RU2812553C2 - Compositions of extracellular vesicles (ev) of plant origin and their use - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to the use of a population of extracellular vesicles (EV) of plant origin in the therapeutic treatment of impaired angiogenesis. Use of a plant-derived population of extracellular vesicles (EVs) derived from a plant selected from the group consisting of orange, lemon, grapes, Anastatica hierochuntica (AH) and Selaginella lepidophylla (SL), wherein the extracellular vesicles (EVs) in the said population are confined to a lipid bilayer membrane and have a diameter ranging from 25 to 350 nm, and a protein content ranging from 1 to 55 ng/10⁹ EV, in the therapeutic treatment of impaired angiogenesis.

EFFECT: above-described plant-derived vesicles used are effective for the therapeutic treatment of impaired angiogenesis.

8 cl, 19 dwg, 1 tbl, 11 ex

Description

Field of technology

The present invention relates to compositions of extracellular vesicles (EVs) of plant origin and their therapeutic applications.

State of the art

Extracellular vesicles (EVs) are a heterogeneous population of particles released by virtually all living cells. They are obtained from almost all types of mammalian cells, as well as from lower eukaryotes, prokaryotes and plants, and purified from body fluids. These include mainly microvesicles released through plasma membrane budding and exosomes derived from the endosomal compartment. Extracellular vesicles are called “particles”, “microparticles”, “nanovesicles”, “microvesicles” and “exosomes” [Yáñez-Mó M. et al., Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles, 2015, May 14; 4: 27066 doi: 10.3402/jev.v4.27066; Lötvall J. et al., Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J. Extracell. Vesicles, 201, Dec. 22; 3: 26913. doi: 10.3402/jev.v3.26913; Harrison P. et al., Extracellular Vesicles in Health and Disease. CRC Press, pages 1-5, 2014].

EVs contain a complex and diverse cargo of cytoplasmic proteins, surface receptors, some lipid-interacting proteins, DNA and RNA molecules. By carrying such cargo, EVs play a key role as mediators of extracellular communication.

EVs derived from edible plants, in their native form, not loaded with exogenous molecules, will be referred to herein as “native EVs”.

Native EVs are known to be effective for the treatment of leukemia [WO2016166716A1] and colitis [Ju S. et al., Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol. Ther., 2013, July; 21(7): 1345-57. doi: 10.1038/mt.2013.64.] by oral administration.

Native nanovesicles derived from grapes, grapefruit, ginger and carrots demonstrate anti-inflammatory effects in chronic bowel inflammation [Zhang M. et al., Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis- associated cancer. Biomaterials. 2016, Sept.;101: 321-40. doi:10.1016/j.biomaterials.2016.06.018; Ju S. et al., Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol. Ther., 2013, July; 21(7): 1345-57. doi:10.1038/mt.2013.64].

WO2017/052267 discloses the use of topically administered native EVs derived from edible plants to promote skin improvement in terms of wrinkle formation, hydration, brightening, epithelial cell proliferation and collagen deposition.

To the best of the inventors' knowledge, the prior art does not disclose the effects of plant-derived EVs on angiogenesis and bacterial viability when applied topically to wounds and skin lesions characterized by ischemia and impaired angiogenesis or increased susceptibility to bacterial infection.

Since EVs inherently protect and transport their cargo to target cells, they represent a useful alternative to synthetic and exogenous particles such as liposomes, cationic nanoparticles, EV-mimetic nanovesicles, and polypeptide-based vesicles for transporting therapeutic agents. EVs can exploit their natural mechanism of action and overcome several limitations of assembled particles, including immunogenicity, toxicity, introduction of exogenous particles, limited cellular uptake, and chemical particle assembly.

In recent years, many methods have been explored to transfer various molecules (RNA, DNA, drugs) into EVs. EV-associated nucleic acids are protected from degrading enzymes present in the microenvironment and can be delivered to target cells. Methods aimed at introducing molecules into EVs include electroporation, sonication, transfection, incubation, cell extrusion, saponin-mediated permeabilization, and freeze-thawing.

WO2017/004526A1 discloses the use of microvesicles derived from grapes and grapefruit as carriers for miR18a and miR17 used as anticancer drugs or for isotopic tracers used for diagnostics.

In order to overcome the limitations and disadvantages of the prior art, the present invention provides a composition comprising a population of plant-derived extracellular vesicles (EVs), as well as a method of loading one or more biologically active molecules into the population of plant-derived extracellular vesicles (EVs) as defined in the accompanying appendix. the claims in independent clauses. Dependent claims identify other advantageous features of the claimed composition and method. The subject matter of the appended claims forms an integral part of the present description.

Detailed Description of the Invention

The present invention relates to a composition comprising a population of plant-derived extracellular vesicles (EVs), wherein the plant-derived extracellular vesicles (EVs) in said population are enclosed in or delimited by a lipid bilayer membrane and are characterized in that they have a diameter of from 10 to 500 nm, protein content in the range from 1 to 55 ng/10 ⁹ EV, RNA content in the range from 10 to 60 ng/10 ¹⁰ EV, and which are further characterized in that they show proangiogenic and antibacterial activity, for use in the therapeutic treatment of a disease or a condition selected from the group including ulcers, dermatitis, corneal lesions, eye diseases, mucosal lesions and infectious lesions.

As used herein, the term “plant-derived extracellular vesicles” or “plant-derived EVs” refers to nanoparticles derived from and derived from plant cells that are confined or embedded in a phospholipid bilayer, and that transport lipids, proteins, nucleic acids and other molecules, originating from cells. Typically, extracellular vesicles have a diameter in the range of 10-1000 nm.

The present invention uses a selected population of plant-derived extracellular vesicles (EVs) that have a diameter from 10 to 500 nm, preferably in the range from 20 to 400 nm, even more preferably in the range from 25 to 350 nm. The plant-derived extracellular vesicles used in the present invention can be native EVs or engineered EVs, as shown in the following examples.

The expressions “protein content” and “RNA content” cover content both within and within the EV membranes used in the present invention.

Examples of EV lipids used in the present invention include, but are not limited to, 24-propylidenecholesterol, beta-sitosterol, campesterol, dipalmitin, eicosanol and/or glycidyl alcohol stearate.

In yet another preferred embodiment of the invention, the EV population is obtained from a plant selected from the group consisting of: the Rutaceae family, such as the genus Citrus; the Rosaceae family, such as Malus pumila; the Vitaceae family such as Vitis vinifera; the Brassicaceae family such as Anastatica hierochuntica; the Selaginellaceae family such as Selaginella lepidophylla; the Asteraceae family such as Calendula officinalis; the Oleaceae family such as Olea europaea; the Xanthorrhoeaceae family such as Aloe vera; the Nelumbonaceae family such as Nelumbo; the Araliaceae family such as subgenus Panax; the Lamiaceae family such as Lavandula; the Hypericaceae family such as Hypericum perforatum; the Pedaliaceae family such as Harpagophytum procumbens; the Ginkgoaceae family such as Ginkgo biloba; the Piperaceae family such as Piper kadsura or Piper futokadsura; family Rubiaceae, such as Hedyotis diffusa. Non-limiting examples of plants of the Citrus genus include lemon, orange, tangerine, clementine, bergamot, pompia.

The scope of the invention includes both compositions containing EVs originating from a single plant species and compositions containing EVs originating from multiple plant species.

The EVs used in the present invention are characterized in that they show proangiogenic and antibacterial activity.

