Layered microneedle patch as well as preparation method and application thereof

Abstract

The invention discloses a layered microneedle patch as well as a preparation method and application thereof, and belongs to the field of biological medicines. Wherein the needle body tip is composed of a therapeutic agent and L-arginine shell modified glycan gel, has excellent biocompatibility and free DNA removal ability, and can deliver deferoxamine and synergistically regulate and control inflammation signals; the upper part of the needle body and the substrate are made of low-molecular-weight hyaluronic acid which is quickly dissolved after being in contact with skin, so that the needle tip is quickly separated from the substrate, the needle tip is retained at a diseased region, and effective deposition of a medicine at the diseased region is ensured; the layered microneedle can slowly release deferoxamine through pH response so as to inhibit ferroptosis; the modified chitosan can efficiently remove free DNA so as to reduce immune inflammation and cooperatively block immune inflammation circulation. The preparation process of the layered microneedle is controllable, the material biocompatibility is excellent, and the layered microneedle has the advantages of efficient targeted delivery, long-acting release and synergistic treatment, so that a feasible strategy is provided for psoriasis treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a layered microneedle patch and a preparation method and application thereof. BACKGROUND

[0002] Psoriasis is a common chronic inflammatory autoimmune skin disease with a global incidence of about 2%~3%. The clinical manifestations are chronic recurrent erythematous rash and silver-white scales on the skin surface. Its pathological mechanism is complex, and the core is the abnormal interaction between keratinocytes (KCs) and infiltrating immune cells (such as lymphocytes, macrophages, and neutrophils), forming a sustained amplification of the inflammatory cascade. At present, although the cytokine targeting therapy of TNF-α and IL-17 can improve the clinical symptoms, 90% of patients still face symptom recurrence after drug withdrawal, and the existing treatment lacks sufficient regulation of the source of inflammation, so it is urgent to explore new treatment strategies.

[0003] Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation, which has been shown to affect immune cell function and regulate inflammatory responses by releasing damage-associated molecular patterns (DAMPs), thereby playing an important role in various self-inflammation, autoimmune and degenerative diseases. In recent years, more and more studies have shown that there is activation of the ferroptosis signaling pathway in the epidermal keratinocytes of psoriasis patients, which is manifested as iron ion accumulation, down-regulation of glutathione peroxidase 4 expression, and significant increase in ferroptosis-specific lipid oxidation activity, and is closely related to the Th22 / Th17 signaling pathway. In addition, in the imiquimod (IMQ)-induced psoriasis model, ferroptosis inhibitors can effectively alleviate psoriasis-like dermatitis, further proving the contribution of ferroptosis to the enhancement of psoriasis inflammatory response. In a recent study, it was shown that abnormal iron metabolism may be an important reason for the occurrence of ferroptosis in psoriasis keratinocytes. During psoriasis, the increase in skin iron-regulating hormones leads to the degradation of iron transport protein (FPN), resulting in abnormal increase in keratinocyte iron content, thereby triggering inflammatory response and abnormal recruitment of neutrophils. Followed by the formation of extracellular traps (NETs) by neutrophils or DAMP signals including extracellular free DNA (cfDNA) released by dead KCs, thereby initiating intracellular immune signaling cascade, forming an inflammation-ferroptosis vicious inflammatory cycle, and further maintaining the chronic inflammatory state of psoriasis. Therefore, the intervention strategy targeting ferroptosis may be a potential breakthrough to break the abnormal inflammatory cycle between KCs and immune cells to alleviate the progression of psoriasis.

[0004] ​Deferoxamine is an FDA-approved small-molecule iron chelator that inhibits ferroptosis by chelating iron ions. However, because deferoxamine is a water-soluble small-molecule drug, it has a short blood circulation time and is easily metabolized by the liver and kidneys, making it difficult to accumulate effectively in the diseased epidermal layer, resulting in low systemic administration efficiency. Furthermore, topical transdermal administration has become a research hotspot in the treatment of skin-related diseases in recent years, but due to the impermeable nature of the stratum corneum barrier and the insufficient permeability of deferoxamine itself, topical deferoxamine therapy still faces many challenges in treating skin diseases.

