Microneedle containing curcumin-zinc MOF and application of microneedle in hair growth promotion

Abstract

The invention relates to an application of a curcumin-zinc organic framework compound as an active ingredient in preparation of a medical instrument for treating alopecia. Through verification, curcumin and zinc ions are prepared into an MOF structure (ZnMOFs), and the MOF structure (ZnMOFs) is encapsulated by gamma-polyglutamic acid (gamma-PGA) to prepare the soluble microneedle patch (ZnMOF-MNs) which is used for promoting hair growth under various adverse conditions. In-vitro studies find that ZnMOFs can protect DPCs from apoptosis caused by zinc deficiency and oxidative stress.

Description

[0001] This application is a divisional application based on the original application with application date of April 2, 2023, application number 202310342919.8, and invention name “Microneedles containing curcumin-zinc MOF and their application in promoting hair growth”. Technical Field

[0002] The invention relates to a medical device made of biomaterials, in particular to a microneedle with curcumin-zinc organic skeleton compound as active ingredient and supplemented with polymer, which is used for promoting hair growth. Background Art

[0003] Hair loss is becoming more and more common in modern life, and it has caused many adverse social and psychological effects. It is reported that up to 85% of men and 40% of women are troubled by hair loss. Hair follicles undergo cyclical changes, including the involuting phase, the resting phase, and the growing phase to produce new hair. Local microenvironmental dysregulation can have a negative impact on the hair cycle, which is an important cause of various types of hair loss. Oxidative stress, which is prevalent in the pathological microenvironment of hair loss, is caused by excessive accumulation of reactive oxygen species (ROS). Nutritional status is another important factor. It is reported that insufficient skin perfusion may reduce the local density of cytokines and nutrients and hinder the transition from the resting phase to the growing phase. According to studies, in clinical hair loss caused by various reasons, the severity and chronicity of the course of the disease are closely related to local or systemic zinc deficiency in the scalp, including diffuse hair loss, telogen effluvium, alopecia areata, etc. In addition, hormones play an important role in the mechanism of androgenic alopecia (AA). They can induce hair follicles from the growth phase to the regression phase by downregulating the Wnt / β-Catenin pathway, promote the apoptosis of dermal papilla cells (DPC) through the prostaglandin D2 pathway, and stimulate local ROS accumulation in the scalp.

[0004] EP0684764A1 discloses an antisense oligomer for the treatment of androgenic alopecia.

[0005] KR1020160065886 discloses a composition for preventing hair loss and promoting hair growth, comprising thionin suberate as an active ingredient which can provide an excellent hair loss prevention effect by inhibiting the entire small molecule action associated with improving hair growth promotion through the androgen receptor signaling pathway (androgen receptor signaling pathway).

[0006] CN104367986A discloses a combined formula for treating and preventing hair loss, which is composed of the following components by mass: 1-10 parts of ginger, 0.5-2 parts of musk, 0.5-2 parts of bezoar, 0.5-3 parts of ophiopogon, 0.5-2 parts of sandalwood, 0.5-5 parts of astragalus, 0.5-5 parts of licorice extract, 0.5-5 parts of moutan bark, 0.5-10 parts of angelica, 0.5-10 parts of salvia miltiorrhiza, 0.5-10 parts of schisandra chinensis, 5-10 parts of polygonum multiflorum, 0.5-10 parts of cypress leaves, 0.5-10 parts of rehmannia glutinosa, 0.5-2 parts of mint, 0.5-10 parts of clematis, 0.5-10 parts of saffron, 0.5-2 parts of vitamin E, 0.5-1 parts of cimetidine, 0.1-2 parts of capsicum tincture, and 0.1-2 parts of skin penetration enhancer. This combination formula has no obvious irritation and can also be used to treat eyebrow and eyelash loss and sparseness. It has been used in 1,670 clinical cases, of which 1,515 were followed up, with an effective rate of 95.7%, and has no toxic side effects, and can be used continuously.

[0007] CN113521231A discloses a medicine for preventing hair loss, reducing scalp inflammation and treating prostatitis. The medicine is prepared from the following raw materials in parts by weight: 200-2000 parts of virgin pumpkin seed oil, 100-800 parts of saw palmetto oil, 50-500 parts of tocotrienols, 100-800 parts of turmeric, 0.1-10 parts of carotenoid vitamins, 0.005-0.1 parts of vitamin D3, 0.1-5 parts of biotin and 1-100 parts of zinc. The medicine of the invention has no toxic side effects, can prevent hair loss symptoms, reduce scalp inflammation and treat prostatitis of mammals.

[0008] CN115252649A discloses a use of a thermophilic bacteria fermentation product in preventing hair loss and / or promoting hair growth, a composition and a preparation thereof. The thermophilic bacteria fermentation product penetrates deeply into the hair follicles to exert its effect, replenishes ATP for the hair follicles, helps activate various metabolic activities in the hair follicles, has significant effects in regulating the key signal pathway Wnt-β catenin signal pathway of hair follicle development and promoting hair regeneration, and can especially prevent the blockage of key signal pathways in the process of hair follicle growth and the decrease in the activity of hair follicle cells caused by androgen stimulation. The product can be used as a product or additive in scalp care products and / or scalp washing products to promote hair growth. Summary of the invention

[0009] One object of the present invention is to provide an organic skeleton compound containing curcumin-zinc, which is used as an active ingredient for promoting hair growth.

[0010] Another object of the present invention is to provide a microneedle containing an organic skeleton compound of curcumin-zinc, which is used to deliver the organic skeleton compound containing curcumin-zinc as an active ingredient to skin tissue, thereby facilitating hair growth.

[0011] Another object of the present invention is to provide an application of an organic skeleton compound containing curcumin-zinc in the preparation of a medical device for treating hair loss.

[0012] Another object of the present invention is to provide an application of an organic skeleton compound containing curcumin-zinc in the preparation of a medical device for treating androgenic alopecia.

