Method for obtaining nanodispersed systems

Abstract

The present invention relates to a method for obtaining nanodispersed systems comprising the steps of: a) mixing a compound C with a fluid A and obtaining a mixture in the form of a solution or dispersion; b) thermostatising the mixture obtained in step a) at a temperature of between -50 ºC and 200 ºC; c) adding a fluid B to the thermostatised mixture until a pressure P between 0 and 300 bar is attained obtaining the mixture AB; wherein molar fraction of fluid B is from 0.01 to 0.8; d) reducing the pressure of the mixture AB obtained in step c) to a pressure lower than or equal to 10 bar, by means of a valve, wherein said valve is heated during depressurization process to a temperature T in the range of 10 ºC to 50 ºC; and e) mixing fluid A with fluid E in which fluid A is miscible and compound C is partially or totally insoluble, controlling the flow rate and the temperature T of fluid A in the range of 10 ºC to 100 ºC.

Description

[0001] METHOD FOR OBTAINING NANODISPERSED SYSTEMS

[0002] DESCRIPTION

[0003] Field of the Invention

[0004] The present invention relates to a method for obtaining nanodispersed systems. In particular, the present invention relates to an improved method that allows said nanodispersed systems in form of nanoparticles such as nanovesicles, nanoemulsions, or nanosuspensions to be obtained with a size lower than 1 pm.

[0005] Background art

[0006] A large number of industrial sectors use or produce products that are supplied as heterogeneous liquid formulations, in which more than one phase exists. The properties and the added value of these disperse systems largely depends on their physico-chemical characteristics: the nature of the various phases present, degree of dispersion of one phase in the other, composition of the system, stability of the system over time, among other parameters. The liposomes, emulsions and suspensions are important heterogeneous liquid formulations with a wide range of applications, e.g. additives and coatings, foodstuffs, drugs and cosmetics. In particular, liposomes are small spherical vesicles made up of a lipid bilayer that encloses an aqueous nucleus. Emulsions are systems made up of small drops of liquid dispersed in another liquid with which they are not miscible, and suspensions are systems formed by solid particles dispersed in a fluid.

[0007] Obtaining nanodispersed systems, such as nanovesicles, nanoemulsions, and nanosuspensions, would help increase the added value of end products in many industrial sectors, e.g. printing inks, cosmetics, powdered paints, drugs, coatings, etc. The obtaining of nanosuspensions of solid substances with high therapeutic activity but with low water solubility is of considerable interest to the pharmaceutical sector, since it would allow said substances to be administered intravenously, transdermally, intranasally or by inhalation. It would also involve greatly increased stability of such disperse systems over time. At present, all the methods of preparation of nanodispersed systems are complex and consume large amounts of energy, since they either require many stages or they must be carried out at high pressures, such as pressures exceeding 100 bar. For example, document WO 99 / 065469 discloses a method in which rapid expansion of a solution of the compound to be suspended is carried out in a compressed fluid and pressures necessary for solubilising significant amounts of the product to be suspended in the liquefied gas are usually in excess of 200 bar.

[0008] On the other hand, document WO 2006 / 079888 A1 discloses method of obtaining micro- and nanodispersed systems in which all the components are mixed in a pressurized container at about 100 bar using a liquid CO2, and then reducing the pressure of the solution giving rise to a fall in temperature increasing supersaturation, such that a compound is separated out in the form of a solid or liquid of micro-, submicro- or nanoscopic size. The size of the disperse phase of the systems obtained depends on the magnitude and speed of the temperature drop, i.e., the greater the temperature drop or the greater speed of temperature drop the smaller the size of the disperse phase will be.

[0009] However, said sudden drop of temperature sometimes causes a clogging of the depressurization valve which leads to decrease of the process yield, and even makes it impossible for the process to be carried out.

[0010] This clogging also makes difficult to keep the depressurization flow constant, thus affecting the batch time and the homogeneity of the particles. Furthermore, this temperature drop also implies a temperature decrease in the stirred reactor and in the piping system, which leads to condensation issues and icing around the equipment, valves and instrumentation. In addition, when preparing nanodispersed systems the temperature drop could lead to the precipitation of some dissolved excipients or actives components of the nanodispersed systems, increasing the batch to batch variability in the product composition. In addition, the high-pressure parts involved and the extreme temperature ranges increase the equipment costs, safety risks, and complexity of construction, among others.

[0011] Thus, there is still considerable interest in research into and development and application of efficient methods for obtaining nanodispersed systems. Detailed description of the invention

[0012] The inventors of the present invention have developed an improved method for obtaining nanodispersed systems that overcome the above-mentioned drawbacks. Thus, an object of the present invention is to provide a process in which a pressure of the pressurized container is much lower than known processes and the flow and piping during depressurization process is heated to avoid clogging of the valve, and that with which it is still possible to obtain nanodispersed systems with a particle size generally lower than 1 pm.

[0013] Definitions

[0014] As used herein, the term “compound C” refers to a substance or mixture of solid or liquid substances, chosen from a drug, for example, small chemical molecules or biologies such as peptide, protein, nucleic acid, excipients such as antioxidants, osmotic agents, preservatives, carbohydrates, waxes, water-soluble and / or swollen polymers, hydrophilic or hydrophobic materials, gelatine, oils, or solvents; explosive, biocide, colorant, pigment, cosmetic, polymer, catalyst, chemical product for agriculture or other substance partially or totally insoluble in fluid E, and liable to be dispersed in a phase that includes fluid E and fluid A. Compound C is soluble in the mixture of fluid A and fluid B to give the solution AB at pressure P and temperature T.

[0015] As used herein, “fluid A” refers to any polar or non-polar solvent or a mixture of both that is miscible with fluid B at pressure P and miscible with fluid E (for example, ethanol as fluid A and water as fluid E) at atmospheric pressure. Preferably, said fluid A can be chosen from the group that includes acetone, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), dichloromethane (DCM), methanol, ethanol, ethyl acetate, toluene or mixtures thereof. Preferably, said fluid A contains at least one additive. Optionally, said fluid A may contain a solid phase, with that solid phase being preferably in the form of particles, which particles would more preferably be in suspension in said fluid A.

[0016] As used herein, “compound D” refers to a compound that can be of the same type tan compound C, and usually is a hydrophilic compound, and thus soluble in water while compound C is lipophilic and soluble in organic solvents.

[0017] As use herein, “fluid E” refers to any polar or non-polar solvent or a mixture thereof which is miscible with fluid A. Preferably, said Fluid E also contains at least one additive, preferably excipients, surfactants, buffer, salts or a hydrophilic active (compound D) compound.

[0018] In a first aspect, the present invention discloses a method for obtaining nanodispersed systems comprising the steps of: a) mixing a compound C with a fluid A and obtaining a mixture in the form of a solution or dispersion; b) thermostatising the mixture obtained in step a) at a temperature of between -50eC and 200eC; c) adding a fluid B to the thermostatised mixture until a pressure P between 0 and 300 bar is attained obtaining the mixture AB, wherein molar fraction of fluid B is from 0.01 to 0.8; d) reducing the pressure of the mixture AB obtained in step c) to a pressure lower than or equal to 10 bar, by means of a valve, wherein said valve is heated during depressurization process to a temperature T in the range of 10eC to 50eC; and e) mixing fluid A with fluid E in which fluid A is miscible and compound C is partially or totally insoluble, controlling the flow rate and the temperature T of fluid A in the range of 10eC to 100eC.

[0019] As mentioned above, said compound C can be a substance or mixture of solid or liquid substances, chosen from a drug, for example, small chemical molecules or biologies such as peptide, protein, nucleic acid, excipients such as antioxidants, osmotic agents, preservatives, carbohydrates, waxes, water-soluble and / or swollen polymers, hydrophilic or hydrophobic materials, gelatine, oils, or solvents; explosive, biocide, colorant, pigment, cosmetic, polymer, catalyst, chemical product for agriculture or other substance partially or totally insoluble in fluid E, and liable to be dispersed in a phase that includes fluid E and fluid A. Compound C is totally or partially soluble in the mixture of fluid A and fluid B to give the solution AB at pressure P and temperature T. Preferably, in step b) the mixture is thermostatised at a temperature between 0eC and 100eC, more preferably between 10eC and 70eC, even more preferably between 10eC and 50eC.

