Local delivery nano preparation for hair follicle diseases and preparation method thereof

Abstract

The invention discloses a local delivery nano preparation for hair follicle diseases and a preparation method of the local delivery nano preparation, and belongs to the technical field of biological medicines. The local delivery nano preparation is a microneedle patch and comprises a backing layer, a bubble layer and a drug-loading needle point layer, the bubble layer is arranged between the backing layer and the drug-loading needle point layer, and the drug-loading needle point layer is made of drug-loading nanoparticles and a water-soluble polymer material. According to the local delivery nano preparation, drug delivery optimization is achieved through a needle body-bubble-backing three-dimensional structure, oral whole-body drug delivery is optimized into local drug delivery of the alopecia areata part, the treatment effect is guaranteed, meanwhile, toxic and side effects of the whole body are avoided, and the compliance of a patient is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a local delivery nano-formulation for hair follicle diseases and its preparation method. Background Technology

[0002] Alopecia areata is an autoimmune disease influenced by environmental factors and has a genetic basis. The immune system attacks hair follicles, leading to hair loss. It is characterized by non-scarring hair loss, where the hair follicles are not destroyed. Under normal physiological conditions, hair follicles cycle through the anagen (growth) phase, catagen (transitional) phase, telogen (resting) phase, and anagen (growth) phase. Hair follicles, which provide nutrients for hair growth, possess relative immune privilege. A local immunosuppressive environment is generated within and around the hair follicle. Once the hair follicle loses its immune privilege, leading to damage to hair stem cells, alopecia areata will occur. The histopathological features of alopecia areata include inflammatory cells—such as antigen-presenting cells and CD8+ cells. + NKG2D + T cells and other cells infiltrate the area surrounding hair follicles in the growth phase, and histocompatibility antigen molecules are abnormally expressed in epidermal cells. The hair follicle channel above the sebaceous gland and the gland itself is preserved, while the follicles below the sebaceous gland shrink and enter the resting phase prematurely.

[0003] Topical steroids, prostaglandin analogs, and calcipotriol have all been used to treat alopecia areata, but their efficacy is limited. In recent years, the mechanism of alopecia areata has been further clarified, revealing that the non-receptor tyrosine protein kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is involved in its development. Small molecule JAK inhibitors can block the JAK-STAT signaling pathway. Tofacitinib was the first JAK inhibitor approved by the U.S. Food and Drug Administration, and oral tofacitinib has been proven effective in treating alopecia areata.

[0004] However, while long-term oral administration of JAK inhibitors can block the JAK-STAT pathway to treat alopecia areata, it leads to systemic exposure and potentially serious adverse reactions. Compared to oral administration, topical administration avoids the drug entering the systemic circulation, effectively reducing systemic adverse reactions. Therefore, topical treatment of alopecia areata, especially local delivery of JAK inhibitors, can improve this deficiency. Traditional topical delivery formulations have low penetration efficiency, making it difficult to reach the hair follicle target site, resulting in limited efficacy.

[0005] Patent CN115887464A discloses a phospholipid-modified calcium carbonate lipid nanoparticle spray, in which the calcium carbonate core provides pH responsiveness and the surface-modified phospholipids provide lipophilicity. However, the skin surface is also weakly acidic, causing the formulation to begin degrading before reaching the hair follicle, making it difficult to achieve a highly effective hair follicle-targeting effect.

[0006] Patent CN115475137A discloses a soluble microneedle with a low molecular weight material forming the solid needle tip. The drug molecules loaded within it have poor stability and diffuse into the backing layer during storage. Furthermore, this layer-by-layer assembly method also leads to drug molecule diffusion during preparation, reducing the effective drug content and thus affecting the product's economics and precise delivery. Simultaneously, during storage, drug molecules continue to diffuse into the backing layer, resulting in a further decrease in effective content and poor stability.

[0007] Therefore, achieving stable delivery of formulations to the hair follicle site and improving delivery efficacy are research challenges and hot topics in the development of hair follicle-targeted drug delivery systems. The industry urgently needs a localized JAK inhibitor formulation that can overcome the skin barrier, precisely deliver drugs to the perifollicular region, and possess stability. Summary of the Invention

[0008] One objective of this invention is to provide a locally delivered nanoformulation of a JAK inhibitor, wherein the formulation is a microneedle patch comprising a backing layer, a bubble layer, and a drug-loaded needle tip layer, wherein the bubble layer is disposed between the backing layer and the drug-loaded needle tip layer, the height of the drug-loaded needle tip layer is 400-800 μm, preferably 600 μm, and the height of the bubble layer is 100-500 μm, preferably 300 μm; The drug-loaded needle tip layer is made of drug-loaded nanoparticles and water-soluble polymer materials, with the mass ratio of drug-loaded nanoparticles to water-soluble polymer materials being 1:5 to 1:1, preferably 1:2.

