WO2023230723A1 - Microemulsion or nanoemulsion formation using supercritical carbon dioxide - Google Patents

Abstract

Disclosed are methods of forming a liquid emulsion by dissolving supercritical carbon dioxide into an oil and water mixture and at least one stabilizer, under conditions above the critical point for carbon dioxide, followed by depressurization of a liquid phase to form the emulsion.

Description

MICROEMULSION OR NANOEMULSION FORMATION USING SUPERCRITICAL CARBON DIOXIDE

Field of the Invention

[0001] The present invention relates to methods of forming a microemulsion or a nanoemulsion using supercritical carbon dioxide.

Background

[0002] A liquid emulsion is a colloidal dispersion consisting of two immiscible liquids such as oil and water, or oil- and water-related liquids, where one of them is dispersed as small droplets in the other. Oil and water emulsions may be either a water-in-oil (W/O) emulsion or an oil-in-water (O/W) emulsion, depending on which liquid is the continuous phase.

[0003] In general, emulsions are thermodynamically unstable, thus surfactants or other stabilizers are needed to make them kinetically more stable (metastable). A number of emulsification technologies and methods are commercially used and many others have been proposed as alternatives to overcome the limitations of current commercial processes.

[0004] At present, several approaches have been developed for the preparation of micro and nano-sized emulsions, which can be classified as high-energy or low-energy methods. High-energy emulsification methods are based on the use of devices to create powerful disruptive forces for size reduction, such as ultrasonication and high-pressure homogenization (HPH). These methods are versatile in that many oils and surfactants can be subjected to emulsification and scalable, but they require a high energy input. For example, HPH applies pressures up to 200 MPa, and a considerable part of energy is dissipated as heat, which is both inefficient and could lead to degradation of heat-sensitive compounds. Moreover, even though emulsions with small droplet size (in the range of micro and nanometers) can be generated, several cycles are usually needed, especially to obtain narrow size distributions.

[0005] On the other hand, low-energy emulsification methods such as spontaneous emulsification and phase inversion temperature methods are based on the spontaneous formation of emulsions when the solution or environmental conditions are altered. They do not demand the use of homogenization equipment, but the need for surfactants and oils with specific physicochemical properties limits the application of these methods considerably.

[0006] There is a need in the art for alternative methods of producing emulsions, which may mitigate disadvantages of prior art processes.

Summary of the Invention

[0007] In one aspect, this disclosure relates to processes to produce water-in-oil (W/O) or oil-in-water (O/W) emulsions using supercritical supercritical carbon dioxide (SCCO2). In some embodiments, the resulting emulsions have droplet sizes in the range of micrometers or smaller, preferably less than 100 pm in diameter, more preferably less than 10 pm, and most preferably less than 1 pm. Nano-sized droplets in the emulsions may be less than 200 nm in diameter. [0008] These W/O or O/W emulsions can be used as delivery systems when loaded with different drugs or bioactive compounds or substances.

[0009] Therefore, in one aspect, disclosed is a process of producing an emulsion, comprising the steps of:

(a) loading a water phase, an oil phase, and at least one stabilizer into a pressure vessel;

(b) introducing CO2 into the pressure vessel and pressurizing the vessel contents at a temperature up to a pressure and temperature above the critical point of CO2;

(c) stirring and then forming the emulsion by depressurization of the liquid phase, while CO2 is released from the emulsion as gas and specific amounts of CO2 at different pressurization rates are introduced into the vessel during depressurization to maintain substantially the same pressure within the vessel; and

(d) collecting the emulsion.

Brief Description of the Drawings

[0010] Figure 1 shows pictures of emulsions formed as described in the Examples (Emulsions A10, Al 1 and A12) at day 0.

[0011] Figure 2 shows pictures of emulsions formed as described in the Examples (Emulsions A12, A13R, A14R and A15R) after 106, 91, 92 and 91 days of storage at 4°C, respectively.

Detailed Description [0012] Disclosed are methods of making an emulsion using supercritical carbon dioxide, and the emulsions themselves, which have long-term stability. Terms used herein have their common art-recognized meanings, unless specifically defined herein.

[0013] In general terms, the process disclosed comprises the steps of solubilizing supercritical CO2 (SCCO2) in an oil-water mixture under pressure, followed by release of gaseous CO2 from the mixture upon depressurization. This leads to the dispersion of the disperse phase into discrete droplets and their subsequent reorganization into monodisperse, microemulsion and nanoemulsion emulsified systems.

