Soluble microneedles, methods of making the same, and use in treating melanoma

Abstract

The application discloses soluble microneedles, a preparation method and application of the soluble microneedles in combination with an anti-PD-L1 antibody for treating superficial melanoma, and belongs to the field of nano delivery and tumor immunotherapy. The microneedles are based on a water-soluble biopolymer material, and only the needle tip is loaded with folic acid modified triptolide liposomes; the liposomes are composed of phospholipids, cholesterol, DSPE-PEG2000-FA and triptolide, and can improve melanoma cell uptake through folic acid receptor mediation. The microneedles penetrate the stratum corneum of the skin to locally release drugs, induce immunogenic cell death by means of triptolide, promote the maturation of dendritic cells and intratumoral CD8 + T cell infiltration. The application adopts a split type drug delivery method, the microneedles are locally applied, and the anti-PD-L1 antibody is separately injected, so that the drug delivery scheme can be flexibly adjusted, antibody inactivation can be avoided, and the systemic toxicity of triptolide can be greatly reduced. In-vivo experiments show that the combination of the two can effectively reverse the tumor immunosuppressive microenvironment, and solve the defects of poor response of a single anti-PD-L1 and great toxic and side effects of systemic administration of triptolide.

Description

Technical Field

[0001] This invention relates to the fields of drug delivery, soluble microneedles, tumor immunotherapy and nanomedicine formulation technology, specifically to a hyaluronic acid soluble microneedle loaded with folic acid-targeting triptolide liposomes, its preparation method and its application in combination with anti-PD-L1. Background Technology

[0002] Melanoma is a highly aggressive malignant tumor. Immune checkpoint blockade therapy, especially PD-1 / PD-L1 axis-related antibody therapy, has become an important strategy for melanoma treatment. However, some tumors have limited response to anti-PD-L1 therapy alone due to insufficient immunogenicity, insufficient antigen presentation, insufficient cytotoxic T cell infiltration, or significant local immunosuppressive environment.

[0003] Tripterygium wilfordii possesses strong antitumor activity and can induce tumor cell stress signals associated with immunogenic cell death. However, it has poor water solubility, nonspecific systemic distribution, a narrow therapeutic window, and is prone to toxic side effects when administered systemically. Liposomes are microparticles formed by encapsulating drugs in a biomembrane-like structure. Liposomes are simple and inexpensive to prepare and offer advantages such as improved biocompatibility, reduced biodegradability, decreased drug toxicity, and sustained-release properties. Using liposomes to support triptolide can improve its bioavailability and enhance its targeting. Liposomes can also improve the formulation properties of triptolide, and folic acid modification can enhance its interaction with tumor cells expressing high levels of folic acid receptors.

[0004] There are three similar solutions in the existing technology, but none of them can achieve the complete combined synergistic system of this invention: 1. Tripterygium wilfordii single-component soluble microneedles: only achieve local delivery, without folic acid-targeting liposome nanocarriers, cannot enhance tumor cell-specific uptake, and does not have the synergistic design for combining immunogenic chemotherapy with anti-PD-L1.

[0005] 2. Folic acid-targeted triptolide nanoliposomes (publication number CN113813233A): administered via intravenous / local injection only, without a percutaneous microneedle delivery carrier, highly invasive, with short local retention time in tumors, and currently only disclosed for the treatment of rheumatoid arthritis.

[0006] 3. Microneedle co-delivery of immune checkpoint inhibitors: Directly encapsulating anti-PD-L1 / PD-1 antibodies onto microneedles makes it impossible to flexibly adjust the antibody dosing cycle and dosage, and large molecular antibodies are easily inactivated during microneedle preparation and drying.

[0007] To address the aforementioned issues, several nanoneedle anti-tumor technologies have been disclosed. Two patents closest to existing patents, CN114096274A and CN114096280A, disclose a combined anti-tumor microneedle and immunotherapy regimen, involving nanocarriers, microneedle delivery, and the combined use of immune checkpoint inhibitors. These patents disclose methods for local drug testing of apoptosis regulators (e.g., deoxyadenosine and triptolide) using microneedles, but the following issues have not yet been adequately resolved: How can we combine the immunogenic chemotherapy effect of triptolide, the local retention / uptake advantage of folic acid-targeted liposomes, the local delivery advantage of microneedles, and the immune amplification effect of anti-PD-L1 into a feasible treatment system with clear experimental support? Summary of the Invention

[0008] The purpose of this invention is to provide a hyaluronic acid soluble microneedle (TP@FA-Lips / dMNs) loaded with folic acid-modified triptolide liposomes, its preparation method, and its application in combination with anti-PD-L1 antibodies for the treatment of superficial melanoma. This invention belongs to the fields of nanomedicine delivery, soluble microneedles, and tumor immunotherapy. This microneedle uses hyaluronic acid as a matrix, with only the tip loaded with folic acid-modified triptolide liposomes. These liposomes are composed of phospholipids, cholesterol, folic acid-modifying components, and triptolide, and can enhance melanoma cell uptake via folic acid receptor mediation. The hyaluronic acid microneedles penetrate the stratum corneum to release the drug locally, inducing immunogenic cell death with the help of triptolide, promoting dendritic cell maturation and intratumoral CD8 activation. + T-cell infiltration. This invention employs a split-drug administration approach, with local microneedle application and separate injection of the anti-PD-L1 antibody. This allows for flexible adjustment of the dosing regimen, avoids antibody inactivation, and significantly reduces the systemic toxicity of triptolide. In vivo experiments show that the combination of the two can effectively reverse the tumor immunosuppressive microenvironment, overcoming the shortcomings of poor response to single anti-PD-L1 drugs and the significant toxic side effects of systemic administration of triptolide.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A soluble microneedle includes a needle tip with a matrix and a backing, the matrix being a water-soluble biopolymer material, and the needle tip containing liposomes of triptolide modified with folic acid, the liposomes including triptolide, phospholipids, cholesterol and folic acid-modified components.

[0010] Compared with existing technologies, soluble microneedles using the above-mentioned technical solution have the following beneficial effects: I. This invention provides a soluble microneedle with folic acid-targeting triptolide liposomes loaded only at the needle tip. It enhances drug uptake by melanoma cells by targeting folic acid receptors. The microneedle matrix is ​​made of water-soluble biopolymer material, which achieves local retention in superficial lesions and reduces the systemic exposure toxicity of triptolide. The transdermal local drug delivery through the microneedle helps to reduce unnecessary systemic exposure of triptolide. Animal experiments showed no significant damage to the hepatobiliary and nephrotoxicity of major organs or blood biochemistry, reducing the risk of liver, kidney / systemic toxicity associated with traditional systemic drug delivery.

[0011] II. Tripterygium wilfordii induced apoptosis in B16F10 melanoma cells and triggered immunogenic cell death-related signals such as CRT exposure, HMGB1 redistribution, and decreased intracellular ATP, further promoting BMDC maturation and thus providing an immune initiation basis for anti-PD-L1 combination therapy. Data from this invention support its ability to induce apoptosis in B16F10 melanoma cells.

