Method for obtaining exosome-like vesicles from microalgae

Abstract

The present invention provides a method for obtaining a composition comprising exosome-like vesicles or biomimetic exosomes from microalgae cells comprising a a)homogenizing microalgae cells by high-pressure, and b) eliminating cell debris from the homogenized solution obtained in step a). The invention also relates to a composition obteined by said method and to cosmetical and medical uses thereof.

Description

[0001] METHOD FOR OBTAINING EXOSOME-LIKE VESICLES FROM MICROALGAE

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention provides a method for producing a composition comprising exosomes from microalgae and uses thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Exosomes are extracellular vesicles that are released from cells upon fusion of an intermediate endocytic compartment, the multivesicular body (MVB), with the plasma membrane.

[0006] Mammalian-derived exosomes, have been extensively studied for their therapeutic potential. These exosomes have shown promise in wound healing, tissue regeneration, and immunomodulation. However, the production and purification of exosomes can be costly and challenging, leading researchers to explore alternative sources.

[0007] Exosome-like vesicles or biomimetic exosomes on the other hand, are a broader category of extracellular vesicles that share similarities with mammalian-derived exosomes but may have different origins or characteristics.

[0008] Exosome-like vesicles have emerged as promising therapeutic agents in various fields, including skincare and medicine. Some vesicles have been isolated from various sources, including plants and microalgae. These organisms produces vesicles that are functionally and structurally similar to mammalian exosomes but may have distinct formation pathways or compositional differences. Exosome-like vesicles are reported with a range in size from 50 to 200 nanometers in diameter and are similar in structure and function to mammalian exosomes. The size of these exosome-like vesicles is typically consistent with that of mammalian exosomes, which is crucial for their potential therapeutic applications and cellular uptake.

[0009] Traditional methods for obtaining extracellular vesicles from microalgae have primarily relied on centrifugation, ultrafiltration, and extrusion techniques. However, these methods often present limitations in terms of scalability, efficiency, and vesicle size control which present significant challenges when it comes to large-scale production. The time-consuming nature of ultracentrifugation and the expensive equipment required for the traditional methods make them less practical for industrial-scale applications.

[0010] Recently it has been described in KR20230016937A a method to obtain large amounts of exosome analogues from microalgae by extrusion, but this method does not present any scalability advantage.Therefore, alternative methods for exosome-like production are needed to scalability challenges and expand their broader application.

[0011] SUMMARY OF THE INVENTION

[0012] The inventors of the present invention have developed a method which is a solution to the longstanding issue of insufficient extracelular vesicles (EV) quantities for skincare applications and therapeutic applications, potentially accelerating the development and implementation of EV-based therapies. In particular, the invention relates to a method for obtaining exosome-like vesicles or biomimetic exosomes and to uses thereof.

[0013] A first aspect of the present invention relates to a method for obtaining a composition comprising exosome-like vesicles from microalgae cells comprising a) homogenizing microalgae cells by high-pressure, and

[0014] b) eliminating cell debris from the homogenized solution obtained in step a). In another aspect, the invention relates to a composition comprising exosome-like vesicles obtained by the method of the invention.

[0015] In another aspect, the invention relates to a cosmetic composition comprising the composition comprising exosome-like vesicles according to the invention and a cosmetically acceptable excipient.

[0016] In another aspect, the invention relates to a cosmetic method for reducing the cosmetic effect of aging and / or photoaging and / or and for the prevention and / or treatment of a non-pathological skin scar which comprises administering the composition comprising exosome-like vesicles according to the invention or the cosmetic composition according to the invention to a subject in need thereof.

[0017] In another aspect, the invention relates to a pharmaceutical composition comprising the composition comprising exosome-like vesicles according to the invention and a pharmaceutically acceptable excipient.

[0018] In another aspect, the invention relates to the composition comprising exosome-like vesicles according to the invention or the pharmaceutical composition according to the invention for use in medicine.

[0019] In another aspect, the invention relates to the composition comprising exosome-like vesicles according to the invention or the pharmaceutical composition according to the invention for use in the prevention and / or treatment of wound healing or a skin scar.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] Figure 1 and 2. Transmission electron microscope image of the microalgae-derived exosome-like vesicles.

[0022] Figure 3. Graph showing the effects of the exosome-like vesicles in wound healing.DETAILED DESCRIPTION OF THE INVENTION

[0023] The authors of the present invention have developed a method for obtaining exosome-like vesicles from microalgae, providing a more efficient and scalable approach and adressing all the limitations of conventional methods, in particular showing scalability, minimal processing, size control and improved efficiency.

[0024] Method for obtaining a composition comprising exosome-like vesicles

[0025] The method of the invention provides a more efficient and scalable approach to harnessing microalgae-derived exosome-like vesicles. The method of the invention shows several advantages due to the homogenization step, addressing the limitations of conventional methods:

[0026] - Scalability: The typical method for exosome-like isolation is by differential centrifugation and ultrafiltration. It can be expensive due to the specialized equipment required and scaling up the process for industrial applications can be challenging.

[0027] - Minimal processing: Following high-pressure treatment, the resulting mixture undergoes elimitation of cell debris from the homogenized solution, for example by low-speed centrifugation to remove cellular debris, preserving the integrity of the extracted vesicles.

[0028] - Size control: This high-pressure method enables control over vesicle size, potentially enhancing their therapeutic and cosmetic efficacy.

[0029] - Improved efficiency: Unlike previously described extrusion methods for microalgae, which needs diluted samples and often result in extruder scalability limitations and blockage, the method of the invention comprising the high-pressure step allows for obtaining larger-scale production of therapeutically relevant vesicles.

[0030] In a first aspect, the invention relates to a method for obtaining a composition comprising exosome-like vesicles from microalgae cells comprising

[0031] a) homogenizing microalgae cells by high-pressure, and

[0032] b) eliminating cell debris from the homogenized solution obtained in step a). The expression “exosome-like vesicles”, or biomimetic exosomes, as used herein relates to relates to small sized lipid-bilayer membrane-enclosed vesicles which enclose the components of the cells from which they derive. The exosome-like vesicles are formed by the reasembly of the cell lipids after cell lysis into closed vesicles. Themembranes of the vesicles contain transmembrane protein and enclose the contents of the cytoplasm which are liberated also as the result of the cell lysis.

[0033] “Microalgae cells”, as used herein, relates to large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms, microscopic algae, typically found in freshwater and marine systems. Culture conditions suitable for the growth of the microalga may be different for each type of microalga. However, those conditions are known by skilled workers and are readily determined. Similarly, the duration of maintenance can differ with the microalgae and with the amount of different molecules from the culture supernatant desired to be prepared. Again, those conditions are well known and can readily be determined in specific situations. As a way of illustrative, non-limitative example, the microalge can be cultivated by any method dislosed in Algal Culturing Techniques, Edited by Robert A. Andersen, Elsevier Academic Press 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA ISBN: 0-12-088426-7, and more particularly by those methods described in Appendix A of the cited reference. In a particular embodiment, the microalga is grown under mixotrophic conditions. In a particular embodiment, the microalga is cultured until an OD 750 nm of 2.5.

[0034] In a particular embodiment, the microalga is cultured in a photobioreactor in a suitable medium, under a suitable luminous intensity, at a suitable temperature. Practically any medium suitable for growing microalgae can be used; nevertheless, illustrative, non-limitative examples of said media include TAP media. The luminous intensity can vary widely, nevertheless, in a particular embodiment, the luminous intensity is comprised between 25 and 150 pmol photons m-2 s-1, particularly 100 pE. The temperature can vary usually between about 17°C and about 30°C, particularly 25°C. The culture can be performed in the absence of aeration or with aeration. In a particular embodiment, the culture is carried out without aeration. In another particular embodiment, the culture is carried out with aeration. In a preferred embodiment, the conditions are maintained until reaching a stationary phase.

[0035] In a particular embodiment, the microalgae is a flexible or thin-cell wall microalgae. “Flexible or thin-cell wall microalgae”, as used herein relates to a group of microalgae which has a low thickness of its wall. The thickness of a cell wall from microalgae can be measured by several methods known in the art, for example by transmission electron microscopy (TEM).

[0036] In a preferred embodiment, the wall thickess of the flexible or thin-cell wall microalgae is below 0.25 pm. In another preferred embodiment, the wall thickness of the flexible or thin-cell wall microalgae is below 0.2 pm. In a preferred embodiment, the thickness of a cell wall is measured in exponential growth phase, particularly at day 5.In a preferred embodiment, the microalgae is from phyllium Chlorophyta, Cyanophyta, Rhodophyta, Haptophyta and Heterokontophyta.

[0037] In a prefered embodiment, the microalgae is the phylum Cyanophyta, more preferably from genus Spirulina (Arthrospira), more preferably Arthrospira platensis (Spirulina spp.) In another preferred embodiment, the microalgae is from phylum Haptophyta, more preferably from genus Isochrysis or Tisochrysis, more preferably Isochrysis galbana. In another preferred embodiment, the microalgae from phyllum Heterokontophyta (also known as Ochrophytes or Stramenopiles) is Nannochloropsis, more preferably Nannochloropsis gaditana. In another preferred embodiment, the microalagae from phylum Rhodopyta is from genus Porphyridium, more particularly is Porphyridium cruentum.