[0014] As used herein, a "microemulsion" refers to an isotropic and thermodynamically stable system made of water, oil, and at least one stabilizer, that has a dispersed domain diameter less than about 100 pm. A "nanoemulsion" has a dispersed domain diameter less than about 1 pm, preferably less than about 200 nm, and more preferably in the range of about 50 to 100 nm.

[0015] As used herein, an "oil" is any nonpolar chemical substance that is composed primarily of hydrocarbons and is hydrophobic and lipophilic. Preferably, the oil is a flowable liquid under ambient conditions (atmospheric pressure and 20° C). More preferably, the oil has a solidification point below about 0°, so that an emulsion may be refrigerated and stored.

[0016] A supercritical fluid is any substance which is at a temperature and pressure above its critical point, where distinct liquid and gas phases do not exist. The critical point for carbon dioxide is 31° C and 73.8 bar (7.4 MPa). Supercritical fluids may display gaseous- and liquid-like properties. For example, SCCO2 has the low viscosity of a gas but the higher density of a liquid. [0017] In one aspect, the process comprises a batch process, where water, oil, and at least one stabilizer are loaded into a high-pressure vessel. These components are preferably pre-heated to a desired temperature. Preferably, the components are added without requiring any previous pre-treatment. Then, carbon dioxide is pumped in to the vessel, preferably as a subcritical liquid, and preferably pre-heated to a desired temperature before entering the vessel. The desired temperature may be an elevated temperature above the critical point. The vessel with pre-heated contents is then pressurized up to the operating conditions above the critical point for carbon dioxide. After stirring, the emulsion is formed by depressurization of the liquid phase, while CO2 is released from the product as gas, so that a ready -to-use liquid emulsion is obtained.

[0018] In preferred embodiments, no organic solvent is used, and the resulting emulsion is thus solvent-free.

[0019] Depressurization of the liquid phase is performed while keeping the pressure inside the vessel substantially constant by simultaneously withdrawing a portion of the liquid and adding more CO2 into the vessel. This is preferred in order to obtain small-size droplets and narrower size distributions. To maintain constant pressure, specific amounts of CO2 at different pressurization rates are introduced into the vessel, while the emulsion is being released and collected at different rates from the bottom of the vessel.

[0020] In another aspect, the process comprises a semi-continuous process, where one of the two phases (water or oil phase) is first pumped into a pressure vessel. This stream comprises at least one stabilizer and any other desired compounds or substances which are soluble in that phase. The second phase is then introduced and put into contact with the first phase inside of the vessel. This second stream may comprise also comprise a stabilizer and any other desired compounds/substances which are soluble in that phase. In some embodiments, the oil phase and CO2 are pumped independently into the vessel. In other embodiments, CO2 and the oil phase are mixed before entering the vessel, due to the high solubility of SCCO2 in oil. The flow rate at which the phases are introduced into the vessel may be selected depending on the physicochemical characteristics of the bulk phase and the compounds/substances dissolved therein. The CO2 may be formed as SCCO2 prior to being pumped into the vessel, or introduced into the vessel as a liquid or gas, and then heated and pressurized to above its critical point. Finally, the SCCO2, water and oil in the vessel are stirred over a certain period of time and the emulsion is formed and collected upon depressurization of the liquid phase while keeping the pressure inside the vessel constant, as described for the batch process, for better fine tuning of droplet characteristics, in terms of size and size distribution.

[0021] In another aspect, the process comprises a continuous process, where the water phase (and compounds/substances soluble therein) and the mixture SCCO2 + oil (and compounds/substances soluble in that phase) are continuously pumped through two lines separately into an inline mixer before entering a pressure vessel, where it is stirred. The stirred bulk mixture coming from the vessel is depressurized, and the emulsion is collected simultaneously at a specific rate, which will be set based on the flow rate of the input streams to keep the pressure inside the system constant.

[0022] In some embodiments of any of the batch, semi-continuous or continuous processes, the operating conditions in the pressure vessel may be in the range above the critical point for carbon dioxide, and below 40 MPa and 70° C, respectively. Preferably, the conditions range in between about 10 to about 30 MPa, and about 40° to about 50° C. [0023] Embodiments disclosed herein may be suitably employed for a large variety of different oils, with different stabilizers, as well as for the production of both O/W and W/O systems. Therefore, the versatility of this invention offers the possibility of potential applications in numerous sectors, including but not limited to cosmetic, food, nutraceutical, pharmaceutical and chemical industries.

