Methods of producing nanoparticles of hydrophobic compounds

Abstract

Methods for producing nanoparticles of hydrophobic compounds, such as EGCG-palmitate, Cannabidiol (CBD), Delta-9-tetrahydrocannabinol (THC-9), quercetin, ivermectin, retinoic acid, curcumin, resveratrol, lycopene, lutein, CoQ10, procyanidin B2, oxyresveratrol, tetrahydropiperine, rapamycin, forskolin, and tetrahydrocurcumin, are described. In some forms, one or more water soluble compounds are used with at least one hydrophobic compound to form the nanoparticles. Nanoparticles produced using the disclosed methods have tightly packed structures assembled by the hydrophobic compounds themselves, without the need for other components, such as surfactant, polymer, encapsulation, or metals. These nanoparticles can significantly increase the water solubility of hydrophobic compounds, allowing increased solubility and bioavailability in their own nanoparticle form.

Description

[0001] ATTORNEY DOCKET NO. AURI 2025-008 PCT

[0002] METHODS OF PRODUCING NANOPARTICLES OF HYDROPHOBIC COMPOUNDS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U. S. Provisional Application No. 63 / 725,740 filed November 27, 2024, U. S. Provisional Application No. 63 / 788,538 filed April 14, 2025, and U. S. Patent Application No. 63 / 896,918 filed October 10, 2025, the entire contents of which are incorporated herein by reference in their entireties.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under R41DC020678 awarded by The National Institute of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] This invention is generally in the field of compositions of hydrophobic compounds and methods of making thereof.

[0008] BACKGROUND OF THE INVENTION

[0009] Hydrophobic molecules with poor water solubility are often associated with low bioavailability. This physical property prevents these molecules from being developed for new drug use. Examples of these hydrophobic molecules include tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), and ivermectin. It is estimated that 70-90% of drug candidates under development stage are poorly soluble, which are associated with low bioavailability, reduced therapeutic effects, and increased dosage that could cause unwanted adverse effects.

[0010] Scientists have made various attempts to increase drug solubility and bioavailability, including methods with nanotechnology. Nanotechnology includes solid lipid nanoparticles, nanostructured lipid carriers, which require solid or solid / liquid fats as earners. Supercritical antisolvent (SAS) methods can increase the solubility of certain molecules, but it is a complicated process with limited use. Other nanotech methods, such as nanoemulsions and nanogels, use either oil and surfactant, or crosslinked polymers. In addition, engineered nanoparticles can be made through metal organic frameworks, carbon nanotubes, mesoporous silica. However, these nanotechnologies involve other ingredients / components, specific engineering methods, and equipment.

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[0013] There is a need for methods for producing nanoparticles of hydrophobic compounds.

[0014] Therefore, it is an object of the present invention to provide methods for producing nanoparticles of hydrophobic compounds.

[0015] SUMMARY OF THE INVENTION

[0016] Described are methods (also referred to herein as “Facilitated Self-Assembling Technology’’ or “FAST’’) for producing nanoparticles of hydrophobic compounds, including hydrophobic drugs, hydrophobic nutraceutical compounds, fluorescent dyes, etc., such as EGCG-palmitate, Cannabidiol (CBD), Delta-9-tetrahydrocannabinol (THC-9), quercetin, ivermectin, retinoic acid, etc.

[0017] Exemplary hydrophobic compounds that can be formulated using the methods include, but are not limited to, cannabis (Cannabidiol (CBD) and tetrahydrocannabinol (THC)), synthetic steroid drugs (medroxyprogesterone acetate and triamcinolone acetonide), azole antifungal drug (Fluconazole), anticancer drug (Paclitaxel), flavonoids (quercetin, EGCG-mono-palmitate “EC16m”), antiparasitic drug (ivermectin), fluorescent dyes (e.g., fluorescein, rhodamine, cyanine, and their derivatives), lipid-soluble vitamins, and other lipid-soluble nutritional supplements, and combinations thereof.

[0018] For example, the hydrophobic compounds that can be formulated using the methods are EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQ10, procyanidin B2, rapamycin, oxyresveratrol, tetrahydropiperine, forskolin, tetrahydrocurcumin, or a fluorescent dye (e.g., Cyanine5), or combinations thereof.For example, the hydrophobic compounds that can be formulated using the methods are retinoic acid and tocotrienols, quercetin, ivermectin (e.g., Ivermectin B1a), rapamycin, curcumin, resveratrol, lycopene, lutein, CoQ10, procyanidin B2, rapamycin, oxyresveratrol, tetrahydropiperine, forskolin, tetrahydrocurcumin. or a fluorescent dye (e.g., Cyanine5), or combinations thereof.

[0019] The methods can produce nanoparticles containing a single hydrophobic compound (such as a hydrophobic drug or a nutraceutical compound) or a combination of two or more hydrophobic compounds. When nanoparticles containing more than one hydrophobic compound are produced, the different hydrophobic compounds can be incorporated into one nanoparticle (also referred to herein as “hybrid nanoparticle”). For example, the hybrid nanoparticles produced using the methods contain (1) one or more hydrophobic drug(s) and / or one or more nutraceutical compound(s); and optionally (2) one or fluorescent organic dye(s) incorporated into one nanoparticle.

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[0022] When the nanoparticles are hybrid nanoparticles, in some forms, the hybrid nanoparticles can contain at least one hydrophobic compound and one or more water-soluble compounds. In some forms, the water-soluble compounds suitable for use in the methods for forming the hybrid nanoparticles have a water solubility of >100 ug / mL, such as >500 ug / mL, >1 mg / mL, >5 mg / mL, or >10 mg / mL. In these forms, the hydrophobic compound(s) can serve as a carrier that incorporates the water soluble compound(s) into the hybrid nanoparticle. As such, the hydrophobic compound(s) and water soluble compound(s) are incorporated into one nanoparticle. For example, the hybrid nanoparticles produced using the methods contain one or more hydrophobic compounds and one or more water-soluble drugs. For example, the hybrid nanoparticles produced using the methods contain one or more hydrophobic compounds and one or more water-soluble nutraceuticals.

[0023] “Nanoparticles” is used to generally refer to nanoparticles formed from a single compound or two or more compounds (such as two or more hydrophobic compounds, or at least one hydrophobic compound and one or more water soluble compounds). When the nanoparticles are formed from two or more compounds, the nanoparticles are also referred to as “hybrid nanoparticles”.

[0024] In some forms, hydrophobic compounds suitable for use in the methods for forming the nanoparticles contain a hydrophobic domain and a hydrophilic domain; however, the overall structure of the compound makes it hydrophobic (e.g., having a water solubility of ≤ 100 μg / mL). Exemplary hydrophobic domains in the hydrophobic compounds include, but are not limited to, aromatic rings and polycyclic systems (e.g., phenyl rings, fused rings, bulky rings, etc.), alkylated or halogenated substituents, esterified / ether linkages with long hydrocarbon chains, steroid backbones, hydrocarbon chains, hydrocarbon rings, and other nonpolar functional groups.

[0025] Exemplary hydrophilic domains in the hydrophobic compounds include, but are not limited to, ionizable groups (e.g., carboxyl, amine, phosphate, and sulfonate), strong hydrogen bond donors / acceptors (e. g. hydroxyl, phenolic, carbonyl groups), polyhydroxy and carbohydrate groups, ethers and polyethers, amides, ureas, quaternary ammonium / zwitterionic groups, other polar groups (e.g., sulfhydryl), and moieties containing such polar group(s).

[0026] As demonstrated using imaging techniques, such as Transmission Electron Microscopy described in the Examples, nanoparticles produced using the methods have tightly packed structures assembled by molecules of the hydrophobic compound(s). The nanoparticles are highly organized with hydrophobic domain of the molecules in the center and the hydrophilic domain of the molecules facing out (e.g., negatively charged). For example, the nanoparticles have a micelle-like 3

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[0028] structure formed by molecules of the hydrophobic compound(s) containing a hydrophobic domain and a hydrophilic domain, where the hydrophobic domain faces inward to form a non-polar core, and the hydrophilic domain faces outward. When molecules of more than one hydrophobic compound form a nanoparticle, the nanoparticle contains the hydrophobic domains of the molecules of different hydrophobic compounds in the center and the hydrophilic domains of the molecules facing out (e.g., negatively charged).

[0029] The surface charge of nanoparticles obtained using the disclosed methods is typically strong (< -20 mV, such as < -30 or -40 mV) and consistent in different batches. When suspended in water or an aqueous solution, the hydrophilic moiety of these self-assembled nanoparticles faces the aqueous phase, forming a negatively charged surface. The nanoparticles can expel each other in water or aqueous solutions such as normal saline and phosphate buffer saline, thereby forming a well dispersed suspension. Thus, the nanoparticles produced using the methods are formed by the hydrophobic compound(s) and optionally water soluble compound(s) alone in a concentrated stock - they are not engineered nor encapsulated, and thus are without the need for other components, such as surfactant, polymer, encapsulation, or metals. For example, the nanoparticles produced using the methods only contain the hydrophobic compounds and optionally (a) water soluble compound(s) and / or (b) a small amount of residual organic solvent(s) used in one or more of the steps, such as in the mixing steps, dilution steps, etc. When the nanoparticles contain residue solvent(s), the amount of the residue solvent or total amount of the residue solvents is <100000 ppm, such as <50000 ppm or <5000 ppm. For example, the nanoparticles contain residue solvent(s) with an amount or a total amount <5000 ppm. Accordingly, the disclosed methods are simple, economical, and fast, without the need of any specialized equipment; and can produce nanoparticles of hydrophobic compounds with consistent size range and stability.

[0030] Nanoparticles of hydrophobic compounds produced using the disclosed methods can significantly increase the water solubility of hydrophobic compounds, allowing increased solubility and bioavailability in their own nanoparticle form. The nanoparticles can be provided in dry form or in liquid form and are stable. The dry powder form of the nanoparticles is stable at room temperature. When provided in dry form, these nanoparticles are easy to suspend in water and other aqueous solutions. The nanoparticles can be used in a variety of formulations, such as oral, topical, nasal, inhalation, injections, etc. For example, EC16 nanoparticles can be used in various formulations, drugs, and consumer products for antiviral / virucidal, anti-biofilm, anti-inflammatory, anti-neurodegeneration, antiaging, and sporicidal purposes.

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[0033] The nanoparticles can be formulated into pharmaceutical compositions or nutraceutical compositions. Optionally, the pharmaceutical and nutraceutical compositions containing the nanoparticles further contain one or more suitable carrier(s) and / or excipient(s). In some forms, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a suitable form, such as a pill, capsule, tablet, dry powder, or liquid form, for oral administration to a subject. For example, the composition is in a liquid form (e.g., a ready-to-drink liquid), dry powder form (e.g., a drink mix), capsule form, or tablet form, that is suitable for oral administration. In some other forms, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a suitable form, such as a dry powder or liquid form, for intranasal or pulmonary administration to a subject.

[0034] In particular, the improved solubility of the hydrophobic compounds formulated into nanoparticles allows for liquid solution or suspension thereof for oral administration or intravenous or subcutaneous injection or infusion. For example, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a liquid form (e.g., a ready-to-drink liquid) or dry powder form (e.g., a drink mix for mixing in water or aqueous beverages) that is suitable for oral administration. For example, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a liquid form or dry powder form contained in an inhaler or a nasal device that is suitable for administration to the airway (e.g., to the lung or within the nose) of a subject.

[0035] In some forms, the method for producing nanoparticles of a hydrophobic compound includes: (i) mixing the hydrophobic compound in a first organic solvent to produce a first mixture. In some forms, hybrid nanoparticles are produced where step (i) includes mixing two or more compounds that includes at least one hydrophobic compound. For example, two or more hydrophobic compounds are mixed in step (i) to produce the first mixture. For example, one or more hydrophobic compound(s) are mixed with one or more water-soluble compound(s) in step (i) to produce the first mixture.

[0036] The first organic solvent can be any suitable non-toxic (e.g., solvents that are

[0037] generally recognized as safe or “GRAS”) organic solvent, such as organic solvents with or without one or more hydroxyl group(s). Suitable organic solvents for use in the method are typically volatile (e.g., organic solvents having a boiling point < 85 °C). Exemplary organic solvents suitable for use in the methods include, but are not limited to, ethanol, isopropanol, glycerol, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, and chloroform, and combinations thereof.

[0038] The mixing in step (i) can be performed for a time period of from about 10 seconds to about 20 minutes, from about 10 seconds to about 15 minutes, from about 10 seconds to about 10

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[0041] minutes, from about 10 seconds to about 5 minutes, from about 30 seconds to about 20 minutes, from about 30 seconds to about 15 minutes, from about 30 seconds to about 10 minutes, from about 30 seconds to about 5 minutes, from about 1 min to about 20 minutes, from about 1 min to about 15 minutes, from about 1 min to about 10 minutes, or from about 1 min to about 5 minutes. For example, the mixing in step (i) is performed for equals to or less than 5 mins, optionally using a stirring bar.

[0042] For example, in step (i), the hydrophobic compound is mixed in a first organic solvent for up to 5 minutes optionally using s stirring bar. For example, in step (i), two or more hydrophobic compounds are mixed in a first organic solvent for up to 5 minutes optionally using s stirring bar. For example, in step (i), one or more hydrophobic compound(s) and one or more water soluble compound(s) are mixed in a first organic solvent for up to 5 minutes optionally using s stirring bar.

[0043] Optionally, following step (i), the method includes a step (a), allowing the first mixture to stabilize at a suitable temperature, such as room temperature (20 to 25°C at 1atm), for a suitable time period to allow self-assembly of the molecules of the hydrophobic compound(s) to form nanoparticles. When one or more water soluble compound(s) are used, the stabilization step (a) also allows the molecules of the water soluble compound(s) to incorporate in the nanoparticles as carried in by the molecules of the hydrophobic compound(s).

[0044] Optionally, the method can further include, following step (i) or step (a), a step (ii) mixing the first mixture with a second organic solvent to produce a second mixture. The second organic solvent can be any suitable non-toxic organic solvent, such as any one of those described above for the first organic solvent. When a second organic solvent is used, the first and second organic solvents can be the same or different. The first mixture and the second organic solvent can be mixed at any suitable volume ratio, depending on the desired nanoparticle concentrations.

[0045] Optionally, following step (ii), the method includes a step (b), allowing the second mixture to stabilize at a suitable temperature, such as room temperature (20 to 25°C at 1atm), for a suitable time period to allow self-assembly of the molecules of the hydrophobic compound(s) to form nanoparticles. When one or more water soluble compound(s) are used, the stabilization step (b) also allows the molecules of the water soluble compound(s) to incorporate in the nanoparticles as carried in by the molecules of the hydrophobic compound(s).

[0046] The afore-mentioned steps can be performed at any suitable temperature, such as room temperature or an elevated temperature of up to 95 °C or up to 200°C, such as at a temperature ranging from about 20°C to 200°C, from about 20°C to about 100°C, from about 20°C to about 95°C, from about 20°C to about 80°C, from about 20°C to about 60°C, from about 20°C to about 40°C, from 6

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[0048] about 20°C to about 35 °C, from room temperature to about 200°C, from about room temperature to about 95°C, from about room temperature to about 35°C, from about 30°C to about 200°C, from about 30°C to about 100°C, or from about 30°C to about 50°C. The specific temperature used in each step can vary depending on the hydrophobic compound(s) and solvents.

[0049] Optionally, these steps are all performed at a constant temperature (also referred to as isothermal condition), such as room temperature or an elevated temperature as described above. For example, the mixing in step (i) and / or (ii) can be performed at a constant room temperature (20 to 25°C at 1atm) or elevated temperature, such as up to 95 °C or up to 200°C.

