Rapidly separated microneedle patch and preparation method and application thereof

Abstract

The invention discloses a fast-separation microneedle patch and a preparation method and application thereof. The microneedle patch comprises a needle tip prepared from a needle tip liquid and a separation backing prepared from a separation base liquid; the needle tip liquid consists of a drug-containing inclusion compound solution, a photoinitiator and a hypotonic agent; the drug-containing inclusion compound is prepared by first covalently linking a β-cyclodextrin conjugated polymer material to an angiogenesis-promoting drug and then encapsulating a 5α-reductase inhibitor; the separation base liquid consists of a polymer material, a gas initiator and a solvent; the gas initiator consists of an acid source and an alkali source; the fast-separation microneedle of the invention has strong separation and penetration-promoting performance, improves the hair follicle microenvironment from multiple aspects to promote hair regeneration, and has broad application prospects in the treatment of androgenic alopecia.

Description

[0001] The present invention claims the priority of the Chinese patent application filed with the Chinese Patent Office on July 24, 2024, with application number 2024109982305 and application name “A microneedle patch for treating androgenic alopecia and its preparation method”, the entire contents of which are incorporated by reference into the present invention. Technical Field

[0002] The present invention belongs to the technical field of pharmaceutical preparations, and more specifically, to a fast-separating microneedle patch with enhanced separation and penetration performance, a preparation method thereof, and an application thereof in treating androgenic alopecia. Background Art

[0003] Androgenic alopecia (AGA) is the most common type of progressive hair loss, with an increasing incidence rate and a trend of younger age, which troubles many patients. The pathogenesis of AGA is complex, among which abnormal local androgen metabolism, microcirculatory disorders and oxidative stress in the scalp are the key causes of AGA: ① Testosterone is converted into high-affinity dihydrotestosterone under the action of 5α-reductase, which further binds to androgen receptors to form hormone receptor complexes, inducing hair follicle atrophy and hair loss; ② The function of blood vessels around the hair follicles is impaired, limiting the self-supply of nutrients; ③ The excessive accumulation of reactive oxygen species (ROS) causes the aging and loss of activity of the central cells that control the growth and development of hair follicles, the dermal papilla cells, causing the hair follicles to enter the resting phase prematurely. However, the existing treatment methods of oral finasteride, topical application of minoxidil and hair transplantation have not fully considered the multiple pathogenic factors of AGA, and there are problems such as large side effects, low efficacy and poor patient compliance. Therefore, how to develop a functional drug delivery system with multiple effects based on the pathogenesis and microenvironment characteristics of AGA is of great significance for the treatment of AGA.

[0004] The lesions of AGA are mainly concentrated in the dermis, which is rich in hair follicles. Local transdermal administration can increase the accumulation of drugs in the lesions compared with oral administration, thereby reducing the dosage and side effects of drugs. Microneedles (MNs), as a new transdermal drug delivery system, have more advantages than traditional transdermal drug delivery preparations in the treatment of AGA: ① Microneedles can break through the stratum corneum barrier in a targeted manner and deliver drugs to the epidermis and superficial dermis without touching the dermal blood vessels and nerves to cause pain. They have the characteristics of efficient drug delivery and good patient compliance; ② The mechanical micro-injury caused by microneedles to the skin can induce the secretion of cytokines such as vascular endothelial growth factor (VEGF), activate the Wnt / β-Catenin signaling pathway to promote hair regeneration, and play a physical therapeutic role. However, there are still some limitations in the actual application of soluble microneedles: ① The needle tip slowly dissolves the drug by absorbing the interstitial fluid of the skin, and the microneedle needs to be applied to the skin for a long time, which reduces the patient's medication compliance and increases the risk of skin irritation; ② The elastic resistance formed by the skin barrier and the sequential resistance of drug diffusion reduce the efficiency of drug delivery; ③ The passive diffusion of the drug limits the diffusion area and penetration depth of the drug in the skin tissue, making it difficult to effectively enrich in the hair follicle tissue to exert its effect. Therefore, it is of great significance to develop a microneedle patch that can be quickly separated, has strong permeability, and high drug delivery efficiency for the treatment of androgenic alopecia. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a rapidly separated microneedle patch and its preparation method and application. The microneedle patch of the present invention co-loads a 5α-reductase inhibitor and a pro-angiogenic drug, which can drive the rapid separation of the needle body and the efficient penetration of the drug through the dual forces of gas production and capillary of the microneedle, and exert multiple effects of inhibiting the production of dihydrotestosterone in the hair follicle microenvironment, promoting angiogenesis and reducing the level of oxidative stress in the hair follicle, and ultimately achieving the effect of promoting hair regeneration.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect of the present invention, there is provided a rapidly detachable microneedle patch, the microneedle patch comprising a needle tip prepared from a needle tip liquid and a separation backing prepared from a separation base liquid;

[0008] The needle tip liquid is composed of a drug-containing inclusion compound solution, a photoinitiator and a hypotonic agent in a volume mass ratio of 1 mL: 0.005 g to 0.01 g: 0.05 g to 0.1 g; the concentration of the drug-containing inclusion compound in the drug-containing inclusion compound solution is 0.25 g / mL to 0.75 g / mL; the drug-containing inclusion compound is prepared by first covalently linking a β-cyclodextrin conjugated polymer material to a pro-angiogenic drug, and then encapsulating a 5α-reductase inhibitor; in the drug-containing inclusion compound, the molar concentration ratio of the 5α-reductase inhibitor to the pro-angiogenic drug is 1:3 to 9; the pro-angiogenic drug is deferoxamine or deferiprone; the 5α-reductase inhibitor is finasteride or dutasteride;

[0009] The separation base liquid is composed of a polymer material, a gas initiator, and a solvent; the gas initiator is composed of an acid source and an alkali source; the mass volume ratio of the polymer material, the acid source, the alkali source, and the solvent is 0.8g-1.2g: 0.1g-0.15g: 0.12g-0.18g: 3mL-4mL.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned rapid separation microneedle patch, comprising the following steps:

[0011] (1) covalently linking a β-cyclodextrin conjugated polymer material to an angiogenic drug, and then encapsulating a 5α-reductase inhibitor to obtain a drug-containing inclusion compound, freeze-drying the compound, and dissolving the compound in ultrapure water to obtain a drug-containing inclusion compound solution; heating the drug-containing inclusion compound solution, a photoinitiator, and a hypotonic agent to 45° C. to 55° C. in a dark environment, and mixing the mixture thoroughly until the mixture is completely dissolved to obtain a needle tip solution;

[0012] (2) adding the polymer material, the acid source and the alkali source into the solvent, sealing the container and placing it at room temperature until the polymer is fully swollen to form a viscous solution, thereby obtaining a separation base liquid;

[0013] (3) adding the needle tip liquid of step (1) to the surface of the microneedle female mold and flattening it, and centrifuging it at room temperature at 3500 rpm to 4500 rpm for 4 min to 6 min;

[0014] (4) scraping off the excess needle tip liquid on the surface of the microneedle negative mold with a scraper, irradiating with ultraviolet light for 30s to 45s, and centrifuging at 3500rpm to 4500rpm for 25min to 35min at room temperature;

[0015] (5) adding the separation base liquid of step (2) to the surface of the microneedle negative mold, spreading it flat until it covers the micropore surface of the microneedle, and centrifuging it at 4° C. to 15° C., 3500 rpm to 4500 rpm, for 4 min to 6 min;

[0016] (6) Dry for more than 2 days to obtain a rapidly detachable microneedle patch.

