PARTICLES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority of US provisional patent application No. 63/159,632 filed on March 11 , 2021 , the specification of which is hereby incorporated by reference.

BACKGROUND

(a) Field

[0002] The subject matter disclosed generally relates to methods for the production of nano- and microscopic particles from bulk biological materials, and compositions comprising the same. More particularly, the methods include using of low-voltage electromotive force in contact with the bulk biological material, which is in contact with a fluid for a time sufficient for release of the particles produced in the liquid. Furthermore, the particles are produced over a range of sizes from nano- to microscopic sizes and comprise, in part or whole, constituents of the bulk biological material used and constituents of the fluid used in contact.

(b) Related Prior Art

[0003] Nanoparticles (NP) and microparticles (MP) are particles having wide spectrum of uses. As used herein, the prefixes “nano” and “micro” are generally used to describe sizes of particles ranging from 1 nanometer to 100 nanometer and 0.1 micrometer to 5 micrometers in average diameter respectively. Most metal or inorganic NPs and MPs are produced either by finer grinding or mechanical attrition, combustion, precipitation, evaporation, sol-gel processes, gas condensation, etc. Processes for their productions are energy-intensive and use of toxic solvents, resulting in high production costs of synthetic NPs and MPs. Also, over the years, as their use in many areas of technology increased, a lot of chemically and physically synthesized NPs and MPs were found to be unhealthy for individuals and dangerous forthe environment. For example, many cosmetics, pharmaceuticals, sunscreens, food products used daily contain inorganic nano- and micro- sized materials for their huge value-adding properties for improved product quality, efficiency and look. The ever-increasing use of nano- and micro-materials in cosmetics and other daily use products, in the context of insufficient regulations and techniques for assessing the biological and environmental impact of these products, are concerns for scientific- and world-bodies because of their accumulated toxicity in body and environment. The use of NPs and MPs is evident in many products, ranging from packaging to paints to food-additives, cosmetics and pharmaceuticals. Moreover, in cosmetics, inorganic NPs (mostly titanium dioxide, zinc oxide, silver, gold, silicate, etc.) coated with organic oils or proteins used in lip, face, eye and dermal care products, make them more bio-available to penetrate into cells to the extent of increased neurotoxicity and DNA damage. Apart from their smaller sizes, aggregation / agglomeration with bio- active substances (like synthetic molecules in age-defying nanosome particles) make then more bio- available. There are obvious health concerns raised by scientific communities with some laboratory and animal-based investigations. There are also evidences of increasing contamination of synthetic nano- and micro- materials in city waters and oceans around the planet. These may be the contributors of a general trend of substantial increase in dermal diseases due to new product, like whitening creams, exposures for last 20 years.

[0004] Replacement of synthetic nano- micro- particles with natural substitutes therefore has huge health and environmental benefit and commercial potentials. The “green” synthesis of NP is increasingly gaining interests due to its natural biological extract basis, easy-scale-up, and very cost- effective methodology where metals or their oxides are incubated with a biological extract to form varieties of NP depending on multitude of factors such as concentration, temperatures, the nature of biological extract, etc. These NPs are currently used in wide range of products ranging from baby products to electronic sensors, to food-additives to pharmaceuticals. Green synthesis employs plant or microbial extracts that are mixed with metal-salts (for example, silver or gold salts) to produce a spectrum of different metallic NPs with a multitude of properties. These processes are quick and scale- up friendly, and do not require use of toxic chemicals, high temperatures or pressures. The metal NP produced using green technologies are believed to be safer for human health and harmful for cancers and pathogens. Moreover, the lack of regulations in many consumer-product classes (for example cosmetics, food additives and packaging) allows private entities to avoid disclosing the use of NPs in their products currently in the market, even though they are synthesized using green-synthesis. Extensive research shows that green-synthesized metallic NP are less cytotoxic for human, but not completely harmless. The ease of green NP synthesis from metals or their oxides and exponential expansion of NP particle usage may not have positive outcome for health and environment in the long run.

[0005] Another rapidly growing area is organic NP and MP. Several organic or polymeric molecules have self-organization or lattice formation properties that generate NP and MP of different shapes, for example, polymeric, polymer conjugates, dendrimers NPs and produce different types of particles, such as micelles, vesicles, liposomes, polymersomes, nanosomes, etc. These classes of particles are widely used as cosmetics, cosmeceuticals (e.g., anti-aging creams), and for biomedical purposes. Again, because of the lesser regulation of disclosure regarding cosmetic ingredients as NP or MP in products, concerns are also raised for long-term health or environment impacts of these organic, yet synthetic particles.

[0006] Biological materials are not homogenous at the tissue and cellular levels. Rather, they are constituted of compartmentalized subcellular materials which are regarded as valuable active ingredients of nutraceutical, therapeutic or cosmeceutical importance. For example, spherical, oval or any other shaped sub-cellular storage compartments constituted of oil-bodies, terpenes, alkaloids, flavonoids, carotenoids, essential oils and pigments, appear as particulate shape in cells, as evident in microscopy studies. Extraction of these storage compartments as active ingredients from biological materials may render those active ingredients soluble in an extraction solvent, and this may be followed by distillation and concentration of active ingredients from the solvent in pure bulk forms. These extraction methods are often not cost effective. Over the last decade different industries, like cosmetics, cosmeceuticals, food-additives and pharmaceuticals realized when formulating the purified active ingredients, the most efficient processes often involved nano-emulsions or nano-carriers that provide better product efficiencies, such as physical appeal, slow release, targeted delivery, easy cellular penetration.

[0007] Virtually all living beings have a physiology that has evolved to work at nano- and micro scales for communications and interactions, such as cell to cell, cell to body, body to environment and environment to cells interactions. Most living cells, from bacteria to human, produce different classes of nano- and micro-sized vesicle type particles called exomere, exosomes, extracellular vesicles, microvesicles, apoptotic bodies, oncosomes, enveloped-viruses, etc, which are constituted of different composition of cellular materials, and act as molecular cargo to deliver different molecules to another cell of the body or other living organism in the environment. In normal human physiology, infections with pathogens, diseases like cancers, heart failure, include situations where these vesicles have tremendous biological implications. The composition of these nano-sized vesicles varies depending on the physiology, organ, infection, immunity, cancer progression, etc. Bacteria, yeast, fungus, protozoa, plants, planktons all produce a basic threshold of particles and/or vesicles of nano- and micro- sizes for normal ecological and physiological homeostasis. A spectrum of all-natural biological particles is non-toxic, non-immunogenic and can be therapeutically used.

[0008] The preferential engulfment (e.g., mostly phagocytosis and pinocytosis) of NPs and

MPs (natural or synthetic) are established by fundamental cellular properties of any living organisms. These engulfed particles exhort deviation from normal cellular signaling networks, especially with synthetic NPs and MPs, and thus raise the concerns for health and environments in long-term.

[0009] Therefore, there is a need for scalable all-natural, non-metal, non-synthetic and green nano- and micro- particle production methods from bulk biological materials.

SUMMARY

[0010] According to an embodiment, there is provided a method for the production of a particle from a biological material comprising step a) : a) contacting the biological material immersed in a polar fluid comprising water or an aqueous solution with an electromotive force provided by a low voltage applied to the biological material through a first and a second electrically conducive pole separated by a distance sufficient for a time sufficient for the electromotive force to cause formation and release of the particle from the biological material into the polar fluid, wherein the particle has a diameter of about 20 nm to about 5000 nm, and wherein the low voltage is from about 0.1 Volt to about 100 Volts, applied at about 0.1 Volt per centimeter to about 30 Volts per centimeter of the distance sufficient.

[0011] The distance sufficient may be from about 0.1 cm to about 30 cm.

[0012] The aqueous solution may comprise a saline, a salt solution, a buffer solution, and combinations thereof.

[0013] The buffer solution may be a phosphate buffer, a citrate buffer, an acetate buffer, a

TRIS buffer, a cacodylate buffer, Good buffer, Sorensen’s buffer, a phosphate-citrate buffer, Barbital buffer, glycine-NaOH buffer, and combinations thereof.

[0014] The polar fluid may be pure water.

[0015] The low voltage may be from about 0.1 Volt to about 15 Volts. The low voltage may be from about 4 to about 20 Volts. The low voltage may be from about 8 to about 35 Volts. The low voltage may be from about 20 to about 55 Volts. The low voltage may be from about 50 to about 100 Volts.

[0016] The electromotive force may be provided periodically, continuously, as a gradient, and combinations thereof.

[0017] The electromotive force may be provided as direct current or as alternating current, and a combination thereof.

[0018] The alternating current may be provided at a frequency of from about 10 Hz to about

60 Hz.

[0019] The polar fluid may be stationary during step a).

[0020] The polar fluid may be flowing during step a).

[0021] The polar fluid may further comprise an additive ingredient to be adsorbed onto a surface of the particle, absorbed into the particle, or a combination thereof. [0022] The additive ingredient may be a molecule. The molecule may be a therapeutic molecule. The therapeutic molecule may be adriamycin, curcumin, doxorubicin, a peptide, an antibiotic, an antifungal, and combinations thereof.

[0023] The biological material may comprise a plant, a plant part, a cultured plant cell, a cultured plant tissue, a cultured plant cell mass, a lower organism, a higher organism, a eukaryotic cell, a complete organ, a part of an organ, a cultured higher organism cell mass, a tissue, and combinations thereof.

[0024] The lower organism may be a fungus, a yeast, a phytoplankton, a protozoan, a bacteria, a seaweed and combinations thereof.

[0025] The biological material may be a live biological material. The live biological material may be metabolically active. The biological material may be a preserved biological material. The preserved biological material may be a frozen biological material, a dried biological material, lyophilized biological material, or a combination thereof.

