Particles encapsulating hydrophilic or amphiphilic biological compounds

Abstract

This invention discloses a method for encapsulating hydrophilic or amphiphilic biological compounds, particles obtained by said method, compositions containing them, and their uses.

Description

Technical Field

[0001] This invention relates to the field of particles encapsulating hydrophilic or amphiphilic biomolecules, compositions containing such particles, methods for preparing such particles and compositions, and their uses.

[0002] Specifically, the present invention relates to the encapsulation of hydrophilic or amphiphilic biological (including bioactive) compounds, which enables delivery of these compounds into living cells (including cells surrounded by cell walls) while maintaining the integrity and activity of these compounds. Background Technology

[0003] The efficacy of many bioactive agents is based on their ability to reach a selected target site and maintain an effective concentration for a sufficiently long time to achieve the desired biological activity. For this purpose, biological compounds, including bioactive compounds or bioactive agents, must be delivered into different types of cells, from mammals, yeast, and bacteria to plant cells and the entire organism. In particular, living cells, especially cell types containing cell walls, such as plant cells, are highly recalcitrant in the intracellular delivery of molecules. Nanoparticle / nanocapsule formulations have been described as an effective method for intracellular delivery, but to our knowledge, they cannot be delivered across cell walls.

[0004] Currently, various methods are available for intracellular delivery, such as chemical transfection (e.g., via PEG-polyethylene glycol), electroporation, biolistic gene gun transfer, and nanoencapsulation. Of these, only the gene gun can penetrate the plant cell wall, but its efficiency is very low.

[0005] Among the most common methods for delivering this molecule, one type is the viral vector, but this method requires complex customization for each target and raises concerns about immunogenicity risks and harmful integration events.

[0006] Currently, various nanoparticle delivery methods are available, such as incorporation or encapsulation within core-shell nanoparticles. Specifically, among non-viral methods, conventional encapsulation techniques that have emerged as promising alternatives are actually lipid-based encapsulation techniques, as lipid particles have not exhibited many problems in packaging hydrophilic or amphiphilic biological compounds. However, a major drawback of conventional lipid-based techniques using lipid particles is their inability to penetrate plant cell walls.

[0007] Furthermore, lipid-based platforms, polymer nanocarriers, and gold nanoparticle methods are characterized by the use of very expensive ingredients, low loading capacity, lack of release efficacy, low stability, and great difficulty in implementing large-scale programs.

[0008] Furthermore, none of the above methods are suitable for macromolecules, such as enzyme complexes, nucleic acid sequences, and large proteins.

[0009] In light of the above, there is an increasing need to overcome the limitations of conventional encapsulation techniques and to find ways to provide solid or liquid hydrophilic active substances (such as proteins or nucleic acid molecules) for their controlled release without losing their activity.

[0010] As mentioned above, there remains a need for new methods for hydrophilic and amphiphilic biological compounds, including bioactive compounds such as DNA, RNA, enzymes, and peptides, that can overcome the limitations of conventional encapsulation techniques and ensure the delivery and controlled release of said molecules within different types of living cells, from plant cells, yeast, and bacteria to animal cells, such as mammalian cells, while maintaining their stability and activity.

[0011] Therefore, the main objective of this invention is to deliver hydrophilic and amphiphilic biological compounds, including bioactive compounds, into different types of cells while maintaining the integrity and activity of these biological compounds. Summary of the Invention

[0012] The inventors discovered that by applying a specific solvent with a specific dielectric constant to a specific protein, the conformation of the protein can be permanently modified to form a shell of the protein base, which also partially contains a bio-hydrophilic or amphiphilic compound, thereby obtaining matrix-type or mold-type particles.

[0013] Therefore, the solution proposed in this paper for the above purpose is a method for encapsulating hydrophilic or amphiphilic biological compounds, which includes the following steps:

[0014] a. A two-phase solution is obtained by dissolving hydrophilic or amphiphilic biological compounds and proteins in water to form a solution, and then mixing the protein solution with the solvent to form a two-phase solution;

[0015] b. Emulsify a two-phase solution to obtain an emulsion; and

[0016] c. Evaporate the solvent from the emulsion;

[0017] This yields particles comprising: (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound.

[0018] The hydrophilic or amphiphilic biological compound is at least partially contained within the shell of the protein base, and

[0019] The solvent in step a has a dielectric constant of 1.5 to 15 at 20-25°C.

[0020] In fact, unbound by any theory, the inventors surprisingly discovered that by using a specific solvent that is immiscible with water and has a specific dielectric constant bound to proteins, it is possible to obtain novel particles, particularly in matrix or mold-like forms, in which biohydrophilic or amphiphilic compounds are encapsulated within a protein-based shell, or at least partially contained within it. The specific solvent, due to its polar properties related to the dielectric constant, can serve as a “nucleation center” for particle formation and, upon evaporation, allows for the acquisition of the specific matrix or mold-like particles of the present invention, in which the biocompound is present both within the particle and along its boundaries. The binding of the specific solvent and the protein allows for specific molecular interactions that distribute the biohydrophilic or amphiphilic compound throughout the particle’s structure, particularly encapsulating it within the shell, and not just at its center. The resulting particles can then cross the cytoplasmic membrane, delivering and subsequently releasing the biocompound in several types of living cells, including the most resistant, such as plant cells containing cell walls.

[0021] The solvent having a dielectric constant of 1.5 to 15 at 20-25°C can be readily selected from physiologically acceptable solvents, and more specifically, acceptable solvents can be selected for pharmaceutical, nutritional, and cosmetic applications, so that the matrix particles of the present invention can be used not only in agricultural chemical applications, but also in the fields of pharmaceuticals, nutritional products, and cosmetics.

[0022] A method for encapsulating hydrophilic or amphiphilic biological compounds to obtain specific particles, said specific particles comprising: (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound.

[0023] The hydrophilic or amphiphilic biological compound is at least partially contained within the shell of the protein base and retains its biological activity.

[0024] The inventors were surprised to discover that, through the method of the present invention, they could obtain specific particles with a small diameter that could penetrate the cell walls of eukaryotic organisms, particularly plant cells, and deliver hydrophilic or amphiphilic biological compounds into living cells, particularly plant cells, while maintaining the integrity and activity of these biological compounds.

[0025] Therefore, in a preferred and advantageous aspect, the present invention relates to particles with diameters in the range of 1 to 60 nm, preferably in the range of 5 nm to 60 nm, and more preferably in the range of 5 nm to 50 nm, as measured by dynamic light scattering (DLS).

[0026] The inventors further discovered that, through the method of the present invention, they can also adjust the particle diameter to obtain specific particles with a larger diameter that can pass through living cells without any cell wall, i.e., eukaryotic cells, especially animal cells, and more particularly mammalian cells, while maintaining the integrity and activity of these biological compounds.

[0027] Therefore, in a further preferred and advantageous aspect, the present invention relates to particles with diameters in the range of 70 to 700 nm, preferably in the range of 100 to 500 nm, more preferably in the range of 100 to 300 nm, and even more preferably in the range of 100 to 200 nm, as measured by dynamic light scattering (DLS).

[0028] According to another aspect, the use of the particles obtained by the method of the present invention for delivering at least one hydrophilic or amphiphilic biological compound within living cells, said living cells comprising cells surrounded by a cell wall is provided.

[0029] According to a further aspect, a composition comprising a plurality of obtained particles is provided.

[0030] Advantageously, the composition has a polydispersity index in the range of 0.05 to 0.7, preferably in the range of 0.2 to 0.6, and most preferably in the range of 0.2 to 0.4, as measured by dynamic light scattering (DLS).

[0031] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the practice or testing of embodiments of the invention, although similar or equivalent methods and materials to those described herein may be used, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be restrictive.

[0032] Further embodiments and the full scope of application of the invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific embodiments, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Attached Figure Description

[0033] The present invention will now be described in detail with reference to the accompanying drawings, specifically:

[0034] Figure 1 This is a schematic diagram illustrating the method of the present invention, which results in the formation of the particles of the present invention (i.e., BSA-WPH Ps). -and e BSA-WPH Ps with an additional chitosan coating + ).

[0035] Figure 2A and 2B These represent different types of particles; Figure 2A This represents the core-shell particle (c-Ps) structure of the prior art; Figure 2B This indicates the matrix-type or mold-type particle (m-Ps) structure of the present invention;

[0036] Figure 3A and 3B It shows: Figure 3A In the freeze-dried BSA-WPH Ps of the present invention - Microscopic images of BSA encapsulated in particles; and Figure 3B In the freeze-dried BSA-WPH Ps of the present invention + Microscopic images of BSA encapsulated in particles;

[0037] Figure 4A The BSA-WPH PS was shown. + After cell treatment, protoplasts cannot be obtained from the formulation when using BSA-WPH PS. - This is possible when processing cells. Figure 4B Display from BSA-WPH PS - Protoplasts isolated from processed cells were analyzed for their cellular delivery.

[0038] Figure 5A and 5B Fluorescence and transmission light microscopy images are provided, showing that the delivery of BSA in the BSA-WPH Ps particles of the present invention depends on the charge of Ps; Figure 5A BSA-WPH Ps + Intracellular delivery cannot be achieved; Figure 5B BSA-WPH Ps - It can indeed achieve intracellular delivery;

[0039] Figure 6 Provides fluorescently labeled BSA-WPHPs for tobacco (Nicotiana tabacum) cells in cell suspension. - Delivery of fluorescence and transmission light microscopy images;

[0040] Figure 7A , 7BFigures 7C and 7C show the characteristics of different particles produced according to Example 1, the particles having a shell containing GFP (with or without nuclear localization signal [NLS]) as a hydrophilic biological compound and different proteins as a protein base, such as WPH (OPTIPEP trade name), fava bean protein isolate, potato protein isolate and soy protein isolate. Figure 7A The z-mean is shown. Figure 7B The quantity is shown (where the quantity distribution shows the relative proportion of particles of different sizes, taking into account their quantity in the solution). Figure 7C The PDI index is displayed.

