Microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor and its application

Abstract

The present invention belongs to the field of new drug research and development, and specifically relates to a microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor and its application. The microneedle has good mechanical strength, can effectively deliver IrNP and bFGF to the epidermis and dermis, IrNP plays a strong anti-oxidative stress role, removes free radicals around hair follicles, bFGF reshapes microvessels around hair follicles and promotes vascularization, the two cooperate with each other to improve the hair follicle microenvironment, promote hair growth, and have good cell compatibility, promote endothelial cell vascularization, and resist damage caused by oxidative stressors.

Description

Technical Field

[0001] The present invention belongs to the field of new drug research and development, and specifically relates to a microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor and an application thereof. Background Art

[0002] Androgenic alopecia, also known as seborrheic alopecia, is a common type of hair loss affected by genetics and hormones (such as testosterone). Androgenic alopecia is caused by the conversion of testosterone into dihydrotestosterone (DHT) in the scalp. DHT increases the production of reactive oxygen species (ROS), causing hair follicles to be exposed to excessive oxidative stress. Free radicals damage hair follicle cells. This damage may lead to apoptosis (cell death) of hair follicle cells, affecting hair growth. At the same time, DHT inhibits angiogenesis around hair follicles, reduces the production of vascular endothelial growth factor (VEGF), and limits the hair follicles' access to necessary nutrients and oxygen. These effects together lead to the deterioration of the hair follicle microenvironment, prolonging the time for hair follicles to enter the resting phase, shortening the growth phase, and making new hair thinner and shorter. Oxidative stress and angiogenesis defects are interrelated, and the accumulation of free radicals can lead to vascularization difficulties and vascular dysfunction. Therefore, oxidative stress and vascularization difficulties in the hair follicle microenvironment lead to a disordered growth environment for the hair follicles, making it difficult for the hair follicles to transition from the resting phase to the growth phase. Under this long-term influence, the hair follicles may be miniaturized, eventually leading to hair loss. In addition, inflammation also plays an important role in AGA, destroying the progressive fibrosis of the sheath around the hair follicles and the normal hair cycle, thereby accelerating the hair loss process (YF Mah´e, JF Michelet, N. Billoni, et al., Androgenetic alopecia and microinflammation, Int. J. Dermatol. 39 (2000) 576–584.). Currently, the conventional drugs for the treatment of androgenic alopecia are mainly finasteride and minoxidil. Their main mechanism is to improve the blood supply around the hair follicles to promote hair growth. However, they will produce some side effects during use, including loss of libido, erectile dysfunction, scalp irritation, itching, and systemic reactions such as weight gain. In conclusion, improving perifollicular oxidative stress and vascular distress is a potential therapeutic strategy for the treatment of androgenetic alopecia. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor and its application.

[0004] The technical scheme adopted by the present invention is as follows: a microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor, which includes a degradable microneedle matrix, in which the microneedle matrix is ​​loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor.

[0005] Preferably, the preparation method comprises the following steps: dissolving the material of the microneedle matrix in a solvent, then adding iridium nanoparticles modified with polyvinyl pyrrolidone and basic fibroblast growth factor to obtain a needle tip solution, adding the needle tip solution to a microneedle mold, then centrifuging the microneedle mold to fill the needle tip solution into the needle cavity, and curing by ultraviolet light to obtain the microneedle matrix.

[0006] Preferably, the material used for the microneedle matrix is ​​gelatin methacrylamide. GelMA (gelatin methacrylamide) is a multifunctional biomaterial derived from collagen, which is widely present in various tissues including skin, and has excellent solubility, biocompatibility and adjustable mechanical properties.

[0007] Preferably, a detachable patch substrate is provided on the back of the microneedle matrix;

[0008] The microneedle preparation method also includes the following steps: after the microneedle mold is centrifuged to fill the needle tip solution into the needle cavity, the excess mixed solution outside the needle cavity is removed with a scraper, and then ultraviolet light curing is performed, and then a patch substrate is attached.

[0009] Preferably, the patch substrate is made of polyvinyl pyrrolidone-k90. PVP-K90 has excellent adhesion and high molecular weight, ensuring that the patch can be firmly attached to the skin surface and provide stable support for the microneedle array. At the same time, its good solubility and biocompatibility make it easy to prepare and safe and reliable.

[0010] Preferably, the method for synthesizing polyvinyl pyrrolidone-modified iridium nanoparticles comprises the following steps: dispersing iridium trichloride in water, adding the solution to an ethanol solution containing polyvinyl pyrrolidone, stirring the solution at room temperature, and removing the solvent to obtain polyvinyl pyrrolidone-modified iridium nanoparticles.

[0011] Use of the microneedles loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor as described above for preparing a preparation for treating hair loss.

[0012] The present invention provides hydrogel microneedles Ir / bFGF-MNs loaded with polyvinyl pyrrolidone-modified iridium nanoparticles (IrNP) and basic fibroblast growth factor (bFGF). Ir / bFGF-MNs are composed of microneedles (MNs) composed of degradable gelatin methacrylate (GelMA) encapsulating polyvinyl pyrrolidone-modified iridium nanoparticles and bFGF and a detachable polyvinyl pyrrolidone (PVP)-k90-based patch substrate.

