Degradable microneedle patch co-loaded with sound-sensitive agent and STING agonist as well as preparation method and application of degradable microneedle patch

Abstract

The invention discloses a degradable microneedle patch co-loaded with a sound sensitive agent and an STING agonist as well as a preparation method and application of the degradable microneedle patch. The invention provides a microneedle raw material composition. The microneedle raw material composition comprises a sound-sensitive agent and an STING agonist, the mass ratio of the sound sensitive agent to the STING agonist is (0.2-1): 1. Furthermore, the invention provides a microneedle patch which comprises a back weighing layer and a needle body array arranged on the back weighing layer, and a microneedle raw material composition is loaded in an aqueous solution of a degradable polymer of the needle body array. According to the invention, co-delivery of the sound-sensitive agent and the STING agonist is utilized, penetration of the drug to the deep part of the tumor tissue can be enhanced through a sonodynamic therapy, more accurate drug release is realized, a stronger immune effect is caused, in-situ tumors are eliminated, growth of far-end tumors is inhibited, and the immunotherapy effect of the drug is synergistically improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tumor treatment, and particularly relates to a degradable microneedle patch co-loaded with a photosensitizer and a STING agonist, a preparation method and application thereof. BACKGROUND

[0002] Breast cancer is one of the most common malignant tumors in women, accounting for 7-10% of systemic malignant tumors in China, and showing an increasing trend year by year. Triple-negative breast cancer (TNBC) is a special type of breast cancer, which is characterized by negative estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in immunohistochemical detection. Due to the lack of these clear treatment targets, traditional endocrine therapy and HER2-targeted therapy are ineffective for TNBC, making it one of the breast cancer subtypes with the highest malignancy and the worst prognosis. The current treatment strategy for TNBC mainly relies on chemotherapy.

[0003] STING agonists can initiate innate and adaptive immune responses, promote dendritic cell maturation, and increase cytotoxic T lymphocyte infiltration and natural killer cell recruitment, thereby killing tumors. Studies have shown that STING agonists can not only inhibit the growth of B16 melanoma, 4T1 breast cancer, CT26 colon cancer, triple-negative breast cancer, skin cancer and B-cell lymphoma, but also inhibit the growth of tumors in untreated distant sites. This means that STING agonists have the potential to become a new type of tumor immunotherapy drug for the treatment of triple-negative breast cancer. However, how to deliver STING agonists to tumor tissues and achieve deep drug delivery in tumor tissues is a key scientific problem that affects the effectiveness of cancer treatment. SUMMARY

[0004] In view of the above existing problems in the prior art, the primary object of the present application is to provide a microneedle raw material composition.

[0005] The second object of the present application is to provide a microneedle comprising the above-mentioned microneedle raw material composition.

[0006] The third object of the present application is to provide a microneedle patch comprising the above-mentioned microneedle and a degradable polymer.

[0007] The fourth object of the present application is to provide the use of the above-mentioned microneedle raw material composition, microneedle and microneedle patch in the preparation of a drug for sonodynamic therapy of tumors.

[0008] In order to achieve the above-mentioned objects, the present application is realized by the following technical solutions: The primary object of the present application is to provide a microneedle raw material composition, which comprises a sonosensitizer and a STING agonist; the mass ratio of the sonosensitizer to the STING agonist is (0.2-1):1.

[0009] The present application utilizes the co-delivery of a sonosensitizer and a STING agonist, which not only enhances the penetration of drugs into deep tumor tissues through sonodynamic therapy (SDT), but also synergistically improves the immunotherapy effect on tumors in combination with the STING agonist. SDT is a new type of physical tumor treatment mode combining low-intensity ultrasound and a sonosensitizer. During SDT, not only can the nanodrug be promoted to penetrate into the deep area of the tumor tissue, but also can induce immunogenic cell death (ICD) of tumor cells; the STING agonist can initiate innate and adaptive immune responses. The sonosensitizer and the STING agonist synergistically induce immature dendritic cells (DCs) to develop into mature dendritic cells, and further present tumor antigens to cytotoxic T cells, ultimately initiating specific killing of tumor cells. The present application provides a raw material composition containing a sonosensitizer and a STING agonist, which solves the technical problems of delivering the STING agonist to the tumor tissue and achieving deep drug delivery of the tumor tissue when it is applied as a microneedle, and the sonosensitizer and the STING agonist synergistically improve the tumor treatment effect.

