Preparation method and application of IP10-fusion cell vesicle microneedle vaccine with T cell activation function

Abstract

The invention discloses a preparation method and application of an IP10-fusion cell vesicle microneedle vaccine with a T-cell activation function, IP10-fusion cell vesicles with the T-cell activation function are prepared into the microneedle vaccine, on the immune activation level, the IP10-fusion cell vesicles have a vesicle antigen presentation function through the chemotactic effect of IP10, and the T-cell activation function of the IP10-fusion cell vesicles is achieved. T cell-mediated anti-tumor immune response can be efficiently excited, and a new path is provided for solving the problem of tumor immune escape; in the aspect of a delivery system, the minimally invasive slow release characteristic of the microneedle not only reduces the acceptance resistance of a patient, but also can maintain lasting immunostimulation, and compared with a traditional injection mode, the compliance and the treatment effect of the vaccine are remarkably improved. In addition, good biocompatibility ensures the safety of clinical application, and has great transformation potential in the field of tumor prevention and treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor vaccines, and particularly relates to a preparation method of an IP10-fused cell vesicle microneedle vaccine with T cell activation function. BACKGROUND

[0002] Vaccination as an efficient and economical prevention strategy has attracted widespread attention in the field of tumor prevention and treatment. However, current breast cancer vaccine research is mostly focused on therapeutic aspects, and the development of preventive vaccines is relatively slow. Among the many tumor vaccine research directions, dendritic cells (DCs) have become a research hotspot due to their strong antigen presentation capacity and immune activation function. The emergence of DC vaccines has opened up new ideas for breast cancer treatment, but the immunosuppressive effect in the tumor microenvironment greatly limits its therapeutic effect. Although DC / tumor fusion cell vaccines can integrate multiple tumor antigens and effectively overcome the immune escape problem of tumor cells, their high cost and low yield make them difficult to be used in large-scale clinical applications. With the deepening of research, extracellular vesicles (EVs) as an emerging carrier have brought new hope for the development of tumor vaccines. EVs have natural biological properties and show great potential in drug delivery and immune activation. Compared with traditional fusion cell vaccines, EV vaccines not only have higher yield and stronger safety, but also have nanoscale size, which makes them more easily penetrate tissues and accumulate in lymph nodes, providing a new direction for the development of breast cancer vaccines. Although EVs show great potential in the development of breast cancer vaccines, their immune stimulation activity is insufficient when used alone, which limits the overall efficacy of the vaccine. Therefore, the rational selection of adjuvants is crucial for stimulating and regulating immune responses. Interferon-inducible protein-10 (IP10) is a cytokine with unique biological activity, which can effectively recruit and activate immune cells, enhance the function of antigen-presenting cells, and thus improve the immune response level of the body to tumor antigens. Therefore, we choose IP10 as an adjuvant, hoping to optimize the immune activation mechanism of the vaccine and enhance the immune effect of the vaccine through the synergistic effect of IP10 and EVs. In terms of vaccine delivery, traditional subcutaneous injection has the disadvantages of high infection risk and poor patient compliance, while some emerging delivery technologies are difficult to be popularized in clinics due to high cost. The emergence of microneedle (MN) technology brings new hope for vaccine delivery. Microneedles are minimally invasive and painless, and have low cost, and can effectively enhance the immunogenicity of vaccines. Preparing vesicle vaccines into hydrogel microneedles for subcutaneous vaccination can not only achieve sustained release of drugs and continuously enhance immune effect, but also highly match the immune characteristics of breast tissue. This innovative delivery method is expected to bring major breakthroughs to the research and development of preventive vaccines for breast cancer, and has immeasurable value and broad prospects in both theoretical research and clinical application. SUMMARY

[0003] Therefore, the purpose of the present application is to improve the preparation method of IP10-fused cell vesicle microneedle vaccine with T cell activation function.

