Self-heating microneedle patch for delivering vaccine and application of self-heating microneedle patch

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a self-heating microneedle patch for delivering vaccines and application of the self-heating microneedle patch. The patch consists of an upper-layer self-heating patch and a lower-layer medicine-carrying microneedle patch, the self-heating patch contains a heating material in a specific ratio, the drug-loaded microneedle patch takes 10kDa hyaluronic acid as a base material, a needle tip is coated with an antigen and / or an immunologic adjuvant, a microneedle is prepared by a molding method, and the self-heating patch can increase the temperature of local skin to 39-45 DEG C. The invention further discloses a preparation method of the patch. The preparation method comprises the steps of preparing the drug-loaded microneedle and the self-heating patch and compounding the drug-loaded microneedle and the self-heating patch. The patch can be used for preparing anti-tumor immunotherapy drugs, can promote recruitment and maturation of dendritic cells and cross presentation of antigens through local heating, can enhance immune response of CD8 + T cells, has the advantages of safe delivery, immunological enhancement, high patient compliance and wide application range, and is remarkable in in-vivo anti-tumor effect and good in clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a self-heating microneedle patch for vaccine delivery and its application. Background Technology

[0002] Cancer is a serious threat to human health. Besides conventional surgery, radiotherapy, and chemotherapy, tumor immunotherapy has achieved remarkable efficacy in the clinical treatment of various cancers. Tumor immunotherapy activates the body's immune system to specifically kill tumor cells and promotes the development of immune memory to prevent tumor recurrence and metastasis. Immune checkpoint blockade therapy, represented by PD-1 / PD-L1 antibodies, has become a highly promising research direction in this field. The US FDA has approved seven antibodies targeting the PD-1 / PD-L1 pathway for the treatment of more than twenty types of cancer, and its indications are constantly expanding; however, the response rate is only about 20%. Therefore, exploring new tumor immunotherapy methods is of great significance.

[0003] Tumor vaccines activate the patient's own immune system, invigorating tumor-specific CD8+ T cells to specifically attack tumors and inhibit their growth and metastasis. Therefore, the application of tumor vaccines has become a significant breakthrough in the treatment of solid tumors. In April 2010, the US FDA officially approved Provenge, the first tumor vaccine for the treatment of prostate cancer. In 2017, Nature magazine reported the results of clinical trials of tumor neoantigen vaccines, demonstrating the great success of personalized vaccines tailored to tumor mutations in the treatment of melanoma. Currently, many tumor vaccine clinical trials are still underway. More importantly, most tumor vaccines are used in combination with PD-1 antibody therapy, exhibiting significant synergistic therapeutic effects. Therefore, the development of novel tumor vaccines is of great significance for clinical tumor immunotherapy.

[0004] Unlike most vaccines that elicit a humoral immune response, tumor vaccines require the activation of tumor antigen-specific CD8+ T cells to exert their immunotherapeutic effect. However, since most cancer patients are in a state of high immunosuppression, gradually shifting the body from immunosuppression to immune activation requires a long-term administration process, often necessitating multiple hospital visits for injections to achieve the desired therapeutic effect. This method of administration presents several challenges for patients, including the inability to administer the medication themselves, requiring professional intervention and increasing the burden on the healthcare system; the inconvenience of multiple injections; and the significant pain during injections, which greatly reduces patient compliance and increases the risk of bloodborne disease transmission. Therefore, exploring effective and more compliant methods of tumor vaccine administration is crucial.

[0005] Currently, the commonly used methods of vaccine administration mainly include the following: (1) Oral vaccines. Oral administration is non-invasive, painless, and safe, suitable for large-scale vaccination. However, the physical barrier of the intestine and the chemical barrier of gastric acid and digestive enzymes limit the oral absorption of vaccines, and there are limitations such as slow onset of action and unstable absorption; (2) Nasal administration. The nasal mucosa is rich in blood vessels, so the drug is absorbed rapidly, which is suitable for emergency treatment. Drugs that enter the blood directly avoid liver metabolism and improve the bioavailability of drugs. However, the absorption of inhaled drugs may be inconsistent due to factors such as nasal secretions and inflammation, and the nasal cavity has limited capacity, so the dosage of a single administration is limited; (3) Sublingual administration. Patients can use it themselves, it has a fast onset of action, is non-invasive, and can avoid the first-pass effect. However, the absorption of drugs is inconsistent due to factors such as saliva secretion, swallowing in the mouth, or sublingual activity; (4) Transdermal administration. It does not require frequent administration, is suitable for drugs that require continuous and stable release, and has the advantage of avoiding the first-pass effect. The skin, as a vital immune organ, contains a large number of immune cells in its epidermis and dermis, primarily dendritic cells (DCs), T cells, and macrophages. Subcutaneous DCs are the main antigen-presenting cells. When foreign antigens enter the skin, they are recognized and taken up by DCs, forming stable antigen peptide-MHC complexes within the cells, which are then transported to the cell membrane. Activated DCs migrate to draining lymph nodes, where the generated antigen peptide-MHC class I complexes are presented to CD8+ T cells, activating them and inducing an immune response. Therefore, percutaneous vaccine delivery can better mobilize subcutaneous immune cells and improve immunogenicity. However, traditional percutaneous drug administration methods, such as intramuscular or subcutaneous injection, generally require professional operation, can cause pain, and are difficult to withdraw, resulting in low compliance and safety. Therefore, exploring safe, effective, and more compliant percutaneous delivery strategies is of great significance for the design of novel tumor vaccines.

