Preparation method of composite film-forming liquid for promoting hair regeneration

Abstract

The invention discloses a preparation method of a composite film-forming liquid for promoting hair regeneration, and belongs to the technical field of regenerative medicine. The specific method is as follows: gelatin-genipin particles containing stem cell-derived active substances, chitosan, polyvinylpyrrolidone, vitamin E and glycerol are utilized to construct a novel composite film-forming solution which has excellent film-forming ability, is antioxidant and slowly releases the active substances. The composite film-forming liquid is easily smeared on the surface of alopecia skin, can quickly form a film and immobilize effective components, promotes repair of damaged hair follicles by neutralizing free radicals, reducing oxidative stress and slowly releasing stem cell-derived active substances, further promotes hair regeneration, and has important clinical practical application significance.

Description

Technical Field

[0001] The invention belongs to the technical field of regenerative medicine, and in particular relates to a preparation method and application of a composite film-forming liquid for promoting hair regeneration. Background Art

[0002] Hair loss is a common hair disease, with an increasing incidence rate worldwide, especially among middle-aged people, and a trend of gradually becoming younger. According to statistics, the incidence rate of hair loss in adult men in China has exceeded 20%, and the incidence rate in women has also reached more than 10%. Hair loss not only affects the patient's appearance and mental health, but may also lead to social problems such as decreased self-confidence and social disorders, and even mental illnesses such as anxiety and depression in severe cases. The causes of hair loss are complex, mainly including genetic factors, hormone imbalance, oxidative stress, malnutrition, environmental pollution, mental stress, and bad living habits.

[0003] At present, the treatments for hair loss mainly include drug therapy, physical therapy and surgical treatment. Among drug treatments, minoxidil and finasteride are commonly used drugs. Minoxidil promotes blood circulation in hair follicles by dilating blood vessels, while finasteride reduces dihydrotestosterone levels by inhibiting 5α-reductase, thereby slowing down hair loss. However, these drugs have certain limitations, such as minoxidil may cause scalp irritation and allergic reactions, and finasteride may cause side effects such as sexual dysfunction. Although physical treatments such as low-energy laser therapy and microneedle therapy can stimulate hair follicle regeneration to a certain extent, their efficacy is very limited and requires long-term persistence. Surgical treatments such as hair transplantation have certain effects, but they are expensive and there is a risk of postoperative infection and scar formation. In general, the main defect of existing treatment methods is that their mechanism of action is single, and it is difficult to comprehensively solve the complex causes and environment of hair loss. For example, drug treatment mainly targets hormone levels and blood circulation, but ignores oxidative stress and the repair of the hair follicle microenvironment. In addition, existing treatments often lack stable control of active ingredients, resulting in large fluctuations in local action concentrations, affecting the treatment effect. Therefore, it is of great clinical significance to develop a new hair regeneration system that can resist oxidation, repair the hair follicle microenvironment, stabilize the local concentration of active ingredients and achieve sustained release of active substances.

[0004] In recent years, with the rapid development of biotechnology, the application of active substances derived from exogenous stem cells in tissue repair and regenerative medicine has become increasingly widespread. Active ingredients such as growth factors and extracellular matrix proteins secreted by stem cells have the function of promoting cell proliferation, differentiation and angiogenesis, and can effectively repair damaged hair follicles and promote hair regeneration. However, active substances derived from exogenous stem cells face problems such as poor stability and easy degradation during application, which limits their efficacy. Therefore, there is an urgent need to develop new technologies that can stabilize active ingredients through immobilization technology and achieve long-term sustained release in the local area of ​​hair loss.