As used herein, the expression “proangiogenic effect” is intended to mean stimulating endothelial cell proliferation or vascular formation by endothelial cells and increasing the release of proangiogenic mediators in vitro or in vivo. Angiogenesis is a fundamental biological process and its deterioration has been implicated in the pathogenesis of a number of diseases, including ischemic ulcers such as pressure ulcers, arterial ulcers, venous ulcers, diabetic ulcers, exudative ulcers, dysmetabolic ulcers, traumatic ulcers, burns, fistulas, psoriasis, keratosis, keratitis , fissures, mucosal injuries (such as traumatic injuries due to prosthesis and the like, diabetic, oral mucosal injuries, decubital, genital mucosal injuries), corneal injuries/eye diseases (including ulcers, traumatic injuries, degenerative injuries, abrasions, chemical injuries, contact lens problems, UV damage, keratitis), dry eye disease, conjunctivitis, dermatitis (including acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, dermatitis herpetiformis), androgenetic alopecia, scabies , cellular damage caused by proapoptotic drugs aimed at treating precancerous lesions (eg, actinic keratoses). Accordingly, the EVs used in the present invention are particularly useful in the treatment of such diseases. Native extracellular vesicles of plant origin with pro-angiogenic action used in the present invention preferably come from Citrus plants: lemon, orange, tangerine, clementine, bergamot, pompia; from the family Rutaceae, such as Citrus; from the Rosaceae family such as Malus pumila; from the family Vitaceae, such as Vitis vinifera; from the family Brassicaceae, such as Anastatica hierochuntica; from Selaginella lepidophylla; from the family Asteraceae, such as Calendula officinalis; from the family Oleaceae, such as Olea europaea; from the family Xanthorrhoeaceae, such as Aloe vera, from the family Nelumbonaceae, such as Nelumbo; from the family Araliaceae, such as subgenus Panax; from the family Lamiaceae, such as Lavandula; from the family Hypericaceae, such as Hypericum perforatum; from the family Pedaliaceae, such as Harpagophytum procumbens; from the family Ginkgoaceae, such as Ginkgo biloba; the Piperaceae family such as Piper kadsura or Piper futokadsura; family Rubiaceae, such as Hedyotis diffusa.

As used herein, the expression "antimicrobial action" is intended to indicate any action capable of killing microbes, microbicidal or capable of inhibiting the growth of microbes, biostatic. Bacterial infections are common and cause diseases and complications of wounds, including lesions of the mucous membrane (such as traumatic injuries due to a prosthesis, etc., diabetic, injuries to the oral cavity, decubital, damage to the genital mucosa), infectious injuries ( such as viral infections, herpes infections, bacterial infections), ulcers (including diabetic, arterial, venous, dysmetabolic, exudative, ischemic ulcers, pressure ulcers), burns, fistulas, corneal injuries/eye diseases (including ulcers, traumatic injuries, degenerative injuries, abrasions, chemical injuries, contact lens problems, UV damage, keratitis), dry eye disease, conjunctivitis, dermatitis (including acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, dermatitis herpetiformis), traumatic ulcers, cellular damage caused by proapoptotic drugs aimed at treating precancerous lesions (eg, actinic keratoses). Accordingly, the EVs used in the present invention are particularly useful in the treatment of such diseases.

The plant-derived extracellular vesicles with antimicrobial activity used in the present invention preferably come from citrus plants: lemon, orange, tangerine, clementine, bergamot, pompia; from the family Rutaceae, such as Citrus; from the Rosaceae family such as Malus pumila; from the family Vitaceae, such as Vitis vinifera; from the family Brassicaceae, such as Anastatica hierochuntica; from Selaginella lepidophylla; from the family Asteraceae, such as Calendula officinalis; from the family Oleaceae, such as Olea europaea; from the family Xanthorrhoeaceae, such as Aloe vera, from the family Nelumbonaceae, such as Nelumbo; from the family Araliaceae, such as subgenus Panax; from the family Lamiaceae, such as Lavandula; from the family Hypericaceae, such as Hypericum perforatum; from the family Pedaliaceae, such as Harpagophytum procumbens; from the family Ginkgoaceae, such as Ginkgo biloba; the Piperaceae family such as Piper kadsura or Piper futokadsura; family Rubiaceae, such as Hedyotis diffusa.

As stated above, the scope of the invention also includes a method of loading one or more negatively charged bioactive molecules into a population of plant-derived extracellular vesicles (EVs) defined above. The resulting EVs loaded with one or more negatively charged bioactive molecules will be referred to hereinafter as “loaded EVs”.

The method of the invention is based on the formation of a bridge using a polycationic substance between negatively charged EVs and negatively charged biologically active molecules. The expression "negatively charged biologically active molecules" includes, but is not limited to, drugs, nucleic acid molecules and fat-soluble molecules such as fat-soluble vitamins. Nucleic acid molecules include, but are not limited to, DNA and RNA molecules, including microRNA, mRNA, tRNA, rRNA, miRNA, regulatory RNA, non-coding and coding RNA, DNA fragments, DNA plasmids). Loaded EVs obtained by the method according to the invention are able to protect their cargo - biologically active molecules from degradation and transfer them to target cells. Biologically active cargo molecules preferably have therapeutic potential.

The method of the invention involves contacting a population of plant-derived extracellular vesicles (EVs) defined above with a polycationic substance and a negatively charged biologically active molecule and incubating them together. After co-incubation, EVs are purified of polycationic material and remaining negatively charged active molecules.

In a first embodiment, the EV is first contacted and co-incubated with a polycationic substance to allow the polycationic substance to bind to the surface of the EV, and then the mixture of EV and polycationic substance is contacted and co-incubated with a negatively charged active molecule.

In a second embodiment, the polycationic substance and the negatively charged active molecules are mixed and then added to the EV.

According to a preferred embodiment, the polycationic substance is selected from the group consisting of protamine, polylysine, cationic dextrans, salts thereof and combinations thereof. A preferred protamine salt is protamine hydrochloride.

As stated above, after loading the EV is cleaned. Suitable purification methods include, but are not limited to, gradient ultracentrifugation, ultrafiltration, diafiltration, tangential flow filtration, sedimentation-based methods, chromatography-based methods, concentration, immunoaffinity capture-based methods, and microfluidic-based isolation methods.

As will be explained in the following experimental section, we loaded EVs with synthetic miRNA molecules and then confirmed by RT-PCR analysis that the miRNA molecules were introduced into EVs. Using quantitative RT-PCR and confocal microscopy, the authors also confirmed that miRNA-loaded EVs are able to efficiently transfer their cargo to target cells. The use of mammalian microRNA, which is absent in plants, allows for effective load assessment. In addition, miRNAs transferred into target cells have been shown to be biologically active and influence the expression of the target miRNA in cells.

As stated above, the loaded EVs produced by the method of the present invention can be used to transport several negatively charged bioactive molecules through the EVs. For example, microRNAs are involved in various important key pathways in both physiological and pathological processes. Several miRNAs, for example, have been described in the literature as being involved in cancer angiogenesis and regenerative processes. As a demonstration of the effectiveness of the method, EVs are effectively loaded with pro-regenerative miRNAs, such as miR-21 and miR-126, and antiogenic and antitumor miRNAs or miRNA inhibitors. Loaded EVs show and enhance efficiency over native EVs.

Accordingly, the method of the present invention can be used to produce loaded EVs with enhanced therapeutic effects, including pro-angiogenic and antibacterial properties, or to add new therapeutic activity to native EVs for pro-regenerative purposes, which include, but are not limited to, anti-angiogenic activities.

On the other hand, the method of the present invention can be used to obtain EVs loaded with specifically modulated biological effects, for example, remote pro-angiogenic effects without affecting antibacterial properties and vice versa.

The method of the present invention can be used to modulate the intrinsic biological effects of EVs in order to obtain loaded EVs with tailored and specifically desired biological activities.

