Honokiol-loaded microneedle as well as preparation method and application thereof

Abstract

The invention provides a honokiol-loaded microneedle as well as a preparation method and application thereof. The invention provides a honokiol-loaded microneedle. The microneedle can effectively improve the transdermal delivery drug effect of honokiol, accelerate the hair growth speed, increase the hair coverage rate, weight and length, increase the number of hair follicles, enhance the proliferation activity of hair follicle cells and improve the blood supply of the hair follicles. The honokiol-loaded microneedle provided by the invention has a good clinical application prospect in promoting hair growth and preventing hair loss.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a microneedle loaded with honokiol and its preparation method and application. Background Art

[0002] In recent years, more and more patients, even at a young age, have begun to suffer from hair loss. For hair loss, there are several treatment techniques, such as hair follicle transplantation, drug treatment, low-level laser therapy, platelet-rich plasma therapy, and stem cell therapy.

[0003] Minoxidil and finasteride are widely used as FDA-approved hair loss treatment drugs. However, due to the barrier effect of the stratum corneum, the penetration rate of topically used minoxidil is low, so the effect can only be observed after long-term use. Oral finasteride is used to treat androgenetic alopecia and is a type II 5α-reductase inhibitor, but its systemic side effects are more obvious, including male sexual dysfunction and suicidal tendencies. Platelet-rich plasma therapy and stem cell therapy are treatment methods that have shown good clinical manifestations. These two treatments mainly promote hair regeneration by promoting the release of growth factors and anti-inflammatory pathways. However, platelet-rich plasma and stem cell therapies are relatively invasive, resulting in poor patient compliance and requiring surgeons to have a high level of skill. In addition, bacterial infection at the surgical site is another issue worthy of consideration in platelet-rich plasma and stem cell therapies.

[0004] Previous studies of the present invention found that honokiol liposomes have a definite effect of promoting hair growth through intraperitoneal injection, and its effectiveness and safety have been proven. However, frequent intravenous application reduces patient compliance and is difficult to widely apply; and the pharmacodynamics of topical honokiol liposomes has not shown efficacy in the research, and the delivery efficiency of topical use is limited and the dosage is uncontrollable. Summary of the Invention

[0005] In view of this, in order to make up for the deficiencies of the prior art, the present invention is specifically proposed.

[0006] The first aspect of the present invention provides a drug-loaded microneedle, and the drug-loaded microneedle includes a microneedle body, and the microneedle body includes a microneedle matrix and honokiol, and the honokiol is loaded on the microneedle matrix.

[0007] In the present invention, a microneedle (MN) refers to a needle-like structure with a diameter of 25 μm - 2000 μm, which has the characteristic of being able to pierce the stratum corneum of the skin without causing obvious pain and is a new type of transdermal drug delivery method. In the present invention, the microneedle can penetrate to the dermal layer.

[0008] In the present invention, the loading modes include, but are not limited to, a mode of mixing the microneedle matrix with honokiol, a mode of coating honokiol on the microneedle matrix, and a mode of designing the interior of the microneedle body as a hollow structure to directly accommodate honokiol.

[0009] Furthermore, the loading mode is selected from the mode of mixing the microneedle matrix with honokiol.

[0010] In the present invention, the honokiol can be honokiol itself or honokiol liposome. The honokiol liposome is formed by encapsulating honokiol in a liposome.

[0011] Furthermore, the honokiol is honokiol liposome.

[0012] In the present invention, the material of the microneedle body is not limited, as long as it can achieve the purpose of transdermal delivery of honokiol, it falls within the protection scope of the present invention.

[0013] Furthermore, the material of the microneedle matrix includes polymer biomaterials.

[0014] In the present invention, the material of the microneedle matrix refers to the core material that constitutes the main structure of the microneedle body.

[0015] In the present invention, the polymer biomaterials have the following advantages: a large drug loading capacity and a controllable drug release effect; b good biocompatibility, which can avoid potential damage to the skin caused by the breakage of the needle tip; c the microneedles can swell subcutaneously without dissolving, thereby increasing the drug loading capacity of the microneedles.