[0012] Third, local ICD chemotherapy and systemic anti-PD-L1 are administered separately, with antibodies not encapsulated in microneedles. This helps avoid potential activity loss of antibodies during microneedle preparation, drying, or storage, and facilitates independent adjustment of dosing frequency and dosage. The dosing frequency and dosage of the two drugs can be adjusted independently to jointly enhance CD8. + T-cell intratumoral infiltration has a better tumor-suppressing effect than single therapy.

[0013] Fourth, hyaluronic acid is naturally biodegradable, minimally invasive, and easy to administer, which helps improve the convenience of administration and potential compliance.

[0014] The water-soluble biopolymer material of the soluble microneedle matrix is ​​selected from any one or a combination of two or more of the following: hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, sodium carboxymethyl cellulose, gelatin, dextran, and alginate.

[0015] Hyaluronic acid is preferably used. As a natural extracellular matrix polysaccharide of the skin, hyaluronic acid exhibits excellent biocompatibility, can be degraded by hyaluronidase, and thus has good biocompatibility, helping to reduce the risk of material residue. Hyaluronic acid can also interact with receptors such as CD44. However, this invention primarily relies on folic acid-modified liposomes for targeting, further enhancing local tissue affinity. It has excellent film-forming properties and mechanical strength suitable for skin puncture, rapidly absorbing water and dissolving to release the liposomes after insertion. Furthermore, hyaluronic acid can also be associated with receptors such as CD44, which are expressed in various tumor cells and tumor microenvironments. Although this invention primarily relies on folic acid-modified liposomes for targeting, the hyaluronic acid matrix helps improve local biocompatibility and skin / tumor local delivery adaptability. Therefore, hyaluronic acid combines the advantages of a soluble microneedle-forming matrix, skin friendliness, biodegradability, and local delivery adaptability.

[0016] The phospholipid is PC-98, lecithin, hydrogenated lecithin, soybean lecithin, phosphatidylcholine or other pharmaceutically acceptable combinations, with PC-98 being the preferred phospholipid. PC-98 constructs a liposome bilayer framework, which enhances the encapsulation ability of hydrophobic triptolide.

[0017] The folic acid modification component is folic acid-modified polyethylene glycol phospholipid (DSPE-PEG2000-FA) or other polyethylene glycol-modified lipids capable of attaching folic acid to the liposome surface. The preferred folic acid modification component is DSPE-PEG2000-FA, which possesses a triple function of liposome membrane anchoring, PEG spacer stabilization, and folic acid receptor targeting. This allows folic acid ligands to be stably displayed on the liposome surface, thereby improving tumor cell uptake and local retention; it differs from ordinary lipids that only serve as structural membrane materials or membrane stabilizers.

[0018] Preferably, the liposome raw material ratio is 8 parts PC-98, 2 parts cholesterol, 1 part DSPE-PEG2000-FA, and 1 part triptolide.

[0019] The liposomes modified with folic acid for triptolide have a particle size of 80-150 nm, with a preferred particle size of 109.5 ± 1.9 nm. The zeta potential is -10 mV to -35 mV, with a preferred zeta potential of -21.37 ± 1.50 mV. The drug loading of triptolide in the liposomes can be 1%-8%, with a preferred drug loading of 3.1%. The encapsulation efficiency can be 35%-75%, with a preferred encapsulation efficiency of 56%. Within this range of drug loading and encapsulation efficiency, it is indicated that triptolide can be effectively encapsulated in the liposomes.

[0020] The molecular weight of hyaluronic acid in the soluble microneedle matrix can be 50-1000 kDa, preferably 200-400 kDa.

[0021] This invention also discloses a method for preparing hyaluronic acid-soluble microneedles, which involves preparing targeted liposomes through thin-film hydration → freeze-drying and concentration → mixing with high-concentration hyaluronic acid to prepare a needle tip solution → centrifugation and layering in a mold. The two-step centrifugation process achieves the layering of the drug-loaded layer and the blank backing layer at the needle tip, effectively preventing the diffusion and loss of liposomes. The entire process is gentle and does not damage the liposome nanostructure and folic acid targeted modification. The process is scalable and batch-stable.

[0022] The specific preparation steps are as follows: S10 Prepare liposome raw materials: Mix according to the following ratio: 8 mg of PC-98; 2 mg of cholesterol; 1 mg of DSPE-PEG2000-FA; 1 mg of triptolide; and 8 mL of organic solvent.

[0023] S20 liposome film formation: PC-98, cholesterol, DSPE-PEG2000-FA and triptolide are dissolved in dichloromethane / methanol, and the organic solvent is removed by rotary evaporation under reduced pressure at 40°C to form a liposome film.

[0024] S30 hydration and homogenization: The crude liposome suspension was obtained by rotating 1 mL of ultrapure water at 37 °C. Then, it was sonicated in an ice-water bath with a 150 W probe for 5 min, with the working mode being 3 seconds on and 2 seconds off. The product was filtered through a 0.45 μm polyethersulfone filter membrane to obtain folic acid-modified triptolide liposomes.

[0025] Preparation of S40 needle tip casting solution: The liposomes modified with folic acid were freeze-dried or concentrated and then redispersed to a redispersible concentration of 6 mg / mL. They were then mixed with an equal volume of 30% hyaluronic acid solution to a concentration of 3 mg / mL, thus obtaining the needle tip casting solution.

[0026] S50 microneedle shaping: S51 needle tip molding: 100μL of needle tip casting liquid was added to the PDMS negative mold, and the needle tip casting liquid was filled into the needle cavity by centrifugation at 3000rpm for 15 minutes to obtain the microneedle tip. S52 backing molding: After removing excess liquid, add 15% hyaluronic acid backing solution and continue centrifugation at 3000 rpm for 10 minutes to obtain microneedle backing. S60 Drying and Demolding: After drying the mold at 37℃ for 12 hours, the mold was naturally demolded to obtain hyaluronic acid soluble microneedles of folic acid-modified triptolide liposomes, which were then stored in the dark.

[0027] The PDMS female mold includes an 11×11 microneedle array, a needle cavity height of 800μm, a needle base of 330×330μm, a backing groove of 15×15mm, and a needle body height of 750μm after molding.

[0028] This invention also discloses the application of hyaluronic acid-soluble microneedles loaded with folic acid-targeting triptolide liposomes in the combined treatment of melanoma with anti-PD-L1 agents. These soluble microneedles can promote the increase of mature dendritic cell (DC)-associated cell populations within tumors and increase CD8+. + T cell infiltration increases intratumoral CD8. + T lymphocyte infiltration enhances the efficacy of anti-PD-L1 immunotherapy. Attached Figure Description

[0029] Figure 1A This is a schematic diagram of the microstructure of the folic acid-modified triptolide liposomes (hereinafter referred to as TP@FA-Lips) in Example 1.

[0030] Figure 1BThis is a flowchart of the preparation process of TP@FA-Lips in Example 1.

