Nanovesicles isolated from persea americana seeds and uses thereof

Abstract

The present invention relates to a nanovesicle isolated from Persea americana seed, to uses of said nanovesicle or use of a composition comprising said nanovesicle for the treatment of wounds and / or burns and / or to prevent or reduce scar formation, and to a method for obtaining said nanovesicle.

Description

[0001] NANO-VESICLES ISOLATED FROM PERSEA AMERICANA SEEDS AND

[0002] ITS USES

[0003] This application claims priority over U.S. Provisional Patent Application No. 63 / 571 ,681 , filed March 29, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The present invention relates to the technical field of cosmetic and / or pharmaceutical products, particularly, it refers to a nano-vesicle isolated from Persea americana seed, a method of obtaining said nano-vesicle and uses of the nano-vesicle for the treatment of wounds and burns and / or for the prevention or reduction of scars.

[0006] BACKGROUND

[0007] Approximately 80,000 children suffer burns annually in Chile, 84% of whom are under the age of 10. Seventy-four percent of these patients have intermediate burns, which involve the epidermis and part of the dermis (Zegers JR, Soucheiron MB No more fireworks. Compendium of the IV International Congress of Hospital Pedagogy of REDLACEH. 2019). Inflammation in wounds is a process mediated by cytokines and chemokines that promote the release of proteases and cytotoxic agents to eliminate bacteria and necrotic tissue; However, acute inflammation in burns is exacerbated and can damage healthy tissue (Rawlingson A. Nitric oxide, inflammation and acute burn injury. Burns. 2003;29(7):631-640; Bird MD, Kovacs EJ. Organ-specific inflammation following acute ethanol and burn injury. J Leukoc Biol. 2008;84(3):607-613).

[0008] Acute treatment includes dressings that promote the dehesion process (removal of necrotic tissue) and protect the wound from bacterial colonization and impacts (Shi C, Wang C, Liu H, et al. Selection of Appropriate Wound Dressing for Various Wounds. Front Bioeng Biotechnol. 2020; 19;8: 182; Quinn RH, Wedmore I, Johnson E, et al. Wilderness Medical Society Practice Guidelines for Basic Wound Management in the Austere Environment. Wilderness Environ Med. 2014;25(3):295-310); however, these dressings do not modulate inflammation or the resulting injuries. Incorporating antibacterial and anti-inflammatory agents into dressings would modulate the process in its acute stage and reduce the number of patients requiring an autologous graft.

[0009] Persea americana, also known as avocado, is a product with high nutritional, cosmetic, and recently therapeutic value due to its complex biochemical profile and ability to regulate metabolic processes. Avocados, especially the pulp, are an excellent source of vitamins A, D, and E, and are known for the treatment of dry or damaged skin. Avocado pulp has even been observed to produce complete epithelialization in a cut or excision wound model in less time than the control (Nayak BS, Raju SS, Chalapathi Rao AV Wound healing activity of Persea americana (avocado) fruit: a preclinical study on rats. J. Wound Care. 2008; 17(3): 123-126).

[0010] In the industry, avocado skin and seeds are by-products currently considered waste, representing up to 30% of the raw material. These by-products contain compounds with antioxidant, antimicrobial, and adsorbent properties that are attractive to the industry. For example, patent document WO 2012 / 042404 A2 discloses an avocado seed extract with antimicrobial, antibacterial, and spore germination-inhibiting effects. Said extract is obtained by grinding the seeds (particles of 0.5 to 2 mm), extracting them with organic solvents such as acetone or hexane, filtering and evaporating the solvent, and finally suspending the dried compounds in acetone for subsequent use as an antimicrobial.On the other hand, patent document US 5,928,659 discloses a cosmetic or dermatological formulation that reduces stretch marks, keratosis and skin lesions that contains unsaponifiable lipids extracted from avocado seeds, where the effectiveness of the composition increases with the addition of zinc and / or copper chelates.

[0011] However, both patent documents mention that the active compounds are obtained through solvent extraction using solvents such as hexane, which is toxic and harmful to the environment. In turn, phenolic compounds and metabolites extracted from plants are often unstable, which limits their half-life and performance.

[0012] In this context, nanovesicles have been isolated from plants or other organisms and have been described as transporters of proteins and metabolites. These can be an alternative to traditional solvent extracts, which have also been used for wound treatment. Exosome-like vesicles from bee pollen, honey, and Apis mellifera royal jelly have been reported to have an antibacterial effect and pro-regenerative activity in an in vitro wound model (Schuh CMAP, Aguayo S, Zavala G, et al. Exosome-like vesicles in Apis mellifera bee pollen, honey and royal jelly contribute to their antibacterial and pro- regenerative activity. J Exp Biol. 2019 Oct 16;222(Pt 20):jeb208702; Leiva-Sabadini C, Alvarez S, Barrera NP, et al. Antibacterial Effect of Honey-Derived Exosomes Containing Antimicrobial Peptides Against Oral Streptococci. Int J Nanomedicine. 2021 Jul 20;16:4891-4900; Álvarez S, Contreras-Kallens P, Aguayo S, et al.Royal jelly extracellular vesicles promote wound healing by modulating underlying cellular responses. Mol Ther Nucleic Acids. 2023 Feb 14;31 :541-552) and the production of a collagen hydrogel with said vesicles for wound treatment (Ramírez OJ, Alvarez S, Contreras-Kallens P, et al. Type I collagen hydrogels as a delivery matrix for royal jelly derived extracellular vesicles. Drug Deliv. 2020 Dec;27(1 ):1308-1318). It has also been described that nano-vesicles derived from the skin of the Aloe vera plant show anti-inflammatory properties and prevent the differentiation of myofibroblasts, so it can be useful for the treatment of wounds (Ramírez O, Pomarada F, Olivaras B, et al. Aloe vera peel-derived nanovesicles display antiinflammatory properties and prevent myofibroblast differentiation. Phytomedicine. 2024 Jan; 122: 155108).

