Suspensions with high concentrations of exosomes and cosmetic uses thereof

Abstract

The present disclosure provides a process for obtaining a concentrated extracellular vesicles suspension. The method can include fermenting tea (camellia sinensis) with a combination of at least one yeast and bacterium to obtain a fermentation product comprising a liquid material; filtering the fermentation product through a filtering device to give a permeate; filtering the permeate through a second filtering device to give a suspension comprising extracellular vesicles; and recovering the suspension of extracellular vesicles.

Description

9&7.52 NQ2SUSPENSIONS WITH HIGH CONCENTRATIONS OF EXOSOMES ANDCOSMETIC USES THEREOFCLAIM OF PRIORITY

[0001] This patent application claims the benefit of priority to French Application Serial No. 2412645, filed November 19, 2024, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The present invention relates to a method of purifying suspensions or obtaining a concentrated extracellular vesicles suspension, to suspensions of extracellular vesicles obtainable by said process and to cosmetic compositions comprising such suspensions, in particular for improving the aesthetic appearance of skin of a human, in one example when affected by weakened skin barrier, and for treating or preventing senescence.BACKGROUND OF THE INVENTION

[0003] The present invention is in the field of cosmetic preparations for improving the aesthetic appearance of the skin.

[0004] Cell-secreted extracellular vesicles (EVs) are vesicles that have an interior aqueous core surrounded by a lipid bilayer membrane and are loaded with proteins, nucleic acids, lipids, and other biomolecules. After being released into the extracellular environment, some of these vesicles are delivered to recipient cells; consequently, the target cell may undergo physiological or pathological changes. Thus, extracellular vesicles as biological nano-carriers, have a pivotal role in facilitating long-distance intercellular communication.

[0005] EVs are produced and released constitutively into the extracellular space by almost all cell types in both prokaryotes and eukaryotes (animals, plants, fungi, yeasts and bacteria) and vary in size between about 50 nm and 500 nm, depending on their precise origin. Owing to their natural origin, EVs present good biocompatibility and low toxicity; they present enhanced stability in the blood due to the evasion of the immune system, and in addition, have limited immunogenicity and are biodegradable.9&7.52 NQ2

[0006] Traditionally, EVs have been classified into three major types, depending on their size and biogenesis prior to secretion: apoptotic bodies, with relatively large size (100-500 nm), variable in structure and composition; they are typically released by cells undergoing apoptosis;- microvesicles, with a diameter size of 100 nm-400 pm; exosomes (EXOs), with a diameter size ranging from 30 to 200 nm; they are intracellularly produced into organelles called multivesicular bodies (MVBs) and ultimately, they are released into the extracellular milieu as a result of MVBs fusion with the plasma membrane.

[0007] Exosomes contain a multitude of substances like proteins, nucleic acids, carbohydrates, lipids and other compounds.

[0008] Several methods are available to characterise EVs, like for example Conventional Western blotting, mass spectrometry, flow cytometry (bead coupled), microfluidics chips, polymerase chain reaction, and nuclear magnetic resonance (NMR). However, none of these technologies is sufficient to characterise EVs: presently, electron microscopy (EM) is the only method that allows determining simultaneously their size, shape, morphology-integrity, interparticle interaction, and spatial relationship with tissues and cells

[0009] A further difficulty in the study of EVs is that no available purification method can strictly separate them based on their size.

[0010] Thus, there remains the need to develop a reliable method of isolation of high enough concentrations of exosomes in a definite size range to be able to observe their activity when adequately formulated, for example as cosmetic formulations.

[0011] As described in W02004 / 0122650, Kombucha (INCI: Saccharomyces / Xylinum / Black Tea Ferment) is a fermented tea which has undergone a microbiological transformation.

[0012] Skin aging is a multi-factorial process 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) factors. The apparent signs of intrinsic ageing include the loss and descent of underlying fat, leading to hollowed cheeks and eye sockets, as well as loss of firmness and sagging skin as the bones shrink away from the skin due to bone loss. Other9&7.52 NQ2 visible signs include thin and dry skin, fine wrinkles, decreased elasticity, aberrant pigmentation, hair greying and hair loss.

[0013] Intrinsic and extrinsic aging are partly due to the process of senescence and represent an inevitable change attributable to the passage of time alone and is represented primarily by physiologic alterations with subtle but undoubtedly important consequences for both healthy and diseased skin and is largely genetically determined. Senescence, and in particular skin senescence, is a continuous process that usually begins in the mid-20s but may not become evident for decades. In a person’s 20s collagen production begins to slow and elastin has a bit less spring. Dead skin cells do not shed as quickly, and turnover of new skin cells decreases. The process of intrinsic skin aging resembles that seen in most internal organs and an explanation is thought to involve decreased proliferative capacity leading to cellular senescence, and altered biosynthetic activity of skin derived cells The intrinsic rate of skin aging in any individual can be dramatically influenced by personal and environmental factors, particularly the amount of exposure to ultraviolet light. For example, photodamage, which considerably accelerates the visible aging of skin, also greatly increases the risk of cutaneous neoplasms: the processes of intrinsic and extrinsic aging are superimposed. It is essential to stress however that the terms aging and cellular senescence cannot be used interchangeably: the term 'aging' encompasses the overall process of becoming older, including the physical and mental decline in our health. Senescence, however, refers to the aging process on a cellular level, focusing on the microscopic changes that occur within our cells throughout our lifetime.