[0005] The present invention aims to provide a novel composite film-forming liquid for promoting hair regeneration, which can enhance the effect of promoting hair regeneration by strengthening the antioxidant effect, supplementing the active substances derived from exogenous stem cells, immobilizing the effective therapeutic ingredients, stabilizing the local concentration of the effective ingredients, and realizing the sustained release of the effective therapeutic ingredients. After the composite film-forming liquid is applied to the surface of the hair loss skin, it can quickly form a film, immobilize and slowly release the effective ingredients, so that it can effectively neutralize free radicals, repair the hair follicle microenvironment, prolong the action time of the active ingredients, reduce the treatment frequency, and better promote hair regeneration. The present invention will provide a safe, effective and convenient composite film-forming liquid for promoting hair regeneration for hair loss patients, and has broad application prospects. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a preparation method and application of a composite film-forming liquid for promoting hair regeneration, aiming to strengthen the immobilization and sustained release of effective active ingredients at the site of hair loss, and further promote hair regeneration by neutralizing free radicals, repairing the hair follicle microenvironment and prolonging the action time of the active ingredients.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0008] The present invention discloses a method for preparing a composite film-forming liquid for promoting hair regeneration, which is characterized by comprising the following steps: S1. collecting stem cell culture supernatant, filtering with a 0.22 micron filter membrane to remove impurities, then filtering and concentrating with a hollow fiber membrane filtration device, then freezing the concentrate at -80°C, and then freeze-drying to obtain freeze-dried stem cell-derived active substance A; S2. The freeze-dried active substance A prepared in S1 is dissolved in alkaline gelatin and acidic gelatin solutions respectively, stirred and mixed evenly, and then genipin is added respectively, stirred and mixed evenly, and then the two mixed solutions are placed in -80°C for freezing, and then freeze-dried to obtain a freeze-dried product B of alkaline gelatin-genipin and a freeze-dried product C of acidic gelatin-genipin containing stem cell-derived active substances respectively; S3. The two freeze-dried products B and C prepared in S2 were ground into powders using a grinder and sieved using a sieve to obtain alkaline gelatin-genipin particles D and acidic gelatin-genipin particles E containing stem cell-derived active substances; S4. The polyvinyl pyrrolidone was added to the chitosan solution, and the mixture was stirred to obtain a mixed solution F; S5. Vitamin E was added to glycerol and stirred to obtain a mixture G; S6. The mixed solution G prepared in S5 was added to the liquid F prepared in S4, and stirred to mix to obtain a mixed solution H; S7. Add the two particles D and E prepared in S3 to the mixed solution H prepared in S6, and stir to mix evenly to obtain a composite film-forming solution for promoting hair regeneration.

[0009] Furthermore, the stem cell in step S1 is one of mesenchymal stem cells, skin stem cells, embryonic stem cells, and iPS cells.

[0010] Furthermore, the culture supernatant in step S1 is the culture fluid obtained after culturing stem cells for 24 to 96 hours.

[0011] Furthermore, the molecular weight cut-off of the filter membrane of the hollow fiber membrane filtration device in step S1 is 1.5 to 5 kDa.

[0012] Furthermore, in the step S2, the concentration of the freeze-dried active substance A when dissolved in the alkaline gelatin solution and the acidic gelatin solution is 5 to 25 mg / mL.

[0013] Furthermore, the concentration of genipin added in step S2 is 0.02-0.08% (w / v, g / mL).

[0014] Furthermore, the mesh size of the sieve in step S3 is 220-500 meshes, and the longest dimension of the particles is 30-70 μm.

[0015] Furthermore, in the step S4, the molecular weight of the polyvinyl pyrrolidone is 20-400 kDa, the deacetylation degree of the chitosan is 90% or more, and the molecular weight of the chitosan is 80-400 kDa.

[0016] Furthermore, in step S7, the two particles D and E are added to the mixed solution H prepared in S6 at a concentration of 20-100 mg / mL.

[0017] Furthermore, in the composite film-forming liquid for promoting hair regeneration in step S7, the concentration of chitosan is 0.5-2% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 2-15% (w / v, g / mL), the concentration of glycerol is 3-10% (v / v), the concentration of vitamin E is 0.1-10% (w / v, g / mL), and the concentrations of the two particles D and E are both 20-100 mg / mL.

[0018] The present invention also discloses the use of a composite film-forming liquid prepared by any of the above-mentioned preparation methods in the preparation of a film-forming agent for promoting hair regeneration.

[0019] Furthermore, the composite film-forming liquid is used in combination with microneedle therapy to enhance the penetration of active substances in the hair follicle area through the skin pores.

[0020] The present invention also discloses a composite film-forming liquid for promoting hair regeneration, characterized in that it is prepared by any of the preparation methods described above; the composite film-forming liquid is applied to hair loss skin to promote hair regeneration.

[0021] Compared with the prior art, the present invention has the following beneficial effects.

