Process and apparatus for encapsulation of hydrophobic molecules

Abstract

The present document describes an apparatus for the encapsulation of a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material, and a process for extracting and encapsulating biological molecules from a bulk multicellular biological material.

Description

File No.: P6501 PC00PROCESS AND APPARATUS FOR ENCAPSULATION OF HYDROPHOBIC MOLECULESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from and the benefit of US Provisional Patent Applications Nos. 63 / 715,819, and 63 / 715,825, filed on November 4, 2024, and No. 63 / 843,627, filed on July 14, 2025, the specifications of which are hereby incorporated by reference in their entireties.BACKGROUND(a) Field

[0002] The subject matter disclosed generally relates to processes for the encapsulation of a hydrophobic molecule into a capsule, and more specifically to processes for the encapsulation of a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material, as well as systems for performing the encapsulation of a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material.(b) Related Prior Art

[0003] Disclosed herein are processes and system for the encapsulation of hydrophobic molecules for formulation developments utilizing molecules from one or a plurality of biological matter to formulate biological capsules encapsulating hydrophobic molecules.

[0004] The delivery of hydrophobic bioactive molecules in water-based media requires emulsification and encapsulation technologies that require surfactant and emulsifier molecules. These molecules make ultra-tiny droplets encapsulating the hydrophobic molecules in a structure that is energetically stable and structurally sufficiently resilient to always be in the state of Brownian Motion or in a colloidal state of suspension in water, without getting stuck to each other, merge, aggregate, precipitate, or flocculate together, or bind to the walls of the container over time. Many commonly used surfactants in emulsification or encapsulation excipients are derived from petroleum-based chemicals and or synthetic molecules, which are often toxic.

[0005] There is a trend for cleaner and greener alternatives to current emulsification and encapsulation systems utilizing these synthetic surfactant and emulsifier. A most preferred solution to this problem would be one completely devoid of any synthetic surfactants or emulsifiers and carrier oils used for encapsulating hydrophobic compounds of interest in current art.SUMMARY

[0006] According to an embodiment, there is provided a system for extracting and encapsulating a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material, the system comprising:File No.: P6501 PC00(a) a vacuum pump;(b) a first vessel, the first vessel having a first outlet, the first vessel being operable to contain and contact the bulk multicellular biological material with a first solvent to make a solvent contacted bulk multicellular biological material, the first vessel being fluidly connected to the vacuum pump, the first vessel being adapted to perform at least 2 cycles of (b-i) and (b- ii):(b-i) using the vacuum pump, selectively creating a negative pressure inside the first vessel containing the solvent contacted bulk multicellular biological material; and(b-ii) returning to atmospheric pressure the first vessel containing the solvent contacted bulk multicellular biological material, wherein (b-i) and (b-ii) are performed for a time sufficient to permeate the first solvent through, and to extract and separate the biological molecules from the solvent contacted bulk multicellular biological material in a mixture of soluble biological molecules in the first solvent;(c) a filter, the filter having a filter inlet and a filter outlet, the filter inlet being fluidly connected to the first outlet of the first vessel through a first connection for receiving the mixture of soluble biological molecules in the first solvent, the filter being operative to separate remaining fragments of the bulk multicellular biological material to produce a filtered mixture of soluble biological molecules in the first solvent;(d) a second vessel, the second vessel having a second inlet and a second outlet, the second inlet being fluidly connected to the filter outlet through a second connection for receiving the filtered mixture of soluble biological molecules in the first solvent, the second vessel being operable for receiving the filtered mixture of soluble biological molecules in the first solvent; and(e) a third vessel, the third vessel having a third inlet and a third outlet, the third inlet being fluidly connected to the second outlet of the second vessel through a third connection for receiving the filtered mixture of soluble biological molecules in the first solvent from the said second vessel and a solution of the hydrophobic molecule in a second solvent, thereby producing a homogeneous mixture, the third vessel being operable to contact the mixture of soluble biological molecules in the first solvent with a solution of the hydrophobic molecule in a second solvent to produce a homogeneous mixture.(f) if the bulk multicellular biological material contains one or a plurality of endogenous hydrophobic molecules of interest for encapsulation which are present along with the solubleFile No.: P6501 PC00 biological molecules in the first solvent, the second solvent may be free of the hydrophobic molecule or the first solvent may comprise the hydrophobic molecule.

[0007] The system may further comprise a fourth vessel having a fourth inlet and a fourth outlet, the fourth inlet being fluidly connected to the third outlet of the third vessel through a fourth connection for receiving the capsule encapsulating the hydrophobic molecule, the fourth vessel being operable to contact the capsule encapsulating the hydrophobic molecule with third solvent to cause formation of the capsule by self-aggregation of the biological molecules and encapsulation of the hydrophobic molecule therein, thereby producing encapsulation of the hydrophobic molecules in the aqueous liquid.

[0008] The system may further comprise a treatment unit, the treatment unit being connected to the fourth vessel for further treatment of the encapsulation of the hydrophobic molecules in the aqueous liquid, the treatment unit being selected from the group consisting of a concentrator, a freezer, a frost-free freezer, an oven, a sprayer and a gel-producing device.

[0009] The system may further comprise a liquid-liquid mixing chamber within the fourth connection, the liquid-liquid mixing chamber having a main inlet, the main inlet being fluidly connected to the fourth outlet of the fourth vessel through a fifth connection for receiving the encapsulation of the hydrophobic molecules in the aqueous liquid; a main outlet, the main outlet being fluidly connected to the fourth inlet of the fourth vessel through a second portion of the fourth connection; an auxiliary inlet, the auxiliary inlet being fluidly connected through a first portion of the fourth connection to the third outlet of the third vessel for receiving the capsule encapsulating the hydrophobic molecule; and a mixing cavity intermediate the main inlet and the main outlet, the auxiliary inlet being fluidly connected to the mixing cavity, wherein the liquid-liquid mixing chamber is operable to mix in the mixing cavity the capsule encapsulating the hydrophobic molecule received from the third vessel through the auxiliary inlet with the encapsulation of the hydrophobic molecules in the aqueous liquid received from the fourth vessel through the main inlet.

[0010] The auxiliary inlet may comprise a plurality of nozzles, the plurality of nozzles being oriented at least partially counter-flow so as to inject the encapsulating molecules and hydrophobic molecules from the third vessel against a flow of the aqueous liquid received from the fourth vessel.File No.: P6501 PC00

[0011] Alternatively, the second solvent is free of the hydrophobic molecule if the bulk multicellular biological material contains one or more endogenous hydrophobic molecules of interest for encapsulation, present in the soluble biological molecules in the first solvent, where the first solvent may comprise the hydrophobic molecule.

[0012] The vacuum pump may be operative to generate a negative pressure of a maximum of -200 kPa.

[0013] The filter may have pore size of from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about 0.45 pm, or about 0.1 pm to about 0.2 pm.

[0014] The filter may be operative to filter molecules having 103- 106Da.

[0015] The first vessel may comprise a vacuum relief valve, the vacuum relief valve being selectively operable to return to atmospheric pressure the first vessel when the first vessel is initially under the negative pressure created by the vacuum pump.

[0016] According to another embodiment, there is provided a process for the encapsulation of a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material comprising the steps of:(a) contacting the bulk multicellular biological material with a first solvent, to obtain a solvent contacted bulk multicellular biological material,(b) negative pressure-solvent extraction of the solvent contacted bulk multicellular biological material comprising at least 2 cycles of (b-i) and (b-ii):(b-i) negative pressure treatment of the solvent contacted bulk multicellular biological material; and(b-ii) return to atmospheric pressure of the solvent contacted bulk multicellular biological material; for a time sufficient to permeate the first solvent through, and extract the biological molecules from the solvent contacted bulk multicellular biological material,(c) separation of the solvent contacted bulk multicellular biological material from the first solvent, to obtain a mixture comprising soluble biological molecules in the first solvent and optionally further comprising an endogenous hydrophobic molecule present in the bulk multicellular biological material; andFile No.: P6501 PC00(d) contacting the mixture of soluble biological molecules, and optionally the endogenous hydrophobic molecule present in the bulk multicellular biological material, in the first solvent with a second solvent free of the hydrophobic molecule, or with a second solvent comprising the hydrophobic molecule and mixing thoroughly to obtain a homogeneous mixture, and(e) contacting the homogeneous mixture with a third solvent to cause formation of the capsule by self-aggregation of the soluble biological molecules and encapsulation of the hydrophobic molecule and / or the endogenous hydrophobic molecule therein, to form a capsule encapsulating the hydrophobic molecule.

[0017] The mixture of soluble biological molecules in the first solvent may comprise terpenoids, fatty-acids, amino acids, peptides, alkaloids, carbohydrates, polyketides, shikimates, phenylpropanoids, polyphenols, and combinations thereof.

[0018] The process may further comprise a further collection step (f) comprising at least one of concentration, freeze-drying, heat-drying, spray-drying and gelling, of the capsule encapsulating the hydrophobic molecule.

[0019] The negative pressure treatment may be at a maximum of -200 kPa.

[0020] The negative pressure treatment may be from about -1 kPa to about -200 kPa.

[0021] In step (c), the separation may be a filtration for removal of a remaining fragment of the bulk multicellular biological material.

[0022] The filtration may be microfiltration, ultrafiltration, or combinations thereof.

[0023] The filtration may be with a filter having pore size of from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about 0.45 pm, or about 0.1 pm to about 0.2 pm.

[0024] The filtration may be with a filter configured for filtration of molecules having 103- 106Da.

[0025] The capsule encapsulating the hydrophobic molecule may comprise a core and an outer shell layer comprised of the soluble biological molecules providing a water compatible particle stability.

[0026] The formation of the capsule encapsulating the hydrophobic molecule may be performed at a temperature of from about 10°C to 80°C.

[0027] The time sufficient for spray-drying the capsule encapsulating the hydrophobic molecule may be from about 5 minutes to about 1 hour, or from about 5 minutes to about 30 minutes,File No.: P6501 PC00 or from about 5 minutes to about 25 minutes, or from about 5 minutes to about 20 minutes, or from about 5 minutes to about 15 minutes, or from about 5 minutes to about 10 minutes; and wherein a time sufficient for freeze-drying is from about 12h to about 24h, or from about 24h to about 36h, or from about 36h to about 48h.

[0028] The freeze-drying may be performed at a temperature of -10°C to about -60°C.

[0029] The freeze-drying may be performed with a freezing excipient.

[0030] The freezing excipient may be sucrose, glucose, dextran, trehalose, lactose, mannitol, maltose, alanine, glycine or a combination thereof.

[0031] The concentration may be performed by microfiltration, ultrafiltration, dead-end filtration, tangential flow filtration, differential centrifugation, or a combination thereof.

[0032] The heat-drying may be performed at a temperature of 30°C to about 60°C.

[0033] The -drying may be performed under vacuum.

[0034] The heat-drying may be performed with a stabilizing excipient.

[0035] The stabilizing excipient may be microcrystalline cellulose, methyl-cellulose, hydroxypropyl methyl cellulose, maltodextrin, gum Arabic, Guar gum, chitosan, pectin, sucrose, lactose, trehalose, Arabic gum, whey protein, polyvinyl alcohol, modified, starch, nut gum, sodium alginate, sodium carboxymethyl cellulose, L-leucine, polyethylene glycol, or a combination thereof.

[0036] The spray-drying may be performed at a temperature of 110°C to about 165°C.

[0037] The spray-drying may be performed with a spray-drying excipient.

[0038] The spray-drying excipient may be from about 10% w / v to about 30% w / v.

[0039] The gelling may be performed with a gelling excipient.

[0040] The gelling excipient may be hyaluronic acid, methyl-cellulose, alginate, pectin, carrageenan, gellan, gelatin, agar, modified starch, methyl cellulose and hydroxypropyl methyl cellulose, xanthan gum, or a combination thereof.

[0041] The wherein step (e) may be performed by mixing the homogeneous mixture with the third solvent at a ratio of from about 1 :3 to 1 :100.

[0042] In step (d) an absolute mass ratio of the soluble biological molecules and the hydrophobic molecule may be from about 1 :10 to 2000:1.

[0043] The first solvent, the second solvent, or both may be a non-aqueous solvent completely miscible with water.File No.: P6501 PC00

[0044] In step (d), the second solvent is free of said hydrophobic molecule. Alternatively, in step (d), the second solvent comprises said hydrophobic molecule.

[0045] The non-aqueous solvent may be a solvent with a polarity index between 3.9 and 8.0.

[0046] The non-aqueous solvent may be ethanol, methanol, acetone, dimethyl sulfoxide, N,N- dimethylformamide, N-methylpyrrolidone, acetonitrile, 2-methoxyethanol, pyridine, 1 ,4-dioxane, tetrahydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.

[0047] The wherein the third solvent is an aqueous solvent.

[0048] The third aqueous solvent may be water or a mixture of water and a non-aqueous solvent.

[0049] The non-aqueous solvent may be ethanol, methanol, acetone, dimethyl sulfoxide, N,N- dimethylformamide, N-methylpyrrolidone, acetonitrile, 2-methoxyethanol, pyridine, 1 ,4-dioxane, tetrahydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.

[0050] The negative pressure treatment may be for about 0.5 second to about 5 seconds.

[0051] The negative pressure treatment may be for about 0.5 second.

[0052] The return to atmospheric pressure may be for about 0.01 second to about 1 seconds.

[0053] The return to atmospheric pressure may be for about 0.01 second.

[0054] In step (b), the time sufficient to permeate the first solvent through may be from about 3 to about 200 cycles of (b-i) and (b-ii).

[0055] The capsule encapsulating the hydrophobic molecule may have a size of about 10 pm or less.

[0056] The capsule encapsulating the hydrophobic molecule may have a size of from about 120 nm to about 10 pm.

[0057] The capsule encapsulating the hydrophobic molecule may have a polydispersity index (PDI) of less than or equal to 0.7 (< 0.7), or less than or equal to 0.3 (< 0.3).

[0058] The hydrophobic molecule may be a molecule having a solubility of < 20 mg / L of water at room temperature.

[0059] The hydrophobic molecule may have a molecular weight of < 3 kDa.

[0060] The hydrophobic molecule may be a pesticide, a fungicide, a herbicide, an antimicrobial, a drug, a nutrient, a hydrophobic plant extract, the endogenous hydrophobic molecules present in bulk multicellular biological material or combinations thereof.File No.: P6501 PC00

[0061] The bulk multicellular biological material may be from a plant, a fungus, an alga, a yeast, and combinations thereof.

[0062] The bulk multicellular biological material may contain one or plurality of endogenous hydrophobic molecules of interest for encapsulation which are present along with the soluble biological molecules, in the first solvent.

[0063] The capsule encapsulating the hydrophobic molecule may be free of an emulsifier, a carrier oil, or combinations thereof.

[0064] According to another embodiment, there is provided a turmeric nanoparticle composition comprising: a plurality of nanoparticles comprising a capsule comprising self-aggregated soluble biological molecules extracted from a turmeric rhizome material, said capsule forming a micelle around a hydrophobic core; said hydrophobic core encapsulating hydrophobic molecules endogenous to said turmeric rhizome material, said hydrophobic molecules comprising curcuminoids, said nanoparticles having a particle size of from about 150 nm to about 250 nm, a polydispersity index (PDI) of less than or equal to 0.3 (< 0.3); and a curcuminoids loading capacity of about 20% to about 30% mass of curcuminoids per total mass of said nanoparticles, and a carrier.

[0065] The turmeric nanoparticle composition may further comprise a freezing excipient, a stabilizing excipient, a spray-drying excipient, a gelling excipient, or a combination thereof.

[0066] The freezing excipient may be sucrose, glucose, dextran, trehalose, lactose, mannitol, maltose, alanine, glycine or a combination thereof.

[0067] The stabilizing excipient may be microcrystalline cellulose, methyl-cellulose, hydroxypropyl methyl cellulose, maltodextrin, gum Arabic, Guar gum, chitosan, pectin, sucrose, lactose, trehalose, Arabic gum, whey protein, polyvinyl alcohol, modified, starch, nut gum, sodium alginate, sodium carboxymethyl cellulose, L-leucine, polyethylene glycol, or a combination thereof.

[0068] The spray-drying excipient may be from about 10% w / v to about 30% w / v of gum Arabic, maltodextrin, or a combination thereof.

[0069] The gelling excipient may be hyaluronic acid, methyl-cellulose, alginate, pectin, carrageenan, gellan, gelatin, agar, modified starch, methyl cellulose and hydroxypropyl methyl cellulose, xanthan gum, or a combination thereof.

[0070] The capsule may have a size of from about 170 nm to about 240 nm.File No.: P6501 PC00

[0071] The said capsule may have a PDI of from about 0.05 to about 0.26.

[0072] The capsule or hydrophobic molecules endogenous to the turmeric rhizome material may be free of an emulsifier, a surfactant, a carrier oil, or combinations thereof.

[0073] The nanoparticles may have a curcuminoids loading capacity of about 25% mass of curcuminoids per total mass of said nanoparticles.

[0074] The nanoparticles may have an antioxidant capacity of from about 2 to about 4 times higher than an equivalent pM concentration of pure curcuminoids as measured by a Ferric Reducing Antioxidant Power (FRAP) assay.

[0075] According to another embodiment, there is provided a method of treating or preventing an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease, comprising administering a turmeric nanoparticle composition of the present invention, to a subject in need thereof.

[0076] According to another embodiment, there is provided a use of the turmeric nanoparticle composition of the present invention, for the treatment or prevention of an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease.

[0077] According to another embodiment, there is provided a turmeric nanoparticle composition of the present invention which may be for use in the treatment or prevention of an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease.

[0078] The inflammatory disease may be arthritis, or inflammatory bowel disease.

[0079] The following terms are defined below.

[0080] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”. Similarly, the word “another” may mean at least a second or more.

[0081] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0082] The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.File No.: P6501 PC00

[0083] It is noted that terms like “preferably”, “commonly”, and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0084] For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0085] The terms “formulation” or “composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Further the formulation / composition refers to the mixture wherein the substances in the mixture do not react with each other but have desirable properties as a mixture. Such term in relation to pharmaceutical composition is intended to encompass any composition made by admixing the formulation of the present invention and an acceptable carrierora pharmaceutically acceptable carrier. By “pharmaceutically acceptable” or “acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0086] The term “capsule” as used herein is intended to mean a membrane which surrounds the hydrophobic molecule. As used herein, the term “encapsulate” is intended to mean to enclose in or as if in a capsule.

[0087] The term “hydrophobic molecule” is intended to mean a molecule that is non-polar and that does not mix with water. In some embodiments, the hydrophobic molecules are defined as insoluble and sparsely soluble molecules with solubility limit less than 20 mg per litre of water at room temperature. According to another embodiment, the hydrophobic molecules may be molecules of molecular weight less than < 3 kDa. According to embodiments, the hydrophobic molecules may be pesticide, a fungicide, a herbicide, an antimicrobial, a drug, a nutrient, a hydrophobic plant extract, or combinations thereof. In embodiments, the hydrophobic molecules may be free of an emulsifier, a carrier oil, or combinations thereof. According to another embodiment, second solvent is free of the hydrophobic molecule if the bulk multicellular biological material contains one or more endogenous hydrophobic molecules of interest for encapsulation, present in the soluble biological molecules in the first solvent, where the first solvent may comprise the hydrophobic molecule.File No.: P6501 PC00

[0088] The term “emulsifier” is intended to mean substance that helps stabilize emulsions, which are mixtures of two immiscible liquids (like oil and water), by reducing the surface tension at the interface between the liquids and preventing them from separating. Emulsifiers are a type of surfactant (surface-active agent). All emulsifiers are surfactants, but not all surfactants are emulsifiers. Surfactants reduce surface or interfacial tension and may have additional roles like detergency or foaming. Natural Emulsifiers are emulsifiers derived from biological sources, often biodegradable and biocompatible. Examples include Lecithin (from soy or egg yolk), casein (milk protein), saponins (from plants), gum arabic, Starches and proteins. These emulsifiers are widely used in food, cosmetics, and pharmaceuticals for their safety and mildness. Synthetic Emulsifiers are man-made chemicals, often more efficient or stable than natural counterparts. Examples include polysorbates (e.g., Tween 20, Tween 80), sodium lauryl sulfate (SLS), PEG-based compounds, Span series (e.g., Span 80). They are common in industrial, cosmetic, and pharmaceutical formulations where higher performance or shelf-stability is needed.

[0089] The term “dispersity”, used herein refers to the measure of the heterogeneity of sizes of molecules or particles in a mixture, particularly in a mixture of particles of the present invention.

