A microneedle patch for regulating tumor lactic acid metabolism and a preparation method and application thereof

Abstract

This invention proposes a microneedle patch for regulating lactate metabolism in tumors, its preparation method, and its application, belonging to the field of microneedle patch technology. It includes a backing layer and a needle tip matrix. The needle tip matrix is ​​composed of calcium carbonate-mineralized cyanobacteria and ZIF-8 nanoparticles loaded with lactate oxidase synergistically integrated into γ-polyglutamic acid microneedles. In this invention, cyanobacteria are mineralized with calcium carbonate, and the resulting calcium carbonate-mineralized cyanobacteria are synergistically integrated with ZIF-8 nanoparticles loaded with lactate oxidase into γ-polyglutamic acid microneedles. The resulting microneedle patch can disrupt calcium metabolism in solid tumors. 2+ It achieves stable and efficient induction of ICD, and systematically eliminates lactate through a three-level synergistic strategy of "extracellular clearance - intracellular consumption - metabolic source blockade". It is further supplemented by photocontrolled oxygen production to reverse the immunosuppressive microenvironment, transforming the tumor from "immune cold" to "immune hot", significantly inhibiting the primary tumor and blocking its distant metastasis, providing a new strategy for precisely regulating tumor metabolism-immune interaction and improving the response of immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of microneedle patch technology, and more specifically, to a microneedle patch for regulating tumor lactate metabolism, its preparation method, and its application. Background Technology

[0002] Tumor recurrence and metastasis remain the leading cause of death in cancer patients. Although immunotherapy has shown revolutionary potential by revitalizing the host's immune system, the clinical response rate in solid tumors remains limited. The core bottlenecks are mainly due to: (1) the highly immunosuppressive tumor microenvironment (ITME); and (2) the inherent low immunogenicity of tumor cells. In solid tumors, the persistent hypoxia caused by rapid proliferation and abnormal angiogenesis not only weakens the function of effector immune cells but also drives tumor cells to reprogram to aerobic glycolysis, producing and accumulating large amounts of lactate, forming an immunosuppressive metabolic niche with both deep hypoxia and high lactate load. This environment significantly weakens the anti-tumor immune response by restricting T cell oxygen-dependent metabolism, inhibiting dendritic cell maturation, promoting Treg amplification, and M2 macrophage polarization, giving tumors a strong immune escape ability.

[0003] To alleviate persistent hypoxia, oxygen carriers or oxygen-generating systems (such as hemoglobin, perfluorinated carbon, calcium peroxide, and catalase) have been extensively explored, but they generally face problems such as limited solubility, uncontrollable release, and rapid in vivo clearance, making it difficult to achieve continuous and precise oxygen supply. Meanwhile, lactate clearance strategies (such as lactate transport blockade or lactate oxidase-catalyzed oxidation) have limited overall efficiency, failing to act simultaneously on both extracellular and intracellular lactate accumulation zones, and making it even more difficult to achieve comprehensive and sustained clearance of tumor lactate. Therefore, constructing a strategy that can achieve sustained and controllable oxygen supply while completely clearing lactate is a key breakthrough for alleviating ITME and restoring immune activity.

[0004] Besides the immunosuppressive microenvironment, the low immunogenicity of tumors also severely hinders the effective recognition and elimination of tumor cells by the immune system, representing another core obstacle to immunotherapy. Immunogenic cell death (ICD) can induce the release of danger-related molecules (DAMPs) from tumors, promoting antigen presentation and serving as an important means to improve the response rate of immunotherapy. However, current methods for inducing ICD (chemotherapy, radiotherapy, phototherapy, etc.) all suffer from significant systemic toxicity, radiation-related tissue damage, and limited light penetration depth, and these methods are all constrained by the tumor microenvironment.

[0005] In recent years, Ca 2+ Ion interference therapy can break down the calcium in tumor cells. 2+Homeostasis, inducing endoplasmic reticulum stress, mitochondrial dysfunction, and multi-pathway synergistic potent ICD, exhibiting unique advantages. However, its efficacy is limited by local tumor calcium. 2+ The calcium metabolism mechanism is a compensatory mechanism for calcium deficiency and tumor cells' "low intake and high excretion".

