COSMETIC SKINCARE COMPOSITION OF KERATINOUS MATERIALS

Abstract

COSMETIC COMPOSITION FOR THE CARE OF KERATINOUS MATERIALS This disclosure relates to a cosmetic composition comprising at least one exogenous active ingredient loaded in extracellular vesicles. This disclosure also relates to a non-therapeutic method for the care of keratinous materials, comprising at least one step of topical application of the composition according to this disclosure in a ready-to-use form to the keratinous materials. Figure for the abstract: none

Description

Title of the invention: COSMETIC CARE COMPOSITION OF KERATINOUS MATERIALS technical field

[0001] This disclosure relates to a cosmetic composition comprising at least one exogenous active ingredient loaded into extracellular vesicles (EVs). This disclosure also relates to a non-therapeutic process for the treatment of keratinous materials, preferably of the skin and / or hair, using the cosmetic composition according to this disclosure. CONTEXT

[0002] The quest for youthful, radiant skin remains a universal consumer aspiration, driving the cosmetics industry toward the development of innovative formulations that promote skin regeneration and combat the signs of aging. At the heart of these advances are active ingredients capable of achieving desirable biological effects, such as stimulating cell proliferation and supporting cell repair mechanisms. However, the efficacy of such active ingredients is often hampered by inherent limitations such as inadequate transdermal absorption, poor stability under exposure to air or light, low solubility in commonly used cosmetic solvents, and the like, which pose significant challenges in their formulation and application.

[0003] Strategies to overcome these obstacles focus on the design of chemically modified derivatives, the creation of an encapsulating agent for the active ingredient, and the development of efficient delivery systems that enhance their stability and ability to reach target sites in the skin, thereby facilitating superior skin regeneration and anti-aging results. Strategies combined with emerging technologies, such as nanotechnology, bioengineering, and precision targeting, hold promise for developing skincare solutions tailored to different skin aging profiles.

[0004] Therefore, it is desirable to develop a cosmetic composition constructed using bioengineering technology, comprising at least one active ingredient loaded into nanoscale vehicles that serve both as an encapsulating agent and as an efficient delivery system to facilitate efficacy in modulating cellular biomarkers associated with skin regeneration and anti-aging. Summary of the invention

[0005] The inventors have discovered that such a need can be met by the cosmetic composition according to the present disclosure. This cosmetic composition, comprising at least one exogenous active ingredient loaded in extracellular vesicles, offers superior efficacy in modulating cellular biomarkers associated with skin regeneration and anti-aging compared to formulations containing the exogenous active ingredient alone, unloaded EVs, or a simple mixture thereof.

[0006] According to a first aspect, the present disclosure relates to a cosmetic composition comprising at least one exogenous active ingredient loaded in extracellular vesicles.

[0007] According to a second aspect, the present disclosure relates to a process for preparing the cosmetic composition according to the first aspect.

[0008] According to a third aspect, the present disclosure relates to a composition comprising at least one cosmetic active ingredient loaded in extracellular vesicles, wherein the extracellular vesicles are preferably derived from human stem cells, and wherein at least one cosmetic active ingredient is preferably a lipophilic and / or hydrophilic active ingredient, more preferably retinol and / or vitamin C.

[0009] According to a fourth aspect, the present disclosure relates to a non-therapeutic process for the treatment of keratinous materials, preferably of the skin and / or hair, comprising at least one step of topical application of the cosmetic composition according to the first aspect or of the cosmetic composition prepared by the process according to the second aspect in a ready-to-use form on the keratinous materials.

[0010] According to a fifth aspect, the present disclosure relates to a non-therapeutic use of the cosmetic composition according to the first aspect or of the cosmetic composition prepared by the process according to the second aspect for the care of keratinous materials, preferably of the skin and / or hair.

[0011] Other subjects and features, aspects and advantages of this disclosure will become even clearer upon reading the detailed description and examples that follow. Brief description of the drawings

[0012] Implementations of this disclosure will now be described, by way of example only, with reference to the attached figure, in which:

[0013] [Fig. 1] The [Fig. 1] represents a diagram illustrating the process of loading vitamin C or retinol into a VE particle.

[0014] [Fig. 2] [Fig. 2] shows (a) the HPLC (high-performance liquid chromatography) standard curve of the VC acquired with linearization of the peak area, and (b) the result of (c) the HPLC assay of VC in VC-VE; (c) the HPLC standard curve of retinol acquired with linearization of the peak area, and (d) the result of the HPLC assay of retinol in VE with retinol diluted 5 times.

[0015] [Fig. 3] [Fig. 3] shows the stimulation effect of (a) VC alone (VC), (b) VE uncharged (VE), (c) VE charged in VC (VC-VE) and (d) uncharged VE + VC (VC+VE) on NHF proliferation. The symbol “+ / - sd” on the figure means + / - Standard deviation.

[0016] [Fig. 4] Figure 4 represents the expression of the NHK gene regulated by non-VEs charged (VE), retinol alone (Retinol), uncharged VE + retinol (Retinol+VE) and retinol-charged VE (Retinol-VE). DETAILED DESCRIPTION OF THE INVENTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by a person skilled in the art in the field covered by this disclosure. Where the definition of a term in this description conflicts with the meaning commonly understood by a person skilled in the art in the field covered by this disclosure, the definition described herein shall apply.

[0018] Throughout this application, all proposed ranges are intended to include each specific range within the given ranges, and a combination of intermediate sub-ranges. Thus, a range from 1 to 5 specifically includes 1, 2, 3, 4 and 5, as well as sub-ranges such as 2 to 5, 3 to 5, 2 to 3, 2 to 4, 1 to 4, etc.