[0004] The specific technical scheme adopted by the present application is as follows: The preparation method of IP10-fused cell vesicle microneedle vaccine with T cell activation function comprises the following steps: (1) isolating and culturing DC from bone marrow to obtain BMDC cell suspension; (2) transfecting tumor cells with IP10 plasmid to obtain 4T1@IP10 cell suspension; (3) precipitating BMDC cells and 4T1@IP10 cells respectively, then culturing the precipitates of BMDC cells and 4T1@IP10 cells after fusion with PEG to obtain fused cell suspension; (4) collecting the fused cells, centrifuging to remove supernatant, resuspending them with PBS, assembling an extruder, adding PBS to open the membrane channels, discarding the PBS, adding the fused cell suspension, extruding multiple times, collecting the liquid into an EP tube, and filtering to obtain uniform EVs@IP10 solution; (5) preparation of GelMa: taking Gelatin and completely dissolving it, adding methacrylic anhydride dropwise to the Gelatin solution, after the dropwise addition is completed, uniformly stirring and mixing in an oil bath, adding PBS and uniformly stirring and mixing again, dialysis, freezing, and drying; (6) preparation of EVs@IP10-containing GelMa solution, defoaming the prepared EVs@IP10-containing GelMa solution, adding it into a microneedle mold, placing it into a preheated vacuum drying oven, vacuumizing and keeping the vacuum state for 1-2 min, closing the vacuum valve, taking out the mold, scraping off the bubbles at the bottom of the liquid, supplementing the liquid, concentrating, ultraviolet curing, drying the microneedles, and demolding to obtain IP10-fused cell vesicle microneedle vaccine.

[0005] Further, step (2) of isolating and culturing DC from bone marrow specifically comprises: collecting bone marrow suspension of 6-8 week old mice, lysing after adding red blood cell lysis solution, washing the cells with PBS, resuspending the cells in RPMI1640 medium for in vitro culture, and changing the medium containing GM-CSF and IL-4 every other day.

[0006] Further, step (3) is to culture after fusion according to the cell number ratio of DC:4T1@IP10 = 2:1.

[0007] Further, in the EVs@IP10-containing GelMa solution prepared in step (6), the mass fraction of GelMa is 10-20% w / v, and the concentration of EVs@IP10 is 1×10 8 / mL.

[0008] Further, a GelMa solution is prepared first: 20% w / v GelMA+ 2.00% w / v LAP, mixed with an equal volume of EVs@IP10 solution to obtain a GelMa solution containing EVs@IP10, at this time the final concentration of the GelMa solution: 10% w / v GelMA+1.00% w / v LAP;wherein the preparation method of the GelMa solution is: place the sample bottle in a constant temperature magnetic stirring water bath, until completely dissolved, ultrasonic defoaming after dissolving, to obtain a clear solution, cool the clear solution to about 37℃, add the EVs@IP10 solution, ultrasonic mixing.

[0009] The application also provides an IP10-fused cell vesicle microneedle vaccine with T cell activation function, which is prepared by the above preparation method.

[0010] The application also provides application of the above IP10-fused cell vesicle microneedle vaccine with T cell activation function in preparation of an anti-tumor product.

[0011] Beneficial effects: The IP10-fused cell vesicle with T cell activation function is made into a microneedle vaccine, in the aspect of immune activation, the IP10-fused cell vesicle can efficiently stimulate T cell-mediated anti-tumor immune response through the chemotaxis of IP10 and the antigen presentation function of the vesicle, and provides a new path to solve the problem of tumor immune escape; in the delivery system aspect, the minimally invasive and sustained-release characteristics of the microneedle not only reduce the resistance of patients to accept, but also maintain long-term immune stimulation, compared with the traditional injection method, significantly improve the compliance and treatment effect of the vaccine. In addition, good biocompatibility ensures the safety of its clinical application, and it has great transformation potential in the field of tumor prevention and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 (A) After PEG-induced cell fusion, observation was performed under a fluorescence inverted microscope (20x, 40x), blue represents DAPI-stained cell nuclei, green represents CESE-labeled 4T1 cell membranes, red represents PKH26-labeled DC cell membranes, and white arrows point to fused cells; (B) DLS detected the particle size distribution of EVs and EVs@IP10 to be about 100-200 nm; (C) DLS detected the ZETA potential of the vesicle vaccine, and the potential values of the two different vesicles were both negative; (D) TEM observed the morphology of the fused cell vesicles (EVs) and the fused cell vesicles loaded with chemokines (EVs@IP10), and the morphologies were both hollow irregular spheres; (E) NTA detected the number of vesicle particles, about 50 vesicles can be squeezed out from one fused cell, and the size of the vesicles is about 100-200 nm.