[0014] W02018 / 004145 discloses an anti-inflammatory composition containing a yeast- derived extracellular vesicle as an active ingredient, and an anti-inflammatory composition containing, as an active ingredient, a food-derived extracellular vesicle containing yeast. The yeast may be Saccharomyces cerevisiae.

[0015] WO2023 / 282508 discloses a cosmetic composition for anti-aging, skin regeneration, skin elasticity improvement or skin-wrinkle reduction containing, as an active ingredient, exosomes derived from Saccharomyces cerevisiae and a lysate thereof.SUMMARY OF THE INVENTION

[0016] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to9&7.52 NQ2 identify required or essential features of the subject matter of the present invention. Nor is this summary intended to be used to limit the scope of the subject matter of the present invention.

[0017] The present inventors have surprisingly found that a concentrated extracellular vesicles (EVs) suspension can be obtained by a process comprising the steps of: i. Fermenting tea (Camellia sinensis) with a combination of at least one yeast and one bacterium to obtain a fermentation product comprising a liquid material and a non-soluble scoby; ii. Optionally pre-cleaning the fermentation product by at least one centrifugation and / or filtration; iii. Filtering the product of step i. or ii. through a 150-450 nm filtering device to give a permeate; iv. Filtering the permeate of step iii. through a 30-80 nm filtering device to give a retentate comprising a suspension of extracellular vesicles; v. Recovering the suspension of extracellular vesicles; and vi. Optionally concentrating the suspension of extracellular vesicles.

[0018] In the priority application FR2412645 the “liquid material” was called “soluble material” and the “non-soluble scoby” was called “non-soluble material”.

[0019] The suspension thus obtained contains a high concentration of extracellular vesicles in the range between about 4.2* 109and 8.8* 1011particles / ml, the highest concentration being the product of the present invention further purified with a phosphate buffer solution (PBS).Compared to other purified EV’s this is about one order of magnitude more particles / ml than is known in W02018 / 004145 (1.3*1010to 1.2*10nparticles / ml) or WO2023282508 (1.0*108to 1.0* 1010particles / ml).

[0020] The mean particle size of the extracellular vesicles thus obtained can be in the range of about 112 nm ± 10 nm at the time of production.

[0021] The present inventors have further surprisingly found that the method above allows the formulation of the EVs in a cosmetically acceptable vehicle at concentrations in the range between 0.05% wt and 10% wt, which corresponds to about 2.1 x 106to 8.8x 1010EVs / ml.9&7.52 NQ2

[0022] At these concentrations, the thus obtained formulations can be used in the cosmetic and medical fields, in particular for improving the aesthetic appearance of the skin, for improving the skin barrier function and for treating and / or preventing senescence.

[0023] Both the foregoing summary and the following detailed description provide examples and are explanatory only. Accordingly, the foregoing summary and the following detailed description should not be considered to be restrictive. Further, features or variations may be provided in addition to those set forth herein. For example, certain aspects may be directed to various feature combinations and sub-combinations described in the detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1 shows the transmission electron microscopy pictures of a commercial Kombucha and of Samples 1 and 2 according to the invention. Arrows indicate structures compatible with extracellular vesiclesDEFINITIONS

[0025] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0026] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.9&7.52 NQ2

[0027] All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0028] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range and includes the exact stated value or range. The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%.

[0029] The term “skin biomarkers” as used herein refers to a measurable indicator of some biological state or condition of skin cells, including without limitation those related to Skin barrier (e.g. CASP14, FLG, IVL, LOR, SPRR1A, SPRR2A, TGM1, CDSN, CLDN1, CLDN4, OCLN, ZO-1, KLK5, KLK7, EGR3, KIF3A, MTA3, ABCA12, CerS3, PNPA1, GBA, SMPD1, ELOVL1, ELOVL4, FASN, SCD, HMGCR, HMGCS1, PPARa), Hydration (e g. AQP3, AQP9, BLMH, HAS1, HAS2, HAS3, HYAL1, PPARG, TRPV3, TRPV4), Dermal Epidermal Junction (e.g. Col IV, Col VII, Col XVII, Col XVIII, nidogen), Extra Cellular Matrix (e.g. Col I, Col III, Col V, Col VI, Col XII, Col XIV, Col 17, DCN, FN, FMOD, PLOD1, BMP1, CLU, POSTN, HSP47, CD44, HAS1, HAS2, MMPs, TIMPs, PXN, VCL, ITGA2, ELN, EMILIN1, EMILIN2, FBN, FBNL, MFAP2, 4, 5, LOXL1), AntiInflammation (e.g. Cytokines, TNFa, PGE2, eicosanoids, leukotriens, IFN, NFkB), both senolytic and senom orphic cellular senescence (P21, IL-6, IL-8, TNFa, b-galctosidase and any other possible markers for senolytic and / or for senomorphic action with other SASP components), Metabolism energetic and mitochondrial activity (e.g. ATP synthesis, membrane potential, mass mitochondrial, fusion / fission) and Adipocytes differentiation (e.g. stimulation of G3PDH activity).