[0006] In recent years, domestic and international research on highly compliant percutaneous vaccine delivery strategies has focused on patches, gels, and microneedles, all of which offer advantages such as convenience, painlessness, avoidance of the first-pass effect, and efficient activation of the immune response. However, patches and gels often limit the percutaneous absorption of vaccines due to the influence of the stratum corneum of the skin and external factors. In contrast, percutaneous microneedles consist of a matrix of regularly arranged microneedles and a base, which can directly pierce the epidermal layer of the skin to efficiently deliver drugs to the subcutaneous layer. In addition, percutaneous microneedle vaccines have other advantages: (1) They are simple and economical, and patients can use them themselves, reducing the need for professional personnel; (2) Most microneedle percutaneous immunizations are administered in solid form, reducing the requirements for low-temperature cold chain during transportation and storage, and reducing transportation and storage costs; (3) They do not stimulate nerve endings and blood vessels in the dermis during use, and there is no obvious pain; (4) They have high social benefits, as microneedle vaccines do not require special medical equipment recycling, reducing resource consumption and environmental pollution; (5) They can directly contact the vaccine with subcutaneous immune cells, improving antigen presentation efficiency. Therefore, based on the above advantages, percutaneous delivery of microneedles is an ideal method for tumor vaccination and has great clinical application prospects.

[0007] However, percutaneous microneedle delivery of tumor vaccines also faces several challenges. First, there's the issue of vaccine stability. Subcutaneously injected antigens are easily degraded by enzymes in the body, leading to poor vaccine efficacy. Second, microneedle-delivered vaccines primarily reside in the epidermis; therefore, efficiently presenting the vaccine to dendritic cells (DCs) mainly distributed in the dermis is crucial. Furthermore, vaccines typically require the addition of adjuvants to synergistically enhance the immune response. In recent years, researchers have incorporated vaccines and adjuvants into microneedles for percutaneous administration. The addition of adjuvants more effectively activates immune cells and significantly improves the systemic immune response. The Caudill research group (Caudill C, Perry JL, Iliadis K, Tessema AT, Lee BJ, Mecham BS, Tian S, DeSimone JM. Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity. Proceedings of the National Academy of Sciences. 2021;118(39):e2102595118.) loaded OVA antigen, CpG oligonucleotide Toll-like receptor agonist, and aluminum adjuvant into polymer microneedles. After transdermal administration, they generated a good cellular immune response and a systemic humoral immune response. The Du team (Du G, Hathout RM, Nasr M, Nejadnik MR, Tu J, KoningRI, Koster AJ, Slütter B, Kros A, Jiskoot W, Bouwstra JA. Intradermalvaccination with hollow microneedles: A comparative study of various protein antigens and adjuvant encapsulated nanoparticles. Journal of Controlled Release. 2017;266:109-18.) co-loaded OVA antigen and polyinosinic-polycytidylic acid adjuvant into polylactic acid microneedles. The adjuvant group significantly enhanced the immune response of CD8+ T and CD4+ T cells. However, in many past studies, microneedle vaccine delivery generally involves directly loading adjuvants and drugs into the microneedles. Due to the limited drug loading space in microneedles, the addition of adjuvants reduces the drug loading rate of the vaccine. Therefore, there is an urgent need to explore novel materials designed to design microneedle systems that can achieve synergistic immune enhancement, ensuring the stability of the vaccine under the skin while generating a stronger immune response.

[0008] Among numerous microneedle materials, hyaluronic acid (HA) possesses several advantages, including excellent biocompatibility, avoidance of subcutaneous adverse reactions, and superior drug release characteristics. Therefore, HA has been widely used in the preparation of microneedles. Hyaluronic acid exhibits good biocompatibility, is a natural component of the skin, has low irritation, a low risk of allergic reactions, and can be naturally degraded by hyaluronidase in the skin without residue. Microneedles prepared using low molecular weight hyaluronic acid possess good mechanical properties and strong skin penetration. Simultaneously, HA microneedles have high drug loading capacity, allowing for the direct encapsulation of nucleic acid drugs or lipid-soluble antigens (such as tumor membrane proteins). Therefore, microneedles prepared with HA offer advantages such as good biocompatibility, high mechanical strength, large drug loading capacity, and improved antigen stability, making them a promising candidate material for microneedles.