[0013] Figure 2 In vitro anti-tumor effect of the vesicular vaccine EVs@IP10: (A) Flow cytometry detection of the chemotaxis of T cells by EVs and EVs@IP10 and statistical chart of the results; (B) Flow cytometry detection and analysis of cells collected on day 0 of co-culture of each experimental group with T cells and statistics; (C) Flow cytometry detection and analysis of cells collected on day 3 of co-culture of each experimental group with T cells and statistics; (D) Flow cytometry detection and analysis of cells collected on day 5 of co-culture of each experimental group with T cells and statistics; (E) Flow cytometry detection results of activated T cells of each experimental group after co-culture with tumor cells for 48 h and statistical chart of the proportion of apoptotic cells.

[0014] Figure 3 Preparation of fusion cell vesicular vaccine microneedles with certain penetration: (A) Nuclear magnetic resonance hydrogen spectrum of Gelatin and GelMa. Green is the nuclear magnetic hydrogen spectrum of Gelatin, red is the nuclear magnetic hydrogen spectrum of GelMa, and the red box circled is the characteristic signal peak of GelMa; (B) Gel morphology formed by different concentrations of GelMA under the action of different concentrations of LAP and the morphology of the prepared microneedles, ① 100 mg GelMA + 2 mL 0.25% w / v LAP ② 200 mg GelMA + 2 mL 0.50% w / v LAP ③ 200 mg GelMA + 2 mL 1.00% w / v LAP ④ 300 mg GelMA + 2 mL 0.75% w / v LAP ⑤ 300 mg GelMA + 2 mL 1.00% w / v LAP ⑥ 400 mg GelMA + 2 mL 1.00% w / v LAP (C) SEM observation of the tip shape of GelMa microneedles; (D) Dissolution curve of GelMA, GelMa reaches degradation equilibrium in 12 days; (E) Swelling curve of GelMA, it can reach swelling equilibrium in about 24 h; (F) Maximum stress, i.e. breaking strength, of the microneedles when they are broken under external force, proving that the microneedles have good penetration ability, and the MNs have sufficient mechanical strength to penetrate the mouse skin; (G) Release curve of MN-EVs@IP10 in 37°C physiological saline; (H) H&E staining of mouse skin after sampling, the red box in the figure is the skin channel formed after the action of the microneedles; (I) In vitro hemolysis results of different experimental groups and statistical chart, the hemolysis rate of each group is <5%, which has biological safety. DETAILED DESCRIPTION

[0015] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0016] Unless otherwise specified, the materials described in the embodiments are all commonly used materials in the art and can be obtained commercially.

[0017] Example 1 Preparation of vesicular vaccine EVs@IP10: 1. Isolation and culture of DCs from mouse bone marrow Bone marrow suspension was collected from mice aged 6-8 weeks. The cells were lysed with red blood cell lysis buffer, washed with PBS, resuspended in RPMI 1640 medium, and cultured in vitro. The medium was changed every other day with medium containing GM-CSF and IL-4.

[0018] IP10 transfection of tumor cells Once the tumor cells reach a plate density of 70%-90%, replace the medium with DMEM without antibiotics or serum. Continue culturing for about 1 hour, then prepare the transfection complex solution, incubate at room temperature for 5 minutes, add the complex solution to the cells, and incubate for 6 hours before replacing the medium again.

[0019] Prepared fused cells (FCs) and observed by fluorescence staining. Collect BMDCs and 4T1 cells and count them using trypan blue staining. Add 4% tissue fixative to each cell pellet, incubate, wash twice with PBS, and discard the supernatant. Prepare CFSE and PKH26 staining solutions. Add CFSE staining solution to the 4T1 pellet and PKH26 staining solution to the DC pellet, mix well, and incubate at 37°C. After staining, stop the staining, wash twice with PBS, and discard the supernatant. Add DAPI staining solution to each cell pellet, incubate at room temperature for 2 min, stop the staining, wash twice with PBS, and discard the supernatant. Mix the cell suspension thoroughly according to a DC:4T1@IP10 ratio of 2:1, centrifuge, and discard the supernatant. Gently scrape the bottom of the centrifuge tube several times until the pellet becomes fine and sandy. Incubate in a 40°C water bath, add preheated PEG, and slowly stir for 1 min. Incubate in a water bath. Add preheated PBS at a constant speed along the centrifuge tube wall, gently invert and mix, centrifuge and discard the supernatant, resuspend the cells in PBS, drop the cell suspension onto a glass slide and air dry appropriately, add an anti-fluorescence quencher, place a coverslip and observe the cell fusion status under a fluorescence microscope.