[0013] The microneedle has good mechanical strength and can effectively deliver IrNP and bFGF to the epidermis and dermis. IrNP plays a strong role in anti-oxidative stress, removes free radicals around hair follicles, and bFGF reshapes the microvessels around hair follicles to promote vascularization. The two synergistically improve the microenvironment of hair follicles and promote hair growth. Ir / bFGF-MNs have good cell compatibility, promote endothelial cell vascularization, and resist damage caused by oxidative stressors. In AGA model mice, after Ir / bFGF-MNs are inserted into the skin, the hydrogel tip degrades and releases IrNP and bFGF, improves oxidative stress in the hair loss area of ​​mice, removes ROS, promotes vascularization near hair follicles, relieves inflammation, reshapes the microenvironment around hair follicles, effectively induces the transition of hair follicle cycle to growth phase, and promotes hair growth. At the same time, the mechanical stimulation caused by MNs can also contribute to the regeneration of skin microvessels. Ir / bFGF-MNs have high hair coverage, thicker diameter, and satisfactory hair regeneration effect. Compared with the minoxidil group, Ir / bFGF-MNs can have a therapeutic effect superior to minoxidil with a lower frequency of administration. In conclusion, the Ir / bFGF-MNs of the present invention can play a therapeutic role in AGA through anti-oxidation and vascularization. The study of Ir / bFGF-MNs in the treatment of AGA provides new ideas for the development of more effective strategies for the treatment of hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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 only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0015] Figure 1 Schematic diagram of bFGF and PVP-modified iridium nanoparticle hydrogel microneedles to improve the microenvironment around hair follicles to treat androgenic alopecia.

[0016] Figure 2Characterization of IrNPs; (A) Physical image of powdered IrNPs, scale bar is 1 cm; (B). Transmission electron microscopy (TEM) image of IrNPs, scale bar is 10 nm; (C) Statistical analysis of the size distribution of IrNPs obtained by TEM (n = 100); (D) Zeta potential of IrNPs (n = 3); (E) UV-Vis absorbance of IrCl3 and IrNPs; (F) Scavenging efficiency of H2O2 by IrNPs of different concentrations (n= 3); (G) Scavenging efficiency of •OH by IrNPs of different concentrations (n= 3); (H) Scavenging efficiency of DPPH• by IrNPs of different concentrations (n= 3); (I) Scavenging efficiency of ABTS• by IrNPs of different concentrations (n= 3).

[0017] Figure 3 Characterization of Ir / bFGF-MNs; (A) Scanning electron microscopy (SEM) image of Ir / bFGF-MNs, scale bar size is 200 μm; (B) Image of Ir / bFGF-MNs under a stereo microscope, scale bar size is 200 μm; (C) Fluorescence microscopy image of Ir / bFGF-MNs containing rhodamine b-labeled bFGF, scale bar size is 100 μm; (D) Degradation of Ir / bFGF-MNs over time (n = 3); (E) Mechanical properties of Ir / bFGF-MNs; (F) IrNPs release curve of Ir / bFGF-MNs (n = 3); (G) bFGF release curve of Ir / bFGF-MNs (n = 3).

[0018] Figure 4 Biocompatibility and cell experiments of Ir / bFGF-MNs; (A) Calcein / PI fluorescence staining of HUVECs on the 3rd day after different treatments, scale bar is 50 μm; (B) DCF fluorescence intensity of HUVECs under different treatments (n = 3); (C) Scale bar is 50 μm, scale bar is 50 μm; (D) Quantitative analysis of tubular formation of HUVECs under different treatments (n = 3); All data are expressed as mean ± SD, compared with the control group, ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0019] Figure 5In vivo AGA treatment efficiency of Ir / bFGF-MNs; (A) Schematic diagram of the establishment of the AGA mouse model by daily application of testosterone solution for 28 days and the treatment strategy used in the model. The control group was only given testosterone solution. In the B-MNs group and the Ir / bFGF-MNs group, MNs were applied 6 times every 4 days, and the minoxidil group was applied minoxidil every day for 21 days; (B) Representative photos of hair regeneration in mice after different treatments; (C) Hair coverage of mice after different treatments (n = 3); (D) Hematoxylin and eosin (HE) staining of the skin of mice with different treatments on the 14th day after hair removal, with a scale of 200 μm; (E) Counting of hair follicles in the hair removal area of ​​mice with different treatments on the 14th day after hair removal (n = 3); (F) SEM images of regenerated hair in the hair removal area of ​​mice with different treatments on the 28th day after hair removal, with a scale of 20 μm; (G) DHT levels in the hair removal area of ​​mice with different treatments on the 14th day after hair removal (n = 4); All data are expressed as mean ± SD, compared with the control group, ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0020] Figure 6 In vivo evaluation of Ir / bFGF-MNs remodeling perifollicular angiogenesis and immune microenvironment; (A) Representative images of Ki-67 immunohistochemical (IHC) staining of mouse depilatory skin tissue on day 14 after different treatments, with a scale bar of 100 μm; (B) Representative images of CD31 immunohistochemical staining of mouse depilatory skin tissue on day 14 after different treatments, with a scale bar of 100 μm; (C) Quantitative analysis of Ki67 IHC staining (n = 3); (D) Quantitative analysis of CD31 IHC staining (n = 3); ELISA was used to evaluate the expression of VEGF (E), TGF-β (F), TNF-α (G), and IL-1β (H) in the depilatory skin of mice on day 14 after depilation (n = 4); All data are expressed as mean ± SD, compared with the control group, ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0021] Figure 7In vivo evaluation of Ir / bFGF-MNs in alleviating perifollicular oxidative stress; (A) Representative images of DHE staining of mouse depilatory skin tissue on day 14 after different treatments to detect ROS. Scale bar = 50 μm; (B) Representative images of 4-HNE IHC staining of mouse depilatory skin tissue on day 14 after different treatments, scale bar size is 100 μm; (C). Quantitative analysis of DHE expression (n = 3); (D). Quantitative analysis of 4-HNE immunohistochemical staining (n = 3); (E) MDA concentration of mouse depilatory skin tissue on day 14 after different treatments (n = 3); All data are expressed as mean ± SD, compared with the control group, ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0022] Figure 8 In vivo degradation of Ir / bFGF-MNs. (A) HE staining of mouse skin sections showing the insertion depth of Ir / bFGF-MNs; in vivo percutaneous administration and the content of Ir (B) and bFGF (C) in the skin after MN administration (n = 3); (D) IVIS image of the dorsal skin of mice on day 11 after administration of rhodamine B-labeled Ir / bFGF-MNs; (E) Quantitative analysis of fluorescence intensity in the dorsal skin of mice (n = 3); detection of Ir (F) and bFGF (G) concentrations in the dorsal skin of mice at different time points after Ir / bFGF-MNs application in the MNs application area (n = 3). DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0024] The main materials used in the following examples are as follows: IrCl 3 ·3H 2O, Polyvinylpyrrolidone (PVP, Mw ~ 55,000), rhodamine B fluorescently labeled bFGF (Qiyue Biology, Q-0096828); Mouse VEGF ELISA Kit (Hangzhou Lianke, EK283), GelMA, Malondialdehyde (MDA) assay kit (TBA method) Nanjing Jiancheng A003-1-2, Mouse Dihydrotestosterone (DHT) ELISA Kit, Jianglai Biology, JL10916; Testosterone Testosterone (Solarbio, IT0110); Minoxidil was purchased from Zhejiang Wansheng Pharmaceutical Co., Ltd.; ROS fluorescent probe DHE (aladdin, R353922).

