WO2016182926A1

WO2016182926A1 - Preparation of nanoemulsions

Info

Publication number: WO2016182926A1
Application number: PCT/US2016/031263
Authority: WO
Inventors: Shuo Zhao
Original assignee: Affinsci Inc.
Priority date: 2015-05-08
Filing date: 2016-05-06
Publication date: 2016-11-17

Abstract

Provided herein are methods and compositions for the preparation of oil-inwater nanoemulsions, nanoemulsions produced thereby, and applications thereof. In particular, methods are provided for preparation of nanoemulsions at ambient temperature and atmosphere pressure (e.g., in a conventional chemical reactor or mixer).

Description

PREPARATION OF NANOEMULSIONS

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U. S. Provisional Patent Application 62/158,568 filed May 8, 2015, which is incorporated by reference in its entirety.

FIELD

Provided herein are methods and compositions for the preparation of oil-in- water nanoemulsions, nanoemulsions produced thereby, and applications thereof. In particular, methods are provided for preparation of nanoemulsions at ambient temperature and atmosphere pressure (e.g., in a conventional chemical reactor or mixer).

BACKGROUND

Conventional techniques for preparing nanoemulsions of cosmetic ingredients, proteins, drugs, etc. include high pressure homogenization, ultrasonic treatment, or a phase inversion temperature process. Existing processes have many disadvantages including: high cost, unsuitability for labile reagents (e.g., material which cannot tolerate heat), inclusion of toxic reagents (e.g., quaternary ammonium salts), inability to produce transparent nanoemulsions, and larger droplet size (e.g., >100 nm).

SUMMARY

In some embodiments, provided herein are methods for preparing a nanoemulsion comprising: (a) combining: (i) an aqueous solution and (ii) a homogeneous mixture comprising a nonionic surfactant and hydrophobic agent to form a nanoemulsion; and (b) stirring the nanoemulsion to reduce average diameter of droplets in the nanoemulsion. In some embodiments, the aqueous solution and the homogeneous mixture are combined at sufficiently slow enough rate to allow a nanoemulsion to form (e.g., the particular rate may depend upon the identity of the components and/or the volume to be added). In some embodiments, the aqueous solution and the homogeneous mixture are combined over the course of greater than 5 minutes (e.g., 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or more, or any rangers therein (e.g., 10-30 minutes)). In some embodiments, the

nanoemulsion is stirred for greater than 1 hour (e.g., 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, or more, or ranges therein (e.g., 2-96 hours)) to reduce average diameter of droplets in the nanoemulsion. In some embodiments, one or both of steps (a) and (b) is performed at ambient temperature and atmospheric pressure. In some embodiments, both steps (a) and (b) are performed at ambient temperature and atmospheric pressure. In some

embodiments, the aqueous solution is distilled water. In some embodiments, the aqueous solution comprises buffer and/or salt. In some embodiments, the

nanoemulsion formed in step (a) comprises an average droplet diameter of 40-150 nm. In some embodiments, the average droplet diameter following step (b) is less than 20 nm. In some embodiments, the average droplet diameter following step (b) is less than 10 nm. In some embodiments, the average droplet diameter following step (b) is less than 5 nm. In some embodiments, the hydrophobic agent is physiologically compatible. In some embodiments, the hydrophobic agent is an oil. In some embodiments, the surfactant is physiologically compatible. In some embodiments, the surfactant is polysorbate 80 (e.g., Tween 80). In some embodiments, the aqueous solution comprises a modifier and/or co-surfactant. In some embodiments, the homogeneous mixture further comprises a modifier and/or co-surfactant. In some embodiments, the modifier is physiologically compatible. In some embodiments, the modifier is a viscosity modifier. In some embodiments, the viscosity modifier is a viscosity -reduction agent. In some embodiments, the viscosity -reduction agent is selected from the group comprising ethyl alcohol, isopropyl alcohol, and propylene glycol. In some embodiments, the co-surfactant is physiologically compatible. In some embodiments, the co-surfactant is PEG-400.

In some embodiments, provided herein are compositions, nanoemulsions, and/or nanodroplets produced by the methods described herein. In some

embodiments, compositions are formulated for administration or delivery to a subject (e.g., human or mammalian subject).

DEFINITIONS

As used herein, the term "nanoemulsion" refers to oil-in-water dispersions comprising nanometer scale oil-phase droplets in an aqueous phase. Nanoemulsions include oil droplets ("nanodroplets") of 200 nm or less diameter (e.g., <180 nm, <160 nm, <140 nm, <120 nm <100 nm, <80 nm, <60 nm, <40 nm, <30 nm, <20 nm, <15 nm, <10 nm, <8 nm, <6 nm, <5 nm, or less) in aqueous solution. As used herein, the term "surfactant" refers to a compound that, when included in a mixture, lowers the surface tension or interfacial tension between two liquids or between a liquid and a solid in the mixture. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and/or dispersants. The term "nonionic surfactant" typically refers to uncharged long-chain (e.g., >8 carbons) alcohols.