[0038] In another particular embodiment, the microalgae is from the phylum Chlorophyta, for example from genus Dunaliella orTetraselmis.

[0039] In another particular embodiment, the microalgae is a green algae, more preferably the microalgae is from genus Chlamydomonas.

[0040] Chlamydomonas, as used herein relates to a genus of green algae consisting of about 325 species all unicellular flagellates, found in stagnant water and on damp soil, in freshwater, seawater, and even in snow as "snow algae". In a more preferred embodiment, the microalga is Chlamydomonas reinhardtii.

[0041] “Chlamydomonas reinhardtii’’, as used herein is a single-cell green alga about 10 micrometres in diameter that swims with two flagella. It has a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and an "eyespot" that senses light.

[0042] In another preferred embodiment, the microalga is from genus Haematococcus. In another preferred embodiment, the microalga is from genus Nannochloropsis. In another preferred embodiment, the microalga is from genus Scenedesmus. In another preferred embodiment, the microalgae is from genus Nannochloropsis. In another preferred embodiment, the microalgae is from genus Phaeodactylum. In another preferred embodiment, the microalgae is from genus Odontella.

[0043] The first step of the method of the invention requires homogenizing microalgae cells by high-pressure.

[0044] “High-pressure honogenization” or HPH or French press , relates to a mechanical cell disruption method which uses high pressure for disruption. Microalgal cells are pumped through a narrow orifice in a valve under high pressure, and the suspension is then released into a low-pressure chamber. Cell wall disruption occurs due to high-pressure impingement of accelerated cellular jet on the stable valve surface and due to a pressure drop-induced shear stress when the cell passes from valve to chamber. Thehomogenization at high pressure can be performed by several methods known in the art, for example by using a high-pressure homogenizer, ultrasound, bead milling or mechanical blenders. In a more preferred embodiment, the homogenization at high pressure is carried out by a high-pressure homogenizer.

[0045] In a preferred embodiment, the homogenization of step a) is carried out at a pressure between 5 MPa-100 MPa, more preferably between 10 MPa-90 MPa, 15 MPa-85 MPa 15 MPa-80 MPa, 20 MPa-70 MPa, 25 MPa-65 MPa, 25 MPa-60 MPa, 25 MPa-55 MPa, 25 MPa-65 MPa, 30 MPa-50 MPa. In another preferred embodiment, the homogenization is carried out ata pressure of 10 Mpa. In another preferred embodiment, the homogenization is carried out at a pressure of 30 Mpa. In another preferred embodiment, the homogenization is carried out at a pressure of 50 Mpa.

[0046] The microalgae cells used in the homogenization step may be for example in the form of a suspension or alternatively in the form of a frozen paste that may be suspended in buffer.

[0047] The second step of the method of the invention requires eliminating cell debris from the homogenized solution obtained in step a).

[0048] “Cell debris”, as used herein, relates to the fragments and leftover material of cells that have been ruptured.

[0049] The elimination of cell debris may be performed by any method known in the art, preferably by a method that preserves the integrity of the exosomes-like vesicles. A person skilled in the art can known whether a method for elimination cell debris from the homogenized solution preserves the integrity of exosomes-like vesicles. The microalgae cells may be removed from the homogenized solution obtained in step a) for example by centrifugation, flocculation, sedimentation or microfiltration. In a preferred embodiment, the elimination of cell cebris is performed by centrifugation at low speed.

[0050] “Low speed”, as used herein relates to a speed between 200-2000 g, preferably 200-400 g, more preferably at 300 g. In a prefered embodiment, the centrifugation can be performed at 300 g for 10 minutes and then 2000 g for another 10 minutes to remove larger cells and debris. Another centrifugation can be performed after the low speed centrifugation, for example a intermediate centrifugation, more particularly a centrifugation step at 10,000 g for 15 minutes. This step can help pellet larger vesicles and some shedding vesicles.

[0051] In another preferred embodiment, the elimination of cell cebris is carried out by filtration, particularly by tangential flow filtration.

[0052] The method of the invention can comprise further steps. In a preferred embodiment, the method of the invention further comprises

[0053] c) adding a surfactant to the fraction free of cell debris, andd) homogenizing the solution resulting from step c).

[0054] “Surfactant”, as used herein relates to a compound that lowers the surface tension or interfacial tension between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents and dispersants. Surfactants have a hydrophobic part and a hydrophilic part. Depending on the nature of the hydrophilic part the surfactants are classified as non-ionic (surfactant with a noncharged but polar hydrophilic part), anionic (when the hydrophilic part contains a negatively charged group), cationic (when the hydrophilic part contains a positively charged group) or amphoteric (when the when the hydrophilic part contains has both cationic and anionic groups). Preferably, the surfactant is non-ionic surfactant.

[0055] The term “non-ionic surfactant” as used herein refers to a compound having a hydrophobic part and a hydrophilic part which allows producing an emulsion.

[0056] Preferably, the surfactant, preferably non-ionic surfactant, is selected from the group consisting of polyoxyl castor oil with 30 to 40 oxyethylene units, in particular polyoxyl 35 castor oil (also known as polyethylene glycol 35 castor oil; marketed as Kolliphor® EL, Cremophor® EL), polyoxyl hydrogenated castor oil with 40 to 60 oxyethylene units, in particular polyoxyl 40 hydrogenated castor oil (also known as polyethylene glycol 40 hydrogenated castor oil; marketed as Cremophor® RH40), polyoxyethylene 20 sorbitan monooleate (also known as polysorbate 80 and marketed as Tween® 80), polyoxyethylene 20 sorbitan monostearate (also known as Polysorbate 60 and marketed as Tween® 60), polyoxyethylene 20 sorbitan trioleate (marketed as Tween® 85), polyoxyethylene 20 sorbitan tristearate (marketed as Tween® 65), polyoxyethylene 20 sorbitan monolaurate (also known as Polysorbate 20), polyoxyethylene 20 sorbitan monopalmitate (also known as Polysorbate 40), sorbitan trioleate, sorbitan tristearate, sorbitan sesquioleate, sorbitan oleate, sorbitan stearate, sorbitan isostearate, sorbitan palmitate, sorbitan laurate, polyethylene glycol hexadecyl ether (marketed as Brij® C10), glyceryl stearate (marketed as Cithrol® GMS40), glyceryl monooleate, glycol stearate, glycol distearate, D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS), polyoxyl steraryl ether with 2 oxyethylene units, polyoxyl castor oil with 2 to 20 oxyethylene units, cetostearyl alcohol, cetyl alcohol, stearyl alcohol, and mixtures thereof. More preferably, the one or more surfactant, preferably non-ionic surfactant, is selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene 20 sorbitan monooleate, polyoxyethylene 20 sorbitan monostearate polyoxyethylene 20 sorbitan trioleate, polyoxyethylene 20 sorbitan tristearate, polyethylene glycol hexadecyl ether, glyceryl stearate, D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS), and mixtures thereof. Still more preferably, the non-ionic surfactant is selected from the group consisting of polyoxyl 35castor oil, polyoxyethylene 20 sorbitan trioleate, polyethylene glycol hexadecyl ether, and mixtures thereof. Still more preferably, the surfactant is selected from the group consisting of polyoxyl 35 castor oil, polyoxyethylene 20 sorbitan monooleate, sorbitan laurate, and mixtures thereof. Even more preferably, the non-ionic surfactant is Polyglyceryl-4 caprate, Polyglyceryl-4 caprylate / caprate, Polyglyceryl-5 caprate, Polyglyceryl-6 caprate, Polyglyceryl-20 sorbitan laurate. Even more preferably, the nonionic surfactant is Polyglyceryl-4 caprate. In another preferered embodiment, the amount of surfactant is below the critical micelle concentration (CMC) so that the membrane of the exosomes can be stabilized minimazing the risk of breaking the membrane.

[0057] The “CMC (critical micelle concentration)” is the concentration of a surfactant in a bulk phase, above which aggregates of surfactant molecules, so-called micelles, start to form. The CMC is an important characteristic for surfactants and it is widely known by a person skilled in the art. The CMC of surfactants can be determined by a number of methods, including tensiometry, conductometry, viscometry, light scattering, fluorimetry, calorimetry, spectrophotometry, and nuclear magnetic resonance (NMR) spectroscopy. The most frequently used methods are tensiometry, fluorimetry, and spectrophotometry.

[0058] What constitutes the CMC will depend on the specific surfactant agent, and technique employed in each case, and can be easily determined by simple experimentation or reference to similar. The CMC of some non-ioinic surfactants are:

[0059] - Polyoxyl 35 Castor Oil (PEG-35 Castor Oil) CMC: - 0.5- 1.5 mM

[0060] - Polyoxyl 40 Hydrogenated Castor Oil (PEG-40 Hydrogenated Castor Oil) CMC: about 1 - 3 mM

[0061] - Polyoxyethylene 20 Sorbitan Monooleate (Polysorbate 80) CMC: about 0.02 - 0.05 mM

[0062] - Polyoxyethylene 20 Sorbitan Monostearate (Polysorbate 60) CMC: about 0.03 - 0.07 mM.

[0063] - Polyoxyethylene 20 Sorbitan Trioleate (Polysorbate 85) CMC: about 0.01 - 0.02 mM.

