Nasal mucosa microneedle vaccine delivery system capable of inducing mucosal immunity as well as preparation method and application of nasal mucosa microneedle vaccine delivery system

Abstract

The invention discloses a nasal mucosa microneedle vaccine delivery system capable of inducing mucosa immunity and a preparation method and application of the nasal mucosa microneedle vaccine delivery system. The administration mode of vaccines is nasal mucosa microneedle vaccine delivery. The soluble layered microneedle is characterized in that a needle body is inspired by a hunting claw, a leopard-claw-shaped needle tip is designed, and the needle tip is bent; the strategy is proved to be capable of inducing potent antigen-specific immune response in mucous membrane tissues. The technology can be widely applied to research and development of novel mucosal vaccines for preventing respiratory tract infection pathogens (such as influenza viruses and the like).

Description

Technical Field

[0001] The present invention relates to the technical field of microneedle administration of vaccines, and in particular to a nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity, and a preparation method and application thereof. Background Art

[0002] The history of human development is a history of human struggle against infectious diseases. Infectious diseases caused by pathogens seriously threaten human health. Vaccination is a powerful means to address the threats of emerging and established infectious diseases, and it is listed as one of the 10 greatest public health achievements of the 20th century.

[0003] The nasal mucosa is the first barrier that airborne pathogens must pass through. Therefore, inducing a strong local immune response at the main entry site of airborne pathogens is key to fighting infection. Nasal mucosal administration involves introducing the vaccine directly into the nasal cavity, targeting the upper respiratory tract, and includes nasal sprays, drops, and inhaled powders. Nasal vaccines are a valuable tool in the field of public health, with advantages such as easy administration, needle-free vaccination, and the ability to induce local and systemic immune responses. Existing respiratory mucosal immunization vaccines have the risk of vaccine particle spillage during nasal spray or aerosol inhalation of vaccines; nasal mucociliary clearance and mucus barriers will hinder the uptake of vaccine antigens, resulting in reduced vaccine efficacy and other challenges.

[0004] Vaccination is considered one of the most effective measures to prevent and control the spread of infectious diseases. Traditional vaccination methods are mainly oral and injection. Oral vaccines are easy to use, but require antigens to pass through the gastrointestinal tract, which will lead to reduced immunogenicity, low absorption and utilization, and side effects in the liver; injection vaccines are subject to many restrictions, such as the need for professional medical staff to operate; injections may cause pain and bleeding, and people who are afraid of injections have poor compliance with vaccination; before injection, the vaccine must be prepared into an unstable liquid that needs to be refrigerated or prepared into a freeze-dried powder for reconstitution; there are risks of needlestick injuries and reuse of needles during injection, which increases the possibility of cross-contamination, etc., resulting in low vaccination rates and insufficient vaccine effectiveness. The development of new vaccine delivery systems is imminent.

[0005] Microneedles use a variety of natural materials and synthetic polymers, with lengths ranging from a few hundred microns to a few millimeters. They have uniform nano or micro structures and can easily penetrate the stratum corneum of the skin in a minimally invasive manner without touching the pain nerves, forming transient pores in the skin. They can effectively deliver drugs to the target area without causing pain and skin damage, and maintain local drug concentrations for a long time, greatly enhancing the efficiency of systemic drug delivery while improving patient compliance. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide a novel nasal mucosal microneedle vaccine delivery system that can induce mucosal immunity.

[0007] The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity of the present invention comprises a backing layer, a needle body and a support rod. The backing layer is a cylinder, and a plurality of rows of "leopard claw-shaped" needle bodies are arranged along the cylinder. The needle bodies are curved, and the support rod is fixed to one end of the backing layer cylinder. The needle body contains a vaccine.

[0008] Preferably, the vaccine is an influenza vaccine, such as a quadrivalent subunit influenza vaccine.

[0009] Preferably, the raw materials for preparing the backing layer include one or more of gelatin, polyvinyl alcohol, chitosan quaternary ammonium salt, polyethylene glycol, silk fibroin, and polyvinyl pyrrolidone.

[0010] Preferably, the needle body is made of a soluble high molecular polymer, including one or more of sodium carboxymethyl cellulose, chitosan quaternary ammonium salt, hyaluronic acid and sodium hyaluronate.