The method of the present invention can be used to produce loaded EVs that contain one or more exogenous molecules, or loaded EVs enriched with biologically active endogenous compounds. Optionally, the method of the present invention can be used to improve EV loading efficiency using any protocol aimed at introducing molecules into EVs, including electroporation, sonication, transfection, incubation, cell extrusion, saponin-mediated permeabilization and freeze-thaw cycles. As an example, we show that protamine-based EV loading associated with electroporation is capable of increasing loading.

The method of the present invention can also be used in combination with a load of plant-derived EVs loaded with fat-soluble molecules.

Accordingly, the present invention embraces loading plant-derived EVs to enhance their native effects on cellular regeneration. The beneficial effects of plant-derived EVs can be enhanced by loading fat-soluble molecules such as antioxidant vitamins. Fat-soluble molecules in their native or modified form are efficiently incorporated into EVs. Thus, plant-derived EVs can be loaded with antioxidant molecules such as vitamins A and E to enhance their beneficial effects.

Native and loaded EVs can be administered via multiple routes depending on the destination. To restore the skin and outer mucous membranes, EVs can be administered topically, while oral administration is preferred to reach the digestive system.

Accordingly, the composition of the present invention, which includes a population of plant-derived EVs described above, wherein the EVs are either native or loaded with exogenous or endogenous negatively charged biologically active molecules, can be provided as a pharmaceutical composition formulated, for example, for topical application, local injection or oral administration, or may be provided as a dietary supplement preparation.

The composition of the invention may further include suitable matrices to cause controlled release of EVs into damaged or diseased tissue, stabilize EVs and/or enhance their therapeutic effects.

Suitable matrices used in the present invention are capable of encapsulating EVs and releasing them in a controlled manner when injected or applied externally, or are capable of acting as an inert carrier of biologically active molecules. Suitable matrices include, but are not limited to, scaffolds, films, hydrogels, hydrocolloids, membranes, foams, nanofibers, gels and sponges. To facilitate delivery of the EV matrix, the composition can be combined with medical devices such as patches, surgical sutures, and gauze.

In general, the compositions of the present invention, formulated for topical use or local injection, are particularly useful for promoting tissue repair when the tissue suffers from impaired angiogenesis or is exposed to bacterial infection. The invention relates to the utility of plant-derived extracellular vesicles as a therapeutic topical treatment to promote therapeutic effects on damaged tissues and cellular repair, for example, when damaged tissues show impaired angiogenesis or are exposed to microbial infections.

Compositions of the present invention comprising native or loaded EVs of plant origin, wherein the EVs have proangiogenic activity, are particularly useful for the therapeutic treatment of ulcers such as pressure ulcers, arterial ulcers, venous ulcers, ischemic ulcers, diabetic ulcers, exudative ulcers, dysmetabolic ulcers, burns, fistulas, fissures, and skin diseases, including psoriasis, dermatitis, acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, dermatitis herpetiformis, keratosis, keratitis, corneal damage/eye diseases (including ulcers, traumatic injuries, degenerative damage, abrasion, chemical damage, contact lens problems, UV damage, keratitis), dry eye disease, conjunctivitis, androgenetic alopecia, scabies.

Compositions of the present invention comprising native or loaded EVs of plant origin, wherein the EVs have antibacterial activity, are particularly useful for the treatment of mucosal injuries (such as traumatic injuries due to prosthesis and the like, diabetic, oral injuries, decubital, genital mucosal injuries ), infectious injuries (such as viral infections, herpes infections, bacterial infections), ulcers (including diabetic, arterial, venous, dysmetabolic, exudative, ischemic ulcers, pressure ulcers), burns, fistulas, fissures, corneal injuries and eye diseases (including ulcers , traumatic injury, degenerative injury, abrasion injury, chemical injury, ultraviolet injury, keratitis), dry eye disease, conjunctivitis, dermatitis (including acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, herpetiformis dermatitis), cellular damage caused by proapoptotic drugs that help treat precancerous lesions (eg, actinic keratosis).

The dosage of the pharmaceutical composition of the present invention may vary depending on various factors, including the potency of the particular compound used, the age of the patient, body weight, general health, sex, nutrition, time of administration, route of administration, rate of excretion, combination of drugs and severity of the particular disease. , which is prevented or treated, and can be accordingly determined by one skilled in the art depending on the condition of the patient, body weight, severity of the disease, form of the drug, route of administration and period of administration.

The pharmaceutical composition of the present invention may be formulated as pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries or suspensions.

The pharmaceutical composition of the present invention, formulated for local delivery, is effective in enhancing tissue regeneration and cell repair. The delivery system ensures local efficacy and controlled release of EVs over time at the site of injury. In addition, the delivery system can also guarantee the stability and storage of the EV drug. Such pharmaceutical compositions for local delivery of native or loaded EVs may contain hydrocolloid/hydrogel matrices suitable for the site and type of injury being treated. The composition is intended to enhance cell and/or tissue repair. Matrix-containing compositions can be adjusted to suit the requirements of the injury of interest (presence of exudate, burn, dry injury, mucosal ulcer, surgical suture). The matrix itself can also support the therapeutic effects of EVs and dressings and enhance the persistence of EVs. The matrices can be solid/gelatinous or liquid at room temperature and preferably include hydrocolloid/hydrogel matrices. Matrices can be created from several compounds (or chemical modifications thereof) and/or combinations thereof and include, but are not limited to, chitosan, gelatin, hydroxyapatite, collagen, cellulose, hyaluronic acid, fibrin, alginate, cyclodextrin, starch, dextran, agarose, chondroitin sulfate, pullulan, protamine, pectin, glycerophosphate and heparin, synthetic polymers such as polyethylene glycol (PEG), polyglycolic acid (PGA), polyvinyl alcohol [PVA], polycaprolactone [PCL], poly(D,L-lactic acid) ( PDLLA), poly(N-isopropylacrylamide) [PNIPAAm], and copolymers such as D,L-lactic acid copolymer (PDLLGA). Such molecules can be used in their native or chemically modified form. Such components can be used separately or in combination.

In addition, the pharmaceutical compositions of the invention formulated for local delivery of native or loaded EVs may contain suitable excipients, preservatives, solvents or diluents, according to conventional methods. Excipients, preservatives, solvents or diluents include, but are not limited to, lactose, agar, dextose, sucrose, glycol, sorbitol, triclosan, benzyl alcohol, mannitol, propylene glycol, xylitol, erythritol, maltitol, starch, parabens, gum arabic, alginate, gelatin , calcium phosphate, calcium silicate, cellulose, methylcellulose, salicylic acid, microcrystalline cellulose, sorbic acid, creolin, polyvinylpyrrolidone, quaternary ammonium cations, citric acid, acetic acid, ascorbic acid, boric acid, alginic acid, methylhydroxybenzoate, glycerin, propylhydroxybenzoate, pyrithione zinc, talc, sulfites, magnesium stearate, benzoic acid, mineral oils, propionic acid, chlorobutanol, fillers, dry diluents, binders, wetting agents, disintegrants, surfactants, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, ethyl oleate, witepsol, macrogol, twin, cocoa butter, lauric fat, glycerogelatin, purified water, oils, waxes, fatty acids, fatty alcohol acids, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols , vitamins, UV blockers, fragrances, dyes, antibiotics, bactericides or antifungals, etc. Such molecules can be used in their native form or with chemical modifications. Such components may be used alone or in combination.

The native or loaded population of EVs, combined with or without a matrix, can also be used as active compounds in a dietary supplement formulation suitable as an edible dietary supplement. The therapeutic properties of EVs may support cell repair in the gastrointestinal tract.

