Hair loss preventing and hair growing essence and preparation method thereof

Abstract

The invention discloses an anti-hair loss and hair growth essence and a preparation method thereof, the anti-hair loss and hair growth essence comprises a solid component A and a liquid component B, the component A comprises a lipid exosome hybrid vesicle freeze-dried powder, and the lipid exosome hybrid vesicle freeze-dried powder comprises lipid exosome hybrid vesicles and a freeze-drying protective agent; the lipidosome and exosome hybrid vesicle is obtained by fusing lipidosome and exosome through a membrane; the freeze-drying protective agent comprises zinc ions, chlorogenic acid and trehalose. The essence has good room-temperature storage stability and transdermal permeability, improves the cell utilization rate of active substances, and has a good prevention and treatment effect on AGA.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an anti-hair loss and hair growth essence and a preparation method thereof. Background Art

[0002] Hair loss has become a health and aesthetic concern for hundreds of millions of people worldwide. It not only affects patients' appearance but can also trigger psychological issues such as anxiety and low self-esteem, significantly reducing quality of life. Based on its pathological mechanism, hair loss is primarily categorized as: androgenetic alopecia (AGA), alopecia areata (AA), telogen effluvium (TE), and cicatricial alopecia. AGA accounts for over 90% of all hair loss cases (clinical studies show that AGA accounts for 95% of male and 70%-90% of female hair loss cases), making it a core pathology that urgently needs to be addressed. Current mainstream treatments include: medications with minoxidil or finasteride (for men), but the former has been shown to cause scalp irritation, exacerbating itching and dandruff, while the latter may cause sexual dysfunction; physical therapy with laser stimulation, but this is expensive and generally ineffective; surgical hair follicle transplantation, which is expensive and can still cause hair loss; and plant extract supplements, which are slow to show results.

[0003] In order to improve the effect of topical anti-hair loss and hair growth agents and reduce their side effects and irritation, the existing technology uses exosomes from certain animal sources as active ingredients and has been found to have significant therapeutic effects. Extracellular vesicles from mesenchymal stem cells (MSCs) can activate dermal papilla cells and promote the transition of hair follicles from the resting phase to the growth phase. This effect has been verified in animal experiments, indicating that these exosomes can effectively promote the regeneration of hair follicles. However, since the biological activity of exosomes is easily inactivated at room temperature in vitro, they are usually stored at -80°C, and therefore are often implemented in animals by injection. In order to improve bioavailability and enable use and storage under daily conditions, the present invention is proposed.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide an anti-hair loss and growth essence and a preparation method thereof. The essence contains liposome-exosome hybrid vesicle freeze-dried powder, has good room temperature storage stability and transdermal permeability, and improves the cellular utilization rate of active substances, and has a good preventive and therapeutic effect on AGA.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides an anti-hair loss and hair growth essence, comprising a solid component A and a liquid component B, wherein the component A comprises liposome exosome hybrid vesicle freeze-dried powder, and the liposome exosome hybrid vesicle freeze-dried powder comprises the following components: liposome exosome hybrid vesicles and a freeze-drying protectant; the liposome exosome hybrid vesicles are obtained by membrane fusion of liposomes and exosomes; the freeze-drying protectant comprises: zinc ions, chlorogenic acid, and trehalose.

[0008] Exosomes contain bioactive substances such as RNA and proteins, which transport substances and transmit information between cells. Existing technologies have demonstrated that certain animal-derived exosomes, particularly those derived from mesenchymal stem cells, carry signaling molecules that can act on hair follicle stem cells in the bulge area, promoting their transition from a resting phase to a growing phase. They provide growth factors (such as VEGF, IGF-1, HGF, FGF, etc.) or deliver proliferation-promoting miRNAs, increasing the number of hair papilla cells. They are rich in pro-angiogenic factors (such as VEGF, FGF, and Angiopoietin-1), which act on the vascular endothelial cells surrounding the hair follicles, stimulating the formation of new blood vessels and increasing the blood supply and nutrient / oxygen supply to the hair follicles, creating favorable conditions for hair growth. They carry antioxidant enzymes (such as SOD and CAT) and related miRNAs, which help scavenge reactive oxygen species and protect hair follicle cells from damage. Therefore, exosomes, as active substances for hair growth and development, possess excellent biocompatibility and can effectively prevent hair loss and promote hair growth in multiple ways.

[0009] The present invention aims to produce an anti-hair loss and hair growth essence for daily use or application via coating. The goal is to improve the room-temperature storage stability of exosomes. Therefore, an essence containing components A and B was developed. Solid component A is the exosome active substance, while liquid component B serves as a carrier for the water-soluble active ingredient and also as a reconstitution medium for solid component A. To further enhance the storage stability of exosomes, the present invention fuses and hybridizes liposomes with exosomes. Exosomes have a membrane similar to that of liposomes, possessing a lipid bilayer. However, exosomes are more sensitive to physical factors such as temperature, external forces, osmotic pressure, and tension, making them susceptible to membrane rupture, leakage of contents, or protein deformation, leading to inactivation. Artificially synthesized liposomes, on the other hand, are highly designable. By fusing and hybridizing designed liposomes with exosomes, the membrane strength and stability can be enhanced, minimizing damage during freeze-drying and storage. Compared to liquid phase, freeze-dried hybrid vesicles are less susceptible to environmental factors, further enhancing stability. Furthermore, the lyoprotectant provided by the present invention is also an important factor in maintaining the activity of the hybrid vesicles. Trehalose, as a conventional lyoprotectant component, replaces water molecules during the drying stage, forms hydrogen bonds with the polar groups of the active substance, maintains its natural structure, and prevents structural collapse and denaturation during the drying process; zinc ions and chlorogenic acid can form amorphous coordination polymers after dehydration, which can play the role of fillers, provide a good physical structure to improve the mechanical strength of the lyophilized powder, and make the lyophilized powder easy to re-dissolve. The reconstituted hybrid vesicles release zinc ions and chlorogenic acid, which can also serve as active ingredients for preventing hair loss and promoting hair growth. Zinc ions have antibacterial activity, while chlorogenic acid has antioxidant, anti-inflammatory, and 5α-reductase activity inhibition effects.

[0010] In some embodiments, the exosomes are derived from bovine colostrum or mesenchymal stem cells.