[0025] Embodiment 1:

[0026] (1) Synthesis of polyvinylpyrrolidone-modified iridium nanoparticles: IrCl 3 Preparation of polyvinylpyrrolidone (PVP)-modified nano-iridium by reduction. Specifically, IrCl 3 · 3H 2 O (37 mg) was added to pure water (50 mL), ultrasonicated until completely dissolved, and then added to an ethanol solution (50 mL) containing PVP (232.5 mg) and stirred at room temperature for 12 hours to obtain a clear and uniform light yellow solution. The above solution was rotary evaporated at 80 ° C for 8 hours to remove the organic solvent, and the remaining solution was freeze-dried to finally obtain a black powder product (IrNP), which was stored at room temperature for later use.

[0027] Polyvinyl pyrrolidone (PVP)-modified nano-iridium was prepared by a one-pot hydrothermal reduction of IrCl 3. The whole process is simple and environmentally friendly, and the final product is a black powder ( Figure 2 A). TEM shows that IrNPs are ultra-small nanoparticles with uniform morphology ( Figure 2 B), the particle size of 100 IrNPs in the TEM image was statistically analyzed, and the average particle size of IrNP nanozymes was 1.32 ± 0.32 nm ( Figure 2 C), which is much smaller than the particle size of other conventional nanoparticles. At the same time, the polydispersity index (PDI) of IrNP nanozyme in aqueous solution is 0.034, which indicates that the synthesized IrNP nanozyme has good monodispersity in water. In addition, the Zeta potential of IrNP nanozyme was measured to be 3.59mV ± 0.32nm ( Figure 2D). With IrCl 3 In contrast, the IrNP nanozyme generated by PVP thermal reduction showed a new peak at 280 nm in the UV-visible spectrum ( Figure 2 E), indicating that PVP has been successfully attached to Ir.

[0028] Through H 2 O 2 The antioxidant capacity of IrNPs was evaluated by the scavenging ability of ·OH, DPPH·, and ABTS·. The antioxidant capacity of IrNPs at different concentrations was tested. 2 O 2 , ·OH, DPPH· and ABTS· clearance rates, each group was repeated 3 times. Figure 2 As shown in FI, the results showed that IrNPs could effectively scavenge hydrogen peroxide (H 2 O 2 ), hydroxyl radicals, DPPH and ABTS, and the free radical scavenging ability was positively correlated with IrNP. 2 O 2 The scavenging rates of DPPH and ABTS were close to 100%, and the scavenging rate of hydroxyl was more than 80%, which proved that IrNP had excellent antioxidant capacity.

[0029] (2) Preparation of microneedles: Microneedles (MNs) were made using polydimethylsiloxane (PDMS) molds. The distance between the centers of each two needle tips of the microneedle was 650 μm, the diameter of the tip bottom was 250 μm, and the height was 600 μm. The microneedles were arranged in a 10*10 structured array, and each needle had a conical tip and a tapered edge.

[0030] Ir / bFGF-MNs were prepared by centrifugation. First, GelMA (1 g) was dissolved in pure water (10 mL), and then IrNP (20 mg) and bFGF (100 ug) were added to the above solution and mixed thoroughly to obtain the needle tip solution for preparing microneedles. Then 100 μL of the mixed solution was added to the microneedle mold. The microneedle mold was then placed in a 50 mL centrifuge tube and centrifuged (5000 rpm, 10 min) to fill the mixture into the needle cavity. Subsequently, the excess mixed solution outside the cavity was removed with a plastic scraper, and UV light was applied for 50 s to solidify the needle tip. Then, 200 ul (40%, w / v) of PVP-K90 solution was added to the back layer of the microneedle mold. According to the above method, the microneedle mold was centrifuged again, and then dried and demolded at room temperature to finally obtain Ir / bFGF-MNs.