[0031] Figure 1C Particle size distribution diagrams of triptolide liposomes (hereinafter referred to as TP@Lips) and TP@FA-Lips.

[0032] Figure 1D The diagram shows the hydrodynamic diameter dimensions of TP@Lips and TP@FA-Lips.

[0033] Figure 1E Zeta potential plots for TP@Lips and TP@FA-Lips.

[0034] Figure 1F Transmission electron microscopy (TEM) image of the nanoscale morphology of TP@FA-Lips.

[0035] Figure 1G This is a graph showing the colloidal stability of TP@FA-Lips in 10% fetal bovine serum (FBS).

[0036] Figure 1H This is a graph showing the colloidal stability of TP@FA-Lips in phosphate-buffered saline (PBS).

[0037] Figure 1I The in vitro release curves of triptolide (hereinafter referred to as TP) from TP@FA-Lips under pH 6.0 and pH 7.4 conditions are shown.

[0038] Figure 1J This is a graph showing the cytotoxicity of TP preparations on B16F10 cells.

[0039] Figure 1K This is a graph showing the cytotoxicity of the blank vector / HA material to B16F10 cells.

[0040] Figure 2A This is a flow cytometry analysis of apoptosis in B16F10 cells after treatment with TP preparations or free TP.

[0041] Figure 2B This is a graph illustrating the quantitative analysis of late-stage apoptosis in melanoma cells.

[0042] Figure 2C Fluorescence microscopy images showing cellular uptake of C6-labeled formulations.

[0043] Figure 2D This is a semi-quantitative analysis diagram of intracellular fluorescence intensity based on ImageJ.

[0044] Figure 2E Flow cytometry histograms of C6 fluorescence intensity in different treatment groups.

[0045] Figure 2F This is a quantitative analysis graph of C6 fluorescence intensity in different treatment groups.

[0046] Figure 3A Fluorescence microscopy images of B16F10 cells after different treatments and CRT exposure.

[0047] Figure 3B The histogram of flow cytometry data for CRT-positive cells.

[0048] Figure 3C A flow cytometry graph showing the quantitative analysis of CRT-positive cells.

[0049] Figure 3D Fluorescence microscopy images showing the localization of HMGB1.

[0050] Figure 3E The flow cytometry histogram shows the fluorescence intensity associated with HMGB1.

[0051] Figure 3F This is a flow cytometry graph showing the quantitative analysis of HMGB1-related fluorescence intensity.

[0052] Figure 3G The graph shows the intracellular ATP concentration in B16F10 cells after different treatments.

[0053] Figure 3H A schematic diagram of the process for culturing BMDCs and stimulating them with conditioned medium containing treated B16F10 cells.

[0054] Figure 3I Flow cytometry atlas of mature BMDCs.

[0055] Figure 3J This is a flow cytometry quantitative analysis diagram of mature BMDCs.

[0056] Figure 4A Photographs of TP@FA-Lips / dMNs.

[0057] Figure 4B Images of microneedle arrays and individual microneedles obtained by scanning electron microscopy (SEM).

[0058] Figure 4C Cross-sectional fluorescence micrographs of TP@FA-Lips / dMNs.

[0059] Figure 4D Force-displacement curves of TP@FA-Lips / dMNs prepared with different hyaluronic acid (HA) concentrations.

[0060] Figure 4EPhotographs of the microneedle insertion and dissolution process on mouse skin (0 minutes and 15 minutes).

[0061] Figure 4F H&E staining images of mouse skin sections after microneedle insertion.

[0062] Figure 4G Cross-sectional images of TP@FA-Lips / dMNs before and after skin insertion / dissolution; Figure 4H The in vitro transdermal cumulative release curves of TP in TP@FA-Lips / dMNs and TP@Lips / dMNs are shown.

[0063] Figure 4I Fluorescence images of skin / tumor sections 1, 4, and 8 hours after administration of C6@FA-Lips / dMNs or C6@Lips / dMNs.

[0064] Figure 4J IVIS fluorescence imaging to show tumor retention at 2, 4, 8, 12 and 24 hours after local administration of DIR-labeled TP@FA-Lips / dMNs or TP@Lips / dMNs.

[0065] Figure 4K A quantitative analysis of fluorescence intensity in tumor slices labeled with C6 and radiation efficiency at the tumor site in in vivo imaging.

[0066] Figure 5A This is a schematic diagram of the treatment regimen for B16F10 tumor-bearing mice.

[0067] Figure 5B The change in body weight of mice after receiving the corresponding treatment.

[0068] Figure 5C Images of the tumor removed at the experimental endpoint.

[0069] Figure 5D This is the tumor growth curve during treatment.

[0070] Figure 5E A semi-quantitative analysis of the weight of the removed tumor, the endpoint tumor volume, and the TUNEL fluorescence intensity.

[0071] Figure 5F Representative images of H&E staining and TUNEL fluorescence staining of tumor sections.

[0072] Figure 6A This is a schematic diagram of the immune mechanism.

[0073] Figure 6B CD80 associated with mature dendritic cells (DCs) in tumors after different treatments + CD86 +Flow cytometry atlas of cell populations.

[0074] Figure 6C CD8 in tumors + Flow cytometry atlas of T cells.

[0075] Figure 6D Representative CRT and CD8 immunofluorescence images of tumor sections.

[0076] Figure 6E For mature DC-associated CD80 in tumors after different treatments + CD86 + Cell population, intratumoral CD8 + A quantitative analysis chart of T cells, CRT positivity, and CD8 positivity rates in tumor sections.

[0077] Figure 7A These are representative H&E staining images of the heart, liver, spleen, lung, and kidney tissues of tumor-bearing mice after different treatments.

[0078] Figure 7B A graph showing plasma ALT and AST levels used to assess liver function.

[0079] Figure 7C Charts showing plasma UREA and CREA levels used to assess renal function.

[0080] Figure 7D Charts of plasma CK and CK-MB levels (measured using an automated biochemical analyzer) used to assess indicators related to myocardial injury.

[0081] Figure 8A Transmission electron microscopy (TEM) images of TP@Lips.

[0082] Figure 8B The staining results of live / dead cells of B16F10 cells after different treatments.

[0083] Figure 8C This shows the skin recovery after microneedles were inserted into the mouse skin.

[0084] Figure 8D The absorbance spectra of hemolysed supernatants after incubation of red blood cells with PBS, ultrapure water, or TP@FA-Lips / dMNs (TP equivalent concentrations of 1 and 10 μg / mL, respectively).

[0085] Figure 8E This is a representative image of red blood cell suspension after an in vitro hemolysis experiment.

[0086] Figure 8F The quantitative hemolysis rate of TP@FA-Lips / dMNs (TP equivalent concentrations of 1 and 10 μg / mL, respectively).

[0087] Figure 8G The tumor inhibition rate is calculated based on the endpoint tumor volume of B16F10 melanoma-bearing mice.