[0011] The prior art (Kim H, Jang Y, Kim EH, Jang H, Cho H, Han G, Song HK, Kim SH and Yang Y (2022) Potential of Colostrum-Derived Exosomes for Promoting Hair Regeneration Through the Transition From Telogen to Anagen Phase. Front. Cell Dev. Biol. 10: 815205.) records that the active substances carried by bovine colostrum exosomes accelerate the transition of the hair cycle from the resting phase to the growth phase by activating the Wnt / β-catenin pathway, promote the proliferation of human hair dermal papilla cells, inhibit dihydrotestosterone (DHT)-induced follicular development arrest, and induce hair regeneration on the back of mice without any related adverse reactions.

[0012] In some embodiments, the liposome satisfies at least one of the following conditions:

[0013] Condition 1: The average particle size of the liposomes is 100-140 nm; for example, it can be 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, or any value between 100 and 140 nm. Liposomes meeting the above particle size range have similar average particle sizes to exosomes, which is conducive to promoting fusion and hybridization between the two.

[0014] Condition 2: The zeta potential of the liposome is -17 to 3mV; for example, it can be -17mV, -15mV, -13mV, -11mV, -9mV, -7mV, -5mV, -3mV, -1mV, 0mV, 1mV, 2mV, 3mV, etc. or any value between -17 and 3mV. Liposomes that meet the above zeta potential range have good fusion with exosomes, and the vesicles obtained after fusion and hybridization can still maintain high activity. Because under physiological conditions, exosomes are naturally negatively charged. If the negative charge of the liposome is too strong, it will lead to charge repulsion and difficulty in fusion. If the positive charge is too strong, it will easily lead to strong charge neutralization with the exosomes, resulting in aggregation and precipitation of the two, rather than controlled fusion, which will destroy the integrity of the exosome membrane or change its biological activity. Under the relatively weak negative charge of -17 to 0mV, through the divalent Zn 2+ The electrostatic force can build a bridge between two negatively charged membrane surfaces (liposomes and exosomes), neutralize the local charge repulsion, shorten the distance between the membranes, and significantly promote fusion; under weak positive charge conditions of 0-3mV, the two can easily fuse spontaneously without strong electrical neutralization.

[0015] In some embodiments, the lyoprotectant further comprises an antioxidant and mannitol; the antioxidant is selected from at least one of ascorbic acid and methionine.

[0016] In some embodiments, the component B comprises the following components in percentage by mass: 0-8% moisturizer, 0-0.2% allantoin, 0-3% sponge spicules, 0.8%-1.5% osmotic pressure regulator, and the balance being pH buffer.

[0017] The liquid component B provided by the present invention uses a buffer solution and an osmotic pressure regulator to maintain its osmotic pressure within an appropriate range, so that it can effectively redissolve the hybrid vesicle freeze-dried powder of component A and maintain its activity for a certain period of time; furthermore, allantoin is added to component B to soften the scalp stratum corneum, and sponge spicules are added to promote the transdermal penetration of the hybrid vesicles, thereby improving the bioavailability of the hybrid vesicles.

[0018] Sponge spicules are the skeletons of multicellular sponges, consisting of glassy, ​​fiber-like silica or calcium spicules. In the skincare field, sponge spicules are described as microcrystalline, needle-like substances used to stimulate skin microcirculation, promote metabolism, and accelerate the natural exfoliation of the aging stratum corneum. The addition of sponge spicules to the present invention can promote the transdermal penetration of hybrid vesicles.

[0019] In some embodiments, the moisturizing agent is selected from at least one of glycerin, propylene glycol, butylene glycol, hydrolyzed collagen, hyaluronic acid or a salt thereof; the hyaluronic acid or a salt thereof can be hyaluronic acid or sodium hyaluronate.

[0020] In some embodiments, the osmotic pressure regulator is at least one of citrate, sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium lactate, mannitol, and sodium phosphate.

[0021] In some embodiments, the pH buffer is PBS buffer or Tris-HCl buffer.

[0022] In some embodiments, the osmotic pressure of component B is 280-320 mOsmol / kg; for example, it can be 280, 285, 290, 295, 300, 305, 310, 315, 320 mOsmol / kg, etc., or any value between 280 and 320 mOsmol / kg; meeting the above osmotic pressure is conducive to the resolubilization of liposome hybrid vesicles and maintaining their activity for a certain period of time.

[0023] In some embodiments, the pH value of component B is 6.5-7.5; for example, it can be 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc. or any value between 6.5 and 7.5; meeting the above pH value is conducive to the redissolution of liposome hybrid vesicles and maintaining their activity for a certain period of time.

[0024] In some embodiments, the method for preparing liposome hybrid vesicles comprises the following steps:

[0025] (1) extracting exosomes to obtain an exosome heavy suspension;

[0026] (2) preparing a blank liposome suspension;

[0027] (3) Zinc salt, chlorogenic acid, trehalose, antioxidants, and excipients are added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension is added and stirred evenly to obtain a freeze-dried mixture, which is repeatedly frozen and thawed 2 to 4 times to obtain a liposome-exosome hybrid vesicle dispersion.

[0028] In some embodiments, step (1) uses ultracentrifugation to extract exosomes from bovine colostrum or mesenchymal stem cells.

[0029] In some embodiments, step (2) specifically comprises: dissolving the liposome matrix in a solvent, volatilizing the solvent by rotary evaporation, adding PBS buffer for hydration, ultrasonic treatment, and extruding through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension.

[0030] In some embodiments, the molar ratio of phospholipid to cholesterol in the liposome matrix is ​​1:0.3 to 0.7, for example, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, etc., or any value between 1:0.3 and 0.7.

[0031] In some embodiments, the phospholipids can be selected from dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylglycerol (DPPG), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylglycerol (DPPG), dilauroylphosphatidylcholine (DLPC ... ), dioleoylphosphatidylglycerol (DOPG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), cardiolipin (CL), phosphatidylethanolamine (PE), dilinoleylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), dimyristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG) or one or more thereof.

[0032] In some embodiments, the liposome matrix further comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0033] In some embodiments, the zinc salt comprises at least one of zinc chloride, zinc acetate, and zinc gluconate.

[0034] In some embodiments, the protein concentration of the exosome resuspension is 50-160 ng / uL, the average particle size of the exosomes is 80-120 nm, and / or the average particle size of the blank liposomes is 100-140 nm.

[0035] In some embodiments, the concentration of phospholipids in the blank liposome suspension is 5-15 mg / mL.

[0036] In some embodiments, the zeta potential of the blank liposome is -17 to 3 mV.

[0037] In some embodiments, the exosome heavy suspension and the blank liposome suspension are mixed at a protein:phospholipid mass ratio of 1:30-60.