Accordingly, the invention also covers an edible preparation containing native or loaded EVs of plant origin, preferably obtained from plants from the Brassicaceae family, such as Anastatica hierochuntica; from Selaginella lepidophylla; from the family Asteraceae, such as Calendula officinalis; from the family Oleaceae, such as Olea europaea; from the family Xanthorrhoeaceae, such as Aloe vera, from the family Nelumbonaceae, such as Nelumbo; from the family Araliaceae, such as Subgenus Panax; from the family Lamiaceae, such as Lavandula; from the family Hypericaceae, such as Hypericum perforatum; from the family Pedaliaceae, such as Harpagophytum procumbens; from the family Ginkgoaceae, such as Ginkgo biloba; the Piperaceae family such as Piper kadsura or Piper futokadsura; family Rubiaceae, such as Hedyotis diffusa.

The dietary supplement preparation can be obtained in several forms for oral administration, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols. In addition, the dietary supplement of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavors, colors, pectic acid and its salt, alginic acid and its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks, etc. Such components may be used alone or in combination.

The dietary supplement preparation of the invention may also contain suitable excipients, preservatives, solvents or diluents known to those skilled in the art. Excipients, preservatives, solvents or diluents include, but are not limited to, lactose, agar, dextose, sucrose, glycol, sorbitol, triclosan, benzyl alcohol, mannitol, propylene glycol, xylitol, erythritol, maltitol, starch, parabens, gum arabic, alginate, gelatin , calcium phosphate, calcium silicate, cellulose, methylcellulose, salicylic acid, microcrystalline cellulose, sorbic acid, creolin, polyvinylpyrrolidone, quaternary ammonium cations, citric acid, acetic acid, ascorbic acid, boric acid, alginic acid, methylhydroxybenzoate, glycerin, propylhydroxybenzoate, pyrithione zinc, talc, sulfites, magnesium stearate, benzoic acid, mineral oils, propionic acid, chlorobutanol, fillers, dry diluents, binders, wetting agents, disintegrants, surfactants, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, ethyl oleate, witepsol, macrogol, twin, cocoa butter, lauric fat, glycerogelatin, purified water, oils, waxes, fatty acids, fatty alcohol acids, fatty acid esters, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, vitamins, UV -blockers, fragrances, dyes, antibiotics, bactericides or antifungals, etc. Such molecules can be used in their native form or with chemical modifications. Such components may be used alone or in combination.

EXAMPLES

The following experimental section is provided for illustration purposes only and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section, reference is made to the accompanying drawings described below.

Figure 1 shows the characteristics of native plant-derived EVs in Experimental Example 1 for EVs derived from a) lemon, B) orange, C) grape, D) Anastatica hierochuntica and E) Selaginella lepidophylla. Representative nanoparticle analysis image (Nanosight) and transmission electron microscopy photographs of EVs (original magnifications ×40,000 and ×120,000) show a size typical of EVs.

Figure 2 shows the protein content of native plant-derived EVs in Experimental Example 1, expressed in nanograms (ng) of protein in 10 ⁸ EVs isolated from apple, lemon, orange, grape, Anastatica hierochuntica and Selaginella lepidophylla.

Figure 3 shows the results of promoting endothelial cell migration in vitro mediated by native plant-derived EVs in Experimental Example 2. The graph shows the percentage of wound healing (mean ± SEM) compared to control cells (CTR) measured by scratch test. Cells are treated with three different doses of native orange-derived EVs: 10,000 EV/cell (EV 10k), 50,000 EV/cell (EV 50k), 100,000 EV/cell (EV 100k). Perform N=4 experiments for each data set indicated, and use 10 μM endothelial growth factor (EGF) as a positive control. Statistical significance is calculated by comparing each condition with CTR. p: * < 0.05; **<0.01; *** <0.005; **** <0.001.

Figure 4 shows the results of the ability of native plant-derived EVs in Experimental Example 2 to promote angiogenesis. Endothelial cells are stimulated with EVs derived from lemon, orange, grape, Anastatica hierochuntica (AH) and Selaginella lepidophylla (SL) (100,000 EV/cell) and tube formation assays are performed. Perform N=4 experiments for each data set, and use 10 μM vascular endothelial growth factor (VEGF) as a positive control. Statistical significance is calculated by comparing each condition with CTR. p: * < 0.05; **<0.01; *** <0.005; **** <0.001.

Figure 5 shows the results of native plant-derived EVs in Experimental Example 2 promoting cell proliferation on hypoxia-stimulated endothelial cells. Endothelial cells are incubated under hypoxic conditions for 24 hours and then treated with three different doses of orange-derived EVs (10,000 (10k) or 30,000 (30k) or 50,000 (50k) or 100,000 (10k) EVs/cell) for another 24 hours. . Proliferation is tested by incorporating BrdU, and analysis is performed comparing fold change versus control cells (CTR). EGF, 10 μM, was used as a positive control. (CTR+). Statistical significance is calculated by comparing each condition with CTR. p: * < 0.05; **<0.01; *** <0.005; **** <0.001.

Figure 6 shows the results of the therapeutic effect in humans in vivo of native plant-derived EVs in Experimental Example 4. Native EVs derived from orange are used to treat skin damage caused by ingenol mebutate (ingenol-3-angelate, picato), used for the local treatment of precancerous lesions, actinic keratosis. Representative images of tissue damage are shown: damage before (Figure 6A) and three days after (Figure 6B) plant-derived EV treatment versus untreated damage before (Figure 6C) and three days after (Figure 6D).

Figure 7 shows the results of EV charge measurements in Experimental Example 5. The Z-potential (mV), an indicator of particle charge, in native orange-derived EVs and EVs engineered with 1.0 μg/mL protamine (EV) was measured. + protamine). The results are obtained from three experiments, repeated three times. p: **** <0.001.

Figure 8 illustrates the method of modifying the EV in Experimental Example 5. The invention consists of using a positively charged molecule (like protamine) as a bridge to bind negatively charged bioactive molecules (eg, microRNA) to concentrate the molecules on the surface of the EV.

Figure 9 shows the results of the presence of miRNAs in loaded EVs in Experimental Example 5. The amplification pattern is obtained by RT-PCR analysis of native orange-derived EVs (EV CTR), EVs engineered with protamine and synthetic human miR-145, miR-221 or miR -223 (EV+PROT+ miR-145/ miR-221/miR-223). miRNA expression is represented as ΔRn, the magnitude of the signal obtained from miRNA amplification versus the number of cycles.

Figure 10 shows the results of protection of engineered molecules (miRNAs) after RNase treatment in Experimental Example 5. Orange-derived EVs engineered with miR-221 were treated with a physiological concentration of RNase (0.2 μg/ml), and expression of quantitative RT- was assessed. PCR in control native EVs loaded with both EVs engineered with protamine and microRNA (EV+PROT+ miR-221), and free miRNA (miR-221). Data are given as initial Ct (A) and percent inhibition relative to untreated samples (B). p: **** <0.001.

Figure 11 shows the entry of EVs into target cells using confocal microscopy in Experimental Example 6. Endothelial cells (TECs) are treated with fluorescently labeled loaded orange-derived EVs (30,000 EV/cell) for various times (30 min, 6 hours) and analyzed using confocal microscopy to detect their invasion of target cells. Representative micrographs of cells treated with stained EVs (EV CTR) or tagged EVs for 30 minutes and 6 hours are shown. EV membrane, microRNA, cell nuclei are stained with PKH26 red, FITC green, DAPI blue, respectively. (Original magnification ×630).