[0019] Throughout this application, the term "including one" shall, unless otherwise stated, be understood as synonymous with "including at least one". Furthermore, the expression "at least one" used in this description is equivalent to the expression "one or more".

[0020] Throughout this application, an embodiment defined with "comprising" or similar shall be understood as encompassing a preferred embodiment defined with "consisting substantially of" and a preferred embodiment defined with "consisting of".

[0021] Apart from the operational examples, or unless otherwise indicated, all numbers expressing quantities of components and / or reaction conditions should be understood as being modified in all cases by the term "approximately", with a meaning classically known in the art, for example, to within 10% of the number indicated (for example, "approximately 10%" means 9% to 11% and "approximately 2%" means 1.8% to 2.2%).

[0022] As used herein, the expression "keratinous material(s)" refers to skin, hair, scalp, eyelashes, eyebrows, body hair, nails, lips or mucous membranes, preferably to skin and / or hair. As used herein, the The term "skin" refers to the outer covering of an animal body, particularly the human body; the outermost layer of skin is called the epidermis, the middle layer below the epidermis is called the dermis, and the lower layer below the dermis is called the hypodermis, in which keratinocytes are the primary cell type found in the epidermis and fibroblasts are an essential component of dermal connective tissue.

[0023] As used herein, the expression "non-covalent interaction" refers to the interaction between molecules or atoms without sharing or transfer of electrons (i.e., through non-covalent bonds), including, but not limited to, hydrogen bonding, van der Waals forces, ionic-dipolar interactions, hydrophilic interactions, lipophilic interactions, metal coordination and ir-ir stacking, preferably hydrophilic / lipophilic interactions.

[0024] As used herein, the term "topical" refers to the application or spreading of the cosmetic composition according to this disclosure onto the surface of a healthy area of ​​keratinous material.

[0025] As used herein, the expression "effective quantity" refers to a quantity of active or actively loaded EV which produces the desired effect through its implementation, in particular the regeneration / anti-aging of keratinous materials.

[0026] As used herein, the expression "cosmetically acceptable medium" refers to a cosmetically acceptable ingredient, composition or vehicle, for example a solid or liquid filler, diluent, excipient, carrier, solvent, buffer or encapsulating material, which can be involved, for example, in the suspension, maintenance of activity and / or the carrying or transport of the EV population to active and suitable for contact with keratinous materials, such as skin, hair or mucous membranes, without causing a toxic or intolerant reaction.

[0027] The term "INCI" is an abbreviation for International Nomenclature of Cosmetic Ingredients, which is a system of names provided by the International Nomenclature Committee of the Personal Care Products Council to describe ingredients in personal care products. EVs loaded with exogenous active material (EVs with active material)

[0028] The composition or cosmetic composition according to this disclosure comprises at least one exogenous active ingredient loaded in extracellular vesicles.

[0029] Extracellular vesicles are membrane-bound vesicles secreted from cells to the outside and are widely present in body fluids. They are produced by many cell types in in vitro culture models and living cells. EVs contain lipids, proteins, and genetic material and organelles, which can be used as communication vectors between different cells and potential biomarkers.

[0030] As used herein, the expressions "extracellular vesicle" or terms "VE" or "exosome" are used interchangeably herein and should be understood as referring to any type of vesicle that can be obtained from a cell in any form whatsoever, for example a microvesicle (for example, any vesicle detached from the plasma membrane of a cell), an exosome (for example, any vesicle derived from the endolysosomal pathway), a microparticle (which can be derived, for example, from platelets), or an ectosome (which can be derived, for example, from neutrophils and monocytes in serum), etc. Furthermore, these expressions should also be understood as referring to extracellular vesicle mimetics, vesicles based on cells and / or cell membranes obtained by cell extrusion, membrane extrusion, vesicle extrusion, or other techniques, etc.It will be clear to those skilled in the art that, in describing cosmetic and scientific uses and applications of EVs, this disclosure normally relates to a plurality of EVs, i.e., an EV population that may include tens, hundreds, thousands, millions, billions, or even trillions of EVs. In a medium for suspension, maintaining activity, and / or carrying or transporting the EV population, the EVs may have a particle concentration of 1E+1 particles / mL to 1E+15 particles / mL, preferably 1E+2 particles / mL to 1E+14 particles / mL, and more preferably 1E+3 particles / mL to 1E+12 particles / mL.For example, EVs may be present in particle concentrations such as 1E+1 particles / mL, 1.5E+3 particles / mL, 3E+7 particles / mL, 1E+8 particles / mL, 2E+9 particles / mL, 1E+10 particles / mL, 1.5E+11 particles / mL, 1E+15 particles / mL, or any higher, lower, or intermediate number. Similarly, the term "population," which may, for example, refer to an EV loaded with a certain active ingredient, should be understood as encompassing a plurality of entities constituting such a population. In other words, an individual EV, when present in a plurality of instances, constitutes a population of EVs. Thus, naturally, this disclosure relates to both individual EVs loaded with active ingredient(s) and populations comprising EVs loaded with active ingredient(s), as will be evident to those skilled in the art.The concentration of EVs when applied can naturally vary considerably depending on the efficiency to be achieved, the application route, the active loading, etc.

[0031] The EVs used according to this disclosure are derived from source cells, preferably human stem cells. As used herein, the expressions "source cell" or "EV source cell" or "EV producing cell" The terms are used interchangeably here and should be understood as referring to any cell type capable of generating EVs under appropriate culture conditions. These conditions encompass various culture systems, including, but not limited to, suspension and adherent cultures. Furthermore, source cells may include those that naturally produce EVs in vivo. A diverse range of cells and cell lines can serve as sources, including, but not limited to, mesenchymal stem cells, stromal cells, and fibroblasts. These can be obtained from various tissues such as bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, amniotic tissue and / or fluid, tooth buds, umbilical cord blood, skin tissue, and so on.Other suitable cell sources include myeloid suppressor cells, polarized M2 macrophages, adipocytes, endothelial cells, fibroblasts, etc. Cell lines of particular interest include mesenchymal stem cells (MSCs) of different origins, such as adipose tissue-derived stem cells (ADSCs), human umbilical cord endothelial cells (HUVECs), endometrial stem cells (ESCs), induced pluripotent stem cells (iPSCs), fibroblasts, endothelial cells, etc.