[0009] Furthermore, the drug-loaded nanoparticles exist in the form of a nano-suspension before being loaded onto the needle tip layer. The raw materials, by mass-volume ratio, include 0.5–2% JAK inhibitor, 8–16% lipid material, 2–8% emulsifier, and the balance being water.

[0010] Preferably, the raw materials for the drug-loaded nanoparticles include, by mass-volume ratio, 0.5-1% JAK inhibitor, 10-12% lipid material, 4-6% emulsifier, and the balance being water.

[0011] More preferably, the raw materials of the drug-loaded nanoparticles include, by mass-volume ratio, 1% JAK inhibitor, 11% lipid material, 5% emulsifier, and the balance being water.

[0012] Furthermore, the JAK inhibitor is tofacitinib.

[0013] Further, the lipid material includes solid lipids and liquid lipids. The solid lipids are selected from lauric acid, stearic acid, glyceryl monostearate, glyceryl distearate, glyceryl behenate, glyceryl polyoxyethylene-8 behenate, glyceryl palmitate, propylene glycol dicaprylate, propylene glycol didecanoate, cetyl palmitate, lauroyl polyoxyethylene-32 glyceryl ester, stearoyl polyoxyethylene glyceryl ester, and glyceryl citrate. The liquid lipids are selected from propylene glycol monocaprylate, medium-chain triglycerides, castor oil, soybean oil, olive oil, oleic acid, squalene, and jojoba oil.

[0014] Furthermore, the mass ratio of the solid lipid to the liquid lipid is 2:1 to 5:1, preferably 4:1.

[0015] Further, the emulsifier includes surfactants and cationic surfactants, wherein the surfactants are selected from Tween 20, Tween 80, poloxamer 188, linoleyl polyoxyethylene-6-glycerol ester, polyethylene glycol succinate, polyvinyl alcohol, polyoxyethylene castor oil, polyoxyethylene lauryl ether, lecithin, and sodium lauryl sulfate; and the cationic surfactants are selected from dodecyl dimethyl ammonium bromide, hexadecyl ammonium bromide, hexadecyl pyridine chloride, benzalkonium chloride, benzyl chloride, dimethyl octadecyl ammonium chloride, and dimethyl heptadecanyl ammonium bromide.

[0016] Furthermore, the cationic surfactant accounts for 10-60% of the emulsifier, preferably 40%.

[0017] Furthermore, the water-soluble polymer material is selected from hyaluronic acid, sodium hyaluronate, polyvinyl alcohol, sodium carboxymethyl cellulose, gelatin, sucrose, and polyvinylpyrrolidone.

[0018] Furthermore, the bubble layer is made of polyvinyl alcohol or a polyvinyl alcohol-sucrose solution.

[0019] Furthermore, the backing layer is made of polyvinyl alcohol, gelatin, and polyvinylpyrrolidone.

[0020] The second objective of this invention is to provide a method for preparing the above-mentioned JAK inhibitor locally delivered nanoformulation, comprising the following steps: Step 1: Heat the lipid material to obtain a molten lipid mixture, add the JAK inhibitor to the molten lipid mixture, stir, and obtain a drug-containing lipid mixture; Step 2: Dissolve the emulsifier in water and heat to obtain an aqueous phase containing the emulsifier; Step 3: Add the aqueous phase from step 2 to the drug-containing lipid mixture from step 1, then perform high-speed shearing under heating conditions, followed by probe ultrasonic treatment under low-temperature conditions to obtain drug-loaded nanoparticles. Step 4: Add the drug-loaded nanoparticles obtained in Step 3 to an aqueous solution of a water-soluble polymer material, assemble the tip of the microneedle array using a template filling method, add a bubble layer solution after drying and deposition, assemble the bubble portion, add a backing material solution after drying and deposition, assemble the backing portion, demold after drying and deposition, and obtain the formulation.

[0021] In one or more embodiments of the present invention, in step 1, the heating temperature is 70~85°C.