[0090] The dispersity E) was formerly referred to as the polydispersity index (PDI) is an unitless number to scale homogeneity of hydrodynamic diameter of a particle population wherein 1.0 refer to highest point of most heterogeneous particles present, 0.05 refers to most homogeneous particles present in the said formulation in water. According to embodiments of the present invention, the dispersity of the size of the particles generated by the process of the present invention is a D value equal to or of less than 0.7 (< 0.7) or equal to or of less than 0.3 (< 0.3). For example, the E) value may be from about 0.05 to about 0.7, or from about 0.1 to about 0.7, or from about 0.15 to about 0.7, or from about 0.2 to about 0.7, or from about 0.25 to about 0.7, or from about 0.3 to about 0.7, or from about 0.35 to about 0.7, or from about 0.4 to about 0.7, or from about 0.45 to about 0.7, or from about 0.5 to about 0.7, or from about 0.55 to about 0.7, or from about 0.6 to about 0.7, or from about 0.65 to about 0.7, or from about 0.05 to about 0.65, or from about 0.1 to about 0.65, or from about 0.15 to about 0.65, or from about 0.2 to about 0.65, or from about 0.25 to about 0.65, or from about 0.3 to about 0.65, or from about 0.35 to about 0.65, or from about 0.4 to about 0.65, or from about 0.45 to about 0.65, or from about 0.5 to about 0.65, or from about 0.55 to about 0.65, or from about 0.6 to about 0.65, or from about 0.05 to about 0.60, or from about 0.1 to about 0.60, or from about 0.15 to about 0.60, or from about 0.2 to about 0.60, or from about 0.25 to about 0.60, or from about 0.3 to about 0.60, or from about 0.35 to about 0.60, or from about 0.4 to about 0.60, or from about 0.45 to about 0.60, or from about 0.5 to about 0.60, or from about 0.55 to about 0.60, or from about 0.05 to about 0.55, or from about 0.1 to about 0.55, or from about 0.15 to about 0.55, or from about 0.2 toFile No.: P6501 PC00 about 0.55, or from about 0.25 to about 0.55, or from about 0.3 to about 0.55, or from about 0.35 to about 0.55, or from about 0.4 to about 0.55, or from about 0.45 to about 0.55, or from about 0.5 to about 0.55, or from about 0.05 to about 0.50, or from about 0.1 to about 0.50, or from about 0.15 to about 0.50, or from about 0.2 to about 0.50, or from about 0.25 to about 0.50, or from about 0.3 to about 0.50, or from about 0.35 to about 0.50, or from about 0.4 to about 0.50, or from about 0.45 to about 0.50, or from about 0.05 to about 0.45, or from about 0.1 to about 0.45, or from about 0.15 to about 0.45, or from about 0.2 to about 0.45, or from about 0.25 to about 0.45, or from about 0.3 to about 0.45, or from about 0.35 to about 0.45, or from about 0.4 to about 0.45, or from about 0.05 to about 0.40, or from about 0.1 to about 0.40, or from about 0.15 to about 0.40, or from about 0.2 to about 0.40, or from about 0.25 to about 0.40, or from about 0.3 to about 0.40, or from about 0.35 to about 0.40, or from about 0.05 to about 0.35, or from about 0.1 to about 0.35, or from about 0.15 to about 0.35, or from about 0.2 to about 0.35, or from about 0.25 to about 0.35, or from about 0.3 to about 0.35, or from about 0.05 to about 0.30, or from about 0.1 to about 0.30, or from about 0.15 to about 0.30, or from about 0.2 to about 0.30, or from about 0.25 to about 0.30, or from about 0.05 to about 0.25, or from about 0.1 to about 0.25, or from about 0.15 to about 0.25, or from about 0.2 to about 0.25, or from about 0.05 to about 0.2, or from about 0.1 to about 0.2, or from about 0.15 to about 0.2, or from about 0.05 to about 0.15, or from about 0.1 to about 0.15, from about 0.05 to about 0.1 , or at least about 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.40, 0.45, 0.5, 0.55, 0.6, 0.65, or 0.7.

[0091] The terms “bulk biological material” and “bulk multicellular biological material” and “biomass” are intended to mean parts and portions of material obtained from a biological entity that was biologically grown. The biological entity is a multicellular entity. In embodiments, the biological material or multicellular biological material may be from any biological origin such as plants, animals, insects and even human. In some aspects of the invention the biological matter includes, but is not limited to cultured plant cells, tissues or plants, for example, but not limited to, callus culture, bioreactor grown or cultured cell-mass or tissue or artificially conditioned cultured plants. In some embodiment, the bulk biological matter includes masses and aggregations of unicellular organisms including but not limited to masses and aggregations of yeasts, fungus, mycorrhizae and other lower phylogenetic organisms. According to another embodiment, bulk multicellular biological material contains one or more of endogenous hydrophobic molecules of interest for encapsulation.

[0092] For example, when the biological material is from plants such as therapeutic plants, edible-plants, medicinal plants, pharmaceutically important plants, cosmetically important plants, or parts thereof. In some embodiment, the bulk biological matter may include macroalgae (for example seaweeds) and microalgae (for example phytoplankton) used for human nutrition and functional food or medicinal or food-additives or agricultural purpose. In other embodiments it may be the whole plant,File No.: P6501 PC00 or parts of a plant (e.g., the flowers, leaves, stems, branches, fruits, fruit skin, seeds, meristem, pulp, and roots), combinations of parts of plants from the same part or from different parts.

[0093] For example, when the biological material is from animals, it may be from the whole animal, or parts of animals, such has the different tissues (e.g., bone tissue, epithelial tissue, cartilage tissue, adipose tissue, nervous tissue, muscle tissue, and connective tissue, and excluding blood cells) or organs tissues (e.g., muscles, bones, stomach, liver, intestine, pancreas, heart, blood vessels, muzzle, windpipe, lungs, kidneys, bladder, brain, nerves spinal cord, eyes, ears, nose, skin, testes, penis ovaries, uterus, vagina, vulva, udder, lymph nodes, and spleen), combinations of parts of plants from the same part or from different parts.

[0094] In some embodiments, the biological matter may include insect and other invertebrates or parts thereof.

[0095] In some embodiments, the bulk biological matter includes multiplicity of individual biological matter or their parts thereof mixed.

[0096] In other aspects of the invention the biological material includes, but is not limited to, preserved biological mass or their parts thereof as frozen, lyophilized, or dried conditions prior to the process of the invention.

[0097] According to embodiments, the bulk multicellular biological material may be separated into fragments of the bulk multicellular biological material, for example, fragments or smaller parts of plant flowers, leaves, stems, branches, fruits, seeds, and roots, or fragments of tissues or organs. For example, plant roots or fruits may be cut into smaller pieces.

[0098] In embodiments, the bulk multicellular biological material may be comprised of cells masses and aggregations of cells of unicellular biological material, such as single cells grown in cell or tissue culture and precipitates of the unicellular biological material.

[0099] In embodiments, the bulk multicellular biological material should not be considered to comprised of biological material, such as cells, or whole tissues or organs that have been disaggregated by means known in the art.

[0100] The term “tissue” is intended to mean any of the distinct types of material of which animals or plants are made, consisting of specialized cells and their products.

[0101] The term “organ” is intended to mean a part of an organism that is typically self- contained and has a specific vital function, such as the heart or liver in humans.

[0102] The term “solvent” is intended to mean a liquid that has the ability to dissolve, suspend, or extract other materials, without chemical change to the material or solvent. According to anFile No.: P6501 PC00 embodiment, a first solvent may be used to contact the bulk multicellular biological material and obtain a mixture of soluble biological molecules, the hydrophobic molecule may be dissolved in a second solvent, and a third solvent may be used to cause formation of the capsules by self-aggregation of the biological molecules and encapsulation of the hydrophobic molecule in the formed capsules. Alternatively, the second solvent may be free of the hydrophobic molecule or the first solvent may comprise the endogenous hydrophobic molecule present in the bulk multicellular biological material.

[0103] The terms “negative pressure” or “negative air pressure” are intended to refer to conditions whereby the air pressure is lower in one place in comparison to another. According to the present invention’s embodiments, the negative air pressure refers to the pressure inside the container containing the solvent contacted dried bulk multicellular biological material, where the air pressure inside this container is lower than the pressure outside the container, causing air to flow into the containerwhen the seal is released. The negative pressure treatment effected on the solvent contacted bulk multicellular biological material may be from about -1 kPa to about -200 kPa.

[0104] The term “vacuum””, as used herein is synonymous with “negative pressure” or “negative air pressure”. As described herein, the term means that vacuum condition is created by removing gaseous phase inside the container, on top of the first solvent contacted bulk multicellular biological material, using a suction devise attached to the container, for example.

[0105] The term “release” is intended to mean that the pressure inside the container is returned to atmospheric pressure and that no negative pressure remains in the container.

[0106] The terms “contact”, “contacting” or “contacted” are intended to refer to the action of physically touching. For example, with respect to the present invention, the biological material is contacted with the first solvent, which may include resuspending, submerging in whole or in part the biological material in the solvent.

[0107] The term “mixture of soluble biological molecules” is intended to refer to the solution of“soluble biological molecules” obtained after removing the multicellular biological material through separation (e.g., filtration such as microfiltration and ultrafiltration) process for clearing from the solution of first solvent.

[0108] The term “microfiltration” is intended to mean the physical filtration process where a fluid is passed through a micrometer (micron) pore-sized from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about 0.45 pm, or about 0.1 pm to about 0.2 pm.

[0109] The term “ultrafiltration” refers to the membrane filtration process in which forces like pressure or concentration gradients lead to a separation through a semipermeable membrane.File No.: P6501 PC00Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate (filtrate). This separation process is used in industry and research for purifying and concentrating macromolecular (103- 106Da) solutions, especially protein solutions.

[0110] The terms “vessel” and “tank” refer to a container having a closing lid and plurality of connection ports, wherein connection ports refer to pipe connection fitting areas to attach pipes to carry and flow liquid in and out of the vessel or tank.

[0111] The term “Level Switch” refers generally to a control switch to measure and control the level of liquid or contents in a said vessel or tank.

[0112] The term “pressure switch” refers to a control switch to monitor and control pressure differences in a fluid flow or gas flow situation in a closed system;

[0113] The term “vacuum switch” refers to a switch control device to monitor specific vacuum pressure.

[0114] The term “position switch” refers to safety switch boxes which are cover guarded for extra precaution for operation.

[0115] The term “Motor” refers to electric motors and motor-pumps refer to fluid flow generating motor operated pump and wherein, vacuum-pump refers to air or gas suction pump.

[0116] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

[0118] Figure 1 is a schematic representation of a system for extracting and encapsulating a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material in accordance with an embodiment of the invention;

[0119] Figure 2 is a cross-section of a mixer used in the system of Figure 1 ;

[0120] Figure 3 is an axonometric view of the mixer used in the system of Figure 1 ;File No.: P6501 PC00

[0121] Figure 4a is a simulation of flow inside the mixer used in the system of Figure 1 ;

[0122] Figure 4b is a simulation of flow inside the mixer used in the system of Figure 1 ;

[0123] Figure 5 is a high-level schematic of the system of Figure 1 showing various steps of an associated process;

[0124] Figure 6 is a detailed schematic of the system of a first portion of the system of Figure 1 ;

[0125] Figure 7 is a detailed schematic of the system of a second portion of the system of Figure 1 .

[0126] Figure 8 is demonstrating encapsulation of curcumin (hydrophobic molecule) and transmission electron microscopy of the capsule

[0127] Figure 9 is showing particle characteristics of encapsulation of a hydrophobic molecule. Each curve represents different encapsulations using different biomass (Table 1).

[0128] Figure 10 is a schematic of the capsule.

[0129] Figure 11A shows freeze dried Turmeric Phyto-Nanoparticle (TPNP) stored at room temperature;

[0130] Figure 11 B shows rehydrated freeze dried TPNP in water (right beaker: water, left beaker: rehydrated TPNP, red laser source bottom right red-dot);

[0131] Figure 11C shows a representative image of TPNP using Transmission Electron Microscopy;

[0132] Figure 11 D shows particle size distribution of TPNP using Dynamic Light Scattering (DLS) where insert represent its monodisperse correlation coefficient curve, a measure of decaying exponential function;

[0133] Figure 11 E shows particle size distribution of TPNP using Tunable Resistive Pulse Sensing (TRPS);

[0134] Figure 11 F shows Zeta potential of TPNP using DLS;

[0135] Figure 11 G shows Zeta potential of TPNP using TRPS;

[0136] Figure 12A shows particle size distribution of curcumin spontaneous aggregation particle (CSAP) in RPMI-1640 media with 0.5% FBS;

[0137] Figure 12B shows particle size distribution of CSAP in RPMI-1640 media without FBS;File No.: P6501 PC00

[0138] Figure 12C shows particle size distribution of CSAP in 0.1x PBS with 0.5% FBS;

[0139] Figure 12D shows particle size distribution of CSAP in 0.1x PBS without FBS;

[0140] Figure 12E shows particle size distribution of TPNP in RPMI-1640 media with 0.5%FBS;

[0141] Figure 12F shows particle size distribution of TPNP in RPMI-1640 media without FBS;

[0142] Figure 13A shows representative HPLC absorbance graphs of 100 ppm of curcumin;

[0143] Figure 13B shows representative HPLC absorbance graphs of 100 ppm of desmethoxycurcumin;

[0144] Figure 13C shows representative HPLC absorbance graphs of 100 ppm of bisdemethoxycurcumin.

[0145] Figure 13D shows representative HPLC chromatograms showing 100 ppm equivalent mass fraction distributions of each of the three species of curcuminoids mixed together: curcumin (Cu), desmethoxycurcumin (DC) and bisdemethoxycurcumin (BDC). The numbers on the Cu peaks are (top to bottom) Retention time in minutes, peak area and peak height respectively and retention time of DC and BDC are also shown.

[0146] Figure 14A shows the relative mass fraction distribution of three species of curcuminoids [curcumin (Cu), desmethoxycurcumin (DC) and bisdemethoxycurcumin (BDC)] in standard Sigma curcumin used. The numbers on the Cu peaks are (top to bottom) Retention time in minutes, peak area and peak height respectively).

[0147] Figure 14B shows the relative abundance of Cu, DC and BDC as mass fraction distribution of curcuminoids in TPNP. The numbers on the Cu peaks are (top to bottom) Retention time in minutes, peak area and peak height respectively).

[0148] Figure 15A shows the absorbance spectra of different dilutions of standard Sigma curcumin.

[0149] Figure 15B shows the absorbance spectra of different dilutions of TPNP in water.

[0150] Figure 15C shows the absorbance spectra of different dilutions of standard curve of curcuminoids using fluorescent spectroscopic method.

[0151] Figure 16 illustrates the results of Ferric Reducing Antioxidant Power (FRAP) for the quantification and detection of Ferric Antioxidant Status on 62.5 pM equivalents of Sigma curcumin, TPNP, N-Acetyl-L-Cysteine (NAC), and gallic acid (GA). The results are presented as percent antioxidant capacity or FRAP.File No.: P6501 PC00

[0152] Figure 17A illustrates a cell viability assay using curcumin spontaneous aggregation particle (CSAP) treatments on THP-1 monocyte cells to determine dose tolerance. Treatment concentrations were expressed as pM curcuminoids.

[0153] Figure 17B illustrates a cell viability assay using TPNP treatments on THP-1 monocyte cells to determine dose tolerance. Treatment concentrations were expressed as pM curcuminoids.

[0154] Figure 17C illustrates a cell viability assay using CSAP treatments on macrophages to determine dose tolerance. Treatment concentrations were expressed as pM curcuminoids.

[0155] Figure 17D illustrates a cell viability assay using TPNP treatments on macrophages to determine dose tolerance. Treatment concentrations were expressed as pM curcuminoids.

[0156] Figure 18A illustrates a cell viability assay to determine tolerance of standard Sigma CSAP and TPNP as indicated in human cardiomyocyte (AC16).

[0157] Figure 18B illustrates a cell viability assay to determine tolerance of standard Sigma CSAP and TPNP as indicated in human blood brain barrier (BBB) cells (hCMEC / D3).

[0158] Figure 19 illustrates the relative abundance of TPNP in sub-cellular localizations of macrophages, (i) Nucleus (Hoechst stain), (ii) F-actin (Phalloidin stain) (iii) TPNP (fluorescence of curcuminoids in green channel), (iv) Merged images of three previous panels.

[0159] Figure 20 illustrates representative FACS data of comparative-bioavailability kinetics of CSAP and TPNP in THP1 macrophages at 0.5h, 2h, 4h 6h, 16h and 24h time points.

[0160] Figure 21A illustrates median fluorescent intensity of gated macrophages positive for curcuminoid fluorescence signal over time.

[0161] Figure 21 B illustrates median fluorescent intensity of gated monocytes positive for curcuminoid fluorescence signal over time.

[0162] Figure 22 illustrates representative FACS data of comparative-bioavailability kinetics of CSAP and TPNP in THP1 monocytes at 0.5h, 2h, 4h 6h, 16h and 24h time points.

[0163] Figure 23 shows immunoblotting of Human Heme Oxygenase-1 (HMOX1) on cells treated with indicated TPNP, CSAP, vehicle control and media only. On left, HMOX1 expression in monocytes (Hemin was used as a positive control), on right HMOX1 expression in macrophages following indicate treatments.

[0164] Figure 24 illustrates the TNF-a concentration (pg 1 100 pl) in the conditioned media of treated macrophages with indicated time and treatment regimes.File No.: P6501 PC00

[0165] Figure 25A illustrates the untargeted total metabolomic analysis using Nano-LC- MS / MS using hierarchical chemical structural ontology annotation - ClassyFire level 5 structural annotation - to identify total molecular fingerprint of TPNP.

[0166] Figure 25B illustrates the untargeted total metabolomic analysis using Nano-LC- MS / MS using hierarchical chemical structural ontology annotation - ClassyFire SubClass structural annotation - to identify total molecular fingerprint of TPNP.

[0167] It will be noted that throughout the appended drawings, like features are identified by like reference numerals.DETAILED DESCRIPTION

[0168] In embodiments there is disclosed a process for the encapsulation of a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material. The process and capsules detailed herein result from the extraction of soluble biological molecules from the bulk multicellular biological material. The present disclosure details a process for the formation of capsules encapsulating a hydrophobic molecule. The process of the present invention includes steps of (a) contacting the bulk multicellular biological material with a first solvent, (b) negative pressure-solvent extraction, (c) separation of the solvent contacted bulk multicellular biological material from the first solvent, to obtain a mixture comprising soluble biological molecules in the first solvent and optionally further comprising an endogenous hydrophobic molecule present in the bulk multicellular biological material, followed by (d) contacting the mixture of soluble biological molecules (and optionally the endogenous hydrophobic molecule present in the bulk multicellular biological material) in the first solvent with a second solvent which is either free of the hydrophobic molecule, or comprises the hydrophobic molecule, and mixing thoroughly to obtain a homogeneous mixture, and (e) contacting the homogeneous mixture with a third solvent to cause formation of capsules by selfaggregation of the biological molecules and encapsulation of the hydrophobic molecule and / or the endogenous hydrophobic molecule therein.Contact with first solvent

[0169] In embodiments, the process of the present invention comprises step (a):(a) contacting the bulk multicellular biological material with a first solvent, to obtain a solvent contacted bulk multicellular biological material.

[0170] In embodiments, the first solvent, may be a non-aqueous solvent completely miscible with water. In embodiments, the non-aqueous solvent is a solvent with a polarity index between 3.9 and 8.0. Examples of suitable solvents include ethanol, methanol, acetone, dimethyl sulfoxide, N,N-File No.: P6501 PC00 dimethylformamide, N-methylpyrrolidone, acetonitrile, 2-methoxyethanol, pyridine, 1 ,4-dioxane, tetrahydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.Negative Pressure-solvent extraction

[0171] In embodiments, the process of the present invention comprises step (b) negative pressure-solvent extraction of the first solvent contacted bulk multicellular biological material. This step comprises at least 2 cycles of (b-i) and (b-ii):(b-i) negative pressure treatment of the solvent contacted bulk multicellular biological material; and(b-ii) return to atmospheric pressure of the solvent contacted bulk multicellular biological material;

[0172] Step (b) is performed for a time sufficient to permeate the first solvent through, and extract the biological molecules from the solvent contacted bulk multicellular biological material.