[0020] Preparation of vesicular vaccine EVs@IP10 Collect fused cells, centrifuge to remove supernatant, and mix 10 μL of cell suspension with 10 μL of trypan blue solution. Add 10 μL of the mixture to a cell counting chamber, observe and count under a microscope, centrifuge to remove supernatant, and resuspend in PBS for later use. Assemble the pusher, add 1 mL of PBS to open the membrane channel, discard the PBS, add the cell suspension, and push back and forth several times to collect the liquid into an EP tube. Filter to obtain a homogeneous EVs@IP10 solution.

[0021] Characterization of EVs@IP10: TEM was used to observe and distinguish the morphology and structure of vesicles; particle size: after diluting the vesicles, the liquid was added to the mark of the cuvette, and the particle size distribution was detected after ensuring that there were no air bubbles; potential: the diluted sample was added to the corresponding potential sample cell and the zeta potential of the vesicles was detected; NTA was used to detect the yield and particle size distribution of vesicle vaccine EVs@IP10.

[0022] The results showed that most cells exhibited simultaneous fluorescence signals of three colors, and 4T1 cells successfully fused with DCs under the influence of 50% PEG. Figure 1 A). DLS results showed that the average particle size of EVs was 104.22 ± 9.06 nm, and the average particle size of EVs@IP10 was 148.38 ± 7.19 nm. Figure 1 B); the average potential of EVs was -19.50 ± 1.28 mV, and the average potential of EVs@IP10 was -17.03 ± 1.56 mV ( Figure 1 C). TEM results showed that EVs and EVs@IP10 had similar morphology and their sizes were basically consistent with the DLS detection results ( Figure 1 D). NTA detection counts vesicles within a given volume, providing a direct numerical quantification. Results showed that 1 mL of EVs@IP10 solution contained 1.6 × 10⁻⁶ vesicles. 8 Based on the concentration of fused cells before extrusion, it can be inferred that one fused cell can extrude approximately 50 vesicles, and the vesicle size remains stable between 100-200 nm. Figure 1 E).

[0023] Transwell assay of chemotaxis of T cells by EVs@IP10: Mouse spleen cells were isolated and T cells were induced, incubated, and the cultured T cells were collected and counted. The cell concentration was adjusted to 5 × 10⁶ cells / year using serum-free RPMI 1640 medium. 6After adding 200 μL of cells to each Transwell chamber, RPMI 1640 complete medium containing IL-2 was added to the lower layer of the Transwell, followed by the addition of PBS, EVs, and EVs@IP10. After incubation in a cell culture incubator for 2 h, the cell suspension was collected. Flow cytometry antibodies PE anti-mouse CD3, APC-Cy7 anti-mouse CD4, and APC anti-mouse CD8 were added. After incubation at 4°C in the dark for 30 min, PBS was added and mixed to terminate the incubation. The cells were centrifuged to remove the supernatant, and then resuspended in 300 μL of PBS. The cells were then filtered through a 200-mesh filter to prepare a single-cell suspension and introduced into flow cytometry tubes for detection using a full-spectrum flow cytometer.

[0024] The results showed that EVs@CXCL10 can effectively chemotize CD3. + CD8 + T cells ( Figure 2 A).

[0025] Flow cytometry analysis of the in vitro antitumor activity of vesicular vaccine EVs@IP10: Activation of T cells by EVs@IP10: The obtained mouse cell suspension was seeded into 6-well plates, and PBS, EVs and EVs@IP10 were added for co-culture. Cells were collected on days 1, 3 and 5 of co-culture for flow cytometry staining. Flow cytometry antibodies were used for staining with FITC anti-mouse CD3, PE anti-mouse CD4 and PE-Cy7 anti-mouse CD8, and the cells were detected by full-spectrum flow cytometry.