[0050] Optionally, a polythermal conditioning may be performed during or after step (i), step (a), step (ii), and / or step (b) to facilitate formation of nanoparticles. When polythermal conditioning is performed, the temperature is varied from hot (a higher temperature) to cold (a lower temperature) or vice versa. For example, during polythermal conditioning, the temperature is varied from about -80°C to about 200°C or any combination of a lower and a higher temperature in the range from about -80°C to about 200°C to facilitate formation of nanoparticles.

[0051] Typically, following step (i), (a), (ii), or (b), a product containing nanoparticles of the hydrophobic compound(s) and optionally one or more water soluble compound(s) is formed. The product can be condensed and dried to provide the nanoparticles in dry powder or semi-dry form. Typically, the dry powder or semi-dry powder of the nanoparticles is in amorphous form.

[0052] The nanoparticles of hydrophobic compound(s) and optionally water soluble compound(s) can be used in a variety of applications, such as new drug development, improvement of existing drugs, increasing bioavailability of active or functional compounds for different administration, such as oral, topical, inhalable, injectable, and nasal, etc.

[0053] BRIEF DESCRIPTION OF THE FIGURES

[0054] Figures 1A-1B are graphs showing the size and distribution of EC 16 nanoparticles prepared using Method I, without (Figure 1A) or with (Figure IB) a food-grade dispersing agent.

[0055] Figures 2A-2B are graphs showing the size and distribution of EC 16 nanoparticles prepared using Method II, without (Figure 2A) or with (Figure 2B) a food-grade dispersing agent.

[0056] Figures 3A-3B are graphs showing the size and distribution of EC 16m nanoparticles prepared using Method III, without (Figure 3A) or with (Figure 3B) a food-grade dispersing agent.

[0057] Figures 4A-4B are graphs showing the size and distribution of CBD nanoparticles prepared using Method III, without (Figure 4A) or with (Figure 4B) a food-grade dispersing agent.

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[0060] Figures 5A-5B are graphs showing the size and distribution of THC-9 nanoparticles (Figure 5A) and EC16 nanoparticles reconstituted from dried powder (Figure 5B) prepared using Method III.

[0061] Figures 6A-6B are graphs showing the size and distribution of quercetin nanoparticles prepared using Method III, without (Figure 6A) or with (Figure 6B) a food-grade dispersing agent.

[0062] Figures 7A-7B are graphs showing the size and distribution of Ivermectin nanoparticles prepared using Method III, without (Figure 7A) or with (Figure 7B) a food-grade dispersing agent.

[0063] Figures 8A-8B are graphs showing the size and distribution of retinoic acid nanoparticles prepared using Method III, without (Figure 8A) or with (Figure 8B) a food-grade dispersing agent.

[0064] Figures 9A-9B are graphs showing the size and distribution of EC 16 nanoparticles in two water-based oral rinse formulations: unflavored oral rinse (Figure 9A) and peppermint oral rinse (Figure 9B).

[0065] Figure 10 shows a representative transmission electron microscopy image of EC16 nanoparticles.

[0066] Figure 11 is a schematic illustrating the in vivo mouse study design.

[0067] Figure 12 is a graph showing the effect of EC 16 nanoparticles on P. gingivalis growth in broth culture in vitro.

[0068] Figures 13A-13B are images showing the effect of EC 16 nanoparticles (Figure 13B) on bone loss in mice with experimental periodontitis compared to a control (Figure 13A). Figure 13C is a bar graph showing the percentage of bone loss in control and EC 16 nanoparticles-treated mice.

[0069] Figure 14A is a bar graph showing that EC 16 nanoparticles decreased pg 16s mRNA expression in mouse model. Figure 14B is a graph showing that EC16 nanoparticles increased Treg count in the mouse model. Figure 14C is a graph showing the expression of IL- 17 in control animals and animals that received EC 16 NPs. Figure 14D is a series of flow cytometry scatter plots showing gating strategy for flow cytometry analysis of head / neck lymph nodes and representative scatter plots for IL-17 and FOX-P3 expression showing Treg and Thl7 populations in control animals and animals that received EC 16 NPs.

[0070] Figures 15A-15D are bar graphs showing that EC 16 nanoparticles reduced inflammatory and senescence markers in the brains of mice with experimental periodontitis: IL1B / ACTIN (Figure 15A), P16 / ACTIN (Figure 15B), P53 / ACTIN (Figure 15C), and p-Tau / GAPDH (Figure 15D).

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[0073] Figure 16A is a graph showing the zeta potential in relation to the position of the EC 16-dopamine hybrid nanoparticles. Figure 16B is a graph showing a histogram of the frequency of zeta potential for the EC16-dopamine hybrid nanoparticles.

[0074] Figure 17A is a graph showing a histogram of the diameter of EC16-dopamine hybrid nanoparticles counted as particles per mL. Figure 17B is a graph showing a histogram of the diameter of EC16-dopamine hybrid nanoparticles, where the volume is reported in nm3.

[0075] DETAILED DESCRIPTION OF THE INVENTION

[0076] I. Methods for Producing Nanoparticles of Hydrophobic Compounds

[0077] Described are methods (also referred to herein as “Facilitated Self-Assembling Technology” or “FAST”) for producing nanoparticles of hydrophobic compounds, including hydrophobic drugs, hydrophobic nutraceuticals (nutritional supplement compounds derived from foods with potential pharmacologic activity), fluorescent dyes, etc. Exemplary hydrophobic compounds that can be formulated using the methods include, but are not limited to, cannabis (Cannabidiol (CBD) and tetrahydrocannabinol (THC)), synthetic steroid drugs (medroxyprogesterone acetate and triamcinolone acetonide), azole antifungal drug (Fluconazole), anticancer drug (Paclitaxel), flavonoids (quercetin, EGCG-mono-palmitate “EC16m”), antiparasitic drug (ivermectin), fluorescent dyes (e.g., fluorescein, rhodamine, cyanine, and their derivatives), lipid-soluble vitamins, and other lipid-soluble nutritional supplements, and combinations thereof.

[0078] For example, the hydrophobic compounds that can be formulated using the methods are EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, rapamycin, oxyresveratrol, tetrahydropiperine, forskolin, tetrahydrocurcumin, or a fluorescent dye (e.g., Cyanine5), or combinations thereof.For example, the hydrophobic compounds that can be formulated using the methods are retinoic acid and tocotrienols, quercetin, ivermectin (e.g., Ivermectin B1a), rapamycin, curcumin, resveratrol, lycopene, lutein, CoQ10, procyanidin B2, rapamycin, oxyresveratrol, tetrahydropiperine, forskolin, tetrahydrocurcumin, or a fluorescent dye (e.g., Cyanine5), or combinations thereof.

[0079] The method can produce nanoparticles containing a single hydrophobic compound (such as a hydrophobic drug or a nutraceutical compound) or a combination of two or more hydrophobic compounds. When nanoparticles containing more than one hydrophobic compound are produced, the different hydrophobic compounds can be incorporated into one nanoparticle (also referred to 9

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[0081] herein as “hybrid nanoparticle”). For example, the nanoparticles produced using the methods contain (1) one or more hydrophobic drug(s) and / or one or more nutraceutical compound(s); and optionally (2) one or fluorescent organic dye(s) incorporated into one nanoparticle.

[0082] For example, the nanoparticles produced using the methods contain one or more hydrophobic drug(s) and one or more nutraceutical compound(s) incorporated into one nanoparticle. For example, the nanoparticles produced using the methods contain one or more hydrophobic drug(s) and one or more fluorescent organic dye(s) incorporated into one nanoparticle. For example, the nanoparticles produced using the methods contain one or more nutraceutical compound(s) and one or more fluorescent organic dye(s) incorporated into one nanoparticle. For example, the nanoparticles produced using the methods contain two or more hydrophobic drug(s). For example, the nanoparticles produced using the methods contain two or more nutraceutical compounds.

[0083] When the nanoparticles are hybrid nanoparticles, in some forms, the hybrid nanoparticles can contain at least one hydrophobic compound and one or more water-soluble compounds. In these forms, the hydrophobic compound(s) can serve as a carrier that incorporates the water soluble compound(s) into the hybrid nanoparticle. As such, the hydrophobic compound(s) and water soluble compound(s) are incorporated into one nanoparticle. For example, the hybrid nanoparticles produced using the methods contain one or more hydrophobic compounds and one or more water-soluble drugs. For example, the hybrid nanoparticles produced using the methods contain one or more hydrophobic compounds and one or more water-soluble nutraceuticals. For example, the hybrid nanoparticles produced using the methods contain one or more hydrophobic compounds and one or more water-soluble neurotransmitters.

[0084] The water soluble compounds used in the methods can be any suitable compounds that have a water solubility of >100 ug / mL, such as >500 ug / mL, >1 mg / niL, >5 mg / niL, or >10 mg / niL. For example, the water soluble compounds used to form the hybrid nanoparticles are water soluble neurotransmitters, such as, glycine, glutamate, gamma-aminobutyric acid (GABA), aspartate, dopamine, norepinephrine, epinephrine, serotonin, histamine, acetylcholine, oxytocin, and somatostatin. For example, the hybrid nanoparticles are formed with EC 16 as the hydrophobic compound and dopamine as the water soluble compound. For example, the hybrid nanoparticles are formed with EC 16 as the hydrophobic compound and GABA as the water soluble compound. Other exemplary water soluble compounds that are suitable for use in the methods include, but are not limited to, water-soluble vitamins, diuretics, antibiotics, beta-blockers, and analgesics / antipyretics.

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[0087] The disclosed methods are simple, economical, and fast, without the need of any non-GRAS material or specialized equipment; and can produce nanoparticles of hydrophobic compounds with consistent size range and stability. Thus, the disclosed methods can be used as a general and versatile platform for producing nanoparticles of hydrophobic compounds and optionally water soluble compounds, particularly small hydrophobic molecules. “Small molecule” generally refers to an organic molecule that is less than about 2500 Da, such as between 100 Da and 2500 Da.

[0088] Typically, small molecules are non-polymeric and / or non-oligomeric.

[0089] The term “hydrophobic,” as used herein, refers to the property of lacking affinity for or repelling water. For example, the more hydrophobic a compound, the more that compound tends to not dissolve in, not mix with, or not be wetted by water. Hydrophobicity can be quantified by measuring a compound’s partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is attained in the organic solvent than in water, the drug is considered hydrophobic. For example, if the organic solvent is octanol, then a positive log P value indicates that the compound is hydrophobic. “Hydrophobic” may also refer to a compound that when applied to a surface, such as glass, forms a contact angle with water, which is greater than the contact angle of water on a surface of glass without the compound.

[0090] In some forms, hydrophobic compounds suitable for use in the methods for forming the nanoparticles contain a hydrophobic domain and a hydrophilic domain; however, the overall structure of the compound makes it hydrophobic (e.g., having a water solubility of ≤ 100 μg / mL). Exemplary hydrophobic domains in the hydrophobic compounds include, but are not limited to, aromatic rings and polycyclic systems (e.g., phenyl rings, fused rings, bulky rings, etc.), alkylated or halogenated substituents, esterified / ether linkages with long hydrocarbon chains, steroid backbones, hydrocarbon chains, hydrocarbon rings, and other nonpolar functional groups.

[0091] Exemplary hydrophilic domains in the hydrophobic compounds include, but are not limited to, ionizable groups (e.g., carboxyl, amine, phosphate, and sulfonate), strong hydrogen bond donors / acceptors (e. g. hydroxyl, phenolic, carbonyl groups), polyhydroxy and carbohydrate groups, ethers and polyethers, amides, ureas, quaternary ammonium / zwitterionic groups, other polar groups (e.g., sulfhydryl), and moieties containing such polar group(s).

[0092] In some forms, one or more water-soluble compound(s) are used together with at least one hydrophobic compound to form hybrid nanoparticles. In these forms, the hydrophobic compound(s) can serve as a carrier that incorporates the water soluble compound(s) into the hybrid nanoparticle.

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[0095] As such, the hydrophobic compound(s) and water soluble compound(s) are incorporated into one nanoparticle.

[0096] As demonstrated using imaging techniques, such as Transmission Electron Microscopy described in the Examples, nanoparticles produced using the methods have tightly packed structures assembled by molecules of the hydrophobic compound(s). The nanoparticles are highly organized with hydrophobic domain of the molecules in the center and the hydrophilic domain of the molecules facing out (negatively charged). For example, the nanoparticles have a micelle-like structure formed by molecules of the hydrophobic compound containing a hydrophobic domain and a hydrophilic domain, where the hydrophobic domain faces inward to form a non-polar core, and the hydrophilic domain faces outward. When molecules of more than one hydrophobic compound form the nanoparticle, the nanoparticle contains the hydrophobic domains of the molecules of different hydrophobic compounds in the center and the hydrophilic domains of the molecules facing out (e.g., negatively charged).

[0097] The surface charge of nanoparticles obtained using the disclosed methods is typically strong (≤ -20 mV, such as ≤ -30 or -40 mV) and consistent in different batches. When suspended in water or an aqueous solution, the hydrophilic moiety of these self-assembled nanoparticles faces the aqueous phase, forming a negatively charged surface. The nanoparticles can expel each other in water or aqueous solutions such as normal saline and phosphate buffer saline, thereby forming a well dispersed suspension. Thus, the nanoparticles produced using the methods are formed by the hydrophobic compound(s) and optionally water soluble compound(s) alone in a concentrated stock - they are not engineered nor encapsulated, and thus are without the need for other components, such as surfactant, polymer, encapsulation, or metals.

[0098] Further, nanoparticles of hydrophobic compounds and optionally with water soluble compound(s) produced using the disclosed methods can significantly increase the water solubility of hydrophobic compounds, allowing increased solubility and bioavailability in their own nanoparticle form. The nanoparticles can be provided in dry form, semi-dry, or in liquid form and are stable. For example, the dry powder form of the nanoparticles is stable at room temperature. When provided in dry form or semi-dry form, these nanoparticles are easy to suspend in water and other aqueous solutions. The nanoparticles, in either a suspension form, semi-dry, or a dry form, can be used in a variety of formulations, such as oral, topical, nasal, inhalation, injections, etc. For example, EC 16 nanoparticles can be used in various formulations, drugs, and consumer products for antiviral / virucidal, anti-biofilm, anti-inflammatory, anti-neurodegeneration, antiaging, and sporicidal purposes.

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[0101] In some forms, the method for producing nanoparticles of a hydrophobic compound includes: (i) mixing the hydrophobic compound in a first organic solvent to produce a first mixture. In some forms, hybrid nanoparticles are produced where step (i) includes mixing two or more compounds that includes at least one hydrophobic compound. For example, two or more hydrophobic compounds are mixed in step (i) to produce the first mixture. For example, one or more hydrophobic compound(s) are mixed with one or more water-soluble compound(s) in step (i) to produce the first mixture. The mixing can be performed using any suitable techniques known in the art, such as using stirring bar and / or shaking, for example, manual shaking or shaking using a suitable device such as orbital shakers, rotators, rockers, etc.

[0102] The mixing in step (i) can be performed for a time period of from about 10 seconds to about 20 minutes, from about 10 seconds to about 15 minutes, from about 10 seconds to about 10 minutes, from about 10 seconds to about 5 minutes, from about 30 seconds to about 20 minutes, from about 30 seconds to about 15 minutes, from about 30 seconds to about 10 minutes, from about 30 seconds to about 5 minutes, from about 1 min to about 20 minutes, from about 1 min to about 15 minutes, from about 1 min to about 10 minutes, or from about 1 min to about 5 minutes. For example, the mixing in step (i) is performed for equals to or less than 5 mins, optionally using a stirring bar.

[0103] For example, in step (i), the hydrophobic compound is mixed in a first organic solvent for up to 5 minutes optionally using s stirring bar. For example, in step (i), two or more hydrophobic compounds are mixed in a first organic solvent for up to 5 minutes optionally using s stirring bar. For example, in step (i), one or more hydrophobic compound(s) and one or more water soluble compound(s) are mixed in a first organic solvent for up to 5 minutes optionally using s stirring bar.