[0013] SUMMARY OF THE INVENTION

[0014] The present invention relates to nano-vesicles obtained from avocado seeds (Persea americana), where said nano-vesicles are extracted by a passive extraction method without the use of toxic solvents, and where said nano-vesicles are useful for the treatment of wounds and burns and / or to prevent or reduce the formation of scars.

[0015] A first object of the present invention corresponds to a nanovesicle isolated from Persea americana seed for use in the treatment of wounds and burns, and / or for the prevention or reduction of scar formation. In a preferred embodiment, the nanovesicle has an average diameter between 50 and 150 nm.

[0016] In one embodiment of the present invention, the nano-vesicle can be used in a pharmaceutical composition or in a cosmetic composition, wherein the composition has a form selected from the group consisting of a patch, dressing, liquid, cream, toner, gel, hydrogel, ointment, suspension, emulsion, paste, lotion, oil, and spray.

[0017] Another object of the present invention is a method for the treatment of wounds and burns, and / or the prevention or reduction of scar formation, which comprises administering an effective amount of nano-vesicles isolated from Persea americana seed to a subject in need thereof.

[0018] In one embodiment of the treatment method of the present invention, the nanovesicles have an average diameter between 50 and 150 nm. Preferably, the isolated nanovesicles reduce fibrosis.

[0019] In another embodiment of the invention, the method of treatment comprises administering to a subject in need thereof an effective amount of a composition comprising, as an active compound, a nano-vesicle isolated from Persea americana seed, wherein said composition may be pharmaceutical or cosmetic, and whose form is selected from the group consisting of a patch, dressing, liquid, cream, tonic, gel, hydrogel, ointment, suspension, emulsion, paste, powder, lotion, oil, and spray.

[0020] Another object of the present invention is a method for obtaining a nano-vesicle isolated from a Persea americana seed, comprising the steps of: a. obtaining a Persea americana seed; b. obtaining a film from the seed; c. incubating the seed film in a buffered solution to obtain a nano-vesicle in suspension; d. centrifuging and filtering the buffered solution containing the nano-vesicle in suspension; and e. isolating the nano-vesicle by a nano-vesicle concentration method, where, preferably, the concentration method is selected from the group consisting of centrifugation, ultracentrifugation, filtration, immunoaffinity and chromatography.

[0021] In one embodiment of the present invention, the seed film has a thickness between 0.1 to 3 mm.

[0022] In one embodiment of the present invention, the buffered solution is selected from the group consisting of phosphate buffered saline, saline, 2-(N-morpholino)ethanesulfonic acid (MES) and a combination thereof, preferably the buffered solution is phosphate buffered saline.

[0023] In another embodiment of the present invention, the ratio of seed film to buffer solution is preferably between 1:2 and 1:20 (w / v). In another embodiment of the present invention, the seed film is incubated in the buffer solution for 0.5 to 24 hours.

[0024] In another embodiment of the present invention, the nano-vesicle isolated by the method of obtaining said nano-vesicle has an average diameter between 50 and 150 nm.

[0025] Another object of the present invention is a nano-vesicle isolated from Persea americana seed for the treatment of wounds and burns, and for the prevention or reduction of scar formation, where said nano-vesicle is obtained according to any of the embodiments of the method for obtaining nano-vesicles described herein.

[0026] The present invention also includes a composition for the treatment of wounds and burns, and for the prevention or reduction of scar formation, comprising the nanovesicle isolated from Persea americana seed according to any of the embodiments of the method for obtaining nanovesicles described herein. Another object of the present invention is a pharmaceutical composition for the treatment of wounds and burns, and for the prevention or reduction of scar formation, where the pharmaceutical composition comprises, as an active compound, a nanovesicle isolated from Persea americana seed, and a pharmaceutically acceptable carrier. In a particular embodiment, the nanovesicle comprised in the pharmaceutical composition has a diameter between 50 and 150 nm.

[0027] Another object of the present invention is a cosmetic composition for the treatment of wounds and burns, and for the prevention or reduction of scar formation, wherein the cosmetic composition comprises, as an active compound, a nanovesicle isolated from Persea americana seed, and a cosmetically acceptable carrier. In a particular embodiment, the nanovesicle comprised in the cosmetic composition has a diameter between 50 and 150 nm.

[0028] The present invention also includes a method for the treatment of wounds and burns, and the prevention or reduction of scar formation, which comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising, as an active compound, a nanovesicle isolated from Persea americana seed and a pharmaceutically acceptable carrier.