[0038] In some embodiments, the final concentrations of zinc salt, chlorogenic acid, trehalose, antioxidant, and excipient in the lyophilized mixture are 0.007 g / mL to 0.035 g / mL, 0.1 g / mL to 0.45 g / mL, 1 to 5 g / mL, 0 to 0.05 g / mL, and 0 to 5 g / mL.

[0039] In a second aspect, the present invention provides a method for preparing the anti-hair loss and hair growth essence according to any of the above embodiments, comprising the following steps:

[0040] (1) freeze-drying the liposome hybrid vesicle dispersion to obtain liposome hybrid vesicle freeze-dried powder, and encapsulating it under a nitrogen atmosphere to obtain component A; the freeze-drying conditions include: freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours;

[0041] (2) Prepare a pH buffer solution, then add the other components in component B, stir and disperse, and defoam to obtain component B.

[0042] The present invention has the following beneficial effects:

[0043] The present invention provides an anti-hair loss and hair growth essence, comprising liposome exosome hybrid vesicle freeze-dried powder and a liquid component. The liposome exosome hybrid vesicle freeze-dried powder has good storage stability at room temperature, can still maintain good activity after reconstitution, and has the effect of significantly promoting the proliferation of human hair follicle dermal papilla cells; the liquid component serves as a reconstitution medium for the liposome exosome hybrid vesicle freeze-dried powder, can maintain the morphology and activity of the hybrid vesicles for a certain period of time, and has the effect of promoting the transdermal absorption of the vesicles; the liposome exosome hybrid vesicle freeze-dried powder and the liquid component synergistically improve the transdermal absorption rate and the bioavailability of the active substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 Transmission electron microscopy (TEM) images of exosomes, blank liposomes, and liposome-exosome hybrid vesicles in Example 1;

[0046] Figure 2 The zeta potential of the lyophilized powder of liposome hybrid vesicles obtained in each specific embodiment of Test Example 2 is left to stand at room temperature, and the curve of the change in zeta potential after reconstitution with the standing time is shown;

[0047] Figure 3 The liposome exosome hybrid vesicle freeze-dried powder obtained in each specific embodiment of Test Example 2 was left to stand at room temperature, and the relationship curve of the change of particle size after reconstitution with the standing time. DETAILED DESCRIPTION

[0048] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0049] In the description of the embodiments of this application, technical terms such as "first" and "second" are only used to distinguish different objects and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features.

[0050] Reference herein to an "embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive with other embodiments.

[0051] In the embodiment of the present application, the term "or / and" is only a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A or / and B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0052] In addition, the character “ / ” in this article generally indicates that the previous and next related objects are in an “or” relationship.

[0053] In the embodiments of the present application, "multiple" means more than two (including two). Similarly, "multiple groups" means more than two groups (including two groups), and "multi-layer" means more than two layers (including two layers), unless otherwise clearly specified and limited.

[0054] In the embodiments of the present application, “at least one” means one or more than one.

[0055] Those skilled in the art may understand the specific meanings of the above terms in the embodiments of this application based on specific circumstances.

[0056] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0057] Example 1

[0058] This embodiment provides an anti-hair loss and hair growth essence, comprising a solid component A and a liquid component B, the ratio of component A to component B being 20 μg:1 mL, component A comprising liposome exosome hybrid vesicle freeze-dried powder 1; component B comprising the following components in percentage by mass: 4% glycerol, 2% propylene glycol, 0.09% allantoin, 1.27% sponge spicules (purchased from Shaanxi Pinhong Biotechnology Co., Ltd.), 0.8% sodium chloride, 0.02% potassium chloride, and the remainder being a phosphate buffer solution with a pH of 7.4 (1 L containing 1.44 g Na2HPO4 and 0.24 g KH2PO4), and the osmotic pressure of component B is 294 mOsmol / kg.

[0059] The preparation method of liposome hybrid vesicle freeze-dried powder 1 is as follows:

[0060] (1) Fresh bovine colostrum was obtained from Guangzhou Suixin Animal Husbandry Co., Ltd. The colostrum was centrifuged at -4°C for 5000 g / 30 min and 12000 g / 1 h to remove milk fat globules, somatic cells and cell debris. The supernatant was taken and then ultracentrifuged at 35000 g / 1 h and 70000 g / 3 h to remove residual milk fat globules and casein. The supernatant was passed through 0.80 μm, 0.45 μm and 0.2 μm filters in sequence. The filtered supernatant was ultracentrifuged at 100000 g for 1 h, and the exosome pellet was resuspended in 1× PBS solution and then ultracentrifuged at 20000 g for 3 h for washing. The exosome pellet was resuspended in 1× PBS solution to obtain an exosome resuspension. Pierce TM A BCA protein assay kit (purchased from Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the total protein concentration of the exosome heavy suspension. Dynamic light scattering (DLS) was used to determine the particle size distribution of the exosome heavy suspension. The protein concentration in the exosome heavy suspension was 125 ng / µL, and the average exosome size was 92 nm.

[0061] (2) The liposome matrix was dissolved in chloroform, and after the solvent was evaporated at 50°C, 1×PBS solution was added and stirred at 50°C for 30 minutes. The mixture was ultrasonically treated at 200W for 20 minutes and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension. The liposome matrix included a complex phospholipid and cholesterol with a molar weight of 31% of the complex phospholipid. The complex phospholipid was a mixture of soybean phosphatidylcholine (DMPC), dilinoleoylphosphatidylethanolamine (DOPE), and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000) in a molar ratio of 5:3:2. The average particle size of the blank liposome was 104 nm, the zeta potential was -12.24±0.62 mV, and the phospholipid concentration was 11.42 mg / mL.

[0062] (3) Zinc chloride, chlorogenic acid, and trehalose were added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension was added and stirred evenly to obtain a mixture to be lyophilized; then the mixture was subjected to repeated freeze-thaw cycles for 3 times, where freeze refers to freezing at -80°C for 30 seconds and thaw refers to melting at room temperature for 5 minutes. The mixture was then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a hybrid vesicle dispersion, which was then freeze-dried (freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0063] Among them, the exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:50; the final concentrations of zinc chloride, chlorogenic acid, and trehalose in the freeze-dried mixture were 0.021 g / mL, 0.27 g / mL, and 3 g / mL.

[0064] Attachment Figure 1 This is a transmission electron microscopy (TEM) image of the exosomes, blank liposomes, and liposome-exosome hybrid vesicles extracted in Example 1. It can be seen from the figure that the exosomes and liposomes have successfully fused, and a spherical hybrid vesicle is obtained.