Figure 12 shows direct transfer of loaded miRNA into target cells and its functionality in Experimental Example 6. Endothelial cells (TECs) cultured with normal medium (CTR), native orange-derived EVs (EVs), loaded orange-derived EVs, protamine engineered and microRNA miR-221 (EV+PROT+mimetic-221) or scrambled miRNA (EV+PROT+scramble) or anti-miR-29a (EV+PROT+anti-mir-29a) (30,000 EV/cell). A) Transfer of microRNA (miR-221) into target cells is assessed by qRT-PCR analysis using RNU6B as housekeeping miRNA and cells cultured without stimuli as control. The data is presented as RQ values and compared with CTR. B) Effect of miRNA on collagen 4A3 target after treatment with EVs engineered with miRNA (anti-mir-29a). Assessment of microRNA activity on its target collagen4 isoform A3 mRNA after 72 hours. Cells are co-incubated with loaded EVs engineered with anti-miR-29a (EV+PROT+anti-mir-29a) (30,000 EV/cell) or normal medium (CTR), and collagen 4A3 expression is assessed by RT-PCR. Data are given as RQ values. The data is presented in the form of RQ values and compared with CTR. p: *** < 0.005.

Figure 13 shows size analysis of loaded EVs engineered with decreasing doses of protamine in Experimental Example 7. Nanosize analysis of control native orange-derived EVs (EV CTR), loaded EVs engineered with protamine (initial dose 1.0 μg/ml) and in smaller doses: 1.0 ng/ml, 0.1 ng/ml, 0.01 ng/ml. After co-incubation with microRNA (miR-221), the EV assay estimates the mean A) and mode B) size of loaded EVs. The data is compared with EV CTR (native EV). p: * < 0.05.

Figure 14 shows the results of miRNA expression in loaded EVs after engineering and incorporation into target cells using low doses of protamine in Experimental Example 7. A) Loaded orange-derived EVs are engineered with a lower dose of protamine (1.0 ng/ml) and miR miRs. -221 and analyzed for the content of exogenous microRNA. Data obtained by RT-PCR analysis are shown in RQ values using RNU6B as the housekeeping gene and normalized to native EVs (EV CTR). p: ** < 0.01. B) Loaded orange-derived EVs are engineered with protamine (1.0 ng/ml) and miR-221 microRNA or scramble and co-incubated with endothelial cells (TECs) for 24 hours. The presence of loaded miRNA is measured in RT-PCR target cells and reported as RQ in cells cultured with normal medium (CTR), normal native EVs (EVs), or loaded EVs engineered using protamine and scrambled miRNA (EV+PROT+scramble ) or miR-221 (EV+PROT+miR-221). p: * < 0.05.

Figure 15 shows the improvement in the therapeutic effect of native plant-derived EVs after engineering with pro-regenerative miRNA in Experimental Example 8. The graph illustrates the enhanced migration of keratinocytes and shows the percentage of wound healing (mean ± SEM) when compared with control cells (CTR). Cells are treated with three different doses of native orange-derived EVs: 10,000 EV/cell (EV 10k), 50,000 EV/cell (EV 50k), 100,000 EV/cell (EV 100k); and a dose of 5000 EV/cell of loaded EVs plus protamine (1.0 ng/ml) (EV + P) and loaded EVs plus protamine and miR-21 (EV + miR-21). EGF (10 μM) was used as a positive control. Perform N=4 experiments for each data set. Statistical significance is calculated by comparing each condition with CTR.

Figure 16 shows the acquisition of new biological functions by loaded EVs after engineering with miRNAs in Experimental Example 9. Loaded orange-derived EVs engineered with multiple anti-angiogenic miRNAs were tested for vascular endothelial cell (TEC) formation using an angiogenesis assay. TECs are cultured with normal medium (CTR), native EVs (EVs), loaded EVs engineered with protamine (EV+c protamine), or loaded with EVs modified with protamine (1.0 ng/ml) and synthetic angiogenic microRNA (anti-miR for pro-angiogenic miRNAs and miR for anti-angiogenic miRNAs). Scrambles are control microRNAs. After 24 hours of treatment, the total length of the vessels is measured, and the percentage of total length compared to normal cells (CTR) is reported. p: * < 0.05, ** < 0.01, *** < 0.005, **** < 0.001.

Figure 17 shows the biological activity results of loaded EVs engineered with two different doses of protamine in Experimental Example 10. Orange-derived EVs were engineered with an initial (1.0 μg/mL) or lower (1.0 ng/mL) amount of protamine and various antiangiogenic microRNAs (anti-imiR-29a, miR-145, miR-221). Loaded EVs are used to treat endothelial cells (TECs), and vascular formation is assessed by comparison with control cells (CTR) and cells cultured with native EVs (EVs). The total length is given as a percentage relative to control cells. p: * < 0.05, *** < 0.005, **** < 0.001.

Figure 18 illustrates the enhancement of molecular internalization using the modification method described in this patent application and the addition of a conventional transfection method in Experimental Example 11. Binding of a negatively charged molecule (such as a microRNA) to an EV increases the number of molecules on the surface of the EV and increases their loading after transfection protocol such as electroporation. Indeed, the increased number of molecules on the EV surface allows for increased loading within the EV following rearrangement at the membrane, which favors the movement of miRNAs into the EV interior.

Figure 19 shows the results of enhanced engineering using a combination of the modification method described in this patent application and the addition of a conventional transfection method in Experimental Example 11. Endothelial cells (TECs) were stimulated for 24 hours, and vascular formation was measured using an angiogenesis assay. Stimuli are normal medium (CTR), native orange-derived EVs (EVs), loaded EVs, engineered using protamine (1.0 ng/ml) and miR-221 microRNA (EV+PROT+miR-221), electroporated miR- 221 EVs (EVs + electroporated miR-221) and electroporated loaded EVs after modification with protamine (1.0 ng/ml) and microRNA miR-221 (EVs + PROT - electroporated miR-221). Vessel formation is assessed as the percentage of vessel formation compared to normal cells (CTR). p: * < 0.05, ** < 0.01.

Materials and methods

Isolation of extracellular vesicles

Extracellular vesicles are isolated from plant sap. The fruits are pressed and the juice is successively filtered using pores in decreasing order to remove fibers. EVs are then purified by ultracentrifugation. For differential ultracentrifugation, the juice is first centrifuged at 1500 g for 30 minutes to remove debris and other contaminants. EVs were then purified by first centrifugation at 10,000 g followed by ultracentrifugation at 100,000 g for 1 hour at 4°C (Beckman Coulter Optima L-90K, Fullerton, CA, USA). The final pellet was resuspended with phosphate buffered saline supplemented with 1% DMSO and filtered through 0.22 micron filters for sterilization. Extracellular vesicles are used or stored at -80°C for long periods of time. Purified EVs are characterized using nanoparticle trajectory analysis and electron microscopy.

Nanoparticle Trajectory Analysis (NTA)

Nanoparticle trajectory analysis (NTA) was used to determine EV size and profile using a NanoSight LM10 system (NanoSight Ltd., Amesbury, UK) equipped with a 405 nm laser and NTA 3.1 analysis software. The Brownian motions of EVs present in a sample exposed to a laser source are recorded by a camera and converted into size and concentration parameters by NTA via the Stokes-Einstein equation. Camera levels for all measurements are set to 16, and five 30-s videos were recorded for each sample. Briefly, purified EVs and engineered EVs were diluted (1:1000 and 1:200, respectively) in 1 ml vesicle-free saline (Fresenius Kabi, Runcorn, UK). NTA settings after data collection are optimized and kept constant across all samples, and each video is then analyzed to measure mean EV, mode, and concentration.

