Modularized combined microneedle patch based on mechanical interlocking tenon-and-mortise structure as well as preparation method and application of modularized combined microneedle patch

Abstract

The invention discloses a modular combined vaccine soluble microneedle patch based on a mechanical interlocking tenon-and-mortise structure as well as a preparation method and application of the modular combined vaccine soluble microneedle patch. According to the combined and spliced micro-needle vaccine patch designed by the invention, synchronous delivery of various vaccines is realized by virtue of a micro-needle technology through fragmented loading and mechanical interlocking'mortise and tenon 'structural design, mutual interference among different vaccines is reduced at the same time, efficient protection can be provided for groups needing multi-vaccine protection by virtue of one-time inoculation, the number of inoculants is reduced, and the immune procedure is simplified; vaccine hesitation is reduced, and inoculation rate is increased; meanwhile, the biosoluble material of the microneedle structure can quickly release antigens in the skin to stimulate immune cells to aggregate so as to enhance the immune effect; the micron-sized needle array (lt; 500 m) acts on the superficial layer of the skin, is painless and can be designed into a self-adhesive patch, so that the inoculation compliance is improved; single-dose inoculation can also reduce medical cost, is suitable for resource-deficient regions, improves accessibility and burdenability, and accelerates establishment of a population immune barrier.

Description

Technical Field

[0001] This invention relates to the field of microneedle drug delivery technology for combination vaccines, specifically to a microneedle transdermal drug delivery system, and particularly to a modular combination vaccine soluble microneedle patch based on a mechanically interlocked "mortise and tenon" structure that achieves modular splicing through a mechanical interlocking structure and is used for loading multiple vaccine antigens in different time zones and regions, as well as its preparation method and application. Background Technology

[0002] In the long history of humankind, the invention and application of vaccines is undoubtedly a major breakthrough in the field of public health. It not only provides humanity with a powerful weapon against infectious diseases but also greatly reduces the threat of disease to human health. As an important component of preventive medicine, the use of vaccines is widely regarded as one of the most brilliant achievements in the field of public health in the 20th century.

[0003] Multivalent vaccines (preventing multiple diseases) and combination vaccines (preventing different subtypes of the same pathogen) offer significant advantages in reducing the number of vaccinations and improving adherence, but their limitations cannot be ignored. In terms of research and development, the difficulty and time required far exceed those of monovalent vaccines. Different antigens can easily interfere with each other when mixed; for example, pertussis antigen may suppress the immune response to polio antigen, requiring repeated adjustments to the ratio and optimization of adjuvants to balance the effect. Simultaneously, the chemical properties of different antigens vary greatly (such as temperature sensitivity and pH), and they may become inactive due to interactions after mixing, requiring extremely high standards for production and storage environments, further increasing the complexity of research and development. In terms of safety, the risks are increased. Vaccines contain more antigens and adjuvants, increasing the probability of adverse reactions such as local redness, swelling, pain, and systemic fever and fatigue compared to monovalent vaccines, especially in infants and young children whose immune systems are not yet fully developed. The complexity of the composition also expands the risk of allergies; if specific components such as yeast are included, allergic individuals will be unable to use the vaccine. The applicable population is limited. Immunocompromised individuals or those receiving immunosuppressant therapy (such as HIV patients or cancer patients) often have poor tolerability and are generally contraindicated; furthermore, it is difficult to adapt to the disease prevalence spectrum of different regions, and may contain antigens of local low-incidence diseases, resulting in resource waste. Production and cost issues are prominent. The production process is complex, requiring simultaneous assurance of the activity, stability, and proportion of each antigen, with stringent quality control standards, leading to low production efficiency and low pass rates; high R&D and production costs make the vaccine price relatively high (e.g., the HPV 9-valent is about 3-4 times that of the 2-valent), limiting accessibility in low-income areas. Immunogenicity and update flexibility are also insufficient. Antigens with strong immunogenicity may "compete" for immune system resources, resulting in insufficient antibody titers for some antigens, reducing protective efficacy; long-term protective efficacy data requires longer verification time; when pathogens mutate, adjusting the formulation is much more difficult than with monovalent vaccines, resulting in a slower response time. These limitations are essentially a trade-off between "complexity" and "safety, efficacy, and accessibility," requiring continuous optimization through technological innovation.