[0024] In some embodiments, the oil may comprise an animal or plant or vegetable oil obtained from different sources used in cosmetic, food, nutraceutical, pharmaceutical and chemical industries. Suitable oils may comprise oil derived from canola, sunflower, olive, or palm plants. In other examples, the oil may comprise an animal oil such as fish oil or chicken liver oil. The oil is preferably a flowable liquid at ambient temperatures.

[0025] In some embodiments, the emulsion is formed with at least one stabilizer, which may act as an emulsifier or a surfactant. In some embodiments, the at least one stabilizer may comprise a surfactant, such as an anionic, cationic, or a nonionic surfactant such as polysorbate (Tween® 20 or Tween® 80) type surfactants. In other examples, the stabilizer may comprise an emulsifier such as lecithin. As is known to those skilled in the art, an emulsifier is a surfactant which is known to stabilize emulsions.

[0026] In other embodiments, the stabilizer may comprise an agent which increases the viscosity of the aqueous phase, which may be added in addition to or in place of a surfactant stabilizer. An aqueous phase with increased viscosity will minimize the mobility of oil droplets thus minimizing coalescence of oil droplets. Aqueous viscosifiers may include natural products such as guar gums or xanthan gums, or synthetic polymeric viscosifiers. [0027] In preferred embodiments, all of the components are biocompatible, such that the emulsion may be used in a food, beverage, nutraceutical or pharmaceutical product, to be ingested or applied by a person.

[0028] Embodiments of the present invention may offer the possibility of different approaches to process design. The methods of the present invention can be carried out in batch, semi-continuous and continuous mode. These approaches to production methods can be relatively easily implemented and can be fine-tuned based on needs, production capacity and the facilities available. In some embodiments, the order of addition of the components forming the emulsion may be varied. The order of addition of the components and the initial location of the stabilizer (either in the water phase or oil phase) influences the droplet size, the stability of the emulsion and its further utilization.

[0029] In some embodiments, the pressurization rate to achieve supercritical conditions and/or to maintain semi-continuous or continuous processing conditions may be between about 5 MPa per minute to about 20 MPa per minute.

[0030] The stability of emulsions and the maximum amount of dispersed phase that can be incorporated depend mainly on the conditions of emulsion medium (ionic strength, pH, temperature, etc.), amount of water, concentration and characteristics of the surfactant stabilizer molecules (head group, tail group, geometry, steric phenomena, etc.) and the concentration and properties of the oil molecules (molar volume, geometry, polarity, chemical composition, etc.). In some embodiments, the dispersed phase comprises about 5% to about 40% by weight of the continuous phase.

[0031] Generally, the term "emulsion stability" refers to the ability of an emulsion to resist changes in its physicochemical properties over time. Emulsions may become unstable and phase separation can occur due to a variety of different physicochemical mechanisms, including flocculation, Ostwald ripening, creaming, coalescence and sedimentation. A large number of stabilizers have been studied and are being commercially used to minimize the phenomena causing emulsion instability. Moreover, a number of methods and technologies are applied in combination with stabilizers to produce emulsions with specific characteristics, primarily to obtain dispersed phase droplets having specific sizes, due to its great influence on the rheology and stability of emulsions. In general, monodisperse small-sized emulsion droplets are preferred, but large-scale emulsification techniques like stirring and homogenization are very energy intensive to achieve those emulsion properties.

[0032] Unlike high-energy methods conventionally used for commercial emulsification, embodiments of the present invention do not require as high an energy input and energy is efficiently utilized. The moderate critical point of carbon dioxide translates to lower energy requirements to work with SCCO2. In addition, SCCO2 has a low viscosity and transport properties similar to those of gases, so friction loss during its pumping throughout the process is minimal. SCCO2 presents a high solvation power and fast diffusion rates. Therefore, unlike conventional processes, most of the energy required for a SCCO2 process is used to pressurize CO2 gas to form SCCO2 and later heat dissipation is avoided upon depressurization to atomize the SCCCh-oil-water mixture. Moreover, the Joule-Thomson effect as a result of the sudden pressure change brings about a decrease in system temperature, which favors the stabilization of emulsion droplets. [0033] In preferred embodiments, and unlike many low-energy methods, the methods disclosed do not require any organic solvent. As a result, additional processing steps to remove the solvent are not required, and the resulting products are solvent-free.

[0034] The use of emulsions as carriers of drugs and other bioactive compounds in pharmaceutical, cosmetic and natural health product industries is well known. Unfortunately, many of these compounds are heat-sensitive, and the use of high temperatures can lead to their degradation or loss of bioactivity. The application of low temperatures makes SCCO2 a suitable technology for the processing of heat sensitive compounds. In addition, the absence of oxygen during the process protects these compounds from oxidation.

