A nanoparticle microneedle loaded with subunit vaccine and a preparation method thereof

Abstract

The application belongs to the field of medicine and particularly relates to a nanoparticle microneedle loaded with a subunit vaccine and a preparation method thereof.The preparation method is as follows: (I) preparing nanoparticles and loading the nanoparticles with antigens and adjuvants; (II) preparing nanoparticle microneedles and forming the nanoparticles at the tip of the microneedles.The preparation method can load the antigens, adjuvants and the like in the nanoparticles or on the surface of the nanoparticles and form the nanoparticles at the tip of the microneedles, so that when the nanoparticle microneedle is pressed on the skin, it can pierce the stratum corneum of the skin, deliver the antigens and adjuvants to the skin, target the lymph nodes, be taken up by the dendritic cells, cross-present the antigens, increase the expression of CCR9 of the dendritic cells, induce the dendritic cells to home to the mesenteric lymph nodes, and thus realize the intestinal mucosal immune protection effect.The application uses the skin route to inoculate the mucosal vaccine and further induces the protection effect of mucosal immunity.

Description

Technical Field

[0001] This invention belongs to the medical field, specifically relating to a nanoparticle microneedle loaded with a subunit vaccine and its preparation method. Background Technology

[0002] Helicobacter pylori and Salmonella can cause intestinal mucosal infections, and mucosal vaccines can trigger a strong protective immune response at the site of pathogen infection. However, the development of mucosal vaccines is technically very challenging because the pathogens colonize the intestinal mucosa. To prevent the colonization of these bacteria by the immune system, it is necessary to stimulate a mucosal immune response and induce effective secretory IgA (sIgA) secretion in order to produce an effective protective effect.

[0003] Previous studies have suggested that due to the tissue-specific activation of immune cells, vaccination via the respiratory mucosa or oral administration is necessary to induce local sIgA production and systemic immune responses. However, the respiratory mucosal microenvironment is complex, antigen molecules are easily degraded by numerous enzymes, and various physiological barriers (such as the bronchial barrier, mucus barrier, immune barrier, and cell membrane barrier) hinder antigen molecules from reaching effector targets. Oral vaccines must withstand the harsh environment of the gastrointestinal tract (such as low pH and intestinal proteases), where protein antigens are easily destroyed, reducing the vaccine's immunizing effect. Precisely because of its low delivery efficiency, most currently marketed oral vaccines are live attenuated vaccines. Large-scale use also poses safety risks such as incomplete pyrogen removal and viral reactivation. Immune cells activated through traditional peripheral injection methods such as intramuscular and subcutaneous injections remain only in the systemic immune system, failing to elicit a mucosal immune response and thus not effectively triggering a dual response of systemic and intestinal mucosal immunity. This is related to the homing behavior of immune cells to specific sites.

[0004] As an important immune organ, the skin contains a large number of antigen-presenting cells, mainly dendritic cells and their immature Langerhans cells, distributed in the epidermis and dermis. These cells can effectively take up, process, and present foreign antigens, and effectively stimulate an immune response after migrating to the draining lymph nodes. Therefore, the active epidermis and dermis are considered one of the best sites for vaccination. However, the most significant obstacle at present is the barrier function of the stratum corneum, which makes it difficult for large molecular antigens of vaccines to penetrate the skin surface and enter the active epidermis and dermis rich in antigen-presenting cells. Furthermore, how to achieve intestinal mucosal immunity through transdermal immunization remains unreported.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a method for preparing nanoparticle microneedles loaded with subunit vaccines, the preparation method comprising the following steps:

[0007] (I) Preparation of nanoparticles:

[0008] Prepare nanoparticles and load them with antigens and adjuvants;

[0009] (II) Preparation of nanoparticle microneedles:

[0010] (II-1) The nanoparticles are mixed with an aqueous solution of polyvinylpyrrolidone to obtain a mixture;

[0011] (II-2) Add the mixture to the microneedle mold and centrifuge to fill the tip of the microneedle mold with the mixture;

[0012] (II-3) Add an aqueous solution of polyvinylpyrrolidone without nanoparticles to the microneedle mold to form a microneedle backing layer;

[0013] (II-4) Dry and demold the microneedle mold to obtain soluble nanoparticle microneedles loaded with subunit vaccine;

[0014] or

[0015] (II-a) Polyethylene glycol diacrylate and dimethyl sulfoxide containing a photoinitiator and the nanoparticles are mixed to obtain a first solution;

[0016] (II-b) Polyethylene glycol diacrylate and dimethyl sulfoxide containing a photoinitiator are mixed to obtain a second solution;

[0017] (II-c) The first solution is added to the microneedle mold, so that the first solution fills the tip of the microneedle mold; the second solution is added to the microneedle mold, so that the second solution fills the backing layer of the microneedle mold;

[0018] (II-d) The microneedle mold is placed under ultraviolet light for cross-linking and curing. After demolding, swellable nanoparticle microneedles loaded with subunit vaccines are obtained.