[0026] The particle may be comprised of

(a) an aggregate of constituents of the biological material comprising DNA, RNA, proteins, lipids, polysaccharides, alkaloids, terpenoids, phenolics, oligosaccharide, shikimate, polyketide, flavonoids, peptides, vitamins, and antioxidants;

(b) an aggregate of membranes constituents of the biological material, cytoplasmic constituents of the biological material, subcellular storage organelles of the biological material, vacuoles, and fat-granules, and combinations thereof.

[0027] The particle may comprise polymeric molecules from the biological material.

[0028] The polymeric molecules may comprise starch, cellulose, hemicellulose, lignin, chitin, proteins, oils, lipid particles, collagen, polysaccharides, pectin, amylopectin, pentosan, glucomannan, agar, inulin, rosin, Aloe Vera mucilaginous extract, alginates, carrageenans, psyllium, xanthan gum, tragacanthin, fucoidan, hyaluronic acid, and combinations thereof.

[0029] The particle may comprise a single layer membranous vesicles-type particle, a multilayer membranous vesicles-type particle, or a combination thereof.

[0030] The membranous vesicles-type particle may comprise a plurality of cellular or sub- cellular constituents from the biological material of its lumen. [0031] The cellular or sub-cellular constituents may comprise DNA, RNA, protein, lipid, polymers, metabolites, and combinations thereof.

[0032] The particle size may have a diameter of from about 20 nm to about 5000 nm.

[0033] The particle size may have a diameter of from about 20 nm to about 1000 nm.

[0034] The method of the present invention may further comprise step b): concentrating the particle from the polar fluid.

[0035] The method of the present invention may further comprise step c): storage of the particle.

[0036] The method of the present invention may further comprise step a’) prior to step a): hydrating the biological material in the polar fluid for a time sufficient to obtain a hydrated biological material.

[0037] The hydrated biological material may be saturated with the polar fluid.

[0038] The pH of the polar fluid may be from about 3 to about 10.

[0039] The pH of the polar fluid may be from about 6 to about 8.

[0040] The temperature of the polar fluid may be from about 4°C to about 65°C.

[0041] The temperature of the polar fluid may be from about 15°C to about 25°C.

[0042] The time sufficient may be from about 1 min to about 60 mins. The time sufficient may be from about 2.5 min to about 15 mins, or from about 1.0 min to about 100 hours.

[0043] The low voltage provided per each centimeter of distance sufficient may be from about

2 Volt/cm to about 20 Volt/cm.

[0044] The first and the second electrically conducive pole may be in physical contact with the biological material.

[0045] The following terms are defined below.

[0046] All references cited herein including publications, patent applications, and patents are hereby incorporated by reference to the same extent as if each had been individually incorporated.

[0047] As used herein, the term “particle” is intended to mean a very-small or microscopic part or particulate portion of a bigger bulk mass of biological-material, which are of regular or irregular shapes constituting subsets or whole of the ingredients of the biological-material which are smaller than 5000 nanometers of average diameter or sizes across the longest edges of the particles.

[0048] As used herein, the term “nano-particle” is intended to mean the size range of the particles from about 20 nm (nanometer) to about 100 nm, or from about 20 nm to about 75 nm, or from about 20 nm to about 50 nm, or from about 20 nm to about 25 nm, or from about 20 nm to about 90 nm, or from about 20 nm to about 80 nm, or from about 20 nm to about 70 nm, or from about 20 nm to about 60 nm, or from about 20 nm to about 50 nm, or from about 20 nm to about 40 nm, or from about 20 nm to about 30 nm, or from about 30 nm to about 100 nm, or from about 30 nm to about 75 nm, or from about 30 nm to about 50 nm, or from about 30 nm to about 90 nm, or from about 30 nm to about 80 nm, or from about 30 nm to about 70 nm, or from about 30 nm to about 60 nm, or from about 30 nm to about 50 nm, or from about 30 nm to about 40 nm, or from about 40 nm to about 100 nm, or from about 40 nm to about 75 nm, or from about 40 nm to about 50 nm, or from about 40 nm to about 100 nm, or from about 40 nm to about 90 nm, or from about 40 nm to about 80 nm, or from about 40 nm to about 70 nm, or from about 40 nm to about 60 nm, or from about 40 nm to about 50 nm, or from about 50 nm to about 100 nm, or from about 50 nm to about 90 nm, or from about 50 nm to about 80 nm, or from about 50 nm to about 70 nm, or from about 50 nm to about 60 nm, or from about 60 nm to about 100 nm, or from about 60 nm to about 90 nm, or from about 60 nm to about 80 nm, or from about 60 nm to about 70 nm, or from about 70 nm to about 100 nm, or from about 70 nm to about 90 nm, or from about 70 nm to about 80 nm, or from about 80 nm to about 100 nm, or from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm,

[0049] As used herein, the term “micro-particle” is intended to mean the size range of particle from about 0.1 pm (micrometer) to about 5 pm, or from about 0.1 pm to about 4.5 pm, or from about 0.1 pm to about 3.5 pm, or from about 0.1 pm to about 2.5 pm, or from about 0.1 pm to about 1.5 pm, or from about 0.1 pm to about 1 .0 pm, or from about 0.1 pm to about 0.5 pm, or from about 0.5 pm to about 5 pm, or from about 0.5 pm to about 4 pm, or from about 0.5 pm to about 3 pm, or from about 0.5 pm to about 2 pm, or from about 0.5 pm to about 1 pm, or from about 1 pm to about 5 pm, or from about 1.0 pm to about 4 pm, or from about 1.0 pm to about 3 pm, or from about 1.0 pm to about 2 pm, or from about 1.5 pm to about 2 pm, , or from about 1.5 pm to about 2.5 pm, , or from about 1.5 pm to about 3 pm, , or from about 1.5 pm to about 3.5 pm, or from about 1.5 pm to about 4.5 pm, or from about 1.5 pm to about 5 pm, or from about 2 pm to about 2.5 pm, or from about 2 pm to about 3 pm, or from about 2 pm to about 4 pm, or from about 2 pm to about 5 pm, or from about 3 pm to about 3.5 pm, or from about 3 pm to about 4 pm, or from about 3 pm to about 4.5 pm, or from about 3 pm to about 5 pm, or from about 4 pm to about 4.5 pm, or from about 4 pm to about 5 pm, or from about 4.5 pm to about 5 pm. [0050] As used herein, “production” means particles formation and release from the bulk biological material into the polar fluid due to provided low-voltage electromotive force through the bulk biological material applied through a first and a second electrically conducive pole for a sufficient duration to make sufficient particles.

[0051] As used herein, “biological material” is intended to mean a mass of matter or its parts thereof that is originating from a once-living organism and/or that is present in a living organism and/or grown biologically. Biological material includes chemical substances present or produced in a living organism, such as biomolecule, biogenic substance produced by a living organism, biotic material, natural material, or natural product, produced by a living organism, biomass (living or dead biological matter, often plants grown as fuel, and which may represent the total mass of living matter in a given environment, or of a given species); cellular component [material and substances of which cells (and thus living organisms) are composed]; tissue (biology), cells, complete or portions of organs, and viable material, capable of living, developing, or germinating under favorable conditions (e.g. spores). Biological matter does not include body fluid (i.e., any liquid originating from inside the bodies of living people, for example blood, plasma, semen, lymph, cerebrospinal fluid, peritoneal fluid, saliva, mucus, and urine).

[0052] 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

[0053] 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:

[0054] Fig. 1A shows a schematic drawing illustrating the basic assembly of parts and materials of the process in accordance with an embodiment of the present invention;

[0055] Fig. 1 B shows a schematic drawing illustrating the basic assembly of parts and materials of the process in accordance with an embodiment of the present invention;

[0056] Fig. 2A shows an example of a turmeric root, which was cleaned and cut into blocks;

[0057] Fig. 2B shows submerging of an assembly of the turmeric root with two conductive wires in water in glass container; [0058] Fig. 2C shows the assembly of B post application of an electromotive-force in accordance with the present invention. The particles obtained due to applied electromotive force are extracted in the water;

[0059] Fig. 2D shows Transmission Electron Microscopic (TEM) analysis of the turmeric root sample pursuant to application of an electromotive-force in accordance with the present invention; and

[0060] Fig. 2E shows TEM analysis of the turmeric root sample pursuant to application of an electromotive-force in accordance with the present invention;

[0061] Fig. 2F shows TEM analysis of the turmeric root sample pursuant to application of an electromotive-force in accordance with the present invention;

[0062] Fig. 2G shows TEM of a turmeric root sample where no electromotive force was applied.

[0063] Fig. 3A shows an example of a ginger root, which was cleaned and cut into blocks, as well as submerging of an assembly of the ginger root with two conductive wire in water in glass container;

[0064] Fig. 3B shows Transmission Electron Microscopic (TEM) analysis of the ginger root sample pursuant to application of an electromotive-force in accordance with the present invention; and

[0065] Fig. 3C shows a larger magnification of the TEM analysis of 3B, of the ginger root sample pursuant to application of an electromotive-force in accordance with the present invention;

[0066] Fig. 3D shows TEM of a ginger root sample where no electromotive force was applied.