[0088] Figure 8H Gating strategies for flow cytometry (FCM) analysis of intratumoral immune cells after different treatments. Detailed Implementation

[0089] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0090] Example 1: Hyaluronic acid-soluble microneedles (hereinafter referred to as TP@FA-Lips / dMNs) loaded with folic acid-modified triptolide liposomes were prepared. The process consisted of two steps: preparation of folic acid-modified triptolide liposomes (hereinafter referred to as TP@FA-Lips) and microneedle forming. The specific preparation steps are as follows. Microstructure of TP@FA-Lips is shown in the figure. Figure 1A For the preparation process, please refer to [link / reference]. Figure 1B .

[0091] S10 TP@FA-Lips Raw Material Preparation: Mix according to the following ratio: 8 mg PC-98; 2 mg cholesterol; 1 mg DSPE-PEG2000-FA; 1 mg triptolide; and 8 mL organic solvent.

[0092] S20 liposome film formation: PC-98, cholesterol, DSPE-PEG2000-FA and triptolide are dissolved in dichloromethane / methanol, and the organic solvent is removed by rotary evaporation under reduced pressure at 40°C to form a liposome film.

[0093] S30 hydration and homogenization: The crude liposome suspension was obtained by rotating 1 mL of ultrapure water at 37 °C. Then, it was sonicated in an ice-water bath with a 150 W probe for 5 min, with the working mode being 3 seconds on and 2 seconds off. The resulting product was filtered through a 0.45 μm polyethersulfone filter membrane to obtain TP@FA-Lips.

[0094] Preparation of S40 needle tip casting solution: TP@FA-Lips was freeze-dried or concentrated and then redispersed. The redispersed concentration of triptolide was 6 mg / mL. It was then mixed with an equal volume of 30% hyaluronic acid solution. The molecular weight of hyaluronic acid was selected to be 200-400 kDa. After mixing, the concentration of triptolide was 3 mg / mL, and the needle tip casting solution was obtained.

[0095] S50 microneedle molding and PDMS female mold selection: The PDMS female mold includes an 11×11 microneedle array, a needle cavity height of 800μm, a needle base of 330×330μm, and a backing groove of 15×15mm.

[0096] S51 needle tip forming: 100μL of needle tip casting liquid is added to the PDMS female mold, and the needle tip casting liquid is filled into the needle cavity by centrifugation at 3000rpm for 15 minutes to obtain the microneedle tip with a needle height of 750μm.

[0097] S52 backing molding: After removing excess liquid, add 15% hyaluronic acid backing solution and continue centrifugation at 3000 rpm for 10 minutes to obtain microneedle backing.

[0098] S60 Drying and Demolding: After drying the mold at 37℃ for 12 hours, the mold was naturally demolded to obtain hyaluronic acid soluble microneedles of folic acid-modified triptolide liposomes, which were then stored in the dark.

[0099] The TP@FA-Lips prepared by the above S10-30 process have the following characteristics: the TP@FA-Lips particle size is 109.5 nm, the zeta potential is -21.37 mV, the drug loading of triptolide is 3.1%, and the encapsulation efficiency is 56%.

[0100] Example 2: In vitro characterization and cell experiments of TP@FA-Lips Laboratory animals and their handling methods: Female C57BL / 6J mice (6 weeks old, 18-22g) were purchased from Shanghai Silex Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed in a specific pathogen-free (SPF) environment at a controlled temperature of 25±1℃, relative humidity of 50±5%, and a 12-hour light / dark cycle, with free access to food and water. During the experiment, the mice's general condition, activity level, weight, and tumor burden were closely monitored, and humane endpoint treatment was performed according to the approved protocol. At the endpoint sample collection, mice were euthanized first by inhaling CO2, followed by cervical dislocation to confirm death. Specifically, CO2 was continuously inhaled until the mouse's breathing stopped and maintained for more than 1 minute, followed by cervical dislocation as a secondary confirmation of death, and finally, tissue was collected.

[0101] Preparation of TP@FA-Lips: TP@FA-Lips were prepared using a thin-film hydration method, following steps S10, S20, and S30 of Example 1. In short, PC-98 (8 mg), cholesterol (2 mg), DSPE-PEG2000-FA (1 mg), and TP (1 mg) were dissolved in 8 mL of a dichloromethane / methanol (1:1 volume ratio) mixture in a round-bottom flask. The mixture was sonicated until completely dissolved, and then the solvent was evaporated under reduced pressure at 40°C to form a uniform lipid film. At 37°C, 1 mL of ultrapure water was added, and the film was hydrated by rotation to obtain a crude liposome suspension. The suspension was sonicated in an ice-water bath using a probe-type sonicator. The product was filtered through a 0.45 μm polyethersulfone (PES) membrane to remove large particles, and the collected filtrate was the TP@FA-Lips. Blank FA-Lips without TP were prepared using the same procedure. The method for preparing non-targeted TP@Lips is to replace DSPE-PEG2000-FA with DSPE-PEG2000.

[0102] Characterization of TP@FA-Lips: The particle size distributions of TP@Lips and TP@FA-Lips were determined using a NanoZS90 instrument at 25°C via dynamic light scattering (DLS) technology (see [reference]). Figure 1C ), fluid dynamic diameter dimensions (see Figure 1D ) and Zeta potential (see Figure 1E (All samples were diluted with ultrapure water first). The morphology of liposomes was observed using transmission electron microscopy. The specific procedure was as follows: 5 μL of the diluted liposome suspension was dropped onto a carbon-coated copper grid, allowed to stand for 2 minutes for adsorption, and then excess liquid was removed with filter paper. The samples were negatively stained with 2% phosphotungstic acid (30 seconds), washed twice with PBS, dried at room temperature, and then imaged using transmission electron microscopy. The results are shown in [link to results]. Figure 1F To assess colloidal stability, TP@FA-Lips were dispersed in a solution containing 10% fetal bovine serum (FBS, see below). Figure 1G ) PBS solution or PBS (pH 7.4, see Figure 1H The samples were placed in a container and stored at 4°C. Particle size and PDI were measured at predetermined time points within 22 days.

[0103] TP content was determined by high-performance liquid chromatography (HPLC). An ODS-3C18 column (5 μm, 4.6 × 250 mm) was used, with acetonitrile / water (40:60, v / v) as the mobile phase and a flow rate of 1.0 mL / min. The detection wavelength was 218 nm, the column temperature was 25 °C, and the injection volume was 20 μL. A standard curve for TP in the range of 0.2–25 μg / mL was established. Liposome samples were diluted 20-fold, filtered through a 0.45 μm polyethersulfone membrane, and then analyzed by HPLC. The in vitro release behavior of TP from TP@FA-Lips was evaluated using dialysis. 1 mL of TP@FA-Lips suspension (containing 0.3 ± 0.025 mg TP) was placed in a dialysis bag, sealed, and immersed in 30 mL of PBS (pH 7.4 or pH 6.0) preheated to the experimental temperature. The release system was incubated at 37 °C with horizontal shaking at 100 rpm. Take 1 mL of the release medium at the predetermined time point and replenish with an equal volume of fresh medium. Filter the sample through a 0.45 μm filter membrane and perform HPLC analysis (see [link to HPLC analysis]). Figure 1I All release experiments were conducted in triplicate.

