Metformin-loaded pegylated two-dimensional phosphorene nanosheet as well as preparation method and application thereof

Abstract

The invention discloses a metformin-loaded pegylated two-dimensional phosphorene nanosheet as well as a preparation method and an application of the metformin-loaded pegylated two-dimensional phosphorene nanosheet. The preparation method comprises the following steps: preparing two-dimensional phosphorene nanosheets from black phosphorus crystals through a liquid phase stripping method; the preparation method comprises the following steps: mixing polyethylene glycol amino with a two-dimensional phosphorene nanosheet to prepare a pegylated two-dimensional phosphorene nanosheet; the preparation method comprises the following steps: mixing metformin with the pegylated two-dimensional phosphorene nanosheet, and removing free metformin, so as to obtain the metformin-loaded pegylated two-dimensional phosphorene nanosheet. The metformin-loaded pegylated phosphorene nanosheet disclosed by the invention has transdermal permeation and anti-oxidative stress capabilities, can deliver a drug into skin tissues, induces angiogenesis of hair follicles and promotes hair regeneration of an androgenetic alopecia animal model by removing excessive ROS (reactive oxygen species) in the surrounding environment of the hair follicles, and is degradable, so that the metformin-loaded pegylated phosphorene nanosheet disclosed by the invention has a good application prospect. The compound is safe and low in toxicity, and has good clinical application prospect and development value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical nanomaterials, and in particular to a PEGylated two-dimensional phosphorene nanosheet loaded with metformin, and a preparation method and application thereof. Background Art

[0002] Androgenetic Alopecia (AGA), also known as seborrheic alopecia, is the most common progressive hair loss. The clinical manifestations are male hair loss involving the forehead, bilateral frontal corners or temples with a receding hairline, known as a "horseshoe" pattern. Female hair loss is mainly diffusely sparse and thin on the top of the head. The pathogenesis of AGA is still unclear, and it is mainly believed to be related to genetics, immune regulation and hormone metabolism disorders. One of its pathological characteristics is that the dermal papilla cells (DPCs) located below the hair follicle stem cells (HFSCs) have a high level of oxidative stress, which increases the sensitivity of androgens to DPCs in the hair loss area of ​​AGA patients. Under the action of type II 5α-reductase, testosterone (TS) is converted into dihydrotestosterone (DHT) to stimulate the hair papilla and cause premature aging of DPCs. Premature DPCs secrete a large amount of negative transforming growth factor β (TGFβ) in response to the increase of DHT, or secrete TGFβ through the paracrine pathway, which promotes the apoptosis of vascular endothelial cells in the hair papilla, causing blood vessels to atrophy, and ultimately leading to hair loss. Therefore, the development of a drug to improve the disorder of the hair follicle microenvironment, that is, inhibiting the oxidative stress of DPCs and promoting their angiogenesis, can provide a new idea for the treatment of AGA.

[0003] Metformin (Met) is a first-line drug for the treatment of type 2 diabetes, but some studies have reported that metformin can promote hair regeneration. In a mouse model of hair loss, metformin activates autophagy, promotes angiogenesis, and accelerates hair regeneration in hair loss mice. Black phosphorus (BP), also known as phosphorene, is a rising star in two-dimensional materials. It has antioxidant properties and can promote hair regeneration by removing excess reactive oxygen around hair follicles and improving the hair follicle microenvironment. It also has a large specific surface area, high drug loading rate, and good biocompatibility. It can also be used as an excellent drug delivery carrier. However, black phosphorus has lone pairs of electrons, is unstable at room temperature, and is easily degraded. Therefore, it is necessary to develop a transdermal drug delivery preparation that can improve drug stability and drug bioavailability. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing metformin-loaded PEGylated two-dimensional phosphorene nanosheets.

[0005] Another object of the present invention is to provide a PEGylated two-dimensional phosphorene nanosheet loaded with metformin.

[0006] Another object of the present invention is to provide an application of the above-mentioned PEGylated two-dimensional phosphorene nanosheet loaded with metformin.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for preparing a PEGylated two-dimensional phosphorene nanosheet loaded with metformin, comprising the following steps:

[0009] S1, mixing black phosphorus crystals (BP) with a sodium hydroxide N-methylpyrrolidone (NMP) solution to prepare two-dimensional phosphorene nanosheets by liquid phase exfoliation;

[0010] S2, mixing the polyethylene glycol amino group with the two-dimensional phosphorene nanosheet obtained in step S1, removing excess polyethylene glycol amino group, and obtaining a PEGylated two-dimensional phosphorene nanosheet;

[0011] S3, mixing metformin (Met) with the PEGylated two-dimensional phosphorene nanosheet obtained in step S2, removing free metformin, and obtaining a PEGylated two-dimensional phosphorene nanosheet loaded with metformin.

[0012] As a preferred embodiment of the method for preparing the nanosheets of the present invention, the method for preparing the saturated sodium hydroxide solution in N-methylpyrrolidone (NMP) described in step S1 comprises the following steps: weighing sodium hydroxide powder and dissolving it in N-methylpyrrolidone at a ratio of sodium hydroxide: N-methylpyrrolidone = 1 to 5 g: 50 mL, preferably at a ratio of 5 g: 50 mL, stirring and mixing to obtain a saturated sodium hydroxide solution in N-methylpyrrolidone (NMP).

[0013] As a preferred embodiment of the preparation method of the present invention, step S1 specifically includes the following steps: mixing black phosphorus crystals with a saturated sodium hydroxide solution in N-methylpyrrolidone (NMP) to obtain a black phosphorus dispersion, ultrasonicating in a water bath at 10-20°C for 12±2h, centrifuging, taking the supernatant, ultrasonicating in an ice bath again for 24±5h, centrifuging a second time, taking the supernatant, centrifuging again to collect the precipitate, and drying to obtain a two-dimensional phosphorene nanosheet; as a more preferred embodiment, the centrifugation is at a speed of 2000±500rpm and at 4°C for 25±5min; the secondary centrifugation is at a speed of 2000±500rpm and at 4°C for 25±5min; the secondary centrifugation is at a speed of 12000±1000rmp and at 4°C for 30±5min.

[0014] Furthermore, the ratio of the black phosphorus crystals to the saturated sodium hydroxide solution in N-methylpyrrolidone is black phosphorus crystals: saturated sodium hydroxide solution in N-methylpyrrolidone = 2-4 mg: 5 mL; preferably 3 mg: 5 mL.

[0015] As a preferred embodiment of the preparation method of the present invention, the sodium hydroxide described in step S1 is saturated sodium hydroxide.

[0016] As a preferred embodiment of the preparation method of the present invention, the size of the two-dimensional phosphorene nanosheet is 100-200 nm and the thickness is 5-7 nm.

[0017] As a preferred embodiment of the preparation method of the present invention, the molecular weight of the polyethylene glycol of the polyethylene glycol amino group described in step S2 is 2000-8000, and more preferably 2000.

[0018] As a preferred embodiment of the method for preparing the nanosheets of the present invention, the mass ratio of the polyethylene glycol amino group to the two-dimensional phosphorene nanosheets in step S2 is 3 to 7:1, more preferably 5:1.

[0019] As a preferred embodiment of the preparation method described in the present invention, step S2 specifically includes the following steps: dispersing polyethylene glycol amino groups in distilled water to obtain a polyethylene glycol amino dispersion; dispersing the two-dimensional phosphorene nanosheets obtained in step S1 in distilled water to obtain a two-dimensional phosphorene nanosheet dispersion; mixing the polyethylene glycol amino dispersion with the two-dimensional borophene nanosheet dispersion as needed, stirring, centrifuging, and washing to obtain PEGylated two-dimensional phosphorene nanosheets.