[0013] Curcumin is extracted from the plant turmeric (Curcuma Longa L.), and as a herbal ingredient, it has been widely used to treat various diseases, including skin diseases, neurological diseases and cancer. As a natural ROS scavenger, curcumin has been shown to have antioxidant and anti-inflammatory effects in skin aging, dermatitis and wound healing. In addition, curcumin analogs have also been reported to have anti-androgenic effects by inhibiting androgen receptor activation and inhibiting the activity of 5-α reductase. In addition, curcumin has strong biological tolerance, with a maximum intake of 3 mg / kg or 12 g / day, and good biosafety, making it a drug with great potential. However, the bioavailability of curcumin is very low, and its low water solubility, low stability and obvious first-pass elimination limit the further application of curcumin. Therefore, in order to give full play to the biological effects of curcumin, the present invention provides a topical transdermal drug delivery system with a protective effect.

[0014] Zinc is present in many enzymes and protein domains, is essential for growth and development, and plays a key role in the regulation of apoptosis and inflammation. Hair is the third highest zinc-rich tissue in the body, and zinc deficiency has a wide range of negative effects on keratinocytes, fibroblasts, microvessels, and the overall health of hair. Clinical studies have shown that zinc deficiency is common in patients with different types of hair loss, and appropriate zinc supplementation is strongly recommended for patients with hair loss problems. However, the biosafe concentration range of zinc is relatively narrow. Traditional oral supplementation methods may cause chronic zinc poisoning, as well as the associated risk of copper deficiency, and should be used with caution. Therefore, local zinc supplementation using a controlled drug release system would be safer for patients with hair loss.

[0015] Metal-organic framework compounds (MOFs) are porous crystalline materials formed by self-assembly of metal ions and organic ligands under certain conditions, with the characteristics of adjustable composition and strong biocompatibility. Previous studies, namely the application of magnesium-containing MOFs (Mg-MOFs) and copper-containing MOFs (Cu-MOFs) in diabetic wound healing models, have proved that MOF structures are very reliable in controlling metal release and maintaining the biological activity of organic substances in complex microenvironments.

[0016] The curcumin-zinc organic skeleton compound provided by the invention is used as an active ingredient and has the function of effectively promoting hair growth.

[0017] An embodiment of a curcumin-zinc organic framework compound, wherein curcumin and zinc ions are self-assembled to form elliptical nanoparticles in an alkaline water environment, wherein the long radius of the particles is 1.5 μm ± 0.2 μm, the short radius is 1.0 μm ± 0.2 μm, and carbon (C), oxygen (O) and zinc (Zn) are uniformly distributed in ZnMOFs.

[0018] Another embodiment of the curcumin-zinc organic framework compound has an overall crystal structure.

[0019] In another embodiment of the curcumin-zinc organic framework compound, the weight ratio of curcumin to zinc ion is 6.21-6.55.

[0020] Exemplarily, the ZnMOF of the present invention is prepared as follows:

[0021] 100 mg ZnSO4·7H2O was dissolved in 10 ml double distilled water, and 120 mg curcumin was first dissolved in 1 ml DMSO and then 9 ml double distilled water was added. The two were mixed thoroughly and the pH value was adjusted to 12. Then, the mixture was placed in a muffle furnace and heated at 120°C for 24 hours. Centrifuged at 12000 rpm at 4°C for 15 minutes, and the orange-yellow powder, namely ZnMOF, was collected.

[0022] Soluble polymer microneedle (MN) patches have been reported as a transdermal drug delivery system, and industrialized products are already available. Microneedles with nanoparticle-encapsulated tips can penetrate the epidermis and deliver drugs to a certain depth under the skin. In addition, studies have shown that using a needle tip with a height of less than 750 μm to penetrate the skin will hardly cause bleeding or pain. Therefore, for the treatment of hair loss, microneedle drug delivery is an effective, safe and patient-friendly transdermal drug delivery method. The microneedle of the present invention has a needle tip of 10×10 array, 260 μm×260 μm×500 μm (length×width×height).

[0023] Exemplarily, the ZnMOF-MN of the present invention is prepared as follows:

[0024] The prepared ZnMOF powder was dissolved in DMSO (for example, the weight-volume ratio of ZnMOF to DMSO was 20:1), and then diluted with water to a concentration of 1 mg / mL. 600 μL of the diluted ZnMOF solution was added to 350 mg of γ-PGA, dispersed at room temperature (for example, ultrasonically shaken for 30 minutes), and then centrifuged (for example, at 4°C, 12,000 rpm, 15 minutes) to obtain a ZnMOF gel.

[0025] The ZnMOF gel was completely filled into the microcavity of the needle tip, and the mold was placed at 40°C to dry overnight to obtain ZnMOF-MN.

[0026] Another specific implementation of ZnMOF-MN uses γ-PGA with a molecular weight distribution of Mw=1000-15000 KDa.

[0027] It has been verified that curcumin and zinc ions are made into MOF structures (ZnMOFs) and encapsulated with γ-polyglutamic acid (γ-PGA) to prepare soluble microneedle patches (ZnMOF-MNs) for promoting hair growth under various adverse conditions. In vitro studies have shown that ZnMOFs can protect dermal papilla cells (DPCs) from apoptosis caused by zinc deficiency and oxidative stress. In addition, ZnMOFs can promote the expression of genes related to DPCs proliferation (PCNA) and Wnt / β-catenin signaling activation (CCND1, AMER3, LEF1, and DDK1), and downregulate the levels of genes related to apoptosis (Caspase-3, Caspase-9) and androgen metabolism-related genes (HSD17B2, SRD5A1).

