Stem cell exosome anti-hair loss and hair growth compound and preparation method thereof

Abstract

The invention relates to the technical field of biology, and discloses a stem cell exosome anti-hair loss and hair growth complex and a preparation method thereof.A hair growth agent is wrapped and loaded in adipose-derived mesenchymal stem cell exosome to form a drug-loaded stem cell exosome; the medicine-carrying stem cell exosome is loaded in cross-linked hydrogel composed of a gentisic acid-cyclodextrin complex and epsilon-polylysine modified hyaluronic acid to form the stem cell exosome anti-hair loss and hair growth complex, and the stem cell exosome anti-hair loss and hair growth complex is directly smeared and externally applied to the scalp surface and acts on the hair follicle infection part in a targeted mode, so that the hair loss prevention and hair growth promotion effect is achieved. The stem cell exosome anti-hair-loss and hair-growth-promoting compound can inhibit bacterial growth, relieve and treat hair follicle inflammation caused by bacterial infection and improve the scalp environment, so that under the condition that inflammation is effectively controlled, the drug-loaded stem cell exosome in the stem cell exosome anti-hair-loss and hair-growth-promoting compound is stably released, damaged hair follicle cells are repaired, hair regeneration is promoted, the exosome treatment effect is prolonged, and the anti-hair-loss and hair-growth-promoting effect is achieved. The purpose of treating alopecia is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a stem cell exosome anti-hair loss and hair growth complex and a preparation method thereof. Background Art

[0002] Folliculitis is a scalp disease mainly caused by bacterial infection of hair follicles. This inflammation can damage the structure and function of hair follicles, causing the deterioration of the scalp environment, affecting the normal function of hair follicles, and causing symptoms such as itching, redness, swelling, and pain on the scalp. Long-term presence may aggravate hair follicle damage, accelerate hair loss, and even form alopecia areata areas. Common treatments for scalp folliculitis caused by bacteria include medication and physical therapy. However, the use of antibiotics, anti-inflammatory drugs and other medications is prone to skin allergies, dryness or other adverse reactions. Long-term use can also easily lead to increased bacterial resistance. Physical therapy has problems such as long treatment cycles and difficulty in curing.

[0003] Stem cell exosomes are a type of extracellular vesicles with a diameter of about 30-150nm secreted by cells. They carry a variety of bioactive substances from donor cells, including DNA, RNA, proteins, etc., into effector cells, effectively regulating the body's cell signal transduction. Studies have shown that exosomes secreted by stem cells can physiologically repair or remove damaged, diseased and aged cells by delivering stem cell-derived bioactive substances to effector cells, and can play anti-inflammatory, immune-regulating, promoting effector cell proliferation, migration and differentiation, and promoting angiogenesis. Therefore, the application of stem cell exosomes in the treatment of scalp folliculitis has great development prospects. However, the stability of stem cell exosomes is easily affected by environmental factors. Due to the complex scalp environment, including oil secretion, bacterial communities and other factors, stem cell exosomes may have been degraded or inactivated before reaching the target area for exosome treatment, making it difficult to effectively deliver stem cell exosomes to hair follicles and deep skin, resulting in a low utilization rate of stem cell exosomes. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a stem cell exosome anti-hair loss and hair growth complex and a preparation method thereof, which can alleviate and treat hair follicle inflammation caused by bacterial infection, reduce damage to scalp tissue and hair follicle cells, facilitate the penetration of exosomes into the deep layers of the scalp, repair damaged hair follicle cells, reshape the scalp environment, and promote hair regeneration.

[0005] A stem cell exosome anti-hair loss and hair growth complex, comprising the following raw materials:

[0006] A 3% to 5% by mass epsilon-polylysine-modified hyaluronic acid solution, a 2% to 4% by mass gentisic acid-cyclodextrin complex solution, and drug-loaded stem cell exosomes, wherein the volume ratio of the epsilon-polylysine-modified hyaluronic acid solution to the gentisic acid-cyclodextrin complex solution is 1:(1 to 2), and the amount of drug-loaded stem cell exosomes added is 0.3% to 2% of the mass of the epsilon-polylysine-modified hyaluronic acid solution;

[0007] The ε-polylysine modified hyaluronic acid solution with a mass fraction of 3% to 5% is prepared by mixing the ε-polylysine modified hyaluronic acid with a phosphate buffer solution;

[0008] The gentisic acid-cyclodextrin complex solution with a mass fraction of 2% to 4% is prepared by mixing the gentisic acid-cyclodextrin complex with a phosphate buffer solution;

[0009] The raw materials for preparing ε-polylysine-modified hyaluronic acid include: 1 to 3 parts by weight of aldehyde-modified hyaluronic acid, 100 to 270 parts by weight of deionized water, 0.1 to 1.5 parts by weight of reaction activation solution, and 0.3 to 1 part by weight of ε-polylysine, wherein the aldehyde-modified hyaluronic acid is obtained by aldehyde-modifying hyaluronic acid with sodium periodate;

[0010] The raw materials for preparing the gentisic acid-cyclodextrin complex include: 12 to 25 parts by weight of gentisic acid, 80 to 130 parts by weight of pretreated amino-β-cyclodextrin solution, and 50 to 90 parts by weight of reaction activation solution, wherein the pretreated amino-β-cyclodextrin solution is obtained by deoxygenating an amino-β-cyclodextrin solution with a mass fraction of 1.5% to 3%;

[0011] Drug-loaded stem cell exosomes are adipose-derived mesenchymal stem cell exosomes that are loaded with hair growth agents.

[0012] Furthermore, the reaction activation solution is prepared by mixing 50% ethanol solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of (150-300): (4-7): (2-5).

[0013] A method for preparing a stem cell exosome anti-hair loss and hair growth complex comprises the following steps:

[0014] S1: Exosome culture and extraction and separation: cryopreserved adipose-derived mesenchymal stem cells are thawed, cultured and revived, and subcultured, and the supernatant is collected. The adipose-derived mesenchymal stem cell exosomes are obtained by ultrafiltration concentration and sucrose density gradient centrifugation;

[0015] S2: Preparation of hair growth agent, dissolving phospholipids and cholesterol in anhydrous ethanol to obtain solution A, dissolving pyrrolidino diaminopyrimidine oxide and adenosine in 1,3-propylene glycol to obtain solution B, heating and stirring glycerol, acetyl tetrapeptide-3, hair growth peptide and solubilizer to obtain solution C, mixing the above solution A, solution B and solution C and homogenizing under high pressure to obtain a hair growth agent;

[0016] S3: Preparation of drug-loaded stem cell exosomes, mixing adipose-derived mesenchymal stem cell exosomes with a phosphate buffer solution to prepare an exosome suspension, mixing the exosome suspension with a hair growth agent, and encapsulating the hair growth agent into adipose-derived mesenchymal stem cell exosomes by incubation, electroporation, extrusion, ultrasound or freeze-thaw treatment to obtain drug-loaded stem cell exosomes;

[0017] S4: Preparation of gentisic acid modified amino-β-cyclodextrin, mixing amino-β-cyclodextrin with deionized water to prepare an amino-β-cyclodextrin solution, deoxygenating the amino-β-cyclodextrin solution to obtain a pretreated amino-β-cyclodextrin solution, adding gentisic acid to the pretreated amino-β-cyclodextrin solution, activating the solution, reacting for a period of time, dialyzing, freezing and drying the obtained solution to obtain a gentisic acid-cyclodextrin complex;

