and the mesoparticle design might further allow for cellspecific targeting of therapeutics by incorporating specific ligands or antibodies on the surface of the nanoparticles. The nano- or meso-particle design could potentially enable the delivery of genes or RNA therapies, extending the potential applications to the burgeoning field of gene therapy. The formulation can further potentially improve the delivery and efficacy of vaccines by protecting the antigen and providing adjuvant effects

The potential applications of the technology extend to animal health, potentially improving the delivery and absorption of veterinary therapeutics and to plant treatments and nutrition.

Example 1

Preparation of an aqueous mesoparticle cannabinoid composition

A mixture of emulsifiers was prepared by combining 10g beta cyclodextrin (Landor Trading Comp.), 10g sucrose ester (Ryoto TM P-1670 from Mitsubishi Chemical Company), and 5g sunflower lecithin (buXtrade). This mixture was then diluted to 800ml with purified water at 25°C.

Separately, an oily mixture was prepared by melting 10g stearic acid, 50g natural cannabis sativa extract, 20g coconut oil (Ekoplaza), 30g C8 MCT (Lus Health Ingredients), 2g hempseed oil (Holland and Barrett), 2g linseed oil (Holland and Barrett), 1g natural tocopherols concentrate (soapqueen.nl), and 1g rosemary extract in order of descending melting point, using a heated stirrer. The temperature during this process did not exceed 75°C and the total process lasted for approximately 30 minutes.

The oily phase and emulsifier solution were then combined using a mixer until the mixture was visibly homogeneous. This mixture was then further processed with 600W of sonication power (U.S. SOLID sonicator) in a 1 L beaker, with a cycle of 10 seconds on and 2 seconds off for 7 minutes and 30 seconds.

The mixture was cooled to room temperature (approximately 20°C) using a water and ice bath before being sonicated for a further 2 minutes with the same conditions.

The final product was filtered using a 200nm filter to remove any larger particles. The resulting filtrate had a mean particle size of 114.8 nm (measured using a Izon Science Apparatus (using TPRS) and a THC concentration of 50 mg/ml.

The filtrate could be preserved using 25% glycerol if not intended for immediate use.

Example 2

Preparation of a dosage form of the composition from Example 1

A strawberry-flavored preparation was made by adding 20ml of the THC filtrate from Example 1 , 200ml of strawberry syrup BP, 1g of potassium sorbate, 0.5g Guar Gum (buXtrade), 150mg Ascorbic Acid (buXtrade), and 50mg Citric Acid (buXtrade) to 1 L of water. After thorough mixing it was divided into dosage forms containing 25 cl of the preparation. The preparation should be consumed within 2 days of preparation.

Example 3 A 25 ml preparation taken from the preparation made according to Example 2, containing 12.5 mg of THC, was opened and rinsed inside a person’s mouth as mouthwash for 40 seconds. Thereafter, the contents of their mouth was spat into a collection cup. An oil layer was visible on top of the solution. This was collected and separated from the aqueous phase/continuous phase by harvesting from the top The THC concentration in the aqueous phase was then measured with HPLC using an Agilent 1100 HPLC with LIV-DAD detector at a flow rate of 0.3 ml/min. There was very little THC left in the water (less than 0,01 % of the total starting amount), which was below the accuracy threshold of the HPLC measurement.

This indicates that all of the THC that was initially present in the water was released and had moved into the oily layer.

Example 4 Preparation of an aqueous mesoparticle auxin composition

A mixture of emulsifiers was prepared by combining 1.25 g beta cyclodextrin (Landor Trading Comp.), 1.36 g sucrose ester (Ryoto TM P-1670 from Mitsubishi Chemical Company), and 0.87 g sunflower lecithin (buXtrade). This mixture was then diluted to 102 ml with purified water at 25°C.