Transmission electron microscopy

Transmission electron microscopy of EVs is performed by loading EVs onto 200 mesh carbon-coated nickel-formvar grids (Electron Microscopy Science, Hatfield, PA) for 20 min. EVs are then fixed with a solution containing 2.5% glutaraldehyde and 2% sucrose, and after repeated washes in distilled water, samples are negatively stained with NanoVan (Nanoprobes, Yaphank, NK, USA) and examined using a Jeol JEM 1010 electron microscope.

Cell culture

Human microvascular endothelial cells (HMECs) are generated by immortalizing 40 primary microvascular endothelial cells in human skin with simian virus. HMECs were cultured in endothelial basal medium supplemented with a bullet kit (EBM, Lonza, Basel, Switzerland) and 1 ml Mycozap CL (Lonza, Basel, Switzerland).

Immortalized human keratinocytes (HaCat) were cultured in DMEM (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2. Cells are seeded at a density of 3.5 x 10 ² cells/cm ² using 1 ml of medium/cm ² and subcultured when cell confluency is 70-80%. Briefly, mattress tubes were washed with HEPES buffered saline, incubated with trypsin for 6 min, and then trypsin was neutralized with medium containing 10% FBS. If the cells are not completely separated within 7 minutes, incubation with trypsin is repeated.

Human kidney cancer cell (TEC)-derived endothelial cells were isolated from clear cell kidney cancer samples using anti-CD105 Ab in combination with magnetic beads by magnetic cell sorting using a MACS system (Miltenyi Biotec, Auburn, CA, USA). TEC cell lines are established and maintained in culture in Endogro basal complete medium (Merck Millipore, Billerica, MA, USA). TECs are tentatively characterized as endothelial cells by morphology, positive staining for vWF antigen, CD105, CD146, and vascular endothelial cadherin, and negative staining for cytokeratin and desmin.

Protein analysis

Proteins were extracted from EVs with RIPA buffer (150 nM NaCl, 20 nM Tris-HCl, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1% Triton X-100, pH 7.8) supplemented with a protease cocktail and phosphatase inhibitors (Sigma- Aldrich, St. Louis, Missouri, USA). Protein content was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following the manufacturer's protocol. Briefly, 10 μl of sample was dispensed into wells of a 96-well plate and total protein concentrations were determined using a linear standard curve established with bovine serum albumin (BSA).

In vitro scratch assay

Keratinocytes (HaCaT) and endothelial cells (HMEC) are seeded at a density of ~50×10 ³ cells/well in 24-well plates in DMEM supplemented with 10% FCS. When cells reach full confluence, they are left in medium without FCS overnight. The next day, scratches are applied with a sterile tip. Before stimulation (t=0), micrographs of the socket are obtained using a Leica microscope (Leica, Wetzlar, Germany). Cells are then stimulated with DMEM with 10% FBS or EGF as a positive control (CTR+) or EVs (10,000 (10k) or 50,000 (50k) or 100,000 (100k) EVs/target cells). The wound healing phenomenon was monitored for 48 hours using a Leica microscope, and images were analyzed using ImageJ software (Bethesda, MD, USA), observing a decrease in wound area in EV-stimulated cells when compared with non-EV-stimulated cells.

In vitro angiogenesis assay

The in vitro formation of capillary-like structures was studied on Matrigel with a reduced content of growth factor (BD Bioscience, Franklin Lakes, NJ, USA) in 24-well plates. HMEC or TEC (25,000 cells/well) are seeded in Matrigel-coated wells in DMEM or EndoGRO MV-VEGF medium, respectively, containing EVs (50,000 or 30,000 EV/target cells). The processing is performed three times. Cell organization on Matrigel was visualized using a Nikon Eclipse TE200. After incubation for 24 hours, phase-contrast images (×10 magnification) were recorded and the total extent of network structures was measured using ImageJ software. The total extent in the visual field is calculated in five randomly selected fields and expressed as a percentage of the corresponding control.

In vitro proliferation assay

HMECs are seeded into 96-well plates at a density of 2000 cells/well and left until confluent.

The culture medium is replaced with DMEM and left overnight. The plate is then sealed in a hypoxic chamber filled with the following gas mixture: 5% CO2, 1% O2, 94% N. The hypoxic chamber is placed in a CO2 incubator for 24 hours. The plate is then removed from the hypoxic chamber and cells are treated in DMEM alone (CTR), with positive control (10 ng/ml EGF, CTR+), increasing doses of native plant-derived EVs (10,000, 30,000, 50,000 and 100,000 EVs/cell). Each condition is performed four times. 10 μl of BrdU labeling solution (BrdU colorimetric assay, Roche) is then added to each well and the plate is incubated overnight. The following procedure is performed according to the manufacturer's instructions for BrdU assay. Measure the absorbance with an ELISA reader at 370 nm. Calculate the average optical density for each condition. Optical density is directly proportional to the proliferation rate. All average absorbance values are normalized to the average of the untreated control (CTR) used as a control sample. The results show the relative proliferation rate when compared to CTR, which is set to 1.

EV charge measurement

The analysis is performed using a Zeta-sizer nanoinstrument (Malvern Instruments, Malvern, UK). All samples are analyzed at 25°C in filtered (cut-off = 200 nm) saline. A zeta potential (slip plane) is generated at a distance x from the particle, indicating the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. A negative zeta potential indicates a high degree of particle dispersion.

Designing EVs with Protamine

EVs were mixed with protamine (1.0 μg/ml) (Sigma-Aldrich, St. Louis, MO) and co-incubated at 37°C for 5–30 min to allow binding to the EV surface. Various doses of protamine are used (1.0 ng/ml, 0.1 ng/ml, 0.01 ng/ml). Negatively charged synthetic miRNA molecules (100 pmol/ml) (miRNA mimics or anti-miR, Qiagen, Hilden, Germany) are then added to the mixture and co-incubated at 37°C for 3 hours. The mixture is diluted with saline and stored at 4°C overnight. Ultracentrifugation at 100,000 g for 2 hours at 4°C (Beckman Coulter Optima L-90K, Fullerton, CA, USA) removes free miRNA and protamine molecules, and the pellet is resuspended in phosphate buffered saline supplemented with 1% DMSO and filtered with using 0.22 micron filters for sterilization.

RNase treatment

EVs were treated with RNase A (Thermo Fisher Scientific, Waltham, MA, USA) using a concentration of 0.2 μg/ml for 30 min at 37°C. RNase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) is used to stop the reaction as described in the manufacturer's protocol, and EVs are washed using ultracentrifugation at 100,000 g for 1 hour at 4°C (Beckman Coulter Optima L-90K, Fullerton , CA, USA).

Confocal microscopy

For EV insertion, they are labeled with the red fluorescent membrane dye PKH26 (Sigma-Aldrich, St. Louis, MO) and engineered with green fluorescent (FITC)-labeled siRNA (Qiagen, Hilden, Germany). Labeled EVs are used to treat TEC seeded in 24-well plates (30,000 cells/well) for various times (30 min, 1 hour, 3 hours, 6 hours, 18 hours, 24 hours). EV uptake was analyzed using a confocal microscope (Zeiss LSM 5 Pascal, Carl Zeiss, Oberkochen, Germany).

RNA extraction

Total RNA was extracted from EVs and cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The RNA concentration of the samples was quantified using a spectrophotometer (mySPEC, VWR, Radnor, PA, USA).

MicroRNA and mRNA analysis using RT-PCR

For microRNA analysis, the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) is used. Briefly, RNA samples are reverse transcribed using the miScript Reverse Transcription Kit, and cDNA is then used to detect and quantify miRNAs of interest. Experiments were performed three times using 3 ng of cDNA for each reaction as described in the manufacturer's protocol (Qiagen). For mRNA analysis, cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Add five nanograms of cDNA to the SYBR GREEN PCR Master Mix (Applied Biosystems) and load PCR into a QuantStudio 12K Flex 96-well real-time PCR (RT-PCR) system (Thermo Fisher Scientific, Waltham, MA, USA) . GAPDH is used as a housekeeping gene. The fold change (Rq) of miRNA expression among all samples is calculated as 2 ^-ΔΔCt compared to control samples.