As used herein, the term "physiologically compatible" refers to a material or agent having the characteristic of being non-toxic and otherwise well-tolerated by biological systems including upon topical or internal administration to a human subject. A substance may be termed physiologically compatible if the benefits of administration or consumption of the substance outweigh any toxicity or negative side effects.

As used herein, the term "atmospheric pressure" refers to the magnitude of the air pressure at sea level, approximately 760 torr. Atmospheric pressure encompasses a range of aout 760 ±10% (e.g., 684 Torr, 692 Torr, 700 Torr, 708 Torr, 716 Torr, 724 Torr, 732 Torr, 740 Torr, 748 Torr, 756 Torr, 764 Torr, 772 Torr, 780 Torr, 788 Torr, 796 Torr, 804 Torr, 812 Torr, 820 Torr, 828 Torr, 836 Torr, or any ranges therebetween), particularly when variation is caused by weather, altitude, or other natural causes.

As used herein, the term "ambient temperature" refers to approximately 18-26

°C (e.g., 18, 19, 20, 21 , 22, 23, 24, 25, 26, or any ranges therebetween). Variation within or around that range is particularly acceptable when the variation is caused by natural conditions or the environmental (e.g., room) temperature, rather than specific heating or cooling of a reaction vessel.

DETAILED DESCRIPTION

Provided herein are methods and compositions for the preparation of oil-in- water nanoemulsions, nanoemulsions produced thereby, and applications thereof. In particular, methods are provided for preparation of nanoemulsions at ambient temperature (e.g., 20-26°C) and atmosphere pressure (e.g., in a conventional chemical reactor or mixer).

In some embodiments, methods generate highly clear and transparent oil-in- water nanoemulsions (e.g., in large quantity (e.g., >5L (e.g., 10 L, 15 L, 20 L, 25 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L or more)). Processes find use in the preparation of delivery vehicles for a variety of cosmetics, personal care materials, medicines, etc.

In some embodiments, processes of preparing nanoemulsions comprise the slow addition an aqueous solution (e.g., water, distilled water, water and salt/buffer, etc.) to a homogeneous mixture comprising a nonionic surfactant (e.g., Tween 80, alkyl glucoside, PEG-400, etc.) and a hydrophobic agent (e.g., insoluble or poorly soluble in water). In other embodiments, a homogeneous mixture (e.g., comprising a nonionic surfactant and a hydrophobic agent) is slowly added to an aqueous solution.

In some embodiments, slow addition (e.g., of aqueous solution to the homogeneous mixture, of the homogeneous mixture to the aqueous solution, etc.) is at a sufficiently low enough rate to allow formation of moderately sized nanodroplets (e.g., 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 80 nm, 60 nm, 40 nm, or any suitable ranges therebetween (e.g., 60-120 nm). In some embodiments in which 100 or fewer grams liquid are added, slow addition is at a rate less than 5 g/min., less than 2 g/min., less than 1 g/min., less than 0.5 g/min., or less than 0.2 g/min. (e.g., 5 g/min., 4 g/min., 3 g/min., 2 g/min., 1 g/min., 0.8 g/min., 0.6 g/min., 0.5 g/min., 0.4 g/min., 0.3 g/min. , <0.2 g/min., 0.1 g/min., 0.08 g/min. , 0.06 g/min., 0.04 g/min., 0.02 g/min., or any suitable ranges therebetween (e.g., 0.05-0.2 g/min, etc.)). In some embodiments in which 100-1000 grams of liquid are added, slow addition is at a rate of 1 -30 g/min (e.g., 1 g/min, 2 g/min, 3 g/min, 4 g/min, 5 g/min, 6 g/min, 7 g/min, 8 g/min, 9 g/min, 10 g/min, 15 g/min, 20 g/min, 25 g/min, 30 g/min, or any suitable ranges therebetween (e.g., 4-20 g/min, etc.)). In some embodiments in which 1 kilogram or more of liquid are added, slow addition is at a rate of 20-200 g/min (e.g., 20 g/min, 30 g/min, 40 g/min, 50 g/min, 60 g/min, 70 g/min, 80 g/min, 90 g/min, 100 g/min, 120 g/min, 140 g/min, 160 g/min, 180 g/min, 200 g/min, or any suitable ranges therebetween (e.g., 40-120 g/min, etc.)).