[0064] - Polyethylene Glycol Hexadecyl Ether (Brij® C10) CMC: about 0.01 - 0.05 mM.

[0065] - Glyceryl Stearate (® GMS40) CMC: - about 0.1 - 0.3 mM.

[0066] - D-a-Tocopherol Polyethylene Glycol 1000 Succinate () CMC: about 0.03 - 0.1 mM.

[0067] - Polyglyceryl esters CMC: 0.5 - 2 mM for polyglyceryl esters.

[0068] - Sorbitan Oleate CMC: about 0.02 - 0.05 mM.

[0069] The concentration of the non-ionic surfactant in the dense fluid composition may be in a range of from about 0.01 % to about 10%, more preferably about 0.01 % to about1 %, based on the total weight / weight of the composition. In a preferred embodiment, the surfactant is added at 0.05 %-5%w / w, more preferably is added at a 0.5% w / w. In a particular embodiment, the amount of surfactant added at 0.05 %-5% w / w, more preferably at a 0.5 %w / w is prior dilution.

[0070] Step d) of the method of the invention requires homogenizing the solution resulting from step c).

[0071] The homogenization of step d) can be performed by several methods known in the art. As a way of illustrative non limitative examples the homogenization of step d) may be carried out by high-pressure, high-shearing or ultrasonication, more preferably by ultrasonication.

[0072] The resulted solution can be further diluted or concentrated to obtain the desired exosome-like vesicles concentration.

[0073] Compositions of the invention

[0074] All the terms, definitions, and embodiments previous disclosed in relation to previous aspects are equally valid for the following aspects and their embodiments.

[0075] In another aspect, the invention relates to a composition comprising exosome-like vesicles obtained by the method of the invention.

[0076] In a preferred embodiment, the exosome-like vesicles of the composition obtained by the method of the invention comprise heat shock proteins, antioxidant enzymes and / or EGF-containing proteins. In a more preferred embodiment, the heat shock protein is HSP60, HSP70 and / or HSP90. In another preferred embodiment, the antioxidant enzymes is thioredoxin, glutathione peroxidase and / or superoxide dismutase.

[0077] In another preferred embodiment, the exosome-like vesicles of the composition obtained by the method of the invention comprises phosphatidylethanolamine, phosphatidylglycerol and / or ceramides. In a preferred embodiment, the phosphatidylethanolamine is PE 34:2 or PE 32:1. In some embodiments, the phosphatidylethanolamine is is found at a concentration of between 1 and 200 ppm. In some embodiments, the phosphatidylethanolamine is PE 34:2 which is found at a concentration of between 1 and 200 ppm.

[0078] In another preferred embodiment, the exosome-like vesicles of the composition obtained by the method of the invention comprises phosphatidylglycerol. In some embodiments, the phosphatidylglycerol is is found at a concentration of between 1 and 500 ppm. In a more preferred embodiment, the phosphatidylglycerol is PG 32:0, PG 34:1 or PG 34:2. In some embodiments, the phosphatidylglycerol is PG 34:2 which is found at a concentration of between 1 and 500 ppm.In another preferred embodiment, the exosome-like vesicles of the composition obtained by the method of the invention comprises ceramides. In a more preferred embodiment, the ceramide is alpha-hydroxy fatty acid-phytosphingosine or non-hydroxy fatty acid-sphingosine. In some embodiments, the ceramide is found at a concentration of 1-200 ppm. In another embodi.ent, the ceramide is non-hydroxy fatty acid-sphingosine which is present at a concentration of between 1 and 200 ppm.

[0079] In a preferred embodiment, the exosomes contain PE 34:2 at a concentration of between 1 and 200 ppm, PG 34:2 at a concentration of between 1 and 500 ppm and non-hydroxy fatty acid-sphingosine at a concentration of between 1 and 200 ppm, In another preferred embodiment, the exosome-like vesicles of the composition obtained by the method of the invention comprise sterols and / or triacylglycerols.

[0080] In a preferred embodiment, the composition comprising exosome-like vesicles obtained by the method of the invention, is characterized by having exosome-like vesicles having a mean size between 100 to 400 nm, more preferaby from 100-200 nm.

[0081] In another preferred embpodiment, the mean size of the exosome-like vesicles obtained by the method of the invention is less than 200 nm. In another preferred embodiment, the mean size of the exosome-like vesicles obtained by the method of the invention is between 150-180 nm preferably between 160-170 nm, more preferably, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, preferably 167 nm.

[0082] The size of the particle a) refers to a mean size. “Mean size” is understood as the average diameter of the vesicle population, moving together in an aqueous medium. The mean size of these systems can be measured by standard methods known by the person skilled in the art and are described, by way of illustration, by TEM, Nanoparticle Tracking Analysis (NTA) or Dynamic Light Scattering (DLS). The mean particle size of various types of particles may differ from the particle size distribution of the particles. For example, a particle can have a mean particle size of about 200 nm while its particle size distribution can generally vary between about 100 nm to about 300 nm. (It should be understood that the particle sizes that are described herein are for particles when they are dispersed in an aqueous medium and the mean particle size is based on the mean of the particle number distribution).

[0083] In another embodiment, the exosome-like vesicles obtained by the method of the invention is characterized by their polidispersity index (PDI). The term “Polydispersity Index” (PDI), often referred to as dispersity (recently recommended by IIIPAC), is used to describe the ‘degree of non-uniformity’ of a distribution, so it is an indicator of the heterogeneity of particle sizes in the sample and is derived from fitting the autocorrelation function. Polydispersity is defined as the weight average divided by the number averagemolecular weight (Mw / Mn), In a preferred embodiment the PDI is less than or equal to 0.4, more preferably less than 0.4. In another preferred embodiment, the PDI is less than or equal to 0.3.

[0084] In another preferred embodiment, the exosome-like vesicles of the composition obtained by the method of the invention shows nanovesicle structure, in particular bilayer composition as well as the size and shape of the nanovesicles.

[0085] In another aspect, the invention relates to a cosmetic composition comprising the composition comprising exosome-like vesicles according to the invention and a cosmetically acceptable excipient.

[0086] The term “cosmetic composition” or “personal care composition”, as used herein, refers to a composition suitable for use in personal hygiene of human beings or animals, or in order to enhance the natural beauty or change the body appearance without affecting the structure or functions of the human or animal body, comprising one or more products providing such effects. If desired, the cosmetic composition provided by the invention can contain, in addition to the active product of the invention, one or more cosmetics or cosmetic products, i.e., substances or mixtures intended to be placed in contact with the external parts of the human or animal body (e.g., epidermis, hair system, nails, lips, etc.) or with the teeth and the buccal mucosa, for the exclusive or main purpose of cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odors.

[0087] “Cosmetically acceptable excipient” or a cosmetically active ingredient is a compound or mixture of compounds that exerts a beneficial effect on the skin such as improving or maintaining the cosmetic qualities of the skin, for example the level of hydration, elasticity, firmness, gloss, tone and texture, among others.

[0088] In a particular embodiment, the cosmetically acceptable excipient is selected from the group consisting of antioxidants, moisturizers, skin conditioning agents, antistatic agents, smoothing agents, soothing agents, emollient agents, astringent agents, antiseborrheic agents, antidandruff agents, tonics, bleaching agents, cleansing agents, keratolytic agents, refatting agents, lifting agents, anti-wrinkle agents, repairing agents, regenerating agents, firming agents, energizing agents, volumizing agents, anti-dark circle agents, anti-puffiness agents, pore minimizing agents, protective agents and mixtures thereof; or antimicrobial agents, anti-inflammatory agents and fungicides .

[0089] An antioxidant is a compound which inhibits reactions promoted by oxygen, thus avoiding oxidation reactions. These compounds also avoid rancidity of the compositions. Non-limiting examples of antioxidants are Camellia sinensis leaf extract, transresveratrol, alpha lipoic acid, nordihydroguaiaretic acid, caffeine, Aloe vera, ascorbylpalmitate, alpha-arbutin, arbutin, Echinacea purpurea extract, niacinamide and astaxanthine.

[0090] A moisturizer is a compound that increases the water content of the skin and helps to keep it soft and smooth. Non-limiting examples of moisturizers are Echinacea purpurea extract, niacinamide and pyroglutamic acid.

[0091] A moisturizer is a compound that increases the water content of the skin and helps to keep it soft and smooth. Non-limiting examples of moisturizers are Echinacea purpurea extract, niacinamide and pyroglutamic acid.

[0092] A skin conditioning agent is a compound that maintains the skin in good condition. Non-limiting examples of skin conditioning agents are Echinacea purpurea extract, Camellia sinensis leaf extract, salicylic acid, biotin, Helichrysum arenarium flower extract, Chamomilla recutita flower extract, Ginkgo biloba leaf extract, Aloe vera, caprylyl glycol, Ranunculus ficaria extract, Centella asiatica extract, Aesculus hippocastanum seed extract, ethyl nicotinate, glycyrrhetinic acid, alpha-arbutin, arbutin, Enantia chlorantha bark extract, astaxanthin, Spilanthes acmella flower extract and benzyl nicotinate.