[0022] 1. The present invention uses chitosan, polyvinyl pyrrolidone and glycerol to construct a composite film-forming liquid for sustained release of stem cell-derived active substances and vitamin E. Applying the film-forming liquid to the hair loss area to form a polymer film is conducive to the local fixation of the hair regeneration-promoting components, and a stable effective concentration is maintained through sustained release. The film-forming property of chitosan alone is insufficient, and the formed thin film has low mechanical properties and uneven structure, and cannot form a homogeneous and stable film in the hair loss area. There is a strong hydrogen bond between polyvinyl pyrrolidone and chitosan, which can significantly improve the film-forming property of the chitosan-based film-forming liquid, improve the tensile strength, and make the film more uniform and stable. Glycerol can be inserted into the molecular chains of chitosan and polyvinyl pyrrolidone, increase the mobility of the molecular chains, and improve the flexibility and ductility of the polymer film. In addition, there is also a hydrogen bond between glycerol and chitosan and polyvinyl pyrrolidone, which enhances the stability and mechanical properties of the film. In addition, the addition of glycerol enhances the moisturizing properties of the composite film, so that dehydration and fragmentation will not occur when used in the hair loss area. Therefore, the present invention uses chitosan, polyvinyl pyrrolidone and glycerol to prepare a film-forming liquid with three interpenetrating polymer chains. The film-forming property is improved by relying on the interaction between the three molecules, and the properties of the film are improved, so that after being applied to the hair loss area, a stable, homogeneous and moisturizing polymer film can be formed to immobilize and slowly release stem cell-derived active substances and vitamin E.

[0023] 2. The composite film-forming liquid prepared by the present invention contains vitamin E. After applying to the hair loss area, the vitamin E is locally fixed and slowly released, accelerating hair regeneration through anti-oxidation and other effects. Oxidative stress is one of the important factors leading to hair follicle aging and hair loss. Vitamin E is a powerful antioxidant that can neutralize free radicals and reduce the damage of oxidative stress to hair follicles. Vitamin E in a free state is insoluble in water and is easily lost. The effective concentration in the hair loss area is low and the effect is limited. The present invention first dissolves vitamin E in glycerol, and then mixes the glycerol containing vitamin E and the mixed solution of chitosan and polyvinyl pyrrolidone evenly, so that vitamin E is uniformly present in the film-forming solution. After applying to the hair loss area, the formed polymer film uniformly contains vitamin E, and the film fixes vitamin E in the hair loss area, especially in the skin pores after microneedle treatment. The sustained release of vitamin E can be achieved through the reticular structure of the membrane and the interaction between molecules. This sustained release effect can prolong the action time of vitamin E and improve its bioavailability and therapeutic effect. In addition, there is a hydrogen bond between chitosan and vitamin E, which is more conducive to the dispersibility and stability of vitamin E on the one hand, and can enhance the stability of the composite film on the other hand. In addition, chitosan also has certain antioxidant properties. When combined with vitamin E, it can produce a synergistic antioxidant effect, which is more conducive to hair regeneration. In addition, chitosan and vitamin E also have the effect of promoting cell regeneration. Chitosan promotes cell proliferation and differentiation through its biological activity, while vitamin E protects cells from damage through antioxidant and repair effects. The combination of the two can more effectively promote the regeneration and repair of hair follicle cells, thereby accelerating hair regeneration.

[0024] 3. The composite film-forming liquid prepared by the present invention contains gelatin-genipin particles carrying stem cell-derived active substances. After being applied to the hair loss area, the gelatin-genipin particles are locally fixed and slowly release stem cell-derived active substances, accelerating hair regeneration through anti-oxidation, hair follicle activation, angiogenesis promotion, hair follicle stem cell activation, etc. The stem cell culture supernatant contains a variety of growth factors and active proteins secreted by stem cells, which can improve the inflammatory microenvironment of the hair loss site and achieve hair regeneration through a variety of biological effects. However, the active substances in the free state are easily inactivated. The present invention first dissolves the freeze-dried stem cell-derived active substances in alkaline gelatin and acidic gelatin solutions respectively. Since the two gelatins are negatively and positively charged, respectively, they can be electrostatically combined with the positively and negatively charged substances in the active substances, thereby reducing the inactivation of the active substances. Afterwards, the present invention uses genipin to moderately cross-link the two gelatin solutions containing active substances to form a hydrogel state, and then freeze-drying, grinding and sieving are performed to obtain alkaline and acidic gelatin-genipin microparticles containing active substances, respectively. These particles can achieve sustained release of active substances under conditions that reduce inactivation. The cross-linking of genipin can improve the stability and degradation resistance of the particles and regulate the sustained release of active substances. Afterwards, the present invention further mixes the two particles into a mixture of chitosan, polyvinyl pyrrolidone, vitamin E and glycerol to form a composite film-forming liquid containing particles. When the composite film-forming liquid is applied to the hair loss area, the particles are transported to the hair loss area, especially to the inside of the skin pores after microneedle treatment, so that they are locally fixed, and the sustained release of active substances is achieved in the deep layers of the skin, so that their biological effects can be exerted more lastingly and hair regeneration can be promoted.