[0104] Optionally, following step (i), the method includes a step (a), allowing the first mixture to stabilize at a suitable temperature, such as room temperature (20 to 25 °C at 1atm), for a suitable time period to allow self-assembly of the molecules of the hydrophobic compound to form nanoparticles. When one or more water soluble compound(s) are used, the stabilization step (a) also allows the molecules of the water soluble compound(s) to incorporate in the nanoparticles as carried in by the molecules of the hydrophobic compound(s). In some forms, the time period for the selfassembly process to complete ranges from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

[0105] Steps (i) and / or (a) can be performed at any suitable temperature, such as room temperature or an elevated temperature of up to 95 °C or up to 200°C, such as at a temperature ranging from about 13

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[0107] 20°C to 200°C, from about 20°C to about 100°C, from about 20°C to about 95°C, from about 20°C to about 80°C, from about 20°C to about 60°C, from about 20°C to about 40°C, from about 20°C to about 35°C, from room temperature to about 200°C, from about room temperature to about 95°C, from about room temperature to about 35 °C, from about 30°C to about 200°C, from about 30°C to about 100°C, or from about 30°C to about 50°C. The specific temperature used in each step can vary depending on the hydrophobic compound(s) and solvents.

[0108] Optionally, steps (i) and (a) are both performed at a constant temperature (also referred to as isothermal condition), such as room temperature or an elevated temperature as described above. For example, the mixing in step (i) can be performed at a constant room temperature (20 to 25°C at 1atm) or elevated temperature, such as up to 95 °C or up to 200°C.

[0109] Optionally, during or after step (i) and / or step (a), polythermal conditioning is performed to facilitate the formation of nanoparticles. When polythermal conditioning is performed, the temperature is varied from hot (a higher temperature) to cold (a lower temperature) or vice versa. The polythermal conditioning can be performed at any suitable temperatures, such as varied temperatures in the range from about -80°C to about 200°C. For example, during polythermal conditioning, the temperature is varied from about -80°C to about 200°C or any combination of a lower and a higher temperature in the range from about -80°C to about 200°C to facilitate formation of nanoparticles.

[0110] The first organic solvent can be any suitable non-toxic (e.g., GRAS solvents) organic solvent, such as organic solvents with or without one or more hydroxyl group(s). Suitable organic solvents for use in the method are typically volatile (e.g., organic solvents having a boiling point < 85 °C). Exemplary organic solvents suitable for use in the methods include, but are not limited to, ethanol, isopropanol, glycerol, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, and chloroform, and combinations thereof. The boiling points of representative exemplary volatile organic solvents are provided in Table 1 below.

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[0113] Table 1. Exemplary Volatile Solvents

[0114] Solvent Name Boiling Point (°C) Common Uses Notes on Toxicity /

[0115] Safety Ethanol 78.4 Pharmaceuticals, GRAS; low toxicity,

[0116] cosmetics, extracts flammable Isopropanol (IP A) 82.6 Disinfectants, lab GRAS; Relatively solvent safe; avoid ingestion Ethyl Acetate 77.1 Extraction, GRAS; Low acute chromatography, toxicity; common in coatings food-grade applications

[0117] Acetone 56.5 Nail polish remover, GRAS; Low chronic cleaning, labs toxicity; rapid evaporation

[0118] 2-Methyltetrahydrofuran 80 Greener alternative Derived from

[0119] (2-MeTHF) to THF renewable resources;

[0120] lower toxicity Methyl acetate 56.9 Fast-evaporating Generally low

[0121] solvent, coatings toxicity and

[0122]

[0123] biodegradable

[0124] For example, the first organic solvent used in step (i) is an organic solvent with one or more hydroxyl group(s), where the first organic solvent with one or more hydroxyl group(s) can be any suitable alcohol or a mixture of two or more alcohols. Examples of alcohols suitable for use in step (i) to form the first mixture with the hydrophobic compound include, but are not limited to, glycerol, ethanol, n-propanol, or isopropanol, or a combination thereof. Other examples of organic solvents suitable for use in step (i) to form the first mixture with the hydrophobic compound include, but are not limited to, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, or chloroform, or a combination thereof, such as a combination of acetone and chloroform, a combination of acetone and / or chloroform and one or more alcohol(s). In some forms, the organic solvent used in step (i) to form the first mixture with the hydrophobic compound is ethanol, isopropanol, glycerol, acetone, ethyl acetate, or chloroform, or a combination thereof.

[0125] Optionally, the method further includes, following step (i) or step (a), a step (ii) mixing the first mixture with a second organic solvent to produce a second mixture. The first mixture and the second organic solvent can be mixed at any suitable volume ratio, depending on the desired nanoparticle concentrations. For example, the first mixture and the second organic solvent can be mixed at a volume ratio from 1:1 to 1:50, from 1:1 to 1:20, or from 1:1 to 1:10, such as 1:9. The mixing can be performed using any suitable techniques known in the art, such as using stirring bar and / or shaking, for example, manual shaking or shaking using a suitable device such as orbital shakers, rotators, rockers, etc.

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[0128] The mixing in step (ii) can be performed for a time period of from about 1 minute to about 30 minutes, from about 1 minute to about 20 minutes, from about 1 minute to about 15 minutes, from about 1 minute to about 10 minutes, from about 2 minutes to about 30 minutes, from about 2 minutes to about 20 minutes, from about 2 minutes to about 15 minutes, from about 2 minutes to about 10 minutes, from about 5 minutes to about 30 minutes, from about 5 minutes to about 20 minutes, from about 5 minutes to about 15 minutes, or from about 5 minutes to about 10 minutes. For example, the mixing in step (ii) is performed for a time period of 5 minutes to 10 minutes, optionally by shaking.

[0129] Optionally, following step (ii), the method includes a step (b), allowing the second mixture to stabilize at a suitable temperature, such as room temperature (20 to 25°C at 1atm), for a suitable time period to allow self-assembly of the molecules of the hydrophobic compound to form nanoparticles. When one or more water soluble compound(s) are used, the stabilization step (b) also allows the molecules of the water soluble compound(s) to incorporate in the nanoparticles as carried in by the molecules of the hydrophobic compound(s). In some forms, in step (b) the time period for the self-assembly process to complete ranges from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

[0130] Steps (ii) and / or (b) can be performed at any suitable temperature, such as room temperature or an elevated temperature of up to 95 °C or up to 200°C, such as at a temperature ranging from about 20°C to 200°C, from about 20°C to about 100°C, from about 20°C to about 95°C, from about 20°C to about 80°C, from about 20°C to about 60°C, from about 20°C to about 40°C, from about 20°C to about 35°C, from room temperature to about 200°C, from about room temperature to about 95°C, from about room temperature to about 35°C, from about 30°C to about 200°C, from about 30°C to about 100°C, or from about 30°C to about 50°C. The specific temperature used in each step can vary depending on the hydrophobic compound(s) and solvents.

[0131] When steps (ii) and / or (b) are included in the method, steps (ii) and / or (b) can be performed at the same temperature as steps (i) and / or (a) or at a different temperature from steps (i) and / or (a). For example, steps (i) and (a) are performed at a first temperature and steps (ii) and (b) are performed at a second temperature, where the first temperature is the same as or different from the second temperature.

[0132] Optionally, steps (ii) and (b) are both performed at a constant temperature (also referred to as isothermal condition), such as room temperature or an elevated temperature as described above.

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[0135] For example, the mixing in step (ii) can be performed at a constant room temperature (20 to 25 °C at 1atm) or elevated temperature, such as a temperature of up to 95 °C or up to 200°C.

[0136] Optionally, during or after step (ii) and / or step (b), polythermal conditioning is also performed to facilitate the formation of nanoparticles. The polythermal conditioning can be performed at any suitable temperature. When polythermal conditioning is performed, the temperature is varied from hot (a higher temperature) to cold (a lower temperature) or vice versa. The polythermal conditioning can be performed at any suitable temperatures, such as varied temperatures in the range from about -80°C to about 200°C. For example, during polythermal conditioning, the temperature is varied from about -80°C to about 200°C or any combination of a lower and a higher temperature in the range from about -80°C to about 200°C to facilitate formation of nanoparticles.

[0137] The second organic solvent can be any suitable non-toxic (e.g., GRAS solvents) organic solvent, such as any one of those described above for the first organic solvent. When a second organic solvent is used, the first and second organic solvents can be the same or different. For example, the second organic solvent used in step (i) is an organic solvent with one or more hydroxyl group(s), where the organic solvent with one or more hydroxyl group(s) can be any suitable alcohol or a mixture of two or more alcohols, such as those described above for the first organic solvent with one or more hydroxyl group(s). Examples of alcohols suitable for use in step (ii) to form the second mixture with the hydrophobic compound include, but are not limited to, glycerol, ethanol, n-propanol, and isopropanol, and a combination thereof. Other examples of organic solvents suitable for use in step (ii) to form the second mixture with the hydrophobic compound include, but are not limited to, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, or chloroform, or a combination thereof, such as a combination of acetone and chloroform, a combination of acetone and / or chloroform and one or more alcohol(s). In some forms, the organic solvent used in step (ii) to form the second mixture with the hydrophobic compound is ethanol, isopropanol, glycerol, acetone, ethyl acetate, or chloroform, or a combination thereof.

[0138] Typically, following step (i), (a), (ii), or (b), a product containing nanoparticles of the hydrophobic compound(s) and optionally one or more water soluble compound(s) is formed. The product contains the nanoparticles, the first organic solvent, and optionally the second organic solvent. The nanoparticles can be present in any desired amount in the product, such as an amount from about 10% (w / v) to about 20% (w / v), from about 5% (w / v) to about 20% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 10% (w / v), from about 1% (w / v) to 17

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[0140] about 5% (w / v), from about 1% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), or from about 0.05% (w / v) to about 2% (w / v), in the product.

[0141] Generally, the nanoparticles can have a size distribution in a range from about 20 nm to about 1000 nm, or from about 40 nm to about 1000 nm, with a majority distributed in a range from about 100 nm to about 1000 nm or from about 50 nm to about 500 nm. In these forms, the nanoparticles can have a mean diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm. For example, the nanoparticles can have a mean diameter ranging from about 100 nm to about 300 nm. The mean diameter of the nanoparticles can be determined using known methods, for example, by using nanoparticle tracking analysis (NTA) or imaging such as TEM. The specific size distribution and mean diameter of the nanoparticles depend on the specific organic solvent used in preparation, the specific hydrophobic compound, the specific water soluble compound, the presence of dispersing agent(s), etc. More specific exemplary size distribution and mean diameters of nanoparticles are described in the Examples below.

[0142] Further, the surface charge of nanoparticles obtained using the disclosed methods is strong (< -20 mV, such as < -30 or -40 mV) and consistent in different batches. When suspended in water or an aqueous solution, a hydrophilic moiety of these self-assembled nanoparticles faces the aqueous phase, forming a negatively charged surface. The nanoparticles can expel each other in water or aqueous solutions such as normal saline and phosphate buffer saline, thereby forming a well dispersed suspension. The nanoparticles generally have a negative zeta potential that equals to or is stronger than -20 mV, indicating the stability of these nanoparticles. For example, the nanoparticles can have a zeta potential that equals to or is stronger than -20 mV or -40 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV, from about -80 mV to about -30 mV or from about -60 mV to about -30 mV, from about -80 mV to about -40 mV, or from about -60 mV to about -40 mV. Methods for measuring the zeta potential of the nanoparticles are known, such as by using a commercial zeta potential analyzer.

[0143] The product is typically stable (e.g., no or minimal flocculation, precipitation, or creaming on the shelf or in fridge for at least 1 week, 2 weeks, 1 month, 3 months, 6 months, or 1 year), and ready for further dilution using water (such as distilled water, deionized water, and / or tap water) or an aqueous solution (such as a normal saline solution) to form an aqueous suspension.

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[0146] The product can be diluted using any suitable amount of water or the aqueous solution to form the aqueous suspension with a desired nanoparticle concentration. The specific amount of water / aqueous solution used to dilute the product depends on the desired concentration of the nanoparticles, the specific solvent, etc. For example, the suspension contains a mixture of glycerol and water / aqueous solution, the amount of glycerol can range from 0.1 wt% to 99.5 wt% and the amount of water / aqueous solution can range from 0.001 wt% to 99.9 wt%, from 0.01 wt% to 99.9 wt%, from 0.1 wt% to 99.9 wt%, from 60 wt% to 99.9 wt%, from 0.001 wt% to 30 wt%, from 0.01 wt% to 30 wt%, from 0.1 wt% to 30 wt%, from 0.001 wt% to 15 wt%, from 0.01 wt% to 15 wt%, or from 0.1 wt% to 15 wt% in the aqueous suspension. The ratio of glycerol:water / aqueous solution can vary from 10:1 to 1:100 or 1:1 to 1:100, such as 1:1, 1:5, 1:10, 1:20, 1:50, or 1:100. For example, the suspension contains water or an aqueous solution, where the water or aqueous solution is present in an amount from 0.001 wt% to 99.9 wt%, from 0.01 wt% to 99.9 wt%, from 0.1 wt% to 99.9 wt%, from 60 wt% to 99.9 wt%, from 0.001 wt% to 30 wt%, from 0.01 wt% to 30 wt%, from 0.1 wt% to 30 wt%, from 0.001 wt% to 15 wt%, from 0.01 wt% to 15 wt%, or from 0.1 wt% to 15 wt% in the aqueous suspension.

[0147] After dilution using water / aqueous solution, the density of the nanoparticles can be measured using known methods. For example, the nanoparticles in an aqueous suspension can have a density ranging from 108to 1012or from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01% (w / v) to 0.1% (w / v) or from 0.01% (w / v) to 0.06% (w / v). The specific density of the nanoparticles in aqueous suspensions depends on the specific organic solvent used in preparation, the aqueous solution used for dilution, the specific hydrophobic compound, the specific water soluble compound, the presence of dispersing agent(s), etc. More specific exemplary densities of nanoparticles in aqueous suspensions are described in the Examples below.

[0148] In some forms, hybrid nanoparticles contain one or more hydrophobic compound(s) and one or more water soluble compound(s) have a density in an aqueous suspension that is significantly higher than nanoparticles formed using the water soluble compound(s) alone. For example, the density of hybrid nanoparticles containing one or more hydrophobic compound(s) and one or more water soluble compound(s) in an aqueous suspension is at least 100 times higher, at least 200 higher, at least 300 higher, at least 500 higher, or at least 1000 higher than nanoparticles formed using the same water soluble compound(s).

[0149] In some forms, the method may further include adding a dispersing agent (which, in some forms, may be considered a food additive) in the water / aqueous solution before or during dilution of 19

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[0151] the product, adding a dispersing agent in the product before dilution, or adding a dispersing agent in the aqueous suspension. Optionally, the dispersing agent may be added in the product after the selfassembly process of the hydrophobic molecules and optionally incorporation of water soluble molecules is completed. The dispersing agent may improve the separation of nanoparticles and / or prevent settling or clumping of the nanoparticles. In some forms, the dispersing agent is considered a food additive. Examples of suitable dispersing agents include, but are not limited to, carbohydrates or salts thereof (e.g., sodium carboxymethyl cellulose) and metaphosphates (e.g., trimetaphosphate, hexametaphosphate, etc.) or salts thereof (e.g., sodium trimetaphosphate, sodium hexametaphosphate, etc. For example, the dispersing agent used in the aqueous suspension is a metaphosphate, such as trimetaphosphate, hexametaphosphate, etc., or a salt thereof, such as sodium trimetaphosphate, sodium hexametaphosphate, etc., for example, sodium hexametaphosphate.