[0010] Preferably, the sonosensitizer is at least one of indocyanine green, indocyanine green carboxylic acid, indocyanine green-thiol, indocyanine green-amino, and indocyanine green-succinimidyl ester; further preferably, the sonosensitizer is at least one of indocyanine green carboxylic acid, indocyanine green-thiol, indocyanine green-amino, and indocyanine green-succinimidyl ester. Under this preferred condition, the sonosensitizer has more excellent water solubility and biological labeling ability.

[0011] Specifically, the indocyanine green carboxylic acid, indocyanine green-thiol, indocyanine green-amino, and indocyanine green-succinimidyl ester are indocyanine green derivatives, which are obtained by replacing the sulfonate group with a corresponding conversion group (such as a carboxylic acid group, a thiol group, an amino group, a succinimidyl ester group, etc.).

[0012] Preferably, the STING agonist is at least one of SR-717 and MSA-2.

[0013] Preferably, the mass ratio of the sonosensitizer to the STING agonist is (0.5-1):1; more preferably, the mass ratio is 1:1.

[0014] Further, the present application also provides a microneedle, which comprises the microneedle raw material composition, a polymer matrix, and a solvent; the proportion of the microneedle raw material composition in the microneedle is 11%-22% in terms of mass percentage.

[0015] The dissolvable microneedle (DMN) has a unique advantage of achieving drug dispersion at the lesion site and effectively improving the skin penetration of the drug as a new transdermal drug delivery system. In addition, the DMN can be quickly dissolved in a physiological environment, avoiding the discomfort caused by the retention of traditional metal microneedles or insoluble microneedles in the local skin, and is more conducive to the rapid recovery of the skin. By using a dissolvable microneedle array with a needle length of 25-2000 μm to load nanoparticles, the stratum corneum barrier can be broken without touching the subcutaneous pain nerves, and after forming a few hundred micrometer pore in the skin, the drug is quickly delivered to the superficial tumor target area, and a high-concentration nano-drug depot is formed in the tumor target area, thereby improving the anti-tumor effect of the drug and avoiding the side effects on other normal tissues caused by systemic administration.

[0016] Preferably, the proportion of the microneedle raw material composition in the microneedle is 15% to 22% by mass percentage; more preferably, the proportion is 16% to 20%.

[0017] Preferably, the polymer matrix is a degradable polymer.

[0018] Preferably, the polymer matrix is at least one of hyaluronic acid, polyvinylpyrrolidone, and polyvinyl alcohol; further preferably, the degradable polymer is at least one of hyaluronic acid and polyvinylpyrrolidone.

[0019] Preferably, the mass ratio of the polymer matrix to the sonosensitizer is 1:(1-15); preferably, the mass ratio of the polymer matrix to the sonosensitizer is 1:(10-15); more preferably, the mass ratio of the polymer matrix to the sonosensitizer is 1:10.

[0020] Further, the present application also provides a preparation method of the microneedle, comprising the following operations: uniformly mixing the microneedle raw material composition, the polymer matrix, and the solvent, adding a microneedle mold, and centrifuging to obtain the microneedle.

[0021] Preferably, each needle body in the microneedle is in a conical shape with a height of 300-1000 μm and a bottom diameter of 100-400 μm; more preferably, each needle body in the microneedle is in a conical shape with a height of 300-500 μm and a bottom diameter of 200-300 μm; further preferably, the height is about 420 μm and the bottom diameter is about 250 μm.

[0022] Further, a third object of the present application is to provide a microneedle patch comprising the microneedle and a backing layer.

[0023] Preferably, the backing layer is obtained by solidification and molding of the polymer matrix.

[0024] Preferably, the concentration of the polymer matrix in the backing layer is (1-2) g / mL; more preferably, the concentration of the polymer matrix is 1.5 g / mL.

[0025] Further, the present application provides a preparation method of the above-mentioned microneedle patch, wherein the backing layer solution is added to the microneedle mold after centrifugation, and then dried to obtain the microneedle patch.

[0026] Specifically, the preparation method of the microneedle patch comprises the following steps: (1) dissolving the polymer matrix in deionized water to obtain a backing layer solution; (2) dissolving the polymer matrix in deionized water, then adding a sonosensitizer and a STING agonist, stirring at room temperature for 2-24 h in the dark to obtain a microneedle solution; (3) filling the microneedle solution into the micropores of the needle body of the microneedle mold by microtransfer molding, removing the excess part outside the micropores, then adding the backing layer solution above the micropores, drying and demolding to obtain the microneedle patch.