[0011] Preferably, the support rod is made of ABS plastic or polyurethane.

[0012] Preferably, the support rod fixing method includes bonding, casting or 3D printing.

[0013] Preferably, the selected backing layer has a diameter of 0.6-2.5 mm, the needle body is in the shape of a "leopard claw", the outer diameter distance from the needle tip to the needle bottom circular surface is 100-500 μm, the height is 100-1000 μm, the diameter of the needle bottom circular surface is 100-500 μm, the needle tip diameter is 1-20 μm, and the spacing between adjacent needle bodies is 200-1500 μm.

[0014] The second object of the present invention is to provide a method for preparing a nasal mucosal microneedle vaccine delivery system that can induce mucosal immunity, comprising the following steps:

[0015] The vaccine and hyaluronic acid are mixed to prepare a hyaluronic acid vaccine solution, which is cast in a microneedle negative mold, and bubbles and excess solution outside the needle tip are removed. The soluble high molecular polymer solution is vacuumed into the mold and dried at 2 to 6°C to form a needle body in the microneedle negative mold; the gelatin solution is cast in a cylindrical negative mold, and the mold is hollowed out and dried at 2 to 6°C to obtain a backing layer; a gelatin and polyvinyl alcohol mixture is coated on the base of the backing layer so that the base and the needle body are completely fitted together, and dried at 2 to 6°C; finally, the needle body, the backing layer and the support rod are bonded together using a gelatin and polyvinyl alcohol mixture, and the mold is demolded after drying at 2 to 6°C. The support rod is fixed to one end of the cylinder of the backing layer to obtain a nasal mucosal microneedle vaccine delivery system that can induce mucosal immunity.

[0016] The third object of the present invention is to provide a nasal mucosal microneedle vaccine delivery system that can induce mucosal immunity for use in vaccines to prevent pathogens that infect the respiratory tract.

[0017] Preferably, the respiratory tract infection pathogen comprises influenza virus.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention constructs a nasal mucosal microneedle vaccine delivery system that can induce mucosal immunity, which can induce the body to produce specific antibodies at the mucosal site and directly establish an immune defense line on the mucosal surface. This technology is widely used in the development of new nasal mucosal microneedle vaccines for pathogens (such as influenza viruses, etc.) that prevent respiratory infections.

[0020] The vaccine provided by the present invention has sufficient mechanical strength to pierce the nasal mucosa, thereby delivering the vaccine into the nasal mucosal tissue and cells, and has high dissolution efficiency and good safety.

[0021] Nasal mucosal microneedle vaccines can not only avoid the stress response caused by intramuscular injection, but also induce the body to produce specific antibodies in the mucosal area, and directly establish an immune defense line on the mucosal surface. The nasal mucosal microneedle vaccine delivery system can enhance the mucosal adhesion of antigens, avoid the clearance of water-soluble antigens by mucus and nasal cilia, prolong the retention time of antigens in the nasal cavity, open the tight junctions between epithelial cells, improve the internalization of antigens by monolayer epithelial cells, and promote the transdermal absorption of antigens; encapsulate antigens to prevent degradation by enzymes in the nasal cavity. This technology can be widely used in the development of new nasal mucosal microneedle vaccines to prevent mucosal infection of pathogens (such as influenza viruses, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the preparation process of the nasal mucosal microneedle vaccine delivery system;

[0023] Figure 2 Optical microscope observation and scanning electron microscope results of the nasal mucosal microneedle vaccine delivery system;

[0024] Figure 3 It is a mechanical characterization diagram of the nasal mucosal microneedle vaccine delivery system and an in vitro fluorescence image of nasal mucosal puncture;

[0025] Figure 4 The in vivo fluorescence imaging results of the nasal mucosal microneedle vaccine delivery system;

[0026] Figure 5 This is the scanning electron microscopy result and H&E staining section of the nasal mucosa after immunization with the nasal mucosal microneedle vaccine delivery system;

[0027] Figure 6The statistical results of serum biochemical indexes after immunization with the nasal mucosal microneedle vaccine delivery system;

[0028] Figure 7 This is the H&E stained section of the main organs of rats after immunization with the nasal mucosal microneedle vaccine delivery system;