Separately, an oily mixture was prepared by melting 1 g stearic acid, 5.11 g auxin (indole-3-acetic acid, Sigma Aldrich), 2 g coconut oil (Ekoplaza), 3g C8 MCT (Lus Health Ingredients), 0.2 g hempseed oil (Holland and Barrett), 0.2 g flackseed (Holland and Barrett), 0.1 g natural tocopherols concentrate (soapqueen.nl), and 0.1g rosemary extract in order of descending melting point, using a heated stirrer. The temperature during this process did not exceed 75°C and the total process lasted for approximately 30 minutes.

The oily phase and emulsifier solution were then combined using a mixer until the mixture was visibly homogeneous. This mixture was then further processed with 600W of sonication power (U.S. SOLID sonicator) in a 1 L beaker, with a cycle of 10 seconds on and 2 seconds off for 7 minutes and 30 seconds. The mixture was cooled to room temperature (approximately 20°C) using a water and ice bath before being sonicated for a further 2 minutes with the same conditions. Residual clumps were removed by sonication for an additional minute.

The final product was filtered using a 2pm polypropylene filter (woven, nonbinder from VWF) to remove any larger particles. These samples were indicated as the meso-fast samples.

For slower release ‘meso-slow’ samples were prepared in an identical way, but now with an oil mixture of 4 g stearic acid, 2 g coconut oil and 2 g medium chain triglycerides to which 5.11 g auxin was added.

Example 5

For testing the release from the auxin-(fast and slow)- meso particles as prepared in Example 4, incubations were performed in waterbaths set to 20° or 37°C. A sample volume of 7.5ml was first distributed into 15 ml falcon tubes on ice water before temperature treatment. To ensure complete increase of temperature, a test sample was measured with a pH probe after 5 and 10 minutes. This indicated that 10 minutes was enough to reach a temperature of 37°C. Samples were incubated for either 12 or 45 minutes and directly cooled on ice water to block further release. Samples were transferred to Amicon® Ultra Centrifugal Filter tubes (30 kDa MWCO, Cat . No. UFC503024, Millipore) and centrifuged for 20 minutes at 2500 ref and 4°C. After centrifugation, the fraction that passed through the filter (hereafter named filtrate) was collected in Eppendorf tubes and immediately stored at -20°C for HPLC measurement. The residual product left in the filter was supplemented with 20% ethanol in a volume equal to the filtrate that passed the column. To this, a volume of 7.5 ml 100% hexane was added and vigorously shaken. Samples were incubated for 30 minutes at 20°C on a shaker. The hexane phase (top) was then transferred to a fresh tube. 7.5 ml of hexane was added to the bottom phase, and 7.5 ml of water was added to the hexane phase, and both tubes were incubated for 30 minutes at 20°C on a shaker. Both hexane phases were combined into a fresh tube and stored at -20°C. Filtrate samples were thawed and thoroughly mixed before use. 200pl of the sample was dispensed into 5ml volumetric and diluted to 5ml using Mobile Phase, after which the sample was thoroughly mixed. 1 ml of the sample was drawn into a 2ml syringe and filtered into a vial, which was repeated to produce a 2^nd sample vial. Three replicates of 8 pl were injected and measured using an Agilent 1100 HPLC with LIV-DAD detector at a flow rate of 0.3ml/min.

Imaged software was used to measure a) the pixel number of the Y axis corresponding with 100 mAll, b) the pixel number of the X axis corresponding with 0.1 min. For the samples, the height of the peak was measured in pixels, as well as the width of the peak at half height. The height of the peak in mAll was calculated by dividing the pixel number of the height peak by the pixel number of 100 mAll and multiplied by 100. The width of the peak was calculated by dividing the pixel number of the peak width at half height by the pixel number of 0.1 min. The Peak area was calculated by multiplying the peak height in mAll by the peak width at half height.

Corrected peak areas were calculated by subtracting the peak area of the samples with the peak area of the samples kept at 4°C for the respective condition. In conclusion, it appears that fast mesoparticles appear to better release their components. Release at 20°C occurs, but is enhanced at 37°C. Slow mesoparticles seem to release their compounds slower or less efficiently, at least at 20°C (see Figure 2).