[0029] BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1 shows a tracking analysis of nanovesicles isolated from Persea americana pits (PaNVs). FIG. 1A shows a graph of the particle concentration per µl, and FIG. 1B shows a graph of the particle size distribution, n = 4. E7 = 10. 7 .

[0031] FIG. 2 shows graphs with results of the carbohydrate impurity assay in pre-isolation PaNVs obtained from Persea americana seed homogenate or laminate (FIG. 2A) and in post-isolation PaNVs obtained from Persea americana seed homogenate or laminate (FIG. 2B). Data are shown in pg / ml. n = 4, Student's t test. FIG. 3 shows the results of the cell damage assay in human dermal fibroblasts as measured by the LDH assay after 24 h of incubation with PaNVs obtained from laminated seeds (FIG. 3A) or with PaNVs obtained from homogenized seeds (FIG. 3B), compared to the untreated control. An increase in LDH above baseline indicates an increase in apoptotic cells. Baseline is 100%. Five doses were analyzed: 7.5 pg, 5 pg, 2.5 pg, 1 pg, and 0.5 pg. Significance was assessed by comparison with the positive control.Data are presented as a percentage of the positive control (%Ctrl positive, untreated cells); n = 4; one-way ANOVA.

[0032] FIG. 4 shows the results of the metabolic activity assay in human dermal fibroblasts measured by the MTT assay after 24 h of incubation with PaNVs obtained from slivered seeds (FIG. 4A) or with PaNVs obtained from homogenized seeds (FIG. 4B), compared to the untreated control. A decrease in MTT indicates a decrease in metabolic activity. The baseline value is 100%. Five doses were analyzed: 7.5 pg, 5 pg, 2.5 pg, 1 pg, and 0.5 pg. Significance was checked compared to the positive control. Data are presented as a percentage of the positive control (% Ctrl positive, untreated cells); n = 4; one-way ANOVA.

[0033] FIG. 5 shows the results of assays to evaluate the anti-inflammatory effects of PaNVs. Graphs of IL-1 (3 (FIG. 5A), IL-6 (FIG. 5B) and TNF-α (FIG. 5C) secretion of the test cell line RAW 264.7 after LPS stimulation, measured by ELISA, are shown. PaNVs were applied at 3 different concentrations: 25, 250 and 2500 PaNVs per cell, n = 4; data were analyzed by one-way ANOVA and Tukey’s post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; the asterisk above the bar indicates the difference with the LPS-stimulated control; the asterisks with line indicate the differences between the respective groups. Indo: Indomethacin.

[0034] FIG. 6 shows the results of assays to evaluate the antibacterial effect of PaNVs on Staphylococcus aureus ATCC 25923. In a microplate assay, S. aureus, at a defined concentration of 0.5 McFarland, was exposed to 5 different concentrations (0.25, 2.5, 25, 250 and 2500 PaNVs / CFU). After 24 hours at 37 °C, the plates were measured with a plate reader at a wavelength of 600 nm (OD600). Data are shown as percentage of the positive control (% Positive Ctrl; Growth Control); n = 4.

[0035] FIG. 7 shows the results of assays to evaluate the effect of PaNVs on myofibroblast differentiation. The antifibrotic activity of PaNVs was assessed by Western blot analysis, quantifying GAPDH-normalized α-SMA and the myofibroblast positive control. The higher the α-SMA, the greater the myofibroblast differentiation in the cell lysate. Fibroblasts were exposed to 250 PaNVs per cell during TGF-β differentiation; n = 3. Data were analyzed by one-way ANOVA and Tukey’s post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Myof: Myofibroblasts. Ctrl: control.

[0036] FIG. 8 shows the results of the evaluation of the anti-contractile effect of PaNVs as measured by a collagen contraction assay. Myofibroblasts were seeded onto collagen type 1 hydrogels together with 250 PaNVs per cell, and the area covered by the gel was evaluated after 24 h. The larger the area covered by the gel, the lower the contraction, n = 4. Data were analyzed by one-way ANOVA and Tukey’s post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CTRL: Control. LPA: lysophosphatidic acid.

[0037] FIG. 9 shows a schematic and the results of the PaNV release assays over time. FIG. 9A shows the schematic of the experimental design. FIG. 9B shows the released nanovesicles (particle release per cm 2 ) for 24 hours on days 1 and 3. n = 3.

[0038] FIG. 10 shows the results of the evaluation of the antibacterial effect of PaNVs released from collagen type 1 hydrogels. Staphylococcus aureus ATCC 25923; S. aureus, at a defined concentration of 0.5 McFarland, was exposed to PaNVs released from collagen type 1 gels. After 24 h at 37 °C, the plaques were measured using a plate reader at a wavelength of 600 nm (OD600). n = 4. Data were analyzed using Student’s t test. *p < 0.05, **p < 0.01. Ctrl: control.

[0039] FIG. 11 shows the results of the evaluation of the effect of PaNVs released from type 1 collagen hydrogels on myofibroblast differentiation. The anti-fibrotic activity of PaNVs was assessed by Western blot analysis, quantifying α-SMA normalized to GAPDH and a positive control of myofibroblasts. Fibroblasts were exposed to 250 PaNVs per cell or to Transwell systems containing PaNVs or control collagen during TGF-β differentiation. n = 3; data were analyzed by one-way ANOVA and Tukey’s post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Ctrl: control; Myof.: Myofibroblasts; PaNV collagen: laminated Persea americana seed nanovesicles in a type 1 collagen hydrogel; Collagen Ctrl: type 1 collagen control without PaNVs.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to a nano-vesicle isolated from Persea americana seed (indistinctly called PaNVs in the present description), uses of said nano-vesicle or a composition comprising said nano-vesicle for the treatment of wounds and burns and / or to prevent or reduce the formation of scars, and a method of obtaining said nano-vesicle.

