A soluble skinning microneedle for delivering enterovirus inactivated vaccine and a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a soluble skin-pasting microneedle for delivering enterovirus inactivated vaccine and a preparation method and application thereof. In the present application, sodium hyaluronate is used as a film-forming material to wrap enterovirus 71 (EV71) and coxsackievirus A16 (CA16) type bivalent inactivated vaccine antigens, forming a needle body layer of soluble microneedle; a base layer is prepared by using pullulan material, and then a microneedle patch with stable overall structure is formed. The above-mentioned application technology is completed in a clean environment, and a sterile vaccine patch that passes the sterility test is obtained. The soluble microneedle described in the present application has good antigen stability under the condition of 2-8 DEG C. In animal experiments, the microneedle is attached to the surface of the mouse skin for skin-pasting immunization, which can induce a strong immune response and show good immunogenicity, thereby providing a new, safe and effective vaccine delivery route for the prevention and control of hand, foot and mouth disease in children.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a soluble transdermal microneedle for delivering an inactivated enterovirus vaccine, its preparation method, and its application. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a highly contagious disease caused by various enteroviruses, with enterovirus type 71 (EV71) and Coxsackie virus A16 (CA16) being the main pathogens. Both EV71 and CA16 belong to the Picornaviridae family, and their single-stranded positive-sense RNA genomes and nucleocapsid structures determine their similar infection and replication patterns. The translation and cleavage of viral polymers after viral uncoating and entry into cells are crucial for the formation of the viral structural proteins VP1, VP2, VP3, VP4, and non-structural proteins. However, different viruses exhibit different characteristics in their infection-immune response patterns with the host during the infection cycle, potentially leading to diversity in the epidemiology and disease progression of HFMD. EV71 is the pathogen causing the highest incidence of neurological complications, and infection can lead to severe cases such as encephalitis, paralysis, myelitis, and meningitis, which can even be fatal in severe cases. CA16 infection usually causes mild clinical symptoms, mainly manifested as blisters or ulcers on the hands, feet and mouth, as well as herpetic pharyngitis and other diseases.

[0003] Hand-foot-and-mouth disease vaccines in whole-virus inactivated form are characterized by intact antigen particles, good immunogenicity, and guaranteed safety. The EV71 monovalent inactivated vaccine administered via intramuscular injection is already on the market, while the EV71 and CA16 bivalent inactivated vaccines administered via intradermal injection have entered clinical trials. Dissolvable microneedles (DMNs), as a new generation of painless, needle-free, portable, and low-cost vaccine delivery platforms, are gradually gaining global attention. The skin is rich in antigen-presenting cells (APCs), such as Langerhans cells (LC) in the epidermis and dendritic cells (DCs) in the dermis. Directly delivering immunogens to these APCs induces an effective immune response. DMNs consist of a microarray with nanoscale needles, which can rapidly penetrate the skin surface to deliver vaccine antigens to areas of the epidermis or dermis rich in immune cells, thereby stimulating a strong immune response. They offer advantages such as controlled release and ease of large-scale immunization. The vaccine delivery method plays a crucial role in the efficiency of vaccine immunization. Compared with intramuscular and subcutaneous injection, intradermal immunization effectively improves post-immunization serum titers and provides higher vaccine protection. However, the discomfort associated with intradermal injections can lead to poor patient compliance. Therefore, soluble transdermal microneedle (DMN) vaccines offer a minimally invasive and painless alternative for delivering vaccine antigens to the epidermis or dermis.

[0004] Materials used in microneedle fabrication can be broadly categorized into two types: naturally derived and synthetic. Common natural materials include hyaluronic acid (HA), silk fibroin, and sodium alginate; synthetic materials include sodium carboxymethyl cellulose, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), and polylactic acid (PLA). However, hyaluronic acid has relatively poor mechanical strength and stability. Meanwhile, the properties of the microneedle backing material also significantly influence microneedle structure formation. An ideal backing material for a transdermal drug delivery system should possess a certain degree of flexibility and mechanical strength. PVA has good flexibility and high plasticity, but its mechanical strength is insufficient for effective skin penetration for drug delivery; PVP is a brittle material, offering good mechanical strength during microneedle fabrication, but it is prone to breakage during drug delivery. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a soluble transdermal microneedle for delivering inactivated enterovirus vaccines, its preparation method, and its application. The soluble transdermal microneedle provided by this invention uses pullulan as a backing material and is used in combination with sodium hyaluronate, which effectively improves the mechanical properties, solubility, and biocompatibility of the microneedle, providing a good material basis for the development of soluble microneedles.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a soluble patch microneedle for delivering an inactivated enterovirus vaccine, comprising microneedles and a backing, wherein the microneedles comprise sodium hyaluronate and enterovirus inactivated antigen encapsulated thereon, and the backing is prepared from pullulan polysaccharide solution.

[0007] Preferably, the enterovirus inactivated antigen is enterovirus 71 inactivated antigen and coxsackievirus A16 inactivated antigen.

[0008] Preferably, the enterovirus is enterovirus 71 strain FY23; and the Coxsackievirus A16 strain is Coxsackievirus A16 strain KM / M08.

[0009] Preferably, the area of ​​each soluble microneedle patch is 144 mm². 2 The dosage of the enterovirus 71 inactivated antigen is 50-100 U / patch, and the dosage of the coxsackievirus A16 inactivated antigen is 50-100 U / patch.