[0018] S5: hyaluronic acid modification treatment, after hyaluronic acid is modified by sodium periodate to obtain aldehyde-modified hyaluronic acid, the aldehyde-modified hyaluronic acid is dissolved in deionized water, after activation treatment, ε-polylysine is added, reacted for a period of time, the obtained solution is dialyzed, frozen and dried to obtain ε-polylysine-modified hyaluronic acid;

[0019] S6: Preparation of stem cell exosome anti-hair loss and hair germination complex: ε-polylysine-modified hyaluronic acid and gentisic acid-cyclodextrin complex are mixed with phosphate buffer solution respectively to prepare ε-polylysine-modified hyaluronic acid solution and gentisic acid-cyclodextrin complex solution; first, the drug-loaded stem cell exosomes are added to the ε-polylysine-modified hyaluronic acid and dispersed by ultrasonic; then, the gentisic acid-cyclodextrin complex solution is added and the mixture is shaken to obtain the stem cell exosome anti-hair loss and hair germination complex.

[0020] Furthermore, step S2: preparing the hair growth agent, specifically comprises the following steps:

[0021] S2.1: 0.3-3 parts by weight of phospholipids and 0.05-0.5 parts by weight of cholesterol are dissolved in 4-10 parts by weight of anhydrous ethanol to obtain solution A, 0.01-0.5 parts by weight of pyrrolidino diaminopyrimidine oxide and 0.01-0.2 parts by weight of adenosine are dissolved in 2-5 parts by weight of 1,3-propylene glycol to obtain solution B, 0.5-3 parts by weight of glycerol, 0.01-0.3 parts by weight of acetyl tetrapeptide-3, 0.01-0.1 parts by weight of hair growth peptide and 1-4 parts by weight of solubilizer are heated and dissolved in a constant temperature water bath at 60-65°C and magnetically stirred for 20-30 minutes to obtain solution C;

[0022] S2.2: After mixing the above solution A, solution B and solution C, continue stirring for 10 to 30 minutes to obtain a mixed solution, and then use a high-pressure homogenizer to homogenize the mixed solution 2 to 3 times under the condition of 60 to 80 MPa to obtain a hair growth agent.

[0023] Further, step S4: preparation of gentisic acid-modified amino-β-cyclodextrin, specifically comprises the following steps:

[0024] S4.1: adding amino-β-cyclodextrin to deionized water to prepare an amino-β-cyclodextrin solution with a mass fraction of 1.5% to 3%, and deoxygenating the amino-β-cyclodextrin solution for 10 to 15 minutes in a water bath heated at 50 to 65° C. and under nitrogen conditions to obtain a pretreated amino-β-cyclodextrin solution;

[0025] S4.2: Add 12 to 25 parts by weight of gentisic acid to 80 to 130 parts by weight of the pretreated amino-β-cyclodextrin solution, stir evenly, and then slowly drop 50 to 90 parts by weight of the reaction activation solution. After the addition of the reaction activation solution, slowly stir and react at 30 to 60° C. under nitrogen for 12 to 24 hours to obtain a mixed solution A;

[0026] S4.3: The obtained mixed solution A is dialyzed in acidified deionized water containing hydrochloric acid for 3 to 5 days, and then frozen and dried to obtain a gentisic acid-cyclodextrin complex.

[0027] Further, step S5: hyaluronic acid modification treatment, specifically comprising the following steps:

[0028] S5.1: After dissolving 1 to 3 parts by weight of hyaluronic acid in 100 to 300 parts by weight of deionized water, add 1 to 3 parts by weight of a 0.5 mol / L sodium periodate solution, and carry out oxidation reaction for 3 to 5 hours in a light-proof condition. After the reaction is completed, add 0.5 to 2 parts by weight of ethylene glycol, and continue stirring for 1 to 3 hours to obtain a post-reaction solution;

[0029] S5.2: dialyzing the reaction solution in deionized water for 3 to 5 days, and freeze-drying to obtain aldehyde-modified hyaluronic acid;

[0030] S5.3: After dissolving 1 to 3 parts by weight of aldehyde-modified hyaluronic acid in 100 to 270 parts by weight of deionized water, slowly drop 0.1 to 1.5 parts by weight of a reaction activation solution, after the addition of the reaction activation solution, slowly stir and react at 30 to 40° C. for 20 to 30 minutes, then add 0.3 to 1 parts by weight of ε-polylysine, stir evenly, and obtain a mixed solution B;

[0031] S5.4: The pH of the mixed solution B is adjusted to 5-6, and the mixture is slowly stirred and reacted at 22-30°C for 8-12 hours. The mixed solution B after the reaction is dialyzed in a weakly acidic aqueous solution with a pH of 5.5 for 3-5 days, and then, it is frozen and dried to obtain ε-polylysine-modified hyaluronic acid.

[0032] Further, step S6: preparation of stem cell exosome anti-hair loss and hair growth complex specifically comprises the following steps:

[0033] S6.1: mixing ε-polylysine-modified hyaluronic acid with a phosphate buffer solution to prepare a 3% to 5% by mass ε-polylysine-modified hyaluronic acid solution, and mixing gentisic acid-cyclodextrin complex with a phosphate buffer solution to prepare a 2% to 4% by mass gentisic acid-cyclodextrin complex solution;

[0034] S6.2: Add the drug-loaded stem cell exosomes to 3% to 5% ε-polylysine-modified hyaluronic acid, where the amount of drug-loaded stem cell exosomes added is 0.3% to 2% of the mass of the ε-polylysine-modified hyaluronic acid solution, and ultrasonically disperse for 10 to 20 minutes. Subsequently, add 2% to 4% gentisic acid-cyclodextrin complex solution, and shake and mix for 10 to 40 minutes to obtain a stem cell exosome anti-hair loss and hair growth complex, wherein the volume ratio of the ε-polylysine-modified hyaluronic acid solution to the gentisic acid-cyclodextrin complex solution is 1:(1 to 2).

[0035] Furthermore, the solubilizer is at least one of polyglycerol ricinoleate, hydrogenated castor oil or caprylic-capric triglyceride.

[0036] Furthermore, the hair growth peptide is at least one of myristoyl pentapeptide-7, myristoyl pentapeptide-16, myristoyl pentapeptide-17, palmitoyl pentapeptide-4, oligopeptide-1, tripeptide-1 or tripeptide-2.

[0037] Furthermore, the phospholipid is at least one of soybean lecithin, egg yolk lecithin or hydrogenated soybean lecithin.

[0038] The present invention has the following advantages:

[0039] 1. In the present invention, amino-β-cyclodextrin is modified by gentisic acid, so that the carboxyl group in gentisic acid reacts with the amino group in amino-β-cyclodextrin to form an amide bond, thereby forming a gentisic acid-cyclodextrin complex. The polyphenol group on gentisic acid gives the gentisic acid-cyclodextrin complex good tissue adhesion, antioxidant and antibacterial properties, and can enhance the adhesion ability of the gentisic acid-cyclodextrin complex on the scalp or hair follicle surface, which helps the accumulation of the gentisic acid-cyclodextrin complex at the infection site, reduces the damage of hair follicle cells by oxidative stress, helps protect the scalp hair follicles from oxidative damage, and can play an antibacterial and anti-inflammatory role, reduce tissue damage caused by inflammation, and effectively relieve and treat scalp hair follicle inflammation caused by bacterial infection.