[0031] Ir / bFGF-MNs were prepared by a simple centrifugation method, and microneedles consisting of a degradable microneedle body (GelMA) and a detachable patch substrate (PVP-K90) were obtained. The distance between the tips of the microneedles was 650μm, the tip height was 600μm, and each Ir / bFGF-MNs contained 8.65 ± 0.76 μg Ir and 8.65 ± 0.76 μg bFGF. SEM showed the conical tip structure of Ir / bFGF-MNs ( Figure 3 A), black Ir was observed to be loaded into the microneedle tip under an optical microscope ( Figure 3 B), Fluorescence micrograph of Ir / bFGF-MNs with rhodamine B labeled bFGF showed that bFGF was also successfully loaded into the microneedle body ( Figure 3 C).

[0032] Comparative Example 1:

[0033] Blank-MNs were prepared according to a procedure similar to step (2) of Example 1, except that IrNP and bFGF were not added to the needle tip solution.

[0034] Test Example 1 In vitro degradation experiment:

[0035] The microneedles were immersed in a PBS solution containing collagenase IV (0.5 mg / mL) and placed in a 37°C water bath. The microneedles were taken out at the predetermined time and freeze-dried. Finally, the dried microneedles were weighed and the degradation rate of the microneedles was calculated according to the following formula (n=3). Degradation rate = (initial microneedle weight - microneedle weight at the predetermined time point) / microneedle weight at the predetermined time point * 100%.

[0036] The main factor involved in gelatin degradation in the skin is matrix metalloproteinases (MMPs). In order to evaluate the degradation behavior of Ir / bFGF-MNs in vitro, collagenase IV (a MMP) was added to the PBS solution to simulate the natural enzymatic degradation process of Ir / bFGF-MNs in vivo. In the PBS solution containing collagenase IV, the degradation rate of microneedles was close to 80% within 24 hours, and the entire degradation process lasted about 72 hours ( Figure 3 D).

[0037] Test Example 2 Microneedle Mechanical Strength Test:

[0038] The microneedle was fixed on a flat plate with the tip facing upward. A universal testing machine (6800, Instron) was used to apply a vertical force to the microneedle at a speed of 10 mm / s in compression mode to test the mechanical strength of the microneedle tip. Three parallel samples were tested in each group.

[0039] The smooth continuous force-displacement curves showed that the compressive force of Ir / bFGF-MNs increased to 4.13 ± 0.83 N ( Figure 3 E), which indicates that Ir / bFGF-MNs have a certain mechanical strength and are able to penetrate the stratum corneum of the skin.

[0040] Test Example 3 In vitro drug release experiment:

[0041] The microneedles were immersed in a PBS solution containing collagenase IV (0.5 mg / mL) and placed in a 37°C water bath. 50 μL of the supernatant was taken at the predetermined time and supplemented with an equal amount of PBS solution. The bFGF content in the supernatant was detected (n=3), and the Ir content in the supernatant was determined by ICP-MS (n=3). The drug release rate of the microneedles was calculated according to the following formula.

[0042] bFGF drug release rate = (bFGF concentration in microneedles - bFGF concentration in the supernatant in which microneedles are immersed) / bFGF concentration in microneedles * 100%.

[0043] IrNP drug release rate = (IrNP concentration in microneedles - IrNP concentration in the supernatant in which the microneedles are immersed) / IrNP concentration in microneedles * 100%.

[0044] In Ir / bFGF-MNs, more than half of IrNPs and bFGF were released from the microneedle tip within 24 hours ( Figure 3 F, 3G), which indicates that Ir / bFGF-MNs have good drug release ability and therapeutic potential.

[0045] Test Example 4 In vitro cell biocompatibility:

[0046] Mouse embryonic fibroblasts (NIH 3T3) and human umbilical vein endothelial cells (HUVEC) were used as model cells for biocompatibility evaluation. The cells were seeded in 96-well culture plates, with 1 × 10 3 cells. After the cells adhered to the wall, different cells were treated differently. The blank MNs group (B-MNs) was treated with Blank-MN immersion solution, and the Ir / bFGF-MNs group was treated with Ir / bFGF-MNs immersion solution. The immersion solutions of Blank-MN and Ir / bFGF-MNs were obtained by immersing the microneedles in PBS containing collagenase IV for 72 hours. At 24 hours and 72 hours, the viability of cells in each group was detected using a CCK-8 kit. At the same time, the live-dead staining kit was used to perform fluorescence staining on the 72-hour cells, and then the fluorescence of the cells was observed using a fluorescence microscope to evaluate the cell biocompatibility of the microneedles.

[0047] After 1 and 3 days of co-culture with NIH 3T3 and HUVEC cells, the cell viability of the blank microneedle group (B-MNs) and the microneedle group loaded with IrNP and bFGF (Ir / bFGF-MNs) did not decrease significantly and remained close to 100%. At the same time, on the third day, live-dead staining was performed on the two groups of cells, and the results showed that there was no significant difference between the cells in the B-MNs and Ir / bFGF-MNs groups and the control group ( Figure 4 A), which shows that Ir / bFGF-MNs have good cell biocompatibility.