[0067] Fig. 4A illustrates an example where small minced or diced pieces of biological material from green butternut squash are enclosed in a flexible mesh-material (cotton fabric) in water;

[0068] Fig. 4B illustrates an example where small minced or diced pieces of biological material from green butternut squash are enclosed in a flexible mesh-material (cotton fabric) in water attached with two conductive wire;

[0069] Fig. 4C illustrates an example where small minced or diced pieces of biological material from green butternut squash are enclosed in a flexible mesh-material (cotton fabric) in water attached with two conductive wire;

[0070] Fig. 4D illustrates the particle concentration as a function of particle size (in nm) of the liquid samples, in accordance with an embodiment of the present invention. The samples are presented in the following order, for each particle size range: “With sample in mesh, no voltage”, “No sample in mesh, with voltage” and “With sample in mesh, with voltage”;

[0071] Fig. 5A illustrates TEM analysis (with inset underneath) of particle from a very young

Canadian maple tree leaf (approximately 3 cm in width and length) sample, in accordance with an embodiment of the present invention;

[0072] Fig. 5B illustrates TEM analysis of particle from a very young Canadian maple tree leaf

(approximately 3 cm in width and length) sample, in accordance with an embodiment of the present invention;

[0073] Fig. 5C illustrates TEM analysis of particle from a very young Canadian maple tree leaf

(approximately 3 cm in width and length) sample, in accordance with an embodiment of the present invention.

[0074] Fig. 6 illustrates a graph of Nanoparticle tracking analysis (NTA) to identify concentration, size distribution and polydispersity of particles from dried cannabis leaf/floral material.

[0075] Fig. 7 illustrates graphical representation of NTA to identify size distribution of particles from carrot root with or without applied electromotive force. The graphical representations show particle size (x-axis, 100 nm per division), relative intensity (y-axis) and relative particle number (z-axis).

[0076] Fig. 8 illustrates graphical representation of NTA to identify size distribution of particles from fresh English cucumberwith or without applied electromotive force. The graphical representations show particle size (x-axis, 100 nm per division), relative intensity (y-axis) and relative particle number (z-axis).

[0077] Fig. 9A illustrates an example of biological material where fresh white lupin flower petals were used. (A) is from a first control assembly where no electromotive force was applied.

[0078] Fig. 9B illustrates an example of biological material where fresh white lupin flower petals were used. (B) represents a second assembly with electromotive force was applied. Scale bar is 3 microns.

[0079] Fig. 9C illustrates an example of biological material where fresh white lupin flower petals were used. (C) represents a third assembly with electromotive force was applied. Scale bar is 3 microns.

[0080] Fig. 9D illustrates a characteristic spectral distribution observed when samples from

9B was examined. [0081] Fig. 9E illustrates a different spectral profile curve obtained when sample from 9C was examined. The spectral curve was skewed to blue range (425 to 500 nm) indicating adsorption of blue dye in the particles formed.

[0082] Fig. 10A shows fluid containing suspended particles after filtration with a 0.45-micron filter for a sample of dried green-tea leaves treated with an electromotive force (right) or not (left). The turbidity of the fluid testifies to the increase presence of particles in the fluid.

[0083] Fig. 10B illustrates the size distribution of particles obtained from a dried green-tea leaves sample not treated with an electromotive force.

[0084] Fig. 10C illustrates the size distribution of particles obtained from a dried green-tea leaves sample treated with an electromotive force.

[0085] Fig. 11 A shows fluid containing suspended particles after filtration with a 0.45-micron filter for a sample of dried lion's mane mushrooms treated with an electromotive force (right) or not (left). The turbidity of the fluid testifies to the increase presence of particles in the fluid.

[0086] Fig. 11 B illustrates the size distribution of particles obtained from a dried lion's mane mushrooms sample not treated with an electromotive force.

[0087] Fig. 11C illustrates the size distribution of particles obtained from a dried lion's mane mushrooms sample treated with an electromotive force.

[0088] It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DETAILED DESCRIPTION

[0089] In embodiments there are disclosed a method for the production of a particle from a biological material comprising step a) : a) contacting the biological material immersed in a polar fluid comprising water or an aqueous solution with an electromotive force provided by a low voltage applied to the biological material through a first and a second electrically conducive pole separated by a distance sufficient for a time sufficient for the electromotive force to cause formation and release of the particle from the biological material into the polar fluid, wherein the particle has a diameter of about 20 nm to about 5000 nm, and wherein the low voltage is from about 0.1 Volt to about 100 Volts, applied at about 0.1 Volt per centimeter (Volts/cm) to about 30 Volts/cm of the distance sufficient. Biological material

[0090] In embodiments, the biological material includes, but not limited to, plants, plant parts

(any plant part, but preferred parts include shoots, roots, leaves, fruits, flowers, seeds, meristems, and pulps), cultured plant cell and tissue (for example callus culture). According to other embodiments, the biological material may include, but is not limited to genetically selected plants, medicinal plants, pharmaceutically important plants, cosmetically important plants, therapeutic plants, edible-plants and any of their parts (preferred parts include shoots, roots, leaves, fruits, flowers, seeds, meristems, and pulps), their bioreactor grown or cultured cell-mass or tissue (for example callus culture).

[0091] Embodiments of biological material also includes but is not limited to lower organism, non-animal lower phylogenetic organisms, for example fungus, yeast, phytoplankton, protozoans, bacteria, seaweed, etc. Other embodiments of the biological material include but are not limited to bioreactor-grown or cultured lower organism, non-animal lower phylogenetic organisms, for example fungus, yeast, phytoplankton, protozoans, bacteria, seaweed, etc., particularly those that may be used in the production of food products. In another embodiment, the biological material used for example, not limited to, cell-mass of lower organism like, yeast, bacteria, fungus or protozoa for the purpose of vaccination. In another embodiment, the biological material used for example, not limited to, genetically engineered or selected cell-mass of lower organism like, yeast, bacteria, fungus or protozoa for the purpose of immunomodulation. In another embodiment, the biological material used for example, not limited to, cell-mass, lower organisms like, yeast, bacteria, fungus or protozoa for the purpose of vaccine-adjuvants. In another embodiment, the biological material used for example, not limited to, edible plants, other plants, sea-weeds or their part thereof for the purpose of allergy desensitization or shots to treat food and pollen allergies. In another embodiment, the biological material used for example, not limited to, plants producing saponin or similar types soapy plants and sea-weeds or their part thereof for the purpose of producing particles with detergent or surfactant properties.

[0092] According to embodiments, the biological material may also include, but is not limited to, tissue or cell-mass from higher organisms such as biological material of animals, birds, fish and human origin. In some embodiment, the biological material includes but is not limited to laboratory grown tissue or cell-mass genetically expressing antigens or peptides or engineered-proteins in cells of plant, animals, birds, fish and human origin. In some embodiment, the biological material includes, but not limited to, laboratory grown tissue or cell-mass genetically expressing antigens or peptides or engineered-proteins from pathogens, like virus or bacterial or fugal or protozoan pathogens.

[0093] In some embodiment, the biological material includes, but not limited to, preserved cell- mass or tissue or parts thereof as frozen, lyophilized, or dried conditions prior to the method of the present invention. In some embodiment, the biological material is a live biological material. In some embodiment, the biological material is metabolically active.

[0094] In use, the biological material may be portioned into small pieces, minced or diced or clumps biological material, which may be provided in a wrapped or enclosed in a non-conductive material, for example in a mesh-material (pore size more than 5000 nanometer), which may be flexible or rigid, where the two electrically-conductive poles are in contact with the biological materials contained in the mesh-material and the above assembly is in contact with a provided fluid, contained in a non-conductive container. Alternatively, small pieces, minced pieces, diced pieces or clumps of biological material 20 may be provided wrapped or enclosed in a flexible or rigid conductive container made of mesh-material (pore size more than 5000 nanometer), where the two conductive poles are in contact with electrically-conductive mesh-material and the above assembly is contained inside a porous (more than 5 micrometer pore size) container, where the above whole assembly is in contact with a provided polar fluid, contained in a non-conductive container

Electromotive force

[0095] In embodiments, the electromotive force is provided by contacting the biological material with the low voltage. According to an embodiment, the electromotive force is provided by low voltage from about 0.1 volt and to about 100 Volts, applied at about 0.1 Volt per centimeter (Volts/cm) to about 30 Volts/cm, of a distance sufficient, and for a time sufficient to cause formation and release of the particle from the biological material into the polar fluid. Preferably, the low voltage is from about 4 Volts to about 20 Volts, or from about 8 Volts to about 35 Volts, or from about 20 Volts to about 55 Volts, or from about 50 Volts to about 100 Volts applied between the first and a second electrically conducive pole separated by a distance sufficient. As used herein, according to an embodiment, the term contacting means that the first and second poles are in contact with the biological material to allow electromotive force to pass through the provided biological materials. In embodiments, electromotive force refers to the force exerted as a result of the applied low voltage through the first and second poles onto molecules of subcellular, intracellular, intercellular, and extracellular structures and assemblies of the biological material having plurality of net charges and thus may gain mobility through the intracellular, intercellular and extracellular spaces as the particles. The contact may be actual physical contact, where the first and second poles touch the biological material. The contact may also be through a container within which the biological material is contained, for example, as illustrated herein, pouches of mesh material, or physical containing enclosing the biological material. According to an embodiment, the biological materials may be put in contact with electromotive force through two conductive poles from inside a closed container, such as a mesh material, having pore with size larger than 5000 nanometers, so that the particles of 5000 nanometres or smaller can pass through the pores into the polar fluid container.