[0152] The dispersing agent, such as a metaphosphate, can be present in any suitable amount, such as from 0.0005% (w / v) to about 5% (w / v), from 0.0005% (w / v) to about 1% (w / v), from about 0.005% (w / v) to about 5% (w / v), from about 0.005% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), from about 0.05% (w / v) to about 1% (w / v), from about 0.005% (w / v) to about 0.5% (w / v), from about 0.01% (w / v) to about 0.5% (w / v), from about 0.1% (w / v) to about 0.5% (w / v), from about 0.005% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), or from about 0.1% (w / v) to about 2% (w / v) of the aqueous suspension. In some forms, the method does not use any dispersing agent.

[0153] The nanoparticles can be provided either as a suspension (nanoparticles suspended in an aqueous carrier), a semi-dry, or a dry form (e.g., dried powder) suitable for various delivery methods, such as oral, nasal, topical, injectable, etc. In some forms, the method further includes a step of condensing and drying the product to produce the nanoparticles in dry powder form.

[0154] Typically, the dry powder or semi-dry powder of the nanoparticles is in amorphous form.

[0155] Nanoparticles in dry powder form are stable and can be stored at room temperature or in fridge for at least 1 week, 2 weeks, 1 month, 3 months, 6 months, or 1 year without significant change of properties. For example, nanoparticles in dry powder form can be reconstituted using water or an aqueous solution to form a reconstituted aqueous suspension. Compared to a fresh aqueous suspension prepared by diluting a fresh product using water or the same aqueous solution, nanoparticles in the reconstituted aqueous suspension have a mean diameter and / or density similar to (within 10%) that of the fresh aqueous suspension at the same nanoparticle concentration.

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[0158] II. Compositions

[0159] The nanoparticles can be formulated into pharmaceutical compositions or nutraceutical compositions for a variety of applications. Exemplary applications of compositions containing the nanoparticles include, but are not limited to, new drug development, improvement of existing drugs, increasing bioavailability of active or functional compounds for different administration, such as oral, topical, inhalable, injectable, and nasal, etc.

[0160] Optionally, the pharmaceutical and nutraceutical compositions containing the nanoparticles further contain one or more suitable carrier(s) and / or excipient(s). In some forms, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a suitable form, such as a pill, capsule, tablet, dry powder, or liquid form, for oral administration to a subject. For example, the composition is in a liquid form (e.g., a ready-to-drink liquid), dry powder form (e.g., a drink mix), capsule form, or tablet form, that is suitable for oral administration. In some other forms, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a suitable form, such as a dry powder or liquid form, for intranasal or pulmonary administration to a subject.

[0161] In particular, the improved solubility of the hydrophobic compounds formulated into nanoparticles allows for liquid solution or suspension thereof for oral administration or intravenous or subcutaneous injection or infusion. For example, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a liquid form (e.g., a ready-to-drink liquid) or dry powder form (e.g., a drink mix for mixing in water or aqueous beverages) that is suitable for oral administration. For example, the composition (e.g., pharmaceutical composition or nutraceutical composition) is in a liquid form or dry powder form contained in an inhaler or a nasal device that is suitable for administration to the airway (e.g., to the lung or within the nose) of a subject.

[0162] A. Airway Administered Compositions

[0163] In some forms, the nanoparticles can be formulated for pulmonary or mucosal administration to the respiratory tract of a subject. The administration can include delivery of the composition to the lungs, nasal, and oral (sublingual, buccal) mucosa. In some forms, the composition contains at least one liquid pharmaceutically acceptable carrier and is in a liquid form, such as an emulsion or a suspension, for intranasal administration, such as a nasal spray.

[0164] In some forms, the nanoparticles are formulated for pulmonary delivery, such as intranasal administration or oral inhalation. Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as suspensions or solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. For administration via the upper respiratory tract, the nanoparticles can be formulated into an aqueous solution, e.g., water or 21

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[0166] isotonic saline, buffered or un-buffcrcd, or as an aqueous suspension, for intranasal administration as drops or as a spray. Such aqueous solutions or suspensions may be isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.

[0167] In some forms, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, poly vinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0168] In some forms, the composition may contain minor amounts of excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might affect or mediate penetration of the nanoparticles in tissues and that the excipients that are present in amount that do not adversely affect penetration of the nanoparticles in tissues. Preferably, the excipients are all food grade ingredients, such as dispersing agents, thickeners, sweeteners, preservatives, and / or flavorings.

[0169] An exemplary composition containing the nanoparticles for intranasal administration contains all-food ingredients: water, EC16 nanoparticles, carboxymethylcellulose sodium (CMC), sodium hexametaphosphate (SHMP), glycerol. Optionally, the EC 16 nanoparticles contained in the oral rinse are in an effective amount to inactivate >99% human coronavirus in 5 min.

[0170] B. Oral Care Compositions

[0171] In some forms, the nanoparticles can be formulated for oral care products, which may have any suitable form, such as powder, paste, gel, suspension, solution, ointment, or tablet.

[0172] Exemplary oral care products formulated using the nanoparticles include, but are not limited to, toothpaste, dental cream, gel or tooth powder, mouthwash, breath freshener, oral sprays, rinses, gargles, gums, tablets, and oral lozenges. Compositions of such oral care products are well known to those of skill, and the nanoparticles can simply be added to such compositions in an effective dose for a desired therapeutic goal (e.g., treating periodontal disease, support brain health, etc.). The 22

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[0174] specific amount of nanoparticles in the oral care compositions depend on the specific hydrophobic compounds, the therapeutic goal, and the form of the composition.

[0175] In addition to the nanoparticles, the oral care products may contain one or more of the following: abrasives, chelating agents, fluoride sources, thickening agents, buffering agents, solvents, humectants, sweetening agents, carriers, and bulking agents. Preferably, the ingredients of the composition are all food grade ingredients.

[0176] In some forms, the oral care compositions include a humectant. The humectant serves to keep toothpaste compositions from hardening upon exposure to air, to give compositions a moist feel to the mouth, and, for particular humectants, to impart desirable sweetness of flavor to toothpaste compositions. The humectant, on a pure humectant basis, can be present in a concentration from about 0% to about 70%, preferably from about 5% to about 25%, by weight of the compositions. Suitable humectants include edible polyhydric alcohols such as glycerin, sorbitol, xylitol, butylene glycol, polyethylene glycol, and propylene glycol, especially sorbitol and glycerin.

[0177] In some forms, flavoring and / or sweetening agents can also be included in the oral care compositions. Suitable flavoring agents include oil of wintergreen, oil of peppermint, oil of spearmint, clove bud oil, menthol, anethole, methyl salicylate, eucalyptol, cassia, 1 -menthyl acetate, sage, eugenol, parsley oil, oxanone, alpha-irisone, marjoram, lemon, orange, propenyl guaethol, cinnamon, vanillin, thymol, linalool, cinnamaldehyde glycerol acetal known as CGA, and mixtures thereof. Elavoring agents are generally used in the composition at levels of from about 0.001% to about 5%, by weight of the composition. Sweetening agents which can be used include sucrose, glucose, saccharin, dextrose, levulose, lactose, mannitol, sorbitol, fructose, maltose, xylitol, saccharin salts, thaumatin, aspartame, D-tryptophan, dihydrochalcones, acesulfame and cyclamate salts, especially sodium cyclamate and sodium saccharin, and mixtures thereof. A composition optionally contains from about 0.1% to about 10% of these agents, preferably from about 0.1% to about 1%, by weight of the composition.

[0178] The pH of the compositions can be adjusted through the use of buffering agents. In some forms, buffering agents can be used to adjust the pH of the compositions to a range of about 4.5 to about 9.5. Buffering agents include monosodium phosphate, trisodium phosphate, sodium hydroxide, sodium carbonate, sodium acid pyrophosphate, citric acid, and sodium citrate.

[0179] In some forms, the nanoparticles are formulated into oral care compositions, e.g., a prescription or over the counter product for use in a home, for travel, at work, in a dental office, at a hospital, etc. Such compositions include, but are not limited to mouthwashes, cleaning solutions, oral sprays, oral rinses, oral lozenges, and the like. For example, the composition containing the 23

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[0181] nanoparticles is in the form of an oral rinse that contains water and optionally one or more food grade ingredients, such as xylitol, and glycerol.

[0182] An exemplary composition containing the nanoparticles for oral care is in the form of an oral rinse containing all-food ingredients: water, EC16 nanoparticles, xylitol, and glycerol.

[0183] Optionally, the EC16 nanoparticles contained in the oral rinse are in an effective amount to treat periodontal disease and / or support brain health of a subject, such as indicated by reduced bacterial load in a saliva or plaque sample of the subject, reduced periodontal bone loss in the subject, and / or reduced inflammatory and / or senescence markers in a biological sample (e.g., blood or serum) of the subject.

[0184] C. Orally Administered Pharmaceutical Compositions

[0185] Pharmaceutical compositions that contain the nanoparticles in a form suitable for oral administration to a mammal are disclosed. The pharmaceutical composition may include one or more suitable carriers and / or one or more suitable excipients for oral administration, such as in solid or liquid forms. Representative carriers and excipients include water, ethanol, polyethylene glycol, propylene glycol, bulking agents (e.g., microcrystalline cellulose, silicon dioxide, etc.), drying agents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, and stabilizing agents, and a combination thereof. Optionally, the ingredients of the composition are all food grade ingredients.

[0186] Oral administration typically involves swallowing, so that the pharmaceutical composition containing the nanoparticles enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the pharmaceutical composition containing the nanoparticles enters the blood stream directly from the mouth.

[0187] Compositions suitable for oral administration include solid compositions such as tablets, capsules, powders, lozenges (including liquid-filled lozenges), chews, dry blend, multi- and nano-particulates, gels, films, ovules, and / or dry powder sprays.

[0188] Liquid compositions for oral administration include suspensions, solutions, syrups, and / or elixirs. Optionally, the liquid compositions are delivered in soft or hard capsules. Liquid compositions for oral administration may also be prepared by the reconstitution of a solid (e.g., a drink mix), for example, from a sachet shortly before ingestion.

[0189] Optionally, the pharmaceutical composition is in the form of a liquid, such as a solution or a suspension, and contains the nanoparticles in an aqueous medium, optionally with one or more suitable excipients for the liquid composition, such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, a suitable oil, one or more emulsifying agents, and / or

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[0192] suspending agents. When provided in a liquid form, the liquid pharmaceutical composition may be a ready-to-drink liquid.

[0193] When in the form of a liquid composition, in addition to the nanoparticles, the liquid composition may contain buffering agent, antioxidants, colorants, flavoring agents, preservatives, or taste-masking agents, or a combination thereof.

[0194] Optionally, the pharmaceutical composition is in a solid form, such as dry powder form, and contains the nanoparticles and one or more suitable excipients for a solid composition, such as one or more drying agent(s). The solid pharmaceutical composition, such as dry powder (e.g., a drink mix), may be added to water or other suitable liquid for oral administration.

[0195] Optionally, the nanoparticles are included in a fast-dissolving and / or fast-disintegrating dosage form. Optionally, the nanoparticles are provided as dry powder form, such as produced by freeze drying, spray drying, or micronization.

[0196] For pill, tablet, capsule, or dry powder composition, in addition to the disclosed nanoparticles, tablets may contain drying agents, disintegrants, binders, diluents, surface active agents, lubricants, glidants, antioxidants, colorants, flavoring agents, preservatives, or taste masking agents, or a combination thereof.

[0197] Examples of suitable drying agents for forming a pill, tablet, capsule, or dry powder composition containing the nanoparticles include, but are not limited to, silica gel and molecular sieves.

[0198] Examples of suitable disintegrants for forming a pill, tablet, capsule, or dry powder composition containing the nanoparticles include, but are not limited to, sodium starch glycolate, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Generally, the disintegrant has a concentration in a range from about 1 wt% to about 25 wt%, from about 5 wt% to about 20 wt% of the composition containing the disclosed nanoparticles.

[0199] Binders are generally used to impart cohesive qualities to a pill or tablet composition.

[0200] Suitable binders for forming a pill, tablet, capsule, or dry powder composition containing the nanoparticles include, but are not limited to, microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methylcellulose.

[0201] Suitable diluents for forming a pill, tablet, capsule, or dry powder composition containing nanoparticles include, but are not limited to, lactose (as, for example, the monohydrate, spray-dried

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[0204] monohydratc or anhydrous form), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystallinc cellulose, and starch.

[0205] Pill, tablet, capsule, or dry powder compositions containing the nanoparticles may also contain surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants, such as silicon dioxide and talc. When present, surface active agents have a concentration in a range from about 0.2 wt% to 5 wt% of the composition.

[0206] Other possible excipients included in a pill, tablet, capsule, or dry powder composition containing the nanoparticles include glidants (e.g. Talc or colloidal anhydrous silica at about 0.1 wt% to about 3 wt% of the composition), antioxidants, colorants, flavoring agents, preservatives, and taste-masking agents. When present, glidants have a concentration in a range from about 0.2 wt% to 1 wt% of the composition.

[0207] Pills or tablets, including the nanoparticles and one or more suitable excipients, may be compressed directly or by roller to form pills or tablets. Dry blends or portions of the blends may alternatively be wet-, dry-, or melt-granulated, melt-congealed, or extruded before tableting. The final pill, tablet, capsule, or dry powder composition may contain one or more layers and may be coated or uncoated.

[0208] Solid compositions containing the nanoparticles for oral administration may be formulated to be immediate and / or to provide modified release. Modified release compositions include delayed, sustained, pulsed, controlled, targeted and / or programmed release formulations.

[0209] D. Orally Administered Nutritional Supplement Compositions

[0210] In some forms, the hydrophobic compounds from which the nanoparticles are prepared can be both endogenous biochemicals and found in many foods. When water soluble compound(s) are used with hydrophobic compound(s), the water soluble compounds can also be endogenous biochemicals and found in many foods. The production of the nanoparticles does not alter the covalent bonding of their constituents. Therefore, the nanoparticles can fall under regulatory guidelines as a nutritional supplement or “nutraceutical” (a food-derived substance with potential pharmacologic activity). Pharmaceutical compositions, including their earners and excipients, disclosed above can be used as nutraceutical compositions. Powder of isolated nanopaiticles, without an excipient, is also a form of the nutraceutical composition, similar to the form of common nutraceuticals such as creatine, provided in bulk to be mixed into liquids. Nutraceutical compositions containing the disclosed nanoparticles in combination or co-administered with any other active ingredients or excipients can be selected for complementary activity in a given health condition.

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[0213] Optionally, in addition to the nanoparticles, the nutraceutical compositions further contain one or more additional active agents. In some forms, the additional active agent(s) in the composition can act additively or collectively with the nanoparticles to achieve a therapeutic or prophylactic treatment goal.

[0214] E. Parenterally Administered Pharmaceutical Compositions

[0215] In some forms, the pharmaceutical compositions containing the nanoparticles are in a form suitable for administration directly into the blood stream, into muscle, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intramuscular, and subcutaneous delivery. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0216] Parenteral compositions containing the disclosed nanoparticles are typically liquid formulations, which may be a solution, a suspension, or an emulsion. In some forms, the parenteral compositions are aqueous solutions that optionally contain excipients such as salts, carbohydrates and pH buffering agents (e.g., from about pH 6.0 to about pH 7.4, from about pH 6.5 to about pH 7.0, but, for some applications, they may be more suitably formulated as a sterile aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

[0217] Exemplary solutions for intravenous or subcutaneous injection or infusion contain the nanoparticles, water, and optionally salts or osmolytes, such as glucose or glycerol, to yield an adequately isotonic solution for systemic administration.

[0218] The preparation of parenteral compositions containing the nanoparticles is typically under sterile conditions, for example, by lyophilization, which can be accomplished using standard pharmaceutical techniques known to those skilled in the art.