[0104] In vitro cytotoxicity and apoptosis: The cytotoxicity of the TP formulation was assessed using the MTT assay. B16F10 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured overnight. The medium was then replaced with complete medium containing FA-Lips, TP@Lips, TP@FA-Lips, or free TP (pre-set concentration). After 48 hours, the medium was removed, and MTT solution (0.5 mg / mL) was added and incubated for 4 hours. Formazan crystals were dissolved in 100 μL LDMSO, and the absorbance was measured at 570 nm using a microplate reader.

[0105] Using the same MTT assay, the cell compatibility of HA was investigated in the range of HA concentrations from 0.001 to 100 μg / mL.

[0106] The MTT assay results above showed that TP@FA-Lips retained the concentration-dependent inhibitory effect of TP on B16F10 cells. Related cytotoxicity results can be found in [link to relevant data]. Figure 1J No significant cytotoxicity was observed in blank FA-Lips and HA materials within the tested concentration range; relevant cell compatibility results can be found in [link to relevant documentation]. Figure 1K .

[0107] Live / dead cell staining was performed using a Calcein / PI cell viability assay kit. B16F10 cells were cultured at 6 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 12-well plates and cultured in complete culture medium for 12 hours. Subsequently, cells were incubated for 24 hours with TP@Lips, TP@FA-Lips, or free TP solution (TP equivalent concentration 0.02 μg / mL). After treatment, cells were washed with PBS and stained with Calcein-AM (0.8 μL / mL) and PI (0.4 μL / mL) for 30 minutes at room temperature in the dark. Fluorescence images of live and dead cells were captured using an EVOS M5000 fluorescence microscope.

[0108] Apoptosis was analyzed using Annexin V-FITC / PI staining. B16F10 cells were cultured at 6 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 cells / mL in 12-well plates (1 mL cell suspension per well) and cultured overnight. Subsequently, cells were treated with free TP, TP@Lips, or TP@FA-Lips (TP equivalent concentration of 0.02 μg / mL) for 24 hours; control cells were replaced with fresh complete culture medium. Cells were collected, washed twice with cold PBS, resuspended in 100 μL of 1× binding buffer, and 5 μL of Annexin V-FITC and 10 μL of LPI were added. Staining was performed for 15 minutes at room temperature in the dark. After adding 400 μL of binding buffer, the samples were immediately analyzed by flow cytometry. Data processing was performed using FlowJo software. Figure 2A The flow cytometry analysis of apoptosis is shown; for quantitative analysis results of late-stage apoptosis in melanoma cells, please refer to [link to relevant documentation]. Figure 2B .

[0109] Cellular uptake experiment: Cellular uptake was assessed using C6-labeled liposomes. B16F10 cells in logarithmic growth phase were loaded at 2 × 10⁻⁶ cells per cell line. 6 Cells were seeded at a density of 2 mL of cell suspension per well in 6-well plates and cultured overnight. Cells were divided into control, C6@Lips, C6@FA-Lips, FA+C6@FA-Lips, and free-C6 groups. For the treatment groups, the final concentration of C6 was 0.02 μg / mL. In the FA blocking groups, cells were pre-incubated for 2 hours in medium containing free FA (final FA concentration 0.02 μg / mL) before receiving C6@FA-Lips treatment. After 2 hours of incubation, cells were washed three times with PBS and stained with Hoechst 33342 for 10 minutes. Cell fluorescence was observed using a fluorescence microscope (IX73, Olympus, Japan). See [link to relevant documentation]. Figure 2C The images were then semi-quantitatively analyzed using ImageJ software; see [link / reference]. Figure 2D .

[0110] For flow cytometry analysis, cells were processed as described above, digested with 0.25% trypsin, and collected by centrifugation at 1000 rpm at 4°C. The cell pellet was washed twice with cold PBS, resuspended in 500 μL PBS, and analyzed by flow cytometry within 30 minutes. Data were processed using FlowJo software; see [link to FlowJo analysis]. Figure 2E and Figure 2F .

[0111] In vitro ICD biomarker analysis and evaluation: ICD-related signals of B16F10 cells after TP treatment. Cell seeding and treatment methods were the same as in apoptosis experiments. For intracellular ATP assays, cells were collected by centrifugation at 10,000 rpm at 4°C 24 hours after treatment. Following the manufacturer's instructions, the ATP luminescence signal in the collected cell samples was measured using an ATP assay kit and expressed as intracellular ATP concentration. In short, 100 μL of ATP assay working solution was added to each well, and the mixture was allowed to equilibrate for 3 minutes to reduce background signal. Subsequently, 100 μL of sample was added, rapidly mixed, and the assay was performed within 30 minutes using a chemiluminescent microplate reader.

[0112] For CRT exposure assays, cells were washed twice with PBS 24 hours after treatment and blocked for 30 minutes at room temperature with a 1:1000 dilution of anti-mouse CD16 / 32Fc blocking solution. Cells were incubated with a 1:800 dilution of anti-CRT primary antibody for 1 hour, washed with PBS, and then incubated with AF488-labeled secondary antibody in the dark for 30 minutes. Cell nuclei were stained with Hoechst 33342 (10 minutes). CRT exposure was observed using a fluorescence microscope; see [link to relevant documentation]. Figure 3A For quantitative analysis by flow cytometry, collect the treated cells, wash twice with cold PBS, process them according to the antibody staining procedure described above, and then perform flow cytometry analysis. See [link to relevant documentation]. Figure 3B and Figure 3C .

[0113] For HMGB1 release assay, treated cells were fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.1% Triton X-100 at room temperature for 20 minutes. Cells were blocked with 1:1000 dilution of anti-mouse CD16 / 32Fc blocking buffer for 30 minutes and incubated with 1:800 dilution of anti-HMGB1 primary antibody for 1 hour. After washing with PBS, cells were incubated with AF488-labeled secondary antibody in the dark for 30 minutes and stained with Hoechst 33342 for 10 minutes. The localization of HMGB1 was observed using fluorescence microscopy; see [link to relevant documentation]. Figure 3D And quantitative analysis was performed by flow cytometry, see [link to relevant documentation] Figure 3E and Figure 3F For quantitative results on intracellular ATP concentration, please refer to [link / reference needed]. Figure 3G .