Electroporation of extracellular vesicles

Electroporation was performed on a Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. For each electroporation, the sample volume is fixed at 200 μL.

In vivo experiments

Ingenol mebutate (ingenol-3-angelate, picato) is used for topical treatment of precancerous lesions caused by actinic keratosis. The medication is applied to actinic keratosis lesions for 3 days, removing the precancerous lesion but causing apoptotic tissue to form. After treatment, native orange-derived EVs are administered topically to one damaged tissue site, while one untreated tissue site from the same patient is used as a control. The effect of plant-derived EVs is assessed 3-7 days after treatment.

Statistical analysis

Data analysis is carried out using Graph Pad Demo software, version 6.01. Results are expressed as mean ± standard error (SEM). One-way analysis of variance (ANOVA) is used to test for statistical differences between groups, while Student's t test is used to compare two samples. The authors used p < 0.05 as the minimum level of significance. p: * < 0.05, ** < 0.01, *** < 0.005, **** < 0.001.

Results/Examples

Example 1

To study the feasibility of the method of the present invention, the inventors used native EVs isolated in pure form from various plants, including lemon, orange, grape, Anastatica hierochuntica and Selaginella lepidophylla. EVs were isolated by microfiltration and differential ultracentrifugation or tangential flow filtration, and they display a size range of 25–350 nm using nanoscale analysis (Figure 1). In addition, all native plant-derived EVs exhibit a round morphology confined by an electron-dense membrane as shown by electron microscopy analysis (Figure 1).

To test the content of plant-derived EVs, the protein content of native EVs isolated from apple, orange, lemon, grape, Anastatica hierochuntica and Selaginella lepidophylla is measured using a BCA assay. The results are shown in Figure 2 and show the heterogeneous protein content for EVs shown in Table 1.

Table 1. Protein content of EVs

Protein, average, ng/E+08 EV SD apple EV 12.06 0.71 EV of grapes 47.93 2.17 EV lemon 63.13 4.55 EV of orange 143.74 49.21 EV AH 210.58 11.05 EV SL 504.58 13.06

Additionally, deeper analysis reveals that native plant-derived EVs contain vesicle-specific proteins such as HSP70, HSP80, glyceraldehyde-3-phosphate dehydrogenase (G3PD), and fructose bisphosphate aldolase 6 (FBA6); and plant proteins such as patellin-3-like and clathrin heavy chain.

In addition, the lipid content of native plant-derived EVs shows a lipid cargo in amounts that vary depending on the plant, including 24-propylidenecholesterol, beta-sitosterene, glycidyl alcohol stearate, dipalmitin, campesterol, eicosanol, eicosane, hexadecane, hexadecanol, octadecanol, octadecanol , tetradecane, tetradecanol, valencene and stearate.

Example 2

The ability of native plant-derived EVs to promote cell migration and angiogenesis is tested. By creating a scratch on a monolayer of endothelial cells, we observed a significantly higher rate of endothelial cell migration using different doses of native plant-derived EVs compared to a negative control (CTR) (Figure 3), demonstrating that native plant-derived EVs can promote endothelial cell migration. cells and support angiogenesis.

In addition, the angiogenesis assay evaluates the ability of native plant-derived EVs to promote vascular formation, confirming their effect on vascular formation in vitro. The results shown in Figure 4 show that the native plant-derived EVs tested significantly promote neovascularization by stimulating endothelial cells, thereby promoting angiogenesis.

In addition, the ability of native plant-derived EVs to promote cellular proliferation of endothelial cells in vitro after hypoxic injury is tested. Various doses of native orange-derived EVs significantly promoted the rate of cell proliferation compared to the negative control (CTR), demonstrating the beneficial effects of EVs on endothelial cells (Figure 5).

Example 3

Native EVs of plant origin are analyzed for their antimicrobial activity. Most pathogenic bacteria associated with infectious lesions in humans require a pH value >6 and their growth is inhibited at lower pH values, ranging from 4 to 5. Application of native plant-derived EVs to the surface of the lesion creates an acidic environment unfavorable for growth and reproduction pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella spp., Proteus spp., Citrobacter spp., S. epidermidis, S. pyogenes, streptococci and enterococci. Application of plant-derived EVs is effective in clearing pathogenic bacteria from contaminated or infected lesions by lowering the pH. Indeed, treatment with native plant-derived EVs restores the average surface pH of the skin (typically ranging from approximately 4.2 to 5.6), regulating external infection by enhancing the skin's natural antimicrobial activity. In addition, lowering pH has been shown to enhance the antimicrobial activity of other drugs against both gram-positive and gram-negative bacteria.

Example 4

Native EVs of plant origin are analyzed for their therapeutic effects in vivo. Native EVs of plant origin are used to treat skin lesions caused by ingenol mebutate (ingenol-3-angelate, picato) in a volunteer. The named substance is a cell death inducer and is used for local treatment of precancerous lesions of actinic keratoses. The results illustrated in Figure 6 show the lesion before (Figure 6A) and after treatment (Figure 6B) for three days with native plant-derived EVs compared to an untreated lesion that is similarly shown before (Figure 6C) and after three days (Figure 6D). Native plant-derived EVs show therapeutic effects in pro-apoptotic lesions induced by ingenol mebutate, promoting tissue regeneration after three days compared to untreated lesions.

Example 5

In order to modify plant-derived EVs in a charge interaction-based manner, native plant-derived EVs are analyzed for their surface charge. Zeta potential analysis is performed on various preparations, showing that native EVs derived from orange display a negative charge of -13.59 ± 1.83 mV (Figure 7). Other native plant-derived EVs show similar negative charges. Interestingly, orange-derived EVs co-incubated with the positively charged linker protamine and washed by ultracentrifugation showed a significant increase in their charge, suggesting modification of their surface (Figure 7).

To investigate the method of loading negatively charged molecules onto the surface of EVs using a positively charged linker, loaded plant-derived EVs modified using protamine were mixed with microRNA molecules, as illustrated in Figure 8. As an example, loaded orange-derived EVs using protamine is mixed with various miRNA mimics (miR-145, miR-221, miR-223), and RT-PCR analysis demonstrates a clear accumulation of miRNAs in loaded EVs relative to control native EVs (Figure 9), suggesting efficient binding of the molecules to EVs. Interestingly, EV-associated miRNAs are protected from degradation by physiological concentrations of RNase present in biological fluids, thus conferring biological resistance. Figure 10A shows complete inactivation of free miRNA by RNase, while EV-associated miRNA is protected from inactivation when compared to native EVs that do not express miRNA. The percentage of miRNA inhibition in all samples is shown in Figure 10B.

Example 6

To understand whether it is possible to efficiently transfer loaded molecules into target cells, the transport of loaded molecules into target cells mediated by loaded plant-derived EVs is analyzed. First, orange-derived EVs are loaded with PKH26 (a red fluorescent dye) and engineered with fluorescent FITC-labeled synthetic microRNA. Loaded EVs were co-incubated with human kidney carcinoma-derived endothelial cells (TECs) for various times (30 min, 1 hour, 3 hours, 6 hours, 18 hours, 24 hours). Confocal microscopy analysis shows that small nucleic acids present on the surface of EVs do not alter their uptake by target cells. Figure 11 shows control cells (CTR), with labeled nuclei, and that treatment with loaded EVs increases the fluorescent signal in target cells after only 30 min of co-incubation with the highest uptake at 6 hours (Figure 11). In addition, efficient transport by loaded EVs is also demonstrated by detection of uptake into target cells. For this purpose, TECs are treated with loaded orange-derived EVs modified with a 221 miRNA mimic and analyzed by RT-PCR after 24 hours (dose 30,000 EVs/cell). As seen in Figure 12A, miRNAs are efficiently transported to target cells via EVs. The functionality of loaded molecules in target cells is also tested. To this end, TEC cells are stimulated with loaded orange-derived EVs engineered with anti-miR-29a, and target gene mRNA expression is measured in target cells using quantitative RT-PCR experiments. The results demonstrate that miRNAs transferred to EV target cells are also functional and induce a significant increase in their target gene collagen 4 A3 (Figure 12B).