[0017] The third aspect of the present invention provides the use of the above-mentioned rapid separation microneedle patch in the treatment of androgenic alopecia.

[0018] Based on the pathogenesis and microenvironmental characteristics of AGA, the present invention constructs a microneedle co-delivery drug system with both rapid separation and efficient penetration promotion for the efficient treatment of AGA. On the one hand, the microneedle patch achieves rapid separation of the microneedle patch by introducing a hypotonic agent into the needle tip and a gas initiator into the separation backing, that is, the microneedle patch generates a capillary effect with the help of the rapid water absorption ability of the hypotonic agent during the drug administration process, accelerates the rise of skin tissue fluid with the needle tip, and then quickly triggers the gas initiator in the separation base fluid to dissolve and produce a gas reaction, and finally the microneedle patch is quickly separated under the shear action of the airflow, driving the drug to diffuse into the deep tissue of the skin, significantly improving the penetration and separation performance of the drug. On the other hand, the microneedle patch is loaded with 5α-reductase inhibitors (such as finasteride, dutasteride, etc.) and angiogenic drugs (such as deferoxamine, deferiprone, etc.) at the same time, which can play a synergistic role. The 5α-reductase inhibitor acts on the 5α-reductase in the hair papilla cells and reduces the intracellular dihydrotestosterone level, activating the hair regeneration signaling pathway, and the angiogenic drug can not only reduce the local oxidative stress and inflammation of the hair follicles, increase the activity of the hair papilla cells, but also improve the microcirculation of the hair follicles by promoting angiogenesis, provide sufficient nutrition supply for the new hair follicles, and finally achieve the reconstruction of the hair follicle microenvironment. Therefore, the fast-separated microneedle patch of the present invention has a wide range of application prospects in the treatment of androgenetic alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Characterization of the four materials in Example 1: (A) H NMR spectra of Gelatin, GelMA, and GelMA-β-CD, and (B) H NMR spectra of GelMA-β-CD and GelMA-β-CD-DFO.

[0020] Figure 2 The invention discloses the structural design concept (A) and the mechanism of rapid separation (B) of the rapid separation microneedle patch.

[0021] Figure 3 The scanning electron micrographs (A) of the four microneedle patches in Example 3 and the H&E staining (B) of the skin after microneedle patch puncture. Among them, G1: blank rapid separation microneedle patch (Blank-RSMN); G2: finasteride single rapid separation microneedle patch (FIN-RSMN); G3: deferoxamine single rapid separation microneedle patch (DFO-RSMN); G4: finasteride and deferoxamine co-loaded rapid separation microneedle patch (FIN-DFO-RSMN).

[0022] Figure 4The effects of different maltose concentrations and cross-linking times on the water absorption and swelling properties of the microneedle patch in Example 3: (A) The volume of skin interstitial fluid absorbed by the microneedle patch prepared with different maltose concentrations (n=4); (B) The volume of skin interstitial fluid absorbed by the microneedle patch prepared with different cross-linking times (n=4).

[0023] Figure 5 This is the separation of different types of microneedle patches in PBS in Example 4.

[0024] Figure 6 The skin retention of different types of microneedle patches in Example 4 at 6h, 12h, and 24h (n=3). Among them, G1: ordinary microneedle patch loaded with finasteride (FIN-MN); G2: separation microneedle patch loaded with finasteride (FIN-SMN); G3: rapid separation microneedle patch loaded with finasteride (FIN-RSMN).

[0025] Figure 7 The DHT levels in hDPCs after treatment with different concentrations of drug-containing inclusion complexes in Example 5 (n=3). Among them, F1: GelMA-β-CD-FIN; F2: GelMA-β-CD-DFO; F3: GelMA-β-CD-DFO / FIN.

[0026] Figure 8 The drug-containing inclusion compound GelMA-β-CD-DFO in Example 6 promotes the proliferation and migration of hDPCs: (A) cell proliferation rate of hDPCs after testosterone treatment (n=6); (B) cell proliferation curve (n=3); (C) scratch healing image; (D) cell migration rate (n=3). Among them, F1: Blank; F2: DFO; F3: GelMA-β-CD; F4: GelMA-β-CD-DFO.

[0027] Fig. 9 The drug-containing inclusion compound GelMA-β-CD-DFO in Example 7 promotes the proliferation and migration of hUVECs: (A) cell proliferation curve (n=3); (B) scratch healing image; (C) cell migration rate (n=3). Among them, F1: Blank; F2: DFO; F3: GelMA-β-CD; F4: GelMA-β-CD-DFO.

[0028] Fig.10 The results of promoting angiogenesis by the drug-containing inclusion compound GelMA-β-CD-DFO in Example 7: (A) optical microscope image of angiogenesis in Matrigel; (B) number of tubular nodes (n=4); (C) length of formed tubes (n=4). F1: Blank; F2: DFO; F3: GelMA-β-CD; F4: GelMA-β-CD-DFO.

[0029] Fig.11 The in vivo pharmacodynamic evaluation of different methods for treating androgenic alopecia in Example 8: (A) hair growth on the back of mice; (B) scanning electron micrograph of hair on the back; (C) hair coverage (n=6); (D) hair follicle growth cycle ratio (n=3). Among them, G1: Control, healthy mice; G2: AGA model mice; G3: Blank-RSMN; G4: FIN-RSMN; G5: DFO-RSMN; G6: FIN-DFO-RSMN; G7: Minoxidil.

[0030] Fig.12 The melanin deposition and hair follicle growth cycle of the mice with androgenic alopecia treated by different methods in Example 8: (A) H&E staining of the skin; (B) melanin deposition rate of the skin (n=3); (C) hair follicle growth cycle (n=3). DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0033] In some embodiments of the present invention, a fast-separation microneedle patch is disclosed, wherein the microneedle patch comprises a needle tip prepared from a needle tip liquid and a separation backing prepared from a separation base liquid; the needle tip liquid is composed of a drug-containing inclusion compound solution, a photoinitiator, and a hypotonic agent in a volume-mass ratio of 1 mL: 0.005 g to 0.01 g: 0.05 g to 0.1 g; the concentration of the drug-containing inclusion compound in the drug-containing inclusion compound solution is 0.25 g / mL to 0.75 g / mL, and the drug-containing inclusion compound is prepared by first covalently linking a pro-angiogenic drug to a β-cyclodextrin conjugated polymer material, and then encapsulating The drug-containing inclusion compound is prepared by combining a 5α-reductase inhibitor; in the drug-containing inclusion compound, the molar concentration ratio of the 5α-reductase inhibitor to the angiogenesis-promoting drug is 1:3-9; the angiogenesis-promoting drug is deferoxamine or deferiprone; the 5α-reductase inhibitor is finasteride or dutasteride; the separation base liquid consists of a polymer material, a gas initiator, and a solvent; the gas initiator consists of an acid source and an alkali source; the mass volume ratio of the polymer material, the acid source, the alkali source and the solvent is 0.8g-1.2g: 0.1g-0.15g: 0.12g-0.18g: 3mL-4mL.