[0004] Combination vaccines are used to prevent diseases caused by different pathogens; multivalent vaccines are used to prevent diseases caused by different serotypes / strains of the same pathogen. Compared to monovalent vaccines, combination vaccines have significant advantages, mainly in the following aspects: ① They can reduce the number of doses, simplify the immunization schedule, and improve vaccination rates and timeliness, especially in cases where children are delayed or missed from vaccination due to special circumstances. ② They can reduce the pain, stress, and discomfort caused by vaccination in infants and young children by reducing the number of doses, thus improving compliance. ③ They can relatively reduce the amount of total adjuvants, preservatives, or stabilizers used in vaccines, reducing the risk of adverse reactions. ④ They can reduce the frequency, time, and lost work costs for parents taking infants and young children to and from vaccination sites, and can also reduce the risk of related infectious diseases for infants, young children, and accompanying persons to some extent. ⑤ They can reduce the workload of vaccination personnel, improve work efficiency, and reduce potential vaccination errors and management risks. ⑥ They can simplify vaccine supply, reduce the difficulty of cold chain storage and storage space, and lower vaccination costs.

[0005] Current vaccines are generally administered via needle injection, which carries risks such as tissue damage, local pain, or inflammation. Patient compliance is poor, and before injection, vaccines must be formulated into unstable liquids that require refrigeration or into freeze-dried powders for reconstitution. There are also risks of needlestick injuries and needle reuse during injection, increasing the possibility of cross-contamination. These factors contribute to low vaccination rates and insufficient vaccine effectiveness. Therefore, there is an urgent need for a new vaccine administration route.

[0006] Microneedle patches, as an innovative transdermal drug delivery technology, offer significant advantages such as safety, painlessness, ease of operation, minimal tissue damage, reduced psychological burden, and improved medication adherence. They are particularly suitable for individuals with a fear of needles and for children. In the field of immunology, microneedles, through precise control of needle length, penetrate the stratum corneum and create microchannels between the epidermis and dermis. This characteristic effectively avoids damage to nerves or blood vessels and provides an ideal route for percutaneous vaccination. Notably, combined microneedle vaccine technology can deliver multiple vaccines simultaneously in a single dose, significantly reducing the frequency of administration and further improving patient adherence, offering a new direction for optimizing vaccination regimens.

[0007] Combination-based microneedle vaccines, an innovative technology in the field of vaccination, combine combination vaccines with microneedle delivery technology. Through segmented loading and a snap-fit ​​design, multiple vaccines can be delivered simultaneously, reducing interference between different vaccines. This allows for the simultaneous delivery of multiple vaccines during the same vaccination process, reducing the number of administrations required in traditional vaccination models and alleviating patient resistance to multiple injections. Furthermore, the painless and minimally invasive nature of microneedles (the micron-scale needle array acts on the superficial layer of the skin, avoiding contact with pain nerves) improves patient compliance, making it particularly suitable for children, the elderly, and other groups requiring multiple vaccine protection. This technology precisely splices multiple pathogen antigens or different serotype antigens of the same pathogen. By leveraging the mechanism of microneedles inducing the aggregation of skin immune cells and directly delivering antigens to immune-active areas, it can stimulate a stronger dual response of humoral and cellular immunity, prolong the activation cycle of immune memory cells, and enhance the persistence of the immune response. It has significant social value in reducing vaccine hesitation, optimizing the allocation of medical resources (such as reducing medical and nursing operation costs, cold chain pressure, and medical waste), and accelerating the construction of herd immunity, providing crucial support for infectious disease prevention and control and the upgrading of public health systems. Summary of the Invention

[0008] This invention aims to overcome the shortcomings of existing technologies and provide a modular microneedle vaccine system and its construction method. This system solves the compatibility problem of different vaccine components through physical isolation and achieves personalized and customizable vaccination through flexible assembly and combination.

[0009] The purpose of this invention is to provide a modular combined vaccine soluble microneedle patch based on a mechanically interlocked "mortise and tenon" structure, its preparation method, and its application. The microneedle patch provided by this invention enables rapid release of vaccines / drugs, meeting the need for rapid delivery. In addition, for vaccines or drugs that require multiple vaccinations or administrations, such as Hib and MenC vaccines, this microneedle patch can deliver multiple components simultaneously in a single vaccination, reducing the number of vaccinations and pain, and achieving multi-immune protection.