Example 6

To determine the effect of released compounds from mesoparticles in C. elegans, the present assay was developed in which the release of compounds from mesoparticles can be quantitatively determined. We used transgenic nematode strains that express various tagged proteins and other components to perform this assay. This transgenic strain allowed us to visualize the degradation of a read-out protein when exposed to the plant hormone auxin (Reference 1). We were able to test the ability of a mesoprotected compound (auxin) to become bioactive by analyzing the degrading signal of the read-out protein. We could compare the meso-protected compound with the unprotected (native) compound. In brief, this system is composed of three components. First, a protein of interest is tagged with a fluorescent protein and an auxin-inducible degron (AID) derived from indole-3-acetic acid (IAA) proteins. Second, the F-box transport inhibitor response 1 protein (TIR1) is expressed under a tissue-specific promoter, allowing protein degradation to occur in only selected tissues. The final component of the system is the plant hormone auxin. In the presence of auxin, TIR1 associates with the AID degron resulting in ubiquitination and rapid degradation of the target protein. This can be detected with a loss of GFP signal in the selected tissue. While auxin is normally provided in the NGM plates on which the nematodes grow, we adapted the protocol to allow us to expose the nematodes at a desired time. Nematodes can be exposed to the normal plant hormone auxin and auxin that has been encapsulated into mesoparticles.

For this study we chose endogenously tagged eGFP::AID::bb/n-1 as the read-out protein and the target for degradation. BBLN-1 is a small coiled-coil protein that acts as a regulator of lumen morphology and is strongly expressed in many tissues in C. elegans and especially in the intestine (Reference 2). We studied two different transgenes of this gene which are identical in expression levels of the read-out GFP but differ in the AID sequence leading to different degradation dynamics. Accordingly, we expected the mib111 allele to degrade at a slower rate than the mib171 allele (Reference 1).

To synchronise nematodes, several plates of gravid adults were washed off and bleached according to standard protocols. Nematodes were hatched overnight in medium without food for 18-22 hours. Hatched L1 synchronised nematodes were grown on standard nematode growth media (NGM) plates with OP50 bacteria for 30 hours at 20°C. Prior to meso-auxin incubation, nematodes were washed off with M9 + Tween (0.05% and added to glass vials as described in paragraph 1.3.

To prevent a stress response in the nematodes as a result of heat treatment, (fast) meso-auxin as prepared in Example 4 + OP50 bacteria (5 pL of 3.8% meso-auxin per 10mL OP50, final meso-auxin concentration of approximately 50 pM) were preincubated in a glass vial at either 20° or 37°C for 30 minutes. After briefly cooling at room temperature, 200 nematodes were added to each vial and were incubated for 60 (BOX817) or 120 minutes (BOX632) at 20°C.

For analysis and quantification, nematodes were collected in centrifuge tubes and gently centrifuged. 3.7 pl of worm pellet was added to 1 pl 50mM sodium azide on 3% agarose slides. Images were obtained with a LD A-Plan 20X/0.30 Ph1 lens on a Zeiss Axioplan 2 microscope with an exposure time of 3000 milliseconds.

Images were analyzed using Imaged software by selecting regions of interest corresponding with the pharynx, intestine or background (Figure S1). Area, Mean, StDev, Min, Max, INTDEN and RAWINTDEN values were collected from Imaged. The mean green fluorescent protein (GFP) intensity was calculated by subtracting the mean fluorescence of a tissue with the mean fluorescence of the background area outside the worm. The corrected intensity was calculated by subtracting the integrated density of the tissue with the total background fluorescence intensity of an equally sized area.

Intensity ratios were calculated by dividing either the mean intensity or the corrected total intensity of the intestine by the corresponding intensity in the pharynx.