[0016] In the present invention, the polymer biomaterials include, but are not limited to, hyaluronic acid, polyvinyl alcohol, polylactic acid, poly(L-lactic acid), poly(glycolic acid), chitosan, and polycaprolactone.

[0017] Furthermore, the polymer biomaterial is selected from hyaluronic acid.

[0018] In the present invention, the hyaluronic acid can be hyaluronic acid itself or a hyaluronate.

[0019] Furthermore, the hyaluronic acid is a hyaluronate.

[0020] In the present invention, the hyaluronates include, but are not limited to, sodium hyaluronate, calcium hyaluronate, and zinc hyaluronate.

[0021] Furthermore, the hyaluronate is selected from sodium hyaluronate.

[0022] Furthermore, the mass ratio of the material of the microneedle matrix to honokiol is 1:1 - 10:1.

[0023] Furthermore, the mass ratio of the material of the microneedle substrate to honokiol is 3:1.

[0024] Furthermore, the material of the microneedle body further includes a body forming agent.

[0025] In the present invention, the body forming agent can form a crystal network, provide mechanical support, and ensure that the needle body remains sharp and has sufficient hardness before piercing the skin. When the body forming agent is mixed with the polymer biomaterial, the body forming agent can reduce the shrinkage of the needle body and avoid blunting of the needle tip.

[0026] Furthermore, the body forming agent includes but is not limited to sucrose, trehalose, and maltose.

[0027] Furthermore, the body forming agent is selected from sucrose.

[0028] Furthermore, the height of the microneedle body is 400 - 800 μm.

[0029] Furthermore, the height of the microneedle body is 600 μm.

[0030] Furthermore, the microneedle body is conical.

[0031] Furthermore, the bottom diameter of the microneedle body is 200 - 300 μm.

[0032] Furthermore, the bottom diameter of the microneedle body is 250 μm.

[0033] Furthermore, the drug - loaded microneedle further includes a microneedle backing.

[0034] In the present invention, the microneedle backing is a base structure that supports the microneedle body, responsible for fixing the needle body, providing mechanical stability, and ensuring the integrity and functionality of the microneedle when piercing the skin.

[0035] In the present invention, the material of the microneedle backing includes but is not limited to polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, hyaluronic acid, polylactic acid, polycarbonate, polyglycolic acid, chitosan, and polycaprolactone.

[0036] Furthermore, the material of the microneedle backing is selected from polyvinylpyrrolidone.

[0037] The second aspect of the present invention provides a pharmaceutical composition, which includes the drug - loaded microneedle described in the first aspect of the present invention.

[0038] Furthermore, the pharmaceutical composition includes its pharmaceutically acceptable carrier and / or excipient.

[0039] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes but is not limited to penetration enhancers, stabilizers, mechanical enhancers, and pH regulators.

[0040] In the present invention, the pharmaceutical composition and at least one additional therapeutic agent or therapy can be administered sequentially, simultaneously, and / or alternately, wherein the at least one additional therapeutic agent or therapy includes, but is not limited to, minoxidil, finasteride, platelet-rich plasma therapy, and stem cell therapy.

[0041] A third aspect of the present invention provides a method for preparing the drug-loaded microneedles described in the first aspect of the present invention, the method comprising preparing a microneedle body material containing a microneedle matrix and honokiol.

[0042] Further, the method includes dissolving the material of the microneedle matrix in a honokiol solution and swelling to form the microneedle body material.

[0043] Further, the concentration of the honokiol solution is 1 - 20 mg / ml.

[0044] Further, the concentration of the honokiol solution is 10 mg / ml.

[0045] Further, the concentration of the material of the microneedle matrix is 3 - 60 mg / ml.

[0046] Further, the concentration of the material of the microneedle matrix is 30 mg / ml.

[0047] Further, the method also includes dissolving a needle body forming agent in the honokiol solution.

[0048] Further, the mass ratio of the needle body forming agent to honokiol is 5:1 - 20:1.

[0049] Further, the mass ratio of the needle body forming agent to honokiol is 10:1.