[0006] Therefore, it is necessary to develop a method that can simultaneously provide abundant Ca. 2+ Drug delivery systems that disrupt the Ca2+ metabolic homeostasis of tumor cells are beneficial for further enhancing Ca2+ metabolism. 2+ Interference therapy has the ability to induce ICD and enhance tumor immunogenicity. Summary of the Invention

[0007] The purpose of this invention is to provide a microneedle patch for regulating lactate metabolism in tumors, its preparation method, and its application. The patch involves mineralizing cyanobacteria with calcium carbonate, resulting in calcium carbonate-mineralized cyanobacteria (PC), which is then synergistically integrated with ZIF-8 nanoparticles (LZH) loaded with lactate oxidase (LOx) into γ-polyglutamic acid microneedles. The resulting microneedle patch can disrupt calcium metabolism within solid tumors. 2+ It achieves stable and efficient induction of ICD, and systematically eliminates lactate through a three-level synergistic strategy of "extracellular clearance-intracellular consumption-metabolic source blockade". It is further supplemented by photocontrolled oxygen production to reverse the immunosuppressive microenvironment, transforming the tumor from "immune cold" to "immune hot", significantly inhibiting the primary tumor and blocking its distant metastasis, providing a new strategy for precisely regulating tumor metabolism-immune interaction and improving the response of immunotherapy.

[0008] The technical problem solved by this invention is achieved by the following technical solution.

[0009] In a first aspect, embodiments of this application provide a microneedle patch for regulating tumor lactate metabolism, comprising a backing layer and a needle tip matrix, wherein the needle tip matrix is ​​formed by the synergistic integration of calcium carbonate mineralized cyanobacteria and ZIF-8 nanoparticles loaded with lactate oxidase into γ-polyglutamic acid microneedles.

[0010] Furthermore, the backing layer material is any one or more of polyvinyl alcohol, polyvinylpyrrolidone, and hyaluronic acid.

[0011] Secondly, embodiments of this application provide a method for preparing the above-mentioned microneedle patch, comprising the following steps: S1: Dissolve zinc acetate dihydrate in deionized water containing lactate oxidase, stir and add 2-methylimidazole, incubate at room temperature for 6-10 h, collect nanoparticles by centrifugation, wash and dry, disperse in hyaluronic acid aqueous solution, stir in the dark for 45-55 h, centrifuge and wash again to obtain ZIF-8 nanoparticles loaded with lactate oxidase. S2: Disperse cyanobacteria in deionized water containing polyvinylpyrrolidone, stir at room temperature and add calcium chloride solution dropwise. After the addition is complete, add sodium carbonate solution and continue stirring for 1-2 hours. After centrifugation and washing, obtain calcium carbonate mineralized cyanobacteria. S3: Polyvinyl alcohol and γ-polyglutamic acid are dissolved in deionized water and stirred continuously at room temperature until viscous. Then, ZIF-8 nanoparticles loaded with lactate oxidase and calcium carbonate mineralized cyanobacteria are dispersed in the γ-polyglutamic acid solution. The resulting mixture is poured into a polydimethylsiloxane microneedle mold and vacuum treated. Polyvinyl alcohol solution is then added and dried at room temperature for 20-30 hours. After demolding, a microneedle patch for regulating tumor lactate metabolism is obtained.

[0012] Furthermore, in step S1, the mass ratio of zinc acetate dihydrate, lactate oxidase, and 2-methylimidazole is 200-300:1-10:400-500, and the centrifugation conditions are centrifugation at 8000 rpm for 5-15 min.

[0013] Furthermore, in step S2, the concentration of polyvinylpyrrolidone is 2-5 mg / mL, and the molar ratio of calcium chloride solution to sodium carbonate solution is 1:1.

[0014] Furthermore, in step S3, the concentration of polyvinyl alcohol is 0.1-0.2 g / mL, and the concentration of γ-polyglutamic acid is 0.6-0.8 g / mL.

[0015] Thirdly, embodiments of this application provide the application of the above-mentioned microneedle patch in the preparation of anti-tumor products.