[0009] Studies have shown that local physical stimulation of the skin has multiple benefits. For example, thermotherapy and cupping can promote blood circulation, reduce inflammation, and enhance immunity; electrostimulation therapy can relieve pain and promote muscle repair. Thermotherapy, by increasing local tissue temperature (usually within the range of 39-45℃), induces a heat stress response, which helps improve the body's ability to recognize and eliminate pathogens. Furthermore, when the body temperature reaches above 38.5℃, the expression of heat shock protein 90 (Hsp90) molecules in immune cells increases. After the body is stimulated by heat, Hsp90 binds to α4 integrin on the cell membrane, putting immune cells into a "combat readiness state" and enhancing their migration ability. Simultaneously, fever can also enhance the release of related immunomodulatory molecules (such as cytokines and nitric oxide) from dendritic cells (DCs), promote neutrophil arrival at the site of infection to exert an immune function; enhance the phagocytic capacity of macrophages and DCs; promote the production of chemokines by vascular endothelial cells; enhance the vascular exudation capacity of lymphocytes; and enhance the function of natural killer cells. It is worth mentioning that our preliminary experiments showed that heat treatment of mouse skin not only promoted DC recruitment but also stimulated cDC1 to produce IL-21, which significantly enhanced the tumor-killing ability of T cells and laid the foundation for subsequent research. In summary, fully utilizing the local thermal effect combined with the advantages of microneedles holds promise for promoting DC recruitment and activation, and for synergistic enhancement of novel tumor vaccines.

[0010] By utilizing the microneedle delivery strategy described above, once dendritic cells (DCs) are recruited to the subcutaneous region, antigens should be promptly presented to the gathered DCs. Therefore, further designing specific receptors on the surface of DCs to target antigens is expected to promote antigen presentation to DCs. Studies have found that the cDC1 subset is the DC with the strongest antigen-presenting capacity, thereby stimulating a robust cytotoxic T lymphocyte response. Therefore, to more effectively exert the anti-tumor effect of the vaccine, we aim to increase the proportion of DC1 cells in the subcutaneous region and enhance their function, delivering tumor antigens to subcutaneous cDC1 cells.

[0011] Current heating strategies mostly revolve around heating tumor sites through photothermal or electrothermal methods, with radiofrequency heating being the primary clinical application. The main aim is to directly kill tumor cells by exploiting their thermal sensitivity. However, these basic research approaches have significant limitations in clinical translation; for example, some solid tumors in vivo are difficult to heat using photothermal methods, limiting their application scope. In contrast, the heating strategy proposed in this study acts on the local drug delivery site, enhancing the local immune response to improve vaccine efficacy. As a vaccine microneedle platform, it can be adapted to various applications. Summary of the Invention

[0012] The purpose of this invention is to address existing problems by providing a self-heating microneedle patch for vaccine delivery and its application.

[0013] This invention is achieved through the following technical solution: The first objective of this invention is to provide a self-heating microneedle patch for delivering vaccines, the self-heating microneedle patch comprising two layers, the upper layer being a self-heating patch and the lower layer being a drug-loaded microneedle patch; The self-heating patch contains a heating material; The drug-loaded microneedle patch shown is made of hyaluronic acid (HA), and its microneedle tips are loaded with antigens and / or immune adjuvants.

[0014] Furthermore, the heating material of the self-heating patch comprises the following components by weight percentage: iron powder 20.0-60.0%, activated carbon 4.0-20.0%, ultra-high molecular weight polyethylene fiber 2.0-6.0%, xanthan gum, gum arabic or carboxymethyl cellulose (CMC) 0.5-5.5%, industrial salt 1.0-4.0%, anionic surfactant NP-4 0.3-1.5%, and water 12.0-28.0%.

[0015] Furthermore, the molecular weight of the hyaluronic acid is 10 kDa.

[0016] Furthermore, the antigen is OVA protein, and the adjuvant is CpG.

[0017] Furthermore, the microneedles are prepared by molding, and the HA solution concentration is 20~30% w / v.

[0018] Furthermore, the microneedles can dissolve and release antigens and / or adjuvants within the skin, while the self-heating patch can raise the local skin temperature to 39-45°C.

[0019] A second objective of this invention is to provide a method for preparing the self-heating microneedle patch, comprising the following steps: (1) Preparation of drug-loaded microneedles: The antigen and / or adjuvant are mixed with HA solution, added into PDMS mold, and obtained by centrifugation, drying and demolding; (2) Preparation of self-heating patch: Mix the heating material evenly and make it into a patch; (3) The self-heating patch is combined with the drug-loaded microneedle patch to obtain the self-heating microneedle patch.

[0020] Further, the concentration of the HA solution in step (1) is 20~30% w / v, the centrifugation speed is 3000~4000 rpm, and the centrifugation time is 30 min.

[0021] A third objective of this invention is to provide the application of the self-heating microneedle patch in the preparation of a medicament for antitumor immunotherapy.

