Curcumin nano coordination polymer for treating androgenetic alopecia as well as preparation method and application of curcumin nano coordination polymer

Abstract

The invention provides a curcumin nano coordination polymer for treating androgenetic alopecia as well as a preparation method and application of the curcumin nano coordination polymer, and belongs to the technical field of nano drugs. Curcumin targets for relieving oxidative stress and activating autophagy are screened through network pharmacology and molecular docking and are verified; a curcumin nano coordination polymer TFC is designed and loaded into a soluble microneedle, and AGA hair regeneration is promoted by reducing oxidative stress and activating hair follicle cell autophagy; the TFC with enzyme-like activity can efficiently remove various free radicals, reduce the oxidative stress level of human hair papilla cells and activate autophagy of the human hair papilla cells. The TFC is loaded in the soluble microneedle to break through the cuticle barrier, and transdermal drug delivery can effectively reduce the oxidative stress level of AGA, activate the autophagy activity of hair follicle cells, enhance the proliferation activity of the hair follicle cells and microangiogenesis, promote the hair regeneration of androgenetic alopecia, and provide a novel treatment strategy for AGA.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-drugs, and specifically relates to a curcumin nano-coordination polymer for treating androgenic alopecia, and a preparation method and application thereof. Background Art

[0002] Hair follicles are the basic units of hair, with strong self-renewal capabilities and periodic growth. The normal hair growth cycle is divided into three stages: growth phase, regression phase and resting phase, of which about 85% of hair is in the growth phase. When the normal hair growth cycle is disturbed, it can lead to hair loss. Androgenic alopecia (AGA) is one of the most common forms of hair loss, characterized by progressive hair loss on the forehead and top of the head, affecting more than 50% of people over 50 years old. AGA affects aesthetics and mental health and reduces the quality of life of patients. AGA is the result of the combined action of multiple factors, including androgens, insufficient microvascularization, oxidative stress, etc. After the androgen testosterone is converted into dihydrotestosterone by 5α-reductase in the hair follicle, its affinity with the androgen receptor is significantly increased, which in turn activates the downstream signaling pathway, resulting in a decrease in the duration of the hair growth phase and a prolonged resting phase. Oral finasteride can reduce the effect of androgens on hair follicles by inhibiting 5α-reductase, but male patients may experience sexual adverse reactions such as decreased libido and erectile dysfunction if they take it for a long time. At the same time, the disorder of hair follicle microenvironment is another important cause of hair loss. Oxidative stress causes aging and apoptosis of hair follicle cells, and insufficient microvascularization also makes the hair follicles lack sufficient nutrient supply, thereby limiting the transition of hair follicles from the resting phase to the growth phase, resulting in slow hair growth. Based on this, topical minoxidil exerts its therapeutic effect by dilating blood vessels to enhance blood supply and promote the transition from the resting phase to the growth phase, but the topical application of minoxidil makes it difficult for the drug to reach the deep subcutaneous area, and long-term local use will produce adverse reactions such as allergic dermatitis. Therefore, it is of great clinical significance to develop highly effective and low-toxic causal treatment drugs based on the pathological characteristics of AGA.

[0003] If the hair follicles in the AGA resting phase are compared to dormant seeds, the hair follicle microenvironment is the soil. AGA hair regeneration requires the transformation of hair follicles from the resting phase to the growth phase and a healthy hair follicle microenvironment, just as seeds need to break dormancy and fertile soil to provide nutrients for germination and growth. Recent studies have shown that the apoptosis of miniaturized hair follicles in the early stage of degeneration in AGA patients is increased and accompanied by severe endogenous autophagy impairment. Autophagy activators can promote the transition of mouse hair from the resting phase to the growth phase and promote hair growth; while the use of autophagy inhibitors can induce apoptosis of hair follicle cells, leading to premature degeneration of hair follicles. Autophagy is an evolutionarily conserved intracellular stress response process that maintains cell homeostasis and adapts to nutrient deficiency and other adverse environmental conditions by clearing redundant, damaged or aged organelles of the cell. The above studies suggest that activating hair follicle autophagy is a potential way to stimulate the transition of hair follicles from the resting phase to the growth phase in the pathological state of AGA. Other studies have confirmed that oxidative stress is an important factor causing the adverse hair follicle microenvironment in AGA. Excessive oxidative stress can cause aging and apoptosis of hair follicle cells, and inhibit the proliferation of keratinocytes and the differentiation of hair follicle stem cells by inducing the secretion of inflammatory factors. Therefore, oxidative stress is the main cause of the deterioration of the hair follicle microenvironment. Based on the "seed-soil" model relationship, we believe that activating hair follicle (seed) autophagy to transform it from the resting phase to the growth phase and alleviating the oxidative stress state of the hair follicle microenvironment (soil) is a potential idea for treating AGA, but no relevant research has been reported so far. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a curcumin nano-coordination polymer for treating androgenic alopecia, and its preparation method and application. The curcumin nano-coordination polymer with enzyme-like activity can efficiently remove various free radicals, reduce the oxidative stress level of human hair papilla cells and activate their autophagy activity, playing a significant cell protection role. Microneedles can reduce the oxidative stress microenvironment at a lower administration frequency, activate hair follicle cell autophagy and promote hair follicle cell proliferation and microangiogenesis, that is, improve the "soil" and "seeds", significantly promote the hair regeneration of AGA, and provide a new treatment strategy with great application prospects for AGA.

[0005] To achieve the above object, the present invention first provides a curcumin nano-coordination polymer for treating androgenic alopecia, comprising curcumin (Cur), Fe 3+ , tannic acid (TA), the curcumin and tannic acid are interlocked, the Fe 3+ It forms metal coordination bonds with the phenolic hydroxyl and carbonyl groups in flavin and tannic acid.

[0006] Preferably, the encapsulation efficiency of the curcumin is 100% and the drug loading is 52%.

[0007] Based on a general inventive concept, the present invention also provides a method for preparing a curcumin nano-coordination polymer, comprising the following steps:

[0008] S1. Tannic acid, curcumin and Fe were stirred vigorously. 3+ Add to 20% acetone solution in sequence and sonicate immediately;

[0009] S2. Use a rotary evaporator to remove acetone from the solution prepared in S1, collect the flavin nano-coordination polymer by high-speed centrifugation, and disperse it evenly with ultrapure water.

[0010] Preferably, in step S1, tannic acid, curcumin and Fe 3+ The concentration ratio is 8:1:2.

[0011] Preferably, the temperature of the high-speed centrifugation in step S1 is 4° C., the speed is 16000 rpm, and the time is 20 min.

[0012] Based on a general inventive concept, the present invention also provides an application of a curcumin nano-coordination polymer for treating androgenic alopecia in the preparation of a drug for treating androgenic alopecia, characterized in that the drug is a microneedle.