[0010] This invention provides a modular combined vaccine soluble microneedle patch based on a mechanically interlocked "mortise and tenon" structure, which is formed by combining multiple vaccine soluble microneedle patches together through a mechanically interlocked structure.

[0011] Preferably, the mechanical interlock is a mortise and tenon structure, a sawtooth structure, or a jigsaw puzzle structure.

[0012] Preferably, the vaccines in the plurality of vaccine soluble microneedle patches can be various available vaccines, such as IIV3, Hib, MenC, and 19F-PS vaccine soluble microneedles.

[0013] Preferably, the soluble microneedle patch includes a backing layer and microneedles disposed on the backing layer.

[0014] Preferably, the backing layer is prepared using a backing layer preparation material, and its size can be 0.6 to 2.5 mm in diameter and 100 to 1000 µm in thickness.

[0015] Preferably, the backing layer is prepared from one or more of the following materials: gelatin, quaternary ammonium salt, polyethylene glycol, silk fibroin, polyvinylpyrrolidone, polyvinyl alcohol, chitosan, and hyaluronic acid.

[0016] Preferably, the microneedles are formed by mixing a biosoluble material with a vaccine, casting the mixture into a microneedle mold, removing air bubbles and excess solution from the needle tip, centrifuging to allow the mixture to enter the mold, and drying to form the microneedles. The diameter of the needle root circular surface is 100–500 µm, and the spacing between adjacent needles is 200–1500 µm.

[0017] The biosoluble material is one or more of the following: hyaluronic acid, carboxymethyl cellulose, gelatin, sucrose, PVP, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyethylene glycol, and sodium hyaluronate.

[0018] The second objective of this invention is to provide a method for preparing a modular combination vaccine soluble microneedle patch assembly based on a mechanically interlocking "mortise and tenon" structure, comprising the following steps:

[0019] (1) First mold pouring

[0020] The material for preparing the backing layer is dissolved in a solvent to form a uniform backing liquid. The backing liquid is then coated onto a negative mold, dried, and removed from the mold to obtain the backing layer.

[0021] (2) Second mold pouring

[0022] The vaccine and biosoluble materials are dissolved in a solvent and mixed to obtain a homogeneous soluble polymer vaccine solution. The soluble polymer vaccine solution is coated onto a negative mold, centrifuged to allow the needle fluid to enter the holes of the negative mold, and then the negative mold is removed, the solution outside the holes is scraped off, and dried to obtain the microneedles.

[0023] (3) Third mold pouring

[0024] A soluble polymer vaccine solution or a soluble polymer vaccine solution without added vaccine is used as an adhesive to coat the microneedles in step (2). The dried backing layer is then attached to the adhesive, centrifuged, dried, and demolded to obtain the combined vaccine soluble microneedle patch assembly.

[0025] Preferably, the centrifugation is performed at 4500 rpm at 4°C.

[0026] Preferably, the drying is performed at 4°C.

[0027] A third objective of this invention is to provide the application of soluble microneedle patches for combination vaccines in vaccine preparation.

[0028] This invention designs a combined splicing microneedle vaccine patch. The combined design integrates multiple antigens into a single needle, providing highly effective protection for groups requiring multiple vaccines without the need for multiple doses, thus reducing the vaccination burden. The biosoluble material of the microneedle structure can rapidly release the vaccine and stimulate the aggregation of immune cells to enhance the immune effect. The micron-scale needle array (<500 µm) acts on the superficial layer of the skin, is painless, and can be designed as a self-adhesive patch, improving vaccination compliance and accessibility. A single dose can reduce medical costs, is suitable for resource-scarce areas, and can also increase vaccination rates to accelerate the establishment of herd immunity, showing significant advantages in public health events. Through "antigen splicing" and matrix regulation, it can be adapted to diverse scenarios, combining practicality and scalability.