[0096] In embodiments, the provided electromotive force is the low voltage which is provided per each centimeter of the distance sufficient between the two poles. For example, the low voltage may be from about 0.1 Volt and about 30 Volts per centimeter of distance between the first and second conductive poles in contact with biological material. For example, from about 0.1 Volts to about 1.0 Volts, or from about 0.1 Volts to about 2.5 Volts, or from about 0.1 Volts to about 5 Volts, or from about 0.1 Volts to about 7.5 Volts, or from about 0.1 Volts to about 10 Volts, or from about 0.1 Volts to about 15 Volts, or from about 0.1 Volts to about 20 Volts, or from about 0.1 Volts to about 25 Volts, or from about 0.1 Volts to about 30 Volts, or from about 1 .0 Volts to about 5 Volts, or from about 1 .0 Volts to about 10 Volts, or from about 1.0 Volts to about 15 Volts, or from about 1.0 Volts to about 20 Volts, or from about 1.0 Volts to about 25 Volts, or from about 5 Volts to about 10 Volts, or from about 5 Volts to about 15 Volts, or from about 5 Volts to about 20 Volts, or from about 5 Volts to about 25 Volts, or from about 5 Volts to about 30 Volts, or from about 10 Volts to about 15 Volts, or from about 10 Volts to about 20 Volts, or from about 10 Volts to about 25 Volts, or from about 10 Volts to about 30 Volts, or from about 20 Volts to about 30 Volts, or about 0.1 Volt, 0.5, 1.0, 2.0 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, or 30 Volts per centimeter of the distance sufficient between the two poles.

[0097] In embodiments, the distance sufficient between the two poles may be from about 0.1 cm to about 30 cm, for example, from about 0.1 cm to about 1 cm, or from about 0.1 cm to about 2 cm, or from about 0.1 cm to about 4 cm, or from about 0.1 cm to about 8 cm, or from about 0.1 cm to about 10 cm, or from about 0.1 cm to about 15 cm, or from about 0.1 cm to about 20 cm, or from about 0.1 cm to about 25 cm, or from about 0.1 cm to about 30 cm, or from about 1 cm to about 2 cm, or from about 1 cm to about 2.5 cm, or from about 1 cm to about 5 cm, or from about 1 cm to about 7.5 cm, or from about 1 cm to about 10 cm, or from about 1 cm to about 12.5 cm, or from about 1 cm to about 15 cm, or from about 1 cm to about 20 cm, or from about 1 cm to about 25 cm, or from about 1 cm to about 30 cm, or from about 2.5 cm to about 5 cm, or from about 2.5 cm to about 7.5 cm, or from about

2.5 cm to about 10 cm, or from about 2.5 cm to about 15 cm, or from about 2.5 cm to about 20 cm, or from about 2.5 cm to about 25 cm, or from about 2.5 cm to about 30 cm, or from about 5 cm to about

7.5 cm, or from about 5 cm to about 10 cm, or from about 5 cm to about 15 cm, or from about 5 cm to about 20 cm, or from about 5 cm to about 25 cm, or from about 5 cm to about 30 cm, or from about

7.5 cm to about 10 cm, or from about 7.5 cm to about 15 cm, or from about 7.5 cm to about 20 cm, or from about 7.5 cm to about 25 cm, or from about 7.5 cm to about 30 cm, or from about 10 cm to about 15 cm, or from about 10 cm to about 20 cm, or from about 10 cm to about 25 cm, or from about 10 cm to about 30 cm, or from about 15 cm to about 20 cm, or from about 15 cm to about 25 cm, or from about 15 cm to about 30 cm, or from about 20 cm to about 25 cm, or from about 20 cm to about 30 cm, or from about 25 cm to about 30 cm, or 0.1 , 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30 cm.

[0098] According to an embodiment, the electromotive force may be provided as periodically, continuously, as a gradient, and combinations thereof. In another embodiment, the electromotive force may be provided as direct current or as alternating current, and combinations thereof. According to another embodiment, the alternating current may be provided at a frequency of from about 10 Hz to about 60 Hz.

[0099] In embodiments, the time sufficient for the electromotive force to cause formation and release of the particle from the biological material into the polar fluid may be from about 1 min to about 2.5 min, or from about 1 min to about 5 min, or from about 1 min to about 7.5 min, or from about 1 min to about 10 min, or from about 1 min to about 15 min, or from about 1 min to about 20 min, or from about 1 min to about 30 min, or from about 1 min to about 1 h, or from about 1 min to about 2 h, or from about 1 min to about 3 h, or from about 1 min to about 4 h, or from about 1 min to about 6 h, or from about 1 min to about 9 h, or from about 1 min to about 12 h, or from about 1 min to about 15 h, or from about 1 min to about 18 h, or from about 1 min to about 21 h, or from about 1 min to about 24 h, or from about 2.5 min to about 15 min, or from about 2.5 min to about 30 min, or from about 2.5 min to about 45 min, or from about 2.5 min to about 1 h, or from about 2.5 min to about 2 h, or from about 2.5 min to about 2 h, or from about 2.5 min to about 3 h, or from about 2.5 min to about 4 h, or from about 2.5 min to about 6 h, or from about 2.5 min to about 9 h, or from about 2.5 min to about 12 h, or from about 2.5 min to about 15 h, or from about 2.5 min to about 18 h, or from about 2.5 min to about 21 h, or from about 2.5 min to about 24 h, or from about 5 min to about 10 min, or from about 5 min to about 20 min, or from about 5 min to about 30 min, or from about 5 min to about 40 min, or from about 5 min to about 60 min, or from about 5 min to about 2 h, or from about 5 min to about 3 h, or from about 5 min to about 4 h, or from about 5 min to about 6 h, or from about 5 min to about 9 h, or from about 5 min to about 12 h, or from about 5 min to about 15 h, or from about 5 min to about 18 h, or from about 5 min to about 21 h, or from about 5 min to about 24 h, or from about 10 min to about 30 min, or from about 10 min to about 60 min, or from about 10 min to about 1.5 h, or from about 10 min to about 2 h, or from about 10 min to about 3 h, or from about 10 min to about 5 h, or from about 10 min to about 10 h, or from about 10 min to about 12 h, or from about 30 min to about 1.5 h, or from about 30 min to about 3 h, or from about 30 min to about 6 h, or from about 30 min to about 9 h, or from about 30 min to about 12 h, or from about 1 h to about 3 h, or from about 1 h to about 6 h, or from about 1 h to about 9 h, or from about 1 h to about 12 h, or from about 1 h to about 15 h, or from about 1 h to about 18 h, or from about 1 h to about 21 h, or from about 1 h to about 24 h, or from about 2 h to about 3 h, or from about 2 h to about 6 h, or from about 2 h to about 9 h, or from about 2 h to about 12 h, or from about 2 h to about 15 h, or from about 2 h to about 18 h, or from about 2 h to about 21 h, or from about 2 h to about 24 h, or from about 3 h to about 6 h, or from about 3 h to about 9 h, or from about 3 h to about 12 h, or from about 3 h to about 12 h, or from about 3 h to about 15 h, or from about 3 h to about 18 h, or from about 3 hh to about 21 h, or from about 3 h to about 24 h, or from about 6 h to about 9 h, or from about 6 h to about 12 h, or from about 6 h to about 15 h, or from about 6 h to about 18 h, or from about 6 h to about 21 h, or from about 6 h to about 24 h, or from about 9 h to about 12 h, or from about 9 h to about 15 h, or from about 9 h to about 18 h, or from about 9 h to about 21 h, or from about 9 h to about 24 h, or from about 12 h to about 15 h, or from about 12 h to about 18 h, or from about 12 h to about 21 h, or from about 12 h to about 24 h, or from about 15 h to about 18 h, or from about 15 h to about 21 h, or from about 15 h to about 24 h, or from about 18 h to about 21 h, or from about 18 h to about 24 h, or from about 21 h to about 24 h, or from about 20 h to about 100 h, or 1 , 2, 2.5, 5, 10,

15, 20, 25, 30, 45, 60 minutes or 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 198, 20, 21 , 22, 23, 24, 25, 50, 100 hours.

[00100] In another embodiment, the time sufficient for the electromotive force to cause formation and release of the particle from the biological material into the polar fluid may need intermittent periods without any voltage applied, referred to as ‘periodically’, which may be from about 1 min to a about 24 h and periodically for plurality of time sufficient of voltage applied described above. As used herein, periodically refers to period longer than 1 minute, and is intended to exclude very short pulsations of current shorter than 1 minute, and especially pulses in the millisecond, microsecond, or shorter time interval.

Fluid

[00101] As used herein, the terms “fluid”, “polar fluid”, or “liquid media” mean a volume of liquid in contact with the biological material. The particles (i.e. , the nanoparticles and microparticles) are released from the biological material into this polar fluid when the electromotive force is applied shortly or thereafter, and for the duration of the application of the electromotive force. In embodiments, the “fluid”, “polar fluid”, or “liquid media” is one that is compatible with the biological sample and will not damage it nor damage the particles produced therein. In embodiments, the polar fluid is water or an aqueous solution. According to an embodiment, the polar fluid is pure water.

[00102] In embodiments, the aqueous solution may contain ingredients necessary to obtain physiologically compatible solution. For example, the aqueous solution may comprise a saline solution, a salt solution, a buffer solution, and combinations thereof. Examples of buffer solution include but are not limited to a phosphate buffer, a citrate buffer, an acetate buffer, a TRIS buffer, a cacodylate buffer, Good buffer, Sorensen’s buffer, a phosphate-citrate buffer, Barbital buffer, glycine-NaOH buffer, and combinations thereof.