[0013] Preferably, the preparation method of the microneedle is: using HA and PVPK30 as carriers, and loading the curcumin nano-coordination polymer into the soluble microneedle by a two-step molding method.

[0014] Preferably, the microneedle is a square patch of 1×1 cm in size, comprising a 10×10 microneedle array, with an average length of 511 μm and a needle distance of 218 μm, and each microneedle contains 32 μg of curcumin nano-coordination polymer.

[0015] Preferably, the method of using the microneedles is: pressing the microneedles on the skin for 5 minutes.

[0016] The mechanism of this regimen for treating androgenic alopecia is as follows:

[0017] Through network pharmacology and molecular docking, we screened and verified the targets of curcumin for alleviating oxidative stress and activating autophagy. We designed curcumin nano-coordination polymer (TFC) and loaded it into soluble microneedles to promote AGA hair regeneration by reducing oxidative stress and activating hair follicle cell autophagy. TFC with enzyme-like activity can efficiently scavenge various free radicals, reduce the oxidative stress level of human hair papilla cells and activate their autophagy. Loading TFC into soluble microneedles to break through the stratum corneum barrier and deliver the drug transdermally can effectively reduce the oxidative stress level of AGA, activate the autophagic activity of hair follicle cells, enhance the proliferation activity of hair follicle cells and microangiogenesis, and promote hair regeneration in androgenic alopecia. In summary, inspired by seed germination, we believe that the two-pronged approach of improving "soil" and "seeds" by delivering TFC transdermally through soluble microneedles to alleviate the oxidative stress level of AGA and promote hair follicle cell autophagy provides a new treatment strategy for AGA.

[0018] Cur-loaded nanoparticles TFC were prepared by a simple one-step mixing. TFC had a uniform structure and good colloidal stability. The encapsulation efficiency of Cur was as high as 100%, and the drug loading was 52%. The doping of TA and Cur gave TFC a broad-spectrum free radical scavenging ability, and the Fe 2+ / Fe 3+ The valence state shuttle of TFC endows nanoparticles with superoxide dismutase and catalase activity, thereby scavenging superoxide anions and decomposing hydrogen peroxide, and generating oxygen to alleviate the hypoxic environment in the lesion site; TFC's extensive regulatory effect on RONS can effectively alleviate cell damage under oxidative stress, and release Cur to activate cell autophagy, thereby exerting a cytoprotective effect. After transdermal delivery by microneedles, TFC is delivered to the hair follicle site deep in the skin, promoting AGA hair regeneration by improving the oxidative stress microenvironment (soil) and activating autophagy of hair follicle cells (seeds). In the AGA mouse model, TFC showed better therapeutic effects than Minoxidil and had excellent biosafety. This scheme proposes a new AGA treatment idea based on the "seed-soil" model relationship between hair follicles and the microenvironment. Based on this idea, it was found that Cur has therapeutic potential for AGA, and a new nano-delivery system Cur was developed to address the bottleneck of Cur's in vivo application, laying a formulation foundation for its clinical application.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) This protocol uses the alleviation of oxidative stress or activation of autophagy as a potential mechanism, and explores the potential targets of Cur in the treatment of androgenetic alopecia through network pharmacology combined with cell experiments. In this protocol, it has been confirmed that Cur has both the chemical activity of scavenging free radicals and the pharmacological effect of alleviating intracellular oxidative stress by inhibiting c-Jun phosphorylation. At the same time, Cur can effectively inhibit the phosphorylation of Akt and mTOR, promote the expression of LC3B, and thus activate the autophagy pathway. Therefore, Cur has both the activity of inhibiting oxidative stress and inducing autophagy, and is a candidate drug for the treatment of AGA.

[0021] (2) Cur-loaded nanoparticles TFC were prepared by a simple one-step mixing. TFC has a uniform structure and good colloidal stability. The encapsulation efficiency of Cur is as high as 100%, and the drug loading is 52%. The doping of TA and Cur makes TFC have a broad-spectrum free radical scavenging ability, and the Fe 2+ / Fe 3+ The valence state shuttling of the nanoparticles endows them with superoxide dismutase and catalase activities, thereby scavenging superoxide anions and decomposing hydrogen peroxide, and generating oxygen to alleviate the hypoxic environment at the lesion site.

[0022] (3) TFC can effectively activate cell autophagy through Cur in its structure, thereby playing a cytoprotective role. The microneedles TFC MN made of TFC have sufficient mechanical properties and strength to break through the stratum corneum barrier to achieve transdermal drug transport. TFC MN promotes hair regeneration in AGA mice by reducing oxidative stress and activating hair follicle cell autophagy, thereby promoting hair follicle cell proliferation and microangiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1The network pharmacology target screening, molecular docking and verification of Cur in alleviating oxidative stress and regulating autophagy in Experimental Example 1, (A) PPI network of Cur alleviating oxidative stress and activating autophagy targets, (B) GO functional enrichment analysis of Cur alleviating oxidative stress and activating autophagy targets, (C) KEGG pathway enrichment analysis of Cur alleviating oxidative stress and activating autophagy targets, (D) Molecular docking of Cur with mTOR, (E) Molecular docking of Cur with c-Jun, (F) Western blot verification of Cur's regulatory effect on oxidative stress and autophagy-related pathway proteins, (G) UV spectroscopy characterization of different concentrations of Cur in clearing DPPH·, (H) Different concentrations of Cur in clearing ABTS· + UV spectroscopy characterization;

[0025] Figure 2 Characterization of the curcumin nano-coordination polymer TFC in Experimental Example 2, (A) particle size distribution of TFC and appearance of TFC solution, (B) zeta potential of TFC, (C) transmission electron microscopy image of TFC and (D) EDS spectrum, (E) encapsulation efficiency and drug loading of Cur in TFC, (F) UV-visible absorption spectra of TFC, Cur and TA, (G) infrared absorption spectra of TFC, Cur and TA;

[0026] Figure 2 The self-assembly mechanism of curcumin nano-coordination polymer TFC in Experimental Example 3, (H) the process of calculating the self-assembly force of TFC by molecular dynamics simulation, (I) the self-assembly mechanism of tannic acid, curcumin and Fe 3+ Conformational analysis of the interactions between (J) tannic acid, curcumin and Fe 3+ Interaction mode and energy analysis, (K) XRD patterns of TFC, Cur, TA and TA / Cur mixture, (L) XPS spectrum of TFC, (M) XPS spectrum of Fe in TFC, (N) Particle size colloidal stability of TFC (n=3);