[0042] All technical and scientific terms used to describe this invention have the same meaning as understood by a person with basic knowledge in the technical field concerned. However, to more clearly define the scope of the invention, a list of the terminology used in this description is included below.

[0043] The term “seed” shall be understood as a synonym for the terms “pit”, “kernel”, “stone” and shall be used interchangeably in this description to refer to the seed of the species Persea americana.

[0044] The term “avocado” shall be understood as a synonym for the terms “avocado” or “cura” and shall be used interchangeably in this description to refer to the fruit or berry of the Persea americana species.

[0045] “Nano-vesicle” should be understood as a vesicle that contains bioactive compounds and that has a diameter on the nanometer scale, preferably with diameters less than 500 nm, more preferably with diameters less than 200 nm.

[0046] The term "subject" shall be understood to mean any mammal, such as humans, mice, rats, rabbits, primates, cats, dogs, among others, without being limited to those mentioned herein. The term "subject" shall be understood as a synonym for the term "individual" and shall be used interchangeably in this description.

[0047] The term "treatment" refers to a set of methods or means used for therapeutic purposes in a subject who has developed or is in the early stages of developing a disease, syndrome, or condition. The treatment may be a treatment of the subject's symptoms for the purpose of alleviating their symptoms, or it may refer to a treatment of the underlying cause of the disease, syndrome, or condition, for the purpose of reversing, delaying, or halting the progression of said disease, syndrome, or condition. Therefore, the nanovesicles of the present invention, as well as the methods described herein, may be used, for example, as therapeutic treatment methods for a specific period of time or continuously, as required by the subject.The term “treatment” also includes preventive treatments that involve treating individuals who are at risk of developing a disease, syndrome, or condition (such as a scar) to avoid or reduce the risk of developing it.

[0048] The term "effective amount" or "therapeutically effective dose" refers to the dose and period of time required to achieve the desired therapeutic result. The effective amount can depend on many factors, such as the stage of progression of the disease or condition, the patient's age, sex, weight, the presence of other medical conditions, the use of other medications simultaneously, race, and other factors.

[0049] The term "wound" refers to an injury or alteration in the integrity of the skin or internal organ that occurs as a result of an aggression, trauma or surgery, which can be classified according to type of wound, origin and severity, depending on the depth and number of layers of the skin or organ affected. For the purposes of the present invention, a wound includes all types of wounds (incisions, lacerations, abrasions, burns, tearing, puncture, scrapes, bites, blisters, but not limited to those mentioned) caused by different types of agents (physical, biological or chemical) with varying degrees of severity (superficial, that is, only passing through the epidermis; deep, that is, passing through the subcutaneous tissue; penetrating, that is, passing through the skin and into underlying tissues or organs; or perforating, that is, passing through the body) and can be an acute or chronic wound.

[0050] The term “burn” refers to an injury to the skin or other internal organ caused by contact with heat (hot liquids, vapors, or objects, fire, etc.), electricity, radiation, or chemicals, and includes all types of burn severity.

[0051] "Constant agitation" refers to the constant and gentle movement of the support containing the sample or tissue that is subjected to the method of obtaining the present invention. This constant agitation is preferably achieved by placing the support in an orbital shaker, which typically has a shaking speed range of 20 to 500 rpm.

[0052] The terms “about” or “approximately,” or the symbol used interchangeably throughout this specification, should be understood as the value or range of a parameter that includes a standard deviation of error according to the method or apparatus used to determine said value or range, and that is within the acceptable tolerance or statistically significant range for the value of said parameter. Such statistically significant range may be, for example, within 30%, 20%, 10% or 5% of the indicated value or range. That is, the mentioned values ​​and ranges are not and need not be exact, and may be approximate, whether equal, lower or higher. The term “between,” when referring to ranges, should be understood as around the lower value and around the upper value mentioned.

[0053] "Room temperature" is understood to mean the temperature range or value found in the environment. A person skilled in the art will understand that such temperature values ​​vary depending on multiple factors such as the seasons, nearby devices or equipment that may be causing an increase or decrease in temperature, among others. Typically, room temperature is approximately 20-25°C; however, it may be higher or lower than this range without affecting the method or the result obtained from said method of the present invention.The present invention includes a nano-vesicle isolated from Persea americana seed for use in the treatment of wounds and / or burns and / or for use in preventing or reducing scar formation, and a method of treating wounds and / or burns and a method of preventing and / or reducing scar formation comprising administering an effective amount of nano-vesicles isolated from Persea americana seed or administering an effective amount of a composition comprising said nano-vesicles isolated from Persea americana seed, to a subject in need thereof.

[0054] The nanovesicles of the present invention have been shown to have antimicrobial, anti-inflammatory, anti-fibrotic, and anti-contractile properties that allow for the treatment of wounds and / or burns and reduce fibrosis to prevent or avoid scar formation. The nanovesicles of the present invention have the ability to reduce fibrosis by decreasing myofibroblast differentiation. In this way, scar formation is prevented or reduced at the site where the nanovesicles are applied.