[0065] Example 2

[0066] The difference from Example 1 is that Component B does not contain sponge spicules. Instead, it comprises the following components, by mass percentage: 4% glycerol, 2% propylene glycol, 0.09% allantoin, 0.8% sodium chloride, 0.02% potassium chloride, and the balance is phosphate buffer at pH 7.4 (1 L contains 1.44 g Na2HPO4 and 0.24 g KH2PO4). The osmotic pressure of Component B is 294 mOsmol / kg. The remainder is the same as in Example 1.

[0067] Example 3

[0068] The difference from Example 1 is that component B does not contain allantoin. Instead, it comprises the following components, by mass percentage: 4% glycerol, 2% propylene glycol, 1.27% sponge spicules (purchased from Shaanxi Pinhong Biotechnology Co., Ltd.), 0.8% sodium chloride, 0.02% potassium chloride, and the balance is phosphate buffer at pH = 7.4 (1 L contains 1.44 g Na2HPO4 and 0.24 g KH2PO4). The osmotic pressure of component B is 294 mOsmol / kg. The remainder is the same as in Example 1.

[0069] Example 4

[0070] The difference from Example 1 is that Component B does not contain allantoin and sponge spicules. Instead, it comprises the following components, by mass percentage: 4% glycerol, 2% propylene glycol, 0.8% sodium chloride, 0.02% potassium chloride, and the balance is phosphate buffer at pH 7.4 (1 L contains 1.44 g Na2HPO4 and 0.24 g KH2PO4). The osmotic pressure of Component B is 294 mOsmol / kg. The remainder is the same as in Example 1.

[0071] Example 5

[0072] The difference from Example 1 is that Component B does not include allantoin, sponge spicules, or moisturizer. Instead, it comprises the following components, by mass percentage: 0.8% sodium chloride, 0.02% potassium chloride, and the remainder, a phosphate buffer solution having a pH of 7.4 (1 L contains 1.44 g Na2HPO4 and 0.24 g KH2PO4). The osmotic pressure of Component B is 292 mOsmol / kg. The remainder is the same as in Example 1.

[0073] Example 6

[0074] The difference from Example 1 is that component A is liposome exosome hybrid vesicle freeze-dried powder 2, and the preparation method is as follows:

[0075] (1) Same as Example 1;

[0076] (2) The liposome matrix was dissolved in chloroform, and after the solvent was evaporated at 50°C, 1×PBS solution was added and stirred at 50°C for 30 minutes. The mixture was ultrasonically treated at 200W for 20 minutes and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension. The liposome matrix included a complex phospholipid and cholesterol with a molar weight of 61% of the complex phospholipid. The complex phospholipid was a mixture of soybean phosphatidylcholine (DMPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000) in a molar ratio of 5:0.8:4. The average particle size of the blank liposome was 117 nm, the zeta potential was 1.88±0.24 mV, and the phospholipid concentration was 11.64 mg / mL.

[0077] (3) Same as Example 1.

[0078] The rest is the same as Example 1.

[0079] Example 7

[0080] The difference from Example 1 is that component A is liposome exosome hybrid vesicle freeze-dried powder 3, and the preparation method is as follows:

[0081] (1) Same as Example 1;

[0082] (2) The liposome matrix was dissolved in chloroform, and after the solvent was evaporated at 50°C, 1×PBS solution was added and stirred at 50°C for 30 minutes. The mixture was ultrasonically treated at 200W for 20 minutes and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension. The liposome matrix included a complex phospholipid and cholesterol with a molar weight of 56% of the complex phospholipid. The complex phospholipid was a mixture of soybean phosphatidylcholine (DMPC), dilinoleylphosphatidylethanolamine (DOPE), and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000) in a molar ratio of 5:4:3.5. The average particle size of the blank liposome was 129 nm, the zeta potential was -16.72±0.41 mV, and the phospholipid concentration was 11.58 mg / mL.

[0083] (3) Same as Example 1.

[0084] The rest is the same as Example 1.

[0085] Example 8

[0086] The difference from Example 1 is that component A is liposome exosome hybrid vesicle freeze-dried powder 4, and the preparation method is as follows:

[0087] (1) Same as Example 1;

[0088] (2) The liposome matrix was dissolved in chloroform, and after the solvent was evaporated at 50°C, 1×PBS solution was added and stirred at 50°C for 30 minutes. The mixture was ultrasonically treated at 200W for 20 minutes and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension. The liposome matrix included a complex phospholipid and cholesterol with a molar weight of 66% of the complex phospholipid. The complex phospholipid was a mixture of soybean phosphatidylcholine (DMPC), dilinoleylphosphatidylethanolamine (DOPE), and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000) in a molar ratio of 5:2:3. The average particle size of the blank liposome was 115 nm, the zeta potential was -6.82±0.37 mV, and the concentration of phospholipid was 11.37 mg / mL.

[0089] (3) Same as Example 1.

[0090] The rest is the same as Example 1.

[0091] Example 9

[0092] The difference from Example 1 is that component A is liposome exosome hybrid vesicle freeze-dried powder 5, and the preparation method is as follows:

[0093] (1) Same as Example 1;

[0094] (2) Same as Example 1;

[0095] (3) Zinc chloride, chlorogenic acid, and trehalose were added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension was added and stirred evenly to obtain a mixture to be lyophilized; then the mixture was subjected to repeated freeze-thaw cycles for 3 times, where freeze refers to freezing at -80°C for 30 seconds and thaw refers to melting at room temperature for 5 minutes. The mixture was then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a hybrid vesicle dispersion, which was then freeze-dried (freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0096] Among them, the exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:50; the final concentrations of zinc chloride, chlorogenic acid, and trehalose in the freeze-dried mixture were 0.007 g / mL, 0.1 g / mL, and 3 g / mL.

[0097] The rest is the same as Example 1.

[0098] Example 10

[0099] The difference from Example 1 is that component A is liposome exosome hybrid vesicle freeze-dried powder 6, and the preparation method is as follows:

[0100] (1) Same as Example 1;

[0101] (2) Same as Example 1;

[0102] (3) Zinc chloride, chlorogenic acid, and trehalose were added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension was added and stirred evenly to obtain a mixture to be lyophilized; then the mixture was subjected to repeated freeze-thaw cycles for 3 times, where freeze refers to freezing at -80°C for 30 seconds and thaw refers to melting at room temperature for 5 minutes. The mixture was then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a hybrid vesicle dispersion, which was then freeze-dried (freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0103] Among them, the exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:50; the final concentrations of zinc chloride, chlorogenic acid, and trehalose in the freeze-dried mixture were 0.035 g / mL, 0.45 g / mL, and 3 g / mL.