[0020] Furthermore, the concentration of the polyethylene glycol amino dispersion is 5±1 mg / mL.

[0021] Furthermore, the concentration of the two-dimensional phosphorene nanosheet dispersion is 1±0.5 mg / mL.

[0022] Furthermore, the volume ratio of the polyethylene glycol amino dispersion to the two-dimensional phosphorene nanosheet dispersion is 1-2:1-2.

[0023] Furthermore, the stirring is carried out at room temperature, away from light, and at 600±100 rpm for 4±1 h.

[0024] Furthermore, the centrifugation is performed at 12000±1000 rpm for 30±5 min.

[0025] Furthermore, the cleaning is cleaning with deoxygenated ultrapure water.

[0026] As a preferred embodiment of the preparation method of the present invention, the mass ratio of metformin to PEGylated two-dimensional phosphorene nanosheets in step S3 is 15-25:1, more preferably 20:1.

[0027] As a preferred embodiment of the preparation method described in the present invention, step S3 specifically includes the following steps: dispersing metformin in distilled water to obtain a metformin dispersion; dispersing the PEGylated two-dimensional phosphorene nanosheets obtained in step S2 in deoxygenated ultrapure water to obtain a PEGylated two-dimensional phosphorene nanosheet dispersion; mixing the metformin dispersion with the PEGylated two-dimensional phosphorene nanosheet dispersion as needed, stirring, centrifuging, and washing to obtain PEGylated two-dimensional phosphorene nanosheets loaded with metformin.

[0028] Furthermore, the concentration of the metformin dispersion is 1±0.5 mg / mL.

[0029] Furthermore, the concentration of the PEGylated two-dimensional phosphorene nanosheet dispersion is 50±10 μg / mL.

[0030] Furthermore, the volume ratio of the metformin dispersion to the PEGylated two-dimensional phosphorene nanosheet dispersion is 1-2:1-2.

[0031] Furthermore, the stirring is carried out at room temperature, away from light, and at 600±100 rpm for 6±1 h.

[0032] Furthermore, the centrifugation is performed at 12000±1000 rpm for 30±5 min.

[0033] Furthermore, the cleaning is cleaning with deoxygenated ultrapure water.

[0034] As a preferred embodiment of the preparation method of the present invention, steps S2 and S3 are both performed under light-proof conditions.

[0035] In a second aspect, the present invention also provides a PEGylated two-dimensional phosphorene nanosheet loaded with metformin prepared by the above preparation method.

[0036] In a third aspect, the present invention also provides the use of the above-mentioned metformin-loaded PEGylated two-dimensional phosphorene nanosheets in the preparation of drugs for treating skin diseases.

[0037] As a preferred embodiment of the use of the nanosheets of the present invention in the preparation of a drug for treating skin diseases, the skin disease is at least one of androgenic alopecia, infection, burns, mechanical trauma, and diabetic dermatosis.

[0038] As a preferred embodiment of the use of the composite hydrogel of the present invention in the preparation of a drug for treating skin diseases, the drug is an external dressing.

[0039] The present invention improves the dispersibility, stability and biocompatibility of two-dimensional phosphorene nanosheets by modifying the two-dimensional phosphorene nanosheets with polyethylene glycol.

[0040] Compared with the prior art, the present invention has the following advantages and effects:

[0041] 1. The two-dimensional phosphorene nanosheets (BP NSs) of the present invention can efficiently remove various types of oxygen free radicals and eventually degrade them into phosphite ions and phosphates that are harmless to organisms. They have excellent biocompatibility and are beneficial to the treatment of diseases.

[0042] 2. The antioxidant property of the two-dimensional phosphorene nanosheets (BP NSs) of the present invention can promote the regeneration of new blood vessels by removing excessive reactive oxygen around hair follicles and cooperating with metformin, thereby improving the microvascular system around hair follicles, reshaping the hair follicle microenvironment, inducing hair regeneration, and being beneficial to anti-AGA.

[0043] 3. The two-dimensional phosphorene nanosheets (BP NSs) of the present invention can reduce the surface energy of nanoparticles, neutralize their surface charge, increase the steric hindrance between particles, prevent nanoparticles from agglomerating, and increase the stability of BP through modification of the amino group of the polymer polyethylene glycol.

[0044] 4. The preparation method of the metformin-loaded PEGylated two-dimensional phosphorene nanosheets of the present invention has the advantages of simple steps and low cost, and can be used for large-scale industrial production.

[0045] 5. The metformin-loaded PEGylated two-dimensional phosphorene nanosheets provided by the present invention are expected to be used as a drug delivery strategy in the multi-directional and multi-dimensional nanobiomedicine field, such as androgenic alopecia, diabetes, infection, burns, mechanical trauma and other diseases mainly with skin lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1SEM, TEM and AFM characterization images of the two-dimensional phosphorene nanosheets (BP NSs) prepared in Example 1; wherein, (a) is the SEM image of BP NSs (scale bar = 200 μm), (b) is the TEM image of BP NSs (scale bar = 50 μm), and (c) is the AFM image of BP NSs (scale bar = 200 μm).

[0047] Figure 2 These are the XRD and XPS characterization graphs of the two-dimensional phosphorene nanosheets prepared in Example 1; wherein (a) is the XRD graph of BP NSs, and (b) is the XPS graph of BP NSs.

[0048] Figure 3 The particle size distribution diagram of the two-dimensional phosphorene nanosheets (BP NSs), PEGylated two-dimensional phosphorene nanosheets (BP-PEG), and PEGylated two-dimensional phosphorene nanosheets loaded with metformin (BP-PEG-Met) prepared in Example 1.

[0049] Figure 4 Zete potential diagrams of BP NSs, BP-PEG and BP-PEG-Met prepared in Example 1.

[0050] Figure 5 This is the UV-visible spectrum of BP NSs and BP-PEG-Met prepared in Example 1.

[0051] Figure 6 This is the TEM characterization picture of BP-PEG-Met prepared in Example 1.

[0052] Figure 7 This is the element mapping diagram of BP-PEG-Met prepared in Example 1.

[0053] Figure 8 This is a graph showing the stability evaluation of BP-PEG-Met prepared in Example 1 after being placed for 7 days.

[0054] Fig. 9 These are the UV-visible spectra of BP NSs and BP-PEG-Met prepared in Example 1 within seven days; wherein, (a) is the UV-visible spectrum of BP NSs within seven days, and (b) is the UV-visible spectrum of BP-PEG-Met within seven days.

[0055] Fig.10 This is the standard curve of Met at 233 nm prepared in Example 1.

[0056] Fig.11 This is a graph showing the in vitro drug release results of the Met solution and BP-PEG-Met prepared in Example 1.

[0057] Fig.12 This is a graph showing the in vitro skin permeation results of Met and BP-PEG-Met prepared in Example 1.

[0058] Fig.13 This is a graph showing the drug skin retention results of Met and BP-PEG-Met prepared in Example 1.

[0059] Fig.14 This is a graph showing the ability of BP NSs and BP-PEG-Met prepared in Example 1 to scavenge ·OH at different concentrations.

[0060] Fig.15 The BP NSs and BP-PEG prepared in Example 1 were used to remove O at different concentrations of BP. 2- Capability results graph.

[0061] Fig.16 This is a graph showing the ability of different concentrations of BP in BP NSs and BP-PEG-Met prepared in Example 1 to scavenge ABTS.