[0034] In some embodiments, the hypotonic agent is a sugar that has fast dissolution properties and produces micro-channels to form a siphon effect, and can be one or more of maltose, sucrose, and lactose, preferably maltose.

[0035] In some of the embodiments, the photoinitiator is composed of a substance that is an active intermediate (free radical or cation) capable of initiating polymerization under ultraviolet or blue light, and may be one or more of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) and lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP).

[0036] In some of the embodiments, the polymer material in the needle tip liquid is one or more of type A gelatin, type B gelatin, methacrylated gelatin, hyaluronic acid, methacrylated hyaluronic acid, silk fibroin, and methacryloylated silk fibroin; preferably, the β-cyclodextrin conjugated polymer material in the needle tip liquid is β-cyclodextrin conjugated methacrylated gelatin; and / or, the polymer material in the separation base liquid is one or more of polyvinyl pyrrolidone K30, polyvinyl pyrrolidone K45, and polyvinyl pyrrolidone K90.

[0037] In some of the embodiments, the needle tip fluid is composed of a drug-containing inclusion complex solution, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and maltose, and the drug-containing inclusion complex in the drug-containing inclusion complex solution is prepared by first covalently linking β-cyclodextrin conjugated methacrylic anhydride gelatin to deferoxamine, and then including finasteride; the molar concentration ratio of finasteride to deferoxamine is 1:6.

[0038] In some of the embodiments, the volume mass ratio of the drug-containing inclusion complex solution, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and maltose is 1 mL: 0.006 g to 0.008 g: 0.06 g to 0.08 g.

[0039] In some embodiments, the acid source is citric acid, tartaric acid or polyacrylic acid, and the alkali source is one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate and magnesium carbonate.

[0040] In some embodiments, the solvent is anhydrous ethanol; and / or the separation substrate liquid is prepared from polyvinyl pyrrolidone K90, citric acid, sodium bicarbonate and anhydrous ethanol in a mass volume ratio of 0.8g~1.2g:0.126g~0.130g:0.14g~0.18g:3.2mL.

[0041] In other embodiments of the present invention, a method for preparing the above-mentioned rapid separation microneedle patch is disclosed, comprising the following steps:

[0042] (1) covalently linking a β-cyclodextrin conjugated polymer material to an angiogenic drug, and then encapsulating a 5α-reductase inhibitor to obtain a drug-containing inclusion compound, freeze-drying the compound, and dissolving the compound in ultrapure water to obtain a drug-containing inclusion compound solution; heating the drug-containing inclusion compound solution, a photoinitiator, and a hypotonic agent to 45° C. to 55° C. in a dark environment, and mixing the mixture thoroughly until the mixture is completely dissolved to obtain a needle tip solution;

[0043] (2) adding the polymer material, the acid source and the alkali source into the solvent, sealing the container and placing it at room temperature until the polymer is fully swollen to form a viscous solution, thereby obtaining a separation base liquid;

[0044] (3) adding the needle tip liquid of step (1) to the surface of the microneedle female mold and flattening it, and centrifuging it at room temperature at 3500 rpm to 4500 rpm for 4 min to 6 min;

[0045] (4) scraping off the excess needle tip liquid on the surface of the microneedle negative mold with a scraper, irradiating with ultraviolet light for 30s to 45s, and centrifuging at 3500rpm to 4500rpm for 25min to 35min at room temperature;

[0046] (5) adding the separation base liquid of step (2) to the surface of the microneedle negative mold, spreading it flat until it covers the micropore surface of the microneedle, and centrifuging it at 4° C. to 15° C., 3500 rpm to 4500 rpm, for 4 min to 6 min;

[0047] (6) Dry for more than 2 days to obtain a rapidly detachable microneedle patch.

[0048] In other embodiments of the present invention, the use of the above-mentioned rapid separation microneedle patch in the treatment of androgenic alopecia is disclosed.

[0049] The present invention is further described below through the accompanying drawings and specific embodiments.

[0050] Example 1 Synthesis and Characterization of GelMA, GelMA-β-CD and GelMA-β-CD-DFO

[0051] 1. Synthesis of GelMA

[0052] 10.00g of type A gelatin was slowly added to 100mL of Dulbecco's Phosphate-Buffered Saline (DPBS) preheated to 50°C and stirred until completely dissolved. Then, the pH of the gelatin solution was adjusted to 9 using a 5M hydrochloric acid solution. Then, 8mL of methacrylic anhydride (MA) was added under magnetic stirring at 500rpm, and gelatin and MA were reacted at 50°C for 3h. Finally, 500mL of DPBS was added to terminate the reaction. The reaction product was collected in a dialysis bag with a molecular weight cutoff of 12kDa to 14kDa, and the dialysis bag was placed in distilled water and dialyzed at 40°C for 1 week to remove unreacted MA and other impurities. After the dialysis, the remaining liquid was heated to 60°C and filtered with a microporous membrane with a pore size of 0.22μm. The filtrate was pre-frozen in a refrigerator overnight, and then freeze-dried in a freeze dryer for 2 days to obtain methacryloyl gelatin (GelMA), which was stored at -20°C for future use.

[0053] 2. Synthesis of GelMA-β-CD

[0054] 1.20 g of GelMA was weighed and dissolved in 12 mL of DPBS (pH = 7.4), and then placed in a magnetic stirrer at 80°C to fully dissolve. 2.40 g of carboxymethyl-β-cyclodextrin (CM-β-CD) was weighed and added to 12 mL of 2-Morpholinoethane-sulphonic acid (MES) buffer (pH = 6), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added in a molar ratio of CM-β-CD carboxyl: EDC: NHS = 1:2:4, and the pH was adjusted to 3.5-4.5, and the reaction was carried out for 30 minutes to activate the carboxyl group. After 12 mL of GelMA solution and 12 mL of CM-β-CD solution were mixed evenly, the pH was adjusted to 8-9 and stirred overnight at 40°C. Unreacted CM-β-CD and residual salts were removed by dialysis for 5 days using a dialysis bag with a molecular weight cutoff of 12 kDa to 14 kDa. After the dialysis, the remaining liquid was heated to 60°C and filtered with a microporous membrane with a pore size of 0.22 μm. The filtrate was pre-frozen in a refrigerator overnight and freeze-dried in a freeze dryer for 2 days to finally obtain β-cyclodextrin conjugated methacryloyl gelatin (GelMA-β-CD), which was stored at -20°C for use.