[0030] The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or9&7.52 NQ2 less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0031] The term “scoby” is an acronym for Symbiotic Culture of Bacteria and Yeast, a living culture responsible for converting sweet tea into tangy kombucha. It assumes the form of a rubbery, pancake-shaped substance that is typically beige or brown in color which separates out during fermentation.

[0032] When percentages of ingredients in compositions are indicated, the standard error applies. For example, 3.0% is to be interpreted as 2.9% to 3.1%

[0033] The term “comprises” and variations thereof is to be interpreted as meaning both “includes”, “substantially consists of and “consists o , and variations thereof.

[0034] If any given chemical entity is generally known in the art to belong to two or more cosmetic functional classes (is for example known to be both a film former and an emollient) and those functional classes are part of the definition of the claimed subject matter, that chemical entity may be present only once in any embodiment of the claimed invention, i.e. it can belong to only one of those two or more functional classes.

[0035] When an embodiment is herein described which further specifies one or more features of the present invention it is intended that this embodiment can be combined with another embodiment further specifying one or more other features of the present invention into a further embodiment specifying those features combined even when such combined embodiment is not explicitly described herein.DETAILED DESCRIPTION OF THE INVENTION

[0036] The present inventors have surprisingly found that a concentrated extracellular vesicles (EVs) suspension can be obtained by a process comprising the steps of: i. Fermenting tea (Camellia sinensis) with a combination of at least one yeast and one bacterium to obtain a fermentation product comprising a liquid material and a scoby; ii. Optionally pre-cleaning the fermentation product by at least one centrifugation and / or filtration;9&7.52 NQ2 iii. Filtering the product of step i. or ii. through a 150-450 nm filtering device to give a permeate; iv. Filtering the permeate of step iii. through a 30-80 nm filtering device to give a retentate comprising a suspension of extracellular vesicles; v. Recovering the suspension of extracellular vesicles; and vi. Optionally concentrating the suspension of extracellular vesicles.

[0037] The fermentation step is carried out in a way well known to the skilled person. The ideal temperature range for fermenting Kombucha is 24-30°C and the brewing time is anywhere from 10-12 days in summer and 2 weeks or longer in winter.

[0038] The fermentation is typically done in the presence of a symbiote of a yeast of the genus Saccharomyces which nidifies within a polysaccharide matrix produced by a xylinum bacterium.

[0039] The exact composition of this symbiose (in particular the proportions of each of the species) varies with the geographical and climatic conditions and depends on the wild local subspecies of yeast and bacteria; nevertheless, mention may be made, among others, of the yeasts Dekkera / Brettanomyces bruxellensis, Hanseniaspora valbyensis Saccharomyces cerevisiae, Saccharomyces ludwigii, Saccharomyces apicalutus species and / or Schizosaccharomyces pombe.

[0040] Similarly, the bacterium may belong to the genera Acetobacter, Liquorilactobacillus and / or Gluconobacter.

[0041] . In the literature on kombucha, this symbiote of yeasts and bacteria is also called"fungus", "long-life winner" and many synonyms thereof.

[0042] In the fermentation, the alcohol produced by the yeast is transformed by the bacterium into various acids (glucuronic, lactic, usnic and especially acetic acids), which give its acidulated taste to kombucha with a final pH of between 2,5 and 4. On the other hand, the xylinum bacterium, termed "acetic bacterium", also uses the sugar present in tea and transforms the sucrose into cellulose microfibrils, thus constituting the support membrane in which the yeast nidifies and develops, the so-called Scoby.

[0043] The products of yeast metabolism are excreted in kombucha and consist of numerous vitamins such as Bl, B2, B3 and B12, cofactors essential to the growth of bacteria.9&7.52 NQ2

[0044] The fermentation product comprises a liquid material and a scoby, which is a nonsoluble rubbery aggregated material. The liquid material comprises extracellular vesicles contaminated by debris and other materials from the fermentation process.

[0045] In the priority application FR2412645 the “liquid material” was called “soluble material” and the “non-soluble scoby” was called “non-soluble material”.

[0046] In order to eliminate the contaminating debris and other undesirable materials from the liquid material a gentle pre-cleaning step is optionally carried out. This is done by centrifugation and or filtration. Multiple repetitions of the pre-cleaning step may be necessary so that the following step can be performed without clogging the filter.

[0047] Centrifugation may be performed with a batch or continuous equipment, generally with a centrifugal force from 1000 xg to 10000 xg.