[0055] Preferably, said nano-vesicle is a vesicle that has an average diameter between 50 - 150 nm, which is why in the present description they are defined as nano-vesicles.

[0056] The nano-vesicle can be incorporated as an active compound into a pharmaceutical or cosmetic composition, where said compositions can have any form suitable for use, such as, for example, a patch, dressing, liquid, cream, tonic, gel, hydrogel, ointment, suspension, emulsion, paste, powder, lotion, oil, aerosol, among others, and can be formulated for rapid, sustained or controlled release of the nano-vesicles at the site where the wound or burn is to be treated, or in the area where it is desired to prevent or reduce the formation of scars. The type of formulation and the concentration of nano-vesicles present in the composition will depend on the type and severity of the wound or burn to be treated or the type of scar that is desired to prevent or reduce its formation, the type of organ where the nano-vesicles or the composition comprising the nano-vesicles will be applied (e.g. skin or internal organs), among other factors.

[0057] Additionally, such compositions may include a pharmaceutically acceptable carrier or a cosmetically acceptable carrier such as an excipient, which should be broadly understood as any component of the composition other than the active compound, and may refer to a diluent, stabilizer, binder, disintegrant, humectant, coating agent and / or colorant, among others, or a mixture thereof. These excipients are known in the state of the art as those reported, for example, in Allen L, Popovich N, Ansel H. (2011). Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. 9th edition. Lippincott Williams & Wilkins, and Rowe R, Sheskey P, Quinn M. (2009). Handbook of Pharmaceutical Excipients. 6th edition. Pharmaceutical Press, without being limited to those mentioned therein.The specific agents and compounds for each of these functions are obvious to a person with average knowledge of the state of the art and can be found in numerous publications such as those mentioned above. The cosmetic composition according to the present invention also comprises excipients suitable for application to the skin or skin appendages of said cosmetic composition.

[0058] Both the nanovesicle and the composition comprising it can be used to treat wounds or burns on the skin or other internal organs and / or to prevent or reduce scarring on the skin or other internal organs. Thus, both the nanovesicle and the composition comprising it can be administered to a subject by any route of administration appropriate for the intended use. Preferably, administration is topical or to the internal organ to be treated, for example, during surgery.

[0059] The present invention also includes a method for obtaining a nanovesicle isolated from Persea americana seed, comprising a first step of obtaining Persea americana seed. Preferably, the fruit pulp is removed (or separated for use in other applications) and the seed or pit is washed with plenty of water and detergent for the time necessary to remove pulp residues and any contaminants present. In a preferred embodiment, said detergent is any common soap. Preferably, the washing time is at least 5 minutes.

[0060] Then, one or more sheets are obtained from the seed by a rolling process. In one embodiment of the invention, the rolling is carried out with the seed submerged in a buffered solution, which can be phosphate-buffered saline (PBS), saline, 2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof, but not limited to those mentioned above. Preferably, PBS is used. In another embodiment of the invention, the sheets obtained have a thickness between 0.1 and 3 mm, or nanovesicles can even be obtained by cutting the seeds in half. However, it is preferable to obtain at least 1 g of sheets to obtain a sufficient quantity of nanovesicles.

[0061] The seed film obtained in the previous step is incubated in a buffered solution that can be phosphate buffered saline (PBS), saline solution, 2-(N-morpholino)ethanesulfonic acid (MES) or a combination thereof, without being limited to those mentioned. Any of these alternatives works adequately to obtain nano-vesicles of Persea americana seeds. To incubate the film, it is immersed in a sufficient amount of buffered solution to at least cover the film. In one embodiment of the present invention, a seed film:buffered solution ratio of 1:2 to 1:20 (w / v) can be used. In another embodiment of the present invention, the incubation time is at least 30 minutes, and it is not necessary to incubate for more than 24 hours, where the incubation temperature is preferably at room temperature, preferably under constant agitation, although the latter is not limiting for obtaining the nano-vesicle.

[0062] With this incubation step, nanovesicles in suspension are obtained. To purify them, a centrifugation step is included, preferably a serial centrifugation, which is carried out at a temperature between 4 and 37 °C, preferably between 4 and 10 °C. This eliminates any plant debris that may be present in the nanovesicle suspension. The suspension, free of plant debris, is then filtered using any filter material such as cellulose acetate filters, with filter sizes between 0.45 and 0.22 pm.

[0063] From the solution resulting from the filtration step, the nanovesicles are isolated using any vesicle concentration method known in the state of the art. For example, centrifugation, ultracentrifugation, filtration, immunoaffinity, chromatography, or others can be used, without being limited to those mentioned herein. In one embodiment of the invention, ultracentrifugation can be performed at 40,000, 75,000 or 100,000xg for 60, 75 or 90 minutes at a temperature between 4 ° C and 10 ° C. With this, isolated nanovesicles are obtained so that they can be used for the treatment of wounds and / or burns and / or to prevent or reduce scar formation, or they can be used to obtain a composition that can be used for the aforementioned purposes.