[0029] Compared with traditional technologies, the present invention has the following advantages:

[0030] The method of this invention is safe and effective. Its unique design not only achieves precise quantitative drug delivery but also significantly enhances the immune response. By integrating combination vaccines into spliced ​​microneedle vaccines, compatibility issues are completely resolved: by physically isolating different vaccine components, mutual interference during storage is fundamentally avoided, ensuring the stability and potency of each vaccine. It offers extremely high flexibility: achieving true "programmable vaccines." Healthcare providers can customize vaccines based on age, region, season, and individual immunization history, like spliced ​​microneedles. Figure 1 This allows for the free combination of required vaccine units, enabling personalized immunization programs. It simplifies the production process: different vaccine units can be produced separately at different times and on different production lines, simplifying process development, making quality control easier, and significantly reducing production complexity and costs. It allows for staggered administration and booster immunization: some units can be used for primary immunization, with units containing a booster dose of antigen added for booster immunization, providing a new paradigm for immunization strategies. It improves the vaccination experience: the single-dose administration after assembly avoids the pain and inconvenience of multiple injections. Attached Figure Description

[0031] Figure 1 This is a diagram illustrating the preparation process of soluble microneedle patches for combination vaccines.

[0032] Figure 2 Design drawings and physical images of the mortise and tenon structure for mechanical interlocking;

[0033] Figure 3 Optical microscope images and scanning electron microscope images of the soluble microneedle patch for the combined vaccine;

[0034] Figure 4 Mechanical characterization of the soluble microneedle patch for the combined vaccine and in vitro fluorescence image of the patch inserted into the skin;

[0035] Figure 5 Image showing in vivo fluorescence imaging results of soluble microneedle patches for combination vaccines;

[0036] Figure 6 Image showing skin irritation results of the soluble microneedle patch for the combination vaccine;

[0037] Figure 7 This image shows the results of a routine blood test after immunization with a soluble microneedle patch containing a combination vaccine.

[0038] Figure 8 The image shows the serum biochemical parameters detected after immunization with the soluble microneedle patch of the combination vaccine.

[0039] Figure 9 H&E stained sections of major organs in mice after immunization with soluble microneedle patches containing a combination vaccine;

[0040] Figure 10 The image shows the results of specific antibody levels in mouse serum after immunization with the soluble microneedle patch of the combination vaccine.

[0041] Figure 11 The image shows the results of ELISPOT assay of specific T cell responses in the spleen after immunization with a soluble microneedle patch of the combination vaccine. Detailed Implementation

[0042] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0043] Example 1: Preparation of soluble microneedle patches for combination vaccines

[0044] This design belongs to the field of microneedle technology for dermal drug delivery. It aims to solve the problems of limited functionality in traditional monolithic microneedle arrays and poor biocompatibility and insufficient assembly stability in adhesive-jointed microneedles. It combines the traditional tenon-and-mortise interlocking principle with the miniaturization requirements of microneedles, achieving modular, adhesive-free, and stable assembly. Figure 2 A. For example Figure 1As shown, in this invention, a 40% hyaluronic acid solution (Shanghai Yuanye Biotechnology Co., Ltd.) was cast into a cylindrical negative mold and dried at 4°C to obtain a backing layer. IIV3, Hib, MenC, 19F-PS, and soluble polymers (each vaccine at 1 mg / mL, with a ratio of 0.4 mg of 40% hyaluronic acid per mL of vaccine) were separately mixed to prepare soluble polymer vaccine solutions. These solutions were then cast into microneedle negative molds. Air bubbles and excess solution outside the needle tip were removed. The molds were then centrifuged at 4500 rpm for 25 min at 4°C to allow the soluble polymer vaccine solutions to be deposited into the molds. The molds were dried at 4°C to form needles within the microneedle negative molds. A suitable amount of soluble polymer vaccine solution was used as an adhesive to coat the needle body. The dried backing layer was then attached to the adhesive. The mixture was centrifuged at 4500 rpm for 5 min at 4°C, dried at 4°C, and demolded to obtain soluble microneedle patches for IIV3, Hib, MenC, and 19F-PS, respectively. The uneven structure on the backing layer, as shown in the image, further enhances the effect. Figure 2 B. The four microneedle patches mentioned above are spliced ​​together to form a single microneedle patch, i.e., a soluble microneedle patch for combination vaccines. Figure 2 C. The selected backing layer has a diameter of 1.3 mm, a height of 700 µm, a needle bottom diameter of 320 µm, and a spacing of 600 µm between adjacent needles.

[0045] Example 2 Morphological Characterization

[0046] The soluble microneedle patch of the combined vaccine prepared in Example 1 was observed under an optical microscope, and its surface morphology was observed under a scanning electron microscope (GeminiSEM 300, Zeiss, Oberkochen, Germany) at 20 KV after being sputter-coated with gold.

