CGAS-STING activated microneedle system and preparation method and application thereof

Abstract

The invention discloses a cGAS-STING activated micro-needle system and a preparation method and application thereof.The cGAS-STING activated micro-needle system comprises a needle tip and a backing which are connected, the needle tip is loaded with a mesoporous photosensitive nano-composite drug, the mesoporous photosensitive nano-composite drug takes hollow Prussian blue nanoparticles as a carrier, the interior of the mesoporous photosensitive nano-composite drug is loaded with a small molecule drug, and the surface of the mesoporous photosensitive nano-composite drug is coated with a coating. The outer layer is coated with natural CD44 ligand polysaccharide; the small molecular medicine is a mixture of hydroxyurea and carotene. The mesoporous photosensitive nano-composite drug can induce death of immunogenic cells through mild photothermal induction, and release an injury-related molecular mode; meanwhile, a cGAS-STING signal channel is activated, secretion of type I interferon is promoted, and dendritic cells are induced to be activated and mature, so that T cell mediated anti-tumor immune response is enhanced. The invention provides an innovative tumor immunotherapy strategy and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a cGAS-STING activated microneedle system and a preparation method and application thereof. Background Art

[0002] Melanoma is a highly invasive malignant tumor. Its rapid spread and metastasis have led to a continuously increasing mortality rate, posing a serious threat to human health. Currently, melanoma is primarily treated clinically with surgery, chemotherapy, and radiotherapy. However, existing treatments struggle to effectively control distant metastasis, contributing to the persistently high mortality rate from melanoma.

[0003] Immunotherapy is widely hailed as a revolutionary anti-cancer strategy, marking a major breakthrough in modern oncology. Dendritic cells (DCs) play an indispensable role in this strategy. As short-lived antigen-presenting cells, DCs serve as critical "sentinels" in the immune system, their primary function being to capture and process antigens, thereby activating T cell-mediated immune responses. However, the effective activation and maturation of DCs remains a major challenge in cancer immunotherapy.

[0004] Immunogenic cell death (ICD) is an effective strategy for activating DCs (DCs) through damage-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs) released by dying tumor cells. Currently, chemotherapy and radiotherapy are the primary approaches for inducing ICD, but their clinical application is hampered by biosafety and spatial and temporal constraints. In contrast, photothermal therapy (PTT) has garnered significant attention due to its non-invasive nature, high controllability, and excellent targeting specificity. However, the efficacy of PTT is affected by the levels of heat shock proteins (HSPs) within tumor cells, and temperature regulation presents challenges: excessively high temperatures (>45°C) can damage surrounding normal tissues, while low temperatures (<45°C) hinder effective tumor cell killing. Currently, HSPs are primarily eliminated through small molecule inhibitors, but these inhibitors often suffer from low delivery efficiency and high acute cytotoxicity, making them difficult to effectively silence HSPs in complex physiological environments.

[0005] In recent years, chemodynamic therapy (CDT) has shown promising application prospects. It utilizes the high levels of hydrogen peroxide (H2O2) in the tumor microenvironment to undergo a Fenton reaction, generating hydroxyl radicals (•OH) that can precisely and efficiently eliminate HSPs, thereby synergizing with mild photothermal therapy (mild-PTT) to induce ICD. Although mild photothermal therapy (mPTT)-induced ICD can promote the activation of DCs, their maturation process still faces significant challenges. Therefore, developing innovative strategies that can effectively activate and promote DC maturation is crucial for enhancing anti-tumor immune responses and improving tumor immunogenicity. Summary of the Invention

[0006] In response to the above problems, the present invention aims to provide a cGAS-STING activated microneedle system and its preparation method and application.

[0007] The technical solutions of the present invention are as follows: On the one hand, a cGAS-STING activated microneedle system is provided, comprising a connected needle tip and a backing, wherein the needle tip is loaded with a mesoporous photosensitive nanocomposite drug, wherein the mesoporous photosensitive nanocomposite drug uses hollow Prussian blue nanoparticles as a carrier, loads a small molecule drug internally, and is coated with a natural CD44 ligand polysaccharide on the outer layer; the small molecule drug is a mixture of hydroxyurea and thapsigargin.

[0008] Preferably, the mass ratio of the hydroxyurea to the thapsigargin is 1-14:1, the small molecule drug accounts for 5%-10% of the mass of the mesoporous photosensitive nanocomposite drug, and the mesoporous photosensitive nanocomposite drug accounts for 5%-20% of the mass of the needle tip after loading the mesoporous photosensitive nanocomposite drug.