[00103] According to an embodiment, such buffers will contain pH adjusting agent that may be chosen from citric acid, lactic acid, hydrochloric acid, boric acid, acetic acid, sodium hydroxide, potassium hydroxide, sulfuric acid, calcium carbonate (CaCC ), ammonium carbonate, ammonium bicarbonate, ammonium citrate, sodium citrate, magnesium carbonate, sodium carbonate, mono, di and/or trisodium phosphate, mono, di and/or tripotassium phosphate, Tris(hydroxymethyl) aminomethane (TRIS), paracetic acid, propionic acid, fumaric acid, sorbic acid, benzoic acid, phenylacetic acid, malic acid, tartaric acid, dehydroacetic acid, amino acids and zwitterions, such as glycine, 2-amino-2methyl-1 ,3-propanediol (AMPD), N-(1 ,1-Dimethyl-2-hydroxyethyl)-3-amino-2- hydroxypropanesulfonic acid (AMPSO), N-Glycylglycine (Gly-Gly), 4-(2-hydroxyethyl)piperazine-1- propanesulfonic acid (EPPS or HEPPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3- (cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), 2-(cyclohexylamino)ethanesulfonic acid (CHES), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES), (2-[2-hydroxy-1 ,1- bis(hydroxymethyl)ethylamino] ethanesulphonic acid) (TES), 2-(N-morpholino)ethanesulfonic acid (MES), N-[Tris(hydroxymethyl)methyl]glycine (Tricine); N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPS) and 3-N-Morpholino propanesulfonic acid (MOPS), piperazie-N,N'- bis[2-hydroxypropanesulphonic]acid (POPSO), and combinations thereof.

[00104] The role of the pH adjusting agent is to adjust the acidity or alkalinity of the polar fluid used in the present invention, or increase the buffer capacity in order to reduce pH variation upon application of the electromotive force. According to an embodiment, the aqueous solution used in the process of the present invention may comprise from about 0.01% to about 5% (w/w), or from about 0.01% to about 4%, or from about 0.01% to about 3%, or from about 0.01% to about 2%, or from about 0.01% to about 1%, or from about 0.01% to about 0.75%, or from about 0.01% to about 0.5%, or from about 0.01% to about 0.25%, or from about 0.01% to about 0.1%, or from about 0.01% to about 0.075%, or from about 0.01% to about 0.05%, or from about 0.01% to about 0.025%, or from about 0.01% to about 0.02%, or from about 0.01% to about 0.015%, or 0.015% to about 5% (w/w), or from about 0.015% to about 4%, or from about 0.015% to about 3%, or from about 0.015% to about 2%, or from about 0.015% to about 1%, or from about 0.015% to about 0.75%, or from about 0.015% to about 0.5%, or from about 0.015% to about 0.25%, or from about 0.015% to about 0.1%, or from about 0.015% to about 0.075%, or from about 0.015% to about 0.05%, or from about 0.015% to about 0.025%, or from about 0.015% to about 0.02%, or 0.02% to about 5% (w/w), or from about 0.02% to about 4%, or from about 0.02% to about 3%, or from about 0.02% to about 2%, or from about 0.02% to about 1%, or from about 0.02% to about 0.75%, or from about 0.02% to about 0.5%, or from about 0.02% to about 0.25%, or from about 0.02% to about 0.1%, or from about 0.02% to about 0.075%, or from about 0.02% to about 0.05%, or from about 0.02% to about 0.025%, or 0.025% to about 5% (w/w), or from about 0.025% to about 4%, or from about 0.025% to about 3%, or from about 0.025% to about 2%, or from about 0.025% to about 1%, or from about 0.025% to about 0.75%, or from about 0.025% to about 0.5%, or from about 0.025% to about 0.25%, or from about 0.025% to about 0.1%, or from about 0.025% to about 0.075%, or from about 0.025% to about 0.05%, or 0.05% to about 5% (w/w), or from about 0.05% to about 4%, or from about 0.05% to about 3%, or from about 0.05% to about 2%, or from about 0.05% to about 1%, or from about 0.05% to about 0.75%, or from about 0.05% to about 0.5%, or from about 0.05% to about 0.25%, or from about 0.05% to about 0.1%, or from about 0.05% to about 0.075%, or 0.075% to about 5% (w/w), or from about 0.075% to about 4%, or from about 0.075% to about 3%, or from about 0.075% to about 2%, or from about 0.075% to about 1%, or from about 0.075% to about 0.75%, or from about 0.075% to about 0.5%, or from about 0.075% to about 0.25%, or from about 0.075% to about 0.1%, or 0.1% to about 5% (w/w), or from about 0.1% to about 4%, or from about 0.1% to about 3%, or from about 0.1% to about 2%, or from about 0.1% to about 1%, or from about 0.1% to about 0.75%, or from about 0.1% to about 0.5%, or from about 0.1% to about 0.25%, or 0.25% to about 5% (w/w), or from about 0.25% to about 4%, or from about 0.25% to about 3%, or from about 0.25% to about 2%, or from about 0.25% to about 1%, or from about 0.25% to about 0.75%, or from about 0.25% to about 0.5%, or 0.5% to about 5% (w/w), or from about 0.5% to about 4%, or from about 0.5% to about 3%, or from about 0.5% to about 2%, or from about 0.5% to about 1%, or from about 0.5% to about 0.75%, or 0.75% to about 5% (w/w), or from about 0.75% to about 4%, or from about 0.75% to about 3%, or from about 0.75% to about 2%, or from about 0.75% to about 1%, or 1% to about 5% (w/w), or from about 1% to about 4%, or from about 1% to about 3%, or from about 1% to about 2%, or 2% to about 5% (w/w), or from about 2% to about 4%, or from about 2% to about 3%, or 3% to about 5% (w/w), or from about 3% to about 4%, or 4% to about 5% (w/w), or 0.01 , 0.025, 0.05, 0.075, 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5% (w/w) of a pH adjusting agent.

[00105] In embodiment, the pH of the polar fluid may be from about 3 to about 10, preferably, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 7, about 6 to about 8, about 6 to about 7.6, about 6 to about 7.4, about 7 to about 8, about 7 to about 7.6, about 7 to about 7.4, about 8 to about 9, about 8 to about 10, about 3, about 4, about 5, about 6, about 7, about 7.6, about 7.4, about 8, about 9, or about 10.

[00106] According another embodiment, the temperature of the polar fluid may be from about 4°C to about 10°C, or from about 4°C to about 15°C, or from about 4°C to about 20°C, or from about 4°C to about 25°C, or from about 4°C to about 30°C, or from about 4°C to about 40°C, or from about 4°C to about 50°C, or from about 4°C to about 65°C, or from about 10°C to about 15°C, or from about 10°C to about 20°C, or from about 10°C to about 25°C, or from about 10°C to about 30°C, or from about 10°C to about 30°C, or from about 10°C to about 40°C, or from about 10°C to about 50°C, or from about 10°C to about 65°C, or from about 15°C to about 25°C, or from about 15°C to about 30°C, or from about 15°C to about 40°C, or from about 15°C to about 50°C, or from about 15°C to about 65°C, or from about 20°C to about 30°C, or from about 20°C to about 40°C, or from about 20°C to about 50°C, or from about 20°C to about 65°C, or from about 25°C to about 30°C, or from about 30°C to about 40°C, or from about 30°C to about 50°C, or from about 30°C to about 65°C, or from about 40°C to about 50°C, or from about 40°C to about 65°C, or from about 50°C to about 65°C, or 4, 10, 15, 20, 25, 30, 40, 50, 65°C.

[00107] According to another embodiment, the polar fluid may include constituents of culture media or defined-culture media for growth and maintenance of metabolically active cells and tissue and parts thereof. In another embodiment, the polar fluid may include constituents of bioreactor culture media for growth and maintenance of metabolically active biological material like, cells and cell-mass.

[00108] According to another embodiment, the polar fluid may be stationary during step a). According to another embodiment, the polar fluid may be flowing during step a). According to another embodiment, the polar fluid may be subjected to alternating cycles of stationary and flowing step a).

Additive ingredients

[00109] According to another embodiment, the polar fluid used in the method of the present invention may further comprises an additive ingredient to be adsorbed onto a surface of the particle, absorbed into the particle, or a combination thereof. Examples of additive ingredients include but are not limited to molecules, such as therapeutic molecule, stabilizing molecules, coloring molecules for the purpose to be adsorbed on or within the produced particles. Without wishing to be bound by theory, it is believed that presence of the additive ingredient within the polar fluid will cause and enhance its absorbance or incorporation within the produced particles.

[00110] Examples of therapeutic molecule include adriamycin, curcumin, doxorubicin, peptides, drugs, antibiotics, antifungal, etc. In embodiments, therapeutic molecules may include any molecule having sizes of about 0.1 Dalton to about 1 kiloDalton (kDa), small planar or non-planar, symmetric or asymmetric, linear or cyclic, mono-cyclic, polycyclic or heterocyclic compounds of therapeutic use. Therapeutic molecules may also include natural, synthetic or bioengineered oligomeric (e.g., 1 kDa to 3 kDa) and polymeric (more than 3 kDa) molecules.

[00111] In another embodiment, additive ingredients may include natural or synthetic peptides, proteins, vitamins, DNA, RNA, lipids, micronutrients, and the likes. According to another embodiment, the additive ingredient may include coloring dyes which are anionic, cationic or neutral, to be adsorbed on, bonded to or within the particles. Coloring dyes which are classified as food-colors or other color compounds and their metallic salts of spectral bands between 375 to 700 nanometer wavelengths of light are of interest.

[00112] In other embodiment, additive ingredients may include soluble minerals, metal-salts or oxides to encapsulate, incorporate or aggregate on or within the produced particles.

[00113] In another embodiment, the particles produced as a result of applied electromotive force constitute a sub-cellular sub-set of components of the biological material used, for example, not limited to, sub-cellular parts, for example vesicles, storage-vacuoles, single membrane, multi membrane vesicles with cytoplasmic constituents or other cellular parts, cell-wall, or aggregates of protein, lipid, carbohydrates, nucleic acid, alkaloids, cell-wall components, etc. or mixtures thereof. In another embodiment, the particles produced as a result of applied electromotive force comprise sub- cellular components of the biological material used, for example, containing the active ingredients of herbal, medicinal and therapeutic values of the biological material known in the art.