[0050] Further, the needle body forming agent includes, but is not limited to, sucrose, trehalose, and maltose.

[0051] Further, the needle body forming agent is selected from sucrose.

[0052] Further, the method also includes combining the microneedle body material with a microneedle backing.

[0053] Further, the method includes adding the microneedle body material to a microneedle mold, forming needles, and combining with the microneedle backing.

[0054] In the present invention, the microneedle mold can achieve high-precision and reproducible production of a microneedle array by replicating the microneedle structure of a master mold.

[0055] Further, the microneedle mold includes, but is not limited to, a metal mold, a PDMS mold, a photoresist mold, and a biodegradable mold.

[0056] Further, the microneedle mold is selected from PDMS molds.

[0057] Further, the method for forming needles includes the centrifugation method and the vacuum pumping method.

[0058] Further, the method for forming needles is selected from the vacuum pumping method.

[0059] Further, the method includes adding the microneedle body material into the PDMS mold, removing air bubbles under negative pressure, concentrating, adding the microneedle body material again, concentrating, adding the microneedle backing material, drying, and demolding.

[0060] Further, the method also includes preparing the microneedle backing.

[0061] Further, the material of the microneedle backing includes but is not limited to polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, hyaluronic acid, polylactic acid, polycarbonate, polyglycolic acid, chitosan, and polycaprolactone.

[0062] Further, the material of the microneedle backing is selected from polyvinylpyrrolidone.

[0063] Further, the material of the microneedle backing is dissolved using an organic solvent.

[0064] In the present invention, the organic solvent includes but is not limited to ethanol and isopropanol.

[0065] Further, the organic solvent is selected from ethanol.

[0066] Further, the ethanol is absolute ethanol.

[0067] Further, the usage concentration of the material of the microneedle backing is 100 - 500 mg / ml.

[0068] Further, the usage concentration of the material of the microneedle backing is 300 mg / ml.

[0069] The fourth aspect of the present invention provides any one of the following applications:

[0070] (1) The application of the drug-loaded microneedles described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of drugs for promoting hair growth;

[0071] (2) The application of the drug-loaded microneedles described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of drugs for preventing hair loss;

[0072] (3) The application of microneedles in enhancing the percutaneous delivery efficacy of honokiol.

[0073] In the present invention, the promotion of hair growth includes, but is not limited to, accelerating the hair growth rate, increasing hair coverage, increasing hair weight, increasing hair length, increasing the number of hair follicles, enhancing the proliferation activity of hair follicle cells, and improving the blood supply of hair follicles.

[0074] In the present invention, the prevention of hair loss includes, but is not limited to, reducing hair loss, maintaining the health of hair follicles, and improving the hair growth environment.

[0075] In the present invention, the improvement refers to that the transdermal delivery efficacy of honokiol can be improved by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 98%.

[0076] Advantages and beneficial effects of the present invention:

[0077] The present invention provides a microneedle loaded with honokiol, a preparation method thereof and an application. The present invention provides a microneedle loaded with honokiol, which can effectively improve the transdermal delivery efficacy of honokiol, accelerate the hair growth rate, increase hair coverage, weight, length, increase the number of hair follicles, enhance the proliferation activity of hair follicle cells, and improve the blood supply of hair follicles. The microneedle loaded with honokiol provided by the present invention has good clinical application prospects in promoting hair growth and preventing hair loss. Description of the Drawings

[0078] Figure 1 It is a schematic diagram of the preparation method of the microneedle.

[0079] Figure 2 It is a morphological diagram of the microneedle under a transmission electron microscope.

[0080] Figure 3 It is a diagram for observing the situation of neonatal hair in mice, (A) a treatment flow chart of each group of mice; (B) a diagram for observing neonatal hair of each group of mice; (C) a neonatal hair coverage map of each group of mice; (D) a neonatal hair net weight map of each group of mice; (E) a neonatal hair length map of each group of mice, *p<0.05, ** p<0.01.

[0081] Figure 4 It is a diagram of HE staining of the skin of each group.