[0055] 3. Synthesis of GelMA-β-CD-DFO

[0056] 728.11 mg of GelMA-β-CD was weighed and dissolved in 3.6 mL of MES buffer (pH = 6), and then EDC and NHS were added in a molar ratio of GelMA-β-CD carboxyl: EDC: NHS = 1:2:4, and the reaction was continued for 30 min to activate the carboxyl group. 100.00 mg of DFO was weighed and dissolved in 1 mL of DPBS (pH = 7.4), and mixed evenly with 3.6 mL of GelMA-β-CD solution, the pH of the reaction system was adjusted to 7, and the reaction was stirred at 40 ° C overnight. Unreacted DFO and residual salts were removed by dialysis for 5 days with a dialysis bag with a molecular weight cutoff of 12 kDa to 14 kDa. After the dialysis, the remaining liquid was heated to 60 ° C, filtered with a microporous membrane with a pore size of 0.22 μm, and the filtrate was placed in a refrigerator for pre-freezing overnight and freeze-dried in a freeze dryer for 2 days to finally obtain GelMA-β-CD-DFO, which was stored at -20 ° C for use.

[0057] 4. H NMR

[0058] Weigh 10.00 mg of Gelatin, GelMA, GelMA-β-CD and GelMA-β-CD-DFO respectively and place them in 1.5 mL centrifuge tubes. Dissolve and mix them with deuterated water and transfer them into NMR tubes for NMR detection. 1 Changes in characteristic peaks on the HNMR spectrum.

[0059] The results are as follows Figure 1 As shown, the spectra of Gelatin, GelMA, GelMA-β-CD and GelMA-β-CD-DFO all have a proton peak at 7.20 ppm (region Ⅰ), which belongs to the hydrogen on the aromatic ring of gelatin, and the spectra of GelMA, GelMA-β-CD and GelMA-β-CD-DFO have increased proton peaks at 5.40 ppm and 5.70 ppm (region Ⅱ), which belong to the hydrogen on the double bond in methacrylamide, proving that MA has successfully modified type A gelatin. In addition, the proton peak at 2.90 ppm (region Ⅳ) of the Gelatin spectrum shows a decrease in the GelMA spectrum. This is because the free amino group on the lysine in gelatin is the modification site of MA, and the expression of the methylene group decreases. The spectra of GelMA-β-CD and GelMA-β-CD-DFO both have proton peaks of hydrogen on the double bond of methacryloyl at 5.40ppm and 5.70ppm (region II), and proton peaks of hydrogen on the glucose ring of CM-β-CD at 4.80ppm and 5.7ppm (region III), proving that CM-β-CD is successfully bound to GelMA. In addition, the spectrum of GelMA-β-CD-DFO also has proton peaks of hydrogen on the glucose ring at 4.80ppm and 5.7ppm (region III), and the proton peak at 3.60ppm (region V) shows an increase, which belongs to the hydrogen on the hydroxyl group in the hydroxamic acid structure, proving that DFO is successfully covalently linked to the GelMA-β-CD skeleton.

[0060] Example 2 Rapidly detachable microneedle patch and preparation method thereof

[0061] Please refer to Figure 2 , which are the structural design ideas (A) and the mechanism of action (B) of the rapid separation microneedle patch of the present invention. This embodiment provides a method for preparing a rapid separation microneedle patch, which specifically includes the following steps:

[0062] 1. Preparation of drug-containing inclusion complex

[0063] 5.00 mg of finasteride (FIN) was added to a GelMA-β-CD or GelMA-β-CD-DFO solution (20%, g / mL) pre-dissolved in 1 mL of DPBS, and stirred at 37°C / 1000 rpm for 6 h to form a drug-containing inclusion solution. Further, the drug-containing inclusion solution was centrifuged at 12000 rpm for 10 min to remove the unincluded finasteride, and the supernatant was filtered through a 0.22 μm microporous membrane. Finally, the filtrate was placed in a freeze dryer and dried for 2 days to obtain a drug-containing inclusion powder GelMA-β-CD / FIN or GelMA-β-CD-DFO / FIN, which was stored in a -20°C refrigerator for later use.

[0064] 2. Preparation of needle tip fluid

[0065] Preparation of finasteride and deferoxamine co-loaded rapid separation microneedle (FIN-DFO-RSMN) tip liquid: 0.50 g GelMA-β-CD-DFO / FIN (drug-containing inclusion complex, finasteride and deferoxamine loading amounts were 8.66±3.34 mg / g and 65.91±6.17 mg / g, respectively), 5.00 mg I2959 (photoinitiator), and 50.00 mg maltose (hypotonic agent) were added to 1 mL ultrapure water, heated to 50°C in the dark, and mixed thoroughly for 1 h until completely dissolved.

[0066] Except for the different drug loadings of the inclusion complexes, the tip fluids of blank rapid separation microneedles (Blank-RSMN, GelMA-β-CD without drug inclusion complex), finasteride single-loaded rapid separation microneedles (FIN-RSMN, GelMA-β-CD-FIN with drug inclusion complex) and deferoxamine single-loaded rapid separation microneedles (DFO-RSMN, GelMA-β-CD-DFO with drug inclusion complex) were prepared according to the preparation method of FIN-DFO-RSMN tip fluid.

[0067] 3. Preparation of separation substrate

[0068] Add 10.00 g PVP K90 (31.25%, w / v), 1.28 g citric acid (4%, w / v), and 1.60 g sodium bicarbonate (5%, w / v) into a centrifuge tube containing 32 mL of anhydrous ethanol solution, and stir with a glass rod. After PVP K90 is fully swollen, store the centrifuge tube in a 4°C refrigerator.

[0069] 4. Preparation of Rapid Separation Microneedle Patches

[0070] (1) Take 300 μL of each of the above different needle tip liquids and add them to the surface of the microneedle negative mold to spread it evenly. Place the negative mold in a centrifuge and centrifuge for 5 minutes at room temperature and 4000 rpm; (2) Scrape off the excess needle tip liquid on the surface of the negative mold and use a UV lamp to irradiate the microneedle negative mold for 30 seconds to cross-link the needle tip liquid raw materials. Then, place the negative mold in a centrifuge and continue to centrifuge for 30 minutes at room temperature and 4000 rpm, then place the microneedle negative mold in a dryer and dry overnight. (3) Add 400 μL of separation substrate liquid to the surface of the negative mold and spread it flat until it covers the surface of the microneedle micropores. Centrifuge at 10°C and 4000 rpm for 5 min. (4) Place the microneedle negative mold in a dryer and dry for 2 days to obtain a blank rapid separation microneedle patch (Blank-RSMN), a finasteride single-loaded rapid separation microneedle patch (FIN-RSMN), a deferoxamine single-loaded rapid separation microneedle patch (DFO-RSMN), and a finasteride and deferoxamine co-loaded rapid separation microneedle patch (FIN-DFO-RSMN).