[0114] BMDC Maturation Experiment: To assess whether treated tumor cells promoted DC maturation, B16F10 cells were seeded and treated with free TP, TP@Lips, or TP@FA-Lips for 24 hours, respectively. The culture medium used during treatment was RPMI 1640 complete medium containing GM-CSF and IL-4. Tumor cell supernatant was collected and co-incubated with immature BMDCs for 24 hours. BMDCs were collected using a cell scraper, blocked with anti-mouse CD16 / 32Fc inhibitor (1:1000 dilution) at room temperature for 30 minutes, followed by centrifugation at 300g for 5 minutes. Cells were stained at 4°C in the dark with FITC-labeled anti-CD11c, APC-labeled anti-CD80, and PE-labeled anti-CD86 antibodies (all 1:1000 dilution) for 30 minutes. For detailed procedures, please refer to [link to relevant documentation]. Figure 3H After centrifugation and washing with PBS, the cells were resuspended in 500 μL PBS, filtered into flow cytometry tubes, and analyzed by flow cytometry. (See [link to relevant documentation]). Figure 3I and Figure 3J Mature BMDC is defined as CD11c. + CD80 + CD86 + cell.

[0115] Example 3: In vitro characterization and animal experiments of TP@FA-Lips / dMNs Preparation of TP@FA-Lips / dMNs: TP@FA-Lips / dMNs were prepared using micromolding and centrifugation, following steps S40, S50, S51, S52, and S60 in Example 1. A PDMS negative mold containing an 11×11 microneedle array with a needle cavity height of 800 μm, a substrate size of 330×330 μm, and a backing groove size of 15×15 mm was used. HA solutions with concentrations of 30% (w / v) and 15% (w / v) were prepared by dissolving HA powder in deionized water, followed by swelling overnight at room temperature.

[0116] TP@FA-Lips were freeze-dried and redispersed to achieve a TP concentration of 6 mg / mL. This suspension was mixed with 30% HA solution at a 1:1 volume ratio to obtain a casting solution with a TP concentration of 3 mg / mL. 100 μL of the casting solution was placed on a PDMS mold and centrifuged at 3000 rpm for 15 minutes at room temperature to fill the needle cavity. Excess liquid was removed from the mold surface. Subsequently, 15% HA solution was added as a backing layer, and the mixture was centrifuged at 3000 rpm for 10 minutes. The mold was dried at 37°C for 12 hours. The dried patch was then peeled off the mold to obtain TP@FA-Lips / dMNs. (See [link to relevant documentation]). Figure 4A and Figure 4B Store the patch in a dry and light-protected environment below 28°C.

[0117] Characterization of TP@FA-Lips / dMNs: The morphology of the microneedles was observed using a scanning electron microscope (JSM6390, JEOL Ltd., Japan). To visually demonstrate the load distribution, C6@FA-Lips were prepared using the same liposome preparation method, with 0.1% (w / v) Nile red added to the backing layer. The distribution of C6-labeled liposomes at the needle tip and the distribution of Nile red in the backing layer were evaluated by acquiring fluorescence images. (See [link to relevant documentation]). Figure 4C .

[0118] The TP loading in microneedles was determined using high-performance liquid chromatography (HPLC). One microneedle patch was completely dissolved in 1 mL of ultrapure water and centrifuged at 12000 rpm for 5 minutes. The supernatant was collected, filtered through a 0.45 μm polyethersulfone (PES) membrane, and analyzed using the HPLC method described above. Three independently prepared batches of samples were analyzed.

[0119] The mechanical strength of microneedles was evaluated using an electronic universal testing machine. TP@FA-Lips loaded microneedles with HA concentrations of 10%, 15%, and 20% (w / v), as well as blank HA microneedles, were prepared. The microneedle patch was fixed with the needle tip facing upwards, and the initial distance between the sensor and the needle tip was set to 2 mm. The sensor vertically compressed the microneedle at a speed of 0.01 mm / s, and force-displacement data were recorded at a sampling rate of 200 points / second. Three patches were tested in each group, and three microneedles at different locations on each patch were measured. (See [reference needed]). Figure 4D .

[0120] The puncture performance of microneedles was evaluated using dorsal skin from hairless female C57BL / 6 mice. TP@FA-Lips / dMNs were applied vertically to the skin for 15 minutes. (See [link to relevant documentation]). Figure 4ESkin tissue from the puncture site was collected, fixed in 4% paraformaldehyde solution at 4°C for 24 hours, then embedded in paraffin, sectioned at 4 μm, and stained with H&E. Skin microchannels were observed using an optical microscope; see [reference needed]. Figure 4F .

[0121] In the solubility test, TP@FA-Lips / dMNs were pressed onto the skin of the back of hairless mice and removed at 0 or 15 minutes after application. The patches were dried at room temperature, cut into single-row strips, and observed under a stereomicroscope. See [link to relevant documentation]. Figure 4G In the skin recovery test, TP@FA-Lips / dMNs were applied for 15 minutes and then removed. Skin images were taken at 0 hours and 1 hour after patch removal.

[0122] In vitro transdermal delivery performance was evaluated using a Franz diffusion cell. Fresh mouse skin was harvested, subcutaneous fat and connective tissue were removed, and the skin was fixed between the donor and recipient compartments of the diffusion cell, with the stratum corneum facing the donor compartment. TP@FA-Lips / dMNs were pressed onto the skin for 5 minutes and secured with breathable medical tape. The recipient compartment contained 18 mL of PBS (pH 7.4), maintained at 37°C, and magnetically stirred at 100 rpm. 1 mL of the recipient solution was sampled at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours, and an equal volume of preheated PBS was added. The samples were filtered through a 0.45 μm filter and analyzed by HPLC. The cumulative transdermal release curve of TP is shown in [reference needed]. Figure 4H Each experiment was repeated three times.

[0123] Hemolysis was assessed using erythrocytes from 6-week-old female mice. Blood (2 mL) was collected via cardiac puncture after euthanasia and diluted with 30 mL PBS. Erythrocytes were collected by centrifugation at 3000 rpm for 5 minutes and washed repeatedly with PBS until the supernatant was almost colorless. The erythrocytes were then resuspended in 6 mL PBS to prepare an erythrocyte suspension. The samples were co-incubated with PBS (negative control), ultrapure water (positive control), or microneedle solutions with equivalent concentrations of 1 and 10 μg / mL TP, respectively. After incubation at 37°C for 2 hours, the samples were centrifuged at 3000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate was calculated as follows: Hemolysis rate (%) = (OD sample - OD negative) / (OD positive - OD negative) × 100 Tumor retention and intratumoral infiltration: Tumor retention and penetration after local microneedle administration were evaluated in mice bearing B16F10 tumors. This model was established by subcutaneous injection of B16F10 cells into the right back near the hip region of mice. The cells were resuspended in cold PBS (concentration 2 × 10⁻⁶). 6Each mouse was injected with 0.1 mL of cell suspension (cells / mL). When the tumor volume reached approximately 100-150 mm², the tumor was treated. 3 At that time, mice were randomly grouped for fluorescence imaging.

[0124] Regarding intratumoral penetration, C6@FA-Lips / dMNs and C6@Lips / dMNs fluorescent microneedles were prepared and applied as described above. Mice were sacrificed 1, 4, and 8 hours after administration, following the experimental animal and treatment methods described in Example 2, and skin and tumor tissue were collected from the administration site. Samples were embedded in OCT embedding medium and sectioned to a thickness of 20 μm using a cryostat at -20°C. Sections were fixed with 4% paraformaldehyde for 5 minutes, washed with PBS, mounted with anti-quenching mounting medium, and observed under a fluorescence microscope (see [reference]). Figure 4I .