[0219] F. Topically Administered Pharmaceutical Compositions

[0220] In some forms, the pharmaceutical compositions containing the nanoparticles are in a form suitable for topical application to the skin. In some forms, topical administration of pharmaceutical composition containing the disclosed nanopaiticles is suitable for local treatment of skin, for example, to improve the appearance of skin, such as by reducing wrinkels. Such topical compositions can be in various forms suitable for direct application to the skin, such as gels, creams, ointments, or patches. Optionally, the topical compositions can be in suitable forms for delivery into the skin, such as using needles or arrays of microneedles.

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[0223] Compositions for topical administration generally contain a dermatologically acceptable carrier and / or excipient that is suitable for application to the skin, has good aesthetic properties, is compatible with the active agents and any other components, and will not cause any untoward safety or toxicity concerns.

[0224] The carrier can be in a wide variety of forms. For example, emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, are useful herein. These emulsions can cover a broad range of viscosities, e.g., from about 100 cps to about 200,000 cps. These emulsions can also be delivered in the form of sprays using either mechanical pump containers or pressurized aerosol containers using conventional propellants. These carriers can also be delivered in the form of a mousse or a transdermal patch. Other suitable topical carriers include anhydrous liquid solvents such as oils, alcohols, and silicones (e.g., mineral oil, ethanol isopropanol, dimethicone, cyclomethicone, and the like); aqueous-based single phase liquid solvents (e.g., hydro-alcoholic solvent systems, such as a mixture of ethanol and / or isopropanol and water); and thickened versions of these anhydrous and aqueous-based single phase solvents (e.g. where the viscosity of the solvent has been increased to form a solid or semi-solid by the addition of appropriate gums, resins, waxes, polymers, and the like). Examples of topical carrier systems useful in the topical compositions are described in the following four references all of which are incorporated herein by reference in their entirety: “Sun Products Formulary” Cosmetics & Toiletries, vol. 105, pp. 122-139 (December 1990); “Sun Products Formulary,” Cosmetics & Toiletries, vol. 102, pp. 117-136 (March 1987); U. S. Pat. No. 5,605,894 to Blank et al., and U. S. Pat. No. 5,681,852 to Bissett.

[0225] Compositions containing the nanoparticles for topical administration may be formulated to be immediate and / or to provide modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations. Thus, the compounds may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the nanoparticles. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.

[0226] G. Cosmetic Compositions

[0227] In some forms, the compositions containing the nanoparticles are formulated as cosmetic compositions, with, optionally, one or more additional active agents, one or more cosmetically acceptable excipients, or one or more cosmetically acceptable excipients, or combinations thereof.

[0228] Carriers and excipients for the cosmetic compositions are formed with materials that are generally recognized as safe. Carriers suitable for dermatological applications, injections,

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[0231] implantations, oral administration, and other suitable routes administered to an individual without causing undesirable biological side effects or unwanted interactions. Representative carriers and excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. The carriers and excipients of the cosmetic composition can contain from about 5% to about 95% by weight of the composition, such as from about 10% to about 90% or from about 15% to about 85% by weight of the composition.

[0232] In some forms, the cosmetic compositions are formulated for topical administration. In some forms, the cosmetic composition contains nanoparticles and one or more dermatologically acceptable carrier(s) and / or excipient(s) as described above for the topical compositions.

[0233] In some forms, the cosmetic compositions are formulated for oral administration. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. The cosmetic compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the cosmetic compositions (as, for example, proteinoid microspheres reported in U. S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U. S. Pat. No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes, Chapter 10, 1979. Other forms provide liquid dosage forms for oral administration, including emulsions, solutions, suspensions, and syrups, which may contain other components such as inert diluents; adjuvants (e.g., wetting agents), emulsifying and suspending agents; and sweetening and flavoring agents.

[0234] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0235] Paragraph 1. A method for producing nanoparticles of one or more hydrophobic compound(s), comprising:

[0236] (i) mixing the one or more hydrophobic compound(s) in a first organic solvent to produce a first mixture, optionally wherein the first organic solvent is a volatile organic solvent.

[0237] Paragraph 2. The method of paragraph 1, wherein step (i) further comprises mixing two or more hydrophobic compounds.

[0238] Paragraph 3. A method for producing hybrid nanoparticles, comprising:

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[0241] (i) mixing one or more water soluble compound(s) and one or more hydrophobic compound in a first organic solvent to produce a first mixture, optionally wherein the first organic solvent is a volatile organic solvent.

[0242] Paragraph 4. The method of any one of paragraphs 1-3, further comprising (a) allowing the first mixture to stabilize at room temperature (20 to 25°C at latm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

[0243] Paragraph 5. The method of any one of paragraphs 1-4, wherein polythermal conditioning is performed during or after step (i) and / or step (a), optionally in a temperature range from about -80°C to about 200°C.

[0244] Paragraph 6. The method of any one of paragraphs 1-5, further comprising (ii) mixing the first mixture with a second organic solvent to produce a second mixture, optionally wherein the second organic solvent is a volatile organic solvent.

[0245] Paragraph 7. The method of paragraph 6, further comprising (b) allowing the second mixture to stabilize at room temperature (20 to 25 °C at 1atm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

[0246] Paragraph 8. The method of paragraphs 6 or 7, wherein polythermal conditioning is performed during or after step (ii) and / or step (b), optionally in a temperature range from about -80°C to about 200°C.

[0247] Paragraph 9. The method of any one of paragraphs 1-8, wherein the first organic solvent and the second organic solvent are independently ethanol, isopropanol, glycerol, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, or chloroform, or combinations thereof.

[0248] Paragraph 10. The method of any one of paragraphs 1-9, wherein following step (i), (a), (ii), or (b), a product comprising nanoparticles of the one or more hydrophobic compound(s) or a product comprising nanoparticles of the one or more water soluble compound(s) and the one or more hydrophobic compound(s) is formed.

[0249] Paragraph 11. The method of paragraph 10, further comprising (c) condensing and drying the product to produce the nanoparticles in dry powder form.

[0250] Paragraph 12. The method of paragraph 10 or 11, wherein the nanoparticles are formed by selfassembling of molecules of the one or more hydrophobic compound(s), and wherein the molecules of the one or more hydrophobic compound(s) comprise a hydrophobic domain and a hydrophilic domain.

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[0253] Paragraph 13. The method of any one of paragraphs 1-12, wherein the one or more hydrophobic compound(s) are one or more hydrophobic drug(s), one or more hydrophobic nutraceutical(s), and / or one or more fluorescent dye(s).

[0254] Paragraph 14. The method of paragraph 13, wherein the one or more hydrophobic compound(s) are selected from the group consisting of EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, oxyresveratrol, tetrahydropiperine, rapamycin, forskolin, and tetrahydrocurcurain.

[0255] Paragraph 15. The method of any one of paragraphs 3-14, wherein the one or more water soluble compound(s) are one or more water soluble neurotransmitter(s).

[0256] Paragraph 16. The method of any one of paragraphs 1-15, wherein the nanoparticles have a median diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm.

[0257] Paragraph 17. The method of any one of paragraphs 10-16, wherein the nanoparticles are present in an amount from about 10% (w / v) to about 20% (w / v), from about 5% (w / v) to about 20% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 10% (w / v), from about 1% (w / v) to about 5% (w / v), from about 1% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), or from about 0.05% (w / v) to about 2% (w / v), in the product.

[0258] Paragraph 18. The method of any one of paragraphs 10-17, wherein the nanoparticles have a density ranging from 108to 1012or from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01 % (w / v) to 0.1 % (w / v). Paragraph 19. The method of any one of paragraphs 10-18, wherein the nanoparticles have a zeta potential less than -20 mV or less than -50 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV.

[0259] Paragraph 20. The method of any one of paragraphs 10-19, further comprising (d) diluting the product with water or an aqueous solution to form an aqueous suspension, wherein the water or aqueous solution is present in an amount from 0.001 wt% to 99.9 wt%, from 0.01 wt% to 99.9 wt%, from 0.1 wt% to 99.9 wt%, from 60 wt% to 99.9 wt%, from 0.001 wt% to 30 wt%, from 0.01 wt%

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[0262] to 30 wt%, from 0.1 wt% to 30 wt%, from 0.001 wt% to 15 wt%, from 0.01 wt% to 15 wt%, or from 0.1 wt% to 15 wt% in the aqueous suspension.

[0263] Paragraph 21. The method of paragraph 20, further comprising (e) adding a dispersing agent in the water or aqueous solution before, during, or after step (d), optionally wherein the dispersing agent is a carbohydrate or salt thereof (e.g., sodium carboxymethyl cellulose), or a metaphosphate (e.g., trimetaphosphate, hexametaphosphate, etc.) or salt thereof (e.g., sodium trimetaphosphate, sodium hexametaphosphate, etc.), or a combination thereof.

[0264] Paragraph 22. The method of paragraph 21, wherein the dispersing agent is present in an amount from 0.0005% (w / v) to about 5% (w / v), from 0.0005% (w / v) to about 1% (w / v), from about 0.005% (w / v) to about 5% (w / v), from about 0.005% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), from about 0.05% (w / v) to about 1% (w / v), from about 0.005% (w / v) to about 0.5% (w / v), from about 0.01% (w / v) to about 0.5% (w / v), from about 0.1% (w / v) to about 0.5% (w / v), from about 0.005% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), or from about 0.1% (w / v) to about 2% (w / v) of the aqueous suspension.

[0265] Paragraph 23. The method of paragraphs 21 or 22, wherein the dispersing agent is sodium hexametaphosphate.

[0266] Paragraph 24. A method for producing a liquid product comprising nanoparticles of one or more hydrophobic compound(s), consisting of:

[0267] (i) mixing the one or more hydrophobic compound(s) in a first organic solvent to produce a first mixture, optionally wherein the first organic solvent is a volatile organic solvent;

[0268] optionally (a) allowing the first mixture to stabilize at room temperature (20 to 25 °C at 1atm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours;

[0269] optionally (ii) mixing the first mixture with a second organic solvent to produce a second mixture, optionally wherein the second organic solvent is a volatile organic solvent; and optionally (b) allowing the second mixture to stabilize at room temperature (20 to 25°C at latm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

[0270] Paragraph 25. The method of paragraph 24, wherein polythermal conditioning is performed during step (i) and / or step (ii), optionally in a temperature range from about -80°C to about 200°C.

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[0273] Paragraph 26. The method of paragraph 24 or 25, wherein the first organic solvent and the second organic solvent are independently ethanol, isopropanol, glycerol, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, or chloroform, or combinations thereof.

[0274] Paragraph 27. The method of any one of paragraphs 24-26, wherein the nanoparticles are formed by self-assembling of molecules of the one or more hydrophobic compound(s), and wherein the one or more hydrophobic compound(s) comprise a hydrophobic domain and a hydrophilic domain. Paragraph 28. The method of any one of paragraphs 24-27, wherein the nanoparticles are present in an amount from about 10% (w / v) to about 20% (w / v), from about 5% (w / v) to about 20% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 10% (w / v), from about 1% (w / v) to about 5% (w / v), from about 1% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), or from about 0.05% (w / v) to about 2% (w / v), in the liquid product.

[0275] Paragraph 29. Nanoparticles formed according to any one of paragraphs 1-28.

[0276] Paragraph 30. The nanoparticles of paragraph 29, wherein the nanoparticles do not contain surfactant, polymer, encapsulation, or metals.

[0277] Paragraph 31. Nanoparticles consisting essentially of one or more hydrophobic compounds and optionally an organic solvent in an amount <100000 ppm, <50000 ppm, or <5000 ppm.

[0278] Paragraph 32. The nanoparticles of paragraph 31, wherein the molecules of the one or more hydrophobic compounds are tightly packed in the form of micelles.

[0279] Paragraph 33. The nanoparticles of paragraph 31 or 32, wherein the molecules of the one or more hydrophobic compounds comprise a hydrophobic domain and a hydrophilic domain, and wherein the hydrophobic domain is in the center of the nanoparticles forming a core and the hydrophilic domain faces outward forming a surface of the nanoparticles.

[0280] Paragraph 34. The nanoparticles of any one of paragraphs 31-33, wherein the one or more hydrophobic compound(s) are one or more hydrophobic drug(s), one or more hydrophobic nutraceutical(s), and / or one or more fluorescent dye(s).

[0281] Paragraph 35. The nanoparticles of any one of paragraphs 31-34, wherein the one or more hydrophobic compound(s) are selected from the group consisting of EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, oxyresveratrol, tetrahydropiperine, forskolin, and tetrahydrocurcumin.

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[0284] Paragraph 36. The nanoparticles of any one of paragraphs 31-35, wherein the nanoparticles have a median diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm.

[0285] Paragraph 37. The nanoparticles of any one of paragraphs 31-36, wherein the nanoparticles have a density ranging from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01% (w / v) to 0.1% (w / v).

[0286] Paragraph 38. The nanoparticles of any one of paragraphs 31-37, wherein the nanoparticles have a zeta potential less than -20 mV or less than -50 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV.

[0287] Paragraph 39. Nanoparticles consisting essentially of one or more hydrophobic compounds, one or more water soluble compounds, and optionally an organic solvent in an amount <100000 ppm, <50000 ppm, or <5000 ppm.

[0288] Paragraph 40. The nanoparticles of paragraph 39, wherein the molecules of the one or more hydrophobic compounds are tightly packed in the form of micelles.

[0289] Paragraph 41. The nanoparticles of paragraph 39 or 40, wherein the molecules of the one or more hydrophobic compounds comprise a hydrophobic domain and a hydrophilic domain, and wherein the hydrophobic domain is in the center of the nanoparticles forming a core and the hydrophilic domain faces outward forming a surface of the nanoparticles.

[0290] Paragraph 42. The nanoparticles of any one of paragraphs 39-41, wherein the one or more hydrophobic compound(s) are one or more hydrophobic drug(s), one or more hydrophobic nutraceutical(s), and / or one or more fluorescent dye(s).

[0291] Paragraph 43. The nanoparticles of any one of paragraphs 39-42, wherein the one or more hydrophobic compound(s) are selected from the group consisting of EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, oxyresveratrol, tetrahydropiperine, forskolin, and tetrahydrocurcumin.

[0292] Paragraph 44. The nanoparticles of any one of paragraphs 39-43, wherein the one or more water soluble compound(s) comprise one or more water soluble neurotransmitters.

[0293] Paragraph 45. The nanoparticles of any one of paragraphs 39-44, wherein the nanoparticles have a median diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such 34

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[0295] as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm.

[0296] Paragraph 46. The nanoparticles of any one of paragraphs 39-45, wherein the nanoparticles have a density ranging from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01% (w / v) to 0.1% (w / v).

[0297] Paragraph 47. The nanoparticles of any one of paragraphs 39-46, wherein the nanoparticles have a zeta potential less than -20 mV or less than -50 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV.

[0298] Paragraph 48. A composition comprising the nanoparticles of any one of paragraphs 29-47.

[0299] Paragraph 49. The composition of paragraph 48, in the form of a dry powder or suspension for intranasal administration, optionally wherein the composition is in the form of a nasal spray.

[0300] Paragraph 50. The composition of paragraph 49, comprising or consisting of water, glycerol, epigallocatechin-3-gallate-palmitates nanoparticles, carboxymethylcellulose sodium, and sodium hexametaphosphate.

[0301] Paragraph 51. The composition of paragraph 48, in the form of a solution or suspension for oral care, optionally wherein the composition is in the form of an oral rinse.

[0302] Paragraph 52. The composition of paragraph 51, comprising or consisting of water, epigallocatechin-3-gallate-palmitates nanoparticles, xylitol, and glycerol.

[0303] Paragraph 53. The composition of paragraph 48, in the form of a dry powder for oral administration, optionally wherein the composition is in the form of a drink mix.

[0304] Paragraph 54. The composition of any one of paragraphs 48-53, wherein the composition is a nutraceutical composition.