[0071] Example 3 Characterization and optimization of fast-detaching microneedle patches

[0072] The four microneedle patches of Blank-RSMN, FIN-RSMN, DFO-RSMN and FIN-DFO-RSMN prepared in Example 2 were characterized as follows.

[0073] 1. The shape of the microneedle patch

[0074] The morphology of the four microneedle patches was observed using a scanning electron microscope. Figure 3 As shown in A, the needle bodies of the four microneedles are all in the shape of quadrangular pyramids, and the needle tips are complete and regularly arranged, indicating that the addition of hypotonic agents (maltose) or gas initiators has little effect on the integrity and appearance of the needle tips.

[0075] 2. Skin puncture performance of microneedle patches

[0076] The microneedle patch was inserted into the rat skin in vitro. After 1 minute of drug administration, the microneedle patch was removed. The skin tissue at the drug administration site was fixed with 4% paraformaldehyde for subsequent skin tissue sectioning and H&E staining. The stained skin tissue was placed under an upright microscope for observation. The results are as follows: Figure 3 As shown in B: All four microneedle patches can effectively puncture the skin tissue, and the penetration depth of the skin is 100μm to 400μm, indicating that each group of microneedle patches has good skin puncture performance, which is conducive to delivering drugs to the skin tissue.

[0077] 3. Water absorption and swelling properties of microneedle patches

[0078] The initial mass of the microneedle patch was measured, and then the microneedle patch was pressed vertically on the skin surface for 1 min, then the microneedle patch was removed and the final mass of the microneedle patch was recorded. The volume of skin interstitial fluid (ISF) extracted by the microneedle patch (V ISF ) to evaluate the effects of maltose concentration and photocrosslinking time on the water absorption and swelling properties of the microneedle patch.

[0079] V ISF =(W t -W0) / ρ (1)

[0080] Among them, W0 and W t are the initial and final weights of the microneedles; ρ is the density of skin ISF and is set to 1 g / mL.

[0081] The results are as follows Figure 4 As shown: The volume of skin interstitial fluid absorbed by the rapidly separated microneedle patch is positively correlated with the concentration of maltose. Compared with the microneedles without maltose, the microneedles containing 50 mg / mL maltose have a 2.51-fold increase in water absorption and swelling capacity, indicating that the addition of maltose to the needle tip fluid can significantly improve the water absorption and swelling properties of the microneedle patch, thereby affecting the gas production and separation effect of the microneedle patch ( Figure 4 The volume of interstitial fluid absorbed by the rapidly detached microneedle patch is inversely proportional to the photocrosslinking time ( Figure 4 In B). In order to take into account both the water absorption and swelling properties and the sustained release properties of the microneedle patch, 50 mg / mL maltose is preferably added when preparing a fast-separated microneedle patch, and the photocrosslinking time is 30 s.

[0082] Example 4 Performance of different microneedle patches

[0083] Preparation of microneedle patches: The needle tip liquid of ordinary microneedle patches does not contain maltose, and the separation base liquid does not contain gas initiators (citric acid and sodium bicarbonate), and the other ingredients are the same as those of rapid separation microneedle patches; the needle tip liquid of separation microneedle patches does not contain maltose, and the separation base liquid and the separation base liquid of rapid separation microneedle patches have the same ingredients. The preparation methods of ordinary microneedle patches and separation microneedle patches are the same as those of rapid separation microneedle patches.

[0084] 1. Separation performance of microneedle patch

[0085] Rhodamine B was used to replace the drug in the needle tip solution, and fluorescently labeled ordinary microneedle patches, separation microneedle patches, and rapid separation microneedle patches were prepared according to the methods of Examples 1 and 2, so as to better observe the separation performance of the microneedles under an optical microscope. The microneedle patches were pasted on the inside of a square dish using double-sided tape, and an appropriate amount of PBS solution was added until the microneedle patches were immersed. The separation process of the microneedle patches was observed using an upright microscope. The results are shown in FIG. Figure 5As shown in the figure: the ordinary microneedle patch gradually absorbed water and swelled to form a hydrogel within 40 seconds, and there was no obvious needle separation phenomenon; the separation microneedle patch gradually produced bubbles at the backing after 10 seconds, and the needle tip was partially separated after 20 seconds. After 40 seconds, the needle tip was basically completely separated under the push of bubbles; the fast separation microneedle patch produced bubbles at both the needle tip and the separation backing after 10 seconds, and the needle tip was completely separated within 30 seconds. This result shows that both the separation microneedle patch and the fast separation microneedle patch can achieve needle separation, and the fast separation microneedle patch promotes the water absorption and swelling of the needle body and the gas production reaction in the separation backing due to the dissolution of maltose and the formation of low osmotic pressure locally on the needle body, which further accelerates the separation of the needle body.

[0086] 2. Transdermal delivery efficiency of microneedle patches

[0087] The drug skin retention at different time points of microneedle patch administration was determined by Franz transdermal diffusion cell to evaluate the transdermal delivery efficiency of common microneedle patches, separation microneedle patches and rapid separation microneedle patches. The ex vivo rat skin was fixed between the donor chamber and the receiving chamber with the epidermis facing up. 7.5 mL of PBS solution (containing 30% PEG 400, pH adjusted to 6.8) was used as the receiving medium, and the medium was continuously stirred in a magnetic stirrer at 250 rpm and a circulating water bath was performed at 32 ° C. Then, the finasteride single-loaded common microneedle patch (FIN-MN), finasteride single-loaded separation microneedle patch (FIN-SMN), and finasteride single-loaded rapid separation microneedle patch (FIN-RSMN) were pierced into the skin, and the microneedle patch was removed after 1 min of administration. Skin tissues were collected 6h, 12h and 24h after administration. After washing the skin tissue, the skin tissue was cut into pieces using scissors and placed in a glass grinding tube, and 1 mL of receiving medium was added to the glass grinding tube to grind the skin tissue thoroughly. After centrifugation and filtration of the ground skin tissue fluid, the drug retention in the skin was determined by high performance liquid chromatography.

[0088] Drug skin retention of different microneedle patches Figure 6 As shown in the figure: the skin retention of the microneedle patch at each time point is the order of rapidly separated microneedle patch > separated microneedle patch > ordinary microneedle patch. At 6 hours after administration, the skin retention of the rapidly separated microneedle patch was 2.54 times that of the ordinary microneedle patch and 1.36 times that of the separated microneedle patch. At 24 hours, the retention of the rapidly separated microneedle patch was still significantly higher than that of the ordinary microneedle patch and the separated microneedle patch, indicating that the rapidly separated microneedle patch can significantly improve the efficiency of transdermal delivery. The main reasons are: (1) accelerating the gas production reaction of the separation backing, forming a local gas vortex field to promote drug penetration into the deep skin; (2) the rapid separation performance of the microneedle patch overcomes the elastic resistance of the skin and increases the dose of drug delivered to the skin through the microneedle patch.