[0047] Figure 3 (A) Figure 3 (B) Scanning electron microscopy results show that the prepared microneedle array structure is complete and orderly arranged, and the needles are all conical in shape with a height of about 600~700 µm.

[0048] Example 3 Mechanical property characterization

[0049] (1) Mechanical strength test: The mechanical strength of the combined vaccine soluble microneedle patch prepared in Example 1 was tested by a microneedle electronic universal testing machine. Specifically, the combined vaccine soluble microneedle patch (hereinafter referred to as microneedle) was placed vertically above the probe of the texture analyzer. After the probe contacted the microneedle (at which point the triggering force reached 0.5 N), the microneedle was squeezed downward at a speed of 30 mm / min until the deformation of the sample reached 40% and then returned. The force-displacement curve was plotted. The probe diameter was 5 mm. The mechanical strength of each microneedle was calculated based on the number of microneedles contacted by the probe.

[0050] Experimental results are as follows Figure 4 (A) shows that each needle of the soluble microneedle patch for the combination vaccine can withstand pressure of more than 0.3 N and can effectively puncture skin tissue.

[0051] (2) In vitro puncture capability test of microneedles: Pig skin was used as a substitute for human skin. The isolated pig skin was thawed, equilibrated with PBS solution for 1 h, and then taken out for use. Paper soaked in PBS solution was placed under the skin, and the skin was placed at 37°C to simulate the in vivo environment. Then, an appropriate amount of FITC was dissolved in HA solution (final concentration was 10 mg / mL) to replace the soluble polymer vaccine solution in Example 1, and FITC-loaded microneedles were prepared according to Example 1. The microneedles were inserted into the skin and the insertion was recorded.

[0052] like Figure 4 (B) shows that microneedles carrying FITC can be effectively inserted into pig skin tissue.

[0053] Example 4: Local fluorescence of Cy3.5-OVA@HA MNs in mice after immunization

[0054] Preparation of Cy3.5-OVA @HA MNs: Cy3.5-labeled OVA (chicken ovalbumin, Cy3.5-OVA) was dissolved in 1 / 10 of the reaction mixture with PBS. Then, 9 / 10 of the reaction mixture with sodium bicarbonate buffer solution at approximately pH 8.3 was added. The mixture was vortexed thoroughly and incubated overnight at 4°C. The next day, unbound Cy3.5 molecules were removed by centrifugation and washing using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. The solution containing the fluorescently labeled OVA, i.e., Cy3.5-OVA, was collected from the centrifuge tube, and the OVA concentration was measured. Cy3.5-OVA was then used to replace the vaccine in Example 1, and the preparation method of Example 1 was followed to obtain Cy3.5-labeled combination vaccine soluble microneedle patches, i.e., Cy3.5-OVA @HA MNs.

[0055] BALB / c female mice were divided into a control group (intramuscular injection group) and a Cy3.5-OVA@HAMNs group. After intramuscular injection of 100 μL of 3 µg Cy3.5-labeled OVA (Cy3.5-OVA) and Cy3.5-OVA@HAMNs soluble microneedle patches (inserted into the skin of the mouse back for 3 min for skin immunization, each patch containing 3 µg OVA), the fluorescence distribution of Cy3.5 at different time points after immunization and on the back was observed using a small animal in vivo imaging system, and the fluorescence signal was observed using an IVIS spectral imaging system.

[0056] Experimental results are as follows Figure 5As shown, the antigen in the intramuscular injection group remained in the mice for about 2 days, while that in the microneedle group remained for about 3 days. This suggests that the soluble microneedle patch of the combination vaccine can significantly prolong the residence time of the vaccine in the body.

[0057] Example 5: Preliminary safety evaluation of mice after immunization with soluble microneedle patches of the combination vaccine.