In some embodiments, processes further comprise the addition of one or more of a modifier (e.g., propylene glycol, isopropyl alcohol, etc.), and/or a co-surfactant (e.g., PEG-400). In some embodiments, a homogeneous mixture comprises: a surfactant, co-surfactant, and hydrophobic agent; surfactant, modifier, and hydrophobic agent; or surfactant, co-surfactant, modifier, and hydrophobic agent. In some embodiments, the aqueous phase comprises: water (e.g., distilled or with buffer and/or salt); water and surfactant (or co-surfactant); water and modifier; or water, modifier, and surfactant (or co-surfactant). In some embodiments, suitable surfactants and co-surfactants for use in the processes, mixtures, and compositions described herein are nonionic. Suitable surfactants (and co-surfactants) include, but are not limited to: polyoxy ethylene glycol alkyl ethers (e.g., CH₃-(CH₂)io i6-(0-C₂H₄)i 25-OH, octaethylene glycol

monododecyl ether, pentaethylene glycol monododecyl ether, etc.), polyoxypropylene glycol alkyl ethers (e.g., CH₃-(CH₂)io-i6-(0-C₃H₆)i-25-OH), glucoside alkyl ethers (e.g., CH₃-(CH₂)io-i6-(0-glucoside)i_3-OH, decyl glucoside, lauryl glucoside, octyl glucoside, etc.), polyoxyethylene glycol octylphenol ethers (e.g., C₈Hi₇-(C₆H₄)-(0- C₂H₄)i_25-OH, Triton X-100 (e.g., polyethylene glycol p-(l, l ,3,3-tetramethylbutyl)- phenyl ether), etc.), polyoxyethylene glycol alkylphenol ethers (e.g., C₉Hi₉-(C₆H )- (0-C₂H4)i_25-OH, nonoxynol-9, etc.), glycerol alkyl esters (e.g., glyceryl laurate, etc.), polyoxyethylene glycol sorbitan alkyl esters (e.g., polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), etc.), sorbitan alkyl esters, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, block copolymers of polyethylene glycol and polypropylene glycol (e.g., poloxamers), polyethoxylated tallow amine (POEA), polyethylene glycols (e.g., PEG- 400, PEG-600, PEG-800, PEG-1000, PEG-2000, etc.), Spartan 20, Spartan 80, etc..

Suitable modifiers include, but are not limited to: propylene glycol, ethanol, isopropanol, butanol, polypropylene glycol, cellulose ethers, etc. Other modifiers that are tolerated in the system and the products and applications utilizing the

nanoemulsions are also contemplated. For example, modifier could also be long chain (greater than or equal to C4) fatty alcohols, such as: tert-butyl alcohol, tert-amyl alcohol, 3-methyl-3-pentanol, ethchlorvynol, octanol, 2-ethylhexanol, 1 -nonanol, 1 - decanol, undecanol, dodecanol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, palmitoleyl alcohol, heptadecyl alcohol, stearyl alcohol, nonadecyl alcohol, arachidyl alcohol, heneicosyl alcohol, behenyl alcohol, eucyl alcohol, lignoceryl alcohol, ceryl alcohol, 1-heptacosanol, montanyl alcohol, 1 -nonacosanol, myricyl alcohol, 1 -dotriacontanol, geddyl alcohol, Cetearyl alcohol, etc. Other alcohols, for example, commonly used sugar alcohol could also be applied. This includes, but not limited to: arabitol, erythritol, glycerol, hydrogenated starch hydrolysates (HSH), isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, sucrose, etc. Other example of modifier could be crown ethers, such as (but not limited to) 12- crown-4, 15-crown-5, 18-crown-6, dibenzo-18-crown-6, and diaza-18-crown-6. In some embodiments, the modifier is a "viscosity modifier" and is included to adjust viscosity (e.g., to increase/decrease viscosity of the homogeneous mixture or nanoemulsion). In some embodiments, the viscosity modifier is a "viscosity reduction agent" and is included to reduce the viscosity (e.g., of the homogeneous mixture, aqueous solution, and/or nanoemulsion).

In some embodiments, suitable hydrophobic agents are oils, lipids, or other compositions that are non-miscible with water or have low miscibility with water. In some embodiments, a hydrophobic agent is an active agent (e.g., having a desired property for which the nanoemulsion is a delivery vehicle). In some embodiments, the hydrophobic agent is the active agent of the nanoemulsion. Suitable hydrophobic agents include, but are not limited to, oils of the type of mineral oils, vegetable oils, animal oils, essential oils, synthetic oils, or mixtures thereof. In some embodiments, a hydrophobic agent is an oil rich in triglycerides, such as safflower oil, soybean oil, sesame oil, camellia oil, cotton seed oil, medium chain triglycerides, their mixtures or mixtures with other vegetable oils. In some embodiments, a hydrophobic agent is a poorly water-soluble drugs, such as: paclitaxel, camptothecin, curcumin, tanshinone HA, capsaicin, cyclosporine, erythromycin, nystatin, itraconazole,

celecoxib, clofazimine, digoxin, oleandrin, nifedipine, and amiodarone, etc. In some embodiments, a hydrophobic agent comprises one or more essential oils, such as those derived from berries (e.g., allspice, juniper, etc.), seeds (e.g., almond, anise, buchu, celery, cumin, nutmeg oil, etc.), bark (e.g., cassia, cinnamon, sassafras, etc.), wood