[0016] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention is the first to synergistically integrate calcium carbonate mineralized cyanobacteria with ZIF-8 nanoparticles loaded with lactate oxidase into γ-polyglutamic acid microneedles. The resulting microneedle patch can achieve "lactic acid-oxygen-Ca" synthesis. 2+ "Multi-dimensional regulation, through the physical penetration of microneedles to break through the tissue barrier, achieves precise delivery of functional components to the tumor site; at the same time, it utilizes the active targeting effect mediated by CD44 receptor to promote the efficient uptake of LZH by tumor cells, thereby reshaping ITME and enhancing tumor immunogenicity, and thus significantly enhancing the systemic anti-tumor immune response." 2. This invention also proposes a systematic strategy for eliminating lactic acid: "extracellular clearance - intracellular consumption - metabolic source blocking": the outer layer of CaCO3 rapidly dissolves in an acidic environment, releasing Ca... 2+ It neutralizes extracellular lactate; intracellular LZH continuously oxidizes lactate to generate H2O2; and, combined with oxygen production under cyanobacterial light, inhibits HIF-1α stability and blocks glycolysis pathways from the source. Furthermore, the metabolically generated H2O2 enhances Ca2+ in the TRPA1 channel.2+ Influx and Ca2+ inhibiting ATP2B1 2+ Outflow, causing lethal calcium to tumor cells 2+ Overload triggers potent immunogenic cell death, achieving a deep linkage between metabolic reprogramming and immune activation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the LZH obtained in Embodiment 1 of the present invention, wherein (a) represents a TEM observation image and (b) represents an elemental composition image; Figure 2 This is a schematic diagram of the PC prepared according to Embodiment 1 of the present invention, wherein (a) represents a TEM observation image and (b) represents an elemental composition image; Figure 3 The image shown is an XRD analysis image of a PC obtained in Example 1 of this invention. Figure 4 This is a schematic diagram of the LZH / PC microneedles prepared in Example 1 of the present invention, where (a) represents a SEM observation image and (b) is an elemental mapping image; Figure 5 This is an analysis chart showing the amount of lactic acid consumed by PC at different concentrations obtained in Example 1 of the present invention; Figure 6 This is an acid-base titration curve of PC prepared in Example 1 of the present invention; Figure 7 This is an analysis chart of the oxygen production of PC prepared in Example 1 of the present invention under light and dark conditions; Figure 8 The graph shows the analysis of LZH obtained in Example 1 of this invention at different concentrations, which consumes lactic acid and generates H2O2. Figure 9 This is a CLSM image of B16F10 cells stained with [Ru(dpp)3]Cl2 in an embodiment of the present invention. Figure 10 In this embodiment of the invention, the Fura-2 AM probe is used to detect intracellular Ca2+. 2+ Horizontal CLSM image; Figure 11 This is a flow cytometry analysis diagram of dendritic cells after different treatments in an embodiment of the present invention; Figure 12This is a quantitative analysis diagram of lactate levels in B16F10 cells after different treatments in this embodiment of the invention, where (a) represents extracellular lactate levels and (b) represents intracellular lactate levels. Figure 13 This is a schematic diagram of the growth of individual tumors in different groups of mice after treatment with B16F10 in an embodiment of the present invention; where (a) is the tumor growth curve of individual mice in different groups; and (b) is the corresponding tumor inhibition rate. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0021] Example 1 This embodiment provides a detailed method for preparing a microneedle patch for regulating tumor lactate metabolism, including the following steps: S1. Preparation of LZH: 200-300 mg of Zn(OAc)₂·2H₂O (i.e., zinc acetate dihydrate, 1 mmol = 219.5 mg) was dissolved in 3 mL of deionized water containing 1-10 mg LOx. The solution was then slowly stirred at room temperature, and during stirring, an aqueous solution containing 400-500 mg of 2-MIM (2-methylimidazole) (2-MIM concentration 160 mg / mL) was rapidly added. As a preferred embodiment, in this example, 219.5 mg of Zn(OAc)₂·2H₂O, 4 mg of LOx, and an aqueous solution containing 480 mg of 2-MIM were mixed to obtain a mixture. This mixture was then incubated at room temperature for 6 h to achieve ZIF-8 crystallization and enzyme encapsulation. After incubation, the product was transferred to a centrifuge and centrifuged at 8000 rpm for 5 min. The resulting nanoparticles were collected and designated as LZ nanoparticles. Then, the collected LZ was washed three times with deionized water to remove residual reactants. After washing, it was dried and refrigerated for later use.

[0022] Furthermore, LZ was modified with HA. The prepared LZ nanoparticles were dispersed in a 10 mg / mL HA aqueous solution and stirred in the dark for 48 h to achieve HA adsorption on the surface of the LZ nanoparticles. After stirring, the nanoparticles were centrifuged at 8000 rpm for 15 min, collected, and washed multiple times with ultrapure water to completely remove unbound HA, thus obtaining LZH.