[0173] In embodiments, the negative pressure treatment is performed at a maximum of -200 kPa. In embodiments, the negative pressure treatment is at from about -1 kPa to about -200 kPa, or from about from about -1 kPa to about -25 kPa, or from about -5 kPa to about -25 kPa, or from about -10 kPa to about -25 kPa, or from about -15 kPa to about -25 kPa, or from about -20 kPa to about -25 kPa, or from about -20 kPa to about -100 kPa, or from about -30 kPa to about -100 kPa, or from about -40 kPa to about -100 kPa, or from about -50 kPa to about -100 kPa, or from about -60 kPa to about - 100 kPa, or from about -70 kPa to about -100 kPa, or from about -80 kPa to about -100 kPa, or from about -90 kPa to about -100 kPa, or from about -100 kPa to about -200 kPa, or from about -110 kPa to about -200 kPa, or from about -120 kPa to about -200 kPa, or from about -130 kPa to about -200 kPa, or from about -140 kPa to about -200 kPa, or from about -150 kPa to about -200 kPa, or from about -160 kPa to about -200 kPa, or from about -170 kPa to about -200 kPa, or from about -180 kPa to about -200 kPa, or from about -190 kPa to about -200 kPa, or about -1 , -2, -3, -4, -5, -6, -7, -8, -9, - 10, -11 , -12, -13, -14, -15, -16, -17, -18, -19, -20, -21 , -22, -23, -24, -25, -26, -27, -28, -29, -30, -31 , -32, -33, -34, -35, -36, -37, -38, -39, -40, -41 , -42, -43, -44, -45, -46, -47, -48, -49, -50, -51 , -52, -53, -54, -55, -56, -57, -58, -59, -60, -61 , -62, -63, -64, -65, -66, -67, -68, -69, -70, -71 , -72, -73, -74, -75, -76, -77, -78, -79, -80, -81 , -82, -83, -84, -85, -86, -87, -88, -89, -90, -91 , -92, -93, -94, -95, -96, -97, -98, -99, -100, -101 , -102, -103, -104, -105, -106, -107, -108, -109, -110, -111 , -112, -113, -114, -115, - 116, -117, -118, -119, -120, -121 , -122, -123, -124, -125, -126, -127, -128, -129, -130, -131 , -132, -133, -134, -135, -136, -137, -138, -139, -140, -141 , -142, -143, -144, -145, -146, -147, -148, -149, -150, - 151 , -152, -153, -154, -155, -156, -157, -158, -159, -160, -161 , -162, -163, -164, -165, -166, -167, -168, -169, -170, -171 , -172, -173, -174, -175, -176, -177, -178, -179, -180, -181 , -182, -183, -184, -185, - 186, -187, -188, -189, -190, -191 , -192, -193, -194, -195, -196, -197, -198, -199, -200 kPa.File No.: P6501 PC00

[0174] In embodiments, the negative pressure treatment may be for about 0.5 second to about 5 seconds, or for about 1 second to about 5 seconds, or from about 2 second to about 5 seconds, or from about 3 second to about 5 seconds, or from about 4 second to about 5 seconds, or for about 0.5 second to about 4 seconds, or from about 1 second to about 4 seconds, or from about 2 second to about 4 seconds, or from about 3 second to about 4 seconds, or for about 0.5 second to about 3 seconds, or from about 1 second to about 3 seconds, or from about 2 second to about 3 seconds, or for about 0.5 second to about 2 seconds, or from about 1 second to about 2 seconds, or for about 0.5 second to about 1 second, or at least 0.5, 1 , 2, 3, 4, or 5 seconds.

[0175] According to some embodiments, the return to atmospheric pressure is for about 0.01 second to about 0.02 second, or for about 0.01 second to about 0.03 second, or for about 0.01 second to about 0.04 second, or for about 0.01 second to about 0.05 second, orfor about 0.01 second to about 0.06 seconds, or for about 0.01 second to about 0.07 seconds, or for about 0.01 second to about 0.08 second, or for about 0.01 second to about 0.09 second, or for about 0.01 second to about 0.1 second, or for about 0.01 second to about 0.11 second, or for about 0.01 second to about 0.12 second, or for about 0.01 second to about 0.13 second, or for about 0.01 second to about 0.14 second, or for about 0.01 second to about 0.15 second, or for about 0.01 second to about 0.16 second, or for about 0.01 second to about 0.17 second, orfor about 0.01 second to about 0.18 second, orfor about 0.01 second to about 0.19 second.

[0176] In embodiments, the time sufficient to permeate the first solvent through is from about 3 to about 200, or from about 4 to about 200, or from about 5 to about 200, or from about 6 to about 200, or from about 7 to about 200, or from about 8 to about 200, or from about 9 to about 200, or from about 10 to about 200, or from about 20 to about 200, or from about 30 to about 200, or from about 40 to about 200, or from about 50 to about 200, or from about 60 to about 200, or from about 70 to about 200, or from about 80 to about 200, or from about 90 to about 200, or from about 100 to about 200, or from about 125 to about 200, or from about 150 to about 200, or from about 175 to about 200, or 3 to about 175, or from about 4 to about 175, or from about 5 to about 175, or from about 6 to about 175, or from about 7 to about 175, or from about 8 to about 175, or from about 9 to about 175, or from about 10 to about 175, or from about 20 to about 175, or from about 30 to about 175, or from about 40 to about 175, or from about 50 to about 175, or from about 60 to about 175, or from about 70 to about 175, or from about 80 to about 175, or from about 90 to about 175, or from about 100 to about 175, or from about 125 to about 175, or from about 150 to about 175, or 3 to about 150, or from about 4 to about 150, or from about 5 to about 150, or from about 6 to about 150, or from about 7 to about 150, or from about 8 to about 150, or from about 9 to about 150, or from about 10 to about 150, or from about 20 to about 150, or from about 30 to about 150, or from about 40 to about 150, or from about 50File No.: P6501 PC00 to about 150, or from about 60 to about 150, or from about 70 to about 150, or from about 80 to about 150, or from about 90 to about 150, or from about 100 to about 150, or from about 125 to about 150, or 3 to about 125, or from about 4 to about 125, or from about 5 to about 125, or from about 6 to about 125, or from about 7 to about 125, or from about 8 to about 125, or from about 9 to about 125, or from about 10 to about 125, or from about 20 to about 125, or from about 30 to about 125, or from about 40 to about 125, or from about 50 to about 125, or from about 60 to about 125, or from about 70 to about 125, or from about 80 to about 125, or from about 90 to about 125, or from about 100 to about 125, or3 to about 100, or from about 4 to about 100, or from about 5 to about 100, or from about 6 to about 100, or from about 7 to about 100, or from about 8 to about 100, or from about 9 to about 100, or from about 10 to about 100, or from about 20 to about 100, or from about 30 to about 100, or from about 40 to about 100, or from about 50 to about 100, or from about 60 to about 100, or from about 70 to about 100, or from about 80 to about 100, or from about 90 to about 100, or 3 to about 90, or from about 4 to about 90, or from about 5 to about 90, or from about 6 to about 90, or from about 7 to about 90, or from about 8 to about 90, or from about 9 to about 90, or from about 10 to about 90, or from about 20 to about 90, or from about 30 to about 90, or from about 40 to about 90, or from about 50 to about 90, or from about 60 to about 90, or from about 70 to about 90, or from about 80 to about 90, or 3 to about 80, or from about 4 to about 80, or from about 5 to about 80, or from about 6 to about 80, or from about 7 to about 80, or from about 8 to about 80, or from about 9 to about 80, or from about 10 to about 80, or from about 20 to about 80, or from about 30 to about 80, or from about 40 to about 80, or from about 50 to about 80, or from about 60 to about 80, or from about 70 to about 80, or 3 to about 70, or from about 4 to about 70, or from about 5 to about 70, or from about 6 to about 70, or from about 7 to about 70, or from about 8 to about 70, or from about 9 to about 70, or from about 10 to about 70, or from about 20 to about 70, or from about 30 to about 70, or from about 40 to about 70, or from about 50 to about 70, or from about 60 to about 70, or 3 to about 60, or from about 4 to about 60, or from about 5 to about 60, or from about 6 to about 60, or from about 7 to about 60, or from about 8 to about 60, or from about 9 to about 60, or from about 10 to about 60, or from about 20 to about 60, or from about 30 to about 60, or from about 40 to about 60, or from about 50 to about 60, or 3 to about 50, or from about4 to about 50, or from about 5 to about 50, or from about 6 to about 50, or from about 7 to about 50, or from about 8 to about 50, or from about 9 to about 50, or from about 10 to about 50, or from about 20 to about 50, or from about 30 to about 50, or from about 40 to about 50, or 3 to about 40, or from about4 to about 40, or from about 5 to about 40, or from about 6 to about 40, or from about 7 to about 40, or from about 8 to about 40, or from about 9 to about 40, or from about 10 to about 40, or from about 20 to about 40, or from about 30 to about 40, or 3 to about 30, or from about 4 to about 30, or from about5 to about 30, or from about 6 to about 30, or from about 7 to about 30, or from about 8 to about 30, or from about 9 to about 30, or from about 10 to about 30, or from about 20 to about 30, or 3 to about 20,File No.: P6501 PC00 or from about 4 to about 20, or from about 5 to about 20, or from about 6 to about 20, or from about 7 to about 20, or from about 8 to about 20, or from about 9 to about 20, or from about 10 to about 20, or 3 to about 10, or from about 4 to about 10, or from about 5 to about 10, or from about 6 to about 10, or from about 7 to about 10, or from about 8 to about 10, or from about 9 to about 10, or 3 to about 9, or from about 4 to about 9, or from about 5 to about 9, or from about 6 to about 9, or from about 7 to about 9, or from about 8 to about 9, or 3 to about 8, or from about 4 to about 8, or from about 5 to about 8, or from about 6 to about 8, or from about 7 to about 8, or 3 to about 7, or from about 4 to about 7, or from about 5 to about 7, or from about 6 to about 7, or 3 to about 6, or from about 4 to about 6, or from about 5 to about 6, or 3 to about 5, or from about 4 to about 5, or 3 to about 4, or at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 cycles of (b-i) and (b -ii).

[0177] According to another embodiment, the step (b) may be performed at temperatures ranging from about 20°C to about 30°C, or from about 21 °C to about 30°C, or from about 22°C to about 30°C, or from about 23°C to about 30°C, or from about 24°C to about 30°C, or from about 25°C to about 30°C, or from about 26°C to about 30°C, or from about 27°C to about 30°C, or from about 28°C to about 30°C, or from about 29°C to about 30°C, or from about 20°C to about 29°C, or from about 21 °C to about 29°C, or from about 22°C to about 29°C, or from about 23°C to about 29°C, or from about 24°C to about 29°C, or from about 25°C to about 29°C, or from about 26°C to about 29°C, or from about 27°C to about 29°C, or from about 28°C to about 29°C, or from about 20°C to about 28°C, or from about 21 °C to about 28°C, or from about 22°C to about 28°C, or from about 23°C to about 28°C, or from about 24°C to about 28°C, or from about 25°C to about 28°C, or from about 26°C to about 28°C, or from about 27°C to about 28°C, or from about 20°C to about 27°C, or from about 21 °C to about 27°C, or from about 22°C to about 27°C, or from about 23°C to about 27°C, or from about 24°C to about 27°C, or from about 25°C to about 27°C, or from about 26°C to about 27°C, or from about 20°C to about 26°C, or from about 21 °C to about 26°C, or from about 22°C to about 26°C, or from about 23°C to about 26°C, or from about 24°C to about 26°C, or from about 25°C to about 26°C, or from about 20°C to about 25°C, or from about 21 °C to about 25°C, or from about 22°C to about 25°C, or from about 23°C to about 25°C, or from about 24°C to about 25°C, or from about 20°C to about 24°C, or from about 21 °C to about 24°C, or from about 22°C to about 24°C, or from about 23°C to about 24°C, or from about 20°C to about 23°C, or from about 21 °C to about 23°C, or from about 22°C to about 23°C, or from about 20°C to about 22°C, or from about 21 °C to about 22°C, or from about 20°C to about 22°C, or about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30°C.SeparationIn embodiments, the process of the present invention comprises a step (c) of separating the first solvent contacted bulk multicellular biological material from the first solvent, to extract and separate theFile No.: P6501 PC00 biological molecules from bulk multicellular biological material. Separation may be achieved by any means known in the art such as e.g., filtration, centrifugation, decantation, aspiration, sieving, pumping, flowing and the combinations thereof.Filtration

[0178] In embodiments, the process of the present invention comprises a step (c) separation of the solvent contacted bulk multicellular biological material from the first solvent, for removal of remaining fragments of the bulk multicellular biological material and to obtain a mixture of soluble biological molecules and optionally further comprising an endogenous hydrophobic molecule present in the bulk multicellular biological material in the first solvent. According to some embodiments, the bulk multicellular biological material may comprise endogenous hydrophobic molecules that may be of interest, and the process of the present invention allows for their encapsulation directly, with or without additional hydrophobic molecules being added exogenously.

[0179] In embodiments, the mixture of soluble biological molecules comprises terpenoids, fatty-acids, amino acids, peptides, alkaloids, carbohydrates, polyketides, shikimates, phenylpropanoids, polyphenols, and combinations thereof.

[0180] In embodiments, the separation is microfiltration, ultrafiltration, or a combination thereof.

[0181] In embodiments, the microfiltration is achieved with a filter having pore size in the micrometer (micron) pore sized from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.9 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.7 pm, or from about 0.1 pm to about 0.6 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about 0.45 pm, or from about 0.1 pm to about 0.4 pm, or from about 0.1 pm to about 0.3 pm, or about 0.1 pm to about 0.22 pm, or about 0.1 pm to about 0.2 pm, 0.2 pm to about 1 pm, or from about 0.2 pm to about 0.9 pm, or from about 0. 2 pm to about 0.8 pm, or from about 0.2 pm to about 0.7 pm, or from about 0.2 pm to about 0.6 pm, or from about 0.2 pm to about 0.5 pm, or about 0.2 pm to about 0.45 pm, or from about 0.2 pm to about 0.4 pm, or from about 0.2 pm to about 0.3 pm, or about 0.2 pm to about 0.22 pm, or 0.22 pm to about 1 pm, or from about 0.22 pm to about 0.9 pm, or from about 0. 22 pm to about 0.8 pm, or from about 0.22 pm to about 0.7 pm, or from about 0.22 pm to about 0.6 pm, or from about 0.22 pm to about 0.5 pm, or about 0.22 pm to about 0.45 pm, or from about 0.22 pm to about 0.4 pm, or from about 0.22 pm to about 0.3 pm, 0.3 pm to about 1 pm, or from about 0.3 pm to about 0.9 pm, or from about 0.3 pm to about 0.8 pm, or from about 0.3 pm to about 0.7 pm, or from about 0.3 pm to about 0.6 pm, or from about 0.3 pm to about 0.5 pm, or about 0.3 pm to about 0.45 pm, or from about 0.3 pm to about 0.4 pm, or 0.4 pm to about 1 pm, or from about 0.4 pm to about 0.9 pm, or from aboutFile No.: P6501 PC000.4 pm to about 0.8 pm, or from about 0.4 pm to about 0.7 pm, or from about 0.4 pm to about 0.6 pm, or from about 0.4 pm to about 0.5 pm, or about 0.4 pm to about 0.45 pm, or 0.45 pm to about 1 pm, or from about 0.45 pm to about 0.9 pm, or from about 0.45 pm to about 0.8 pm, or from about 0.45 pm to about 0.7 pm, or from about 0.45 pm to about 0.6 pm, or from about 0.45 pm to about 0.5 pm, or 0.5 pm to about 1 pm, or from about 0.5 pm to about 0.9 pm, or from about 0.5 pm to about 0.8 pm, or from about 0.5 pm to about 0.7 pm, or from about 0.5 pm to about 0.6 pm, or 0.6 pm to about 1 pm, or from about 0.6 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm, or from about 0.6 pm to about 0.7 pm, 0.7 pm to about 1 pm, or from about 0.7 pm to about 0.9 pm, or from about 0.7 pm to about 0.8 pm, or 0.8 pm to about 1 pm, or from about 0.8 pm to about 0.9 pm, or 0.8 pm to about 1 pm, or at least about 0.1 , 0.2, 0.22, 0.3, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 pm.

[0182] In embodiments, the ultrafiltration is the membrane filtration process in which forces like pressure or concentration gradients lead to a separation through a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate (filtrate). In embodiments, the filtration is with a filter configured for filtration of molecules having 103- 106Da. This separation process is used in industry and research for purifying and concentrating macromolecular (103- 106, or 104- 106, 105- 106, 103- 105, or 104- 105, 104- 106, 104- 105Da) solutions, especially protein solutions. In certain aspects of the invention, the ultrafiltration is achieved with a suitable ceramic filter, a metallic filter, a membrane filter or hollow fiber membrane filter. According to an embodiment, the membrane microfiltration filter may be for example a 0.1 pm membrane filter. According to an embodiment, the membrane filter may be in the configuration of dead-end flow or cross-flow or spiral-wound flow or multi-membrane assembly flow or the combination thereof.Addition of hydrophobic molecules

[0183] Next, the process of the present invention comprises step (d):(d) contacting the mixture of soluble biological molecules in the first solvent with a solution of hydrophobic molecules in a second solvent and mixing thoroughly to obtain a homogeneous mixture thereof.

[0184] In embodiments, as indicated above, the first solvent may optionally comprise one or more endogenous hydrophobic molecule present in the bulk multicellular biological material. Therefore, in step (d), the second solvent may be a second solvent that is free of the hydrophobic molecule (i.e., to encapsulate an endogenous hydrophobic molecule) or the second solvent may be a second solvent comprising the hydrophobic molecule (in other words, the second solvent would contain an hydrophobic molecule being added exogenously, to encapsulate the hydrophobic molecule alone, orFile No.: P6501PC00 to encapsulate the hydrophobic molecule in combination with one or more endogenous hydrophobic molecule).

[0185] As used herein, hydrophobic molecule are molecules that are non-polar and that do not mix with water. In some embodiments, the hydrophobic molecules are defined as insoluble and sparsely soluble molecules with solubility limit less than 20 mg per litre of water at room temperature. According to another embodiment, the hydrophobic molecules may be molecules of molecular weight less than < 3 kDa. According to embodiments, the hydrophobic molecules may be pesticide, a fungicide, a herbicide, an antimicrobial, a drug, a nutrient, a hydrophobic plant extract, an endogenous hydrophobic molecules present in bulk multicellular biological material or combinations thereof. In embodiments, the hydrophobic molecules may be free of an emulsifier, a carrier oil, or combinations thereof.

[0186] According to embodiments, in step (d), an absolute mass ratio of the soluble biological molecules and the hydrophobic molecule is from about 1:10 to 2000:1. An absolute mass ratio of the soluble biological molecules and the hydrophobic molecule is from about 1:10 to about 2:10, or from about 1:10 to about 3:10, or from about 1:10 to about 4:10, or from about 1:10 to about 5:10, or from about 1:10 to about 6:10, or from about 1 :10 to about 7:10, or from about 1 :10 to about 8:10, or from about 1:1 to about 9:10, or from about 1 :10 to about 20:1 , or from about 1 :10 to about 30:1, or from about 1:10 to about 40:1, or from about 1:10 to about 50:1, or from about 1:10 to about 60:1, or from about 1:10 to about 70:1, or from about 1 :10 to about 80:1 , or from about 1 :10 to about 90:1 , or from about 1:10 to about 100:1 , 1:10 to about 200:1 , or from about 1:10 to about 300:1 , or from about 1:10 to about 400:1, or from about 1 :10 to about 500:1 , or from about 1 :10 to about 600:1, or from about 1:10 to about 700:1, or from about 1:10 to about 800:1, or from about 1:10 to about 900:1, or from about 1 :10 to about 1000:1 , or from about 1 :10 to about 1100:1 , 1 :10 to about 1200:1 , or from about 1:10 to about 1300:1, or from about 1:10 to about 1400:1, or from about 1:10 to about 1500:1, or from about 1:10 to about 1600:1, or from about 1:10 to about 1700:1, or from about 1:10 to about 1800:1, or from about 1:10 to about 1900:1, or from about 1:10 to about 2000:1, or from about 1:1 to about 10:1, orfrom about 1:1 to about 20:1, orfrom about 1:1 to about 30:1, orfrom about 1:1 to about 40:1, or from about 1:1 to about 50:1, or from about 1:1 to about 60:1 , or from about 1 :1 to about 70:1, or from about 1 : 1 to about 80: 1 , or from about 1:1 to about 90: 1 , or from about 1 : 1 to about 100:1, 1:1 to about 200:1, orfrom about 1:1 to about 300:1, orfrom about 1:1 to about 400:1, orfrom about 1:1 to about 500:1, orfrom about 1:1 to about 600:1, orfrom about 1:1 to about 700:1, orfrom about 1:1 to about 800:1, orfrom about 1:1 to about 900:1, orfrom about 1:1 to about 1000:1, orfrom about 1:1 to about 1100:1, 1:1 to about 1200:1, orfrom about 1:1 to about 1300:1, orfrom about 1:1 to about 1400:1, orfrom about 1:1 to about 1500:1, orfrom about 1:1 to about 1600:1, orfrom about 1:1 toFile No.: P6501PC00 about 1700:1, or from about 1:1 to about 1800:1, or from about 1:1 to about 1900:1, or from about 1:1 to about 2000:1 orfrom about 10:1 to about 20:1, orfrom about 10:1 to about 30:1, or from about 10:1 to about 40:1, orfrom about 10:1 to about 50:1, orfrom about 10:1 to about 60:1, orfrom about 10:1 to about 70:1, orfrom about 10:1 to about 80:1, orfrom about 10:1 to about 90:1, orfrom about 10:1 to about 100:1, or from about 10:1 to about 200:1, or from about 10:1 to about 300:1, or from about 10:1 to about 400:1 , or from about 10:1 to about 500:1 , or from about 10:1 to about 600:1 , or from about 10:1 to about 700:1, orfrom about 10:1 to about 800:1, orfrom about 10:1 to about 900:1, or from about 10:1 to about 1000:1, orfrom about 10:1 to about 1100:1, orfrom about 10:1 to about 1200:1, orfrom about 10:1 to about 1300:1, orfrom about 10:1 to about 1400:1, orfrom about 10:1 to about 1500:1, orfrom about 10:1 to about 1600:1, orfrom about 10:1 to about 1700:1, orfrom about 10:1 to about 1800:1, orfrom about 10:1 to about 1900:1, orfrom about 10:1 to about 2000:1, orfrom about 100:1 to about 200:1, or from about 100:1 to about 300:1, or from about 100:1 to about 400:1, orfrom about 100:1 to about 500:1, orfrom about 100:1 to about 600:1, orfrom about 100:1 to about 700:1 , orfrom about 100:1 to about 800:1, orfrom about 100:1 to about 900:1 , orfrom about 100:1 to about 1000:1, orfrom about 100:1 to about 100:1100, orfrom about 100:1 to about 1200:1, orfrom about 100:1 to about 1300:1, orfrom about 100:1 to about 1400:1 , orfrom about 100:1 to about 1500:1, orfrom about 100:1 to about 1600:1, orfrom about 100:1 to about 1700:1 , orfrom about 100:1 to about 1800:1, orfrom about 100:1 to about 1900:1, orfrom about 100:1 to about 2000:1, or about 1:10, 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1100:1, 1200:1, 1300:1, 1400:1, 1500:1, 1600:1, 1700:1, 1800:1, 1900:1 or 2000:1.