[0082] Figure 5 It is an immunofluorescence map of the skin of each group on the 14th day; (A) Ki-67 immunofluorescence map; (B) CD31 immunofluorescence map. Detailed Embodiments

[0083] The present invention will be further described below in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention and does not limit the present invention in any other form. Any person skilled in the relevant art may use the disclosed technical content to make equivalent changes to equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0084] Example 1

[0085] 1. Experimental materials

[0086] 1.1. Required equipment:

[0087] Vacuum degassing device (Model: EFL-VDM-001): Assist in better eliminating the influence of air bubbles on the micro-needle forming process during micro-needle preparation. The device includes ① oil-free vacuum pump; ② 500 ml sealed vacuum tank (equipped with a vacuum gauge).

[0088] 1.2. Micro-needle mold model:

[0089] EFL-MMN-600, material: PDMS, number of needles per sheet: 20*20, needle pitch: 550 um, needle bottom diameter: 250 um, needle height: 600 um, shape of micro-needle patch: square (side length 14.5 mm).

[0090] 1.3. Experimental reagents:

[0091] Absolute ethanol 2021081602 Chengdu Kelong Chemical Co., Ltd.

[0092] 4% Paraformaldehyde tissue fixative 22011938 Biosharp

[0093] Xylene 10023418 Sinopharm Chemical Reagent Co., Ltd.

[0094] HE staining kit BL700B Biosharp

[0095] Tissue autofluorescence quencher G1221 Wuhan Sevier Biotechnology Co., Ltd.

[0096] CD31 antibody, product number: GB11063-2-50, Wuhan Sevier Biotechnology Co., Ltd.

[0097] Ki-67 antibody, product number: GB121141-50, Wuhan Sevier Biotechnology Co., Ltd.

[0098] β-Catenin antibody, product number: GB12015-50, Wuhan Sevier Biotechnology Co., Ltd.

[0099] DAPI BS097 Biosharp

[0100] Anti-fluorescence fading sealing medium S2100 China Solebao Company

[0101] Sodium hyaluronate: Product number: 9067-32-7, Manufacturer: Beijing Puxitang Biotechnology Co., Ltd.

[0102] Sucrose product number: 57-50-1, manufacturer: Beijing Puxitang Biotechnology Co., Ltd.

[0103] Honokiol liposomes were obtained from Chengdu Jinrui Jiye Biotechnology Co., Ltd.

[0104] 2. Experimental methods

[0105] 2.1. Preparation of Sodium Hyaluronate Microneedles

[0106] Compared with traditional metal and silicon-based microneedles, microneedles based on biomaterials such as hydrogels have the following advantages: a. They have a large drug loading capacity and controllable drug release effect; b. The needle body material is composed of polymer biomaterials, which has good biocompatibility and avoids potential damage to the skin caused by needle tip breakage; c. Microneedles prepared with cross-linked polymer biomaterials can achieve subcutaneous swelling of microneedles without dissolving, thereby increasing the drug loading capacity of microneedles. Therefore, this study used hydrogel material sodium hyaluronate and molds produced by EFL to prepare microneedles. The mold model is EFL-MMN-600. Sodium hyaluronate (HA) was prepared into a solution with a mass volume ratio of 30 mg / mL, and microneedles were prepared by vacuum method. The vacuum defoaming device used a vacuum pump with model EFL-VDM-001. The HA solution with a concentration of 30 mg / mL was filled into the microneedle mold, vacuumed to -0.07MPa, and maintained at room temperature for 10 minutes. The mold was taken out and the excess needle body solution on the upper layer was scraped off, and then the backing material was added to the surface of the mold and placed in a desiccator overnight. According to previous literature, when the backing material is 300 mg / mL PVP, the patch formed is complete in shape after drying, easy to peel off, and has no problems such as bubbles, broken needles, and empty needles. Therefore, this study uses 300 mg / mL PVP as the backing material for microneedles.