[0048] Pre-filtration may be performed to remove debris and big particles with a dead-end filter or tangential flow filter. It is well understood that the pore size of the filter will be higher than the pore size of the biggest filter described in the invention, for example 250 micrometer, 16 micrometers, 10 micrometers, and up to 450 nanometers. The preferred material of the prefilter is borosilicate glass or polyether sulphone. Notwithstanding, any other choice of inert material is possible to pre-filter the liquid.

[0049] The next step is filtration of the liquid material of the fermentation product through a 150-450 nm filtering device to separate out the scoby. The filtering device used in this step is commonly known to the skilled person. The liquid material is collected as the (first) permeate, whereas the (first) retentate, i.e. the scoby, is discarded.

[0050] The permeate thus obtained is subjected to a second filtration through a finer, 30-80 nm filtering device. The filtering device used in this step is commonly known to the skilled person. In this case, what is retained is the (second) retentate comprising extracellular vesicles suspended in the medium (i.e. water), whereas the (second) permeate is discarded.

[0051] This is followed by an optional step of concentration of the suspension of extracellular vesicles thus obtained, for example by lyophilization. In one embodiment during concentration the volume of the suspension is reduced to between 1 / 3 and 1 / 50 of its original volume, preferably to 1 / 5 of its original volume.9&7.52 NQ2

[0052] Optionally, the (concentrated) suspension of extracellular vesicles thus obtained is treated to microbiologically stabilize it. In one embodiment stabilization is achieved by addition of a polyol, preferably glycerol, and a preservative.

[0053] The suspension thus obtained contains a high concentration of extracellular vesicles in the range between about 4.2* 109and 8.8* 1011particles / ml, the highest concentration being the product of the present invention further purified with PBS. Compared to other purified EV’s this is about one order of magnitude more particles / ml than is known in W02018 / 004145 (1.3*1010to 1.2*10nparticles / ml) or WO2023282508 (1.0*108 to 1.0*1010particles / ml).

[0054] The present inventors have further surprisingly found that the method above allows the formulation of the EVs in a cosmetically acceptable vehicle at relatively high concentrations in the range between 0.05% wt and 10% wt, which corresponds to about 2.1 x 106to 8.8x 1010EVs / ml.

[0055] The thus obtained suspensions can be formulated with cosmetically acceptable excipients to obtain cosmetic compositions, in particular cosmetic compositions suitable for dermal application.

[0056] The cosmetic composition is in particular water-based or emulsion-based.

[0057] In embodiments where the carrier is emulsion-based, the composition may be in the form of a water-in-oil, an oil-in-water, a water-in-oil-in-water or an oil-in-water-in-oil emulsion.

[0058] In embodiments where the carrier is water-based, water may be present at a level of about 40% or more, about 45% or more, about 50% or more, about 55% or more, or about 60% or more by weight of the composition.

[0059] The water-based carrier may be 100%) water or it may comprise components other than water. These may be components known for use in cosmetic formulations. They may include, but are not limited to, agents such as water-soluble moisturising agents, conditioning agents, anti-microbials, humectants and / or other water- soluble skin care actives.9&7.52 NQ2

[0060] In another embodiment, the carrier may be oil or wax based. The oil may be natural oil or synthetic oil, but preferably is natural oil such as a vegetable oil or a nut oil. The oil may be liquid or solid. The wax is preferably a natural wax.

[0061] Combinations of one or more oils and / or one or more waxes may be used.

[0062] Liquid oils that can be mentioned include avocado oil, Camellia oil, turtle bean oil, macadamia nut oil, com oil, mink oil, olive oil, Canoga oil, egg yolk oil, sesame seed oil, Persic oil, wheatgerm oil, Camellia sasanqua oil, castor oil, linseed oil, safflower oil, sunflower oil, grapeseed oil, apricot oil, shea oil, sweet almond oil, cotton oil, evening primrose oil, palm oil, perilla oil, hazelnut oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, rapeseed oil, alfalfa oil, Chinese tung tree wood oil, Japanese tung tree wood oil, jojoba oil, germ oil, poppyseed oil, pumpkin oil, blackcurrant oil, millet oil, barley oil, quinoa oil, rye oil, candlenut oil, passionflower oil, musk rose oil, triglycerine, glyceryl trioctanoate, and glyceryl triisopalmitate.

[0063] Solid oils / fats that can be mentioned include cocoa butter, coconut butter, horse fat, hardened coconut oil, palm oil, beef tallow, mutton tallow, hardened beef tallow, palm kernel oil, lard, Japan wax kernel oil, hardened oil, Japan wax, shea butter, and hardened castor oil.

[0064] Waxes that can be mentioned include beeswax, candelilla wax, carnauba wax, lanolin, lanolin acetate, liquid lanolin, sugar cane wax, fatty acid isopropyl lanolin, hexyl laurate, reduced lanolinjojoba wax, hard lanolin, polyoxyethylene (POE), lanolin alcohol ether, POE lanolin alcohol acetate, lanolin fatty acid polyethylene glycol, and POE hydrogenated lanolin alcohol ether. In one embodiment the carrier is not lanolin based.