[0025] 4. The composite film-forming liquid proposed in the present invention is easy to use and does not require the use of special devices. It can form a film and take effect by evenly applying it on the hair loss area after microneedle treatment. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail in conjunction with the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] The present invention uses gelatin-genipin particles, chitosan, polyvinyl pyrrolidone, vitamin E and glycerol containing stem cell-derived active substances to construct a novel composite film-forming liquid with excellent film-forming ability, anti-oxidation, and slow release of active substances. The composite film-forming liquid is easily applied to the surface of hair loss skin, can quickly form a film, immobilize the effective ingredients, and promote the repair of damaged hair follicles by neutralizing free radicals, reducing oxidative stress, and slow release of stem cell-derived active substances, thereby further promoting hair regeneration.

[0028] Embodiment 1.

[0029] The culture supernatant of mesenchymal stem cells cultured for 24 hours was collected, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (membrane molecular weight cutoff 1.5 kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 5 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.02% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved with a 220-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 70 μm. Polyvinyl pyrrolidone with a molecular weight of 20 kDa was added to a chitosan solution with a deacetylation degree of 90% and a molecular weight of 80 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0030] The concentration of chitosan in this composite film-forming liquid 1# is 2% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 15% (w / v, g / mL), the concentration of glycerol is 10% (v / v), the concentration of vitamin E is 10% (w / v, g / mL), and the concentration of both particles is 20 mg / mL.

[0031] At the same time, three control composite membrane-forming solutions without chitosan, polyvinyl pyrrolidone and glycerol were prepared while other conditions remained unchanged, and they were named control composite membrane-forming solutions 2#, 3# and 4#, respectively.

[0032] 150 μl of each of the composite film-forming liquid 1# and three control composite film-forming liquids 2#, 3# and 4# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm), and the membrane area retention rate of each group on the 14th day was calculated to evaluate the differences in membrane stability among the groups.

[0033] The experiment found that on the 14th day, the film area retention rate of composite film-forming liquid 1# was 100%, while the control composite film-forming liquid 2# did not form a film after application, and the film area retention rates of 3# and 4# were 36% and 44% respectively. This experiment shows that the addition of chitosan, polyvinyl pyrrolidone and glycerol has a significant effect on the film-forming stability of the composite film, with chitosan having the greatest impact, followed by polyvinyl pyrrolidone, and glycerol having the least impact. The composite film-forming liquid with all three components added at the same time has the best film-forming stability in the hair loss area.

[0034] Embodiment 2.

[0035] The culture supernatant of the skin stem cells was collected after 96 hours of culture, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (molecular weight cutoff 5kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 25 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.08% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved with a 500-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 30 μm. Polyvinyl pyrrolidone with a molecular weight of 400 kDa was added to a chitosan solution with a deacetylation degree of 98% and a molecular weight of 400 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0036] The concentration of chitosan in the composite film-forming liquid 1# is 0.5% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 2% (w / v, g / mL), the concentration of glycerol is 3% (v / v), the concentration of vitamin E is 0.1% (w / v, g / mL), and the concentration of both particles is 100 mg / mL.

[0037] At the same time, six control composite film-forming solutions containing chitosan concentration of 0.4% (w / v, g / mL) or 2.5% (w / v, g / mL), polyvinyl pyrrolidone concentration of 1.5% (w / v, g / mL) or 15.5% (w / v, g / mL), and glycerol concentration of 2.5% (v / v) or 10.5% (v / v) were prepared, while other conditions remained unchanged. They were named control composite film-forming solutions 2-7#, respectively.

[0038] 150 μl of each of the composite film-forming liquid 1# and six control composite film-forming liquids 2-7# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm). The membrane area retention rate and hair area coverage rate of the hair loss area of ​​each group on the 14th day were calculated to evaluate the differences in membrane stability and hair regeneration promotion among the groups.

[0039] The experiment found that on the 14th day, the film area retention rate of composite film-forming liquid 1# was 100%. The film area retention rate of the control composite film-forming liquid 2# was 74%, and 3# could not be applied due to the excessively high viscosity of the composite film-forming liquid caused by the high concentration of chitosan. The film area retention rate of the control composite film-forming liquid 4# was 78%, and 5# could not be applied due to the excessively high viscosity of the composite film-forming liquid caused by the high concentration of polyvinyl pyrrolidone. The film area retention rate of the control composite film-forming liquid 6# was 80%, and 7# could not be applied due to the excessively high viscosity of the composite film-forming liquid caused by the high concentration of glycerol. This experiment shows that the concentrations of chitosan, polyvinyl pyrrolidone and glycerol used in the present invention are in the optimal range for application to the hair loss area and film stability.