[0009] Preferably, the average particle size of the mesoporous photosensitive nanocomposite drug is 150 nm to 200 nm.

[0010] Preferably, the thickness of the natural CD44 ligand polysaccharide is 8.6 nm to 12.8 nm.

[0011] Preferably, the natural CD44 ligand polysaccharide is any one or more of hyaluronic acid, chondroitin sulfate, and dermatan sulfate.

[0012] Preferably, the length of the needle tip is 650µm to 1250µm, and the thickness of the backing is 20µm.

[0013] In another aspect, a method for preparing any of the above-described cGAS-STING activated microneedle systems is provided, comprising the following steps: S1: preparing hollow Prussian blue nanoparticles, and mixing and encapsulating the hollow Prussian blue nanoparticles with the small molecule drug to obtain a mesoporous photosensitive nanodrug; S2: preparing a natural CD44 ligand polysaccharide aqueous solution, and dispersing the mesoporous photosensitive nanodrug in the natural CD44 ligand polysaccharide aqueous solution, so that the natural CD44 ligand polysaccharide is coated on the surface of the mesoporous photosensitive nanodrug to obtain the mesoporous photosensitive nanocomposite drug; S3: mixing the mesoporous photosensitive nanocomposite drug with the needle tip material to obtain a mixed solution; S4: adding the mixed solution into a microneedle mold and drying to obtain a microneedle mold filled with needle tips; S5: preparing a backing material solution, adding the backing material solution into the microneedle mold filled with needle tips, and drying to obtain the cGAS-STING activated microneedle system.

[0014] Preferably, in step S1, the hollow Prussian blue nanoparticles are obtained by etching solid Prussian blue nanoparticles with hydrochloric acid.

[0015] Preferably, in step S3, the needle tip material is biodegradable material 1, and the biodegradable material 1 is any one or more of γ-polyglutamic acid, sodium alginate, gelatin, chitosan, and polyvinyl alcohol; in step S5, the backing material used in the backing material solution is biodegradable material 2, and the biodegradable material 2 is any one or more of polyvinyl alcohol, gelatin, hyaluronic acid, and polyvinyl pyrrolidone.

[0016] On the other hand, the present invention also provides the use of any of the above-mentioned cGAS-STING activated microneedle systems in the preparation of drugs for treating tumors, wherein the tumor is melanoma.

[0017] The beneficial effects of the present invention are: The mesoporous photosensitive nanocomposite drug described in this invention utilizes hollow Prussian blue (HMPB) nanoparticles as a carrier, loaded with hydroxyurea (HU) and thapsigargin (TG), and integrated into a cGAS-STING-activating microneedle system, enabling precise delivery to tumor sites. The mesoporous photosensitive nanocomposite drug induces immunogenic cell death (ICD) through gentle photothermal stimulation, releasing damage-associated molecular patterns (DAMPs). Simultaneously, it activates the cGAS-STING signaling pathway, promoting the secretion of type I interferon, inducing the activation and maturation of dendritic cells (DCs), and thereby enhancing T cell-mediated anti-tumor immune responses. In summary, the cGAS-STING-activating microneedle system described in this invention provides an unprecedented solution for activating and promoting DC maturation and improving immunotherapy efficacy, making a significant contribution to the continued development of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Schematic diagram of the preparation process of the cGAS-STING activated microneedle system in Example 1; Figure 2 Schematic diagram of the morphological characteristics of the nanoparticles in each step of Example 1; wherein a is a TEM image of solid Prussian blue nanoparticles, and b is a TEM image of the mesoporous photosensitive nanocomposite drug; Figure 3 This is the SEM image of the GAS-STING activated microneedle system in Example 1; Figure 4 Schematic diagram of the Zeta potential and infrared spectrum results of each material during the preparation process of Example 1; wherein a is the Zeta potential diagram and b is the infrared spectrum diagram; Figure 5 Graph showing the evaluation results of the killing effect of the microneedle system of Example 1, Comparative Example 2, and Comparative Example 3 on B16F10 cells; Figure 6 Schematic diagram of the in vitro immune activation effect test results of the microneedle system of Example 1, Comparative Example 2, and Comparative Example 3; wherein a is a schematic diagram of the INF-β production results, and b is a statistical diagram of the DCs maturation status; Figure 7 These are graphs showing the in vivo antitumor effects of the microneedle systems of Example 1, Comparative Example 2, and Comparative Example 3 in B16F10 tumor-bearing mice; wherein, a is a statistical graph showing the changes in tumor volume in B16F10 tumor-bearing mice within 14 days of treatment, and b is a statistical graph showing the tumor inhibition rate in B16F10 tumor-bearing mice after 14 days of treatment. DETAILED DESCRIPTION