Example 7

To further explore the use of a positively charged linker, different doses of protamine are evaluated to load plant-derived EVs. Positively charged molecules such as protamine can form micelles around negatively charged molecules such as microRNA. Orange-derived EVs are then incubated with decreasing doses of protamine and the negatively charged microRNA molecule miR-221-3p. EV size analysis is performed using Nanosight, which measures the average and typical size of loaded EVs. The results show that the initial amount of protamine (1.0 μg/ml) causes an increase in both average and typical size with a significant difference in the average (Figure 13). However, this change in EV size is absent when EVs are co-incubated with lower doses of protamine (1.0 ng/ml, 0.1 ng/ml, 0.01 ng/ml). The results suggest that an excessive initial dose of protamine results in the formation of micelles larger than those of typical native EVs present in the EV preparation. To confirm that lower doses of positively charged linker are sufficient to allow interaction with negatively charged molecules, the expression of loaded miRNA molecules in loaded plant-derived EVs is measured and their transport to target cells is assessed. qRT-PCR analysis of loaded orange-derived EVs engineered with a signature miRNA (miR-221-3p) using a lower dose of protamine (1.0 ng/ml) shows that the miRNA efficiently binds to EVs (Figure 14a). Moreover, loaded orange-derived EVs modified with a lower dose of protamine (1.0 ng/ml) were able to efficiently transfer miRNAs to target cells, as demonstrated by RT-PCR analysis of target cells treated with loaded EVs (Figure 14b).

Example 8

As a representative example of a modification method to further improve the native activity of plant-derived EVs, modification of plant-derived EVs is used to improve their native activity while promoting wound healing by keratinocytes. In these experiments, loaded orange-derived EVs are engineered with miR-21 microRNA using protamine as a positively charged linker. Human keratinocytes are treated with three different doses of native EVs, loaded EVs co-incubated with protamine alone (EV+P) as a control, and loaded EVs with protamine and miR-21 (EV+miR-21). EV+P and EV+miR-21 are used at an intermediate dose (50k). The measurement of wound healing under each condition is used as a parameter of EV activity. The chart in Figure 15 shows that EV+P promotes wound healing as well as the same dose of native EVs, while EV+miR-21 promotes a statistically significant increase in wound healing as well as double the dose of native EVs (EV 100k) . The results indicate that the modification method can be used to enhance the therapeutic effects of native plant-derived EVs.

Example 9

The modification method can also be used to alter the biological activity of native plant-derived EVs. Plant-derived EVs can be engineered with negatively charged molecules to provide different or novel biological effects. As a representative example, orange-derived EVs were modified with various anti-angiogenic miRNAs and their ability to inhibit angiogenesis was assessed by an in vitro angiogenesis assay on TEC cells. In particular, loaded EVs are engineered with anti-angiogenic miRNA mimics (miR-221, miR-223, miR-145) and anti-miRNAs for proangiogenic miRNAs (miR-29, miR-126, miR-31), and their effect on the formation of vessels on endothelial cells 24 hours after treatment (Figure 16). The results show that loaded orange-derived EVs engineered with anti-angiogenic molecules are able to significantly inhibit endothelial cell angiogenesis in vitro.

Example 10

The therapeutic effects of loaded plant-derived EVs are evaluated using different doses of positively charged linker. As a representative example, loaded orange-derived EVs were engineered with two different doses of protamine (1 μg/ml, 1 ng/ml) and three different doses of anti-angiogenic miRNAs (anti-mir-29, miR-145 and miR-221-3p). Loaded EVs are used to stimulate endothelial cells (TECs) for 24 hours, and their activity is assessed by an angiogenesis assay. The results shown in Figure 17 show that the activity of loaded EVs engineered with a lower dose of protamine is equal to or more effective, demonstrating the applicability of different doses of a positively charged linker for the effective modification of native plant-derived EVs.

Example 11

The method of modifying plant-derived EVs with negatively charged molecules can be further improved by transfection protocols. Indeed, the association of negatively charged molecules (e.g., miRNA) with the EV surface through a positively charged linker (e.g., protamine) may facilitate their entry into EVs. The close proximity of negatively charged molecules to EVs can enhance their loading after a transfection protocol such as electroporation, as illustrated in Figure 18. The positively charged linker, by forming a bridge between negatively charged EVs and negatively charged molecules, concentrates the molecules on the EV surface and favors inward rotation (Figure 18 ). Membrane rearrangement produced by strategies other than electroporation, such as sonication, mechanical cell extrusion, saponin-mediated permeabilization, and freeze-thaw cycles, are also indicated for EV loading.

To test this hypothesis, orange-derived EVs were modified using protamine and miR-221-3p, and their ability to inhibit vascular formation was assessed. Figure 19 shows that the use of a transfection protocol, such as electroporation, on loaded EVs is able to increase their inhibitory activity on vascular formation on endothelial cells.

Claims (8)

1. The use of a population of plant-derived extracellular vesicles (EVs) obtained from a plant selected from the group consisting of orange, lemon, grape, Anastatica hierochuntica (AH) and Selaginella lepidophylla (SL), wherein the extracellular vesicles (EVs) in said population are lipid limited bilayer membrane and have a diameter ranging from 25 to 350 nm, and a protein content ranging from 1 to 55 ng/10 ⁹ EV, in the therapeutic treatment of impaired angiogenesis. 2. The use of claim 1, wherein the population is used in the therapeutic treatment of impaired angiogenesis in a disease or condition selected from the group consisting of ulcers, psoriasis, dermatitis, acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, Dermatitis herpetiformis, keratosis, keratitis, corneal injury, traumatic injury, degenerative injury, abrasion, chemical injury, contact lens problem, UV damage, keratitis, conjunctivitis, dry eye disease, androgenetic alopecia, itching and mucosal injury. 3. Use according to claim 2, wherein the ulcers are selected from the group consisting of pressure ulcers, arterial ulcers, venous ulcers, diabetic ulcers, ischemic ulcers, exudative ulcers, dysmetabolic ulcers, traumatic ulcers, burns, fistulas, fissures. 4. Use according to claim 2, wherein the mucosal injuries are selected from the group consisting of traumatic injuries due to a prosthesis, diabetic, oral, decubital or genital mucosal injuries. 5. Use according to any one of claims 1 to 4, wherein the EVs are native EVs or EVs loaded with one or more negatively charged pro-regenerative bioactive molecules selected from the group consisting of drugs, nucleic acids and lipophilic vitamins. 6. Use according to claim 1, wherein the EVs are loaded with one or more negatively charged pro-regenerative nucleic acids selected from microRNA, mRNA, tRNA, rRNA, miRNA, regulatory RNA, non-coding and coding RNA, DNA fragments and DNA plasmids. 7. Use according to any one of claims 1 to 6, wherein the EVs are formulated in a pharmaceutical composition for topical use, local injection or oral administration, or formulated as a dietary supplement preparation. 8. Use according to any one of claims 1 to 7, wherein the EVs are obtained from orange.