Freezing microneedle loaded with hair follicle stem cells as well as preparation method and application of freezing microneedle

Abstract

The invention relates to a frozen microneedle loaded with hair follicle stem cells as well as a preparation method and application of the frozen microneedle, and relates to the technical field of frozen microneedles. The freezing microneedle comprises a substrate and a plurality of needle bodies distributed on the substrate in an array mode, the substrate is prepared from a substrate solution, the needle bodies are prepared from hair follicle stem cells and an anti-freezing mixed solution, the substrate solution is a sucrose-PBS solution, and the anti-freezing mixed solution comprises sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and a sucrose-PBS solution. According to the anti-freezing mixed solution prepared by the preparation method provided by the invention, the transdermal mechanical strength of the microneedle can be improved while the living cell loading of the frozen microneedle is ensured, and the frozen microneedle loaded with the hair follicle stem cells prepared by the scheme can realize a minimally invasive cell therapy for promoting hair growth.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of frozen microneedle technology, in particular to a frozen microneedle loaded with hair follicle stem cells and a preparation method and application thereof. BACKGROUND

[0002] Microneedle (MN) technology combines the advantages of traditional intradermal injection and the convenience of transdermal drug delivery, providing a convenient, minimally invasive and efficient transdermal drug delivery method. Microneedles are composed of micrometer-sized needles arranged in an array. Microneedles penetrate the stratum corneum barrier of the skin to form micrometer-sized pores without touching pain nerves. The loaded drugs penetrate the surrounding skin tissue through the micropores to achieve minimally invasive, painless and efficient transdermal drug delivery.

[0003] Currently reported microneedle transdermal drug delivery materials include nanoparticles, proteins, etc. However, living tissues or cells are very sensitive to the surrounding environment, and therefore, it is necessary to develop MNs that can ensure cell viability and stemness and have high mechanical strength. Frozen microneedles are a new transdermal drug delivery technology that retains the demolding and skin penetration ability of traditional microneedles. They are prepared by adding a liquid with good mechanical strength after freezing into a specially designed mold after freezing. Currently, frozen microneedles have been widely used in various fields such as tumor treatment, immunotherapy, wound healing, etc. due to their characteristics of being able to load drugs, nanoparticles, active substances and even living cells, as well as their good skin penetration mechanical strength.

[0004] Currently, frozen microneedles loaded with living cells can preserve a certain viability of the cells after freezing. However, due to the lack of antifreeze technology, the survival rate of the cells is still relatively low. Therefore, the solution used to prepare the frozen microneedles usually contains a certain concentration of cryopreservation agent (CPA) to enhance the freezing tolerance of the solution and reduce the damage of ice crystal formation to the cryopreserved cells. In the prior art, Chenjie Xu team published an article entitled "Cryomicroneedles for transdermal cell delivery" in Nature Biomedical Engineering, which introduced a cryoMNs patch specially designed for packaging and transporting living cells to the skin. A 5% DMSO + 100 mM sucrose solution was selected as the cryopreservation agent, and dendritic cells were loaded into the microneedle mold. The microneedles were prepared by stepwise cryomicro-molding and had biocompatibility, which could promote a series of minimally invasive cell delivery for cell therapy. However, the highest survival rate of the cells was only about 50%, and the cell survival rate was still relatively low. Therefore, how to design and develop a cell-loaded frozen microneedle with good cell survival and good mechanical strength is a problem that needs to be solved in the field of medical regeneration. SUMMARY

[0005] The application aims to solve the above problems, and provides a frozen microneedle loaded with hair follicle stem cells, a preparation method and application thereof.

[0006] The first aspect of the application provides a frozen microneedle loaded with hair follicle stem cells, which adopts the following technical solution:

[0007] The frozen microneedle loaded with hair follicle stem cells comprises a substrate and a plurality of needle bodies arranged on the substrate in an array, the substrate is prepared from a substrate solution, and the needle bodies are prepared from hair follicle stem cells and an antifreezing mixed solution.

[0008] Preferably, the substrate solution is a sucrose-PBS solution, and the antifreezing mixed solution comprises sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and sucrose-PBS solution.