[0019] Preferably, in step (I), the method for preparing the nanoparticles is as follows:

[0020] (I-1) The antigen aqueous solution is mixed with an organic solution containing a polymeric carrier and an adjuvant, and then sonicated to obtain a primary emulsion;

[0021] (I-2) The primary emulsion is added to a polyvinyl alcohol aqueous solution and homogenized to obtain a secondary emulsion;

[0022] (I-3) Add polyvinyl alcohol aqueous solution to the complex emulsion again, stir and centrifuge to wash (the purpose is to remove organic solvent and allow it to evaporate) to obtain nanoparticles loaded with antigen and adjuvant.

[0023] or

[0024] (Ia) An organic solution containing a polymeric carrier, adjuvant and surfactant is mixed with an aqueous solution of surfactant (such as poloxamer F127) and ultrasonically treated to obtain an emulsion.

[0025] (Ib) The emulsion is sequentially subjected to rotary evaporation under reduced pressure, washing, and freeze-drying. Before immunization, an antigen aqueous solution is added and stirred to obtain nanoparticles loaded with antigen and adjuvant.

[0026] Preferably, the antigen is one or more of recombinant Helicobacter pylori protein HpaA, recombinant Helicobacter pylori protein UreB1, or Salmonella protein; the polymeric carrier is one of polylactic-co-glycolic acid (PLGA) or gelatin; and the adjuvant is all-trans retinoic acid (atRA) or a combination of all-trans retinoic acid (atRA) and oligodeoxynucleotide chain (CpG).

[0027] Preferably, in step (I-1), the volume ratio of the antigen aqueous solution to the adjuvant organic solution is 1:3 to 1:10; the concentration of the antigen in the antigen aqueous solution is 0.1 to 5 mg / mL; the concentration of the adjuvant in the organic solution is 0.01 to 5 mg / mL, and the concentration of the polymer carrier is 5 to 40 mg / mL; the power of the ultrasonic treatment is 20 to 200 W, the working time in each ultrasonic cycle is 3 to 5 s, the interval time is 3 to 5 s, and the total ultrasonic time is 1 to 30 min.

[0028] Preferably, in step (I-2), the concentration of the polyvinyl alcohol aqueous solution is 1%; the rotation speed of the homogenizer is 7400-7600 r / min, and the homogenization time is 14-16 min.

[0029] Preferably, in step (Ia), the concentration of the polymeric carrier in the organic solution is 5–40 mg / mL, the concentration of the adjuvant is 0.1–15 mg / mL, and the concentration of the surfactant is 0.1–10 mg / mL; the concentration of the F127 aqueous solution is 0.5 wt.%; and the power of the ultrasonic treatment is 120 W.

[0030] Preferably, in step (Ib), the concentration of the antigen aqueous solution is 4 mg / mL, and the stirring time is 2–12 h.

[0031] Preferably, in step (II-a), each 1 mL of the first solution contains 0.99 mL of the polyethylene glycol diacrylate and 0.01 mL of the dimethyl sulfoxide; the dimethyl sulfoxide contains 10 mg / mL of the photoinitiator and 0.2–1 mg of the nanoparticles.

[0032] Preferably, in step (II-d), the wavelength of the ultraviolet light is 365 nm, and the power is 1–5 W / cm². 2 The cross-linking and curing time is 2 to 3 minutes.

[0033] Based on the same technical concept, another aspect of the present invention is to provide nanoparticle microneedles loaded with subunit vaccines obtained by the above preparation method.