[0062] Fig.17 This is a graph showing the ability of different concentrations of BP in BP NSs and BP-PEG-Met prepared in Example 1 to scavenge DPPH.

[0063] Fig.18 These are the cytotoxicity test results of BP NSs, Met and BP-PEG-Met prepared in Example 1; wherein, (a) is the cytotoxicity test result of BP NSs, (b) is the cytotoxicity test result of Met, and (c) is the cytotoxicity test result of BP-PEG-Met.

[0064] Fig.19 This is a graph showing the live-dead staining experimental results of BP NSs, Met and BP-PEG-Met prepared in Example 1 on human skin fibroblasts (HSF).

[0065] Fig. 20 This is a graph showing the experimental results of using reactive oxygen species probes of different concentrations of BP NSs prepared in Example 1 to test the antioxidant effect of BP on HSF cells.

[0066] Fig.21 This is a graph showing the results of a flow cytometry experiment of different concentrations of BP NSs prepared in Example 1 to detect the antioxidant capacity of BP on HSF cells.

[0067] Fig. 22The results of the effects of the JC-1 probes of Met and BP-PEG-Met prepared in Example 1 on the mitochondrial membrane of HSF cells are shown in the figure; wherein, (a) is a fluorescence image of BP on mitochondrial membrane potential (scale bar = 100 μm), and (b) is a quantitative image of mitochondrial membrane potential (n = 3) **p<0.01, ***p<0.001 vs. Con.

[0068] Fig.23 This is a graph showing the concentration-dependent uptake analysis of BP NSs prepared in Example 1 on HSF cells (scale bar = 25 μm).

[0069] Fig.24 This is a graph showing the time-dependent uptake analysis of BP NSs prepared in Example 1 on HSF cells (scale bar = 25 μm).

[0070] Fig.25 The uptake mechanism of BP prepared in Example 1 on HSF cells; wherein, (a) is a fluorescent image of BP uptake by HSF cells (scale bar = 25 μm), and (b) is a quantitative analysis diagram of BP uptake by HSF cells (n = 3) **p<0.01, ***p<0.001 vs. Con.

[0071] Fig.26 The intracellular distribution of BP prepared in Example 1 in HSF cells; wherein, (a) is a fluorescence image of co-localization of BP NSs with endoplasmic reticulum, lysosomes and mitochondria, and (b) is the co-localization coefficient of BP NSs with endoplasmic reticulum, lysosomes and mitochondria (n=3).

[0072] Fig. 27 Schematic diagram of the administration of Met and BP-PEG-Met prepared in Example 1 in animal experiments.

[0073] Fig.28 This is a graph showing the experimental results of the effects of Met and BP-PEG-Met prepared in Example 1 on animal hair growth after administration.

[0074] Fig.29 The figures are the results of evaluating the quality of new hair of animals after treatment with Met and BP-PEG-Met prepared in Example 1; wherein, (a) is a statistical graph of the coverage area of ​​new hair (n=5), (b) is a statistical graph of the weight of new hair (n=5), (c) is a statistical graph of the length of new hair (n=5), and (d) is a SEM characterization graph of new hair (**p<0.01, ***p<0.001 vs. TS).

[0075] Fig.30The results of the effects of Met and BP-PEG-Met prepared in Example 1 on the hair follicle cycle of animals after treatment (scale bar = 200 μm); (a) is the HE staining result, and (b) is the statistical diagram of the number of hair follicles.

[0076] Fig.31 This is a diagram showing the results of Ki67 and PCNA immunohistochemical staining experiments on the proliferation of hair follicle cells in animals after administration of Met and BP-PEG-Met prepared in Example 1 (scale bar = 200 μm).

[0077] Fig.32 The results of immunofluorescence staining of 4-HNE, a lipid peroxidation product in skin tissue, for the antioxidant effect on the hair follicle microenvironment of animals after administration of Met and BP-PEG-Met prepared in Example 1; wherein, (a) is a fluorescence image of 4-HNE in skin tissue (scale bar = 50 μm), and (b) is a fluorescence quantitative statistical graph of 4-HNE in skin tissue (n = 3) **p < 0.01 vs. TS.

[0078] Fig.33 This is a statistical graph of the content of malondialdehyde (MDA), a lipid peroxidation product, in skin tissue on day 28 after administration of Met and BP-PEG-Met prepared in Example 1 (n=5). ***p<0.001 vs. TS.

[0079] Fig.34 The results of immunofluorescence analysis of vascular endothelial growth factor (VEGF) in skin tissue after administration of Met and BP-PEG-Met prepared in Example 1; wherein, (a) is an immunofluorescence image of vascular endothelial growth factor (VEGF) in skin tissue (scale bar = 50 μm), and (b) is a fluorescence quantitative statistical graph of VEGF in skin tissue (n = 3) **p<0.01, ***p<0.001 vs. TS.

[0080] Fig.35 The results of CD31 immunofluorescence analysis of VEGF after administration of Met and BP-PEG-Met prepared in Example 1; wherein, (a) is the CD31 immunofluorescence image of VEGF (scale bar = 50 μm), and (b) is the quantitative statistical graph of CD31 fluorescence of VEGF (n = 3) ***p < 0.001 vs. TS.

[0081] Fig.36 This is a graph showing the changes in animal body weight during the treatment with Met and BP-PEG-Met prepared in Example 1.

[0082] Fig.37 This is a graph showing the results of blood biochemical analysis of animals after administration of Met and BP-PEG-Met prepared in Example 1.

[0083] Fig.38 This is the HE staining result of pathological detection of animal organs after administration of Met and BP-PEG-Met prepared in Example 1 (scale bar = 200 μm). DETAILED DESCRIPTION

[0084] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0085] Sources of materials in the following embodiments: black phosphorus crystals: purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd.; metformin hydrochloride: purchased from Nantong Feiyu Biotechnology Co., Ltd.; PEG-NH2 (MW: 2000): purchased from Xi'an Ruixi Biotechnology Co., Ltd.; N-methylpyrrolidone: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; sodium hydroxide: purchased from Shanghai McLean Biochemical Technology Co., Ltd.; dialysis bag MD34 (3500D): purchased from Beijing Solebow Technology Co., Ltd.; FITC-PEG-NH2: purchased from Xi'an Ruixi Biotechnology Co., Ltd.; fetal bovine serum: purchased from Thermo Fisher Scientific, USA; pH7.4 PBS: purchased from the United States FGF-2: purchased from Wuhan Pronocell Life Science Co., Ltd.; recombinant human insulin solution: purchased from Wuhan Pronocell Life Science Co., Ltd.; L-glutamine: purchased from Thermo Fisher Scientific, USA; sodium pyruvate: purchased from Thermo Fisher Scientific, USA; CCK8 kit: purchased from Tongren Chemical Research Institute; Lyso-Tracker: purchased from Shanghai MedChemExpress; ER-Tracker: purchased from Shanghai Beibo Biotechnology Co., Ltd.; Mito-Tracker: purchased from Thermo Fisher Scientific; live cell / dead cell double staining kit: purchased from Beijing Annolon Biotechnology Co., Ltd.; DMEM / F12: purchased from Thermo Fisher Scientific, USA; anhydrous ethanol: purchased from Guangzhou Bell Biotechnology Co., Ltd.; xylene: purchased from Guangzhou Bell Biotechnology Co., Ltd.; 4% paraformaldehyde: purchased from Beijing White Shark Easy Technology Co., Ltd.; Ki67 antibody: purchased from Shanghai Abcam Trading Co., Ltd.; PCNA antibody: purchased from Cell Signaling Technology (CST), USA; CD31 antibody: purchased from Shanghai Abcam Trading Co., Ltd.; VEGF antibody: purchased from Beijing Bioson Biotechnology Co., Ltd.; 4-HNE antibody: purchased from Shanghai Abcam Trading Co., Ltd.; depilatory cream: purchased from China Reckitt Benckiser Home Chemical Co., Ltd.; DAB colorimetric solution: purchased from Fuzhou Maixin Biotechnology Co., Ltd.; minoxidil: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; testosterone: purchased from Beijing Solebow Technology Co., Ltd.; hematoxylin-eosin staining: purchased from Beijing White Shark Easy Technology Co., Ltd.; sodium citrate repair solution: purchased from Beijing White Shark Easy Technology Co., Ltd.; MDA kit: purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; goat serum: purchased from Beijing Solebow Technology Co., Ltd.; Leica 819 blade: purchased from Ernst Leitz, Germany; histochemistry rabbit two-step kit: purchased from Zhongshan Jinqiao Biotechnology Co., Ltd.; antibody diluent: purchased from Shanghai Gene Technology Co., Ltd.; neutral gum: purchased from Beijing White Shark Easy Technology Co., Ltd.