[0032] Preferably, the EVs used according to this disclosure are adipose tissue-derived stem cell (ADSC) EVs, such as those supplied by the vendor Echo Biotech under the name ADSC EV.

[0033] Preferably, the EVs used according to this disclosure have a size in the range of about 10 to 1000 nm, preferably about 20 to 500 nm, and more preferably about 30 to 200 nm.

[0034] As used herein, the terms “active,” “active agent,” or “cosmetic active” are used interchangeably and should be understood as referring to any molecular agent that can be used for beauty benefits, such as hydration, nourishment, skin brightening, skin firming, anti-wrinkle, repair, regeneration, whitening, antioxidant, anti-aging, and / or anti-inflammatory efficacy, etc. The actives described herein encompass a wide variety of cosmetic actives, including (i) organic compounds with cosmetic activity that are normally synthesized by chemical synthesis, (ii) compounds of natural origin that can, for example, be obtained by purification from natural sources, and (iii) essentially any type of active agent that can be loaded into EVs.This disclosure is naturally also applicable to other active agents without departing from the essence of the invention, as would be clear to a person skilled in the art.

[0035] As used here, the expression "exogenous active" refers to an active that comes from outside the EVs, as opposed to the "endogenous active" which comes from inside the EVs, such as an active generated by the metabolism of the EVs themselves.

[0036] In particular, the exogenous active ingredient used according to this disclosure is a lipophilic active ingredient, such as retinol and / or its derivatives, and / or a hydrophilic active ingredient, such as vitamin C and / or its derivatives, wherein the lipophilic active ingredient is primarily loaded in the membrane zone, specifically between the bilipid layers, of the VEs, while the hydrophilic active ingredient is primarily loaded within the intravesicular space defined by the bilipid layer structure of the VEs, as shown in [Fig. 1]. The stabilization of these exogenous active ingredients is achieved through non-covalent interactions, particularly hydrophilic / lipophilic interactions, ensuring their efficacy and longevity in the designated delivery system.

[0037] In particular, in the cosmetic composition or composition according to this disclosure, the exogenous active ingredient has a concentration in the extracellular vesicles of at least 0.01 qM, preferably from 0.1 qM to 10,000 qM, more preferably from 1 qM to 8,000 qM, and most preferably from 1 qM to 5,000 qM when the extracellular vesicles have a particulate concentration of 1E+11 particles / mL. The concentration of the exogenous active ingredient in the extracellular vesicles increases or decreases proportionally to the particulate concentration of the extracellular vesicles.For example, if the composition or cosmetic composition is concentrated 10 times so that the extracellular vesicles have an increased particle concentration from 1E+11 particles / mL to 1E+12 particles / mL, the concentration of the exogenous active ingredient in the extracellular vesicles increases proportionally from at least 0.01 qM @ 1E+11 particles / mL of extracellular vesicles to at least 0.1 qM @ 1E+12 particles / mL of extracellular vesicles.

[0038] Preferably, in the composition or cosmetic composition according to this disclosure, the exogenous active ingredient loaded into the extracellular vesicles has a concentration of at least 0.001 qM, preferably from 0.01 qM to 10,000,000 qM, more preferably from 0.1 qM to 5,000,000 qM, and most preferably from 1 qM to 1,000,000 qM.

[0039] Preferably, the EVs loaded with exogenous active according to the present disclosure have a size in the range of about 10 to 1000 nm, preferably about 20 to 500 nm, and more preferably about 30 to 200 nm.

[0040] Processes for engineering or loading EVs with exogenous actives include, but are not limited to: (i) pre-incubation, i.e., culturing EV source cells in the presence of active agents as metabolic components or modified as such under cell culture conditions that promote incorporation metabolic of metabolic components. Appropriate examples of such conditions include culturing EV source cells under low oxygen (hypoxic conditions), exposure to cytokines and / or other forms of cellular stress, e.g. exposure to agents such as bafilomycin; (ii) coincubation, i.e. loading active agents into EVs by directly exposing the EVs (and not the EV source cells) to culture media including the active agents, to allow direct incorporation into the EVs as such.Non-limiting examples of such processes include loading based on the conjugation of an active agent to, for example, lipids such as a sphingolipid, ceramide, cholesterol, phospholipid, or fatty acid, or a ganglioside such as GM1, or a sterol and / or peptide or protein (such as a conventional VE protein or a peptide / protein that interacts with an VE protein and thus an VE), or any other type of suitable metabolic component; (iii) electroporation and / or mixing of the active agent with a transfection reagent (e.g., liposomes, cell-penetrating peptides, cationic polymers such as PEI, lipid nanoparticles, etc.). Preferably, the process for loading VEs with exogenous actives used according to this disclosure is electroporation.