[0040] 2. In the present invention, the ε-polylysine-modified hyaluronic acid obtained by aldehyde treatment of hyaluronic acid and then modification with ε-polylysine not only has good antibacterial activity and biocompatibility, but also, in the ε-polylysine-modified hyaluronic acid, the positively charged ε-polylysine can provide a targeting effect, promote the ε-polylysine-modified hyaluronic acid to aggregate on the surface of the negatively charged bacterial cell membrane, and interact electrostatically with the bacterial surface, thereby promoting the disappearance of inflammation and killing of bacteria.

[0041] 3. In the present invention, the drug-loaded stem cell exosomes are loaded into a cross-linked hydrogel composed of a gentisic acid-cyclodextrin complex and ε-polylysine-modified hyaluronic acid to form a stem cell exosome anti-hair loss and hair growth complex. The stem cell exosome anti-hair loss and hair growth complex is directly applied to the scalp surface for external application. With the help of the targeting characteristics of the ε-polylysine-modified hyaluronic acid component in the stem cell exosome anti-hair loss and hair growth complex, the stem cell exosome anti-hair loss and hair growth complex can directly act on the infected part of the hair follicle, inhibit bacterial growth, and assist in reducing inflammation. At the same time, with the help of the antioxidant effect of the gentisic acid-cyclodextrin complex component in the stem cell exosome anti-hair loss and hair growth complex, The antibacterial and anti-inflammatory properties are beneficial to further relieve and treat follicular inflammation caused by bacterial infection and reduce damage to scalp tissue and hair follicle cells. In this way, the scalp environment is improved and follicular inflammation is relieved through the synergistic combination of gentisic acid-cyclodextrin complex and ε-polylysine-modified hyaluronic acid. In addition, when scalp follicular inflammation is effectively controlled, the drug-loaded stem cell exosomes loaded in the stem cell exosome anti-hair loss and hair growth complex can be stably released, which is beneficial for the drug-loaded stem cell exosomes to penetrate into the deep layers of the scalp, repair damaged hair follicle cells, reshape the scalp environment, promote hair regeneration, prolong the therapeutic effect of exosomes, and achieve the purpose of treating hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The flowchart is a method for preparing the stem cell exosome anti-hair loss and hair growth complex in an embodiment of the present invention.

[0043] Figure 2 The graph shows the antibacterial and antimicrobial effects of the stem cell exosome anti-hair loss and hair growth complex prepared in the examples and comparative examples.

[0044] Figure 3 This is a result diagram of the skin condition of the hair loss area on the back of mice after applying the stem cell exosome anti-hair loss and hair growth complex prepared in the example and the comparative example.

[0045] Figure 4 This is a graph showing the results of hair growth on the back of mice after applying the stem cell exosome anti-hair loss and hair growth complex prepared in the examples and comparative examples.

[0046] Figure 5 This is a fluorescence intensity curve diagram after fluorescence imaging detection and quantitative normalization of the stem cell exosome anti-hair loss and hair growth complex prepared in Comparative Example 7 after absorption by the back skin of mice. DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] Example 1

[0049] A method for preparing a stem cell exosome anti-hair loss and hair growth complex, such as Figure 1 As shown, the specific steps include:

[0050] S1: Exosome culture and extraction and separation,

[0051] S1.1: Place the cryopreserved adipose-derived mesenchymal stem cells and basal culture medium in a 35°C water bath and slowly thaw, then centrifuge, discard the supernatant, resuspend the cell pellet, inoculate the obtained cell suspension into a culture bottle, and place the culture bottle in a carbon dioxide incubator for 3 days to obtain resuscitated adipose-derived mesenchymal stem cells;

[0052] S1.2: Subsequently, a subculture medium made of basal culture medium, fetal bovine serum, sodium heparin, and vitamin E is added to the culture bottle, and the adipose-derived mesenchymal stem cells in the culture bottle are subcultured for 3 days;

[0053] S1.3: Then, the culture flask is transferred to a biosafety cabinet, and the supernatant is collected. The supernatant is concentrated by ultrafiltration and centrifuged using a 30% sucrose density gradient, and the concentrate is collected to obtain adipose-derived mesenchymal stem cell exosomes;

[0054] S2: Preparation of hair growth agent,

[0055] S2.1: 3 parts by weight of soybean lecithin and 0.5 parts by weight of cholesterol were dissolved in 6 parts by weight of anhydrous ethanol to obtain solution A, 0.5 parts by weight of pyrrolidino diaminopyrimidine oxide and 0.2 parts by weight of adenosine were dissolved in 5 parts by weight of 1,3-propylene glycol to obtain solution B, 3 parts by weight of glycerol, 0.3 parts by weight of acetyl tetrapeptide-3, 0.1 parts by weight of palmitoyl pentapeptide-4 and 4 parts by weight of hydrogenated castor oil were heated and dissolved in a constant temperature water bath at 65° C. and magnetically stirred for 20 min to obtain solution C;

[0056] S2.2: After mixing the above solution A, solution B and solution C, continue stirring for 30 minutes to obtain a mixed solution, and then use a high-pressure homogenizer to homogenize the mixed solution three times at 80 MPa to obtain a hair growth agent;

[0057] S3: Preparation of drug-loaded stem cell exosomes,

[0058] S3.1: Mix the adipose-derived mesenchymal stem cell exosomes with phosphate buffer solution to prepare an exosome suspension with a mass fraction of 3%;

[0059] S3.2: Mixing an exosome suspension with a mass fraction of 3% with a hair growth agent, wherein the mass ratio of the exosome suspension to the hair growth agent is 100:4, and using a needle ultrasound instrument to perform ultrasonic treatment on the mixed solution of the exosome suspension and the hair growth agent, wherein the ultrasonic treatment conditions are as follows: the voltage range is 500 V, the frequency range is 2 KHz, the number of cycles range is 10 times, the ultrasonic output time in the cycle is 5 s, and the interval time range is 10 s;

[0060] S3.3: Subsequently, the mixed solution of the exosome suspension after ultrasonic treatment and the hair growth agent is placed in an ultrafiltration tube with a molecular weight cutoff range of 100 KDa, and ultrafiltration and washing are performed three times using physiological saline to obtain drug-loaded stem cell exosomes;

[0061] S4: Preparation of gentisic acid modified amino-β-cyclodextrin,

[0062] S4.1: adding amino-β-cyclodextrin to deionized water to prepare a 3% amino-β-cyclodextrin solution, heating the amino-β-cyclodextrin solution in a water bath at 65° C. under nitrogen conditions, deoxygenating the amino-β-cyclodextrin solution for 15 minutes to obtain a pretreated amino-β-cyclodextrin solution;