(e.g., camphor, cedar, rosewood, sandalwood, agarwood, etc.), rhizome (e.g., galangal, ginger, etc.), leaves (e.g., basil, bay leaf, buchu, cinnamon, common sage, eucalyptus, guava, lemon grass, melaleuca, oregano, patchouli, peppermint, pine, rosemary, spearmint, tea tree, thyme, tsuga, wintergreen, etc.), resin (e.g., benzoin, copaiba, frankincense, myrrh, etc.), flowers (e.g., cannabis, chamomile, clary sage, clove, scented geranium, hops, hyssop, jasmine, lavender, manuka, marjoram, orange, rose, ylang-ylang, etc.), peel (e.g., bergamot, grapefruit, lemon, lime, orange, tangerine, etc.), root (e.g., valerian, etc.). In some embodiments, a hydrophobic agent is peptide or protein drug such as: insulin, leuprorelin, bortezomib, goserelin, bivalirudin, eptifibatide, glatiramer, liraglutide, telaprevir, boceprevir, icatibant, etc.

In some embodiments, the active agent is the hydrophobic agent. In some embodiments, an additional active agent is added. In some embodiments, an active agent is dissolved or placed in solution in a hydrophobic carrier (e.g., vegetable oil, etc.). In embodiments described throughout, reagents, solutions, mixtures, etc. are mixed, stirred, shaken, or otherwise combined. Unless otherwise specified, combination of reagents occurs passively (e.g., by diffusion), or reagents are actively combined by mechanical intervention (e.g., stirring, agitating, etc.) or other processes (e.g., soni cation, etc.). In some embodiments in which stirring is employed, the rate of stiring is at least 1 rpm and not exceeding 5000 rpms (e.g., 1 rpm, 2 rpms, 5 rpms, 10 rpms, 20 rpms, 50 rpms, 100 rpms, 200 rpms, 500 rpms, 1000 rpms, 2000 rpms, 2500 rpms, 3000 rpms, 4000, rpms, 5000 rpms or any suitable ranges therebetween). In some embodiments, sonication is employed (e.g., when initially mixing the reagents to form the homogeneous mixture). In some embodiments, vigorous stirring (e.g., 1000-2000 rpms, about 1500 rpms, etc.) is employed during the addition of aqueous phase to the homogeneous mixture. In some embodiments, gentle stirring (e.g., 100 to 600 rpms (e.g., 100 rpms, 200 rpms, 300 rpms, 400 rpms, 500 rpms, 600 rpms or any suitable ranges therebetween (e.g., 200-500 rpms))) is employed (e.g., to induce a reduction in nanodroplet size).

Formation of homogeneous mixture (step Oa)

In some embodiments, formation of nanoemulsion according to the embodiments described herein utilizes a homogeneous mixture comprising at least a hydrophobic agent (which is mixed with an aqueous solution (e.g., in a subsequent step)).

In some embodiments, methods comprise combining (e.g., mixing) a nonionic surfactant (e.g., Tween 80), a co-surfactant (e.g., PEG-400), and/or a modifier (e.g., propylene glycol isopropyl alcohol, etc.) with a hydrophobic agent (e.g., a hydrophobic agent having a desired property (e.g., cosmetic property, wellness- promotion property, therapeutic/prophalactic property, etc.). In some embodiments, the nonionic surfactant, co-surfactant, and/or modifier are combined into a homogeneous mixture before adding the hydrophobic agent. In some embodiments, the nonionic surfactant, co-surfactant, modifier, and hydrophobic agent, and added together and then homogenized. In some embodiments, nonionic surfactant, co- surfactant, modifier, and/or hydrophobic agent are mixed (e.g., stirred) or allowed to mix at ambient temperature and atmospheric pressure to provide homogeneous mixture. Formation of an aqueous solution (step Ob)

In some embodiments, formation of nanoemulsion according to the embodiments described herein utilizes an aqueous solution (which is mixed with a homogeneous mixture comprising a hydrophobic agent (e.g., in a subsequent step)). In some embodiments, the aqueous solution described in embodiments herein comprises, consists, or consists essentially of water (with common salt, metal, and oxide impurities) or distilled water. In some embodiments, the aqueous solution further comprises one or more suitable buffers, salts, etc. The aqueous solution is not limited by the type or presence of cations (e.g., (NH₄+, Ca , Mg , Na , Fe , Fe , etc.), anions (CI^", Br^", C0₃ ^2", P0₄ ^3", S0₄ ^2", etc.), buffers (e.g., TRIS, MOPS, HEPES, etc.), or other solutes in the aqueous solution, unless an embodiment specifies otherwise.