[0028] ZnMOF-MNs were tested in the wound healing model and AA model of C57 / BL6 mice, two representative pathological conditions that cause hair growth obstruction. The process of wound skin repair and hair regeneration in mice treated with ZnMOF-MNs was significantly accelerated by the observation of shortened scab shedding period and increased number of hair follicles. In the AA model, the hair growth rate of mice treated with ZnMOF-MNs increased significantly. In addition, in both models, an increase in the number of microvessels (CD31+) and an increase in the proportion of actively proliferating DPCs (PCNA+) were observed in the skin tissue treated with ZnMOF-MNs, suggesting that ZnMOF-MNs successfully improved the adverse microenvironment and promoted hair regeneration. Therefore, ZnMOF-MNs have been shown to effectively promote hair growth in hair loss problems caused by a variety of conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Synthesis and characterization diagram of ZnMOF; wherein, a is a schematic diagram of the synthesis of ZnMOF microspheres, b is a FE-SEM image of ZnMOF, c is a SEM image of ZnMOF, d is a C element spectrum of ZnMOF, e is a Zn element spectrum of ZnMOF, f is an O element spectrum of ZnMOF, g is an XRD spectrum of ZnMOF powder, h is an absorption spectrum of ZnMOF, i is the absorption intensity of ZnMOF solutions of different concentrations at 430nm, and j is a cell viability result diagram of DPCs after treatment with ZnMOF solutions of different concentrations (0, 2.5, 5, 7.5 and 10 μg / mL) for 48 hours;

[0030] Figure 2The synthesis and characterization of ZnMOF-MN; a is a schematic diagram of the synthesis process of ZnMOF-MNs, and b is a photo of the microneedle patch (from left to right: BMN, 0.1 mg / mL ZnMOF-MN and 1 mg / mL ZnMOF-MN), c is the SEM image of ZnMOF-MN (top view), d is the recovery process of ZnMOF-MN after puncture in the back skin of C57 / BL6 mice, e is the bright field (BF) and fluorescence (FL) microscope images of the skin of untreated C57 / BL6 mice (Ctrl) and the skin after puncture with BMN or ZnMOF-MN (ZnMOF shows green fluorescence signal), f is the release curve of ZnMOF released by ZnMOF-MN in PBS environment within 24 hours, g is the SEM image of ZnMOF-MN needle tip at each time point under 75% humidity and room temperature conditions. The average size of the MN needle tip in the figure is: base diameter 260.38±3.02μm, height 466.23±11.72μm;

[0031] Figure 3Figure 2 is the experimental results of the in vitro antioxidant, anti-apoptotic and anti-androgen abilities of ZnMOF and ZnMOF-MN; wherein, a is a DPCs cell viability detection diagram after pretreatment with 5μg / mL ZnMOF solution, BMN extract or ZnMOF-MN extract (~6.46μg / mL ZnMOF) for 24 hours and then incubated with 3mM H2O2 for 30 minutes, * represents p < 0.05 compared with the blank control group, b is a DPCs cell viability detection diagram after co-treatment of cells with 5μg / mL ZnMOF solution, BMN extract or ZnMOF-MN extract (~6.46μg / mL ZnMOF) and 5μM TPEN for 24 hours, c is a bar chart of the dead cell counts in each group in Figure d, and d is a 5μg / mL Figure 5. Dead cell staining of cells treated with ZnMOF solution, BMN extract or ZnMOF-MN extract (~6.46μg / mL ZnMOF) and 5μM TPEN for 24 hours. e is a representative picture of cell apoptosis detected by flow cytometry. f is a picture of cell apoptosis rate detected by flow cytometry. g is a picture of 100μM DHT and 20μM curcumin, 2.5μg / mL ZnMOF, BMN extract or ZnMOF-MN extract (~3.23μg / mL The transcriptomic expression levels of DDK1, CCND1, AMER3, LEF1, HSD17B2, SRD5A1, Caspase-3 and Caspase-9 genes in DPCs were detected by RT-qPCR after treatment with ZnMOF for 24 h. The RQ values ​​(relative quantitative value) of DPCs in the DHT group and BMN extract group were compared with those in the blank control group (treated with 0.1% dimethyl sulfoxide only). The RQ values ​​of DPCs treated with 20 μM curcumin, 2.5 μg / mL ZnMOF and ZnMOF-MN extract were compared with those in the DHT group, n=3, # represents p<0.05 compared with the blank control group, * represents p<0.05 compared with the DHT group;

[0032] Figure 4Figure 2 is a graph showing the results of hair growth evaluation and histological research in the wound healing model of C57BL / 6 mice; wherein, a is a representative photographic image of wound skin treated differently on day 0, day 3, day 5, day 7, day 9, day 11, day 13, day 15 and day 17, and the blank control group (Ctrl) is untreated mice; b is a statistical graph of the area rate of the wound surface in each group on day 0, day 3, day 5, day 7, day 9 and day 11, and c is a statistical graph of the area rate of the wound surface in each group on day 17. (Hair has been re-covered) H&E color (upper row), anti-CD31 immunohistochemical staining (middle row) and anti-Ki67 immunohistochemical staining (lower row) results of each group, d is the quantitative analysis result of the total number of hair follicles per visual field of each group after H&E, e is the quantitative analysis result of the capillary density per visual field of each group after anti-CD31 staining, f is the quantitative analysis result of the proportion of actively proliferating cells per visual field of each group after anti-Ki67 staining, n=3, *p<0.05, NS indicates no significance;

[0033] Figure 5 Figure 2 is the establishment of the mouse androgenic alopecia model and the results of histological research, wherein a is a schematic diagram of the process of establishing the AA model in C57 / BL6 mice, b is the results of anti-CD31 immunofluorescence staining and anti-Ki67 immunofluorescence staining of skin specimens in each group, c is the quantitative analysis result of the capillary density in each field of view in each group after anti-CD31 immunofluorescence staining, d is the quantitative analysis result of the proportion of actively proliferating cells in each field of view in each group after anti-Ki67 immunofluorescence staining, n=3, *p<0.05;

[0034] Figure 6 The figure is a result diagram of hair growth evaluation in the androgenic alopecia model of C57BL / 6 mice; wherein, a is a result diagram of hair loss area and hair growth in each group on day 1, day 4, day 7, day 10, day 13, day 16 and day 18, b is a figure of the overall situation of mice in different groups on day 18, and c is a figure of analysis of hair growth rate (coverage) in each group on day 7, day 10, day 13, day 16 and day 18, n=3, *p<0.05, NS indicates no significance;

[0035] Figure 7 These are images of the wound area and hair growth in each group of the mouse wound healing model on day 0, day 3, day 5, day 7, day 9, day 11, day 13, day 15 and day 17. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0037] The sources of the main materials used in the following embodiments of the present invention are as follows:

[0038] γ-PGA (MW = 1000-15000) was purchased from Cetex Biotechnology Co., Ltd. (China).