[0187] In embodiments, the second solvent may be a non-aqueous solvent completely miscible with water. In embodiments, the non-aqueous solvent is a solvent with a polarity index between 3.9 and 8.0. Examples of suitable solvents include ethanol, methanol, acetone, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, 2-methoxyethanol, pyridine, 1,4- dioxane, tetrahydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.Capsule formation

[0188] Next, the homogeneous mixture from step (d) is introduced into step e:(e) contacting the homogeneous mixture with a third solvent to cause formation of the capsule by self-aggregation of the biological molecules and encapsulation of the hydrophobic molecule and / or the endogenous hydrophobic molecule therein, to form a capsule encapsulating the hydrophobic molecule.File No.: P6501PC00

[0189] In embodiments, the capsule encapsulating the hydrophobic molecule comprises a core and an outer shell layer comprised of the soluble biological molecules providing a water compatible particle stability.

[0190] According to an embodiment, step (e) may be performed by mixing the homogeneous mixture with the third solvent at a ratio of from about 1:3 to 1:100, or from about 1:3 to about 1:10, or from about 1 :3 to about 1 :20, 1 :3 to about 1 :30, 1 :3 to about 1 :40, 1 :3 to about 1 :50, 1 :3 to about 1 :60, 1:3 to about 1:70, 1:3 to about 1:80, 1:3 to about 1:90, 1:3 to about 1:100, from about 1:10 to about 1:100, or from about 1:20 to about 1:100, or from about 1:30 to about 1:100, or from about 1:40 to about 1:100, or from about 1:50 to about 1:100, or from about 1:60 to about 1:100, or from about 1:70 to about 1:100, or from about 1:80 to about 1:100, or from about 1:90 to about 1:100, or from about 1:10 to about 1:90, or from about 1:20 to about 1:90, or from about 1:30 to about 1:90, or from about1 :40 to about 1 :90, or from about 1 :50 to about 1 :90, or from about 1 :60 to about 1 :90, or from about1:70 to about 1:90, or from about 1:80 to about 1:90, or from about 1:10 to about 1:80, or from about1 :20 to about 1 :80, or from about 1 :30 to about 1 :80, or from about 1 :40 to about 1 :80, or from about1 :50 to about 1 :80, or from about 1 :60 to about 1 :80, or from about 1 :70 to about 1 :80 , or from about1:10 to about 1:70, or from about 1:20 to about 1:70, or from about 1:30 to about 1:70, or from about1 :40 to about 1 :70, or from about 1 :50 to about 1 :70, or from about 1 :60 to about 1 :70, or from about1:10 to about 1:60, or from about 1:20 to about 1:60, or from about 1:30 to about 1:60, or from about1:40 to about 1:60, or from about 1:50 to about 1:60, or from about 1:10 to about 1:50, or from about1 :20 to about 1 :50, or from about 1 :30 to about 1 :50, or from about 1 :40 to about 1 :50, or from about1:10 to about 1:40, or from about 1:20 to about 1:40, or from about 1:30 to about 1:40, or from about1:10 to about 1:30, or from about 1:20 to about 1:30, or from about 1:10 to about 1:20, or 1:3, 1:10. 1:20, 1:30, 1:40, 1:50, 1:60, 1:70. 1:80, 1:90 or 1:100.

[0191] In embodiments, the third solvent may be an aqueous solvent. In embodiments, the third solvent is water or a mixture of water and a non-aqueous solvent. In embodiments, the nonaqueous solvent is completely miscible with water. In embodiments, the non-aqueous solvent is a solvent with a polarity index between 3.9 and 8.0. Examples of suitable solvents include ethanol, methanol, acetone, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, 2- methoxyethanol, pyridine, 1,4-dioxane, tetrahydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.

[0192] In embodiments, the formation of the capsule encapsulating the hydrophobic molecule is performed at a temperature of from about 10°C to about 80°C, or from about 15°C to about 80°C, or from about 20°C to about 80°C, or from about 25°C to about 80°C, or from about 30°C to about 80°C, or from about 35°C to about 80°C, or from about 40°C to about 80°C, or from about 45°C to aboutFile No.: P6501 PC0080°C, or from about 50°C to about 80°C, or from about 55°C to about 80°C, or from about 60°C to about 80°C, or from about 65°C to about 80°C, or from about 70°C to about 80°C, or from about 75°C to about 80°C, or from about 10°C to about 75°C, or from about 15°C to about 75°C, or from about 20°C to about 75°C, or from about 25°C to about 75°C, or from about 30°C to about 75°C, or from about 35°C to about 75°C, or from about 40°C to about 75°C, or from about 45°C to about 75°C, or from about 50°C to about 75°C, or from about 55°C to about 75°C, or from about 60°C to about 75°C, or from about 65°C to about 75°C, or from about 70°C to about 75°C, or from about 10°C to about 70°C, or from about 15°C to about 70°C, or from about 20°C to about 70°C, or from about 25°C to about 70°C, or from about 30°C to about 70°C, or from about 35°C to about 70°C, or from about 40°C to about 70°C, or from about 45°C to about 70°C, or from about 50°C to about 70°C, or from about 55°C to about 70°C, or from about 60°C to about 70°C, or from about 65°C to about 70°C, or from about 10°C to about 65°C, or from about 15°C to about 65°C, or from about 20°C to about 65°C, or from about 25°C to about 65°C, or from about 30°C to about 65°C, or from about 35°C to about 65°C, or from about 40°C to about 65°C, or from about 45°C to about 65°C, or from about 50°C to about 65°C, or from about 55°C to about 65°C, or from about 60°C to about 65°C, or from about 10°C to about 60°C, or from about 15°C to about 60°C, or from about 20°C to about 60°C, or from about 25°C to about 60°C, or from about 30°C to about 60°C, or from about 35°C to about 60°C, or from about 40°C to about 60°C, or from about 45°C to about 60°C, or from about 50°C to about 60°C, or from about 55°C to about 60°C, or from about 10°C to about 55°C, or from about 15°C to about 55°C, or from about 20°C to about 55°C, or from about 25°C to about 55°C, or from about 30°C to about 55°C, or from about 35°C to about 55°C, or from about 40°C to about 55°C, or from about 45°C to about 55°C, or from about 50°C to about 55°C, or from about 10°C to about 50°C, or from about 15°C to about 50°C, or from about 20°C to about 50°C, or from about 25°C to about 50°C, or from about 30°C to about 50°C, or from about 35°C to about 50°C, or from about 40°C to about 50°C, or from about 45°C to about 50°C, or from about 10°C to about 45°C, or from about 15°C to about 45°C, or from about 20°C to about 45°C, or from about 25°C to about 45°C, or from about 30°C to about 45°C, or from about 35°C to about 45°C, or from about 40°C to about 45°C, or from about 10°C to about 40°C, or from about 15°C to about 40°C, or from about 20°C to about 40°C, or from about 25°C to about 40°C, or from about 30°C to about 40°C, or from about 35°C to about 40°C, or from about 10°C to about 35°C, or from about 15°C to about 35°C, or from about 20°C to about 35°C, or from about 25°C to about 35°C, or from about 30°C to about 35°C, or from about 10°C to about 30°C, or from about 15°C to about 30°C, or from about 20°C to about 30°C, or from about 25°C to about 30°C, or from about 10°C to about 25°C, or from about 15°C to about 25°C, or from about 20°C to about 25°C, or from about 10°C to about 20°C, or from about 15°C to about 20°C, or from about 10°C to about 15°C, or 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80°C.File No.: P6501 PC00

[0193] In embodiments, the capsule encapsulating the hydrophobic molecule may have a size of about 10 pm or less. The capsule encapsulating the hydrophobic molecule may have a size of from about 120 nm to about 10 pm, or from about 120 nm to about 1000 nm, or from about 200 nm to about 1000 nm, or from about 300 nm to about 1000 nm, or from about 400 nm to about 1000 nm, or from about 500 nm to about 1000 nm, or from about 600 nm to about 1000 nm, or from about 700 nm to about 1000 nm, or from about 800 nm to about 1000 nm, or from about 900 nm to about 1000 nm, or from about 1000 nm to about 1000 nm, or from about 2 pm to about 10 pm, or from about 3 pm to about 10 pm, or from about 4 pm to about 10 pm, or from about 5 pm to about 10 pm, or from about 6 pm to about 10 pm, or from about 7 pm to about 10 pm, or from about 8 pm to about 10 pm, or from about 9 pm to about 10 pm, or from about 2 pm to about 9 pm, or from about 3 pm to about 9 pm, or from about 4 pm to about 9 pm, or from about 5 pm to about 9 pm, or from about 6 pm to about 9 pm, or from about 7 pm to about 9 pm, or from about 8 pm to about 9 pm, or from about 2 pm to about 8 pm, or from about 3 pm to about 8 pm, or from about 4 pm to about 8 pm, or from about 5 pm to about 8 pm, or from about 6 pm to about 8 pm, or from about 7 pm to about 8 pm, or from about 2 pm to about 7 pm, or from about 3 pm to about 7 pm, or from about 4 pm to about 7 pm, or from about 5 pm to about 7 pm, or from about 6 pm to about 7 pm, or from about 2 pm to about 6 pm, or from about 3 pm to about 6 pm, or from about 4 pm to about 6 pm, or from about 5 pm to about 6 pm, or from about 2 pm to about 5 pm, or from about 3 pm to about 5 pm, or from about 4 pm to about 5 pm, or from about 2 pm to about 4 pm, or from about 3 pm to about 4 pm, or from about 2 pm to about 3 pm, or about 120 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm.

[0194] The capsule encapsulating the hydrophobic molecule may have a polydispersity index (PDI) equal to or of less than 0.7 (< 0.7), as defined above.Collection

[0195] According to an embodiment, the process of the present invention may further comprise collection step (f):(f) comprising at least one of concentration, freeze-drying, heat-drying, spray-drying and gelling, of the capsule encapsulating the hydrophobic molecule.Concentration

[0196] In embodiments, concentration may be performed by microfiltration, ultrafiltration, dead-end filtration, tangential flow filtration, differential centrifugation, or a combination thereof.Spray dryingFile No.: P6501 PC00

[0197] In embodiments, a time sufficient for spray-drying the capsule encapsulating the hydrophobic molecule is from about 5 minutes to about 1 hour, or from about 5 minutes to about 30 minutes, or from about 5 minutes to about 25 minutes, or from about 5 minutes to about 20 minutes, or from about 5 minutes to about 15 minutes, or from about 5 minutes to about 10 minutes.

[0198] The spray-drying may be performed at a temperature of 110°C to about 165°C, or from about 120°C to about 165°C, or from about 130°C to about 165°C, or from about 140°C to about 165°C, or from about 150°C to about 165°C, or from about 160°C to about 165°C, or from about 110°C to about 160°C, or from about 120°C to about 160°C, or from about 130°C to about 160°C, or from about 140°C to about 160°C, or from about 150°C to about 160°C, or from about 110°C to about 150°C, or from about 120°C to about 150°C, or from about 130°C to about 150°C, or from about 140°C to about 150°C, or from about 110°C to about 140°C, or from about 120°C to about 140°C, or from about 130°C to about 140°C, or from about 110°C to about 130°C, or from about 120°C to about 130°C, or from about 110°C to about 120°C, or 110, 120, 130, 140, 150, 160, or 165°C.

[0199] The spray-drying may be performed with a spray-drying excipient. For example, the spray-drying excipient may be from about 10% w / v to about 30% w / v. For example, from about 10% w / v to about 30% w / v of gum arabic, maltodextrin, gelatin, cellulose derivatives or a combination thereof.Freeze Drying

[0200] In embodiments, the time sufficient for freeze-drying is from about 12h to about 24h, or from about 24h to about 36h, or from about 36h to about 48h.

[0201] In embodiments, freeze-drying is performed at a temperature of -10°C to about -60°C, or from about -20°C to about -60°C, or from about -30°C to about -60°C, or from about -40°C to about -60°C, or from about -50°C to about -60°C, or from about -10°C to about -50°C, or from about -20°C to about -50°C, or from about -30°C to about -50°C, or from about -40°C to about -50°C, or from about -10°C to about -40°C, or from about -20°C to about -40°C, or from about -30°C to about -40°C, or from about -10°C to about -30°C, or from about -20°C to about -30°C, or from about -10°C to about -30°C, or about -10. -20, -30, -40, -50, or -60°C.

[0202] In embodiments, the freeze-drying may be performed with a freezing excipient. For example, the freezing excipient may be sucrose, glucose, dextran, trehalose, lactose, mannitol, maltose, alanine, glycine or a combination thereof. For example, about 10% w / w of any of these freezing excipients, or their combinations.Heat DryingFile No.: P6501 PC00

[0203] In embodiments, the heat-drying is performed at a temperature of 30°C to about 60°C, or from about 40°C to about 60°C, or from about 50°C to about 60°C, or from about 30°C to about 50°C, or from about 40°C to about 50°C, or from about 30°C to about 40°C, or 30, 40, 50, or 60°C.

[0204] In embodiments, the heat-drying may be performed under vacuum.

[0205] In embodiments, the heat-drying may be performed with a stabilizing excipient. For example, the stabilizing excipient may be microcrystalline cellulose, methyl-cellulose, hydroxypropyl methyl cellulose, maltodextrin, gum Arabic, Guar gum, chitosan, pectin, sucrose, lactose, trehalose, Arabic gum, whey protein, polyvinyl alcohol, modified, starch, nut gum, sodium alginate, sodium carboxymethyl cellulose, L-leucine, polyethylene glycol, or a combination thereof.Gelling

[0206] In embodiments, the gelling may be performed with a gelling excipient. For example, the gelling excipient may be hyaluronic acid, methyl-cellulose, alginate, pectin, carrageenan, gellan, gelatin, agar, modified starch, methyl cellulose and hydroxypropyl methyl cellulose, xanthan gum or a combination thereof.System

[0207] According to another embodiment, a system for extracting and encapsulating a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material is provided.

[0208] As best shown in Figure 1 , the system 10 comprises a vacuum pump 30, a first, second, third and fourth vessels 14, 16, 18, 20 and a filter 22. The first vessel 14 is used to contain the multicellular biological material 24 and to receive the first solvent through a first inlet 26. Both the multicellular biological material 24 and the first solvent come into contact with each other in a first cavity 28 of the first vessel 14. To ensure that the first solvent sufficiently impregnates the multicellular biological material 24, a source of negative pressure, such as a vacuum pump 30 is connected to the first vessel 14 so that it is operable to create a negative pressure (vacuum) inside the first vessel 14. Using the vacuum pump 30, the first vessel 14 can subject the mix of the first solvent and of the multicellular biological material 24 to an alternation of negative pressure, up to a maximum of -200 kPa, and atmospheric pressure. This cycle is performed at least twice, and for a time sufficient to permeate the first solvent through and to extract and separate the biological molecules from a resulting solvent contacted bulk multicellular biological material in a mixture of soluble biological molecules in the first solvent. Once the first vessel is under negative pressure it can be returned to the atmospheric pressure either by stopping the vacuum pump 30, or by using a vacuum relief valve 32 connected to the first cavity 28 of the first vessel 14 and to the atmosphere. Using this vacuum relief valve 32, it isFile No.: P6501 PC00 possible to keep the vacuum pump 30 continuously running while alternatively subjecting the material inside the first vessel 14 to the negative pressure when the vacuum relief valve 32 is closed or to the atmospheric pressure when the vacuum relief valve 32 is open. The resulting mixture of soluble biological molecules in the first solvent as well as some bulk multicellular biological material is evacuated through a first outlet 34.

[0209] The first outlet 34 is fluidly connected to a filter inlet 36 of the filter 22 through a first connection 38. The filter 22 receives the mixture of soluble biological molecules in the first solvent and separates remaining fragments of the bulk multicellular biological material. A filtered mixture of soluble biological molecules in the first solvent exits the filter 22 through a filter outlet 40, itself fluidly connected to a second inlet 42 of the second vessel 16 at through a second connection 44.

[0210] The filter 22 has pore size of from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about 0.45 pm, or about 0.1 pm to about 0.2 pm. The filter 22 is operative to filter molecules having 103- 106Da.

[0211] The second vessel 16 receives the filtered mixture of soluble biological molecules in the first solvent.

[0212] This second outlet 46 is fluidly connected at a third inlet 48 of the third vessel 18 through a third connection 50.

[0213] The third vessel 18 receives the filtered mixture of soluble biological molecules in the first solvent from the second vessel 16, as well as a solution of hydrophobic molecule in a second solvent. This mixture and this solution are put in contact and mixed within the third vessel, thereby producing a homogeneous mixture which, when ready, exits the third vessel 18 through a third outlet 52. According to an embodiment, if the bulk multicellular biological material contains one or a plurality of endogenous hydrophobic molecules of interest for encapsulation which are present along with the soluble biological molecules in the first solvent, the second solvent may be free of the hydrophobic molecule or may comprise exogenous hydrophobic molecule.

[0214] A third outlet 52 of the third vessel 18 is fluidly connected at a fourth inlet 54 of the fourth vessel 20 through a fourth connection 56.

[0215] The fourth vessel 20 is used to receive and store a third solvent. The liquids of third vessel 18 having the homogeneous mixture and a third solvent placed in vessel 20 where both are placed into contact through a liquid-liquid mixing chamber 60. This contact causes formation of capsules by self-aggregation of the biological molecules and encapsulation of the hydrophobic molecule therein. It is therefore within the liquid-liquid mixing chamber 60 that the capsule encapsulating of hydrophobic molecule takes place.File No.: P6501 PC00

[0216] Once the encapsulation of hydrophobic molecule is accomplished, the fourth vessel 20 may be used with an internal or external treatment unit 58 which is used for treatments selected from the group consisting of a concentrator, a freezer, a frost-free freezer, an oven, a spray-dryer and a gelproducing device.

[0217] The system 10 may also be equipped with of a liquid-liquid mixing chamber 60 that intercepts the fourth connection 56. Details of the example of liquid-liquid mixing chamber are best shown in Figure 2, now concurrently referred to. This example of liquid-liquid mixing chamber 60 is equipped with a main inlet 62, a main outlet 64, auxiliary inlets 66 and a mixing cavity 68. When the liquid-liquid mixing chamber 60 is used, a first portion 56a of the fourth connection 56 leaving the third vessel 18 is connected to the auxiliary inlets 66. A second portion 56b of the fourth connection 56 interconnects the main outlet 64 to the fourth vessel 20. A fourth outlet 70 of the fourth vessel 20 is fluidly connected to the main inlet 62 through a fifth connection 72.

[0218] The liquid-liquid mixing chamber60 is operable to mix in its mixing cavity 68 the mixture from vessel 18 (soluble biological molecules in the first solvent and the hydrophobic molecule received from the third vessel 18) with the third solvent received from the fourth vessel 20, thereby enriching the encapsulation of the hydrophobic molecules.

[0219] The auxiliary inlets 66 comprises a plurality of nozzles 74 that are oriented at least partially against the flow (e.g. counter-flow) of the third solvent from the fourth vessel 20 to inject the liquid received from the third vessel 18.