[0027] Figure 3 The in vitro RONS scavenging ability and enzyme-like activity of the curcumin nanocomposite polymer TFC in Experimental Example 4 are characterized. (A) Schematic diagram showing the RONS scavenging ability and enzyme-like activity of TFC, (B) UV spectra of different concentrations of TFC scavenging DPPH·, (C) quantitative analysis of the scavenging rate of DPPH· (n=3), (D) ABTS· scavenging rate of different concentrations of TFC + UV spectroscopy characterization of (E)ABTS· +(F) MB UV absorbance characterization of ·OH scavenging effect of different concentrations of TFC (n=3), (G) ERS graph of ·OH scavenging by different concentrations of TFC, (H) Quantitative analysis of ·NO scavenging rate by different concentrations of TFC (n=3), (I) ·O2 - Quantitative analysis of clearance rate (n=3), (J) Clearance of ·O2 by different concentrations of TFC - ERS diagram, (K) dynamic curve of dissolved oxygen produced after TFC decomposes H2O2, (L) oxygen produced by TFC decomposing H2O2 at different concentrations;

[0028] Figure 4 The intracellular free radical scavenging activity of TFC in Experimental Example 5, (A) TFC cytotoxicity (n=3), (B) TFC cell uptake fluorescence image and (C) flow cytometry fluorescence intensity and (D) quantitative analysis (n=3), (E) TFC scavenging human hair papilla cell RONS fluorescence image and fluorescence quantitative results, including (F) total RONS, (G)·O2 - , (H)·OH / ONOO- and (I)·NO (n=3), (J) is the level of MDA significantly alleviated after TFC addition;

[0029] Figure 5 The protective effect of TFC on cells under oxidative stress in Experimental Example 6, (A) Effects of different concentrations of TFC on cell viability after H2O2 treatment under oxidative stress (n=3), (B) fluorescence image of live / dead cell staining and (C) quantification of live / dead cell ratio (n=3), (D) fluorescence image of cell autophagy structure, (E) transmission electron microscopy image of cell autophagy structure, (F) protein expression level of LC 3 and (G) semi-quantitative analysis (n=3);

[0030] Figure 6 Preparation and characterization of TFC MN in Experimental Example 7, (A) schematic diagram of TFC MN preparation process, (B) appearance of TFC MN, (C) micrograph of TFC MN, (D) scanning electron microscope image of TFC MN, (E) mechanical property test of TFC MN, (F) TFC MN after insertion into ex vivo mouse skin, (G) ex vivo mouse skin after TFC MN treatment, (H) H&E image of mouse skin after TFC MN treatment;

[0031] Figure 7TFC in Experimental Example 7 In vivo pharmacodynamic evaluation of MN, (A) Schematic diagram of AGA modeling and dosing regimens of each group, (B) Photos of hair regeneration of mice in each group on days 1, 7, 14, 21, and 28 after hair removal, (C) Days of darkening of the skin of mice in each group after hair removal (n=5), (D) Hair coverage of mice in each group on day 28 after hair removal (n=5), (E) Length of new hair of mice in each group on day 28 after hair removal (n=5), (F) H&E staining of the skin of mice in each group on day 14 after hair removal, (G) Quantitative determination of hair follicle length of mice in each group on day 14 after hair removal (n=3), (H) Epidermal thickness of mice in each group on day 14 after hair removal (n=3), (I) DHE detection of ROS, Ki67, CD31, and LC in the skin of mice in each group on day 14 after hair removal Fluorescence micrographs of 3Ⅱ and quantitative determination of (K) DHE, (L) Ki67, (M) CD31, and (N) LC 3Ⅱ (n=3), (O) changes in body weight of mice in each group (n=5), (P) H&E sections of heart, liver, spleen, lung, and kidney of mice in each group, (Q) iron content in skin, heart, liver, spleen, lung, and kidney of mice in normal group and TFC MN group (n=3);

[0032] Figure 8 Schematic diagram of the construction and mechanism of action of TFC. (A) is the schematic diagram of the construction of TFC; (B) is the schematic diagram of RONS scavenging ability and enzyme-like activity; (C) is the mechanism of action of TFC in the treatment of AGA. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0037] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0038] Related reagents: Curcumin was purchased from Bailingwei Technology Co., Ltd., tannic acid, ABTS, and DHE were purchased from Shanghai McLean Company, ferric chloride hexahydrate and PVPK30 were purchased from Sinopharm Group, sodium hyaluronate was purchased from Shanghai Yuanye Company, testosterone was purchased from Shanghai Aladdin Company, DPPH was purchased from Shanghai Jixing Company, hydroxyl free radical detection kit and superoxide anion detection kit were purchased from Nanjing Jiancheng Bioengineering Institute, nitric oxide kit and DCFH-DA were purchased from Sigma Company, USA, DAF-FM DA, live-dead staining detection kit, and lipid oxidation (MDA) detection kit were purchased from Shanghai Bio-Sky Co., Ltd., human dermal papilla cells were purchased from Shanghai Boquaner Biotechnology Co., Ltd., DMEM culture medium was purchased from Gibco, fetal bovine serum was purchased from Suzhou Yikesai Co., Ltd., trypsin and penicillin-streptomycin were purchased from Beijing Solebow Co., Ltd., MTT was purchased from White Shark Co., Ltd., HPF was purchased from Shanghai Maokang Co., Ltd., autophagy detection kit was purchased from Jiangsu Keyi Co., Ltd., anti-LC3 antibody was purchased from Wuhan Mitaka Co., Ltd., anti-p-Akt antibody, anti-Akt antibody, anti-p-mTOR antibody, anti-mTOR antibody, anti-LC3B antibody, and anti-pc-Jun antibody were purchased from Hangzhou Huaan Biotechnology Co., Ltd., anti-Jun antibody was purchased from Cell Signaling Technology, USA, anti-Ki67 antibody and anti-CD31 antibody were purchased from Abcam, and minoxidil gel was purchased from Bausch and Lomb.

[0039] Related instruments: Malvern Zeta Sizer Nano (Nano ZS, Malvern Instruments, UK) was used to determine its particle size distribution, polydispersity index (PDI) and Zeta potential. High performance liquid chromatography (HPLC) (1260infinityⅡ, Agilent) was used to determine the encapsulation efficiency (EE%) and loading efficiency (LE%) of curcumin. Transmission electron microscopy (TEM) (TitanG2-F20, FEI) was used to characterize its morphology and elemental spectrum. The ultraviolet-visible (UV-vis) spectrum was measured by an ultraviolet-visible spectrophotometer (UV-2600, Shimadzu). The infrared spectrum was measured by an infrared spectrometer (Spectrum Two, PerkinElmer). X-ray diffractometer (Advance D8 X, Bruker) and X-ray electron spectrometer (ESCALAB250Xi, Thermo Fisher) were used to characterize its crystal form, elemental composition and valence state, respectively. The stability of TFC in H2O, Tris (10 mM, pH 7.4) and DMEM (10% FBS) was measured by monitoring the particle size and PDI after 72 h of incubation. The absorbance was measured by a multifunctional microplate reader (Infinite M200PRO, TECAN). Cell fluorescence was observed and measured by confocal fluorescence microscopy (LSM900, Zeiss), fluorescence microscopy (Cytation 5, BioTek) and flow cytometer (CytoFLEX, Beckman Coulter). Scanning electron microscopy (Regulus8230, Hitachi) was used to observe the morphology of microneedles. Inductively coupled plasma optical emission spectrometer (iCAP PRO X, Thermo Fisher) was used to determine Fe.