[00114] Therefore, and according to another embodiment, the particle may be comprised of (a) an aggregate of constituents of the biological material comprising of primary metabolites (for example, DNA, RNA, proteins, lipids, polysaccharides), and secondary metabolites (for example, alkaloids, terpenoids, phenolics, oligosaccharide, shikimate, polyketide, flavonoids, peptides, etc.), vitamins, and antioxidants; and (b) an aggregate of membranes constituents of the biological material, cytoplasmic constituents of the biological material, subcellular storage organelles of the biological material, vacuoles, and fat-granules, and combinations thereof.

[00115] Also, the particle may comprise polymeric molecules from the biological material. The polymeric molecules comprise starch, cellulose, hemicellulose, lignin, chitin, proteins, oils, lipid particles, collagen, polysaccharides, pectin, amylopectin, pentosan, glucomannan, agar, inulin, rosin, Aloe Vera mucilaginous extract, alginates, carrageenans, psyllium, xanthan gum, tragacanthin, fucoidan, hyaluronic acid, and combinations thereof.

[00116] According to another embodiment, the particle may also comprise a single layer membranous vesicles-type particle, a multilayer membranous vesicles-type particle, or a combination thereof. The membranous vesicles-type particle may comprise a plurality of cellular or sub-cellular constituents from the biological material of its lumen. The cellular or sub-cellular constituents may comprise DNA, RNA, protein, lipid, polymers, metabolites, and combinations thereof. In another embodiment, the particles comprise a mixture of sub-cellular components of the biological material used and additive molecules in the fluid for example, not limited to, therapeutic or stabilizing molecules, coloring molecules for the purpose to be absorbed on or within the produced particles.

Particle sizes / diameter

[00117] In embodiments, the size and/or diameter of the particles obtained from the method of the present invention may as detailed above, and particularly from about 20 nm to about 5000 nm, and preferably from about 20 nm to about 100 nm, or from about 20 nm to about 200 nm, or from about 20 nm to about 300 nm, or from about 20 nm to about 400 nm, or from about 20 nm to about 500 nm, or from about 20 nm to about 600 nm, or from about 20 nm to about 700 nm, or from about 20 nm to about 800 nm, or from about 20 nm to about 900 nm, or from about 20 nm to about 1000 nm, or from about 20 nm to about 2000 nm, or from about 20 nm to about 3000 nm, or from about 20 nm to about 4000 nm, or from about 20 nm to about 5000 nm, or from about 100 nm to about 200 nm, or from about 100 nm to about 300 nm, or from about 100 nm to about 400 nm, or from about 100 nm to about 500 nm, or from about 100 nm to about 600 nm, or from about 100 nm to about 700 nm, or from about 100 nm to about 800 nm, or from about 100 nm to about 900 nm, or from about 100 nm to about 1000 nm, or from about 200 nm to about 300 nm, or from about 200 nm to about 400 nm, or from about 200 nm to about 500 nm, or from about 200 nm to about 600 nm, or from about 200 nm to about 700 nm, or from about 200 nm to about 800 nm, or from about 200 nm to about 900 nm, or from about 200 nm to about 1000 nm, or from about 300 nm to about 400 nm, or from about 300 nm to about 500 nm, or from about 300 nm to about 600 nm, or from about 300 nm to about 700 nm, or from about 300 nm to about 800 nm, or from about 300 nm to about 900 nm, or from about 300 nm to about 1000 nm, or from about 400 nm to about 500 nm, or from about 400 nm to about 600 nm, or from about 400 nm to about 700 nm, or from about 400 nm to about 800 nm, or from about 400 nm to about 900 nm, or from about 400 nm to about 1000 nm, or from about 500 nm to about 600 nm, or from about 500 nm to about 700 nm, or from about 500 nm to about 800 nm, or from about 500 nm to about 900 nm, or from about 500 nm to about 1000 nm, or from about 600 nm to about 700 nm, or from about 600 nm to about 800 nm, or from about 600 nm to about 900 nm, or from about 600 nm to about 1000 nm, or from about 700 nm to about 800 nm, or from about 700 nm to about 900 nm, or from about 700 nm to about 1000 nm, or from about 800 nm to about 900 nm, or from about 800 nm to about 1000 nm, or from about 900 nm to about 1000 nm, or from about 750 nm to about 1000 nm, or from about 750 nm to about 1500 nm, or from about 750 nm to about 2000 nm, or from about 750 nm to about 3000 nm, or from about 1000 nm to about 1500 nm, or from about 1000 nm to about 2000 nm, or from about 1000 nm to about 3000 nm, or from about 1500 nm to about 2000 nm, or from about 1500 nm to about 2500 nm, or from about 1500 nm to about 3000 nm, or from about 1500 nm to about 4000 nm, or from about 1500 nm to about 5000, or from about 2000 nm to about 3000 nm, or from about 2000 nm to about 4000 nm, or from about 2000 nm to about 5000 nm or from about 3000 nm to about 4000 nm, or from about 3000 nm to about 5000 nm, or from about 4000 nm to about 5000 nm.

[00118] Implementations:

[00119] Several embodiments of the present invention provide natural particles for several implementations.

[00120] In one implementation of the invention the particles can be used as ingredients of cosmetics or cosmeceuticals. The present invention provides a way to produce particles for use in cosmetics or cosmeceuticals from herbal, medicinal and therapeutic plants, seaweeds or parts thereof, any cell-mass, lower organism like, yeast, bacteria, fungus, phytoplankton, blue-green algae, other algae etc. Applications of these particles of the present invention in the cosmetics or cosmeceutical industry can be for multiple purposes, for example superior bioavailability of active ingredients, pigments, antimicrobial properties, formulation, prolonged shelf-life, all-natural, non-toxic, organic, etc.

[00121] In another implementation the particles obtained from the process of the present invention may have properties for topical or dermal application as all-natural, non-toxic and organic particles. The present invention opens possibilities for the production of particles from biological materials, for example, but not limited to, herbal, medicinal and therapeutic plants, sea-weeds or parts thereof, any cell-mass, lower organism like, yeast, bacteria, fungus, phytoplankton, blue-green algae, other algae etc. Other examples of applications of particles of this invention can be sunscreen, or other topical uses or containing UV absorbing particles, or antioxidant or water-holding properties or combination thereof of particles produced from herbal, medicinal and therapeutic plants, seaweeds or their part thereof, any cell-mass, lower organism like, yeast, bacteria, fungus, phytoplankton, blue- green algae, other algae etc. The particles also can be used as antimicrobials for skin-care and oral hygiene.

[00122] In another implementation, the particles obtained from the process of the present invention may be used as food additives as all-natural, non-toxic and organic particles. The present invention facilitates production of particles for use in food items from herbal, medicinal and therapeutic plants, seaweeds or their parts thereof, any cell-mass, lower organism like, yeast, bacteria, fungus, phytoplankton, blue-green algae, other algae etc. as antimicrobial, food coloring, increasing food selflife and ingredients for multiple food formulations. The particles of the present invention can be used as ingredients to improve food preservation, to improve bioavailability of active components of the source biomass, to improve other product qualities. The particles can also be used as ingredients of beverages or supplements or nutraceuticals, etc. The particles of present invention can also be used for food safety, food preservation, food packaging, food storage, antimicrobials, food formulations, etc. [00123] In another implementation the particles obtained from the process of the present invention may be used as all-natural, non-toxic, non-ionic biosurfactants / bio-detergents, organic particles to replace synthetic surfactants or detergents, which are proven to be toxic to life and environment. The particles can be used as ingredients in multiple daily-care products, for example, but not limited to, toothpaste, bodywash, shampoo, soaps, kitchen/bath/surface cleaners, industrial cleaners, soil and water body restorations, etc. Natural renewable sources of the biological materials may include but are not limited to, plants, seaweeds or parts thereof, yeast, bacteria, fungus, phytoplankton, blue-green algae, other algae, etc. with ingredients having properties of ionic or nonionic biosurfactants / bio-detergents.

[00124] In another implementation the particles obtained from the process of the present invention may contain antigens, peptides, proteins, glycoproteins, multiple metabolites produced from cell-mass of pathogenic lower organism like, yeast, bacteria, fungus or protozoa or other biological materials for example, genetically engineered eukaryotic cell-mass or organisms or other lower organism like, yeast, bacteria, fungus or protozoa, etc. for the purpose of immunomodulation, vaccination and allergy desensitization. The particles produced according to the present invention can be used for manufacturing of particulate-vaccine containing specific epitopes, engineered peptides, engineered biological molecules already expressed in the biological material used. These particles can also be used as vaccine-adjuvants or immunomodulation. Another example of the use of these particles are for the purpose of allergy desensitization to treat food and pollen allergies. The particles of this invention may also be used for humans, animals, birds / poultry, fish etc.

[00125] In another implementation the particles of the present invention may have agriculturally important properties, like bioinsecticide, biopesticides, biofungicides, biofertilizers, etc. to replace their synthetic counterparts which are often disruptors of ecosystem.

[00126] In any implementation of the invention the particles obtained from the method of the invention, as obtained in examples presented here, are homogenous, structurally defined and near monodispersed. They are not, and they are not to be referred to as or used as cellular debris, as opposed to the highly irregular, polydispersed cellular debris formed during processes such as electroporation using pulse electric generators, particularly very short pulses in the microsecond time range.

[00127] The process of the present invention is distinct from processes of electroporation where electroporation in the art generally refer to introducing external material into cells using high voltage electric pulses for very short durations, for example, from about 8 to 10 milliseconds or even microsecond range pulses. The process of the present invention is distinct from processes of electroporation where electroporation seeks to insert functional molecules using electromotive force into living cells to manipulate cellular and physiological functions, while the process of the present invention is concerned with production of particles from the biological material.