[0041] Figure 8A and 8B Fluorescence and transmission light microscopy images of nanoparticles and tobacco cells with intact plant cell walls are provided; specifically, Figure 8A A basic fluorescence image of the nanoparticles is provided, thus serving as a control and intended to examine background fluorescence as untreated cells; Figure 8B Optical microscopy images of empty nanoparticles (NPs) with WPH and no GFP suspended in the shells of tobacco cells with intact plant cell walls are provided.

[0042] Figure 9 Fluorescence and transmission light microscopy images of the bioavailability of unencapsulated GFP (naked GFP) suspended in tobacco cells with intact plant cell walls are provided.

[0043] Figure 10 Fluorescence and transmitted light microscopy images are provided, showing the GFP-WPH NPs of this invention encapsulated in tobacco cells with intact plant cell walls. - Intracellular delivery of GFP protein in particles; this figure shows that after GFP is delivered into the cell through the plant cell wall, it maintains its biological integrity (i.e., GFP fluorescence retention) throughout the process.

[0044] Figure 11 Fluorescence and transmitted light microscopy images are provided, showing intracellular delivery of GFP protein with nuclear localization signal (NLS), said GFP protein encapsulated in the GFP-WPH NPs of the present invention. - The particles were suspended overnight in tobacco cells with intact plant cell walls, then washed and observed through an optical microscope equipped with a fluorescence filter, as well as the nuclear localization of signals generated by biological activity, i.e., the interaction between NLS and the host cell's natural intracellular transport mechanisms.

[0045] Figure 12 This invention provides broad bean-GFPNPs encapsulated in tobacco cells with intact plant cell walls, which are suspended in these cells. -Fluorescence and transmission light microscopy images of intracellular delivery of GFP in particles;

[0046] Figure 13A and Figure 13B It provides, in particular, tobacco cells suspended in plants with intact cell walls. Figure 13A ) and in cell callus tissue ( Figure 13B The soybean-GFP NPs of the present invention are encapsulated in the present invention. - Fluorescence and transmission light microscopy images of intracellular delivery of GFP in (GFP-SP NPs-) particles;

[0047] Figure 14A provides an optical microscopic image of the bioavailability of naked DNA (naked p-RAP) suspended in tobacco cells;

[0048] Figure 14B shows DNA-WPH Ps of the present invention suspended in tobacco cells and encapsulated in them. - DNA in the particles (pRAP-NPs (DNA-WPH Ps)) - Optical microscope images of the bioavailability of the material;

[0049] Figure 15A Optical microscopy images of the bioavailability of empty cellulase-GFP Ps nanoparticles are provided.

[0050] Figure 15B Tobacco for use in callus tissue is provided, encapsulated in the cellulase-GFP Ps of the present invention. - Optical microscopic image of the bioavailability of 0.5 mg / ml cellulase in the particles;

[0051] Figure 16 A proposed scheme for the formation of enzymes (cellulase and amylase)-WPH-Ps of the present invention;

[0052] Figure 17 Provided are standalone cellulases, cellulases dissolved in whey protein, and encapsulated cellulases of the present invention - WPH Ps, when in contact with sugar. - Comparison of enzyme activities between (NPs) and cellulase-NPs that were encapsulated and then ruptured. Activity was tested using the phenol-sulfuric acid method.

[0053] Figure 18 This invention provides, when in contact with sugar, standalone amylase, amylase dissolved in whey protein, and encapsulated amylase-WPH Ps of the present invention. - Comparison of enzyme activities between encapsulated and re-crushed amylase-NPs. Activity was tested using the phenol-sulfuric acid method;

[0054] Figure 19Optical microscopic images of the basal fluorescence of untreated macrophages J77A.1 are provided; and

[0055] Figure 20 Nanoparticles (NPs) containing WPH and GFP in their shells are provided (GFP-WPH Ps) - Optical microscope image of the J77A.1 cell suspension treated with [the treatment]. Detailed Implementation

[0056] Therefore, the present invention relates to a method for encapsulating hydrophilic or amphiphilic biological compounds, comprising the following steps:

[0057] a. A two-phase solution is obtained by dissolving hydrophilic or amphiphilic biological compounds and proteins in water to form a protein solution, and then mixing the protein solution with a solvent to form a two-phase solution.

[0058] b. Emulsify a two-phase solution to obtain an emulsion;

[0059] c. Evaporate the solvent from the emulsion;

[0060] This results in particles containing (i) a shell of protein groups and (ii) hydrophilic or amphiphilic biological compounds.

[0061] The hydrophilic or amphiphilic biological compound is at least partially contained within the shell of the protein base.

[0062] The solvent in step a has a dielectric constant of 1.5 to 15 at 20-25°C.

[0063] In this invention, the following terms are used:

[0064] - "Bio-compound" or "hydrophilic or amphiphilic bio-compound" refers to biological macromolecules, including proteins and nucleic acids and combinations thereof. The bio-compound can be a bioactive compound or a non-bioactive compound. The bioactive compound refers to a compound that produces biological or chemical changes in an organism or living organism or its cells, including but not limited to mammals, vascular plants, non-vascular plants (eukaryotic algae, bryophytes), fungi, yeasts, and prokaryotes (bacteria, cyanobacteria, etc.). In some embodiments, the bio-hydrophilic or amphiphilic compound has a microwave energy (MW) of less than 1,000 Da;

[0065] - "Hydrophilic compound or hydrophilic biological compound" means a biological compound that, when introduced into water at a concentration of at least 1% by weight, at least 5% by weight, or at least 10% by weight, produces a homogeneous solution visible to the naked eye.

[0066] - "Core-shell particles" refer to particles in which biological compounds are encapsulated only in the core, not in the shell, or even on the surface of the particle;

[0067] - "Matrix-type particles" or "mold-type particles" refer to particles in which the active ingredients are distributed not only in the core but also in the structure of the particle, especially in the shell of the protein base, and some biological compounds may be exposed on the surface.

[0068] - "Protein hydrolysate" refers to all hydrolysates of proteins prepared by using proteolytic enzymes, microorganisms containing suitable proteolytic activity or acid hydrolysis, or any combination thereof, and having an effect on improving serum lipid distribution. Commercially available hydrolysates can be used, or hydrolysates can be prepared. In some embodiments, the molecular weight of the hydrolysate is 300-100,000 Da, 500-50,000 Da;

[0069] - In some implementations, "whey protein" means a product containing at least 80%, 85%, or 90% whey protein;

[0070] - A "living cell" is a cell that is capable of: - responding to changes in its environment, - growing and developing throughout its life cycle, - reproducing or replicating itself, - carrying out metabolism, - maintaining homeostasis, or keeping its internal environment unchanged regardless of external changes, - passing on its characteristics to its offspring.

[0071] Specifically, living cells can be selected from "cells without cell walls" (e.g., animal cells, certain microorganisms, and protoplasts) and "cells with cell walls" (typical plant cells, certain microorganisms); more specifically, they can be selected from prokaryotes, such as bacteria and archaea; more specifically, such as bacterial cells and yeast cells, as well as eukaryotes, such as eukaryotic microorganisms, including plant cells, fungi, and animal cells, especially mammalian cells.

[0072] "Z-potential" is the scientific term for the electromotive force in colloidal systems. In colloid chemistry literature, it is usually represented by the Greek letter ζ, hence the name zeta potential. The zeta potential is a measure of the magnitude of repulsion or attraction between particles. It is an indicator of the strength of the interaction between colloidal particles, and its measurement is used to assess the stability of colloidal systems. In aqueous media, the pH of a sample affects its zeta potential. For example, if a base is added to a suspension with a negative zeta potential, the particles tend to acquire more negative charge. If enough acid is added to the suspension, the charge will be neutralized. Further addition of acid will lead to the accumulation of positive charge.

[0073] - "Approximately" refers to +10%.

[0074] - "comprises", "comprising", "includes", "including", "having" and their variations are intended to mean "including but not limited to".

[0075] - "composed of" means "including and limited to".

[0076] - "Exemplary" means "used as an embodiment, example, or illustration". Any implementation described as "exemplary" is not necessarily to be construed as superior to or advantageous to other implementations, and / or as excluding combinations of features of other implementations.

[0077] - "Optional" is intended to mean "provided in some embodiments but not in others". Any particular embodiment of the invention may include a number of "optional" features unless these features conflict with each other.

[0078] - The singular forms “a,” “an,” and “the” include the plural, unless the context clearly specifies otherwise. For example, the terms “compound” or “at least one compound” can include a variety of compounds, including mixtures thereof.

[0079] - "Method" means the manner, means, techniques and procedures used to accomplish a given task, including but not limited to those manner, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or those manner, means, techniques and procedures that can be easily developed from those manner, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0080] - "Treatment" includes eliminating, substantially inhibiting, slowing down or reversing the development of the disease, substantially improving the clinical or aesthetic symptoms of the disease, or substantially preventing the occurrence of the clinical or aesthetic symptoms of the disease.