[0034] The beneficial effects of this invention are as follows:

[0035] The preparation method described in this invention can load antigens and adjuvants onto the interior or surface of nanoparticles, and shape the nanoparticles at the tip of a microneedle. When the nanoparticle microneedle is pressed onto the skin, it can pierce the stratum corneum, delivering the antigen and adjuvant to the skin, targeting the lymph nodes, where they are then taken up by dendritic cells. Antigen cross-presentation occurs, and the expression of CCR9 on dendritic cells is increased, inducing dendritic cell homing to the mesenteric lymph nodes, thereby achieving a protective effect against intestinal mucosal immunity. In other words, this invention uses a skin route to administer a mucosal vaccine, thereby inducing a protective effect against mucosal immunity. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is an electron microscope image of the nanoparticles obtained in Example 1.

[0038] Figure 2 This is an electron microscope image of the nanoparticles obtained in Example 3.

[0039] Figure 3 This is a schematic diagram of the structure of nanoparticles.

[0040] Figure 4 This is a particle size distribution diagram of the nanoparticles obtained in Example 1.

[0041] Figure 5 Scanning electron microscope image of the nanoparticle microneedles obtained in Example 1.

[0042] Figure 6This is a schematic diagram of the structure of nanoparticle microneedles.

[0043] Figure 7 This is a photograph of nanoparticles loaded with fluorescent dye being delivered to the back of a mouse (after shaving).

[0044] Figure 8 This is a photograph showing the result of applying fluorescent dye-loaded nanoparticles directly to the back of a mouse (after shaving).

[0045] Figure 9 This is the result of antibody IgA detection in vaginal irrigation fluid after immunizing mice.

[0046] The attached figures are labeled as follows:

[0047] 1-Nanoparticle; 2-Antigen; 3-Adjuvant; 4-Needle tip; 5-Backing layer. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] Example 1

[0050] This embodiment provides a method for preparing nanoparticle microneedles loaded with subunit vaccines, the preparation method comprising the following steps:

[0051] (I) Preparation of nanoparticle 1:

[0052] (I-1) Mix 0.3 mL of HpaA aqueous solution (concentration 0.1 mg / mL) with 1.5 mL of trichloromethane organic solution containing polymer carrier PLGA (concentration 5 mg / mL) and atRA (concentration 0.01 mg / mL), and then sonicate at 20 W for 30 min (3 s interval between each cycle, 3 s working time) to obtain the primary emulsion;

[0053] (I-2) The primary emulsion was added dropwise to 10 mL of a 1 wt.% polyvinyl alcohol aqueous solution, and stirred for 16 min at a rate of 7400 r / min using a homogenizer to obtain a secondary emulsion;

[0054] (I-3) Add 0.1 wt.% of polyvinyl alcohol aqueous solution to the emulsion again, stir overnight at a rate of 600 r / min, wash the nanoparticles three times with pure water by centrifugation under a centrifugal force of 16000 g, and freeze-dry to obtain nanoparticles 1 loaded with antigen 2 and adjuvant 3.

[0055] (II) Preparation of nanoparticle microneedles:

[0056] (II-1) The nanoparticles 1 are mixed with an aqueous solution of polyvinylpyrrolidone (30%, w / v, molecular weight 3500) to obtain a mixture;

[0057] (II-2) The mixed solution is added to the polydimethylsiloxane (PDMS, 1×1cm) microneedle mold, so that each microneedle patch contains about 20μg of HpaA and 25μg of ATRA. The mixture is filled into the tip 4 of the microneedle mold by high-speed centrifugation.

[0058] (II-3) Add an aqueous solution of polyvinylpyrrolidone without nanoparticle 1 to the microneedle mold to form a microneedle backing layer;

[0059] (II-4) After the microneedle mold is thoroughly dried in a drying oven, it is demolded and stored in a refrigerator at 4°C to obtain soluble nanoparticle microneedles loaded with subunit vaccines (HpaA and atRA are loaded at the tip 4 sites).

[0060] Example 2

[0061] This embodiment provides a method for preparing nanoparticle microneedles loaded with subunit vaccines, the preparation method comprising the following steps:

[0062] (I) Preparation of nanoparticle 1:

[0063] (I-1) Mix 0.3 mL of HpaA aqueous solution (concentration of 5 mg / mL) with 1.5 mL of trichloromethane organic solution containing polymer carrier PLGA (concentration of 40 mg / mL) and atRA (concentration of 5 mg / mL), and then sonicate at 200 W for 1 min (5 s interval between each cycle, 5 s working) to obtain the primary emulsion.