[0064] The following examples are intended to illustrate the invention and its preferred embodiments, but under no circumstances should they be considered to restrict the scope of the invention, which will be defined by the tenor of the claims appended hereto.

[0065] EXAMPLES

[0066] Example 1. Obtaining and characterizing Persea americana seed nano-vesicles (PaNVs).

[0067] Persea americana pits were collected and isolated. The pits were washed with detergent (soap) and rinsed with water. The cleaned pits were immersed in phosphate-buffered saline (PBS) to prevent oxidation. The pits were then kept submerged in said buffer before being sliced ​​using stainless steel blades, resulting in thin pit sheets, with thicknesses between 0.1 and 3 mm. These sheets were placed in PBS at a ratio of 1:2 (grams of seed sheets: ml of PBS) and incubated for 12 hours at room temperature on an orbital shaker to release the nanovesicles. Subsequently, plant debris was removed by sequential centrifugation (500, 1000, 1500 and 2500 g for 15 minutes each) at 4 °C, and then the extract free of plant debris was filtered through a 0.22 pm filter. From the resulting extract, nano-vesicles were isolated by ultracentrifugation (100.000xg for 90 min at 4°C) or filtration (10,000 MWCO, 4500xg). Nanovesicles isolated from Persea americana pits (PaNVs) were resuspended in 150 pl of PBS and stored at -80°C until use.

[0068] The concentration of PaNVs was subsequently measured by nanoparticle tracking analysis. As shown in FIG. 1A, the average concentration obtained was 5.24 x 10 7 PaNVs per pl. Additionally, PaNV diameter was assessed using the same technique. FIG. 1 B shows a graph showing that PaNVs range in diameter from 50 to 150 nm.

[0069] The yield of PaNVs was also evaluated using the method described above, but instead of slicing the pit, the pit was homogenized using a grater. The PaNVs obtained using both methods were compared as described below.

[0070] Carbohydrate impurities are known to alter the stability and therapeutic effect of PaNVs. To determine the carbohydrate content of the PaNVs obtained by both methods, a total carbohydrate analysis kit (Sigma, MAK104) was used. Prior to isolation, it was observed that PaNVs obtained from the pit homogenate had a higher amount of impurities than PaNVs obtained from the pit laminate (FIG. 2A). After isolation, no carbohydrate impurities were detected in the PaNVs obtained from the laminate, but this was not the case in the PaNVs obtained from the homogenate. The presence of an undetectable concentration or a carbohydrate concentration lower than 1 pg / ml or 2.15 x 10' 9 pg / PaNVs is a desirable feature of the present invention (FIG. 2B).

[0071] Example 2. Cytocompatibility of PaNVs with human dermal fibroblasts.

[0072] Cellular damage caused by PaNVs obtained by both methods was assessed using a lactate dehydrogenase assay kit (LDH Cytotoxicity Kit, Roche). LDH in culture is directly proportional to cellular damage, as it is not usually released by the cell except during apoptosis or necrosis. 5000 cells (human dermal fibroblasts) were subjected to 5 different protein concentrations (7.5 pg, 5 pg, 2.5 pg, 1 pg and 0.5 pg) of pre-isolation supernatant or with PaNV solution isolated after 24 h of incubation. Untreated cells and those subjected to 2.5% Triton X-100 served as positive and negative controls. FIG. 3A shows the results obtained for isolated PaNVs obtained from the laminated pit and FIG. 3B shows the results for the isolated PaNVs obtained from the homogenized stone.Additionally, metabolic activity was measured in human dermal fibroblasts using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colohmetic assay. Cell mitochondria convert soluble MTT (yellow) to insoluble formazan (purple), which is directly proportional to the cellular metabolic activity. Cells were incubated with 0.5 mg / ml MTT reagent for 2 h, and formazan formation was subsequently measured. Untreated cells and cells subjected to 2.5% Triton X-100 served as positive and negative controls. FIG. 4A shows the results obtained for isolated PaNVs obtained from the laminated pit, and FIG. 4B shows the results for isolated PaNVs obtained from the homogenized pit.

[0073] As a result of both experiments, it was observed that rolling Persea americana seeds produces a supernatant with cytocompatible PaNVs. No increase in cell damage was observed (FIG. 3A), and metabolic activity remained comparable to that of the untreated control at all concentrations tested. Homogenization of Persea americana seeds produces a cytotoxic supernatant (FIG. 3B), whose cytotoxicity remains significant after PaNV isolation at concentrations of 7.5, 5, 2.5, and 1 pg. 0.5 pg of pre-isolation supernatant and post-isolation PaNV solution did not show an increase in cell damage, but a decrease in metabolic activity (FIG. 4B).

[0074] The influence of PaNV isolation methods on PaNV cytotoxicity was also assessed. The effect of isolating PaNVs from laminated pits by ultracentrifugation or filtration (10,000 MWCO) was compared. PaNVs obtained by either of these methods were observed to be cytocompatible with human dermal fibroblasts. No increase in cell damage was observed, and metabolic activity remained comparable to untreated controls at all concentrations tested (results not shown).

[0075] Based on these results, the analysis of PaNVs obtained from laminated stones was continued. Example 3. Functional analysis to determine the therapeutic range of PaNVs.