[0058] Female BALB / c mice (approximately 20 g in weight) aged 5–8 weeks were selected as animal models to evaluate the safety of the soluble microneedle patch of the combination vaccine. The mice were randomly divided into 7 groups of 6 mice each: Control group, IIV3 MN group, Hib MN group, MenC MN group, 19F-PC MN group, IIV3+Hib+MenC+19F-PC MN group, and IIV3+Hib+MenC+19F+PCMN group. On days 0, 14, and 28, mice were immunized with 30 μL of the corresponding vaccine containing 3 µg, 2.5 µg, 2.5 µg, and 2 µg of antigen, respectively, in the IIV3+Hib+MenC+19F-PC IM group. In the IIV3 MN group, Hib MN group, MenC MN group, 19F-PC MN group, and the IIV3+Hib+MenC+19F-PC MN group (the soluble microneedle patch of the combined vaccine from Example 1, containing 3 μg, 2.5 µg, 2.5 µg, and 2 µg of IIV3, Hib, MenC, and 19F-PS), skin immunization was performed by inserting a microneedle patch into the back of the mouse, with antigen concentrations of 3 μg, 2.5 µg, 2.5 µg, and 2 µg, respectively. The control group was inoculated with an equal volume of PBS.

[0059] The IIV3 MN group, Hib MN group, MenC MN group, and 19F-PC MN group used soluble microneedle patches of IIV3, Hib, MenC, and 19F-PC, respectively.

[0060] 1. Skin irritation

[0061] The hair on the backs of mice was shaved off, and mice with intact skin and no redness or swelling after hair removal were selected for skin irritation testing. Subsequently, the soluble microneedle patch of the combination vaccine was inserted into the skin of the mice, and photographs were taken at 0, 5, 10, 15, 20, and 30 minutes after the microneedles were removed for record-keeping.

[0062] Experimental results are as follows Figure 6As shown in the figure (IIV3+Hib+MenC+19F-PC MN), the microchannels pierced by the microneedles in the skin gradually disappeared after 30 minutes, and no adverse phenomena such as edema, erythema or lumps occurred during the process. This suggests that the soluble microneedle patch of the combination vaccine is a safe material and will not cause skin irritation.

[0063] 2. Complete blood count (CBC)

[0064] On day 42 post-immunization, blood was collected from the orbital venous plexus of mice and placed into anticoagulant tubes containing EDTA. The levels of RBC, WBC, and PLT were analyzed using a fully automated blood routine analyzer.

[0065] Experimental results are as follows Figure 7 As shown, the RBC, WBC, and PLT levels in each group were all within the normal range, indicating that the soluble microneedle patch of the combination vaccine is non-toxic.

[0066] 3. Blood biochemistry tests for liver and kidney toxicity

[0067] Blood was collected from the orbital venous plexus of mice, left to stand at room temperature for 2 h, and centrifuged at 3000 g for 10 min. 150 µL of serum was collected and the levels of ALT, AST, CK, LDH, BUN, and CR were analyzed using a fully automated blood biochemistry analyzer.

[0068] Experimental results are as follows Figure 8 As shown, ALT, AST, CK, LDH, BUN, and CR in each group were all within the normal range, indicating that the soluble microneedle patch of the combination vaccine has no hepatotoxicity or nephrotoxicity.

[0069] 4 H&E slices

[0070] On day 42 after immunization, the mice were euthanized, and tissues were surgically removed after euthanasia. Pathological sections were prepared from the major organs. The specific operation is as follows: (1) Sampling: After obtaining the heart, liver, spleen, lung and kidney of the mouse through surgery, cut the appropriate size with a scalpel and put it into the embedding box; soak and fix it in 4% paraformaldehyde for 2-3 days in time, and rinse the tissue with running water to remove excess paraformaldehyde; (2) Dehydration: preheat the dehydrator in advance, set the dehydration program, and put the rinsed tissue block into the dehydrator for dehydration; (3) Embedding: preheat the embedding machine, immerse the tissue block in paraffin for embedding, demold after cooling, and seal for preservation; (4) Slicing: precool the tissue block in advance, slice continuously, put it into the slicer, flatten the slice and pick it up, dry it in a 56℃ oven for later use; Dewaxing: put the dried paraffin slice into xylene I solution (15 min), xylene II solution (15 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% ethanol (5 min), 90% (5 min), 80% (5 min), double distilled water (5 min) in the following order. Hematoxylin eosin (HE) staining preparation: The dewaxed sections were stained according to the method provided by the H&E staining kit, mounted with neutral resin, and photographed and observed under an optical microscope.