[0029] Figure 8 This is the result of sIgA in the proximal and distal mucosal lavage fluids of Wistar rats immunized with the nasal mucosal microneedle vaccine delivery system loaded with quadrivalent subunit influenza vaccine;

[0030] Fig. 9 This is the specific antibody result after Wistar rats were immunized with quadrivalent subunit influenza vaccine loaded with nasal mucosal microneedle vaccine delivery system;

[0031] Fig.10 This is the result of HI titer after Wistar rats were immunized with quadrivalent subunit influenza vaccine loaded with nasal mucosal microneedle vaccine delivery system;

[0032] Fig.11 Elispot was used to detect HA protein-specific T cell responses in the spleen after immunization with quadrivalent subunit influenza vaccine loaded into the nasal mucosal microneedle vaccine delivery system;

[0033] Fig.12 Loading the nasal mucosal microneedle vaccine delivery system with quadrivalent subunit influenza vaccine after immunization with CD4 + CD8 + T cell cytokine level results DETAILED DESCRIPTION

[0034] Example 1 Preparation of nasal mucosal microneedle vaccine

[0035] like Figure 2 As shown, the nasal mucosal microneedle vaccine of this embodiment includes a backing layer 1, a needle body 2 and a support rod 3. The backing layer is cylindrical, and two rows of "leopard claw-shaped" needle bodies are arranged along the cylindrical direction of the cylinder. It is inspired by the cheetah's claws. Its curved shape enhances the adhesion of the nasal mucosa. The bionic "leopard claw" shape is distributed in the backing layer, and the support rod is fixed to one end of the backing layer cylinder. The selected backing layer has a diameter of 0.6mm, and the needle body is in the shape of a "leopard claw". The outer diameter distance from the needle tip to the needle bottom circular surface is 500μm, the height is 500μm, the diameter of the needle bottom circular surface is 256μm, the needle tip diameter is 1μm, and the spacing between adjacent needle bodies is 800μm.

[0036] like Figure 1As shown, the present invention mixes a quadrivalent subunit influenza vaccine with hyaluronic acid to prepare a hyaluronic acid vaccine solution with a mass fraction of 30%, casts it in a microneedle negative mold, removes bubbles and excess solution outside the needle tip, vacuumizes the soluble polymer solution into the mold, dries at 2 to 6°C, and forms a needle body in the microneedle negative mold. Dissolve polyvinyl alcohol in 90°C distilled water for 4 hours to obtain polyvinyl alcohol with a volume fraction of 10%; dissolve methacrylic gelatin in distilled water at room temperature to obtain methacrylic gelatin with a volume fraction of 10%, cast a mixed solution of 10% methacrylic gelatin and 10% polyvinyl alcohol in a volume ratio of 1:1 in a cylindrical negative mold, evacuates the mold, dries at 2 to 6°C, crosslinks at 365nm ultraviolet for 40s, refrigerates and dries at 4°C, and demolds to obtain a backing layer. A 10% methacryloyl gelatin and 10% polyvinyl alcohol mixed solution with a volume ratio of 1:1 was applied to the base of the backing layer, so that the base and the two rows of needle bodies were completely fitted, and dried at 2-6°C to make the needle bodies adhere to the backing layer. Finally, a 10% methacryloyl gelatin and 10% polyvinyl alcohol mixed solution with a volume ratio of 1:1 was used to bond the two parts of the needle body and the support rod to the backing layer, so that the support rod was fixed to one end of the cylinder of the backing layer, and the IIV4@HA Mns nasal mucosal microneedle vaccine delivery system was obtained after drying at 2-6°C.

[0037] Example 2 Morphology Characterization

[0038] The IIV4@HA Mns nasal mucosal microneedle vaccine delivery system prepared in Example 1 was observed by optical microscopy, and its surface morphology was observed by scanning electron microscopy (GeminiSEM 300, zeiss, Oberkochen, Germany) at 20 KV after gold spraying.

[0039] like Figure 2 (A) Optical microscope observation and Figure 2 (B) As shown in the scanning electron microscopy results, the prepared microneedle array has a complete structure and orderly arrangement. The needles are all "leopard claw-shaped", with a height of 500μm and a needle spacing of 800μm.