[0022] Preferably, in step 2, the heating temperature is 70~85℃.

[0023] Preferably, in step 3, the high-speed shearing is performed at 70~85℃ and a rotation speed of 2000~10000rpm for 10~30 minutes; the probe ultrasound is performed at 0~10℃ and a power of 400~800W for 10~60 minutes.

[0024] Preferably, in step 4, the template filling method is a vacuum filling method.

[0025] A third objective of this invention is to provide the application of the above-mentioned JAK inhibitor local delivery nanoformulation in the preparation of drugs for the prevention and / or treatment of alopecia areata.

[0026] This invention constructs soluble bubble microneedles loaded with lipid nanoparticles, achieving highly efficient hair follicle targeting. Compared with existing technologies, the drug delivery system proposed in this invention has the following advantages: (1) Lipid materials and needle materials have high biocompatibility, so the formulation has high safety; (2) Lipid materials form a low-crystallinity core, which avoids drug leakage and has higher stability; (3) Since microneedles can form microchannels on the skin surface with the help of puncture force, the lipid nanoparticle drug delivery system can overcome the physiological barrier of the skin surface. The needle body forms a bubble structure inside. The bubble structure ensures that the microneedles break preferentially at a specific location (bubble) after piercing the skin, so that the drug-loaded lipid nanoparticles are accurately retained around the hair follicle bulb. (4) Lipid nanoparticles have a positive charge on their surface, making them more likely to interact with cells that have a negative charge on their surface, resulting in higher cellular uptake efficiency. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure for local delivery of nano-formulations according to the present invention.

[0028] Figure 2 The particle size distribution of the lipid nanoparticles in the examples is shown.

[0029] Figure 3The image shown is a transmission electron microscope (TEM) image of the lipid nanoparticles in the example.

[0030] Figure 4 The data provided are the 3-month stability data of the lipid nanoparticles in the examples.

[0031] Figure 5 The release behavior of lipid nanoparticles and drug molecules in the drug-loaded microneedles is illustrated in this example.

[0032] Figure 6 This example illustrates the drug distribution of drug-loaded microneedles at different sites within the skin.

[0033] Figure 7 In this example, the drug-loaded microneedles are used for cellular uptake in dermal sheath cells.

[0034] Figure 8 The cytotoxicity of the drug-loaded microneedles is shown in the examples.

[0035] Figure 9 The efficacy of drug-loaded microneedles is shown in the examples. Detailed Implementation

[0036] Local drug delivery for alopecia areata is essentially drug delivery to the hair follicles, which are evenly distributed across the skin surface, extending into the dermis. The infundibulum of the hair follicle has a diameter of approximately 150 μm, and there is a space for movement between the hair and the follicular epithelial cells. Hair movement can both secrete sebum metabolites and promote the penetration of external particles. The infundibulum of the hair follicle is weakly acidic, gradually becoming neutral as it extends deeper into the hair shaft. The raised portion of the hair follicle contains sebaceous glands, which are highly lipophilic. Therefore, constructing lipid-based nanoparticle drug delivery systems (such as liposomes, lipid nanoemulsions, and lipid nanoparticles) can improve the efficiency of drug delivery to the periphery of the hair follicle. Among these, lipid nanoparticles are suitable for therapeutic drugs requiring long-term use due to their high stability, encapsulation efficiency, and good sustained-release effect.

[0037] Traditional soluble microneedles suffer from non-specific drug distribution at the needle-backing interface due to the diffusion effect of the matrix material, resulting in low drug enrichment at the needle tip and affecting drug delivery accuracy. Furthermore, the materials of soluble microneedles are sensitive to temperature and humidity; during storage, drug molecules migrate to the backing, causing a decrease in the effective drug content at the needle tip.

[0038] To address the aforementioned issues, this invention focuses on resolving problems such as low drug loading capacity and unstable temperature and humidity environment in soluble microneedles. By utilizing lipid nanoparticles and soluble microneedles, a soluble bubble microneedle system is constructed. The drug delivery is optimized through a three-dimensional structure of "needle body-bubble-backing". This optimizes oral systemic drug delivery to local drug delivery at the alopecia areata site, ensuring therapeutic efficacy while avoiding systemic toxicity and improving patient compliance, thus finding a balance between safety and effectiveness.