[0089] Example 5 Study on the performance of GelMA-β-CD-DFO / FIN in inhibiting 5α-reductase activity

[0090] Human dermal papilla cells (hDPCs) were cultured at 1×10 5 Cells / well were inoculated into 12-well plates, and the culture medium was removed after overnight culture. Subsequently, the drug was diluted with serum-free DMEM culture medium containing 75 μM testosterone to prepare GelMA-β-CD-FIN solution, GelMA-β-CD-DFO solution and GelMA-β-CD-DFO / FIN containing a series of drug concentrations. Among them, the concentrations of finasteride (Finasteride, FIN) were 0, 2.5 μM, 5 μM, 10 μM, 20 μM, the concentrations of DFO were 0, 15 μM, 30 μM, 60 μM, 120 μM, and the molar ratio of FIN and DFO was 1:6. Different solution-containing culture media were added to a 12-well plate, the cells were incubated for 24 hours, and then the cells were washed with PBS. Subsequently, 500 μL of methanol solution was added to each well of cells to extract dihydrotestosterone from the cells, and the dihydrotestosterone content in the cells was determined using an ELISA kit. The results are as follows Figure 7 As shown: After hDPCs cells were treated with testosterone, the average content of dihydrotestosterone in the cells increased from 153.2ng to 213.7ng, indicating that testosterone can be converted into dihydrotestosterone under the catalysis of 5α-reductase in the cells; the content of dihydrotestosterone in cells treated with GelMA-β-CD-FIN and GelMA-β-CD-DFO / FIN decreased significantly, and showed a certain positive correlation with the concentration of FIN, while the content of dihydrotestosterone in cells treated with GelMA-β-CD-DFO was not significantly different from that in the testosterone group. These results show that FIN can be successfully taken up by hDPCs, inhibit the activity of 5α-reductase in cells, and significantly reduce the conversion of testosterone to dihydrotestosterone.

[0091] Example 6 Study on the performance of GelMA-β-CD-DFO / FIN in promoting proliferation and migration of dermal papilla cells

[0092] 1. Cell proliferation assay

[0093] hDPCs were cultured at 5×10 3The number of cells / well was inoculated in a 96-well plate and cultured overnight. The culture medium was discarded, and the culture medium containing different concentrations of drug solution was added to incubate the cells: ① DMEM complete culture medium; ② DMEM culture medium containing 75μM testosterone + DFO; ③ DMEM culture medium containing 75μM testosterone + GelMA-β-CD; ④ DMEM culture medium containing 75μM testosterone + GelMA-β-CD-DFO. Among them, the concentrations of DFO, GelMA-β-CD and GelMA-β-CD-DFO were calculated based on the DFO content, which were 0, 30μM, 60μM, 90μM, 120μM, 150μM, 180μM, 210μM and 240μM, respectively. After incubation for 24h, the cell survival rate was determined using the CCK8 detection kit, and the cell proliferation rate was calculated.

[0094] The cell proliferation rates of different groups were shown in Figure 8 As shown in A and B in the figure: At concentrations of 30μM to 240μM, the cell proliferation rates of the three treatment groups were all positive as a whole. Compared with the DFO and GelMA-β-CD groups, covalently linking DFO to GelMA-β-CD to form GelMA-β-CD-DFO promoted cell proliferation. When the DFO concentration was 90μM, the cell proliferation rates were 1.72 times and 1.55 times that of DFO and GelMA-β-CD, respectively.

[0095] 2. Cell scratch test

[0096] hDPCs were divided into 5×10 5 The cells were inoculated into a 6-well plate at a density of 100 μM / well. When the cells were completely fused into a monolayer, the cells were gently scratched along a straight line with a 200 μL pipette, and then washed twice with PBS to remove the scratched cells. Different culture media were added to the cells for incubation: ① DMEM culture medium; ② DMEM culture medium containing free DFO; ③ DMEM culture medium containing GelMA-β-CD; ④ DMEM culture medium containing GelMA-β-CD-DFO. Among them, the concentrations of DFO, GelMA-β-CD and GelMA-β-CD-DFO were calculated based on the DFO content, with a concentration of 120 μM. At 0h, 6h, 12h and 24h after incubation, photos were taken with an upright microscope to record the cell migration in the scratch area. The scratch area was measured using Image J software, and the scratch healing rate was calculated according to Formula 2.

[0097] Scratch healing rate (%) = (initial scratch area - final scratch area) / initial scratch area × 100 (2)

[0098] Cell scratch healing results Figure 8As shown in C and D: The rate of cell migration to the scratch area in the GelMA-β-CD-DFO group was faster than that in the DFO and GelMA-β-CD groups, and the ability to promote cell migration was stronger; at 24 hours, the scratch healing rate was GelMA-β-CD-DFO (50.6%) > DFO (39.4%) > GelMA-β-CD (29.1%) > Blank (18.7%). Among them, the scratch healing rate of GelMA-β-CD-DFO was 1.29 times and 1.74 times that of DFO and GelMA-β-CD, respectively, indicating that the polymer is expected to promote hair regeneration by enhancing cell migration.

[0099] Example 7 Study on the performance of GelMA-β-CD-DFO in promoting angiogenesis

[0100] 1. Cell proliferation assay

[0101] Human umbilical vein endothelial cells (hUVECs) were cultured at 5×10 3 The cells were inoculated into 96-well plates at a density of 100 μM / well and placed in a constant temperature incubator containing 5% CO2 and 37°C for overnight culture. The culture medium was discarded and drug solutions containing different concentrations were added and incubated for 24 hours: ① DMEM / F12 culture medium; ② DMEM / F12 culture medium containing free DFO; ③ DMEM / F12 culture medium containing GelMA-β-CD; ④ DMEM / F12 culture medium containing GelMA-β-CD-DFO. Among them, the concentrations of DFO, GelMA-β-CD and GelMA-β-CD-DFO were calculated based on the DFO content, which were 0, 30μM, 60μM, 90μM, 120μM, 150μM, 180μM, 210μM and 240μM, respectively. After culturing for 24 hours, the cell survival rate was determined using the CCK8 detection kit and the cell proliferation rate was calculated.

[0102] The cell proliferation rate results were as follows Fig. 9As shown in A: The cell proliferation rate curve incubated with DFO showed a negative growth trend, indicating that free DFO had strong cytotoxicity at high concentrations; on the contrary, the cell proliferation rates treated with GelMA-β-CD and GelMA-β-CD-DFO were mostly positive, and at a concentration of 30μM to 180μM, they showed a certain dose-effect relationship, and there was no significant difference between the GelMA-β-CD and GelMA-β-CD-DFO groups. GelMA-β-CD has a good effect on promoting the proliferation of hUVECs cells, which may be related to the three-dimensional network structure it forms providing action sites for cell proliferation; when DFO is covalently linked to GelMA-β-CD to form GelMA-β-CD-DFO, the cell proliferation rate curve showed a positive growth trend, indicating that GelMA-β-CD-DFO prevents DFO from being released locally, thereby reducing the cytotoxicity of DFO.