[0026] After extracting mouse spleen cells, they were activated with PBS, EVs, and EVs@IP10, and then co-incubated with pre-cultured 4T1 cells. All cells were collected and centrifuged, resuspended in 1× Binding buffer, and labeled as PBS, EVs, and EVs@IP10. 5 μL Annexin V-FITC and 5 μL PI were added to each tube, mixed well, and incubated at room temperature. Cells were then filtered into flow cytometry tubes. 5 μL Annexin V-FITC and 5 μL PI were added to each tube, mixed well, and incubated at room temperature for 15 min. Cells were then collected using flow cytometry.

[0027] The results showed that co-incubating vesicles with mouse spleen-derived cells for 7 days and detecting the induction of T cell frequency on days 1, 3, and 5 revealed that the EVs@CXCL10 group induced T cells with significantly higher efficiency than the EVs group and the PBS group. Figure 2 (BD), the apoptosis rate in the EVs@IP10 group was significantly higher than that in other groups, reaching as high as 48.90 ± 0.596%. This indicates that EVs@IP10-activated T cells have a significant killing effect on tumor cells.Figure 2 E).

[0028] Example 2 Preparation of IP10-fusion cell vesicle microneedle vaccine with T cell activation function: 1. Preparation and Verification of GelMa Weigh 5.0 g of Gelatin into a round-bottom flask, add 50 mL of 1×PBS, and stir in an oil bath at 50°C until the Gelatin is completely dissolved. Using a syringe, draw 5 mL of MA (methacrylic anhydride) and attach it to a microfluidic device. Add the MA solution dropwise to the dissolved Gelatin solution at a uniform rate. After all the MA has been added, stir in an oil bath at a uniform rate for 3 hours. Terminate the reaction by adding 150 mL of PBS and stirring for 1 hour. Dialyze the completed solution for 7 days and then dry it in a vacuum freeze dryer.

[0029] Trace amounts of GelMa (methacrylic acid esterified gelatin) and Gelatin were weighed, placed separately into EP tubes, dissolved in deuterated water (D2O), and then added to NMR sample tubes for detection. The results showed that when Gelatin reacted with methacrylic anhydride (MA), the structure of MA contained an olefinic double bond (H2C=C(CH3)-), and its proton signal showed a double peak in the 1H NMR spectrum. The results showed a new double peak at 5.1-5.6 ppm, proving that Gelatin and MA were successfully modified to prepare GelMa (methacrylic acid esterified gelatin). Figure 3 A).

[0030] Swelling and degradation experiments of GelMa: Weigh GelMa into a brown sample vial and add 1% w / v LAP solution to achieve a concentration of 10% w / v. Place the vial in a temperature-controlled magnetically stirred water bath and stir until the material is completely dissolved. Pour the liquid into a mold and irradiate it under 365 nm UV light to solidify the gel. Then, place the mold in a vacuum drying oven to obtain a dried gel sample.

[0031] Swelling experiment. Record the dry weight of each sample as W. d Place the sample into a labeled centrifuge tube, add physiological saline, and incubate at 37°C in a shaker. Remove the hydrogel at 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours. Blot dry with lens paper, weigh, and record the weight as W. S1 ...W S72 The swelling ratio SR is calculated according to the formula: SR = (W s - W d ) / W d ×100%, plot the swelling curve.

[0032] Degradation experiment. Three samples were prepared at each time point, and the dry weight of each sample was recorded as M.t Place the samples into labeled centrifuge tubes, add physiological saline, and incubate at 37°C in a constant temperature shaker. Each day, remove one set of hydrogels for drying, weigh, and record the weight as M. d1 ...M d17 The remaining mass percentage of the hydrogel is calculated using the formula M. d / M t ×100%, and select appropriate time points to plot degradation curves.

[0033] The results showed that, according to the degradation curve, GelMa reached degradation equilibrium in 12 days, and the degradation rate was >50%. Figure 3 D). According to the swelling curve, the swelling rate of GelMa reaches 400%, and swelling equilibrium is reached in 24 hours. Figure 3 E).