[0027] The present application constructs a biologically responsive polymer microneedle drug release system, i.e. a microneedle patch co-loaded with a sonosensitizer and a STING agonist. The microneedle patch can be used for transdermal drug delivery to treat tumors and inhibit metastasis. The microneedle patch uses soluble drug-loaded microneedles to improve drug solubility, realizes local and efficient drug delivery through transdermal drug delivery, and realizes deep penetration of drugs into tumors through external ultrasound stimulation. The sonodynamic therapy (SDT) realized under the action of external ultrasound can induce immunogenic cell death (ICD) by increasing intracellular ROS, and release cell damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1). DAMPs can recruit and activate dendritic cells (DCs) and enhance the anti-tumor response of cytotoxic T cells. At the same time, in the process of SDT, the intratumoral responsive release of the STING agonist and the deep penetration of the tumor tissue region can be promoted, thereby activating the cGAS-STING signaling pathway, recruiting and activating TANK binding kinase 1 (TBK1), further phosphorylating interferon regulatory factor 3 (IRF3), and inducing the expression of type I interferon. Type I IFN produced by DCs can bind to interferon receptors on the surface of itself or adjacent DCs or other immune cells in a paracrine or autocrine manner, and can synergize with SDT to promote the maturation of DCs, thereby promoting the infiltration of cytotoxic T lymphocytes and the secretion of cytokines, and ultimately eliminating the primary tumor and inhibiting the growth of the distal tumor.

[0028] Further, the fourth object of the present application is to provide the use of the above-mentioned microneedle raw material composition, microneedle and microneedle patch in the preparation of a drug for sonodynamic therapy of tumors.

[0029] Preferably, the tumor is at least one of melanoma, breast cancer, colon cancer, skin cancer, B-cell lymphoma. Preferably, the tumor is at least one of triple negative breast cancer, melanoma and squamous cell carcinoma; more preferably, the tumor is triple negative breast cancer.

[0030] Compared with the prior art, the present application has the following beneficial effects: (1) The present application first combines a sonosensitizer and a STING agonist to prepare a microneedle raw material composition, which can enhance the penetration of the microneedle raw material composition into the deep part of the tumor tissue through sonodynamic therapy, and the combination of the sonosensitizer and the STING agonist can also synergistically improve the immunotherapy effect on the tumor.

[0031] (2) The present application first prepares a soluble microneedle loaded with a sonosensitizer and a STING agonist, which has simple preparation method, mild reaction condition, uniform product morphology and low production cost, and is suitable for industrial scale-up production.

[0032] (3) The microneedle patch prepared by the present application can realize rapid dissolution, and can realize more precise drug release by transdermal administration on the upper epidermis of the tumor site, with smaller drug dosage and better biocompatibility, which provides a new technical method for precise treatment of TNBC, and has important reference significance for improving the treatment effect of TNBC patients.

[0033] (4) The present application combines a sonosensitizer and a STING agonist into a microneedle to form a solid, which can be stored at room temperature for a long time, and the sonosensitizer and the STING agonist are released through the microneedle patch, which not only significantly reduces the drug dosage and side effects of the sonosensitizer and the STING agonist, but also can cause stronger immune effect and better curative effect.

[0034] (4) The microneedle patch prepared by the present application can realize SDT under the action of exogenous ultrasound, which can induce ICD by increasing intracellular ROS, release DAMPs, recruit and activate DCs, and enhance the anti-tumor response of cytotoxic T cells.

[0035] (5) The microneedle patch prepared by the present application can promote the intratumoral responsive release of the STING agonist and the deep penetration of the tumor tissue region, thereby activating the cGAS-STING signaling pathway and inducing the expression of type I interferon. At the same time, it synergistically promotes the maturation of DCs, and then promotes the infiltration of cytotoxic T lymphocytes and the secretion of cytokines, finally eliminates the in situ tumor and inhibits the growth of the distal tumor. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1The preparation flowchart of the microneedle patch loaded with sound sensitizer ICG and STING agonist SR-717 prepared in the application and the schematic diagram of the antitumor mechanism thereof.

[0037] Figure 2 The optical picture of the microneedle prepared in Example 1.

[0038] Figure 3 The scanning electron microscope (SEM) picture (cross-sectional view) of the microneedle prepared in Example 1.

[0039] Figure 4 The scanning electron microscope (SEM) picture (top-down view) of the microneedle prepared in Example 1.

[0040] Figure 5 The inverted fluorescence microscope picture of the microneedle prepared in Example 1.

[0041] Figure 6 The drug release behavior of the microneedle prepared in Example 1 in PBS buffer solution.

[0042] Figure 7 The intracellular CRT and extracellular and intracellular HMGB1 content of 4T1 tumor cells in each treatment group.