[0081] - "Catonic polymers" refers to cationic polymers derived from both natural and synthetic sources.

[0082] - Use "chitosan derivatives" in which one or more hydroxyl groups and / or one or more amino groups have been modified (e.g., acetylated, alkylated or sulfonated chitosan, thiolized derivatives).

[0083] Throughout this application, various embodiments of the invention may be presented in the form of scope. It should be understood that the scope description is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of the invention. Therefore, the scope description should be considered as having specifically disclosed all possible sub-scopes and individual numerical values ​​within that scope. For example, a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 3, 4, 5, and 6. This applies to any width of scope.

[0084] Whenever this document indicates a range of values, it means any referenced number (fraction or integer) included within the indicated range. The phrases “range between the first and second indicated values” and “range between the first and second indicated values” are used interchangeably in this document and mean including the first and second indicated values ​​and all fractions and integers in between.

[0085] According to preferred and advantageous aspects, the encapsulation method of the present invention is non-destructive and reversible, at least for industrial enzymes.

[0086] Advantageously, the encapsulation method of the present invention can be carried out using acceptable chemicals such as pharmaceuticals, food, and cosmetics.

[0087] The method includes step a: mixing the protein solution and the solvent.

[0088] The solvent in step a has a dielectric constant of 1.5 to 15, preferably 1.8 to 6.02, at 20-25°C.

[0089] Preferably, the solvent in step a, having a dielectric constant of 1.5-15 at 20-25°C, is selected from the group consisting of ethyl acetate (6.02), dichloromethane (8.93), pentane (1.84), chloroform (4.81), 1,4-dioxane (2.25), benzene (2.27), toluene (2.38), n-pentane (1.84), n-hexane (1.88), and cyclohexane (2.02).

[0090] More preferably, the solvent in step a has a dielectric constant of 1.8 to 6.02 at 20-25°C.

[0091] More preferably, the solvent in step a is ethyl acetate, which has a dielectric constant of 6.02.

[0092] All described dielectric constants are provided by the supplier and are ultimately verified using known techniques such as dielectric probes, oscilloscopes, or microwave dielectric meters.

[0093] Preferably, the protein:solvent weight ratio of the solvent in step a, which has a dielectric constant of 1.5 to 15 at 20-25°C, is in the range of 10:0.5 to 8:1.5.

[0094] More preferably, the weight ratio of protein to solvent in step a is 9:1.

[0095] In an advantageous and preferred embodiment, the method further includes step e: adding a cationic polymer to coat the particles, thereby encapsulating the particles.

[0096] Preferably, the cationic polymer is a cationic polysaccharide.

[0097] Non-limiting examples of cationic polysaccharide polymers include: cationic cellulose and hydroxyethyl cellulose; cationic starch and hydroxyalkyl starch; cationic polymers based on arabinose monomers (e.g., those that can be derived from arabinose plant gums); cationic polymers derived from xylose polymers found in materials such as wood, straw, cottonseed hulls, and corn cobs; cationic polymers derived from fucose polymers (found as a component of seaweed cell walls); cationic polymers derived from fructose polymers (e.g., inulin found in some plants); cationic polymers based on acidic sugars (such as galacturonic acid and glucuronic acid); cationic polymers based on amino sugars (such as galactosamine and glucosamine); cationic polymers based on 5- and 6-membered ring polyols; cationic polymers based on galactose monomers found in plant gums and mucilages; cationic polymers based on mannose monomers (e.g., those found in plants, yeast, and red algae); and cationic polymers based on galactomannan copolymers (called guar gum, obtained from the endosperm of guar beans).

[0098] More preferably, the cationic polymer is a cationic polysaccharide, and even more preferably, the cationic polysaccharide is chitosan or a derivative thereof.

[0099] Advantageously, chitosan or its derivatives are characterized by having a low molecular weight, such as less than 90 kDa, less than 75 kDa, less than 50 kDa, less than 30 kDa or less than 15 kDa.

[0100] More advantageously, derivatives of chitosan are used, wherein one or more hydroxyl groups and / or one or more amino groups have been modified (e.g., acetylated, alkylated or sulfonated chitosan, thiolated derivatives) to increase the solubility of chitosan or to increase its adhesiveness.

[0101] In an advantageous and preferred embodiment, the method includes step e, prior to step d, of drying and evaporating the emulsion: adding a cationic polymer to coat the particles, thereby encapsulating the particles.

[0102] In another advantageous and preferred embodiment, the method includes step e after step d of drying and evaporating the emulsion: adding a cationic polymer to coat the particles, thereby encapsulating the particles.

[0103] According to the method of the present invention, there is a step a: forming a two-phase solution by dissolving a hydrophilic or amphiphilic biological compound and a protein in water to form a protein solution.

[0104] Preferably, the hydrophilic or amphiphilic biological compound in step a is a biological compound selected from the group consisting of oligonucleotides, nucleic acids, proteins, peptides, hormones, enzymes, or any combination thereof.

[0105] More preferably, the hydrophilic or amphiphilic biological compound is selected from BSA (bovine serum albumin), GFP (green fluorescent protein), DNA, antisense RNA, messenger RNA, CRISPR enzyme, CRISPR-guided RNA complex, cellulase, and amylase.

[0106] In an advantageous embodiment, the hydrophilic or amphiphilic biological compound of step a is in a suspension. Preferably, the hydrophilic or amphiphilic biological compound of step a is a suspension in a solvent.

[0107] Advantageously, the protein in step a may be an extract selected from animal proteins, plant proteins, or algal proteins. The protein extract may be selected from: purified proteins, concentrated proteins, isolated protein fractions, protein hydrolysates, or any combination thereof.

[0108] Preferably, the protein in step a is selected from whey protein, soy protein, pea protein, fava bean protein, and potato protein, or any combination thereof.

[0109] More preferably, the protein in step a is in the form of protein hydrolysate.

[0110] In fact, the inventors have noted that hydrolyzed proteins can be used in the method of the present invention to obtain the specific particles because the low molecular weight of the hydrolyzed proteins allows for smaller particle sizes within the nanoparticle range. According to this preferred embodiment, advantageously, in the case of plant cell membranes, the particles of the present invention are more readily permeable.

[0111] More preferably, the protein in step a is whey protein, fava bean protein, and soy protein, and more preferably, it is a whey protein hydrolysate.

[0112] Preferably, step a is performed using an ultrasonic device and lasts from 5 seconds to 30 minutes, more preferably from 1 minute to 15 minutes, and even more preferably from 1 minute to 10 minutes.

[0113] More preferably, the duration of step a, in which the solvent is mixed with the protein using an ultrasonic device, is selected from the group consisting of 1 minute, 3 minutes, 2 minutes, and 5 minutes.

[0114] More preferably, the duration of step a, in which the solvent is mixed with the protein using an ultrasonic device, is 5 minutes.

[0115] More preferably, step a, mixing the solvent with the protein, is performed by sonicating the solution for 5 minutes using a Microson Ultrasonic Cell Disruptor XL at 10W.

[0116] Advantageously, the mixing in step a is carried out at 5°C (e.g., on ice) to avoid overheating of the solution.

[0117] In an advantageous embodiment, step a of mixing the solvent with the protein is performed using a high-shear homogenizer.

[0118] Advantageously, the mixing in step a is performed using a combination of a high-shear homogenizer and an ultrasonic instrument.

[0119] The method according to the invention includes step b: emulsifying the two-phase solution obtained in step a to obtain an emulsion.

[0120] Preferably, the emulsification in step b is performed using an ultrasonic device for a duration of 5 seconds to 30 minutes, more preferably 1 minute to 15 minutes, and more preferably 1 minute to 10 minutes, including any range therein.

[0121] More preferably, the duration of emulsification in step b is selected from a group consisting of 1 minute, 3 minutes, 2 minutes, and 5 minutes.

[0122] More preferably, the emulsification in step b is performed using an ultrasonic instrument for 5 minutes.

[0123] More preferably, emulsification in step b is performed by sonicating the solution for 5 minutes using a Microson Ultrasonic Cell Disruptor XL at 10W power.

[0124] Advantageously, the emulsification in step b is carried out at 5°C (e.g., on ice) to avoid overheating of the solution.

[0125] In some advantageous embodiments, the emulsification in step b is performed using a high-shear homogenizer.

[0126] Advantageously, the emulsification in step b is performed using a combination of a high-shear homogenizer and an ultrasonic instrument.

[0127] These conditions enable the formation of finer particles. In fact, the inventors believe that by adjusting the power and time of the ultrasonic treatment in step b, it is possible to obtain particle diameters in different ranges, as shown below.

[0128] According to the method of the present invention, in order to form and stabilize the NPs structure, step c is required: evaporating the solvent from the emulsion.

[0129] In an advantageous embodiment, the evaporation of the solvent in step c is accomplished by using a nitrogen stream, a nitrogen stream in the dark, an evaporator, a rotary evaporator (e.g., a circulating evaporator, a falling film evaporator, a rising film evaporator, a rising and falling film plate evaporator, a multi-effect evaporator, a stirred thin film evaporator stream) or any combination thereof.

[0130] Preferably, the solvent evaporation in step c is accomplished by a nitrogen gas flow in the dark.

[0131] According to preferred and advantageous embodiments, the method of the present invention further includes step d: drying the evaporated particles in step c, the particles comprising (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound, wherein the hydrophilic or amphiphilic biological compound is at least partially contained in the protein-based shell in step c; or further including drying the coated particles in step e, the particles comprising (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound; and (iii) a polysaccharide coating encapsulating the particles, wherein the hydrophilic or amphiphilic biological compound is at least partially contained in the protein-based shell.