[0040] Example 3 Mechanical Properties Characterization

[0041] (1) Mechanical strength test: The IIV4@HA Mns nasal mucosal microneedle vaccine delivery system prepared in Example 1 was tested for its mechanical strength using an electronic universal testing machine for microneedles. Specifically, the microneedle was vertically placed above the probe of a texture analyzer. After the probe contacted the microneedle (the trigger force reached 0.5 N at this time), the microneedle was squeezed downward at a speed of 30 mm / min until the deformation of the sample reached 40% and then returned, and a force-displacement curve was plotted. The diameter of the probe was 5 mm, and the mechanical strength of each microneedle was calculated based on the number of microneedles contacted by the probe.

[0042] The experimental results are as follows Figure 3 (A) shows that each nasal mucosal microneedle can withstand a pressure of more than 0.1N and can effectively puncture the nasal mucosal tissue.

[0043] (2) In vitro puncture depth test of pig nasal mucosa: Prepare a soluble microneedle loaded with rhodamine B, attach the microneedle vaccine to the isolated pig nasal mucosa tissue and press for 30 seconds to allow the microneedle to penetrate into the isolated pig nasal mucosa tissue. Quickly fix the isolated pig nasal mucosa tissue after microneedle action on a glass slide with the action site facing upwards and place it under a fluorescence microscope.

[0044] The experimental results are as follows Figure 3 (B) shows that the microneedles loaded with rhodamine can puncture the pig nasal mucosa in vitro, and the fluorescence microscopy results show that it can effectively puncture the nasal mucosa.

[0045] Example 4 Cy5.5-IIV4@HA Fluorescence of the local nasal cavity of rats after immunization with Mns

[0046] The vaccine (quadrivalent subunit influenza vaccine or IIV4@HA Mns nasal mucosal microneedle vaccine delivery system) was fluorescently labeled with Cy5.5 to prepare a nasal mucosal microneedle vaccine delivery system loaded with Cy5.5-IIV4. The Cy5.5 fluorescent probe was mixed with the IIV4@HA Mns nasal mucosal microneedle vaccine delivery system or the quadrivalent subunit influenza vaccine, and the pH was adjusted to 7.4, and then incubated at 4°C overnight. The unbound Cy5.5 molecules were removed by centrifugal washing with an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and the liquid in the ultrafiltration centrifuge tube was finally collected, which was the Cy5.5-labeled IIV4@HA Mns nasal mucosal microneedle vaccine delivery system or the quadrivalent subunit influenza vaccine, namely Cy5.5-IIV4@HA MNs and Cy5.5-IIV4. The protein concentration of Cy5.5-IIV4@HA MNs was determined by using a Micro-BCA protein quantitative detection kit and the concentration was calculated. The subsequent preparation method was the same as in Example 1. Wistar rats (female) were divided into a control group and a Cy5.5-IIV4@HA MNs group. The control group was immunized by intranasal drops, with a vaccine (Cy5.5-IIV4) at a dose of 3 μg / rat, and 50 μL was dripped into each rat; the Cy5.5-IIV4@HA MNs group was immunized intranasally, with a vaccine at a dose of 3 μg per rat, and 50 μL was dripped into each rat. After the rats were anesthetized with isoflurane, the rats were placed in a supine position, and the microneedles penetrated 5 to 10 mm into the rat's nasal cavity. After the microneedles were dissolved, they were removed. The fluorescence distribution of the respiratory system of Cy5.5-IIV4@HA MNs and Cy5.5-IIV4 at different time points after intranasal immunization was observed by a small animal in vivo imaging system, and the fluorescence signals were observed and counted by an IVIS spectral imaging system.

[0047] The experimental results are as follows Figure 4 The in vivo imaging of small animals showed local fluorescence in the nasal cavity after immunization. The results showed that compared with the control group, the nasal mucosal microneedles could significantly prolong the residence time of the vaccine in the nasal cavity.

[0048] Example 5 Preliminary evaluation of the safety of rats after immunization with IIV4@HA Mns

[0049] Female 8-10-week-old Wistar rats (weighing about 250g) were selected as model animals to evaluate the safety of IIV4@HA Mns nasal mucosal microneedle vaccine. The rats were randomly divided into 4 groups, 6 in each group, namely Control group (injection of normal saline), HA Mns group (nasal administration of hyaluronic acid), IIV4@HA Mns group (nasal administration of IIV4@HA Mns), and IIV4 IM group (intramuscular injection of quadrivalent subunit influenza vaccine). After the rats were anesthetized with isoflurane, the rats were placed in the supine position, and the microneedles were inserted into the rats' nasal cavity 5-10mm, and removed after the microneedles dissolved. The experimental groups were inoculated with 3μg of vaccine in the nasal cavity, and the control group was inoculated with the same amount of normal saline, and each was dripped or injected with 50μL.