[0086] Embodiment 1:

[0087] The preparation of PEGylated two-dimensional phosphorene nanosheets loaded with metformin comprises the following steps:

[0088] S1. Mix 30 mg of black phosphorus crystals with 50 ml of saturated sodium hydroxide NMP (N-methylpyrrolidone) solution to obtain a black phosphorus dispersion, and prepare two-dimensional phosphorene nanosheets by liquid phase exfoliation. The liquid phase exfoliation method is as follows: ultrasonicate the black phosphorus dispersion at a 1:1 drop-to-space ratio probe in a 10°C water bath for 12 hours, centrifuge at 2000 rpm at 4°C for 20 minutes, take the supernatant, ultrasonicate again at a 10°C water bath at a 1:1 drop-to-space ratio probe for 24 hours, centrifuge at 2000 rpm at 4°C for 20 minutes, take the supernatant, centrifuge at 12000 rpm at 4°C for 30 minutes, collect the precipitate, and freeze-dry to obtain two-dimensional phosphorene nanosheets (BP NSs).

[0089] S2, 10mg polyethylene glycol amino (polyethylene glycol molecular weight is 2000) and 2mL distilled water are formulated into 5mg / mL polyethylene glycol amino dispersion; the two-dimensional phosphorene nanosheets obtained in step S1 are centrifuged at 12000rpm for 30min, the supernatant is removed, and after washing three times with deoxygenated ultrapure water, 1mg of the precipitate is taken, and 1mL of distilled water is added to prepare a two-dimensional phosphorene nanosheet dispersion, the polyethylene glycol amino dispersion and the two-dimensional borophene nanosheet dispersion are mixed in a volume ratio of 1:1, and stirred at room temperature, protected from light, and 600rpm for 4h to make the dispersions uniformly mixed, centrifuged at 12000rpm for 30min to remove the supernatant, washed three times with deoxygenated ultrapure water, and the precipitate was resuspended in deoxygenated ultrapure water to a fixed volume of 1mg / mL to obtain PEGylated two-dimensional phosphorene nanosheets (BP-PEG), which were stored at 4°C;

[0090] S3. Prepare metformin into a 1 mg / mL metformin solution with distilled water, prepare the PEGylated two-dimensional phosphorene nanosheets obtained in step S2 into a 50 μg / mL dispersion, mix the metformin solution and the PEGylated two-dimensional phosphorene nanosheet dispersion in a volume ratio of 1:1, stir at room temperature, avoid light, and 600 rpm for 6 hours, centrifuge at 12000 rpm, remove the supernatant, wash three times with deoxygenated ultrapure water, and resuspend in 1 mL of deoxygenated ultrapure water to obtain metformin-loaded PEGylated two-dimensional phosphorene nanosheets (BP-PEG-Met), which are stored at 4°C.

[0091] Comparative Example 1:

[0092] The method for preparing PEGylated two-dimensional phosphorene nanosheets loaded with metformin is different from Example 1 in that in step S1, 20 mg, 30 mg, 50 mg, and 70 mg of black phosphorus crystals are respectively added to 50 mL of saturated sodium hydroxide NMP (N-methylpyrrolidone) solution to obtain a black phosphorus dispersion. Among the two-dimensional phosphorene nanosheets prepared by liquid phase exfoliation, the nanosheets prepared by 30 mg of black phosphorus crystals and 50 mL of saturated sodium hydroxide NMP (N-methylpyrrolidone) solution have better effect and higher black phosphorus loading rate.

[0093] Effect Example 1: Characterization of BP-PEG-Met

[0094] 1. The two-dimensional phosphorene nanosheets (BPNSs) prepared in Example 1 were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscope (AFM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Figure 1 As shown in Figure 2, BP NSs were observed to be a lamellar structure with a smooth surface under SEM and TEM, and its particle size distribution was about 200 nm, while the results of AFM showed that the thickness of the prepared BP NSs was less than 7 nm, which indicated the successful preparation of single-layer BP NSs; Figure 2 As shown in the figure, the characteristic crystal planes 020 and 040 of BPNSs are located at 2θ=16.7° and 34.7° respectively; the XPS measurement data show that there are two peaks at 133.83eV and 138.98eV, representing the P2p 2 / 3 and P2p 1 / 2 The binding energy of BP NSs was slightly red-shifted compared with that of BP crystal, confirming that the monolayer BP NSs prepared by liquid phase exfoliation did not change the original crystal structure of BP crystal, which indicated the successful preparation of BP NSs.

[0095] 2. The particle size, PDI and Zeta potential of BP NSs, BP-PEG and BP-PEG-Met prepared in Example 1 were measured. The results are as follows: Figure 3 and Figure 4As shown, the particle size of BP is 198.16±9.23nm, the PDI coefficient is 0.22, and the Zeta potential is -26.25±0.10mV; after modification with PEG-NH2, the particle size of BP-PEG increases to 217.76±1.58nm, the PDI coefficient is 0.21, and the Zeta potential is -12.08±2.66mV; when BP-PEG is loaded with metformin, the particle size of BP-PEG-Met increases again to 224.5±1.32nm, the PDI coefficient is 0.13, and the Zeta potential increases to -19.53±3.12mV; the changes in particle size and Zeta potential indicate that Met is successfully loaded on the BP nanosheets, and the PDI coefficient is less than 0.2, indicating that the prepared BP-PEG-Met has a concentrated particle size distribution.

[0096] 3. The BP NSs and BP-PEG-Met prepared in Example 1 were tested by UV-visible absorption spectroscopy. The results are as follows: Figure 5 As shown, the UV-visible spectrum of BP NSs is a smooth curve with absorption between 200-800nm; BP-PEG-Met not only retains the absorption region of BP NSs at 200-800nm, but also has a characteristic absorption peak of Met at 233nm, confirming the successful construction of the BP-PEG-Met drug delivery system.

[0097] 4. The BP-PEG-Met prepared in Example 1 was characterized by TEM and Mapping analysis. Figure 6 As shown in Figure 2, it can be observed that small black round particles of BP-PEG-Met are evenly distributed on the monolayer of BPNSs, such as Figure 7 As shown in Figure 2, three different elements are distributed in the entire BP-PEG-Met system, among which the P element comes from BP ( Figure 7 c), N element comes from Met( Figure 7 d), confirming that Met was successfully loaded onto BPNSs. In addition to P and N elements, O elements ( Figure 7 b), which indicates that BP is partially oxidized.