[0048] Test Example 5 In vitro anti-oxidative stress:

[0049] The in vitro antioxidant capacity of the microneedles was evaluated using a reactive oxygen species detection kit, and the intracellular ROS was detected by a DCFH-DA probe to evaluate the antioxidant capacity of the microneedles. HUVEC were seeded in a 96-well culture plate, and 1 × 10 4 cells / well. When all cells were attached, serum-free DMEM medium containing DCFH-DA (50 μM) was added and incubated for 30 min, and then the cells were washed three times with serum-free cell culture medium to fully remove DCFH-DA that did not enter the cells. Then Rosup reactive oxygen stimulator (1 uL) provided by the kit was added to each well, and the cells were treated with serum-free DMEM medium containing Blank-MN soaking solution or Ir / bFGF-MNs soaking solution or both, incubated in the dark for 1 hour, and finally washed the cells three times with PBS, and the microplate reader was set to 488nm excitation wavelength and 525nm emission wavelength to detect the fluorescence intensity of cell DCF.

[0050] Under in vitro oxidative stress stimulation, the 2′,7′-dichlorofluorescein (DCF) fluorescence intensity of HUVEC cells co-incubated with Ir / bFGF-MNs was significantly weakened compared with the control group and B-MNs group ( Figure 4 B), indicating that IrNPs in Ir / bFGF-MNs can still alleviate oxidative stress in vitro.

[0051] Test Example 6 In vitro tube formation experiment:

[0052] 50 μL of Matrigel (Corning, USA) was evenly coated on a 24-well plate and incubated at 37°C for 30 minutes to polymerize for 1 hour. HUVEC were plated at 2 × 10 5The cells were seeded in a well plate at a density of 10 cells / well. The cells were treated with a medium containing Blank-MN soaking solution or Ir / bFGF-MNs soaking solution or neither of them, incubated for 8 hours, and HUVEC cells were stained with calcein-AM. The HUVEC tube formation was observed using a fluorescence microscope (Nikon). Finally, the number of cell tubes in each group was counted and analyzed (n = 3).

[0053] HUVEC tube formation is an important step in the angiogenesis process and affects the effect of hair regeneration. Therefore, the angiogenic effect of Ir / bFGF-MNs was studied by HUVEC tube formation experiment. Compared with the control group, it can be seen that endothelial cells treated with Ir / bFGF-MNs have obvious tube formation, and B-MNs alone also have a tendency to form tubes ( Figure 4 C). However, by counting the number of tubes, it was found that the number of tubes in the Ir / bFGF-MNs group was much higher than that in the control group and the B-MNs group ( Figure 4 D).

[0054] Experimental Example 7 Animal Experiment on Ir / bFGF-MNs in Treating Male Baldness:

[0055] The therapeutic effect of Ir / bFGF-MNs was verified in AGA male C57 BL / 6 mice. All procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) and strictly followed the relevant guidelines of animal ethics. First, 7-8 week-old male C57 BL / 6 mice were housed in a new environment for 1 week. Mice with no damage on the back were selected, and the hair on the back of the mice was shaved with an electric shaver, and the hair was removed with a depilatory cream, and the remaining hair on the back was cleaned with warm water, and this area was designated as the depilatory area. Subsequently, different treatments were applied to the AGA mice ( Figure 5 A), 24 mice were randomly divided into 4 groups, namely, control group, B-MNs group, Ir / bFGF-MNs group and minoxidil group. The androgenic alopecia (AGA) mouse model was established. Specifically, testosterone was dissolved in ethanol solution (50%) to prepare 0.2% testosterone solution (w / v). The testosterone solution was applied topically to the hair removal area of ​​each group of mice once a day at a dose of 0.1 mL / cm 2, used continuously for 28 days. At the same time, each group received corresponding treatment. The Control group did not receive any treatment; the B-MNs group was treated with B-MNs; the Ir / bFGF-MNs group was treated with Ir / bFGF-MNs, and the minoxidil group was treated with local application of minoxidil solution to the hair removal area for 21 days. The microneedle treatment time was 1, 5, 9, 13, 17 and 21 days after hair removal. The method of microneedle patch is: press the microneedle vertically on the skin of the hair removal area so that the needle body is inserted into the skin, and remove the microneedle patch substrate after 10 minutes. The minoxidil group used 5% (w / v) minoxidil solution topically applied to the hair removal area daily, with a dose of 0.1 mL / cm 2 On days 1, 14, 21, and 28, photos were taken to record the hair growth in the hair loss area on the back of the mice, and Image J software was used to analyze the hair coverage in the hair loss area on the back of the mice. On day 28, a scanning electron microscope was used to observe the microstructure of the regenerated hair.

[0056] On the 14th day, the skin tissues of the hair loss area of ​​each group of mice were collected, fixed with conventional 4% paraformaldehyde, embedded in paraffin, and 4 μm thick sections were obtained by microtome. After dewaxing and rehydration, these sections were finally stained with hematoxylin-eosin (HE). The levels of Ki67, CD 31, and 4-HNE in the hair loss area were detected by immunohistochemistry. In short, the tissue samples first needed to be fixed, embedded, sliced, and dewaxed, followed by antigen retrieval and blocking to reduce nonspecific binding. Next, specific primary antibodies (anti-Ki67, anti-CD 31, and anti-4-HNE antibodies) and secondary antibodies were used for incubation. Finally, the sections were sealed and observed under a microscope.

[0057] The oxidative stress level of skin tissue was assessed by DHE staining. Skin tissue was embedded in OCT compound and frozen, and then the frozen tissue blocks were cut into 10 μm thick sections. The sections were fluorescently stained with 2.5 mM DHE, and then the cell nuclei were stained with DAPI, and the images were taken with a fluorescence microscope (Nikon, Japan), and the quantitative fluorescence intensity was analyzed using Image J software.