[0103] 2. Cell scratch test

[0104] hUVECs were cultured at 5 × 10 5 Each well was inoculated in a 6-well plate. When the cells were completely fused into a monolayer, the hUVECs cells were gently scratched in a straight line with a 200 μL pipette, and then washed twice with PBS to remove the scratched cells. Different culture media were added to the cells for incubation: ① DMEM / F12 culture medium; ② DMEM / F12 culture medium containing free DFO; ③ DMEM / F12 culture medium containing GelMA-β-CD; ④ DMEM / F12 culture medium containing GelMA-β-CD-DFO. Among them, the concentrations of DFO, GelMA-β-CD and GelMA-β-CD-DFO were calculated based on the DFO content, and the concentration was 120 μM. At 0, 6h, 12h and 24h after incubation, the cells were photographed with an upright microscope to record the cell migration in the scratch area. The scratch area was measured using Image J software, and the scratch healing rate was calculated according to Formula 2.

[0105] Cell migration results Fig. 9 As shown in B and C: The migration rate of hUVECs in the GelMA-β-CD-DFO group to the scratch area was faster than that in the DFO and GelMA-β-CD groups, and the ability to promote cell migration was stronger ( Fig. 9 B); at 24h, the cell scratch healing rates of the GelMA-β-CD-DFO group were 1.45 times and 1.50 times those of the DFO and GelMA-β-CD groups, respectively ( Fig. 9 C in the figure). This is mainly because GelMA-β-CD-DFO reduces the toxicity of DFO to cells, and the three-dimensional network structure formed by GelMA-β-CD-DFO and GelMA-β-CD polymer materials can provide adhesion sites for cell proliferation and promote cell migration to a certain extent.

[0106] 3. In vitro angiogenesis assay

[0107] hUVECs were resuspended in DMEM / F12, DMEM / F12 containing free DFO, DMEM / F12 containing GelMA-β-CD, and DMEM / F12 containing GelMA-β-CD-DFO without serum. The concentrations of DFO, GelMA-β-CD, and GelMA-β-CD-DFO were all calculated based on the DFO content, which was 120 μM. 4 hUVECs were incubated in a constant temperature incubator at 37°C and 5% CO2 for 4 hours, and then the formation of tubules was observed using an inverted microscope. Fig.10 As shown in the figure, compared with the control group, DFO group, and GelMA-β-CD group, hUVECs treated with GelMA-β-CD-DFO formed a complete network structure on the matrix gel after 4 hours, and the number and total length of tubular structure connections increased significantly. These results indicate that GelMA-β-CD-DFO has a good angiogenic effect, which is beneficial to reversing the microcirculatory disorder around the hair follicles and providing sufficient nutrition for hair follicle regeneration.

[0108] Example 8 In vivo pharmacodynamic evaluation of microneedle patch

[0109] 1. Establishment of mouse model of androgenic alopecia

[0110] Male C57BL / 6 mice (18 g to 22 g) aged 6 to 8 weeks were used as experimental animals and were adaptively raised for one week. The AGA model was induced by subcutaneous injection of testosterone solution on the back. Except for the control group that did not receive any treatment, 0.1 mL of 5 mg / mL testosterone solution was subcutaneously injected into the back skin of each mouse every day for 28 consecutive days.

[0111] 2. Pretreatment before administration

[0112] After the AGA model was successfully established, 7% chloral hydrate was injected intraperitoneally for anesthesia and ear tags were made. The hair of 2 cm × 3 cm on the back of all mice was shaved, and the long hair on the back was shaved with an electric pet shaver, and then the remaining hair roots were gently removed with depilatory cream, and then the depilatory cream was gently wiped off with a wet towel.

[0113] 3. Experimental Grouping and Drug Administration

[0114] Healthy mice were used as the positive control group, and AGA model mice were randomly divided into 7 groups, with 6 mice in each group. The specific experimental groups are as follows: (1) positive control group (Control); (2) AGA model group (Model); (3) blank rapid separation microneedle patch group (Blank-RSMN); (4) FIN single rapid separation microneedle patch group (FIN-RSMN); (5) DFO single rapid separation microneedle patch group (DFO-RSMN); (6) FIN and DFO co-loaded rapid separation microneedle patch group (FIN-DFO-RSMN); (7) minoxidil group (Minoxidil). The mice in the Control group and the Model group were not given any medication and were raised normally. The mice in the microneedle patch treatment group were given medication once every three days, and the mice in the minoxidil group were given medication once a day.

[0115] 4. Experimental results

[0116] (1) Evaluation of hair regeneration and hair morphology in mice

[0117] The change in mouse skin color can reflect the growth cycle of hair follicles: pink indicates that the hair follicles have entered the resting phase, and black indicates that the hair follicles have entered the growth phase. During the entire treatment process, the changes in the skin color and hair growth of the mice's backs were observed and recorded every two days. After the experiment was terminated, Image J software was used to quantitatively analyze the back hair coverage of each group of mice, and the back hair of the mice was collected and the hair morphology was observed using a scanning electron microscope.

[0118] Hair growth in mice Fig.11As shown in A: The back skin of the untreated Control group mice was light black, indicating that the hair follicles were mainly in the growth phase, and melanin was deposited in the hair follicles; after continuous subcutaneous injection of testosterone for one month, the back skin of the mice was light pink, indicating that the hair follicles of the mice entered the resting phase, the hair follicles atrophied, and the model was successfully established. The Model group mice did not show obvious melanin deposition on the back skin within 7 days, and until the 16th day, their back hair had not grown completely, and 74.3% of the area grew shorter vellus hair. Compared with the untreated model group mice, each modeling mouse group showed different degrees of melanin deposition on the 7th day after being treated with different microneedle preparations or minoxidil; on the 10th day, the melanin deposition on the back skin of the mice increased significantly, indicating that most of the hair follicles were in the growth phase at this time, and the hair began to grow rapidly; on the 13th day, a relatively dense hair area appeared on the back of the mice, and the hair growth was obvious. Image J software was used to calculate the hair coverage rate of each group of mice, and the order was Control (100%) > FIN-DFO-RSMN (99.9%) > FIN-RSMN (96.5%) > DFO-RSMN (93.06%) > Minoxidil (86.11%) > Blank-RSMN (79.7%) > Model (66.95%).