[0005] Based on this, the present invention develops a layered microneedle system that can efficiently deliver deferoxamine to the site of skin lesions to inhibit ferroptosis and synergistically regulate DAMPs inflammatory signals, which is of great significance for improving the treatment effect of psoriasis and reducing the recurrence rate. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide a layered microneedle patch, its preparation method and application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A layered microneedle patch includes a microneedle array and a substrate; the needle body of the microneedle array includes a needle tip portion and a support portion, the needle tip portion is composed of a gel matrix containing a therapeutic agent and modified chitosan; the support portion and the substrate are composed of low molecular weight hyaluronic acid; wherein, the support portion dissolves upon contact with skin tissue fluid, causing the needle tip portion to separate from the substrate and remain within the skin tissue.

[0009] The modified chitosan is L-arginine-modified chitosan; the molar ratio of L-arginine to chitosan is 1:1~3; and the degree of deacetylation of the chitosan is 80%~95%.

[0010] The therapeutic agents include ferroptosis inhibitors.

[0011] The ferroptosis inhibitor comprises an iron chelating agent and / or an antioxidant; the iron chelating agent is selected from at least one of deferoxamine, deferasirox, and deferoxone; the antioxidant is selected from at least one of ferrostatin-1, liproxstatin-1, and vitamin E.

[0012] When using or preparing the layered microneedle patch of the present invention, other active ingredients can be added into the microneedles, or used in combination with other active ingredients. The therapeutic agents thus include at least one of methotrexate, cyclosporine, acitretin, azathioprine, hydroxyurea, glucocorticoids, anti-tumor necrosis factor-α inhibitors, IL-12 / 23 antibodies, IL-17A antibodies, and IL-23p19 antibodies.

[0013] In the gel matrix, the mass ratio of the modified chitosan to the therapeutic agent is 1:(0.8~1.2).

[0014] The low molecular weight hyaluronic acid has a molecular weight of 8000 Da to 10000 Da.

[0015] A method for preparing the layered microneedle patch includes the following steps:

[0016] 1) Provide PDMS microneedle molds;

[0017] 2) Prepare a needle tip gel solution containing a therapeutic agent and modified chitosan;

[0018] 3) Prepare a base solution for low molecular weight hyaluronic acid;

[0019] 4) Layered molding: The tip gel solution is filled into the tip cavity of the PDMS microneedle mold, degassed under vacuum, and then dried after centrifugation; then the base solution is injected into the mold to cover the tip gel solution, degassed under vacuum, and then dried after centrifugation.

[0020] 5) Demolding to obtain the layered microneedle patch.

[0021] The preparation method of the modified chitosan includes the following steps:

[0022] a) Activation: In a buffer system, the carboxyl group of L-arginine was activated using EDC and NHS;

[0023] b) Grafting: Add the chitosan solution to the activation system of step a), and adjust the pH to a weakly acidic range to carry out the grafting reaction;

[0024] c) Purification: The reaction product of step b) is dialyzed and dried to obtain the modified chitosan.

[0025] The use of the aforementioned layered microneedle patch in the preparation of a medicament for treating psoriasis.

[0026] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0027] 1. Highly efficient targeted delivery: In the layered structure design, the rapid dissolution of hyaluronic acid in the basal layer enables the separation of the needle tip from the basal layer, avoiding drug loss; the needle tip layer is directly retained at the lesion site, solving the problems of insufficient transdermal delivery of deferoxamine and poor accumulation of systemic drug delivery.

[0028] 2. Long-lasting release and synergistic treatment: The needle tip layer composed of modified chitosan and deferoxamine has the characteristic of pH-responsive release, which can uniformly release deferoxamine drug molecules in an acidic environment, significantly prolonging the drug release time and maintaining the effective drug concentration at the lesion site; at the same time, modified chitosan not only serves as a carrier for deferoxamine, but also has excellent cfDNA / NETs clearance ability, playing a synergistic anti-inflammatory role.

[0029] 3. Excellent biocompatibility of materials: Modified chitosan and hyaluronic acid are both biocompatible materials, non-irritating to the skin, and suitable for long-term local administration; deferoxamine is an FDA-approved small molecule iron chelating agent with clinical application value.

[0030] 4. Layered microneedle patches are prepared using a layered casting method, which is simple to operate. The release rate and separation time can be controlled by adjusting the ratio of modified chitosan to deferoxamine and the molecular weight of hyaluronic acid. It has high repeatability and is easy to scale up for production. Attached Figure Description

[0031] Figure 1 The diagram shows the results of verifying the DNA binding efficiency of modified chitosan (CS: chitosan; CA1: modified chitosan (1:1); CA2: modified chitosan (1:2); CA3: modified chitosan (1:3)).