[0220] In Figure 3, 401 is an inlet tube fitting (preferably round to square transition) for liquid flow from the fourth vessel 20, 402 is an outlet tube fitting (preferably square to round transition), 403 is an inlet sanitary tube flange connection (inlet from fourth vessel 20), 404 is an outlet sanitary tube flange connection (outlet to fourth vessel 20), 405s are solvent inlet ports from third vessel 18 (56, 56a), 406s removable plates, 407s are injection nozzles (preferably removable) delivering solvent from vessel 18 through 405s and 408 is cavity of liquid-to-liquid mixing chamber 60.

[0221] Figures 4a and 4b depict two velocity heat-map showing two different liquid velocity cap situations (top and bottom) of 1 meter per second (figure 4a) and 2 meter per second (Figure 4b) respectively as they are occurring inside the liquid-liquid mixer 60. The highest velocity gradient is represented as red and low velocity is represented as green. In these simulations, the spray nozzles 74 are fitted with removeable metal needles having nominal internal diameter of 0.838 millimeter.

[0222] Figure 5 is a schematic representation of system 10 showing the first vessel 14, the filter 22, the second vessel 16, the third vessel 18, the liquid-liquid mixer 60 and the fourth vessel 20. This schematic describes the position of the units, fluid flow (solid arrows) and gaseous atmosphereFile No.: P6501 PC00 flow (dashed arrow), wherein vessels 14, 16, 18 and 20 are vessels or tanks capable of holding liquid and built with stainless steel or ceramic or glass or other metal alloy of desired capacity and equipped with closing lids. The system 10 is operated sequentially in steps and fluid flow is controlled by a variety of pumps and valves (V-L-1 to V-L-9) having plurality of sensors, switches, pressure transducers and other electronic parts to connect with a controller 76 for operating and coordinating the flow of fluid into and out of the vessels. The atmosphere or gaseous flow is controlled by a variety of valves (V-R-1 to V-R-6) also equipped with sensors, switches, pressure transducers and other electronic parts to connect with the controller 76. Communication between the electronic component and the controller may be through wired or wireless communication.

[0223] The process occurs as sequential operation wherein, to start the operation (Step 1), one or plurality of biological matter of choice are added into first vessel 14 followed by addition of first solvent, through V-L-1 , sufficient to contact all the biological matter. First vessel 14 is a closed vacuum tight vessel and using closed lid valve controls generation of mild vacuum can be achieved through a vacuum pump and capable of attaining multiple cycles of mild vacuum of maximum of - 200 kPa followed by release vacuum to atmospheric pressure (+101.325 kPa) while the biological matter and first solvent are present and in contact, wherein in some embodiments the generated vacuum may draw to a connected auxiliary sealed tank through the vacuum pump to contain atmospheric air and vapor in close containment. In some embodiments the atmospheric air of first vessel 14 is replaced by nitrogen or carbon-dioxide. While the process in Step 1 operates the V-L-1 , V-L-2 and V-R-2 remain close wherein the V-R-1 remains open connected to vacuum pump.

[0224] In step 2 of the process, the first solvent having biological encapsulant materials dissolved in first vessel 14 is filtered through filter 22, equipped with microfiltration and ultrafiltration filters, so as to provide the filtered biological encapsulant materials that is devoid of any particulate matter of the original bulk biological matter to second vessel 16. While the process in Step 2 operates the V-L-1 , V-L-4 and V-R-1 remain close wherein the V-L-2, V-L-3, V-R-2, V-R-3 and V-R-4 remain open.

[0225] In step 3 of the process, the first solvent having biological encapsulant materials dissolved is pumped from second vessel 16 to third vessel 18 to be mixed with the hydrophobic molecules to be encapsulated already dissolved in first solvent or a second solvents according to the specifications of this invention. While the process in Step 3 operates, the V-L-3 and V-L-6 remain close wherein the V-L-4, V-L-5, V-R-4, V-R-5 remain open.

[0226] In step 4 of the process, a third solvent of aqueous solution is stored in fourth vessel 20, for the preparation of encapsulation of the hydrophobic molecules in third solvent aqueous liquidFile No.: P6501 PC00 in process Step 5. While the process in Step 4 operates, the V-L-7 and V-L-8 remain close wherein the V-L-9 and V-R-6 remain open.

[0227] In step 5 of the process of the invention, the liquids from the third vessel 18 and the fourth vessel 20 mix through liquid-liquid mixer 60. The liquid-liquid mixer 60 delivers all the liquid content of third vessel 18 to pre-existing third solvent aqueous liquid of fourth vessel 20 in a circular close loop of piping connection wherein the circular close loop of piping between liquid-liquid mixer 60 and fourth vessel 20 creates one directional aqueous liquid flow forming the encapsulation. While the process in Step 5 operates, the V-L-4, V-L-5, V-L-9 remain close wherein the V-L-6, V-L-7, V-L-8, V- R-5 and V-R-6 remain open and wherein all the content of third vessel 18 mixes with the third solvent aqueous content of fourth vessel 20.

[0228] Figures 6 and 7 are now concurrently referred to. First vessel 14 comprises a lid 101 to make the vessel vacuum tight. The first vessel 14 is also equipped with a plurality of pressure / vacuum relief valve 102 and a perforated container 103 within vessel 14 for containing the biological matters in contact with the first solvent. It may also be a centrifuge. In one embodiment, 105 is one or plurality of ports connected to first vessel 14 to connect a plurality of liquid flow piping for the functional operations, wherein 106 is a safety relief valve that connects to 107 vent piping. Drip leg 108 collects condensate which is vented to the exterior through vent 109. Motor 110 is capable of mild rotating or shaking 103. Process valve 111 controls liquid flow from first vessel 14. Pneumatic solenoid valve 112 controls liquid flow control. 113 is a motor pump capable of forcing liquid flow to a specified direction, 114 is a pressure gauge, 115 is a safety valve, 116 is a sight glass to visualize the liquid, 117 is filter comprising of one or multiple liquid filters capable of microfiltration and ultrafiltration, 118 is flowmeter, 119s are pipe flanged connection connections of 117.

[0229] Second vessel 16 comprises of a 201 lid, 202 plurality of pressure / vacuum relief valve, wherein 203 is a safety relief valve to connect to 204 vent piping, 205 drip leg to collect condensate and 206 is a pipe connection fordisplacement of atmosphere when liquid is transferred from one vessel to another vessel, 207 is liquid inlet from 117 filter.

[0230] Third vessel 18 comprises a 301 lid, 302 plurality of pressure / vacuum relief valve, 303 is one or plurality of ports connected to 300 to connect plurality of liquid flow piping for the functional operations, 303 and 304 are liquid inlet connection ports, 305 is a liquid outlet port, 306 is a process valve, 307 is a pneumatic solenoid valve, 308 is a motor pump to direct flow and flowrate to 400, 309 is a flowmeter, 310 is a safety valve, 311 pipe connection to 400, 312 is a safety relief valve to connect to 313 drip leg to collect condensate and 314 vent to exterior.File No.: P6501 PC00

[0231] Liquid-liquid mixer 60 comprises liquid flow main inlet 401 , 403 from fourth vessel 20 and liquid flow main outlet 402, 404

[0232] Fourth vessel 20 comprises lid 501 , a plurality of pressure / vacuum relief valve 502, liquid outlet 503 running from fourth vessel 20 to liquid-liquid mixer 60. 504 is a process valve, 505 is a pneumatic solenoid valve, 506 is a motor pump to drive liquid flow and flowrate to 400, 507 is a flowmeter, 508 is pipe connection to 403, 509 is a pipe connection liquid flow outlet from 400 to vessel 20, 510 is a safety relief valve, 511 is a drip leg to collects condensate, 512 is a vent connection, 513 is a pipe connection for displacement of atmosphere when liquid is transferred from one vessel to another vessel, 514 is a valve for safety loop connections and 515 is a pneumatic solenoid valve.

[0233] In some embodiments of this invention multiple parts are connected to machine or computer readable media which may be used for the purpose of storing data, computer reading of data, controlling, identifying, isolating and feedback controls of valves, pumps, flowmeters and pressure / vacuum. The data collected and stored in computer readable media may be using random access memory (RAM) or removable data storage devices like removable drive or disk or data storage network or local area network (LAN).

[0234] The first vessel 14 is capable of being vacuum or air tight and can withstand as a mild vacuum chamber when all the ports and connections of the vessel are closed, which is connected to a vacuum pump to create mild vacuum in the vessel to a maximum of negative 200 kilopascal (kPa) or negative 59 inches of mercury (inHg) and wherein the vacuumed atmosphere is transported to auxiliary sealed tank to contain air and vapor of this atmosphere in close containment. In some other embodiments, atmospheric air present in the first vessel 14 is replaced by nitrogen or carbon dioxide. In some embodiments 103 is a container inside vessel 14 which is perforated, act as holder of the biological materials to gain contact with the first solvent and can be a shaker or centrifugation which is powered by a motor 110. In some other embodiments, the first vessel 14 release the vacuum created and back to the atmospheric pressure and wherein the vacuum / release cycles may be repeated for plurality of times. The biological material in first vessel 14 is fully in contact with the first solvent, wherein the vessel is closed and operated to create the mild vacuum in the vessel in the presence of contacted biological materials and the first solvent experiencing mild vacuum. This is followed by the release of the vacuum to normal atmospheric pressure and repeat the cycle of vacuum and release for plurality of time sufficient to release extracted biological molecules from the contacted biological materials as constituents of encapsulation into the first solvent as dissolved molecules. Once the biological molecules released into the first solvent, it is being collected to second vessel 16 through plurality of filtration devices (117) to clarify the liquid devoid of any particulate matter from the biological materials, wherein the filtration devices are microfilters and ultrafilters. Microfilters are filters which are capableFile No.: P6501 PC00 of removing micron sized particles from a liquid and ultrafilters remove particles of submicron scales from a liquid. In embodiments of the process once the biological molecules remain dissolved in the first solvent and filtered using filtration devices 117 to remove particles of micron and submicron sizes, the liquid is stored in second vessel 16. In some embodiments, the process vacuum / release cycles and filtration devices 117 are controlled or programmed using machine or computer readable system and connected to appropriate valves, pressure gauges and flowmeter to collect data as well as to control operational process. In some embodiments the process may be manual or combinations of manual and computer readable operations.

[0235] The encapsulation operation occurs in fourth vessel 20, using constituents of the third vessel 18. The third vessel 18 contains the extracted biological molecules in first solvent from second vessel 16 mixed with one or plurality of hydrophobic molecules to be encapsulated in second solvent. In some embodiments the one or plurality of hydrophobic molecules to be encapsulated is dissolved in first solvent or second solvent which are completely mixable to the first solvent.

[0236] The first solvent is a non-aqueous solvent having property of completely mixing with water and wherein the nonaqueous solvent is selected from plurality of solvents with polarity index between 3.9 and 8.0 (wherein polarity index of water is 10.2 and polarity index of pentane, a nonpolar solvent, is 0). In some embodiments the first solvent is ethanol; methanol; acetone; Dimethyl Sulfoxide; N,N-Dimethylformamide; N-Methylpyrrolidone; Acetonitrile; 2-Methoxyethanol; Pyridine; 1 ,4-Dioxane; Tetrahydrofuran; n-Propyl Alcohol; Isopropyl Alcohol; or combinations thereof.

[0237] The one or plurality of hydrophobic molecules to be encapsulated is dissolved in a second solvent which may be the first solvent or a plurality of solvents which are completely mixable with the first solvent and wherein the nonaqueous solvent is selected from plurality of solvents with polarity index between 3.9 and 8.0 (wherein polarity index of water is 10.2 and polarity index of pentane, a nonpolar solvent, is 0). The second solvent may be ethanol; methanol; acetone; Dimethyl Sulfoxide; N,N-Dimethylformamide; N-Methylpyrrolidone; Acetonitrile; 2-Methoxyethanol; Pyridine; 1 ,4-Dioxane; Tetrahydrofuran; n-Propyl Alcohol; Isopropyl Alcohol; or combinations thereof.

[0238] System 10 achieves encapsulation of hydrophobic molecules to be compatible in an aqueous solution by assembling biological encapsulation agent molecules as homogeneous particle in the liquid-to-liquid mixer 60. The mixture of the biological encapsulation agent molecules and the hydrophobic molecules in third vessel 18 in the solvent in the proportions, is pumped (308) from the outlet connection port (305) of the third vessel 18 through pipe connection 311 into the solvent inlet ports of the mixer 60, where aqueous solution (third solvent) from fourth vessel 20 pumped into mixer 60 through outlet connection (503 and 508) using a motor pump (506) so that the aqueous solution (third solvent) passes through liquid-to-liquid mixer 60 and mixed with liquid of third vessel 18 and flowFile No.: P6501 PC00 back to fourth vessel 20 through pipe connection 509 creating a circular flow out of fourth vessel 20 and into the same vessel, wherein the liquid of vessel flow into liquid-to-liquid mixer 60 in one direction and ultimately collected in fourth vessel 20. In embodiments of different examples, the flow-rates of third solvent aqueous liquid from fourth vessel 20 flowing into the mixer 60 may vary depending on the character of the hydrophobic molecules to be encapsulated and wherein the flow-rate of liquid from third vessel 18 into the mixer 60 also vary depending on a specific process embodiment. In some embodiments the inlet flow-rate of liquid into mixer 60 from fourth vessel 20 may be 0.1 meter per second to 10 meter per second, preferably 1 to 2 meter per second. Similarly, in some embodiments the flow-rate of liquid from third vessel 18 into mixer 60 may vary from 0.1 meter per second to 10 meter per second, preferably 1 to 2 meter per second.Turmeric nanoparticles composition

[0239] According to another embodiment, a turmeric nanoparticle composition is provided where the bulk multicellular biological material (turmeric roots) contains one or plurality of hydrophobic molecules (curcuminoids) of interest for encapsulation. These curcuminoids are enriched along with the soluble biological molecules in the first solvent. According to embodiments, the second solvent may therefore be free of any exogenous hydrophobic molecule, or it may comprise a different hydrophobic molecule. In embodiments, the turmeric nanoparticle composition comprises a plurality of nanoparticles comprising a capsule. The capsule comprises self-aggregated soluble biological molecules extracted from a turmeric rhizome material. The capsule forms a micelle around a hydrophobic core; which encapsulates hydrophobic molecules endogenous to the turmeric rhizome material. The hydrophobic molecules comprise curcuminoids. The nanoparticles have a particle size of from about 150 nm to about 250 nm. The nanoparticles have a polydispersity index (PDI) of less than or equal to 0.3 (< 0.3). The nanoparticles have a curcuminoids loading capacity of about 20% to about 30% mass of curcuminoids per total mass of said nanoparticles. The turmeric nanoparticle composition comprises an acceptable carrier.

[0240] In embodiments, the capsule orthe hydrophobic molecules endogenous to the turmeric rhizome material are free of an emulsifier, a carrier oil, or combinations thereof.

[0241] As used herein, the term “turmeric rhizome material” is intended to refer to the underground stem (rhizome) of the turmeric plant (Curcuma longa). It may be harvested and used fresh, or dried, and often ground into a yellow-orange powder. It is rich in curcuminoids (like curcumin) and essential oils, and is widely used in spices, traditional medicine, cosmetics, and dietary supplements due to its color, flavor, and bioactive properties. As used herein, the turmeric rhizome material is a bulk multicellular biological material.File No.: P6501 PC00Curcuminoids loading capacity

[0242] As used herein, the term “curcuminoids” are a group of bioactive polyphenolic compounds found primarily in the turmeric plant (Curcuma longa), which belongs to the ginger family (Zingiberaceae). They are responsible for turmeric's bright yellow-orange color and much of its therapeutic activity. The main types of curcuminoids are curcumin, desmethoxycurcumin (DMC) and disdemethoxycurcumin (BDMC).

[0243] In embodiments, the nanoparticles have curcuminoids loading capacity of about 20% to about 30% mass of curcuminoids per total mass of said nanoparticles. In embodiments, the nanoparticles have a curcuminoids loading capacity of about 25% mass of curcuminoids per total mass of said nanoparticles.ExcipientsFile No.: P6501 PC00

[0244] The turmeric nanoparticle composition of the present invention may comprise excipients. For example, it may contain a freezing excipient, a stabilizing excipient, a spray-drying excipient, a gelling excipient, or a combination thereof.Freeze Drying

[0245] In embodiments, the freezing excipient may be sucrose, glucose, dextran, trehalose, lactose, mannitol, maltose, alanine, glycine or a combination thereof. For example, about 10% w / w of any of these freezing excipient, or their combinations.Stabilizing

[0246] In embodiments, the stabilizing excipient may be microcrystalline cellulose, methylcellulose, hydroxypropyl methyl cellulose, maltodextrin, gum Arabic, Guar gum, chitosan, pectin, sucrose, lactose, trehalose, Arabic gum, whey protein, polyvinyl alcohol, modified, starch, nut gum, sodium alginate, sodium carboxymethyl cellulose, L-leucine, polyethylene glycol, or a combination thereof.Spray drying

[0247] In embodiments, the spray-drying excipient may be from about 10% w / v to about 30% w / v. For example, the spray-drying excipient may be from about 10% w / v to about 30% w / v of gum arabic, maltodextrin, gelatin, cellulose derivatives or a combination thereof.Gelling

[0248] In embodiments, the gelling excipient may be hyaluronic acid, methyl-cellulose, alginate, pectin, carrageenan, gellan, gelatin, agar, modified starch, methyl cellulose and hydroxypropyl methyl cellulose, xanthan gum or a combination thereof.Particle sizes

[0249] In embodiments, the nanoparticles of the turmeric nanoparticle may have a particle size of from about 150 nm to about 250 nm, or from about 170 nm to about 240 nm.Polydispersity index (PDI)

[0250] In embodiments, the nanoparticles of the turmeric nanoparticle may have a PDI of less than or equal to 0.3 (< 0.3), for example of from about 0.05 to about 0.26.Antioxidant capacity

[0251] In embodiments, the nanoparticles of the turmeric nanoparticle may have an antioxidant capacity 2 to 4 times higher than an equivalent pM concentrations of pure curcuminoids as measured by a Ferric Reducing Antioxidant Power (FRAP) assay. The antioxidant capacity may beFile No.: P6501 PC00 from about 2 to about 4, or about 2 to about 3 times higher than an equivalent pM concentration of pure curcuminoids as measured by a Ferric Reducing Antioxidant Power (FRAP) assay.

[0252] A composition according to the invention may also comprise an acceptable diluent, excipient, or carrier or pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art that is compatible with other ingredients in the composition, that is compatible with the method of delivery of the composition, and that is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form, powder form (such as, but not limited to, lyophilized or encapsulated), capsule form or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, or a suitable buffer, and optionally comprise one or more additives to improve solubility and / or stability. Reconstitution to produce a suspension may be effected in a buffer at a suitable pH to ensure the viability of the turmeric nanoparticles. Dry powders may also include additives to improve stability and / or carriers to increase bulk / volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be formulated for delivery of the turmeric nanoparticles to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, time release, or other suitable technologies for delivery of the turmeric nanoparticles and / or compound of the present invention. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present turmeric nanoparticles.Use of the turmeric nanoparticle composition

[0253] In embodiments, there is provided a method of treating or preventing an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease, comprising administering a turmeric nanoparticle composition according to the present invention, to a subject in need thereof.

[0254] According to another embodiment, there is provided a turmeric nanoparticle composition according to the present invention for use in the treatment of an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease.

[0255] The inflammatory disease may be arthritis, or inflammatory bowel disease.EXAMPLE 1EXAMPLE ENCAPSULATION #1

[0256] Now referring to Figure 8, which is an example of encapsulation using system 10 according to the sequential operational steps to achieves encapsulation of hydrophobic molecules.File No.: P6501 PC00Dried organic brewing waste was obtained, and they were not grinded or powdered. The dried organic brewing waste was run in system 10 to demonstrate encapsulation of a fluorescent hydrophobic molecule as an example embodiment. The fluorescent hydrophobic molecule used was curcumin (Empirical Formula: C21H20O6) with molecular weight of 368.385 g / mol and it is almost insoluble in water (less than 8 pg / L at 20 °C).

[0257] In step 1 of the process, dried brewing waste are added into first vessel 14 followed by addition of first solvent (95% ethanol) sufficient to contact all the biological matter. First vessel 14 is then closed, and a mild vacuum of -138 kPa was achieved through a vacuum pump. 50 cycles of mild vacuum of -138 kPa for 2 seconds followed by release vacuum to atmospheric pressure (101 .325 kPa) while the dried organic brewing waste and solvent are present in contact.

[0258] In step 2 of the process, the first solvent having biological encapsulant materials dissolved in first vessel 14 is filtered through filter 22, equipped with microfiltration (2.5 micron followed by 0.45 micron, further followed by 0.1 micron), so as to provide the filtered biological encapsulant.

[0259] In step 3 of the process, the first solvent having biological encapsulant materials dissolved therein is pumped from second vessel 16 to third vessel 18 to be mixed with the hydrophobic curcumin to be encapsulated, already dissolved in a second solvent - 99% ethanol at the concentration of 368.38 pg / ml or 1 mM according to the specifications of this invention. In the third vessel 18, a 3- part volume of first solvent (having biological encapsulant materials dissolved therein) and a 2-part volume of curcumin dissolved in the second solvent, are mixed.