To study the release of meso-auxin in vivo, we exposed synchronised nematodes in the L3 stage of development to meso-auxin and analyzed the signal of the read-out protein over time. Upon exposing the BOX632 strain to meso-auxin, we did not observe any degradation or change in the read-out protein after 30 or 60 minutes. After 120 minutes, we observed a statistically significant degradation compared to controls in the meso-auxin treated samples. This was independent of whether they were heat- treated and exposed to 37°C (Figure 3a-b). This timescale matches the previously described degradation of GFP of this allele when using conventional auxin exposure, where 120 minutes of auxin exposure led to a strong depletion of GFP (Reference 1). Our results suggests that over time, auxin is released from mesoparticles even without treatment at 37°C. We speculate that this is due to changes in the environment in which the mesoparticles are present. Specifically, differences in pH or presence of enzymes in either the bacteria food source or nematodes can result in mesoparticle breakdown or release from the particles. We did not observe a statistically significant decrease in the pharyngeal GFP levels, indicating that loss of GFP is specific for the intestine and caused by the tissue specificity of the auxin-inducible degradation system (Figure S2). Together, we conclude that meso-auxin particles can release their contents leading to degradation of eGFP::AID::bb/n-1 read-out signal in vivo.

When exposing the BOX817 strain to meso-auxin, we did not observe degradation of GFP in the intestine after 30 minutes of presence. In contrast, we observed a statistically significant reduction in mean GFP intensity in the intestine after 60 minutes of meso-auxin exposure in nematodes treated at 37°C compared to non-treated controls (Figure 4a-b). We did not observe a significant difference in GFP intensity between controls and treated at 20°C. Furthermore, statistical comparisons between meso-auxin treated nematodes at 20°C and 37°C showed a trend at lower GFP intensity in the 37°C population. These results indicate that increased temperature at 37°C leads to enhanced release of auxin from the mesoparticles. After 90 minutes we observed degradation of GFP in the presence of meso-auxin, independent of temperature. This enforces our previous findings that auxin can be released from mesoparticles already at 20°C. In summary, our data confirms the notion that release of auxin from mesoparticles leads to in vivo degradation of GFP in the intestine, and release from the particles is enhanced at 37°C.

Claims

1 . Method for the preparation of an aqueous mesoparticle composition comprising a lipophilic compound, comprising the steps of: a. Providing an emulsifier or a blend of emulsifiers in powder form; b. Mixing one or more oils at a temperature above 40°C where all oils have become liquid, wherein said oils differ in melting temperature and which mixture comprises at least a sufficient amount of medium chain triglycerides to enable the composition formed in step g to have a partly liquid oil phase at temperatures around about 4°C; c. Adding the hydrophobic or amphiphilic compound in any hydrophobic solvent to the oil mixture; d. Optionally letting the mixture cool down to room temperature; e. Adding the emulsifier powder and water to the oil mixture and letting the mixture emulsify, under optional agitation and heating to 30-40°C; f. Subjecting the emulsified mixture to a sonication and optionally mixing or fluidisation treatment until the average particle size of the mixture remains stable; g. Cooling down the sonicated mixture allowing sufficient time for crystallisation; and h. Optionally, a second sonication treatment while keeping the mixture cold.

2. Method according to claim 1 , wherein the emulsifier is a blend of emulsifiers, preferably wherein said emulsifiers are non-toxic emulsifiers, more preferably wherein said blend comprises lecithin and sugar-based emulsifiers, such as sucrose ester and/or cyclodextrin.

3. Method according to claim 2, wherein the emulsifier is a blend comprising sucrose ester, cyclodextrin and lecithin, preferably sunflower lecithin.

4. Method according to claim 3, wherein the amount of lecithin is such, that in the final sonified mixture from step g. the concentration of lecithin is less than 5%, preferably less than 2%, more preferably less than 1 %.

5. Method according to claim 3 or 4, wherein the amount of sugar-based emulsifiers is at least two times the amount of lecithin, preferably at least four times.