[0063] S4.2: Add 25 parts by weight of gentisic acid to 130 parts by weight of pretreated amino-β-cyclodextrin solution, stir evenly, and then slowly drip 70 parts by weight of reaction activation solution. After the addition of the reaction activation solution is completed, slowly stir and react at 30°C under nitrogen for 24 hours to obtain a mixed solution A, wherein the reaction activation solution is prepared by mixing a 50% ethanol solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 150:7:5;

[0064] S4.3: dialyzing the obtained mixed solution A in acidified deionized water containing hydrochloric acid for 4 days, and then freezing and drying to obtain a gentisic acid-cyclodextrin complex;

[0065] S5: Hyaluronic acid modification treatment,

[0066] S5.1: After dissolving 3 parts by weight of hyaluronic acid in 150 parts by weight of deionized water, add 3 parts by weight of a 0.5 mol / L sodium periodate solution, and carry out oxidation reaction for 5 hours in a dark environment. After the reaction is completed, add 2.4 parts by weight of ethylene glycol, and continue stirring for 3 hours to obtain a post-reaction solution;

[0067] S5.2: dialyzing the reaction solution in deionized water for 4 days, and freeze-drying to obtain aldehyde-modified hyaluronic acid;

[0068] S5.3: After dissolving 3 parts by weight of formaldehyde-modified hyaluronic acid in 270 parts by weight of deionized water, 1.5 parts by weight of reaction activation solution was slowly added dropwise. After the addition of the reaction activation solution, the mixture was slowly stirred at 40° C. for 20 minutes. Subsequently, 1 part by weight of ε-polylysine was added and stirred evenly to obtain a mixed solution B.

[0069] S5.4: adjusting the pH of the mixed solution B to 5.5, slowly stirring and reacting at 30° C. for 8 h, dialyzing the mixed solution B after the reaction in a weakly acidic aqueous solution with a pH of 5.5 for 4 days, and then freezing and drying to obtain ε-polylysine-modified hyaluronic acid;

[0070] S6: Preparation of stem cell exosome anti-hair loss and hair growth complex,

[0071] S6.1: mixing ε-polylysine-modified hyaluronic acid with a phosphate buffer solution to prepare a 5% by mass ε-polylysine-modified hyaluronic acid solution, and mixing gentisic acid-cyclodextrin complex with a phosphate buffer solution to prepare a 3% by mass gentisic acid-cyclodextrin complex solution;

[0072] S6.2: Add the drug-loaded stem cell exosomes to a 5% by mass ε-polylysine-modified hyaluronic acid solution, where the added amount of the drug-loaded stem cell exosomes is 2% of the mass of the ε-polylysine-modified hyaluronic acid solution. Ultrasonic dispersion is performed for 20 minutes. Subsequently, a 3% by mass gentisic acid-cyclodextrin complex solution equal in volume to the ε-polylysine-modified hyaluronic acid solution is added. Shake and mix for 40 minutes to obtain a stem cell exosome anti-hair loss and hair growth complex.

[0073] Example 2

[0074] A method for preparing a stem cell exosome anti-hair loss and hair growth complex, such as Figure 1 As shown, the specific steps include:

[0075] S1: Exosome culture and extraction and separation,

[0076] S1.1: Place the cryopreserved adipose-derived mesenchymal stem cells and basal culture medium in a 35°C water bath and slowly thaw, then centrifuge, discard the supernatant, resuspend the cell pellet, inoculate the obtained cell suspension into a culture bottle, and place the culture bottle in a carbon dioxide incubator for 3 days to obtain resuscitated adipose-derived mesenchymal stem cells;

[0077] S1.2: Subsequently, a subculture medium made of basal culture medium, fetal bovine serum, sodium heparin, and vitamin E is added to the culture bottle, and the adipose-derived mesenchymal stem cells in the culture bottle are subcultured for 3 days;

[0078] S1.3: Then, the culture flask is transferred to a biosafety cabinet, and the supernatant is collected. The supernatant is concentrated by ultrafiltration and centrifuged using a 30% sucrose density gradient, and the concentrate is collected to obtain adipose-derived mesenchymal stem cell exosomes;

[0079] S2: Preparation of hair growth agent,

[0080] S2.1: 3 parts by weight of soybean lecithin and 0.5 parts by weight of cholesterol were dissolved in 6 parts by weight of anhydrous ethanol to obtain solution A, 0.5 parts by weight of pyrrolidino diaminopyrimidine oxide and 0.2 parts by weight of adenosine were dissolved in 5 parts by weight of 1,3-propylene glycol to obtain solution B, 3 parts by weight of glycerol, 0.3 parts by weight of acetyl tetrapeptide-3, 0.1 parts by weight of palmitoyl pentapeptide-4 and 4 parts by weight of hydrogenated castor oil were heated and dissolved in a constant temperature water bath at 60° C. and magnetically stirred for 30 min to obtain solution C;

[0081] S2.2: After mixing the above solution A, solution B and solution C, continue stirring for 10 minutes to obtain a mixed solution, and then use a high-pressure homogenizer to homogenize the mixed solution three times at 60 MPa to obtain a hair growth agent;

[0082] S3: Preparation of drug-loaded stem cell exosomes,

[0083] S3.1: Mix the adipose-derived mesenchymal stem cell exosomes with phosphate buffer solution to prepare an exosome suspension with a mass fraction of 3%;

[0084] S3.2: Mixing an exosome suspension with a mass fraction of 3% with a hair growth agent, wherein the mass ratio of the exosome suspension to the hair growth agent is 100:4, and using a needle ultrasound instrument to perform ultrasonic treatment on the mixed solution of the exosome suspension and the hair growth agent, wherein the ultrasonic treatment conditions are as follows: the voltage range is 500 V, the frequency range is 2 KHz, the number of cycles range is 10 times, the ultrasonic output time in the cycle is 5 s, and the interval time range is 10 s;

[0085] S3.3: Subsequently, the mixed solution of the exosome suspension after ultrasonic treatment and the hair growth agent is placed in an ultrafiltration tube with a molecular weight cutoff range of 100 KDa, and ultrafiltration and washing are performed three times using physiological saline to obtain drug-loaded stem cell exosomes;

[0086] S4: Preparation of gentisic acid modified amino-β-cyclodextrin,

[0087] S4.1: adding amino-β-cyclodextrin to deionized water to prepare a 3% amino-β-cyclodextrin solution, heating the amino-β-cyclodextrin solution in a water bath at 60° C. under nitrogen conditions, deoxygenating the amino-β-cyclodextrin solution for 10 minutes to obtain a pretreated amino-β-cyclodextrin solution;

[0088] S4.2: Add 25 parts by weight of gentisic acid to 130 parts by weight of pretreated amino-β-cyclodextrin solution, stir evenly, and then slowly drip 70 parts by weight of reaction activation solution. After the addition of the reaction activation solution is completed, slowly stir and react for 12 hours at 60° C. under nitrogen to obtain a mixed solution A, wherein the reaction activation solution is prepared by mixing a 50% ethanol solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 150:7:5;

[0089] S4.3: dialyzing the obtained mixed solution A in acidified deionized water containing hydrochloric acid for 4 days, and then freezing and drying to obtain a gentisic acid-cyclodextrin complex;

[0090] S5: Hyaluronic acid modification treatment,

[0091] S5.1: After dissolving 3 parts by weight of hyaluronic acid in 150 parts by weight of deionized water, add 3 parts by weight of a 0.5 mol / L sodium periodate solution, and carry out oxidation reaction for 3 hours in a dark environment. After the reaction is completed, add 2.4 parts by weight of ethylene glycol, and continue stirring for 1 hour to obtain a post-reaction solution;

[0092] S5.2: dialyzing the reaction solution in deionized water for 4 days, and freeze-drying to obtain aldehyde-modified hyaluronic acid;

[0093] S5.3: After dissolving 3 parts by weight of formaldehyde-modified hyaluronic acid in 270 parts by weight of deionized water, 1.5 parts by weight of reaction activation solution was slowly added dropwise. After the addition of the reaction activation solution, the mixture was slowly stirred at 30° C. for 30 minutes. Subsequently, 1 part by weight of ε-polylysine was added and stirred evenly to obtain a mixed solution B.