[0125] Regarding tumor retention, DIR@FA-Lips / dMNs and DIR@Lips / dMNs were prepared by replacing TP with DIR. Fluorescent microneedles were applied to hair-removed tumor areas, pressed for 2 minutes, and fixed with pressure-sensitive tape for 12 hours. Fluorescence images were acquired using an IVIS imaging system at 2, 4, 8, 12, and 24 hours post-administration, with an excitation wavelength of 748 nm, an emission wavelength of 780 nm, and an exposure time of 2 seconds. For tumor retention details, see [link to IVIS imaging]. Figure 4J .

[0126] Quantitative analysis was performed on the above results regarding intratumoral penetration and tumor retention. Quantitative results on the fluorescence intensity of tumor sections in the C6-labeled group and the radiation efficiency of the tumor site in in vivo imaging of the DIR-labeled group are provided below. Figure 4K .

[0127] In vivo anti-tumor efficacy: A B16F10 tumor-bearing mouse model was established using the method described above. When the tumor volume reached approximately 50 mm²... 3Mice were randomly divided into 7 groups (n=5 per group): Model group, Blank-dMNs group, Free-TP group, Anti-PD-L1 group, TP@Lips / dMNs group, TP@FA-Lips / dMNs group, and TP@FA-Lips / dMNs + Anti-PD-L1 combination therapy group. The Model group received no treatment. The Blank-dMNs group received one Blank-dMNs patch. The Free-TP group received TP via gavage (720 μg / kg). The Anti-PD-L1 group received anti-PD-L1 via intraperitoneal injection (5 mg / kg). The TP@Lips / dMNs and TP@FA-Lips / dMNs groups received one corresponding Microneedle patch per treatment. The combination therapy group received one TP@FA-Lips / dMNs patch and an injection of 5 mg / kg Anti-PD-L1. For microneedle drug delivery, place the patch on the hairless tumor area, press for 2 minutes, and fix it with pressure-sensitive tape for 12 hours.

[0128] Free TP and microneedle treatment were administered on days 1, 3, 5, 7, and 9. Anti-PD-L1 was administered on days 1, 4, 7, and 10. Mouse body weight and tumor volume were measured every two days. On day 11, mice were euthanized according to the experimental animal and treatment methods described in Example 2, tumors were excised, and weighed and measured. The tumor growth inhibition rate was calculated based on the endpoint tumor volume, and the combined tumor inhibition rate of PD-L1 and soluble microneedle treatment was experimentally calculated to be 75.06%.

[0129] For tumor histological analysis, two mice were randomly selected from each group at the experimental endpoint. Tumors were excised, washed with cold PBS, fixed in 4% paraformaldehyde at 4°C for 24 hours, and then embedded in paraffin, sectioned at 4 μm, and stained with H&E. Paraffin sections were dewaxed, hydrated, subjected to antigen retrieval with citrate buffer (pH 6.0), and permeabilized with proteinase K (20 μg / mL, 37°C, 20 min) before fluorescent TUNEL staining. Sections were incubated with TdT reaction solution at 37°C for 60 min, stained with Hoechst 33342, mounted with anti-quenching mounting medium, and imaged using a fluorescence microscope.

[0130] See the schematic diagram of the treatment regimen in the above in vivo anti-tumor experiment. Figure 5A See [reference needed] for mouse weight changes during treatment. Figure 5B The image of the tumor removed at the experimental endpoint can be found in [link to image]. Figure 5C Tumor growth curve during treatment (see attached image). Figure 5D Semi-quantitative analysis of resected tumor weight, endpoint tumor volume, and TUNEL fluorescence intensity is available in [reference needed]. Figure 5E Representative images of H&E staining and TUNEL fluorescence staining of tumor sections can be found in [link to image]. Figure 5F The above results indicate that local administration of TP@FA-Lips / dMNs can enhance the anti-tumor effect of TP at the tumor site and strengthen the anti-PD-L1 therapeutic effect.

[0131] Analysis of intratumor immune cells: Tumor sections were subjected to CRT and CD8 immunofluorescence staining. After dewaxing, hydration, antigen retrieval, and serum blocking, the sections were incubated overnight at 4°C with either anti-CRT antibody (1:800) or anti-CD8 antibody (1:2000). After washing with PBS, the sections were incubated with fluorescent secondary antibody at room temperature in the dark for 1 hour. Cell nuclei were stained using Hoechst 33342, and the sections were mounted with anti-quenching mounting medium. Fluorescence images were acquired using a fluorescence microscope.

[0132] Intratumoral immune cells were analyzed by flow cytometry. Tumor-bearing mice were treated as described above, and three mice from each group were selected for immunoassay on day 8 after treatment. Tumors were excised and cut into small pieces, placed in serum-free DMEM medium, and mechanically dissociated in 6-well plates. The cell suspension was filtered through a 40 μm cell sieve and centrifuged at 300 g for 5 minutes. Cells were resuspended in PBS and counted. For each sample, 1 × 10⁶ cells were collected. 6 Cells were blocked for 30 minutes with an anti-mouse CD16 / 32Fc blocker (Fcblock), followed by centrifugation at 300g for 5 minutes. For analysis of mature dendritic cells (DCs) within the tumor, cells were placed in 100μL of staining solution, and PC5.5-anti-CD45, APC-anti-CD80, and PE-anti-CD86 antibodies were added, respectively. The cells were incubated at 4°C in the dark for 30 minutes. During analysis, the cell population was delineated to include viable CD45 cells. + CD80 + CD86 + Cells. For T cell analysis, cells were stained with PC5.5-anti-CD45, FITC-anti-CD3, APC-anti-CD4, and PE-anti-CD8 antibodies under the same conditions, and CD8 antibodies were also used. + T cells were delimited to live CD45 cells. + CD3 + CD8 + Cells. After staining, cells were centrifuged at 500g for 5 minutes, washed twice with PBS, incubated with 5 μL DAI for 5 minutes, filtered, and transferred to flow cytometry tubes for analysis. Data were processed using FlowJo software.

[0133] See the results of tumor immune mechanism and immune cell detection. Figures 6A to 6ESpecifically, a schematic diagram illustrating the mechanism by which TP@FA-Lips / dMNs, in combination with anti-PD-L1, induce an anti-tumor immune response can be found in [link to diagram]. Figure 6A Mature DC-associated CD80 in tumors after different treatments + CD86 + See the flow cytometry atlas for cell populations. Figure 6B Intratumoral CD8 + Flow cytometry atlas of T cells can be found in [link to atlas]. Figure 6C Representative images of tumor sections obtained via CRT and CD8 immunofluorescence are shown below. Figure 6D Mature DC-related CD80 + CD86 + Cell population, intratumoral CD8 + Quantitative analysis of T cell, CRT positivity rate, and CD8 positivity rate can be found in [link to relevant documentation]. Figure 6E .