[0020] The present invention is further described below with reference to the accompanying drawings and examples. It should be noted that, in the absence of conflict, the embodiments in this application and the technical features in the embodiments can be combined with each other. It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The use of similar words such as "include" or "comprising" in the present invention means that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0021] On the one hand, the present invention provides a cGAS-STING activated microneedle system, comprising a connected needle tip and a backing, wherein the needle tip is loaded with a mesoporous photosensitive nanocomposite drug, wherein the mesoporous photosensitive nanocomposite drug uses hollow Prussian blue nanoparticles as a carrier, loads a small molecule drug inside, and is coated with a natural CD44 ligand polysaccharide on the outer layer; the small molecule drug is a mixture of hydroxyurea and thapsigargin.

[0022] In this invention, the cGAS-STING-activating microneedle system delivers the mesoporous photosensitive nanocomposite drug to the tumor site through the needle tip. Relying on the interaction between the natural CD44 ligand polysaccharide and CD44, it is selectively internalized by tumor cells. Under irradiation with light in the range of 635-900 nm (preferably 808 nm), the mesoporous photosensitive nanocomposite drug induces ICD and releases damage-associated molecular patterns (DAMPs) to activate DCs. Simultaneously, HU and TG work together to activate the cGAS-STING pathway and promote DC maturation. Together, these factors enable this invention to enhance immune responses, thereby utilizing immunotherapy for cancer treatment.

[0023] In a specific embodiment, the mass ratio of hydroxyurea to thapsigargin is 1-14:1, the small molecule drug accounts for 5%-10% of the mass of the mesoporous photosensitive nanocomposite drug, and the mesoporous photosensitive nanocomposite drug accounts for 5%-20% of the mass of the needle tip after loading the mesoporous photosensitive nanocomposite drug. Alternatively, the mass ratio of hydroxyurea to thapsigargin is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, or 14:1, and the mesoporous photosensitive nanocomposite drug accounts for 5%, 8%, 10%, 15%, or 20% of the mass of the needle tip after loading the mesoporous photosensitive nanocomposite drug.

[0024] In the above examples, hydroxyurea and thapsigargin are used in a mass ratio of 1-14:1, and a small molecule drug is used that accounts for 5%-10% of the mass of the mesoporous photosensitive nanocomposite drug. This allows the mesoporous photosensitive nanocomposite drug to contain sufficient amounts of hydroxyurea and thapsigargin. Hydroxyurea can effectively damage nuclear DNA and inhibit its repair, thereby increasing the abundance of cytoplasmic DNA. At the same time, thapsigargin can deplete calcium ions in the endoplasmic reticulum, thereby activating STIM1 protein and relieving its inhibitory effect on STING protein translocation, ultimately activating the cGAS-STING signaling pathway and promoting dendritic cell maturation.

[0025] In a specific embodiment, the average particle size of the mesoporous photosensitive nanocomposite drug is 150 nm to 200 nm. Alternatively, the average particle size of the mesoporous photosensitive nanocomposite drug is 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. In this embodiment, the small-sized mesoporous photosensitive nanocomposite drug can more easily and quickly enter tumor cells, allowing the drug to better exert its efficacy.

[0026] In a specific embodiment, the thickness of the natural CD44 ligand polysaccharide is 8.6 nm to 12.8 nm. Alternatively, the thickness of the natural CD44 ligand polysaccharide is 8.6 nm, 9.2 nm, 9.8 nm, 10.4 nm, 11.0 nm, 11.6 nm, 12.2 nm, or 12.8 nm. In this embodiment, a natural CD44 ligand polysaccharide of sufficient thickness can better interact with the CD44 of tumor cells to mediate the selective internalization of the nanomedicine, thereby delivering the core containing the composite drug into the tumor cells, thereby promoting the composite drug to better exert its efficacy.

[0027] In a specific embodiment, the natural CD44 ligand polysaccharide is any one or more of hyaluronic acid, chondroitin sulfate, and dermatan sulfate.

[0028] In a specific embodiment, the needle tip has a length of 650µm to 1250µm, and the backing has a thickness of 20µm. In this embodiment, the sufficient needle tip length can effectively penetrate the stratum corneum of the skin and deliver a sufficient amount of the compound drug to the tumor site, thereby achieving a therapeutic effect.