[0093] An antistatic agent is a compound that reduces static electricity by neutralizing electrical charge on a surface. Non-limiting examples of antistatic agents are niacin, acrylate copolymer and benzyl nicotinate.

[0094] A smoothing agent is a compound that decreases skin roughness or irregularities, thus achieving an even skin surface. Non-limiting examples of smoothing agents are niacin, Spirulina maxima extract, niacinamide and Centella asiatica extract.

[0095] A soothing agent is a compound that helps alleviate discomfort of the skin or of the scalp. Non-limiting examples of soothing agents are methyl nicotinate, Centella asiatica extract and Echinacea purpurea extract.

[0096] An emollient agent is a compound that softens the skin. Non-limiting examples of emollient agents are Camellia sinensis leaf extract, Panax ginseng root extract, glyceryl stearate and caprylyl glycol.

[0097] An astringent agent is a compound that contracts the skin. A non-limiting example of an astringent agent is Camellia sinensis leaf extract.

[0098] An antiseborrheic agent is a compound that helps control sebum production. Nonlimiting examples of antiseborrheic agents are biotin and Enantia chlorantha bark extract.

[0099] An antidandruff agent is a compound that helps control dandruff. A non-limiting example of an antidandruff agent is salicylic acid.

[0100] A tonic is a compound that produces a feeling of well-being on skin and hair. Nonlimiting examples of tonics are Echinacea purpurea extract, Camellia sinensis leaf extract, Panax ginseng root extract, methyl nicotinate and Centella asiatica extract.A bleaching agent is a compound that lightens skin tone or corrects imperfections in the skin’s pigmentation by lessening the concentration of melanin. Non-limiting examples of bleaching agents are glabridin, alpha-arbutin and arbutin.

[0101] A keratolytic agent is a compound that helps to eliminate the dead cells of the stratum corneum. A non-limiting example of a keratolytic agent is salicylic acid.

[0102] A refatting agent is a compound that replenishes the lipids of the hair or of the top layers of the skin. A non-limiting example of a refatting agent is C(6)-ceramide.

[0103] A lifting agent is a compound that that lifts the skin. A non-limiting example of a lifting agent is acrylate copolymer.

[0104] An anti-wrinkle agent is a compound that can reduce fine lines on the skin. Nonlimiting examples of anti-wrinkle agents are ascorbyl palmitate and niacinamide.

[0105] A repairing agent is a compound that helps the body to restore skin alterations, such as scars. Non-limiting examples of repairing agents are Centella asiatica extract and Echinacea purpurea extract.

[0106] A regenerating agent is a compound that helps in skin self-generation by means of mechanisms such as cell stimulation. A non-limiting example of a regenerating agent is Centella asiatica extract.

[0107] A firming agent is a compound that provides firmness to the skin. Non-limiting examples of firming agents having suitable 5D, 5P and bH values are biotin and niacinamide.

[0108] An energizing agent is a compound that provides in a combined manner brightness and astringency to the skin. Non-limiting examples of energizing agents are trans-resveratrol, Panax ginseng root extract, Camellia sinensis leaf extract.

[0109] A volumizing agent is a compound that stimulates skin adipocytes. A non-limiting example of a volumizing agent is Centella asiatica extract.

[0110] An anti-dark circle agent is a compound that reduces pigmented areas under the eyes. Non-limiting examples of anti-dark circle agents are caffeine, Camellia sinensis leaf extract and Gingko biloba leaf extract.

[0111] An anti-puffiness agent is a compound that reduces swelling, generally in the area around the eye. Non-limiting examples of anti-puffiness agents are caffeine, Aesculus hippocastanum seed extract and Chamomilla recutita flower extract.

[0112] A pore minimizing agent is a compound that helps to clean out pores and make them look smaller. A non-limiting example of a pore minimizing agent is Ranunculus ficaria extract.

[0113] A protective agent is a compound that protects the skin against external agents. Non-limiting examples of protective agents are trans-resveratrol, Centella asiatica extract and niacinamide.An antimicrobial agent is a compound that helps to control the growth of microorganisms on the skin. Non-limiting examples of antimicrobial agents are Camellia sinensis leaf extract, ferulic acid, hexyl resorcinol, Enantia chlorantha bark extract and Spilanthes camel la flower extract.

[0114] A fungicide is a compound that helps to control the growth of fungi on the skin. A non-limiting example of a fungicide values is pyraclostrobin.

[0115] An anti-inflammatory agent is a compound that reduces inflammation. A nonlimiting example of an anti-inflammatory agent is glabridin.

[0116] In another aspect, the invention relates to a pharmaceutical composition comprising the composition comprising exosome-like vesicles according to the invention and a pharmaceutically acceptable excipient.

[0117] “Pharmaceutical composition”, as used herein, relates to compositions and molecular entities that are physiologically tolerable and do not typically produce an allergic reaction or a similar unfavorable reaction as gastric disorders, dizziness and suchlike, when administered to a human or animal. Preferably, the term "pharmaceutically acceptable" means it is approved by a regulatory agency of a state or federal government or is included in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0118] “Pharmaceutically acceptable excipient”, as used herein relates to a vehicle, diluent or adjuvant that is administered with the active ingredient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and similars. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 21st Edition, 2005; or “Handbook of Pharmaceutical Excipients”, Rowe C. R.; Paul J. S.; Marian E. Q., sixth Edition.

[0119] Appropriate amounts of a composition of the invention can be formulated with pharmaceutically acceptable excipients and / or carriers to obtain a pharmaceutical composition for use in the medical uses of the invention.

[0120] Suitable pharmaceutically acceptable vehicles include, for example, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethyl cellulose, polyvinylpyrrolidone and similars.

[0121] The pharmaceutical compositions containing the compound of formula (I) as defined above, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof foruse according to the invention can occur at any pharmaceutical form of administration considered appropriate for the selected administration route, for example, by systemic (e.g intravenous, subcutaneous, intramuscular injection), oral, parenteral or topical administration, for which it will include the pharmaceutically acceptable excipients necessary for formulation of the desired method of administration. Additionally, it is also possible to administer the composition comprising the compound of formula (I) as defined above, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof for use according to the invention intranasally or sublingually which allows systemic administration by a non-aggressive mode of administration. Also, intraventricular administration may be adequate. A preferred route of delivery is oral.

[0122] Those skilled in the art are familiar with the principles and procedures discussed in widely known.

[0123] Where necessary, the composition of the invention is comprised in a composition also including a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0124] In cases other than intravenous administration, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, gel, polymer, or sustained release formulation. The composition can be formulated with traditional binders and carriers, as would be known in the art. Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers having well established functionality in the manufacture of pharmaceuticals. Various delivery systems are known and can be used to administer a composition of the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.

[0125] Solid dosage forms for oral administration may include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. At these solid dosage forms, the active compounds can be mixed with at least one inert excipient such as sucrose, lactose or starch. Such dosage forms can alsocomprise, as in normal practice, additional substances other than inert diluents, e.g. lubricating agents such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can be prepared with enteric coatings.

[0126] Liquid dosage forms for oral administration may include emulsions, solutions, suspensions, syrups and elixirs pharmaceutically acceptable containing inert diluents commonly used in the technique, such as water. Those compositions may also comprise adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening agents, flavoring and perfuming agents.

[0127] Injectable preparations, for example, aqueous or oleaginous suspensions, sterile injectable may be formulated according with the technique known using suitable dispersing agents, wetting agents and / or suspending agents. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvents or suspending media.

[0128] For topical administration, the composition of the invention can be formulated as creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid bars, emulsions, microemulsions and similars which may be formulated according to conventional methods that use suitable excipients, such as, for example, emulsifiers, surfactants, thickening agents, coloring agents and combinations of two or more thereof.

[0129] If desired, the combination, the compositoin or pharmaceutical composition for use acording to the invention is incorporated in a fabric, a non-woven fabric or a medical device. Illustrative examples of said fabric, non-woven fabric or medical device include but are not limited to bandages, gauzes, t-shirts, panty hose, socks, underwear, girdles, gloves, diapers, sanitary napkins, dressings, bedspreads, towelettes, adhesive patches, non-adhesive patches, occlusive patches, microelectric patches and face masks.

[0130] In some embodiments, the compositions, cosmethic composition or pharmaceutical composition of the invention, can be added to suitable contact layer dressings, including, for example, thin, non-adherent sheets placed on an area to protect tissue from for example, direct contact with other agents or dressings applied to the treatment site. In some embodiments, contact layers can be deployed to conform to the shape of the area of the skin treatment site and are porous to allow the skin augmentation composition to pass through for absorption onto the skin treatment site.

[0131] Elastic Bandages: suitable elastic bandages can include dressings that stretch and conform to the body contours. In certain embodiment, the fabric composition can include for example, cotton, polyester, rayon or nylon. In certain other embodiments, the elasticbandage can for example, provide absorption as a second layer or dressing, to hold a cover in place, to apply pressure or to cushion a treatment site.

[0132] Foams: suitable foam dressings can include sheets and other shapes of foamed polymer solutions (including polyurethane) with small, open cells capable of holding liquid solution of the skin augmentation composition. Exemplary foams can be for example, impregnated or layered in combination with other materials. In certain embodiment, the absorption capability can be adjusted based on the thickness and composition of the foam. In certain other embodiments, the area in contact with the treatment site can be non-adhesive for easy removal. In yet another embodiment, the foam can be used in combination with an adhesive border and / or a transparent film coating that can serve as an anti-infective barrier.

