Soluble microneedle patch loaded with immunogen cell death inducer as well as preparation method and application of soluble microneedle patch

Abstract

The invention discloses a soluble microneedle patch loaded with an immunogen cell death inducer as well as a preparation method and application of the soluble microneedle patch. The preparation method comprises the following steps: synthesizing methacrylated hyaluronic acid, synthesizing an ICD inducer PKHB1 polypeptide, preparing a drug-loaded microneedle patch and the like. The microneedle patch can penetrate through a biological membrane to directly deliver a medicine to a specific area, so that the bioavailability of the medicine is improved, adverse reactions are relieved, the compliance of a patient is improved, and the microneedle patch is efficient and convenient. And the limitations of frequent operation, large trauma, easy infection and the like existing in the administration mode of subconjunctival injection are avoided. The synthesis steps are simple and rapid, the used raw materials are cheap, common and easy to purchase, the product is more economical and practical, the economic burden of patients can be effectively relieved, and the method is suitable for popularization. HAMA is used as a raw material, an ICD inducer is loaded, and the constructed soluble drug-loaded MN patch for eyes provides a novel ocular surface drug delivery mode which is minimally invasive, painless, convenient and effective.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a soluble microneedle patch loaded with an immunogenic cell death inducer, and a preparation method and application thereof. Background Art

[0002] Herpes simplex keratitis (HSK) is a serious infectious corneal disease caused by herpes simplex virus type 1 (HSV-1) infection and is the leading cause of corneal blindness. The increasing prevalence of antiviral drug resistance, the side effects of hormonal drugs, and the easy recurrence after surgery make it urgent to develop new methods for the treatment of HSK. After HSV-1 invades the host, it suppresses the host's immune response through various mechanisms, replicates in large quantities, and spreads rapidly. However, at this stage, there is still a lack of sufficient research in this field of enhancing immunity to treat HSK.

[0003] Immunogenic cell death (ICD) is a form of cell death that enhances the host's immune capacity. By exposing or releasing endogenous immunogenic biological factors, namely damage-associated molecular patterns (DAMPs), antigen-presenting cells are recruited to the ICD site, stimulating the uptake, processing and presentation of antigens associated with dead cells, activating CTL-driven adaptive immune responses, and establishing long-term immune memory. As an emerging treatment method, the research on inducing ICD is mainly focused on the field of tumors, but related research in the fields of ophthalmology and infection is still in a relatively early stage. Currently, ICD inducers used in clinical practice, such as alkylating agents, oxaliplatin, anthracyclines, and X-rays, all have serious toxic side effects. Therefore, finding safe and effective ICD inducers, improving the body's immunogenic response and reducing the toxic side effects of the drug itself on the body, is the key to immunotherapy. Recently, American scholars designed a peptide called PKHB1 based on the C-terminal structure-activity relationship of thrombospondin-1, which has been proven to induce ICD in a variety of tumor cells. The previous research of our research group revealed the effective role of PKHB1 in infectious eye diseases for the first time in the world, but the subconjunctival injection method still has limitations such as frequent operation, high trauma, and easy infection. Therefore, it is very important to find a new ocular surface drug delivery method that is minimally invasive, painless, convenient and effective. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide an ophthalmic soluble microneedle patch loaded with immunogenic cell death inducer and having biodegradability, physicochemical stability and controllable release, and a preparation method thereof, in view of the deficiencies in the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer comprises the following steps: (1) Synthesis of methacryloyl hyaluronic acid; Hyaluronic acid and methacrylic anhydride are subjected to a methacrylation reaction, and the methacrylated hyaluronic acid powder is obtained by purification and freeze-drying; (2) Synthesis of ICD inducer PKHB1 peptide; After resin swelling, amino acid condensation, deprotection, cleavage, and purification, high-purity polypeptides are obtained. (3) Preparation of drug-loaded microneedle patches; The methacryloyl hyaluronic acid is placed in the initiator and shaken to mix, and the ICD inducer PKHB1 polypeptide is added at the same time. After centrifugation to remove bubbles, the mixture is dropped into the mold and demoulded after curing.