[0023] S2, Preparation of PC: 1×10 8 PCC7942 cells (cyanobacterial cells) were dispersed in 1.5 mL of deionized water containing polyvinylpyrrolidone (PVP) at a concentration of 2 mg / mL. The mixture was then stirred at room temperature, during which 0.2 mL of 0.33 M calcium chloride aqueous solution was added dropwise. After the addition was complete, stirring continued for 20 min, followed by the addition of an equimolar amount of sodium carbonate solution to initiate CaCO3 precipitation on the cyanobacterial surface. The mixture was then stirred at room temperature for 1 h to ensure uniform distribution of the mineralized deposits. After stirring, the mixture was transferred to a centrifuge and centrifuged at 10,000 rpm for 1 min. The calcium carbonate-mineralized cyanobacteria were collected and washed three times with phosphate-buffered saline to remove unbound ions and loosely attached mineralized residues.

[0024] S3. Preparation of LZH / PC microneedles: Weigh 1.5 g PVA and 1.3 g γ-PGA, and dissolve them in 10 mL and 2 mL of deionized water, respectively. Stir continuously at room temperature until a homogeneous pregel solution is formed. Then, uniformly disperse the LZH and PC obtained in steps S1 and S2 in the γ-PGA pregel solution, stir, pour into a PDMS microneedle mold, and perform vacuum treatment for 20 min to ensure complete filling of the needle cavity. Repeat the vacuum filling process multiple times to achieve a uniform and dense loading within the needle tip. Then, remove excess solution from the mold surface and add PVA pregel solution to form a backing layer. Dry the assembled microneedle patch at room temperature for 24 hours, and then carefully demold to obtain the final LZH / PC microneedles.

[0025] Example 2 The steps in this embodiment are basically the same as those in Embodiment 1. The only difference is that in step S3, LZH is not added, only PC is added, and the resulting microneedles are denoted as PC microneedles.

[0026] Example 3 The steps in this embodiment are basically the same as those in Embodiment 1. The only difference is that in step S3, PC is not added, only LZH is added, and the resulting microneedles are denoted as LZH microneedles.

[0027] Performance verification: I. Please refer to Figures 1-4 First, the LZH, PC, and LZH / PC prepared in Example 1 were observed. Figure 1 It can be seen that the prepared LZH exhibits a typical rhombic dodecahedral structure with uniform particle size, regular morphology, clear edges, and good crystallinity, consistent with the characteristic morphology of ZIF-8 crystals. Furthermore, the highly uniform distribution of C, N, and Zn elements within the particles indicates that the organic ligands of ZIF-8 are uniformly bonded to the metal center, with no elemental segregation or phase separation, and a complete crystal structure. Additionally, from... Figures 2-3 It can be seen that the distribution of C, O, and Ca highly coincides with the rod-shaped morphology, confirming that calcium carbonate successfully adhered to the surface of cyanobacteria, achieving mineralization of the cyanobacteria. Furthermore, from... Figure 4 It can be seen that the surface of the microneedle matrix is ​​smooth and dense, with no obvious pores or defects, indicating that the PC matrix and LZH are uniformly dispersed in the microneedles without obvious phase separation or component agglomeration, which verifies that the composition of the microneedles is completely consistent with the design.

[0028] Furthermore, to verify the ability of PC to neutralize extracellular lactate, a lactate assay kit was used to verify the lactate scavenging ability of PC. The experimental results are as follows: Figure 5 As shown, with increasing PC concentration, lactic acid consumption exhibited a significant dose-dependent increase, demonstrating PC's ability to neutralize lactic acid. Furthermore, as... Figure 6 As shown, when PC was added to PBS with a pH of 6.5, which simulates the tumor microenvironment, the pH of the system increased rapidly, indicating that it has a significant acid neutralization ability.

[0029] refer to Figure 7 Under alternating light (red light) and darkness conditions, the dissolved oxygen concentration in the system exhibited periodic fluctuations, with an overall upward trend over time; indicating that light can trigger PC to circulate oxygen production.

[0030] Furthermore, this step also verified the ability of LZH to consume lactic acid, and the consumption of lactic acid and the production of hydrogen peroxide were measured using a lactic acid assay kit and a hydrogen peroxide detection kit. The experimental results are as follows: Figure 8 As shown, with the treatment concentration increasing from 16.7 μg / mL to 500 μg / mL, both lactic acid consumption and H2O2 generation in the system exhibited a significant dose-dependent increasing trend, and the differences between the two groups of indicators reached extremely significant levels (***, p<0.001). This result indicates that LZH has a good ability to consume lactic acid.