[0076] The problem associated with healing essentially involves the following processes:

[0077] I. exacerbated inflammation in the acute stage of the wound,

[0078] II. Bacterial infections with Staphylococcus aureus,

[0079] III. persistence of microfibroblasts in fibrotic tissue,

[0080] IV. Contraction of microfibroblasts present in fibrotic tissue.

[0081] Anti-inflammatory effect of PaNVs

[0082] The anti-inflammatory effects of PaNVs were assessed using a standard anti-inflammatory assay. The ATCC RAW 264.7 test cell line (mucinous macrophages) was used for the assay. RAW 264.7 cells were stimulated with LPS to secrete pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6. PaNVs were added to the test system at concentrations of 25, 250, and 2500 vesicles per cell. Untreated LPS and indomethacin-treated cells served as controls. After 24 h of LPS stimulation, cell supernatants were collected and analyzed by ELISA (Duoset, R&D Systems). Results were normalized to 100,000 cells.

[0083] As seen in FIGS. 5A, 5B, and 5C, all three proinflammatory cytokines tested showed a reduction at at least one concentration of PaNVs. At no dose, an increase in inflammation was observed. A reduction was also observed at all doses, even at 25 PaNVs / cell. The most effective dose was 2500 PaNVs / cell for all three cytokines tested, which, in the case of IL-6, showed an even more pronounced effect than that of indomethacin.

[0084] Effect of PaNVs on bacterial growth

[0085] The antibacterial effects of PaNVs were tested using the ATCC Staphylococcus aureus strain 25923. In a microplate assay, S. aureus at a defined concentration of 0.5 McFarland was exposed to 5 different concentrations of isolated PaNVs (0.25, 2.5, 25, 250, and 2500 PaNVs / CFU). After 24 h at 37 °C, plaques were measured on a plate reader at a wavelength of 600 nm. S. aureus without PaNVs (growth control) and ampicillin (20 pg / ml, antibiotic control) served as controls. In addition, internal sterility controls included media without bacteria as well as the isolated PaNVs without bacteria. No concentration of PaNVs promoted bacterial growth, and the minimum inhibitory concentration was found to be 2500 PaNVs per CFU (FIG. 6).

[0086] Effect of PaNVs on myofibroblast differentiation

[0087] The antifibrotic effect of PaNVs was evaluated in human dermal fibroblasts purchased from Thermo Fisher (accession no. C0045C). In the presence of TGF-β, dermal fibroblasts differentiate into myofibroblasts, the major cell type responsible for burn contraction. In burns, several cell types secrete TGF-β. Terminally differentiated myofibroblasts express alpha-smooth muscle actin (α-SMA), and inhibition of myofibroblast differentiation is observed in the absence of increased α-SMA. When incubated with 250 PaNVs per cell, TGF-β-stimulated fibroblasts showed a significant reduction in α-SMA expression. No differences in the effect were observed between groups (FIG. 7). These results were confirmed by confocal microscopy, using phalloidin to visualize actin and α-SMA filaments, along with cell morphology in bright field (results not shown).

[0088] Effect of PaNVs on myofibroblast contractility

[0089] In addition to differentiation, myofibroblast contractility is another parameter to be measured. In a collagen contraction assay, collagen gels were seeded with cells and PaNVs and incubated overnight. In fibrous 3D constructs, myofibroblasts remodel the matrix and contract the hydrogel, as occurs in the wound (simplified). Untreated cells in a collagen construct, as well as cells treated with lysophosphatidic acid (LPA) (maximal contraction stimulation) and Y27632 (non-contraction control), served as controls. PaNVs were observed to significantly reduce the contraction of collagen gels (FIG. 8). Based on the data presented in this example, doses that can be used are between 1 pg to 100 pg of total PaNV protein per wound area (cm 2 ).

[0090] Example 4. Compatibility of PaNVs with biomaterials

[0091] PaNVs can be released from collagen matrices

[0092] To evaluate whether PaNVs retain their functionality after release and whether the proposed support material has any effect (positive or negative), the PaNVs were subjected to biomaterial release studies. The biomaterial used for these studies was type 1 collagen (Ramírez O, Pomarada F, Olivaras B, et al. Aloe vera peel-derived nanovesicles display anti-inflammatory properties and prevent myofibroblast differentiation. Phytomedicine. 2024 Jan;122:155108). Prior to functional assays, a 24-hour release rate was established. Type 1 collagen gels were loaded with 1.5 x 10 9 PaNVs and release were quantified by time-tracking nanoparticle analysis. A release rate of 2 x 10 was observed. 8 and 3.5 x 10 8 per cm 2(FIG. 9).

[0093] Antibacterial effects of PaNVs released from collagen type 1 hydrogels

[0094] PaNVs were tested in an in vitro delivery system called "transwell". These transwell systems allow contact with only one side of the biomaterial, simulating the release into a wound bed. PaNVs are released into the medium in contact with the transwell and exert their effects.