[0260] In step 4 of the process, a third solvent of aqueous solution (0.1x PBS, pH 7.4) is stored in fourth vessel 20, for the preparation of encapsulation of the hydrophobic curcumin in third solvent aqueous liquid in process Step 5.

[0261] In step 5 of the process, one volume part of liquid mix from the third vessel 18 and 10 volume part of the fourth vessel 20 mix through liquid-liquid mixer60. The liquid-liquid mixer60 delivers all the liquid content of third vessel 18 to pre-existing third solvent aqueous liquid (O.IxPBS, pH7.4) of fourth vessel 20 in a circular close loop of piping connection wherein the circular close loop of piping between liquid-liquid mixer 60 and fourth vessel 20 creates one directional aqueous liquid flow forming the encapsulation.

[0262] The resultant formulation of encapsulated curcumin was analysis using fluorescent spectroscopic analysis. Curcumin is inherently fluorescent (excitation at 420 nm and emission at 570 nm). The above curcumin formulation was subjected to a separation of particles from the soluble parts using a 300 KDa spin-filter. The spin filtration was done at room temperature with centrifugal force of 2000 rpm for 30 minutes. The flow through from the filter having no particles (i.e., indicating free nonFile No.: P6501 PC00 encapsulated curcumin) which were collected and the volume was measured. The holding part of the spin-filter having particles was collected and volume adjusted to the original volume by adding the same volume of water as the volume measured for flow through. The samples of flow through and the particles were diluted 10 times in 80% ethanol for spectroscopic analysis (excitation at 420 nm and emission at 570 nm). The dilution in 80% ethanol disrupted all the particles so that encapsulated curcumin was liberated into the solution for fluorescent spectroscopic measurements of relative fluorescent units (RFU). The left panel of Figure 8 shows RFU of the flow-through representing free non-encapsulated curcumin present (RFU 1206.33) and encapsulated curcumin present (RFU 38816.33) in the formulation, which is about 32 time less than the formulation, indicating more than 95% encapsulation. At 95% encapsulation the absolute mass ratio of encapsulant molecules to curcumin is about 55:1 .

[0263] The right panel of the Figure 8 is a transmission electron microscopic photograph of the above resulted curcumin encapsulated formulation.EXAMPLE 2EXAMPLE ENCAPSULATION #2

[0264] Table 1 below shows examples of encapsulation using system 10 according to the sequential operational steps to achieves encapsulation of hydrophobic molecules. Dried hulled whole hemp seed, dried whole rosemary aerial parts and dried ginger roots were obtained (they were not grinded or powdered). Three samples of encapsulating materials were extracted from input biomaterials as shown in Table 1 and run separately in system 10 to demonstrate encapsulation of a fluorescent hydrophobic molecule in these encapsulants. The fluorescent hydrophobic molecule used was fluorescein (free acid, Empirical Formula: C20H12O5) with molecular wight of 332.3 g / mol and almost insoluble in water (50 mg / L at 20 °C).

[0265] In embodiments of the examples different biological materials, the parameters of the process and encapsulation are shown in the following table.File No.: P6501 PC00Table 1 - Examples of encapsulation.

[0266] In this example embodiments, the first solvent is 95% ethanol, the Second solvent is a mixture of DMSO: ethanol (90:10, v / v), the third Solvent is 0.1x phosphate-buffered saline (PBS) containing 13.7 mM NaCI, 0.27 mM KCI, 1 mM of Na2HPC>4 and 0.18 mM KH2 O4 in water, pH=7.4.

[0267] In step 1 of the process biological matter of choice are added into first vessel 14 followed by addition of first solvent of first solvent (95% ethanol) sufficient to contact all the biological matter. First vessel 14 is then closed, and a mild vacuum of -138 kPa was achieved through a vacuum pump. 50 cycles of mild vacuum of -138 kPa for 2 seconds followed by release vacuum to atmospheric pressure (101.325 kPa) while the biological matter and solvent are present in contact.

[0268] In step 2 of the process, the first solvent having biological encapsulant materials dissolved in first vessel 14 is filtered through filter 22, equipped with microfiltration (5 micron followed by and 0.22 micron), so as to provide the filtered biological encapsulant.

[0269] In step 3 of the process, the first solvent having biological encapsulant materials dissolved therein is pumped from second vessel 16 to third vessel 18 to be mixed with the hydrophobic fluorescein to be encapsulated already dissolved in a second solvent (90% DMSO + 10% ethanol at the concentration of 0.75 mg / ml) according to the specifications of this invention.

[0270] In step 4 of the process, a third solvent of aqueous solution (0.1x PBS, pH 7.4) is stored in fourth vessel 20, for the preparation of encapsulation of the hydrophobic Fluorescein in third solvent aqueous liquid in process Step 5.

[0271] In step 5 of the process, the liquids from the third vessel 18 and the fourth vessel 20 mix through liquid-liquid mixer60. The liquid-liquid mixer60 delivers all the liquid content of third vessel 18 to pre-existing third solvent aqueous liquid (O.IxPBS, pH7.4) of fourth vessel 20 in a circular close loop of piping connection wherein the circular close loop of piping between liquid-liquid mixer 60 and fourth vessel 20 creates one directional aqueous liquid flow forming the encapsulation.

[0272] The resultant formulation of encapsulated fluorescein was analysis using fluorescent spectroscopic analysis (excitation at 450 nm and emission at 520 nm). The above fluorescein capsule formulation was subjected to a separation of particles from the soluble parts using a 300 KDa spin-File No.: P6501 PC00 filter. The spin filtration was done at room temperature with centrifugal force of 2000 rpm for 30 minutes. The flow through from the filter having no particles (i.e., indicating free non-encapsulated fluorescein) which were collected and the volume was measured. The holding part of the spin-filter having particles was collected and volume adjusted to the original volume by adding the same volume of water as the volume measured for flow through. The samples of flow through and the particles were diluted 650 times and 10,000 times respectively in 80% ethanol for suitable fluorescent-spectroscopic reading range capacity. The dilution in 80% ethanol disrupted all the particles so that encapsulated fluorescein was liberated into the solution for fluorescent spectroscopic measurements of relative fluorescent units (RFU). Relative concentrations were derived using proper dilutions to compare with a standard curve using fluorescein dissolved in 80% ethanol. Based on the RFU and dilution factors the absolute mass ratio of fluorescein encapsulated in the capsule was deduced as shown in Table 1 .

[0273] Figure 9 shows the particle size data samples of Table 1

[0274] Figure 10 shows a schematic of the capsule or encapsulated hydrophobic molecule using biological encapsulant materials from the source biological material. The resultant formulation of particles which became ultra-tiny capsules to protect the hydrophobic molecules from surrounding water-based suspension. An outer shell layer forms as water compatible encapsulation or capsule with a core holding the hydrophobic molecules.EXAMPLE 3TURMERIC PHYTO-NANOPARTICLES ENCAPSULATING CURCUMINOIDS

[0275] Curcuminoids are bioactive polyphenols that are derived from turmeric (Curcuma Longa rhizomes). They have gained substantial interest, as therapeutic benefits of curcuminoid APIs have been demonstrated in the prevention and treatment of diseases [1], Despite the immense pharmacological potential of curcuminoids, their poor pharmacokinetic properties (low water solubility, low absorption and rapid metabolism) are major challenges to harness their beneficial effects. Curcuminoids have potent anti-inflammatory and antioxidant properties, making them attractive for managing chronic inflammatory diseases, cardiovascular health and cancer [2], Although inflammation is a natural response by the immune system in the event of injury or infection, chronic low-grade inflammation leads to tissue damage and contributes to the development and progression of many chronic diseases [3], Chronic, low-grade inflammations are driven by environmental pollutants, poor lifestyle choices, stress and obesity. For example, free radical (FR) damage and inflammation are major contributors to cellular injury and disease development. FRs are highly reactive molecules that are produced by healthy or diseased cells. At elevated levels, and if cellular antioxidant defenses are unable to neutralize them, FRs can damage cell membranes, proteins, DNA, trigger mutations,File No.: P6501 PC00 promote cell death and tissue injury. Curcuminoids have been shown to act as potent cyto protectants through multiple molecular mechanisms: (i) Scavenging free radicals [4], (ii) upregulating endogenous antioxidant enzymes and pathways (e.g., Heme oxygenase (HMOX1) [5] and (iii) suppressing inflammatory cytokines such as TNFoc [6], The beneficial effects of curcuminoids in conditions such as general inflammation, metabolic disorders, wound healing, hepatotoxicity, cardiotoxicity, inflammatory bowel disease, multiple drug resistance and immune modulation [7, 8], gut microbiota modulation and enhancement of gastrointestinal barrier function [9 ,10], Therefore, advancing the pharmacokinetic profile of curcuminoids through innovative delivery systems remains pivotal to fully realize their therapeutic potential across a spectrum of chronic and inflammatory diseases.

[0276] While curcuminoids hold immense therapeutic promise, their clinical translation has been significantly hindered due to poor bioavailability due to low solubility and extensive first-pass metabolism. Varieties of curcuminoid encapsulation technologies are shown to improve metabolic stability, biocompatibility and absorption, for example, liposomes, micelles, and lipid nanoparticles [11 ,12],

[0277] All current nano- or micro- emulsion or encapsulation as curcuminoid delivery systems need specialized emulsifying agents, many of them are shown to be toxic for health, such as surfactants or emulsifiers. Many reports indicating these surfactants, emulsifiers, stabilisers and their combinations currently are related to negative health and environmental effects and more significantly unknown effects for long term small but daily exposures which may accumulate to irreversible health damage.

[0278] In this example, the bioavailability and anti-inflammatory effects of an all-natural, emulsion-free, surfactant-free, carrier-oil free Turmeric Phyto-Nanoparticle (TPNP), produced directly from whole turmeric rhizome, according to a method of the present invention. Notably, the entire mass of TPNP is composed of bioactive molecules exclusively derived from whole turmeric rhizome.Materials and methods

[0279] THP-1 cell culture: THP-1 human monocytes were expanded from frozen stocks originally purchased from the American Type Tissue Culture Collection (ATCC) using plating media containing Roswell Park Memorial Institute-1640 (RPMI-1640; ThermoFisher Scientific® CAT#A1049101 ) supplemented with 10% FBS (ThermoFisher Scientific® CAT#12483020). Cells were maintained at 37°C and the media was replaced every 3-4 days. THP-1 proliferative monocytes remain as suspension of cells which were differentiated into adherent macrophages by diluting 10nM PMA (VWR®, CAT#10187-494) directly in RPMI1640-10% FBS media. After treatment with PMA, media was replenished with PMA-free RPMI-1640-10% FBS media and incubated for 24hrs for differentiation.File No.: P6501 PC00Treatments were then applied to monocytes and differentiated macrophage cell cultures in RPMI-1640 with 0.5% FBS.

[0280] Turmeric phyto-nanoparticle and control standard curcuminoids spontaneous aggregating particles: Turmeric phyto-nanoparticles (TPNP) were manufactured as sterile aqueous suspension in 0.1x PBS (pH7.4) with 15% ethanol as follows: TPNP are manufactured as micelle-type structures enriched with curcuminoids directly from turmeric rhizomes. The standard curcumin used was purchased from Sigma® [curcumin (C1386) powder]. To evaluate comparative cellular assays, standard curcumin is spontaneously aggregated as nanoparticles.

[0281] TPNP manufacturing: In this example embodiments, the first and second solvent is 95% ethanol, the third Solvent is 0.1x phosphate-buffered saline (PBS) containing 13.7 mM NaCI, 0.27 mM KCI, 1 mM of Na2HPC>4 and 0.18 mM KH2PO4 in water, pH=7.4.

[0282] In step 1 of the process, turmeric root are added into first vessel 14 followed by addition of first solvent of first solvent (95% ethanol) sufficient to contact all the biological matter. First vessel 14 is then closed, and a mild vacuum of about -140 kPa was achieved through a vacuum pump. 50 cycles of mild vacuum of -140 kPa for 2 seconds followed by release vacuum to atmospheric pressure (101 .325 kPa) while the biological matter and solvent are present in contact.

[0283] In step 2 of the process, the first solvent having biological encapsulant materials dissolved and hydrophobic molecules (curcuminoids) from the turmeric roots dissolved in first vessel 14 is filtered through filter 22, equipped with microfiltration (5 micron followed by a 0.22 micron filter), so as to provide the filtered biological encapsulant and endogenous hydrophobic molecules (curcuminoids).

[0284] In step 3 of the process, the first solvent having biological encapsulant materials and endogenous hydrophobic molecules (curcuminoids) from the turmeric roots dissolved therein is pumped from second vessel 16 to third vessel 18 to be mixed. As second solvent 95% ethanol is used as diluent without any exogenous hydrophobic molecules, according to the specifications of this invention.

[0285] In step 4 of the process, a third solvent of aqueous solution (0.1x PBS, pH 7.4) is stored in fourth vessel 20, for the preparation of encapsulation of the endogenous hydrophobic curcuminoids in the third solvent aqueous liquid in process step 5.

[0286] In step 5 of the process, the liquids from the third vessel 18 and the fourth vessel 20 are mixed through liquid-liquid mixer 60. The liquid-liquid mixer 60 delivers all the liquid content of the third vessel 18 to pre-existing third solvent aqueous liquid (O.IxPBS, pH7.4) of fourth vessel 20 in aFile No.: P6501 PC00 circular close loop of piping connection wherein the circular close loop of piping between liquid-liquid mixer 60 and fourth vessel 20 creates one directional aqueous liquid flow forming the encapsulation.

[0287] Curcuminoids Standard Spontaneous Aggregating Particle (CSAP) formation in cell treatment media: the cell culture media used for treatment was RPMI-1640 supplemented with 0.5% Fetal Bovine Serum (FBS). The latter media was spin-filtered (300 kDa filters) to remove particles like extracellular vesicles and protein aggregates present in FBS. Sigma® curcumin powder was dissolved in 95% ethanol to make a stock solution (1.5 mM), which was used to further prepare 50 pM curcuminoids equivalent CSAP in 300 kDa-filtered RPMI-1640 (0.5% FBS) media following incubation at 37 °C for 30 min with intermittent mixing and CSAP colloidal formation was observed. Similarly, CSAP formation in aqueous media of RPMI-1640 and 0.1x PBS, with or without 0.5% FBS were evaluated.

[0288] Nanoparticle characterization: Nanoparticle properties were determined using dynamic light scattering (DLS) and Tunable Resistive Pulse Sensing (TRPS) using Zetasizer® Nano ZSP (Malvern®) and qNano Gold (Izon®) instruments respectively. TPNP were diluted 10 times or 15 times (in 0.1x PBS) and 10,000 times (in deionized water) for DLS and TRPS respectively to measure hydrodynamic diameter, count per ml and zeta potential.

[0289] Total curcuminoid quantitation using fluorescent spectroscopy and HPLC: For fluorescent spectroscopy Synergy H4 Hybrid Reader (Biotek®) was used. Standard curcumin (Sigma®) powder was measured and dissolved in 80% ethanol to make two stocks of 1.5 mM curcuminoids equivalent and serially diluted to 1 nM,10 nM, 100 nM, and 1000 nM in 80% ethanol to produce a standard curve with excitation at 420 nm and emission capture at 570 nm. TPNP samples were 3125-times diluted in 80% ethanol for the above quantitation.

[0290] The fluorescent spectroscopy method was further validated with HPLC. To quantitate if three curcuminoids present in standard curcumin (Sigma®) powder and TPNP formulation using HPLC, a ternary isocratic chromatographic method, coupled with optical diode array detection monitoring compound-specific wavelengths was used. In brief, stock solutions of the three individual curcuminoid standards (Curcumin, Desmethoxycurcumin and Bisdemethoxycurcumin) at 1000 pg / mL were prepared in acetone, followed by dilution to 100 pg / mL (100 ppm) in acetonitrile and used to perform HPLC method optimization and to acquire optical diode array detector (DAD) spectra for the individual compounds. Further validation was performed by mixing three individual curcuminoids standards at 100 pg / mL and used for daily instrument calibration. The three reference standards obtained for this work had a purity >98%. The chromatographic separation of the three curcuminoids was optimized using an isocratic method, which proved to be the most reliable. A final ternary isocratic method consisting of 30% acetonitrile, 25% methanol and 45% water, supplemented with 0.1%File No.: P6501 PC00 phosphoric acid was employed to separate the three curcuminoids sufficiently and to minimize secondary interaction of the curcuminoids with the nanocarrier. DAD setting was scanning range 210- 800 nm, with 1 nm increments, data acquisition was done with Masslynx™ 4.2 with TargetLynx™.

[0291] Transmission electron microscopy (TEM): TPNPs were directly deposited as 5 or 10 pl onto 200 mesh formvar / carbon grids and allowed to incubate at room temperature for 10 minutes before removing the excess liquid by wicking with filter paper. The grid was then sequentially washed twice for two minutes by floating sample-side down on two separate 100 pl of ddF (0.22 pm filtered) droplets on parafilm. Contrast staining was done by floating sample-side down on 1 % uranyl acetate (Fisher Scientific Company®) droplet for 1 minute. Grids were imaged with a STEM3+ detector on a Scios 2 DualBeam (Thermo Scientific®) at 80,000x magnification and 30 kV accelerating voltage. Preparation, imaging, and processing were conducted at the Microscopy and Microanalysis facility at the University of New Brunswick, Fredericton campus (Biology Department).

[0292] Antioxidant capacity: The total antioxidant capacity was determined according to the recommended procedures of the manufacturer of the Ferric Reduction Antioxidant Potential (FRAP) assay kit (ThermoFisher®, Cat # EIAFECL2). In brief, Ferrous chloride [10mM] was diluted in 1X acetate buffer to prepare Fe2+standards of 0, 31.25, 62.5, 125, 250, 500 and 1000 (pM) by serial dilution. All samples were diluted in 1X assay buffer. An ascorbic acid positive control was diluted to 250 pM as assay control to find if the chemistry worked. Fresh stock of 62.5 pM of each of Curcumin (Sigma®), N-Acetyl-L-Cysteine (NAC, Amresco® Cat#0108-25G) and gallic acid (GA) were made in 1X assay buffer as references. A fresh Fe2+standard curve was prepared for each assay. The absorbance was read at 560nm using a Synergy H4 Hybrid Reader (Biotek®).

[0293] Immunoblotting and Confocal microscopy: The immunoblotting method is as decribed in

[0013] following treatments as indicated in the result sections below. Confocal microscopy is as described in

[0014] with minor modifications. In brief, THP-1 cells were seeded at a density of 1x105cells / well on sterilized coverslips placed in 6-well plates and differentiated to macrophages. Hoechst 33258 (ThermoFisher®, Cat#62249) was used at a 0.5pL:10 OOOpL dilution to stain nucleus for 1 minute and finally mounted I sealed on a microscope slide using PermaFluor™ mounting media (Thermo Fisher®, Cat#TA030FM).

[0294] Flow cytometry: THP-1 cells were treated as indicated in the result sections below followed by fixing in 2% formalin in FACS buffer (0.1% BSA in 1x PBS) and subsequently washed twice with 1X PBS. Cells were then gated and stored (75,000 cells) using the Gallios™ 10-color Flow Cytometer (Beckman Coulter®) and curcuminoid fluorescence was assessed in the FL2+ channel.File No.: P6501 PC00

[0295] Cell viability assay: Cell viability assay was performed as per supplier recommendation of PrestoBlue™ Cell Viability Reagent (Invitrogen™). Spectral fluorescence was measured using a Synergy H4 Hybrid Reader (Biotek™) with excitation and emission wavelengths of 560 and 590 nm respectively.

[0296] ELISA: For TNF-a measurement in the cell culture media ELISA MAX™ Standard-set kit (BioLegend®) was used according to the manufacturer’s protocol and absorbances were read using a Synergy H4 Hybrid Reader (Biotek®).Results

[0297] Turmeric Phyto-Nanoparticle (TPNP) characterization

[0298] Each batch of TPNP was stored at 4°C to 8°C for three months and batch-to-batch hydrodynamic diameter (nm) and Polydispersity index (PDI) were verified after 10X dilutions in 0.1x PBS (Table 2) using dynamic light scattering (DLS).scattering measurements for different batches of liquid samples and rehydrated freeze-dried samples at different time points.

[0299] The above TPNPs were also dried using vacuum assisted freeze-drying (-40°C) with sucrose (10% w / v, as cryo-drying excipient) to a powder form (Figure 11A) and immediately sealed in multiple air tight containers, stored at room temperature in dark and dry environment. For rehydration, 0.1 g of the dried TPNPs was dissolved in 1 mL of distilled water followed by a further 10X dilution in water (Figure 11 B, Table 2). The freeze-dried powders were found to increase their size after rehydration but still maintained excellent homogeneity (Table 2) for up to 2 years. To visualize TPNPs morphology, Transmission Electron Microscopy (TEM) was performed (Figure 11C) and these phyto- nanoparticles were found to be spherically shaped. The average hydrodynamic diameter distribution of the TPNP shown in Figure 11 D was measured using DLS after 10X dilution in O.IxPBS and a single peak (177 ± 0.964 nm) was observed, demonstrating particle size homogeneity (PDI= 0.111). The uniformity of particle population was also shown by correlation coefficient curve (Figure 11 D insert).