[0034] Preparation of gels and microneedles formed under the action of different concentrations of GelMA and LAP Different concentrations of GelMa were crosslinked with different concentrations of LAP: ① 5% w / v GelMa + 0.25% w / v LAP; ② 10% w / v GelMa + 0.50% w / v LAP; ③ 10% w / v GelMa + 1.00% w / v LAP; ④ 15% w / v GelMa + 0.75% w / v LAP; ⑤ 15% w / v GelMa + 1.00% w / v LAP; ⑥ 20% w / v GelMa + 1.00% w / v LAP. GelMa solutions were prepared according to the following six concentrations and then formed into gels and microneedles. After demolding, the gel state was observed against a black background. The results showed that the gels prepared in groups ③-⑥ all had complete morphology, and all could produce well-formed microneedles. Therefore, concentration ③ was selected as the concentration for subsequent experiments. Figure 3 B).

[0035] Preparation of fusion cell vesicle microneedle vaccine: Preparation of GelMa solution containing EVs@IP10: Cool the prepared GelMa solution to approximately 37°C, add 1 mL of EVs@IP10 solution, and sonicate to mix, obtaining 2 mL of GelMa solution containing EVs@IP10. The final concentration of the GelMa solution containing EVs@IP10 at this point is: 10% w / v GelMA + 1.00% w / v LAP + 1×10⁻⁶ 8 / mL EVs@IP10; After defoaming the prepared GelMa solution containing EVs@IP10, the solution was added to a microneedle mold, vacuumed, concentrated, and then cured under ultraviolet light. The microneedles were then dried and demolded to obtain the EVs@IP10-loaded microneedles, which are the IP10-fusion cell vesicle microneedle vaccines with T-cell activation function.

[0036] SEM observation of the tip morphology of blank microneedles (MN, microneedles formed by GelMA and LAP) and microneedles loaded with EVs@IP10 (MN-EVs@IP10) showed that both types of microneedles had relatively complete microneedle arrays and tips, and compared with MN-EVs@IP10, the tips of MN showed a certain degree of offset. Figure 3 C). Texture analysis of MN and MN-EVs@IP10 yielded parameters such as breaking force and hardness. MN exhibited a breaking force of 58.96 N, a hardness of 62.73 N, a maximum adhesion force of -0.04 N, and an adhesion strength of 0.0036 mJ. MN-EVs@IP10 showed a breaking force of 100.74 N, a hardness of 119.18 N, a maximum adhesion force of -0.00 N, and an adhesion strength of 0.0039 mJ. This demonstrates that both microneedles possess certain compressive strength and rigidity, making them suitable for penetrating skin or tissue. Figure 3 F).

[0037] Blank microneedles (MN) and microneedles loaded with EVs@IP10 (MN-EVs@IP10) were immersed in physiological saline, placed in a shaker, and liquid was aspirated at different time points. Then, 200 μL of chromogenic working solution was added for incubation. Protein concentration was detected by microplate reader, and drug release concentration was calculated according to formula to plot release curve.

[0038] The provesial concentration was calculated to be 678.125 μg / mL using the BCA standard curve. Protein concentrations at each time point were then measured and calculated. To subtract the influence of GelMa on concentration measurements, the OD values ​​of blank microneedles were subtracted at the same time points to obtain the release curve. In the first 8 hours, the drug release concentration was low and remained relatively stable. From 24 hours onwards, the release concentration showed a slow upward trend. After 72 hours, the drug release concentration increased rapidly, reaching approximately 80% around day 5, and then stabilized. Figure 3 G).

[0039] H&E staining to verify microneedle skin puncture results: Mice underwent hair removal treatment, microneedles were pressed onto the skin, and after removal, the mouse skin was photographed. The microneedled mouse skin was then fixed and placed in an embedding cassette. The skin tissue was dehydrated and prepared into paraffin blocks. Paraffin tissue sections were then dried and dewaxed using a slide dewaxing machine.

[0040] H&E staining was performed, followed by rehydration and dewaxing of the tissue. Hematoxylin dye, differentiation solution, and eosin dye were added to the slide in sequence, and the slide was gradually rinsed with running water. After the tissue was completely dried, it was mounted, observed under a microscope, and photographed.

[0041] The results showed that after the microneedles were removed, regularly arranged micropores were visible to the naked eye on the skin. After H&E staining, it was clear that the skin treated by the microneedles had formed obvious channels. Figure 3 H).