[0022] Furthermore, the self-heating microneedle patch promotes the recruitment, maturation, and antigen cross-presentation of dendritic cells through local heating, thereby enhancing the CD8+ T cell immune response.

[0023] The present invention has the following advantages over the prior art: 1. This invention uses 10 kDa low molecular weight hyaluronic acid (HA) as the substrate for microneedle preparation. This material is a natural component of the skin, has good biocompatibility, low subcutaneous irritation, low risk of allergies, and can be naturally degraded by hyaluronidase in the skin without residue, avoiding adverse subcutaneous reactions. Simultaneously, microneedles prepared from low molecular weight HA have high mechanical strength (maximum single-needle withstand force up to 2.018 N), effectively penetrating the epidermal layer to form micropores; they also have a large drug loading capacity, directly encapsulating protein and nucleic acid-based tumor vaccine antigens, and improving antigen stability under the skin, reducing in vivo enzyme degradation of the vaccine, and ensuring the effectiveness and safety of transdermal vaccine delivery.

[0024] 2. This invention combines a self-heating patch with drug-loaded microneedles. The self-heating patch can rapidly raise the local skin temperature to a target temperature range of approximately 39°C and maintain it stably. Local thermal stimulation can induce the production of heat shock protein 70 / 90 (Hsp70 / 90) by skin and immune cells. On the one hand, this promotes the recruitment, migration, and responsiveness of cDC1 cells to the chemokine CCL21; on the other hand, it enhances the endocytic capacity of cDC1 cells for tumor antigens and upregulates the expression of immune-related cytokines such as IL-21. The combined use of thermal stimulation and hyaluronic acid microneedles can also synergistically improve the cross-presentation capacity of cDC1 cells, promote CD8+ T cell proliferation and IFN-γ secretion, and strengthen the tumor antigen-specific cellular immune response, solving the problem that traditional microneedle vaccine delivery is difficult to efficiently activate dermal immune cells.

[0025] 3. Addressing the limitation of drug delivery space caused by the direct co-loading of adjuvants and vaccines in traditional microneedles, this invention induces endogenous heat shock protein 70 / 90 through localized thermal stimulation to exert an adjuvant effect. Heat shock protein can synergistically enhance antigen presentation by DC1 cells and T cell activation by regulating IL-21 expression. This strategy can produce a synergistic effect with adjuvants (such as CpG) encapsulated within the microneedles, further enhancing the strength of the immune response while ensuring vaccine drug delivery rates.

[0026] 4. The self-heating microneedle patch of this invention is a transdermal drug delivery formulation. The microneedles only pierce the epidermal layer of the skin, without stimulating nerve endings and blood vessels in the dermis, resulting in no significant pain. Furthermore, it is simple to operate, allowing patients to use it themselves without the need for professional medical personnel, reducing the burden on the medical system and avoiding the inconvenience of multiple injections, thus significantly improving medication adherence in cancer patients. In addition, the microneedles deliver the drug in a solid form, reducing the requirements for low-temperature cold chain during vaccine transportation and storage, thereby reducing cold chain transportation and storage costs. After use, the microneedles do not require special medical equipment for recycling, reducing resource consumption and environmental pollution, resulting in significant social benefits.

[0027] 5. The self-heating patch of the present invention acts only on the local skin area where the vaccine is administered transdermally. It enhances the efficacy of the vaccine by activating the local immune response. Unlike traditional photothermal, electrothermal, and radiofrequency heating methods that directly act on the tumor site, it does not need to consider the tissue penetration problem of solid tumors. It has a wider range of applications and can be used as a universal vaccine microneedle platform. It is compatible with the transdermal delivery of various tumor vaccines and has good prospects for clinical translation and promotion.

[0028] 6. The self-heating microneedle patch of the present invention exhibited excellent in vivo antitumor activity in the B16-OVA tumor treatment model, significantly increasing serum IgG antibody levels in mice and activating humoral immunity; simultaneously promoting the infiltration of CD8+ T cells and IFN-γ secretion in tumor tissue, strengthening tumor-specific cellular immunity, effectively inhibiting tumor growth, and prolonging the survival of tumor-bearing mice. Furthermore, this self-heating microneedle patch can also improve the memory of CD4+ T and CD8+ T cells in the spleen of tumor-bearing mice, enabling the body to generate long-term tumor immune memory, providing an immune basis for preventing tumor recurrence and metastasis.