[0098] Effect Example 2: Stability Evaluation of BP-PEG-Met

[0099] 1. The BPNSs and BP-PEG-Met suspensions prepared in Example 1 were diluted with deoxygenated ultrapure water, and then left at room temperature for one, three, five, and seven days before measuring their particle sizes, PDI, and UV-visible absorption spectra. Figure 8As shown in the figure, with the increase of observation days, the particle size of BP-PEG-Met increased, but no agglomeration occurred; and the PDI coefficients of the seven days were all less than 0.3, indicating that the particle size distribution range of BP-PEG-Met was narrow; it was confirmed that the BP-PEG-Met system was uniform and had the potential for biomedical applications. Fig. 9 As shown, with the increase of observation days, the OD value of BP decreased significantly, indicating that a large amount of BP was degraded; while the OD value of BP-PEG-Met did not change significantly within seven days, indicating that only part of BP was degraded, and the degradation rate was lower than that of BP, confirming that the constructed BP-PEG-Met system has good stability.

[0100] Effect Example 3: Performance Evaluation of BP-PEG-Met

[0101] 1. The Met solution prepared in Example 1 and BP-PEG-Met were subjected to drug release test. Fig.10 It can be seen that the standard curve of Met at 233nm is y=0.077x-0.0017, R 2 is 0.9997; the drug release results are Fig.11 It can be seen that the drug release of Met is very fast, with an explosive release of 79.18±4.05% within 1 hour, followed by a slow release, and the cumulative release after 6 hours reached 94.33±0.07%; the cumulative release of BP-PEG-Met reached 86.45±5.45% within 1 hour, and the cumulative drug release after 2.5 hours reached 92.81±4.96%; it was confirmed that black phosphorus did not affect the release of Met, and BP-PEG-Met had good drug sustained-release properties.

[0102] 2. The Met solution prepared in Example 1 and BP-PEG-Met were subjected to in vitro skin permeation tests. The results are as follows: Fig.12 As shown in Figure 2, there was no difference in the in vitro cumulative permeation of Met and BP-PEG-Met before 2 h. After 8 h, the cumulative permeation amount of Met per unit area was 305.05±0.19 μg / cm 2 The cumulative permeation per unit area of ​​BP-PEG-Met was 390.55±34.27μg / cm 2 , confirming that under the action of BP, the BP-PEG-Met drug delivery system can enter the skin through a transcellular pathway, increasing the in vitro skin permeability of Met; Fig.13 It can be seen that the skin retention amount of the Met group was 27.84±0.02μg, and the skin retention amount of the BP-PEG-Met group was 35.06±0.07μg, indicating that BP-PEG-Met increased the retention amount of Met in the skin, confirming its better skin penetration effect.

[0103] 3. Scavenging of OH and O by BP and BP-PEG-Met prepared in Example 1 2- , ABTS, and DPPH capabilities were tested, and the results were as follows Fig.14 , Fig.15 , Fig.16 , Fig.17 As shown in Figure 2, BP and BP-PEG-Met at concentrations of 5-80 μg / mL both have antioxidant capacity, and with the increase of BP concentration, the antioxidant capacity of ABTS, DPPH, ·OH, and O 2- The scavenging ability of BP-PEG-Met was also enhanced; and compared with BP, BP-PEG-Met had better antioxidant effect.

[0104] Effect Example 4: In vitro evaluation of BP-PEG-Met

[0105] The BP-PEG-Met prepared in Example 1 was evaluated using human skin fibroblast cell line (HSF) as a test object.

[0106] Human skin fibroblasts were cultured in DMEM / F12 medium containing 10% FBS, 100mM L-glutamine, 100mM sodium pyruvate, 1mg / mL hydrocortisone, 10mg / mL insulin, and 5ng / mL bFGF. The culture conditions were 37°C, 5% CO2, and 95% relative humidity. Fresh medium was replaced every 24h. Cells were observed under a microscope to grow to 70-80% before passage. The following cell culture conditions are the same as those here unless otherwise specified.

[0107] 1. Safety evaluation of BP-PEG-Met prepared in Example 1 at the HSF cell level: HSF cells were cultured at 1×10 3 Cells were inoculated at a density of 100 μg / mL in each well of a 96-well plate and cultured for 24 h. The culture medium was discarded and different groups were treated respectively: 1. Control group; 2. Different concentrations of Met (2.5, 5, 10, 20, 40 mM) were added; 3. Different concentrations of BP (1.25, 2.5, 5, 10, 20 μg / mL) were added; 4. Different concentrations of BP-PEG-Met (1.25, 2.5, 5, 10, 20 μg / mL) were added. After incubation for 24 h, the cell viability was detected using the CCK8 kit. The results are shown in Figure 2. Fig.18 As shown, BP has no cytotoxicity to HSF cells in the concentration range of 1.25-20 μg / mL; Met has no cytotoxicity to HSF cells at a concentration of 2.5-40 mM. Referring to the concentration ranges of the two, BP-PEG-Met has no cytotoxicity to HSF cells in the concentration range of 1.25-20 μg / mL, and the cell survival rate is as high as over 85%.

[0108] 2. The biosafety of BP-PEG-Met prepared in Example 1 at the HSF cell level was tested by live-dead staining experiment: HSF cells were cultured at 1×10 5 The cells were inoculated at a density of 10 μg / mL per well in a 12-well plate and cultured for 24 h. The culture medium was discarded. Except for the Control group, the HSF cells in other groups were incubated with BP (10 μg / mL), Met (20 mM), and BP-PEG-Met (10 μg / mL) for 24 h, washed three times with PBS, stained with calcein acetoxymethyl ester (AM) and propidium iodide (PI), and observed under a confocal fluorescence microscope. The results are shown in Fig.19 As shown, the cell viability of each group was good and no toxic damage was caused to the cells, which confirmed that BP-PEG-Met had good biocompatibility and was beneficial to the survival and proliferation of cells.

[0109] 3. Evaluation of the antioxidant capacity of the BPNSs prepared in Example 1: The antioxidant capacity evaluation includes imaging observation under a confocal microscope and detecting the fluorescence intensity of DCFH-DA in HSF cells by flow cytometry.

[0110] HSF cells were cultured at 1×10 5 The cells were seeded on a 12-well plate at a density of 1.50 μg / mL per well. After overnight attachment, the cells were pretreated with 900 μM H2O2 for 30 min, and then H2O2 was removed. DMEM culture medium containing different concentrations of BP (0, 5, 10, 20, 40, 80 μg / mL) was added, and incubated for 4 h. The culture medium was discarded, and the cells were washed with PBS. The cells were incubated with DMEM culture medium containing 2,7-dichlorodihydrofluorescein diacetate (DCFH-HA) probe at 37°C for 20 min. The culture medium was discarded, and the cells were washed three times with PBS. Alternatively, DAPI was added and incubated for 10 min. DAPI was recovered, and after washing with PBS, 4% paraformaldehyde was added for fixation for 15 min. The fixative was discarded, and the residual fixative was washed with PBS. After sealing with an anti-fluorescence quencher, the imaging results were observed under a confocal microscope, or the cells were blown off the well plate, the cell suspension was collected and blown evenly, and the fluorescence intensity of DCFH-DA in HSF cells was detected by flow cytometry. The results are shown in Figure 2. Fig. 20 , Fig.21 As shown, compared with the Control group, the green fluorescence intensity in the HSF cells of the BP group weakened, confirming that the ROS level in the HSF cells decreased, and as the BP concentration increased, the weaker the green fluorescence in the HSF cells, the lower the ROS level; ROS was quantified by cell flow cytometry, and the expression of ROS decreased with the increase of BP concentration, which was consistent with the results of the fluorescence images, confirming that BP has a concentration-dependent antioxidant capacity at the cellular level and can reduce excessive reactive oxygen in the hair follicle microenvironment.