[0040] In addition, the experiment found that on the 14th day, the hair area coverage rate of the hair loss area of ​​the composite film-forming liquid 1# was 100%, while the hair area coverage rate of the hair loss area of ​​the control composite film-forming liquid 2# was 70%, the hair area coverage rate of the hair loss area of ​​the control composite film-forming liquid 4# was 72%, and the hair area coverage rate of the hair loss area of ​​the control composite film-forming liquid 6# was 75%. This part of the experiment shows that the concentrations of chitosan, polyvinyl pyrrolidone and glycerol used in the present invention are also in the optimal range for promoting hair regeneration in the hair loss area.

[0041] Embodiment three.

[0042] The culture supernatant of embryonic stem cells was collected at 60 hours of culture, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (membrane molecular weight cutoff 3kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 15 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.05% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved with a 360-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 40 μm. Polyvinyl pyrrolidone with a molecular weight of 210 kDa was added to a chitosan solution with a deacetylation degree of 95% and a molecular weight of 240 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. A mixed solution of vitamin E and glycerol was added to a mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed to obtain a mixed solution. Afterwards, the above two particles were added to the mixed solution of chitosan, polyvinyl pyrrolidone, vitamin E and glycerol, and the concentration of the two particles was 60 mg / mL. Stirring and mixing was uniform to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0043] The concentration of chitosan in this composite film-forming liquid 1# is 1.25% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 8.5% (w / v, g / mL), the concentration of glycerol is 6.5% (v / v), the concentration of vitamin E is 5.05% (w / v, g / mL), and the concentration of both particles is 60 mg / mL.

[0044] At the same time, four control composite membrane-forming solutions containing chitosan with a molecular weight of 79 kDa or 405 kDa and polyvinyl pyrrolidone with a molecular weight of 19 kDa or 403 kDa were prepared respectively, while other conditions remained unchanged, and were named control composite membrane-forming solutions 2-5# respectively.

[0045] 150 μl of each of the composite film-forming liquid 1# and four control composite film-forming liquids 2-5# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm), and the membrane area retention rate of each group on the 14th day was calculated to evaluate the differences in membrane stability among the groups.

[0046] The experiment found that on the 14th day, the film area retention rate of composite film-forming liquid 1# was 100%. The film area retention rate of control composite film-forming liquid 2# was 82%, and 3# was unable to be applied due to the excessive molecular weight of chitosan, which made the viscosity of the composite film-forming liquid too high. The film area retention rate of control composite film-forming liquid 4# was 85%, and 5# was unable to be applied due to the excessive molecular weight of polyvinyl pyrrolidone, which made the viscosity of the composite film-forming liquid too high. This experiment shows that the molecular weights of chitosan and polyvinyl pyrrolidone used in the present invention are in the optimal range for application to the hair loss area and film stability.

[0047] Embodiment 4.

[0048] The culture supernatant of iPS cells cultured for 48 hours was collected, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (molecular weight cutoff 4kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 11 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.04% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved with a 300-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 54 μm. Polyvinyl pyrrolidone with a molecular weight of 320 kDa was added to a chitosan solution with a deacetylation degree of 92% and a molecular weight of 190 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0049] The concentration of chitosan in this composite film-forming liquid 1# is 1.55% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 6% (w / v, g / mL), the concentration of glycerol is 5% (v / v), the concentration of vitamin E is 4% (w / v, g / mL), and the concentration of both particles is 85 mg / mL.

[0050] At the same time, a control composite film-forming solution was prepared, which contained two types of particles at a concentration of 105 mg / mL while other conditions remained unchanged and was named control composite film-forming solution 2#.

[0051] 150 μl of composite film-forming liquid 1# and control composite film-forming liquid 2# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm), and the membrane area retention rate of each group on the 14th day was calculated to evaluate the differences in membrane stability among the groups.

[0052] The experiment found that on the 14th day, the film area retention rate of the composite film-forming liquid 1# was 100%, while the film area retention rate of the control composite film-forming liquid 2# was 71%. This experiment shows that when the concentration of both particles exceeds 100 mg / mL, the film stability is significantly reduced. Therefore, the concentration of the two particles used in the present invention is the optimal range for application to the hair loss area and film stability.

[0053] Embodiment five.