[0029] 7. The self-heating microneedle patch of the present invention can significantly promote the delivery of tumor antigens to draining lymph nodes within 8 hours after administration, enabling the antigens to quickly come into contact with immune cells in the lymph nodes, accelerating the initiation of the body's immune response, and further improving the onset speed of the vaccine's immune effect. This solves the problem of low efficiency in the transport of vaccine antigens to the epidermis and lymph nodes in traditional microneedle delivery of vaccines. Attached Figure Description

[0030] Figure 1 Schematic diagram of PDMS mold; Figure 2 The results represent the basic characterization of the self-heating microneedle patch; Figure 3 The effects of different treatments (10k, 200k HA and heat treatment) on cDC1 cell maturation markers and PD-L1 expression; Figure 4 The effects of different treatments (10k, 200k HA and heat treatment) on the transcriptional levels of DC1 cell-related cytokines and heat shock proteins; Figure 5 The effect of heating on DC1 cell endocytosis; Figure 6 The effects of hyaluronic acid and heat treatment on cross-presentation in DC1 cells; Figure 7 The effects of heat treatment on the migration ability and responsiveness of DC1 cells to the chemokine CCL21; Figure 8 The effect of self-heating microneedle patches on local cDC1 recruitment and cytokine expression in mouse skin; Figure 9 Imaging results of antigen delivery in mouse lymph nodes at different time points after administration of fluorescent protein-labeled microneedles; Figure 10 Effects of heating and heat shock protein inhibitors on the OVA endocytosis capacity of DC1 cells; Figure 11 The results of flow cytometry analysis of the effects of heating and heat shock protein inhibitors on endocytic antigens in DC1 cells; Figure 12The effects of heating and heat shock protein inhibitors on IL-21 expression levels in DC1 cells; Figure 13 The effects of CpG and heat shock protein inhibitors on the expression level of MHC I complex in DC1 cells OVA257–264; Figure 14 The effects of CpG, heat shock protein inhibitors and IL-21 neutralizing antibodies on cross-presentation in heat-treated DC1 cells; Figure 15 This is the result of in vivo antitumor activity assay of the self-heating microneedle vaccine in the B16-OVA tumor treatment model. Detailed Implementation

[0031] To further explain the present invention, the following specific embodiments are described.

[0032] Example 1: Preparation of self-heating microneedle patches 1.1 Preparation of drug-loaded microneedles Hyaluronic acid (HA) with a molecular weight of 10 kDa was dissolved in deionized water to prepare a 25% w / v HA solution. OVA protein (2 mg / mL) and CpG (0.6 mg / mL) were added to the HA solution, mixed well, and then poured into a specially made PDMS mold. The mold was centrifuged at 3500 rpm for 30 min to completely fill the mold cavity. The mold was then evacuated again to remove air bubbles. HA solution was added and the mold was centrifuged again. This process was repeated several times until the drug-containing solution was completely in the mold cavity. Excess drug solution was scraped off, and blank hyaluronic acid solution was added as a backing. After drying, the mold was demolded to obtain the drug-loaded microneedle patch. 1.2 Preparation of self-heating patches: Weigh the following components by weight percentage: 50.0% iron powder, 15.0% activated carbon, 4.0% ultra-high molecular weight polyethylene fiber, 3.0% xanthan gum, gum arabic or carboxymethyl cellulose (CMC), 2.0% industrial salt, 1.0% anionic surfactant NP-4, and 25.0% water. Mix the components evenly and press them into a self-heating patch.

[0033] 1.3 Composite of self-heating microneedle patches: The self-heating patch is then attached to the backing layer of the drug-loaded microneedle patch to obtain the self-heating microneedle patch.

[0034] 1.4 Basic Characterization Study of Microneedles and Natural Patch 1.4.1 Characterization of microneedle morphology and structure: HA microneedles were prepared using a molding method, and a clear array of microneedles could be observed. Figure 2 A); Under a 4× microscope, the shape of the microneedles is clearly visible (scale bar 10:1). Figure 2B); Microneedles were prepared by molding using an HA solution containing Rhodamine B. The microneedle array structure was observed using a microscope and confocal microscopy, and the microneedle array structure was clearly visible. Figure 2 D、 Figure 2 E and Figure 2 F).

[0035] 1.4.2 Microneedle skin penetration: Microneedles were applied with a certain force to the back of the hairless rat skin. Local skin sections were excised and stained with hematoxylin and eosin (HE). This allowed observation of the micropores formed by the microneedles in the rat skin. Figure 2 (C) This demonstrates that microneedles have effective skin penetration capabilities.

[0036] 1.4.3 Microneedle in vivo solubility: HA microneedles were applied to the backs of hair-removed mice with a certain force, and the dissolution of the needle tips was observed at different times. Figure 2 (G) It can be seen that HA microneedles have good solubility in mice, which can achieve effective drug release.

[0037] 1.4.4 Mechanical properties of microneedles: The mechanical properties of HA were measured using a texture analyzer. Figure 2 H), the maximum force that the microneedle can withstand is 2.018 N / needle, which is enough to pierce the surface of the skin.

[0038] 1.4.5 Heating performance of self-heating patches: The heating pack was exposed to air, and its surface temperature was measured at different times under natural conditions to study its heating performance. Figure 2 I) The heating pack rapidly heats up to 50°C within 10 minutes and can maintain a temperature above 40°C for 30 minutes.