[0043] Figure 8 The supernatant ATP release of 4T1 tumor cells in each treatment group.

[0044] Figure 9 The cGAS-STING immune pathway activation in BMDCs in each treatment group.

[0045] Figure 10 The schematic diagram of the co-culture experiment process of 4T1 and BMDC cells in each treatment group.

[0046] Figure 11 The proportion of mature BMDCs (CD80 and CD86 double positive) in each treatment group.

[0047] Figure 12 The proportion of mature BMDCs in each group after co-culture.

[0048] Figure 13 The supernatant IFN-β concentration in each group after co-culture.

[0049] Figure 14 The proportion of immature BMDC cells in each group before co-culture.

[0050] Figure 15 The schematic diagram of the co-culture experiment process of BMDCs and CD3+ T cells in each treatment group.

[0051] Figure 16The proliferation of CD4+ and CD8+ T cells in each treatment group.

[0052] Figure 17 The proliferation of CD4+ T cells after co-culture in each treatment group.

[0053] Figure 18 The proliferation of CD8+ T cells after co-culture in each treatment group.

[0054] Figure 19 The blood routine of tumor-bearing mice treated with the microneedle patch prepared in Example 1 for different times (the shaded area is the normal reference range value).

[0055] Figure 20 The liver and kidney function of tumor-bearing mice treated with the microneedle patch prepared in Example 1 for different times (the shaded area is the normal reference range value). DETAILED DESCRIPTION

[0056] The present application will be further described in conjunction with the specification and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0057] Preparation of microneedle patch containing photosensitizer and STING agonist In this example, the microneedle patch was prepared according to the mass ratio of photosensitizer and STING agonist as 1:1 (preparation schematic diagram as shown in Figure 1 ).

[0058] (1) Dissolve polyvinylpyrrolidone (PVP) in deionized water to prepare a backing layer solution with a concentration of 1.5 g / mL.

[0059] (2) Take 100 mg of photosensitizer ICG, 100 mg of STING agonist SR-717 and 1000 mg of hyaluronic acid, and add them to 10 mL of deionized water to avoid light and stir for 24 h to become a uniform gel-like mixture of dark green, obtaining a microneedle raw material composition.

[0060] (3) Transfer the above composition to a 10 mL beaker by micro-transfer molding method, then add the microneedle mold to the beaker; then put the beaker into a centrifuge tube and centrifuge at a speed of 5000 revolutions per minute for 5 min to obtain a needle body mixture; after centrifugation, remove the mold in the beaker and remove the part outside the micropores, and the microneedle is obtained.

[0061] (4) Then add the backing layer solution above the micropores, and then put the mold into the oven at 37°C and dry overnight in the dark. Finally, gently peel the formed microneedle patch from the mold, and the prepared microneedle patch is named as ICG&SRDMNs.

[0062] Preparation of microneedle patch without a sonosensitizer and a STING agonist (1) Dissolve PVP in deionized water to prepare a backing layer solution with a concentration of 1 g / mL.

[0063] (2) Take 1000 mg of hyaluronic acid and add it to 10 mL of deionized water, stir in the dark for 24 h, and become a transparent and uniform gel mixture to obtain a microneedle raw material composition.

[0064] (3) Transfer the above composition to a 10 mL beaker, and then add the microneedle mold (the same as in Example 1) into the beaker; then put the beaker into a centrifuge tube and centrifuge at a speed of 5000 rpm for 5 min to obtain a needle body mixture; after centrifugation, remove the mold in the beaker, and remove the part outside the micropores, and the microneedle is obtained.

[0065] (4) Then add the backing layer solution above the micropores, and then put the mold into the oven at 37°C and dry overnight in the dark. Finally, gently peel the formed microneedle patch from the mold, and the prepared microneedle patch is named as Free DMNs.

[0066] Preparation of microneedle patch containing only a sonosensitizer (1) Dissolve PVP in deionized water to prepare a backing layer solution with a concentration of 1 g / mL.

[0067] (2) Take 100 mg of sonosensitizer ICG (ICG accounts for 10% of the mass of hyaluronic acid) and 1000 mg of hyaluronic acid and add them to 10 mL of deionized water, stir in the dark for 24 h, and become a dark green uniform gel mixture to obtain a microneedle raw material composition.

[0068] (3) Transfer the above composition to a 10 mL beaker, and then add the microneedle mold (the same as in Example 1) into the beaker; then put the beaker into a centrifuge tube and centrifuge at a speed of 5000 rpm for 5 min to obtain a needle body mixture; after centrifugation, remove the mold in the beaker, and remove the part outside the micropores, and the microneedle is obtained.