[0010] Preferably, in the soluble patch microneedles, the dose of enterovirus 71 inactivated antigen is 50-70 U / patch, and the dose of coxsackievirus A16 inactivated antigen is 50-70 U / patch.

[0011] Preferably, the tip length of the microneedle is 800µm; the solvent of the pullulan solution includes phosphate buffer; and the concentration of the pullulan solution is 180-220mg / mL.

[0012] Preferably, the final concentration of sodium hyaluronate in the microneedles is 280-320 mg / ml.

[0013] This invention provides a method for preparing the soluble transdermal microneedles described in the above technical solution, comprising the following steps: Enterovirus inactivated antigen and sodium hyaluronate solution are mixed to obtain vaccine injection solution; The vaccine needle solution is injected into a microneedle mold to obtain a microneedle mold filled with microneedles; The pullulan polysaccharide solution is injected into the microneedle mold filled with microneedles, covering the upper layer of the formed microneedle body so that it completely covers the bottom of all the needles. It is then dried at 35-37℃ for 3.5-4 hours, demolded, and the soluble skin-penetrating microneedles are obtained.

[0014] Preferably, after the vaccine needle solution is injected into the microneedle mold, it is subjected to vacuuming and centrifugation in sequence; before drying, the microneedle mold containing pullulan polysaccharide solution is subjected to vacuuming.

[0015] This invention provides the application of the soluble transdermal microneedles described in the above technical solution, or the soluble transdermal microneedles prepared by the preparation method described in the above technical solution, in vaccines or medical devices for the prevention of hand-foot-mouth disease.

[0016] Beneficial effects: The soluble transdermal microneedles provided by this invention use sodium hyaluronate (SH) as the film-forming material for encapsulating antigens. Compared with HA, SH exhibits improved mechanical strength and stability due to its lower free radical sensitivity and superior mechanical properties. Pullulan possesses low hygroscopicity and good chemical stability, and can form microneedles with the required mechanical strength for skin insertion without the addition of other additives. This invention selects pullulan as a backing material and uses it in combination with sodium hyaluronate, which can effectively improve the mechanical properties, solubility, and biocompatibility of the microneedles, providing a good material basis for the development of soluble microneedles.

[0017] The soluble transdermal microneedles provided by this invention also have the following advantages: First, its delivery method is convenient and simple to operate. Immunization is performed through a skin patch, eliminating the need for traditional injection procedures, which has significant advantages in primary healthcare institutions and children's immunization.

[0018] Second, it has a good immunization effect. The two-component inactivated vaccine antigens of EV71 and CA16 can simultaneously induce an immune response against two major pathogenic enteroviruses, effectively preventing the occurrence and spread of hand-foot-mouth disease.

[0019] Third, it is safe, needle-free and painless. After the soluble microneedles penetrate the skin, the vaccine antigen components dissolve with the biological material, and there is no obvious pain during the vaccination process. It avoids the risk of needle prick injury and cross-infection, and improves vaccine compliance.

[0020] Fourth, it offers precise delivery and high antigen release efficiency. Microneedles can deliver vaccines directly to areas of the skin epidermis or dermis rich in antigen-presenting cells (APCs), promoting antigen uptake and initiation of the immune response, while also possessing excellent controllable release characteristics.

[0021] Fifth, it aligns with modern vaccine development trends. This invention combines soluble microneedle technology with mature inactivated vaccine processes to create a low-cost, easy-to-use novel vaccine platform with promising prospects for industrialization and clinical translation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 Flowchart for the preparation of a bivalent inactivated soluble microneedle vaccine loaded with EV71-CA16; Figure 2 This is a schematic diagram of the structure of a soluble microneedle vaccine loaded with EV71-CA16 bivalent inactivated viral antigen. Figure 3 Microscopic image of the microneedle morphology of a soluble microneedle vaccine; Figure 4 Microscopic images of microneedle morphology of soluble microneedle vaccines with different curing times and the evaluation results of solubility of soluble microneedle vaccines after skin application to mice; Figure 5 The results of the evaluation of the cytotoxicity of sodium hyaluronate, a matrix material for soluble microneedle vaccines, to Vero cells; Figure 6 The results of the stability evaluation of the EV71-CA16 bivalent inactivated soluble microneedle vaccine; Figure 7 Results of regulatory changes in immune factors in Balb / c mice immunized with EV71-CA16 bivalent inactivated vaccine via intramuscular injection and skin graft. Figure 8 The results represent the protective effect induced by INF-γ translational enhancement in mice after immunization with the soluble microneedle vaccine. Figure 9 The results of the immunogenicity evaluation of the EV71-CA16 bivalent inactivated soluble microneedle vaccine; Figure 10 Micronail morphology diagrams under a microscope for soluble microneedle vaccines in Example 10 where the sodium hyaluronate in the needle tip layer was replaced with polyvinylpyrrolidone and polyvinyl alcohol, respectively. Figure 11 The dissolution of microneedles after immunization with a mouse back patch using a soluble microneedle vaccine in Example 11, in which pullulan polysaccharide in the back liner was replaced with sodium hyaluronate and polyvinyl alcohol, respectively. Figure 12 The microneedles prepared in Examples 12 and 13 are those with different concentrations of sodium hyaluronate as the tip layer and different concentrations of pullulan polysaccharide as the backing layer. Detailed Implementation

[0024] The present invention provides a soluble patch microneedle for delivering an inactivated enterovirus vaccine, comprising microneedles and a backing, wherein the microneedles comprise sodium hyaluronate and enterovirus inactivated antigen encapsulated thereon, and the backing is prepared from pullulan polysaccharide solution.