[0094] S5.4: adjusting the pH of the mixed solution B to 5.5, slowly stirring the solution at 22° C. for 12 h, dialyzing the mixed solution B in a weakly acidic aqueous solution with a pH of 5.5 for 4 days, and then freezing and drying the solution to obtain ε-polylysine-modified hyaluronic acid;

[0095] S6: Preparation of stem cell exosome anti-hair loss and hair growth complex,

[0096] S6.1: mixing ε-polylysine-modified hyaluronic acid with a phosphate buffer solution to prepare a 5% by mass ε-polylysine-modified hyaluronic acid solution, and mixing gentisic acid-cyclodextrin complex with a phosphate buffer solution to prepare a 3% by mass gentisic acid-cyclodextrin complex solution;

[0097] S6.2: Add the drug-loaded stem cell exosomes to a 5% by mass ε-polylysine-modified hyaluronic acid solution, where the added amount of the drug-loaded stem cell exosomes is 2% of the mass of the ε-polylysine-modified hyaluronic acid solution. Ultrasonic dispersion is performed for 10 minutes. Subsequently, a 3% by mass gentisic acid-cyclodextrin complex solution equal in volume to the ε-polylysine-modified hyaluronic acid solution is added. Shake and mix for 20 minutes to obtain a stem cell exosome anti-hair loss and hair growth complex.

[0098] Example 3

[0099] A method for preparing a stem cell exosome anti-hair loss and hair growth complex, such as Figure 1 As shown, the specific steps include:

[0100] S1: Exosome culture and extraction and separation,

[0101] S1.1: Place the cryopreserved adipose-derived mesenchymal stem cells and basal culture medium in a 35°C water bath and slowly thaw, then centrifuge, discard the supernatant, resuspend the cell pellet, inoculate the obtained cell suspension into a culture bottle, and place the culture bottle in a carbon dioxide incubator for 3 days to obtain resuscitated adipose-derived mesenchymal stem cells;

[0102] S1.2: Subsequently, a subculture medium made of basal culture medium, fetal bovine serum, sodium heparin, and vitamin E is added to the culture bottle, and the adipose-derived mesenchymal stem cells in the culture bottle are subcultured for 3 days;

[0103] S1.3: Then, the culture flask is transferred to a biosafety cabinet, and the supernatant is collected. The supernatant is concentrated by ultrafiltration and centrifuged using a 30% sucrose density gradient, and the concentrate is collected to obtain adipose-derived mesenchymal stem cell exosomes;

[0104] S2: Preparation of hair growth agent,

[0105] S2.1: 1 part by weight of soybean lecithin and 0.1 part by weight of cholesterol were dissolved in 4 parts by weight of anhydrous ethanol to obtain solution A, 0.1 part by weight of pyrrolidino diaminopyrimidine oxide and 0.06 part by weight of adenosine were dissolved in 2 parts by weight of 1,3-propylene glycol to obtain solution B, 1 part by weight of glycerol, 0.1 part by weight of acetyl tetrapeptide-3, 0.1 part by weight of palmitoyl pentapeptide-4 and 1 part by weight of hydrogenated castor oil were heated and dissolved in a constant temperature water bath at 65°C and magnetically stirred for 30 minutes to obtain solution C;

[0106] S2.2: After mixing the above solution A, solution B and solution C, continue stirring for 30 minutes to obtain a mixed solution, and then use a high-pressure homogenizer to homogenize the mixed solution three times at 80 MPa to obtain a hair growth agent;

[0107] S3: Preparation of drug-loaded stem cell exosomes,

[0108] S3.1: Mix the exosomes of adipose-derived mesenchymal stem cells with phosphate buffer solution to prepare an exosome suspension with a mass fraction of 1.5%;

[0109] S3.2: Mixing an exosome suspension with a mass fraction of 1.5% with a hair growth agent, wherein the mass ratio of the exosome suspension to the hair growth agent is 100:1, and using a needle ultrasound instrument to perform ultrasonic treatment on the mixed solution of the exosome suspension and the hair growth agent, the ultrasonic treatment conditions are as follows: the voltage range is 500V, the frequency range is 2KHz, the number of cycles range is 10 times, the ultrasonic output time in the cycle is 5s, and the interval time range is 10s;

[0110] S3.3: Subsequently, the mixed solution of the exosome suspension after ultrasonic treatment and the hair growth agent is placed in an ultrafiltration tube with a molecular weight cutoff range of 100 KDa, and ultrafiltration and washing are performed three times using physiological saline to obtain drug-loaded stem cell exosomes;

[0111] S4: Preparation of gentisic acid modified amino-β-cyclodextrin,

[0112] S4.1: adding amino-β-cyclodextrin to deionized water to prepare an amino-β-cyclodextrin solution with a mass fraction of 1.5%, and deoxygenating the amino-β-cyclodextrin solution for 15 minutes in a water bath heated at 65° C. under nitrogen conditions to obtain a pretreated amino-β-cyclodextrin solution;

[0113] S4.2: 12 parts by weight of gentisic acid are added to 80 parts by weight of the pretreated amino-β-cyclodextrin solution, and after stirring evenly, 50 parts by weight of the reaction activation solution is slowly dripped in. After the reaction activation solution is dripped in, the mixture is slowly stirred and reacted at 30° C. under nitrogen for 24 hours to obtain a mixed solution A, wherein the reaction activation solution is prepared by mixing a 50% ethanol solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 150:7:5;

[0114] S4.3: dialyzing the obtained mixed solution A in acidified deionized water containing hydrochloric acid for 4 days, and then freezing and drying to obtain a gentisic acid-cyclodextrin complex;

[0115] S5: Hyaluronic acid modification treatment,

[0116] S5.1: After dissolving 1 part by weight of hyaluronic acid in 100 parts by weight of deionized water, add 1 part by weight of a 0.5 mol / L sodium periodate solution, and carry out oxidation reaction for 5 hours in a dark environment. After the reaction is completed, add 0.5 parts by weight of ethylene glycol, and continue stirring for 3 hours to obtain a post-reaction solution;

[0117] S5.2: dialyzing the reaction solution in deionized water for 4 days, and freeze-drying to obtain aldehyde-modified hyaluronic acid;

[0118] S5.3: After dissolving 1 part by weight of formaldehyde-modified hyaluronic acid in 100 parts by weight of deionized water, 0.5 parts by weight of reaction activation solution was slowly added dropwise. After the addition of the reaction activation solution, the mixture was slowly stirred at 40° C. for 20 minutes. Subsequently, 0.5 parts by weight of ε-polylysine was added and stirred evenly to obtain a mixed solution B.