[0071] Experimental results are as follows Figure 9 As shown, no abnormalities were observed in the heart, liver, spleen, lungs, and kidneys of mice in each group, suggesting that the soluble microneedle patch of the combined vaccine does not have adverse effects on the internal organs of mice and is safe.

[0072] Example 6 Humoral immune response of soluble microneedle patches containing combination vaccines in mice

[0073] 1. Mouse grouping and immunization

[0074] The immunization grouping and procedure for mice were as described in Example 5. Mouse serum was collected at 14, 28, and 42 days for relevant immune index detection.

[0075] After blood was collected from the animals, the serum was obtained by centrifugation, and specific antibodies were detected using indirect ELISA. The detailed steps are as follows:

[0076] (1) Coating: Dilute each antigen stock solution to the corresponding concentration with PBS, add 100µL of diluted antigen to each well, incubate overnight at 4℃, discard the liquid in the well on the second day, wash 6 times with 0.1% PBST, with an interval of 30 s each time, and pat dry vigorously.

[0077] (2) Sealing: Add 100 µL of 2.5% skim milk powder to each well for sealing. After 2 h at 37℃, wash 6 times and pat dry.

[0078] (3) Add primary antibody: use 0.1% PBST to serially dilute the serum sample 100 times, add 100 µL to each well of the ELISA plate, incubate at 37℃ for 2 h, and wash the plate 6 times with 0.1% PBST.

[0079] (4) Incubation with secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG was diluted 5000 times with 0.1% PBST as secondary antibody. 100 µL was added to each well and incubated at 37℃ for 1 h. The mixture was washed 6 times with 0.1% PBST and then patted dry.

[0080] (5) Add 100 µL of TMB substrate to each well, incubate at room temperature in the dark for 15 min, terminate the reaction with stop solution, and then measure the OD value at 450 nm using a microplate reader. The OD value of positive serum was used as the reference value. 450 Value (P) / Negative serum OD 450 The highest serum dilution with P / N>2 was used as the ELISA antibody titer in this experiment.

[0081] The results are as follows Figure 10 As shown, the antibody level in the soluble microneedle patch group of the combined vaccine was higher than that in the control group, suggesting that the soluble microneedle patch group of the combined vaccine can induce the body to produce higher humoral immunity.

[0082] Example 7: Cellular Immune Response in Mice Using Combined Soluble Microneedle Patches

[0083] (1) Isolation of mouse lymphocytes: The mice treated in Example 5 were euthanized by cervical dislocation and disinfected by immersing them in 75% alcohol for about 2 minutes. The spleen was dissected in a clean bench and placed in serum-free RPMI 1640 medium for preservation. Then the spleen was removed, cut into pieces and placed in a cell mesh sieve. About 4-5 mL of mouse lymphocyte separation solution was added in batches and the spleen was gently ground with the syringe core until it was completely ground into a cell suspension.

[0084] Transfer the cell suspension to a 15 mL centrifuge tube, slowly add 2 mL of serum-free RPMI 1640 medium along the tube wall, centrifuge at 800 g for 30 min at room temperature, slowly aspirate the white layer cells (i.e., lymphocytes), wash with approximately 10 mL of serum-containing RPMI 1640 medium, centrifuge at 300 g for 10 min, discard the supernatant, repeat three times, and finally count the cells. Adjust the concentration to 5 × 10⁻⁶ cells by adding an appropriate amount of RPMI 1640 medium. 5 / mL, to obtain mouse lymphocytes.

[0085] (2) Enzyme-linked immunospot assay (ELISPOT):

[0086] ① Coating: Add 20 μL of 70% ethanol to each well of the ELISPOT plate for pre-wetting. After drying, add 200 μL of PBS to each well for washing and then dry again. Add 50 μL of diluted coating solution to each well and coat overnight at 4°C.

[0087] ② Blocking: The next day, pour out the coating solution and wash three times with PBS. Add 200 μL of diluted blocking solution to each well and block at room temperature for 1 h.

[0088] ③ Cell seeding and stimulation: After 1 h, pour out the blocking solution, wash with RPMI 1640 medium, pat dry, and then inoculate with 5×10⁻⁶ cells / mL. 5 Cells were seeded in each well at a concentration of 100 μL / well, and gently pipetted to ensure even distribution. Each antigen was added to the well at its optimal concentration as a stimulant. The wells were then covered with a cover plate, wrapped in aluminum foil, and incubated at 37°C with 5% CO2 for 24 hours.