[0104] The rest is the same as Example 1.

[0105] Example 11

[0106] This embodiment provides an anti-hair loss and hair growth essence, comprising a solid component A and a liquid component B, the ratio of component A to component B being 20 μg:1 mL, component A comprising liposome exosome hybrid vesicle freeze-dried powder 7; component B comprising the following components in percentage by mass: 3% glycerol, 2% sodium hyaluronate, 0.2% allantoin, 3% sponge spicules (purchased from Shaanxi Pinhong Biotechnology Co., Ltd.), 0.8% sodium chloride, 0.02% potassium chloride, and the remainder being a phosphate buffer solution with a pH of 7.4 (1 L containing 1.44 g Na2HPO4 and 0.24 g KH2PO4), and the osmotic pressure of component B is 295 mOsmol / kg.

[0107] The preparation method of liposome hybrid vesicle freeze-dried powder 7 is as follows:

[0108] (1) Fresh bovine colostrum was obtained from Guangzhou Suixin Animal Husbandry Co., Ltd. The colostrum was centrifuged at -4°C for 5000 g / 30 min and 12000 g / 1 h to remove milk fat globules, somatic cells and cell debris. The supernatant was taken and then ultracentrifuged at 35000 g / 1 h and 70000 g / 3 h to remove residual milk fat globules and casein. The supernatant was passed through 0.80 μm, 0.45 μm and 0.2 μm filters in sequence. The filtered supernatant was ultracentrifuged at 100000 g for 1 h, and the exosome pellet was resuspended in 1× PBS solution and then ultracentrifuged at 20000 g for 3 h for washing. The exosome pellet was resuspended in 1× PBS solution to obtain an exosome resuspension. Pierce TM A BCA protein assay kit (purchased from Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the total protein concentration of the exosome heavy suspension. Dynamic light scattering (DLS) was used to determine the particle size distribution of the exosome heavy suspension. The protein concentration in the exosome heavy suspension was 58 ng / µL, and the average exosome size was 102 nm.

[0109] (2) The liposome matrix was dissolved in chloroform, and after the solvent was evaporated at 50°C, 1×PBS solution was added and stirred at 50°C for 30 minutes. The mixture was ultrasonically treated at 200W for 20 minutes and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension. The liposome matrix included a complex phospholipid and cholesterol with a molar weight of 31% of the complex phospholipid. The complex phospholipid was a mixture of soybean phosphatidylcholine (DMPC), dilinoleoylphosphatidylethanolamine (DOPE), and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000) in a molar ratio of 5:3:2. The average particle size of the blank liposome was 107 nm, the zeta potential was -12.82±0.55 mV, and the concentration of phospholipid was 14.24 mg / mL.

[0110] (3) Zinc acetate, chlorogenic acid, trehalose, ascorbic acid, and mannitol were added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension was added and stirred evenly to obtain a mixture to be lyophilized; then the mixture was frozen and thawed three times, where freezing refers to freezing at -80°C for 30 seconds and thawing refers to melting at room temperature for 5 minutes. The mixture was then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a hybrid vesicle dispersion, which was then freeze-dried (freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0111] Among them, the exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:30; the final concentrations of zinc acetate, chlorogenic acid, trehalose, ascorbic acid, and mannitol in the freeze-dried mixture were 0.021 g / mL, 0.27 g / mL, 5 g / mL, 0.05 g / mL, and 2.5 g / mL.

[0112] Example 12

[0113] This embodiment provides an anti-hair loss and hair growth essence, comprising a solid component A and a liquid component B, the ratio of component A to component B being 20 μg:1 mL, component A comprising liposome exosome hybrid vesicle freeze-dried powder 8; component B comprising the following components in percentage by mass: 1% glycerol, 1% hydrolyzed collagen (purchased from Hubei Langbowan Biotechnology Co., Ltd.), 0.2% allantoin, 3% sponge bone needles (purchased from Shaanxi Pinhong Biotechnology Co., Ltd.), 0.8% sodium chloride, 0.02% potassium chloride, and the remainder being a phosphate buffer solution with a pH of 7.4 (1 L containing 1.44 g Na2HPO4 and 0.24 g KH2PO4), and the osmotic pressure of component B is 291 mOsmol / kg.

[0114] The preparation method of liposome hybrid vesicle freeze-dried powder 7 is as follows:

[0115] (1) Fresh bovine colostrum was obtained from Guangzhou Suixin Animal Husbandry Co., Ltd. The colostrum was centrifuged at -4°C for 5000 g / 30 min and 12000 g / 1 h to remove milk fat globules, somatic cells and cell debris. The supernatant was taken and then ultracentrifuged at 35000 g / 1 h and 70000 g / 3 h to remove residual milk fat globules and casein. The supernatant was passed through 0.80 μm, 0.45 μm and 0.2 μm filters in sequence. The filtered supernatant was ultracentrifuged at 100000 g for 1 h, and the exosome pellet was resuspended in 1× PBS solution and then ultracentrifuged at 20000 g for 3 h for washing. The exosome pellet was resuspended in 1× PBS solution to obtain an exosome resuspension. Pierce TM A BCA protein assay kit (purchased from Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the total protein concentration of the exosome heavy suspension. Dynamic light scattering (DLS) was used to determine the particle size distribution of the exosome heavy suspension. The protein concentration in the exosome heavy suspension was 157 ng / µL, and the average exosome size was 88 nm.

[0116] (2) The liposome matrix was dissolved in a mixed solution of chloroform and methanol (v:v=3:1), and after the solvent was evaporated at 50°C, 1×PBS solution was added and stirred for hydration at 50°C for 30 minutes. The mixture was ultrasonically treated at 200W for 20 minutes and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension; the liposome matrix included a composite phospholipid and cholesterol with a molar weight of 45% of the composite phospholipid, and the composite phospholipid was a mixture of soybean phosphatidylcholine (DMPC) and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000) with a molar ratio of 6:5. The average particle size of the blank liposome was 138 nm, the zeta potential was 0.2±0.47 mV, and the concentration of phospholipid was 14.24 mg / mL.

[0117] (3) Zinc gluconate, chlorogenic acid, trehalose, methionine, and mannitol were added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension was added and stirred evenly to obtain a mixture to be lyophilized; then the mixture was frozen and thawed three times, where freezing refers to freezing at -80°C for 30 seconds and thawing refers to melting at room temperature for 5 minutes. The mixture was then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a hybrid vesicle dispersion, which was then freeze-dried (freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0118] Among them, the exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:60; the final concentrations of zinc gluconate, chlorogenic acid, trehalose, methionine, and mannitol in the freeze-dried mixture were 0.021 g / mL, 0.27 g / mL, 1 g / mL, 0.05 g / mL, and 5 g / mL.