[0039] A heating pack was applied to the hairless skin on the back of mice, and the skin temperature at the application site was continuously recorded at different times. Figure 2 J), it can be seen that the heating pack can quickly raise the local body temperature of mice to about 39°C, reaching the expected temperature of action.

[0040] Example 2: Performance Testing of Self-Heating Microneedle Patches 2.1 Effects of micro-heating on the maturation and function of DC1 (1) Effects of heating and high and low molecular weight HA on cDC1 maturation Experimental protocol: DC1 cells were induced and treated with heat, 10 kJ, 200 kJ, and LPS for 24 h after induction (heat treatment was 41 °C for 30 min, repeated every 8 h, for a total of 3 treatments). The expression of different maturation markers CD80, CD86, MHC II, CD40, and the immunosuppressive molecule PD-L1 was detected by flow cytometry.

[0041] Experimental results and conclusions: such as Figure 3 As shown, sodium hyaluronate of different molecular weights did not significantly affect the maturation of DC1 cells. Heat treatment (41℃) enhanced the effect of sodium hyaluronate with a molecular weight of 200 kJ. Furthermore, heat treatment (41℃) did not affect the maturation level of mature DC1 cells, but it reduced the expression of PD-L1 in mature DC1 cells. This is consistent with previous DC cell reports, which showed that heat treatment (41℃) had no significant effect on the maturation of DC1 cells.

[0042] (2) Effects of different molecular weight HA and heat treatment on the expression levels of cytokines in DC1 cells Experimental protocol: DC1 cells were induced and treated with heat, 10 kJ, 200 kJ, and LPS for 24 h after induction (heat treatment was 41 °C for 30 min, once every 8 h, for a total of 3 treatments). The transcriptional levels of cytokines such as IL-21, Hsp70, Hsp90, CXCL10, and IL-6, as well as heat shock proteins, were detected by qPCR.

[0043] Experimental Results and Conclusions: The results are as follows Figure 4 As shown, heat treatment can significantly increase the expression of HSP70 and HSP90 in DC1 cells. When sodium hyaluronate and heat treatment are applied simultaneously, the expression of IL-21 in DC1 cells is significantly increased, while heat treatment significantly reduces the expression of CXCL10 in DC1 cells. This indicates that the effect of heat treatment on the secretion of cytokines by DC1 cells is specific.

[0044] (3) Effect of heating on DC1 cell endocytosis Experimental protocol: After heat treatment, cDC1 cells were added with OVA-RhB fluorescent label, and the endocytosis of OVA protein by cells was observed and quantitatively detected by confocal microscopy.

[0045] Experimental Results and Conclusions: Micro-heat treatment does not affect the maturation of DC1 cells. Figure 5 However, it promotes the migration ability of DC1 cells and their response to the chemokine CCL21, and increases the expression of HSP70, HSP90 and IL-21 in DC1 cells; microthermal treatment increases the endocytosis of OVA protein in DC1 cells, and the main receptor for its endocytosis is the TLR2 receptor.

[0046] (4) Effects of hyaluronic acid and heat treatment on cross-presentation of DC1 cells Experimental protocol: DC1 cells were induced and co-incubated with 10 kDa HA and 100 ng / ml OVA protein for 12 h. DC1 cells were then co-cultured with CFSE-stained CD8+ T cells extracted from the spleen and lymph nodes of OT-1 mice for 3 days. Flow cytometry was used to detect CD8+ T cell proliferation, and ELISA was used to detect IFN-γ secretion levels. Figure 6 As shown, the effects of micro-heating and different materials on cross-presentation are illustrated.

[0047] Experimental results and conclusions: Heat treatment can enhance the cross-presentation capacity of DC1 cells, resulting in increased CD8+ T cell proliferation and IFN-γ secretion; hyaluronic acid treatment can significantly enhance the ability of DC1 cells to stimulate CD8+ T cells.

[0048] 2.2 Effect of heating on DC migration Experimental protocol: A cell migration experimental model was used, and the induced DC1 cells (10 6 The cells were added to the upper chamber of the cell culture chamber ( / well), and the lower chamber was filled with chemokine CCL21 (100 ng / ml) and LPS (1 μg / ml). The experimental group was heat-treated at 41℃, while the control group was not heated. After 12 h of migration culture, the cells were fixed, stained with crystal violet, and the number of cells that migrated to the lower chamber was observed and counted under a microscope (corresponding results figure, comparison of Control / Heat / CCL21 / Heat +CCL21 groups).

[0049] Experimental Results and Conclusions: Heat treatment promotes the migration of mature DC1 cells. Figure 7 Furthermore, it can enhance the responsiveness of DC1 cells to the chemokine CCL21, demonstrating that heat stimulation can improve the migration ability of DC1 cells, which is beneficial for antigen presentation and the initiation of immune response.