[0133] Gauzes Non-Woven dressings: suitable gauze dressings and woven dressings can include, dry woven or non-woven sponges and wraps with varying degrees of absorbency. Exemplary fabric composition can include cotton, polyester or rayon. In certain embodiments, gauzes and non-woven dressing can be available sterile or non-sterile in bulk and with or without an adhesive border. Exemplary gauze dressings and woven dressings can be used for moderate to slow release of the skin augmentation composition and covering a variety of wound treatment sites.

[0134] Hydrogels (Amorphous): suitable amorphous hydrogel dressings can include formulations of water, polymers and other ingredients with no shape, designed to donate moisture and to maintain a moist healing environments and or to rehydrate the skin treatment site while concomitantly releasing a therapeutically effective amount of the skin augmentation composition. In some embodiments, hydrogels can be used in combination with a secondary dressing cover.

[0135] Hydrogel Impregnated Dressings: suitable impregnated hydrogel dressings can include gauzes and non-woven sponges, ropes and strips saturated with an amorphous hydrogel. Amorphous hydrogels can include for example, formulations of water, polymers and other ingredients with no shape, designed to donate moisture to a dry treatment site and to maintain a moist healing environment while concomitantly releasing a therapeutically effective amount of the skin augmentation composition.

[0136] Hydrogel Sheets: suitable hydrogel sheets can include for example, three-dimensional networks of cross-linked hydrophilic polymers that are insoluble in water and interact with aqueous solutions by swelling. Exemplary hydrogels are highly conformable and permeable and can release varying amounts of the skin augmentation composition depending on their composition. In some embodiments, the hydrogel is non-adhesive against the skin treatment site or treated for easy removal. The released rate of the skin augmentation composition from the hydrogel can be adjusted depending onthe chemical affinity of the hydrogel for the composition. Generally, the released composition provides an amount of each active agent in the range of about 0.01 mg / cm2 to about 10 mg / cm2 of skin treated.

[0137] All the terms, definitions, and embodiments previous disclosed in relation to previous aspects are equally valid for the present aspects and their embodiments.

[0138] Cosmetic uses of the invention

[0139] All the terms, definitions, and embodiments previous disclosed in relation to previous aspects are equally valid for the following aspects and their embodiments.

[0140] The use of microalgae-derived exosome-like vesicles in skincare offers several advantages:

[0141] a) Rich bioactive content: These vesicles contain a diverse array of bioactive molecules, such as pigments, polyunsaturated fatty acids, antioxidants or antimicrobial compounds, which can modulate key processes in skincare. b) Sustainable production: Microalgae can be cultivated efficiently and are seen as highly productive crops when compared with terrestrial plants. This allows providing a renewable and eco-friendly source of therapeutic vesicles.

[0142] c) Biocompatibility: Generally recognized as safe, they exhibit low toxicity levels.

[0143] Thus, in another aspect, the invention relates to a cosmetic method for reducing the cosmetic effect of aging and / or photoaging and / or for the prevention and / or treatment of non-pathological skin scar which comprises administering the composition comprising exosome-like vesicles according to the invention or the cosmetic composition according to the invention to a subject in need thereof.

[0144] In another aspect, the invention relates to a cosmetic method for skin care which comprises administering the composition comprising exosome-like vesicles according to the invention or the cosmetic composition according to the invention to a subject in need thereof.The term “prevention”, “preventing” or “prevent”, as used herein, relates to avoid the appearance of a particular condition. The prevention may be complete (e.g. the total absence of the condition). The prevention may also be partial, such that for example the occurrence of a scar in a subject is less than that which would have occurred without the administration of the composition of the present invention.

[0145] “Skin care”, as used herein, relates to the care of sensitive, irritable skin, having tendency to pimple, itching and redness, lichenification, xeriss, redness, blistering, oozing, crusting, scaling, thickening and color change. In a particular aspect, the cosmetic composition of the invention is for reducing the cosmetic effects of aging. In another particular aspect, the cosmetic composition of the invention is for anti-wrinkletreatment of the skin. In a preferred embodiment, the cosmetic condition is selected from the group consisting of premature skin aging, chronological aging, photoaging and cutaneous senescence.

[0146] In additional embodiments, the composition or cosmetic composition according to the invention are useful for improving the condition and aesthetic appearance of skin affected by aging, particularly matured or maturing skin, by anyone of the following methods: reducing dermatological signs of chronological aging, photo-aging, hormonal aging, and / or actinic aging; preventing and / or reducing the appearance of lines and / or wrinkles; reducing the noticeability of facial lines and wrinkles, facial wrinkles on the cheeks, forehead, perpendicular wrinkles between the eyes, horizontal wrinkles above the eyes, and around the mouth, marionette lines, and particularly deep wrinkles or creases; preventing, reducing, and / or diminishing the appearance and / or depth of lines and / or wrinkles; improving the appearance of suborbital lines and / or periorbital lines; reducing the appearance of crow's feet; rejuvenating and / or revitalizing skin, particularly aging skin; reducing skin fragility; ameliorating the effects of estrogen imbalance; preventing and / or treating skin atrophy; preventing, reducing, and / or treating hyperpigmentation; minimizing skin discoloration; improving skin tone, radiance, clarity and / or tautness; preventing, reducing, and / or ameliorating skin sagging; improving skin firmness, plumpness, suppleness and / or softness; improving procollagen and / or collagen production; improving skin texture and / or promoting retexturization; improving skin barrier repair and / or function; improving the appearance of skin contours; restoring skin luster and / or brightness; minimizing dermatological signs of fatigue and / or stress; resisting environmental stress; replenishing ingredients in the skin decreased by aging and / or menopause; improving communication among skin cells; increasing cell proliferation and / or multiplication; increasing skin cell metabolism decreased by aging and / or menopause; retarding cellular aging; improving skin moisturization; enhancing skin thickness; increasing skin elasticity and / or resiliency; enhancing exfoliation; improving microcirculation; decreasing and / or preventing cellulite formation; thickening skin tissue (i.e., building the epidermis and / or dermis layers of the skin and where applicable the keratinous layers of the nail and hair shaft), preventing and / or retarding atrophy of mammalian skin, preventing and / or retarding the appearance of spider vessels and / or red blotchiness on mammalian skin, preventing and / or retarding the appearance of dark circles under the eye of a mammal, preventing and / or retarding sallow-colored mammalian skin, preventing and / or retarding sagging of mammalian skin, softening and / or smoothing lips, hair and nails of a mammal, preventing and / or relieving itch of mammalian skin, regulating skin texture (e.g. wrinkles and fine lines), and improving skin color (e.g. redness, freckles) and any combinations thereof.In a more preferred embodiment, the skincare comprises reducing the cosmetic effect of aging and / or photoaging such as thin and dry skin, wrinkles, reduced skin smoothness, decreased elasticity and / or firmness, reduced skin radiance, changes in facial contours or aberrant pigmentation. In another preferred embodiment, the method for reducing the cosmetic effect of aging and / or photoaging comprises reducing thin and dry skin, reducing wrinkles, increasing skin smoothness, increasing elasticity and / or firmness, increasing skin radiance and / or reducing changes in facial contours or aberrant pigmentation.

[0147] “Skin aging”, or cosmetic effect of aging, as used herein relates to a multi-factorial process of the skin that affects nearly every aspect of its biology and function; it is driven by both intrinsic (e.g., time, genetic factors, hormones) and extrinsic (e.g., UV exposure, pollution, cigarette smoke) factors. Skin aging is also produced by senescence. The cosmetic adverse effects of aging relates to characteristics of intrinsic or chronological aging and include as a way of illustrative non limitative examples, visible signs such as thin and dry skin, fine wrinkles, decreased elasticity, aberrant pigmentation, hair graying and hair loss.

[0148] The term “photoaging” relates to the processes due to the prolonged exposure of the skin to ultraviolet radiation or HEV (also known as high energy visible light, or blue light) which result in the premature aging of the skin, and it presents the same physical characteristics as aging, such as and not restricted to, flaccidity, sagging, changes to the color or irregularities in the pigmentation, abnormal and / or excessive keratinization.

[0149] In another aspect, the invention relates to a cosmetic method for prenventing and / or treatment a non-pathological skin scar which comprises administering the composition comprising exosome-like vesicles according to the invention or the cosmetic composition according to the invention to a subject in need thereof.

[0150] “Non-pathological skin scar”, as used herein relates to a scar not causing a pathology. In a preferred embodiment, the non-pathological scar is a flat scar, pervasive scar or atrophic scar.

[0151] “Flat scar” or cicatix, as used herein relats to a mark left on the body from a healed injury.

[0152] “Pervasive (stretched) scar”, as used herein, relates to a scar that appears when a thin line of surgical scar is gradually stretched and widened, usually occurring within 3 weeks after surgery . Such scars are typically flat, pale, soft, asymptomatic scars and are often seen after knee or shoulder surgery. Skin stretch streak (abnormal streak) after pregnancy is a variant of a widespread scar. In the skin stretch streak, there is damage to the dermis and subcutaneous tissue, but the epidermis is not broken. In mature diffusescars, there are no bumps, thickening, or nodules, thereby distinguishing diffuse scars from hypertrophic scars.