[0111] 4. Detection of mitochondrial membrane potential of HSF cells using Met and BP-PEG-Met prepared in Example 1: HSF cells were cultured at 1×10 5 The cells were seeded at a density of 1 / 40 μg / cm2 per well in a 12-well plate. When the cells were 90% full of the wells, the cells in different groups, except the control group, were pretreated with 20 mM Met and BP-PEG-Met for 4 h, and then stained with a mitochondrial membrane potential detection kit (JC-1). The green fluorescence signal of JC-1 monomers and the red fluorescence of JC-1 aggregates were observed under a confocal microscope. The results are shown in Table 1. Fig. 22 As shown in the figure, in normal HSF cells, the strong red fluorescence showed that the mitochondrial membrane potential was at a high level. After Met treatment, the red fluorescence weakened and the green fluorescence enhanced, confirming that Met significantly reduced the mitochondrial membrane potential. Compared with the Met group, the mitochondrial membrane potential of BP-PEG-Met group increased, suggesting that BP played an antioxidant role, reduced the ROS level of cells, rescued the decrease of mitochondrial membrane potential caused by Met, and had a protective effect on HSF cells.

[0112] 5. Evaluation of the cellular uptake of BP prepared in Example 1: BP was labeled with fluoresceinisothiocyanate (FITC) and Nile red (Nile) as fluorophores, and the uptake of BP-FITC and BP-Nile in human skin fibroblasts was observed.

[0113] HSF cells were cultured at 1×10 5 The cells were seeded on a 12-well plate at a density of 1.5 μg / mL on a slide. After 24 hours, the culture medium was discarded, and DMEM culture medium containing 50 μg / mL BP-FITC was added, and the well plate was placed in a 37°C, 5% CO2 incubator for incubation in the dark for 1, 2, and 4 hours. DMEM culture medium containing 25 μg / mL, 50 μg / mL, and 100 μg / mL BP-FITC was added, and the well plate was placed in a 37°C, 5% CO2 incubator for incubation in the dark for 4 hours. The culture medium was discarded, the well plate was washed three times with PBS, DAPI was added and incubated in the dark for 10 minutes to stain the cell nucleus, DAPI was discarded, the well plate was washed three times with PBS, 4% paraformaldehyde was added for fixation for 15 minutes, and the well plate was washed with PBS to remove the residual fixative. After sealing with an anti-fluorescence quencher, the uptake of BP by HSF cells over time and concentration was observed under a confocal fluorescence microscope. The results are shown in FIG. Fig.23 , Fig.24As shown in the figure, the fluorescence intensity of BP-FITC gradually increased with the extension of incubation time, showing time dependence. In addition, as the concentration of BP-FITC (25-100 μg / ml) gradually increased, the fluorescence intensity of BP-FITC increased, showing concentration dependence. It was confirmed that BP-FITC was successfully taken up by HSF cells and had the characteristics of time and concentration dependence.

[0114] HSF cells were cultured at 1×10 5 The cells were seeded on a 12-well plate at a density of 1.5 5 μg / mL per well. After 24 h, the culture medium was discarded and the cells were washed twice with PBS. Except for the Control group, DMEM culture medium containing MβCD (5 mM), HS (0.2 M), CPZ (10 μg / mL) and EIPA (10 μg / mL) was added, and pretreated at 37°C for 30 min. BP-Nile was then added to a total BP concentration of 50 μg / mL. The well plate was placed in a 37°C, 5% CO2 incubator and incubated in the dark for 4 h. The culture medium was discarded and the cells were washed three times with PBS to remove the residual reagents. The subsequent DAPI staining of the cell nucleus to observe under a confocal fluorescence microscope was consistent with the above steps. The results are shown in Fig.25 As shown in the figure, after treatment with inhibitors, the intensity of BP-Nile in HSF cells was weakened. After treatment with MβCD and EPIA, the intensity of BP-Nile in HSF cells was significantly weakened, confirming that BP-Nile mainly enters HSF cells through the endocytosis pathway mediated by caveolin and macropinocytosis.

[0115] 6. Evaluation of the in vivo distribution of BP prepared in Example 1 in HSF cells: HSF cells were cultured at 1×10 5 The cells were seeded on a slide in a 12-well plate at a density of 1.50 μg / mL. After 24 hours, the culture medium was discarded and the cells were incubated at 37°C for 30 minutes with DMEM culture medium containing mitochondrial fluorescent probe (Mito-Tracker), endoplasmic reticulum fluorescent probe (ER-Tracker) and lysosome probe (Lyso-Tracker). The fluorescent probes of each organelle were then recovered and replaced with DMEM culture medium containing 50 μg / mL BP-FITC for 4 hours in the dark. The culture medium was discarded and the cells were washed three times with PBS. The subsequent steps of DAPI staining of cell nuclei and observation under a confocal fluorescence microscope were consistent with the above. The results are shown in Fig.26 As shown, the green fluorescence of BP-FITC and the red fluorescence representing the three organelles are co-localized, confirming that BP is distributed in the endoplasmic reticulum, lysosomes and mitochondria.

[0116] Effect Example 5: In vivo efficacy evaluation of BP-PEG-Met against androgenic alopecia

[0117] To verify the therapeutic effect of BP-PEG-Met in vivo, 200 μL of 5 mg / mL testosterone (TS) ethanol solution was applied daily to the depilated area on the back of male C57BL / 6 (7-week-old) mice to obtain the AGA mouse model.

[0118] There are five groups in this experiment, namely, control group (no treatment after hair removal), TS group (5 mg / ml, 200 μl), MXD group (250 mg / kg), Met group (2 mg / ml, 100 μl), BP-PEG-Met group (2 mg / mL, 200 μl). Fig. 27 As shown, except for the Control group, all groups applied testosterone ethanol solution (5 mg / ml, 200 μl) on the hair removal area every day until the end of the experiment; the drug-treated groups: minoxidil group (MXD group), Met group, and BP-PEG-Met group were topically administered once every other day until the end of the experiment.

[0119] After the administration, the quality of the new hair of the mice was evaluated: the color of the mouse skin and the condition of the new hair were recorded on the 1st, 7th, 14th, 21st and 28th days after hair removal; on the 28th day after hair removal, the new hair of the mice was shaved and collected, and the weight of the new hair of each group was weighed with an analytical balance; then, 5 hairs were randomly selected from different groups, and the length of the new hair was measured with a vernier caliper; finally, the coverage of the new hair was measured with ImageJ; and the new hair of each group on the 28th day was selected to observe the structure of the hair surface under a scanning electron microscope. The results are as follows Fig.28 , Fig.29 shown.

[0120] The analysis and evaluation of the hair follicle cycle transition in mouse skin tissue includes HE staining experiments and Ki67 and PCNA immunohistochemical staining experiments.