[0020] Preferably, pressure P in step c) is less than 200 bar, more preferably less than 100 bar, even more preferably less than 75 bar, 50 bar, 25 bar, 20 bar, 15 bar, 10 bar, or 5 bar or any range therebetween.

[0021] Optionally, after step c) the process of the present invention further comprises the steps of: c1 ) mixing a compound D with fluid E and obtaining a mixture in the form of a solution or dispersion; and c2) thermostating the mixture obtained in step c1 ) at temperature between -20eC and 100eC;

[0022] As mentioned above, compound D refers to a compound that can be of the same type than compound C, and usually is a hydrophilic compound, and thus soluble in water while compound C is lipophilic and soluble in organic solvents.

[0023] Preferably, said fluid A refers to any polar or non-polar solvent, selected from the group comprising acetone, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), dichloromethane (DCM), methanol, ethanol, ethyl acetate, toluene or mixtures thereof. More preferably fluid A is ethanol or acetone or a mixture of polar and non-polar solvents, and even more preferably fluid A is ethanol.

[0024] Preferably, said fluid A further comprises one or more additives, such as polysorbates, quaternary ammonium surfactant, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), polyethilenglycols (PEGs), sterol derivatives and conjugates, lecithin, ascorbyl palmitate, tocopherol, or any other antioxidant or preservative, phospholipids, medium or large-chain triglycerides, fatty acids, silicate compounds, organosilane compounds, carbocyanine dyes, succinyl ester compounds, sterols, alkylpolyglucosides or any other ionic or non-ionic surfactant.

[0025] Preferably, said fluid B is a fluid selected from the group comprising CO2, ethane, propane, hydrochlorofluorocarbons (e.g. HCFC-2) or hydrofluorocarbons (e.g. HCF-134A), which are gases at atmospheric pressure and are miscible or partially miscible with fluid A at pressure P in the mixing reactor. More preferably, fluid B is CO2.

[0026] As mentioned above, “fluid E” refers to any polar or non-polar solvent or a mixture thereof which is miscible with fluid A. Preferably, said Fluid E also contains at least one additive, preferably excipients, surfactants, buffer, salts or a hydrophilic active (compound D) compound.

[0027] Preferably, molar fraction of fluid B in step c) is from 0.02 to 0.8, more preferably from 0.05 to 0.6, and even more preferably from 0.1 to 0.3.

[0028] Preferably, temperature control in step f) is done by heating means, more preferably by external heating means, such as heating gun, electrical jacket, coiled jacket with thermostatic fluid, or other heating means known to the skilled person.

[0029] The disperse phases of the systems thus obtained present a narrow volumetric distribution of sizes and a mean associated sphere diameter of less than 1 pm, preferably less than 500 nm, more preferably less than 200 nm.

[0030] The method for obtaining nanodispersed systems of the present invention may comprise a further step of tangential flow filtration (TFF), for example to purify suspensions from non-entrapped molecules and organic solvents or to change the media to specific buffers for different intended uses. In addition, concentration of the obtained nanodispersed systems can be increased eliminating a part of the media. TFF is a very versatile technique in which recirculation of the product under pressure avoids the clogging of the filters, allowing filtrations that, with another type of filter, would be non-viable. TFF technique consists of a pressurised recirculation current and a permeated liquid flow that passes through the membranes. As indicated above, with the choice of the appropriate membrane, the substance of interest may or may not be able to pass through the membrane. In both cases, the compound will undergo a process of purification / concentration to a greater or lesser extent.

[0031] In a second aspect, the present invention discloses a method for obtaining nanodispersed systems comprising the steps of: a) mixing a compound C with a fluid A and obtaining a mixture in the form of a solution or dispersion; b) thermostatising the mixture obtained in step a) at a temperature of between -50eC and 200eC; c) adding a fluid B to the thermostatised mixture until a pressure P between 0 and 300 bar is attained obtaining the mixture AB; wherein molar fraction of fluid B is from 0.01 to 0.8; d) reducing the pressure of the mixture AB obtained in step c) to a pressure lower than or equal to 10 bar, by means of a valve, wherein said valve is heated during depressurization process to a temperature T in the range of 10eC to 50eC; e) mixing fluid A with fluid E in which fluid A is miscible and compound C is partially or totally insoluble, controlling the flow rate and the temperature T of fluid A in the range of 10eC to 100eC; and f) purifying / concentrating the obtained nanodispersed system by tangential flow filtration (TFF).

[0032] Preferably, the membrane size in the TFF is between 30 Da and 500,000 Da. Different types of membranes are available for TFF. Preferably, the TFF membrane is made of modified Polyethersulfone (mPES). Also preferably, the transmembrane pressure in the TFF process is between 1 and 40 psi, preferably between 5 and 20 psi. In addition, the use of tangential flow filtration does not need to undergo chemical or physical processes (such as heating processes) for the purification thereof and that might reduce the quality thereof due to the degradation of the possible thermolabile substances that make it up.

[0033] Preferably, the method of the present invention comprises at least one diafiltration cycle. Preferably, the method of the present invention comprises at least one concentration step. Preferably, the method comprises at least one diafiltration cycle and at least one concentration step. Preferably, the concentration factor in said at least one concentration step is from 1 .5 to 25 and at least 4 diafiltration cycles.

[0034] Preferably, molar fraction of fluid B in step c) is from 0.02 to 0.8, more preferably from 0.05 to 0.6, and even more preferably from 0.1 to 0.3. One advantage of the method of the present invention is the utilization of pressure values lower than those of the preceding techniques. Another advantage of the method is that the equipment design requirements are lower in terms of design pressure, improving the safety of the process and reducing the equipment costs.

[0035] Other advantages are reduction of material (less fluid material), lower environmental impact, less amount of CO2is needed per batch, volume of the high-pressure reactor used to mix compound C with fluid A is not limited to a molar fraction of the fluid since an expanded solution is not needed. Thus, the same reactor size can produce different volumes of product with the same process parameters. In addition, the process temperature is easier to control, and so the process is more robust in terms of reproducibility. The batch time is also more controlled and reproducible because clogging is avoided, and it is easier to keep the flow rates constant.

[0036] Figure 1 shows an installation for carrying out the method of the present invention.

[0037] Figure 2 shows overlay of sample 1 (lines), sample 2 (separated lines), sample 3 (line and dot), and sample 4 (line and two dots) size distributions by intensity, measured through DLS at day 1 .

[0038] Figure 3 shows overlay of sample 1 (lines), sample 2 (separated lines), sample 3 (line and dot), sample 4 (line and two dots), and sample 5 (dots) size distributions by intensity, measured through DLS at day 5 (samples 1 , 2 and 5) and 6 (samples 3 and 4).

[0039] Figure 4 shows an image of the macroscopical appearance of ChokCTAB nanovesicles (molar ratio 1 :1 ) prepared at different XCO2 (molar ratio of CO2inside the vessel, which is the quantity in mol of CO2divided by the total quantity in mol) by the method of invention, taken 16 days after production.

[0040] Figure 5 shows representative images of the microscopical appearance of ChokCTAB nanovesicles (molar ratio 1 :1 ) prepared at different XCO2 by the method of invention, taken 7 days after production. Figure 6 shows images of the macroscopical appearance of DPPC:cholesterol:chol- PEG400-RGD prepared at different XCO2 by the method of invention, taken 7 days after production.

[0041] Figure 7 shows representative images of the microscopical appearance of DPPC:cholesterol:chol-PEG400-RGD nanovesicles prepared at different XCO2 by the method of invention, taken 7 days after production.

[0042] Figure 8A shows the macroscopical appearance of ChokCPC nanovesicles (molar ratio 1 :1 ) containing cholecalciferol. DELOS (#DELOS), diafiltrated (#DIAF) and concentrated (#CONC) samples 1 month after production, and 8B shows representative cryo-TEM images of #DELOS sample 16 days after production.