[00128] Additional steps

[00129] In embodiments, pursuant to the production of the particles in the polar fluid, the method of the present invention may comprise a further step b), which comprises concentrating the particle from the polar fluid. Such a step comprises any suitable step that allows concentration of the particles, such as affinity chromatography, column chromatography, purification and sub-division using ultrafiltration chromatography, size-exclusion chromatography, and other methods known in the art. Such step may also comprise steps of lyophilization or other steps of drying before use or storage of the produced particles.

[00130] Next, according to an embodiment, the particles may be used as such, or they may be subjected to a storage step, which may involve lyophilization, cold storage or even freezing of the produced particles.

[00131] Also, according to another embodiment, the method of the present invention may further comprise step a’) prior to step a), which involves hydrating the biological material in the polar fluid for a time sufficient to obtain a hydrated biological material. In embodiments, as used herein the term “hydrated biological material” is intended to mean that the biological material is saturated with the polar fluid.

[00132] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE 1

EXAMPLE PROCESS 1

[00133] Referring now to the drawings, and more particularly to Fig. 1A, which is a schematic drawing of a basic assembly 10 of the parts and materials to be used in the method of the present invention. In one embodiment, there a provided biological material 20 which is in contact with two electrically conductive poles (30, 40, respectively) and the above assembly 10 is in contact with a provided fluid 50, contained in a non-conductive container 60. Referring now to the drawings, and more particularly to Fig. 1 B, where alternatively, the biological material 20 as provided is in contact with two electrically-conductive poles 30, 40, respectively and provided biological materials 20 is contained inside a porous (e.g., more than 5 micrometer pore size), flexible or rigid, non-conductive filter-system 70, which is in contact with a provided fluid 50, contained in a non-conductive container 60. [00134] Small pieces, minced pieces, diced pieces or clumps of biological material 20 may be provided wrapped or enclosed in a flexible or rigid nonconductive container 70 made of mesh-material (e.g., pore size more than 5000 nanometer).

[00135] To illustrate a bioreactor setting, the biological material 20 may be provided grown, maintained or cultured in the bioreactor, where the biological material 20 is in an enclosed system in a flexible or rigid conductive mesh-material and the above assembly is contained inside the bioreactor which is filled with a polar fluid.

EXAMPLE 2 EXAMPLE PROCESS 2

[00136] Now referring to Fig. 2. Fig. 2A shows a turmeric root cleaned and cut into almost equal volume blocks, which were washed in distilled water (250 ml) for 10 minutes each, changing the water 3 times to remove debris and free floating materials, followed by contacting with two electrically- conductive poles at the opposite sides of each of the turmeric root tissue blocks, followed by submerging the assembly (Fig. 2B) in mineral water (75 ml, pH 7.8, containing 0.12 mM NaCI, 0.9 mM CaC , 0.03 mM KCI, and 0.36 mM MgC ) contained in glass container. A first turmeric root block was subjected to an electromotive force of 24 volt DC, at a distance of approximately 5 cm between the first and second conductive pole (i.e. about 4.8 volt/cm) for 5 minutes in one polarity, and then 5 minutes in the opposite polarity, whereas the second turmeric root block was not provided with any electromotive force for 10 minutes. The particles produced due to the electromotive force applied were suspended in the water (left container, of Fig. 2C). The fluid from both containers were left to rest overnight at room temperature (20°C) to allow the bigger particles to precipitate to the bottom. The next morning, top half of the liquid was carefully collected for microscopic analysis. The particles released into the water due to applied electromotive force appeared colloidal in nature. To observe the particle size and shapes, the samples were analyzed with transmission electron microscopy (TEM) (Fig. 2D, 2E and 2F). In Figs. 2D and 2E, the particles with different electron densities were observed ranging approximately from 30 nm to 200 nm in size, and were accompanied with microparticle of approximately 1 pm or larger (Fig. 2F). TEM of control sample (no electromotive force provided) did not show any such populations of particles. Rather, irregular shaped materials (Fig. 2G) originated from the provided biological material were observed. Scale-bar = 200 nanometer or 0.2 pm (Figs. 2D, E), 0.5 pm (Figs. 2F, 2G).

EXAMPLE 3

EXAMPLE PROCESS 3 [00137] Now referring to Fig. 3. Fig. 3A shows a ginger-root used to produce particles in similar settings and conditions as for the turmeric root example described above in Example 2, with exception of a distance of approximately 4 cm between the first and second conductive pole (i.e., about 6 volt/cm). The top half of the liquid is collected after sedimentation overnight at 20°C. This supernatant part was examined by TEM. Particles of approximate size between 20 to 50 nanometers were most abundantly observed (Figs. 3B and 3C), accompanied by some nano-filamentous materials and larger particles. The TEM of the control sample (no electromotive force provided) did not show any such populations of particles, and only rather irregular shaped materials (Fig. 3D) which originated from the provided biological material were observed. Scale-bar = 0.5 micrometer (Figs. 3B, 3D), 200 nanometers (Fig. 3C).

EXAMPLE 4 EXAMPLE PROCESS 4

[00138] Now referring to Fig. 4. In Fig. 4, small pieces, minced, diced or clumps of biological materials were provided wrapped or enclosed in a “mesh-material” - a metallic screen porous material which is illustrated in Figs. 4B and 4C. In this example green butternut squash was used. Garden grown butternut squash was harvested when the skin was green. The skin was peeled-off, the fruit tissue was diced in approximately in 3 mm ³ pieces, which were washed in distilled water (250 ml) for 10 minutes changing the water 3 times to remove debris and free floating materials, followed by bagging them (approximately 5 grams each) in a flexible mesh-material (cotton fabric), where the two electrically-conductive poles were in contact with the biological materials contained in the mesh- material and connected at a distance of approximately 3.5 cm between the first and second conductive pole and the above assembly was in contact with provided buffered water (75 ml water with 1 mM phosphate buffer at pH 7.4), contained in a glass container. An electromotive force of 8.0 volts AC for 10 minutes (i.e., about 2.3 Volt/cm) was provided (Fig. 4C), whereas the control did not receive any current (Fig. 4A). To test the possibility of particles generated from the flexible bagging mesh-material (without any diced butternut squash enclosed), the same conditions of 8.0 volts AC electromotive force was applied (Fig. 4B) for 10 minutes (i.e., about 2.3 Volt/cm). The particles produced with and without the electromotive force were collected in water. The fluid from the containers were left to rest overnight at room temperature (20°C) such that larger particles sedimented and precipitated at the bottom. The next morning the top half of the liquid was carefully collected and stored in a refrigerator at 6°C for one week without disturbing the liquid. One milliliter of liquid from the top of each sample (Figs. 4A, 4B, and 4C) were used to perform Nanoparticle tracking analysis (NTA) to identify concentration, size distribution and polydispersity of particles present. The samples were diluted 10 times with distilled water prior to NTA. The bottom graph (Fig. 4D) shows the graphical report. The high abundance of particles between 20 to 200 nm were observed only in the sample provided with both biological material and electromotive force, whereas electromotive force without biological material did not produce any significant number of particles and biological sample without any electromotive force provided also did not produce significant number of particles.

EXAMPLE 5 EXAMPLE PROCESS 5

[00139] Now referring to Fig. 5. Avery young Canadian maple leaf (approximately 3 cm in width and length) was used as biological material. The individual leaves were washed in distilled water (250 ml) for 10 minutes changing the water 3 times to remove debris and free-floating materials, followed by rolling the leaf to a cylindrical shape, where the two conductive poles (wires) were used to contact the ends of the rolled leaf at a distance of approximately 2.75 cm between the first and second conductive pole, and the above assembly was in contact with provided water (10 mL, pH 7.8, containing 0.12 mM NaCI, 0.9 mM CaCb, 0.03 mM KCI, and 0.36 mM MgCb), contained in a small glass container. An electromotive force of 8.0 Volts AC (i.e., about 2.9 Volt/cm) for 2.5 minute was provided. Control samples did not receive any electromotive force. The fluid of both the sample container were left to rest overnight at room temperature (20°C) so that bigger particles sedimented and precipitated at the bottom and the next morning the top half of the liquid was carefully collected and stored in a refrigerator at 6°C for one week without disturbing the liquid. One milliliter of liquid from the top of each samples was further collected from undisturbed liquids refrigerated at 6°C for one more week. This supernatant part was examined by TEM, where average 100 nanometer sized spherical particles (Figs. 5A, 5B) were observed in the sample provided with an electromotive force, whereas the control sample (without electromotive force) did not produce such particles (Fig. 5C). Scale-bar = 0.5 micrometer (Figs. 5A, 5B, and 5C), 200 nanometers (insert of Fig. 5A, at the lower panel).

EXAMPLE 6 EXAMPLE PROCESS 6

[00140] Now referring to Fig. 6. Dried cannabis leaf/floral material (1 g, purchased from Cannabis-NB) was cut into pieces (approximately 2 mm ³ ) which were then soaked in distilled water (3 ml) overnight to absorb moisture to saturation, followed by washing with 10 ml of water 3 times to remove debris and free-floating materials followed by removing excess water by blotting using filter paper. 0.29 g each of the above material was placed in two containers having two electrically conductive poles at the opposite side-walls of the container, at a distance of 0.4 cm apart. The material in the containers was submerged in water (1.0 ml each pH 7.8, containing 0.12 mM NaCI, 0.9 mM CaC , 0.03 mM KCI, and 0.36 mM MgC ) and the conductive poles of the first container are provided with an electromotive force of 8.0 volt AC (i.e., about 20 Volt/cm) for 5 minutes [(+) volt] whereas the second container was not provided with any electromotive force for 5 minutes [(-) volt]. The fluid from both the container were collected and filtered through 0.45-micron syringe filter to remove bulk material suspended and Nanoparticle tracking analysis (NTA) was performed to identify concentration, size distribution and polydispersity of particles present in the liquid. The average particle concentration and size distributions of three technical replicates is presented in Fig. 6. The sample which was treated in the same conditions for 5 minutes but without any electromotive force, did not produce any significant number of particles.