[0040] Animal experiment: Male C57 / BL6 mice (6 weeks old) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. C57 / BL6 mice were used in experimental studies after one week of adaptive breeding at the Department of Experimental Animals of Central South University. Animal experiments were approved by the Central South University Experimental Animal Welfare Ethics Committee.

[0041] All research results are expressed as mean ± SD. GraphPad Prism 8.0 software was used for Student's t test and one-way ANOVA test. p<0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0042] Example 1 Preparation of curcumin nano-coordination polymer TFC

[0043] 20 μL of tannic acid (40 mg / mL), 100 μL of curcumin (5 mg / mL) and 40 μL of FeCl3·6H2O (10 mg / mL) were added to 5 mL of 20% acetone solution in sequence under vigorous stirring and immediately sonicated. Then, acetone was removed from the solution using a rotary evaporator. Finally, TFC was collected by high-speed centrifugation (4°C, 16000 rpm, 20 minutes) and dispersed evenly with ultrapure water.

[0044] Its construction diagram is as follows Figure 8 As shown in A.

[0045] Comparative Example 1 Preparation of tannic acid-iron nanoparticles (TF) without curcumin

[0046] 80 μL of tannic acid (40 mg / mL) and 80 μL of FeCl3·6H2O (10 mg / mL) were added to ultrapure water under stirring, and the reaction solution was collected by high-speed centrifugation (4°C, 16000 rpm, 20 minutes) to obtain TF, which was then evenly dispersed with ultrapure water.

[0047] Experimental Example 1: Exploring the mechanism of Cur regulating oxidative stress and cellular autophagy

[0048] This study used the potential mechanism of alleviating oxidative stress or activating autophagy to screen the potential targets of Cur for the treatment of androgenetic alopecia by network pharmacology. The results showed that a total of 21 genes (red) were directly related to Cur ( Figure 1 A). Among them, Jun is an important target of the oxidative stress pathway, and inhibiting its phosphorylation can effectively alleviate oxidative stress. Akt1 and mTOR are important targets of the autophagy pathway. Akt1 is upstream of mTOR, and inhibiting its phosphorylation can effectively activate autophagy and promote the expression of downstream LC3B. GO enrichment analysis and KEGG pathway enrichment analysis were further performed on 21 genes directly regulated by Cur. GO enrichment analysis showed that Cur is mainly involved in multiple processes, including response to endogenous stimulus, transcription factor binding, positive regulation of gene expression, positive regulation of macromolecule metabolic process, cellular response to chemical stimulus, etc. ( Figure 1B). KEGG analysis showed that the pathways mainly involved in Cur included Pathways in cancer, Prostate cancer, Kaposi sarcoma-associated herpesvirus infection, FoxO signaling pathway, Colorectal cancer, Endocrine resistance, etc. ( Figure 1 C). Cur was molecularly docked and visualized with Jun and mTOR, which have higher adjacency, and the binding energy and mode of action were obtained. The results showed that Cur and the target proteins Jun and mTOR were freely and stably bound to each other mainly through van der Waals forces and hydrogen bonds, thus exerting pharmacological effects. The binding energies were -6.92 kcal / mol and -5.62 kcal / mol, respectively. Figure 1 D, E). Subsequently, we verified the above mechanism at the cellular level and confirmed the regulatory effect of Cur on c-Jun, Akt, mTOR and LC3B through WB experiments ( Figure 1 F). The results showed that Cur could effectively inhibit the phosphorylation of c-Jun under oxidative stress. At the same time, Cur could effectively inhibit the phosphorylation of Akt and mTOR, promote the expression of LC3B, and thus activate the autophagy pathway. It is worth noting that Cur itself has a strong free radical scavenging ability, and can effectively scavenge DPPH· and ABTS· at the test tube level within the concentration range of 25μg / ml. + ( Figure 1 G, H). Therefore, the oxidative stress regulation ability of Cur is attributed to its dual role of pharmacological activity and chemical activity. The above results confirm the multiple activities of Cur in inhibiting oxidative stress and inducing cell autophagy, and have the potential for the treatment of AGA.

[0049] Experimental Example 2 Characterization of Curcumin Nano-Coordination Polymer TFC

[0050] Dynamic light scattering showed that the hydrated particle size of TFC was about 248 nm and the PDI was 0.141, indicating that the particle size distribution was relatively concentrated and the dispersion was good ( Figure 2 A). The surface potential of TFC is -42mV ( Figure 2 B), which may be attributed to the abundant carboxyl groups in the TA / Cur structure. The high negative charge on the surface of the nanoparticles is conducive to the negative electric repulsion between particles, increasing the colloidal stability and making it less likely to aggregate. TEM transmission electron microscopy showed that TFC was a spherical structure with a particle size of about 90nm in the dry state ( Figure 2 C). Further analysis of the elemental composition of TFC by TEM and energy dispersive spectrometer (EDS) showed that TFC contained three elements: C, O, and Fe ( Figure 2C, D). HPLC analysis showed that the encapsulation efficiency of Cur was 100% and the drug loading was 52% ( Figure 2 E).

[0051] UV spectrum analysis shows that TFC has a characteristic absorption peak of Cur at 426nm and a characteristic absorption peak of TA at 276nm ( Figure 2 F). It is worth noting that due to the ligand-metal charge transfer (LMCT) effect, the absorption peak at 426nm in TFC becomes broadened and the ligand-metal charge transfer absorption peak appears at 490-550nm, proving that Cur and Fe 3+ TA has a characteristic absorption peak at 213nm, but this peak appears red-shifted in the TFC structure. TFC also has a ligand-metal charge transfer absorption peak at 600-800nm, proving that TA and Fe 3+ In addition, TFC shows a new absorption peak at 310nm, which may be attributed to the formation of Fe-O coordination bonds in the TFC system. Figure 2 G), 3434cm -1 The stretching vibration of Cur phenolic hydroxyl group is 1715 cm -1 The C=O stretching vibration of the TA ester group is 1505 cm -1 The C=O and C=C vibrations of Cur are at 1200 cm -1 is the C-OH stretching vibration of TA, indicating that TFC contains Cur and TA. In addition, the 3500cm -1 The characteristic absorption peak of the phenolic hydroxyl group of Cur disappears, and the absorption peak of the phenolic hydroxyl group of TA decreases from 3361 cm -1 Move to 3434cm -1 , and 590cm -1 New Fe-O stretching vibration peaks appeared at the positions, which confirmed the coordination effect between TA / Cur and iron ions.