[0043] Figure 9 shows the macroscopical appearance of the liposomal nanovesicular systems composed of DPPC, cholesterol, chol-PEG400-RGD at a molar ratio 10:6.5:0.5 with 0.04 mg / mL of MKC. TFF samples presented a more opalescent appearance as expected because of the more concentration of membrane components after the TFF concentration step.

[0044] Figure 10 shows: A) Stability over time in terms of particle size (S) and size PDI (PDI) over time (t) up to 28 days (d) of ChokMKC nanovesicles (6 mg / mL) containing 25 % of image of ChokMKC nanovesicles (6 mg / mL) containing 25 % of CBD. In black arrows, nanoemulsions are highlighted. C) Stability over time in terms of S and PDI over time up to 28 days of ChokMKC nanovesicles (2 mg / mL) containing 15 % of CBD. D) Representative cryo-TEM image of ChokMKC nanovesicles (2 mg / mL) containing 15 % of CBD. In white arrows, nanoemulsions are highlighted.

[0045] Figure 11 shows: A) Stability over time in terms of particle size (S) and size PDI (PDI) over time (t) up to 28 days (d) of ChokCPC nanovesicles (2 mg / mL) containing 25 % of CBD. In bars is represented particle size, in point size PDI. B) Representative cryo- TEM image of ChokCPC nanovesicles (2 mg / mL) containing 25 % of CBD. In black arrows, nanoemulsions are highlighted. C) Stability over time in terms of S and PDI over time up to 28 days of ChokCPC nanovesicles (4 mg / mL) containing 15 % of CBD. D) Representative cryo-TEM image of ChokCPC nanovesicles (4 mg / mL) containing 15 % of CBD. In black arrows, nanoemulsions are highlighted. E) Stability over time in terms of S and PDI over time up to 28 days of ChokCPC nanovesicles (4 mg / mL) containing 5 % of CBD. D) Representative cryo-TEM image of ChokCPC nanovesicles (4 mg / mL) containing 5 % of CBD. In white arrows, nanoemulsions are highlighted.

[0046] Figure 12 shows a representative cryo-TEM image of DOPC:cholesterol:CPC liposomes at a molar ratio 10:2:2 with 0.87 mg / mL of Miglyol 812. In black arrows, nanoemulsions are highlighted.

[0047] Figure 13 shows representative images of the microscopical appearance of ChokMKC nanovesicles prepared at XCO2= 0.2 by the method of the invention.

[0048] Figure 14 shows representative images of the microscopical appearance of ChokCPC nanovesicles prepared at XCO2= 0.37 and concentrated 10-fold by the method of the invention.

[0049] Preferred embodiment of the present invention

[0050] A detailed description is provided below of a preferred embodiment of the method of the invention implemented on the equipment shown in schematic form in Figure 1 .

[0051] This equipment includes a tank 1 that contains Fluid A with compound C, tank 2 that contains the fluid B, connected to a pump 3 that supplies said fluid until a given at high pressure P. The addition of fluid B to the mixing reactor vessel 5 containing mixture of fluid A with compound C can be carried out at the top part through the valve 4. Valve 7 controls the addition to the mixing reactor 5 of an inert gas that is in a tank 6 to the mixing reactor 5. The mixture AB at pressure P is thermostatised and depressurized through valve 9 and the temperature during depressurization process is maintained by heating device 8. The mixture AB passing through valve 9 is mixed into fluid E that can contain compound D in tank 10, where the nanodispersed systems are obtained. EXAMPLE 1 : OBTAINING A NANOSUSPENSION OF CHOLESTEROL:CTAB NANOVESICLES IN WATER BY THE METHOD OF THE INVENTION.

[0052] Materials and methods

[0053] Materials

[0054] Cholesterol (Choi) was purchased from Alco (Panreac) and Cetyltrimethylammonium bromide (CTAB) was purchased from Sigma-Aldrich. Ethanol (EtOH) was purchased from Scharlab. Carbon dioxide and nitrogen were supplied by Carburos Metalicos SA. The water used was pretreated with a MilliQ Advantage A10 water purification system (Millipore).

[0055] Equipment Configuration

[0056] As shown in Figure 1 , the configuration consists of a high-pressure vessel with an external fluid heating jacket to maintain the temperature, and a temperature controller and a pressure indicator controller to monitor both temperature and pressure. A thermostatic syringe pump (model 260D, ISCO Inc., Lincoln, US) is employed to pump pressurized CO2inside the vessel through two valves until reaching working pressure. A variable speed stirrer inside the vessel ensures the homogeneity of the organic phase and CO2mixture. For depressurization, mixing is stopped and pressurized nitrogen is introduced into the vessel to push the pressurized solution. The pressure of nitrogen is adjusted by a Pressure Adjustment Valve and introduced from a pressurized reservoir to the vessel. Using a depressurization micrometric valve, the solution contained in the vessel is depressurized into an aqueous phase placed in a collector at atmospheric pressure. A temperature controller placed in the depressurization line towards the aqueous phase is employed to measure the fluid temperature after depressurization. The depressurization line was heated with a heating gun in some of the experiments during depressurization, to avoid a marked temperature decrease.

[0057] Preparation of nanovesicles

[0058] The procedure includes three steps. First, the organic solution containing the vesicleforming components (e.g., Choi and CTAB) solubilized in ethanol are loaded at atmospheric pressure in the vessel; then, liquid compressed CO2is injected into the vessel to form a CO2-expanded solution with all the components dissolved, at a CO2molar fraction XCO2= 0.16 or 0.7, working temperature Tw = 40eC, and working pressure Pw = 25 or 85 bar; Finally, the CO2-expanded solution is depressurized into an aqueous solution. Nitrogen (N2) at a working pressure of Pw = 100 bar is injected into the vessel to eject the CO2-expanded solution, keeping a constant pressure inside the vessel during depressurization. The solution temperature after depressurization (Td) is measured through a thermometer placed in the depressurization line before the aqueous phase. At the same time, in some cases, this very same depressurization line between the depressurization valve and the aqueous phase container is warmed with a heat gun to avoid any undesired temperature decrease during depressurization. Depending on the experiment conditions, homogeneous and stable colloidal dispersions of vesicles in water with EtOH 10 % (v / v) were obtained. Vesicles were stored at room temperature for further characterization.

[0059] Characterization of nanovesicles

[0060] The size and colloidal stability features of all produced vesicles, including mean particle size, particle size distribution (or polydispersity index, Pdl), and apparent - potential (calculated with the Helmholtz-Smoluchowski approximation), were analyzed using a dynamic light scattering (DLS) and electrophoretic light scattering (ELS) analyzer combined with non-invasive backscatter technology (NIBS) (Malvern Zetasizer Ultra, Malvern Instruments, U.K). All reported values correspond to the average result of three consecutive measurements performed at 25 °C on the same sample using the Zetasizer Software. Size data, which corresponds to the average of the three measurements, was based on intensity size-distribution.

[0061] Results

[0062] Vesicles comprising Choi and CTAB were prepared using the the method of invention. Aiming to evaluate the effect of the temperature in the depressurization line on the quality of the produced vesicles, different production conditions were evaluated:

[0063] 1. High XCO2without heating the depressurization line while depressurizing: Significant temperature (Td) decrease takes place

[0064] 2. High XCO2heating the depressurization line while depressurizing: Td is controlled and not decreased

[0065] 3. Low XCO2without heating the depressurization line while depressurizing: Td is controlled and not decreased

[0066] The main differences among the parameters of these three procedures and their five samples can be found in Table 1 .

[0067] Table 1 . Summary of the main operating parameters that differ among the five nanovesicle samples produced.

[0068] Table 2. Summary of results obtained from DLS and ELS characterization of all five samples. The first methodology was employed to obtain two batches (samples 1 and 2) with different membrane components concentrations (either 6 or 5 mg / mL). Both were depressurized at a high flow rate (30 g / min) and, since there was no heating on the exit line while depressurizing, they reached temperatures as low as 5eC during depressurization. The temperature decrease is caused by the rapid evaporation of the compressed CO2when it is decompressed to atmospheric pressure. Low temperatures decrease the solubility of the processed components, which causes their precipitation in the depressurization line, and causes. This precipitation clogged the depressurization line, thus practically stopping the depressurization process. Consequently, the size distribution was heterogeneous (Table 2) and stability of the nanovesicles obtained was compromised. In addition, the total production time was higher than for the other conditions evaluated.