In some embodiments, the aqueous solution further comprises a surfactant, co- surfactant, and/or modifier. In some embodiments, one or more of a surfactant, co- surfactant, and/or modifier are mixed with water and any other suitable solutes to generate an aqueous solution. In some embodiments herein that refer to an aqueous solution, an aqueous mixture (e.g., water and one or more undissolved components (e.g., surfactant, co-surfactant, and/or modifier) is used. Formation of nanoemulsion (step 1)

In some embodiments, an aqueous solution (e.g., water, distilled water, water and buffer/salt) is added to the homogeneous mixture (e.g., at ambient temperature (e.g., 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, and any suitable ranges therein (e.g., 20-26 °C, 22-24 °C)) and atmosphere pressure (e.g., 684-836 Torr (e.g., 684 Torr, 700 Torr, 716 Torr, 732 Torr, 748 Torr, 764 Torr, 780 Torr, 796 Torr, 812 Torr, 828 Torr, 836 Torr, or ranges therebetween)) ). In some embodiments, the aqueous solution is added with stirring, shaking, or other mechanical mixing. In some embodiments, the aqueous solution is added (e.g., dropwise) over the course of at least 5 minutes (e.g., 5 mia, 10 mia, 15 mia, 20 mia, 25 mia, 30 mia, 35 mia, 40 mia, 45 mia, 50 mia, 55 mia, 60 mia, 80 mia, 100 mia, 120 mia, >120 mia, or any suitable ranges therein (e.g., 20-40 mia, etc.). In some embodiments, aqueous solution is added at a suitable rate, as described herein (e.g., <5 g/mia, <2 g/mia, <1 g/mia, <0.5 g/mia, <0.2 g/min, etc.). In some embodiments, a fixed volume of aqueous solution is added (e.g., to achieve a particular ratio (e.g., with respect to another ingredient (e.g., surfactant, co-surfactant, modifier, hydrophobic agent, etc.)). In some embodiments, aqueous solution is slowly added to the homogeneous mixture until a nanoemulsion forms (e.g., without concern as to the volume of the water added).

In other embodiments, the homogeneous mixture is added to an aqueous solution (e.g., water, distilled water, water and buffer/salt). In some embodiments, the homogeneous mixture is added with stirring, shaking, or other mechanical mixing. In some embodiments, the aqueous solution is added (e.g., drop wise) over the course of at least 5 minutes (e.g., 5 min., 10 min., 15 min., 20 min., 25 min., 30 min., 35 min., 40 min., 45 min., 50 min., 55 min., 60 min., 80 min., 100 min., 120 min., >120 min., or any suitable ranges therein (e.g., 20-40 min., etc.)). In some embodiments, the homogeneous mixture is added at a suitable rate, as described herein (e.g., <5 g/min., <2 g/min., <1 g/min., <0.5 g/min., <0.2 g/min, etc.). In some embodiments, a fixed volume of homogeneous mixture is added to a fixed volume of homogeneous mixture. In some embodiments, homogeneous mixture is slowly added to a fixed volume of aqueous solution until a nanoemulsion forms (e.g., without concern as to the volume of the homogeneous mixture added).

In some embodiments, the end product of the above-described mixing of the homogenous mixture and aqueous solution is a nanoemulsion. In some embodiments, the nanoemulsion is translucent. In some embodiments, the end product of the initial addition/mixing of the homogenous mixture and aqueous solution is not transparent. In some embodiments, the translucency but not transparency of the nanoemulsion indicates moderately-sized nanodroplets (e.g., 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 80 nm, 60 nm, 40 nm, or any suitable ranges therebetween (e.g., 60-120 nm). In some embodiments, a nanoemultion produced by slow addition (with mixing/stirring) of aqueous solution to homogenous mixture (or vice versa) exhibits moderately-sized mean or median nanodroplets. In some embodiments, such a nanoemulsion is produced in less than 1 hour (e.g., 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or any suitable ranges therein) of combining aqueous solution and homogenous mixture.

In some embodiments, the nanoemulsions comprising moderately-size nanodroplets are termed first-stage nanoemulsions (e.g., having not yet been exposed to further mixing/stirring to produce small (e.g., <40 nm) or ultrasmall (e.g., <10 nm) nanodroplets). Reduction of nanodroplet size (step 2)

In some embodiments, following the combining of an aqueous solution and homogeneous mixture to produce a nanoemulsion (e.g., of moderately-size nanodroplets, first-stage nanoemulsion, etc.), continued mixing (e.g., stirring, agitation, etc.) of the nanoemulsion at ambient temperature and atmospheric pressure. In some embodiments, mixing is continued in the same vessel. In some embodiments, mixing is continued in a different vessel. In some embodiments, continued mixing produces a highly clear and transparent. In some embodiments, the transparency of the nanoemulsion indicates significant reduction of nanodroplet size (e.g., small (e.g., <40 nm) or ultrasmall (e.g., <10 nm) nanodroplets). In some embodiments, following initial formation of the nanoemulsion (e.g., translucent nanoemulsion, moderately- sized nanodroplets, etc.) continued mixing is carried out for at least 30 minutes (e.g., 30 min., 45 min., 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or more, or any suitable ranges therein (e.g., >4 hours, 2-24 hours, etc.)). In some embodiments, mixing is carried out by vigorous stirring. In some embodiments, mixing is carried out by moderate intensity stirring. In some embodiments, mixing is carried out by gently stirring. Other embodiments