[0153] “Atrophic scar” or depressed scar”, as used herein relates to a flat and depressed below the surrounding skin. Atrophic scars are generally small and often round and recessed, i.e., have an inverted center. Atrophic scarring can be the result of surgery, trauma, and common symptoms such as acne vulgaris and chicken pox (chicken pox).

[0154] The cosmetic composition can be administered at any route, for example, by systemic (e.g. intravenous, subcutaneous, intramuscular injection), oral, parenteral or topical administration, preferabkty by topical route. Additionally, it is also possible to administer the cosmetic compositions of the invention as defined above intranasally or sublingually which allows systemic administration by a non-aggressive mode of administration. In a particular embodiment, the cosmetic composition is topically administered. In another particular embodiment is intradermically administered.

[0155] The cosmetic composition of the invention is administered in a cosmetic effective amount.

[0156] The term “cosmetic effective amount”, as used herein, relates to the sufficient amount of a compound (i.e. of the composition of the invention) to provide the desired effect and it will generally be determined, by among other causes, the characteristics of the compound itself and the cosmetic effect to be achieved. The dosage for obtaining a cosmetic effective amount it will also depend on a range of factors, such as, for example, age, weight, sex or tolerance of the animal, preferably a mammal and more preferably human.

[0157] Medical uses of the invention

[0158] All the terms, definitions, and embodiments previous disclosed in relation to previous aspects are equally valid for the following aspects and their embodiments.

[0159] In another aspect, the invention relates to a composition comprising exosome-like vesicles according to the invention or the pharmaceutical composition according to the invention for use in medicine.

[0160] In another aspect, the invention relates to the composition comprising exosome-like vesicles according to the invention or the pharmaceutical composition according to the invention for use in the prevention and / or treatment of wound healing or a skin scar.

[0161] The term “prevention”, “preventing” or “prevent”, as used herein, relates to the administration of a composition according to the invention or of a medicament comprising said composition to a subject who has not been diagnosed as possibly having the disease, but who would normally be expected to develop said disease or be at increased risk for said disease. The prevention intends to avoid the appearance of said disease. The prevention may be complete (e.g. the total absence of a disease). The preventionmay also be partial, such that for example the occurrence of a disease in a subject is less than that which would have occurred without the administration of the composition of the present invention. Prevention also refers to reduced susceptibility to a clinical condition.

[0162] The term “treatment”, as used herein, refers to any type of therapy, which is aimed at terminating, preventing, ameliorating or reducing the susceptibility to a clinical condition as described herein. In a preferred embodiment, the term treatment relates to prophylactic treatment (i.e. a therapy to reduce the susceptibility to a clinical condition), of a disorder or a condition as defined herein. Thus, “treatment,” “treating,” and their equivalent terms refer to obtaining a desired pharmacologic or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder. That is, “treatment” includes (1) preventing the disorder from occurring or recurring in a subject, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or, at least, symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter.

[0163] Alternatively, the invention relates to a method for preventing and / or treating would healing or a skin scar which comprises administering the composition comprising exosome-like vesicles according to the invention or the pharmaceutical composition according to the invention to a subject in need thereof.

[0164] Alternatively, the invention relates to the use of a composition comprising exosome-like vesicles according to the invention or a pharmaceutical composition according to the invention for the preparation of a medicament for the prevention and / or treatment of wound healing or a skin scar.

[0165] Wound healing as used herein relate to is an intricate process in which the skin repairs itself after injury. Healing is the interaction of a complex cascade of cellular events that generates resurfacing, reconstitution, and restoration of the tensile strength of injured skin. Healing is a systematic process, traditionally explained in terms of 4 overlapping steps. Particularly, the pharmaceutical composition or combination of the invention is useful in the proliferation and maturation steps of wound healing.The term "wound" used in the present context means any wound (see below for a classification of wounds) and in any particular phase of the healing process, including the phase before having started any healing or even before a specific wound is produced, such as a surgical incision (prophylactic treatment). Wounds are typically classified in one of four stages depending on the depth of the wound. Thus, stage I wounds are those limited to the epithelium, stage II wounds are those extending to the dermis, stage III wounds are those extending to the subcutaneous tissue and stage IV wounds are those in which the bone is exposed.

[0166] Examples of wounds which can be prevented and / or treated according to the present invention are, for example, open wounds and closed wounds. Open wounds which can be treated with the compositions of the invention include, but are not limited to, burns caused cold or heat, incisions, ulcers, lacerations, abrasions, acne, bite wounds, punctures or gunshot wounds or closed wounds such as contusions or hematomas, lesions of the blood and lymphatic vessels such as Buerger’s disease, lymphedema and ulcus cruris, post-surgery wounds such as wounds after a skin transplant and sutured wounds, decubitus ulcer, pressure ulcer, diabetic ulcer, postherpetic ulcers and lesions by irradiation. Closed wounds which can be treated with the compositions of the invention include, but are not limited to, contusions or hematomas.

[0167] Aseptic wounds, contused wounds, incised wounds, lacerated wounds, nonpenetrating wounds (i.e., wounds in which there is no discontinuity of the skin, but there is lesion in the underlying structures), open wounds, penetrating wounds, perforating wounds, punctured wounds, septic wounds, subcutaneous wounds, etc can also be treated according to the present invention. Examples of sores are decubitus ulcers, aphthae, chrome ulcers, cold ulcers, pressure ulcers, etc. Examples of ulcers are, for example, peptic ulcer, duodenal ulcer, gastric ulcer, gouty ulcer, diabetic ulcer, hypertensive ischemic ulcer, stasis ulcer, ulcus cruris (venous ulcer), sublingual ulcer, submucosal ulcer, symptomatic ulcer, trophic ulcer, tropical ulcer and venereal ulcer, for example caused by gonorrhea (including urethritis, endocervicitis and proctitis). The conditions related to wounds or sores which can be successfully treated according to the invention are burns, anthrax, tetanus, gaseous gangrene, scarlatina, erysipelas, sycosis barbae, folliculitis, impetigo contagiosa or bullous impetigo, etc. There is frequently an overlapping between the use of the terms "wound" and "ulcer" and "wound" and "sore" and, moreover, the terms are often used randomly. Therefore, as has been previously mentioned, in the present context the term "wound" includes the terms "ulcer", "lesion"', "sore" and "infarction" and the terms are used indistinctly unless otherwise indicated.

[0168] The types of wounds to be treated according to the invention also include i) general wounds, such as, for example, surgical, traumatic, infectious, ischemic, thermal,chemical and bullous wounds; ii) specific wounds of the oral cavity, such as, for example, wounds after extractions, endodontic wounds especially in relation to the treatment of cysts and abscesses, ulcers and bacterial, viral or autoimmune lesions, mechanical, chemical, thermal, infectious and lichenoid wounds; herpetic ulcers, aphthous stomatitis, acute necrotizing ulcerative gingivitis and burning mouth syndrome are specific examples; and iii) wounds on the skin, such as, for example, neoplasias, burns (for example, chemical or thermal burns), lesions (bacterial, viral, autoimmune), bite wounds and surgical incisions. Another way to classify the wounds is as i) small loss of tissue due to surgical incisions, minor abrasions and minor bite wounds, or as ii) significant loss of tissue. This latter group includes ischemic ulcers, pressure sores, fistulas, lacerations, severe bite wounds, thermal burns and wounds in donor site (in soft and hard tissues) and infarctions.

[0169] Another type of wounds that can be treated according to the invention include diabetic foot wounds, caused by the disease of the arteries that irrigate the foot and often complicated by periferic nerve damage and infection, that can produce injuries, ulcers and atrophy of skin and gangrene.

[0170] Another further type of wounds treatable by the invention are varicose ulcer wounds, that are a type of venous ulcer characterized by the loss of continuity of the skin on a region with varicose conditions that can appear after a simple trauma.

[0171] In a preferred embodiment the composition of the invention is used for the treatment of a wound in which the wound is selected from the group consisting of an aseptic wound, a contused wound, an incised wound, a lacerated wound, a nonpenetrating wound, an open wound, a penetrating wound, a perforating wound, a punctured wound, a septic wound, a subcutaneous wound, an ischemic ulcer, a pressure ulcer, a fistula, a bite wound, a thermal burn wound, a diabetic foot wound and a donor site wound.

[0172] In another preferred embodiment, the composition of the invention or the pharmaceutical composition of the invention are used for the healing of a wound wherein the wound is selected from the group consisting of an aseptic wound, a contused wound, an incised wound, a lacerated wound, a non-penetrating wound, an open wound, a penetrating wound, a perforating wound, a punctured wound, a septic wound, a subcutaneous wound, an ischemic ulcer, a pressure sore, a fistula, a bite wound, a thermal burn, a donor site wound, a diabetic foot wound and a varicose ulcer wound.

[0173] As used in the present invention “healing” of a wound refers to the physiological process in which the wounded (damaged) area returns to its normal state. If it refers to an open wound, the healing refers to the process by which the skin or mucosa again forms a continuous barrier by means of the increase of connective tissue and of epithelialcells. The person skilled in the art will appreciate that, after the healing, the wounded area can comprise scar tissue which is not identical to the surrounding tissue. The use of the composition of the invention can prevent or reduce the formation of scar or reduce the unpleasant appearance of the scar tissue formed during the process of healing.