[0069] The second methodology was also employed to obtain two batches (samples 3 and 4) produced at different depressurization flow rates (either 30 or 10 g / min). Both had a membrane components concentration of 5 mg / mL. Thanks to the effect of heating the exit line while depressurizing the temperature did not decrease below 18eC for any of the two batches, avoiding the temperature-induced precipitation of the processed components. This allowed for a fluid depressurization process without block formation or delay in the production time. Although the total production time is shorter when depressurizing at 30 g / min than at 10 g / min, the size distribution and stability of the nanovesicles obtained in the latter case were better than those obtained in the former one (see Table 2).

[0070] The differences among samples 1 , 2, 3, and 4 can be observed in Figure 2, where the size distributions of these four samples on day 1 after production have been overlayed. Samples 1 and 2, which were produced without heating the exit line during depressurization (procedure 1 ), present a broad size distribution. On the contrary, samples 3 and 4, which were produced by heating the exit line during depressurization (procedure 2), show some differences: sample 3, produced at a high flow rate, has a larger average size and a broader size distribution, while sample 4, produced at a low flow rate, shows a narrow size distribution with a smaller average size. These results suggest that both heating the exit line while depressurizing and setting a lower flow rate improve the quality of the vesicles obtained. The two methodologies described above employed a high XCO2, which can be useful in enhancing the solubility of certain components. However, it causes a marked temperature decrease that can promote the precipitation of the components while depressurizing if there is no temperature control implemented.

[0071] The differences among all five samples can be observed in Figure 3, where their size distributions from day 5 have been overlayed. Samples 1 and 2 (procedure 1 ), as well as sample 3 (procedure 2), show a double peak size distribution and a moderately high Pdl. On the contrary, sample 4 (procedure 2) keeps showing a narrow size distribution and a good Pdl. Finally, sample 5 (procedure 3) shows the narrowest size distribution and best Pdl. These results suggest that the fact of reducing the XCO2is beneficial for improving the quality of the produced vesicles. Moreover, with a low XCO2the temperature does not decrease as much as to require heating during the depressurization process.

[0072] These results confirm that controlling the depressurization temperature is a critical parameter for obtaining stable monodisperse systems.

[0073] EXAMPLE 2: OBTAINING A NANOSUSPENSION OF CHOLESTEROL:CTAB NANOVESICLES IN WATER BY THE METHOD OF THE INVENTION AT LOWER PRESSURE

[0074] Materials and methods

[0075] Materials

[0076] The materials used to produce the nanovesicles were as disclosed in Materials and Methods section of Example 1 .

[0077] Preparation of nanovesicles

[0078] The production of the formulations was performed as disclosed in Materials and Methods section of Example 1 . In the case of this Example 2, liquid compressed CO2is injected into the vessel to form a CO2-expanded solution with all the components dissolved, at a CO2molar fraction XCO2= 0 - 0.54, working temperature Tw = 45eC, and working pressure Pw = 0 to 100 bar. Characterization of nanovesicles

[0079] The characterization of the nanovesicles was performed as disclosed in Materials and Methods section of Example 1. In addition, cryogenic transmission electron microscopy (cryoTEM) was used to examine the morphology and structure of lipid- based vesicles since this technique allows the preservation of water presence in samples and, therefore, their vesicle structures.

[0080] Through a quick sample freezing using liquid ethane (C2H6), the water content can vitrify without ice crystals formation allowing the pass of the electron beam without interferences. Cryogenic temperatures (- 196eC) using liquid N2must be maintained during the overall process to avoid a phase transition of the vitrified water into crystalline ice and consequently sample damage.

[0081] Mainly, images were acquired in the Servei de Microscopia of the UAB (Barcelona) by using a JEOL JEM-2011 transmission electron microscope (JEOL LTD, Tokyo, Japan) operating at 200 kV under low-dose conditions. For sample vitrifying a controlled environment vitrification system (CEVS) Leica EMCPC (Leica Microsystems, Germany) was used, and 5 - 10 pL of sample were deposited in a copper grid coated with a lacey carbon film, eliminating the water excess blotting it with filter paper (Whatman® grade 1 ). After that, a Leica EM GP automatic plunge freezer (Leica, Wetzlar, Germany) was used to quickly plunge the grid into liquid C2H6 just above its freezing point (- 179eC) to induce immediately sample vitrifying. For sample transference and insertion into the microscope, a cryo-transfer system Gatan 626 (Gatan, Pleasanton, USA) with controlled temperature of - 196eC with liquid N2 was used. Finally, images were recorded on a CCD Gatan 895 USC 4000 camera (Gatan, Pleasanton, USA) and analyzed with the Digital Micrograph 1.8 software. No image processing was applied except for background subtraction.

[0082] Results

[0083] The systems of cholesterokCTAB (molar ratio 1 :1 ) in water / EtOH 10 % (v / v) at 3 mg / mL were produced by DELOS methodology (Merlo-Mas, J. et al. Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method. The Journal of Supercritical Fluids 173 (2021 ) 10520). Aiming to evaluate the effect of the CO2molar fraction (XCO2) and the pressure of the expanded solution (Pw) on the quality of the produced vesicles, we designed a screening of Pw conditions that can be found in Table 3.

[0084] The Samples 1 -6 listed in Table 3 were characterized in terms of particle size, size PDI by DLS and morphology of the nanostructures obtained by cryoTEM. The characterization is reported in Table 4 and in Figure 4 and Figure 5. All the samples presented an average particle size between 68 and 83 nm, and size PDI of 0.14 - 0.20, indicating the obtention of nanometric and homogeneous structures.

[0085] The characterization by cryoTEM showed the existence of small unilamellar nanovesicles in all the conditions, with a notable difference when using or not CO2. On the one hand, Samples 1 -5 (XCO2= 0.02 to 0.54) presented structures surrounded by aqueous environments either for the outside and the inside of the bilayer, and are isolated without touching each other. On the other hand, Sample 6 (XCO2= 0.00) presented nanovesicles that have been aggregated in clusters of material.

[0086] From these results, it can be concluded that DELOS methodology using CO2starting from Pw= 5 bar (XCO2= 0.02) allows the preparation of higher homogeneous nanovesicles than DELOS methodology without using CO2 (Pw = 0 bar, XCO2 = 0.00).

[0087] Table 3: Summary of the main parameters that differ among the six samples produced. The rest of process parameters were kept constant (molar ratio ChokCTAB = 1 :1 ; dispersant media = EtOH 10 % (v / v); depressurization time = 4 min; N2pressure = 100 bar). Table 4: Summary of results obtained from DLS, ELS, and cryoTEM characterization of all six samples.

[0088] EXAMPLE 3: OBTAINING A NANOSUSPENSION OF DPPC:CHOL IN WATER BY THE METHOD OF THE INVENTION

[0089] Materials and methods

[0090] Materials

[0091] 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, MW 734.04, purity 99 %) was obtained from CordenPharma (Plankstadt, Germany). Cholesterol was obtained from Fragon (Barcelona, Spain). Miristalkonium chloride (MKC, MW 368.05, purity 99.2 %) was acquired from US Biological Life Science (Salem, USA). The cholesterol- PEG400-RGD was obtained from Ambiopharm Inc (Beech Island, USA). Ethanol (EtOH) was purchased from Scharlab (Barcelona, Spain). Dimethyl sulfoxide (DMSO, ACS reagent, purity > 99.9 %) was obtained from Sigma-Aldrich (Madrid, Spain). The water was always pre-treated with a Milli-Q® Advantage A10 water purification system (Millipore Iberica, Madrid, Spain).

[0092] Preparation of nanovesicles

[0093] The production of the nanovesicles was performed as disclosed in Materials and Methods section of Example 1 .

[0094] In the case of this Example 3, liquid compressed CO2is injected into the vessel to form a CO2-expanded solution with all the components dissolved, at a CO2molar fraction XCO2= 0- 0.56, working temperature Tw = 45eC, and working pressure Pw = 0 to 85 bar. Characterization of the vesicles

[0095] The characterization of the formulations was performed as disclosed in Materials and Methods section of Example 1 , and also including microscopic analysis by cryoTEM as disclosed in Materials and Methods section of Example 2.