[0121] HE staining: On the 28th day after depilation, mice were killed by dislocation, the skin of the depilated area on the back of the mice was removed, and the subcutaneous fat was removed. After fixing in 4% paraformaldehyde for 48 hours, the fixed skin tissue was taken out, repaired and placed in an embedding box, rinsed with running water to wash off the paraformaldehyde on the surface, and dehydrated in 70% ethanol (overnight), 80% ethanol (2h), 90% ethanol (2h), 95% ethanol (overnight), 100% I ethanol (2h), 100% II ethanol (2h) in sequence. After dehydration, the tissue was immersed in xylene for 2h, embedded in an embedding machine, and sliced ​​to a thickness of 4μm. The cut specimens were placed in xylene I (20 min), xylene II (20 min), 100% I ethanol (5 min), 100% II ethanol (5 min), 95% ethanol (5 min), 90% ethanol (5 min), 80% ethanol (5 min), and 70% ethanol (5 min) for hydration, and washed three times with PBS, each time for 5 min. Hematoxylin was added to the tissue sample, rinsed with running water after 15 seconds, placed in PBS for 5 minutes, and then stained with eosin for 30 seconds. After washing three times with PBS, it was naturally dried in a fume hood. After the specimens were dried, they were soaked in xylene for 20 minutes to remove unknown substances on the specimens, and finally sealed with neutral resin. The morphology of hair follicles in different groups was observed by a pathology scanner and the number of hair follicles was recorded. The results are as follows Fig.30 shown.

[0122] Ki67 and PCNA immunohistochemical staining: After dewaxing and hydrating the paraffin sections according to the above method, wash them with PBS three times, 5 minutes each time, and then use sodium citrate to perform antigen repair in a pressure cooker, boil for 30 seconds, take them out, cool them naturally to room temperature, wash them with PBS three times, 5 minutes each time, wash away the sodium citrate remaining on the specimen, then use an immunohistochemical pen to draw circles outside the tissue, incubate them with 3% catalase at 37°C for 20 minutes, wash them with PBS three times, wash away the residual catalase, and then block them with 10% goat serum at 37°C for 20 minutes, shake off the goat serum, and add a certain proportion of Ki67 antibody or PCNA antibody to the tissue sample, and incubate them in a wet box at 4°C overnight. After the primary antibody is recovered the next day, wash it with PBS three times, 10 minutes each time, wash away the primary antibody that is not bound to the tissue, and then add the secondary antibody of the same species as the primary antibody, incubate it at 37°C for 20 minutes, and then wash away the secondary antibody that is not bound to the primary antibody with PBS. DAB staining was added under a microscope, and the color development time was observed. After the color development was completed, the slices were placed in PBS to terminate the reaction. After the unreacted DAB dye was washed off with PBS, the slices were stained with hematoxylin for 15 seconds, gently rinsed with running water, washed once with PBS, and placed in 70% ethanol (5 seconds), 80% ethanol (5 seconds), 90% ethanol (5 seconds), 95% ethanol (5 minutes), 100% I ethanol (5 minutes), and 100% II ethanol (5 minutes) for dehydration. The slices were soaked in xylene for 20 minutes to wash off the unknown substances stuck on the specimens, and finally sealed with neutral resin. The sealed slices were placed under a pathology scanner to observe the expression of Ki67 or PCNA in different groups. The results are shown in Figure 2. Fig.31 shown.

[0123] Evaluation of oxidative stress around mouse hair follicles: The evaluation included 4-HNE immunofluorescence staining and measurement of MDA content in skin tissue.

[0124] 4-HNE immunofluorescence staining: After dewaxing, hydrating, and antigen repairing the tissue samples according to the aforementioned method, a certain proportion of diluted 4-HNE antibody was added to the tissue samples and incubated in a humidified box at 4°C overnight. After recovering the primary antibody on the second day, wash three times with PBS, 10 minutes each time, add a secondary antibody of the same species as the primary antibody, incubate at 37°C in the dark for 20 minutes, wash away the secondary antibody that is not bound to the primary antibody with PBS, add DAPI, and seal the slices with an anti-fluorescence quencher. The sealed slices were observed under a confocal fluorescence microscope for the expression of 4-HNE in different groups. The results are as follows: Fig.32 shown.

[0125] Determination of MDA content in skin tissue: On the 28th day of hair removal, mice were killed, and the skin of the hair removal area on the back of the mice was removed and stored in a -80°C refrigerator. Take the skin tissue from the -80°C refrigerator, weigh a certain amount of skin tissue into a 2mL EP tube, add appropriate amount of cell lysis solution, protease inhibitors, and put in 3 grinding beads, then put it into a pre-cooled grinder, grind the skin tissue at a frequency of 65Hz for 2min, take out the grinding beads, ultrasonicate in an ice water bath for 2min, then centrifuge at 4°C, 12000rpm for 15min, take the supernatant, and determine the MDA content in the skin tissue according to the instructions of the MDA kit. The results are as follows Fig.33 shown.

[0126] Evaluation of angiogenesis around mouse hair follicles: The evaluation included CD31 and VEGF immunofluorescence staining experiments.

[0127] CD31 and VEGF immunofluorescence staining: After dewaxing, hydrating, and antigen repairing the tissue samples according to the above method, add a certain proportion of platelet endothelial cell adhesion factor (CD31) antibody or vascular endothelial growth factor (VEGF) antibody diluted to the tissue sample and incubate it in a wet box at 4°C overnight. After recovering the primary antibody on the second day, wash three times with PBS, each time for 10 minutes, add a secondary antibody of the same species as the primary antibody, incubate at 37°C in the dark for 20 minutes, wash away the secondary antibody that is not bound to the primary antibody with PBS, add DAPI, and seal the slices with an anti-fluorescence quencher. The sealed slices were observed under a confocal fluorescence microscope for the expression of CD31 or VEGF in different groups. The results are as follows. Fig.34 , Fig.35 shown.

[0128] The results of the in vivo efficacy evaluation of the product prepared in Example 1 against AGA showed that after the BP-PEG-Met group was treated, the coverage area and weight of the newly grown hair were significantly higher than those of the TS group and the Met group; the length of the newly grown hair in the BP-PEG-Met group was higher than that of the TS group and was comparable to that of the Control group; the SEM images also showed that the newly grown hair in the BP-PEG-Met group was thicker and the scales were complete; and the HE staining results showed that the number of hair follicles in the BP-PEG-Met group was the largest, higher than that of the positive control group MXD, and the volume of the hair bulb became larger, indicating that the hair at this stage was in the growth phase of the hair cycle; in the immunohistochemical experiments of Ki67 and PCNA, the positive expression of the BP-PEG-Met group was more obvious; and in the 4-HNE immunofluorescence staining experiment, the Met group and The expression level of 4-HNE in the BP-PEG-Met group was reduced, which confirmed that Met and BP-PEG-Met had antioxidant capacity, and the 4-HNE level in the BP-PEG-Met group decreased most significantly, indicating that BP enhanced the antioxidant capacity of the BP-PEG-Met drug delivery system; the results of MDA content determination in skin tissue were consistent with the results of 4-HNE immunofluorescence staining, confirming that BP-PEG-Met has a good ability to scavenge ROS in vivo and can reshape the environment around hair follicles to a certain extent; CD31 and VEGF immunofluorescence staining results showed that after BP-PEG-Met treatment, the positive VEGF expression around the hair follicles increased, and the positive expression of VEGF at the hair papilla also increased significantly, and CD31 expression was consistent with the results of VEGF immunofluorescence. These results show that the prepared transdermal drug delivery preparation BP-PEG-Met has the effect of improving the growth of new hair in AGA mice.