[0134] Biosafety assessment: Biocompatibility was assessed by monitoring body weight, performing histological analysis of major organs, and detecting plasma biochemical parameters. Body weight was recorded every two days during treatment. At the end of the experiment, two mice were randomly selected from each group and euthanized according to the experimental animal and treatment methods described in Example 2. Heart, liver, spleen, lung, and kidney tissues were collected and fixed, embedded, sectioned, and H&E stained as described above (see [link to relevant documentation]). Figure 7A .

[0135] At the end of the experiment, whole blood samples were collected from mice in each group and centrifuged at 10,000 rpm for 5 minutes at 4°C to obtain plasma. Plasma biochemical parameters were measured using a fully automated biochemical analyzer (F20, Shenzhen Raydu Life Science Co., Ltd., China). Liver function was assessed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), see [link to relevant documentation]. Figure 7B Kidney function is assessed using urea (UREA) and creatinine (CREA), see [link to relevant documentation]. Figure 7C Cardiac injury-related markers include creatine kinase (CK) and creatine kinase isoenzyme MB (CK-MB). See [link to relevant documentation]. Figure 7D .

[0136] Example 4: Supplementary Characterization and Validation Results To further illustrate the technical effects of the above embodiments, supplementary characterization and verification were performed according to the methods described in Embodiments 2 and 3. For the transmission electron microscopy morphology of the non-targeted TP@Lips, please refer to... Figure 8A The results of live / dead cell staining of B16F10 cells after different treatments are shown in the attached table. Figure 8B For skin recovery after microneedle insertion into mice, please refer to [link / reference]. Figure 8CThe absorbance spectra, representative images, and quantitative hemolysis rates of the hemolysin supernatant after incubation of red blood cells with PBS, ultrapure water, or TP@FA-Lips / dMNs solution are shown in the attached images. Figure 8D , Figure 8E and Figure 8F For tumor inhibition rate calculated based on endpoint tumor volume, please refer to [link / reference]. Figure 8G For flow cytometry gating strategies for analyzing intratumoral immune cells after different treatments, please refer to [link to relevant documentation]. Figure 8H The above supplementary results corroborate the foregoing embodiments, illustrating the morphological characteristics, local drug delivery safety, antitumor effect, and immunoassay method of the microneedle delivery system of the present invention.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A soluble microneedle, comprising a needle tip with a matrix and a backing, characterized in that: The matrix is ​​made of water-soluble biopolymer material, and the needle tip contains liposomes of triptolide modified with folic acid. The liposomes include triptolide, phospholipids, cholesterol and folic acid modified components.

2. The soluble microneedles according to claim 1, characterized in that: The water-soluble biopolymer material of the soluble microneedle matrix is ​​selected from any one or a combination of two or more of the following: hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, sodium carboxymethyl cellulose, gelatin, dextran, and alginate. The phospholipids are PC-98, lecithin, hydrogenated lecithin, soybean lecithin, phosphatidylcholine, or other pharmaceutically acceptable combinations. The folic acid modification component is DSPE-PEG2000-FA or other polyethylene glycol-modified lipids that can link folic acid to the surface of liposomes.

3. The soluble microneedles according to claim 2, characterized in that: The water-soluble biopolymer material of the soluble microneedle matrix uses hyaluronic acid, phospholipid PC-98, and folic acid modification component DSPE-PEG2000-FA.

4. The soluble microneedles according to claim 3, characterized in that: The liposome raw material ratio is 8 parts PC-98, 2 parts cholesterol, 1 part DSPE-PEG2000-FA, and 1 part triptolide.

5. The soluble microneedles according to claim 3, characterized in that: The particle size of folic acid-modified triptolide liposomes is 80-150 nm, and the zeta potential is -10 mV to -35 mV; the drug loading of triptolide in the liposomes can be 1%-8%, and the encapsulation efficiency can be 35%-75%; the molecular weight of hyaluronic acid in the soluble microneedle matrix can be 50-1000 kDa.

6. The soluble microneedle according to claim 5, characterized in that: The liposomes modified with folic acid for triptolide have a particle size of 109.5 nm and a zeta potential of -21.37 mV; the drug loading of triptolide in the liposomes is 3.1%, and the encapsulation efficiency can be 56%; the molecular weight of hyaluronic acid in the soluble microneedle matrix can be 200-400 kDa.

7. A method for preparing hyaluronic acid soluble microneedles according to any one of claims 3, characterized in that... Includes the following steps: S10 Prepare liposome raw materials: Mix according to the following ratio: 8mg PC-98, 2mg cholesterol, 1mg DSPE-PEG2000-FA, 1mg triptolide and 8mL organic solvent. S20 liposome film formation: PC-98, cholesterol, DSPE-PEG2000-FA and triptolide are dissolved in dichloromethane / methanol, and the organic solvent is removed by rotary evaporation under reduced pressure at 40°C to form a liposome film; S30 hydration and homogenization: 1 mL of ultrapure water was used for rotary hydration at 37 °C to obtain a crude liposome suspension. Then, the suspension was sonicated in an ice-water bath with a 150 W probe for 5 min, with the working mode being 3 seconds on and 2 seconds off. The resulting product was filtered through a 0.45 μm polyethersulfone filter membrane to obtain folic acid-modified triptolide liposomes. Preparation of S40 needle tip casting solution: The liposomes modified with folic acid were freeze-dried or concentrated and then redispersed. The redispersed concentration of triptolide was 6 mg / mL. It was then mixed with an equal volume of 30% hyaluronic acid solution. After mixing, the concentration of triptolide was 3 mg / mL, and the needle tip casting solution was obtained. S50 microneedle shaping: S51 needle tip molding: 100μL of needle tip casting liquid was added to the PDMS negative mold, and the needle tip casting liquid was filled into the needle cavity by centrifugation at 3000rpm for 15 minutes to obtain the microneedle tip. S52 backing molding: After removing excess liquid, add 15% hyaluronic acid backing solution and continue centrifugation at 3000 rpm for 10 minutes to obtain microneedle backing. S60 Drying and Demolding: After drying the mold at 37℃ for 12 hours, the mold was naturally demolded to obtain hyaluronic acid soluble microneedles of folic acid-modified triptolide liposomes, which were then stored in the dark.

8. The method for preparing hyaluronic acid-soluble microneedles according to claim 6, characterized in that: The PDMS female mold includes an 11×11 microneedle array, a needle cavity height of 800μm, a needle base of 330×330μm, a backing groove of 15×15mm, and a needle body height of 750μm after molding.

9. The use of the soluble microneedles according to any one of claims 1-6 in combination with anti-PD-L1 agents for the treatment of melanoma.

10. The application of the soluble microneedles according to claim 9 in combination with anti-PD-L1 agents for the treatment of melanoma, characterized in that: The soluble microneedles can promote the maturation of dendritic cells (DCs) at the tumor site and increase intratumoral CD8. + T lymphocyte infiltration enhances the efficacy of anti-PD-L1 immunotherapy.

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