[0107] 2.2. Preparation of Honokiol Liposome Composite Hyaluronic Acid Microneedles

[0108] (1) Preparation of needle material: Prepare a 10 mg / ml concentration of honokiol liposome solution. Use an electronic balance to accurately weigh 0.15 g of sodium hyaluronate powder and 0.5 g of sucrose granules, then dissolve the weighed sample in 5 mL of honokiol liposome solution and place at 4°C to dissolve until it is completely swollen to prepare a needle material with a concentration of 30 mg / mL.

[0109] (2) Preparation of the backing material: Accurately weigh 3 g of PVPK-60 using an electronic balance, and then dissolve the weighed sample in 10 mL of absolute ethanol to prepare a PVP solution with a concentration of 300 mg / mL.

[0110] (3) Preparation steps: Pour 200 μL of the needle material into the microneedle mold, evacuate to -0.07 MPa, and maintain for 10 min at room temperature. Then take out the mold and scrape off the excess needle material on the upper layer, place it in an incubator at 37 °C for 4 - 5 hours, add the needle material again, place it in an incubator at 37 °C for 4 - 5 hours, add the backing material, and then place it in an incubator at 37 °C overnight for drying, with the humidity maintained at 20%. After natural demolding, magnolol liposome microneedles are obtained.

[0111] A schematic diagram of the preparation method of the microneedles is as Figure 1 shown.

[0112] 2.3. In vitro simulated skin puncture experiment

[0113] Use 5 layers of stacked Parafilm paraffin film to simulate skin with different thicknesses, and place it on a smooth and flat tabletop. Press the microneedles vertically on the paraffin film for 1 minute and then pull them out. Use a high-definition microscope to observe the number of penetrated layers of the sealing film and the number of holes left, and calculate the penetration rate of each layer.

[0114] 2.4. Experimental animals

[0115] In this study, SPF-grade 6-week-old female C57BL / 6J mice were used. Before the experiment, they were first fed adaptively at the SPF level for 1 week, maintaining a normal circadian rhythm of light every day, freely ingesting food and water, with the environmental temperature at 22 - 25 °C and the humidity at about 55 - 65%.

[0116] 2.5. Experimental grouping and drug administration

[0117] In this experiment, 40 mice were randomly divided into 4 groups (n = 10). Among them, 4 mice in each group were used for photographing and recording observations until the end of the experiment; another 6 mice in each group were sacrificed after the treatment ended for HE staining and immunofluorescence and other experiments.

[0118] Magnolol liposome microneedle group (abbreviated as MN-HNK or Lip-HNK+MN): On the 1st day after hair removal, 1 piece of Lip-HNK+MN was given to the depilated area. The treatment method was to press the magnolol liposome microneedles on the depilated area with the thumb for 30 s, then stay for 5 min, and remove the microneedle patch after 5 min. The drug was administered once every 2 days, for a total of 7 times.

[0119] Sodium hyaluronate microneedle group (Blank-MN): On the 1st day after hair shaving, sodium hyaluronate microneedle treatment was given to the depilated area. Press the sodium hyaluronate microneedle against the depilated area with the thumb for 30 s, then leave it for 5 min, and remove the microneedle patch after 5 min. Administer the drug once every 2 days, for a total of 7 times.

[0120] Honokiol liposome application group (referred to as HNK or Lip-HNK): Starting from the 1st day after hair shaving, apply honokiol liposome solution to the depilated area every day until the end of the experiment.

[0121] Control group (Control): Starting from the 0th day after hair removal, apply normal saline to the depilated area every day until the end of the experiment.

[0122] 2.6. Observation of hair regrowth

[0123] On the 0th, 7th, 14th, 21st, and 28th days after hair removal, take pictures to record skin pigmentation and hair growth, and use a vernier caliper to measure the covered area of newly grown hair on the 17th and 21st days of the experiment, and calculate the regeneration hair coverage rate. After that, use a small animal hair clipper to shave off the newly grown hair in the depilated area of each group, collect and weigh it.