In some embodiments, co-surfactant and modifier are present in a

homogeneous mixture (or in an aqueous solution) to reduce viscosity. In some embodiments, this is particularly important for the preparation of nanoemulsions at ambient temperature (e.g., to save cost, for nanoemulsions made of reagents (e.g., hydrophobic agents) that do not tolerate elevated temperatures, etc.), because heat cannot be added to the system to reduce viscosity; although the embodiments are not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice such embodiments. However, in some embodiments, for the ingredients (e.g., hydrophobic agents) that can tolerate the higher than room temperature, only one nonionic surfactant (e.g., no co-surfactant and/or modifier) is used, for example, in preparation of the homogenous mixture, first-stage nanoemulsion, and/or final transparent nanoemulsion product. In some embodiments, the use of elevated temperature (e.g., 30°C or greater

(e.g., >30°C, >35°C, >40°C, >45°C, >50°C, >55°C, >60°C, >65°C, >70°C, or more), etc.) and atmosphere pressure (or elevated pressure (e.g., >800 Torr, >810 Torr, >820 Torr, >830 Torr, >840 Torr, >850 Torr, >875 Torr, >900 Torr, >950 Torr, >1000 Torr, >1100 Torr, >1200 Torr, 1300 Torr, 1400 Torr, 1500 Torr) allows for formation of the first stage nanoemulsion with a single surfactant (e.g., no co-surfactant and/or modifier). This translucent nanoemulsion is then stirred at ambient temperature (or at elevated temperature) and atmosphere pressure (or elevated pressure) for a time ranging from hours (e.g., 2-20 hours) to days (e.g., 1 to 6 days) to generate the highly clear and transparent nanoemulsion. In some embodiments, this single-surfactant formulation provides a very simple nanoemulsion system for further modification including combination with other ingredients.

Formulation

In some embodiments, the compositions described herein are provided as pharmaceutical, therapeutic, skin-care, hair-care, beauty, health, and/or wellness compositions. Compositions can be administered in a number of ways depending upon whether local or systemic administration is desired. Administration can be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional carriers; aqueous, powder, or oily bases;

thickeners; and the like can be necessary or desirable. Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. Compositions and formulations for parenteral, intrathecal or intraventricular administration can include sterile aqueous solutions that can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Formulations, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the appropriate industries. Compositions can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, oil-based, or mixed media. Suspensions can further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers. Compositions can be formulated and used as foams.

Dosing and administration regimes may be tailored by a clinician or other individual skilled in the appropriate field, based upon well-known considerations including, but not limited to, the desired level of effect, the practical level of effect obtainable, side effects, cost, etc. Dosing may be once per day or multiple times per day for one or more consecutive days.

EXAMPLES

Example 1

Preparation of the black raspberry essential oil oil-in-water nanoemulsion of black raspberry essential oil

The mixture of black raspberry essential oil (lOOmg), Tween 80 (0.22g), PEG-

400 (0.13g) and propylene glycol (0.1 lg) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 2 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 5 days, a highly clear and transparent naonemulsion was obtained.

Example 2

The mixture of black raspberry essential oil (200mg), Tween 80 (0.44g), PEG- 400 (0.09g) and propylene glycol (0.20g) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 4 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 2 days, a highly clear and transparent nanoemulsion was obtained. Example 3

The mixture of black raspberry essential oil (lOOmg), Tween 80 (0.20g), PEG- 400 (0.1 Og) and isopropyl alcohol (91% in water, 0.1 Og) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 4 hours, a highly clear and transparent nanoemulsion was obtained.

Example 4

Preparation of the oil-in-water nanoemulsion of pink grapefruit essential oil

The mixture of pink grapefruit essential oil (lOOmg), Tween 80 (0.20g), PEG- 400 (0.1 Og) and isopropyl alcohol (91% in water, 0.1 Og) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 2 days, a highly clear and transparent naonemulsion was obtained.

Example 5

Preparation of the oil-in-water nanoemulsion of pink grapefruit essential oil

The mixture of pink grapefruit essential oil (lOOmg), Tween 80 (0.20g), PEG- 400 (0.05g) and isopropyl alcohol (91% in water, 0.1 Og) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 3 hours, a highly clear and transparent naonemulsion was obtained. Example 6

Preparation of the oil-in-water nanoemulsion of mango essential oil

The mixture of mango essential oil (lOOmg), Tween 80 (0.20g), PEG-400 (0. lOg) and isopropyl alcohol (91% in water, 0. lOg) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 3 days, a highly clear and transparent naonemulsion was obtained.

Example 7

Preparation of the oil-in-water nanoemulsion of mango essential oil

The mixture of mango essential oil (lOOmg), Tween 80 (0.20g), PEG-400 (0.06g) and isopropyl alcohol (91% in water, 0. lOg) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 4 hours, a highly clear and transparent naonemulsion was obtained.