[0064] (I-2) The primary emulsion was added dropwise to 10 mL of a 1 wt.% polyvinyl alcohol aqueous solution, and stirred for 14 min at a rate of 7600 r / min using a homogenizer to obtain a secondary emulsion;

[0065] (I-3) Add 0.1 wt.% of polyvinyl alcohol aqueous solution to the emulsion again, stir overnight at a rate of 600 r / min, wash the nanoparticles three times with pure water by centrifugation under a centrifugal force of 16000 g, and freeze-dry to obtain nanoparticles 1 loaded with antigen 2 and adjuvant 3.

[0066] (II) Preparation of nanoparticle microneedles:

[0067] (II-a) Polyethylene glycol diacrylate and dimethyl sulfoxide containing photoinitiator 2959 and nanoparticle 1 are mixed to obtain a first solution; wherein each 1 mL of the first solution contains 0.99 mL of polyethylene glycol diacrylate (molecular weight 450) and 0.01 mL of dimethyl sulfoxide; the dimethyl sulfoxide contains 10 mg / mL of photoinitiator 2959 and 0.2 mg of nanoparticle 1;

[0068] (II-b) Polyethylene glycol diacrylate and dimethyl sulfoxide containing photoinitiator 2959 are mixed to obtain a second solution; wherein each 1 mL of the second solution contains 0.99 mL of the polyethylene glycol diacrylate (molecular weight 450) and 0.01 mL of the dimethyl sulfoxide; the dimethyl sulfoxide contains 10 mg / mL of the photoinitiator 2959;

[0069] (II-c) Take 75 μL of the first solution and add it to a microneedle mold made of polydimethylsiloxane (PDMS). Degas under vacuum for 5 min to fill the tip 4 of the microneedle mold with the first solution. Continue to add the second solution to the microneedle mold and degas under vacuum for 2 min to fill the backing layer of the microneedle mold with the second solution.

[0070] (II-d) Cover the microneedle mold with a piezoelectric transducer and expose it to ultraviolet light (365nm, 1W / cm²). 2 After 2 minutes, a cross-linking polymerization reaction was initiated. After demolding, swellable nanoparticle microneedles loaded with subunit vaccines were obtained (HpaA and atRA were loaded at the tip 4).

[0071] Example 3

[0072] This embodiment provides a method for preparing nanoparticle microneedles loaded with subunit vaccines, the preparation method comprising the following steps:

[0073] (I) Preparation of nanoparticle 1:

[0074] (Ia) A chloroform organic solution containing gelatin (5 mg / mL), atRA and CpG (0.1 mg / mL), and surfactant DMAB (0.1 mg / mL) was mixed with a 0.5 wt.% F127 aqueous solution and ultrasonicated at 120 W to obtain an emulsion.

[0075] (Ib) The emulsion was subjected to rotary evaporation under reduced pressure, and the particles were washed with pure water by centrifugation. After freeze-drying, the particles were stored in a 4°C refrigerator. Before immunization, UreB1 aqueous solution was added, and the particles were washed and freeze-dried after slow stirring for 2 hours to obtain nanoparticles 1 loaded with antigen 2 and adjuvant 3.

[0076] (II) Preparation of nanoparticle microneedles:

[0077] (II-1) The nanoparticles 1 are mixed with an aqueous solution of polyvinylpyrrolidone (30%, w / v, molecular weight 3500) to obtain a mixture;

[0078] (II-2) The mixed solution is added to the polydimethylsiloxane (PDMS, 1×1cm) microneedle mold, so that each microneedle patch contains about 20μg of HpaA and 25μg of ATRA. The mixture is filled into the tip 4 of the microneedle mold by high-speed centrifugation.

[0079] (II-3) Add an aqueous solution of polyvinylpyrrolidone without nanoparticle 1 to the microneedle mold to form a microneedle backing layer;

[0080] (II-4) After the microneedle mold is thoroughly dried in a drying oven, it is demolded and stored in a refrigerator at 4°C to obtain soluble nanoparticle microneedles loaded with subunit vaccines (HpaA and atRA are loaded at the tip 4 sites).

[0081] Example 4

[0082] This embodiment provides a method for preparing nanoparticle microneedles loaded with subunit vaccines, the preparation method comprising the following steps:

[0083] (I) Preparation of nanoparticle 1:

[0084] (Ia) A chloroform organic solution containing gelatin (40 mg / mL), atRA and CpG (15 mg / mL), and surfactant DMAB (10 mg / mL) was mixed with a 0.5 wt.% F127 aqueous solution and sonicated at 120 W to obtain an emulsion.