[0052] Experimental Example 3: Investigation of the Self-Assembly Mechanism of Curcumin Nano-Coordination Polymer TFC

[0053] In order to further study the self-assembly mechanism of TFC, molecular dynamics simulation was used to analyze the TA, Cur, Fe 3+ The interaction between the three Figure 2 H). Cluster analysis of the TA and Cur complex was performed, and the dominant conformation was quantitatively optimized. The optimized TA and Cur molecules were interlocked and bound very tightly, indicating that the two interacted mainly through intermolecular nesting and tight stacking ( Figure 2 I) Adding Fe 3+ After that, Fe 3+The position of is between the carbonyl and phenolic hydroxyl groups of the TA-Cur complex, and the overall complex formed has a stable interaction. The interaction mode and energy analysis show that the interaction between TA and Cur molecules is strong, mainly through hydrogen bonding, π-π stacking and hydrophobic interaction, and the binding energy is -27.119Kcal / mol ( Figure 2 J) Fe 3+ The addition of Fe further enhanced the interaction between TA and Cur molecules, and the binding energy increased to -43.853 Kcal / mol, which was attributed to the 3+ The strong attraction to the phenolic hydroxyl and carbonyl groups further intensifies the stacking of TA and Cur molecules. 3+ It can form strong metal bonds with the phenolic hydroxyl and carbonyl groups in TA and Cur molecules, with a distance of about The binding energy is -485.721 Kcal / mol. The above results show that TFC self-assembles mainly through coordination bonds, hydrogen bonds, π-π stacking and hydrophobic interactions.

[0054] The crystal structure of TFC was analyzed by XRD, and it was found that Cur had an obvious sharp crystal peak in the range of 7-30°, while this peak disappeared in TFC ( Figure 2 K), indicating that Cur exists in an amorphous state in TFC. XPS characterization spectrum shows that TFC contains three elements: C, O, and Fe ( Figure 2 L), while the fine spectrum of Fe found that Fe 2p3 / 2 and 2p1 / 2 have two strong binding energy peaks at 711 and 724 eV respectively ( Figure 2 M), indicating that Fe 3+ Partially reduced, with Fe in the structure 3+ and Fe 2+ Through detailed spectrum analysis of Fe, the Fe in the TFC structure 3+ About 89.69%, Fe 2+ About 10.31% ( Figure 2 M). The colloidal stability of TFC was studied and it was found that the particle size of TFC did not change significantly after incubation in ultrapure water, Tris buffer (10 mM pH 7.4) and DMEM (10% FBS) for 72 h ( Figure 2 N), confirming the stability of the nanoparticles.

[0055] Experimental Example 4 Investigating the in vitro RONS scavenging ability and enzyme-like activity characterization of curcumin nanocomposite polymer TFC

[0056] In the TFC structure, Cur and TA are both natural reducing agents, and Fe 2+ / Fe 3+The valence state shuttling endows the nanoparticles with multi-enzyme potential to scavenge free radicals ( Figure 3 A) For example, Fe 2+ The site is responsible for removing superoxide anions by mimicking superoxide dismutase and removing hydroxyl radicals through redox reactions; while Fe 3+ The RONS scavenging ability of TFC was evaluated in vitro.

[0057] First, DPPH· and ABTS· + To investigate the antioxidant capacity of TFC, 20 μL of TFC with different concentrations of DPPH were mixed with 180 μL of acetic acid-sodium acetate buffer (100 mM, pH 5.5) and 200 μL of DPPH (0.3 mM) to make the final concentrations of TFC 0, 1, 2.5, 5, 12.5, 25, 50 μg / mL, and reacted in the dark for 30 minutes, and the absorbance of DPPH at 517 nm was measured. + : 7.4 mM A ABTS and 2.6 mM ammonium persulfate were mixed at a ratio of 1:1 (v:v) and placed at 4°C overnight to obtain ABTS. + 50 μL of each concentration of TFC was mixed with 200 μL of ABTS· + Mix well to make the final concentration of TFC 0, 1, 2.5, 5, 12.5, 25, 50 μg / mL, react for 30 minutes in the dark, and measure ABTS· + Absorbance at 734nm. OH: H2O2-Mn 2+ A Fenton-like system produces hydroxyl radicals, and 40 μL MB (100 μg / mL) is added and mixed with different concentrations of TFC to make the final concentration of TFC 0, 1, 2.5, 5, 12.5, 25, and 50 μg / mL, respectively. The mixture is reacted in a 37°C water bath for 30 minutes, and the absorbance of MB at 662 nm is measured. In addition, each concentration of TFC is mixed with FeSO4, DMPO, and H2O2 to make the final concentration of TFC 0, 25, and 50 μg / mL. After 5 minutes of reaction, EPR test analysis is performed. ·NO: A nitric oxide detection kit is used to measure the absorbance of the reaction solution at 540 nm, and the scavenging ability of each final concentration of TFC (0, 1, 2.5, 5, 12.5, 25, and 50 μg / mL) on ·NO is analyzed. ·O2 -:The absorbance of the reaction solution at 550nm was measured using a superoxide anion detection kit to analyze the scavenging ability of each final concentration of TFC (0, 1, 2.5, 5, 12.5, 25, 50μg / mL) on ·O2-. In addition, each concentration of TFC was mixed with xanthine solution, xanthine oxidase solution, and DMPO to make the final concentration of TFC 0, 25, and 50μg / mL, and EPR test analysis was performed after incubation for 10 minutes. H2O2: TFC was diluted to 0, 25, 50, and 100μg / mL, mixed with equal volumes of H2O2 (4M) and incubated in a 37℃ water bath for 30 minutes to observe the generation of oxygen, and a dissolved oxygen meter was used to monitor the dissolved oxygen content in the solution within 0-10 minutes.