[0054] The culture supernatant of mesenchymal stem cells was collected at 72 hours of culture, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (membrane molecular weight cutoff 2kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 20 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.07% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved with a 250-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 63 μm. Polyvinyl pyrrolidone with a molecular weight of 100 kDa was added to a chitosan solution with a deacetylation degree of 96% and a molecular weight of 325 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed evenly. Afterwards, the above two particles were added to the mixed solution of chitosan, polyvinyl pyrrolidone, vitamin E and glycerol, wherein the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances was 55 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances was 75 mg / mL, and stirred and mixed evenly to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0055] The concentration of chitosan in this composite film-forming liquid 1# is 0.95% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 13% (w / v, g / mL), the concentration of glycerol is 7.1% (v / v), the concentration of vitamin E is 6.3% (w / v, g / mL), the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances is 55 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances is 75 mg / mL.

[0056] At the same time, two control composite film-forming solutions containing vitamin E at concentrations of 0.08% (w / v, g / mL) and 11% (w / v, g / mL) were prepared while other conditions remained unchanged and were named control composite film-forming solutions 2 and 3#.

[0057] 150 μl of composite film-forming liquid 1# and control composite film-forming liquids 2 and 3# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm). The membrane area retention rate and hair area coverage rate of the hair loss area of ​​each group on the 14th day were calculated to evaluate the differences in membrane stability and hair regeneration promotion among the groups.

[0058] The experiment found that on the 14th day, the film area retention rate of composite film-forming liquid 1# was 100%, while the film area retention rate of control composite film-forming liquid 2# was 81%. The control composite film-forming liquid 3# had too high a viscosity of the composite film-forming liquid due to the high concentration of vitamin E, and could not be evenly applied. This part of the experiment shows that the concentration of vitamin E used in the present invention is in the optimal range for application to the hair loss area and film stability.

[0059] In addition, the experiment found that on the 14th day, the hair area coverage rate of the hair loss area of ​​the composite film-forming liquid 1# was 100%, while the hair area coverage rate of the hair loss area of ​​the control composite film-forming liquid 2# was 70%. This part of the experiment shows that the concentration of vitamin E used in the present invention is also in the optimal range for promoting hair regeneration in the hair loss area.

[0060] Embodiment six.

[0061] The culture supernatant of the skin stem cells was collected after culturing for 48 hours, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (molecular weight cutoff 4.5 kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at concentrations of 12 mg / mL and 20 mg / mL, respectively, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.03% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved by a 400-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 38.5 μm. Polyvinyl pyrrolidone with a molecular weight of 275 kDa was added to a chitosan solution with a deacetylation degree of 91% and a molecular weight of 110 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed evenly. Afterwards, the above two particles were added to the mixed solution of chitosan, polyvinyl pyrrolidone, vitamin E and glycerol, wherein the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances was 77 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances was 62 mg / mL, and stirred and mixed evenly to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0062] The concentration of chitosan in this composite film-forming liquid 1# is 1.1% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 9% (w / v, g / mL), the concentration of glycerol is 4% (v / v), the concentration of vitamin E is 0.9% (w / v, g / mL), the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances is 77 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances is 62 mg / mL.

[0063] At the same time, two control composite membrane-forming solutions were prepared, namely, alkaline gelatin-genipin particles containing stem cell-derived active substances at a concentration of 19 mg / mL and acidic gelatin-genipin particles containing stem cell-derived active substances at a concentration of 19 mg / mL, while other conditions remained unchanged. They were named control composite membrane-forming solutions 2 and 3#.

[0064] 150 μl of composite film-forming liquid 1# and control composite film-forming liquids 2 and 3# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm), and the hair area coverage of the hair loss area of ​​each group on the 14th day was calculated to evaluate the differences in promoting hair regeneration among the groups.

[0065] The experiment found that on the 14th day, the hair area coverage rate of the hair loss area of ​​the composite film-forming liquid 1# was 100%, while the hair area coverage rates of the hair loss area of ​​the control composite film-forming liquids 2 and 3# were 31 and 33%, respectively. This experiment shows that the particle concentration used in the present invention is in the optimal range for promoting hair regeneration in the hair loss area.

[0066] Embodiment seven.

[0067] The culture supernatant of embryonic stem cells was collected at 48 hours of culture, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (membrane molecular weight cutoff 3.5kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 13 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.06% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances. The two freeze-dried products were ground into powders by a grinder, and sieved with a 340-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 41 μm. Polyvinyl pyrrolidone with a molecular weight of 170 kDa was added to a chitosan solution with a deacetylation degree of 97% and a molecular weight of 290 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed evenly. Afterwards, the above two particles were added to the mixed solution of chitosan, polyvinyl pyrrolidone, vitamin E and glycerol, wherein the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances was 30 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances was 77 mg / mL, and stirred and mixed evenly to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0068] The concentration of chitosan in this composite film-forming liquid 1# is 0.7% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 11% (w / v, g / mL), the concentration of glycerol is 6% (v / v), the concentration of vitamin E is 7.6% (w / v, g / mL), the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances is 30 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances is 77 mg / mL.