[0009] By adopting the above technical solution, sodium alginate (SA) is a natural polymer material extracted from kelp, has excellent biocompatibility, non-toxic biodegradability and abundant storage, and has a mild gel formation condition, which can effectively avoid the inactivation of active substances. Polyvinyl alcohol (PVA) as a non-permeable cryoprotective agent, the hydroxyl group (-OH) in the molecule can form a hydrogen bond with water molecules, reducing the ability of water molecules to arrange into ice crystals, thereby reducing ice crystal damage (a fatal factor for cells) during the freezing process. And through repeated freezing-thawing treatment, a three-dimensional network structure hydrogel can be formed, which has excellent water absorption, film forming property and mechanical strength. Sodium alginate can increase the mechanical properties of the antifreezing solution while ensuring the biocompatibility of the loaded cells. The unique secretory signal group of the activated hair follicle stem cells can up-regulate the expression of the Wnt / β-catenin signaling pathway, and the mechanical stimulation of the microneedle can synergistically promote the formation of blood vessels around the hair follicle, thereby promoting the activation of hair follicle stem cells in advance, thereby accelerating hair growth. Sucrose-PBS solution as a permeable protective agent protects cells through the dual mechanisms of "extracellular dehydration + vitrification", and forms a complete protection system with dimethyl sulfoxide to reduce intracellular ice crystals and maintain cell activity, thereby achieving the simultaneous loading of live cells and improving the transdermal mechanical strength of the microneedle.

[0010] Preferably, the volume percentage of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and sucrose-PBS solution in the antifreezing mixed solution is (2-3):(2-3):(4-6):(88-92).

[0011] Preferably, the volume percentage of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and sucrose-PBS solution in the antifreezing mixed solution is 2.5:2.5:5:90.

[0012] By adopting the above technical solution, the component ratio is preferably selected to improve the performance of the frozen microneedle.

[0013] Preferably, the needle body is in the shape of a pyramid with the tip located on the side away from the base, the needle body is arranged in a 10*10 array, the distance between two adjacent needle bodies is 0.6 mm, the bottom surface diameter of a single needle body is 400 μm, the height is 1200 μm, and the size of the base is 15 mm*15 mm*2 mm.

[0014] The second aspect of the present application provides a preparation method of the above-mentioned frozen microneedle loaded with hair follicle stem cells, comprising the following steps:

[0015] S1, configuring an anti-freezing mixed solution: first, mixing sodium alginate and polyvinyl alcohol in a certain proportion to obtain a sodium alginate-polyvinyl alcohol mixed solution, then adding the sodium alginate-polyvinyl alcohol mixed solution and dimethyl sulfoxide into a sucrose-PBS solution in a certain proportion, and mixing to obtain an anti-freezing mixed solution;

[0016] S2, configuring a hair follicle stem cell resuspension solution: resuspending the hair follicle stem cells in the anti-freezing mixed solution prepared in S1 to obtain a hair follicle stem cell resuspension solution;

[0017] S3, preparing a hair follicle stem cell-loaded frozen microneedle: adding the hair follicle stem cell resuspension solution prepared in S2 into a PDMS groove mold, centrifuging to remove air, adding a base solution to completely cover the groove of the PDMS groove mold, sucking off the excess liquid, centrifuging again, then primary freezing, demolding after molding, gradient cooling treatment after demolding, and obtaining the hair follicle stem cell-loaded frozen microneedle after the treatment.

[0018] Preferably, in the above-mentioned preparation method technical solution, the density of the hair follicle stem cells in S2 is 1×10 6 cells mL -1 .

[0019] Preferably, in the above-mentioned preparation method technical solution, the step S3 is specifically as follows: S3, preparing a hair follicle stem cell-loaded frozen microneedle: adding the hair follicle stem cell resuspension solution prepared in S2 into a PDMS groove mold, centrifuging at a speed of 2000 rpm for 3 min to remove air, so that the cells are concentrated at the tip of the needle body, sucking off the excess liquid, adding a base solution to completely cover the groove of the PDMS groove mold, sucking off the excess liquid, centrifuging at a speed of 2000 rpm for 3 min again, then primary freezing, demolding after molding, gradient cooling treatment after demolding, and obtaining the hair follicle stem cell-loaded frozen microneedle after the treatment.