[0029] In a specific embodiment, the needle tip is a polygonal pyramid structure, preferably a quadrangular pyramid structure. In this embodiment, the polygonal pyramid structure has higher mechanical properties than a circular cone, making it more conducive to skin penetration; the polygonal pyramid surface can form a "wedge-shaped" drug storage structure, which is conducive to the adhesion of solid drugs; the dissolution rate is related to the surface area, and the polygonal pyramid dissolves faster than the circular cone; and it has a more "geometric" and regular structure.

[0030] It should be noted that, in addition to loading the mesoporous photosensitive nanocomposite drug, the needle tip of the cGAS-STING activated microneedle system of the present invention may also contain various adjuvants to enhance the drug's ability to target tumor cells and the therapeutic effect of the photosensitive nanocomposite drug.

[0031] In another aspect, the present invention further provides a method for preparing any of the above-described cGAS-STING activated microneedle systems, comprising the following steps: S1: preparing hollow Prussian blue nanoparticles, and mixing and encapsulating the hollow Prussian blue nanoparticles with the small molecule drug to obtain a mesoporous photosensitive nanodrug; In this step, a small molecule drug is encapsulated in hollow Prussian blue nanoparticles using a one-pot method. In a specific embodiment, the encapsulation reaction temperature is 10°C to 40°C, and the encapsulation reaction time is ≥12 hours. Alternatively, the encapsulation reaction temperature is 10°C, 20°C, 30°C, or 40°C. In this embodiment, the selected encapsulation reaction parameters enable sufficient reaction between the hollow Prussian blue nanoparticles and the small molecule drug, thereby promoting the effective encapsulation of the small molecule drug within the hollow nanoparticles to obtain a mesoporous photosensitive nanodrug.

[0032] In addition, after encapsulation in this step, the mesoporous photosensitive nano-drug precipitate can be collected by centrifugation, washed three times with deionized water, and finally vacuum-dried to obtain the mesoporous photosensitive nano-drug.

[0033] In a specific embodiment, the hollow Prussian blue nanoparticles are obtained by etching solid Prussian blue nanoparticles with hydrochloric acid. Alternatively, the solid Prussian blue nanoparticles are dissolved in a hydrochloric acid solution containing PVP and stirred until completely dissolved; then, the solution is heated at 120°C to 140°C for more than 4 hours and centrifuged to obtain the hollow Prussian blue nanoparticles.

[0034] It should be noted that the above embodiment is only a preferred method for obtaining hollow Prussian blue nanoparticles of the present invention, and other existing technologies for obtaining hollow Prussian blue nanoparticles in the prior art are also applicable to the present invention.

[0035] In a specific embodiment, the solid Prussian blue nanoparticles are prepared using a hydrothermal method. Optionally, potassium ferricyanide (K3[Fe(CN)6]) and polyvinyl pyrrolidone (PVP) are used as precursors and successfully synthesized via solvothermal method, specifically comprising the following sub-steps: dissolving polyvinyl pyrrolidone (PVP) in deionized water and stirring until the solution is clear to obtain a PVP solution; dissolving potassium ferricyanide (K3[Fe(CN)6]) in deionized water to obtain a potassium ferricyanide solution; mixing the potassium ferricyanide solution and the PVP solution to undergo a coordination reaction to obtain a mixed solution; and heating the mixed solution at 60°C to 80°C for at least 20 hours to obtain the solid Prussian blue nanoparticles.

[0036] It should be noted that the method for preparing solid Prussian blue nanoparticles in the above embodiment is only a preferred preparation method of the present invention, and other methods for preparing solid Prussian blue nanoparticles in the prior art are also applicable to the present invention.

[0037] S2: preparing a natural CD44 ligand polysaccharide aqueous solution, and dispersing the mesoporous photosensitive nanodrug in the natural CD44 ligand polysaccharide aqueous solution, so that the natural CD44 ligand polysaccharide is coated on the surface of the mesoporous photosensitive nanodrug to obtain the mesoporous photosensitive nanocomposite drug; In a specific embodiment, ultrasonic dispersion is used for dispersion. Optionally, the frequency of the ultrasonic dispersion is 15kHz~25kHz, and the time of the ultrasonic dispersion is ≥15min. Optionally, the frequency of the ultrasonic dispersion is 15kHz, 16kHz, 17kHz, 18kHz, 19kHz, 20kHz, 21kHz, 22kHz, 23kHz, 24kHz or 25kHz. In this embodiment, the mesoporous photosensitive nanomedicine can be fully dispersed into the natural CD44 ligand polysaccharide aqueous solution by ultrasonic dispersion, so that the natural CD44 ligand polysaccharide fully wraps the mesoporous photosensitive nanomedicine, thereby finally obtaining a photosensitive nanocomposite drug with sufficient thickness of natural CD44 ligand polysaccharide.