[0039] This invention designs a drug delivery system targeting hair follicles. The invention proposes a "bubble microneedle-lipid nanoparticle" synergistic delivery system, achieving deep targeting of hair follicles through the following three technological breakthroughs: constructing a nanocarrier with high encapsulation efficiency and sustained-release properties using lipid materials; modifying the nanoparticle surface with surfactants to enhance nanoparticle stability and delivery capability; and ensuring that the bubble structure ensures preferential breakage of the needle at a predetermined depth (the bubble) after insertion into the skin, precisely retaining the lipid nanoparticles around the hair follicle bulb.

[0040] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0043] This embodiment provides lipid nanoparticles loaded with tofacitinib, which exist in the form of a nanosuspension before being loaded into the needle tip layer, and contain the following components: 1% (w / v) of tofacitinib; 10% (w / v) of lipid material, including 8% (w / v) glyceryl monostearate and 2% (w / v) oleic acid; 4% (w / v) of emulsifier, including 2% (w / v) of poloxamer 188, 1% (w / v) of polyethylene glycol succinate vitamin E and 1% (w / v) of benzalkonium chloride; water.

[0044] This embodiment also provides a method for preparing the above-mentioned lipid nanoparticles, including the following steps: 1) Weigh 800 mg of glyceryl monostearate and 200 mg of oleic acid, heat the lipid material at 80 °C to obtain a molten lipid mixture, add 100 mg of tofacitinib to the molten lipid mixture, stir, and wait for tofacitinib to dissolve in the lipid mixture; 2) Dissolve 200 mg poloxamer 188, 100 mg vitamin E succinate polyethylene glycol ester and 100 mg benzalkonium chloride in 10 mL of water and heat to obtain an aqueous phase containing 4% (w / v) surfactant; 3) Add the aqueous phase from step 2) to the lipid mixture containing tofacitinib from step 1); then perform high-speed shearing at 80°C for 10 minutes at 8000 rpm, followed by ultrasonic treatment at 4°C with a power of 400W for 20 minutes, and finally bring the volume to 10 mL to obtain a lipid nanoparticle suspension loaded with tofacitinib. Example 2

[0045] This embodiment provides lipid nanoparticles loaded with tofacitinib, which exist in the form of a nanosuspension before being loaded into the needle tip layer, and contain the following components: 0.5% (w / v) tofacitinib; 12% (w / v) of lipid material, including 6% (w / v) palmitate glyceryl stearate, 3% (w / v) lauroyl polyoxyethylene-32 glyceryl ester and 3% medium-chain triglycerides; 5% (w / v) of emulsifier, including 2% (w / v) of poloxamer 188, 2% (w / v) of Tween 80 and 1% (w / v) of cetyl ammonium bromide; water.

[0046] This embodiment also provides a method for preparing the above-mentioned lipid nanoparticles loaded with tofacitinib, including the following steps: 1) Weigh 600mg palmitose glyceryl, 300mg lauroyl polyoxyethylene-32 glyceryl ester and 300mg medium chain triglycerides, heat the lipid material at 82℃ to obtain a molten lipid mixture, add 50mg tofacitinib to the molten lipid mixture, stir until tofacitinib dissolves in the lipid mixture; 2) Dissolve 200 mg poloxamer 188, 200 mg Tween 80 and 100 mg cetyl ammonium bromide in 10 mL of water and heat to obtain an aqueous phase containing 5% (w / v) surfactant; 3) Add the aqueous phase from step 2) to the lipid mixture containing tofacitinib from step 1); then perform high-speed shearing at 82°C for 15 minutes at 8000 rpm, followed by ultrasonic treatment at 4°C with a power of 800W for 10 minutes, and finally bring the volume to 10 mL to obtain a lipid nanoparticle suspension loaded with tofacitinib. Example 3

[0047] This embodiment provides lipid nanoparticles loaded with tofacitinib, which exist in the form of a nanosuspension before being loaded into the needle tip layer, and contain the following components: 0.5% (w / v) tofacitinib; 12% (w / v) of lipid material, including 6% (w / v) stearic acid, 2% (w / v) stearoyl polyoxyethylene glycerol ester, 2% lauric acid and 2% squalene; 6% (w / v) of emulsifier, including 1% (w / v) of poloxamer 188, 3% (w / v) of Tween 80 and 2% (w / v) of dodecyl dimethyl ammonium bromide; water.