[0032] Figure 2 The results of hemolysis tests for three modified chitosans;

[0033] Figure 3 This is a schematic diagram of the fabrication process of a layered microneedle patch according to the present invention;

[0034] Figure 4 Design drawings for the male mold of the layered microneedle patch and a microscope image of the actual microneedle;

[0035] Figure 5 A schematic diagram showing the Evans blue staining of the needle tip of the layered microneedle patch of the present invention;

[0036] Figure 6 Schematic diagram and results of mechanical force testing for layered microneedle patches;

[0037] Figure 7 This describes the inhibitory effect of the layered microneedle patch of the present invention on the skin lesions on the back of psoriasis mice. Detailed Implementation

[0038] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Example 1: Synthesis of Modified Chitosan

[0040] (1) Preparation of MES buffer: Weigh 1.77 g of 2-morpholinoethanesulfonic acid (MES, molecular weight 195.24), dissolve it in 400 mL of deionized water, stir magnetically until completely dissolved, and prepare a 0.023 mol / L MES buffer; adjust the pH to 6.0~7.0 with 1 mol / L HCl or NaOH solution, and keep it at room temperature.

[0041] (2) Activation reaction: The weighed L-arginine (L-Arg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (molar ratio of 1:2:2) were added to the above MES buffer in sequence and stirred magnetically at room temperature (300 r / min) for 0.5 h to ensure that the activating reagents were completely dissolved and the carboxyl group of L-arginine was activated.

[0042] (3) Grafting reaction: Weigh 1 g of chitosan (degree of deacetylation of 80% ~ 95%), dissolve it in 100 mL of 1% (v / v) acetic acid solution, and stir magnetically (200 r / min) until completely dissolved to form a 1% (w / v) chitosan solution. Slowly add the chitosan solution dropwise to the activation system in step (2) above. After the addition is complete, adjust the pH of the reaction system to 5.0~6.0 with 1 mol / L HCl, and react at room temperature with magnetic stirring (300 r / min) for 48 hours.

[0043] (4) Purification: After the reaction was completed, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. Deionized water was used as the dialysis medium (solid-liquid ratio 1:50, w / v), and dialysis was performed at 4°C for 72 hours. The deionized water was replaced every 8 hours until the dialysate tested negative for chloride ions by 0.1 mol / L AgNO3 (to exclude unreacted EDC / NHS). After dialysis, the retentate was transferred to a lyophilized bottle and then dried in a freeze dryer to obtain white, fluffy arginine-modified chitosan. The product was then sealed and stored at -20°C.

[0044] Three modified chitosans with L-arginine to chitosan molar ratios of 1:1, 1:2, and 1:3 were synthesized using this method and named CA1, CA2, and CA3, respectively. Furthermore, the other chemical substances used in the above synthesis steps were commercially available products.

[0045] Example 2: Verification of the efficiency of three modified chitosans in binding to DNA molecules

[0046] The main reagents for this test are: calf thymus DNA (ftDNA) solution and GelGreen staining solution, and the test is completed through the following experimental steps.

[0047] (1) Preparation of ftDNA / GelGreen fluorescent complex: Take 1 mg / mL ftDNA solution and mix it with 1X GelGreen working solution at a volume ratio of 1:1 (e.g., 50 μL ftDNA + 50 μL GelGreen), and incubate at 25℃ in the dark for 10 min to form ftDNA / GelGreen fluorescent complex (at this time, the final concentration of ftDNA in the system is 0.5 mg / mL).

[0048] (2) Binding reaction of modified chitosan to ftDNA: Gradients were set according to the mass ratio of ftDNA to modified chitosan: 0.25, 0.5, 1, 2, 4 (a total of 5 concentration groups), and 2 control groups were set up, namely positive control ( That is, only ftDNA / GelGreen complex (unmodified chitosan) and blank control ( This refers to 1X GelGreen staining solution only (without ftDNA). Specific procedures: Add 50 μL of ftDNA / GelGreen complex (containing 25 μg ftDNA) to a 2 mL centrifuge tube. Calculate and add the corresponding volume of modified chitosan stock solution according to the mass ratio (e.g., for group 1, add 25 μg modified chitosan, i.e., 12.5 μL of 2 mg / mL stock solution). Add PBS buffer to a total reaction volume of 200 μL and gently mix. Incubate at 37°C (150 rpm) in the dark for 4 h to ensure complete reaction.