[0058] On the 14th day, mice were euthanized to obtain skin tissue from the back hair removal area. The same weight of skin tissue was weighed in each group, and then the skin tissue was cut into pieces on ice and put into centrifuge tubes, and pre-cooled steel beads were added. At the same time, 1 mL of pre-cooled PBS was added to each centrifuge tube, and a high-throughput tissue grinder (SCIENTZ-24) was used for grinding. Finally, the centrifuge tube was centrifuged at 4°C (10,000 g, 30 min), and the supernatant was collected. The contents of VEGF, TGF-β1, TNF-α, IL-β and DHT in the supernatant were detected according to the corresponding Elisa detection kits.

[0059] The supernatant of the skin tissue of the depilated area on the back of mice on the 14th day was extracted, and the contents of MDA and SOD in the supernatant were determined according to the instructions provided by the MDA detection kit and the SOD detection kit.

[0060] Based on the above experiments, the following results can be found:

[0061] The hair removal area of ​​the control group of mice remained pink until day 28, with no obvious signs of darkening, which also indirectly indicates the successful establishment of AGA mice. In contrast, the hair removal area of ​​the Ir / bFGF-MNs group began to show darkening of the skin and signs of hair growth on day 14 after the application of Ir / bFGF-MNs, similar to the minoxidil group, while the B-MN group also showed diluted hair growth on day 21 ( Figure 5 B), which shows that Ir / bFGF-MNs can effectively promote hair regeneration, and the mechanical stimulation of microneedles also has a positive effect on hair regeneration. Statistical analysis of the hair coverage rate of each group on the 28th day ( Figure 5 C, Figure S), the results showed that Ir / bFGF-MNs had the best hair coverage, which also showed that Ir / bFGF-MNs was slightly better than the conventional drug minoxidil in treating AGA at a lower administration frequency. Furthermore, from the results of HE staining, hair follicles were observed in the subcutaneous tissue of the depilatory area on the 14th day, and the number of growing hair follicles in the Ir / bFGF-MNs group was significantly higher than that in the other groups ( Figure 5 D, 5E), the diameter of the hair follicles was also larger than that of the other groups (Figure), which was consistent with the hair coverage rate in the Ir / bFGF-MNs group. At the same time, the SEM results of the hair in the hair removal area of ​​each group showed that the regenerated hair in the Ir / bFGF-MNs group was thicker and had more complete scales, which was similar to the regenerated hair in the minoxidil group, while the regenerated hair in the control group and the B-MN group was thinner and the scales were relatively incomplete ( Figure 5 F). The above results indicate that the IrNP and bFGF released from the microneedles can activate the telogen follicles in the hair loss area, promote the enlargement of hair follicles, activate the growth of hair follicles, and thus promote the regeneration of hair. In addition, on the 28th day, the DHT in the hair loss area of ​​each group was detected. The DHT content of the Ir / bFGF-MNs and minoxidil groups was much lower than that of the control group and the B-MN group ( Figure 5G), which suggests that Ir / bFGF-MNs may also play a role in resisting the increase of DHT, thereby alleviating the damage caused by DHT to hair follicle cells. Finally, on the 14th and 28th days, the changes in the thickness of the epidermis and skin of each group were measured (Figure) to evaluate the effects of microneedle administration on the skin. On the 14th day, the epidermal thickness of the B-MNs group, Ir / bFGF-MNs group and minoxidil group increased to varying degrees compared with the control, which may be caused by the transformation of hair follicles from the resting phase to the growth phase, while the epidermal thickening of the B-MNs and Ir / bFGF-MNs groups was more obvious, which may be caused by re-epithelialization during the healing process after microneedle puncture. However, on the 28th day, the epidermal thickness of the B-MNs and Ir / bFGF-MNs groups returned to near normal levels, indicating that the epidermal changes caused by microneedle puncture are reversible. Similarly, the changes in skin thickness in each group were basically consistent with the changes in epidermal thickness, which also means that microneedling does not cause obvious damage to the skin. In summary, Ir / bFGF-MNs showed good therapeutic effect in promoting hair regeneration in the androgenetic alopecia animal model, and compared with minoxidil, it can reduce the frequency of medication, and is a potential candidate drug for the effective, convenient and safe treatment of AGA.