[0039] Curcumin (B20614, HPLC ≥ 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China).

[0040] DHT and ZnSO4·7H2O were purchased from Beijing Solebao Biotechnology Co., Ltd. (China).

[0041] The microneedle mold was purchased from Taizhou Microchip Technology Co., Ltd. (China), and the needle tip was a 10 × 10 array, 260 μm × 260 μm × 500 μm (length × width × height) MN needle tip.

[0042] Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) and fetal bovine serum (FBS) were purchased from Gibco (UK).

[0043] The various test methods used in the following examples of the present invention are specifically described as follows:

[0044] 1) Cell culture

[0045] Primary dermal papilla cells (DPCs) from C57 / BL6 mice were purchased from iCell Bioscience Co., Ltd. (China). The cells were cultured in DMEM / F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin and cultured in an incubator at 37°C with 5% CO2. The cells used in this study were all at passage 3.

[0046] 2) Preparation of ZnMOF and ZnMOF gel

[0047] 100 mg ZnSO4·7H2O was dissolved in 10 ml double distilled water, and 120 mg curcumin was first dissolved in 1 ml DMSO and then 9 ml double distilled water was added. The two were mixed thoroughly and the pH value was adjusted to 12 with 10 mol / L KOH. The mixture was heated at 120 °C in a muffle furnace for 24 hours. Centrifuged at 12000 rpm at 4 °C for 15 minutes, and the orange-yellow powder, i.e. ZnMOF, was collected and washed twice with 95% ethanol and double distilled water respectively for later use.

[0048] When making ZnMOF gel, first dissolve 20 mg ZnMOF in 1 ml DMSO and then dilute to 1 mg / mL with deionized water. Add 600 μL of the diluted ZnMOF solution to 350 mg γ-PGA. Ultrasonicate at room temperature for 30 minutes to fully disperse. Centrifuge at 12000 rpm for 15 minutes at 4°C to remove bubbles. When preparing blank microneedles (BMN), replace 600 μL of the diluted ZnMOF solution with an equal amount of deionized water.

[0049] 3) Preparation of microneedles

[0050] The ZnMOF hydrogel was completely filled into the microcavity of the needle tip and then centrifuged for 5 minutes (3000 rpm, room temperature) to prepare ZnMOF-MN. The excess gel around was wiped off with a cotton swab. The filled mold was placed in an oven at 40°C for overnight drying before demolding.

[0051] 4) Microneedle hygroscopicity test

[0052] The dried MN patch was placed in a sealed jar at a humidity of 75%. Every 5 minutes, the tip morphology was recorded by taking photos using a scanning electron microscope (JCM-5000, NeoScope, USA).

[0053] 5) ZnMOF release curve

[0054] Two pieces of ZnMOF-MN were immersed in 2 mL of PBS, and the absorbance of the supernatant (extract) at 430 nm was measured by a spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 8 h, 18 h, and 24 h. Then, using the calibration curve, the corresponding concentration of ZnMOF in the supernatant was calculated based on the absorbance. The release rate was calculated according to the following equation:

[0055]

[0056] c t represents the ZnMOF concentration detected at time t, and m0 represents the total amount of ZnMOF in the microneedle, and its value is calculated by the following equation:

[0057]

[0058] m represents the total weight of two sheets of dried ZnMOF-MN.

[0059] When preparing the ZnMOF-MN extract required for in vitro experiments, 2 pieces of ZnMOF-MN were immersed in 40ml DMEM / F-12 culture medium. According to the release curve results, 57.86% was released in 2 hours. The mass of two pieces of ZnMOF-MN was 257.8±7.59mg. Therefore, the ZnMOF concentration in the extract after immersion for 2 hours was about 6.46μg / mL. When preparing the blank microneedle extract, blank microneedles were used for immersion, and the rest remained unchanged.

[0060] 6) Skin puncture test

[0061] C57 / B16 mice were anesthetized with isoflurane and their back skin was depilated. Then, ZnMOF-MN was quickly and vertically pressed into the exposed skin and held for 1 minute. After the patch was removed, skin photos were recorded every 5 minutes until the holes disappeared. Then, the skin was peeled off and vertically embedded in OCT mounting medium. 8 μm thick frozen sections were prepared and placed on slides for observation and recording using a fluorescence microscope (Leica DM 2500, Germany).

[0062] 7) In vitro cytotoxicity assay

[0063] Cell viability was detected using a CCK-8 kit (Dojindo Laboratories, Mashiki, Japan) to reflect cytotoxicity. Every 3500 DPCs were inoculated into one well of a 96-well plate, and after overnight attachment, the original culture medium was replaced with a ZnMOF culture medium containing no ZnMOF or ZnMOF at different concentrations (0μg / mL, 2.5μg / mL, 5μg / mL, 7.5μg / mL, and 10μg / mL) and cultured at 37°C for 48 hours. During the test, 100μl of DMEM / F-12 culture medium containing 10% CCK-8 was added to each well. After incubation at 37°C for two hours in the dark, the absorbance at 450nm was detected on a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), and the results were statistically analyzed and plotted. DPCs cultured in DMEM / F-12 containing 0.1% DMSO were blank controls.