[0305] More specific exemplary nanoparticles and method for producing the nanoparticles are described in the Examples below.

[0306] Examples

[0307] Example 1. Preparation of Nanoparticles of Hydrophobic Compounds using Facilitated Selfassembling Technology (FAST) to Increase the Solubility and Bioavailability Thereof Materials and Methods

[0308] Epigallocatechin-3-Gallate-Palmitates (EC16) and Epigallocatechin-3-Gallate-Mono-Palmitate (EC 16m) were obtained from Camellix, LLC (Evans, GA, USA). Delta-9-Tetrahydrocannabinol (THC-9, 06-722-453), Quercetin hydrate, 95% (AC174070100), Ivermectin (AAJ6277703), and Retinoic Acid, All Trans Isomer (MP021902695) were purchased from Thermo 35

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[0310] Fisher Scientific Inc. (Waltham, MA, USA). Cannabidiol (CBD) powder was purchased from Amazon.com. Both unflavored and peppermint flavored oral rinse were provided by International Nutrition, Inc. (Middle River, MD, USA).

[0311] Preparation of Nanoparticles

[0312] Nanoparticles of the compounds were prepared using FAST with three methods (I, II and III). Poly thermal conditioning was used in one of the methods and single organic solvent with hydroxyl groups was used in another method. Methods I and II were used for EC 16 nanoparticle preparation and Method III was used in EC 16m and other nanoparticle preparations. All nanoparticle stocks at 1 % were stabilized in pure glycerol for further dilution with sterilized double distilled water. In addition, a food grade dispersing agent was used in the water nanosuspensions of some nanoparticles as described.

[0313] An exemplary method is outlined below:

[0314] 1. The hydrophobic compound, such as EC 16 or another hydrophobic compound described herein, was mixed in a first organic solvent with hydroxyl group(s) (-OH) at 10-20%.

[0315] 2. The mixture from step 1 was added to a second organic solvent with hydroxyl groups at 1:9 ratio (mixture: second organic solvent), followed by gentle mixing. The EC16 concentration was diluted to 1% or 2%.

[0316] 3. The mixture from step 2 was stabilized in room temperature (22 °C) for 12 hours to allow self-assembling to complete.

[0317] EC 16 nanoparticle stock of 1% or 2% in the organic solvents was obtained. Characterization was performed by dilution of the EC16 stock (30 to 60 billion nanoparticles per ml) to 0.01% before ZetaView analysis. EC16 nanoparticles under 1 pm in size (median size 150 ± 100 nm) with size ranging from 100 to 400 nm were generated. The stability of the EC 16 nanoparticles was also evaluated.

[0318] The oral rinse formulations were mixed directly with the EC16 nanoparticle stock to 0.01%. EC16 nanoparticle powder was obtained by centrifugation of 0.1% EC16 water nanosuspension at 13,400 RPM for 45 minutes, followed by washing with purified water twice before final centrifugation. Subsequently, pellets were collected and dried. The EC 16 nanoparticle powder was then reconstituted with water for analysis.

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[0321] Structures of the exemplary hydrophobic compounds are illustrated below.

[0322] Quercetin Ivermectin

[0323] I3CU H3c t 'CH3

[0324] CHs O

[0325]

[0326] Retinoic Acid

[0327] Evaluation of Particle Size Distribution

[0328] ZetaView nanoparticle tracking analysis was performed according to a method described previously

[0012] , The particle size distribution and concentration were measured using the ZetaView

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[0330] x20 (Particle Metrix, Meerbusch, Germany) and corresponding software. The measuring range for particle diameter was 10-2000 nm. All samples were diluted by the same volume of lx PBS and then loaded into the cell. Particle information was collected from the instrument at 11 different positions across the cell, with two cycles of readings. Standard operating method was set to a temperature of 23 °C, a sensitivity of 70, a frame rate of 30 frames per second, and a shutter speed of 100. The post-acquisition parameters were set to a minimum brightness of 20, a maximum area of 1000, a minimum area of 10, and a trace length of 15

[0030] .

[0331] Electron microscopy imaging of EC 16 nanoparticles.

[0332] The 1% EC16 nanoparticle stock was diluted with PBS to 0.01% and fixed in 4% paraformaldehyde and 2% glutaraldehyde. After mixing, 5 pl of the sample was removed and transferred to a Formvar / Copper 200 mesh grid and allowed to dry for 15 minutes. The excess solution was then removed using filter paper. Particles were negatively stained by the addition of 5 pl of 2% aqueous uranyl acetate. Multiple images were captured from each sample in a JEM 1400Flash Transmission Electron Microscope (JEOL, Peabody, MA) at 120kV, using a Gatan OneView Digital Camera (Gatan Inc., Pleasanton, CA).

[0333] Results

[0334] Both Methods I and II simplified Method III, and can produce EC16 nanoparticles with high stability, consistent with Method III. All three methods are simple, economical, require a short time (<30 min), and little equipment.

[0335] Exemplary ZetaView data from EC 16 nanoparticles and CBD nanoparticles are: Fl 8 (FSAM Method III) 0.2% EC16 nanoparticles in saline - Median size: 173 nm; and CBD (0.06% in water) nanoparticles - Median size: 206 nm.

[0336] EC 16 and EC 16m Nanoparticles

[0337] Figures 1A-1B show the size distribution of EC16 nanosuspensions prepared using Method I and with a food grade dispersing agent. The median size of the EC 16 nanoparticles was 152.5 ± 78.8 nm, with a range of 95 to 218 nm. At 0.01 % w / v ECI 6, the density of the nanoparticles was 3.2xl09particles / ml, and the Zeta potential was -60.11 ± 0.59 mV (Figure 1A). When the dispersing agent was added to the water suspension, the median size of the nanoparticles became 163.8 ± 104.2 nm, ranging from 74 to 435 nm. At 0.01% w / v EC16, the density of the nanoparticles was 2.4xl09particles / ml, and the Zeta potential was -58.08 ± 0.55 mV (Figure IB).

[0338] When EC 16 nanoparticles were prepared by Method II, the median size of the nanoparticles was 128.1 nm, ranging from 66 to 143.9 nm. At 0.01% EC16, the density of the nanopaiticles is IxlO10particles / ml and the Zeta potential was -56.65 mV (Figure 2A). With the addition of the 38

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[0340] dispersing agent, the median size of the nanoparticles was 147.7 nm, ranging from 57.8 to 162.1 nm. At 0.01% EC 16, the density of the nanoparticles is 6.5x109particles / ml, and the Zeta potential was -55.22 mV (Figure 2B).

[0341] Method III was used for EC 16m preparation (Figures 3A-3B). The median size of EC 16m nanoparticles was 115.9 nm, ranging from 30 to 120.9 nm. At 0.02% EC16m, the density of the nanoparticles was 2.3xlO10particles / ml, with Zeta potential of -50.33 mV (Figure 3A). With the addition of a dispersing agent, the median size of the nanoparticles was 154.9 nm, ranging from 65 to 182.5 nm. At 0.03% EC16m, the density of nanoparticles was 4.8xl09particles / ml, and the Zeta potential was -60.56 mV (Figure 3B).

[0342] CBD Nanoparticles

[0343] The median size of CBD nanoparticles is 206 nm, ranging from 147 to 438 nm. At 0.06% CBD, the density of the nanoparticles was 4.7x108particles / ml and the Zeta potential was -51 mV (Figure 4A). With the inclusion of 1% w / v of the dispersing agent, the median size of nanoparticles was 232.3 nm, ranging from 69 to 271.9 nm. At 0.06% CBD, the density of nanoparticles was 4.7x108 particles / ml and the Zeta potential was -48.09 mV (Figure 4B).

[0344] THC-9 and Reconstituted EC16 Nanoparticles

[0345] The median size of the THC-9 nanoparticles was 232.3 nm, ranging from 149 to 605 nm. At 0.01% CBD the density of the nanoparticles is 2.2xl08particles / ml, and the Zeta potential was -38 mV (Figure 5A). The median size of the water-reconstituted EC16 nanoparticles was 141.4 nm, ranging from 49 to 178.9 nm. At 0.01% EC16 the density of nanoparticles was 1010particles / ml and Zeta potential was -56 mV (Figure 5B).

[0346] Size and Distribution of Quercetin Nanoparticles

[0347] The median size of quercetin nanoparticles was 163.8 nm, ranging from 40 to 269.8 nm. At 0.02% w / v quercetin the density of the nanoparticles was 3.2xl09particles / ml, and the Zeta potential was -62 mV (Figure 6A). With 1% w / v of the dispersing agent, the median size of nanoparticles was 184.9 nm, ranging from 69.6 to 256 nm. At 0.02% w / v quercetin the density of nanoparticles was 1.9xl09particles / ml and the Zeta potential was -38.4 mV (Figure 6B).

[0348] Size and Distribution of Ivermectin Nanoparticles

[0349] The median size of ivermectin nanoparticles was 176 nm, ranging from 115 to 420.7 nm. At 0.02% w / v ivermectin the density of the nanoparticles is 3.5xl08particles / ml with Zeta potential of -52.32 mV (Figure 7A). With 1% w / v of the dispersing agent the median size of nanoparticles was 160.6 nm, ranging from 98 to 344.5 nm. At 0.02% w / v ivermectin, the density of nanoparticles was 5xl08particles / ml and the Zeta potential was -53.92 mV (Figure 7B).

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[0352] Size and Distribution of Retinoic Acid Nanoparticles

[0353] The median size of retinoic acid nanoparticles was 147 nm, ranging from 75.5 to 341.5 nm. At 0.02% w / v retinoic acid, the density of nanoparticles was 3.7xl08particles / ml, and the Zeta potential was -48.34 mV (Figure 8A). With 1% w / v of the dispersing agent the median size of nanoparticles was 162.8 nm, ranging from 120.3 to 407.3 nm. At 0.02% w / v retinoic acid the density of nanoparticles was 7.2xl08particles / ml and the Zeta potential was -53.99 mV (Figure 8B).

[0354] Size and Distribution of EC 16 Nanoparticles in Water-Based Oral Rinse The median size of the nanoparticles in unflavored oral rinse formulation was 176.7 nm, ranging from 81 to 268 nm. At 0.01% w / v EC16 the density of the nanoparticles is 1.6xl09particles / ml and the Zeta potential was -39 mV (Figure 9A). In the peppermint flavored oral rinse formulation, the median size of nanoparticles was 121.6 nm, ranging from 103 to 126.9 nm. At 0.01% w / v EC16, the density of nanoparticles was 8.6xl08particles / ml with Zeta potential of -51.54 mV (Figure 9B).

[0355] Transmission Electron Microscopy Imaging of EC16 Nanoparticles Figure 10 shows a representative TEM image of an EC16 nanoparticle suspension fixed in 4% paraformaldehyde and 2% glutaraldehyde. The particle sizes ranged from approximately 100 nm to >300 nm, which is consistent with the EC 16 nanoparticles prepared by Method III.

[0356] Discussion

[0357] Three FAST methods were used to prepare nanoparticles of hydrophobic compounds. Further, an FDA approved, commonly used food additive dispersing agent was used in the suspension. Figure 1A shows the size distribution of EC 16 nanoparticles in water suspension; Figure IB is the size distribution of the water suspension with the dispersing agent at 1% w / v. There was no statistical difference in median size between the two formulations, but the dispersing agent altered the size range and resulted in smaller particles around 74 nm (10.1%) and 3.6% of larger particles around 435 nm; compared to the EC16 nanosuspension without the dispersing agent, which was ranging from 95 to 218 nm. Both nanosuspensions had excellent stability with zeta potentials at about -60 mV (a Zeta Potential value above +30 mV or below -30 mV is generally considered stable). Therefore, FAST Method I was suitable for preparing EC16 nanoparticles.

[0358] FAST Method II is a simplified method derived from Method III. As shown in Figure 2, the EC16 nanoparticles showed a median size of 128.1 ± 65.9 nm, with a range of 66 to 143.9 nm (Figure 2A). The dispersing agent resulted in a slightly larger median size of 147.7 ± 63.8 nm, with 40

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[0360] similar size range 57.8 to 162.1 nm (Figure 2B). The dispersing agent had little effect on the nanoparticle size, and the charges were similar (Zeta potential -56.65 ± 0.65 mV vs. -55.22 ± 0.88 mV), although slightly lower than those for particles produced using Method I. Also, both Method II suspensions had a narrower size range than that of EC 16 nanosuspensions produced using Method I (Figures 1A-1B), showing that Method II can be used for producing EC 16 nanoparticles with smaller range in size.

[0361] Method III was used to prepare EC 16m nanoparticles, as well nanosuspensions for all other compounds. Method III produced EC 16m nanoparticles with a narrow range (30 nm to 120.9 nm) (Figure 3A), compared to the nanoparticles in dispersing agent suspension (65 nm to 182.5 nm) (Figure 3B). The median size of particles was smaller in the suspension without dispersing agent (115.9 ± 57.5 nm) in comparison to the suspension with the dispersing agent (154.9 ± 77.7 nm). Interestingly, the EC 16 nanosuspension without dispersing agent had significantly more nanoparticles than the nanosuspension with the dispersing agent (2.3x1010vs. 4.6x109 / ml).

[0362] However, the EC16 nanosuspension with the dispersing agent maybe more stable, with Zeta potential exceeding -60 mV, although both nanosuspensions were stable, with Zeta potentials greater than -50 mV.

[0363] For CBD nanoparticles (Figures 4A-4B), the dispersing agent reduced the range of particle size (69 to 271 nm vs. 147 to 438 nm), but increased the median size (232.3 ± 135.3 vs. 206 ± 103.4 nm) without statistical differences. Interestingly, the two CBD nanosuspensions with 0.06% CBD had identical particle density of 4.7x108particles / ml, and a potentially high stability with a Zeta potential around -50 mV (Figures 4A-4B). Compared to the EC16 nanoparticles, the CBD nanoparticles were larger in diameter, leading to a lower particle density. The stability of the two compounds in the nanosuspensions were similarly high.

[0364] A noticeable difference was found in the nanosuspension of THC-9 without the dispersing agent. As shown in Figure 5A, the median size of the particles (232.3 ± 151.3 nm) was similar to that of CBD. However, unlike the other compounds tested, the size distribution was discontinued, with four discrete subpopulations (Figure 5A). Although the nanosuspension was stable with a Zeta potential at -38 ± 0.51 mV, this charge was lower than that of other compounds. However, the THC-9 sample was in a methanol solution at a very low concentration, which was different from other compounds that were obtained in powdered form.

[0365] To test if the nanoparticle powder produced by FAST method can be easily reconstituted in water, an EC 16 nanosuspension at 0.1% was condensed and dried, producing a white powder. The dry EC 16 nanoparticles were then reconstituted in double distilled water as a nanosuspension. At 41

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[0367] 0.01% EC16, the density of the particles reached 1x1010particles / ml, with median size of 141.4 ± 105.4 nm and size range from 49 to 178.9 nm (Figure 5B). The size distribution and Zeta potential was comparable to that of EC 16 nanoparticles prepared by Method II (Figures 2A-2B). This result demonstrates that EC 16 nanoparticles can be condensed to a powder form and reconstituted in aqueous suspensions or in dry delivery forms. This process can be used for other compounds when dry powder form is desired.

[0368] Figures 6A-6B demonstrate that quercetin was suitable for nanosuspension preparation using Method III. The results from the two nanosuspensions show that the dispersing agent caused the suspension to have a less negative charge, potentially reducing stability (Zeta potential of -38.4 mV vs. -62 mV) and a decreased particle density (1.9xl09vs. 3.2xl09particles / ml at 0.02%), compared to the nanosuspension without the dispersing agent.