[0069] (4) Then add the backing layer solution above the micropores, and then put the mold into the oven at 37°C and dry overnight in the dark. Finally, gently peel the formed microneedle patch from the mold, and the prepared microneedle patch is named as ICG@DMNs.

[0070] Preparation of Comparative Example 3 microneedle patches containing a STING agonist (1) PVP was dissolved in deionized water to obtain a backing layer solution with a concentration of 1 g / mL.

[0071] 100 mg of STING agonist SR-717 (SR-717 accounts for 10% of the mass of hyaluronic acid) and 1000 mg of hyaluronic acid were added to 10 mL of deionized water and stirred in the dark for 24 h to form a milky white uniform gel mixture, obtaining a microneedle raw material composition.

[0072] (3) The above composition was transferred to a 10 mL beaker, and the microneedle mold (same as Example 1) was added to the beaker; then the beaker was placed in a centrifuge tube and centrifuged at a speed of 5000 rpm for 5 min to obtain a needle body mixture; after centrifugation, the mold in the beaker was removed, and the part outside the micropores was removed, and the microneedle was obtained.

[0073] (4) The backing layer solution was then added above the micropores, and then the mold was placed in a 37°C oven and dried in the dark overnight. Finally, the formed microneedle patch was gently peeled off from the mold, and the prepared microneedle patch was named SR@DMNs.

[0074] Test Example 1 Microstructure of microneedle patch ICG&SR DMNs The microneedle patch ICG&SR DMNs prepared in Example 1 was subjected to optical, scanning electron microscope (SEM), inverted fluorescence microscope image, and drug release behavior in PBS buffer solution.

[0075] Figure 2 The optical picture of the microneedle patch prepared in Example 1, the image result showed that the microneedle patch was a transparent rectangle of 8x8 mm, composed of 100 (10x10) conical needles.

[0076] Figure 3 The scanning electron microscope (SEM) photo (cross-sectional view) of the microneedle patch prepared in Example 1, Figure 4 The scanning electron microscope photo (top-down view) of the microneedle patch prepared in Example 1. The image result showed that all the microneedles were conical, the needle-like structure of the DMNs was complete, sharp, and arranged in order. The height was about 420 μm, the bottom diameter was about 250 μm, and the needle pitch was 250 μm.

[0077] Figure 5 The inverted fluorescence microscope image of the microneedle patch prepared in Example 1. Since the ICG&SR@DMNs microneedle patch contains fluorescent dye ICG, the inverted fluorescence microscope was used for imaging analysis of the microneedle patch, and the result showed that the red fluorescence was uniformly distributed in the DMNs, confirming that ICG was uniformly distributed in the microneedles. In addition, Figure 5The size of the microneedles observed under a microscope was consistent with the results obtained from SEM.

[0078] Figure 6 Drug release behavior of the microneedles prepared in Example 1 in PBS buffer solution. The solutions of the microneedles in contact with PBS for different time were detected for the release of ICG and SR-717. The content of ICG in the microneedles was detected by ultraviolet spectrophotometer, and the content of SR-717 in the microneedles was detected by high performance liquid chromatography. The results showed that the ICG&SR@DMNs released about 70% of ICG and SR-717 simultaneously within 3 min, and had stable drug release behavior.

[0079] Test Example 2: Therapeutic effect of different microneedles and microneedle patches prepared therefrom on breast cancer The microneedle patches prepared in the above examples were tested for their therapeutic effect on breast cancer in the following order.

[0080] I. Effect of different drug-loaded microneedles on ICD after ultrasound stimulation 1. Experimental materials and grouping The mouse breast cancer cells (4T1) were placed in RPMI-1640 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, and cultured in a humidified environment at 37°C and 5% CO2.

[0081] Ultrasound (US) stimulation can induce immunogenic cell death (ICD) of tumor cells. In order to evaluate the ICD of different drug-loaded microneedle patches after ultrasound wave (2w / cm 2 , 1 MHZ, 50% duty cycle) stimulation, a grouping experiment was performed.

[0082] The groups were: Control + US group (Free DMNs + US group), SR@DMNs + US group, ICG@DMNs + US group, ICG&SR@DMNs + US group.

[0083] 2. Experimental method The lysate and culture supernatant of tumor cells after treatment in each group were collected, and the changes in classical damage-associated molecular patterns (DAMPs) were detected by ATP detection kit and WB.