6. Method according to any of claims 3-6, wherein the ratio between sucrose ester, cyclodextrin and lecithin is 2 : 2 : 1.

7. Method according to any of the previous claims, wherein the oil mixture comprises oils or fats that are non toxic.

8. Method according to any of the previous claims, wherein the oil mixture comprises at least one oil with a melting point above 50°C, preferably above 60°C.

9. Method according to any of the previous claims, wherein the oil mixture comprises an oil with a melting point in between room temperature and body temperature .

10. Method according to any of the previous claims, wherein the oil mixture comprises stearic acid, coconut oil and medium chain triglycerides.

11. Method according to any of the previous claims, wherein the oil mixture, when mixed with the lipophilic compound comprises the components in a ratio of stearic acid : coconut oil : medium chain triglycerides : solvent with lipophilic compound of 1 : 2 : 3 : 5.

12. Method according to any of the previous claims, wherein a non-toxic antioxidant is added to the oil mixture, preferably wherein said antioxidant is a blend of antioxidants, more preferably wherein said antioxidant or blend of antioxidants in total does not exceed the amount of 10% of the oil mixture, preferably does not exceed the amount of 5% of the oil mixture.

13. Method according to claim 12, wherein said blend of antioxidants comprises linseed oil, hempseed oil, tocopherol and/or rosemary extract; preferably where it comprises linseed oil, hempseed oil, tocopherol and rosemary extract, preferably in a ratio of 2 : 2 : 2 : 1 .

14. Method according to any of the previous claims, wherein the ratio of oil to emulsifiers is from 3.0 to 5.0, more preferable from 3.2 to 4.0, more preferably about 3.5

15. Method according to any of the previous claims, wherein the water is food-grade water.

16. Method to any of the previous claims, wherein the mesoparticles in the sonicated mixture will have a mean particle size of 10 - 600 nm, preferably of 50 - 150 nm and more preferably of 80 - 130 nm, most preferably about 110 nm.

17. Method according to any of the previous claims, wherein glycerol is added to the sonicated mixture, more preferably wherein the concentration of glycerol is more than 20%, preferably more than 25%.

18. Method according to any of the previous claims, wherein the lipophilic compound is a plant-based extract in oil.

19. Method according to claim 18, wherein the plant-based extract is an extract of Cannabis sativa, preferably, wherein said extract comprises a cannabinoid, more preferably, wherein said extract comprises a cannabinoid chosen from the group consisting of A9-tetrahydrocannabinol (THC), A9-tetrahydrocannabinolic acid (A9- THCA or THCA), A9-tetrahydrocannabiorolic acid (A9-THCA-C1 or THCA-C1), A9-tetra-hydrocannabiorcol (A9-THCO-C1 or THCO-C1), A9-tetrahydrocanna- biorcolic acid (A9-THCOA or THCOA), A9-tetra-hydrocannabivarin (A9-THCV or THCV), A9-tetrahydrocannabivarinic acid (A9-THCVA or THCVA), trihydroxy-A9- tetrahydro-cannabinol (TRIOH-THC), A10-tetrahydro-cannabinol (A10-THC), tetrahydro-cannabiphorol (THCP), THC-0 acetate (THCO), hexa-hydrocannabinol (HHC), 10-oxo- A6a-tetrahydrocannabinol (OTHC), A8-tetra-hydrocannabinol (A8- THC), A8-tetrahydrocannabinolic acid (A8-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiorcol (CBDC1), cannabidiol-C4 (CBDC4), cannabidiol dimethyl ether (CBDD), cannabidiol monomethyl ether (CBDM), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), nabilone, nabiximol, anandamide, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerolic acid A monomethykether (CBGAM), canna-bigerovarin (CBGV), cannabigerovarinic acid (CBGVA), cannabigerol mono-methylether (CBGM), cannabinol (CBN), cannabinolic acid (CBNA), cannabdiorcol (CBN-C1), cannabinol-C2 (CBN-C2), cannabivarin (CBN-C3), cannabinol-C4 (CBN-C4), cannabinodivarin (CBNDC3), cannabinol methylether (CBNM-C5), cannabichromene (CBC), cannabichromenc acid (CBCA), cannabichromanon (CBCN-C5), cannabicoumaronone (CBCON-C5), cannabi-chromanone-C3 (CBCN-C3), cannabichromevarin (CBCV), cannabichromevarinic acid (CBCVA), cannabielsoin (CBE-C5), cannabigelndol-C3 (OH-iso-HHCVC3), C3-canna- bielsoicacid B (CBEA-C3 B), cannabifuran (CBF), dehydrocannabifuran (DCBF- C5), cannabifuran (CBF-C5), dehydrocannabifuran (DCBF or CBFD), cannabicyclol (CBL-C5), cannabicyclovarin (CBLV-C3), cannabitriol (CBT), cannabitriolvarin (CBTV), cannabiripsol (CBR), cannabinodivarin (CBV or CBVD), 2-arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE), isotetrahydrocannabinol, isotetrahydrocannabivarin, palmitoylethanolamide, epigallo- catechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG).