[0095] Type 1 collagen hydrogels were placed with 1.5 x 10 9PaNv and non-PaNVs in transwell systems. S. aureus ATCC 25923 was adjusted to 0.5 McFarland in 600 µl of BHI broth per well. Transwell systems were placed in the wells and the OD was measured after 24 hours. S. aureus at 0.5 McFarland (positive control), 20 µM ampicillin (negative control), 250 PaNVs / CFU and broth without bacteria served as controls. The plates were incubated for 24 hours at 37 °C and subsequently measured at OD600 using a microplate reader. Type 1 collagen reduced bacterial growth by approximately 25%, whereas PaNVs released from type 1 collagen reduced bacterial growth by 60% (FIG. 10). Effect of PaNVs released from collagen type 1 hydrogels on myofibroblast differentiation

[0096] Transwell systems were also used to evaluate the effect of PaNVs on myofibroblast differentiation. PaNVs released continuously into the system showed the same effect as those added to the medium. PaNVs released from a biomaterial prevented myofibroblast differentiation (FIG. 11).

[0097] Conclusions

[0098] During the development of the present invention, different pre- and post-isolation steps were analyzed in the method for preparing PaNVs obtained from Persea americana seeds. Rolling of Persea americana seeds and isolation of PaNVs by ultracentrifugation or filtration resulted in homogeneous, cytocompatible nanovesicles free of carbohydrate impurities.

[0099] Functionally, PaNVs showed anti-inflammatory effects by reducing the release of IL-1 (3, IL-6 and TNFa, starting at a dose of 25 nano-vesicles per cell, with the most effective dose being 2500 nano-vesicles per cell. PaNVs inhibited bacterial growth with a similar dose schedule, starting with 25 nano-vesicles per CFU, with 2500 being the most effective dose. Myofibroblast differentiation and contraction was inhibited by PaNVs. According to the results, PaNVs were observed to be compatible with type 1 collagen and exhibit a stable release. These released PaNVs maintained their function and were effective in reducing bacterial growth and myofibroblast differentiation.

Claims

CLAIMS 1. A method for obtaining a nano-vesicle isolated from a Persea americana seed, comprising the steps of: a) obtaining a Persea americana seed; b) obtaining a film from the seed; c) incubating the seed film in a buffered solution to obtain a nano-vesicle in suspension; d) centrifuging and filtering the buffered solution containing the nano-vesicle in suspension; and e) isolating the nano-vesicle by a nano-vesicle concentration method selected from the group consisting of centrifugation, ultracentrifugation, filtration, immunoaffinity, and chromatography.

2. The method according to claim 1, wherein the seed layer has a thickness between 0.1 to 3 mm.

3. The method according to claim 1, wherein the buffered solution is selected from the group consisting of phosphate buffered saline, saline, 2-(N-morpholino)ethanesulfonic acid (MES) and a combination thereof.

4. The method according to claim 3, wherein the buffered solution is phosphate buffered saline.

5. The method according to any of claims 1 to 4, wherein the ratio of seed film to buffered solution is between 1:2 to 1:20 (w / v).

6. The method according to any one of claims 1 to 5, wherein the seed sheet is incubated in the buffered solution for 0.5 to 24 hours.

7. The method according to any one of claims 1 to 6, wherein the isolated nanovesicle has an average diameter between 50 and 150 nm.

8. A nano-vesicle isolated from Persea americana seed for use in the treatment of wounds and burns, and for the prevention or reduction of scar formation.

9. The nano-vesicle according to claim 8, for use in a pharmaceutical composition or in a cosmetic composition.

10. The nano-vesicle according to claim 9, wherein the composition has a form selected from the group consisting of a patch, dressing, liquid, cream, tonic, gel, hydrogel, ointment, suspension, emulsion, paste, lotion, oil, and spray.

11. The nano-vesicle according to claim 8, characterized in that it has an average diameter between 50 and 150 nm.

12. A method for the treatment of wounds and burns, and the prevention or reduction of scar formation, comprising administering an effective amount of nano-vesicles isolated from Persea americana seed to a subject in need thereof.

13. The method according to claim 12, wherein the nano-vesicles have an average diameter between 50 and 150 nm.

14. The method according to any of claims 12 or 13, wherein the nanovesicles reduce fibrosis.

15. The method according to any one of claims 12 to 14, wherein the nanovesicles are administered in a composition whose form is selected from the group consisting of a patch, dressing, liquid, cream, tonic, gel, hydrogel, ointment, suspension, emulsion, paste, powder, lotion, oil, and spray.

16. A nano-vesicle isolated from Persea americana seed for the treatment of wounds and burns, and for the prevention or reduction of scar formation, characterized in that it is obtained according to the method of any of claims 1 to 7.

17. A composition for the treatment of wounds and burns, and for the prevention or reduction of scar formation comprising the nanovesicle isolated from Persea americana seed according to claim 16.

18. A pharmaceutical composition for the treatment of wounds and burns, and for the prevention or reduction of scar formation, wherein the pharmaceutical composition comprises, as an active compound, a nano-vesicle isolated from Persea americana seed, and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, characterized in that the isolated nano-vesicle has a diameter between 50 to 150 nm.

20. A cosmetic composition for the treatment of wounds and burns, and for the prevention or reduction of scar formation, wherein the pharmaceutical composition comprises, as an active compound, a nano-vesicle isolated from Persea americana seed, and a cosmetically acceptable carrier.

21. The cosmetic composition according to claim 20, characterized in that the isolated nano-vesicle has a diameter between 50 to 150 nm.

22. A method for the treatment of wounds and burns, and the prevention or reduction of scar formation, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising, as an active compound, a nano-vesicle isolated from Persea americana seed and a pharmaceutically acceptable carrier.

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