[0124] 2.7. Hematoxylin-eosin staining of skin tissue

[0125] Take the skin tissue in the treatment area and fix it with 4% paraformaldehyde general tissue fixative for more than 24 h. HE staining method and steps: (1) Immerse the section in xylene for 5 - 10 min; (2) Immerse the section in xylene for 5 - 10 min; (3) 100% alcohol for 1 min; (4) 100% alcohol for 1 min; (5) 95% alcohol for 1 min; (6) 95% alcohol for 1 min; (7) 90% alcohol for 1 min; (8) 80% alcohol for 1 min; (9) Wash with tap water for 1 min; (10) Immerse in hematoxylin staining solution for 10 - 15 min; (11) Wash with tap water for 30 sec - 1 min; (12) Differentiate with 1% hydrochloric acid alcohol for 30 sec; (13) Rinse with running water for more than 15 min; (14) Stain with 1% eosin alcohol for 3 - 5 min; (15) Differentiate with 90% or 95% alcohol for 30 sec; (16) 95% alcohol for 30 sec - 1 min; (17) 95% alcohol for 30 sec - 1 min; (18) 95% alcohol for 30 sec - 1 min; (19) 100% alcohol for 1 min; (20) 100% alcohol for 1 - 2 min; (21) Dimethyl carbonate xylene for 1 min; (22) Xylene for 1 - 2 min; (23) Xylene for 1 - 2 min; (24) Xylene for 1 - 2 min; (25) Seal with neutral gum.

[0126] 2.8. Immunofluorescence

[0127] Paraffin sections of the dorsal skin of mice were taken to analyze the immunofluorescence expression of Ki-67 and CD31.

[0128] 1) Dewaxing of paraffin sections: sequentially place the sections in environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - environmentally friendly dewaxing solution for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and wash with distilled water.

[0129] 2) Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (PH8.0) in a microwave oven for antigen repair. Heat on medium heat for 8 minutes, stop for 8 minutes, and switch to medium-low heat for 7 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After cooling naturally, place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. (The repair solution and repair conditions are determined according to the tissue).

[0130] 3) Draw circles: After the slices are slightly dried, use a histochemical pen to draw circles around the tissue (to prevent the antibodies from flowing away).

[0131] 4) Serum blocking: Add BSA in the circle and incubate for 30 minutes. (If the primary antibody is from goat, add donkey serum).

[0132] 5) Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in a certain proportion of PBS on the slices, and incubate the slices flat in a humidified box at 4°C overnight. (Add a small amount of water in the humidified box to prevent the antibody from evaporating).

[0133] 6) Add secondary antibody: Place the slide in PBS (PH7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add a secondary antibody labeled with the corresponding species of the primary antibody in a certain ratio in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0134] 7) DAPI counterstaining of cell nuclei: After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 10 minutes away from light.

[0135] 8) Autofluorescence quenching: After the slices are slightly dried, add autofluorescence quencher in the circle for 5 minutes and rinse with running water for 10 minutes.

[0136] 9) Sealing: Place the slides in PBS (PH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time. After the sections are slightly dried, seal them with anti-fluorescence quenching sealing medium.

[0137] 10) Microscopic examination and photography: The sections were placed under a scanner to collect images or photographed under a fluorescence microscope. (The ultraviolet excitation wavelength of DAPI is 330 - 380 nm, the emission wavelength is 420 nm, emitting blue light; the excitation wavelength of FITC is 465 - 495 nm, the emission wavelength is 515 - 555 nm, emitting green light; the excitation wavelength of CY3 is 510 - 560, the emission wavelength is 590 nm, emitting red light. The excitation wavelength of CY5 is 608 - 648 nm, the emission wavelength is 672 - 712. The cell nuclei stained by DAPI are blue under ultraviolet excitation, and the positive expression is the red light and green light of the corresponding fluorescein labeling. CD31 quantification refers to counting the number of the highest microvessels under a 400× field of view, and three fields of view were selected for each section (n = 3).

[0138] 3. Experimental results

[0139] 3.1. Morphology and height of microneedles

[0140] The results are as Figure 2 shown. The prepared honokiol liposome microneedle arrays in this study are neat, the needle shape is intact, the honokiol liposome - microneedles are composed of a 20×20 needle array, each needle has a smooth appearance and a sharp tip. The height of the conical microneedles is 600 μm, and the bottom needle diameter is 250 μm.