[0119] S5.4: adjusting the pH of the mixed solution B to 5.5, slowly stirring and reacting at 30° C. for 8 h, dialyzing the mixed solution B after the reaction in a weakly acidic aqueous solution with a pH of 5.5 for 4 days, and then freezing and drying to obtain ε-polylysine-modified hyaluronic acid;

[0120] S6: Preparation of stem cell exosome anti-hair loss and hair growth complex,

[0121] S6.1: mixing ε-polylysine-modified hyaluronic acid with a phosphate buffer solution to prepare a 3% by mass ε-polylysine-modified hyaluronic acid solution, and mixing gentisic acid-cyclodextrin complex with a phosphate buffer solution to prepare a 3% by mass gentisic acid-cyclodextrin complex solution;

[0122] S6.2: Add the drug-loaded stem cell exosomes to a 3% mass fraction of ε-polylysine modified hyaluronic acid solution, the added amount of the drug-loaded stem cell exosomes is 1% of the mass of the ε-polylysine modified hyaluronic acid solution, ultrasonically disperse for 20 minutes, then add a 3% mass fraction of gentisic acid-cyclodextrin complex solution equal to the volume of the ε-polylysine modified hyaluronic acid solution, shake and mix for 40 minutes to obtain a stem cell exosome anti-hair loss and hair growth complex.

[0123] Comparative Example 1

[0124] Compared with Example 1, the difference of Comparative Example 1 is that step S4 is removed, the gentisic acid-cyclodextrin complex in step S6.1 is replaced by amino-β-cyclodextrin, amino-β-cyclodextrin is mixed with phosphate buffer solution to prepare an amino-β-cyclodextrin solution with a mass fraction of 3%, and the gentisic acid-cyclodextrin complex solution in step S6.2 is replaced by amino-β-cyclodextrin solution. The other steps remain unchanged to prepare a stem cell exosome anti-hair loss and hair growth complex, which is recorded as Comparative Example 1.

[0125] Comparative Example 2

[0126] Compared with Example 1, the difference of Comparative Example 2 is that steps S5.3-S5.4 are removed, the ε-polylysine-modified hyaluronic acid in step S6.1 is replaced with aldehyded hyaluronic acid, the aldehyded hyaluronic acid is mixed with a phosphate buffer solution to prepare a aldehyded hyaluronic acid solution with a mass fraction of 5%, and the ε-polylysine-modified hyaluronic acid solution in step S6.2 is replaced with an aldehyded hyaluronic acid solution. The other steps remain unchanged to prepare a stem cell exosome anti-hair loss and hair growth complex, which is recorded as Comparative Example 2.

[0127] Comparative Example 3

[0128] Compared with Example 1, the difference of Comparative Example 3 is that steps S5.1-S5.2 are removed, the aldehyded hyaluronic acid in step S5.3 is replaced with hyaluronic acid, and the other steps remain unchanged to prepare a stem cell exosome anti-hair loss and hair growth complex, which is recorded as Comparative Example 3.

[0129] Comparative Example 4

[0130] Compared with Example 1, the difference of Comparative Example 4 is that the step of adding drug-loaded stem cell exosomes in step S6.2 is removed, and the ε-polylysine-modified hyaluronic acid solution and the gentisic acid-cyclodextrin complex solution are directly mixed in a volume ratio of 1:1, and the other steps remain unchanged to prepare a stem cell exosome anti-hair loss and hair growth complex, which is recorded as Comparative Example 4.

[0131] Comparative Example 5

[0132] Compared with Example 1, the difference of Comparative Example 5 is that step S6.2 is removed, and the drug-loaded stem cell exosomes and the cross-linker (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) are added to the 5% mass fraction of ε-polylysine-modified hyaluronic acid, and the added amounts of the drug-loaded stem cell exosomes and the cross-linker are 2% and 0.5% of the mass of the ε-polylysine-modified hyaluronic acid solution, respectively. Ultrasonic dispersion is performed for 10 to 20 minutes and stirring is performed for 2 hours to obtain a stem cell exosome anti-hair loss and hair growth complex. The other steps remain unchanged and are recorded as Comparative Example 5.

[0133] Comparative Example 6

[0134] Compared with Example 1, the difference of Comparative Example 6 is that step S6.2 is removed, and the drug-loaded stem cell exosomes and the cross-linker (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) are added to a gentisic acid-cyclodextrin complex solution with a mass fraction of 3%, and the added amounts of the drug-loaded stem cell exosomes and the cross-linker are 2% and 0.5% of the mass of the gentisic acid-cyclodextrin complex solution, respectively. Ultrasonic dispersion is performed for 10 to 20 minutes and stirring is performed for 2 hours to obtain a stem cell exosome anti-hair loss and hair growth complex. The other steps remain unchanged and are recorded as Comparative Example 6.

[0135] Comparative Example 7

[0136] Compared with Example 1, the difference of Comparative Example 7 is that the drug-loaded stem cell exosomes are labeled with a lipophilic PKH26 dye (red) to obtain PKH26-labeled drug-loaded stem cell exosomes, and the drug-loaded stem cell exosomes in step S6.2 are replaced with PKH26-labeled drug-loaded stem cell exosomes, and the other steps remain unchanged to prepare a stem cell exosome anti-hair loss and hair growth complex, which is recorded as Comparative Example 7.

[0137] Antibacterial test: Malassezia furfur was activated and cultured, and the activated Malassezia furfur was added into sterile water at a concentration of 10 7cfu / mL of Malassezia suspension, take 10 portions of Malassezia suspension with a volume of 0.5mL each, and inject the 10 portions of Malassezia suspension into 10 culture dishes filled with rapeseed oil liquid culture medium respectively. After the Malassezia suspension is evenly applied, 9 culture dishes are randomly taken out from the above 10 culture dishes, and 3 Oxford cups are placed on these 9 culture dishes. Subsequently, 0.12mL is taken from each of the stem cell exosome anti-hair loss and hair growth complex in Examples 1-3 and Comparative Examples 1-6 and injected into the Oxford cups of nine culture dishes respectively. The three Oxford cups on the same culture dish are injected with the stem cell exosome anti-hair loss and hair growth complex of the same component, and the remaining culture dish is added with sterile water as a control group. Then, all the above culture dishes are placed in a 37°C constant temperature incubator for 48 hours. After the culture is completed, the culture dishes are taken out to observe whether an antibacterial zone appears, and a vernier caliper is used to measure the average diameter of the antibacterial zone on each culture dish. The antibacterial test results are as follows. Figure 2 shown.

[0138] Depend on Figure 2 It can be seen that in the antibacterial test, the culture dishes added with the stem cell exosome anti-hair loss and hair growth complex in Examples 1-3 have antibacterial circle diameters greater than 18 mm, indicating that the stem cell exosome anti-hair loss and hair growth complex in Examples 1-3 have significant inhibitory effects on Malassezia, and the ε-polylysine-modified hyaluronic acid and gentisic acid-cyclodextrin complex components in the stem cell exosome anti-hair loss and hair growth complex have good antibacterial and antibacterial effects, and can play an antibacterial and antibacterial role on Malassezia that is prone to cause skin diseases such as pityrosporum fungus, seborrheic dermatitis and folliculitis.