[0048] This embodiment also provides a method for preparing the above-mentioned lipid nanoparticles loaded with tofacitinib, including the following steps: 1) Weigh 600mg stearic acid, 200mg stearoyl polyoxyethylene glycerol ester, 200mg lauric acid and 200mg squalene. Heat the lipid material at 85℃ to obtain a molten lipid mixture. Add 50mg tofacitinib to the molten lipid mixture and stir until tofacitinib dissolves in the lipid mixture. 2) Dissolve 100 mg poloxamer 188, 300 mg Tween 80 and 200 mg dodecyl dimethyl ammonium bromide in 10 mL of water and heat to obtain an aqueous phase containing 6% (w / v) surfactant; 3) Add the aqueous phase from step 2) to the lipid mixture containing tofacitinib from step 1); then perform high-speed shearing at 85°C for 15 minutes at 10,000 rpm, followed by ultrasonic treatment at 4°C with a power of 800W for 20 minutes, and finally bring the volume to 10 mL to obtain a lipid nanoparticle suspension loaded with tofacitinib. Example 4

[0049] Evaluation of the properties of tofacitinib lipid nanoparticles Particle size distribution and morphological characteristics The lipid nanoparticle suspension sample obtained in Example 3 was diluted 50-fold, and its hydrodynamic diameter (particle size), polydispersity index (PDI), and zeta potential were measured using a BeNano 90 Zeta (Better BT, China, software version 3.0) nanoparticle size analyzer. The results are as follows: Figure 2 As shown, the particle size is 212 nm, the PDI is 0.15, and the zeta potential is 20.58 mV.

[0050] Transmission electron microscopy (TEM, Japan, HT770) was used to examine the morphology of the lipid nanoparticles. 10 μL of sample was deposited on a carbon-coated 300-mesh copper grid and negatively stained with 2% (w / v) phosphotungstic acid. Figure 3 As shown, the lipid nanoparticles exhibit a uniform, spherical shape.

[0051] stability The above-mentioned tofacitinib lipid nanoparticles were placed in a 4°C refrigerator, and their particle size, PDI, zeta potential, and encapsulation efficiency were measured at 1 day, 7 days, 14 days, 30 days, and 90 days. The results are as follows: Figure 4 As shown, all indicators remained stable over the three months. Example 5

[0052] Preparation and in vitro evaluation of soluble bubble microneedles Preparation of soluble bubble microneedles Take 10 mL of the lipid nanoparticle suspension prepared in Example 3, add hyaluronic acid to dissolve it, and make its concentration reach 20% (w / v) to obtain the drug-loaded needle tip solution; prepare a 10% polyvinyl alcohol solution as the bubble layer solution and a 20% polyvinylpyrrolidone solution as the backing layer solution. In this example, a 10×10 array of polydimethylsiloxane mold is used, and the needle tip of the microneedle is assembled using a vacuum template filling method, as follows: 400 μL of needle tip solution is filled into the surface of the microneedle mold, and it is kept under a vacuum of -0.1 MPa for 1 min, then atmospheric pressure is restored, the mold is removed, surface bubbles are removed, and excess needle tip solution outside the needle cavity is removed. Dry and deposit at an ambient temperature of 40°C, and then add 100 μL of bubble layer solution to assemble the bubble part. After a second drying, add 230 μL of backing layer solution to assemble the backing part. After drying, demold to obtain the bubble microneedle formulation.

[0053] Release behavior The direct release method was used to investigate the rapid release characteristics of lipid nanoparticles from soluble microneedles in a simulated physiological environment. 30 mL of release medium (pH 7.4, 10 mM PBS) was added to a 50 mL centrifuge tube, which was then placed in a shaker at 120 rpm and 32 °C. One drug-loaded microneedle was added, and 1 mL of medium was collected at 30, 60, 120, and 240 s, with the same volume of fresh medium added. The collected medium was dissolved in methanol, and the tofacitinib content was determined by HPLC.

[0054] The slow release characteristics of tofacitinib from lipid nanoparticles were investigated using the dialysis bag method. A dialysis bag with a molecular weight cutoff of 3500 Da was selected and preheated for 1 h in release medium (pH 7.4, 10 mM PBS) at 32°C before use. A drug-loaded microneedle was placed in the dialysis bag, clamped at both ends, and placed in 25 mL of release medium at 32°C. The dialysis bag was rotated at 300 rpm. 1 mL of release medium was collected at 0.5, 1, 2, 4, 8, 12, and 24 h, filtered through a 0.45 μm filter membrane, and the drug concentration was determined by HPLC.