[0041] In particular, the engineering or loading of EVs with at least one exogenous active ingredient can be carried out using the process according to this disclosure, which includes the steps of: 1) culturing source cells in a serum-free culture medium to allow the release of extracellular vesicles; 2) collecting and centrifuging the culture medium from step 1) to obtain a supernatant comprising the extracellular vesicles; 3) filtering the supernatant from step 2) to remove residual cells and cellular debris and obtain purified extracellular vesicles; 4) optionally, concentrating and size-fractionating the purified extracellular vesicles from step 3) to obtain concentrated extracellular vesicles;5) preparation of a solution of at least one exogenous active ingredient in an electroporation buffer and combination of the purified extracellular vesicles from step 3) or the concentrated extracellular vesicles from step 4) with the solution to obtain a mixture comprising the extracellular vesicles and at least one exogenous active ingredient; 6) electroporation of the mixture comprising the extracellular vesicles and at least one exogenous active ingredient from step 5) to obtain electroporated extracellular vesicles; and 7) optionally, filtration, washing and concentration of the electroporated extracellular vesicles to obtain extracellular vesicles loaded with at least one exogenous active ingredient.

[0042] Preferably, the source cells used in step 1) of the process according to this disclosure are human stem cells as described above.

[0043] Preferably, the electroporation in step 6) of the process according to this disclosure can be carried out using voltages in the range of 20 V / cm to 1000 V / cm, preferably 50 V / cm to 800 V / cm, more preferably 100 V / cm to 500 V / cm, and most preferably 150 V / cm to 300 V / cm. The capacitance of the electroporation step is normally in the range of 20 pF to 500 pF, preferably 30 pF to 400 pF, more preferably 50 pF to 300 pF, and most preferably 100 pF to 200 pF, although these parameters may vary considerably depending on various factors such as the EV source cells, any genetic or chemical modification of the EV, the nature of the active agent, etc.

[0044] The inventors discovered that cosmetic compositions containing an effective amount of active EVs can achieve superior regenerative / anti-aging bioefficacy. Without wishing to develop a specific theory, it is believed that the bilipid layer structure and transmembrane protein of EVs facilitate improved transdermal penetration and targeted delivery of chemical actives to specific cell types within the skin or hair follicles, thus significantly increasing the bioavailability of these actives. Furthermore, encapsulating these actives within EVs enhances their stability, particularly for those prone to decomposition or oxidation in the presence of atmospheric oxygen, light radiation, or incompatible ingredients frequently encountered in cosmetic formulations.

[0045] In particular, an effective amount of EV to active may be in the range of 0.001% by weight to 20% by weight, for example from 0.05% by weight to 10% by weight, or from 0.1% by weight to 5% by weight on a dry basis, relative to the total weight of the cosmetic composition. Retinol and / or its derivatives

[0046] The cosmetic composition or composition according to this disclosure may include retinol and / or its derivatives, in which at least a portion of the retinol and / or its derivatives is loaded into extracellular vesicles.

[0047] As used herein, the term "retinol" refers to all isomers of retinol, including all-trans retinol, 13-cis retinol, 11-cis retinol, 9-cis retinol, and 3,4-didehydroretinol. Retinol derivatives according to this disclosure are selected from retinoic acid, tretinoin, retinaldehyde, an ester of retinol or retinoic acid, including, for example, the palmitate, acetate, propionate, butyrate, hexanoate, heptanoate, caprylate, and stearate esters of retinol or retinoic acid, or a synthetic retinoid such as, but not limited to, adapalene, bexarotene, and tazarotene.

[0048] In one embodiment, retinol and / or its derivatives used according to this disclosure are retinol. In one embodiment, retinol and / or its derivatives used according to this disclosure are all-trans retinol.

[0049] By way of example, the retinol used according to this disclosure is sold by SIGMA under the trade name Retinol, by BASF under the trade name Retinol 10SU, or by DSM Nutritional Products under the trade name Retinol CB 50.

[0050] Preferably, the retinol and / or its derivatives used according to this disclosure are retinol that may be commercially available from the supplier SIGMA under the trade name Retinol.

[0051] In particular, at least some, preferably all, of the retinol and / or its derivatives present in the composition or cosmetic composition according to this disclosure is loaded into extracellular vesicles, mainly in the membrane zone, specifically between the bilipid layers, of the extracellular vesicles, and is stabilized by at least one lipophilic interaction.

[0052] Preferably, in the composition or cosmetic composition according to this disclosure, retinol and / or its derivatives have a concentration in the extracellular vesicles of at least 0.01 pM, preferably from 0.1 pM to 5,000 pM, more preferably from 1 pM to 3,000 pM, and most preferably from 1 pM to 1,000 pM when the extracellular vesicles have a particulate concentration of 1E+11 particles / mL. The concentration of retinol and / or its derivatives in the extracellular vesicles increases or decreases proportionally to the particulate concentration of the extracellular vesicles.For example, if the cosmetic composition or composition is concentrated 10 times so that the extracellular vesicles have an increased particle concentration from 1E+11 particles / mL to 1E+12 particles / mL, the concentration of retinol and / or its derivatives in the extracellular vesicles increases proportionally from at least 0.01 pM @ 1E+11 particles / mL of extracellular vesicles to at least 0.1 pM @ 1E+12 particles / mL of extracellular vesicles.

[0053] Preferably, in the composition or cosmetic composition according to this disclosure, retinol and / or its derivatives loaded in the extracellular vesicles have a concentration of at least 0.001 pM, preferably from 0.01 pM to 1,000,000 pM, more preferably from 0.1 pM to 500,000 pM, and most preferably from 1 pM to 100,000 pM.

[0054] In particular, the cosmetic composition according to this disclosure may include an effective amount of extracellular vesicles loaded with retinol and / or its derivatives (retinol VE).