[0089] ④ Add antibody and color development: Decant cells and culture medium, wash 3 times with PBS, then wash 5 times with 1×Washing Buffer and pat dry. Add 100 μL / well of diluted biotin-labeled detection antibody, seal the plate, incubate at room temperature for 2 h, wash 5 times with 1×Washing Buffer, incubate at room temperature for 1 h, wash 5 times with 1×Washing Buffer, pat dry, add 100 μL / well of AEC color development solution, react at room temperature in the dark for 25 min, after the reaction is complete, pour out the AEC color development solution, wash 3 times with sterile deionized water, pat dry, place the plate in a cool place at room temperature and let it air dry naturally before closing the base.

[0090] ⑤ Counting: Count the spots on the ELISPOT plate, record various parameters of the spots, and perform statistical analysis.

[0091] Experimental results are as follows Figure 11 The results, plotted with the number of spots secreted by each antigen stimulation, showed that the soluble microneedle patch of the combined vaccine induced a higher T-cell immune response than intramuscular injection. In mice, the number of spots formed by spleen cells secreting IFN-γ and IL-4 after microneedle immunization and intramuscular injection were significantly different compared to the control group.

Claims

1. A modular combination vaccine dissolvable microneedle patch based on mechanical interlocking "mortise and tenon" structure, characterized in that, The application discloses a combined vaccine soluble microneedle patch.

2. The combination vaccine soluble microneedle patch of claim 1, wherein, The mechanical interlocking structure is a mortise-tenon structure, a zigzag structure or a jigsaw puzzle concave-convex structure.

3. The combination vaccine soluble microneedle patch of claim 1, wherein, The vaccine is an IIV3, Hib, MenC or 19F-PS vaccine.

4. The combination vaccine soluble microneedle patch of claim 1, wherein, The soluble microneedle patch comprises a backing layer and microneedles arranged on the backing layer.

5. The combination vaccine soluble microneedle patch of claim 4, wherein, The backing layer is prepared from a backing layer preparation material and has a size of preferably 0.6-2.5 mm in diameter and 100-1000 µm in thickness.

6. The combination vaccine soluble microneedle patch of claim 5, wherein, The backing layer preparation material is one or more of gelatin, quaternary ammonium salt, polyethylene glycol, silk fibroin, polyvinylpyrrolidone, polyvinyl alcohol, chitosan and hyaluronic acid.

7. The combination vaccine soluble microneedle patch of claim 4, wherein, The microneedle is prepared by casting a biologically soluble material and a vaccine into a microneedle negative mold, removing bubbles and excess solution from the needle tip, centrifuging the mixed solution into the mold, drying and forming the microneedle.

8. The combination vaccine soluble microneedle patch of claim 7, wherein, The biologically soluble material is one or more of hyaluronic acid, carboxymethyl cellulose, gelatin, sucrose, PVP, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyethylene glycol and sodium hyaluronate.

9. A method of manufacturing a modular combination vaccine dissolvable microneedle patch based on mechanical interlocking "mortise and tenon" structure of any of claims 1-8, characterized in that, The method comprises the following steps: (1) first-time mold filling The preparation material of the backing layer is dissolved into a uniform backing liquid with a solvent, the backing liquid is coated on the negative mold, the negative mold is taken out after drying and demolding, and the backing layer is obtained; (2) second-time mold filling The vaccine and the biologically soluble material are dissolved with a solvent and mixed into a uniform soluble high molecular polymer vaccine solution, the soluble high molecular polymer vaccine solution is coated on the negative mold, the needle body liquid is centrifuged into the holes of the negative mold, then the negative mold is taken out, the solution outside the holes is scraped off, and the microneedle is obtained after drying; (3) third-time mold filling The soluble high molecular polymer vaccine solution or the soluble high molecular polymer vaccine solution without the vaccine is used as an adhesive and is coated on the microneedle in step (2), the dried backing layer is attached to the adhesive, and the microneedle patch is obtained after centrifugation and drying and demolding; Preferably, the centrifugation is carried out at 4500 rpm at 4℃. Preferably, the drying is carried out at 4℃.

10. The combined vaccine soluble microneedle patch according to any one of claims 1-8 is used for preparing a vaccine.

Images