[0132] Advantageously, the drying of the particles in step d is selected from the group consisting of spray drying, granulation, coagulation, freeze drying, or any combination thereof.

[0133] Preferably, the drying in step d is performed by freeze-drying the particles obtained in step c.

[0134] Freeze-drying was chosen to stabilize BSANP formulations. This method was selected from numerous options (e.g., spray drying and fluidized bed drying) because it is the gentlest technique suitable for handling valuable molecules that are highly unstable to harsh conditions or transport.

[0135] In a specific implementation, the drying in step d is carried out after adding low molecular weight molecules (e.g., maltodextrin glucose equivalent (DE)19 and glycerol) to the solution to avoid damage to the particle structure caused by high pressure.

[0136] Unbound by any theory, the inventors surprisingly observed that adding low molecular weight molecules, particularly maltodextrin, to the solution prior to the drying step could maintain particle stability. In fact, maltodextrin deposits around the particles obtained after step c of the method of the present invention, and they act as fillers, thereby preventing the stress generated during freezing and subsequent freeze-drying from damaging the particle structure.

[0137] In advantageous and preferred embodiments, the method has an encapsulation rate of 65% to 95%, 60% to 90%, 70% to 90%, 70% to 85%, 75% to 80%, or 75% to 90%, including any range therebetween. In some embodiments, the method has an encapsulation rate of 80% to 100%.

[0138] In specific implementations, the method of the present invention allows for the acquisition of particles with diameters ranging from 1 to 60 nm, 5 nm to 60 nm, more preferably 5 nm to 50 nm, or any range therein.

[0139] In another specific embodiment, the method of the present invention allows for the acquisition of particles with diameters ranging from 70 to 700 nm, preferably from 100 to 500 nm, more preferably from 100 to 300 nm, and even more preferably from 100 to 200 nm, including any range therebetween.

[0140] The possibility of obtaining different particle diameters depends on the ultrasonic parameters used in emulsification in step b, especially if the solution is sonicated for at least 5 minutes, which can result in finer particles.

[0141] Furthermore, by adjusting the duration of ultrasonic treatment, it is possible to obtain larger particle diameters, such as those described in this article.

[0142] According to a second aspect, the present invention provides particles obtainable by the above method, comprising:

[0143] (i) the shell of the protein base; and

[0144] (ii) hydrophilic or amphiphilic biological compounds; and

[0145] The hydrophilic or amphiphilic biological compound is at least partially contained within the shell of the protein base.

[0146] In an advantageous embodiment, the particles obtained by the method of the present invention can have a diameter ranging from 1 to 60 nm, preferably from 5 nm to 60 nm, more preferably from 5 nm to 50 nm, including any range therebetween.

[0147] The specific structure of the particle comprises (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound, wherein the hydrophilic or amphiphilic biological compound is at least partially contained within the protein-based shell, having a total diameter of less than 60 nm, such that the particle is capable of crossing the cell membrane in several types of cells (particularly living cells, including the most stubborn cells, such as plant cells surrounded by cell walls), delivering and subsequently releasing the biological compound.

[0148] Advantageously, the plant cells are in the form of a cell suspension or callus tissue.

[0149] On the other hand, when considering mammalian cells, particularly mouse macrophages, larger particles can be used because these types of cells lack plant cell membranes and cell walls and are less resistant.

[0150] Therefore, for these types of mammalian cells, only the specific structure of the particles of the present invention, which comprises (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound, wherein the hydrophilic or amphiphilic biological compound is at least partially contained within the protein-based shell, is a key feature. This allows the delivery of the hydrophilic and amphiphilic biological compounds into the mammalian cell while simultaneously protecting the molecules they carry to the site of action.

[0151] Therefore, in another advantageous embodiment, the particles obtainable by the method of the present invention have a diameter in the range of 70 to 700 nm, preferably 100 to 500 nm, more preferably 100 to 300 nm, and even more preferably 100 to 200 nm, including any range therebetween.

[0152] According to the present invention, particles obtainable by the method of the present invention comprise (i) a protein-based shell, the protein-based shell comprising at least a portion of a hydrophilic or amphiphilic biological compound contained in the protein-based shell.

[0153] The protein contained in the shell of the protein base can be an extract selected from animal proteins, plant proteins, or algal proteins. Protein extracts can be selected from: purified proteins, concentrated proteins, isolated protein fractions, protein hydrolysates, or any combination thereof.

[0154] Preferably, the shell of the protein base comprises a protein selected from whey protein, soy protein, pea protein, fava bean protein, and potato protein, or any combination thereof.

[0155] More preferably, the protein is in the form of a protein hydrolysate.

[0156] In fact, the inventors noted that hydrolyzed proteins can be used in the method of the present invention to obtain the specific particles, and due to the low molecular weight of the hydrolyzed proteins, this allows for smaller particle sizes within the nanoparticle range. These particles are surprisingly more easily permeated, particularly in the case of plant cell membranes.

[0157] Even more preferably, the shell of the protein base comprises a protein selected from whey protein hydrolysate and fava bean protein isolate; and even more preferably, the shell of the protein base comprises hydrolyzed whey protein.

[0158] Plant, animal, or microbial proteins and / or mixtures thereof can be used as protein sources for hydrolysates. Suitable plant protein sources include, for example, soy protein, wheat protein, wheat gluten, corn protein, oat protein, rye protein, rice protein, rapeseed or low-erucic acid rapeseed protein (canola protein), barley protein, flaxseed protein, potato protein, pea protein, lupin protein, sunflower protein, hemp protein, fava bean protein, and buckwheat protein.

[0159] In some implementations, the protein is of animal origin. Suitable animal protein sources are, for example, milk proteins, such as casein and whey protein, as well as their fractions, ovalbumin, collagen, and gelatin.

[0160] Preferably, the protein can be used as a source of hydrolysis products in various commercially available purified or unpurified forms.

[0161] Advantageously, materials containing these proteins and other major components (such as carbohydrates) are used as sources of hydrolysis products.

[0162] In an advantageous embodiment, the protein extract is a plant protein. In some embodiments, the plant protein is extracted from potatoes, peas, soybeans, chickpeas, quinoa, wheat, lentils, broad beans, or beans.

[0163] In another preferred embodiment, the protein extract is an animal protein (e.g., a mammal, bird, or insect).

[0164] Preferably, the protein is whey protein.

[0165] In a preferred embodiment, a shell comprising at least a portion of a protein-based amphiphilic or hydrophilic biological compound at least partially surrounds at least 25% of the total surface area of ​​the biological compound, preferably at least 35%, more preferably at least 50%, or still more preferably at least 75%.

[0166] Preferably, the protein-based shell is in the form of a matrix structure, thereby obtaining matrix-type particles or mold-type particles, which comprise (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound, wherein the hydrophilic or amphiphilic biological compound is at least partially contained in the protein-based shell.

[0167] In an advantageous embodiment, the content of the protein-based shell contained in the particles is 0.1% to 99% (w / w), 0.1% to 90% (w / w), 0.1% to 50% (w / w), 0.1% to 30% (w / w), 0.5% to 30% (w / w), and any range therein.

[0168] In another advantageous embodiment, the content of the protein-based shell depends on the final concentration of the desired encapsulating hydrophilic or amphiphilic biological compound. If a low concentration of the hydrophilic or amphiphilic biological compound is required, the content of the protein-based shell can be increased to 99.9%.

[0169] According to the present invention, the particles obtainable by the method of the present invention further comprise (ii) a hydrophilic or amphiphilic biological compound, wherein the hydrophilic or amphiphilic biological compound is at least partially contained in the shell of the protein base.

[0170] Preferably, the hydrophilic or amphiphilic biological compound is a hydrophilic compound selected from the group consisting of oligonucleotides, nucleic acids, proteins (e.g., enzymes), peptides, hormones, or any combination thereof.

[0171] Preferably, the hydrophilic or amphiphilic biological compound is selected from BSA (bovine serum albumin), GFP (green fluorescent protein), DNA, antisense RNA, messenger RNA, CRISPR enzyme, CRISPR enzyme-guided RNA complex, cellulase, and amylase.

[0172] The aforementioned hydrophilic or amphiphilic compounds are effective for use in a variety of fields, including but not limited to pharmaceutical, cosmetic, biotechnological, agricultural (e.g., herbicides, pesticides, fertilizers), diagnostic and / or diagnostic applications.

[0173] In a preferred embodiment, the bio-hydrophilic or amphiphilic compound is a genome-modifying molecule, such as a gene silencing molecule, a gene substitution and / or gene insertion molecule, or a molecule that targets and modifies the genome, or a messenger RNA encoding such a molecule. Genome-modifying molecules can be CRISPR ribonucleoproteins (RNPs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and oligonucleotides (e.g., small interfering RNA (siRNA) and short hairpin RNA (shRNA)). Each possibility represents a separate embodiment. In some embodiments, the bio-compound is at least one CRISPR element, such as RNPs and / or guide RNA (gRNA), or DNA encoding such an element. RNPs can comprise RNA-guided nucleases and guide RNA (sgRNA). Non-limiting examples of RNA-guided nucleases include: Cas9 (SpCas9, StCas9, SaCas9, ScCas9, and dead Cas9), CasX, CasY, Cas-Phi, Cas12a (Cpf1), Cas13, Cas14, and MAD7.