[0119] Comparative Example 1

[0120] The difference from Example 1 is that exosome lyophilized powder is used instead of liposome exosome hybrid vesicle lyophilized powder 1 as component A, and the rest is the same as Example 1.

[0121] The exosome preparation method is as follows:

[0122] Zinc chloride, chlorogenic acid, and trehalose were dissolved in 1×PBS solution to obtain a lyoprotectant solution, and then the exosome heavy suspension obtained in step (1) of Example 1 was added. After stirring evenly, the solution was extruded through a polycarbonate membrane with a pore size of 200 nm to obtain an exosome dispersion, which was then freeze-dried (freezing at -10°C for 40 min, freezing at -50°C for 3 h, and then drying at -20°C and 10 Pa vacuum for 15 h) to obtain exosome freeze-dried powder.

[0123] The volume ratio of the lyoprotectant solution to the exosome resuspension was the same as the volume ratio of the liposome suspension to the exosome dispersion in Example 1; the final concentrations of zinc chloride, chlorogenic acid, and trehalose in the exosome dispersion were 0.021 g / mL, 0.27 g / mL, and 3 g / mL, respectively.

[0124] Comparative Example 2

[0125] The difference from Example 1 is that the lyoprotectant includes trehalose, mannitol and polyethylene glycol 1000; step (3) is as follows: the lyoprotectant is added to the blank liposome suspension and stirred to dissolve, and then the exosome heavy suspension is added and stirred evenly to obtain a mixture to be lyophilized; then repeated freeze-thaw cycles are performed 3 times, where freezing refers to freezing at -80°C for 30 seconds and thawing refers to melting at room temperature for 5 minutes, and then the hybrid vesicle dispersion is obtained by extruding through a polycarbonate membrane with a pore size of 200 nm, and then freeze-drying (freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10Pa vacuum for 15 hours) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0126] Among them, the exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:50; the final concentrations of polyethylene glycol 1000, trehalose, and mannitol in the freeze-dried mixture were 0.1 g / mL, 5 g / mL, and 5 g / mL.

[0127] The rest is the same as Example 1.

[0128] Comparative Example 3

[0129] The difference from Example 1 is that chlorogenic acid is not added as a freeze-drying protective agent, and step (3) is as follows: zinc chloride and trehalose are added to the blank liposome suspension and stirred to dissolve, and then the exosome heavy suspension is added and stirred evenly to obtain a mixture to be freeze-dried; then the freeze-thaw cycle is repeated 3 times, where freezing refers to freezing at -80°C for 30s and thawing refers to melting at room temperature for 5min, and then the hybrid vesicle dispersion is obtained by extruding through a polycarbonate membrane with a pore size of 200nm, and then freeze-drying (freezing at -10°C for 40min, freezing at -50°C for 3h, and then drying at -20°C and 10Pa vacuum for 15h) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0130] The exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:50; the final concentrations of zinc chloride and trehalose in the freeze-dried mixture were 0.021 g / mL and 3 g / mL respectively.

[0131] The rest is the same as Example 1.

[0132] Comparative Example 4

[0133] The difference from Example 1 is that zinc chloride is not added as a freeze-drying protective agent, and step (3) is as follows: chlorogenic acid and trehalose are added to the blank liposome suspension and stirred to dissolve, and then the exosome heavy suspension is added and stirred evenly to obtain a mixture to be freeze-dried; then, the freeze-thaw cycle is repeated 3 times, where freezing refers to freezing at -80°C for 30s and thawing refers to melting at room temperature for 5min, and then the hybrid vesicle dispersion is obtained by extruding through a polycarbonate membrane with a pore size of 200nm, and then freeze-drying (freezing at -10°C for 40min, freezing at -50°C for 3h, and then drying at -20°C and 10Pa vacuum for 15h) to obtain liposome exosome hybrid vesicle freeze-dried powder.

[0134] The exosome heavy suspension and the blank liposome suspension were mixed at a protein:phospholipid mass ratio of 1:50; the final concentrations of chlorogenic acid and trehalose in the freeze-dried mixture were 0.27 g / mL and 3 g / mL respectively.

[0135] The rest is the same as Example 1.

[0136] Test Example 1: Fusion Efficiency Test

[0137] The fusion efficiency of exosomes and liposomes under different conditions was tested using a fluorescence spectrometer using the fluorescence resonance energy transfer (FRET) method. The principle is to prepare liposomes so that they carry a nitrobenzoxadiazole (NBD) fluorescent donor group and a rhodamine (Rho) fluorescent acceptor group. When the liposomes fuse with the exosomes, the distance between the two fluorescent groups becomes greater, resulting in a decrease in fluorescence intensity. The fluorescent group-carrying liposomes were prepared as follows: 1% of the total molar weight of the total complex phospholipids, Rho-PE, and 1% of NBD-PE were added to replace the equivalent amount of DPPE-PEG2000. Liposomes and hybrid vesicle dispersions were prepared using the same method as in the previous example.

[0138] The test conditions are to excite the sample at 460 nm and measure the fluorescence intensity emitted at 588 nm. Fusion efficiency = (F0-Ft) / (F0-Fmax)×100%, where Ft is the fluorescence intensity of the hybrid vesicle dispersion; F0 is the fluorescence intensity of the lyophilized mixture; and Fmax is the fluorescence intensity after the addition of Triton (maximum fusion fluorescence intensity). The fusion efficiency of different examples is shown in Table 1.

[0139] Table 1

[0140]

[0141]

[0142] The addition of Triton can completely destroy the membrane structure and force all lipids to fuse and mix at the molecular level, thereby obtaining a 100% fusion fluorescence intensity. However, since the fluorescent groups are not completely evenly distributed under natural fusion (including freeze-thaw), and not all liposomes undergo complete fusion, the fusion efficiency is usually less than 100%. As shown in Table 1, since no zinc salt is added to Comparative Examples 2 and 4, their fusion efficiency is significantly lower than that of Example 1, indicating that Zn 2+ It plays a bridging role, attracting exosomes and liposomes, both of which are negatively charged, through electrostatic attraction, thereby reducing the electrostatic repulsion between the two and promoting fusion.