[0038] S3: mixing the mesoporous photosensitive nanocomposite drug with the needle tip material to obtain a mixed solution; In a specific embodiment, the needle tip material is a biodegradable material 1, and the biodegradable material 1 is any one or more of γ-polyglutamic acid, sodium alginate, gelatin, chitosan, and polyvinyl alcohol.

[0039] It should be noted that the needle tip material is mainly considered in terms of its water solubility, mechanical properties, and biocompatibility. The materials in the above embodiments are only preferred materials of the present invention. Other needle tip materials suitable for organisms in the prior art can also be applied to the present invention.

[0040] S4: adding the mixed solution into a microneedle mold and drying to obtain a microneedle mold filled with needle tips; S5: preparing a backing material solution, adding the backing material solution into the microneedle mold filled with needle tips, and drying to obtain the cGAS-STING activated microneedle system.

[0041] In a specific embodiment, the backing material used in the backing material solution is a second biodegradable material, and the second biodegradable material is any one or more of polyvinyl alcohol, gelatin, hyaluronic acid, and polyvinyl pyrrolidone.

[0042] It should be noted that the backing material is mainly used to fix the needle tip and fit the skin, and its main considerations are fit to the skin, flexibility and non-allergenicity. The materials in the above embodiments are only preferred materials for the present invention. Other backing materials suitable for organisms in the prior art can also be applied to the present invention.

[0043] In the above embodiment, steps S4-S5 are a two-step method commonly used to prepare a microneedle system. The microneedle mold used can use a common PDMS mold. The mold cavity of the microneedle mold includes a groove portion and an inverted conical blind hole connected below the groove portion and distributed in an array.

[0044] In a specific embodiment, the drying temperature in steps S4-S5 is 20°C-40°C, and the drying time is ≥20h.

[0045] In another aspect, the present invention further provides use of any of the above-described cGAS-STING activated microneedle systems in the preparation of drugs for treating tumors, wherein the tumor is melanoma.

[0046] Example 1 A cGAS-STING activated microneedle system, such as Figure 1 As shown, it is prepared by the following steps: (1) 3 g of PVP was weighed and dissolved in a hydrochloric acid (HCl, 40 mL, 0.01 M) solution and stirred under magnetic stirring for 30 min. Subsequently, 131.72 mg of K3[Fe(CN)6] was added and stirred for another 30 min. The resulting mixed solution was transferred to an 80 °C oven and allowed to stand for 20 h. After centrifugation to collect the precipitate, solid Prussian blue nanoparticles were obtained, which were designated as PB NPs. (2) 100 mg of PVP was weighed and dissolved in 20 mL of 1 M HCl solution and stirred until clear. Subsequently, 20 mg of the solid Prussian blue nanoparticles were added and stirred for 4 h. The resulting mixture was transferred to a high-pressure reactor and allowed to stand in an oven at 140 °C for 4 h. The product was collected by centrifugation to obtain hollow Prussian blue nanoparticles, which were designated as HMPB NPs. (3) HU and TG (mass ratio 2:1) were loaded into the cavity of HMPB NPs by in situ encapsulation, and further coated with hyaluronic acid (HA, and HTHP:HA = 1:2). Finally, the precipitate was washed with ethanol and centrifuged repeatedly until the supernatant was clear and colorless. The product was dried to obtain a mesoporous photosensitive nanocomposite drug, which was recorded as HTHPH NPs. (4) Weigh 1.3 g of γ-PGA and dissolve it in 2 mL of deionized water. Stir at room temperature until it is completely dissolved to obtain a γ-PGA pregel solution. Disperse HTHPH NPs evenly in the γ-PGA pregel solution. (5) The γ-PGA pregel solution containing HTHPH NPs was evenly added dropwise to the PDMS microneedle mold and placed in a vacuum drying oven. After 20 min of vacuum drying, the mold was taken out. This operation was repeated several times until the needle tip area of the microneedle mold was completely filled. (6) Weigh 1.5 g of PVA and dissolve it in 10 mL of deionized water. Stir at room temperature until it is completely dissolved to obtain a PVA pregel solution. Remove the excess liquid in the mold of step (5), add the PVA pregel solution as a backing material on its surface, and then place it in a room temperature drying oven for 24 hours. Then demold it to obtain the cGAS-STING activated microneedle system, which is recorded as HTHPH MNs.