[0174] In a preferred embodiment, the biological hydrophilic or amphiphilic compound is an enzyme. The enzyme may be selected from the group consisting of glycoses (including cellulase, amylase, pectinase, and lactase), proteases, lipases, phytases, laccases, polymerases, and nucleases.

[0175] In an advantageous and preferred embodiment, a hydrophilic or amphiphilic biological compound is provided as the hydrolysis product. As described below, hydrolyzed proteins can be used due to their low molecular weight, which allows for smaller particle sizes.

[0176] In an advantageous embodiment, the hydrophilic or amphiphilic biological compound is in a suspension. Preferably, the hydrophilic or amphiphilic biological compound is a suspension in a solvent.

[0177] Advantageously, the particles contain 1% to 80% (w / w), 1% to 70% (w / w), 1% to 60% (w / w), 1% to 50% (w / w), 1% to 40% (w / w), 2% to 40% (w / w), 5% to 40% (w / w), 10% to 70% (w / w), 10% to 40% (w / w), 15% to 40% (w / w), 25% to 40% (w / w), or 1% to 35% (w / w). Hydrophilic or amphiphilic biological compounds in the following proportions: (w / w), 1% to 25% (w / w), 1% to 20% (w / w), 1% to 15% (w / w), 1% to 10% (w / w), 5% to 70% (w / w), 5% to 55% (w / w), 5% to 35% (w / w), 5% to 25% (w / w), 5% to 20% (w / w), 5% to 15% (w / w), or 5% to 10% (w / w).

[0178] In an advantageous embodiment of the invention, the particles contain 0.1% to 20% of a hydrophilic or amphiphilic biological compound.

[0179] In another preferred embodiment, the concentration of the hydrophilic or non-hydrophilic biological compound in the particles is from about 0.01 mg / g to 500 mg / g.

[0180] Preferably, the concentration of the hydrophilic or amphiphilic biological compound in the particles is 0.01 mg / g to 250 mg / g, about 1 mg / g to 100 mg / g, about 1 mg / g to 50 mg / g, about 1 mg / g to 30 mg / g, or about 1 mg / g to 5 mg / g, or any range therein, relative to 1 g of the protein-based shell.

[0181] In an advantageous and preferred embodiment, the concentration of the hydrophilic or amphiphilic biological compound in the particles is from 0.01 mg / g to 300 mg / g relative to 1 g of the protein-based shell, and preferably, the concentration of the hydrophilic or amphiphilic biological compound in the particles is from 4 mg / g to 220 mg / g relative to 1 g of the protein-based shell.

[0182] According to the present invention, the particles obtainable by the method of the present invention may further contain (iii) a cationic polymer, thereby forming a coating that encapsulates the particles.

[0183] The cationic polymer is able to interact with at least a portion of the shell of the protein base (e.g., through electrostatic interaction) to form a coating.

[0184] Preferably, the cationic polymer is a cationic polysaccharide.

[0185] Non-limiting examples of cationic polysaccharide polymers include: cationic cellulose and hydroxyethyl cellulose; cationic starch and hydroxyalkyl starch; cationic polymers based on arabinose monomers (e.g., those that can be derived from arabinose plant gums); cationic polymers derived from xylose polymers found in materials such as wood, straw, cottonseed hulls, and corn cobs; cationic polymers derived from fucose polymers (found as a component of seaweed cell walls); cationic polymers derived from fructose polymers (e.g., inulin found in some plants); cationic polymers based on acidic sugars (such as galacturonic acid and glucuronic acid); cationic polymers based on amino sugars (such as galactosamine and glucosamine); cationic polymers based on 5- and 6-membered ring polyols; cationic polymers based on galactose monomers found in plant gums and mucilages; cationic polymers based on mannose monomers (e.g., those found in plants, yeast, and red algae); and cationic polymers based on galactomannan copolymers (called guar gum, obtained from the endosperm of guar beans).

[0186] More preferably, the cationic polymer is a cationic polysaccharide; even more preferably, the cationic polysaccharide is chitosan or a derivative thereof; and even more preferably, it is chitosan.

[0187] Advantageously, chitosan or its derivatives are characterized by having a low molecular weight, such as less than 90 kDa, less than 75 kDa, less than 50 kDa, less than 30 kDa or less than 15 kDa.

[0188] More advantageously, derivatives of chitosan are used, wherein one or more hydroxyl groups and / or one or more amino groups have been modified (e.g., acetylated, alkylated or sulfonated chitosan, thiolated derivatives) to increase the solubility of chitosan or to increase its adhesiveness.

[0189] More advantageously, the purpose of using chitosan derivatives is to increase the solubility of chitosan or to increase its adhesiveness.

[0190] In a preferred and advantageous embodiment of the invention, the particles obtainable by the method of the invention comprise (i) a protein-based shell, (ii) a hydrophilic or amphiphilic biological compound; and the hydrophilic or amphiphilic biological compound is completely contained within the protein-based shell.

[0191] In another preferred and advantageous embodiment of the invention, the particles obtainable by the method of the invention comprise (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound; and the hydrophilic or amphiphilic biological compound is partially contained within the protein-based shell and partially within the particle.

[0192] In a preferred embodiment, the amphiphilic or hydrophilic biological compound, which is at least partially contained in the shell of the protein base, accounts for at least 75% of the total surface area of ​​the compound, preferably at least 50%, more preferably at least 35%, or still more preferably at least 25%.

[0193] In another preferred embodiment, the amphiphilic or hydrophilic biological compound, at least partially contained in the shell of the protein base, accounts for 0.1% to 25% of the total surface area of ​​the compound.

[0194] As described above, the specific combination of solvent and protein selected allows for specific interactions that distribute the biohydrophilic or amphiphilic compound throughout the entire structure of the particle, not just in the particle's core. This enables the particles of the present invention to cross cell membranes and deliver and subsequently release biocompounds in several types of living cells, such as bacterial cells, mammalian cells, and even the most stubborn cells, such as plant cells.

[0195] Therefore, according to one aspect, the present invention provides the use of particles with diameters in the range of 1 to 60 nm, obtainable by the method of the present invention, for delivering hydrophilic or amphiphilic biological compounds to particularly resistant organisms, especially plant cells, and thus increasing the bioavailability of the hydrophilic or amphiphilic biological compounds.

[0196] According to another embodiment, the present invention provides non-therapeutic cosmetic uses of particles obtainable by the method of the present invention, the particles having a diameter in the range of 70 to 700 nm, for delivering hydrophilic or amphiphilic biological compounds to animal cells, preferably mammalian cells, more preferably macrophages, and even more preferably mouse cells, thereby increasing the bioavailability of the hydrophilic or amphiphilic biological compounds.

[0197] Furthermore, the present invention relates to a method for treating pathologies resolved by hydrophilic or amphiphilic biological compounds of the particles of the present invention, the method comprising the step of delivering particles of the present invention comprising an effective amount of the hydrophilic or amphiphilic biological compound.

[0198] Due to their ability to internalize, increase bioavailability, and thus deliver the hydrophilic or amphiphilic biological compounds, the particles of the present invention can be used in methods of treating pathologies addressed by hydrophilic or amphiphilic biological compounds, when applied to living organisms such as plants, bacteria, or animals (preferably mammals) in appropriate amounts to treat the specific pathology.

[0199] Therefore, the present invention also relates to particles for delivering at least one hydrophilic or amphiphilic biological compound for treating pathologies addressed by the hydrophilic or amphiphilic biological compound.

[0200] According to another aspect, the present invention provides a composition comprising a plurality of particles.

[0201] Advantageously, the composition is selected from the group consisting of pharmaceutical compositions, agricultural chemical compositions, edible compositions and cosmetic compositions.

[0202] In fact, the compositions of the present invention are suitable for use in the diet, nutrition, therapeutic or pharmaceutical purposes of mammals.

[0203] Depending on the desired route of administration, the compositions of the present invention can be used in a variety of formulations.

[0204] The compositions of the present invention may be in solid, liquid or semi-liquid form.

[0205] Preferably, the composition of the present invention is in liquid form.

[0206] More preferably, the liquid form of the composition of the invention is stable at 3 to 10°C, and more preferably, it is stable at 4°C.

[0207] Even more preferably, the composition of the invention in liquid form is an aqueous suspension.

[0208] In another advantageous and preferred embodiment, the composition of the invention is in solid form, preferably in powder form, and more preferably, the powder is stable in a temperature range of 4°C to 25°C.

[0209] Advantageously, the composition comprises 1% to 80% (w / w), 1% to 50% (w / w), or 1% to 25% (w / w) of the particles of the present invention.

[0210] Preferably, the powder contains 0.5 mg / g to 500 mg / g of the particles of the present invention.

[0211] In another embodiment, the powder has a content of the particles of the present invention from 0.01 mg to 200 mg / g.

[0212] In a particularly advantageous embodiment, the polydispersity index of the composition of the invention is in the range of about 0.05 to 0.7, preferably in the range of 0.2 to 0.6, and more preferably in the range of 0.2 to 0.3, as measured by dynamic light scattering (DLS).

[0213] Advantageously, the compositions described herein have a zeta potential in the range of about 0 mV to 100 mV as measured by dynamic light scattering (DLS).

[0214] Preferably, the composition of the present invention has a zeta potential of 15-80 mV, more preferably 15-40 mV, and even more preferably 15-30 mV.

[0215] In this embodiment, the particles have a positive surface charge and are coated with polysaccharides (e.g., chitosan), thus allowing for cell-specific interactions during delivery.