[0069] At the same time, two control composite film-forming solutions were prepared in which the freeze-dried active substance was dissolved in the gelatin solution at a concentration of 4 mg / mL and 26 mg / mL, respectively, while other conditions remained unchanged. They were named control composite film-forming solutions 2 and 3#.

[0070] The experiment found that when the freeze-dried active substance was dissolved in the gelatin solution at a concentration of 26 mg / mL, the subsequent freeze-dried product was not formed and could not be ground due to the high concentration of the active substance, so the 3# film-forming solution could not be successfully prepared.

[0071] 150 μl of composite film-forming liquid 1# and control composite film-forming liquid 2# were evenly applied to the hair loss area on the back of the androgenic alopecia mouse model (initial area 10 mm×10 mm), and the hair area coverage of the hair loss area of ​​each group on the 14th day was calculated to evaluate the differences in promoting hair regeneration among the groups.

[0072] The experiment found that on the 14th day, the hair area coverage rate of the hair loss area of ​​the composite film-forming liquid 1# was 100%, while the hair area coverage rate of the hair loss area of ​​the control composite film-forming liquid 2# was 82%. This part of the experiment shows that the concentration of the freeze-dried active substance used in the present invention dissolved in the gelatin solution is in the optimal range for promoting hair regeneration in the hair loss area.

[0073] Embodiment eight.

[0074] The culture supernatant of iPS cells cultured for 24 hours was collected, filtered with a 0.22 micron filter membrane to remove impurities, and then filtered and concentrated with a hollow fiber membrane filtration device (molecular weight cutoff 2.6 kDa), and then the concentrate was placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried stem cell-derived active substances. After that, the prepared freeze-dried active substances were dissolved in alkaline gelatin and acidic gelatin solutions at a concentration of 22 mg / mL, stirred and mixed evenly, and then genipin was added respectively, and the final concentration of genipin was 0.05% (w / v, g / mL), stirred and mixed evenly, and then the two mixed solutions were placed at -80°C for freezing, and then freeze-dried to obtain freeze-dried products of alkaline gelatin-genipin and freeze-dried products of acidic gelatin-genipin containing stem cell-derived active substances, respectively. The two freeze-dried products were ground into powders by a grinder, and sieved with a 250-mesh sieve to obtain alkaline gelatin-genipin particles and acidic gelatin-genipin particles containing stem cell-derived active substances, respectively, and the longest dimension of the particles was 63 μm. Polyvinyl pyrrolidone with a molecular weight of 330 kDa was added to a chitosan solution with a deacetylation degree of 92% and a molecular weight of 220 kDa, and stirred and mixed to obtain a mixed solution. Vitamin E was added to glycerol, and stirred and mixed to obtain a mixed solution. The mixed solution of vitamin E and glycerol was added to the mixed solution of chitosan and polyvinyl pyrrolidone, and stirred and mixed evenly. Afterwards, the above two particles were added to the mixed solution of chitosan, polyvinyl pyrrolidone, vitamin E and glycerol, wherein the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances was 66 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances was 72 mg / mL, and stirred and mixed evenly to obtain a composite film-forming solution 1# for promoting hair regeneration.

[0075] The concentration of chitosan in this composite film-forming liquid 1# is 1.5% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 5% (w / v, g / mL), the concentration of glycerol is 8% (v / v), the concentration of vitamin E is 4.2% (w / v, g / mL), the concentration of alkaline gelatin-genipin particles containing stem cell-derived active substances is 66 mg / mL, and the concentration of acidic gelatin-genipin particles containing stem cell-derived active substances is 72 mg / mL.

[0076] At the same time, two control composite film-forming solutions were prepared in which genipin was dissolved in gelatin solution at concentrations of 0.01% (w / v, g / mL) and 0.09% (w / v, g / mL), respectively, while other conditions remained unchanged. They were named control composite film-forming solutions 2 and 3#.

[0077] The experiment found that when genipin was dissolved in gelatin solution at a concentration of 0.01% (w / v, g / mL), the genipin concentration was too low, resulting in insufficient cross-linking, and the subsequent freeze-dried product was not formed and could not be ground, so the 2# film-forming solution could not be successfully prepared.