[0025] The soluble transdermal microneedles provided by this invention use sodium hyaluronate (SH) as the film-forming material for encapsulating antigens. Compared with HA, SH exhibits improved mechanical strength and stability due to its lower free radical sensitivity and superior mechanical properties. Furthermore, SH possesses good biocompatibility and biodegradability, and does not trigger significant inflammatory reactions after entering the skin, making it an ideal choice for microneedle matrix materials. Pullulan possesses low hygroscopicity and good chemical stability, and can form microneedles with the required mechanical strength for skin insertion without the addition of other additives. This invention selects pullulan as a backing material and uses it in combination with sodium hyaluronate, which effectively improves the mechanical properties, solubility, and biocompatibility of the microneedles, providing a good material basis for the development of soluble microneedles.

[0026] The soluble transdermal microneedle immunization method provided by this invention is easy to operate and stable to store. When stored at 4°C, the antigen of the finished vaccine product remains at 99.6% after 60 days, exhibiting good immunogenicity. Its immunization effect is superior to that of EV71-CA16 bivalent inactivated vaccine for intramuscular immunization.

[0027] In one embodiment, the enterovirus inactivated antigen is enterovirus 71 inactivated antigen and coxsackievirus A16 inactivated antigen. In another embodiment, the enterovirus is enterovirus 71 strain FY23; the coxsackievirus A16 is coxsackievirus A16 strain KM / M08.

[0028] The present invention does not have special requirements for the preparation method of the inactivated enterovirus antigen; any method well known to those skilled in the art can be used. As one embodiment, the present invention amplifies the EV71 working strain and the CA16 working strain separately using human diploid cells (KMB17). After the cytopathic effect reaches 80%, the viral fluid is collected, and after ultrafiltration concentration, purification and inactivation, sterile filtration, and testing to obtain EV71 and CA16 vaccine stock solutions. Al(OH)3 adjuvant is added according to the specified ratio to prepare the EV71-CA16 bivalent inactivated vaccine finished solution.

[0029] As one implementation method, the area of ​​each soluble microneedle patch is 144 mm². 2The dosage of the enterovirus 71 inactivated antigen is 50-100 U / patch, and the dosage of the coxsackievirus A16 inactivated antigen is 50-100 U / patch. In another embodiment, the dosage of the soluble patch microneedles is 50-70 U / patch for both enterovirus 71 inactivated antigen and coxsackievirus A16 inactivated antigen. The soluble patch microneedles provided by this invention achieve good immunogenicity with a dosage of 50 U of EV-A71 inactivated antigen and 50 U of CV-A16 inactivated antigen per patch.

[0030] In one embodiment, the microneedle has a tip length of 800 µm; the pullulan solution is dissolved in phosphate buffer; and the concentration of the pullulan solution is 180-220 mg / mL. In another embodiment, the pullulan solution has a concentration of 200 mg / mL. In one embodiment, the final concentration of sodium hyaluronate in the microneedles is 280-320 mg / ml. In another embodiment, the final concentration of sodium hyaluronate in the microneedles is 300 mg / ml.

[0031] This invention provides a method for preparing the soluble transdermal microneedles described in the above technical solution, comprising the following steps: Enterovirus inactivated antigen and sodium hyaluronate solution are mixed to obtain vaccine injection solution; The vaccine needle solution is injected into a microneedle mold to obtain a microneedle mold filled with microneedles; The pullulan polysaccharide solution is injected into the microneedle mold filled with microneedles, covering the upper layer of the formed microneedle body so that it completely covers the bottom of all the needles. It is then dried at 35-37℃ for 3.5-4 hours, demolded, and the soluble skin-penetrating microneedles are obtained.

[0032] In one embodiment, the drying time is 3.8 hours. In another embodiment, after the vaccine needle solution is injected into the microneedle mold, it is sequentially subjected to vacuuming and centrifugation; before drying, the microneedle mold containing pullulan polysaccharide solution is subjected to vacuuming. Vacuuming eliminates air bubbles within the mold.

[0033] This invention uses sodium hyaluronate as a film-forming material to encapsulate the enterovirus 71 (EV71)-Coxsackievirus A16 (CA16) bivalent inactivated vaccine antigen, forming a soluble microneedle layer. A pullulan polysaccharide base layer is prepared, resulting in a microneedle patch with a stable overall structure. All the above techniques are performed in a clean environment, yielding vaccine patches that pass sterility testing. The soluble microneedles described in this invention exhibit good antigen stability at 2-8°C. In animal experiments, applying the microneedles to the skin of mice for transdermal immunization induced a strong immune response, demonstrating good immunogenicity and providing a novel, safe, and effective vaccine delivery route for the prevention and control of hand-foot-mouth disease in children.