[0058] After incubation with different concentrations of TFC, the color of DPPH gradually faded, and the characteristic absorption peak at 517 nm also gradually decreased ( Figure 3 B). Through quantitative analysis, it was confirmed that TFC showed concentration-dependent scavenging activity against DPPH·, and the scavenging rate reached 97.42% at a concentration of 50 μg / mL ( Figure 3 C) TFC vs ABTS + Similar results were also shown for the removal of ABTS· + The absorbance at 734 nm gradually decreased. The effect of 50 μg / mL TFC on ABTS· + The clearance rate reached 83.51% ( Figure 3 D, E). ·OH is considered to be the most oxidative free radical. Methylene blue (MB) was used as an indicator to detect the scavenging effect of TFC on ·OH. The color of the solution and the absorbance of MB at 662 nm increased with the increase of TFC concentration ( Figure 3 F), indicating that ·OH was removed by TFC. ESR results also showed that the signal intensity of ·OH gradually decreased after incubation with different concentrations of TFC ( Figure 3 G). Similarly, after incubation of ·NO with different concentrations of TFC, its absorbance at 540 nm also gradually decreased, showing good ·NO scavenging ability ( Figure 3 H).

[0059] Subsequently, we tested the ability of TFC to scavenge superoxide anions and decompose hydrogen peroxide to evaluate the enzyme-like activity of the nanoparticles. - It is an active free radical that is removed by superoxide dismutase in the body. The results show that TFC can effectively remove O2 - , the clearance rate at a concentration of 50 μg / mL reached 71% ( Figure 3 I, J). Hydrogen peroxide is a common reactive oxygen molecule in the body that can destroy macromolecular substances in the body and is degraded by catalase in the body. Similarly, TFC can decompose hydrogen peroxide and generate oxygen ( Figure 3K, L). As the concentration of TFC increases, the generation rate and amount of dissolved oxygen in the solution increase. The schematic diagram of TFC's RONS scavenging ability and enzyme-like activity is shown in Figure 8 As shown in Figure B, the above results confirm that TFC has a broad-spectrum RONS scavenging ability and can simulate the activity of superoxide dismutase and catalase to convert active free radicals into oxygen, thereby alleviating the hypoxic environment in the lesion site.

[0060] Experimental Example 5 Study on the intracellular free radical scavenging activity of curcumin nanocomplex polymer TFC

[0061] After confirming the RONS scavenging ability of TFC at the test tube level, its cell biological activity was further explored. Dermal hair papilla cells are specialized fibroblasts that control the hair growth cycle. Their proliferation is very important for the morphogenesis of hair follicles, and oxidative stress can lead to senescence and apoptosis of hair papilla cells. Therefore, dermal hair papilla cells were used as a model for research. HHDPC cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, with 5% CO2 in the incubator and a culture temperature of 37°C. Within the concentration range of 50μM TFC (measured in Cur), after TFC was incubated with cells for 24 hours, the cell survival rate was above 90%, indicating the biological safety of the nanoparticles ( Figure 4 A). Using the characteristics of Cur autofluorescence, the cellular uptake of TFC was observed by confocal microscopy. As the incubation time increased, the fluorescence signal in the cells gradually increased, indicating a time-dependent dynamic uptake of nanoparticles ( Figure 4 B). Flow cytometry results also confirmed that cells have high efficiency in taking up TFC, and the fluorescence signal reached a plateau after 4 hours ( Figure 4 C, D).

[0062] Subsequently, H2O2 was used to induce cellular oxidative stress and the scavenging activity of TFC on intracellular RONS was evaluated. DCFH-DA, DHE, HPF and DAF-FM DA fluorescent probes were used to detect intracellular general radicals, ·O2 - , ·OH / ONOO- and ·NO levels, HHDPC cells were seeded in 24-well plates for overnight adherent culture, 40μM TFC was added for incubation for 12h, the drug-containing medium was discarded, and 500μM H2O2 was added for incubation for 24h. DCFH-DA (10μM), DHE (5μM), HPF (20μM), and DAF-FM DA (5μM) probes were added to the cells and incubated for 30, 30, 45, and 30 minutes, respectively, and the fluorescence was observed under a fluorescence microscope. Similarly, according to the above operation, HHDPC cells were seeded in 6-well plates for overnight adherent culture, and then the drugs were administered and the cells were collected, and the probe fluorescence level was analyzed by flow cytometry. In addition, the lipid oxidation (MDA) detection kit was used and the instructions were followed to detect the intracellular malondialdehyde level.

[0063] Fluorescence microscopy results showed that after H2O2 treatment, the levels of various free radicals increased significantly, indicating that the intracellular oxidative stress state ( Figure 4 E). TFC treatment has a significant regulatory effect on various free radicals. Flow cytometry fluorescence quantification further confirmed that TFC can significantly reduce the fluorescence intensity of various free radicals caused by H2O2 ( Figure 4 FI). When cells are under oxidative stress, lipid oxidation can occur, and malondialdehyde (MDA) is a lipid oxidation product that causes oxidative stress. Therefore, measuring the intracellular MDA level can reflect the state of oxidative stress. The results of the MDA kit showed that the cellular MDA level increased significantly after H2O2 treatment. However, the addition of TFC significantly alleviated the MDA level ( Figure 4 J). The above results confirmed that TFC retained its broad-spectrum free radical scavenging ability at the cellular level.

[0064] Experimental Example 6 Investigating the protective effect of curcumin nano-coordination polymer TFC on cells under oxidative stress

[0065] Oxidative stress in the androgenic alopecia area can lead to excessive senescence of hair papilla cells and atrophy of hair follicles, accompanied by damage to endogenous autophagy, which delays hair growth and promotes the transition from growth phase to regression phase. In view of the excellent free radical scavenging ability of TFC, hydrogen peroxide was used to construct an oxidative stress model, and the MTT method was used to determine the protective effect of TFC on human dermal hair papilla cells under oxidative stress conditions ( Figure 5 A). HHDPC cells were seeded in 96-well plates for overnight adherent culture, and 0, 10, 20, and 40 μM TFC were added for 12 h, and 0, 100, 200, 300, 400, 500, 600, 700, and 800 μM H2O2 were added for 24 h, and MTT solution was added to each well for 4 h. The absorbance at 490 nm was measured to calculate the relative cell survival rate.

[0066] As the concentration of H2O2 increased, the cell survival rate gradually decreased, indicating cell damage under oxidative stress. H2O2-induced cell death can be effectively alleviated by TFC in a dose-dependent manner. In the concentration range of 100-500μM H2O2, 40μM TFC can effectively protect cells, and the cell survival rate remains above 95%. To further clarify the protective effect of TFC on cells, 500μM H2O2 was selected to stimulate cells, and 40μM TFC was used to rescue cells. The live / dead cell staining results showed that the number of fluorescent cells increased significantly after H2O2 stimulation, and the quantitative results showed that the cell death rate reached 36%. However, after TFC treatment, the number of live cells increased significantly, and the cells were in good condition ( Figure 5B, C). The above results indicate that TFC can provide significant protection for human dermal papilla cells under oxidative stress conditions.