[0055] The results are as follows Figure 5As shown, over 80% of the lipid nanoparticles loaded in the microneedles were rapidly released within the first 60 seconds, reaching over 95% at 4 minutes, which can be considered as complete release. However, the release rate of tofacitinib loaded in the lipid nanoparticles was slow; in a hydrophilic environment, the slow release of tofacitinib could last up to 24 hours.

[0056] In vitro transdermal experiments The pathway and ability of lipid nanoparticles to penetrate the skin were investigated using an isolated pig ear model. A sample with an area of ​​1.5 × 1.5 cm was used. 2 Pig ear skin was placed in a Franz diffusion cell, and the receiving chamber was filled with 4 mL of pH 7.4 PBS receiving solution and the temperature was set to 32°C. The rotation speed was set to 600 rpm. At selected time points, all the receiving solution was collected, and the same volume of fresh receiving solution was added. The collected receiving solution was analyzed by HPLC to determine the amount of drug that had penetrated the skin. After the analysis, the stratum corneum was separated using differential ablation, and the amount of drug in the stratum corneum was determined. The remaining skin tissue was homogenized, and the amount of drug in the skin was determined. The experimental group used the soluble microneedle formulation prepared by the method described in Example 5 above, while a control group was set up using lipid nanoparticles prepared by the method described in Example 3 above.

[0057] The results are as follows Figure 6 As shown, the drug concentration in the skin was higher than that in the control group, while the amount of drug in the stratum corneum was lower than that in the control group, indicating that the microneedle formulation improves its delivery efficiency, reduces waste, and has a higher follicle targeting effect.

[0058] Cellular uptake Hair follicle dermal sheath cells were seeded in 35 mm laser confocal dishes and 12-well cell culture plates and cultured at 37°C in 5% CO2. After cell attachment, the culture medium was carefully discarded. For the experimental group, a soluble microneedle formulation prepared as described in Example 5 was added to DMEM medium (50 μg / mL) containing soluble bubble microneedle tip extract. After a given incubation time, the cells were removed and washed three times with PBS to remove uninternalized lipid nanoparticles. Detection was performed using laser confocal microscopy and flow cytometry. Qualitative experiments included Hoechst 33258 staining of cell nuclei.

[0059] The results are as follows Figure 7 As shown, the cellular uptake of lipid nanoparticles in the microneedles is time-dependent, with higher cellular uptake efficiency observed during longer incubation periods, allowing them to successfully enter the cells and reach the target site.

[0060] Cytotoxicity The in vitro cytotoxicity of soluble bubble microneedles was measured using the tetramethylazoazol blue colorimetric method. NIH / 3T3 cells were cultured at 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of cells / mL into 96-well plates. The culture plates were incubated overnight at 37°C in 5% CO2. For the experimental group, soluble microneedles prepared according to the method described in Example 5 were added, along with the tip extract of soluble bubble microneedles (concentration 37.5–900 μg / mL), and co-incubated with the cells for 24 h. Subsequently, 20 μL of MTT (4 mg / mL) was added to each well, and incubation continued for 4 h. Then, 200 μL of DMSO was added, and the absorbance was immediately recorded at 570 nm using a microplate reader. Cell viability for each group was calculated using the following formula: Cell viability (%) = (As - Ab) / ((Ac - Ab)) × 100% Where As, Ab, and Ac represent the absorbance of the experimental group, blank group, and control group, respectively.

[0061] The results are as follows Figure 8 As shown, at a concentration <150.0 μg / mL, the cell viability of NIH / 3T3 was higher than 85%, indicating that the lipid nanoparticles are safe and non-toxic. Example 6

[0062] Efficacy evaluation of soluble bubble microneedles A patchy alopecia wereata model was established by applying imiquimod cream to the back of the neck of 6-8 week old C3H mice. The mice were randomly divided into two groups (n=3). In the treatment group, medication was administered starting after the alopecia areata model was established. A soluble microneedle formulation prepared as described in Example 5 was applied to the alopecia areata area on the back of the neck twice weekly for 3 weeks. The control group mice received no treatment after the alopecia areata developed.

[0063] The results are as follows Figure 9 As shown, stable hair regeneration was observed in the treatment group at the application site, and complete hair growth was observed after 3 weeks of treatment. The self-made microneedles reversed the alopecia areata symptoms that had been established in C3H mice.