[0174] In another preferred embodiment, the composition comprising exosome-like vesicles according to the invention or the pharmaceutical composition according to the invention is for use in the treatment of a skin scar.

[0175] “A skin scar”, as used herein relats to a dermal fiber replacement tissue that results when a wound heals by healing rather than by regeneration. The final appearance is greatly influenced by the time to complete healing 2-3 weeks after wounding. Once a scar is formed, it undergoes a number of characteristic macroscopic and microscopic changes during the maturation process, completing on average one year later.

[0176] In a preferred embodiment, the skin scar is a pathological skin scar.

[0177] “Pathological skin scar”, as used herein relates to scar characterized by the presence of overabundant collagens whose structure and organization are also different from those in unwounded skin. This causes scar tissues to lose some functions performed by normal skin.

[0178] In a prefered embodiment, the pathological skin scar is selected from the group conssting of hypertrophic scar, contracture scar and keloid scar.

[0179] “Hypertrophic scar”, as used herein (raised in red or dark color) are scars that remain elevated within the boundaries of the original lesion and generally regress naturally after the initial injury. Hypertrophic scars are hard, raised, red, ugly, tender and atrophy. They typically occur after burns in the trunk and limbs. Clinically and histologically, hypertrophic and keloid scars are very similar, but unlike keloids, hypertrophic scars expand by pushing the boundaries of the scars, while keloids in the surrounding tissue. Invade. Hypertrophic scars mature and flatten over time. Keloids usually persist in an indeterminate form without treatment. Hypertrophic scars, like keloids, show whorled hyaline collagen bundles with more blood vessels and cells than normal scars.

[0180] “Contracture scar”, as used herein relates to the result of a contractile woundhealing process occurring in a scar that has already been reepithelialized and adequately healed. When a scar forms, the new tissue tends to be tighter and thicker than the skin. Sometimes, this tightening makes it difficult to move. Any scar that limits movement is called a contracture scar. Burns, except for minor ones, often cause a contracture scar.

[0181] “Keloid scar”, as used herein relares to a benign fibrous growths in the dermis that occur after skin trauma. Keloid scars bulge above the skin surface and extend beyond the boundaries of the original wound. These scars are persistent and do not regress overtime. Keloids can be painful. The degree of scarring is not directly proportional to the severity of the original wound

[0182] The pharmaceutical composition of the invention for use in the medical treatments of the invention should be administered in a pharmaceutical effective amount.

[0183] The expression “pharmaceutical effective amount”, as used herein, is understood as an amount capable of providing a therapeutic effect, and which can be determined by the person skilled in the art by commonly used means. The amount of the combination of the invention or the pharmaceutical compositions according to the invention will vary depending upon the subject and the particular mode of administration. Those skilled in the art will appreciate that dosages may also be determined with guidance from Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman and Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.

[0184] The appropriate dosage of the active principle or principles within the combination or pharmaceutical composition will depend on the type of disease to be treated, the severity and course of the disease, whether the composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the peptide or polypeptide, and the discretion of the attending physician. The amount of the combination of the invention or the pharmaceutical compositions according to the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg / kg per day; more preferably about 0.5 to about 100 mg / kg per day. A suitable dosage level may be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg / kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

[0185] The composition or pharmaceutical composition for use according to the invention can be administered at any route, for example, by systemic (e.g. intravenous, subcutaneous, intramuscular injection), oral, parenteral or topical administration, preferably by topical route.Several drug delivery systems are known and can be used to administer the combinations, pharmaceutical compositions, cosmetic compositions of the invention, including, for example, encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules and similars. The required dosage can be administered as a single unit or in a sustained release form.

[0186] Sustainable-release forms and appropriate materials and methods for their preparation are described in, for example, "Modified-Release Drug Delivery Technology", Rathbone, M. J. Hadgraft, J. and Roberts, M. S. (eds.), Marcel Dekker, Inc., New York (2002), "Handbook of Pharmaceutical Controlled Release Technology", Wise, D. L. (ed.), Marcel Dekker, Inc. New York, (2000). In one embodiment of the invention, the orally administrable form of a combination or pharmaceutical composition of the invention is in a sustained release form further comprises at least one coating or matrix. The coating or sustained release matrix include, without limitation, natural polymers, semisynthetic or synthetic water-insoluble, modified, waxes, fats, fatty alcohols, fatty acids, natural semisynthetic or synthetic plasticizers, or a combination of two or more of them.

[0187] ***

[0188] The invention will be described by way of the following examples, which are to be considered as merely illustrative and not limitative of the scope of the invention.

[0189] EXAMPLES

[0190] Example 1. Exosome preparation

[0191] Chlamydomonas frozen paste (C. reinhardtii) was suspended in buffer and gently stirred with a mechanical stirrer. Typical cell count is in the order of 108cells / mL (measured with a Neubauern chamber) . This mixture was then homogenized using a high-pressure homogenizer (FBF Homolab 2.20) and centrifuged to remove cell debris. Starts with centrifugation at low speeds (300 g for 10 minutes and then 2000 g for another 10 minutes) to remove larger cells and debris. Follow up with a centrifugation step at 10,000 g (intermediate centrifugation) for 15 minutes. This step can help pellet larger vesicles and some shedding vesicles. When surfactant is added (typically 0.5%), a second homogenization is done after centrifugation. The homogeneous solution was further diluted 10% with buffer to obtain the desired exosome concentration.To verify the applicability of the method to different microalgae, exosome analogues were prepared using Dunaliella, Spirulina, Isochrysis and Tetraselmis, following the same procedure as for Chlamydomonas.

[0192] Example 2. Exosome characterization

[0193] 2-.1 Transmission electron microscope (TEM): The morphology of the prepared exosomes was verified using a transmission electron microscope (TEM, staining agent: uranyl acetate), with the results presented in Figure 1-2. As shown in Figures, the microalgae-derived exosome-like analogues measures between 100 to 200 nm in size and display a nanovesicle structure.

[0194] 2.2. Nanoparticle tracking analysis (NTA)

[0195] The size and concentration of nanovesicles (10% diluted samples, as described in previous section) were determined using nanoparticle tracking analysis (NTA), and the results are presented in Table 1 below.

[0196]

[0197] As indicated in the Table, the sizes and concentrations of all the samples were found to be at similar levels, confirming that the present invention is applicable to various types of microalgae.

[0198] 2.3.- Lipidomic and proteomic analysis: A lipidomic and proteomic analysis were conducted to characterize some of the exosomes. The lipids and proteins were isolated from the exosomes, followed by their analysis using HPLC-MS, while proteins were lysed, digested into peptides, and analyzed using HPLC-MS / MS.

[0199] These biomimetic vesicles are rich in ceramide and sterols, which are both lipids critical for exosome formation and are both highly beneficial for skin health. Also, high amounts of triacylglycerols were found in the samples. These are essential for the synthesis and function of ceramides, and play a critical role in maintaining the integrity and health of the skin barrier

[0200] Example 3-. Efficacy from In Vitro Studies (wound healing)The study concerns the determination of the in vitro capability of an active to promote wound healing, induced in an in vitro experimental model represented by human skin fibroblast in monolayer.

[0201] The human fibroblast cells were put in well-plate filled with Dulbecco's modification of Eagle medium (DMEM) supplemented with Fetal Bovine Serum 10%. Cells were incubated at 37°C and 5% CO2 until full confluence. An artificial wound was mechanically created in the cell monolayer by sliding the tip of a pipette on the cell monolayer. For each experimental condition 3 replicas were performed. The repairing effect of the test item was determined by acquisition of photographic images of the cell cultures at TO, T2H, T4H, T6H and T8H after the wound creation. At each experimental time, cells were visually checked using an optical microscope Optika at 40X and pictures with camera (Primo Cam HD5) were collected. Pictures were analyzed with Imaged software. The distance between the wound margins for each picture were measured in pm and were recorded in 3 points of the wound length. The statistical variation on wound margins were analyzed by T-test.

[0202] Cells were exposed at a solution containing 1% (v / v) from the 10% diluted samples. The results (see Figure 3, % reduction of the distance vs time) show that the treatment of cell cultures with the test items significantly promotes wound repair.

[0203] Example 4- Efficacy from in vivo studies (anti-aging)

[0204] The study was carried out on 40 (44 included) healthy Caucasian female subjects, aged between 35 and 55 years old, phototype from II to IV (no specific repartition), with normal to dry skin, showing mild to moderate signs of chrono / photoaging such as light to moderate dark spots, fine lines / wrinkles and loss of skin face elasticity.

[0205] The study is performed according to the half-face method: volunteers will apply the same amount of each of the products (active and placebo) according to a randomization scheme.

[0206] The study checks are fixed at baseline (TO), after 14 (T14) and 28 (T28) days of use. INTRUMENTAL EVALUATIONS

[0207] • Skin profilometry (TO, T14, T28)

[0208] Skin surface is quantitatively assessed by Primos 3D (GFMesstechnik GmbH). Primos 3D is a non-contact in vivo skin measurement device based on structured light projection. In conjunction with a comprehensive 3-D measurement and evaluation software, the sensor allows to evaluate skin surface properties (i.e. wrinkle depth, volume, roughness etc.). This study foresees the following evaluation:Wrinkle depth on crow’s feet wrinkles.