[0050] 2.3 Effects of self-heating microneedle patches on DC1 cell recruitment, cytokine expression levels, and antigen lymph node migration in mouse local skin (1) Local immune cell recruitment and cytokine expression detection Experimental protocol: The prepared self-heating microneedle patch (empty microneedles without OVA protein and CpG) was applied to the back of hair-removed mice and fixed in place for 30 min to ensure complete dissolution of the microneedles and effectiveness of the self-heating patch. After 1 h, the mice were sacrificed, and the skin at the microneedle application site was cut off, rinsed in PBS solution, minced, and digested with collagenase I (0.5 mg / ml), collagenase II (0.5 mg / ml), collagenase IV (1 mg / ml), DNase I (0.02 mg / ml), and hyaluronidase (1 mg / ml) at 37°C in a shaker for 40 min. After grinding and filtration, the skin cells were collected and flow cytometry was performed to detect the proportion of DC1 cells in the local skin. In the qPCR experiment, the cut skin was rinsed with PBS, ground in liquid nitrogen, and RNA was extracted from the skin tissue using an RNA extraction kit. The expression of cytokines and heat shock proteins in the local skin tissue was studied by qPCR.

[0051] Experimental results and conclusions: such as Figure 8 As shown, self-heating microneedles promote the recruitment of DC1 cells in the local skin and the expression of IL-6, IL-1β, IFN-β, IL-21, Hsp70, and Hsp90, demonstrating that self-heating microneedle patches can effectively recruit immune cells and upregulate the expression of immune-related molecules in the local skin.

[0052] Figure 8 In the middle: a. Local cDC1 recruitment in mouse skin after application of self-heating microneedles; b. Expression of cytokines in mouse skin after application of self-heating microneedles.

[0053] (2) Lymph node imaging at different time points after administration of fluorescent protein microneedles Experimental protocol: Self-heating microneedle patches loaded with RhB-OVA fluorescently labeled antigen were prepared and administered transdermally to the back of mice. In vivo imaging technology was used to image the lymph nodes of mice at 0.5 h, 4 h, 6 h, and 8 h after administration, and the fluorescence intensity in the lymph nodes was quantitatively detected (attached images of lymph nodes at different times after administration of fluorescent protein microneedles, comparison of OVA-Rhb MN / Heat-OVA-Rhb MN group).

[0054] Experimental Results and Conclusions: A self-heating microneedle patch was prepared by combining self-heating patches and microneedles, and RhB-OVA was encapsulated in it. The drug was administered to the back of mice, and the results were as follows: Figure 9 As shown, in vivo imaging revealed that self-heating microneedle patches promoted lymph node delivery of OVA within 8 hours, indicating that the delivery of antigens by self-heating microneedle patches is beneficial to improving the response level of immune cells and the immune effect of antigens.

[0055] 2.4 Heat shock protein adjuvant effect (1) Effects of heating and HSP inhibitors on DC1 cells Experimental protocol: DC1 cells after induction were seeded on the bottom of a confocal dish and allowed to adhere overnight. After starvation for 2 h, OVA-RhB was added and co-incubated for 2 h. HSP70 and 90 inhibitors were used for treatment. Confocal microscopy was used to detect antigen endocytosis and the fluorescence intensity was quantified.

[0056] Experimental results and conclusions: such as Figure 10 As shown, heating can promote the endocytosis of OVA by DC1 cells, and the stimulation effect of heating can be eliminated by using heat shock protein inhibitors, indicating that heat shock proteins can play a role in the endocytosis process of DC1 cells.

[0057] (2) Flow cytometry analysis of the effects of heating and inhibitor treatment on the endocytosis of antigens in DC1 cells Experimental protocol: DC1 cells after induction were starved for 2 h, then co-incubated with OVA-FITC for 2 h, and treated with HSP70 and 90 inhibitors. Flow cytometry was used to detect antigen endocytosis.

[0058] Experimental results and conclusions: such as Figure 11 As shown, flow cytometry analysis further demonstrates that HSP70 and 90 generated during the heating process participate in the endocytosis of antigens by DC1 cells.

[0059] (3) Effects of heating and heat shock protein inhibitors on IL-21 expression Experimental protocol: DC1 cells were induced, and after heat treatment and treatment with different concentrations of Hsp70 and Hsp90 inhibitors, the expression level of IL-21 was detected by qPCR.

[0060] Experimental results and conclusions: such as Figure 12 As shown, heating can significantly increase IL-21 expression, and HSP70 and 90 inhibitors can significantly inhibit this effect, indicating that heat shock proteins play a role upstream of IL-21. (4) Effects of CpG and heat shock protein inhibitors on the expression of OVA257–264 (SIINFEKL) MHC I complex in DC1 cells Experimental protocol: DC1 cells were induced and co-incubated with inhibitors and 100 ng / ml OVA protein for 12 h (heat treatment at 41℃ for 30 min). The expression level of OVA257–264 (SIINFEKL) MHC I complex in DC1 cells was detected by flow cytometry.