[0065] Ester oils that can be mentioned include C 12-C15 alcohols benzoate, tridecyl salicylate, dibutyl adipate, isopropyl myristate, cetyl octoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyloleate, hexyldecyl dimethyl octoate, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, 12-hydroxy cholesteryl stearate, di-2-ethylhexylic acid ethyleneglycol, dipentaerythritol fatty acid ester, N-alkylglycol monoisostearate, neopentylglycol dicaprate, diisostearyl malate, glyceryl di-2-heptyl undecanate, tri-methylol propane tri-2-ethylhexyl acid, tri-methylol propane triisostearate, pentaerythritol tetra-2-ethylhexyl acid, glyceryl tri- 2- ethyl -hexanoate, tri-methylol propane triisostearate, cetyl-2-ethylexanoate, 2-ethylhexyl- palmitate, glycerine trimyristate, glyceride tri-2-heptyl undecatoic acid, methyl ester of castor9&7.52 NQ2 oil fatty acid, oleate oil, acetoglyceride, palmitate-2-heptyl undecyl, diisopropyl adipate, N- lauroyl-L-glutamic acid-2-octyldodecil ester, di-2-heptylundecyl adipate, di-2- ethylhexyl sebacate, myristate-2-hexyldecyl, palmitate-2-hexyldecyl, adipate-2-hexyldecyl, diisopropyl sebacate, and succinate-2-ethylhexyl.

[0066] Higher fatty acids that can be mentioned include lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, 12-hydroxy-stearic acid, undecylenic acid, lanolin fatty acid, isostearic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid. Higher alcohols of straight / branched chain that can be mentioned include lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol, monostearyl glycerine ether (batyl alcohol), 2-decyltetradecinol, lanolin alcohol, cholesterol, phytosterol, hexyldodecanol, isostearyl alcohol, octyl dodecanol.

[0067] The cosmetic composition of the invention may be provided in any form suitable for topical application to the skin. Typical formulation types of the present invention are oil- in-water emulsions, water-in-oil emulsions, multiple emulsions (Water / Oil / Water or Oil / Water / Oil), microemulsions, nanoemulsions, suspensions, hydrodispersions, gels, ointments, pastes, aerosols, foams, sprays, aqueous gels, powders, foundations, transdermal patches, creams or masks.

[0068] The cosmetic composition of the invention may be provided in a form for enteral or parenteral administration, in particular intradermal or subcutaneous administration.

[0069] The cosmetic composition of the invention will generally further comprise other ingredients or excipients which will be well known to those skilled in the art.

[0070] The cosmetic composition of the invention may further comprise one or more humectants, including but not limited to glycerin, propylene glycol, propanediol, butylene glycol, pentylene glycol, hexylene glycol, hexanediol, dipropylene glycol, polyethylene glycol, sorbitol, sodium hyaluronate, urea, xylitol, lactitol, fructose, glucose, mannose, xylose, honey, pyrrolidone, and carboxylic acid and salts thereof. When present, the one or more humectants may be present in the cosmetic composition in an amount of from about 0.01 % to about 20% by weight of the composition, e.g. from about 0.1 % to about 10%>, or about 0.5% to about 7% by weight of the composition.9&7.52 NQ2

[0071] The cosmetic composition of the invention may further comprise one or more emollients, including but not limited to PPG- 15 stearyl ether, ethylhexyl stearate, cetyl dimethicone, octyldodecanol, PPG-20 methyl glucose ether, isopropyl myristate, isopropyl paltimate, isopropyl laurate, isodecyl laurate, isodecyl neopentanoate, isohexadecane, pentaerythrityl tetraisostearate, caprylic / capric triglyceride, canola oil, sunflower oil (H. annus), olive oil (Olea europea), cottonseed oil (Gossypium herbaceum), jojoba oil (Simmondsia chinensis), shea butter (Butyrospermum parkii), cocoa butter (Theobroma cacao), cupuacu butter {Theobroma grandiflorum), avocado oil (Persea gratissima), liquid paraffin, dimethicone, phenyl trimethicone, cyclopentasiloxane, dimethiconol , sodium hyaluronate, bisaccharide gum, isononyl isononoate, carnauba wax and / or petrolatum. When present, the one or more emollients may be present in the cosmetic composition in an amount of from about 0.01 % to about 20%> by weight of the composition, e.g. from about 0.1 %) to about 10%), or about 0.5%> to about 7%> by weight of the composition. The cosmetic composition may further comprise one or more emulsifiers , including but not limited to steareth-2, steareth-21 , steareth- 10, ceteareth-5, ceteareth-20, cetearyl glucoside, oleth- 10, glyceryl stearate, polyglycerol-3 oleate, polyglyceryl-3 methylglucose distearate, sodium stearate, PEG- 12 oleate, PEG-2 stearate, PEG- 12 stearate, PEG- 100 stearate, cetyl alcohol, cetearyl alcohol, potassium cetyl phosphate, cetearyl olivate, sorbitan olivate, PEG-80 sorbitan, sorbitan oleate, and / or sorbitan palmitate. In embodiments where one or more emulsifiers are present in the cosmetic composition, the one or more emulsifiers may be present in an amount of about 0.1 % to about 10%> by weight of the composition, about 0.25% to about 7.5% by weight of the composition, or about 0.5% to about 6%> by weight of the composition. In one embodiment where one or more emulsifiers are present in the cosmetic composition, the one or more emulsifiers are present in an amount of about 0.5% to about 5% by weight of the composition.