[0064] 8) In vitro antioxidant test

[0065] The cell viability of DPCs was determined using a CCK-8 kit in the same manner as described previously. For the experimental groups, cells were treated with 5 μg / mL ZnMOF solution, BMN extract, or ZnMOF-MN extract (containing approximately 6.46 μg / mL ZnMOF) for 24 hours, and then 3 mM H2O2 was added and incubated for 30 minutes before detecting cell activity. DPCs cultured in DMEM / F-12 containing 0.1% DMSO were blank controls.

[0066] 9) In vitro zinc deficiency model experiment

[0067] The cell viability of DPCs was determined using a CCK-8 kit in the same manner as described previously. Zinc in the culture medium was chelated with 5 μM TPEN (N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine) to simulate a zinc-deficient microenvironment. For the experimental group, 5 μg mL -1 Cell viability was detected after treating cells with ZnMOF solution, BMN extract or ZnMOF-MN extract (containing about 6.46 μg / mL ZnMOF) for 24 hours. DPCs cultured in DMEM / F-12 containing 0.1% DMSO served as the blank control group.

[0068] 10) In vitro dead cell staining

[0069] Propidium iodide (PI, Dojindo Laboratories, Mashiki, Japan) was used for dead cell staining. Every 20,000 DPCs were inoculated into one well of a 24-well plate, and the original culture medium was replaced with 5 μM TPEN culture medium after overnight attachment. For the experimental group, cells were treated with 5 μg / mL ZnMOF solution, BMN extract or ZnMOF-MN extract (containing approximately 6.46 μg / mL ZnMOF) for 24 hours. DPCs cultured in DMEM / F-12 containing 0.1% DMSO were the blank control group. Dead cells stained with red PI were photographed by fluorescence microscopy (Leica DM 2500, Germany). The number of dead cells per field of view under high magnification was automatically counted by Image J 1.53c (National Institutes of Health, USA).

[0070] 11) In vitro apoptosis assay

[0071] Cell apoptosis was detected using Annexin V-FITC assay (556547, BD, USA). DPCs were incubated with 5 μM TPEN for 24 h, and then 5 μg / mL ZnMOF solution, BMN extract, or ZnMOF-MN extract was added to the corresponding groups. DPCs cultured in DMEM / F-12 (0.1% DMSO) without TPEN incubation were set as the control group. After 24 h of treatment, the cells were collected with trypsin (0.25%) and washed twice with PBS. Then 5 μL Annexin V-FITC, 5 μL PI, and 500 μL binding buffer were added to each group and gently mixed. After incubation in the dark for 15 minutes, the fluorescence signal of apoptotic cells was detected by flow cytometry (Beckman-Colter, USA). The sum of the cell proportions in the upper right corner (UR) and the lower right corner (LR) in the flow cytometry result graph is the total apoptosis rate.

[0072] 12) RNA purification and RT-qPCR

[0073] 6-well plates were inoculated with 100,000 cells per well. DPCs cultured in DMEM / F-12 containing 0.1% DMSO were used as the control group. For the experimental group, 100 μM DHT was added to the cells, and then 20 μM curcumin, 2.5 μg / mL ZnMOF solution, BMN extract or diluted ZnMOF-MN extract (containing approximately 3.23 μg / mL ZnMOF) were added for 24 hours. Then, DPCs were collected for total RNA extraction using an RNA purification kit (C10310, EZBioscience Co., Ltd., USA) as instructed. Reverse transcription was performed using 4×Ezscript RT Mix II ((B0004DP, EZBioscience) according to the instructions to synthesize complementary DNA (cDNA). RT-qPCR detection was performed using 2×STBR Green qPCR Mix (ROX2 Plus) (A0001-R2, EZBioscience) and an RT-qPCR instrument (Stratagene, La Jolla, CA, USA) according to the instructions. The housekeeping gene GAPDH was used as an internal reference. The detection was repeated three times. The mouse primer sequences used in RT-qPCR (Sangon Biotech (Shanghai) Co., Ltd.) are shown in Table 1.

[0074] Table 1

[0075]

[0076] 13) In vivo wound healing experiment

[0077] In this study, we used male C57BL / 6 mice (6-8 weeks) for wound healing model establishment. All animal research protocols were approved by the Institutional Animal Care and Experimental Committee of Shanghai Jiao Tong University School of Medicine. Fifteen mice were randomly divided into three groups (control group, BMN group, and ZnMOF-MN group). The mice were anesthetized for a short time using an air isoflurane anesthesia machine, and the back skin of each mouse (n=3) was depilated for surgical modeling to form a circular full-thickness skin wound with a diameter of about 9 mm (the inner diameter of the circular peeler was 10 mm). After modeling, mice in the BMN group and ZnMOF-MN group were treated with MN patch once on the same day. The mice in the control group did not receive other treatments. Each mouse was housed in a cage with sufficient water and food. The wound surface of each group of mice was photographed on days 0, 3, 5, 7, 9, 11, 13, 15, and 17 after wound treatment, and the wound area was calculated using ImageJ 1.53c software (National Institutes of Health, USA). The wound area rate was measured using the following formula:

[0078]

[0079] 14) Hair regrowth experiment

[0080] In this study, we used male C57BL / 6 mice (6-8 weeks) to establish an androgenic alopecia model. All animal research protocols were approved by the Institutional Animal Care and Experimental Committee of Shanghai Jiao Tong University School of Medicine. Twenty mice were randomly divided into four groups (control group, model group, BMN group, and ZnMOF-MN group). Mice in the model group, BMN group, and ZnMOF-MN group were subcutaneously injected with DHT (2 mg / kg), and mice in the control group were injected with an equal amount of normal saline once a day for two weeks. Afterwards, 4 cm on the back of the mice in the resting phase were gently shaved with an electric shaver. -2 The area of ​​​​(2cm×2cm) was shaved again with a depilatory cream (the shaving day was the 1st day). On the 1st, 4th, 7th, and 10th days, BMN or ZnMOF-MN was used on the depilatory area. The specific operation was to press the MN patch for 1 minute and then remove it, and use 4 patches continuously. The skin and hair growth of the area were photographed and recorded.