Example 8

Preparation of the oil-in-water nanoemulsion of coconut essential oil

The mixture of coconut essential oil (lOOmg), Tween 80 (0.20g), PEG-400 (0. lOg) and isopropyl alcohol (91% in water, 0. lOg) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 4 hours, a highly clear and transparent naonemulsion was obtained. Example 9

Preparation of the oil-in-water nanoemulsion of vanilla essential oil

The mixture of vanilla essential oil (lOOmg), Tween 80 (0.20g), PEG-400 (0.1 Og) and isopropyl alcohol (91% in water, 0.1 Og) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 4 hours, a highly clear and transparent naonemulsion was obtained.

Example 10

Preparation of the oil-in-water nanoemulsion of orange oil

The mixture of orange oil (lOOmg), Tween 80 (0.21g), PEG-400 (0.24g) and isopropyl alcohol (91% in water, 0.1 lg) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 1.5 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure overnight, a highly clear and transparent naonemulsion was obtained.

Example 11

Preparation of the oil-in-water nanoemulsion of orange oil

The mixture of orange oil (lOOmg), Tween 80 (0.21g), PEG-400 (0.05g) and isopropyl alcohol (91% in water, 0.1 Og) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 2 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 4 hours, a highly clear and transparent naonemulsion was obtained.

Example 12

Preparation of the oil-in-water nanoemulsion of orange oil

The mixture of orange oil (190mg), Tween 80 (0.29g), PEG-400 (0.29g) and ethyl alcohol (absolute, 0.22g) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (distilled) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 3.2 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 4 hours, a highly clear and transparent naonemulsion was obtained.

Example 13

Preparation of the oil-in-water nanoemulsion of orange oil

The mixture of orange oil (1.0 g) and Tween 80 (2.0 g) was stirred at 50 °C and atmosphere pressure for 5 min. Water (distilled) was added very slowly at 50 °C in 30 min. and atmosphere until the total mass of the mixture reached 20 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure overnight, a highly clear and transparent naonemulsion was obtained. Naoemulsion droplet size is at 4.9±1.1 nm (method is DLS, Dynamic Light Scattering).

Example 14

Preparation of the oil-in-water nanoemulsion of orange oil

The mixture of orange oil (29.0 g) and alkyl glucoside (C8-C16, 50% wt aqueous solution, 58 g) was stirred at 50 °C and atmosphere pressure for 15 min.

Water (distilled) was added very slowly at 50 °C in 60 min. and atmosphere until the total mass of the mixture reached 575 g. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure overnight, a highly clear and transparent naonemulsion was obtained.

Example 15

Preparation of the oil-in-water nanoemulsion of Clofazimine (e.g., for development of anti-tuberculosis drug using an easy-to-use inhalation delivery system)

The mixture of Clofazimine (100 mg) in soybean oil (1.0 g) was stirred at 50 °C for 2 days. PEG-400 (0.1 g) were added and stirred at 50 °C and atmosphere pressure for 1 hour. The mixture of Tween 80 (4.0 g) and water (distilled, 4.0 g) was stirred at 50 °C in 15 min. The above mentioned mixture of Clofazimine, soybean oil and PEG-400 was added very slowly at 50 °C in 30 min. The mixture was stirred at ambient temperature and atmosphere pressure overnight. The mixture was centrifuged and the highly clear and transparent nanoemulsion top was separated. The HPLC results showed the concentration of Clofazimine in this nanoemulsion is 0.45 mg/mL. Naoemulsion droplet size is at 6.5±1.1 nm (method is DLS, Dynamic Light

Scattering).

Example 16

Preparation of the oil-in-water nanoemulsion of Paclitaxel (Taxol as trade name for anti-cancer medicine)

Paclitaxel (22 mg) was dissolved in the mixture of Tween 80 (0.88 g), PEG- 400 (1.23 g) and ethyl alcohol (absolute, 0.6 g) by sonication for 5 min. Then, to this solution, water (distilled) was added very slowly at ambient temperature and atmosphere. When the total mass reached 3 g, a translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure overnight, a highly clear and transparent naonemulsion was obtained.

Example 17

Preparation of the oil-in-water nanoemulsion of peppermint oil

The mixture of peppermint oil (40.3g), Tween 80 (81.4g), PEG-400 (14.2g) and propylene glycol (14.0g) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (filtered) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 400 g.

Potassium sorbate (0.46 g, 24.9% aqueous solution) was added at rt. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 2 days, a highly clear and transparent naonemulsion was obtained. Table 1. Results of particle size measurement'

Time Particle size (percentage)

15 min. 19.7 ± 7.8 nm (91.8%) 588 ± 224 nm (3.7%) 4400 ± 936 nm (4.5%)

1 h 13.7 ± 3.6 nm (84.1%) 1722 ± 556 nm (8.0%) 4155 ± 995 nm (7.9%)

20 h 16.2 ± 4.4 nm (89.9%) 2825 ± 123 6nm (10.1%)

48 h 15.8 ± 5.1 nm ( 100%)

Instrument: Malvern Zetasizer Nano-S90

Example 18

Preparation of the oil-in-water nanoemulsion of lavender oil

The mixture of lavender oil (286g), Tween 80 (572g), PEG-400 (98g) and propylene glycol (198g) was stirred at ambient temperature and atmosphere pressure for 10 min. Water (filtered) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 2.8kg. Potassium sorbate (8g) was added at rt. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 20 h, a highly clear and transparent naonemulsion was obtained.