[0031] II. Based on the three types of microneedles prepared in Examples 1-3, experimental groups were set up, denoted as LZH, PC, PC+L, LZH / PC, and LZH / PC+L, respectively. PBS was used as a blank control. Here, L indicates treatment with 660 nm red light for 20 min. The specific scheme is as follows: 1) B16F10 cells were spaced at 1.0 × 10⁶ cells per well. 5 Cells were seeded at different densities in 12-well plates and cultured for 12 hours. Subsequently, the cells were transferred to fresh culture medium (final concentration 100 μg / mL) containing different formulations (LZH, PC, PC+L, LZH / PC, or LZH / PC+L). -1 The cells were incubated together for 2 hours. Then, 30 μM oxygen-sensitive probe [Ru(dpp)3]Cl2 was added to each well and incubated for 20 minutes. After staining, the cells were washed three times with PBS (three replicates per experiment) and imaged using CLSM. The oxygen-sensitive probe [Ru(dpp)3]Cl2 showed red fluorescence under hypoxic conditions and was quenched by oxygen under hyperoxic conditions. The experimental results are as follows: Figure 9 As shown, the signal intensity of the probe showed significant differences among the different treatment groups: strong red fluorescence was observed in the Control group, LZH group, PC group and LZHM / PC group, while the red fluorescence signal of the PC+L group and LZH / PC+L group (light treatment group) decreased significantly, indicating that under light conditions, the LZH / PC nanoplatform can effectively promote oxygen production in PC, thereby alleviating intracellular hypoxia.

[0032] 2) Experimental results are from Figure 10 As shown, the intracellular calcium ion concentrations (Ca) in the control group and the LZH-treated group were significantly different. 2 + The fluorescence signal was extremely weak; a small amount of Ca was visible in the PC-treated group. 2+ The signal strength of the PC+L group was slightly enhanced; while the Ca signal of the LZH / PC group was enhanced. 2+ The fluorescence intensity increased significantly in the LZH / PC+L group, and the Ca... 2+ The signal reached its strongest point, indicating that LZH / PC combined light treatment can effectively induce intracellular Ca2+. 2+ The elevated levels, and the effect being significantly superior to that of PC alone, PC+L, and LZH / PC without light treatment, indicate that photoactivated LZH / PC can efficiently trigger intracellular calcium overload, providing direct fluorescent evidence for its subsequent biological effects.

[0033] 3) Select several 3-4 week old C57BL / 6 mice, euthanize them, disinfect the skin, remove the femur and tibia, and carefully remove the surrounding muscle tissue. Briefly immerse the bones in 75% ethanol for 5-10 seconds, wash thoroughly with PBS, and then cut the bone ends to expose the medullary cavity. Wash the bone marrow cells with RPMI 1640 medium and collect the cell pellet by centrifugation at 500 g for 5 minutes at 4°C. Resuspend the cell pellet in RPMI 1640 medium, add 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF), and culture in a humidified incubator at 37°C with 5% CO2 to induce dendritic cell differentiation. On day 9, harvest immature dendritic cells and culture at 2.5 × 10⁶ cells per well. 5 B16F10 tumor cells were seeded at a density of 2.5 × 10⁶ cells / well in the lower chamber of a Transwell co-culture system, with 1 mL of RPMI 1640 medium per well. 5 Dendritic cells (number of cells) were seeded in the upper culture dish and co-cultured for 12 hours. Subsequently, B16F10 cells were treated in the dark with PBS, LZH, PC, PC+L, LZH / PC, and LZH / PC+L for 30 minutes each, where L indicates 635 nm laser irradiation for 15 minutes, and co-cultured for another 12 hours. After incubation, dendritic cells were collected from the lower culture dish, blocked with bovine serum albumin (BSA), and stained in the dark with fluorescently labeled anti-CD11c (FITC), anti-CD80 (PE), and anti-CD86 (APC) antibodies for 30 minutes. Each experiment was performed in triplicate, and the maturation status of dendritic cells was analyzed by flow cytometry. The experimental results are as follows: Figure 11 As shown, compared with the control group, the LZH, PC, PC+L, and LZH / PC groups all upregulated CD80 to varying degrees. + With CD86 + The expression level of CD80 was increased, and the proportion of double-positive cells gradually increased with treatment; among them, the CD80 expression level in the LZH / PC+L group was higher. + and CD86 + The highest proportion of double-positive cells (approximately 21.4%) was observed, significantly higher than all other treatment groups, suggesting that the LZH / PC+L treatment combination most effectively promotes cell activation and maturation, and CD80 levels. + With CD86 + The expression level was significantly improved, and there was a clear synergistic enhancement effect.