[0096] Results

[0097] The systems of DPPC:cholesterol:chol-PEG400-RGD (molar ratio 10:6.5:0.5) in water / EtOH 5 % (v / v) / DMSO 1 .25 % (v / v) at 1 .2 mg / mL with MKC at 0.04 mg / mL as an additive were produced by DELOS methodology. Aiming to evaluate the effect of the CO2molar fraction (XCO2) and the pressure of the expanded solution (Pw) on the quality of the produced vesicles, we designed a screening of Pw conditions that can be found in Table 5.

[0098] The Samples 1-9 listed in Table 5 were characterized in terms of particle size, size PDI by DLS and morphology of the nanostructures obtained by cryoTEM. The characterization is reported in Table 6 and in Figure 6 and Figure 7. The primary size of Sample 5 was not measured by DLS due to its high aggregation. The other samples presented an average particle size between 108.9 and 140 nm, and size PDI of 0.10 - 0.20, indicating the obtention of nanometric and homogeneous structures.

[0099] The characterization by cryoTEM showed the existence of small unilamellar nanovesicles in all the conditions, except in Sample 5, with a notable difference when using CO2over Pw = 5 bar. On the one hand, Samples 1-3 and 6-9 (XCO2= 0.08 to 0.56) presented structures surrounded by aqueous environments either for the outside and the inside of the bilayer, and are isolated without touching each other. On the other hand, Samples 4-5 (XCO2< 0.01 ) presented nanovesicles that have been aggregated in clusters of material, as can be seen in Figure 7.

[0100] From these results, it can be concluded that DELOS methodology using CO2starting from Pw= 5 bar (XCO2= 0.02) allows the preparation of higher homogeneous nanovesicles than DELOS methodology without using CO2(Pw = 0 bar). Table 5. Summary of the main parameters that differ among the five samples produced. The rest of process parameters were kept constant (DPPC:cholesterol:chol-PEG400- RGD molar ratio 10:6.5:0.5; dispersant media = EtOH 5 % / DMSO 1.25 % (v / v); depressurization time = 4 min; N2 pressure = 100 bar).

[0101] Table 6. Summary of results obtained from DLS, ELS, and cryoTEM characterization of all five samples.

[0102] EXAMPLE 4: OBTAINING A HIGHLY CONCENTRATED AND PURIFIED NANOSUSPENSIONS BY THE METHOD OF THE INVENTION

[0103] After demonstration of the efficient production of vesicular systems at different conditions by DELOS methodology, their post-processing was also evaluated. The post-processing of nanovesicles obtained through the DELOS methodology is necessary for several reasons: 1 ) to obtain purified vesicles with the encapsulated active compound, eliminating any free fraction that may remain; 2) to evaluate the encapsulation efficiency of the active compound in the formulation by comparing the diafiltered and non-diafiltered samples; 3) to change the medium in which the nanovesicles are found, if necessary, by extracting the organic solvent and / or replacing the medium with buffers or solutions containing sugars, thus adjusting the pH and osmolality to the desired specifications; and 4) to concentrate the samples, both of the active compound and the nanovesicles, to make the systems viable for their application, such as reaching a minimum concentration to observe therapeutic effects during in vivo administration, which is not possible to achieve using only the DELOS methodology due to the process limitations regarding the solubility of membrane components in the organic phase.

[0104] Two different nanosuspensions were taken as an example: i) Diafiltration followed by concentration of cholesterol and cetylpyridinium chloride nanovesicles containing cholecalciferol ii) Concentration followed by diafiltration of a liposomal nanovesicular system

[0105] Materials and methods

[0106] Materials

[0107] The materials used to produce the nanovesicles were as disclosed in Materials and Methods section of Example 1 .

[0108] Further, additional materials and reagents were used to produce the nanovesicles of Example 4: i) Cetylpyridinium chloride (CPC, purity > 98 %) and cholecalciferol (purity >98 % (HPLC)) were from Sigma-Aldrich, Merk (Darmstadt, Germany). ii) 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, MW 734.04, purity 99 %) was obtained from CordenPharma (Plankstadt, Germany). Miristalkonium chloride (MKC, MW 368.05, purity 99.2 %) was acquired from US Biological Life Science (Salem, USA). The cholesterol-PEG400-RGD was obtained from Ambiopharm Inc (Beech Island, USA). Dimethyl sulfoxide (DMSO, ACS reagent, purity > 99.9 %) was obtained from Sigma-Aldrich (Madrid, Spain). The water was always pretreated with a Milli-Q® Advantage A10 water purification system (Millipore Iberica, Madrid, Spain). D-(+)-glucose (glucose, MW 180.16, purity > 99.5 %), was supplied from Sigma-Aldrich (Saint Louis, USA).

[0109] To perform the HPLC analysis, the materials used were the following: i) HPLC analysis of the nanovesicles containing cholecalciferol was performed using Ultra HPLC supergradient, ACS methanol > 99.9 % purchased from PanReac AppliChem (Barcelona, Spain). Optima® LC / MS Grade 2-Propanol > 99.9 % was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Formic acid > 98 % was acquired from Merck (Darmstadt, Germany). The water used was pre-treated with a MilliQ® Advantage A10 water purification system (Millipore Iberica, Madrid, Spain). ii) Ethanol (EtOH; HPLC grade, ref. 34852-M), dimethyl sulfoxide (DMSO; ACS reagent; purity 99.9 %, ref. 276855) and trifluoroacetic acid (TFA; purity > 99 %, ref. 302031 ) were purchased from Sigma-Aldrich (Madrid, Spain). Methanol (MeOH; LC / MS Grade, ref. 9822) was obtained from J.T. Baker (New Jersey, USA). Formic acid (HCOOH; purity 98 - 100 %, ref. 100264) was acquired from Merck Millipore (Massachusetts, USA). Acetonitrile (ACN; LC / MS Grade, ref. A955-212) and glacial acetic acid (AcOH; ref. 10384970) were provided from Fischer Scientific (Sant Cugat del Valles, Spain). Carbon dioxide (CO2; purity, 99.9 %) and nitrogen (N2) was supplied from Carburos Metalicos (Cornelia de Llobregat, Spain). Ultrapure Type I water (H2O), was purified using Milli-Q® Advantage A10 water purification equipment (Millipore Iberica, Madrid, Spain). Water for injection (WFI; ref. SH31191) was purchased from HyClone Laboratories (Utah, USA).

[0110] Preparation of nanovesicles

[0111] The production of the nanovesicles was performed as disclosed in Materials and Methods section of Example 1 . i) Nanovesicles loaded with cholecalciferol were prepared with cholesterol and the quaternary ammonium surfactant cetylpyridinium chloride (CPC) at a molar ratio of 1 :1 and a theoretical concentration of 3 mg / mL with cholecalciferol at a theoretical concentration of 0.2 mg / ml (dispersant media H2O / EtOH 10 % (v / v)). ii) Liposomal nanovesicular system was prepared with DPPC:cholesterol:chol- PEG400-RGD at molar ratio of 10:6.5:0.5 with 0.04 mg / mL of MKC as an additive, and a theoretical concentration of 1 .24 mg / mL (dispersant media H2O / EtOH 5 % / DMSO 1 .25 % (v / v)).

[0112] Diafiltration and concentration of vesicular systems

[0113] The nanovesicular system obtained by DELOS methodology is submitted to a purification step based on tangential flow filtration (TFF) using a AKTA FLUX S System from Cytiva (Massachusetts, USA). Hollow fiber columns were purchased from Repligen (Massachusetts, USA). TFF system was set up following manufacturer’s recommendations. Hollow fiber columns were previously hydrated with water (2 mL / cm2). i) Nanovesicle formulation loaded with cholecalciferol was diafiltrated 5 cycles in water and concentrated 8-fold, using a C04-E300-05-N hollow fiber column, setting the feed flow rate at 60 mL / min and the transmembrane pressure at 5-10 psi during all the process. ii) Liposomal formulation was concentrated 7.5-fold and diafiltrated 6 cycles in glucose solution (5 % w / v), using D04-E300-10-N hollow fiber column, setting the feed flow rate at 159 mL / min and the transmembrane pressure at 5-10 psi during all the process.