Table 2. Results of particle size measurement'

Time Particle size (percentage)

30 min. 19.0 ± 5.5 nm (92.8%) 718 ± 195 nm (3.1%) 4853 ± 703 nm (4.1%)

20 h 16.4 ± 3.6 nm (100%)

instrument: Malvern Zetasizer Nano-S90

Example 19

Preparation of the oil-in-water nanoemulsion of lemon oil The mixture of lemon oil (40.0g), Tween 80 (81. Og), PEG-400 (14.2g) and propylene glycol (28.5g) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (filtered) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 400 g. Potassium sorbate (0.96 g, 24.9% aqueous solution) was added at rt. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 2 days, a highly clear and transparent naonemulsion was obtained. Table 3. Results of particle size measurement'

Time Particle size (percentage)

5 min. 19.3 ± 5.0 nm (65.9%) 418 ± 141 nm (31.6%) 5186 ± 483 nm (2.5%)

12 h 14.6 ± 3.4 nm (65.8%) 375 ± 114 nm (32%) 4155 ± 995 nm (7.9%)

48 h 12.0 ± 3.8 nm ( 100%)

*Instrument: Malvern Zetasizer Nano-S90

Example 20

Preparation of the oil-in-water nanoemulsion of tea tree oil

The mixture of tea tree oil (40g), Tween 80 (80g), PEG-400 (14g) and propylene glycol (28g) was stirred at ambient temperature and atmosphere pressure for 5 min. Water (filtered) was added very slowly in 30 min. at ambient temperature and atmosphere until the total mass of the mixture reached 400 g. Potassium sorbate (1.12 g) was added at rt. A translucent nanoemulsion was formed. Further stirring of this translucent nanoemulsion at ambient temperature and atmosphere pressure for 7 days, a highly clear and transparent naonemulsion was obtained.

Table 4. Results of particle size measurement*

Time Particle size (percentage)

30 min. 18.3 ± 5.0 nm (86.9%) 347 ± 94.4 nm (9.5%) 5131 ± 524 nm (3.6%)

3 d 15.2 ± 2.8 nm (61.3%) 437 ± 82.3 nm (38.7%)

7 d 14.2 ± 2.5 nm (100%)

Instrument: Malvern Zetasizer Nano-S90

Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:

1. A method for preparing a nanoemulsion comprising:

(a) combining: (i) an aqueous solution and (ii) a homogeneous mixture comprising a nonionic surfactant and hydrophobic agent to form a nanoemulsion; and

(b) stirring the nanoemulsion to reduce average diameter of droplets in the nanoemulsion.

2. The method of claim 1 , wherein the aqueous solution and the homogeneous mixture are combined at a sufficiently slow enough rate to allow formation of the nanoemulsion.

3. The method of claim 1 , wherein the nanoemulsion is stirred for greater than 1 hour to reduce average diameter of droplets in the nanoemulsion.

4. The method of claim 1 , wherein one or both of steps (a) and (b) is performed at ambient temperature and atmospheric pressure.

5. The method of claim 4, wherein steps (a) and (b) are performed at ambient temperature and atmospheric pressure.

6. The method of claim 1 , wherein the aqueous solution is distilled water.

7. The method of claim 1 , wherein the aqueous solution comprises buffer and/or salt.

8. The method of claim 1 , wherein the nanoemulsion formed in step (a) comprises an average droplet diameter of 40-150 nm.

9. The method of claim 1 , wherein the average droplet diameter following step (b) is less than 20 nm.

10. The method of claim 1 , wherein the average droplet diameter following step (b) is less than 10 nm.

1 1. The method of claim 1 , wherein the average droplet diameter following step (b) is less than 5 nm.

12. The method of claim 1 , wherein the hydrophobic agent is

physiologically compatible.

13. The method of claim 12, wherein the hydrophobic agent is an oil.

14. The method of claim 1 , wherein the surfactant is Tween 80.

15. The method of claim 1 , wherein the aqueous solution comprises a modifier and/or co-surfactant.

16. The method of claim 1 , wherein the homogeneous mixture further comprises a modifier and/or co-surfactant.

17. The method of claim 15 or 16, the modifier is a viscosity modifier.

18. The method of claim 17, wherein the viscosity modifier is a viscosity- reduction agent.

19. The method of claim 18, wherein the viscosity-reduction agent is selected from the group comprising ethyl alcohol, isopropyl alcohol, and propylene glycol.

20. The method of claim 15 or 16, the co-surfactant is PEG-400.

21. A composition comprising a nanoemulsion produced by the method of one or claims 1 -20. A composition comprising a nanodroplet from a composition of claim