[0081] 15) Histological experiments and immunofluorescence staining

[0082] In the wound healing test, mice were killed on the 17th day (cervical dislocation method). With the wound as the center, a circular full-thickness skin with a diameter of 6 mm was cut as a specimen for subsequent histological experiments. The specimens were immersed in 4% paraformaldehyde for 24 hours, dehydrated and embedded in wax blocks, and then prepared into 5 μm thick sections for histological staining. Some sections were stained with hematoxylin and eosin (H&E) to show the morphology of hair follicles and to count the number of hair follicles. Some sections were used for immunohistochemical detection of CD31+ cells and Ki67+ cells. Specifically, the tissue sample sections were placed in rabbit monoclonal anti-CD31 antibody (ab182981, Abcam, 1:400) or rabbit monoclonal anti-Ki67 antibody (ab15580, Abcam (1:200)) and incubated overnight at 4°C. The sections were then incubated with HRP-labeled polymer anti-rabbit antibody at 37°C. The staining was performed using a 3,3′-diaminobenzidine substrate kit (Boster, Wuhan, China). ImageJ 1.53c software (National Institutes of Health, USA) was used to calculate the hair follicle density, capillary density, and Ki67+ cell ratio of wound samples.

[0083] Immunofluorescence staining was used to detect CD31+ cells and Ki67+ cells in the AA model. Mice were sacrificed by cervical dislocation on day 18, and a 1 cm -2 Full-thickness skin (1 cm × 1 cm) was used as specimens for immunofluorescence detection. The specimens were fixed by immersion in 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin blocks, and then cut into 5 μm thick sections for immunofluorescence staining. The tissue sample sections were cleared with 0.1% Triton X-100 (Sigma-Aldrich), blocked with 5% goat serum in PBS, and incubated with rabbit monoclonal anti-CD31 antibody (ab305267, Abcam, 1:50) or rabbit monoclonal anti-Ki67 antibody (ab15580, Abcam, 1:200) and incubated overnight at 4°C. Finally, the sections were washed with PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI).

[0084] ImageJ (National Institutes of Health) was used to calculate the hair follicle density, capillary density, and Ki67+ cell proportion of wound samples.

[0085] 16) In vitro cell migration assay

[0086] The scratch test was used to evaluate cell migration. When the DPCs adhered to the dish and reached more than 90% of the bottom area, a scratch was made on the monolayer with a 1000 μL pipette tip. For the experimental groups, cells were treated with 5 μg / mL ZnMOF, BMN extract, or ZnMOF-MN extract (containing approximately 6.46 μg / mL ZnMOF). The control group was treated with DPCs in culture medium containing only 0.1% DMSO. Images were collected at 0 h and 24 h after the scratch. The area of ​​the scratched area was automatically measured by Image J 1.53c (National Institutes of Health, USA).

[0087] Subsequently, 44 mice were randomly divided into different treatments (control group, Cur-MOF solution (20 μg / mL) group, γ-PGA / ε-PLL powder group, and Cur-MOF@γ-PGA / ε-PLL powder group). Mice were housed in individual cages with ample food and water. Images of the wound area were taken on days 0, 1, 3, 5, 7, 10, 12, and 14 after burn injury.

[0088] 17) Statistical analysis

[0089] All quantitative data are presented as mean ± standard deviation. Unpaired Student's t test was used for both groups. Statistical analysis was performed using SPSS (Statistical Package for Social Sciences) 26.0, and bilateral P < 0.05 was considered statistically significant. GraphPad Prism 8.0 was used for graphics. Image J software (NIH, Bethesda, MD, USA) was used for image analysis and statistics.

[0090] Example 1 Synthesis and characterization of ZnMOF

[0091] ZnMOF is an elliptical nanoparticle formed by the self-assembly of curcumin and zinc ions in an alkaline water environment. Figure 1 a). Field emission scanning electron microscopy (FE-SEM) image ( Figure 1 b) and scanning electron microscopy images ( Figure 1 c) shows that the long radius of the ZnMOF microspheres is about 1.5 μm and the short radius is about 1 μm. The elemental map shows the uniform distribution of carbon (C), oxygen (O) and zinc in ZnMOFs ( Figure 1 d-1f).

[0092] The crystal structure of ZnMOF powder was analyzed by X-ray diffraction (XRD). Figure 1 g) is consistent with the reported literature, that is, this embodiment successfully synthesized ZnMOF with an overall crystal structure. In addition, the absorption spectrum of the ZnMOF solution shows a characteristic absorption peak at 430nm ( Figure 1h and 1i), which is also consistent with the absorption spectrum of curcumin. Figure 1 j, at ~10 μg mL -1 No obvious cytotoxicity of ZnMOF to mouse DPCs was observed at the ZnMOF concentration of 1.5 wt %. Therefore, a stable and biocompatible metal-organic framework compound composed of zinc and curcumin was successfully prepared.

[0093] Example 2 Synthesis and Characterization of ZnMOF Microneedles (ZnMOF-MN)