[0078] After the composite film-forming liquid 1# and the control composite film-forming liquid 3# were formed into films, L929 cell culture fluid was added and extracted at 37°C for 24h. L929 cells were cultured with these two extracts for 3 days, and the relative proliferation rates of these two groups relative to the normal cell culture group were detected using the CCK-8 kit. The experiment found that the relative cell proliferation rate of composite film-forming liquid 1# was 96%, while the relative cell proliferation rate of the control composite film-forming liquid 3# was 62%, which was caused by the excessively high concentration of genipin used. Therefore, the concentration of genipin dissolved in the gelatin solution used in the present invention is the optimal range for the successful preparation of the composite film-forming liquid and the low in vitro cytotoxicity of the film.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a composite film-forming liquid for promoting hair regeneration, characterized in that The method comprises the following steps: S1. collecting stem cell culture supernatant, filtering with a 0.22 micron filter membrane to remove impurities, then filtering and concentrating with a hollow fiber membrane filtration device, then freezing the concentrate at -80°C, and then freeze-drying to obtain freeze-dried stem cell-derived active substance A; S2. The freeze-dried active substance A prepared in S1 is dissolved in alkaline gelatin and acidic gelatin solutions respectively, stirred and mixed evenly, and then genipin is added respectively, stirred and mixed evenly, and then the two mixed solutions are placed in -80°C for freezing, and then freeze-dried to obtain a freeze-dried product B of alkaline gelatin-genipin and a freeze-dried product C of acidic gelatin-genipin containing stem cell-derived active substances respectively; S3. The two freeze-dried products B and C prepared in S2 were ground into powders using a grinder and sieved using a sieve to obtain alkaline gelatin-genipin particles D and acidic gelatin-genipin particles E containing stem cell-derived active substances; S4. The polyvinyl pyrrolidone was added to the chitosan solution, and the mixture was stirred to obtain a mixed solution F; S5. Vitamin E was added to glycerol and stirred to obtain a mixture G; S6. The mixed solution G prepared in S5 was added to the liquid F prepared in S4, and stirred to mix to obtain a mixed solution H; S7. Add the two particles D and E prepared in S3 to the mixed solution H prepared in S6, and stir to mix evenly to obtain a composite film-forming solution for promoting hair regeneration.

2. The method for preparing the composite film-forming liquid for promoting hair regeneration according to claim 1, characterized in that: The stem cells in step S1 are one of mesenchymal stem cells, skin stem cells, embryonic stem cells, and iPS cells; the culture supernatant is the culture fluid obtained after culturing stem cells for 24 to 96 hours; and the filtration membrane of the hollow fiber membrane filtration device has a molecular weight cutoff of 1.5 to 5 kDa.

3. The method for preparing the composite film-forming liquid for promoting hair regeneration according to claim 1, characterized in that: In the step S2, the concentration of the freeze-dried active substance A when dissolved in alkaline gelatin and acidic gelatin solutions is 5-25 mg / mL; the concentration of the added genipin is 0.02-0.08% (w / v, g / mL).

4. The method for preparing the composite film-forming liquid for promoting hair regeneration according to claim 1, characterized in that: The mesh size of the sieve in step S3 is 220-500 meshes, and the longest dimension of the particles is 30-70 μm.

5. The method for preparing the composite film-forming liquid for promoting hair regeneration according to claim 1, characterized in that: In the step S4, the molecular weight of the polyvinyl pyrrolidone is 20-400 kDa, the deacetylation degree of the chitosan is 90% or more, and the molecular weight of the chitosan is 80-400 kDa.

6. The method for preparing the composite film-forming liquid for promoting hair regeneration according to claim 1, characterized in that: In the step S7, the two particles D and E are added to the mixed solution H prepared in S6 at a concentration of 20-100 mg / mL.

7. The method for preparing the composite film-forming liquid for promoting hair regeneration according to claim 1, characterized in that: In the composite film-forming solution for promoting hair regeneration in step S7, the concentration of chitosan is 0.5-2% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 2-15% (w / v, g / mL), the concentration of glycerol is 3-10% (v / v), the concentration of vitamin E is 0.1-10% (w / v, g / mL), and the concentrations of the two particles D and E are both 20-100 mg / mL.

8. Use of the composite film-forming liquid prepared by the preparation method according to any one of claims 1 to 7 in preparing a film-forming agent for promoting hair regeneration.

9. The use according to claim 8, characterized in that: The composite film-forming liquid is used in combination with microneedle therapy to enhance the penetration of active substances in the hair follicle area through the skin pores.

10. A composite film-forming liquid for promoting hair regeneration, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7; the composite film-forming liquid is applied to hair loss skin to promote hair regeneration.