[0216] In another embodiment, the composition of the present invention has a zeta potential of -15 to -80 mV, more preferably -15 to -40 mV, and even more preferably -20 to -30 mV.

[0217] In this embodiment, the particles have a negative surface charge and are not coated with polysaccharides (e.g., chitosan), thus allowing for different interactions with cells during delivery.

[0218] It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually, or in any suitable sub-combination, or appropriately provided in any other described embodiments of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment would not function without these elements.

[0219] As described above and as claimed in the following claims, various embodiments and aspects of the invention are experimentally supported in the following examples.

[0220] Experimental Section

[0221] Referring now to the following embodiments, which, together with the above description, illustrate some implementations of the invention in a non-limiting manner.

[0222] The listed materials were used to implement the following embodiments: (Whey hydrolyzed protein) was supplied by Deimos Srl (Italy), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC), low molecular weight chitosan and acetic acid were purchased from Sigma-Aldrich (Saint Louis, MO, US), and green fluorescent protein (GFP) was produced and purified in-house.

[0223] To implement the following examples, the listed particle size and surface charge analysis methods were used: Z-mean (average diameter), ζ-potential (charge), and polydispersity index (PDI); Dynamic light scattering (DLS) principle was employed using a Malvern Zetasizer (Nano-ZS; Malvern Instruments, Worcestershire, UK) at 25°C. The samples were diluted 80-fold prior to analysis to avoid multiple scattering effects.

[0224] Example 1 - Production of particles of the present invention, the particles comprising a protein-based shell, various hydrophilic or amphiphilic biocompounds, and optionally further coating with chitosan as a polysaccharide.

[0225] 1.1 Producing the particles of the present invention, the particles comprising a shell of protein-based WPH and BSA (bovine serum albumin) as a hydrophilic biological compound.

[0226] BSA was selected as the model hydrophilic protein, and a fluorescently labeled version (FTIC-BSA) was constructed to track the protein's localization in plant cells.

[0227] like Figure 1 As shown, to produce particles with a protein shell (i.e., whey protein hydrolysate (WPH)) and a hydrophilic biocompound (i.e., BSA (BSA-Ps)), 630 mg of whey protein hydrolysate (WPH) and 270 mg of bovine serum albumin (BSA) labeled with FITC or Alexa Fluor were dissolved in 90 ml of deionized water under continuous stirring (for at least 1 hour). After complete dissolution, the protein solution was mixed with ethyl acetate at a ratio of 9:1 (protein solution / ethyl acetate) to form a two-phase solution. The solution was sonicated for 5 minutes at 10 W (Microson Ultrasonic Cell Disruptor XL) to produce a fine emulsion. To avoid overheating of the solution, the test tubes were placed on ice during sonication. At the end of the process, the ethyl acetate was removed using a nitrogen stream in the dark. The BSA-WPH Ps were lyophilized to obtain dried particles containing a shell of WPH protein and a BSA biohydrophilic compound, which is at least partially contained in the shell of the protein base.

[0228] The specific type of encapsulation and encapsulating agent depends in particular on the chemical-physical properties of the biological compound to be encapsulated, such as its affinity for polar, aqueous chemicals. Specifically, this type of encapsulation is generally known and preferred for lipophilic biological compounds.

[0229] Unlike cases where the active ingredient to be encapsulated is a lipophilic molecule, this case involves a hydrophilic molecule, namely BSA, which is contained within a shell composed of WPH, thus creating matrix-type particles in which the target molecule is distributed throughout the particle structure.

[0230] In fact, at least partially enclosing a biological compound, the shell is in the form of a matrix structure or a mold structure. Among other chemical interactions, the specific type of encapsulation depends on the chemical-physical properties of the active ingredient to be encapsulated, such as its hydrophobic / lipophilic and / or hydrophilic / lipophobic values. Specifically, this paper provides mold forms of such protein-based shells for novel encapsulation of hydrophilic or amphiphilic molecules.

[0231] Figure 2A A schematic diagram of a known core-shell particle structure in the prior art is shown, while Figure 2B A schematic diagram of the matrix-type or mold-type particle structure of the present invention is shown.

[0232] 1.2 Production of the particles of the present invention, the particles comprising a protein-based shell of WPH, a further coating of BSA (bovine serum albumin) as a hydrophilic biological compound and a polysaccharide (i.e., chitosan).

[0233] To impart a positive charge to the surface of BSA-WPH Ps, 10 ml of the pre-formed BSA-WPH Ps solution was mixed with 2.8 ml of a 1% chitosan solution. The 1% chitosan solution was prepared using low-molecular-weight chitosan characterized by low viscosity. The chitosan was dissolved in warm deionized water containing 1% acetic acid and stirred for several hours until completely dissolved, thus obtaining chitosan-coated BSA-WPH particles (chitosan-BSA-WPH Ps or BSA-WPH Ps). + ).

[0234] Therefore, two BSA-WPH Ps with different surface charges were produced to test whether this would affect the uptake of Ps by plant cells.

[0235] BSA-WPH Ps without chitosan coating - In this case, the main population of particles has a diameter of 32.6 ± 13 nm and a negative surface charge of -18 ± 3.6 mV. A layer of chitosan (BSA-WPH Ps) was added. + After that, the average size of NPs increased slightly to 36.8±11.5 nm, and the surface positive charge was 47.8±7.3.

[0236] Then, the negatively charged and positively charged BSA-WPH Ps were freeze-dried, and the crystallization characteristics of the resulting powder can be observed in… Figure 3A In this context, it represents negatively charged particles, namely BSA-WPH Ps. - , Figure 3B Represents positively charged particles, namely BSA-WPH Ps + The bright yellow color in the image is due to the presence of the FITC mark on BSA. This example confirms that the particles are formed from BSA.

[0237] Example 2 - Using the positively charged BSA-WPH Ps of the present invention + With negatively charged BSA-WPH Ps - Cellular testing for particle delivery

[0238] BSA-WPH Ps in lyophilized powder form obtained according to Example 1 - and BSA-WPH Ps + Particles were used to determine the delivery of BSA-FITC to suspensions of tobacco protoplasts and intact tobacco cells. Measurements of BSA-FITC at 25°C were performed using a Malvern Zetasizer (Nano-ZS; Malvern Instruments, Worcestershire, UK) based on the principle of dynamic light scattering (DLS). - With a size of 32.6nm, BSA-WPH Ps + The size is 37nm.

[0239] Figure 4A A comparison is shown that, when using positively charged nanoparticles BSA-WPH Ps + After treatment, protoplasts could not be obtained, so a further coating with chitosan was used, while BSA-WPH Ps was applied. - When processing cells, protoplasts may be obtained.

[0240] Figure 4B The results show that the negatively charged particles BSA-WPH Ps - (Therefore, it was not coated with a further chitosan coating) and is the only particle internalized by the tobacco protoplast, thus the tobacco protoplast is a plant cell with a cell membrane but no cell wall. Due to the lack of a cell wall and an exposed plasma membrane, this type of cell has increased permeability, similar to animal cells in this sense, and even allows negatively charged particles to enter.

[0241] Figure 5A These are optical microscope images showing the use of positively charged BSA-WPH Ps in tobacco cells with cell membranes and cell walls. + Delivery of BSA-FITC resulted in the observed fluorescence located extracellularly, as shown in the image, thus confirming that BSA-WPH Ps... + It was not internalized, but Figure 5B These are optical microscope images showing the use of negatively charged BSA-WPH Ps. - BSA-FITC was delivered, and it was observed that BSA was successfully internalized within tobacco plant cells.

[0242] Therefore, it can be concluded that in the case of intact tobacco plant cells with cell membranes and cell walls, positively charged particles (and thus with an additional chitosan coating on the shell) (BSA-WPH Ps) + Not only can it not pass through the cell membrane, but it also cannot pass through the cell wall. The negatively charged nanoparticles BSA-WPH Ps... - It can internalize biological hydrophilic compounds, especially BSA.

[0243] Example 3 - Delivery of the particles of the present invention in plant cells with intact plant cell walls, the particles comprising a protein-based shell of WPH and BSA (bovine serum albumin) as a hydrophilic biological compound.

[0244] The plant species tobacco was used to detect the delivery of encapsulated biological compounds (i.e., BSA).

[0245] Tobacco cells, such as those obtained from seedlings, can be propagated in suspension cultures where they retain their intact cell walls. The particles (BSA-WPH Ps) obtained in Example 1 will be used in this invention. - Encapsulated and unencapsulated BSA were added to such a cell suspension. The BSA was then conjugated with a fluorescent dye for observation using a fluorescence microscope (as described in Example 1). Figure 6 The top two horizontal lines provide information using BSA-WPH Ps - Representative images of the two experiments (biological replicas) conducted. The bottom row shows an unencapsulated BSA control from one of these experiments.

[0246] These experiments confirmed the presence of the particles of the present invention, namely BSA-WPH Ps. - These particles can enter plant cells and penetrate the cell wall, thus leading to the internalization of biohydrophilic compounds, particularly BSA, within these cells. Furthermore, in this embodiment, the inventors noted that 5-50 nm particles and negative charges were successfully and optimally internalized within plant cells.

[0247] Example 4 - Size distribution and polydispersity (PDI) analysis of the particles of the present invention, the particles comprising a protein-based shell made of different proteins and GFP (green fluorescent protein) as a hydrophilic biological compound.