20. Method according to any of the previous claims, wherein said extract comprises THC or a blend with THC.

21. Method according to any of the previous claims, wherein an additional step of diluting the sonicated mixture obtained in step g. is performed to obtain a diluted composition, preferably, wherein the mixture is diluted with water, more preferably wherein the mixture is diluted in such a way that the dilution comprises between 0.001 % and 5% of the lipophilic compound, preferably between 0.005% and 1 %, more preferably between 0.01 % and 0.5%, more preferably between 0,02 % and 0.1 %.

22. Method according to claim 21 , wherein the water is food-grade.

23. Method according to claim 21 or 22 wherein further a stabiliser is added to the composition, preferably wherein said stabiliser is a food grade stabiliser, more preferably wherein said stabiliser is a gum, more preferably wherein said stabiliser comprises guar gum and/or xanthan gum, more preferably wherein the concentration of guar gum and/or xanthan gum in the diluted composition is between 0.01 and 0.05%, more preferably about 0.02%.

24. Method according to any of claims 21-23, wherein further a preservative is added to the composition, preferably wherein said preservative is a food grade preservative, more preferably, wherein said preservative is chosen from the group consisting of ascorbic acid, sodium ascorbate, isoascorbic acid, sodium isoascorbicate, citric acid, sorbic acid, calcium sorbate, benzoic acid, potassium benzoate, acetic acid, erythorbic acid, sodium erythorbate, ethyl lauroyl arginate, long-chain glycolipids from Dacryopinax spathularia MLICL 53181 , methyl-p- hydroxybenzoate, nisin, sulphurous acid, dimethyl dicarbonate, ascorbyl palmitate and blends thereof.

25. Method according to claim 24, wherein said preservative comprises ascorbic acid, citric acid or sorbic acid or a blend thereof, preferably wherein the ascorbic acid, if present, is present at a concentration between 0.01% and 0,1 %, preferably about 0.05%, and wherein the citric acid, if present, is present at a concentration between 0.005% and 0.05%, preferably at about 0.01 %, and wherein the sorbic acid, if present, is present at a concentration between 0.05% and 0.5%, preferably about 0,1 %.

26. Method according to any of claims 21-25, wherein further a flavouring compound is added, preferably a food grade flavouring compound.

27. Method according to any of the previous claims in which panthenol is added to the oil mixture, preferably wherein panthenol is added to an amount between 0.5 and 5% of the oil mixture, more preferably an amount between 1 and 3% of the oil mixture.

28. Aqueous mesoparticle composition provided by a method according to any of claims 1-27.

29. Pharmaceutical composition comprising the aqueous mesoparticle composition of claim 28.