[0072] The cosmetic composition of the invention may further comprise one or more surfactants, including but not limited to , anionic surfactants (e.g. sodium lauryl sulphate, sodium laureth sulphate, ammonium laureth sulphate, disodium laureth sulfosuccinate and sodium C12- 15 pareth- 12 carboxylate), amphoteric / zwitterionic surfactants (e.g. cocamidopropyl betaine, sodium cocoamphoacetate and cocamidopropyl hydroxysultaine), non-ionic surfactants (e.g. cocamide DEA, cocamide MEA, decyl glucoside, lauryl glucoside), and cationic surfactants (e.g. cetrimonium chloride, behentrimonium chloride and benzalkonium chloride). In embodiments where one or more surfactants are present in the9&7.524NQ2 cosmetic composition, the one or more surfactants may be present in an amount of from about 0.1 %) to about 10% by weight of the composition, e.g. from about 0.25% to about 7.5%) by weight of the composition, or about 0.5% to about 6% by weight of the composition. In one embodiment where one or more surfactants are present in the cosmetic composition, the one or more surfactants are present in an amount of from about 0.5% to about 5%) by weight of the composition.

[0073] The cosmetic composition of the invention may further comprise one or more preservatives, including but not limited to, 2 -bromo-2nitropropane- 1 ,3-diol (bronopol), benzyl alcohol, benzoic acid, sodium benzoate, diazolidinyl urea, imidazolidinyl urea, methyl paraben, phenoxyethanol, ethyl paraben, propyl paraben, sodium methyl paraben, sodium dehydroacetate, dehydroacetic acid, polyhexamethylenebiguanide hydrochloride, isothiazolone, chlorhexidine digluconate, chlorphensin, a sorbate such as potassium sorbate and / or sodium propyl paraben. In one embodiment, the cosmetic composition of the invention does not comprise parabens. In embodiments where one or more preservatives are present in the cosmetic composition, the one or more preservatives may be present in an amount of from about 0.001 % to about 10%> by weight of the composition, e.g. from about 0.01 % to about 8% by weight of the composition, or about 0.1 %) to about 5% by weight of the composition. In one embodiment where one or more preservatives are present in the cosmetic composition, the one or more preservatives are present in an amount of from about 0.05%> to about 8% by weight of the composition.

[0074] The cosmetic composition of the invention may further comprise one or more chelating agents or sequestering agents including but not limited to, ethylenediamine tetraacetic acid (EDTA) and salts thereof (e.g. dipotassium EDTA, disodium EDTA or tetrasodium EDTA), sodium phytate, trisodium ethylene diamine disuccinate, and / or tetrasodium glutamate diacetate. In embodiments where one or more chelating agents are present in the cosmetic composition, the one or more chelating agents may be present in an amount of from about 0.001 %) to about 10%> by weight of the composition, e.g. from about 0.01 %) to about 8%o by weight of the composition, or about 0. 1 %> to about 5%> by weight of the composition. In one embodiment where one or more chelating agents are present in the cosmetic composition, the one or more chelating agents are present in an amount of from about 0.05%) to about 8%> by weight of the composition.9&7.52 NQ2

[0075] The cosmetic composition of the invention may further comprise one or more vitamins. For example, the cosmetic composition may further comprise vitamin B, vitamin B 1 to vitamin B 12, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, derivatives thereof, provitamins thereof (e.g. pro-vitamin B5 (panthenol)), or combinations thereof. In embodiments where one or more vitamins are present in the cosmetic composition, the one or more vitamins may be present in an amount of about 0.0001 %> to about 50%> by weight of the composition, about 0.001 %> to about 10%> by weight of the composition, about 0.01 % to about 8%) by weight of the composition, or about 0. 1 % to about 5%> by weight of the composition. In one embodiment where one or more vitamins are present in the cosmetic composition, the one or more vitamins are present in an amount of from about 0.1 %> to about 5%) by weight of the composition. In one embodiment where one or more vitamins are present, the vitamin is vitamin C and / or vitamin E.

[0076] The cosmetic composition of the invention may further comprise one or more antioxidants. These may be different to the polyphenolic antioxidant agents already present in the composition. In one embodiment where one or more such additional antioxidants are present in the cosmetic composition, the one or more additional antioxidants are present in an amount of from about 0.1 %> to about 5%> by weight of the composition.