[0034] Based on the above advantages, the present invention provides the application of the soluble transdermal microneedles described in the above technical solution or the soluble transdermal microneedles prepared by the preparation method described in the above technical solution in the preparation of vaccines or medical devices for the prevention of hand-foot-mouth disease.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a soluble patch microneedle for delivering an inactivated enterovirus vaccine, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Preparation Example The enterovirus 71 FY23 strain (GenBank ID: EU812515.1), Coxsackievirus A16 KM / M08 strain (GenBank ID: MN046208.1), and KMB17 cells used in this embodiment of the invention were all provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0037] Enterovirus 71 FY23 strain is disclosed in the literature: [Liu L, Zhang Y, Wang J, Zhao H, JiangL, Che Y, Shi H, Li R, Mo Z, Huang T, Liang Z, Mao Q, Wang L, Dong C, Liao Y, Guo L, Yang E, Pu J, Yue L, Zhou Z, Li Q. Study of the integrated immune response induced by an inactivated EV71 vaccine. PLoS One. 2013;8(1):e54451.doi: 10.1371 / journal.pone.0054451. Epub 2013 Jan 23. PMID: 23372725; PMCID: PMC3553120.]; Coxsackie virus A16 type KM / M08 strain is disclosed in the literature: [Fan S, Liao Y, Lian Y, Jiang G, Jiang L, Dong C, Yang E, Wang L, Xu 10.1038 / s41541-019-0108-6. Erratum in: NPJ Vaccines. 2019 Nov 4;4:47. doi: 10.1038 / s41541-019-0141-5. PMID: 30937186; PMCID: PMC6437170.]; KMB17 cells were published in the literature: [Dong, C., Wang, J., Liu, L., Zhao, H., Shi, H., Zhang, Y., … Li, Q. (2010). Optimized development of a candidate strain of inactivated EV71 vaccine and analysis of its immunogenicity in rhesus monkeys.] Human Vaccines , 6 (12), 1028–1037. https: / / doi.org / 10.4161 / hv.6.12.12982.

[0038] Example 1: Preparation of EV71-CA16 bivalent inactivated vaccine solution Enterovirus 71 strain FY23 and Coxsackievirus A16 strain KM / M08 were inoculated into human embryonic lung diploid cells (KMB17) and cultured at 37°C and 5% CO2. Viral solutions were collected after complete cytopathic effect (CPE) was observed under a microscope. The EV71 and CA16 virus harvest solutions were then purified by 0.65µm filtration, 100KD ultrafiltration, and Sepharose 6FF column chromatography to remove host proteins, nucleic acids, and other impurities. Formaldehyde was added to the EV71 and CA16 virus concentrates to a final concentration of 150µg / ml. The solutions were inactivated at 37°C ± 0.5°C for 12 days to ensure complete inactivation while maintaining immunogenicity. After inactivation and passing a virus-free test, the solutions were concentrated using 100KD ultrafiltration based on antigen content to obtain the EV71 or CA16 vaccine stock solution. The EV71-CA16 bivalent inactivated vaccine solution can be prepared by adding aluminum hydroxide (Al(OH)3) adjuvant to bring the final aluminum concentration to 0.14-0.21 mg / ml.

[0039] Example 2 Preparation of soluble microneedle-loaded EV71-CA16 bivalent inactivated vaccine (flowchart shown) Figure 1 ): 1. In a clean bench within a Class A clean area with an environmental pressure ≥10 MPa, the high, medium, and low dose EV71-CA16 bivalent inactivated vaccine solutions obtained in Example 1 were mixed with sodium hyaluronate solution, respectively; the final concentration of sodium hyaluronate after mixing was 300 mg / ml for each solution. The mixture was placed in a magnetic stirrer and stirred continuously for 30 minutes to ensure thorough mixing of the vaccine antigen and carrier. This yielded a homogeneous vaccine needle solution suitable for microneedle presentation.

[0040] 2. Inject high, medium, and low doses of vaccine needle solution into polydimethylsiloxane (PDMS) microneedle molds, 200 µl / patch. The EV71 and CA16 viral antigen content is 100 U / 100 U / patch for the high-dose group (i.e., both EV71 and CA16 viral antigen content are 100 U / patch, the same applies below), 70 U / 70 U / patch for the medium-dose group, and 50 U / 50 U / patch for the low-dose group. Vacuum treatment is performed to remove air bubbles from the mold, followed by centrifugation at 4000 rpm for 2 minutes in a horizontal centrifuge. The vacuuming and centrifugation steps are repeated 2 to 3 times to ensure the needle solution fully fills the mold cavity and eliminates all air bubbles, obtaining a complete microneedle structure.

[0041] 3. Take pullulan polysaccharide separately, dissolve it in 2 mmol / L phosphate buffer (PBS) to a final concentration of 200 mg / mL, add it to a stirring container and stir under magnetic stirring for 4 h, then sonicate to remove air bubbles to obtain microneedle backing solution.

[0042] 4. Inject the backing solution into the PDMS microneedle mold, covering the upper layer of the formed microneedle body, ensuring complete coverage of the bottom of all needles, and continue vacuuming to remove residual air bubbles from the solution. Figure 2 The mold was dried at 37°C for 3.8 hours to allow the backing portion to solidify and set. After drying, the mold was removed to obtain a soluble microneedle patch with a complete structure. Figure 3 (The left image shows the microneedle body, and the right image shows the backing of the needle body), indicating that high, medium, and low dose groups of EV71-CA16 bivalent inactivated soluble microneedle vaccine patches were successfully prepared.

[0043] Example 3: Evaluation of the skin-penetrating solubility of EV71-CA16 bivalent inactivated soluble microneedle vaccine in mice. The EV71-CA16 bivalent inactivated soluble microneedle vaccine patch from Example 2 (taking the medium-dose group as an example) was used to evaluate its penetration and solubility in mouse skin. Different time points were set after immunization with the vaccine patch to observe the degree of microneedle solubility in the skin. Ten 5-6 week old female BALB / c mice were selected for each group. Before the experiment, the skin on the back of the mice was shaved using a shaving tool and depilatory cream, and then disinfected with ethanol cotton balls. The skin was allowed to dry naturally before use.