[0114] Characterization of the nanovesicles

[0115] The characterization of the formulations was performed as disclosed Materials and Methods section in Example 1. Furthermore, determination by HPLC analysis was done to quantify their concentration.

[0116] I) Determination of cholesterol and cholecalciferol by HPLC analysis

[0117] The concentration of cholecalciferol and cholesterol from the nanovesicular systems was quantified by HPLC.

[0118] For both cholecalciferol and cholesterol standards, two separate stock standard solutions were separately prepared in methanol; and after, 5 different standard solutions of lower concentration were obtained by dilution of the previous stock solutions in methanol.

[0119] As for samples preparation, 1 mL of each sample was dissolved into a 10 mL volumetric flask with methanol. Then, the flask was introduced into an ultrasonic bath for 5 minutes. After, it was ensured that samples were transparent after their ultrasonication, and eventually, they were filtrated with a Hamilton syringe, needles and the 0.20 pm (PTFE) filters into HPLC vials of 1 .5 mL.

[0120] As for samples analysis, a volume of 20 pL of each sample was injected into the HPLC system. The analyses were carried out using an Agilent 1260 Infinity II chromatographic system (Santa Clara, CA, USA), connected to both a Diode Array Detector (DAD) and an Evaporative Light Scattering (ELS) Detector. The software OpenLab was used for instrument control and data analysis. Cholecalciferol was detected with the ELS Detector, and cholesterol was detected by the DAD detector at a 206 nm wavelength.

[0121] A Symmetry® C18 5 pm 4.6x150 mm (Waters) was used to separate sample components before detection. The column temperature was set to 25 °C. Two mobile phases, A: methanol-water (95:5, v:v) and B: isopropyl alcohol-formic acid (99.9:0.1 , v:v), were used in a gradient elution mode (see Table 7). Mobile phase B was vacuum filtered through a PVDF membrane (0.45 pm pore diameter. The total running time was of 15 minutes. Quantification was performed by integration of the peak area of the corresponding analyte and interpolation of the peak area in cholecalciferol and cholesterol calibration standard curve, respectively.

[0122] Table 7. Gradient table regarding the HPLC analytical method for the cholecalciferol and cholesterol analysis.

[0123] II) Determination of DPPC, cholesterol, chol-PEG400-RGD and MKC by HPLC analysis

[0124] Quantitative analysis of membrane nanovesicle components was performed by reversed-phase high-performance liquid chromatography-evaporative light scattering detector (RP-HPLC-ELSD) using an HPLC equipment 1260 Infinity II from Agilent Technologies (Santa Clara, USA), composed of a quaternary pump, an automatic injector, an ELSD, and a column oven where the column was placed. The ELSD is coupled to the HPLC equipment to allow component quantification and also determination of purity degree. All modules were controlled using the OpenLab CDS ChemStation software.

[0125] The sample preparation consisted on the solubilization of liposome components by diluting nanoliposomal formulations in pure MeOH. Sample:solvent ratio was precisely leveled up to 1 :5 for samples containing 1 .2 mg / mL lipid (#DELOS samples), and 1 :10 for others containing 9 mg / mL lipid (concentrate #TFF prototypes), by using volumetric flasks. Then, samples were sonicated 10 min to well solubilize the vesicle membrane components.

[0126] Results i) Diafiltration followed by concentration of cholesterol and cetylpyridinium chloride nanovesicles containing cholecalciferol

[0127] The nanovesicular system composed of cholesterol and the quaternary ammonium surfactant CPC at a molar ratio 1 :1 was used to integrate the lipophilic active molecule cholecalciferol by DELOS methodology (sample #DELOS). This formulation was further purified by diafiltration in water to remove the EtOH present in the dispersant media (sample #DIAF) and concentrated to increase the concentration of cholecalciferol (sample #CONC).

[0128] As shown in Figure 8A, all samples (#DELOS, #DIAF and #CONC) presented a macroscopic appearance of opalescent whitish stable dispersions. In Table 8 is represented the physicochemical characterization of the samples 1 and 6 months after production, all of them showing stable values of hydrodynamic diameter, size PDI and apparent -Pot after 6 months. The results indicated the obtention of small and homogeneous nanovesicles for the three samples, which confirmed that the postprocessing of the sample by TFF, either diafiltration or concentration, did not affect the physicochemical properties of the vesicular systems. The apparent -Pot was also similar in the three samples, above the 30 mV, pointing to the colloidal stability of the system. Table 8. Physicochemical characterization of ChokCPC nanovesicles containing cholecalciferol 1 month after production and 6 months after production.

[0129] With respect to the morphology of the nanovesicles obtained, #DELOS samples was characterized by cryo-TEM (Figure 8B). The sample presented the coexistence of unilamellar small vesicles (left image) and lipophilic emulsions as opaque spherical globules (right image). The emulsion droplet had the same size than nanovesicles, so it was not possible to differentiate them by DLS.

[0130] To corroborate that with the removal of EtOH by diafiltration the composition of the nanovesicles and cholecalciferol remained stable, and that they were further concentrate by TFF, HPLC analysis was performed to determine the concentration of cholecalciferol as the active loaded molecule and cholesterol as a representative component of the vesicles (Table 9).

[0131] After diafiltration process, HPLC analysis showed that the sample’s concentration of cholecalciferol and cholesterol remained stable, from 0.15 (#DELOS) to 0.16 mg / mL (#DIAF) for cholecalciferol and from 1.4 (#DELOS) to 1.5 mg / mL (#DIAF) for cholesterol. These results pointed to the correct self-assembly of the vesicles, since the concentration of cholesterol as control of the membrane components was maintained, and the loading of the cholecalciferol inside the nanovesicles for the same reason, it was not removed as a free fraction of the sample.

[0132] The following concentration was also confirmed by HPLC analysis of both components. In the case of cholecalciferol, the concentration factor comparing the #DELOS and #CONC samples was of 6.6-fold. In the case of cholesterol, the concentration factor was the same, 6.6-fold. Together, the concentration values obtained by HPLC indicated the concentration of the vesicular system containing cholecalciferol.

[0133] Table 9. HPLC characterization of cholecalciferol and cholesterol concentration of ChokCPC nanovesicles containing cholecalciferol. ii) Concentration followed by diafiltration of a liposomal nanovesicular system

[0134] The liposomal nanovesicular system composed of DPPC, cholesterol, chol-PEG400- RGD at a molar ratio 10:6.5:0.5 with 0.04 mg / mL of MKC was produced by duplicate using DELOS methodology, samples #DELOS 1 , and DELOS 2. The samples were further concentrated to increase the concentration of its membrane components (DPPC, cholesterol, chol-PEG400-RGD) and purified by diafiltration in glucose 5 % w / w in water to remove the EtOH and DMSO present in the dispersant media, obtaining samples #TFF 1 and #TFF 2, respectively.

[0135] As shown in Figure 9, all samples (#DELOS, and #TFF) presented a macroscopic appearance of opalescent whitish stable dispersions. In Table 10 is represented the physicochemical characterization of the samples, all of them showing stable values of hydrodynamic diameter, size PDI and apparent -Pot. The results indicated the obtention of small and homogeneous nanovesicles for the four samples, which confirmed that the post-processing of the sample by TFF did not affect the physicochemical properties of the vesicular systems. The apparent -Pot was also similar in the four samples, above the 30 mV, pointing to the colloidal stability of the system. Table 10. Physicochemical characterization of liposomal nanovesicular system from DELOS and after TFF.

[0136] To corroborate that liposomal nanovesicular system can be concentrated by TFF, HPLC analysis was performed to determine the concentration of DPPC, cholesterol, chol-PEG400-RGD, and MKC (Table 11 ).

[0137] After concentration and diafiltration process, HPLC analysis showed that the sample’s concentrations of DPPC, cholesterol, chol-PEG400-RGD, and MKC of #TFF samples increased by a factor between 7.20 ± 0.04-fold to 8.1 ± 0.5-fold, respect to #DELOS samples, depending on the component.