[0062] The growth phase of hair promotes cell proliferation, and Ki 67 is the most common cell proliferation marker. According to the immunohistochemical staining results of skin tissue on day 14, the hair loss area treated with Ir / bFGF-MNs showed the highest Ki67 expression level in hair follicles, followed by the minoxidil group ( Figure 6 A, 6C), indicating that Ir / bFGF-MNs can enhance cell proliferation of hair follicles in the growth phase. In addition, the angiogenesis in the microenvironment around the hair follicles was also studied. CD31 immunohistochemical staining showed that compared with the control group, the angiogenesis in the hair loss area of ​​the Ir / bFGF-MNs group increased significantly, and the angiogenesis in the hair loss area of ​​the minoxidil group also increased to varying degrees, indicating that Ir / bFGF-MNs can effectively improve the difficulty of vascularization around the hair follicles, and the effect is better than minoxidil ( Figure 6 B, 6D), Interestingly, there was also a small amount of angiogenesis in the B-MNs group, which may be caused by the mechanical stimulation of the microneedles. VEGF plays a central role in the angiogenesis process and reflects the situation of angiogenesis. On day 14, compared with the control group, the VEGF levels in the skin tissue of the B-MNs group, the Ir / bFGF-MNs group, and the minoxidil group were significantly increased, among which the VEGF level in the Ir / bFGF-MNs group was the highest ( Figure 6E). This may be related to the fact that Ir / bFGF-MNs treatment resists the damage caused by DHT and improves the vascular microenvironment of hair follicles. By increasing the expression of VEGF, it promotes angiogenesis around hair follicles, thereby promoting hair regeneration. TGF-β is a hair growth inhibitor induced by DHT. It induces apoptosis of endothelial cells, leads to degeneration of microvessels around hair follicles, induces fibrosis of hair follicles, inhibits hair from entering the growth phase from the resting phase, and is one of the key factors in the pathogenesis of AGA. The experimental results showed that compared with the control group, the expression of TGF-β in the hair loss area of ​​mice in the Ir / bFGF-MNs group was significantly lower than that in other groups ( Figure 6 F), which suggests that Ir / bFGF-MNs may accelerate the growth phase of hair follicles and promote hair regeneration by reducing TGF-β expression and inhibiting hair follicle fibrosis and microvascular degeneration. Local inflammation is also a risk factor for the destruction of the hair follicle microenvironment. Therefore, studying the changes in inflammatory factors is of great significance for understanding the stability of the hair follicle immune microenvironment. Next, the expression levels of TNF-α and IL-1β in the hair removal area on the 14th day were studied ( Figure 6 G, 6H) to evaluate the immune microenvironment of hair follicles in AGA mice. Compared with the control group, the expression of TNF-α and IL-1β was lower in the Ir / bFGF-MNs group and the minoxidil group. In addition, the B-MNs group also showed a slightly lower level than the control group, which may be due to the improvement of the microenvironment around the hair follicles caused by angiogenesis. This result fully demonstrates that Ir / bFGF-MNs can improve the immune microenvironment of hair follicles in AGA mice and promote hair regeneration.

[0063] Oxidative stress is one of the key factors that cause serious damage to the microenvironment around hair follicles, mainly manifested by direct damage to hair follicle cells, disruption of hair follicle growth cycle, and induction of inflammatory response and fibrosis. These effects lead to impaired hair follicle function, ultimately causing sparse hair and hair loss, seriously affecting the health and regeneration of hair. Therefore, the antioxidant capacity of Ir / bFGF-MNs in vivo was further studied on the 14th day. Dihydroethidium (DHE) fluorescence staining showed that the intracellular ROS level in the hair loss area of ​​the Ir / bFGF-MNs group was significantly reduced compared with the other groups ( Figure 7 A, 7C). Meanwhile, immunohistochemical staining of 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation marker, showed that the expression of Ir / bFGF-MNs group in the hair removal area was also darker than that of other groups ( Figure 7 B, 7D). Furthermore, the levels of SOD and MDA (lipid peroxidation product) in the hair loss area of ​​the Ir / bFGF-MNs group were significantly increased, while the level of MDA (lipid peroxidation product) was significantly decreased, indicating that Ir / bFGF-MNs can effectively alleviate the oxidative stress in the hair loss area ( Figure 7E, S). Compared with the control group, the DHE signal, 4-HNE expression and MDA level in the minoxidil group decreased, while the SOD level increased, which may be related to the fact that minoxidil promotes angiogenesis and increases the antioxidant activity of hair follicle cells. In conclusion, these findings indicate that Ir / bFGF-MNs can effectively reshape the oxidative microenvironment around the hair follicles in the hair loss area and promote hair regeneration.

[0064] Test Example 8 Puncture test:

[0065] In order to evaluate the skin insertion effect, the microneedle was pressed into the skin of the mouse back at a vertical uniform speed and then removed. Then the skin of the experimental area was taken and stained according to the previous HE staining method. The penetration of the microneedle into the skin was observed under a microscope, and the penetration depth of the microneedle was evaluated using image J. Figure 8 As shown in A, the results show that Ir / bFGF-MNs can smoothly penetrate the stratum corneum of the skin and successfully break through the skin barrier for drug delivery.

[0066] Test Example 9 In vivo penetration test:

[0067] In order to detect the permeability of microneedles through the skin, Ir / bFGF-MNs were inserted into the back skin of mice by hand pressure. After 10 minutes, the substrate layer was removed, and IrNP (2 mg / mL) and bFGF (2 ug / mL) solutions were smeared on the back skin surface of mice. The control group did not receive any treatment. After 30 minutes, the back skin of mice was washed with PBS, the surface moisture was absorbed with filter paper, and then a part of the skin was dissolved with aqua regia, and finally the Ir content of each group was determined by ICP-MS. The tissue supernatant was extracted from a part of the skin, and the bFGF content of each group was detected using the bFGF Elisa detection kit.

[0068] It can be observed that the levels of Ir and bFGF detected in the skin of the Ir / bFGF-MNs group were much higher than those of the other groups ( Figure 8 B, 8C), which shows that it is difficult for Ir and bFGF to penetrate the skin directly when applied on the skin surface. In sharp contrast, microneedles can effectively deliver Ir and bFGF to the skin. Overall, Ir / bFGF-MNs can break through the stratum corneum of the skin and effectively deliver Ir and bFGF to the subcutaneous tissue, successfully achieving efficient transdermal drug delivery.

[0069] Test Example 10 Degradation of microneedles in vivo:

[0070] Mice were anesthetized with isoflurane, and then the hair on the back skin of mice was removed using an electric shaver and depilatory cream. Rhodamine B-stained microneedles were applied to the back skin of mice. On days 0, 3, 6, and 10 after microneedle application, the fluorescence signal of the back skin of mice was detected using a live animal fluorescence imaging system (PerkinElmer, IVIS Spectrum), and the quantitative fluorescence intensity was analyzed using Image J software.