[0034] Furthermore, the lactate levels inside and outside the treated B16F10 cells were analyzed, and the results are as follows: Figure 12As shown, both LZH / PC and LZH / PC+L significantly reduced extracellular lactate levels, with the LZH / PC+L group showing a greater reduction. This may be attributed to the enhanced oxygen production by cyanobacteria under light conditions, which increased the activity of lactate oxidase (LOx). Intracellular lactate levels also showed a similar trend, with the LZH / PC+L group significantly lower than the LZH / PC group, further validating the hypothesis that light-controlled oxygen production can enhance lactate clearance.

[0035] 4) Inoculate B16F10 tumor cells onto RPMI 1640 medium. Select several female C57BL / 6 mice of the same age and inoculate them with 2×10⁶ cells. 6 A B16F10 melanoma tumor model was established by subcutaneous injection of B16F10 tumor cells into the mammary adipose tissue of each mouse. When the tumor volume reached approximately 100 mm³ (defined as day 0), the mice were randomly assigned to six treatment groups (n=5 per group). From day 1 to day 7, tumor-carrying mice received intravenous injections of PBS, LZH, PC, PC+L, LZH / PC, and LZH / PC+L. All nanoparticle formulations were administered at an equivalent dose of 10 mg / kg in a total injection volume of 100 μL. Tumor size and body weight were recorded every two days throughout the treatment period. Tumor volume (V) was calculated using the formula:

[0036] Tumor inhibition rate was determined based on changes in tumor volume relative to the control group. At the end of the experiment, mice were euthanized, tumors were removed, weighed, and photographed. Experimental results are as follows: Figure 13 As shown, the tumor volume in the Control group increased rapidly over time, reaching nearly 1300 mm³ by day 15. The LZH, PC, PC+L, and LZH / PC groups all exhibited varying degrees of tumor growth inhibition. The LZH / PC+L group showed significant tumor growth inhibition, with its tumor volume even decreasing in the later stages, demonstrating the best effect among all treatment groups. Further analysis of the inhibition rate revealed that the LZH / PC+L group had the highest tumor inhibition rate, significantly higher than all other treatment groups, exhibiting a synergistic and enhanced anti-tumor effect. The Control group showed almost no inhibitory effect, and the inhibitory effects of the other treatment groups were progressively weaker than those of the LZH / PC+L group. In conclusion, LZH / PC+L exhibited the strongest tumor growth inhibition effect in vivo, significantly superior to single drugs or other combination therapies, confirming the highly effective anti-tumor potential of this synergistic strategy.

[0037] Based on the above verification, the microneedle patch provided by this invention can be applied to the regulation of solid tumors. Its regulatory principle is as follows: the physical penetration of the microneedles breaks through the tissue barrier, achieving precise delivery of functional components to the tumor site; simultaneously, the active targeting effect mediated by the CD44 receptor promotes the efficient uptake of LZH by tumor cells. Specifically, the CaCO3 shell of PC rapidly dissolves in the acidic tumor microenvironment, releasing a large amount of Ca. 2+ Simultaneously, it rapidly consumes extracellular lactic acid through acid-base neutralization, effectively alleviating calcium deficiency. 2+ The problem of lactate deficiency and lactate accumulation. Secondly, LZH enters tumor cells under the active uptake of HA-CD44, releasing LOx to continuously oxidize intracellular lactate and generate H2O2, thereby enhancing the Ca2+ mediated by TRPA1 channels. 2+ Influx and inhibition of ATP2B1-driven Ca2+ 2+ efflux, achieving severe cancer in tumor cells. 2+ Overload induces potent immunogenic cell death and promotes antigen cross-presentation, significantly enhancing tumor immunogenicity. Furthermore, cyanobacteria can continuously produce oxygen under light, not only improving LOx oxidation efficiency, inhibiting HIF-1α stability at its source, and blocking the glycolysis-lactic acid production pathway, but also promoting macrophage phenotype conversion from M2 to M1, further alleviating ITME. Based on these characteristics, those skilled in the art can apply them to anti-tumor products according to clinical needs.