[0139] Folliculitis model construction test: 11 male mice, all 5 weeks old and weighing 18-20 g, were selected. After 7 days of adaptive feeding, the back of each mouse was depilated with paraffin, and the depilatory area was 2.5 cm × 2.5 cm. 30 μL of 10% paraffin was applied to the depilatory area on the back of each mouse. 7 cfu / mL of Malassezia suspension was applied 3 times a day for 5 consecutive days to establish an animal model of folliculitis.

[0140] 9 mice were randomly selected from 11 mice, and the stem cell exosome anti-hair loss and hair growth complex in Examples 1-3 and Comparative Examples 1-6 were applied to the hair loss areas on the backs of the 9 mice, respectively, with an application amount of 0.1 mL each time, and the application was continued for 5 days. One of the remaining two mice was randomly selected as the control group and no treatment was performed.

[0141] After 5 consecutive days of application, the back skin conditions of the mice in the control group and the mice applied with the stem cell exosome anti-hair loss and hair growth complex of Examples 1-3 and Comparative Examples 1-6 were observed and evaluated. The skin condition evaluation criteria were: erythema, edema / papules, epidermal exfoliation / scratches, and scales (dryness), which were divided into four levels: none, mild, moderate, and severe, and recorded as 0, 1, 2, and 3 points respectively. The skin condition results are shown in the figure. Figure 3 As shown; 15 days later, 5 hairs were randomly plucked from the hair loss area on the back of the control group mice and the mice coated with the stem cell exosome anti-hair loss and hair growth complex of Examples 1-3 and Comparative Examples 1-6, and the hair length of the hair loss area on the back of each mouse was measured, and the average value (mm) was taken. The hair growth results are shown in Figure 4 shown.

[0142] Depend on Figure 3 , 4 It can be seen that the use of the stem cell exosome anti-hair loss and hair growth complex in Examples 1 and 3 to treat mice suffering from folliculitis caused by bacteria can effectively improve the inflammation and promote hair growth, indicating that the drug-loaded stem cell exosomes, ε-polylysine-modified hyaluronic acid and gentisic acid-cyclodextrin complex components in the stem cell exosome anti-hair loss and hair growth complex can effectively control the hair follicle inflammation caused by bacteria and promote hair growth. Among them, the ε-polylysine-modified hyaluronic acid and gentisic acid-cyclodextrin complex components have more significant effects on relieving and improving inflammation, while the drug-loaded stem cell exosome components have more significant effects on promoting hair growth.

[0143] The last remaining mouse in the folliculitis animal model was smeared with the stem cell exosome anti-hair loss and hair growth complex in Comparative Example 7, with an application amount of 0.1 mL. On the 0.5th day, 1st day, 2nd day, 3rd day, 5th day and 7th day after smearing, the back of the mouse was subjected to fluorescence imaging detection using a small animal living imager. Before the first detection, the hair loss area on the back of the mouse was rinsed with a phosphate buffer solution to remove the unabsorbed fluorescent substance attached to the surface of the back of the mouse. Subsequently, the fluorescence intensity results after the fluorescence imaging detection were quantified and normalized using Spectrum Living ImageJ4.0 software. The detection results are shown in Figure 2. Figure 5 shown.

[0144] Depend on Figure 5 It can be seen that after the stem cell exosome anti-hair loss and hair growth complex labeled with PKH26 was applied to the back of the mouse, the drug-loaded exosomes in the stem cell exosome anti-hair loss and hair growth complex could be stably and slowly released from the surface of the mouse's back skin and absorbed into the mouse's back skin, so that the drug-loaded exosomes remained effective for at least 5 days, which was beneficial to the treatment of stem cell exosomes and improved the utilization rate of stem cell exosomes.

[0145] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention. Parts not described in detail in this specification belong to the prior art known to those skilled in the art.

Claims

1. A stem cell exosome anti-hair loss and hair growth complex, characterized in that: Including the following raw materials: A 3% to 5% by mass epsilon-polylysine-modified hyaluronic acid solution, a 2% to 4% by mass gentisic acid-cyclodextrin complex solution, and drug-loaded stem cell exosomes, wherein the volume ratio of the epsilon-polylysine-modified hyaluronic acid solution to the gentisic acid-cyclodextrin complex solution is 1:(1 to 2), and the amount of drug-loaded stem cell exosomes added is 0.3% to 2% of the mass of the epsilon-polylysine-modified hyaluronic acid solution; The ε-polylysine modified hyaluronic acid solution with a mass fraction of 3% to 5% is prepared by mixing the ε-polylysine modified hyaluronic acid with a phosphate buffer solution; The gentisic acid-cyclodextrin complex solution with a mass fraction of 2% to 4% is prepared by mixing the gentisic acid-cyclodextrin complex with a phosphate buffer solution; The raw materials for preparing ε-polylysine-modified hyaluronic acid include: 1 to 3 parts by weight of aldehyde-modified hyaluronic acid, 100 to 270 parts by weight of deionized water, 0.1 to 1.5 parts by weight of reaction activation solution, and 0.3 to 1 part by weight of ε-polylysine, wherein the aldehyde-modified hyaluronic acid is obtained by aldehyde-modifying hyaluronic acid with sodium periodate; The raw materials for preparing the gentisic acid-cyclodextrin complex include: 12 to 25 parts by weight of gentisic acid, 80 to 130 parts by weight of pretreated amino-β-cyclodextrin solution, and 50 to 90 parts by weight of reaction activation solution, wherein the pretreated amino-β-cyclodextrin solution is obtained by deoxygenating an amino-β-cyclodextrin solution with a mass fraction of 1.5% to 3%; Drug-loaded stem cell exosomes are adipose-derived mesenchymal stem cell exosomes that are loaded with hair growth agents.

2. The stem cell exosome anti-hair loss and hair growth complex according to claim 1, characterized in that: The reaction activation solution is prepared by mixing 50% ethanol solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of (150-300): (4-7): (2-5).