[0047] Example 2 Different from Example 1, the mass ratio of HU to TG in step (3) of this example is 14:1.

[0048] Example 3 Different from Example 1, the mass ratio of HU to TG in step (3) of this example is 1:1.

[0049] Example 4 Different from Example 1, the natural CD44 ligand polysaccharide used in step (3) of this example is chondroitin sulfate.

[0050] Example 5 Different from Example 1, the natural CD44 ligand polysaccharide used in step (3) of this example is dermatan sulfate.

[0051] Example 6 Different from Example 1, the needle tip material used in step (4) of this example is sodium alginate.

[0052] Example 7 Different from Example 1, the needle tip material used in step (4) of this example is chitosan.

[0053] Example 8 Different from Example 1, the backing material used in step (6) of this example is gelatin.

[0054] It should be noted that the above embodiments are only some embodiments of the present invention. The cGAS-STING activated microneedle system of the present invention can be successfully prepared by changing the mass ratio of hydroxyurea and thapsigargin, changing the proportion of small molecule drugs, changing the proportion of mesoporous photosensitive nanocomposite drugs, changing the amount of natural CD44 ligand polysaccharide, changing the needle tip material, changing the backing material, etc.

[0055] Comparative Example 1 Different from Example 1, this comparative example does not include step (2), and the solid Prussian blue nanoparticles prepared in step (1) are directly used in step (3).

[0056] Comparative Example 2 Different from Example 1, hydroxyurea and thapsigargin were not added in step (3) of this comparative example, and the product obtained in step (3) was recorded as HPH NPs.

[0057] Comparative Example 3 Different from Example 1, in step (3) of this comparative example, only hydroxyurea was encapsulated, and thapsigargin was not encapsulated. The product obtained in step (3) was recorded as HHPH NPs.

[0058] Test Example 1 The morphology of the mesoporous photosensitive nanocomposite drug prepared in each example and each comparative example was observed using a transmission electron microscope (TEM). The morphology of the mesoporous photosensitive nanocomposite drug in Example 1 is shown in FIG. Figure 2 As shown. Figure 2 It can be seen that the average particle size of the mesoporous photosensitive nanocomposite drug of the present invention is about 190 nm, and the surface presents a regular hexahedral structure.

[0059] The morphology of the cGAS-STING activated microneedle system prepared in each embodiment and each comparative example was observed using a scanning electron microscope (SEM). The morphology of the cGAS-STING activated microneedle system in Example 1 is as follows: Figure 3 As shown. Figure 3 It can be seen that the cGAS-STING activated microneedle system obtained in Example 1 presents a neatly arranged quadrangular pyramidal needle tip structure.

[0060] Test Example 2 The PB (solid Prussian blue nanoparticles obtained in step (1)), HMPB (hollow Prussian blue nanoparticles obtained in step (2)), HHP (nanoparticles encapsulated with hydroxyurea but not encapsulated with thapsigargin and not coated with HA in step (3)), HTHP (nanoparticles encapsulated with hydroxyurea and thapsigargin but not coated with HA in step (3)), and HTHPH (mesoporous photosensitive nanocomposite drug in step (3)) in each step of Example 1 were detected using a Zeta potential analyzer and an infrared spectrometer, wherein the detection results are as follows: Figure 4 shown.

[0061] from Figure 4 It can be seen that the Zeta changes with each further treatment, which indirectly proves that the target object is successfully loaded in each step; for example, the Zeta potential of HTHP is positive (-17.8mV), and after HA is modified on its surface, the Zeta potential of the nanodrug HTHPH becomes -29.1mV, indicating that HA is successfully modified on the HTHP nanoparticles; at the same time, the infrared spectrum results also show that HU and TG are successfully encapsulated into the hollow Prussian blue nanoparticles, and HA is successfully modified on the HTHP nanoparticles.

[0062] Similarly, the nanoparticles of each comparative example were tested. The test results showed that comparative examples 2-4 also successfully prepared the target products, while comparative example 1 used solid Prussian blue nanoparticles but failed to successfully encapsulate HU and TG on the particles.