[0020] Preferably, in the above-mentioned preparation method technical solution, in S3, the primary freezing temperature is (-20℃), the freezing time is 2 h, and the gradient cooling treatment step after demolding is: freezing at (-80℃) for 2 h and then freezing at (-196℃) for 2 h, and obtaining the hair follicle stem cell-loaded frozen microneedle after the treatment.

[0021] By adopting the technical scheme, the steps and parameters of the preparation method are optimized, and the performance of the frozen microneedle is improved.

[0022] The third aspect of the application provides use of the frozen microneedle loaded with hair follicle stem cells in preparation of a microneedle for promoting hair growth.

[0023] To sum up, the present application has at least one of the following beneficial technical effects:

[0024] 1. Sodium alginate (SA) has excellent biocompatibility, non-toxic biodegradability and abundant storage, and the sodium alginate gel forming conditions are mild, which can effectively avoid the inactivation of active substances, polyvinyl alcohol (PVA) as a non-permeable cryoprotectant, the hydroxyl group (-OH) in the molecule can form a hydrogen bond with water molecules, reducing the ability of water molecules to arrange into ice crystals, thereby reducing ice crystal damage (a fatal factor for cells) during freezing, and through repeated freezing-melting treatment, a three-dimensional network structure of hydrogel can be formed, which has excellent water absorption, film forming property and mechanical strength, and sodium alginate can increase the mechanical properties of the antifreeze solution while ensuring the biocompatibility of the loaded cells, and the unique secretory signal group of the activated hair follicle stem cells can up-regulate the expression of the Wnt / β-catenin signaling pathway, which synergistically promotes the formation of blood vessels around the hair follicle and promotes the early activation of hair follicle stem cells, thereby accelerating hair growth, and sucrose-PBS solution as a permeable protective agent protects cells through the dual mechanisms of "extracellular dehydration + vitrification", and forms a complete protection system with dimethyl sulfoxide to reduce intracellular ice crystals and maintain cell activity, thereby achieving the simultaneous loading of living cells and the improvement of the transdermal mechanical strength of the frozen microneedle.

[0025] 2. The frozen microneedle prepared by the method provided by the present application shows excellent hair growth promoting performance in mouse experiments. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a real photo of the frozen microneedle loaded with hair follicle stem cells prepared by the embodiment of the present application, wherein Figure 1 a is a whole real photo of the frozen microneedle, Figure 1 b is a real photo of the needle body part of the frozen microneedle;

[0027] Figure 2 is a structure diagram of the PDMS mold used in the present application, wherein Figure 2 a is a top view of the mold, Figure 2 b is a sectional view of the mold;

[0028] Figure 3 is a real photo of the frozen microneedle prepared by the comparative example of the present application;

[0029] Figure 4is the hair growth experiment operation diagram in the application example, wherein Figure 4 a is the mouse transdermal operation diagram, Figure 4 b is the skin state diagram after transdermal administration of the mouse;

[0030] Figure 5 is the hair growth comparison diagram in the application example;

[0031] Figure 6 is the thawed cell staining diagram of the frozen microneedle prepared in Example 1 of the application.

[0032] BRIEF DESCRIPTION OF DRAWINGS: 1, mold; 2, groove; 3, microneedle preparation groove. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with examples and drawings. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application. Those skilled in the art can modify or replace equivalently on the basis of understanding the technical scheme of the present application without departing from the spirit and scope of the technical scheme of the present application, which should be covered within the protection scope of the present application.

[0034] The information of reagents, instruments and equipment used in the following examples is as follows: all reagents and chemicals are used as received without further treatment, and other specific conditions not mentioned are carried out according to conventional conditions or manufacturer's recommended conditions, and the reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market.

[0035] Reagents: neutral protease II (purchased from Solarbio official website, the concentration is 0.2%, dissolved in pbs solution), 0.25% trypsin cell digestion solution (purchased from Biyun Tian official website, used directly), sucrose-PBS (5%, can be configured by yourself, or can be purchased regularly), the rest of the reagents are purchased from conventional channels.