[0061] Experimental results and conclusions: such as Figure 13As shown, heating and CpG can increase the expression level of OVA257–264 (SIINFEKL) MHC I complex in DC1 cells after OVA peptide stimulation. Heat shock protein inhibitors can completely suppress the stimulating effect of heating, indicating that heat-generated heat shock proteins play a role in antigen presentation. (5) Effects of CpG, heat shock protein inhibitors, and IL-21 neutralizing antibodies on cross-presentation of DC1 after heat treatment Experimental protocol: DC1 cells were induced and co-incubated with inhibitors and 100 ng / ml OVA protein for 12 h (heat treatment at 41℃ for 30 min). DC1 cells were then co-cultured with CFSE-stained CD8+ T cells extracted from the spleen and lymph nodes of OT-1 mice for 3 days. Flow cytometry was used to detect the proliferation of CD8+ T cells.

[0062] Experimental results and conclusions: such as Figure 14 As shown, heating and CpG can promote the stimulation of CD8+ T cells by DC1 cells. Heat shock protein inhibitors and IL-21 neutralizing antibodies have inhibitory effects, indicating that heat-treated DC1 cells produce heat shock proteins that exert an adjuvant effect through IL-21.

[0063] Example 3: Study on the antitumor activity of self-heating microneedle vaccine (therapeutic B16-OVA model) Experimental protocol: On day 0, B16-OVA cells were used to induce a tumor model in the right back of mice. On day 4, tumor treatment was performed by injecting OVA protein, heating OVA protein microneedles, heating CBP-12-OVA microneedles (CBP-12 is a DC1 targeting peptide developed by our research group in the past), and heating OVA-CpG microneedles. The drugs were administered once every three days for a total of four times. The tumor volume was recorded every other day. On day 21, the mice were sacrificed, and their lymph nodes, spleen, and tumor tissue were collected for relevant index detection and evaluation and anti-tumor level assessment.

[0064] Experimental results and conclusions: such as Figure 15 As shown, self-heating microneedles significantly improve humoral immunity and inhibit tumor growth; self-heating microneedles can improve the memory of spleen T cells in tumor-bearing mice; self-heating microneedles can promote the infiltration of tumor CD8+ T cells and the production of IFN-γ.

[0065] Figure 15 In the middle: A. B16-OVA prevention experiment; B. Tumor volume and mouse serum IgG levels; C. Memory status of CD4+ T cells and CD8+ T cells in the spleen; D. The level of IFN-γ secreted by CD8+ T cells in the spleen, draining lymph nodes, and tumors; E. Tumor CD8+ T cell infiltration status; F. The proportion of DC1 in bilateral lymph nodes of mice.

[0066] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A self-heating microneedle patch for delivering vaccines, characterized in that, The self-heating microneedle patch consists of two layers: an upper self-heating patch and a lower drug-loaded microneedle patch. The self-heating patch contains a heating material; The drug-loaded microneedle patch shown is made of hyaluronic acid, and its microneedle tips contain antigens and / or immune adjuvants.

2. The self-heating microneedle patch according to claim 1, characterized in that, The heating material of the self-heating patch comprises the following components by weight percentage: iron powder 20.0-60.0%, activated carbon 4.0-20.0%, ultra-high molecular weight polyethylene fiber 2.0-6.0%, xanthan gum, gum arabic or carboxymethyl cellulose 0.5-5.5%, industrial salt 1.0-4.0%, anionic surfactant NP-4 0.3-1.5%, and water 12.0-28.0%.

3. The self-heating microneedle patch according to claim 1 or 2, characterized in that, The hyaluronic acid has a molecular weight of 10 kDa.

4. The self-heating microneedle patch according to claim 1, characterized in that, The antigen is OVA protein, and the adjuvant is CpG.

5. The self-heating microneedle patch according to claim 1, characterized in that, The microneedles were prepared by molding, and the HA solution concentration was 20-30% w / v.

6. The self-heating microneedle patch according to claim 1, characterized in that, The microneedles can dissolve and release antigens and / or adjuvants within the skin, while the self-heating patch can raise the local skin temperature to 39-45°C.

7. A method for preparing a self-heating microneedle patch as described in any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Preparation of drug-loaded microneedles: The antigen and / or adjuvant are mixed with HA solution, added into PDMS mold, and obtained by centrifugation, drying and demolding; (2) Preparation of self-heating patch: Mix the heating material evenly and make it into a patch; (3) The self-heating patch is combined with the drug-loaded microneedle patch to obtain the self-heating microneedle patch.

8. The method according to claim 7, characterized in that, The concentration of the HA solution in step (1) is 20-30% w / v, the centrifugation speed is 3000-4000 rpm, and the centrifugation time is 30 min.

9. The use of a self-heating microneedle patch as described in any one of claims 1 to 6 in the preparation of a medicament for antitumor immunotherapy.

10. The application according to claim 9, characterized in that, The self-heating microneedle patch promotes the recruitment, maturation, and antigen cross-presentation of dendritic cells through local heating, thereby enhancing the CD8+ T cell immune response.

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