[0038] In summary, this invention provides a microneedle patch for regulating tumor lactate metabolism, its preparation method, and its application. It is the first to synergistically integrate calcium carbonate-mineralized cyanobacteria with ZIF-8 nanoparticles loaded with lactate oxidase into γ-polyglutamic acid microneedles. The resulting microneedle patch can achieve the "lactic acid-oxygen-Ca" process. 2+ "Multi-dimensional regulation, through the physical penetration of microneedles to break through the tissue barrier, achieves precise delivery of functional components to the tumor site; at the same time, it utilizes the active targeting effect mediated by CD44 receptor to promote the efficient uptake of LZH by tumor cells, thereby reshaping ITME and enhancing tumor immunogenicity, and thus significantly enhancing the systemic anti-tumor immune response." This invention also proposes a systematic strategy for eliminating lactic acid: "extracellular clearance - intracellular consumption - metabolic source blockade": the outer layer of CaCO3 rapidly dissolves in an acidic environment, releasing Ca2+. 2+ It neutralizes extracellular lactate; intracellular LZH continuously oxidizes lactate to generate H2O2; and, combined with oxygen production under cyanobacterial light, inhibits HIF-1α stability and blocks glycolysis pathways from the source. Furthermore, the metabolically generated H2O2 enhances Ca2+ in the TRPA1 channel. 2+ Influx and Ca2+ inhibiting ATP2B1 2+ Outflow, causing lethal calcium to tumor cells 2+Overload triggers potent immunogenic cell death, achieving a deep linkage between metabolic reprogramming and immune activation.

[0039] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A microneedle patch for regulating tumor lactic acid metabolism, characterized in that, It includes a backing layer and a tip matrix, wherein the tip matrix is ​​composed of calcium carbonate mineralized cyanobacteria and ZIF-8 nanoparticles loaded with lactate oxidase synergistically integrated into γ-polyglutamic acid microneedles.

2. The microneedle patch of claim 1, wherein, The backing layer is made of any one or more of polyvinyl alcohol, polyvinylpyrrolidone, and hyaluronic acid.

3. A method for preparing a microneedle patch for regulating tumor lactate metabolism as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Dissolve zinc acetate dihydrate in deionized water containing lactate oxidase, stir and add 2-methylimidazole, incubate at room temperature for 6-10 h, collect nanoparticles by centrifugation, wash and dry, disperse in hyaluronic acid aqueous solution, stir in the dark for 45-55 h, centrifuge and wash again to obtain ZIF-8 nanoparticles loaded with lactate oxidase. S2: Disperse cyanobacteria in deionized water containing polyvinylpyrrolidone, stir at room temperature and add calcium chloride solution dropwise. After the addition is complete, add sodium carbonate solution and continue stirring for 1-2 hours. After centrifugation and washing, obtain calcium carbonate mineralized cyanobacteria. S3: Polyvinyl alcohol and γ-polyglutamic acid are dissolved in deionized water and stirred continuously at room temperature until viscous. Then, ZIF-8 nanoparticles loaded with lactate oxidase and calcium carbonate mineralized cyanobacteria are dispersed in the γ-polyglutamic acid solution. The resulting mixture is poured into a polydimethylsiloxane microneedle mold and vacuum treated. Polyvinyl alcohol solution is then added and dried at room temperature for 20-30 hours. After demolding, a microneedle patch for regulating tumor lactate metabolism is obtained.

4. The production method according to claim 3, characterized by, In step S1, the mass ratio of zinc acetate dihydrate, lactate oxidase, and 2-methylimidazole is 200-300:1-10:400-500, and the centrifugation conditions are centrifugation at 8000 rpm for 5-15 min.

5. The preparation method according to claim 4, characterized in that, In step S2, the concentration of polyvinylpyrrolidone is 2-5 mg / mL, and the molar ratio of the calcium chloride solution to the sodium carbonate solution is 1:

1.

6. The preparation method according to claim 5, characterized in that, In step S3, the concentration of polyvinyl alcohol is 0.1-0.2 g / mL, and the concentration of γ-polyglutamic acid is 0.6-0.8 g / mL.

7. The use of the microneedle patch as described in claim 1 in the preparation of antitumor products.

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