[0036] Animal source: 6-8 week old C57 mice from Chongqing Medical University Animal Center, fed in SPF generation room, free access to food and water source.

[0037] The chemical reagent abbreviations in the present application are as follows:

[0038] Sodium alginate (SA), polyvinyl alcohol (PVA), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), polydimethylsiloxane (PDMS), and the rest are understood as the general industry cognition.

[0039] I. Example

[0040] Example 1

[0041] The embodiment discloses a frozen microneedle loaded with hair follicle stem cells, comprising a substrate and a plurality of needle bodies arranged in an array on the substrate, the substrate is prepared from a substrate solution, the size of the substrate is 15mm*15mm*2mm, the needle body is prepared from the hair follicle stem cells and an anti-freezing mixed solution, the needle body is in the shape of a pyramid and the tip is located on the side away from the substrate, the needle bodies are arranged in a 10*10 array, the distance between two adjacent needle bodies is 0.6mm, the bottom surface diameter of a single needle body is 400μm, and the height is 1200μm.

[0042] The substrate solution is a 5% sucrose-PBS solution, and the anti-freezing mixed solution comprises sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and a sucrose-PBS solution, and the volume percentage of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and the sucrose-PBS solution in the anti-freezing mixed solution is 2.5:2.5:5:90.

[0043] The actual picture of the frozen microneedle is shown in Figure 1 , wherein, Figure 1 a is the overall actual picture of the frozen microneedle, Figure 1 b is the actual picture of the needle body part of the frozen microneedle.

[0044] The embodiment also discloses a preparation method of the above-mentioned frozen microneedle loaded with hair follicle stem cells, comprising the following steps:

[0045] S1, configuring an anti-freezing mixed solution: first, mixing sodium alginate and polyvinyl alcohol in a certain proportion to obtain a sodium alginate-polyvinyl alcohol mixed solution, then adding the sodium alginate-polyvinyl alcohol mixed solution and dimethyl sulfoxide into a sucrose-PBS solution in a certain proportion, and obtaining the anti-freezing mixed solution after mixing, and 400μL of the anti-freezing mixed solution is configured in the embodiment.

[0046] S2, configuring a hair follicle stem cell suspension: resuspending the hair follicle stem cells in the anti-freezing mixed solution configured in S1 to obtain the hair follicle stem cell suspension.

[0047] Specifically, first, the hair follicle stem cells are obtained, a C57 mouse is executed by decapitation, the whisker pad is cut off and soaked in 75% alcohol, the hair shaft containing the convex part is separated under a stereoscope, and the hair follicle dermal sheath is removed again under a stereoscope under sterile conditions, and then the hair follicle stem cell suspension is obtained after being digested by 0.25% trypsin, filtered and washed, and then the obtained hair follicle stem cells are resuspended in 400μL of the anti-freezing mixed solution prepared in S1 at a density of 1×10 6 cells mL -1 to obtain the hair follicle stem cell suspension.

[0048] S3, preparation of hair follicle stem cell loaded frozen microneedle: the hair follicle stem cell suspension prepared in S2 is added to the PDMS groove mold, centrifuged to remove air, and a base solution is added to completely cover the grooves of the PDMS groove mold, the excess liquid is sucked off, centrifuged again, then primary frozen, demolded after molding, and gradient cooling treatment is performed, and the hair follicle stem cell loaded frozen microneedle is obtained after treatment.

[0049] Specifically, referring to Figure 2 , the PDMS mold 1 is prepared from polydimethylsiloxane (PDMS), and the mold is a cylindrical groove mold with a diameter of 24 mm. The top surface of the mold 1 has a groove 2, and the inner bottom of the groove 2 is provided with a tapered microneedle preparation groove 3. The microneedle preparation grooves 3 are arranged in a 10*10 array. The size of the groove 2 is 15mm*15mm*2mm. The distance between the edge of the groove 2 and the microneedle preparation groove 3 is 2.8mm. The distance between the adjacent two microneedle preparation grooves 3 is 0.6mm. The bottom surface diameter of a single microneedle preparation groove 3 is 400μm, and the depth of the microneedle preparation groove 3 is 1200μm.