[0042] In vitro hemolysis experiment: Blood was collected from the eyes of mice and washed with PBS until the supernatant was clear. Red blood cell suspension and samples were added to EP tubes, and positive and negative control tubes were prepared. After incubation at 37°C, the tubes were centrifuged. Hemolysis was observed in each experimental group. The OD value of the supernatant at 540 nm was measured using an ELISA reader. The hemolysis rate was calculated using the formula: Hemolysis rate = (OD value of experimental group - OD value of negative group) / (OD value of positive group - OD value of negative group) × 100%.

[0043] The results showed that after co-incubation with red blood cells, no significant hemolysis occurred in the supernatant of any of the materials. Calculations of the hemolysis rate for each experimental group revealed that none exceeded 1%. According to internationally accepted biocompatibility assessment criteria, a material with a hemolysis rate of 5% or less in an in vitro hemolysis experiment is generally considered to have good blood compatibility. Therefore, the microneedle material prepared in this application can be considered to have good biocompatibility. Figure 3 I).

[0044] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing an IP10-fusion cell vesicle microneedle vaccine with T-cell activation function, characterized in that, Includes the following steps: (1) Isolate and culture DCs from bone marrow to obtain BMDC cell suspension; (2) Tumor cells were transfected with IP10 plasmid to obtain 4T1@IP10 cell suspension; (3) BMDC cells and 4T1@IP10 cells were precipitated separately, and then the BMDC cell precipitate and 4T1@IP10 cell precipitate were fused with PEG and cultured to obtain a fused cell suspension; (4) Collect fused cells, centrifuge to remove supernatant, resuspend them in PBS, assemble the pusher and add PBS to open the membrane channel, discard the PBS and add fused cell suspension, push repeatedly to collect the liquid into EP tube, filter to obtain a homogeneous EVs@IP10 solution. (5) Preparation of GelMa: Take gelatin and dissolve it completely. Add methacrylic anhydride dropwise to the gelatin solution. After the addition is complete, stir the mixture evenly in an oil bath. Add PBS and stir evenly again. Dialyze, freeze, and dry. (6) Prepare a GelMa solution containing EVs@IP10. After defoaming the prepared GelMa solution containing EVs@IP10, add it to the microneedle mold and place it in a preheated vacuum drying oven. After vacuuming, maintain the vacuum state for 1-2 minutes. After closing the vacuum valve, remove the mold, scrape off the air bubbles at the bottom of the liquid, replenish the liquid, concentrate, cure under ultraviolet light, dry the microneedles, and demold to obtain the IP10-fusion cell vesicle microneedle vaccine.

2. The preparation method according to claim 1, characterized in that, Step (1) isolating and culturing DCs from bone marrow: collect bone marrow suspension from mice aged 6-8 weeks, add red blood cell lysis buffer to lyse the cells, wash the cells with PBS, resuspend the cells in RPMI 1640 medium for in vitro culture, and change the medium every other day with medium containing GM-CSF and IL-4.

3. The preparation method according to claim 1, characterized in that, Step (3) involves culturing cells after fusion with PEG at a cell number ratio of DC:4T1@IP10 = 2:

1.

4. The preparation method according to claim 1, characterized in that, In step (6), the GelMa solution containing EVs@IP10 has a mass fraction of 10-20% w / v and a concentration of EVs@IP10 of 1×10⁻⁶. 8 / mL.

5. The preparation method according to claim 4, characterized in that, First, prepare the GelMa solution: 20% w / v GelMA + 2.00% w / v LAP, and mix it with an equal volume of EVs@IP10 solution to obtain a GelMa solution containing EVs@IP10. At this point, the final concentration of the GelMa solution is: 10% w / v GelMA + 1.00% w / v LAP. The GelMa solution is prepared as follows: place the sample vial in a constant temperature magnetically stirred water bath until it is completely dissolved. After dissolution, defoam it by sonication to obtain a clear solution. Cool the clear solution to about 37°C and add the EVs@IP10 solution, then mix by sonication.

6. An IP10-fusion cell vesicle microneedle vaccine with T-cell activation function, characterized in that, It is prepared by the preparation method according to any one of claims 1-5.

7. The application of the IP10-fusion cell vesicle microneedle vaccine with T cell activation function as described in claim 1 in the preparation of anti-tumor products.

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