[0006] Specifically, in step (1), the methacryloyl hyaluronic acid is prepared by the following steps: S1-1 Raw material preparation: Hyaluronic acid is used as the main raw material; methacrylic anhydride is used as the reaction reagent for methacrylation; deionized water is used as the solvent; sodium hydroxide solution is used as the catalyst to adjust the pH value of the reaction environment; S1-2 Reaction environment preparation: Dissolving hyaluronic acid: Weigh hyaluronic acid solid powder, place in deionized water, and stir continuously under low temperature conditions to ensure complete dissolution to form a uniform solution; Adjusting pH value: maintaining a low temperature environment, adding sodium hydroxide solution, and adjusting the pH value of the solution to 8.0-9.0 to provide an alkaline environment suitable for the reaction of methacrylic anhydride; S1-3 Methacrylation reaction: Adding methacrylic anhydride: maintaining a low temperature environment, slowly drop methacrylic anhydride into the solution of step S1-2, and fully stir; keep the solution at pH 8.0-9.0 and stirring the reaction at low temperature for more than 24 hours to ensure sufficient methacrylation.

[0007] Furthermore, step (1) further includes post-reaction treatment in step S1-4: Precipitation and purification: adding excess ethanol to the solution obtained in step S1-3 to precipitate the methacrylylated hyaluronic acid, and then dissolving it in deionized water. The polymer solution is dialyzed with deionized water for 3-5 days to remove unreacted methacrylic anhydride and small molecule by-products; Freeze drying: The dialyzed solution is freeze dried to finally obtain purified methacryloyl hyaluronic acid powder.

[0008] Specifically, in step (2), the ICD inducer PKHB1 polypeptide is synthesized by the following steps: S2-1: Weigh 0.6-1g of resin and add 1.5 - 2ml / g dichloromethane, shake for 30-45 minutes to make it swell; S2-2: After the product obtained in S2-1 is filtered, Fmoc-Leu-OH, dimethylformamide, ethyl diisopropylamine and condensation reagent are added in sequence, treated with nitrogen for 0.5-1 hour, washed with DMF 2-4 times, and tested with ninhydrin; S2-3: Add 20-30% piperidine / DMF solution, shake for 15-20 minutes, wash with DMF, and detect with ninhydrin; repeat steps S2-2 and S2-3, wash with methanol and drain to obtain resin peptide; S2-4: Dry the resin peptide, add cutting solution, shake, filter, centrifuge and wash to obtain crude peptide; S2-5: Purification of crude peptide.

[0009] Furthermore, step (2) obtains a PKHB1 polypeptide with a purity of >95%.

[0010] Specifically, in step (3), the drug-loaded microneedle patch is prepared by the following steps: S3-1: prepare initiator standard solution; S3-2: Preparation of a mixed solution of methacryloyl hyaluronic acid and ICD inducer PKHB1 polypeptide: Take 50-80 mg of methacryloyl hyaluronic acid and put it into a glass bottle. At the same time, add 1-2 mL of the initiator standard solution obtained in step S3-1 and shake to mix. Then add 250-300 µg of the ICD inducer PKHB1 polypeptide prepared in the above process to the above container, stir and dissolve at room temperature for 1 hour; centrifuge to remove bubbles in the system, sterilize with a sterile syringe filter, and store at 4°C away from light; S3-3: Preparation of drug-loaded microneedles: Clean the polydimethylsiloxane mold and dry it for later use; add the mixed solution obtained in step S3-2; centrifuge to remove bubbles, gently scrape the bubbles on the colloid surface of the pinhole with the gun tip, and repeat 2-3 times; heat in an oven at 25-35°C for 3-5 hours, take it out and add the mixed solution obtained in step S3-2 again, filling it up to above the mold plane; heat in an oven at 25-35°C for 3-5 hours again; demold it after light curing to obtain a patch.

[0011] Specifically, in step S3-1, take 10 mL of PBS and add it to a brown bottle containing the initiator; heat and dissolve it in a water bath at 40-50°C for 10-20 minutes, oscillating several times during the process to obtain a 0.20-0.30% (w / v) initiator standard solution, which is then stored away from light.