[0050] The hair follicle stem cell suspension prepared in S2 is added to the microneedle preparation groove of the PDMS groove mold, and centrifuged at a speed of 2000rpm for 3min to remove air, so that the cells are concentrated at the tip of the needle body. The excess liquid is sucked off using sterile filter paper, then 300μL of base solution is added to completely cover the grooves of the PDMS groove mold, the excess liquid is sucked off using sterile filter paper, and then centrifuged again at a speed of 2000rpm for 3min. Then the mold is frozen as a whole under the condition of (-20℃) for 2h for primary freezing. After molding, demolding is performed, and gradient cooling treatment is performed after demolding. The gradient cooling treatment steps are: frozen for 2h at (-80℃) and then frozen for 2h at (-196℃). The hair follicle stem cell loaded frozen microneedle is obtained after treatment.

[0051] Example 2

[0052] The present embodiment discloses a hair follicle stem cell loaded frozen microneedle and a preparation method thereof. The difference from example 1 is that the volume percentage of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and sucrose-PBS solution in the antifreeze mixture is 3:3:6:88, and the rest is consistent with example 1.

[0053] Example 3

[0054] The present embodiment discloses a hair follicle stem cell loaded frozen microneedle and a preparation method thereof. The difference from example 1 is that the volume percentage of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide and sucrose-PBS solution in the antifreeze mixture is 2:2:4:92, and the rest is consistent with example 1.

[0055] Example 3

[0056] Comparative Example 1

[0057] A frozen microneedle loaded with hair follicle stem cells and a preparation method thereof, which is different from Example 1 in that no sodium alginate and polyvinyl alcohol are added in the antifreezing mixture, and the rest is consistent with Example 1.

[0058] Prepared according to the preparation method provided in Example 1, with reference to Figure 3 , the primary freezing cannot be demolded, and the needle body is damaged during demolding, so it can be concluded that the addition of sodium alginate and polyvinyl alcohol increases the mechanical strength of the frozen microneedle.

[0059] III. Application Examples

[0060] The frozen microneedles prepared in Examples 1-3 of the present application were subjected to mouse experiments.

[0061] Experimental method: with reference to Figure 4 , the back skin of C57 mice was depilated on the first day of the 7th week (49 days after birth, at which time the mouse hair follicles entered the resting period), and the depilated area skin was treated with HFSC-MNs or Blank-MNs on the 1st, 3rd, 5th, and 7th days after depilation. The control group was treated with topical minoxidil at the same time, and the blank group of mice was not treated after anesthesia, and the hair growth of the depilated area of the back of each group of mice was recorded by taking pictures on the 8th, 11th, 14th, 17th, and 21st days after depilation; here, HFSC-MNs refers to the transdermal administration group of the frozen microneedle loaded with hair follicle stem cells prepared according to Examples 1-3, and Blank-MNs refers to the frozen microneedle group prepared according to the preparation method of Example 1 but not loaded with hair follicle stem cells, Figure 4 a is a mouse transdermal operation diagram, Figure 4 b is a skin state diagram after transdermal administration of mice.

[0062] Figure 5 is a comparison diagram of the hair growth rate of the frozen microneedle of Example 1 and other groups of mice, as can be seen from the diagram, compared with other groups, the hair follicles of the back depilated area of the HFSC-MNs group of mice entered the growth period first, and the hair grew the fastest. The hair growth of the Blank-MNs group of mice was significantly accelerated compared with the blank group, showing a similar growth rate to the minoxidil group; Blank-MNs and HFSC-MNs can promote the formation of blood vessels around the hair follicles through mechanical stimulation, and HFSC-MNs can up-regulate the expression of the Wnt / β-catenin signaling pathway through the loaded activated hair follicle stem cells, which synergistically promote the activation of hair follicle stem cells, thereby accelerating hair growth.

[0063] IV. Cell Activity Detection

[0064] The frozen microneedle prepared by using the preparation method of Example 1 was used to dye the cells after thawing, and the results are shown in FIG. 1, wherein the red color is dead cells, and the green color is live cells. Figure 6 As can be seen from the figure, the live cell loading performance of the frozen microneedle prepared by using the antifreezing mixed solution and the preparation method provided in the application is better, and the cell survival rate is higher.