[0209] Sa parameter related to skin smoothness on crow’s feet wrinkles.

[0210] • Skin elasticity and firmness (TO, T14, T28)

[0211] The measurement of skin elasticity is based on the suction method using a negative pressure mechanically deforming the skin (Cutometer® method). A Negative pressure (450 mbar) is created in the device and the skin is drawn into the aperture of the probe for 2 seconds and after a defined time (2 seconds) released again. Inside the probe, the penetration depth is determined by a non-contact optical measuring system. The optical measuring system consists of a light source and a light receptor, as well as two prisms facing each other, which project the light from transmitter to receptor. The light intensity varies due to the penetration depth of the skin. The resistance of the skin to the negative pressure and its ability to return into its original position are displayed as curves (penetration depth in mm / time) in real time during the measurement. The used device is the Cutometer® MPA 580, Courage+Khazaka, electronic GmbH). Skin elasticity is measured in the cheek. RO and R2 parameters are measured.

[0212] • Skin radiance (TO, T14, T28)

[0213] Skin radiance (ability to reflect the light) is measured using a spectrophotometer / colorimeter CM 700D (Konica Minolta) by means of the 8° gloss value. When light reach a surface, it is reflected at the equal but opposite angle from the light source; this is called specularly reflected light. This specular component is reflected as if reflected by a mirror. The light that is not specularly reflected, but scattered in many directions, is called diffuse reflectance. The sum of the specular reflectance plus the diffuse reflectance is called the total reflectance. For objects with shiny surfaces, the specularly reflected light is relatively strong and the diffused light is weaker. On rough surfaces with a low gloss, the specular component is weak, and the diffused light is stronger. The measuring geometry d:8° features an optical device which provides diffuse illumination (Ulbricht sphere). The light (Xenon lamp) is projected into a sphere. The interior of the sphere is coated with a white highly reflecting substance (barium sulphate, ceramic, special plastic) which reflects the light manifold. A shutter, an optical element inside the sphere, prevents the directional rays from reaching the measuring sample directly. The sample is positioned at an opening of the sphere and is illuminated from all directions with a close to perfect diffuse light. Through an opening at the top of the sphere the sensor is viewing the surface being measured with an angle of 8° to the vertical. In order to prevent reflection of specular light from the sample surface, the instrument feature a gloss trap. When the trap which is arranged with an angle of -8° to the viewingopening, is open, the light which would otherwise be reflected from the interior wall of the sphere, will be eliminated and can therefore not illuminate the sample. The relation between directional and diffuse reflection allows calculating the gloss component. The measuring system including gloss is named di:8° whilst the measuring system excluding gloss is described as de:8°.

[0214]

[0215] * Statistically significant variation (Student t test vs TO).

[0216] $Statistically significant variation (Student t test: Product active vs Placebo).

[0217] Example 5- Comparative data of the method with and without surfactant.

[0218] Exosomes like vesicles were obtained by different methods'! -4.

[0219] Method 1 comprises homogenization and centrifugation.

[0220] Method 2 comprises adding a surfactant to the biomass, homogenization and centrifugation.

[0221] Method 3 comprises adding a surfactant to the biomass, homogenization, centrifugation and a further homogenization.

[0222] Method 4 comprises homogenization, centrifugation, adding surfactant and homogenization.

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[0229]

[0230] Table 2.

[0231] The comparative evaluation of different process sequences demonstrated that the inclusion of the surfactant after centrifugation and thus after homogenization, followed by a second homogenization (method 4), resulted in the smallest and most uniform vesicles. Accordingly, method 4 was chosen as the optimized process.

[0232] Example 6- Encapsulation of compounds for skin delivery

[0233] a) Encapsulation step

[0234] Exosome-like vesicles were obtained from Chlamydomonas following the procedure previously described (method 4). Phycocyanin was selected as a fluorescent marker to evaluate the loading capacity of exosome-like vesicles and to assess the skin delivery performance of these vesicles compared to conventional liposomes.

[0235] The encapsulation of phycocyanin was performed during the second homogenization step, by adding the compound together with the surfactant prior to homogenization. This approach promotes efficient incorporation of the compound while maintaining vesicle integrity. Alternatively, encapsulation can also be carried out after vesicle formation, using a gentle homogenization step to facilitate compound incorporation. In both cases, mild conditions were maintained to preserve vesicle structure and compound stability.

[0236] b) Transepidermal penetration studyThree different samples (phycocyanin in buffer, encapsulated in liposomes and encapsulated in exosome-like vesicles) were applied on the surface of the cultured tissue for 24 h. Tissues were then rinsed several times with 25 mL PBS and dried on a sterile absorbent paper. After that, tissues were immediately included in Optimal Cutting Temperature (OCT) compound, cut in sections of 20 pm and placed on a slide. Slides with tissues sections were mounted with DAPI and a coverslip and observed under a fluorescence (confocal) microscope for the localization of stained nuclei and phycocyanin-labelled active present in tested products. Images were acquired and quantitative analysis of the activity was performed using Imaged software. After 24 hours of treatment on reconstructed full-thickness skin models, the fluorescence signal corresponding to phycocyanin was detected in both the epidermis and dermis layers for all formulations. Results were expressed in table 3 as the ratio of dermal to epidermal delivery.

[0237]

[0238] Table 3.

[0239] This study confirmed the successful incorporation of the compound into both vesicle types, demonstrating that the process enables efficient loading of hydrophilic molecules while maintaining their physicochemical properties and vesicle stability. Furthermore, the results indicate that exosome-like vesicles achieve deeper skin penetration, reaching the dermal layer 2.5 times more efficiently than liposomes, while remaining less retained in the epidermis. In contrast, liposomes tend to accumulate in the upper epidermal region, limiting deep delivery.

Claims

34CLAIMS1- A method for obtaining a composition comprising exosome-like vesicles from microalgae cells comprisinga) homogenizing microalgae cells by high-pressure,b) eliminating cell debris from the homogenized solution obtained in step a), c) adding a surfactant to the fraction free of cell debris, andd) homogenizing the solution resulting from step c).2- The method according to claim 1 wherein the homogenization of step a) is carried out at a pressure between 5 MPa-100 MPa.3- The method according to claim 2 wherein the homogenization of step a) is carried out at a pressure selected from the group consisting of about 10 MPa, about 30 MPa and about 50 MPa.4- The method according to any one of claims 1 to 3, wherein the elimination of cell debris is carried out by centrifugation at low speed or by filtration.5- The method according to claim 4 wherein the filtration is tangential flow filtration. 6- The method according to any one of claims 2 to 5, wherein the surfactant is a nonionic surfactant.7- The method according to claim 6, wherein the surfactant is added at 0.05 %-5%. 8- The method according to claim 7, wherein the surfactant is added at 0.5%.9- The method according to any one of claims 1 to 8, wherein the homogenization of step d) is carried out by high-pressure, high-shearing or ultrasonication.10- The method according to any one of claims 1 to 9, wherein the microalgae is a flexible or thin-cell wall microalgae.11- The method according to claim 10, wherein the microalgae is from a phylum selected from the group consisting of Chlorophyta, Cyanophyta, Rhodophyta, Haptophyta and Heterokontophyta .12- The method according to claim 11 , wherein the microalgae is from a genus selected from the group consisting of Chlamydomonas, Dunaliella, Spirulina, Tetraselmis and Isochrysis.3513- The method according to claim 12, wherein the microalgae is from the genus Chlamydomonas.14- The method according to claim 13 wherein the microalgae is C. reinhardtii.15- Composition comprising exosome-like vesicles obtained by the method according to any one of claims 1 to 14.16- Composition comprising exosome-like vesicles according to claim 15, characterized by having a mean size of between 100 to 400 nm.

17. Composition comprising exosome-like vesicles according to any of claims 15 or 16, characterized by having a Polydispersity Index of less than 0.4.18- A cosmetic composition comprising the composition comprising exosome-like vesicles according to any one of claims 15 to 17 and a cosmetically acceptable excipient.19- A cosmetic method for reducing the cosmetic effects of aging and / or photoaging and / or for the prevention and / or treatment of non-pathological skin scar which comprises administering the composition comprising exosome-like vesicles according to any one of claims 15 to 17 or the cosmetic composition according to claim 18to a subject in need thereof.20- The cosmetic method according to claim 19, wherein the method for reducing the cosmetic effect of aging and / or photoaging comprises reducing thin and dry skin, reducing wrinkles, increasing skin smoothness, increasing elasticity and / or firmness, increasing skin radiance and / or reducing changes in facial contours or aberrant pigmentation.21- A pharmaceutical composition comprising the composition comprising exosome-like vesicles according to any one of claims 15 to 17 and a pharmaceutically acceptable excipient.22- The composition comprising exosome-like vesicles according to any one of claims 15 to 17 or the pharmaceutical composition according to claim 21 for use in medicine.23- The composition comprising exosome-like vesicles according to any one of claims 15 to 17 or the pharmaceutical composition according to claim 21 for use in the prevention and / or the treatment of wound healing or a skin scar.

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