[0063] Test Example 3 The microneedle systems prepared in Example 1 and Comparative Examples 2-3 were tested for their cancer cell killing effects. Specifically, B16F10 cells were divided into 6 groups and treated differently: Control group (no treatment), HPH group (i.e., treated with the microneedle system prepared in Comparative Example 2), HPH+L group (i.e., first treated with the microneedle system prepared in Comparative Example 2, and then subjected to 808nm laser irradiation treatment), HHPH group (i.e., treated with the microneedle system prepared in Comparative Example 3), HTHPH group (i.e., treated with the cGAS-STING activated microneedle system prepared in Example 1), and HTHPH+L group (i.e., first treated with the cGAS-STING activated microneedle system prepared in Example 1, and then subjected to 808nm laser irradiation treatment). The killing effects of nanomedicines of different concentrations and types on cancer cells were then evaluated using the CCK-8 method. The results are shown in FIG. Figure 5 shown.

[0064] from Figure 5 It can be seen that at a nanoparticle concentration of 100 μg / mL, the nanodrug HTHPH+L killed over 83% of B16F10 cells after 24 hours of treatment. However, nanodrugs HPH, HPH+L, HHPH, and HTHPH struggled to reach this level (Nanodrug HPH killed approximately 15% of B16F10 cells, Nanodrug HPH+L killed approximately 32% of B16F10 cells, and Nanodrug HHPH killed approximately 75% of B16F10 cells), indicating that the nanodrug HTHPH+L has a significant killing effect on tumor cells.

[0065] Test Example 4 The microneedle systems prepared in Example 1 and Comparative Examples 2-3 were tested for in vitro immune activation effects. B16F10 cells were divided into 6 groups for different treatments: Control group, HPH group, HPH+L group, HHPH group, HTHPH group, and HTHPH+L group to evaluate the in vitro immune activation effects. The results are shown in Figure 2. Figure 6 shown.

[0066] from Figure 6As can be seen from the results, the Control and HPH groups had little effect in inducing immune activation, with INF-β production at a low level. In contrast, the HHPH group, after the addition of HU, exhibited a certain degree of immune activation, with INF-β production 3.1 times that of the control group. When HU and TG were further introduced, INF-β expression increased to 3.6 times that of the control group, indicating that the combined delivery system has an enhanced activation capacity for the cGAS-STING signaling pathway. Furthermore, the level of INF-α released by tumor cells treated with the HTHPH+L group was 4.1 times that of the control group, further verifying that the photothermal / chemotherapy synergistic therapy strategy described in the present invention can significantly induce cells to release immune activation factors.

[0067] from Figure 6 As can be seen in Figure 2, statistical analysis of DC maturation by flow cytometry showed that there was essentially no significant change in the DC maturation rate after treatment in the control and HPH groups. In contrast, the HPH+L, HHPH, and HTHPH groups increased the DC maturation rate by 1.9 times, 2.0 times, and 2.4 times that of the control group, respectively, demonstrating a moderately stimulating effect. Particularly notably, treatment with the HTHPH+L group increased the DC maturation rate by 3.4 times that of the control group, demonstrating that the combined treatment strategy provided by the present invention has significant technical effectiveness in activating anti-tumor immunity and promoting DC maturation.

[0068] In summary, the relevant experimental results fully demonstrate that the HTHPH+L combination strategy proposed in the present invention can effectively activate the cGAS-STING signaling pathway and significantly promote the maturation of DCs, and has potential application value in anti-tumor immunotherapy.

[0069] Test Example 5 The microneedle systems prepared in Example 1 and Comparative Examples 2-3 were tested for their in vivo anti-tumor effects. Specifically, B16F10 tumor-bearing mice were used to evaluate the in vivo anti-tumor effects of different microneedle systems. 3 Under the condition of , B16F10 tumor-bearing mice were randomly divided into 6 groups for different treatments, namely: Control group, HPH group, HPH+L group, HHPH group, HTHPH group and HTHPH+L group, and treated with different nanomedicines. The results are shown in the figure. Figure 7 shown.

[0070] from Figure 7As can be seen, the tumor volume in the control group increased dramatically during treatment, reaching more than seven times its original size on day 14. In contrast, tumor growth in the HPH and HPH+L groups was inhibited to a certain extent, and the final tumor volumes of these two groups were 3.9 times and 2.2 times the corresponding initial tumor volumes, respectively. Notably, the introduction of HU and TG significantly enhanced the anti-tumor effects of the HTHPH and HTHPH+L groups, which is consistent with the in vitro experimental results mentioned above. The tumor inhibition rate in the HTHPH+L group reached as high as 88.99%.