[0055] The inventors discovered that cosmetic compositions comprising an effective amount of retinol EVs have a unique impact on keratinocyte behavior. Compared to those comprising retinol alone, uncharged EVs, or a mixture thereof, cosmetic compositions comprising retinol EVs can achieving superior efficiency in regulating the expression of keratinocyte-related genes, such as upregulated CEACAM6, KRT19, RASAI, IL-1B, PLAT and downregulated DSC1, DSG1, FABP5, which may be associated with increased cell adhesion and migration, increased cell turnover, improved wound healing and decreased keratinocyte differentiation. Vitamin C and / or its derivatives

[0056] The cosmetic composition or composition according to this disclosure may include vitamin C and / or its derivatives, in which at least a portion of the vitamin C and / or its derivatives is loaded into extracellular vesicles.

[0057] Vitamin C, also known as ascorbic acid, is particularly prevalent in the L-form since it can be extracted from natural products. The vitamin C derivatives according to this disclosure are selected from hydrophilic salts, esters, ethers, and sugars of vitamin C.

[0058] In a particular embodiment, the vitamin C derivatives according to this disclosure are in the form of a monosaccharide ester of ascorbic acid or a phosphorylated metal salt of ascorbic acid.

[0059] The monosaccharide esters of ascorbic acid that can be used in the invention are, in particular, glycosyl, mannosyl, fructosyl, fucosyl, galactosyl, N-acetylglucosamine, and N-acetylmuramic derivatives of ascorbic acid and mixtures thereof, such as 6-O-[3-D-galactopyranosyl L-ascorbic acid. These latter compounds and their preparation methods are described in particular in documents EP-A-487 404, EP-A-425 066, and J-05213736.

[0060] For its part, the metallic salt of phosphorylated ascorbic acid can be chosen from ascorbyl phosphates of an alkali metal, ascorbyl phosphates of an alkaline earth metal and ascorbyl phosphates of a transition metal, such as magnesium, sodium, potassium, calcium or zinc; such as magnesium ascorbyl phosphate.

[0061] By way of example, the vitamin C used according to this disclosure is sold by DSM Nutritional Products under the trade name Ascorbic Acid Fine Powder®, by Northeast General Pharmaceutical Factory under the trade name Ascorbic Acid EP / BP / USP / FCC / E300® or by CSPC Weisheng Pharmaceutical under the trade name Vitamin C 100 Mesh®.

[0062] Preferably, the vitamin C and / or its derivatives used according to this disclosure are vitamin C that may be commercially available from the supplier CSPC Weisheng Pharmaceutical under the trade name Vitamin C.

[0063] In particular, at least some, preferably all, of the vitamin C and / or its derivatives present in the composition or cosmetic composition according to this disclosure is loaded into extracellular vesicles, particularly within the intravesicular space defined by the bilipid layer structure of the extracellular vesicles and is stabilized by at least one hydrophilic interaction.

[0064] Preferably, in the composition or cosmetic composition according to this disclosure, vitamin C and / or its derivatives have a concentration in the extracellular vesicles of at least 0.01 µm, preferably from 0.1 µm to 10,000 µm, more preferably from 1 µm to 8,000 µm, and most preferably from 1 µm to 5,000 µm when the extracellular vesicles have a particulate concentration of 1E+11 particles / mL. The concentration of vitamin C and / or its derivatives in the extracellular vesicles increases or decreases proportionally to the particulate concentration of the extracellular vesicles.For example, if the composition or cosmetic composition is concentrated 10 times so that the extracellular vesicles have an increased particle concentration from 1E + 11 particles / mL to 1E + 12 particles / mL, the concentration of vitamin C and / or its derivatives in the extracellular vesicles increases proportionally from at least 0.01 qM @ 1E + 11 particles / mL of extracellular vesicles to at least 0.1 qM @ 1E + 12 particles / mL of extracellular vesicles.

[0065] Preferably, in the composition or cosmetic composition according to this disclosure, vitamin C and / or its derivatives loaded in the extracellular vesicles have a concentration of at least 0.001 qM, preferably from 0.01 qM to 10,000,000 qM, more preferably from 0.1 qM to 5,000,000 qM, and most preferably from 1 qM to 1,000,000 qM.

[0066] In particular, the cosmetic composition or composition according to this disclosure may include an effective amount of extracellular vesicles loaded with vitamin C and / or its derivatives (VC-VE).

[0067] The inventors discovered that the cosmetic composition comprising an effective amount of VC-VE has a unique impact on the behavior of fibroblasts, and can thus achieve superior efficacy in promoting cell proliferation, compared to those comprising vitamin C alone, uncharged VE, or one of their mixtures. Cosmetic composition

[0068] The cosmetic composition according to this disclosure comprises at least one type of EV loaded with exogenous actives (i.e., a population of EVs loaded with one or more desired active agents) optionally formulated with at least one cosmetically acceptable medium. In particular, more than one type of EV population may be included in a cosmetic composition, for example, in cases where overall bioefficacy is desirable. The at least one cosmetically acceptable medium may be chosen from the group comprising any ingredient, composition, or vehicle cosmetically acceptable, for example a solid or liquid filler, diluent, excipient, carrier, solvent, buffer or encapsulating material, which may be involved for example in the suspension, maintenance of activity and / or carrying or transport of the EV population to active and suitable for contact with skin or mucous membrane without causing a toxic or intolerant reaction.

[0069] In one embodiment, the cosmetic composition according to this disclosure may be in the form of a suspension in which extracellular vesicles (EVs) loaded with exogenous active ingredients are suspended in a cosmetically acceptable vehicle. EVs normally have a short in vitro half-life, which can be prolonged by low-temperature storage (such as 4°C or -80°C) or treatment with a specific storage medium (such as polyethylene glycol (PEG), trehalose, etc.). Freezing or lyophilization can significantly prolong the in vitro storage time of extracellular vesicles and maintain their biological activity. In another embodiment, the cosmetic composition according to this disclosure may be in the form of a lyophilized powder suitable for reconstitution into a suspension by the addition of a compatible diluent or buffer before use.In another embodiment, the cosmetic composition according to this disclosure may be in the form of a frozen composition suitable for thawing into a suspension before use. Preferably, relative to the total volume of the cosmetic composition in the form of a suspension or a suspension obtained after thawing or reconstitution, the active ingredients have a particle concentration of 1E+1 particles / mL to 1E+15 particles / mL, preferably from 1E+2 particles / mL to 1E+14 particles / mL, and more preferably from 1E+3 particles / mL to 1E+12 particles / mL.