[0248] like Figure 7A , 7BAs shown in Figures 7C, these figures report the z-mean, number (i.e., the true average diameter of the particles to be considered in this type of study), and PDI index of different particles produced according to Example 1, which have a shell containing GFP (with or without nuclear localization signal [NLS]) as a hydrophilic biological compound and various proteins such as WPH (OPTIPEP), broad bean protein isolate, and soybean protein isolate.

[0249] The analysis was performed on a 4 ml volume of the reaction solution of the particles of the present invention, and the mixture was subjected to sonication for different durations, specifically 1.5 min, 3 min, and 5 min.

[0250] from Figure 7B As can be seen, the average diameter of the particles of the present invention is always kept below the desired value of 60 nm, thus making them small enough to easily enter plant cells.

[0251] from Figure 7C As can be seen, the PDI index of the particles of the present invention is always maintained in the range of 0.2 to 0.6, preferably in the range of 0.2 to 0.3. This is the range required to ensure the stability of the particles in solution to avoid aggregation. Therefore, when delivered in plant cells, the delivery analysis is reproducible.

[0252] Example 5 - Delivery analysis of the particles of the present invention, the particles comprising a shell of WPH protein base and GFP (green fluorescent protein) as a hydrophilic biological compound.

[0253] To examine protein delivery via the particles of the present invention, whey protein hydrolysate was used as the protein constituting the shell, and the particles of the present invention, GFP-WPH Ps, were produced according to Example 1. The obtained GFP-WPH Ps - The particles have a particle size of approximately 60 nm, therefore they are GFP-WPH NPs. - Nanoparticles. Selected suspended tobacco cells were treated with the following formulations;

[0254] - Untreated cells (background fluorescence state, Figure 8A );

[0255] - Empty nanoparticles (NPs) with WPH in the shell but no GFP;

[0256] - Unencapsulated GFP NPs;

[0257] - The GFP-WPH NPs encapsulated in this invention; and

[0258] - The present invention comprises encapsulated GFP-WPH NPs with nuclear localization.

[0259] For each sample, the fluorescence of plant cells was examined by optical fluorescence microscopy at 24 and 48 hours, and the results are summarized in Table 1:

[0260] Table 1

[0261]

[0262]

[0263] The evidence summarized in Table 1 is further illustrated in Figures 8, 9, 10, and 11.

[0264] Specifically, Figure 8A The background showing the fluorescence signal from untreated cells is shown; no fluorescence was observed. The fact that no fluorescence was observed is an important data point because sometimes, cells can still produce autofluorescence when under harsh conditions (e.g., low nutrient content in the suspension medium) and therefore cannot serve as a control in experiments. In light of this, this experiment can be used as a blank. Figure 8B The results of treating a tobacco cell suspension with empty nanoparticles (NPs) are shown, in which no fluorescence was observed.

[0265] Figure 9 The results show the effects of treating a tobacco cell suspension with unencapsulated GFP (naked GFP). No fluorescence was observed, confirming that this molecule (i.e., unencapsulated GFP) cannot enter the cell but needs to be delivered intracellularly via cargo; otherwise, it will not enter the plant cell and thus cannot be internalized.

[0266] Figure 10 Displayed GFP-WPH NPs encapsulated - Results of the treatment of tobacco cell suspension. The latter was successfully absorbed by the cells, which confirms that the nanoparticles of the present invention can penetrate the cell membrane and cell wall of plant cells when encapsulated by the method of the present invention.

[0267] Figure 11 This invention demonstrates GFP-WPH NPs encapsulated with nuclear localization. - The results of treating the tobacco cell suspension showed that the latter was successfully absorbed by the cells, demonstrating that when encapsulated using the method of the present invention, the particles of the present invention (containing a protein-containing shell and a biocompound) can penetrate the cell membrane, cell wall, and even reach the cell nucleus.

[0268] Example 6 - Delivery analysis of particles of the present invention, the particles comprising a shell of broad bean isolate and soybean protein protein and GFP (green fluorescent protein) as a hydrophilic biological compound.

[0269] To test the protein delivery of the nano-encapsulated particles of the present invention, GFP was encapsulated with broad bean protein isolate and soybean protein isolate, selected as the shells for the protein base, and NPs encapsulated with broad bean-GFP and soybean-GFP were produced according to Example 1.

[0270] The obtained broad bean-GFP Ps - With a particle size of 67±26 nm, they are therefore broad bean-GFP NPs nanoparticles. The obtained soybean-GFP Ps - With a particle size of 67±26 nm, it is therefore a soybean-GFP NP nanoparticle.

[0271] Tobacco cells were selected from cell suspensions and solid callus tissues and encapsulated broad bean-GFP NPs. - Nanoparticle treatment. The results are presented in... Figure 12 The figure shows that the nanoparticles were successfully absorbed by the cells and that they exhibited fluorescence within the cells, thus confirming the possibility of using the same encapsulation method of the present invention with a different protein-based shell.

[0272] Figure 13A and 13B Soybean-GFP NPs in cell suspension and callus tissue are shown separately. - This demonstrates the internalization of GFP with a shell containing different protein bases.

[0273] Example 7 - Delivery analysis of the particles of the present invention, wherein the particles comprise a shell of cellulase + WPH protein base and GFP (green fluorescent protein) as a hydrophilic biological compound.

[0274] To test the delivery of GFP protein and enzyme through the particles of the present invention, 0.5 mg / ml of cellulase was used together with WPH as the protein in the shell, and the particles of the present invention, GFP-cellulase Ps, were produced according to Example 1.

[0275] The obtained cellulase-GFP Ps have a particle size of approximately 60-65 nm, and are therefore cellulase-GFP NPs. - Nanoparticles.

[0276] Tobacco solid callus was selected and treated in suspension with empty nanoparticles and encapsulated cellulase-GFP WPHNPs. Results showed... Figure 15A and Figure 15BIn the study, it was shown that nanoparticles encapsulated with 0.5 mg / ml cellulase and GFP as a hydrophilic biological compound on the shell were successfully absorbed by cells and exhibited fluorescence within the cells, while empty nanoparticles did not exhibit any fluorescence. This demonstrates the possibility of using the same encapsulation method as in this invention to encapsulate enzymes with a shell having a protein as the protein base.

[0277] Example 8 - Delivery analysis of particles of the present invention, the particles comprising a protein-based shell of WPH and DNA as a hydrophilic biological compound.

[0278] To test and demonstrate DNA delivery via the particles of the present invention, whey protein hydrolysate was used as the protein in the shell, and the particles of the present invention, DNA-WPH-Ps, were produced according to Example 1. - .

[0279] The obtained DNA-WPH Ps have a particle size of 55±37, and are therefore DNA-WPH NPs nanoparticles.

[0280] The delivered DNA consists of a plasmid containing the GFP gene (which has a nuclear localization signal), located below a strong promoter in plant cells that can be used to trigger expression. Tobacco cells in suspension were selected and treated with the following formulation:

[0281] - Unencapsulated pRAP (Figure 14A)

[0282] -The encapsulated pRAP NPs (DNA-WPH Ps) of this invention - (Figure 14B).

[0283] For each sample, cell fluorescence was examined by optical fluorescence microscopy at 24 and 48 hours. Figure 14A shows no signal when the cell suspension was treated with unencapsulated DNA, while Figure 14B shows pRAP-NPs (DNA-WPH Ps). - The encapsulated DNA expressing GFP was successfully absorbed by the cell and exhibited fluorescence within the cell, thus demonstrating the possibility of using the same encapsulation method as in this invention with different biohydrophilic compounds. The arrows indicate nuclear localization of intracellular fluorescence, a result of DNA expression.

[0284] Example 9 - Evaluation of the retention of cellulase activity after encapsulating the present invention particles containing a protein base of WPH and cellulase as a hydrophilic biological compound.

[0285] To test and demonstrate the retention of enzyme activity after the encapsulation process of the particles of the present invention, whey protein hydrolysate was used as the protein in the shell, and the particles of the present invention were produced according to Example 1, containing cellulase-WPH-Ps, as follows. Figure 16 As shown in the diagram.

[0286] The obtained cellulase-WPH Ps - With a particle size of 48 to 60 nm and a standard deviation of 6 nm, it is therefore a cellulase-WPH NPs. - Nanoparticles.

[0287] The following samples were prepared and subjected to a phenol-sulfuric acid method test directly on starch to examine the activity of cellulase before and after encapsulation in the granules of the present invention:

[0288] - Cellulase alone;

[0289] - Cellulase dissolved in whey protein;

[0290] - NPs of whey protein encapsulating cellulase, namely the cellulase-WPH Ps of this invention. - ( Figure 17 NPs in the middle); and

[0291] - The enzyme activity is preserved after encapsulation, while the NPs are broken (broken NPs).

[0292] from Figure 17 As can be seen, by comparing the sample called "enzyme" and the sample called "enzyme dissolved in whey protein", the activity of cellulase remained unchanged after the dissolution process, thus proving that cellulase did not lose any activity after being dissolved in WPH protein alone.

[0293] also, Figure 17 The results show that, by comparing a sample called “enzyme” with a sample called “NPs” (the cellulases of the present invention – WPHPs –), the activity of the cellulase is only slightly reduced, thus confirming that the encapsulation method of the present invention can produce nanoparticles that retain enzyme activity even after encapsulation.

[0294] also, Figure 17 The whey protein NPs encapsulating cellulase (i.e., the particulate cellulase-WPHPs of the present invention) are shown. - ( Figure 17 "NPs" in the text) and broken nanoparticles ( Figure 17 The “broken NPs” in the text, where the enzyme is thought to be released in the supernatant and break down starch molecules, both show that they retain the same activity.