[0141] 3.2. In vitro simulated transdermal experiment

[0142] The puncture results show that the honokiol liposome microneedles can pierce through 4 layers of paraffin sealing film, indicating good mechanical properties and the ability to break through the skin barrier.

[0143] 3.3. Gross appearance of neonatal hair in mice

[0144] The skin pigmentation and hair growth were photographed and recorded on the 0th, 7th, 14th, and 21st days after hair removal ( Figure 3 ), where Figure A is the treatment flow chart of each group of mice, and the neonatal hair coverage area was measured with a vernier caliper on the 14th and 21st days (the measured neonatal hair coverage area does not include the area where only pigmentation occurred without hair growth), and the regenerated hair coverage rate was calculated. On the 17th and 21st days, the neonatal hair in the hair removal area of each group was shaved off with a small animal hair clipper, collected and weighed, and the average length was calculated. The results in Figure B show that the skin of the Lip - HNK+MN group became darker more significantly on the 14th day and had the highest hair coverage rate on the 21st day; Figure C shows that the hair coverage rate of the HNK - MN group was significantly higher than that of the control group, and there was no significant difference between the HNK group, the Blan - MN group and the control group; Figure D shows that there was a significant difference in the net weight of neonatal hair between the HNK - MN group and the control group, and there was no significant difference between the HNK group, the Blan - MN group and the control group; Figure E shows that there was a significant difference in the length of neonatal hair between the HNK - MN group and the control group, and there was no significant difference between the HNK group, the Blan - MN group and the control group.

[0145] 3.4. HE staining results

[0146] Skin tissues of each group were taken at 0, 7, 14, 21, and 28 days respectively for HE staining. The results were as follows Figure 4 shown. On the 7th day, there was no obvious difference in the number of hair follicles among each group. On the 14th and 21st days, the number of hair follicles in the skin tissues of the control group and the sodium hyaluronate microneedle group was less. The skin tissues of the honokiol liposome microneedle group were rich in hair follicles, densely arranged, with an obvious stratum corneum, and no obvious edema or inflammatory reaction in the stroma. Although the honokiol liposome group also had an increasing trend, there was no statistical significance compared with the honokiol liposome microneedle group.

[0147] 3.5. Immunofluorescence

[0148] Samples were taken, dehydrated, embedded, sectioned, and dewaxed to water, and then processed according to the steps of antigen repair, fluorescence quenching, blocking, adding antibodies, etc. Three fields of view were randomly selected for each section within each group for photographing. The same exposure time was used during photographing to keep the light source stable. The results were as follows Figure 5 shown. Figure A shows a large amount of positive expression of Ki-67 in the hair follicle part of the honokiol liposome microneedle group, and Figure B shows that the expression of CD31 is also significantly higher than that of other groups.

[0149] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A drug-loaded microneedle, characterized in that, The drug-loaded microneedle includes a microneedle body, and the microneedle body includes a microneedle matrix and honokiol, and the honokiol is loaded on the microneedle matrix; Preferably, the loading includes a loading mode of mixing the microneedle matrix with honokiol, a loading mode of coating honokiol on the microneedle matrix, and a loading mode of designing the inside of the microneedle body as a hollow structure to directly accommodate honokiol; Preferably, the loading is selected from the loading mode of mixing the microneedle matrix with honokiol.

2. The drug-loaded microneedle according to claim 1, wherein The honokiol is honokiol liposome.

3. The drug-loaded microneedle according to claim 1, wherein The material of the microneedle matrix includes a polymer biomaterial; Preferably, the polymer biomaterial includes hyaluronic acid, polyvinyl alcohol, polylactic acid, poly-L-lactic acid, polyglycolic acid, chitosan, and polycaprolactone; Preferably, the polymer biomaterial is selected from hyaluronic acid.