[0070] The present invention also relates to skin / hair care products such as creams, lotions, gels, serums, mousses, emulsions, ointments, pastes, powders, liniments, sunscreens, shampoos, conditioners, etc., comprising the cosmetic composition according to this disclosure, in order to deliver beauty benefits, such as promoting skin regeneration, inducing hair growth, etc., and / or alleviating skin / hair problems, such as hair loss, poor hair volume, dry skin, wrinkles, fine lines, creases, furrows, and / or skin creases, etc. Additional ingredients

[0071] According to various embodiments, the cosmetic compositions of this disclosure are proposed for application to keratinous materials, such as skin and / or hair. In accordance with these embodiments, the cosmetic compositions of this disclosure may comprise various ingredients commonly used in skincare compositions for keratinous materials, such as other active agents, humectants, fatty substances, thickening agents, stabilizers, anti-seborrheic agents, other vitamins and provitamins including panthenol, sequestrants, pH correctors, chelating agents, colorants, plasticizers, acidifying agents, opacifiers, pearlescent or pearlescent agents, antioxidants, hydroxy acids, perfumes, preservatives and other multifunctional additives.

[0072] A non-exhaustive list of these ingredients can be found in US patent application publication No. 2004 / 0170586. Further examples of these additional ingredients can be found in the International Cosmetic Ingredient Dictionary and Handbook (9th ed. 2002).

[0073] A person skilled in the art shall ensure that the optional additional ingredients and / or their quantity are selected so that the advantageous properties of the composition according to this disclosure are not, or are not substantially, adversely affected by the envisaged addition.

[0074] These ingredients can be selected in various ways by a person skilled in the art to prepare a composition that has the desired properties, for example, consistency or fragrance. In particular, the ingredients, if used, and their quantities are specifically determined according to their product / application, for example, lotion, shampoo, rinse-out conditioner, emulsion, and the like.

[0075] These ingredients (if present) may be present in the cosmetic composition in an amount ranging from 0.001% to 99.9%, for example from 0.1% to 90%, relative to the total weight of the cosmetic composition. Preparation and use

[0076] The cosmetic composition of this disclosure can be prepared by dispersing the extracellular vesicles loaded with at least one exogenous active ingredient obtained via the process according to this disclosure as described in the section "Exogenously Loaded VEs (Active-Loaded VEs)" in a cosmetically acceptable medium. However, it is understood that a person skilled in the art may choose the preparation process, based on their general knowledge, taking into account the nature of the constituents used, for example, their solubility or dispersibility in the vehicle, and the intended application of the compositions or the necessary product.

[0077] According to one embodiment, the cosmetic composition comprising at least one exogenous active ingredient loaded into extracellular vesicles according to this disclosure can be used for the care of keratinous materials, preferably skin and / or hair. Thus, the present invention relates to a non-therapeutic use of the cosmetic composition according to this disclosure for the care of keratinous materials, preferably skin and / or hair. This use can be manifest in the form of a process for the treatment of keratinous materials, preferably of the skin and / or hair, comprising at least one step of topical application of the cosmetic composition of this disclosure in a ready-to-use form on keratinous materials, preferably the skin and / or hair.

[0078] According to one embodiment, the composition comprising at least one cosmetic active ingredient loaded into extracellular vesicles according to this disclosure can be used to modulate cellular biomarkers associated with the regeneration and / or anti-aging of keratinous materials, preferably of the skin and / or hair. Thus, the present invention also relates to the non-therapeutic use of the composition according to this disclosure to modulate cellular biomarkers associated with the regeneration and / or anti-aging of keratinous materials, preferably of the skin and / or hair.

[0079] The cosmetic composition according to this disclosure can be applied by any means allowing a uniform distribution of the composition on keratinous materials.

[0080] The invention will be illustrated in more detail by the following examples, which present particularly advantageous embodiments.

[0081] Although the numerical ranges and parameters that represent the broad scope of this disclosure are approximations, the numerical values ​​presented in the specific examples are reported as accurately as possible. However, every numerical value inherently contains some errors that necessarily result from the standard deviation found in its respective measurements. EXAMPLES

[0082] The following examples are given by way of illustration of the present invention and shall not be construed as limiting the scope.

[0083] The main raw materials used, their trade names and suppliers are listed in Table 1 below. Other materials not specified here were each commercially available.

[0084] [Tables 1] INCI Name Trade Name / Supplier Name Ascorbic acid Vitamin C CSPC Weisheng Pharmaceutical Retinol Retinol Sigma ND ADSC EV Echo Biotech

[0085] I. Preparation of EVs using adipose tissue-derived stem cells loaded with exogenous active ingredients 1. Culture of adipose tissue-derived stem cells (ADSCs) in commercial serum-free culture media (Pricella®) for 48 hours. 2. Collection of the conditioned media from step 1) and centrifugation at a centrifugal force of 3000 g for 30 min at 4 °C to isolate and purify the ADSC VEs. 3. Filtration of the ADSC EVs from step 2) with a 0.45 pm and 0.22 pm filter (Steritop TM, Millipore) to remove residual cells and debris. 4. Concentration and size fractionation of purified ADSC VEs using an ultra-transparent tube (Millipore) with a 100 kDa cutoff membrane. 5. Preparation of separate solutions of vitamin C (400 pg / mL) and retinol (20 pg / mL) in electroporation buffer (phosphate-buffered saline (PBS)). 6. Combination of concentrated ADSC EVs with a vitamin C or retinol solution and delicate mixing to obtain an EV and active mixture. 7. Electroporation of the VE-active mixture from step 6) using parameters predetermined (250 V, 125 pF, 10 pulses, 2 s intervals). 8. Transfer of electroporated ADSC EVs into a 100 kD ultrafiltration tube, wash 3 times with PBS to remove unattached actives, and concentration of electroporated ADSC EVs to approximately 1 mL volume using PBS, continuous flow collection during the washing process to quantify unattached vitamin C or retinol.