[0084] 3. Experimental results The results are shown in Figure 7 and Figure 8As shown, compared with the Control + US group and the SR@DMNs + US group without loading the sound-sensitive agent ICG, the intracellular CRT content of 4T1 cells in the ICG@DMNs + US group and the ICG&SR@DMNs + US group was obviously increased after SDT treatment, the intracellular and extracellular HMGB1 contents were obviously decreased, and the ATP content in the cell supernatant was obviously increased.

[0085] II. Verification of activation of cGAS-STING immune pathway of BMDC by drug-loaded microneedles SR-717 as a STING agonist can activate the cGAS-STING signaling pathway, recruit and activate TANK-binding kinase 1 (TBK1), further phosphorylate interferon regulatory factor 3 (IRF3), and induce the expression of type I interferon.

[0086] This part studies the activation of the cGAS-STING pathway in BMDC by SR-717.

[0087] 1. Experimental materials and grouping According to different drug loading and ultrasonic treatment, the groups are divided as follows: Control group: Free DMNs group; Free SR-717 group: A treatment group with only SR-717; SR@DMNs group: Same as Comparative Example 3.

[0088] ICG&SR@DMNs group: Same as Example 1.

[0089] 2. Experimental method Primary BMDCs were extracted from mouse bone marrow and induced to culture dendritic cells (BMDCs) in vitro to establish a co-culture system of BMDCs and 4T1 cells.

[0090] The extraction steps of mouse bone marrow-derived dendritic cells (BMDCs) are as follows: Step 1: After anesthetizing and euthanizing the mice by cervical dislocation, completely immerse the mice in 75% alcohol for 10-15 minutes. Step 2: Remove the sterilized mice from a Class II biosafety cabinet and place them on a sterile dissecting board. Surgically remove the skinned hind limbs, immerse them in 75% alcohol for 5-8 seconds, and then quickly wash them three times with sterile, pre-cooled PBS. Step 3: Place the hind limbs in a 10cm cell culture dish, pour in an appropriate amount of pre-cooled sterile PBS buffer, remove the muscle tissue from the hind limbs using surgical scissors and forceps, and remove the femur and tibia to place them in a new 1cm cell culture dish. First, immerse a 0cm cell culture dish in pre-chilled PBS solution, taking care not to break the femur and tibia during muscle tissue removal. Second, cut both ends of the femur and tibia with surgical scissors to expose the medullary cavity. Rinse the medullary cavity with an appropriate amount of pre-chilled serum-free 1640 medium using a 1ml syringe needle. Collect the rinse contents and filter through a 70μm sterile filter to remove muscle and bone fragments. Third, centrifuge at 1000rpm and 4℃ for 6 minutes, add 2ml of red blood cell lysis working solution, incubate at room temperature for 3-5 minutes, add 10ml of pre-chilled PBS to stop lysis, centrifuge at 1000rpm and 4℃ for 6 minutes, discard the supernatant, and wash once with PBS. Fourth, centrifuge at 1000rpm and 4℃ for 6 minutes, discard the supernatant, resuspend the cells in an appropriate amount of pre-prepared BMDC medium, count the cells, and plate them.

[0091] Primary mouse bone marrow cells were extracted and designated as day 0. First, on day 5, 4T1 cells were digested, resuspended, and counted. At a 4T1:BMDC ratio of 2:1, 4T1 cells were seeded into 0.4 μm pore polycarbonate membrane chambers nested within 6-well plates, and 1 ml of culture medium was added for culture. Then, on day 6, after cell adhesion, cells were divided into groups (Control + US group, SR@DMNs + US group, ICG@DMNs + US group, and ICG&SR@DMNs + US group). Sonication was performed at 2 weeks / cm². 2 1 MHz, 50% duty cycle. Chambers containing tumor 4T1 cells and culture medium were placed into the corresponding BMDC wells of a cell culture incubator and co-cultured for 24 hours. The chambers were then removed, the old culture medium collected from each well, and the BMDCs in each well were digested with pre-warmed trypsin. After 3 minutes, culture medium was added to stop the digestion, and the mixture was centrifuged at 1000 rpm for 6 minutes. The supernatant was discarded, and an appropriate amount of pre-prepared protein lysis buffer was added. p-STING, p-TBK1, and p-IRF3 were then analyzed by Western blotting using GADPH as an internal control to verify the cGAS-STING immune pathway.

[0092] 3. Experimental Results like Figure 9As shown, compared to the Control group, all treatment groups exhibited significant levels of phosphorylated proteins activating downstream pathways. Drug-loaded microneedles, upon uptake by tumor cells, released the STING agonist SR-717, which effectively activated the cGAS-STING immune pathway in BMDCs. Therefore, microneedles loaded with the STING agonist SR-717 and / or the sonosensitive agent ICG both have an activating effect on the cGAS-STING immune pathway in BMDCs.