[0071] As time went on, the fluorescence signal of the mouse back skin gradually weakened. Within 3 days, the microneedle fluorescence signal was greatly weakened and almost disappeared on the 10th day ( Figure 8 D, 8E). This indicates that Ir / bFGF-MNs were mostly degraded in vivo within 3 days and almost completely degraded by day 10.

[0072] Experimental Example 11 Drug Metabolism of Microneedles in Vivo

[0073] Mice were anesthetized with isoflurane, and then the hair on the back skin of mice was removed using an electric shaver and depilatory cream to obtain mice with depilated back skin. Ir / bFGF-MNs were vertically pressed into the back skin of mice by hand pressure, and the substrate layer was removed after 10 minutes. On days 0, 3, 6, and 10 after the application of Ir / bFGF-MNs, the skin on the back of mice with microneedles was taken, and the content of Ir and bFGF in the skin tissue was detected using ICP-MS and bFGF kits in the same way.

[0074] The levels of Ir and bFGF in the back skin of mice decreased over time ( Figure 8 F, 8G), which is similar to the results of the fluorescence signal changes in the mouse back skin, indicating that most of the Ir and bFGF in the mouse skin were metabolized and utilized on the 3rd day. This result shows that after Ir / bFGF-MNs enter the skin, as it degrades, the released drugs will be absorbed and utilized by the skin, thereby exerting the therapeutic effects of Ir and bFGF. In short, Ir / bFGF-MNs have sufficient mechanical strength and good degradation ability in the body for transdermal drug delivery to the skin.

[0075] There is currently no effective cure for AGA. Current studies have shown that the main cause of the disease is the imbalance of various factors in the hair follicle microenvironment, including oxidative stress, insufficient angiogenesis, inflammatory response and dysregulation of cell signaling pathways. Improving oxidative stress around hair follicles, regulating inflammatory responses and promoting angiogenesis can help restore the homeostasis of the hair follicle microenvironment and promote hair regeneration. IrNP has a strong antioxidant and anti-inflammatory function, while bFGF plays an important role in promoting angiogenesis. By utilizing the transdermal delivery ability of microneedles, IrNP and bFGF are loaded into hydrogel microneedles with excellent biocompatibility, and the drugs are delivered to the microenvironment around the hair follicles in the form of microneedles. At the same time, the mechanical stimulation of the microneedles is also conducive to stimulating angiogenesis. By evaluating the hair regeneration in the hair loss area of ​​AGA mice, the oxidative stress, inflammatory regulation and angiogenesis of the skin tissue in the hair loss area were further studied, proving that Ir / bFGF-MNs can effectively regulate the dysregulated microenvironment around the hair follicles, promote angiogenesis around the hair follicles, alleviate inflammation and significantly reduce oxidative stress, which may provide a favorable environment for hair follicle growth. Therefore, Ir / bFGF-MNs are expected to promote hair regeneration by effectively regulating the microenvironment around hair follicles, providing a potential new approach for the treatment of androgenic alopecia.

[0076] In summary, the present invention provides an Ir / bFGF-MNs formed by a hydrogel, having a degradable needle tip and a detachable patch substrate, and having good mechanical strength to penetrate the stratum corneum to release IrNP and bFGF. Ir / bFGF-MNs reshapes the microenvironment around the hair follicle by simultaneously resisting oxidative stress and promoting angiogenesis, thereby playing a role in treating AGA. Compared with the commercial drug minoxidil, Ir / bFGF-MNs improves the microenvironment of the hair follicle with a lower dosing frequency, mechanical stimulation of the microneedles and release of IrNP and bFGF, achieving better quality regenerated hair and more satisfactory hair regeneration effects. At the same time, the application of Ir / bFGF-MNs does not cause irreversible damage to the skin, has good biocompatibility, and can be fully degraded in the body. In summary, it is believed that Ir / bFGF-MNs is expected to become an innovative and promising strategy for the treatment of AGA, with potential benefits in terms of treatment outcomes and patient portability and safety.

[0077] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor, characterized in that: The invention comprises a degradable microneedle matrix, wherein the microneedle matrix is ​​loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor; the preparation method comprises the following steps: dissolving the material used for the microneedle matrix in a solvent, then adding the polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor to obtain a needle tip solution, adding the needle tip solution to a microneedle mold, then centrifuging the microneedle mold to fill the needle tip solution into the needle cavity, and curing by ultraviolet light to obtain the microneedle matrix; the material used for the microneedle matrix is ​​gelatin methacrylamide.

2. The microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor according to claim 1, characterized in that: A detachable patch substrate is provided on the back of the microneedle matrix; The microneedle preparation method also includes the following steps: after the microneedle mold is centrifuged to fill the needle tip solution into the needle cavity, the excess mixed solution outside the needle cavity is removed with a scraper, and then ultraviolet light curing is performed, and then a patch substrate is attached.

3. The microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor according to claim 2, characterized in that: The patch substrate is made of polyvinyl pyrrolidone-k90.

4. The microneedle loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor according to claim 1, characterized in that: The method for synthesizing polyvinyl pyrrolidone-modified iridium nanoparticles comprises the following steps: dispersing iridium trichloride in water, adding the solution to an ethanol solution containing polyvinyl pyrrolidone, stirring the solution at room temperature for reaction, and removing the solvent to obtain polyvinyl pyrrolidone-modified iridium nanoparticles.

5. Use of the microneedles loaded with polyvinyl pyrrolidone-modified iridium nanoparticles and basic fibroblast growth factor as claimed in any one of claims 1 to 4 for preparing a preparation for treating hair loss.

Images