[0143] In Examples 1 and 9-10, varying amounts of zinc salt and chlorogenic acid were added, which affected the fusion efficiency of exosomes and liposomes. Specifically, the greater the amount of zinc salt added, the higher the fusion efficiency. In Examples 1 and 6-8, different liposomes were used. As the zeta potential of the liposomes increased, the fusion efficiency first increased and then decreased. The lower the negative charge and the lower the electrostatic repulsion, the more favorable the fusion of the two. However, when the zeta potential approaches 0, the fusion kinetics decrease, making fusion difficult. In summary, the fusion of exosomes and liposomes is affected by the liposome potential and zinc ions. A liposome potential within an appropriate range is beneficial for improving the fusion efficiency of the two.

[0144] Test Example 2 Hybrid vesicle storage stability test

[0145] The liposome exosome hybrid vesicle freeze-dried powder was left to stand at room temperature for several periods of time (0 days, 15 days, 30 days, 90 days, and 180 days) before being reconstituted and the changes in the particle size and zeta potential of the hybrid vesicles were detected. The reconstitution procedure was as follows: the hybrid vesicle freeze-dried powder was dispersed in 1× PBS at a solid-liquid ratio of 1μg:1mL, incubated at 37°C for 0.5h, and then sampled for detection. The results are shown in the attached figure. Figure 2 and 3 As shown, the points less than 0 d correspond to the hybrid vesicle dispersions obtained after repeated freezing and thawing, and 0 d refers to the hybrid vesicle dispersions immediately redissolved after freeze-drying.

[0146] As time goes by, the absolute value of the Zeta potential of the hybrid vesicles usually decreases gradually, while the particle size increases gradually. This change is a typical sign of decreased stability and reflects the occurrence of particle aggregation and precipitation.

[0147] As can be seen from the figure, after freeze-drying and reconstitution, the potential and particle size of the hybrid vesicle dispersions of all embodiments changed to some extent. Compared to the control examples without zinc salt and chlorogenic acid as lyoprotectants and without liposomes, the changes in the examples with zinc salt and chlorogenic acid added and hybridization were lower, indicating that the addition of zinc salt and chlorogenic acid, as well as hybridization with liposomes, helps protect the morphology and biological activity of the hybrid vesicles during the freeze-drying process. The potential change rates before and after freeze-drying and after 180 days were 4.24% and 11.73% for Example 1, 15.86% and 60.98% for Comparative Example 1, 12.44% and 40.31% for Comparative Example 2, 10.02% and 29.24% for Comparative Example 3, and 18.42% and 63.08% for Comparative Example 4. The zinc ions complex with chlorogenic acid to form a coordination polymer, which can act as a filler, providing a good physical structure, improving the mechanical strength of the lyophilized powder, and facilitating its reconstitution. Comparative Example 4 did not add zinc salt, and chlorogenic acid did not pass the Zn 2+ The complexed state appears as a crystalline state, which causes greater damage to the hybrid vesicles during the freeze-drying process, so both change rates are the highest.

[0148] Test Example 3 Transdermal Absorption Test

[0149] In vitro transdermal experiments were performed using a vertical diffusion cell, with nude mouse skin as the model (abdominal skin, subcutaneous fat and blood vessels removed). The receiving solution was a PBS solution, and the skin sheet was fixed between the supply cell and the receiving cell, with the skin layer facing upward, and equilibrated for 20 minutes. For each sample, components A (15 days after freeze-drying) and B were mixed and allowed to stand for several times (0 h, 0.5 h, 2 h, 4 h), then added to the supply cell. The liquid in the supply cell was stirred intermittently, and the receiving solution was taken after 8 hours using Pierce TMThe BCA protein assay kit was used to quantify the total protein concentration in the receiving fluid and calculate the cumulative permeation per unit area. Each experiment was conducted in triplicate, and the results were calculated as the arithmetic mean. The cumulative permeation per unit area of ​​the skin patch is Q = c × V / A, where c is the total protein concentration in the receiving fluid, V is the total volume of the receiving fluid, and A is the permeation area. The results of testing after mixing components A and B for 0 h are listed in Table 2. The results of testing after mixing components A and B for different periods of time in Example 1 are listed in Table 3.

[0150] Table 2

[0151]

[0152] The results in Table 2 show that the composition of component B has a certain impact on the transdermal penetration of the active substance. Specifically, the presence of allantoin softens the stratum corneum, thereby facilitating transdermal penetration. The sponge's micron-sized needle-like structure facilitates penetration into the stratum corneum, stimulating skin microcirculation and promoting the penetration of the active substance. However, excessive amounts of both are detrimental to maintaining the stability of the hybrid vesicles, leading to vesicle rupture and leakage of the contents, preventing transdermal delivery via lipids, thereby reducing protein penetration. In the comparative example, vesicles ruptured during the freeze-drying process, resulting in a decrease in the number and function of exosomes capable of transdermal delivery, leading to a decrease in transdermal protein penetration.

[0153] Table 3

[0154]

[0155] As can be seen from Table 3, the transdermal permeation rate of each example decreases with the increase of the standing time, but it can still maintain a high transdermal permeation rate when used within 0.5 hours after the components are mixed. The above shows that the component B provided by the present invention can effectively reconstitute the hybrid vesicle freeze-dried powder, maintain its stability for a certain period of time, and promote the transdermal absorption of the active substance in the hybrid vesicles.

[0156] Test Example 4 Skin irritation test

[0157] The test was conducted according to the human skin patch test method in the "Safety Technical Specifications for Cosmetics" (2015 edition). Twenty volunteers aged 25 to 45 years who met the test requirements were selected as subjects (10 males and 10 females). The test sample, a mixture of each essence component A and component B, was added to the patch tester at a dosage of 0.020 ± 0.005 g. A blank control consisted of no substance added. The patch tester containing the test substance was applied to the flexor side of the subject's forearm using non-irritating tape. Gently press the patch with the palm of your hand to evenly adhere to the skin. The patch was applied every 24 hours. After removing the patch, the skin reaction was observed 30 minutes after the indentation disappeared. If a suspicious reaction or positive reaction was detected, the patch test was discontinued for that subject. If a negative reaction was detected, the patch test was repeated as described above at the same test site for a total of six replicates. The reaction level and number of subjects were recorded according to the criteria in Table 3. The results are listed in Table 4.

[0158] Table 3

[0159] grade Identification standards 0 Negative reaction: no irritation, no erythema 1 Suspected reaction: mild erythema 2 Weak positive reaction: erythema 3 Strong positive reaction: erythema, papules, blisters 4 Very strong positive reaction: severe Fuzhong, bullae

[0160] Table 4

[0161]

[0162]

[0163] Examples 1, 9, 11, and 12 provided by the present invention are non-irritating to human skin, while Example 10 is slightly irritating.