[0012] Specifically, in step S3-3, after curing with 405nm UV light for 30-60 seconds, tweezers were used to demold it, and a patch with a diameter of 2-3mm was successfully obtained. The conical microneedle patch had an array of 5*5, a height of 200-300μm, a bottom width of 100-200μm, a needle tip distance of 300-400µm, and a base thickness of 1-2mm, and was stored at 4°C.

[0013] Furthermore, the soluble microneedle patch loaded with immunogenic cell death inducer prepared by the above preparation method is also within the protection scope of the present invention.

[0014] Furthermore, the present invention also claims to protect the use of the above-mentioned soluble microneedle patch loaded with immunogenic cell death inducers for ocular drug delivery. Beneficial Effects

[0015] (1) The microneedle patch of the present invention can penetrate the biological membrane and deliver the drug directly to a specific area to improve the bioavailability of the drug, reduce adverse reactions, and improve patient compliance. It is efficient and convenient. The drug can be loaded by physical adsorption, chemical binding or embedding in the microneedle material, and can be released in a controlled manner. It avoids the limitations of the subconjunctival injection method, such as frequent operation, high trauma, and easy infection. The synthesis steps of the present invention are simple and fast, and the raw materials used are not only cheap but also common and easy to purchase, making the product more economical and affordable, which can effectively reduce the economic burden of patients and is suitable for promotion.

[0016] (2) The methacryloyl hyaluronic acid (HAMA) of the present invention is a chemically modified hyaluronic acid, into which a methacryloyl group is introduced. As a derivative of hyaluronic acid, HAMA retains the good biocompatibility and biodegradability of hyaluronic acid, and at the same time has many properties superior to natural hyaluronic acid, such as adjustable mechanical properties, physicochemical stability and enhanced drug delivery. The present invention uses HAMA as a raw material, loads an ICD inducer, and constructs a soluble drug-loaded MN patch for the eye, which provides a new type of ocular surface drug delivery that is minimally invasive, painless, convenient and effective.

[0017] (3) The microneedle patch of the present invention induces the occurrence of ICD, which can trigger multiple immune responses, including the recognition of antigens by innate immunity and the activation of T cells, forming a comprehensive anti-infection barrier. The stimulated immune response is not limited to immediate pathogen clearance, but can also promote the formation of long-term immune memory and improve the body's resistance to reinfection with the same pathogen. It has a good therapeutic effect on infectious diseases and can effectively combat their recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 This is the preparation process and application of Example 1 of the present invention.

[0020] Figure 2 It is a schematic diagram and a physical diagram of Example 1 of the present invention.

[0021] Figure 3 This is a Fourier transform infrared spectrum diagram of Example 2 of the present invention.

[0022] Figure 4 This is a stress-displacement curve diagram of Example 3 of the present invention.

[0023] Figure 5 This is the in vitro drug release diagram of Example 4 of the present invention.

[0024] Figure 6 This is the in vitro cytotoxicity test of Example 5 of the present invention.

[0025] Figure 7 This is the whole-body biosafety test on mice according to Example 6 of the present invention.

[0026] Figure 8 This is the local biological safety test of mouse eyes according to Example 6 of the present invention. DETAILED DESCRIPTION

[0027] The present invention can be better understood with reference to the following examples. Example 1

[0028] Weigh 2.0g of HA solid powder, place it in 100mL of deionized water, and stir continuously at low temperature (4°C) to ensure complete dissolution to form a uniform solution. Maintain a low temperature environment, add 1M sodium hydroxide solution, and adjust the pH of the solution to 8.0-9.0 to provide an alkaline environment suitable for the reaction of methacrylic anhydride. Maintain a low temperature environment, slowly add 4mL of MA, and stir well. Keep the solution stirring and reacting at pH 8.0-9.0 and low temperature for 24 hours to ensure sufficient methacrylation. Add excess ethanol to precipitate HAMA. Then dissolve it in deionized water, and dialyze the polymer solution with deionized water for 3 days to remove unreacted methacrylic anhydride and small molecule by-products. The dialyzed solution is freeze-dried to finally obtain purified HAMA powder.