[0049] (3) Complex separation and supernatant collection: The reaction mixture was transferred to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm and 4℃ for 10 min to allow the modified chitosan-ftDNA complex to precipitate completely. Then, 100 μL of each sample was transferred to a black 96-well plate (Corning), with 3 replicates per sample. Fluorescence intensity was detected using a VICTOR Nivo multimode microplate reader (PerkinElmer): excitation wavelength 485 nm, emission wavelength 590 nm; detection temperature: 25℃; 3 tests were performed per well, and the average value was taken. The fluorescence values ​​(A) of the supernatant for each concentration group and the fluorescence values ​​(A) of the positive control were recorded. ) and blank control fluorescence value ( ).

[0050] (4) Calculation of binding efficiency: The binding efficiency of modified chitosan to DNA is calculated using the formula (1-(A-)). ) / ( - The evaluation is performed by multiplying the value by 100%, where A is the fluorescence intensity of the supernatant after adding modified chitosan. The fluorescence intensity represents the blank control (GelGreen only). The fluorescence intensity is that of the positive control (ftDNA / GelGreen complex).

[0051] Figure 1 The graph shows the DNA binding efficiency validation results for three modified chitosans (CS: chitosan; CA1: L-arginine to chitosan molar ratio 1:1; CA2: L-arginine to chitosan molar ratio 1:2; CA3: L-arginine to chitosan molar ratio 1:3). The results show that modified chitosan binds to DNA more efficiently than unmodified chitosan, with CA1 and CA2 exhibiting higher DNA binding efficiency compared to CA3.

[0052] Example 3: Hemolysis test to evaluate the blood compatibility of three modified chitosans

[0053] Mouse blood (3 mL) was resuspended in 30 mL PBS. Red blood cells (RBCs) were separated from the serum by centrifugation at 3000 rpm for 10 min. The purified RBCs were resuspended in 2% PBS. RBCs treated with 1% Triton-X and PBS served as positive and negative controls, respectively. Different molar ratios of modified chitosan were added to the RBC suspension at concentrations of 62.5, 125, 250, 500, 1000, and 2000 μg / mL. The resulting RBC suspensions were incubated at 37°C for 3 h, followed by centrifugation at 3000 rpm for 10 min. The absorbance of the supernatant was measured at 545 nm using a multi-wall plate reader. The hemolysis rate was calculated using the formula:

[0054] Hemolysis rate (%) = (OD value of test group) - (OD value of negative group) / (OD value of positive group) × 100%

[0055] Figure 2 The image shows the hemolysis test results for three modified chitosans. The study found that when the concentration reached 2 mg / mL, the hemolysis rate of CA1 was higher than 5%. Conversely, the hemolysis rates of CA2 and CA3 were both lower than 5%, indicating good blood compatibility. This is because the positive charge of CA1 generates a strong electrostatic interaction with red blood cells, thus inducing hemolysis; while the lower levels of positive charge in CA2 and CA3 prevent red blood cell rupture.

[0056] Example 4: Preparation of layered microneedle patches

[0057] Given that CA2 has excellent DNA binding ability and good biocompatibility, this invention further selects CA2 (with a molar ratio of L-arginine to chitosan of 1:2) to further prepare microneedles.

[0058] (1) Preparation of polydimethylsiloxane (PDMS) microneedle mold: A 12 mm × 12 mm microneedle array was designed using Solidworks modeling software. The array contained 81 conical microneedles arranged in a 9 × 9 layout. The height h of each microneedle was 1.2 mm and the bottom diameter r was 0.6 mm. The microneedle master mold was then obtained using a 3D printer. The PDMS precursor and curing agent were then mixed in a 10:1 ratio and the master mold was replicated in reverse. The master mold was placed in a constant temperature oven for curing and then carefully demolded from the master mold to obtain a precisely replicated PDMS microneedle array. After 1 hour of UV irradiation, the array was used for subsequent microneedle fabrication.