[0094] Soluble ZnMOF-MN is made of γ-PGA, which is widely used due to its excellent biodegradability, bioactivity and certain mechanical strength after drying. Figure 2 As shown in a, microneedles were made using a commercial polydimethylsiloxane (PDMS) microneedle mold. Figure 2 c is the SEM image of the prepared microneedles. When blank microneedles (BMN) were prepared, an equal volume of ddH2O was used to replace the ZnMOF solution. Figure 2 As shown in (b), with the increase of ZnMOF concentration, the orange-yellow color becomes more prominent, indicating that curcumin is stably present during the production process. The top-down SEM image shows the morphological characteristics of ZnMOF-MN, that is, the microneedle tips of the tetrahedral pyramid are arranged in an orderly manner (10×10 array), with an average height of 466.23μm±11.72μm ( Figure 2 g). In vivo studies have shown that the tips of these cones are sharp enough to penetrate the skin and create temporary perforations that heal within 30 minutes without bleeding ( Figure 2 d). In addition, by detecting the fluorescence signal of curcumin under the skin (excitation wavelength 423nm; emission wavelength 527nm) (fluorescence is visible at about 400μm under the skin, Figure 2 e), further confirming that ZnMOF can achieve transdermal delivery. In order to measure the release curve of ZnMOF in an aqueous environment, ZnMOF-MN was immersed in phosphate buffered saline (PBS), and the absorbance of the supernatant at 430 nm was monitored over time to detect the release amount of ZnMOF. Figure 2 As shown in f, the release rate of ZnMOF was 57.86% at 2 h and 84.14% at 24 h. For the hygroscopicity evaluation, the ZnMOF-MN patch was placed in a sealed jar with humidity maintained at 75%. The morphology of the MN tip was recorded every 5 min, and 90% of the tip was dissolved after 25 min ( Figure 2 g). Therefore, the soluble ZnMOF-MN of this example was proved to be a stable and effective drug release and transdermal delivery platform.

[0095] Example 3 In vitro verification of the antioxidant, anti-apoptotic and anti-androgen effects of ZnMOF

[0096] In order to verify the antioxidant effect of ZnMOF and ZnMOF-MN in this example, DPC was incubated with 5 μg / mL ZnMOF solution, BMN extract or ZnMOF-MN extract for 24 hours, and then treated with H2O2 for 30 minutes to simulate excessive ROS stress. Finally, the cell viability was determined by CCK-8 method.

[0097] Both ZnMOF solution and ZnMOF-MN extract showed strong antioxidant capacity as they greatly reversed the decline in cell viability caused by excessive ROS ( Figure 3 a).

[0098] In order to simulate the microenvironment of zinc deficiency, this example uses 5 μM TPEN to chelate zinc ions in the culture medium to induce cell apoptosis. The decrease in cell viability in the TPEN and BMN groups determined by CCK-8 suggests this phenomenon ( Figure 3 b). At the same time, the study showed that treatment with ZnMOF solution (5 μg / mL) and ZnMOF MN extract (~6.46 μg / mL ZnMOF) for 24 hours can greatly restore the cell viability loss caused by TPEN ( Figure 3 b). Propidium iodide (PI) can cross the dead cell membrane and bind to nucleic acids, and was therefore used to label dead cells in this study. Figure 3 As shown in Figure d, TPEN treatment induced significant cell apoptosis, which was manifested by morphological changes (cells were free and reduced in size) and PI staining. Quantitative studies of PI-positive cells showed that under zinc-deficient conditions, the number of dead DPCs decreased significantly from about 105 cells / field of view in the TPEN group to about 4 cells / field of view in the ZnMOF solution group and about 3 cells / field of view in the ZnMOF extract group. The number of dead cells in the latter two groups was not different from that in the control group (about 2 cells / field of view) ( Figure 3 c). In addition, flow cytometry analysis also showed that the increase in the total apoptosis rate (the sum of the percentage of cells in the UR and LR quadrants) triggered by TPEN was also inhibited by treatment with ZnMOF solution or ZnMOF-MN extract ( Figure 3 e and Figure 3 f). Therefore, ZnMOF and ZnMOF-MN were demonstrated to be effective in reversing the adverse biological effects of zinc deficiency through local zinc supplementation.

[0099] Similarly, dihydrotestosterone (DHT) infiltration is another important cause of poor hair follicle status, especially in AA patients. As the active form of testosterone, DHT can stimulate DPCs and produce chronic inflammation, leading to cell apoptosis and hair cycle decline. In this study, we used DHT treatment to simulate the AA microenvironment and evaluated the changes in gene transcription levels in DPCs by fluorescence quantitative PCR technology (RT-qPCR) to analyze the therapeutic effects of 2.5μg / mL ZnMOF solution and ZnMOF-MN extract (~3.23μg / mL ZnMOF) treatment on DHT-related adverse effects. Figure 3 As shown in (g), DHT treatment for 24 hours significantly upregulated apoptosis-related genes, including Caspase-3 and Caspase-9. Treatment with curcumin, ZnMOF solution, and ZnMOF-MN extract was able to alleviate these negative effects and increase the expression of PCNA in DPCs (indicating active cell proliferation). In addition, the downregulated Wnt / β-catenin signaling pathway is also responsible for the delayed hair growth in AA. To confirm the effect of ZnMOF on DHT-induced Wnt-β-catenin downregulation, we evaluated the expression of related genes in DPCs after DHT treatment. CCND1 is a Wnt target gene, while LEF1 and AMER3 are key mediators of the Wnt / β-catenin signaling pathway. As Figure 3 As shown in g, in DPCs exposed to curcumin, ZnMOF solution, or ZnMOF-MN extract, the levels of genes indicating Wnt / β-catenin signaling activation (CCND1, LEF1, and AMER3) were significantly increased, while the expression of Wnt / α-catenin antagonist (DDK1) was significantly decreased ( Figure 3 g). Therefore, ZnMOF and ZnMOF-MN were demonstrated to counteract the negative effects of DHT and restore the activation of the Wnt / β-catenin signaling pathway in DPCs under the AA microenvironment.

[0100] In this example, we also observed a significant decrease in HSD17B2 and SRD5A1 in DPCs treated with curcumin, ZnMOF solution or ZnMOF-MN extract ( Figure 3 g). This suggests that ZnMOF-MN may improve the AA microenvironment by reducing androgen production and activation. Thus, ZnMOF and ZnMOF-MN were shown to have therapeutic effects on DHT-induced apoptosis and Wnt / β-catenin inactivation, and reduced the expression of androgen production-related genes at the transcriptional level.