[0065] In summary, the frozen microneedle prepared by using the antifreezing mixed solution and the preparation method provided in the application can realize the guarantee of live cell loading of the frozen microneedle while improving the transdermal mechanical strength of the microneedle, and the use of the scheme to prepare the frozen microneedle loaded with hair follicle stem cells can realize the minimally invasive cell therapy for promoting hair growth.

Claims

1. A cryo-microneedle loaded with hair follicle stem cells, characterized in that: It includes a base and multiple needles arrayed on the base, the base being prepared from a base solution, and the needles being prepared from a mixture of hair follicle stem cells and an antifreeze solution.

2. The cryo-microneedle according to claim 1, characterized in that: The base solution is a sucrose-PBS solution, and the antifreeze mixture includes sodium alginate, polyvinyl alcohol, dimethyl sulfoxide, and sucrose-PBS solution.

3. The cryo-microneedle according to claim 2, characterized in that: The volume percentages of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide, and sucrose-PBS solution in the antifreeze mixture are (2-3): (2-3): (4-6): (88-92).

4. The cryo-microneedle according to claim 3, characterized in that: The volume percentages of sodium alginate, polyvinyl alcohol, dimethyl sulfoxide, and sucrose-PBS solution in the antifreeze mixture are 2.5:2.5:5:

90.

5. The cryo-microneedle according to claim 1, characterized in that: The needle is cone-shaped with its tip located away from the base. The needles are arranged in a 10*10 array, with a spacing of 0.6 mm between two adjacent needles. The bottom diameter of a single needle is 400 μm and its height is 1200 μm. The base has dimensions of 15 mm * 15 mm * 2 mm.

6. The method for preparing cryo-microneedles loaded with hair follicle stem cells according to any one of claims 1-5, characterized in that: Includes the following steps: S1. Preparation of antifreeze mixture: First, mix sodium alginate and polyvinyl alcohol in a certain proportion to obtain sodium alginate-polyvinyl alcohol mixture. Then, add sodium alginate-polyvinyl alcohol mixture and dimethyl sulfoxide to sucrose-PBS solution in a certain proportion. After mixing, the antifreeze mixture is obtained. S2. Prepare hair follicle stem cell resuspension: Resuspend hair follicle stem cells in the antifreeze mixture prepared in S1 to obtain hair follicle stem cell resuspension. S3. Preparation of hair follicle stem cell loaded cryo-microneedles: Add the hair follicle stem cell resuspension prepared in S2 into the PDMS groove mold, centrifuge to remove air, add base solution to completely cover the groove of the PDMS groove mold, remove excess liquid, centrifuge again, and then perform the first freezing. After molding, demold and then perform gradient cooling treatment to obtain cryo-microneedles loaded with hair follicle stem cells.

7. The preparation method according to claim 6, characterized in that: The density of hair follicle stem cells in S2 is 1×10⁻⁶. 6 cells mL -1 .

8. The preparation method according to claim 6, characterized in that: S3. Preparation of hair follicle stem cell-loaded cryo-microneedles: Add the hair follicle stem cell resuspension prepared in S2 to the PDMS groove mold, centrifuge at 2000 rpm for 3 min to remove air, so that the cells are concentrated at the tip of the needle, remove excess liquid, add base solution to completely cover the groove of the PDMS groove mold, remove excess liquid, centrifuge at 2000 rpm for 3 min, and then perform initial freezing. After molding, demold and perform gradient cooling treatment to obtain cryo-microneedles loaded with hair follicle stem cells.

9. The preparation method according to claim 6, characterized in that: In S3, the initial freezing temperature is (-20℃) and the freezing time is 2h. The gradient cooling process after demolding is as follows: freezing at (-80℃) for 2h and then freezing at (-196℃) for 2h. After the process, frozen microneedles loaded with hair follicle stem cells are obtained.

10. The use of the cryo-microneedles according to any one of claims 1-5 in the preparation of microneedles that promote hair growth.

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