[0050] 1. Irritation of nasal mucosal tissue

[0051] On the third day after the rats were immunized, they were euthanized and their nasal cavities were cut open to expose the nasal septum and bilateral turbinate bones. The nasal mucosal tissues were slowly peeled off, rinsed with saline and fixed with 4% glutaraldehyde. The nasal mucosal tissues were processed according to the scanning electron microscopy sample preparation method, and the morphological changes of nasal cilia were observed under a scanning electron microscope.

[0052] On the third day after rat immunization, the rat nasal tissue was surgically obtained after euthanasia and fixed by immersion in 4% paraformaldehyde; after routine decalcification, it was dehydrated, paraffin-embedded, sliced, and dewaxed; and then hematoxylin eosin (HE) staining and slide preparation: according to the method provided by the H&E staining kit, the dewaxed sections were stained, sealed with neutral gum, and photographed under an optical microscope.

[0053] 2. Blood biochemical indicators

[0054] After centrifugation at 3000 g for 10 min, 150 μL of serum was collected and analyzed for ALT, AST, TP, TBIL, BUN, and CR levels using an automatic blood biochemical analyzer.

[0055] 3. Histopathological observation of major organs

[0056] On the third day after rat immunization, tissues were surgically removed after euthanasia, and pathological sections of major organs were prepared. The specific operations are as follows: (1) Sampling: After surgically obtaining the rat heart, liver, spleen, lungs, and kidneys, cut them into appropriate sizes with a scalpel and place them in an embedding box; promptly place them in 4% paraformaldehyde for immersion and fixation for 2 to 3 days, and rinse the tissues with running water to remove excess paraformaldehyde; the nasal tissues were routinely decalcified before proceeding to the subsequent steps; (2) Dehydration: Preheat the dehydrator in advance, set the dehydration program, and place the rinsed tissue blocks in the dehydrator for dehydration; (3) Embedding: Preheat the embedding machine, immerse the tissue blocks in paraffin for embedding, and cool Then demould and seal for storage; (4) Sectioning: pre-cool the tissue block in advance, slice it continuously, put it into the slide spreader, flatten the slices and pick them up, dry them in a 56℃ oven for later use; Dewaxing: put the dried paraffin sections into xylene I solution (15min), xylene II solution (15min), anhydrous ethanol I (10min), anhydrous ethanol II (10min), 95% ethanol (5min), 90% (5min), 80% (5min), double distilled water (5min) in the following order. Hematoxylin eosin (HE) staining preparation: stain the dewaxed sections according to the method provided by the H&E staining kit, seal the sections with neutral gum, and take pictures under an optical microscope for observation.

[0057] The experimental results are as follows Figure 5 (A) shows that on day 0 after immunization, the surface of the HA Mns group and the IIV4@HA Mns group was slightly messy, and no cilia were observed to fall off. On day 3 after immunization, the morphology of nasal cilia was not significantly different from that of the normal group. The cilia were neatly arranged, evenly distributed, and long and dense. Figure 5 (B) shows that the nasal tissue showed no signs of irritation to the nasal cilia by the nasal mucosal microneedle vaccine delivery system.

[0058] The experimental results are as follows Figure 6 The results showed that there were no significant differences in blood biochemical indicators among the groups of the present invention, indicating that the nasal mucosal microneedle vaccine delivery system had no obvious liver and kidney toxicity.

[0059] The experimental results are as follows Figure 7 HE staining of sections of heart, liver, spleen, lung and kidney in each group showed no obvious abnormalities, suggesting that the nasal mucosal microneedle vaccine delivery system has no obvious toxicity.