[0059] (2) Preparation of modified chitosan-deferoxamine gel: Mix modified chitosan and deferoxamine at a mass ratio of 1:1, add 1% acetic acid solution, and stir magnetically at 35°C for 3 hours until completely dissolved to form a uniform modified chitosan-deferoxamine gel (mass concentration 5%).

[0060] (3) Preparation of hyaluronic acid solution: Dissolve low molecular weight hyaluronic acid (molecular weight 8000~10000Da) in deionized water and stir until completely dissolved to obtain a hyaluronic acid solution with a mass concentration of 15%. Remove the bubbles under negative pressure and set aside for use.

[0061] (4) Layered molding and microneedle formation: The modified chitosan-deferroamine gel from step (2) is injected into the tip cavity of the PDMS microneedle mold, vacuum degassing for 10-15 minutes, and centrifuged at 3500 rpm to completely fill the tip with the solution, and dried at 37°C for 1-2 hours; continue to inject the hyaluronic acid solution from step (3) into the mold, vacuum degassing for 10-15 minutes, centrifuged at 3500 rpm for 5 minutes, and dried at 37°C for 24 hours. After demolding, the layered microneedle patch is obtained.

[0062] (5) Microneedle morphology observation: The prepared microneedles were peeled off and photographed for observation.

[0063] Figure 3 This is a schematic diagram of the fabrication process of layered microneedle patches; Figure 4 The images show the design drawing of the positive mold for the layered microneedle patch and a microscopic image of the actual microneedles. Observation under an Mshot-MZ62 optical microscope reveals that the tips of the layered microneedles are sharp and conical, with an intact tip structure, which facilitates the microneedles' penetration of the stratum corneum into the dermis.

[0064] Example 5: Verification of the layered structure of layered microneedles

[0065] To verify the layered structure of the layered microneedles and confirm the successful loading of modified chitosan-deferamide into the tip layer, modified chitosan-deferamide gel labeled with Evans blue was used, and layered microneedles with Evans blue-stained tips were obtained according to the experimental steps for preparing layered microneedles. The obtained layered microneedles were then observed under an optical microscope.

[0066] like Figure 5 Observation using an Mshot-MZ62 optical microscope revealed that the tip layer of the layered microneedles was uniformly stained with Evans blue, and the separation from the upper substrate was obvious, indicating that the Evans blue-labeled modified chitosan-deferamine gel was successfully loaded and uniformly distributed within the tip.

[0067] Example 6: Mechanical strength test of layered microneedles

[0068] The layered microneedle patch is pressed onto a stainless steel fixture. The initial gauge between the microneedle tip and the stainless steel fixture is set to 2 mm. A metal sensor, perpendicular to the microneedle, approaches the microneedle downwards at a speed of 0.1 mm / s to apply pressure. As the metal sensor contacts the microneedle tip and begins to apply pressure, the microneedle gradually bends. The stress and displacement data of the microneedle during the compression process are recorded in real time through a data acquisition system connected to an electronic universal testing machine. This data is converted into stress-displacement curves, and analysis of these curves visually reveals the elastic and plastic deformation stages of the microneedle, as well as its final fracture point. The first abrupt change in the curve typically indicates the critical point where the microneedle transitions from an elastic to a plastic state, i.e., the location where permanent deformation begins. Detailed analysis of this abrupt change point allows for the precise calculation of the maximum load-bearing capacity of the microneedle, i.e., the maximum pressure the microneedle can withstand before losing its elastic recovery ability.

[0069] like Figure 6 As shown, the microneedle breaking force of the layered microneedles is approximately 0.35 N, which is sufficient to break the stratum corneum of the skin (0.058 N). Moreover, the breaking points of the microneedle tips are relatively uniform, all breaking at a compression of about 0.5 mm, indicating that the microneedles can deliver drugs subcutaneously.

[0070] Example 7: Evaluation of the therapeutic effect of layered microneedle patches in a mouse model of psoriasis.

[0071] The layered microneedle patch prepared in Example 4 was used to treat a mouse model of psoriasis induced by imiquimod ointment. Six- to eight-week-old male BALB / c mice were used in the experiment and divided into a normal group, a model group, a deferoxamine application group, and a layered microneedle group. Treatment began three days after imiquimod ointment induction, with layered microneedle treatment administered every three days; all treatment regimens were implemented 6 hours after imiquimod ointment administration. Mice were sacrificed on the eighth day, and dorsal skin lesions were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for observation.