[0044] During the experiment, the EV71-CA16 bivalent inactivated soluble microneedle vaccine patch was aligned with the treatment area. The microneedles were applied vertically with constant pressure using the thumb to ensure complete penetration into the skin. The microneedle patch was secured with medical tape, and the patch was removed at 15, 25, 35, and 45 minutes. The dissolution state of the microneedles was then observed under a microscope. Results are shown below. Figure 4 In the figure, a represents the vaccine microneedles that were not fully demolded after drying for 3 hours during preparation; b to d represent the vaccine microneedles that were fully demolded after drying for 3.8 hours during preparation; and e to h represent the microneedle dissolution results after the microneedles were peeled off after skin application at 15 min, 25 min, 35 min, and 45 min, respectively.

[0045] The results showed that insufficient curing time led to needle tip bending after demolding. Figure 4(a) The microneedles prepared by this invention using sodium hyaluronate combined with pullulan can successfully pierce mouse skin and exhibit good intradermal solubility. After 15 minutes of skin application, the needle tips showed slight dissolution; after 35 minutes, most of the needles had undergone significant degradation; and after 45 minutes, dissolution was essentially complete, with no visible needle residue. The microneedles removed at different time points remained intact without breakage, indicating that the microneedles are structurally stable, flexible, and not prone to breakage, possessing good biomechanical properties. No obvious redness, swelling, or exudation was observed during the microneedle insertion process, demonstrating that this soluble microneedle system has excellent biocompatibility and feasibility for intradermal delivery.

[0046] Example 4: Cytotoxicity detection of sodium hyaluronate, a matrix material for soluble microneedle vaccines. To verify the biocompatibility and safety of the prepared sodium hyaluronate (SH) soluble microneedle material, this embodiment uses an in vitro cytotoxicity test, the specific method of which is as follows: Vero cells in the logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates, with 180 µl of cell suspension per well. Cells were incubated at 37°C with 5% CO2 for 6–24 h to allow cell adhesion. Cells were then administered drugs, divided into SH and control groups. 100 µl of drug was added to each well. SH was administered at final concentrations of 70 mg / 100 µl, 60 mg / 100 µl, 50 mg / 100 µl, 40 mg / 100 µl, 30 mg / 100 µl, and 15 mg / 100 µl. The control group received PBS solution. Each concentration was used in triplicate. Cells were incubated at 37°C with 5% CO2. After 24 h, the supernatant was carefully aspirated, and 90 µl of fresh culture medium was added, followed by 10 µl of MTT solution. Cells were incubated for another 4 h. Then, the supernatant was aspirated, and 110 µl of dimethyl sulfoxide (DMSO) solution was added to each well. The plates were shaken at low speed for 10 min to dissolve any crystals. The OD value of each well was measured at 490 nm using an ELISA reader. Cell viability % was calculated as OD. exp / OD con ×100%, where OD exp Represents the OD value of the SH group, OD con This represents the OD value of the blank control group. Results are shown below. Figure 5 .

[0047] The results showed that the OD values ​​of cells treated with microneedle extract at different concentrations were not significantly different from those of the blank control group and the negative control group. The cell viability remained above 90%, and there were no obvious morphological changes, cell rupture, shrinkage or other toxic phenotypes. This indicates that the sodium hyaluronate soluble microneedle material used has no obvious toxic effect on cells, has good biocompatibility, and is suitable as a delivery carrier for soluble microneedle vaccines.

[0048] Example 5: Stability evaluation of EV71-CA16 bivalent inactivated soluble microneedle vaccine To evaluate the storage stability of the EV71-CA16 bivalent inactivated soluble microneedle vaccine prepared in this invention, the high-dose group of EV71-CA16 bivalent inactivated soluble microneedle vaccine patches from Example 2 were stored at 37°C, 25°C, 4°C, and -20°C, respectively. On days 0, 7, 14, 30, 60, and 90 after storage, samples were taken, and the microneedles were dissolved in 100 µl of sterile PBS buffer. The EV-A71 and CV-A16 antigen contents in the solution were quantitatively detected using an ELISA kit, and the optical density (OD) values ​​were recorded. 450 And calculate the relative antigen retention rate.

[0049] For CA16 ELISA antigen detection, samples and standards were serially diluted 2-fold beforehand. Samples and standards of appropriate dilution were added separately to ELISA plates coated with rabbit anti-CA16 polyclonal antibody. The plates were incubated at 37°C for 2 hours, then washed 5 times, blotted dry, and 100 µl of enzyme conjugate (HRP-labeled rabbit anti-CA16 polyclonal antibody) was added to each well. The plates were then incubated at 37°C for 1 hour. After incubation, the plates were washed 5 times, blotted dry, and 100 µl of chromogenic solution (single-component TMB) was added to each well. The plates were then incubated at 37°C in the dark for 10 minutes. After development, 50 µl of stop solution was added to each well to terminate the development. The OD value of each well was read at 450 nm using an ELISA reader. The antigen content of the samples was calculated using the double parallel line method with the standards.

[0050] For EV71 ELISA antigen detection, samples and standards were serially diluted 2-fold beforehand. Samples and standards of appropriate dilution were added to ELISA plates coated with rabbit anti-EV71 polyclonal antibody. The plates were incubated at 37°C for 2 hours, then washed 5 times, blotted dry, and 100 µl of biotin-labeled goat anti-EV71 polyclonal antibody was added to each well. The plates were then incubated at 37°C for 1 hour. After incubation, the plates were washed 5 times, blotted dry, and 100 µl of enzyme conjugate (avidin-labeled HRP) was added to each well. The plates were incubated at 37°C for 0.5 hours. After incubation, the plates were washed 5 times, blotted dry, and 100 µl of chromogenic solution (single-component TMB) was added to each well. The plates were then incubated at 37°C in the dark for 10 minutes. After chromogenic development, 50 µl of stop solution was added to each well to stop the development. The OD value of each well was read using a microplate reader at 450 nm. The antigen content of the samples was calculated using the double parallel line method with standards. The results are shown below. Figure 6 .