[0060] Example 6 Mucosal immune response of nasal mucosal microneedle vaccine delivery system in rats

[0061] 1. Rats Grouping and Immunization

[0062] Wistar rats weighing 250-300 g were randomly divided into three groups, with 6 rats in each group, namely the Control group, IIV4@HA Mns group, and IIV4 IM group. The Control group was not treated. The rats in the IIV4@HAMns group were anesthetized with isoflurane and nasally inoculated with a 3 μg dose of vaccine (IIV4@HAMns). IIV4 IM is a quadrivalent subunit influenza vaccine injected intramuscularly. Each rat was inoculated with a 3 μg dose of vaccine by intramuscular injection. On the 28th day after immunization, the serum, lavage fluid, and spleen tissue of the immunized and control groups were collected for detection of relevant immune indicators.

[0063] ELISA was used to detect the IgA antibody levels in the nasal, bronchial lung lavage fluid and vaginal lavage fluid of rats. The nose, trachea and lungs were removed, and 0.5 mL of sterile PBS rinsing solution was drawn with a 1 mL syringe, inserted into the nostrils, the entrance of the lung bronchus, and the vaginal opening, and repeatedly rinsed three times, and collected in EP tubes, and stored at -70 ° C for inspection.

[0064] The ELISA experimental steps are as follows: (1) Prepare H1N1 HA protein before the experiment, dilute it to a concentration of 5 μg / mL with coating buffer, and coat a 96-well ELISA plate at 100 μL / well at 4°C overnight. (2) Wash the coated plate with PBST, 3 min / time, 250 μL / well, repeat the wash 3 times, and then pat dry. (3) Block the plate with PBS containing 0.05% Tween 20 and 1% bovine serum albumin (BSA), 150 μL / well, and block at 37°C for 2 h. (4) After blocking, discard the liquid in the wells, add 250 μL of washing solution to each well, let it stand for 1 min, then discard it, repeat the wash 3 to 5 times, and pat dry. (5) Dilute rat serum with blocking solution starting from 1:40, dilute lung lavage fluid starting from 1:2, dilute 2-fold (set up duplicate wells), set up blank control wells, and incubate at 37°C for 2 h. (6) Wash the plate with PBST, 3 min / time, 250 μL / well, repeat washing 3 times, and then pat dry. (7) Dilute the horseradish peroxidase (HRP) labeled sheep anti-mouse IgA antibody with blocking solution at a dilution of 1:2000, 100 μL / well, incubate at 37°C for 1 hour. (8) Wash the plate with PBST, 3 min / time, 250 μL / well, repeat washing 3 times, and then pat dry. (9) Add the color developing solution TMB, 100 μL / well, incubate at room temperature, keep away from light, and stop color development when the color turns blue. (10) Add 100 μL of stop solution to each well to stop the reaction, and measure the experimental results within 15 minutes. (11) Read the optical density (OD) at a wavelength of 450 nm using an enzyme reader. The highest serum dilution when P / N>2 was taken as the ELISA antibody titer of this experiment, with the positive serum OD450 value (P) / negative serum OD450 value (N).

[0065] The experimental results are as follows Figure 8 The results showed that after microneedle immunization of rat nasal mucosa, the proximal mucosa (nasal lavage fluid and lung lavage fluid) and distal mucosa (vaginal lavage fluid) of rats produced certain sIgA antibodies compared with the control group. The sIgA concentrations of both vaccination methods were higher than those of unvaccinated rats. The experimental results showed that the nasal mucosal microneedle vaccine delivery system can induce sIgA antibodies in rat mucosa.

[0066] Example 7 Humoral immune response of nasal mucosal microneedle vaccine delivery system in rats

[0067] Blood was drawn from the animals in Example 6 and then centrifuged to obtain serum. For H1N1, H3N2, BV, and BY specific antibodies, the method used was indirect ELISA, and the detailed steps were the same as in Example 6.

[0068] The experimental results are as follows Fig. 9 The results showed that rats produced certain specific antibodies after nasal mucosal microneedle immunization and intramuscular injection. The above experimental results show that rats can produce certain levels of H1N1, H3N2, BV, and BY specific antibodies after immunization with nasal mucosal microneedle vaccines. Compared with the control group, rat serum produced certain IgG antibodies, with significant differences. The experimental results show that the nasal mucosal microneedle vaccine delivery system can induce IgG antibodies in rat serum.