[0086] IL Characterization of EVs loaded with exogenous active material and unloaded

[0087] 2.1 Morphology, size and particle concentration

[0088] Cryo-TEM (using a transmission electron microscope, Talos F200C G2) was performed to visualize the morphology of VE materials loaded with exogenous actives and unloaded. Cryo-TEM images of unloaded VEs, vitamin C-loaded VEs (VC-VEs), and retinol-loaded VEs (Retinol VEs) showed that the VEs before and after loading with different actives have a round or approximately round morphology, with a size range of approximately 30 to 200 nm in diameter. In particular, compared to unloaded VEs, the images showed that VC-VEs have internal padding, while retinol VEs have a thicker membrane. This could explain why hydrophilic actives will be primarily loaded within the intravesicular space of the VEs as they cross the membrane, while lipophilic actives will be primarily allocated to the membrane zone.

[0089] A nanoparticle tracking analysis (NTA, ZetaView®) was performed to obtain the size distribution profile of the EVs and their concentration, as summarized below. The results showed that the EVs after loading had a size range of approximately 30 to 200 nm in diameter. The initial particle concentration was measured to be around 1E+11 particles / mL.

[0090] 2.2 Characterization of assets: verification of exogenous assets loaded into EVs and measurement of the concentration of active ingredients in EVs

[0091] HPLC (high-performance liquid chromatography, instrument model, column parameters: Cl8 column, ID 4.6 x 2250 mm, charge particle size: 5 pm. For VC analysis, mobile phase A: 0.1% phosphoric acid in water, mobile phase B: methanol, gradient elution with a volumetric flow rate of 1 mL / min, column temperature: 25 °C, detection wavelength: 245 nm, and injection volume: 10 pL; injection mode: automatic injection. For retinol analysis, mobile phase: methanol, gradient elution with a volumetric flow rate of 1 mL / min, column temperature: 25 °C, detection wavelength: 328 nm / 325 nm, and injection volume: 10 pL; injection mode: automatic injection) was used to quantify the concentration of active ingredients in the VE with the The following protocol applies: 1. Plotting of standard curves for the active ingredients (vitamin C and retinol). The vitamin C standard was diluted with 0.1% phosphoric acid to 7.8 pg / mL, 15.6 pg / mL, 31.2 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, and 500 pg / mL, and the diluted vitamin C standard samples were measured by HPLC. The retinol standard was diluted with 100% methanol to 0.625 pg / mL, 1.25 pg / mL, 3.9 pg / mL, 7.8 pg / mL, 15.6 pg / mL, and 31.2 pg / mL, and the diluted retinol standard samples were measured by HPLC. The HPLC standard curve was acquired with linearization of the peak area. 2. Ultrafiltration of EVs with active membrane that could trap EVs on top of the membrane with the media they pass through. 3. Detection of free active substances in media by HPLC, without active signal detected. 4. Lysis of the active EVs by methanol to extract the EVs and detection again by HPLC, with a clear active signal detected, indicating that all the actives have been loaded into the EVs and encapsulated by them. 5. Calculation of the concentration of active ingredients in EVs by fitting the standard curves.

[0092] The HPLC standard curves of VC and retinol, and the HPLC measurement curves of their corresponding forms in the VE were shown in [Fig. 2]. The concentration of VC in the VE was calculated to be 440 pg / mL (2500 p,M VC at 1E+11 particles / mL of VE) by fitting the VC standard curve (see [Fig.2](a)-(b)). The retinol concentration in the VE was calculated to be 34.5 µg / mL (120 µM retinol at 1E+11 particles / mL of VE) by fitting the retinol standard curve (see [Fig.2](c)-(d)).

[0093] III. In vitro bioefficacy evaluation of active EVs compared to the active ingredient alone, uncharged EVs, and active EVs+

[0094] 3.1 VC-VE: The NHF 2D (normal human fibroblast) assay was used to Evaluate the effectiveness of the capacity to promote the proliferation of VC-VE according to the following protocol: 1. Cell spreading: spreading or seeding human fibroblasts onto a tissue culture dish or plate to allow cells to adhere and proliferate in a controlled two-dimensional space; 2. Cell culture: culturing cells in a growth medium with 2% FBS and maintaining the culture medium at 37°C in a humidified incubator with a 5% CO2 atmosphere for 24 hours; 3. Stimulation: replacement of culture media with test media (medium with 2% FBS plus test samples at corresponding doses, media with 10% FBS alone as positive control, media with 2% FBS alone as negative control) and storage for 3 additional days; on day 4, disposal of media and measurement of cell number; establishment of cultured cells in media with 2% FBS as control, measurement of the effect of uncharged VE, VC-VE, VC alone, uncharged VE+VC and normalization to that without VE treatment (in media with 2% FBS).