[0051] The results showed that the antigen retention rate of the microneedles was above 99.4% after 90 days of storage at -20℃ and 4℃, indicating good antigen activity. Storage at 25℃ showed a decrease in antigen stability, with a retention rate of approximately 90%. At 37℃, antigen degradation was significant, with the antigen retention rate decreasing to about 3% after 90 days. These results demonstrate that the soluble microneedle vaccine prepared in this invention exhibits good antigen stability under low-temperature conditions, meeting the requirements for routine vaccine storage, transportation, and preservation.

[0052] Example 6: Expression profile of innate immune signaling molecules in local tissues at the immunization site of soluble microneedle vaccine The soluble microneedle vaccine from the medium-dose group of the EV71-CA16 bivalent inactivated vaccine in Example 2 and the medium-dose group of the EV71-CA16 bivalent inactivated vaccine solution from Example 1 were used to immunize mice via skin grafting (DMNs-EV71+CA16) and intramuscular injection (IM-EV71+CA16), respectively. Skin grafting of DMNs-EV71+CA16 was completed 45 minutes after application. The IM-EV71+CA16 group underwent two immunizations (days 0 and 28), with each injection containing 70U of EV71 antigen and 70U of CA16 antigen. Skin tissue samples from the immunization sites were homogenized, and total RNA was extracted from these samples using Trizol. Quantitative analysis was performed using a one-step q-RT-PCR method on a thermal cycler. Primers for cytokine detection are shown in Table 1.

[0053] Table 1 Primers for Cytokine Detection

[0054] See results Figure 7 ,in, express P <0.05, express P <0.001, express P <0.0001. Results showed that at 12h and 24h post-immunization, the IFN-γ level in the DMNs-EV71+CA16 group did not increase significantly, but at 48h, it increased significantly and was significantly higher than that in the IM-EV71+CA16 group; the IM-EV71+CA16 group showed an increasing trend from 12h to 24h, and decreased at 48h. The IFN-β levels in both the DMNs-EV71+CA16 and IM-EV71+CA16 groups showed an increasing trend. IL-5, IL-7, and IL-9 all showed a decreasing trend in the DMNs-EV71+CA16 group.

[0055] EV71 or CA16 antigens bind to intracellular pattern recognition receptors (PRRs), inducing innate or adaptive immune responses, in which the activation and transduction of signaling molecules play a crucial role. This invention selected factors associated with the activation of antigen-presenting cells (APCs), particularly the activation of the NF-κB signaling pathway and activating factors of T cell proliferation, for analysis. The results showed that RANKL, LTa3, LIGHT, and 4-1BBL were all upregulated to varying degrees. Figure 7 These results indicate that immunization can activate antigen-presenting cells in the epidermis, thereby inducing a strong immune response.

[0056] Example 8: Protective effect of specific INF-γ translation enhancement in mice after immunization with soluble microneedle vaccine. IFN-γ specific ELISApot assay: Mice were immunized with either skin grafts (DMNs-EV71+CA16) or intramuscular injections (IM-EV71+CA16) using the method described in Example 7. Splenic lymphocytes were isolated by Ficoll-Paque density gradient centrifugation and resuspended in RPMI 1640 medium containing 10% fetal bovine serum. Cells were cultured at 2 × 10⁶ cells / well. 5 Each 96-well ELISA Pot plate pre-coated with anti-human IFN-γ antibody was inoculated with 50 µl (5.0 CCID) of EV71 or CVA16 antigen. 50 Cells were stimulated with a concentration of 10 μg / mL, and triple-well plates were used. PHA (100 µl, 10 μg / mL) served as a positive control, and the culture medium as a negative control. Cells were incubated at 37°C and 5% CO2 for 36 h. Cells were then removed, and the plate was sequentially incubated with the secondary antibody and substrate solution before cell removal to complete the staining process and form spots. Spot counting was performed using an automated ELISpot plate reader. Results are shown below. Figure 8 ,in, express P <0.05, express P <0.0001.

[0057] To a certain extent, the specific IFN-γ response in the spleen of immunized mice can reflect the intensity of the immune response. This invention detected the levels of neutralizing antibodies and the IFN-γ and IL-4 responses in the spleen of mice on day 28 after the initial immunization (day 0) and booster immunization (day 28). Seven days after the second immunization, the specific IFN-γ response reached a relatively high level (e.g., Figure 8 (As shown). The IFN-γ of the DMNs-EV71+CA16 group was higher than that of the IM-EV71+CA16 group.