[0101] Example 4 In vivo study of C57 / BL6 mouse wound model: hair growth assessment and histological study

[0102] Skin damage, especially dermal damage, is a common external cause of hair loss. In order to evaluate the therapeutic effect of ZnMOF-MN in hair loss caused by skin damage, we excised a 9mm diameter circular full-thickness skin on the back of C57 / BL6 mice (male, 6-8 weeks) as a wound model. The mice in the control group (Ctrl) were only modeled without other treatments, while the mice in the BMN group or ZnMOF-MN group were treated with BMN or ZnMOF-MN.

[0103] This example records the photos of the wound surface of each group of mice on days 0, 3, 5, 7, 9, 11, 13, 15 and 17 for further analysis ( Figure 4 a and Figure 7 ).like Figure 4 As shown in (b), the wound area of ​​the ZnMOF-MN group shrank much faster than that of the control group before scab detachment (day 11). On day 9, the average remaining wound area ratios of the Ctrl, BMN, and ZnMOF-MN groups were 72.16%, 67.93%, and 23.55%, respectively. The increased cell migration observed in DPCs treated with the ZnMOF-MN extract also confirmed its ability to accelerate wound healing (Figure S2). On day 17, the wounds of all groups were healed and covered with regenerated hair, but H&E staining of skin samples showed that the number of hair follicles in the ZnMOF-MN group doubled compared with the control and BMN groups ( Figure 4 c and Figure 4 d). In addition, immunohistochemical staining images of CD31 ( Figure 4 c) also showed that the capillaries around the hair follicles in the ZnMOF-MN group were more densely distributed ( Figure 4 e). Similarly, the proportion of Ki67-positive cells in the ZnMOF-MN group was almost five times that of the other two groups ( Figure 4 c and Figure 4 f). These results suggest that ZnMOF-MNs have a promoting effect on hair growth during wound healing by accelerating wound closure, regulating microvessels, and stimulating cell proliferation.

[0104] Example 5 In vivo study of the male pattern baldness model in C57 / BL6 mice: evaluation of hair growth and histological study

[0105] AA is the most common type of hair loss and is closely related to internal abnormalities such as hormone fluctuations and immune disorders. We established an AA mouse model with slight adjustments based on the method reported in the literature to confirm the therapeutic effect of ZnMOF-MN in the AA microenvironment. The flow chart is shown in the figure. Figure 5As shown in a. We injected male C57 / BL6 mice (6-8 weeks) subcutaneously with 2 mg / kg DHT for 14 consecutive days. Mice treated with an equal volume of normal saline (NS) injection served as the control group (Ctrl) and were depilated together with other mice on day 1. In the experimental groups, mice were treated with BMNs or ZnMOF-MNs in the exposed skin on days 1, 4, 7, and 10. For mice in the model group, no additional intervention was performed. Fluorescent images of anti-CD31 staining of skin samples collected on day 18 suggested that the decrease in capillary density caused by DHT treatment was significantly improved in the ZnMOF-MN group ( Figure 5 b and Figure 5 c). Similarly, the decreased Ki67 positivity (representing proliferating DPCs) in the model and BMN groups was also enhanced by ZnMOF-MN treatment ( Figure 5 b and Figure 5 d).

[0106] We recorded photographs of the skin in the depilated area on days 1, 4, 7, 10, 13, 16, and 18 to assess hair growth ( Figure 6 a). On day 18, we observed that hair regeneration was delayed in the model group and BMN group mice ( Figure 6 a and Figure 6 b). In contrast, the hair coverage of mice treated with ZnMOF-MNs reached 90%, which was significantly higher than that of the model group and BMN group, and there was no significant difference in growth compared with the normal control group ( Figure 6 a to Figure 6 c).

[0107] It can be seen that the MN of this example can reverse the adverse effects of DHT on DPCs, positively affect the microenvironment, and show a significant therapeutic effect in improving hair regeneration in AA.

Claims

1. An application of a curcumin-zinc organic framework compound as an active ingredient in the preparation of a medical device for treating hair loss; The curcumin-zinc organic skeleton compound has an overall crystal structure, and the weight ratio of curcumin to zinc ion is 6.21-6.

55.

2. The use according to claim 1, Features The curcumin-zinc organic skeleton compound is an elliptical nanoparticle formed by self-assembly of curcumin and zinc ions in an alkaline water environment. The long radius of the particle is 1.5 μm±0.2 μm, and the short radius is 1.0 μm±0.2 μm.

3. The use according to claim 1, Features The hair loss is androgenic alopecia.

4. The use according to claim 1, Features The preparation method of the curcumin-zinc organic framework compound is as follows: 100mg ZnSO 4 7H 2 O was dissolved in 10 ml of double distilled water, 120 mg of curcumin was first dissolved in 1 ml of DMSO and then 9 ml of double distilled water was added. The two were fully mixed and the pH value was adjusted to 12. Then, the mixture was placed in a muffle furnace and heated at 120°C for 24 hours. Centrifuged at 12000 rpm at 4°C for 15 minutes, and the orange-yellow powder was collected.

5. A microneedle, Features The invention comprises a soluble polymer and an active ingredient for promoting hair growth, wherein the active ingredient is the curcumin-zinc organic skeleton compound as claimed in claim 1.

6. The microneedle according to claim 5, Features The needle tips are arranged in a 10×10 array, and each needle tip has a length×width×height of 260 μm×260 μm×500 μm.

7. The microneedle according to claim 5, Features Prepared by the following method: The obtained curcumin-zinc organic framework compound powder was dissolved in DMSO and then diluted with water to a concentration of 1 mg / mL. 600 μL of the diluted curcumin-zinc organic framework compound solution was added to 350 mg of γ-PGA, dispersed at room temperature and centrifuged to obtain a ZnMOF gel; The ZnMOF gel was completely filled into the microcavity of the needle tip, and the mold was placed at 40°C to dry overnight to obtain ZnMOF-MN.

8. The microneedle according to claim 5, Features The soluble polymer is γ-PGA, and the molecular weight distribution is Mw=1000-15000 KDa.

Images