[0067] It has been confirmed that in addition to alleviating oxidative stress, Cur also has the activity of inducing autophagy. Therefore, it is speculated that TFC can also protect cells by promoting autophagy of Cur. To verify this hypothesis, the nanoparticles (TF) without Cur encapsulation prepared in comparative example 1 were used as a control to systematically characterize cell autophagy. MDC (an eosinophilic fluorescent dye) was used as a probe to trace intracellular autophagosomes. After HHDPC was planted in a 6-well plate for overnight adherent culture, 40 μM TFC and an equivalent amount of TF were administered and incubated for 12 hours, and blank culture medium was added and incubated for 24 hours. The cells were collected and the cell autophagy structure was observed by transmission electron microscopy. In addition, the autophagy detection kit was used to detect autophagosomes according to the instructions, the fluorescence was observed by fluorescence microscopy, and the expression level of LC3 protein was analyzed by protein immunoblotting.

[0068] The increased accumulation of MDC-labeled vacuoles indicated enhanced autophagic activity, usually presenting as bright spots. Fluorescence microscopy results showed that a small number of bright green spots were observed in both the control group and the TF group, while a large number of aggregated bright green spots were observed in the TFC group ( Figure 5 D), indicating that TFC induced an increase in the level of cell autophagy. Subsequently, the intracellular autophagic structure was observed by transmission electron microscopy, and it was found that the control group and TF group contained a small amount of membrane structures containing contents, and the autophagic level was low. After TFC treatment, a large number of membrane structures containing contents were observed ( Figure 5 E), indicating an increase in the level of autophagy. LC3, as a marker protein for autophagy, is widely used in the detection of autophagy. When autophagy occurs, LC3 is cleaved to generate cytoplasmic LC3Ⅰ, and then LC3Ⅰ is connected with phosphatidylethanolamine to enhance the conversion to LC3Ⅱ. Therefore, the expression level of LC3Ⅱ is positively correlated with the level of autophagy. WB results showed that the conversion level of LC3Ⅰ to LC3Ⅱ was low in the control group and TF group. The conversion of LC3Ⅰ to LC3Ⅱ in the TFC group was enhanced, and the expression level of LC3Ⅱ protein was high ( Figure 5 F, G). The above experiments all confirmed that TFC can effectively activate cell autophagy through Cur in its structure, thereby playing a cytoprotective role.

[0069] Experimental Example 7 Evaluation of the efficacy of curcumin nano-coordination polymer microneedle TFC MN in the mouse AGA model

[0070] Preparation of curcumin nano-coordination polymer microneedles TFC MN: To achieve effective transdermal penetration of TFC, biocompatible HA and PVPK30 were used as carriers and TFC was loaded into soluble microneedles by a two-step molding method to break through the skin barrier ( Figure 6A). TFC MN is a 1×1 cm square patch with no bubbles or cracks in the base layer and contains a 10×10 microneedle array ( Figure 6 B). The digital micrograph shows that the microneedle tip is sharp, the needle body is intact and conical, the array is neatly arranged, and the drug is evenly distributed in the needle body ( Figure 6 C). The scanning electron microscopy image further clearly confirmed that the microneedle array was neat, the needle shape was complete and conical, the average length of the microneedles was 511 μm, and the needle distance was 218 μm ( Figure 6 D). Content determination showed that each microneedle contained 32μg TFC (Cur about 16.5μg). The biomechanical test force-displacement curve showed that no breaking turning point appeared in the TFC MN during continuous pressing ( Figure 6 E). When the displacement is 0.4 mm, the force on each needle reaches 1 N, which meets the minimum 0.09 N / needle required for puncturing the stratum corneum of the skin. After pressing the TFC MN on the ex vivo skin for 5 minutes, there are no microneedles on the backing layer ( Figure 6 F), indicating that the microneedles were completely dissolved. Obvious dot-like holes were visible on the mouse skin, and the insertion rate was 97% ( Figure 6 G). H&E sections show that the holes corresponding to the shape of the microneedles are inserted into the skin at a depth of about 115 μm, which is much greater than the thickness of the stratum corneum (~20 μm). Figure 6 H). The above results indicate that TFC MN has sufficient mechanical properties and strength to break through the stratum corneum barrier and achieve transdermal drug transport.

[0071] To evaluate the in vivo efficacy of TFC MN, the AGA model was established by topical application of testosterone. The administration schedule was as follows Figure 7 As shown in A, on day 0 of the experiment, a 2×2 cm area on the back of 7-week-old C57 / BL6 mice was depilated using a shaver and depilatory cream. The experiment was divided into a normal group, an AGA group, a minoxidil group, a blank microneedle group (Blank MN group), and a TFC MN group. Starting from day 1 of the experiment, except for the normal group, the other groups were treated with 0.1 mL / cm 2 A 0.2% testosterone solution (prepared with 50% ethanol) was applied to the depilatory area at a dose of 100 mg / kg for 28 consecutive days. The normal group did not receive any treatment. In the minoxidil group, 50 μL of 2.2% minoxidil gel was applied to the depilatory area on the back of each mouse until the 13th day after depilation. For the blank microneedle group and the TFC MN group, the mice were given blank microneedle patches and TFC MN patches every 3 days after depilation until the 13th day after depilation. Hair growth was recorded every 7 days, and the number of days required for the skin to begin to darken was recorded. On the 28th day after depilation, the length of the new hair of the mice was measured, and the coverage of the new hair of the mice was measured using Image J software.

[0072] The skin of the normal group turned pink after hair removal, and began to turn black after one week, and then gradually grew hair ( Figure 7 B). Changes in the hair follicle cycle can be preliminarily determined based on changes in skin color. Pink skin indicates that the hair follicles are in the resting phase. When the skin begins to darken and melanin is deposited, it indicates that the hair follicles begin to enter the growth phase. The earlier the skin turns darker, the earlier the hair grows. Therefore, the number of days it takes for the skin to darken after hair removal is an important indicator for evaluating hair growth.

[0073] The skin of the AGA model group was pink within three weeks after hair removal. In the fourth week, only a small area grew hair, and the rest of the skin remained pink, indicating that the model was successfully established. After the AGA model mice were treated with the positive control drugs Minoxidil and TFCMN, both showed a certain effect of promoting hair growth ( Figure 7 B). Interestingly, Blank MN also showed some efficacy, which may be attributed to the mechanical stimulation of the microneedles on the skin, which caused the regeneration of skin tissue and microvessels, creating favorable conditions for hair growth. The number of days required for the skin to darken after hair removal in the TFC MN group was about 12 days, which was significantly earlier than that in the AGA group (23.50 days), the Minoxidil group (16.50 days), and the Blank MN group (18.50 days) ( Figure 7 C). The results of hair coverage on the 28th day showed that the TFCMN group (85.48%), Blank MN group (75.31%), Minoxidil group (73.06%) and AGA group (38.28%) all had significant differences, and the TFC MN group showed the most obvious difference ( Figure 7 D). The results of hair length on day 28 showed that the hair length of the TFC MN group (8.49 mm) was significantly longer than that of the AGA model group (4.43 mm) and the Blank MN group (4.85 mm) ( Figure 7 E).