[0119] The TEM images of hair were taken using a scanning electron microscope to observe the hair morphology. Fig.11 As shown in B, compared with the Control group, the hair scales in the Model group were incomplete and sparse, and the diameter was significantly reduced. The hair morphology of the other drug-treated groups restored the hair structure to a certain extent and was more complete. Among them, the hair scales in the FIN-DFO-RSMN group were complete and dense, and the diameter was significantly increased, which was almost indistinguishable from the Control group, indicating that FIN combined with DFO had the best effect in treating AGA.

[0120] (2) Melanin deposition in mouse hair and hair follicle growth cycle

[0121] H&E staining was performed on the back skin of mice to calculate the melanin deposition and hair follicle growth cycle of the skin. Hair growth is accompanied by changes in melanin deposition and hair follicle growth cycle. This is a complex physiological process, which mainly includes three stages: growth phase, regression phase and resting phase. Under normal physiological conditions, hair follicles are mainly maintained in the growth phase, and melanin is deposited at the root of the hair follicle to ensure normal hair growth. Under pathological conditions, hair follicles tend to gradually transition to the regression phase and eventually enter the resting phase, with hair follicles atrophying and hair falling off.

[0122] The results are as follows Fig.12As shown: Compared with the healthy mice group, the melanin deposition rate and the proportion of hair follicles in the growth phase of AGA model mice were significantly reduced; after drug treatment, the melanin deposition rate and the proportion of hair follicles in the growth phase of AGA mice increased to a certain extent, and the proportion of hair follicles in the growth phase was FIN-DFO-RSMN (88.9%) > Minoxidil (80.6%) > FIN-RSMN (77.2%) > Control (73.2%) > DFO-RSMN (69.5%) > Model (26.3%). Among them, the FIN-DFO-RSMN group and the FIN-RSMN group were the most obvious.

[0123] These results further indicate that the rapidly detached microneedle patch co-loaded with FIN and DFO can significantly promote the transition of hair follicles from the resting phase to the growth phase in AGA mice, and its effect in promoting hair regeneration is better than that of single drug treatment and commercially available minoxidil.

[0124] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0125] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A quick-detachable microneedle patch, characterized in that: The microneedle patch includes a needle tip prepared from a needle tip liquid and a separation backing prepared from a separation base liquid; The needle tip liquid is composed of a drug-containing inclusion compound solution, a photoinitiator and a hypotonic agent in a volume mass ratio of 1 mL: 0.005 g to 0.01 g: 0.05 g to 0.1 g; the concentration of the drug-containing inclusion compound in the drug-containing inclusion compound solution is 0.25 g / mL to 0.75 g / mL; the drug-containing inclusion compound is prepared by first covalently linking a β-cyclodextrin conjugated polymer material to a pro-angiogenic drug, and then encapsulating a 5α-reductase inhibitor; in the drug-containing inclusion compound, the molar concentration ratio of the 5α-reductase inhibitor to the pro-angiogenic drug is 1:3 to 9; the pro-angiogenic drug is deferoxamine or deferiprone; the 5α-reductase inhibitor is finasteride or dutasteride; The separation base liquid is composed of a polymer material, a gas initiator, and a solvent; the gas initiator is composed of an acid source and an alkali source; the mass volume ratio of the polymer material, the acid source, the alkali source, and the solvent is 0.8g-1.2g: 0.1g-0.15g: 0.12g-0.18g: 3mL-4mL.

2. The rapid-detachable microneedle patch according to claim 1, characterized in that: The hypotonic agent is one or more of maltose, sucrose and lactose.

3. The rapid-detachable microneedle patch according to claim 1, characterized in that: The photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and / or phenyl-2,4,6-trimethylbenzoyl lithium phosphonate.

4. The rapid-detachable microneedle patch according to claim 1, characterized in that: The polymer material in the needle tip liquid is one or more of type A gelatin, type B gelatin, methacrylated gelatin, hyaluronic acid, methacrylated hyaluronic acid, silk fibroin, and methacrylated silk fibroin.

5. The rapid-detachable microneedle patch according to claim 1, characterized in that: The β-cyclodextrin conjugated polymer material in the needle tip liquid is β-cyclodextrin conjugated methacrylate acylated gelatin; and / or, The polymer material in the separation base liquid is one or more of polyvinyl pyrrolidone K30, polyvinyl pyrrolidone K45 and polyvinyl pyrrolidone K90.

6. The rapid-detachable microneedle patch according to claim 1, characterized in that: The needle tip liquid consists of a drug-containing inclusion complex solution, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and maltose. The drug-containing inclusion complex in the drug-containing inclusion complex solution is prepared by first covalently linking β-cyclodextrin conjugated methacrylic acid acylated gelatin to deferoxamine, and then including finasteride. The molar concentration ratio of finasteride to deferoxamine is 1:

6.

7. The rapid-detachable microneedle patch according to claim 6, characterized in that: The volume mass ratio of the drug-containing inclusion compound solution, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and maltose is 1 mL: 0.006 g to 0.008 g: 0.06 g to 0.08 g.

8. The rapid-detachable microneedle patch according to any one of claims 1 to 7, characterized in that: The acid source is citric acid or tartaric acid; the alkali source is one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, and magnesium carbonate; and the solvent is anhydrous ethanol.

9. The rapid-detachable microneedle patch according to claim 8, characterized in that: The separation substrate liquid is prepared from polyvinyl pyrrolidone K90, citric acid, sodium bicarbonate and anhydrous ethanol in a mass volume ratio of 0.8g-1.2g:0.126g-0.130g:0.14g-0.18g:3.2mL.

10. The method for preparing the rapid separation microneedle patch according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) covalently linking a β-cyclodextrin conjugated polymer material to an angiogenic drug, and then encapsulating a 5α-reductase inhibitor to obtain a drug-containing inclusion compound, freeze-drying the compound, and dissolving the compound in ultrapure water to obtain a drug-containing inclusion compound solution; heating the drug-containing inclusion compound solution, a photoinitiator, and a hypotonic agent to 45° C. to 55° C. in a dark environment, and mixing the mixture thoroughly until the mixture is completely dissolved to obtain a needle tip solution; (2) adding the polymer material, the acid source and the alkali source into the solvent, sealing the container and placing it at room temperature until the polymer is fully swollen to form a viscous solution, thereby obtaining a separation base liquid; (3) adding the needle tip liquid of step (1) to the surface of the microneedle female mold and flattening it, and centrifuging it at room temperature at 3500 rpm to 4500 rpm for 4 min to 6 min; (4) scraping off the excess needle tip liquid on the surface of the microneedle negative mold with a scraper, irradiating with ultraviolet light for 30s to 45s, and centrifuging at 3500rpm to 4500rpm for 25min to 35min at room temperature; (5) adding the separation base liquid of step (2) to the surface of the microneedle negative mold, spreading it flat until it covers the micropore surface of the microneedle, and centrifuging it at 4° C. to 15° C., 3500 rpm to 4500 rpm, for 4 min to 6 min; (6) Dry for more than 2 days to obtain a rapidly detachable microneedle patch.

Images