[0300] TPNPs was also analyzed using Tunable Resistive Pulse Sensing (TRPS) based particle analysis. Figure 11 E shows TRPS of TPNPs diluted 10,000X in deionized water and found toFile No.: P6501 PC00 contain about 9.58 x 1010particles I mL counts with mean particle diameter 176 ± 67.9 nm whereas mode particle diameter was 132 nm.

[0301] Both DLS and TRPS were also used to measure the surface charge (mV) or Zeta potential of those particles. In DLS, TPNP showed a net surface charge (zeta potential) of -0.189 mV in an ionic environment O.IxPBS (15X dilution in O.IxPBS) (Figure 11 F).

[0302] Diluted TPNPs (10.000X dilution in deionized water) was used to measure zeta potential using TRPS producing a mean value of -15.0 mV and mode -11.7 mV, respectively (Figure 11G). The discrepancies of zeta potential value of DLS data in comparison to TRPS data may be due to the presence of sufficient buffering salt from 0.1x PBS (pH7.4) used as diluent in DLS which may neutralize charges, whereas 10.000X dilution in deionized water did not have sufficient buffering salt.

[0303] Curcuminoids Standard Spontaneous Aggregating Particle (CSAP) formation in cell treatment media:

[0304] Curcuminoids are poorly water soluble and form unstable colloidal particles or aggregates in aqueous media like PBS and RPMI-1640 cell growth media

[0015] , Thus, whether curcumin used here as pharmacological reference standard could produce particles by spontaneous aggregation in cell treatment media as described in the method section was tested. Hydrodynamic diameter of the CSAP formed in the cell treatment media (RPMI-1640 + 0.5% FBS) exhibited an average size of 122.1 ± 0.6 nm and PDI of 0.259 in DLS measurements (Figure 12A). To find if the same stock of Sigma curcumin when added to different aqueous media produce same or different sized spontaneous aggregations, the following conditions were verified using DLS and observed that: (i) in RPMI-1640 without any FBS, average diameter was 940.4 ± 98.52 nm and PDI=0.214 (Figure 12B), (ii) in 0.1x PBS with 0.5% FBS, average diameter was 120.2 ± 1 .6 nm) and PDI=0.112 (Figure 12C), (iii) in 0.1x PBS, average diameter was 1280.0 ± 71.48 nm and PDI= 0.214 (Figure 12D). These results indicate that the presence of complex biological fluids containing proteins, lipids and metabolites stabilizes the curcuminoid colloidal particle, resulting in smaller particle sizes in those aqueous environments.

[0305] TPNP in cell treatment media:

[0306] To evaluate whether TPNPs in cell-treatment media (RPMI-1640 with 0.5% FBS) changed particle size, TPNPs were diluted to make 50 pM curcuminoids equivalent in cell-treatment media. It was found that the average hydrodynamic diameter increased to 429.3 nm ± 14.6 nm with PDI = 0.225 (Figure 12E), whereas TPNP diluted in RPMI-1640 without FBS showed diameter of 140.6 ± 2.35 nm), PDI=0.179 (Figure 12F). These results may suggest that complex biological molecules present in FBS may form biological corona around the TPNPs.File No.: P6501 PC00

[0307] Pharmaceutical equivalent curcuminoids loading of TPNP:

[0308] Curcuminoids loading capacity of TPNP and concentration of curcuminoids in standard Sigma curcumin were determined using High Performance Liquid Chromatograph equipped with an optical diode array detector (HPLC-DAD) with three individual curcuminoid standards (curcumin, desmethoxycurcumin and bisdemethoxycurcumin). HPLC-DAD analysis was also carried out for validation of a rapid spectroscopic assays developed.

[0309] Curcumin, desmethoxycurcumin, and bisdemethoxycurcumin were individually measured in HPLC and absorbance maximum at 429 nm, 423 nm and 419 nm, were determined respectively (Figure 13A, 13B and 13C). Standard HPLC mass-fraction distribution of the three individual curcuminoid mixed together (100 ppm each) are shown in Figure 13D. HPLC results indicated that Sigma curcumin standard is composed of three curcuminoids (curcumin, Desmethoxycurcumin and Bisdemethoxycurcumin) and its actual pharmaceutical equivalent curcuminoids’ concentrations were verified. Relative_mass fraction distribution of three curcuminoids of Sigma curcumin standard and TPNP are shown in Figure 14A and 14B respectively. The pharmaceutical equivalent total curcuminoids loading capacity of TPNP samples was found to be 2.03 mM by HPLC method.

[0310] For a rapid and inexpensive 96-well assay, the standard Sigma curcumin forming CSAP and TPNP is prepared in 0.1x PBS and scanned from 250 nm to 800 nm at 1 nm interval to identify maximum absorbance wavelength. The absorbance maximum was found to be about 430 nm for both the samples (Figure 15A, 15B), meaning the highest absorbing light to standard curcumin and TPNP is about 420-430 nm, but was found to be not very sensitive. A more sensitive rapid assay was developed using fluorescent spectroscopic method to quantify total curcuminoids in samples, which was further validated HPLC. For fluorescent spectroscopy assay the standard Sigma curcumin stock and TPNP were diluted in 80% ethanol followed by measurement with fluorescent excitation at 420 nm and emission capture at 570 nm. Based on HPLC spectra, 420 nm was selected as a common excitation wavelength for all three curcuminoids, as it provides near-maximal absorbance for all three species and is suitable for use in fluorescence spectroscopy assays. The rationale of using 80% ethanol as a dilutant for fluorescent spectroscopy is to liberate all curcuminoids from the TPNP and CSAP into the solution for spectral measurements and to limit self-aggregating particles. Using fluorescent excitation at 420 nm and emission capture at 570 nm a pharmaceutical equivalent curcuminoids concentration standard curve was built (Figure 15C) for further batch-to-batch curcuminoid load measurements.

[0311] Using fluorescent based method, the pharmaceutical equivalent curcuminoids concentration of TPNP samples used in in vitro work was measured to be 1.91 mM, whereas 2.03 mMFile No.: P6501 PC00 by HPLC method. Validation by HPLC-DAD showed that fluorescent spectroscopy (combined with 80% ethanol for particles disruption) measured total curcuminoid content with a margin of error within 6%, supporting its use as a reliable method for quantifying total curcuminoids in the TPNP sample batches used in this study.

[0312] The dry weight of TPNP samples were determined to be 2877.6 pg / mL and found to contain 715 pg / mL total curcuminoid (Sum of curcumin, desmethoxycurcumin and bisdemethoxycurcumin concentrations determined by HPLC-DAD), indicating about 24.85% curcuminoid loading capacity (mass x mass) in the TPNP samples.

[0313] The particle concentration of TPNP was 9.58 x 1010 / mL, with a corresponding dry weight of 2877.6 pg / ml. Based on these values, the average dry weight of a single TPNP was estimated to be approximately 30.29 fg (femtogram), of which ~7.5 fg corresponds to curcuminoids; the remaining mass consists of molecules derived from the same turmeric rhizomes.

[0314] Antioxidant capacity

[0315] Curcuminoids and other polyphenols are known to exhibit potent antioxidant capacity. The measurement of antioxidant capacity by Ferric Reducing Antioxidant Power (FRAP) assay for TPNP and standard Sigma curcumin was performed, where NAC (N-acetycysteine) and Gallic Acid (GA) served as reference antioxidants. To calculate % antioxidant capacity, 62.5 pM curcuminoids (Sigma) was used as 100% reference point and compared with (i) TPNP (curcuminoid equivalent 62.5 pM), (ii) 62.5 pM NAC and (iii) 62.5 pM GA, as shown in Figure 16. The TPNP suspension medium or vehicle showed no antioxidant activity. The antioxidant capacity observed at the 62.5 pM curcuminoid equivalent dose of TPNP, approximately 2.6 times greater than that of 62.5 pM standard Sigma curcumin (Table 3), may be attributed to the presence of other polyphenols sourced from the biomass of turmeric rhizome, which comprises approximately 75% of the total mass.Table 3 - Reference-based total antioxidant capacity. All to reference of 62.5 pM Curcuminoids Standard and the values of optical density (CD) corresponding to Fe2+concentrations.

[0316] Dose tolerance of pharmaceutical equivalent concentration CSAP and TPNP in THP- 1 monocyte / macrophages cells:File No.: P6501 PC00

[0317] Titration of CSAP and TPNP doses were performed to identify LD50 (Figure 17). As shown in Figure 17A and 17B, THP-1 monocytes were treated with varying doses of standard curcuminoids (forming CSAP in RPMI-1640-supplemented with 0.5% FBS) as well as TPNP in cell culture media followed by cellular viability analysis, which was measured by resazurin method. In monocytes, the LDsoof the standard curcuminoids-based CSAP was approximately 9.24 pM, whereas that of TPNP was unexpectedly higher, at 12.0 pM. A similar cell viability study was conducted in THP- 1 macrophages (differentiated from THP-1 monocytes as described in above) by exposing the differentiated cells to varying doses of CSAP and TPNP, and observed an LD50 of 10.3 pM for both CSAP and TPNP treatments (Figure 17C and 17D).

[0318] Two sub-lethal pharmaceutical doses curcuminoid equivalents of CSAP (3.75 pM and 7.5 pM) and TPNP (2.5 pM and 5.0 pM) were chosen to evaluate cellular survival in other non-immune cell types: human cardiomyocyte (AC16) and human blood brain barrier (BBB) cells (hCMEC / D3) as shown in Figure 18A & 18B respectively. Human AC16 cardiomyocytes were unexpectedly found to be less tolerant to high CSAP doses of 7.5 pM in comparison to 3.75 pM whereas human Blood Brain Barrier cells and AC16 cardiomyocytes were found to tolerate well TPNP at 3.75 pM and 5.0 pM curcuminoids.

[0319] Bioavailability of CSAP and TPNP in THP1 monocytes and macrophages:

[0320] The internalization and accumulation of TPNP were observed using confocal microscopy where TPNP localization (autofluorescence of curcumin detected in the GFP channel), DAPI staining for nucleus (blue) and F-actin (Phalloidin-staining, red) were performed on THP1 macrophages and found to be comparatively enriched into nucleus than cytoplasm (Figure 19). CSAP and TPNP curcuminoids doses of 7.5 pM and 5.0 pM were selected to evaluate cellular uptake kinetics using Fluorescence-Activated Cell Sorting (FACS). Higher dose of CSAP (7.5 pM curcuminoid) were selected to obtain optimal FACS fluorescence signal to differentiate between curcuminoid positive and unstained cells with minimum background. As shown in Figure 20, THP-1 macrophages were treated with standard pure curcuminoids derived CSAP (7.5 pM curcuminoid) and TPNP (5.0 pM curcuminoid). TPNP (5.0 pM curcuminoid) was unexpectedly internalized rapidly by these cells, accumulating in 47.5% of cells (median fluorescent intensity = 527) within 30 min of exposure and progressively accumulating in 95.3% of cells (median fluorescent intensity = 1271) at 16 h whereas standard pure curcuminoids derived CSAP (7.5 pM curcuminoid) dose accumulating in only about 8.05% of cells (median fluorescent intensity = 277) within 30 min of exposure and reaching 28.8% of cells (median fluorescent intensity = 423) at 2 h of exposure followed by progressively less cells containing standard pure curcuminoids as shown Figure 21A and Table 4.File No.: P6501 PC00Table 4 - Summary of Macrophage FACS data of percent of gated cells in the FL2 channel (curcuminoid positive) with indicated time of treatments with indicated CSAP and TPNP dose. PVDL- veh refer to the liquid vehicle without any TPNP.

[0321] Similarly, THP-1 monocytes were treated with CSAP (7.5 pM curcuminoid) and TPNP (5.0 pM curcuminoid) pharmaceutical doses for up to 24 h, see Figure 21 B. FACS analysis demonstrated the accumulation / internalization of TPNP (5.0 pM curcuminoid) in monocytes reaching 16.9% cells (median fluorescent intensity = 739) after 4 h of exposure and highest at 59.4% of cells (median fluorescent intensity= 1397) after 24 h of exposure, whereas standard pure curcuminoids derived CSAP (7.5 pM curcuminoid) dose accumulating to 2.85% (median fluorescent intensity= 313) in 4h and highest about 15.4% of cells (median fluorescent intensity = 659) after 24h of exposure (Figure 22 and Table 5). TPNP was internalized more rapidly than CSAP in both macrophages and monocytes, which may indicate that TPNP formulation is better for cellular bioavailability compared to CSAP.File No.: P6501 PC00Table 5 - Summary of monocyte FACS data of percent of gated cells in the FL2 channel (curcuminoid positive) with indicated time of treatments with indicated CSAP and TPNP dose.Cellular pharmacodynamic anti-inflammatory effects of TPNP:

[0322] Cellular pharmacodynamic is an estimation of pharmacological actions on living cells and its manifestation of the actions of the pharmaceutically important compounds.

[0323] Anti-inflammatory enzyme HMOX1 expression increases with treatments of TPNP in THP-1 monocytes and macrophages:

[0324] Increased expression of HMOX1 correlates with anti-inflammatory effects and promotion of wound healing in damaged tissues. HMOX1 enzyme expression is evaluated using immunoblotting in THP-1 monocytes (Figure 23, left). HMOX1 enzyme expression were evaluated with treatments (4.5 h) of TPNP (5.0 pM curcuminoid) doses and standard pure curcuminoids derived CSAP curcuminoid dose of (7.5 pM). No HMOX1 enzyme expression was observed in control treatments, but relatively higher expression was observed with TPNP (5.0 pM) than standard pure curcuminoids derived CSAP (7.5 pM curcuminoid). The total MemCode protein stain (bottom) confirms equal protein loading across samples.File No.: P6501 PC00

[0325] Similarly, THP-1 macrophages were treated for 6 h with TPNP curcuminoid doses (5.0 pM) and standard pure curcuminoids derived CSAP (7.5 pM curcuminoid) and immunoblotted for HMOX1 enzyme expression. As shown in Figure 23, right, TPNP induce more HMOX1 enzyme expression in macrophages at lower curcuminoid dose in comparison to higher dose of standard pure curcuminoids derived CSAP. Respective vehicle treated and untreated control cells showed basal level of HMOX1 enzyme expression, with the total MemCode protein stain (bottom) confirming uniform protein loading across samples.

[0326] Dampening of LPS induced inflammation with treatments of TPNP in THP1 macrophages: Curcuminoids are known to downregulate levels of TNF-a. THP-1 macrophages responded to lipopolysaccharide (LPS) stimulation by quickly releasing TNF-a in the media

[0016] , THP- 1 macrophages were pre-treated with 5 pM curcuminoid TPNP or CSAP followed by LPS treatment (50 ng / mL) at 0.5 h to induce inflammatory responses for a period of 1 .5 h and 4 h. As shown in Figure 24, the concentrations of TNF-a detected in the conditioned media demonstrates thatTNF-a is a rapidly released pro-inflammatory cytokine, measurable within 1.5 h of LPS exposure. Treatment with 5pM curcuminoid dose of TPNP or CSAP on LPS challenged macrophages reduced total secretion of TNF- a in comparison to media and vehicle controls. Unexpectedly, TPNP demonstrated superior reduction in TNF-a secretion when compared to CSAP at the timepoint of 1 .5 h (p=0.0393) and 4 h (p=0.0003).

[0327] To identify total molecular fingerprints of TPNP a NanoLC-MS / MS based profiling was performed. Structural annotation was conducted using ClassyFire, an automated structure-based classification tool integrating multiple biomolecular databases for natural products. Metabolite annotation was conducted using the opensource Sirius 4 CSI: Fingerid software. The .mgf file of spectral data, exported from MzMine3, was imported into Sirius 4. Default parameters were used for identification, using “Orbitrap” as the type of data and the following database were used: BioDataBase, Biocys, KNApSAcK, Natural products, Plantcys, YMDB and GNPS. The ClassyFire framework organizes molecular entities into a hierarchical ontology: (i) Kingdom level: organic and inorganic compounds, (ii) Superclass: 26 organic and 5 inorganic categories, (iii) Class: 764 structural categories, (iv) SubClass: 1 ,729 subdivisions encompassing >10,000 compounds, (v) Levels 5-11 : finer resolution based on chemical weight, spanning 910 to 2 entities.

[0328] NanoLC-MS / MS analysis of TPNP confirmed the presence of phospholipids, fatty acids, curcuminoids, terpenoids, etc in TPNP. The figure 25A and 25B represent two bottom hierarchical ontology: SubClass and level-5. Figure 25A shows distribution of top seven categories of molecules by percentage of signal peak intensity area after ClassyFire level 5 structural annotation showing predominance of curcuminoids signals. Similarly, top seven categories of molecules present in TPNP based on ClassyFire SubClass structural annotation is shown in Figure 25B.File No.: P6501 PC00Discussion

[0329] TPNPs are not plant-derived exosomes, vesicles or any sub-cellular structures originally synthesized within plant cells and tissues. Instead, they are manufactured as micelle-type structures enriched with curcuminoids directly from turmeric rhizomes, containing 24.85% curcuminoids by mass, with the remaining mass composed of endogenous molecules originally present in turmeric rhizomes.

[0330] This novel class of all-natural, emulsion-free, surfactant-free, carrier-oil free Turmeric Phyto-Nanoparticle (TPNP) was evaluated alongside with Curcuminoids Spontaneous Aggregating Particles (CSAP), which formed in cell culture media under emulsion-free, surfactant-free, and carrier- oil-free conditions. Both TPNP and CSAP exhibited sub-micron hydrodynamic diameters. The rational is to evaluate equivalent curcuminoid dose-based potential of TPNP with a similar nanoparticle made with standard pure curcuminoid. To confirm nanoparticles average hydrodynamic diameters two methods of biological nanoparticle analysis (DLS and TRPS) were performed and hydrodynamic diameters of TPNP were observed to be the same using both the methods. TPNP demonstrated stability as dried powder after freeze-drying and storage at room temperature. Powdered TPNP increase their size after rehydration but maintain excellent population homogeneity. The presence of complex biological materials such as FBS was found to influence the effective hydrodynamic diameters of both TPNP and CSAP which may be due to interaction of FBS ingredients or formation of a biocorona. The pharmaceutical equivalent curcuminoids contents of each particle type was quantified using High-Performance Liquid Chromatography, with individual curcuminoid standards (curcumin, desmethoxycurcumin, and bisdemethoxycurcumin), as well as HPLC-validated fluorescence spectroscopy for total curcuminoid concentration for batch-to-batch analysis. Curcuminoids and other plant polyphenol are known scavenger of free radicals. At same pharmaceutical equivalent curcuminoids concentration, antioxidant capacity of TPNP was observed to be surprisingly and significantly higher in comparison to standard Sigma curcuminoids. The higher antioxidant capacity of TPNP may be attributed to other endogenous molecules from turmeric rhizome contributing to the unexpectedly improved antioxidant capacity.

[0331] Curcuminoids are known for dose specific altered outcome, for example cancer cells are susceptible to a certain dose whereas similar curcuminoid dose generally exerts protective effects in healthy cells. Here, THP-1 monocytes and macrophages treated with CSAP and TPNP exhibited cytotoxic effects at higher pharmaceutical equivalent curcuminoid concentration with comparable LD5o doses observed in monocytes, while TPNP was better tolerated in macrophages.

[0332] Cellular bioavailability

[0017] kinetics is the final dynamic process-stage in which active compounds to internalized to engage target cellular signaling pathways and serves as a useful tool forFile No.: P6501 PC00 readily assessing compound bioavailability. Most hydrophobic drug candidates (both natural and synthetic) suffer from poor bioavailability, prompting the development of delivery technologies such as nanoemulsions. Curcuminoids are fluorescent green and can be detected using standard GFP filters / or channels of fluorescent spectroscopy, microscopy and FACS, which are ideal for curcuminoid quantitation. FACS analysis showed that while both cell types internalized TPNP and CSAP, uptake was slower and less efficient in monocytes. In contrast, macrophages internalized TPNP in 95% of cells (median fluorescent intensity = 1271) compared to only 5.58% of cells (median fluorescent intensity= 236) for CSAP at 16hr.

[0333] Turmeric extracts and curcuminoids are attributed to possess immunomodulatory and anti-inflammatory / anti-oxidant effects. Monocytes to macrophages differentiation dynamics and further pro-inflammatory M1 or anti-inflammatory M2 macrophage polarization is essential for tissue repair pathways in the event of infection and damage. Increased expression of HMOX1 correlates with the M2 phenotype

[0018] , which has been shown to exert anti-inflammatory effects and promote wound healing in damaged tissues. Both TPNP and CSAP induced HMOX1 protein expression, with macrophages showing stronger responses than monocytes.