[0129] Effect Example 6: In vivo safety evaluation of BP-PEG-Met

[0130] The safety evaluation includes evaluating the changes in mouse body weight, blood sample testing, and HE staining analysis of organs after drug administration.

[0131] The weight changes of mice in each group during the experiment were statistically analyzed. Fig.36 As shown, the weight of mice increased slowly over time, and there was no significant difference in body weight between the BP-PEG-Met group and the Con group with normal diet and free activities, indicating that the drug dosage was within a safe range during the entire administration period and did not cause harm to the mice.

[0132] After the experiment, blood samples were collected from each group of mice for testing and analysis. The test indicators included the concentration of red blood cells (RBC), white blood cells (WBC) and platelets (PLT). The results are as follows: Fig.37As shown in the figure, compared with the blank control group Control, the relevant indicators of all the drug delivery groups were within a reasonable range; further biochemical analysis was performed on the blood of mice, and the evaluation indicators included blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin test (TBIL), creatinine (CREA), and albumin (ALB). Compared with the blank control Control group, no abnormalities were found in the relevant parameters of all the drug delivery groups, confirming that the BP-PEG-Met drug delivery system has good biosafety and does not cause functional damage to the liver and kidneys of mice.

[0133] After the experiment, paraffin sections were prepared for the main organs (heart, liver, spleen, lung, and kidney) of each group of mice according to the above method, and the conditions of the organs in different groups were observed by pathological scanner. The results are as follows Fig.38 As shown, the cardiomyocytes of each group were intact, the liver cells had normal morphology, no inflammation or fibrosis occurred, no inflammatory response occurred in the spleen and lungs, and no inflammatory infiltration or glomerular atrophy appeared in the kidneys, confirming that the BP-PEG-Met drug delivery system has good in vivo biosafety.

[0134] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It is particularly pointed out that for those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a PEGylated two-dimensional phosphorene nanosheet loaded with metformin, characterized in that: The following steps are involved: S1, mixing black phosphorus crystals with a sodium hydroxide solution in N-methylpyrrolidone to prepare two-dimensional phosphorene nanosheets by liquid phase exfoliation; S2, mixing the polyethylene glycol amino group with the two-dimensional phosphorene nanosheet obtained in step S1, removing excess polyethylene glycol amino group, and obtaining a PEGylated two-dimensional phosphorene nanosheet; S3, mixing metformin with the PEGylated two-dimensional phosphorene nanosheet obtained in step S2, removing free metformin, and obtaining the PEGylated two-dimensional phosphorene nanosheet loaded with metformin.

2. The method for preparing the metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to claim 1, characterized in that: Preparation method of saturated sodium hydroxide solution in N-methylpyrrolidone described in step S1 The method comprises the following steps: weighing sodium hydroxide powder and dissolving it in N-methylpyrrolidone in a ratio of sodium hydroxide to N-methylpyrrolidone = 1-5 g: 50 mL, stirring and mixing, and preparing a saturated sodium hydroxide N-methylpyrrolidone solution; Step S1 specifically includes the following steps: mixing black phosphorus crystals with a saturated sodium hydroxide solution in N-methylpyrrolidone to obtain a black phosphorus dispersion, ultrasonicating in a water bath at 10-20° C. for 12±2 h, centrifuging, taking the supernatant, ultrasonicating again in an ice bath for 24±5 h, centrifuging a second time, taking the supernatant, centrifuging again to collect the precipitate, and drying to obtain a two-dimensional phosphorene nanosheet.

3. The method for preparing the metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to claim 2, characterized in that: The ratio of the black phosphorus crystals to the saturated sodium hydroxide solution in N-methylpyrrolidone in step S1 is black phosphorus crystals: saturated sodium hydroxide solution in N-methylpyrrolidone = 2-4 mg: 5 mL; The centrifugation is performed at a speed of 2000±500rpm and a temperature of 4°C for 25±5min; the secondary centrifugation is performed at a speed of 2000±500rpm and a temperature of 4°C for 25±5min; the third centrifugation is performed at a speed of 12000±1000rpm and a temperature of 4°C for 30±5min.

4. The method for preparing the metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to claim 1, characterized in that: The molecular weight of the polyethylene glycol of the polyethylene glycol amino group described in step S2 is 2000 to 8000; The mass ratio of the polyethylene glycol amino group to the two-dimensional phosphorene nanosheets in step S2 is 3 to 7:1; The mass ratio of metformin to PEGylated two-dimensional phosphorene nanosheets in step S3 is 15 to 25:

1.

5. The method for preparing metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to claim 1, characterized in that: The molecular weight of the polyethylene glycol of the polyethylene glycol amino group described in step S2 is 2000; The mass ratio of the polyethylene glycol amino group to the two-dimensional phosphorene nanosheet described in step S2 is 5:1; The mass ratio of metformin to PEGylated two-dimensional phosphorene nanosheets described in step S3 is 20:

1.

6. The method for preparing the metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to any one of claims 1 to 5, characterized in that: Step S2 specifically includes the following steps: dispersing polyethylene glycol amino groups in distilled water to obtain polyethylene glycol amino dispersion; dispersing the two-dimensional phosphorene nanosheets obtained in step S1 in distilled water to obtain a two-dimensional phosphorene nanosheet dispersion; mixing the polyethylene glycol amino dispersion with the two-dimensional borophene nanosheet dispersion as needed, stirring, centrifuging, and washing to obtain polyethylene glycol-modified two-dimensional phosphorene nanosheets; Step S3 specifically includes the following steps: dispersing metformin in distilled water to obtain a metformin dispersion; dispersing the PEGylated two-dimensional phosphorene nanosheets obtained in step S2 in deoxygenated ultrapure water to obtain a PEGylated two-dimensional phosphorene nanosheet dispersion; mixing the metformin dispersion with the PEGylated two-dimensional phosphorene nanosheet dispersion as needed, stirring, centrifuging, and washing to obtain PEGylated two-dimensional phosphorene nanosheets loaded with metformin.

7. The method for preparing the metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to claim 6, characterized in that: The concentration of the polyethylene glycol amino dispersion described in step S2 is 5±1 mg / mL; The concentration of the two-dimensional phosphorene nanosheet dispersion in step S2 is 1±0.5 mg / mL; The volume ratio of the polyethylene glycol amino dispersion to the two-dimensional phosphorene nanosheet dispersion in step S2 is 1-2:1-2; The stirring in step S2 is stirring at room temperature, away from light, and 600±100 rpm for 4±1 h; The centrifugation in step S2 is performed at 12000±1000 rpm for 30±5 min; The cleaning described in step S2 is cleaning with deoxygenated ultrapure water; The concentration of the metformin dispersion described in step S3 is 1±0.5 mg / mL; The concentration of the PEGylated two-dimensional phosphorene nanosheet dispersion in step S3 is 50±10 μg / mL; The volume ratio of the metformin dispersion to the PEGylated two-dimensional phosphorene nanosheet dispersion in step S3 is 1-2:1-2; The stirring in step S3 is stirring at room temperature, away from light, and 600±100 rpm for 6±1h; The centrifugation in step S3 is performed at 12000±1000 rpm for 30±5 min; The cleaning described in step S3 is cleaning with deoxygenated ultrapure water.

8. A PEGylated two-dimensional phosphorene nanosheet loaded with metformin, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 7.

9. Use of the metformin-loaded PEGylated two-dimensional phosphorene nanosheets according to claim 8 in the preparation of drugs for treating skin diseases.

10. The use according to claim 9, characterized in that: The skin disease is at least one of androgenic alopecia, infection, burns, mechanical trauma, and diabetic dermatosis.

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