[0058] Example 9: Evaluation of the Immunogenic Potency of EV71-CA16 Bivalent Inactivated Soluble Microneedle Vaccine This invention evaluates the immunogenicity of soluble microneedles as a mediated delivery of a bivalent inactivated soluble microneedle vaccine of EV71-CA16 by assessing the immunogenicity produced by different immunization routes. Neutralizing antibody detection was performed according to a standard protocol, including: mixing diluted serum with a solution containing 500-1000 CCIDs. 50 Equal volumes of culture medium were mixed with EV71 or CA16 in 96-well plates and incubated for 2 hours. Vero cells were then added to the wells, and the results were evaluated after 7 days. The highest dilution was considered the neutralizing titer. Five- to six-week-old Balb / c mice were immunized with an intramuscular injection group (DMNs), a soluble microneedle immunization group (IM), and an aluminum hydroxide adjuvant control group (Control) according to an immunization schedule of 0 (primary immunization) and 28 days (booster immunization). Serum was collected from the tail vein at 14, 28, and 42 days for serum neutralization evaluation.

[0059] Solution vaccine groups: High-dose group: EV71 antigen content 100U / 100µl, CA16 antigen content 100U / 100µl; Medium-dose group: EV71 antigen content 70U / 100µl, CA16 antigen content 70U / 100µl; Low-dose group: EV71 antigen content 50U / 100µl, CA16 antigen content 50U / 100µl; 0.15mg / ml AL(OH)3 adjuvant group.

[0060] Patch-based vaccine groups: High-dose group: EV71 antigen content 100U per patch, CA16 antigen content 100U per patch; Medium-dose group: EV71 antigen content 70U per patch, CA16 antigen content 70U per patch; Low-dose group: EV71 antigen content 50U per patch, CA16 antigen content 50U per patch; 0.15mg / ml AL(OH)3 adjuvant group.

[0061] Serum antibody titer testing, such as Figure 9 As shown, where, express P <0.05, express P <0.01, express P<0.0001. Results showed that in the intramuscular injection immunization group, the average seroconversion rate of EV71 antibodies in mice on day 14 after primary immunization was less than 50% (1:8 was used as the threshold for vaccine efficacy evaluation); the seroconversion rate of CA16 antibodies was also less than 50% (1:4 was used as the threshold for vaccine efficacy evaluation). When booster immunization was performed on day 28 after primary immunization, the seroconversion rate of EV71 in the intramuscular injection group was less than 80% on day 42; the seroconversion rate of CA16 in the intramuscular injection group was also less than 80%. In contrast, in the transdermal immunization group, the EV71 antibody titer and CA16 antibody titer were both 1:4 on day 14 after primary immunization; when booster immunization was performed on day 28 after primary immunization, the EV71 antibody titer and CA16 antibody titer were both 1:64 on day 42, with a seroconversion rate of 100% in both groups; the seroconversion rates in the intramuscular injection groups were all less than 80%. Serum neutralization results showed that the immune response induced by the soluble microneedle transdermal immunization route was significantly higher than that induced by the intramuscular injection route.

[0062] From the perspective of different immunization routes, both intramuscular injection and transdermal administration produced immune effects, but the transdermal immunization route had a higher titer than the intramuscular immunization route. These results suggest that transdermal immunization of Balb / c mice with the EV71-CA16 bivalent inactivated vaccine can induce higher titers of antibodies in the mouse serum.

[0063] Example 10 In Example 2, the sodium hyaluronate (SH) in the needle tip layer of the low-dose group of soluble microneedle vaccines was replaced with polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA), respectively. The three soluble microneedle vaccines were administered via transdermal patch immunization using the method described in Example 3. The microneedle status was observed under a microscope, and the results are shown in [Figure 3]. Figure 10 In this context, A represents the result of immunization with a complete microneedle patch prepared using sodium hyaluronate as the needle tip layer in Example 2; B represents the result of immunization with a patch prepared using polyvinylpyrrolidone as the needle tip layer; and C represents the result of immunization with a patch prepared using polyvinyl alcohol as the needle tip layer.

[0064] The results showed that the mechanical strength of the tip layer of the soluble microneedles prepared by PVP was insufficient, making them prone to breakage during drug administration and unable to penetrate the skin. The microneedles prepared by PVA were also prone to breakage when the injection angle was less than 90 degrees. In contrast, the soluble microneedles prepared with sodium hyaluronate (SH) selected in this invention exhibited suitable mechanical strength, good toughness, and uniform solubility at the needle tip, providing excellent conditions for the fabrication of soluble microneedles.

[0065] Example 11 In Example 2, pullulan polysaccharide in the backing layer of the low-dose group of soluble microneedle vaccines was replaced with sodium hyaluronate (SH) and polyvinyl alcohol (PVA), respectively. The three soluble microneedle vaccines were administered via transdermal patch immunization using the method described in Example 3. The microneedle status was observed under a microscope, and the results are shown in [Figure 3]. Figure 11In this case, A represents the complete microneedles prepared using pullulan polysaccharide from Example 2 as the dorsal layer, which were applied to the back of mice for immunization, and from... Figure 11 In diagram D, the microneedles are completely absorbed and dissolved; in diagram B, sodium hyaluronate is used as the backing layer. The results after skin grafting immunization in mice show that the mouse skin was not punctured by the needle tip layer. Figure 11 Image E shows that the needle tip layer is relatively soft, causing all the needle tips to be flattened after external pressure. Image C shows the results of immunizing mice with polyvinyl alcohol as the dorsal layer. The image shows that some needle tips penetrated the mouse skin, but the overall penetration efficiency of the patch was not high. Figure 11 The image in middle F shows the microneedles after skin grafting, where some needle tips are mechanically broken.

[0066] The results showed that the soluble microneedle backing layer prepared by SH dissolved too quickly, resulting in rapid melting during drug delivery and hindering effective transdermal administration. The soluble microneedle backing layer prepared by PVA was prone to breakage when the drug delivery angle was less than 90 degrees. In contrast, the soluble microneedles prepared by pullulan combined with sodium hyaluronate selected in this invention exhibited suitable mechanical strength, provided better support for transdermal drug delivery at the needle tip, and possessed favorable fabrication conditions for soluble microneedles.