[0164] Test Example 5 Hair Follicle Dermal Papilla Cell Proliferation Test

[0165] Human hair follicle dermal papilla cells (HFDPC, passage 5) were seeded in a 96-well plate at a density of 5000 cells / well and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was then replaced. The test was divided into a non-stimulation test and a DHT stimulation test. The non-stimulation test was performed by adding a culture medium with a final concentration of 1 μg / mL of the test sample and culturing for 24 hours. The DHT stimulation test was performed by adding a culture medium with a DHT (dihydrotestosterone) concentration of 30 μM for 24 hours, followed by replacement with a culture medium with a final concentration of 1 μg / mL of the test sample and continuing to culture for 24 hours. CCK-8 solution was added, and the absorbance at a wavelength of 450 nm was measured using a microplate reader, and the cell proliferation rate was calculated, as shown in Table 5. The blank group was a blank culture medium without the test sample. The test group had a final concentration of 1 μg / mL of the test sample, which was obtained by adding component A (15 days after freeze-drying) of each specific embodiment to the culture medium.

[0166] Table 5

[0167]

[0168] As shown in Table 5, the hybrid vesicles provided by the present invention can promote the proliferation of human hair follicle dermal papilla cells and even overcome the cell cycle arrest induced by DHT (DHT can induce human hair follicle dermal papilla cells to shorten the growth phase and prolong the resting phase, which is an important pathogenic factor of AGA).

[0169] In summary, the present invention provides an anti-hair loss and hair growth essence, which has good room temperature storage stability and transdermal penetration performance, which is beneficial to improving the cellular utilization rate of active substances. In addition, the essence can overcome DHT-induced cycle arrest and has a proliferation-promoting effect on human hair follicle dermal papilla cells, and has a good preventive and therapeutic effect on AGA.

[0170] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An anti-hair loss and hair growth essence, characterized in that: The anti-hair loss and hair growth essence includes a solid component A and a liquid component B, wherein the component A includes liposome exosome hybrid vesicle freeze-dried powder, and the liposome exosome hybrid vesicle freeze-dried powder includes the following components: liposome exosome hybrid vesicles and a freeze-drying protective agent; the liposome exosome hybrid vesicles are obtained by membrane fusion of liposomes and exosomes; the freeze-drying protective agent includes zinc ions, chlorogenic acid and trehalose.

2. The anti-hair loss and hair growth essence according to claim 1, characterized in that The exosomes are derived from bovine colostrum or mesenchymal stem cells.

3. The anti-hair loss and hair growth essence according to claim 2, characterized in that The liposome satisfies at least one of the following conditions: Condition 1: The average particle size of the liposome is 100-140 nm; Condition 2: The zeta potential of the liposome is -17 to 3 mV.

4. The anti-hair loss and hair growth essence according to claim 1, characterized in that: The freeze-drying protective agent further comprises an antioxidant and mannitol; the antioxidant is selected from at least one of ascorbic acid and methionine.

5. The anti-hair loss and hair growth essence according to any one of claims 1 to 3, characterized in that: The component B comprises the following components in terms of mass percentage: 0-8% moisturizing agent, 0-0.2% allantoin, 0-3% sponge spicules, 0.8%-1.5% osmotic pressure regulator, and the balance is pH buffer solution.

6. The anti-hair loss and hair growth essence according to claim 5, characterized in that: The moisturizing agent is selected from at least one of glycerin, propylene glycol, butylene glycol, hydrolyzed collagen, hyaluronic acid or its salt; and / or, the osmotic pressure regulator is at least one of citrate, sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium lactate, mannitol and sodium phosphate; And / or, the pH buffer is PBS buffer or Tris-HCl buffer; and / or, the osmotic pressure of component B is 270 to 350 mOsmol / kg; And / or, the pH value of component B is 6.5-7.

5.

7. The anti-hair loss and hair growth essence according to claim 1 or 3, characterized in that: The method for preparing liposome hybrid vesicles comprises the following steps: (1) extracting exosomes to obtain an exosome heavy suspension; (2) preparing a blank liposome suspension; (3) Zinc salt, chlorogenic acid, trehalose, antioxidants, and excipients are added to the blank liposome suspension and stirred to dissolve, and then the exosome resuspension is added and stirred evenly to obtain a freeze-dried mixture, which is repeatedly frozen and thawed 2 to 4 times to obtain a liposome-exosome hybrid vesicle dispersion.

8. The anti-hair loss and hair growth essence according to claim 7, characterized in that: Step (1) extracting exosomes from bovine colostrum or mesenchymal stem cells by ultracentrifugation; And / or, step (2) specifically comprises: dissolving the liposome matrix in a solvent, volatilizing the solvent by rotary evaporation, adding PBS buffer for hydration, ultrasonic treatment, and extruding through a polycarbonate membrane with a pore size of 200 nm to obtain a blank liposome suspension.

9. The anti-hair loss and hair growth essence according to claim 8, characterized in that: The molar ratio of phospholipid to cholesterol in the liposome matrix is ​​1:0.3-0.7; And / or, the zinc salt includes at least one of zinc chloride, zinc acetate, and zinc gluconate; and / or, the protein concentration in the exosome heavy suspension is 50 to 160 ng / uL, and the average particle size of the exosomes is 80 to 120 nm; and / or, the average particle size of the blank liposomes is 100 to 140 nm; and / or, the concentration of phospholipids in the blank liposome suspension is 5 to 15 mg / mL; and / or, the zeta potential of the blank liposome is -17 to 3 mV; and / or, the exosome heavy suspension and the blank liposome suspension are mixed at a protein:phospholipid mass ratio of 1:30 to 60; And / or, the final concentrations of zinc salt, chlorogenic acid, trehalose, antioxidant, and excipient in the lyophilized mixture are 0.007 g / mL to 0.035 g / mL, 0.1 g / mL to 0.45 g / mL, 1 to 5 g / mL, 0 to 0.05 g / mL, and 0 to 5 g / mL.

10. The method for preparing the anti-hair loss and hair growth essence according to claim 1, wherein: The method for preparing the liposome exosome hybrid vesicle freeze-dried powder comprises the following steps: (1) freeze-drying the liposome hybrid vesicle dispersion to obtain liposome hybrid vesicle freeze-dried powder, and encapsulating it under a nitrogen atmosphere to obtain component A; the freeze-drying conditions include: freezing at -10°C for 40 minutes, freezing at -50°C for 3 hours, and then drying at -20°C and 10 Pa vacuum for 15 hours; (2) Prepare a pH buffer solution, then add the other components in component B, stir and disperse, and defoam to obtain component B.

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