[0369] The results also demonstrate that nanosuspensions of ivermectin had identical Zeta potentials (Figures 7A-7B). The particle size distributions were different. In the nanosuspension with the dispersing agent, only 14.2% of particles had diameters greater than 200 nm, while the counterpart has more than 35% particles with diameters greater than 200 nm. This effect of the dispersing agent resulted in significantly more particles (5xl08 / ml) in the presence of the dispersing agent compared to the suspension without the agent (3.5xl08 / ml) at 0.02% w / v ivermectin (Figures 7A-7B).

[0370] Retinoic acid has a hydrocarbon chain and 6-carbin ring, similar to EC 16, EC 16m, CBD, and THC. Despite that the carboxylic acid group is charged at pH 7, retinoic acid is hydrophobic with poor water solubility. The results demonstrate that retinoic acid self-assembled into nanoparticles using FAST Method III (Figures 8A-8B). The dispersing agent gave a similar Zeta potential (-54 mV) to that of nanosuspension without the dispersing agent (-48.34 mV). Further, the particle density almost doubled (7.2xl08vs. 3.7xl08 / ml) at 0.02% concentration, by shifting the distribution from larger to smaller particles as shown in Figures 8A-8B.

[0371] To determine the feasibility of using ECI 6 nanoparticles in oral care products, 0.05% and 0.005% w / v of EC 16 nanoparticles were initially tested in an unflavored oral rinse product containing erythritol, provided by International Nutrition, Inc. Both concentrations were stable and compatible with the oral rinse with particle size range of 40.7 to 251.4 nm, and median size of 170.6 ± 97.3 nm (data not shown). To further investigate the feasibility of using EC16 nanoparticles in oral rinse, the 1% stock was directly added to two oral rinse products containing xylitol. One oral rinse also contains natural peppermint flavor. As shown in Figure 9A, the unflavored oral rinse with EC 16 nanoparticles had a similar size distribution with the previously tested unflavored 42

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[0373] product, with more than 60% particles under 200 nm, resulting in a high density of 1.6x109particles / ml at 0.01% EC16. The peppermint oral rinse with EC16 nanoparticles was highly stable with Zeta potential of -51.54 mV. Further, the particle range was narrow, with most particles at around 100 to 130 nm. These results demonstrate that EC 16 nanoparticles can be easily incorporated into aqueous products with high stability.

[0374] To investigate the EC 16 nanoparticle structure after the self-assembling process, transmission electron microscopy was performed after EC 16 nanoparticles in suspension was fixed in 4% paraformaldehyde and 2% glutaraldehyde. Figure 10 shows seven nanoparticles with high polydispersity, with diameters ranging from approximately 100 nm to >300 nm. Other compounds are expected to have similar particle structures and characteristics.

[0375] Conclusions

[0376] In summary, Facilitated Self-assembling Technology (FAST) allow compounds with poor solubility and bioavailability to form nanoparticles by self-assembling to either suspend in an aqueous formulation or be in a dried powder form for a variety of delivery methods such as oral, nasal, topical, injectable, etc. Methods based on FAST can be used in drug development and drug improvement, as well as for healthcare, disease control and prevention, cosmetic, and consumer products.

[0377] References

[0378] 1. Ogden J., et al. Nanotechnology approaches to solving the problems of poorly water-soluble drugs. Drug Discov 2005, 6:71-76.

[0379] 2. Kumari, L., et al. Advancement in Solubilization Approaches: A Step towards Bioavailability Enhancement of Poorly Soluble Drugs. Life (Basel) 2023, 13:1099.

[0380] 3. Bhalani, D. V., et al. Bioavailability Enhancement Techniques for Poorly Aqueous Soluble Drugs and Therapeutics. Biomedicines 2022, 10:2055.

[0381] 4. Frank, N., et al. Feasibility Study of Developing a Saline-Based Antiviral Nanoformulation Containing Lipid-Soluble EGCG: A Potential Nasal Drug to Treat Long COVID. Viruses 2024, 16:196.

[0382] 5. Frank, N., et al. Evaluation of Novel Nasal Mucoadhesive Nanoformulations Containing Lipid-Soluble EGCG for Long COVID Treatment. Pharmaceutics 2024, 16:791.

[0383] 6. Frank, N., et al. Evaluation of Aqueous Nanoformulations of Epigallocatechin-3-Gallate-Palmitate (EC 16) Against Human Coronavirus as a Potential Intervention Drug. Biomed J Sei & Tech Res 2023, 50:2023.

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[0386] 7. Hurst, B. L., et al. Epigallocatechin-3-Gallate (EGCG) Inhibits Sars-Cov-2 Infection in Primate Epithelial Cells. Microbiol Infect Dis 2021, 5: 1-6.

[0387] 8. Frank, N., et al. Potential Therapeutic Use of EGCG-Palmitate Nanoparticles for Norovirus Infection. Biomed J Sci & Tech Res 2024, 59:2024.

[0388] 9. Rebelatto, E. R. L., et al. An update of nano-based drug delivery systems for cannabinoids: Biopharmaceutical aspects & therapeutic applications. International Journal of Pharmaceutics 2023, 635:122727.

[0389] 10. Tomou, E. M., et al. Recent Advances in Nanoformulations for Quercetin Delivery.

[0390] Pharmaceutics 2023, 15:1656.

[0391] 11. Maiara Callegaro Velho, M. C., et al. Ivermectin-Loaded Mesoporous Silica and Polymeric Nanocapsules: Impact on Drug Loading, In Vitro Solubility Enhancement, and Release Performance. Pharmaceutics 2024, 16:325.

[0392] 12. Napoli, R. F. J., et al. Advances and challenges in retinoid delivery systems in regenerative and therapeutic medicine. Nature Communications 2020, 11: 4265.

[0393] Example 2. Preparation of Nutraceutical Nanoparticles using FAST to Increase the Solubility and Bioavailability Thereof

[0394] Materials and Methods

[0395] Nanoparticles of the compounds were prepared using the FAST method as described in Example 1 above.

[0396] Results

[0397] Nutraceuticals, such as curcumin, resveratrol, etc., are known for their poor solubility and bioavailability. Formation of Quercetin nanoparticles formed using FAST are described in Example 1 above. Six additional nutraceuticals with high demand were also studied using FAST platform. The data presented in Table 2 below demonstrates that all of them formed nanoparticles with various particle size and zeta potential. Table 3 shows the modified surface charge and particle size of the nanopartides of these nine nutraceutical compounds.

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[0400] Table 2. Characterization of Nanoparticles of 9 Nutraceutical Compounds

[0401] Compound Median Size nm Zeta Potential mV Density / ml, 0.01%

[0402] Curcumin 136.6 ± 104.9 -33.72 + 0.93 4.4x 108

[0403] Resveratrol 174.0 ± 76.76 -31.75 + 1.86 0.8x108

[0404] Lycopene 184.9 ± 156.2 -20.50 + 0.87 1.7xl07

[0405] Lutein 141.7 + 53.4 -24.54 + 1.18 1.3xl09

[0406] CoQlO 315.3 + 131.8 -26.8 + 1.11 1.2xl07

[0407] Procyanidin B2 144.2 ± 120.4 -71.59 + 0 4.5x108

[0408] Quercetin 163.8 + 102.2 -62 + 1.0 1.6 xlO9

[0409] EGCG-palmitate 152.5 + 78.8 -60.11+ 0.59 3.2 xlO9

[0410] (EC 16)

[0411] Rapamycin 147.7 ± 89.1 -44.28 + 1.79 2.8 x 109

[0412]

[0413] Table 3. Characterization of Nanoparticles of 9 Nutraceutical Compounds with Modified Surface Charge and Size

[0414] Compound Median Size nm Zeta Potential mV Density / ml, 0.01%

[0415] Curcumin 166.4 ± 57.3 -45.71 ± 1.53 4.2x 108

[0416] Resveratrol 134.2 ± 55.8 -44.43 ± 1.89 1.3x108

[0417] Lycopene 149 ± 74.9 -38.86 ± 0.87 2.7x108

[0418] Lutein 154.2 ± 66.7 -32.63 ± 0.13 3.0x108

[0419] CoQlO 232.3 ± 151.3 -42.81 ± 1.71 2.3xl07

[0420] Procyanidin B2 144.2 ± 120.4 -71.59 ± 0 4.5x108

[0421] Quercetin 163.8 ± 102.2 -62 ± 1.0 1.6 xlO9

[0422] EGCG-palmitate 152.5 ± 78.8 -60.11± 0.59 3.2 xlO9

[0423]

[0424] (EC 16)

[0425] Example 3. Preparation of Hybrid Nanoparticles using FAST

[0426] Materials and Methods

[0427] Hybrid nanoparticle preparation

[0428] Cy5 and EC16 were dissolved in an organic solvent, followed by mixing in another organic solvent according to FAST method at 1:999 ratio (Cy5: EC16). The hybrid nanoparticles were purified by centrifugation method to eliminate free Cy5, free EC16, and Cy5 nanoparticles. The resulting Cy5 / EC 16 hybrid nanoparticles were analyzed for the size and zeta potential.

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[0431] Binding analysis of hybrid nanoparticles

[0432] The Cy5 / EC16 NPs were then diluted in cell culture medium DMEM at 0.1 % EC16 and 0.0001% Cy5 in hybrid NP form. To RAW cells, the hybrid NPs were added to the cell monolayer for 1 h and 2 h prior to fluorescence microscopy.

[0433] Results

[0434] The resulting hybrid Cy5 / EC16 nanoparticles had a median size of 141.9 nm with a range from 40 nm to 157 nm, whereas the Cy5 nanoparticles had a mean median of 204.6 nm with a range from 165.5 nm to 418 nm. The zeta potential of the hybrid Cy5 / EC16 nanoparticle was -43.10 mV, whereas the Cy5 nanoparticle zeta potential was -20.42 mV. Thus, the hybrid Cy5 / EC16 nanoparticles had a smaller size and stronger surface charge than the Cy5 nanoparticles. The hybrid Cy5 / EC16 nanoparticles were also stable.

[0435] The fluorescent images (data not shown) demonstrated that the Cy5 / EC16 nanoparticles successfully attached to RAW cells with increased intensity of incubation time. The control showed that no Cy5 NP was present.

[0436] Additional exemplary hybrid nanoparticles were synthesized using the same hybrid nanoparticle preparation method.

[0437] The characteristics of the exemplary hybrid nanoparticles, including median size, zeta potential, and density, are summarized in Table 4.

[0438] Table 4. Characterization of Hybrid Nanoparticles

[0439] Compounds Type of Median Size Zeta Potential Density / ml,

[0440] NPs nm mV 0.01%

[0441] EC16-Resveratrol Hybrid396.2 + 64.1 -53.29 + 0.67 lx 1010

[0442] EC16-Curcumin Hybrida85.0 + 36.9 -57.22 + 0.87 2.5.3xlO10

[0443] EC16-Res-Cur Hybrid399.9 + 79.8 -51.23 + 1.25 5.7xl09

[0444] EC 16 / Resveratrol Mixb180.0 + 155.2 -57.02 + 1.37 2.3xl09EC16 / Curcumin Mixb198.8 + 115.8 -67.45 + 1.17 5.0xl09

[0445] EC 16 / Res / Cur Mixb201.4 ± 117.7 -62.32 ± 0.92 4.7xl09

[0446] EC16-Cy5 Hybrid3141.9 + 86.8 -43.1 + 1.13 6.1 xlO10

[0447]

[0448] (Fluorescent)

[0449] aHybrid nanoparticles were produced by co-processing of more than one hydrophobic compounds to form a single nanoparticle. Thus, hybrid nanoparticles each contain molecules of two or more hydrophobic compounds incorporated into one nanoparticle.

[0450] bMix nanoparticles are produced by mixing two or more populations of nanoparticles that were independently formed using a single hydrophobic compound. Thus, the particles in the mix nanoparticles each contain molecules of a single hydrophobic compound.

[0451] These results demonstrate that hybrid nanoparticles of multiple hydrophobic compounds can be made via the FAST platform.

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[0454] Example 4. Treatment of Periodontitis with Nanoparticles Prepared using FAST Materials and Methods

[0455] Antimicrobial efficacy of EC16 NPs

[0456] Porphyromonas gingivalis (Pg, strain 381, ATCC 33277) and Fusobacterium nucleatum (Fn, ATCC 49256) were cultured anaerobically (10% H2, 10% CO2, 80% N2) in Difco™ Anaerobe Broth (BD Biosciences) at 37°C using an anaerobic glove box. Growth curves were established over 1-4 days to determine the log phase. Streptococcus gordonii DL1 (ATCC 10558) were grown under both aerobic and anaerobic conditions, with all experiments conducted anaerobically to match the polymicrobial consortium environment. The three strains were cocultured on sterile extracted human tooth root surfaces to simulate the oral environment. Initially,. S', gordonii was incubated on the root surface for 2 hours, followed by the addition of P. gingivalis and F. nucleatum at a 1:10 ratio (aerobes to anaerobes) for 12 hours. Consortium maturity was defined by a 12-hour co-culture.

[0457] EC16 NPs were used as the testing article. EC16 NPs (0.02-0.08% w / v) and vehicle controls (water / glycerol) were applied on 24-hour multispecies biofilms of Sg, Fn, and Pg grown anaerobically on sterile extracted human tooth root (6 / concentration) surfaces for 24 h as described in Method section in ref. 41, page 11, prior to EC 16 NP formula treatment and data collection.

[0458] The biofilm biomass was measured using crystal violet assay, OD600, during a 38 hour incubation with EC16 nanoparticles.

[0459] Mouse study design

[0460] Adult mice subjected to ligature-induced periodontitis were randomly divided into 2 groups (n-5 / group); control group (water) and intervention group (EC16 NPs in water at 0.02% w / v). Oral gavage with Pg was applied every other day for 21 days before tissue and sample collection for further analysis (Figure 11). After oral gavage of Cy5 only and Cy5 / EC16 (1:999) hybrid NPs, images were captured at 1, 8, 24, and 48 hours to determine the biodistribution of nanopartides. Brain IE-1, P16, P53, and p-Tau levels were determined via western blot.

[0461] Periodontitis outcome measurement

[0462] Pg load was measured via qPCR / CFU. Inflammatory bone loss indicative of periodontitis (PD) was confirmed by assessing volumetric alveolar bone loss by MicroCT using a SkyScan 1272 system with software including NRecon reconstruction®, CT An ®, and CTvol (Figures 13A-13C). Draining lymph nodes for inflammatory markers and T cell populations was assessed using flow cytometry (Figures 14A-14D). Excised gingiva was profiled for inflammatory cytokines (e.g.,

[0463] 47

[0464] 45801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCT

[0465] IL1B, IL6, and TNFa) using qPCR using immunostaining, and confocal microscopy. Brain IL- Ip, TNF-a, IL-6 levels were determined via western blot.

[0466] Safety measurement

[0467] At the end of the treatment period, a body weight chart was generated, portions of liver, gingival and brain tissues were fixed, stained, and analyzed at the Histology Core Lab for potential toxicity signs.

[0468] Results

[0469] In an in vitro study on P. gingivalis (Pg), bacteria stopped growing after one dose of EC 16 nanoparticles in the culture (Figure 12). In a mouse model, EC16 NPs reduced bacterial load, bone loss, and inflammation. EC 16 NPs, produced via the FAST platform, reduced alveolar bone loss by approximately 50% (micro-CT) (Figures 13A-13C) and reduced Pg load to baseline with EC16 NP treatment (Figure 14A).

[0470] Additional animals were administered Cy5 / EC16 hybrid NPs, Cy5 dye alone or PBS by oral gavage and then live animal imaging was performed using IVIS™ Bioluminescence Imaging to track the biodistribution of Cy5 / EC16 NP. Cy5 / EC16 hybrid NP were present in the brains of mice 24 hours after oral gavage while only traces of the Cy5 NPs alone were detected (data not shown).