[0334] Tumor Necrosis Factor Alpha (TNF-a) is a rapid-response cytokine released by immune cells under conditions of stress, infection, or injury. It is widely regarded as a master regulator of inflammation, initiating a cascade of inflammatory signaling pathways that underlie many chronic diseases. Therefore, suppression or inhibition of TNF-a release presents a promising strategy for the development of anti-inflammatory therapeutics. Upon LPS stimulation, macrophages produced TNF- a, which was significantly reduced by TPNP pre-treatment compared to CSAP at the same pharmaceutical equivalent curcuminoids dose.

[0335] TPNPs unexpectedly increased the intracellular delivery efficiency of active curcuminoids, suggesting that all-natural nanoparticle formulations may offer enhanced therapeutic potential without the adverse effects associated with synthetic emulsifying excipients commonly used in nano-curcuminoid delivery systems. TPNPs may be proposed as safer to conventional nanoemulsions and lipid-based nanocarrier.

[0336] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.File No.: P6501 PC00REFERENCES1 . Jabczyk M, Nowak J, Hudzik B, Zubelewicz-Szkodziriska B. Curcumin in Metabolic Health and Disease. Nutrients. 2021 Dec 11 ;13(12):4440. doi: 10.3390 / nu13124440. PMID: 34959992; PMCID: PMC8706619.2. Peng Y, Ao M, Dong B, Jiang Y, Yu L, Chen Z, Hu C, Xu R. Anti-Inflammatory Effects of Curcumin in the Inflammatory Diseases: Status, Limitations and Countermeasures. Drug Des Devel Ther. 2021 Nov 2;15:4503-4525. doi: 10.2147 / DDDT.S327378. PMID: 34754179; PMCID: PMC8572027.3. Cifuentes M, Verdejo HE, Castro PF, Corvalan AH, Ferreccio C, Quest AFG, Kogan MJ, Lavandero S. 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Heme oxygenase-1 overexpression exacerbates heart failure with aging and pressure overload but is protective against isoproterenol-induced cardiomyopathy in mice. Cardiovasc Pathol. 2014 Jul- Aug;23(4):231-7. doi: 10.1016 / j.carpath.2014.03.007. Epub 2014 Apr 5. PMID: 24813593.14. Ghosh A, Kuppusamy H, Pilarski LM. Aberrant splice variants of HAS1 (Hyaluronan Synthase 1) multimerize with and modulate normally spliced HAS1 protein: a potential mechanism promoting human cancer. J Biol Chem. 2009 Jul 10;284(28):18840-50. doi: 10.1074 / jbc.M109.013813. Epub 2009 May 18. PMID: 19451652; PMCID: PMC2707203.15. Kang, S.; Kim, M.; Kim, H.; Hong, J. Enhancement of Solubility, Stability, Cellular Uptake, and Bioactivity of Curcumin by Polyvinyl Alcohol. Int. J. Mol. Sci. 2024, 25, 6278. https: / / doi.org / 10.3390 / iims2511627816. Takashiba S, Van Dyke TE, Amar S, Murayama Y, Soskolne AW, Shapira L 1999. 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Claims

File No.: P6501 PC00CLAIMS :1 . A system for extracting and encapsulating a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material, the system comprising:(a) a vacuum pump;(b) a first vessel, said first vessel having a first outlet, said first vessel being operable to contain and contact said bulk multicellular biological material with a first solvent to make a solvent contacted bulk multicellular biological material, said first vessel being connected to said vacuum pump, said first vessel being adapted to perform at least 2 cycles of (b-i) and (b-ii):(b-i) using the vacuum pump, selectively creating a negative pressure inside said first vessel containing said solvent contacted bulk multicellular biological material; and (b-ii) returning to atmospheric pressure said first vessel containing said solvent contacted bulk multicellular biological material, wherein (b-i) and (b-ii) are performed for a time sufficient to permeate said first solvent through, and to extract and separate said biological molecules from said solvent contacted bulk multicellular biological material in a mixture of soluble biological molecules in said first solvent;(c) a filter, said filter having a filter inlet and a filter outlet, said filter inlet being fluidly connected to said first outlet of said first vessel through a first connection for receiving said mixture of soluble biological molecules in said first solvent, said filter being operative to separate remaining fragments of said bulk multicellular biological material to produce a filtered mixture of soluble biological molecules in said first solvent;(d) a second vessel, the second vessel having a second inlet and a second outlet, said second inlet being fluidly connected to said filter outlet through a second connection for receiving said filtered mixture of soluble biological molecules in said first solvent, said second vessel being operable for receiving said filtered mixture of soluble biological molecules in said first solvent; and(e) a third vessel, said third vessel having a third inlet and a third outlet, said third inlet being fluidly connected to said second outlet of said second vessel through a third connection for receiving said filtered mixture of soluble biological molecules in said first solvent from theFile No.: P6501 PC00 said second vessel and a solution of the hydrophobic molecule in a second solvent, thereby producing a homogeneous mixture, said third vessel being operable to contact said mixture of soluble biological molecules in said first solvent with a solution of said hydrophobic molecule in a second solvent to produce said homogeneous mixture.(f) if the bulk multicellular biological material contains one or a plurality of hydrophobic molecules of interest for encapsulation which are present along with the soluble biological molecules in the first solvent, the second solvent is free of the hydrophobic molecule or the first solvent comprises the hydrophobic molecule.

2. The system of claim 1 , further comprising a fourth vessel having a fourth inlet and a fourth outlet, said fourth inlet being fluidly connected to the third outlet of the third vessel through a fourth connection for receiving said capsule encapsulating said hydrophobic molecule, the fourth vessel being operable to contact said capsule encapsulating said hydrophobic molecule with a third solvent to cause formation of the capsule by self-aggregation of the biological molecules and encapsulation of the hydrophobic molecule therein, thereby producing encapsulation of said hydrophobic molecules in said aqueous liquid.

3. The system of claim 2, further comprising a treatment unit, said treatment unit being connected to said fourth vessel for further treatment of said encapsulation of said hydrophobic molecules in said aqueous liquid, the treatment unit being selected from the group consisting of a concentrator, a freezer, a frost-free freezer, an oven, a sprayer and a gel-producing device.

4. The system of any one of claims 1 to 2, further comprising a liquid-liquid mixing chamber within said fourth connection, said liquid-liquid mixing chamber having a main inlet, said main inlet being fluidly connected to said fourth outlet of said fourth vessel through a fifth connection for receiving said encapsulation of said hydrophobic molecules in said aqueous liquid; a main outlet, said main outlet being fluidly connected to said fourth inlet of said fourth vessel through a second portion of said fourth connection; an auxiliary inlet, said auxiliary inlet being fluidly connected through a first portion of said fourth connection to said third outlet of said third vessel for receiving said capsule encapsulating said hydrophobic molecule; and a mixing cavity intermediate said main inlet and said main outlet, said auxiliary inlet being fluidly connected to said mixing cavity,File No.: P6501 PC00 wherein said liquid-liquid mixing chamber is operable to mix in said mixing cavity said capsule encapsulating said hydrophobic molecule received from said third vessel through said auxiliary inlet with said encapsulation of said hydrophobic molecules in said aqueous liquid received from said fourth vessel through said main inlet.

5. The system of claim 4, wherein said auxiliary inlet comprises a plurality of nozzles, said plurality of nozzles being oriented at least partially counter-flow so as to inject said encapsulating molecules and said hydrophobic molecules received from said third vessel against a flow of said aqueous liquid received from said fourth vessel, or wherein said second solvent is free of said hydrophobic molecule if the bulk multicellular biological material contains one or more endogenous hydrophobic molecules of interest for encapsulation, present in the soluble biological molecules in the first solvent, where the first solvent may comprise the hydrophobic molecule.

6. The system of claim 1 wherein said vacuum pump is operative to generate a negative pressure of a maximum of -200 kPa.

7. The system of claim 1 , wherein said filter has pore size of from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about0.45 pm, or about 0.1 pm to about 0.2 pm.

8. The system of claim 1 , wherein said filter is operative to filter molecules having 103- 106Da.

9. The system of claim 1 , wherein said first vessel comprises a vacuum relief valve, said vacuum relief valve being selectively operable to return to atmospheric pressure said first vessel when said first vessel is initially under the negative pressure created by said vacuum pump.

10. A process for the encapsulation of a hydrophobic molecule into a capsule formed from biological molecules from a bulk multicellular biological material comprising the steps of:(a) contacting said bulk multicellular biological material with a first solvent, to obtain a solvent contacted bulk multicellular biological material,(b) negative pressure-solvent extraction of said solvent contacted bulk multicellular biological material comprising at least 2 cycles of (b-i) and (b-ii):(b-i) negative pressure treatment of said solvent contacted bulk multicellular biological material; andFile No.: P6501 PC00(b-ii) return to atmospheric pressure of said solvent contacted bulk multicellular biological material; for a time sufficient to permeate said first solvent through, and extract said biological molecules from said solvent contacted bulk multicellular biological material,(c) separation of said solvent contacted bulk multicellular biological material from said first solvent, to obtain a mixture comprising soluble biological molecules in the first solvent and optionally further comprising an endogenous hydrophobic molecule present in the bulk multicellular biological material; and(d) contacting said mixture of soluble biological molecules, and optionally the endogenous hydrophobic molecule present in the bulk multicellular biological material, in said first solvent with second solvent free of the hydrophobic molecule, or with a second solvent comprising said hydrophobic molecule and mixing thoroughly to obtain a homogeneous mixture, and(e) contacting said homogeneous mixture with a third solvent to cause formation of said capsule by self-aggregation of said soluble biological molecules and encapsulation of said hydrophobic molecule and / or the endogenous hydrophobic molecule therein, to form a capsule encapsulating said hydrophobic molecule.

11. The process of claim 10, wherein said mixture of soluble biological molecules comprises terpenoids, fatty-acids, amino acids, peptides, alkaloids, carbohydrates, polyketides, shikimates, phenylpropanoids, polyphenols, and combinations thereof.

12. The process of claim 10 or 11 , further comprising a further collection step (f) comprising at least one of concentration, freeze-drying, heat-drying, spray-drying and gelling, of said capsule encapsulating said hydrophobic molecule.

13. The process of any one of claims 10 - 12, wherein said negative pressure treatment is at a maximum of -200 kPa.

14. The process of any one of claims 10 - 12, wherein said negative pressure treatment is from about -1 kPa to about -200 kPa.

15. The process of any one of claims 10 - 14, wherein in step (c), said separation is a filtration for removal of a remaining fragment of said bulk multicellular biological material.File No.: P6501 PC0016. The process of claim 15, wherein filtration is microfiltration, ultrafiltration, or combinations thereof.

17. The process of any one of claims 15 - 16, wherein filtration is with a filter having pore size of from about 0.1 pm to about 1 pm, or from about 0.1 pm to about 0.8 pm, or from about 0.1 pm to about 0.5 pm, or about 0.1 pm to about 0.45 pm, or about 0.1 pm to about 0.2 pm.

18. The process of any one of claims 16 - 16, wherein filtration is with a filter configured for filtration of molecules having 103- 106Da.

19. The process of claim 11 , wherein said capsule encapsulating said hydrophobic molecule comprises a core and an outer shell layer comprised of said soluble biological molecules providing a water compatible particle stability.

20. The process of any one of claims 10 - 19, wherein formation of said capsule encapsulating said hydrophobic molecule is performed at a temperature of from about 10°C to 80°C.

21. The process of claim 12, wherein a time sufficient for spray-drying said capsule encapsulating said hydrophobic molecule is from about 5 minutes to about 1 hour, or from about 5 minutes to about 30 minutes, or from about 5 minutes to about 25 minutes, or from about 5 minutes to about 20 minutes, or from about 5 minutes to about 15 minutes, or from about 5 minutes to about 10 minutes; and wherein a time sufficient for freeze-drying is from about 12 h to about 24h, or from about 24h to about 36h, or from about 36h to about 48h.

22. The process of claim 12, wherein said freeze-drying is performed at a temperature of -10°C to about -60°C.

23. The process of claims 12, 21 and 22, wherein said freeze-drying is performed with a freezing excipient.

24. The process of claim 23, wherein said freezing excipient is sucrose, glucose, dextran, trehalose, lactose, mannitol, maltose, alanine, glycine or a combination thereof.File No.: P6501 PC0025. The process of claim 12, wherein said concentration is performed by microfiltration, ultrafiltration, dead-end filtration, tangential flow filtration, differential centrifugation, or a combination thereof.

26. The process of claim 12, wherein said heat-drying is performed at a temperature of 30°C to about 60°C.

27. The process of claim 26, wherein said heat-drying is performed under vacuum.

28. The process of claims 12, 26 and 27, wherein said heat-drying is performed with a stabilizing excipient.

29. The process of claim 28, wherein said stabilizing excipient is microcrystalline cellulose, methylcellulose, hydroxypropyl methyl cellulose, maltodextrin, gum Arabic, Guar gum, chitosan, pectin, sucrose, lactose, trehalose, Arabic gum, whey protein, polyvinyl alcohol, modified, starch, nut gum, sodium alginate, sodium carboxymethyl cellulose, L-leucine, polyethylene glycol, or a combination thereof.

30. The process of claim 12, wherein said spray-drying is performed at a temperature of 110°C to about 165°C.31 . The process of claims 12 and 30, wherein said spray-drying is performed with a spray-drying excipient.

32. The process of claim 31 , wherein said spray-drying excipient is from about 10% w / v to about 30% w / v.

33. The process of claim 12, wherein said gelling is performed with a gelling excipient.

34. The process of claim 33, wherein said gelling excipient is hyaluronic acid, methyl-cellulose, alginate, pectin, carrageenan, gellan, gelatin, agar, modified starch, methyl cellulose and hydroxypropyl methyl cellulose, xanthan gum, or a combination thereof.

35. The process of any one of claims 10 - 34, wherein step (e) is performed by mixing said homogeneous mixture with said third solvent at a ratio of from about 1 :3 to 1 :100.File No.: P6501 PC0036. The process of any one of claims 10 - 35, wherein in step (d) an absolute mass ratio of said soluble biological molecules and said hydrophobic molecule is from about 1 :10 to 2000:1.

37. The process of any one of claims 10 - 36, wherein said first solvent, said second solvent, or both is a non-aqueous solvent completely miscible with water.

38. The process of any one of claims 10 - 36, wherein in step (d), said second solvent is free of said hydrophobic molecule or comprises said hydrophobic molecule.

39. The process of claim 37, wherein said non-aqueous solvent is a solvent with a polarity index between 3.9 and 8.0.

40. The process of claim 37, wherein said non-aqueous solvent is ethanol, methanol, acetone, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, 2-methoxyethanol, pyridine, 1 ,4-dioxane, tetra hydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.41 . The process of any one of claims 10 - 39, wherein said third solvent is an aqueous solvent.

42. The process of claim 41 , wherein said third aqueous solvent is water or a mixture of water and a non-aqueous solvent.

43. The process of claim 42, wherein said non-aqueous solvent is ethanol, methanol, acetone, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, 2-methoxyethanol, pyridine, 1 ,4-dioxane, tetra hydrofuran, n-propyl alcohol, isopropyl alcohol, or combinations thereof.

44. The process of any one of claims 10 - 43, wherein said negative pressure treatment is for about 0.5 second to about 5 seconds.

45. The process of claim 44, wherein said negative pressure treatment is for about 0.5 second.

46. The process of any one of claims 10 - 45, wherein said return to atmospheric pressure is for about 0.01 second to about 1 seconds.

47. The process of claim 46, wherein said return to atmospheric pressure is for about 0.01 second.File No.: P6501 PC0048. The process of any one of claims 44 - 46, wherein in step (b), said time sufficient to permeate said first solvent through is from about 3 to about 200 cycles of (b-i) and (b-ii).

49. The process of any one of claims 1 - 47, wherein said capsule encapsulating said hydrophobic molecule have a size of about 10 pm or less.

50. The process of claim 48, wherein said capsule encapsulating said hydrophobic molecule have a size of from about 100 nm to about 10 pm.51 . The process of claim 49, wherein said capsule encapsulating said hydrophobic molecule have a polydispersity index (PDI) of less than or equal to 0.7 (< 0.7), or less than or equal to 0.3 (< 0.3).

52. The process of any one of claims 10 - 50, wherein said hydrophobic molecule is a molecule having a solubility of < 20 mg / L of water at room temperature.

53. The process of any one of claims 10 - 49, wherein said hydrophobic molecule has a molecular weight of < 3 kDa.

54. The process of any one of claims 10 - 48, wherein said hydrophobic molecule is a pesticide, a fungicide, a herbicide, an antimicrobial, a drug, a nutrient, a hydrophobic plant extract, an endogenous hydrophobic molecules present in bulk multicellular biological material or combinations thereof.

55. The process of any one of claims 10 - 51 , wherein said bulk multicellular biological material is from a plant, a fungus, an alga, a yeast, and combinations thereof.

56. The process of any one of claims 10 to 54, wherein said bulk multicellular biological material comprises one or a plurality of said endogenous hydrophobic molecule of interest for encapsulation which are present along with the soluble biological molecules, in the first solvent.

57. The process of any one of claims 10 - 55, wherein said capsule encapsulating said hydrophobic molecule are free of an emulsifier, a carrier oil, or combinations thereof.

58. A turmeric nanoparticle composition comprising:File No.: P6501 PC00 a plurality of nanoparticles comprising a capsule comprising self-aggregated soluble biological molecules extracted from a turmeric rhizome material, said capsule forming a micelle around a hydrophobic core; said hydrophobic core encapsulating hydrophobic molecules endogenous to said turmeric rhizome material, said hydrophobic molecules comprising curcuminoids, said nanoparticles having a particle size of from about 150 nm to about 250 nm, a polydispersity index (PDI) of less than or equal to 0.3 (< 0.3); and a curcuminoids loading capacity of about 20% to about 30% mass of curcuminoids per total mass of said nanoparticles, and a carrier.

59. The turmeric nanoparticle composition of claim 58, further comprising a freezing excipient, a stabilizing excipient, a spray-drying excipient, a gelling excipient, or a combination thereof.

60. The turmeric nanoparticle composition of claim 59, wherein said freezing excipient is sucrose, glucose, dextran, trehalose, lactose, mannitol, maltose, alanine, glycine or a combination thereof.

61. The turmeric nanoparticle composition of claim 59, wherein said stabilizing excipient is microcrystalline cellulose, methyl-cellulose, hydroxypropyl methyl cellulose, maltodextrin, gum Arabic, Guar gum, chitosan, pectin, sucrose, lactose, trehalose, Arabic gum, whey protein, polyvinyl alcohol, modified, starch, nut gum, sodium alginate, sodium carboxymethyl cellulose, L-leucine, polyethylene glycol, or a combination thereof.

65. The turmeric nanoparticle composition of claim 59, wherein said spray-drying excipient is from about 10% w / v to about 30% w / v of gum Arabic, maltodextrin, or a combination thereof.

63. The turmeric nanoparticle composition of claim 59, wherein said gelling excipient is hyaluronic acid, methyl-cellulose, alginate, pectin, carrageenan, gellan, gelatin, agar, modified starch, methyl cellulose and hydroxypropyl methyl cellulose, xanthan gum, or a combination thereof.

64. The turmeric nanoparticle composition of any one of claims 58 - 63, wherein said capsule have a size of from about 170 nm to about 240 nm.

65. The turmeric nanoparticle composition of any one of claims 58 - 64, wherein said capsule have a PDI of from about 0.05 to about 0.26.File No.: P6501 PC0066. The turmeric nanoparticle composition of any one of claims 58 - 65, wherein said capsule or said hydrophobic molecules endogenous to said turmeric rhizome material are free of an emulsifier, a surfactant, a carrier oil, or combinations thereof.

67. The turmeric nanoparticle composition of any one of claims 56 - 64, wherein said nanoparticles have a curcuminoids loading capacity of about 25% mass of curcuminoids per total mass of said nanoparticles.

68. The turmeric nanoparticle composition of any one of claims 56 - 65, wherein said nanoparticles have an antioxidant capacity of from about 2 to about 4 times higher than an equivalent pM concentration of pure curcuminoids as measured by a Ferric Reducing Antioxidant Power (FRAP) assay.69 A method of treating or preventing an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease, comprising administering a turmeric nanoparticle composition of any one of claims 56 - 68, to a subject in need thereof.

70. Use of the turmeric nanoparticle composition of any one of claims 56 - 68, for the treatment or prevention of an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease.71 . The turmeric nanoparticle composition of any one of claims 56 - 68, for use in the treatment or prevention of an inflammatory disease, a cardiovascular disease, or a neurodegenerative disease.

72. The method of claim 69, the use of claim 70 or the turmeric nanoparticle composition of claim 70, wherein said inflammatory disease is arthritis, or inflammatory bowel disease.

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