[0035] Physicochemical properties of SCCO2 (viscosity, diffusivity and density) can be tuned by moderate changes in pressure and temperature. These tunable properties modulate the solubility of the components forming part of the system (SCCCh-water-oil- surfactant equilibrium), which in turn, has a strong influence in droplet size and size distribution of the emulsion. The dissolution of SCCO2 in the liquid phase (water + oil) brings about a decrease in the interfacial tension between water and oil, promoting better mixing between both fluids, which will enhance the formation of smaller and more homogeneous drops upon depressurization. Therefore, some embodiments allow finetuning of the rheology and stability of the emulsions formed through moderate changes in processing conditions. Moreover, unlike high-energy methods, which generally require several cycles to obtain small droplet sizes and narrow size distribution, the present invention offers the possibility of obtaining droplets in the range of nanometers in a single step by simple tuning of operating conditions. [0036] Conventionally, the order of addition of the components and the initial location of the surfactant (either in the water phase or oil phase) influences the stability of the emulsion. Embodiments disclosed herein may provide versatility in terms of component addition. In some embodiments, the process can be carried out in batch mode where the order of addition has no influence over emulsion properties. Alternatively, a continuous or semi-continuous mode can be employed if the order of addition is important for the production of stable emulsions.

[0037] Supercritical fluid technology is well-known for extraction purposes; however, it has not been previously applied for the purpose of emulsion formation. Furthermore, this invention offers the possibility of integrating the methods into multipurpose plants. Therefore, a facility using SCCO2 processing (for example, SCCO2 extraction processes) could implement the methods of this invention making minor modifications to existing processing equipment.

Examples:

[0038] The following examples are intended to illustrate specific embodiments of the invention described herein, and not be limiting of the claimed invention in any way.

[0039] The following coded emulsions were obtained in batch mode. The oil-to-water ratio employed was 10% (w/w) and soy lecithin was used as a stabilizer emulsifier. Processing conditions are specified in terms of pressure, temperature and pressurization rate.

[0040] Emulsions were stored at 4°C and the particle size (Ps) and size distribution (in terms of poly dispersity index, Pdl) were determined over time. The emulsions produced from this invention were stable after 91-106 days, as may be seen in Figure 2, where the emulsions do not display any phase separation. The particle size of droplets was in many examples in the range of nanometer scale (about 200 nm or smaller). The processing conditions allow fine tuning of the size and size distribution of droplets formed. [0041] Emulsions were kept in capped cylindrical lab vials and an aliquot from the top and bottom of the vials were collected for each analysis.

Utilization of different surfactants:

Table 1: Ps (nm) and Pdl of emulsions produced from the addition of different surfactants at 30 MPa, 40°C, 12 MPa/min and using sunflower oil.

[0042] Application of different pressures:

Table 2: Ps (nm) and Pdl of emulsions produced using lecithin as surfactant and sunflower oil at 40°C, 12 MPa/min and two different pressures.

[0043] Application of different temperatures:

Table 3: Ps (nm) and Pdl of emulsions produced using lecithin and sunflower oil at 30 MPa, 12 MPa/min and two different temperatures.

[0044] Application of different pressurization rates: Table 4: Ps (nm) and Pdl of emulsions produced using lecithin and sunflower oil at 30 MPa, 40°C and two different pressurization rates.

[0045] Utilization of different oils:

Table 5: Ps (nm) and Pdl of emulsions produced at 30 MPa, 40°C, 12 MPa/min using lecithin and two different oils: chicken liver oil (fatty acid composition contains saturated and monounsaturated fatty acids) and sunflower oil (high concentration of polyunsaturated fatty acids).

[0046] Addition of Coenzyme Q10 (CoQlO) to the emulsion for its use as a carrier of bioactive compounds:

Table 6: Ps (nm) and Pdl of emulsions produced at 30 MPa, 40°C, 12 MPa/min with the addition of different concentrations of CoQlO to the oil and using lecithin as surfactant.

[0047] As may be seen in Figure 1 and Figure 2, the emulsions are visibly stable after greater than 90 days in storage at 4° C.

References

[0048] The following references are indicative of the level of skill and knowledge in the art.

Aslanidou, D., Karapanagiotis, I., Panayiotou C. (2016). Tuneable textile cleaning and disinfection process based on supercritical CO2 and Pickering emulsions. Journal of Supercritical Fluids, 118, 128-139.

Chen, J., Azhar, U., Wang, Y., Liang, J., Geng, B. (2019). Preparation of fluoropolymer materials with different porous morphologies by an emulsion template method using supercritical carbon dioxide as a medium. RSC Advances, 9, 11331-11340.