[0471] Mice on drinking water with 0.02% EC 16 NPs had increased Treg population in draining submandibular and cervical lymph nodes in the neck and reduced inflammatory and senescence markers in the brains (Figures 14B-14D and 15A-15D).

[0472] The body weight was recorded during the 21 day-period with no statistical difference was found at the end of study.

[0473] These results demonstrate that EC 16 nanoparticles controlled periodontal disease and reduced brain markers of inflammation and cell arrest.

[0474] Example 5. Preparation of Hybrid Nanoparticles with Water-Soluble Compounds using FAST

[0475] Methods

[0476] Hybrid nanoparticles produced using a hydrophobic compound (EC16) and a water-soluble compound (dopamine or GABA) were produced using the FAST method. The zeta potential and diameter of these hybrid nanoparticles were measured using the sample and instrument parameters described in Table 5 and Table 6, respectively.

[0477] 48

[0478] 45801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCT

[0479] Table 5. Sample and instrument parameters used to measure the zeta potential of EC 16-dopamine hybrid nanoparticles

[0480] Parameters

[0481] Electrolyte PBS

[0482] pH 7.0

[0483] Conductivity 26.50 uS / cm

[0484] Laser Wavelength 520 nm

[0485] Filter Wavelength Scatter

[0486]

[0487] Temperature 22.98°C

[0488] Table 6. Sample and instrument parameters used to measure the diameter of EC16-dopamine hybrid nanoparticles

[0489] Parameters

[0490] Electrolyte PBS

[0491] pH 7.0

[0492] Conductivity 26.00 uS / cm

[0493] Laser Wavelength 520 nm

[0494] Filter Wavelength Scatter

[0495]

[0496] Temperature 23.06°C

[0497] Results

[0498] The results of the measurement of the zeta potential and the diameter of the EC16-dopamine hybrid nanoparticles are shown in Figures 16A-16B and Figures 17A-17B. The results of the measurement of the zeta potential of the hybrid nanoparticles shown in Figures 16A-16B are summarized in Table 7. The results of the measurement of the diameter of the hybrid nanoparticles shown in Figures 16A-16B are summarized in Table 8.

[0499] Table 7. Results from analysis of the zeta potential of EC16-dopamine hybrid nanoparticles Mobility -1.89 ± 0.05 um / sec / V / cm, @ 25°C: -1.96 um / sec / V / cm

[0500] ZP Factor 13.3 (Smoluchowski)

[0501] Zeta Potential @ 25°C: -25.17 + 0.67 mV

[0502] Zeta Potential -25.17 mV FWHM 10.15 (SL1 / 2)

[0503] Distribution

[0504] Concentration 3.2E+7 particles / mL

[0505] Dilution Factor 100

[0506] Original 3.2E+9 particles / mL

[0507]

[0508] Concentration

[0509] 49

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[0511] Table 8. Results from analysis of the diameter of EC16-dopamine hybrid nanoparticles Number Concentration Volume

[0512] Median (X50) 187.2 nm 187.2 nm 289.0 nm

[0513] StdDev 86.0 nm 85.9 nm 305.6 nm

[0514] Concentration 3.2E+7 particles / mL

[0515] Dilution Factor 100

[0516] Original 3.2E+9 particles / mL

[0517]

[0518] Concentration

[0519] Nanoparticles were produced using dopamine alone, GABA alone, dopamine with EC16, or GABA with EC16. The median size and zeta potential of the nanoparticles are shown in Table 9.

[0520] These results demonstrate that when water soluble compounds are incorporated in the nanoparticles with a hydrophobic compound (i.e., hybrid nanoparticles), the density is significantly increased compared to nanoparticles formed using the water soluble compound alone.

[0521] Table 9. Characterization of hybrid nanoparticles formed with water-soluble compounds Compound Median Size ± SD nm Zeta Potential ± SD Density / ml at 0.02% Dopamine 188.1 ± 176.2 -23.4 + 1.89 mV 2.6 xlO7

[0522] EC16 / Dopamine 1:1 146.1 ± 97.3 -39.62 ± 1.02 mV 8.1 x 109

[0523] GABA 155.6 ± 2582 -37.1 ± 2.42 mV 1.2 x 107

[0524] EC16 / GABA 1:1 177.6 ± 136.7 -34.72 ± 0.85 mV 1.7 x 1010

[0525]

[0526] 45801369.1

Claims

ATTORNEY DOCKET NO. AURI 2025-008 PCTCLAIMSI claim:

1. A method for producing nanoparticles of one or more hydrophobic compound(s), comprising:(i) mixing the one or more hydrophobic compound(s) in a first organic solvent to produce a first mixture, optionally wherein the first organic solvent is a volatile organic solvent.

2. The method of claim 1, wherein step (i) further comprises mixing two or more hydrophobic compounds.

3. A method for producing hybrid nanoparticles, comprising:(i) mixing one or more water soluble compound(s) and at least one hydrophobic compound in a first organic solvent to produce a first mixture, optionally wherein the first organic solvent is a volatile organic solvent.

4. The method of any one of claims 1-3, further comprising (a) allowing the first mixture to stabilize at room temperature (20 to 25 °C at 1atm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

5. The method of any one of claims 1-3, wherein polythermal conditioning is performed during or after step (i), optionally in a temperature range from about -80°C to about 200°C.

6. The method of any one of claims 1-3, further comprising (ii) mixing the first mixture with a second organic solvent to produce a second mixture, optionally wherein the second organic solvent is a volatile organic solvent.

7. The method of claim 6, further comprising (b) allowing the second mixture to stabilize at room temperature (20 to 25 °C at 1atm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

8. The method of claims 6, wherein polythermal conditioning is performed during or after step (ii), optionally in a temperature range from about -80°C to about 200°C.

9. The method of any one of claims 1-3, wherein the first organic solvent is ethanol, isopropanol, glycerol, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, or chloroform, or combinations thereof.

10. The method of any one of claims 1-3, wherein following step (i), a product comprising nanoparticles of the one or more hydrophobic compound(s) is formed.5145801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCT11. The method of claim 10, further comprising (c) condensing and drying the product to produce the nanoparticles in dry powder form.

12. The method of claim 10, wherein the nanoparticles are formed by self-assembling of molecules of the one or more hydrophobic compound(s), and wherein the molecules of the one or more hydrophobic compound(s) comprise a hydrophobic domain and a hydrophilic domain.

13. The method of any one of claims 1-3, wherein the one or more hydrophobic compound(s) are one or more hydrophobic drug(s), one or more hydrophobic nutraceutical(s), and / or one or more fluorescent dye(s).

14. The method of claim 13, wherein the one or more hydrophobic compound(s) are selected from the group consisting of EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin B 1 a), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, oxyresveratrol, tetrahydropiperine, rapamycin, forskolin, and tetrahydrocurcumin.

15. The method of claim 3, wherein the one or more water soluble compound(s) comprise one or more water soluble neurotransmitter(s).

16. The method of any one of claims 1-3, wherein the nanoparticles have a median diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm.

17. The method of claim 10, wherein the nanoparticles are present in an amount from about 10% (w / v) to about 20% (w / v), from about 5% (w / v) to about 20% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 10% (w / v), from about 1% (w / v) to about 5% (w / v), from about 1% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), from about 0.01 % (w / v) to about 2% (w / v), from about 0.01 % (w / v) to about 1 % (w / v), from about 0.05% (w / v) to about 5% (w / v), or from about 0.05% (w / v) to about 2% (w / v), in the product.

18. The method of claim 10, wherein the nanoparticles have a density ranging from 108to 1012or from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01% (w / v) to 0.1% (w / v).

19. The method of claim 10, wherein the nanoparticles have a zeta potential less than -20 mV or less than -50 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV.5245801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCT20. The method of claim 10, further comprising (d) diluting the product with water or an aqueous solution to form an aqueous suspension, wherein the water or aqueous solution is present in an amount from 0.001 wt% to 99.9 wt%, from 0.01 wt% to 99.9 wt%, from 0.1 wt% to 99.9 wt%, from 60 wt% to 99.9 wt%, from 0.001 wt% to 30 wt%, from 0.01 wt% to 30 wt%, from 0.1 wt% to 30 wt%, from 0.001 wt% to 15 wt%, from 0.01 wt% to 15 wt%, or from 0.1 wt% to 15 wt% in the aqueous suspension.

21. The method of claim 20, further comprising (e) adding a dispersing agent in the water or aqueous solution before, during, or after step (d), optionally wherein the dispersing agent is a carbohydrate or salt thereof (e.g., sodium carboxymethyl cellulose), or a metaphosphate (e.g., trimetaphosphate, hexametaphosphate, etc.) or salt thereof (e.g., sodium trimetaphosphate, sodium hexametaphosphate, etc.), or a combination thereof.

22. The method of claim 21, wherein the dispersing agent is present in an amount from 0.0005% (w / v) to about 5% (w / v), from 0.0005% (w / v) to about 1% (w / v), from about 0.005% (w / v) to about 5% (w / v), from about 0.005% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), from about 0.05% (w / v) to about 1% (w / v), from about 0.005% (w / v) to about 0.5% (w / v), from about 0.01% (w / v) to about 0.5% (w / v), from about 0.1% (w / v) to about 0.5% (w / v), from about 0.005% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), or from about 0.1% (w / v) to about 2% (w / v) of the aqueous suspension.

23. The method of claims 21 or 22, wherein the dispersing agent is sodium hexametaphosphate.

24. A method for producing a liquid product comprising nanoparticles of one or more hydrophobic compound(s), consisting of:(i) mixing the one or more hydrophobic compound(s) in a first organic solvent to produce a first mixture, optionally wherein the first organic solvent is a volatile organic solvent;optionally (a) allowing the first mixture to stabilize at room temperature (20 to 25 °C at 1 atm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 15 hours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours;optionally (ii) mixing the first mixture with a second organic solvent to produce a second mixture, optionally wherein the second organic solvent is a volatile organic solvent; and optionally (b) allowing the second mixture to stabilize at room temperature (20 to 25°C at latm) for a time period from 0.1 hour to 15 hours, from 0.5 hour to 15 hours, from 1 hour to 155345801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCThours, from 2 hours to 15 hours, from 5 hours to 15 hours, from 2 hours to 12 hours, or from 5 hours to 12 hours.

25. The method of claim 24, wherein polythermal conditioning is performed during step (i) and / or step (ii), optionally in a temperature range from about -80°C to about 200°C.

26. The method of claim 24, wherein the first organic solvent and the second organic solvent are independently ethanol, isopropanol, glycerol, acetone, ethyl acetate, methyl acetate, 2-methyltetrahydrofuran, or chloroform, or combinations thereof.

27. The method of claim 24, wherein the nanoparticles are formed by self-assembling of molecules of the one or more hydrophobic compound(s), and wherein the one or more hydrophobic compound(s) comprise a hydrophobic domain and a hydrophilic domain.

28. The method of claim 24, wherein the nanoparticles are present in an amount from about 10% (w / v) to about 20% (w / v), from about 5% (w / v) to about 20% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 10% (w / v), from about 1% (w / v) to about 5% (w / v), from about 1% (w / v) to about 2% (w / v), from about 0.1% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 1% (w / v), from about 0.05% (w / v) to about 5% (w / v), or from about 0.05% (w / v) to about 2% (w / v), in the liquid product.

29. Nanoparticles formed according to any one of claims 1-3 and 24.

30. The nanoparticles of claim 29, wherein the nanoparticles do not contain surfactant, polymer, encapsulation, or metals.

31. Nanoparticles consisting essentially of one or more hydrophobic compounds and optionally an organic solvent in an amount <100000 ppm, <50000 ppm, or <5000 ppm.

32. The nanoparticles of claim 31, wherein the molecules of the one or more hydrophobic compounds are tightly packed in the form of micelles.

33. The nanoparticles of claim 31, wherein the molecules of the one or more hydrophobic compounds comprise a hydrophobic domain and a hydrophilic domain, and wherein the hydrophobic domain is in the center of the nanoparticles forming a core and the hydrophilic domain faces outward forming a surface of the nanoparticles.

34. The nanoparticles of claim 31, wherein the one or more hydrophobic compound(s) are one or more hydrophobic drug(s), one or more hydrophobic nutraceutical(s), and / or one or more fluorescent dye(s).

35. The nanoparticles of claim 31, wherein the one or more hydrophobic compound(s) are selected from the group consisting of EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol 5445801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCT(CBD), tetrahydrocannabinol (THC), ivermectin (c.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, oxyresveratrol, tetrahydropiperine, forskolin, and tetrahydrocurcumin.

36. The nanoparticles of claim 31, wherein the nanoparticles have a median diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm.

37. The nanoparticles of claim 31, wherein the nanoparticles have a density ranging from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01% (w / v) to 0.1% (w / v).

38. The nanoparticles of claim 31, wherein the nanoparticles have a zeta potential less than -20 mV or less than -50 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV.

39. Nanoparticles consisting essentially of one or more hydrophobic compounds, one or more water soluble compounds, and optionally an organic solvent in an amount <100000 ppm, <50000 ppm, or <5000 ppm.

40. The nanoparticles of claim 39, wherein the molecules of the one or more hydrophobic compounds are tightly packed in the form of micelles.

41. The nanoparticles of claim 39, wherein the molecules of the one or more hydrophobic compounds comprise a hydrophobic domain and a hydrophilic domain, and wherein the hydrophobic domain is in the center of the nanoparticles forming a core and the hydrophilic domain faces outward forming a surface of the nanoparticles.

42. The nanoparticles of claim 39, wherein the one or more hydrophobic compound(s) are one or more hydrophobic drug(s), one or more hydrophobic nutraceutical(s), and / or one or more fluorescent dye(s).

43. The nanoparticles of claim 39, wherein the one or more hydrophobic compound(s) are selected from the group consisting of EGCG-mono-palmitate, tocotrienols, quercetin, cannabidiol (CBD), tetrahydrocannabinol (THC), ivermectin (e.g., Ivermectin Bia), retinoic acid, paclitaxel, fluconazole, medroxyprogesterone acetate, triamcinolone acetonide, curcumin, resveratrol, lycopene, lutein, CoQlO, procyanidin B2, oxyresveratrol, tetrahydropiperine, forskolin, and tetrahydrocurcumin.5545801369.1ATTORNEY DOCKET NO. AURI 2025-008 PCT44. The nanoparticles of claim 39, wherein the one or more water soluble compound(s) comprise one or more water soluble neurotransmitters.

45. The nanoparticles of claim 39, wherein the nanoparticles have a median diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm, such as ranging from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm, or from about 200 nm to about 300 nm.

46. The nanoparticles of claim 39, wherein the nanoparticles have a density ranging from 108to 1011nanoparticles / mL, as measured using an aqueous solution of the nanoparticles at a concentration ranging from 0.01% (w / v) to 0.1% (w / v).

47. The nanoparticles of claim 39, wherein the nanoparticles have a zeta potential less than -20 mV or less than -50 mV, such as in a range from about -80 mV to about -20 mV or from about -60 mV to about -20 mV.

48. A composition comprising the nanoparticles of any one of claims 31-47.

49. The composition of claim 48, in the form of a dry powder or suspension for intranasal administration, optionally wherein the composition is in the form of a nasal spray.

50. The composition of claim 49, comprising or consisting of water, glycerol, epigallocatechin-3-gallate-palmitates nanoparticles, carboxymethylcellulose sodium, and sodium hexametaphosphate.

51. The composition of claim 48, in the form of a solution or suspension for oral care, optionally wherein the composition is in the form of an oral rinse.

52. The composition of claim 51, comprising or consisting of water, epigallocatechin-3-gallate-palmitates nanoparticles, xylitol, and glycerol.

53. The composition of claim 48, in the form of a dry powder for oral administration, optionally wherein the composition is in the form of a drink mix.

54. The composition of claim 48, wherein the composition is a nutraceutical composition.5645801369.1

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