[0069] The experimental results are as follows Fig.10 The results showed that the HI antibody titer was detected by hemagglutination inhibition experiment by taking rat serum at different time points. The results showed that the HI antibody titer was still high 24 weeks after immunization.

[0070] Example 8 Cellular immune response of nasal mucosal microneedle vaccine delivery system in rats

[0071] 1. Isolation of Rat Splenic Lymphocytes

[0072] After removing the eyeballs of the treated rats in Example 6 and taking blood, the rats were de-whitened and killed, and soaked in 75% alcohol for a few minutes. The rats were placed in a clean bench and the spleen was removed. 5 to 8 mL of rat lymphocytes were added to a sterile culture dish for separation. The rat spleen was placed on a 200-mesh screen, and the spleen was gently ground using a syringe piston. The ground single cells were separated through the screen. The cells in the culture dish were blown and mixed, transferred to a 15 mL centrifuge tube, and then 1 mL of 1640 culture medium was gently added along the tube wall on the upper layer. Centrifuge at 800g at room temperature for 20 to 30 minutes, and a lower centrifugal speed was set (the speed was 3). Take out the white layer of cells in the middle, add 1640 culture medium, and centrifuge at 400g for 10 minutes. Discard the supernatant, add 1640 culture medium, and centrifuge at 300g for 10 minutes. Discard the supernatant, add complete culture medium to resuspend the cells, count them, and obtain rat spleen lymphocytes.

[0073] 2. Enzyme-linked Immunospot Assay

[0074] (1) Wetting: Add 25 μL of 70% ethanol to each well to pre-wet and pat dry. Add 200 μL / well of sterile 1xPBS and wash twice. According to the instructions, add 50 μL / well of coating solution (coating antibody) diluted with sterile 1xPBS and coat overnight at 4°C. (2) The next day, pour out the coating solution (coating antibody) and wash three times with sterile 1xPBS. Add 200 μL / well of diluted blocking solution and block at room temperature for 1 hour. Pour out the blocking solution, no need to wash, and directly add the test cells for ELI SPOT detection. (3) Take out the blocked plate and prepare to add cells. If it is a pre-coated plate or the blocking has been done before, first add 200 μL of RPMI-1640 culture medium and let it stand at room temperature for 10 minutes, then pour it out. Inoculate 1x10 6 cells, 100 μL / well, the distribution of cells in the wells should be as uniform as possible. HA protein is used as a stimulator and added to the stimulator experimental wells at a final concentration of 10 μg / mL. (4) Cover the plate and place it in a carbon dioxide incubator at 37°C for 18 to 24 hours. (5) Pour the cells and culture medium in the wells, add 200 μL / well 1xPBS, wash twice, wash 5 times with PBST, and dry on absorbent paper for the last time. Add 100 μL of diluted biotin-labeled detection antibody to each well, seal the plate, and incubate at room temperature for 2 hours. Wash 5 times with PBST, and dry on absorbent paper for the last time. Add 100 μL of diluted avidin to each well, seal the plate, and incubate at room temperature for 1 hour. Wash 5 times with PBST, and dry on absorbent paper for the last time. Prepare the colorimetric solution (AEC) according to the colorimetric solution preparation method in the solution preparation, add 100 μL of colorimetric solution to each well, seal the plate, and incubate at room temperature in the dark for 30 minutes. Wash, dry, and read the plate.

[0075] 3. Intracellular cytokine staining (ICS)

[0076] Before preparing the working solution, slowly invert to mix. Dilute Perm / Wash Buffer (10x) with deionized water, that is, 10mL Perm / Wash Buffer (10x) and add 90mL deionized water. It is recommended to prepare it before use. During the experiment, the buffer needs to be placed on ice or at 2-8℃. Add 2μL of activator and protein transport inhibitor to 1mL of cell culture medium and mix thoroughly. Continue to culture for 4-6 hours, and should not exceed 12 hours in the culture medium.