[0074] The growth of skin hair follicles and the changes in skin thickness caused by them can also reflect the changes in hair growth, so we evaluated them through H&E staining sections. On the 14th day after depilation, the skin of the depilated area on the back of each group of mice was immersed in 4% paraformaldehyde and liquid nitrogen, and pathological sections were made and observed after staining with H&E and DHE working solutions. In addition, the skin sections of each group fixed with paraformaldehyde were added with the corresponding primary antibodies (anti-Ki67, anti-CD31, anti-LC3Ⅱ) and incubated overnight at 4°C, and then the secondary antibodies were added. DAPI was used to stain the cell nucleus, and finally the anti-fluorescence quenching sealing agent was added to seal the sections and observed under a fluorescence microscope.

[0075] The results of H&E sections on the 14th day showed that the hair follicles in the AGA group were fewer and in the resting phase ( Figure 7F). The Minoxidil group and the Blank MN group contained some miniaturized telogen follicles, while the TFC MN group contained a large number of follicles, which were enlarged and in the growth phase, and the length of the follicles was significantly longer than that of the other groups ( Figure 7 F, G). In addition, the thickness of the epidermis in each treatment group increased significantly, and the thickness of the epidermis in the BlankMN and TFC MN groups was significantly higher than that in the Minoxidil group, which may be due to the mechanical stimulation of the microneedles accelerating epidermal regeneration ( Figure 7 H). The results of dermal thickness measurements showed that the dermal thickness of the TFC MN group also increased significantly ( Figure 7 I).

[0076] After confirming the efficacy, the therapeutic mechanism of TFC MN in vivo was further explored. The level of superoxide anion in skin tissue was measured by DHE probe. After being oxidized by superoxide anion, DHE probe can be embedded in DNA and emit red fluorescence. Therefore, the fluorescence intensity of DHE can reflect the level of superoxide anion. Compared with other groups, TFC MN effectively reduced the level of superoxide anion and relieved oxidative stress in the AGA site ( Figure 7 J, K). Ki67 is a cell proliferation marker that can be used to evaluate the proliferation activity of hair follicle cells. Compared with the AGA group, the cell proliferation of each treatment group was significantly enhanced, among which the TFC MN group had the most obvious effect ( Figure 7 J, L). CD31 is an endothelial cell marker that reflects the level of angiogenesis. Angiogenesis can increase blood flow to the site of AGA disease, enhance nutrient supply, and help hair regeneration. Similarly, among all treatment groups, TFC MN showed the strongest ability to promote angiogenesis ( Figure 7 J, M), which may be attributed to the improvement of the oxidative stress microenvironment of the hair follicles by nanoparticles, which is conducive to angiogenesis. In addition, the expression level of LC3Ⅱ in the TFC MN group was significantly upregulated, proving that it activated the autophagy activity of hair follicle cells and promoted the transformation of AGA hair follicles from the resting phase to the growth phase ( Figure 7 J, N).

[0077] The mechanism of action of TFC in treating AGA is shown in the figure Figure 8 As shown in C, the above evidence shows that TFC MN promotes hair regeneration in AGA mice by reducing oxidative stress and activating hair follicle cell autophagy, promoting hair follicle cell proliferation and microangiogenesis.

[0078] Experimental Example 8 Investigating the in vivo safety of TFC MN

[0079] Finally, the in vivo safety of TFC MN was evaluated. The body weight was recorded every 7 days after the mice were depilated. The mice were killed on the 28th day after depilation, and the heart, liver, spleen, lung, and kidney were fixed in 4% paraformaldehyde overnight and processed for H&E sections. In addition, about 0.1 g of skin, heart, liver, spleen, lung, and kidney tissues were taken, 1 mL of aqua regia was added, and the tissues were heated in a 60°C water bath overnight to digest the tissues. The digestion solution was centrifuged at 1000g for 10 minutes, the supernatant was taken and diluted with an appropriate amount of ultrapure water, and the iron content in the tissues was analyzed by ICP-OES.

[0080] During the experimental period, each treatment group had no significant effect on the body weight of mice, and no significant damage to the main organs ( Figure 7 O, P). In addition, no iron accumulation was found in various tissues and organs after TFC MN treatment ( Figure 7 Q). The above results demonstrate the in vivo safety of TFC MN.

[0081] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A curcumin nano-coordination polymer for treating androgenic alopecia, characterized in that: Including curcumin, Fe 3+ , tannic acid, the curcumin and tannic acid are interlocked, the Fe 3+ It forms metal coordination bonds with the phenolic hydroxyl and carbonyl groups in flavin and tannic acid.

2. The curcumin nano-coordination polymer according to claim 1, characterized in that The encapsulation rate of the curcumin is 100%, and the drug loading is 52%.

3. A method for preparing a curcumin nano-coordination polymer for treating androgenic alopecia as claimed in claim 1, characterized in that: The following steps are involved: S1. Tannic acid, curcumin and Fe were stirred vigorously. 3+ Add them into 20% acetone solution in sequence and sonicate them immediately; S2. Use a rotary evaporator to remove acetone from the solution prepared in S1, collect the flavin nano-coordination polymer by high-speed centrifugation, and disperse it evenly with ultrapure water.

4. The preparation method according to claim 2, characterized in that: In step S1, tannic acid, curcumin and Fe 3+ The concentration ratio is 8:1:

2.

5. The preparation method according to claim 2, characterized in that: The temperature of the high-speed centrifugation in step S1 is 4° C., the speed is 16000 rpm, and the time is 20 min.

6. Use of a nano-coordination polymer of curcumin for treating androgenic alopecia as claimed in any one of claims 1 to 2 or a nano-coordination polymer of curcumin for treating androgenic alopecia obtained by the preparation method of any one of claims 3 to 5 in preparing a drug for treating androgenic alopecia, characterized in that: The drug is a microneedle.

7. The use according to claim 6, characterized in that: The preparation method of the microneedle is: using HA and PVP K30 as carriers, and loading the curcumin nano-coordination polymer into the soluble microneedle through a two-step molding method.

8. The use according to claim 6, characterized in that: The microneedle is a square patch of 1×1 cm in size, comprising a 10×10 microneedle array, with an average length of 511 μm and a needle distance of 218 μm, and each microneedle contains 32 μg of curcumin nano-coordination polymer.

9. The use according to claim 6, characterized in that: The method of using the microneedle is: press the microneedle on the skin for 5 minutes.

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