[0071] It should be noted that the above test examples are only test results of some embodiments of the present invention. Other embodiments of the present invention have similar effects to Example 1 and all have strong anti-tumor effects.

[0072] In summary, the present invention combines localized photothermal therapy with systemic immune activation, inducing ICD in tumor cells under mild photothermal conditions. This effectively releases tumor-associated antigens and danger signaling molecules, promotes the activation of DCs, and further enhances the anti-tumor immune response in vivo. Compared with existing technologies, the present invention represents a significant advancement.

[0073] The above description is merely a representative embodiment of the present invention and does not constitute any form of limitation to the present invention. Any technical personnel familiar with the present invention who, without departing from the scope of the technical solution of the present invention, makes some changes or modifications to the embodiments disclosed above using the technical contents disclosed above are equivalent embodiments of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A cGAS-STING activated microneedle system comprising a connected needle tip and a backing, characterized in that: The needle tip is loaded with a mesoporous photosensitive nanocomposite drug, which uses hollow Prussian blue nanoparticles as carriers, internally loads small molecule drugs, and is coated with natural CD44 ligand polysaccharide on the outer layer; the small molecule drug is a mixture of hydroxyurea and thapsigargin.

2. The cGAS-STING activated microneedle system according to claim 1, characterized in that The mass ratio of the hydroxyurea to the thapsigargin is 1-14:1, the small molecule drug accounts for 5%-10% of the mass of the mesoporous photosensitive nanocomposite drug, and the mesoporous photosensitive nanocomposite drug accounts for 5%-20% of the mass of the needle tip after loading the mesoporous photosensitive nanocomposite drug.

3. The cGAS-STING activated microneedle system according to claim 1, characterized in that The average particle size of the mesoporous photosensitive nanocomposite drug is 150nm to 200nm.

4. The cGAS-STING activated microneedle system according to claim 1, characterized in that The thickness of the natural CD44 ligand polysaccharide is 8.6nm-12.8nm.

5. The cGAS-STING activated microneedle system according to claim 1, characterized in that The natural CD44 ligand polysaccharide is any one or more of hyaluronic acid, chondroitin sulfate, and dermatan sulfate.

6. The cGAS-STING activated microneedle system according to any one of claims 1 to 5, characterized in that: The length of the needle tip is 650µm to 1250µm, and the thickness of the backing is 20µm.

7. The method for preparing the cGAS-STING activated microneedle system according to any one of claims 1 to 6, wherein: The following steps are involved: S1: preparing hollow Prussian blue nanoparticles, and mixing and encapsulating the hollow Prussian blue nanoparticles with the small molecule drug to obtain a mesoporous photosensitive nanodrug; S2: preparing a natural CD44 ligand polysaccharide aqueous solution, and dispersing the mesoporous photosensitive nanodrug in the natural CD44 ligand polysaccharide aqueous solution, so that the natural CD44 ligand polysaccharide is coated on the surface of the mesoporous photosensitive nanodrug to obtain the mesoporous photosensitive nanocomposite drug; S3: mixing the mesoporous photosensitive nanocomposite drug with the needle tip material to obtain a mixed solution; S4: adding the mixed solution into a microneedle mold and drying to obtain a microneedle mold filled with needle tips; S5: preparing a backing material solution, adding the backing material solution into the microneedle mold filled with needle tips, and drying to obtain the cGAS-STING activated microneedle system.

8. The method for preparing the cGAS-STING activated microneedle system according to claim 7, characterized in that: In step S1, the hollow Prussian blue nanoparticles are obtained by etching solid Prussian blue nanoparticles with hydrochloric acid.

9. The method for preparing the cGAS-STING activated microneedle system according to claim 7 or 8, characterized in that: In step S3, the needle tip material is biodegradable material 1, and the biodegradable material 1 is any one or more of γ-polyglutamic acid, sodium alginate, gelatin, chitosan, and polyvinyl alcohol; in step S5, the backing material used in the backing material solution is biodegradable material 2, and the biodegradable material 2 is any one or more of polyvinyl alcohol, gelatin, hyaluronic acid, and polyvinyl pyrrolidone.

10. Use of the cGAS-STING activated microneedle system according to any one of claims 1 to 6 in the preparation of a drug for treating tumors, wherein the tumor is melanoma.

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