[0138] Table 11. HPLC quantification of DPPC, cholesterol, chol-PEG400-RGD, and MKC of liposomal nanovesicular system for #DELOS and #TFF samples.

[0139] The results showed in Table 11 confirmed the concentration increase of liposomal nanovesicular system by TFF. EXAMPLE 5: OBTAINING A NANOEMULSION BY THE METHOD OF THE INVENTION

[0140] Besides vesicular systems, DELOS methodology can be also used to obtain other nanostructures, in particular, nanoemulsions. Emulsion are stabilized dispersions of oily and aqueous phases. They present a stabilized core phase surrounded by the other phase. In the present example there are presented oily nanoemulsions stabilized in aqueous formulations.

[0141] Two different nanosuspensions were taken as an example: i) Nanoemulsion systems composed of cholesterol and quaternary ammonium surfactants with cannabidiol ii) Liposomal nanovesicular systems with medium chain triglycerides

[0142] Materials and methods

[0143] Materials

[0144] The materials used to produce the formulations were as disclosed in Materials and Methods section in Example 1 . Further, additional materials and reagents were used to produce the nanovesicles of Example 5. i) Sodium chloride (NaCI, purity >99.5 %) was acquired from Thermo Fisher Scientific (Waltham, MA, USA), sodium hydrogen phosphate (Na2HPO4, purity >99.0 %) and sodium phosphate monobasic dihydrate (NaH2PO4-2H2O, purity >99.0 %) were acquired from Merck KGaA (Darmstadt, Germany). Cannabidiol was purchased from CBD Cure (Barcelona, Spain). ii) 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC, purity 100 %) was obtained from CordenPharma (Plankstadt, Germany). Miglyol 812 (medium chain triglycerides C8- C10) was from Acofarma (Terrassa, Spain).

[0145] Preparation of nanosupension systems

[0146] The production of the formulations was performed as disclosed in Materials and Methods section in Example 1 . i) Formulations containing cannabidiol were prepared with cholesterol and a quaternary ammonium surfactant at a molar ratio 1 :1 (dispersant media PBS 100 mM pH 7.4 / EtOH 10 % (v / v)). MKC and CPC were the surfactants tested to produce the formulations. Different concentration of membrane components and cannabidiol were used as described in Table 12.

[0147] Table 12. Composition of ChokMKC and ChokCPC formulations containing cannabidiol (CBD). ii) Liposomal formulation was prepared with DOPC:cholesterol:CPC at molar ratio of 10:2:2 with 0.87 mg / mL of Miglyol 812 as an additive, and a theoretical concentration of 5 mg / mL (dispersant media H2O / EtOH 10 % (v / v)).

[0148] Characterization of the nanoemulsions

[0149] The characterization of the formulations was performed as disclosed in Materials and Methods section in Example 1 .

[0150] Results i) Nanoemulsion systems composed of cholesterol and quaternary ammonium surfactants with cannabidiol. The systems of ChokMKC and ChokCPC (molar ratio 1 :1 ) in PBS 100 mM pH 7.4 with 10 % of EtOH (v / v) with CBD at different concentrations were produced by DELOS methodology.

[0151] The different formulations listed in Table 12 were characterized in terms of particle size, size PDI by DLS and morphology of the nanostructures obtained by cryo-TEM. The characterization is reported in Figure 10 for the ChokMKC systems and in Figurel 1 for the ChokCPC systems. All the samples presented particle size between 100 and 275 nm, and size PDI of 0.5 - 0.2, indicating the obtention of nanometric and homogeneous structures. The characterization by cryo-TEM showed the coexistence of two types of nanostructures. On the one hand, there were nanovesicles, which are structures surrounded by aqueous environments either for the outside and the inside of the bilayer, which is reflected in the cryo-TEM images as a thin black circle line. The aqueous lumen inside the vesicles shows the same color than the outside environment since they are both aqueous phases. On the other hand, emulsions are dispersions, in this particular case, of an oily phase stabilized in the aqueous environment, which is reflected in cryo-TEM images as black solid dots or spheres. Thus, DELOS methodology allows the preparation of different types of nanostructures depending on the components used to prepare them. ii) Liposomal nanovesicular system with medium chain triglycerides

[0152] The liposomal nanovesicular system composed of DOPC, cholesterol, CPC at a molar ratio 10:2:2 with 0.87 mg / mL of Miglyol 812 was successfully produced by DELOS methodology. The physicochemical characteristics of the formulations were evaluated, obtaining a hydrodynamic diameter of 131 ± 1 nm, a size PDI of 0.46 ± 0.02 and an apparent -Pot of 84 ± 4 mV. Thus, the characterization indicated the obtention of a homogeneous sample.

[0153] Furthermore, the liposomal formulation was also evaluated by cryo-TEM, showing the presence of both, vesicular structures and nanoemulsions. As in the last example, both types of structures can be differentiated by cryo-TEM due to its different structure, where nanoemulsions are seen as black spheres. In this particular case, the emulsions are seen isolated as showed in Figure 12, but also attached to the vesicles forming a combined structure.

[0154] From these results, it can be deduced that different types of nanostructures can be formed using DELOS methodology. EXAMPLE 6: OBTAINING A NANOSUSPENSION OF CHOLESTEROL:MKC NANOVESICLES IN PBS BY THE METHOD OF THE INVENTION AT DIFERENT PRESSURES AND XCO2

[0155] Materials and methods

[0156] Materials

[0157] Cholesterol (Choi) was purchased from Alco (Panreac), miristalkonium chloride (MKC) was purchased from USBiologicals, and Phospate-Buffered Saline (PBS) was purch from Sigma-Aldrich. Ethanol (EtOH) was purchased from Scharlab. Carbon dioxide and nitrogen were supplied by Carburos Metalicos SA. The water used was pretreated with a MilliQ Advantage A10 water purification system (Millipore).

[0158] Preparation of nanovesicles

[0159] The production of the formulations was performed as disclosed in Materials and Methods section of Example 1 . In the case of this Example 6, liquid compressed CO2is injected into the vessel to form a CO2- solution with all the components dissolved, at a CO2molar fraction XCO2= 0.05 or 0.50, working temperature Tw = 35eC, and working pressure Pw = 10 to 85 bar.

[0160] Characterization of nanovesicles

[0161] The characterization of the nanovesicles was performed as disclosed in Materials and Methods section of Example 1 and Example 2.

[0162] Results

[0163] The systems of ChokMKC (molar ratio 1 :1 ) in PBS 150 mM pH 7.4 with EtOH 10 % (v / v) at 1.2 and 3 mg / mL were produced by DELOS methodology. Aiming to evaluate the effect of the CO2molar fraction (XCO2) and the pressure of the solution (Pw) on the quality of the produced vesicles, we designed a screening of Pw conditions that can be found in Table 13. Table 13. Summary of the main parameters that differ among the nine samples produced. The rest of process parameters were kept constant (molar ratio ChokMKC = 1 :1 ; dispersant media = PBS 150 mM with EtOH 10 % (v / v); N2 pressure = 100 bar).

[0164] The Samples 1 -9 listed in Table 13 were characterized in terms of particle size, size PDI and Z-Potential, 12 days after production. CryoTEM analysis was also performed for Sample 8 and Sample 9. The size characterization results are reported in Table 14 and representative CryoTEM images in Figure 13. All the samples presented an average particle size between 83 and 101 nm, and size PDI of 0.05 - 0.16, indicating the obtention of nanometric homogeneous structures. The characterization of the morphology by cryoTEM of Sample 8 and Sample 9 (Figure 13) showed the existence of small unilamellar nanovesicles. Table 14: Summary of Particle size, size Pdl and Z potential results obtained from DLS and ELS of ChokMKC nanovesicles samples obtained by the method of the invention. From these results, it can be concluded that homogeneous ChokMKC nanovesicles were successfully obtained with DELOS methodology, in a controlled and robust manner, using working pressures from 9 to 86 bar and XCO2 from 0.05 to 0.5. Hydrodynamic diameters ranging from 83 to 101 nm and low size dispersity (PDI between 0.05 and 0.16) were obtained. Besides, CryoTEM imaging of Samples 8 and 9, obtained at XCO2» 0.2 and 30 bar, confirmed the presence of small, unilamellar and well-formed nanovesicles, with no signs of aggregated structures.