[0067] Example 12 The final concentration of sodium hyaluronate in the low-dose group of soluble microneedle vaccines in Example 2 was adjusted to 200 mg / ml, 300 mg / ml, and 400 mg / ml as the needle tip layer. The three soluble microneedle vaccines were administered via transdermal immunization using the method described in Example 3. The microneedle status was observed under a microscope, and the results are shown in [Figure 3]. Figure 12 In Figures A, B, and C, the state of the soluble microneedle tip layer prepared with sodium hyaluronate at a final concentration of 200 mg / ml after demolding.

[0068] The results showed that the soluble microneedle tip layer prepared with sodium hyaluronate at a final concentration of 200 mg / ml was too soft, resulting in an unformed tip and numerous vacuoles after demolding. Conversely, the tip layer prepared with sodium hyaluronate at a final concentration of 400 mg / ml was too hard, making it prone to breakage during demolding. In contrast, the soluble microneedles prepared using 300 mg / ml sodium hyaluronate as the tip layer combined with 200 mg / ml pullulan as the backing layer exhibited suitable mechanical strength, good transdermal drug delivery, and demonstrated favorable fabrication conditions for soluble microneedles.

[0069] Example 13 The final concentration of pullulan polysaccharide in the low-dose group soluble microneedle vaccine of Example 2 was adjusted to 100 mg / ml, 200 mg / ml, and 300 mg / ml as a backing layer. The three soluble microneedle vaccines were administered via skin grafting using the method of Example 3. The microneedle status was observed under a microscope, and the results are shown in [Figure 3]. Figure 12 In the diagram, D represents the state of the soluble microneedle backing layer prepared with pullulan at a final concentration of 100 mg / ml after demolding; E represents the state of the soluble microneedle backing layer prepared with pullulan at a final concentration of 200 mg / ml after demolding; and F represents the state of the soluble microneedle backing layer prepared with pullulan at a final concentration of 300 mg / ml after demolding.

[0070] The results showed that the soluble microneedle backing layer prepared with pullulan at a final concentration of 100 mg / ml was too soft, and its rigidity was insufficient to support demolding during unmolding, leading to air bubbles in the microneedles. Conversely, the soluble microneedle backing layer prepared with pullulan at a final concentration of 300 mg / ml was too hard, resulting in a brittle and bent microneedle that could not adhere to the skin for insertion. In contrast, the soluble microneedles prepared using 200 mg / ml pullulan as the backing layer and 300 mg / ml sodium hyaluronate as the tip layer exhibited suitable mechanical strength, provided good support for transdermal drug delivery, and demonstrated favorable fabrication conditions for soluble microneedles.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A soluble patch microneedle for delivering an inactivated enterovirus vaccine, comprising microneedles and a backing, characterized in that, The microneedles comprise sodium hyaluronate and enterovirus inactivated antigen encapsulated therein, and the backing material comprises pullulan polysaccharide solution; the enterovirus inactivated antigen is enterovirus 71 inactivated antigen and coxsackievirus A16 inactivated antigen; the concentration of the pullulan polysaccharide solution is 180-220 mg / mL; and the final concentration of sodium hyaluronate in the microneedles is 280-320 mg / mL.

2. The soluble transdermal microneedle according to claim 1, characterized in that, The enterovirus is Enterovirus 71 strain FY23; the Coxsackievirus A16 strain is Coxsackievirus A16 strain KM / M08.

3. The soluble transdermal microneedle according to claim 1 or 2, characterized in that, The area of ​​each of the soluble microneedles is 144 mm². 2 The dosage of the enterovirus 71 inactivated antigen is 50-100 U / patch, and the dosage of the coxsackievirus A16 inactivated antigen is 50-100 U / patch.

4. The soluble transdermal microneedle according to claim 3, characterized in that, In the soluble microneedles, the dose of enterovirus 71 inactivated antigen is 50-70 U / patch, and the dose of coxsackievirus A16 inactivated antigen is 50-70 U / patch.

5. The soluble transdermal microneedle according to claim 1, characterized in that, The microneedle has a tip length of 800µm; the solvent for the pullulan polysaccharide solution includes phosphate buffer.

6. The method for preparing the soluble transdermal microneedles according to any one of claims 1 to 5, characterized in that, Includes the following steps: Enterovirus inactivated antigen and sodium hyaluronate solution are mixed to obtain vaccine injection solution; The vaccine needle solution is injected into a microneedle mold to obtain a microneedle mold filled with microneedles; The pullulan polysaccharide solution is injected into the microneedle mold filled with microneedles, covering the upper layer of the formed microneedle body so that it completely covers the bottom of all the needles. It is then dried at 35-37℃ for 3.5-4 hours, demolded, and the soluble skin-penetrating microneedles are obtained.

7. The preparation method according to claim 6, characterized in that, After the vaccine needle solution is injected into the microneedle mold, it is subjected to vacuuming and centrifugation in sequence; before drying, the microneedle mold containing pullulan polysaccharide solution is subjected to vacuuming.

8. The use of the soluble transdermal microneedles according to any one of claims 1 to 5, or the soluble transdermal microneedles prepared by the preparation method according to claim 6 or 7, in the preparation of vaccines or medical devices for the prevention of hand-foot-mouth disease.

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