[0095] The results of evaluating VC alone (VC), uncharged EVs (VE), VC-charged EVs (VC-VE), and uncharged EVs + VC (VE+VC) with the 2D NHF assay (normal human fibroblast) have been summarized and shown in Figures 3(a)-(d), respectively. The results demonstrated that VC-VE had a significantly greater promoting effect on NHF proliferation than VC alone, uncharged EVs, and VC+ uncharged EVs.

[0096] 3.2 Retinol VE: The 2D NHK (normal human keratinocyte) assay was used to evaluate the efficacy of retinol EVs by quantifying key gene expression levels related to skin regeneration according to the following protocol: 1. Cell spreading: spreading or seeding human keratinocytes onto a tissue culture dish or plate to allow cells to adhere and proliferate in a controlled two-dimensional space; 2. Cell culture: culturing cells in a growth medium containing essential nutrients, vitamins, minerals, and acids amino acids and growth factors at 37°C in a humidified incubator with a 5% CO2 atmosphere; 3. Cell processing: after 3 days of culture, replacement of culture media with test media (culture media supplemented with CaCl2) with / without test samples (uncharged EV, retinol alone, uncharged EV + retinol, or EV with retinol) at high or low dose as listed in Table 2 below and incubation of cells for 24 hours; at the end of the treatments, lysis of cells to extract RNA (ribonucleic acid), followed by RT-qPCR (real-time polymerase chain reaction); and obtaining the PCR results with the processed data.

[0097] [Tables2] Retinol Dose (pM) VE (parts / mL) VE with retinol High 12 1E+10 Retinol 12 qM to 1E+10 parts / mL of VE Low 4 3E+9 Retinol 4 qM to 3E+9 parts / mL of VE

[0098] The main levels of gene expression regulated by uncharged EVs (EVs), retinol alone (Retinol), uncharged EVs + retinol (Retinol+EVs) and retinol-charged EVs (Retinol-EVs) have been summarized and shown in [Fig.4].

[0099] Compared to retinol alone, uncharged EVs or uncharged EVs + retinol, retinol EVs showed an overall improved tendency to regulate representative regenerative genes, i.e. increased gene expression related to cell adhesion and migration (see [Fig.4](a): CEACAM6), increased gene expression related to cell renewal (see [Fig.4](b): KRT19, 4(c): RASAI), improved gene expression related to wound healing (see [Fig.4](d): IL-1B, 4(e): PLAT), and decreased gene expression related to keratinocyte differentiation (see [Fig.4](f): DSC1, 4(g): DSG1, 4(h): FABP5).

[0100] The preceding description illustrates and describes the disclosure. Furthermore, the disclosure presents and describes only the preferred embodiments, but as mentioned above, it should be understood that it can be used in various other combinations, modifications, and various other environments, and that it may undergo changes or modifications in the scope of the inventive concepts as expressed herein, in accordance with the teachings above and / or the skills or knowledge of the relevant art. The embodiments described herein are further intended to explain the best known means to the applicant and to enable others in the art to use the disclosure in such or other embodiments, and with the various modifications required by the specific applications or uses of the latter.

[0101] Accordingly, the description is not intended to limit the invention to the form disclosed herein. The appended claims are also intended to be interpreted as including other embodiments.

Claims

Demands

1. Cosmetic composition, comprising at least one exogenous active ingredient loaded in extracellular vesicles, the extracellular vesicles not being derived from human embryonic stem cells.

2. Cosmetic composition according to claim 1, wherein the extracellular vesicles are derived from human stem cells.

3. Cosmetic composition according to claim 2, wherein, relative to the total volume of the cosmetic composition in the form of a suspension or a suspension obtained after thawing or reconstitution, the extracellular vesicles have a particle concentration of 1E+1 particles / mL to 1E+15 particles / mL, preferably of 1E+2 particles / mL to 1E+14 particles / mL, and more preferably of 1E+3 particles / mL to 1E+12 particles / mL.

4. Cosmetic composition according to any one of the preceding claims, wherein the exogenous active ingredient is a lipophilic and / or a hydrophilic active ingredient.

5. Cosmetic composition according to claim 4, wherein the lipophilic active ingredient is retinol and / or its derivatives.

6. Cosmetic composition according to claim 4, wherein the hydrophilic active ingredient is vitamin C and / or its derivatives.

7. A method for preparing the cosmetic composition according to any one of claims 1 to 6, comprising the steps of: 1) culturing source cells in serum-free culture media to allow the release of extracellular vesicles; 2) collecting and centrifuging the culture media from step 1) to obtain a supernatant comprising the extracellular vesicles; 3) filtering the supernatant from step 2) to remove residual cells and cell debris and obtain purified extracellular vesicles; 4) optionally, concentrating and size-fractionating the purified extracellular vesicles from step 3) to obtain concentrated extracellular vesicles; 5) preparing a solution of at least one exogenous active ingredient in an electroporation buffer and combining the purified extracellular vesicles from step 3) or the vesicles

8. concentrated extracellular from step 4) with the solution to obtain a mixture comprising the extracellular vesicles and at least one exogenous active ingredient; 6) electroporation of the mixture comprising the extracellular vesicles and at least one exogenous active ingredient from step 5) to obtain electroporated extracellular vesicles; 7) optionally, filtration, washing and concentration of the electroporated extracellular vesicles to obtain extracellular vesicles loaded with at least one exogenous active ingredient; and 8) optionally, dispersion of the extracellular vesicles loaded with at least one exogenous active ingredient in a cosmetically acceptable medium to obtain the cosmetic composition. Composition, comprising at least one cosmetic active ingredient loaded in extracellular vesicles, the extracellular vesicles not being derived from human embryonic stem cells, wherein the extracellular vesicles are preferably derived from human stem cells, and wherein at least one cosmetic active ingredient is preferably a lipophilic and / or hydrophilic active ingredient, more preferably retinol and / or vitamin C.

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