[0093] III. Validation of Immune Activation in In Vitro Cell Experiments Using Drug-Loaded Microneedles Immature tumor cells (BMDCs) lack immune function, and their membrane surface shows low expression of molecules such as CD80 and CD86. After stimulation and activation, BMDCs gradually mature, accompanied by the expression of their membrane maturation markers. Therefore, this experiment constructed an in vitro co-culture system of tumor cells and BMDCs and investigated the effect of drug-loaded microneedle sonodynamic therapy on the maturation of BMDCs and the verification of their immune function.

[0094] 1. Experimental Materials and Grouping Based on different drug loading and ultrasound treatment, they are grouped as follows: Control group (PBS control group): A control group containing only phosphate-buffered saline (PBS) and without any microneedle treatment was prepared. Control + US group (PBS + US group): A control group containing only PBS was prepared, and ultrasound (2w / cm) was applied. 2 (1MHz, 50% duty cycle); SR-717 group: Prepare a treatment group containing only SR-717; SR-717 + US group: A treatment group containing only SR-717 was prepared and subjected to ultrasound (2w / cm). 2 1MHz, 50% duty cycle).

[0095] The processing of the SR@DMNs group, SR@DMNs + US group, ICG&SR@DMNs group, and ICG&SR@DMNs + US group is as described above.

[0096] 2. Experimental Methods As per the process Figure 10 As shown, primary BMDCs were extracted from mouse bone marrow and induced for culture. They were then co-cultured with 4T1 tumor cells from different treatment groups. Flow cytometry was used to detect the CD11c positivity rate and the proportion of CD80 and CD86 double positivity, maturation markers on the surface of BMDCs.

[0097] The specific procedures are as follows: On day 5, 4T1 cells were seeded into plate chambers. On day 6, culture media were prepared and the cells were divided into PBS control group and PBS + US group, and various drug treatment groups (SR-717 group, SR-717 + US group, SR@DMNs group, SR@DMNs + US group, ICG&SR@DMNs group, ICG&SR@DMNs + US group). Tumor cells were processed, and the chambers containing tumor 4T1 cells and culture media were placed into the corresponding BMDC wells of the plate and cultured in a cell culture incubator for 24 hours (sonication was performed 6 hours after drug administration). On day 7, the chambers were removed, the supernatant of each group was collected, and the IFN-β concentration of the supernatant of each group was detected according to the ELISA instructions; BMDCs of each group were collected, counted, and centrifuged at 1000 rpm for 6 minutes, and the supernatant was discarded. The cells were then processed at 10... 6 One cell per system: Resuspend cells in 100 μl of pre-chilled FACS buffer, add 0.25 μg of Fc receptor blocking nonspecific antibody, block and incubate on ice for 10-15 minutes, then add Zombie Aqua™ Fixable Viability Kit working solution for cell viability staining, incubate at room temperature for 15 minutes, and wash once; resuspend cells in 100 μL of pre-chilled FACS buffer, add 0.25 μg of Brilliant Violet421™ anti-mouse CD45 antibody, FITC anti-mouse CD11c antibody, APC anti-mouse CD80 antibody, and PE anti-mouse CD86 antibody to each well, incubate on ice for 30 minutes, add 1 ml of pre-chilled FACS buffer to stop, centrifuge at 1000 rpm, 4°C for 6 minutes, discard the supernatant, resuspend in 100 μl of FACS buffer, and analyze by flow cytometry.

[0098] Meanwhile, a 6-well plate system was set up without nanomedicine treatment or co-culture. The plates were cultured under the same conditions and flow cytometry was performed on day 7 to detect the differences in BMDC maturity among the groups before co-culture.

[0099] 3. Experimental Results The results are as follows Figure 11 and Figure 12As shown, compared to the Control group and the Control + US group, all drug treatment groups (SR-717 group, SR-717 + US group, SR@DMN group, SR@DMNs + U group, ICG&SR@DMNs group, ICG&SR@DMNs + US group) showed varying degrees of BMDC activation. SR-717 exerted its immune stimulating effect, and the proportion of CD80 and CD86 double-positive mature BMDCs was significantly increased. Among them, the combination of SR-717 and sonodynamic therapy (ICG&SR@DMNs + US group) was more significant. In addition to exerting the immune stimulating effect of SR-717 stimulating microneedles, it also induced tumor immunogenic cell death in tumor cells (as above). Figure 7 and 8 As shown), it promotes more BMDCs to turn towards maturation, and also further increases their IFN-β secretion (as shown). Figure 13 Meanwhile, validation testing revealed no significant difference in BMDC maturity among the groups before co-cultivation. Figure 14 ).