[0295] This evidence demonstrates that the biological compound (i.e., cellulase) is exposed on the surface rather than physically enclosed in the core of the particle, thus proving that the biological compound is at least partially contained in the protein-shell, and therefore the matrix structure of the particles of the present invention differs from that of core-shell particles.

[0296] Example 10 - Evaluation of the retention of amylase activity after encapsulating the particles of the present invention containing a protein base of WPH and an amylase as a hydrophilic biological compound.

[0297] To test and demonstrate the retention of enzyme activity after the encapsulation process of the particles of the present invention, whey protein hydrolysate was used as the protein in the shell, and the granular amylase-WPH-Ps of the present invention was produced according to Example 1. - And in Figure 16 The scheme is illustrated schematically.

[0298] The obtained amylase-WPH Ps have a particle size of 40 to 56 nm, therefore they are amylase-WPH Ps. - Nanoparticles.

[0299] The following samples were prepared and subjected to a phenol-sulfuric acid method test directly on starch to examine the activity of amylase before and after encapsulation in the granules of the present invention:

[0300] -Amylase alone;

[0301] -Amylase dissolved in whey protein;

[0302] - NPs of whey protein encapsulating amylase, namely the amylase-WPH Ps of this invention. - ( Figure 18 (NPs in the text);

[0303] - Broken NPs.

[0304] from Figure 18 As can be seen, by comparing the sample labeled "enzyme" and the sample labeled "enzyme dissolved in whey protein," the activity of amylase decreased significantly after the dissolution process, thus indicating that the activity of amylase was not preserved when dissolved in WPH protein. In fact, the enzyme appears to interact with the protein itself (i.e., WPH), as evidenced by the decreased activity in the sample containing both enzyme and protein in solution.

[0305] also, Figure 18 The results showed that by comparing samples referred to as "enzymes" and those referred to as "NPs" (the amylase-WPH Ps of this invention), - In the sample, the amylase activity was reduced but still present because the enzyme site was protected, thus confirming that the encapsulation method of the present invention successfully preserved enzyme activity after encapsulating the particles of the present invention, the particles comprising a shell containing them and proteins (i.e., WPH).

[0306] The NPs of whey protein encapsulating amylase, namely the particulate amylase-WPH Ps of the present invention. Figure 18The “NPs” in the text show lower activity relative to broken NPs, where the enzyme is thought to be released in the supernatant and break down starch molecules.

[0307] This evidence demonstrates that the biological compound, namely amylase, is partially exposed on the surface of the particles of the present invention (partially contained in the protein shell) and partially inside the shell.

[0308] Example 11: Delivery analysis of the particles of the present invention, the particles comprising a shell of WPH protein base and GFP (green fluorescent protein) as a hydrophilic biological compound.

[0309] To test protein delivery in mammalian cells via the particles of the present invention, whey protein hydrolysate was used as the protein in the shell, and the particles of the present invention, GFP-WPH Ps, were produced according to Example 1. - The obtained GFP-WPHPs - The particles have a size of approximately 60-70 nm, therefore they are GFP-WPH NPs. - Nanoparticles. Using GFP-WPH NPs - J774A.1 (mouse macrophage) cells were treated with suspension and observed under a fluorescence optical microscope after 30 minutes.

[0310] Specifically, Figure 19 The results show the results using an untreated cell suspension as a fluorescence baseline reference. Figure 20 The results of treating cell suspensions with GFP-containing nanoparticles (NPs) are shown. From Figure 20 It can be observed that GFP-WPH NPs - The nanoparticles were successfully absorbed by cells, which confirms that they can be absorbed by mammalian cells when encapsulated using the method of the present invention.

[0311] Although the invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many substitutions, modifications, and variations will be readily apparent. Therefore, it is intended to include all such substitutions, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0312] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually incorporated herein by reference. Furthermore, any reference or designation of any reference in this application should not be construed as an admission that such reference is prior art to the invention. The section headings used should not be construed as necessary limitations.

Claims

1. A method for encapsulating hydrophilic or amphiphilic biological compounds, comprising the following steps: a. A two-phase solution is obtained by dissolving hydrophilic or amphiphilic biological compounds and proteins in water to form a solution, and then mixing the protein solution with the solvent to form a two-phase solution. b. Emulsify the two-phase solution to obtain an emulsion; and c. Evaporate the solvent from the emulsion. This yields particles comprising: (i) a protein-based shell and (ii) a hydrophilic or amphiphilic biological compound, the particles carrying a negative charge. The hydrophilic or amphiphilic biocompounds are not only distributed at the center of the particle, but are also at least partially contained within the shell of the protein base. The solvent in step a has a dielectric constant of 1.5-15 at 20-25°C, and the solvent is selected from the group consisting of ethyl acetate, n-hexane, and cyclohexane. In step a, the hydrophilic or amphiphilic biological compound is selected from the group consisting of BSA, GFP, DNA, antisense RNA, messenger RNA, CRISPR enzyme, CRISPR-guided RNA complex, cellulase, and amylase. The protein in step a is selected from whey protein, soy protein, pea protein, broad bean protein, potato protein, or any combination thereof.

2. The method according to claim 1, wherein in step a, the solvent has a dielectric constant of 1.8 to 6.02 at 20-25°C.

3. The method according to claim 1, wherein in step a, the weight ratio of protein to solvent in the solvent is from 10:0.5 to 8:1.

5.

4. The method according to claim 3, wherein the weight ratio of protein to solvent in the solvent is 9:

1.

5. The method according to claim 1, wherein the protein in step a is in the form of a protein hydrolysate.

6. The method of claim 1, wherein the emulsification in step b is performed by sonicating the solution for 5 minutes.

7. The method according to claim 1, further comprising step d: drying the particles evaporated in step c.

8. The method according to claim 7, wherein the drying of the particles in step d is selected from the group consisting of spray drying, granulation, agglomeration, freeze drying, or any combination thereof.

9. The method according to claim 8, wherein the drying in step d is performed by freeze drying.

10. The method of claim 1, wherein, measured by dynamic light scattering, the particles evaporated after step c have an average diameter of 1-60 nm.

11. The method of claim 10, wherein the particles evaporated after step c have an average diameter of 5-60 nm.

12. The method of claim 10, wherein the particles evaporated after step c have an average diameter of 5-50 nm.

13. The method of claim 1, wherein, measured by dynamic light scattering, the particles evaporated after step c have an average diameter of 70-700 nm.

14. The method of claim 13, wherein the particles evaporated after step c have an average diameter of 100-500 nm.

15. The method of claim 13, wherein the particles evaporated after step c have an average diameter of 100-300 nm.

16. The method of claim 13, wherein the particles evaporated after step c have an average diameter of 100-200 nm.

17. Particles obtainable by the method of claim 1, comprising: (i) a protein-based shell, wherein the protein-based shell comprises a protein selected from whey protein, soy protein, pea protein, fava bean protein, and potato protein, or any combination thereof; and (ii) A hydrophilic or amphiphilic biological compound selected from the group consisting of BSA, GFP, DNA, antisense RNA, messenger RNA, CRISPR enzyme, CRISPR-guided RNA complex, cellulase and amylase. The hydrophilic or amphiphilic biocompounds are not only distributed at the center of the particle, but are also at least partially contained within the shell of the protein base. The particles carry a negative charge.

18. The particles of claim 17, measured by dynamic light scattering, have a diameter in the range of 1 to 60 nm.

19. The particles according to claim 18, wherein the diameter is in the range of 5 nm to 60 nm.

20. The particles according to claim 18, wherein the diameter is in the range of 5 nm to 50 nm.

21. The particles according to claim 17, measured by dynamic light scattering, have a diameter in the range of 70-700 nm.

22. The particles according to claim 21, wherein the diameter is in the range of 100-500 nm.

23. The particles according to claim 21, wherein the diameter is in the range of 100-300 nm.

24. The particles according to claim 21, wherein the diameter is in the range of 100-200 nm.

25. The particle of claim 17, wherein the shell of the protein base comprises a protein in the form of a protein hydrolysate.

26. The particles according to claim 17 or 25, wherein the shell of the protein base comprises a protein selected from whey protein hydrolysate and fava bean protein isolate.

27. The particles of claim 26, wherein the shell of the protein base comprises hydrolyzed whey protein.

28. Use of the particles according to claim 17 for delivering hydrophilic or amphiphilic biological compounds in plant cells.

29. The non-therapeutic cosmetic use of the particles according to claim 17, for delivering at least one hydrophilic or amphiphilic biological compound within animal cells.

30. The non-therapeutic cosmetic use of the particles according to claim 29, wherein the animal cells are mammalian cells.

31. A composition comprising a plurality of particles according to claim 17.

32. The composition according to claim 31, measured by dynamic light scattering, has a zeta potential of 15-80 mV.

33. The composition according to claim 32, having a zeta potential of 15-40 mV.

34. The composition according to claim 32, having a zeta potential of 20-30 mV.

35. The composition according to claim 31, measured by dynamic light scattering, has a zeta potential of -15 to -80 mV.

36. The composition according to claim 35, having a zeta potential of -15 to -40 mV.

37. The composition according to claim 35, having a zeta potential of -20 to -30 mV.

38. The composition according to claim 31, measured by dynamic light scattering, has a polydispersity index of 0.05 to 0.

7.

39. The composition according to claim 38, having a polydispersity index of 0.2 to 0.

6.

40. The composition according to claim 38, having a polydispersity index of 0.2 to 0.3.

Images