[0077] The specific operation is as follows: (1) Fixation / Permeabilization: remove the supernatant, vortex and resuspend the cell pellet, add 250μL of fixation / permeabilization solution, mix well, and incubate at 4℃ for 20 minutes. (2) Directly add 1mL 1x Perm / Wash Buffer, mix well, centrifuge at 500g for 5min, and discard the supernatant. (3) Remove the supernatant, vortex and resuspend the cell pellet, add 1ml 1x Perm / Wash Buffer, mix well, centrifuge at 500g for 5min, and discard the supernatant. (Repeat step 2) (4) Remove the supernatant, add 100μL 1x Perm / Wash Buffer to resuspend the cells (5) Add flow cytometry antibodies (IFN-γ, IL-2) for intracellular labeling, and incubate at 4℃ for 30min. At the same time, add 50μL BSB and incubate with specific antibodies. (6) Add 1mL 1x Perm / Wash Buffer, mix well, centrifuge at 500g for 5min, and discard the supernatant. (7) Remove the supernatant, resuspend the cell pellet by vortexing, add 2 mL of 1x Perm / Wash Buffer, mix well, centrifuge at 500 g for 5 min, and discard the supernatant. (8) Remove the supernatant, resuspend the cell pellet by vortexing, add 350 μL of PBS and detect on the analyzer.

[0078] The experimental results are as follows Fig.11 The results showed that the number of spots secreted by H1N1 influenza HA protein was plotted, and the nasal mucosal microneedle induced a higher T cell immune response than intramuscular injection. After microneedle immunization and intramuscular injection, the number of spots formed by spleen cells secreting INF-γ and IL-4 was significantly different from that of the control group. Fig.12 CD4+ T cells in spleen cells + T cells and CD8 + The secretion of IFN-γ and IL-2 cytokines in T cells increased to varying degrees. Compared with the control group, CD 8 + There were significant differences in the levels of IL-2 cytokine secreted by T cells.

Claims

1. A nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity, characterized in that: It includes a backing layer, a needle body and a support rod. The backing layer is a cylinder. Several rows of "leopard claw-shaped" needle bodies are arranged along the cylinder. The needle bodies are curved. The support rod is fixed to one end of the backing layer cylinder. The needle body contains a vaccine.

2. The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1, characterized in that: The vaccine is an influenza vaccine, preferably a quadrivalent subunit influenza vaccine.

3. The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1, characterized in that: The raw materials for preparing the backing layer include one or more of gelatin, polyvinyl alcohol, chitosan quaternary ammonium salt, polyethylene glycol, silk fibroin, and polyvinyl pyrrolidone.

4. The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1, characterized in that: The needle body is made of soluble high molecular polymer, including one or more of sodium carboxymethyl cellulose, chitosan quaternary ammonium salt, hyaluronic acid and sodium hyaluronate.

5. The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1, characterized in that: The support rod is made of ABS plastic or polyurethane.

6. The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1 or 5, characterized in that: The support rod fixing method includes bonding, casting or 3D printing.

7. The nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1, characterized in that: The selected backing layer has a diameter of 0.6-2.5 mm, the needle body is in the shape of a "leopard claw", the outer diameter distance from the needle tip to the needle bottom circular surface is 100-500 μm, the height is 100-1000 μm, the diameter of the needle bottom circular surface is 100-500 μm, the needle tip diameter is 1-20 μm, and the spacing between adjacent needle bodies is 200-1500 μm.

8. A method for preparing the nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity according to claim 1, comprising the following steps: The vaccine and hyaluronic acid are mixed to prepare a hyaluronic acid vaccine solution, which is cast in a microneedle negative mold, and bubbles and excess solution outside the needle tip are removed. The soluble high molecular polymer solution is vacuumed into the mold and dried at 2 to 6°C to form a needle body in the microneedle negative mold; the gelatin solution is cast in a cylindrical negative mold, and the mold is hollowed out and dried at 2 to 6°C to obtain a backing layer; a gelatin and polyvinyl alcohol mixture is coated on the base of the backing layer so that the base and the needle body are completely fitted together, and dried at 2 to 6°C; finally, the needle body, the backing layer and the support rod are bonded together using a gelatin and polyvinyl alcohol mixture, and the mold is demolded after drying at 2 to 6°C. The support rod is fixed to one end of the cylinder of the backing layer to obtain a nasal mucosal microneedle vaccine delivery system that can induce mucosal immunity.

9. Use of the nasal mucosal microneedle vaccine delivery system capable of inducing mucosal immunity as claimed in claim 1 in preventing respiratory tract infection pathogen vaccines.

10. The use according to claim 9, characterized in that: The respiratory tract infection pathogens include influenza viruses.

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