Ciaglia, E., Montella, F., Trucillo, P., Ciardulli, M.C. Di Pietro, P., Amodio, G., Remondelli, P., Vecchione, C., Reverchon, E., Maffulli, N., Puca, A. A., Della Porta, G. (2019). A bioavailability study on microbeads and nanoliposomes fabricated by dense carbon dioxide technologies using human-primary monocytes and flow cytometry assay. International Journal of Pharmaceutics, 570, 118686.

Junior, S.J.H., Ract, J.N.R., Gioielli, L.A., Vitolo, M. (2018). Conversion of triolein into mono- and diacylglycerols by immobilized lipase. Arabian Journal for Science and Engineering, 43, 2247-2255.

Trupej, N., Novak, Z., Knez, Z., Slugovc, C., Kovacic, S. (2017). Supercritical CO2 mediated functionalization of highly porous emulsion-derived foams: SCCO2 absorption and epoxidation. Journal of CO2 Utilization, 21, 336-341 Definitions and Interpretation

[0049] The description of the present invention has been presented for purposes of illustration and description, but it is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention.

[0050] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to combine, affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not such connection or combination is explicitly described. In other words, any element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility between the two, or it is specifically excluded.

[0051] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation. The terms “preferably,” “preferred,” “prefer,” “optionally,” “may,” and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.

[0052] The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The term "comprising" or "comprises" means "including but not limited to".

[0053] The term "about" means any value within 10% or 5% or 2% or 1% of the stated value, or any value within the accuracy limits of a measuring device by which the value may be measured.

[0054] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

[0055] As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited, and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio.

Claims

1. A process of producing an emulsion, comprising the steps of

(a) loading a water phase, an oil phase, at least one stabilizer into a pressure vessel;

(b) introducing CO2 into the pressure vessel and pressurizing the vessel contents at a temperature up to a pressure and temperature above the critical point of CO2;

(c) stirring and then withdrawing a liquid phase, wherein the emulsion is formed by depressurization of the liquid phase, releasing CO2 from the emulsion; and

(d) collecting the emulsion.

2. The process of claim 1 wherein additional CO2 is introduced into the vessel during withdrawal of the liquid phase, to maintain substantially the same pressure within the vessel.

3. The process of claim 1 or 2 wherein the process does not use an organic solvent.

4. The process of claim 1, 2 or 3, wherein the operating conditions are below 40 MPa and 70° C.

5. The process of claim 4 wherein the operating conditions are between about 10 to about 30 MPa, and/or about 40° C to about 50° C.

6. The process of any one of claims 1-5 which is a batch, semi-continuous, or continuous process.

7. The process of any one of claims 1-6 wherein the emulsion dispersed domain diameter is less than about 100 pm, 1 pm, 200 nm, or in the range of 50 to 100 nm.

8. The process of claim 6 or 7, wherein the process is semi-continuous, wherein the water phase and the oil phase are separately pumped in to the pressure vessel, and SCCO2 is pumped into the pressure vessel either separately or together with either the water phase or the oil phase, and the emulsion is formed by depressurizing the liquid phase while keeping the pressure inside the vessel substantially constant by adding additional SCCO2.

9. The process of claim 6 or 7, wherein the process is continuous and wherein the water phase, the oil phase, and SCCO2 are continuously pumped into the vessel through at least two lines separately and mixed together before entering a pressure vessel.

10. The process of claim 8 or 9, wherein the SCCO2 is mixed with the oil phase prior to mixing with the water phase.

11. The process of any one of claims 1-10 wherein the at least one stabilizer comprises at least one surfactant.

12. The process of claim 11 wherein the at least one surfactant comprises a non-ionic surfactant.

13. The process of claim 11 wherein the at least one surfactant comprises lecithin.

14. The process of any one of claims 1-10 wherein the at least one stabilizer comprises an aqueous viscosifier.

15. The process of any one of claims 11-13 wherein the at least one surfactant is mixed with either or both the water phase or oil phase, prior to mixing the two phases together

16. The process of any one of claims 1-15, comprising the further step of adding a bioactive compound to at least one of the water phase or the oil phase before formation of the emulsion.

17. An emulsion formed by dissolving SCCO2 into an oil and water mixture and at least one stabilizer, under conditions above the critical point for carbon dioxide, followed by depressurization to form the emulsion.

18. The emulsion of claim 17 which is a nanoemulsion consisting essentially of biocompatible substances.

19. The emulsion of claim 17 or 18 further comprising a bioactive substance.