[0100] IV. Verification that activated BMDCs promote T cell proliferation 1. Experimental Materials and Grouping As mentioned before, the groups were divided into: Control group (PBS control group), PBS + US group (Control + US group), SR-717 group, SR-717 + US group, ICG&SR@DMNs group, and ICG&SR@DMNs + US group.

[0101] 2. Experimental Methods Further steps include setting up and constructing a BMDC and T cell co-culture system, such as the workflow. Figure 15 As shown.

[0102] CD3+ T cells were sorted, and progeny cells were detected by CFSE labeling. BMDCs and CD3+ T cells in each treatment group were co-cultured directly for 72 hours. CD4+ and CD8+ T cells were then enclosed by flow cytometry to detect the proportion of progeny T cells.

[0103] 3. Experimental Results like Figure 16 , Figure 17 and Figure 18As shown, compared with the Control group and the PBS + US group, all drug treatment groups (SR-717 group, SR-717 + US group, ICG&SR@DMNs group, ICG&SR@DMNs + US group) promoted T cell proliferation to varying degrees. Among them, the BMDC in the combined SR-717 and sonodynamic therapy group (ICG&SR@DMNs + US group) had a more significant effect, and its more mature BMDC further promoted T cell proliferation.

[0104] V. In vivo toxicity of drug-loaded microneedles 1. Experimental methods and grouping 4T1 cells (1.0×10⁻⁶) 6 Cells were suspended in 100 µL of phosphate-buffered saline solution and subcutaneously injected into the back of BALB / c mice to establish an animal tumor model (triple-negative breast cancer). When the tumor size reached 100 mm... 3 In this experiment, the ICG&SR@DMNs microneedle patches prepared in Example 1 were applied to the tumor sites of mice. Treatment was administered once every other day for a total of three treatments, with five mice in each group participating in the experiment. The other group, designated as the control group, received no treatment after tumor modeling.

[0105] The mice were then subjected to routine blood tests on days 7, 14, and 21.

[0106] 2. Experimental Results like Figure 19 As shown, compared with the control group, the blood routine test indicators of mice in each group, such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), and mean platelet volume (MPV), were all within the normal range and showed no significant differences. The ICG&SR@DMNs microneedle patch did not have a significant adverse effect on the hematopoietic function of mice, and also preliminarily verified the advantages of the microneedle patch in terms of low toxicity and side effects.

[0107] There were no significant differences in serum urea nitrogen (BUN), creatinine (CREA), uric acid (UA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in mice, and all were within the normal reference range. Figure 20 This indicates that the microneedle patch has the advantages of low toxicity and few side effects.

[0108] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A microneedle raw material composition, characterized by, The sonosensitizer and the STING agonist; the mass ratio of the sonosensitizer to the STING agonist is (0.2~1):

1.

2. The microneedle raw material composition according to claim 1, wherein The sonosensitizer is at least one of indocyanine green, indocyanine green carboxylic acid, indocyanine green-thiol, indocyanine green-amino, and indocyanine green-succinimidyl ester.

3. The microneedle raw material composition according to claim 1, wherein The STING agonist is at least one of SR-717 and MSA-2.

4. A microneedle, characterized by, The microneedle raw material composition, a polymer matrix, and a solvent according to any one of claims 1~3; the microneedle raw material composition accounts for 11%~22% of the microneedle in terms of mass percentage.

5. The microneedle of claim 4, wherein the microneedle is a microneedle array. The polymer matrix is at least one of hyaluronic acid, polyvinylpyrrolidone, and polyvinyl alcohol.

6. The microneedle of claim 4, wherein the microneedle is a microneedle array. The mass ratio of the sonosensitizer to the polymer matrix is 1:(10~15).

7. A method of making the microneedle of any one of claims 4-6, wherein, The microneedle raw material composition, a polymer matrix, and a solvent according to any one of claims 1~3 are uniformly mixed, added to a microneedle mold, and centrifuged to obtain the microneedle.

8. A microneedle patch, characterized by, The microneedle and a backing layer according to any one of claims 4~6.

9. Use of the microneedle raw material composition according to any one of claims 1~3, the microneedle according to any one of claims 4~6, or the microneedle patch according to claim 8 in the preparation of a drug for the sonodynamic treatment of tumors.

10. Use according to claim 9, characterized in that, The tumor is at least one of melanoma, breast cancer, colon cancer, skin cancer, and B-cell lymphoma.

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