3. A method for preparing the stem cell exosome anti-hair loss and hair growth complex according to claim 1, characterized in that: The steps include: S1: Exosome culture and extraction and separation: cryopreserved adipose-derived mesenchymal stem cells are thawed, cultured and revived, and subcultured, and the supernatant is collected. The adipose-derived mesenchymal stem cell exosomes are obtained by ultrafiltration concentration and sucrose density gradient centrifugation; S2: Preparation of hair growth agent, dissolving phospholipids and cholesterol in anhydrous ethanol to obtain solution A, dissolving pyrrolidino diaminopyrimidine oxide and adenosine in 1,3-propylene glycol to obtain solution B, heating and stirring glycerol, acetyl tetrapeptide-3, hair growth peptide and solubilizer to obtain solution C, mixing the above solution A, solution B and solution C and homogenizing under high pressure to obtain a hair growth agent; S3: Preparation of drug-loaded stem cell exosomes, mixing adipose-derived mesenchymal stem cell exosomes with a phosphate buffer solution to prepare an exosome suspension, mixing the exosome suspension with a hair growth agent, and encapsulating the hair growth agent into adipose-derived mesenchymal stem cell exosomes by incubation, electroporation, extrusion, ultrasound or freeze-thaw treatment to obtain drug-loaded stem cell exosomes; S4: Preparation of gentisic acid modified amino-β-cyclodextrin, mixing amino-β-cyclodextrin with deionized water to prepare an amino-β-cyclodextrin solution, deoxygenating the amino-β-cyclodextrin solution to obtain a pretreated amino-β-cyclodextrin solution, adding gentisic acid to the pretreated amino-β-cyclodextrin solution, activating the solution, reacting for a period of time, dialyzing, freezing and drying the obtained solution to obtain a gentisic acid-cyclodextrin complex; S5: hyaluronic acid modification treatment, after hyaluronic acid is modified by sodium periodate to obtain aldehyde-modified hyaluronic acid, the aldehyde-modified hyaluronic acid is dissolved in deionized water, after activation treatment, ε-polylysine is added, reacted for a period of time, the obtained solution is dialyzed, frozen and dried to obtain ε-polylysine-modified hyaluronic acid; S6: Preparation of stem cell exosome anti-hair loss and hair germination complex: ε-polylysine-modified hyaluronic acid and gentisic acid-cyclodextrin complex are mixed with phosphate buffer solution respectively to prepare ε-polylysine-modified hyaluronic acid solution and gentisic acid-cyclodextrin complex solution; first, the drug-loaded stem cell exosomes are added to the ε-polylysine-modified hyaluronic acid and dispersed by ultrasonic; then, the gentisic acid-cyclodextrin complex solution is added and the mixture is shaken to obtain the stem cell exosome anti-hair loss and hair germination complex.

4. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 3, characterized in that: Step S2: Preparation of hair growth agent, specifically comprising the following steps: S2.1: 0.3-3 parts by weight of phospholipids and 0.05-0.5 parts by weight of cholesterol are dissolved in 4-10 parts by weight of anhydrous ethanol to obtain solution A, 0.01-0.5 parts by weight of pyrrolidino diaminopyrimidine oxide and 0.01-0.2 parts by weight of adenosine are dissolved in 2-5 parts by weight of 1,3-propylene glycol to obtain solution B, 0.5-3 parts by weight of glycerol, 0.01-0.3 parts by weight of acetyl tetrapeptide-3, 0.01-0.1 parts by weight of hair growth peptide and 1-4 parts by weight of solubilizer are heated and dissolved in a constant temperature water bath at 60-65°C and magnetically stirred for 20-30 minutes to obtain solution C; S2.2: After mixing the above solution A, solution B and solution C, continue stirring for 10 to 30 minutes to obtain a mixed solution, and then use a high-pressure homogenizer to homogenize the mixed solution 2 to 3 times under the condition of 60 to 80 MPa to obtain a hair growth agent.

5. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 4, characterized in that: Step S4: Preparation of gentisic acid-modified amino-β-cyclodextrin, specifically comprising the following steps: S4.1: adding amino-β-cyclodextrin to deionized water to prepare an amino-β-cyclodextrin solution with a mass fraction of 1.5% to 3%, and deoxygenating the amino-β-cyclodextrin solution for 10 to 15 minutes in a water bath heated at 50 to 65° C. and under nitrogen conditions to obtain a pretreated amino-β-cyclodextrin solution; S4.2: Add 12 to 25 parts by weight of gentisic acid to 80 to 130 parts by weight of the pretreated amino-β-cyclodextrin solution, stir evenly, and then slowly drop 50 to 90 parts by weight of the reaction activation solution. After the addition of the reaction activation solution, slowly stir and react at 30 to 60° C. under nitrogen for 12 to 24 hours to obtain a mixed solution A; S4.3: The obtained mixed solution A is dialyzed in acidified deionized water containing hydrochloric acid for 3 to 5 days, and then frozen and dried to obtain a gentisic acid-cyclodextrin complex.

6. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 5, characterized in that: Step S5: hyaluronic acid modification treatment, specifically comprising the following steps: S5.1: After dissolving 1 to 3 parts by weight of hyaluronic acid in 100 to 300 parts by weight of deionized water, add 1 to 3 parts by weight of a 0.5 mol / L sodium periodate solution, and carry out oxidation reaction for 3 to 5 hours in a light-proof condition. After the reaction is completed, add 0.5 to 2 parts by weight of ethylene glycol, and continue stirring for 1 to 3 hours to obtain a post-reaction solution; S5.2: dialyzing the reaction solution in deionized water for 3 to 5 days, and freeze-drying to obtain aldehyde-modified hyaluronic acid; S5.3: After dissolving 1 to 3 parts by weight of aldehyde-modified hyaluronic acid in 100 to 270 parts by weight of deionized water, slowly drop 0.1 to 1.5 parts by weight of a reaction activation solution, after the addition of the reaction activation solution, slowly stir and react at 30 to 40° C. for 20 to 30 minutes, then add 0.3 to 1 parts by weight of ε-polylysine, stir evenly, and obtain a mixed solution B; S5.4: The pH of the mixed solution B is adjusted to 5-6, and the mixture is slowly stirred and reacted at 22-30°C for 8-12 hours. The mixed solution B after the reaction is dialyzed in a weakly acidic aqueous solution with a pH of 5.5 for 3-5 days, and then, it is frozen and dried to obtain ε-polylysine-modified hyaluronic acid.

7. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 6, characterized in that: Step S6: Preparation of stem cell exosome anti-hair loss and hair growth complex, specifically comprising the following steps: S6.1: mixing ε-polylysine-modified hyaluronic acid with a phosphate buffer solution to prepare a 3% to 5% by mass ε-polylysine-modified hyaluronic acid solution, and mixing gentisic acid-cyclodextrin complex with a phosphate buffer solution to prepare a 2% to 4% by mass gentisic acid-cyclodextrin complex solution; S6.2: Add the drug-loaded stem cell exosomes to 3% to 5% ε-polylysine-modified hyaluronic acid, where the amount of drug-loaded stem cell exosomes added is 0.3% to 2% of the mass of the ε-polylysine-modified hyaluronic acid solution, and ultrasonically disperse for 10 to 20 minutes. Subsequently, add 2% to 4% gentisic acid-cyclodextrin complex solution, and shake and mix for 10 to 40 minutes to obtain a stem cell exosome anti-hair loss and hair growth complex, wherein the volume ratio of the ε-polylysine-modified hyaluronic acid solution to the gentisic acid-cyclodextrin complex solution is 1:(1 to 2).

8. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 4, characterized in that: The solubilizer is at least one of polyglycerol ricinoleate, hydrogenated castor oil or caprylic-capric triglyceride.

9. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 4, characterized in that: The hair growth peptide is at least one of myristoyl pentapeptide-7, myristoyl pentapeptide-16, myristoyl pentapeptide-17, palmitoyl pentapeptide-4, oligopeptide-1, tripeptide-1 or tripeptide-2.

10. The method for preparing a stem cell exosome anti-hair loss and hair growth complex according to claim 4, characterized in that: The phospholipid is at least one of soybean lecithin, egg yolk lecithin or hydrogenated soybean lecithin.

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