4. The drug-loaded microneedle according to claim 3, wherein, The hyaluronic acid includes hyaluronic acid itself or a hyaluronate; Preferably, the hyaluronate includes sodium hyaluronate, calcium hyaluronate, and zinc hyaluronate; Preferably, the hyaluronate is selected from sodium hyaluronate.

5. The drug-loaded microneedle according to claim 1, wherein The mass ratio of the material of the microneedle matrix to honokiol is 1:1 - 10:

1.

6. The drug-loaded microneedle according to claim 5, wherein, The mass ratio of the material of the microneedle matrix to honokiol is 3:

1.

7. The drug-loaded microneedle according to claim 1, wherein The drug-loaded microneedle further includes a microneedle backing; Preferably, the material of the microneedle backing includes polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, hyaluronic acid, polylactic acid, polycarbonate, polyglycolic acid, chitosan, and polycaprolactone; Preferably, the material of the microneedle backing is selected from polyvinylpyrrolidone.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the drug-loaded microneedle according to any one of claims 1 - 7; Preferably, the pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient thereof.

9. A method for preparing the drug-loaded microneedles according to any one of claims 1-7, characterized in that, The method includes preparing a microneedle body material containing a microneedle matrix and honokiol; Preferably, the method includes dissolving the material of the microneedle matrix in a honokiol solution and swelling to form the microneedle body material; Preferably, the concentration of the honokiol solution is 1 - 20 mg / ml; Preferably, the concentration of the honokiol solution is 10 mg / ml; Preferably, the concentration of the material of the microneedle matrix is 3 - 60 mg / ml; Preferably, the concentration of the material of the microneedle matrix is 30 mg / ml; Preferably, the method further includes dissolving a needle body forming agent in the honokiol solution; Preferably, the mass ratio of the needle body forming agent to honokiol is 5:1 - 20:1; Preferably, the mass ratio of the needle body forming agent to honokiol is 10:1; Preferably, the needle body forming agent includes sucrose, trehalose, and maltose; Preferably, the needle body forming agent is selected from sucrose; Preferably, the method further includes combining the microneedle body material with the microneedle backing; Preferably, the method includes adding the microneedle body material into a microneedle mold, forming needles, and combining with the microneedle backing; Preferably, the microneedle mold includes a metal mold, a PDMS mold, a photoresist mold, and a biodegradable mold; Preferably, the microneedle mold is selected from a PDMS mold; Preferably, the method of forming needles includes a centrifugation method and a vacuum pumping method; Preferably, the method of forming needles is selected from the vacuum pumping method; Preferably, the method includes adding the microneedle body material into a PDMS mold, removing air bubbles under negative pressure, concentrating, adding the microneedle body material for the second time, concentrating, adding the microneedle backing material, drying, and demolding; Preferably, the method further includes preparing the microneedle backing; Preferably, the material of the microneedle backing includes polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, hyaluronic acid, polylactic acid, polycarbonate, polyglycolic acid, chitosan, polycaprolactone; Preferably, the material of the microneedle backing is selected from polyvinylpyrrolidone; Preferably, the material of the microneedle backing is dissolved using an organic solvent; Preferably, the organic solvent includes ethanol and isopropanol; Preferably, the organic solvent is selected from ethanol; Preferably, the ethanol is anhydrous ethanol; Preferably, the usage concentration of the material of the microneedle backing is 100 - 500 mg / ml; Preferably, the usage concentration of the material of the microneedle backing is 300 mg / ml.

10. Any of the following applications: (1) The application of the drug-loaded microneedle according to any one of claims 1 - 7 or the pharmaceutical composition according to claim 8 in the preparation of a drug for promoting hair growth; Preferably, the promotion of hair growth includes accelerating the hair growth rate, increasing the hair coverage rate, increasing the hair weight, increasing the hair length, increasing the number of hair follicles, enhancing the proliferative activity of hair follicle cells, and improving the blood supply of hair follicles; (2) The application of the drug-loaded microneedle according to any one of claims 1 - 7 or the pharmaceutical composition according to claim 8 in the preparation of an anti-hair loss drug; (3) The application of microneedles in enhancing the transdermal delivery efficacy of honokiol.

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