[0077] The cosmetic composition of the invention may further comprise one or more sunscreen agents, including but not limited to inorganic sunscreen agents (e.g. microfine titanium dioxide, microfine zinc oxide, iron oxides, talcs and / or boron nitride) and organic sunscreen agents (e.g. p-aminobenzoic acids, esters and derivatives thereof (e.g. 2- ethylhexyl p-dimethyl-aminobenzoate), methoxycinnamate esters (e.g., 2-ethylhexyl p- methoxycinnamate, 2-ethoxyethyl p-methoxycinnamate or a, -di-(p-methoxycinnamoyl)-a'- (2ethylhexanoyl)-glycerin), benzophenones (e.g. oxybenzone), dibenzoylmethanes (e.g. 4- (tert-butyl)-4'-methoxydibenzoylmethane), 2-phenylbenzimidazole-5 sulfonic acid and salts thereof, alkyl- , diphenyl acrylates (e.g. alkyl a-cyano- , -diphenylacrylates such as octocrylene) triazines (such as 2,4,6-trianilino-(p-carbo-2-ethyl-hexyl-l -oxy)-l ,3,5 triazine), and / or camphor derivatives (such as methylbenzylidene camphor). In embodiments where one or more sunscreen agents are present in the cosmetic composition, the one or more sunscreen agents may be present in an amount of from about 0.01 to about 10% by weight of the composition.9&7.52 NQ2

[0078] The cosmetic composition of the invention may further comprise one or more pH adjusting agents, including but not limited to potassium hydroxide, sodium hydroxide, aminomethyl propanol, sodium citrate and / or triethanolamine. The cosmetic composition of the invention may have a pH from about 3 to about 10, e.g. from about 4 to about 8, or from about 5 to about 7. In embodiments where one or more pH adjusting agents are present in the cosmetic composition, the one or more pH adjusting agents may be present in an amount of from about 0.01 to about 10% by weight of the composition.

[0079] The cosmetic composition of the invention may further comprise one or more thickeners or gelling agents. For example, when the cosmetic composition is in the form of a gel, the cosmetic composition may comprise one or more thickeners or gelling agents. Examples of thickeners / gelling agents that can be used in the present invention include, but are not limited to, acrylic acid polymers (e.g. available commercially under the trade name Carbopol or Ultrez (Lubrizol) , modified celluloses (e.g. hydroxyethylcellulose available commercially under the trade name Natrosol from Hercules) hydroxypropylmethyl cellulose, amine oxides, block polymers of ethylene oxide and propylene oxide ( e.g. those available from BASF Wyandotte under the trade name "Pluronic"), PVM, MA, decadiene crosspolymer (e.g. available under the trade name Stabilez 60), ethoxylated fatty alcohols, salt (e.g. magnesium chloride, sodium chloride), Aristoflex AVC, phthalic acid amide, xanthan gum, sodium polyacrylate, polyvinyl alcohols, fatty alcohols, and / or alkyl galactmanans (e.g. available under the trade name N-Hance from Hercules). In embodiments where one or more thickeners / gelling agents are present in the cosmetic composition, the one or more thickeners / gelling agents may be present in an amount of about 0.01 to about 10% by weight of the composition.

[0080] The cosmetic compositions of the invention may further comprise one or more perfumes and / or colouring agents.

[0081] In another aspect of the invention, there is provided a method of cosmetic treatment of a skin condition comprising the step of applying the cosmetic composition according to the invention onto the skin of a subject to improve its aesthetic appearance.

[0082] The term “improving aesthetic appearance” as used herein means an improvement of any attribute or characteristic of skin, including without limitation reducing dermatological signs of chronological aging, photo-aging, hormonal aging, and / or actinic aging; preventing9&7.52 NQ2 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; preventing and / or reversing of loss of glycosaminoglycans and / or collagen; ameliorating the effects of estrogen imbalance; preventing 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; and any combinations thereof.

[0083] Additionally, the compositions comprising the extracellular vesicles of the present invention can be used to treat or prevent senescence as distinguished from aging.EXAMPLES

[0084] The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations to the scope of this invention. Various other aspects, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present invention or the scope of the appended claims.

[0085] Samples obtained according to the claimed invention were further processed as follows:9&7.52 NQ2Sample 1 : concentration to 1 / 40 of the initial volume and purified with PBS;Sample 2: concentration to 1 / 5 of the initial volume without further purification.The samples were compared with commercially-available Kombucha fermented products.EXAMPLE 1

[0086] Sample 1 tested at 0.5% showed an increase of Collagen Type I and Involucrin expression of 30% and 62% respectively (Table I), whereas the filtered Kombucha commercial product NG Kombuchka™ tested at the same dose in the medium did not show any increase.Table I: Involucrin on Normal Human Keratinocytes and Collagen expression on Normal Human FibroblastsNG Kombuchka ™ is sold by Sederma s = significantly different vs. non-treated sample (P<0.05) ns = not significantly different vs. non-treated sample (P<0.05)These results show that the Kombucha extracellular vesicles contained in Sample 1 can help promote skin barrier integrity via involucrin increase and delivers anti-aging benefits by increasing the synthesis of collagen in skin cells. This activity is absent in commercially available samples, whose processing reduces the amounts of vesicles present.EXAMPLE 2Senescence Biomarkers on Normal Human Dermal Fibroblasts