[0029] Weigh 0.6g of resin, add 1.5ml / g of dichloromethane, and shake for 30 minutes to swell it. After the obtained product is filtered, Fmoc-Leu-OH, dimethylformamide, ethyl diisopropylamine and condensation reagent are added in sequence, nitrogen treatment for 0.5 hours, DMF washing 3 times, and ninhydrin detection. Add 20% piperidine / DMF solution, shake for 15 minutes, DMF washing, ninhydrin detection; repeat the above, wash with methanol and dry to obtain resin peptide; dry the resin peptide, add cutting solution, shake, filter, centrifuge and wash to obtain crude peptide; purify the crude peptide to obtain PKHB1 polypeptide with a purity of >95%.

[0030] Take 10mL PBS and add it to the brown bottle containing the initiator LAP (containing 0.025g LAP); heat and dissolve in a 40-50℃ water bath for 15 minutes, shake several times during the period, and successfully prepare a 0.25% (w / v) initiator standard solution. Take 50mg HAMA and put it into a glass bottle. At the same time, add 1mL of the initiator standard solution to the above container and shake to mix. Then add 270µg PKHB1 polypeptide to the above container and stir and dissolve at room temperature for 1 hour. Centrifuge (3min*3000rpm) to remove bubbles in the system, sterilize with a 0.22μm sterile syringe filter, drop into the polydimethylsiloxane (PDMS) mold, centrifuge for 10min*3000rpm to remove bubbles, and gently scrape the bubbles on the surface of the pinhole with the gun tip, repeat 2-3 times. Heat in a 30℃ oven for 4 hours, take it out and drop the drug-loaded HAMA solution again, try to fill it up to the surface of the mold. Heat in a 30℃ oven again for 4 hours. After UV405nm light curing for 30 seconds, the microneedle patch was demoulded with tweezers to successfully obtain a soluble drug-loaded MN patch for eye use. Finally, it was applied to the ocular surface by pressing with fingers. The preparation process and principle are as follows Figure 1 As shown. The tapered arrow with a height to matrix diameter ratio of 2:1 is most suitable for tissue penetration, so the actual object we prepared is as follows Figure 2 As shown in the figure, the patch is composed of cone-shaped microneedles with an array of 5*5, a height of 200-300μm, a bottom width of 100-200μm, a needle tip distance of 300-400µm, and a base thickness of 1-2mm. The microneedle patch reversibly physically penetrates the corneal epithelium, effectively increases the retention time of the drug on the ocular surface, significantly improves the bioavailability of the drug, and provides a new strategy and method for the clinical treatment of anterior segment diseases. Example 2

[0031] In a dry environment, the ATR accessory was placed in the optical path of the spectrometer to scan the air background. Then, the PKHB1 peptide, blank microneedle patch, and drug-loaded microneedle patch were placed on the crystal surface of the ATR accessory in turn. The Nicoleti S5 Fourier transform infrared spectrometer was used to test the chemical composition of the samples in the measurement range of 400-4000 cm -1.like Figure 3 As shown, PKHB1 at 2960 cm -1 、1625cm -1 、801cm -1 、722cm -1 The characteristic peak appears at the wavelength. Blank-MN is at 1405cm -1 、1030cm -1 、947cm -1 The characteristic peaks appeared at the wavelength, which was consistent with the HAMA detection in the literature. In the MN patch loaded with PKHB1, all the typical peaks of PKHB1 and HAMA were seen, indicating that PKHB1 was successfully encapsulated in the MN patch. Example 3

[0032] Place the blank microneedle patch and the drug-loaded microneedle patch with the needle tip facing up on the horizontal platform of the electronic universal material testing machine. Before starting the measurement, adjust the sensor probe to about 1 cm away from the needle tip. Run the program to adjust the sensor probe measurement speed to 0.05 mm / s and the measurement direction to vertically downward. After starting the measurement, record the relationship between deformation and force, and then remove the sample. Figure 4 As shown in the figure, the stress-displacement curve is complete and there is no missing part, which indicates that the microneedle did not break during the test, but stably bent under constant pressure. According to the literature, it takes 0.5N of insertion force for the microneedle to enter the corneal barrier. The microneedle we prepared has the mechanical strength to be inserted into the cornea. Example 4