[0165] EXAMPLE 7: OBTAINING A NANOSUSPENSION OF CHOLESTEROL:CPC NANOVESICLES IN WATER BY THE METHOD OF THE INVENTION

[0166] Materials and methods

[0167] Materials

[0168] Cholesterol (Choi) was purchased from Alco (Panreac), cetylpyridinium chloride (CPC) was purchased from Sigma-Aldrich. Ethanol (EtOH) was purchased from Scharlab. Carbon dioxide and nitrogen were supplied by Carburos Metalicos SA. The water used was pretreated with a MilliQ Advantage A10 water purification system (Millipore).

[0169] Preparation of nanovesicles

[0170] The production of the formulations was performed as disclosed in Materials and Methods section of Example 1 . In the case of this Example 7, liquid compressed CO2is injected into the vessel to form a CO2- solution with all the components dissolved, at a CO2molar fraction XCO2= 0.18 to 0.37, and working temperature Tw = 45eC.

[0171] Characterization of nanovesicles

[0172] The characterization of the nanovesicles was performed as disclosed in Materials and Methods section of Example 1 and Example 2.

[0173] Results

[0174] The systems of ChokCPC (molar ratio 1 :1 ) in water with EtOH 10 % (v / v) at 3 mg / mL were prepared using the method of invention. Aiming to evaluate the effect of the CO2molar fraction (XCO2) and the pressure of the solution (Pw) on the quality of the produced vesicles, we designed a screening of Pw conditions that can be found in Table 15.

[0175] Table 15. Summary of the main parameters that differ among the three samples produced. The rest of process parameters were kept constant (molar ratio ChokCPC = 1 :1 ; dispersant media = water with EtOH 10 % (v / v); Flow rate 10 g / min, Tw = 45eC, N2 pressure = 100 bar).

[0176] The Samples 1 -3 listed in Table 15 were characterized in terms of particle size, size PDI by DLS. The characterization is reported in Table 16 and in Figure 14. All samples presented an average particle size between 54 and 65 nm, and size PDI of 0.17 - 0.22, indicating the obtention of nanometric and homogeneous structures.

[0177] Table 16: Summary of results obtained from DLS and ELS of the samples.

[0178] Sample 1 was characterized by CryoTEM after a 10-fold concentration and diafiltration in pure water. Figure 14 showed the existence of small unilamellar nanovesicles.

[0179] From these results, it can be concluded that nanovesicles composed of cholesterol and cetylpyridinium chloride (CPC) at 1 :1 molar ratio were successfully produced using the DELOS methodology in aqueous medium with 10 % EtOH (v / v), achieving hydrodynamic diameters between 53 and 65 nm and size dispersity indices (PDI) from 0.17 to 0.22, indicating nanoscale and relatively monodisperse systems. All tested conditions (XCO2from 0.18 to 0.37) led to stable nanostructures with high positive zeta potential values (83-97 mV), ensuring good colloidal stability. The cryoTEM analysis of Sample 1 , produced at XCO2= 0.37 and concentrated 10-fold, confirmed the formation of well- defined, small unilamellar vesicles, with no visible aggregates after purification.

Claims

1. CLAIMS1 . Method for obtaining nanodispersed systems comprising the steps of: a) mixing a compound C with a fluid A and obtaining a mixture in the form of a solution or dispersion; b) thermostatising the mixture obtained in step a) at a temperature of between -50eC and 200eC; c) adding a fluid B to the thermostatised mixture until a pressure P between 0 and 300 bar is attained obtaining the mixture AB; wherein molar fraction of fluid B is from 0.01 to 0.

8. d) reducing the pressure of the mixture AB obtained in step c) to a pressure lower than or equal to 10 bar, by means of a valve, wherein said valve is heated during depressurization process to a temperature T in the range of 10eC to 50eC; and e) mixing fluid A with fluid E in which fluid A is miscible and compound C is partially or totally insoluble, controlling the flow rate and the temperature T of fluid A in the range of 10eC to 100eC.

2. Method, according to claim 1 , wherein said compound C can be a substance or mixture of solid or liquid substances, chosen from the list comprising a drug, for example, small chemical molecules or biologies such as peptide, protein, nucleic acid, excipients such as antioxidants, osmotic agents, preservatives, carbohydrates, waxes, water-soluble and / or swollen polymers, hydrophilic or hydrophobic materials, gelatine, oils, or solvents; explosive, biocide, colorant, pigment, cosmetic, polymer, catalyst, chemical product for agriculture or other substance partially or totally insoluble in fluid E, and liable to be dispersed in a phase that includes fluid E and fluid A. Compound C is soluble in the mixture of fluid A and fluid B to give the solution AB at pressure P and temperature T.

3. Method, according to claim 1 or 2, wherein in step b) the mixture is thermostatised at a temperature between 0eC and 100eC, more preferably between 10eC and 70eC, even more preferably between 10eC and 50eC.

4. Method, according to any of the preceding claims, wherein pressure P in step c) is less than 200 bar, more preferably less than 100 bar, even more preferably less than75 bar, 50 bar, 25 bar, 20 bar, 15 bar, 10 bar, or 5 bar.

5. Method, according to any of the preceding claims, wherein after step c) the method further comprises the steps of: c1 ) mixing a compound D with fluid E and obtaining a mixture in the form of a solution or dispersion; c2) thermostating the mixture obtained in step c1) at temperature between -20eC and 100eC; and6. Method, according to claim 5, wherein, compound D is of the same type than compound C, and is a hydrophilic compound, and soluble in water.

7. Method, according to any of the preceding claims, wherein said fluid A is acetone, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), dichloromethane (DCM), methanol, ethanol, ethyl acetate, toluene or mixtures thereof.

8. Method, according to claim 7, wherein fluid A is ethanol.

9. Method, according to any of the preceding claims, wherein fluid A further comprises one or more additives, selected from the group comprising such as polysorbates, quaternary ammonium surfactant, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), polyethilenglycols (PEGs), sterol derivatives and conjugates, lecithin, ascorbil palmitate, tocopherol, or any other antioxidant or preservative, phospholipids, medium or large-chain triglycerides, fatty acids, silicate compounds, organosilane compounds carbocyanine dyes, succinyl ester compounds, sterols, alkylpolyglucosides or any other ionic or non-ionic surfactant.

10. Method, according to any of the preceding claims, wherein said fluid B is fluid selected from the group comprising CO2, ethane, propane, hydrochlorofluorocarbons (e.g. HCFC-2) or hydrofluorocarbons (e.g. HCF-134A), which are gases at atmospheric pressure and are miscible or partially miscible with fluid A at pressure P in the mixing reactor.11 . Method, according to any of the preceding claims, wherein said fluid E is any polaror non-polar solvent or a mixture thereof which is miscible with fluid A.

12. Method, according to claim 11 , wherein said Fluid E also contains at least one additive, preferably excipients, surfactants, buffer, salts or an hydrophilic active (compound D) compound.

13. Method, according to any of the preceding claims, wherein molar fraction of fluid B in step c) is from 0.02 to 0.8, preferably from 0.05 to 0.6.

14. Method, according to any of the preceding claims, wherein said valve is heated by external heating means.

15. Method, according to any of the preceding claims, wherein the disperse phases of the nanodispersed systems obtained present a narrow volumetric distribution of sizes and a mean associated sphere diameter of less than 1 pm, preferably less than 500 nm, more preferably less than 200 nm.

16. Method, according to any of the preceding claims, wherein said method comprise a further step of tangential flow filtration (TFF).

17. Method, according claim 16, wherein the membrane size of the filter in the TFF is between 30 Da and 500,000 Da.

18. Method, according to claim 16 and 17, wherein the transmembrane pressure in the TFF process is between 1 and 40 psi, preferably between 5 and 20 psi.

19. Method, according to claims 16-18, wherein the concentration factor in said at least one concentration step is from 1 .5 to 25.

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