EXAMPLE 7 EXAMPLE PROCESS 7

[00141] Now referring to Fig. 7. A fresh carrot root was cut into pieces (approximately 2.5 mm ³ ) followed by washing with 25 ml of water 3 times to remove debris and free-floating materials followed by removing excess water using filter paper to soak. 0.6 grams each of the above material was placed in two containers each having two electrically conductive poles at the opposite sidewalls of the container at a distance of 0.4 cm apart. The material in the containers was submerged in water (1.0 ml each, pH 7.8, containing 0.12 mM NaCI, 0.9 mM CaC , 0.03 mM KCI, and 0.36 mM MgC ) in those containers. The conductive poles of the first container was provided with an electromotive force of 8.0 volt AC (i.e. about 20 Volt/cm) for 4 minutes [(+) volt] whereas the second container was not provided with any electromotive force but incubated for 4 minutes [(-) volt]. The fluid from both the container were collected and filtered through 0.45-micron syringe filter to remove bulk material suspended and subjected to NTA to identify concentration, size distribution and polydispersity of particles present. Particle size distribution-graphs of two technical replicates are presented. The sample without any electromotive force did not produce any significant particles.

EXAMPLE 8 EXAMPLE PROCESS 8

[00142] Now referring to Fig. 8. Minced parts of fresh English cucumber (biomass used here are material between the green skin and central soft seed-areas) was cut into pieces (approximately 2.5 mm ³ ) followed by washing with 20 ml of water 3 times to remove debris and free-floating materials followed by removing excess water using filter paper. 0.45 grams each of the above material was placed in two containers each having two electrically conductive poles at the opposite side-walls of the container at a distance of 0.4 cm apart. The material in the containers was submerged in 0.1 mM phosphate buffer, pH 7.4 (1.0 ml each) in those containers. The conductive poles of the first container was provided with an electromotive force of 8.0-volt AC (i.e. about 20 Volt/cm) for 6 minutes [(+) volt] whereas the second container was not provided with any electromotive force but incubated for 6 minutes [(-) volt]. The fluid from both the container were collected and filtered through 0.45-micron syringe filter to remove bulk material suspended and subjected to NTA to identify concentration, size distribution and polydispersity of particles present. Particle size distribution-graphs of three technical replicates is presented. The sample without any electromotive force was analyzed with NTA in the same conditions did not produce any significant particles.

EXAMPLE 9 EXAMPLE PROCESS 9

[00143] Now referring to Figs. 9A-E. The fresh flowers were collected from local forest area. Immediately the individual flower petals were cut with fine tip scissor from the base (close to sepal and pedicle). The petals were washed in distilled water (150 ml) for 10 minutes changing the water 3 times to remove debris and free-floating materials. Three cotton fabric pouch each containing 5 petals (equal sized) were prepared. For each pouch, two conductive poles were in contact with the pouch at approximately 2 cm apart. Each of the above assemblies were in contact with 10 ml of saline water (0.9 % NaCI w/v in water), separately in three glass containers. The first and second assemblies were provided with 10 ml of saline water and the third assembly was provided with 10 ml of saline water containing blue-food-color (triarylmethane disodium salt, E133 / CI-42090) at 0.1 % (v/v) final concentration in saline. The first assembly was a control and did not receive any electromotive force. An electromotive force of 8.0 volt (i.e. , about 4 Volt/cm) for two minutes was applied to the second and third assemblies. The fluid was collected and filtered through 0.45-micron syringe filters. For the third sample, 3 ml of 0.45-micron filtered fluid was subjected to buffer exchange with saline (without color) to remove excess blue color using 300 kDa spin filter. The buffer exchange was performed 3 times to concentrate the sample to 3-5% of the original volume at each round. Finally, the volume was made to a total of 3 ml with saline and collected from spin filters. A 10-pl aliquot from the above three samples were examined with dark field microscopy for hyperspectral analysis of the particles using CytoViva™ microscope. Representative photographs represent particles formed.

[00144] Fig. 9A shows the control, while Figs. 9B and 9C show the particles formed in the second and third assembly with electromotive force in saline. In Fig. 9C (third assembly) blue-food coloring was mixed saline after removing the excess blue dye using spin filters produce (C). Scale bar is 3 microns. Significantly more particles were produced in (B) in comparison to control (A). There were less particles observed in (C), which may be due to particle loss during buffer exchange. Representative particles were selected to collect the spectral data and profile. A characteristic spectral distribution (D) was observed when samples from (B) was examined. A different spectral profile curve was obtained (E) when sample (C) was examined where the spectral curve was skewed to blue range (425 to 500 nm) indicating adsorption of blue dye in the particles formed.

EXAMPLE 10 EXAMPLE PROCESS 10

[00145] Now referring to Figs.10A-C. Dried green-tea leaves ( Camellia sinensis) were sourced from local organic store, washed three times with distilled water to remove free floating debris followed by soaking in distilled water, at room temperature, to the 100% moisture absorption capacity of those leaves. Excess water was removed by placing the green-tea leaves material onto absorbent filter paper. The green-tea leaves material was frozen at -8°C until used. Next, two samples of 4.0 grams each of the green-tea leaves material were thawed at room temperature and placed in two containers having two electrically conductive poles at the opposite sidewalls of the container, spaced 1.5 cm apart. The green-tea leaves material in the containers was submerged in 15 mL of 0.1 mM phosphate buffer, pH 7.4. The conductive poles of the first container was provided with an electromotive force of 15 volt DC (i.e. about 10 Volt/cm) from the first pole, for 5 minutes followed by reversing the pole and providing an electromotive force of 25 volt DC (i.e. about 16.66 Volt/cm) from the second pole for 10 minutes [(+) volt]. The second container was not provided with any electromotive force, but incubated for a total of 15 minutes [(-) volt]. No significant increase in temperature of the fluid was noticed when electromotive force was applied. The fluids from each container were collected and filtered through 0.45-micron syringe filter to remove bulk green-tea leaves material suspended, and they were subjected to dynamic light scattering (DLS, Zetasizer Nano) to identify size distribution and polydispersity index (PDI) of particles present. Particle size distribution-graphs of three technical replicates is presented is presented in Figs 10B [(-) volt] and 10C [(+) volt].

[00146] Fig. 10A shows suspended particles after filtration with a 0.45-micron syringe filter: without [(-) volt,] on left and with [(+) volt] right the electromotive force applied, respectively. The visual turbidity of the (+) volt sample on the right indicating significantly high number of particles suspended in the liquid of [(+) volt] samples. Table 1 above shows the Z-average and PDI for three technical replicates of the samples of Fig. 10A in DLS analysis. Z-average is nanometer size of mean hydrodynamic size of particles based on dynamic light scattering (DLS) measurement and PDI is polydispersity index. Fig. 10B and 10C shows size distributions of particles (not concentrations of particles) from three technical replicates. The results show that particles are produced after application of the electromotive force according to the process of the present invention. The process of the present invention results in an important production of particles compared to no electromotive force application (i.e., compare the intensity levels from Fig. 10B versus Fig. 10C), and the particles are homogenous, structurally defined and near monodispersed. The process of the present invention is a mild process, where no heat, pressure, or distillation is applied, which may be rapidly deployed for any suitable biological material.

EXAMPLE 11 EXAMPLE PROCESS 11

[00147] Now referring to Figs.11A-C. Dried lion's mane mushrooms ( Hericium erinaceus) fruiting-bodies were sourced from local organic store and stored at -8°C until used. Two samples of 1.5 grams each of the above dried lion's mane mushrooms material was thawed to room temperature and placed in two containers having two electrically conductive poles at the opposite sidewalls of the container, spaced 1.5 cm apart. The material in the containers was submerged in 15 mL of 0.08x phosphate buffer, pH 7.4. The conductive poles of the first container was provided with an electromotive force of 25 volt DC (i.e. about 16.66 Volt/cm) for 8 minutes followed by reversing the pole and providing 25 volt DC (i.e. about 16.66 Volt/cm) for 8 minutes [(+) volt] whereas the second container was not provided with any electromotive force but incubated fora total of 16 minutes [(-) volt]. No significant increase in temperature of the fluid was noticed when electromotive force was applied. The fluids from each container were collected and filtered through 0.45-micron syringe filter to remove bulk green-tea leaves material suspended, and they were subjected to dynamic light scattering (DLS, Zetasizer Nano) to identify size distribution and polydispersity index (PDI) of particles present. Particle size distribution-graphs of three technical replicates is presented is presented in Figs 1 1 B [(-) volt] and 11C [(+) volt]. [00148] Fig. 11 A shows suspended particles after filtration with a 0.45-micron syringe filter: without [(-) volt,] on left and with [(+) volt] right the electromotive force applied, respectively. The visual turbidity of the (+) volt sample on the right indicating significantly high number of particles suspended in the liquid of [(+) volt] samples. Table 2 above shows the results for three technical replicates of samples of Fig. 11 A in DLS analysis. Z-average is nanometer size of mean hydrodynamic size of particles based on DLS measurement and PDI is polydispersity index. Fig. 11 B and 11C shows size distributions of particles (not concentrations of particles) (three technical replicate). The results show that particles are produced after application of the electromotive force according to the process of the present invention. The process of the present invention results in an important production of particles compared to no electromotive force application (i.e. , compare the intensity levels from Fig. 11 B versus Fig. 11 C), and the particles are homogenous, structurally defined and near monodispersed. The process of the present invention is a mild process, where no heat, pressure, or distillation is applied, which may be rapidly deployed for any suitable biological material.

[00149] 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.