[0087] Intracellular assay for P21 and SASP factors release (IL6 and IL8) was measured on normal human dermal fibroblasts in triplicates. Cells were subjected to oxidative stress toinduce senescence by chronic exposure to hydrogen peroxide (H2O2) for 1 hour per day over 8 to 9 consecutive days.Following each H2O2 exposure, cells were treated with Sample 2 for 24 hours and compared with commercial Yunnan Kombucha as indicated in the Table below. P21 protein marker expression was measured with ELISA assay kits. The secretome associated with senescence (SASP proteins) was measured by the content of pro- inflammatory cytokines IL-6 and IL-8 by using ELISA assay kits.Sample 2, tested at 3%, showed (see Table II) a significant decrease of IL6, IL8 and P21 intracellular content. By comparison, a commercial Yunnan Kombucha, showed a much lower decrease of the same markersTab IL IL6 release, IL8 release and P21 intracellular content on normal human fibroblastsa All changes are significantly different (P<0.05) versus non treated control b All changes are significantly different (P<0.05) versus Yunnan KombuchaSample 2 according to the invention showed (see Table II) a significant decrease (P <0.05) of IL6, IL8 and P21 intracellular content compared to the non-treated sample (baseline 0%) and a commercial sample (Yunnan Kombucha). This shows that Sample 2 allows to modulate cell senescence for antiaging & longevity benefits.

Claims

CLAIMS1. Process for obtaining a concentrated extracellular vesicles suspension comprising the steps of: i. Fermenting tea (Camellia sinensis) with a combination of at least one yeast and one bacterium to obtain a fermentation product comprising a liquid material and a scoby; ii. Optionally pre-cleaning the liquid material by centrifugation and / or filtration; iii. Filtering the product of step i. or ii. through a 150-450 nm filtering device to give a permeate; iv. Filtering the permeate of step iii. through a 30-80 nm filtering device to give as retentate a suspension comprising extracellular vesicles; v. Recovering the suspension of extracellular vesicles; and vi. Optionally concentrating the suspension of extracellular vesicles.

2. Process according to claim 1, wherein the suspension of extracellular vesicles is concentrated to 1 / 3 to 1 / 50 of its original volume, preferably 1 / 5.

3. Process according to any of the preceding claims, additionally comprising the step of stabilizing the concentrated extracellular vesicles suspension.

4. Process according to claim 3, wherein the concentrated extracellular vesicles suspension is stabilized by addition of a polyol, preferably glycerol, and a preservative, preferably a sorbate and / or a benzoate.

5. Process according to any of the preceding claims, wherein the yeast is Dekkera / Brettanomyces bruxellensis. Hanseniaspora valbyensis Saccharomyces cerevisiae, Saccharomyces ludwigii, Saccharomyces apicalutus species and / o Schizosaccharomyces pombe.

6. Process according to any of the preceding claims, wherein the bacterium belongs to the genera Acetobacter, Liquorilactobacillus and / or Gluconobacter.

7. Process according to any of the preceding claims, wherein the bacterium is at least Acetobacter xylinum and the yeast is at least Saccharomyces cerevisiae.

8. A suspension of of extracellular vesicles obtainable by the process of any of claims 1 to 7.

9. A suspension according to claim 8, wherein the concentration of extracellular vesicles is between about 4.2* 109and 8.8* 1011particles / ml.

10. A cosmetic or pharmaceutical composition comprising a suspension according to any of claims 8 to 9 in a cosmetically or pharmaceutically acceptable medium.

11. Cosmetic composition according to claim 10 for improving the aesthetic appearance of skin of a human or animal body.

12. Cosmetic composition according to claim 10 for improving the barrier function of the skin of a human or animal body.

13. Cosmetic or pharmaceutic composition according to claim 10 for use in treating or preventing cellular senescence.

14. Cosmetic or pharmaceutic composition according to any of claims 10 to 13, wherein the composition is in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion (Water / Oil / Water or Oil / Water / Oil), a microemulsion, a nanoemulsion, a solution, a suspension, a hydrodispersion, a gel, an ointment, a paste, an aerosol foam, a spray, an aqueous gel, a powder, a foundation, a transdermal patch, a cream or a mask.

15. Cosmetic or pharmaceutic composition according to any of claims 10 to 13, wherein the composition is for enteral or parenteral administration, in particular intradermal or subcutaneous administration.

16. Method of cosmetic or medical treatment of a skin condition comprising the step of applying the cosmetic or pharmaceutical composition according to anyone of claims 10 or 14 onto the skin of a subject.

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