Claims

1. A JAK inhibitor locally delivered nanoformulation, characterized in that, The preparation is a microneedle patch, comprising a backing layer, a bubble layer and a drug-loaded needle tip layer, the bubble layer is arranged between the backing layer and the drug-loaded needle tip layer, the height of the drug-loaded needle tip layer is 400-800 μm, and the height of the bubble layer is 100-500 μm. The drug-loaded needle tip layer is made of drug-loaded nanoparticles and water-soluble polymer materials, and the mass ratio of drug-loaded nanoparticles to water-soluble polymer materials is 1:5~1:

1.

2. The JAK inhibitor locally delivering nanoformulation according to claim 1, wherein, The raw materials of the drug-loaded nanoparticles include 0.5~2% JAK inhibitor, 8~16% lipid material, 2~8% emulsifier, and the balance is water.

3. The JAK inhibitor locally delivering nanoformulation according to claim 2, wherein, The JAK inhibitor is tofacitinib.

4. The JAK inhibitor locally delivering nanoformulation according to claim 2, wherein, The lipid material includes solid lipid and liquid lipid, the solid lipid is selected from lauric acid, stearic acid, glyceryl monostearate, glyceryl distearate, glyceryl behenate, polyoxyethylene-8 glyceryl behenate, glyceryl palmitostearate, propylene glycol dicaprylate, propylene glycol didecanoate, cetyl palmitate, lauroyl polyoxylethylene-32 glycerides, stearoyl polyoxylethylene glycerides, and glyceryl citrate; the liquid lipid is selected from propylene glycol monocaprylate, medium-chain triglyceride, castor oil, soybean oil, olive oil, oleic acid, squalene, and jojoba oil; and the mass ratio of the solid lipid to the liquid lipid is 2:1~5:

1.

5. The JAK inhibitor locally delivering nanoformulation of claim 2, wherein, The emulsifier includes a surfactant and a cationic surfactant, the surfactant is selected from Tween 20, Tween 80, poloxamer 188, linoleoyl polyoxylethylene-6 glycerides, vitamin E succinate polyethylene glycol ester, polyvinyl alcohol, polyoxylethylene castor oil, polyoxylethylene lauryl ether, lecithin, and sodium dodecyl sulfate; and the cationic surfactant is selected from dodecyl dimethyl ammonium bromide, hexadecyl ammonium bromide, cetyl pyridinium chloride, benzalkonium chloride, benzethonium chloride, dimethyloctadecyl ammonium chloride, and dimethylheptadecyl ammonium bromide; and the proportion of the cationic surfactant in the emulsifier is 10~60%.

6. The JAK inhibitor locally delivering nanoformulation of claim 1, wherein, The water-soluble polymer material is selected from hyaluronic acid, sodium hyaluronate, polyvinyl alcohol, sodium carboxymethyl cellulose, gelatin, sucrose, and polyvinyl pyrrolidone.

7. The JAK inhibitor topical delivery nanoformulation of claim 1, wherein, The bubble layer is made of polyvinyl alcohol solution or polyvinyl alcohol-sucrose solution.

8. The JAK inhibitor local delivery nanoformulation of claim 1, wherein, The backing layer is made of polyvinyl alcohol, gelatin, and polyvinyl pyrrolidone.

9. A method of preparing a topical delivery nanoformulation of a JAK inhibitor according to any one of claims 1-8, characterized in that, The method comprises the following steps: Step 1, heating the lipid material to obtain a molten lipid mixture, adding the JAK inhibitor into the molten lipid mixture, and stirring to obtain a drug-containing lipid mixture; Step 2, dissolving the emulsifier in water, heating to obtain an emulsifier-containing water phase; Step 3, adding the water phase of step 2 into the drug-containing lipid mixture of step 1, then performing high-speed shearing under heating, and then performing probe ultrasonic treatment under low-temperature conditions to obtain drug-loaded nanoparticles; Step 4, adding the drug-loaded nanoparticles obtained in step 3 into a water solution of water-soluble polymer materials, using a template filling method to assemble the needle tip part of the microneedle array, adding a bubble layer solution after drying and depositing to assemble the bubble part, adding a backing material solution after drying and depositing to assemble the backing part, and demolding after drying and depositing to obtain the preparation.

10. Use of the topical delivery nanofonnulation of a JAK inhibitor according to any one of claims 1 to 8 for the manufacture of a medicament for the prevention and / or treatment of alopecia areata.

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