[0072] Figure 7 The appearance of the mouse backs showed that, compared with the model control group and the deferoxamine application group, the layered microneedling treatment group had significantly improved skin lesions; in addition, Figure 7 The H&E staining results of tissue sections showed that the epidermal thickness of mice in the layered microneedle treatment group was significantly lower than that in the model group and the deferoxamine application group, and the reduction was more significant, indicating that the present invention has a significant therapeutic effect on the back lesions of psoriatic mice.

[0073] In this invention, the modified chitosan is made by modifying L-arginine with chitosan, and has excellent cfDNA / NETs scavenging ability and biocompatibility. It can be used as a carrier of deferoxamine and synergistically block the inflammatory cycle.

[0074] The needle support portion and base surface of the microneedle array are made of low molecular weight hyaluronic acid, which has rapid water solubility and can dissolve quickly after contact with the skin, achieving rapid separation of the needle tip from the base surface, avoiding the needle tip from falling off with the base surface, and ensuring effective deposition of drugs at the lesion site.

[0075] In this invention, the needle tip layer composed of modified chitosan-deferoxamine has the characteristic of pH-responsive release, which can uniformly release deferoxamine drug molecules in an acidic environment, significantly prolonging the drug release time and maintaining the effective drug concentration at the lesion site.

Claims

1. A layered microneedle patch, characterized in that: It includes a microneedle array and a substrate; the needle body of the microneedle array includes a needle tip portion and a support portion, the needle tip portion is composed of a gel matrix containing a therapeutic agent and modified chitosan; the support portion and the substrate are composed of low molecular weight hyaluronic acid; wherein, the support portion dissolves upon contact with skin tissue fluid, causing the needle tip portion to separate from the substrate and remain in the skin tissue.

2. The layered microneedle patch as described in claim 1, characterized in that: The modified chitosan is L-arginine-modified chitosan; the molar ratio of L-arginine to chitosan is 1:1~3; and the degree of deacetylation of the chitosan is 80%~95%.

3. The layered microneedle patch as described in claim 1, characterized in that: The therapeutic agents include ferroptosis inhibitors.

4. A layered microneedle patch as described in claim 3, characterized in that: The ferroptosis inhibitor comprises an iron chelating agent and / or an antioxidant; the iron chelating agent is selected from at least one of deferoxamine, deferasirox, and deferoxone; the antioxidant is selected from at least one of ferrostatin-1, liproxstatin-1, and vitamin E.

5. A layered microneedle patch as described in claim 3, characterized in that: The therapeutic agents also include at least one of methotrexate, cyclosporine, acitretin, azathioprine, hydroxyurea, glucocorticoids, anti-tumor necrosis factor-α inhibitors, IL-12 / 23 antibodies, IL-17A antibodies, and IL-23p19 antibodies.

6. A layered microneedle patch as described in claim 1, characterized in that: In the gel matrix, the mass ratio of the modified chitosan to the therapeutic agent is 1:(0.8~1.2).

7. A layered microneedle patch as described in claim 1, characterized in that: The low molecular weight hyaluronic acid has a molecular weight of 8000 Da to 10000 Da.

8. A method for preparing a layered microneedle patch as described in any one of claims 1 to 7, characterized in that, Includes the following steps: 1) Provide PDMS microneedle molds; 2) Prepare a needle tip gel solution containing a therapeutic agent and modified chitosan; 3) Prepare a base solution for low molecular weight hyaluronic acid; 4) Layered molding: The tip gel solution is filled into the tip cavity of the PDMS microneedle mold, degassed under vacuum, and then dried after centrifugation; then the base solution is injected into the mold to cover the tip gel solution, degassed under vacuum, and then dried after centrifugation. 5) Demolding to obtain the layered microneedle patch.

9. The method as described in claim 8, characterized in that, The preparation of the modified chitosan includes the following steps: a) Activation: In a buffer system, the carboxyl group of L-arginine was activated using EDC and NHS; b) Grafting: Add the chitosan solution to the activation system of step a), and adjust the pH to a weakly acidic range to carry out the grafting reaction; c) Purification: The reaction product of step b) is dialyzed and dried to obtain the modified chitosan.

10. The use of a layered microneedle patch as described in any one of claims 1 to 7 in the preparation of a medicament for treating psoriasis.

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