[0033] Accurately weigh a certain amount of PKHB1 to prepare a series of standard solutions with concentrations of 50, 100, 250, 500, and 1000 µg / mL. The solvent is PBS phosphate buffer containing 25% ethanol. The characteristic absorption peak of amino acids containing aromatic rings is at 280nm, so the absorbance value of each concentration is measured at a wavelength of 280nm. Use a UV-visible spectrophotometer to measure the absorbance value of each concentration at a wavelength of 280nm, fit the obtained absorbance value with the corresponding concentration, and obtain the standard curve of PKHB1. Figure 5 As shown. The absorbance values ​​of PKHB1 solutions of different concentrations at 280nm are linearly correlated with the concentration. The linear regression equation is: Y=0.001089*X-0.01164, R 2 =0.996, good linear relationship.

[0034] To further evaluate the drug release behavior of the drug-loaded microneedles, the microneedle patch loaded with 600µg of drug was immersed in 10ml PBS phosphate buffer and 25% ethanol solution was added to facilitate the dissolution of the drug. The container was placed in a shaker with the temperature set to 37°C and shaken at a constant speed of 100 rpm. 1mL of solution was taken at a predetermined time point and replaced with an equal amount of fresh matrix solution. The collected solution was measured for its absorbance at a wavelength of 280nm. After comparison with the standard curve, the release curve of PKHB1 was obtained by quantitative processing, and its cumulative release amount ( Figure 5 ). Example 5

[0035] HCEC cells and VERO cells were cultured in 96-well plates, and the experiment was carried out when the cell density reached about 80%. The supernatant was removed, and a direct contact experiment (the microneedle patch was placed on the well plate) was used to detect cell viability using CCK8. After culturing for 12h, 24h and 36h in a 37°C incubator containing 5% CO2, the culture medium was aspirated and 100mL of fresh culture medium containing 10mL of CCK-8 working solution was added to each well, and the incubation continued for 2 hours. Finally, the OD value of each well was measured at 450nm using an enzyme reader, and the OD value of the treated cells was compared with the OD value of the untreated cells to obtain the percentage of cell activity at different times of microneedle patch treatment. Figure 6 As shown, the viability of both cell types was observed to be greater than 90% after 36 h of incubation, indicating that the microneedles did not affect cell viability in vitro. Example 6

[0036] In order to study the biosafety of the microneedle patch on mice, the microneedle patch was inserted into the mouse cornea by gently pressing the thumb, and the mouse cornea was observed using sodium fluorescein at 0h, 3h, 6h, 12h, and 24h, and the weight of the mice was weighed at 5 days, 10 days, and 15 days. Figure 7-8 As shown, after using the microneedle patch, the weight of the mice did not change and the cornea completely recovered within 24 hours, indicating that the invention has good biosafety.

[0037] The present invention provides a soluble microneedle patch loaded with an immunogenic cell death inducer and a method for preparing and applying the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer, characterized in that: The steps include: (1) Synthesis of methacryloyl hyaluronic acid; Hyaluronic acid and methacrylic anhydride are subjected to a methacrylation reaction, and the methacrylated hyaluronic acid powder is obtained by purification and freeze-drying; (2) Synthesis of ICD inducer PKHB1 peptide; After resin swelling, amino acid condensation, deprotection, cleavage, and purification, a high-purity polypeptide is obtained; (3) Preparation of drug-loaded microneedle patches; The methacryloyl hyaluronic acid is placed in the initiator and shaken to mix, and the ICD inducer PKHB1 polypeptide is added at the same time. After centrifugation to remove bubbles, the mixture is dropped into the mold and demoulded after curing.

2. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 1, characterized in that: In step (1), the methacryloyl hyaluronic acid is prepared by the following steps: S1-1 Raw material preparation: Hyaluronic acid is used as the main raw material; methacrylic anhydride is used as the reaction reagent for methacrylation; deionized water is used as the solvent; sodium hydroxide solution is used as the catalyst to adjust the pH value of the reaction environment; S1-2 Reaction environment preparation: Dissolving hyaluronic acid: Weigh hyaluronic acid solid powder, place in deionized water, and stir continuously under low temperature conditions to ensure complete dissolution to form a uniform solution; Adjusting pH value: maintaining a low temperature environment, adding sodium hydroxide solution, and adjusting the pH value of the solution to 8.0-9.0 to provide an alkaline environment suitable for the reaction of methacrylic anhydride; S1-3 Methacrylation reaction: Adding methacrylic anhydride: maintaining a low temperature environment, slowly drop methacrylic anhydride into the solution of step S1-2, and fully stir; keep the solution at pH 8.0-9.0 and stirring the reaction at low temperature for more than 24 hours to ensure sufficient methacrylation.

3. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 1, characterized in that: Step (1) further includes post-reaction treatment of steps S1-4: Precipitation and purification: adding excess ethanol to the solution obtained in step S1-3 to precipitate the methacrylylated hyaluronic acid, and then dissolving it in deionized water. The polymer solution is dialyzed with deionized water for 3-5 days to remove unreacted methacrylic anhydride and small molecule by-products; Freeze drying: The dialyzed solution is freeze dried to finally obtain purified methacryloyl hyaluronic acid powder.

4. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 1, characterized in that: In step (2), the ICD inducer PKHB1 polypeptide is synthesized by the following steps: S2-1: Weigh 0.6-1g of resin and add 1.5 - 2ml / g dichloromethane, shake for 30-45 minutes to make it swell; S2-2: After the product obtained in S2-1 is filtered, Fmoc-Leu-OH, dimethylformamide, ethyl diisopropylamine and condensation reagent are added in sequence, treated with nitrogen for 0.5-1 hour, washed with DMF 2-4 times, and tested with ninhydrin; S2-3: Add 20-30% piperidine / DMF solution, shake for 15-20 minutes, wash with DMF, and detect with ninhydrin; repeat steps S2-2 and S2-3, wash with methanol and drain to obtain resin peptide; S2-4: Dry the resin peptide, add cutting solution, shake, filter, centrifuge and wash to obtain crude peptide; S2-5: Purification of crude peptide.

5. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 4, characterized in that: Step (2) obtains a PKHB1 polypeptide with a purity of >95%.

6. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 1, characterized in that: In step (3), the drug-loaded microneedle patch is prepared by the following steps: S3-1: prepare initiator standard solution; S3-2: Preparation of a mixed solution of methacryloyl hyaluronic acid and ICD inducer PKHB1 polypeptide: Take 50-80 mg of methacryloyl hyaluronic acid and put it into a glass bottle. At the same time, add 1-2 mL of the initiator standard solution obtained in step S3-1 and shake to mix. Then add 250-300 µg of the ICD inducer PKHB1 polypeptide prepared in the above process to the above container, stir and dissolve at room temperature for 1 hour; centrifuge to remove bubbles in the system, sterilize with a sterile syringe filter, and store at 4°C away from light; S3-3: Preparation of drug-loaded microneedles: Clean the polydimethylsiloxane mold and dry it for later use; add the mixed solution obtained in step S3-2; centrifuge to remove bubbles, gently scrape the bubbles on the colloid surface of the pinhole with the gun tip, and repeat 2-3 times; heat in an oven at 25-35°C for 3-5 hours, take it out and add the mixed solution obtained in step S3-2 again, filling it up to above the mold plane; heat in an oven at 25-35°C for 3-5 hours again; demold it after light curing to obtain a patch.

7. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 6, characterized in that: In step S3-1, take 10 mL of PBS and add it to the brown bottle containing the initiator; heat and dissolve it in a 40-50°C water bath for 10-20 minutes, shaking it several times during the process, to obtain a 0.20-0.30% (w / v) initiator standard solution, which is stored away from light.

8. The method for preparing a soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 6, characterized in that: In step S3-3, after curing with 405nm UV light for 30-60 seconds, tweezers were used to demold it, and a patch with a diameter of 2-3mm was successfully obtained. The conical microneedle patch with an array of 5*5, a height of 200-300μm, a bottom width of 100-200μm, a needle tip distance of 300-400µm, and a base thickness of 1-2mm was stored at 4°C.

9. A soluble microneedle patch loaded with an immunogenic cell death inducer prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the soluble microneedle patch loaded with an immunogenic cell death inducer according to claim 9 for ocular drug delivery.

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