[0085] (Ib) The emulsion was subjected to rotary evaporation under reduced pressure, and the particles were washed with pure water by centrifugation. After freeze-drying, the particles were stored in a 4°C refrigerator. Before immunization, UreB1 aqueous solution was added, and the particles were washed and freeze-dried after slow stirring for 12 hours to obtain nanoparticles 1 loaded with antigen 2 and adjuvant 3.

[0086] (II) Preparation of nanoparticle microneedles:

[0087] (II-a) A first solution is obtained by mixing polyethylene glycol diacrylate and dimethyl sulfoxide containing photoinitiator 2959 and nanoparticle 1; wherein each 1 mL of the first solution contains 0.99 mL of polyethylene glycol diacrylate (molecular weight 700) and 0.01 mL of dimethyl sulfoxide; the dimethyl sulfoxide contains 10 mg / mL of photoinitiator 2959 and 1 mg of nanoparticle 1;

[0088] (II-b) Polyethylene glycol diacrylate and dimethyl sulfoxide containing photoinitiator 2959 are mixed to obtain a second solution; wherein each 1 mL of the second solution contains 0.99 mL of the polyethylene glycol diacrylate (molecular weight 700) and 0.01 mL of the dimethyl sulfoxide; the dimethyl sulfoxide contains 10 mg / mL of the photoinitiator 2959;

[0089] (II-c) Take 75 μL of the first solution and add it to a microneedle mold made of polydimethylsiloxane (PDMS). Centrifuge at high speed to fill the tip 4 of the microneedle mold with the first solution. Continue to add the second solution to the microneedle mold and degas under vacuum for 2 min to fill the backing layer of the microneedle mold with the second solution.

[0090] (II-d) Cover the microneedle mold with a piezoelectric transducer and expose it to ultraviolet light (365nm, 5W / cm). 2 After 2 minutes, a cross-linking polymerization reaction was initiated. After demolding, swellable nanoparticle microneedles loaded with subunit vaccines were obtained (HpaA and atRA were loaded at the tip 4).

[0091] Test and Verification Example (I): Morphology and Structure of Nanoparticles

[0092] Example 1: Nanoparticles were prepared using a W / O / W double emulsion method. The nanoparticles were then imaged using electron microscopy. Figure 1 As shown in the figure, the results indicate that the nanoparticles described in Example 1 were successfully prepared.

[0093] Example 3: Nanoparticles were prepared using an emulsification-drying method. The nanoparticles were then imaged using an electron microscope. Figure 2 As shown in the figure, the results indicate that the nanoparticles described in Example 3 have a uniform projection morphology and stable structure, and the nanoparticles were successfully prepared.

[0094] A schematic diagram of the nanoparticle structure is shown below. Figure 3 As shown, the preparation method of the present invention enables nanoparticles to encapsulate antigen 2 and adjuvant 3 inside or adsorb them on the surface to form a composite system.

[0095] (II) Particle Size Distribution

[0096] The nanoparticles obtained in Example 1 were dispersed, and the particle size distribution was measured using a Malvern particle size analyzer. Figure 4 As shown in the figure, the results indicate that the obtained nanoparticles have a uniform particle size distribution.

[0097] (III) Nanoparticle Microneedle Morphology

[0098] The nanoparticle microneedles obtained in Example 1 were characterized using scanning electron microscopy, such as... Figure 5As shown in the figure, the results indicate that the nanoparticles and microneedles have regular morphology and are uniformly arranged, indicating that the preparation was successful.

[0099] A schematic diagram of the structure of nanoparticle microneedles is shown below. Figure 6 As shown, the preparation method of the present invention enables nanoparticles to be located at the tip of a microneedle, facilitating skin injection for vaccine administration.

[0100] (iv) Mouse experiments

[0101] After labeling the antigen with Cy7 fluorescent molecules, Cy7 fluorescently labeled nanoparticle microneedles were prepared according to the method described in Example 1, and delivered to the back of mice (after shaving) (after wiping off the skin surface), as shown. Figure 7 As shown, in vivo imaging (IVISR) images reveal that mice receiving microneedle delivery exhibited higher photon intensity, larger areas of diffused dye, and longer dye retention time within the skin tissue. In contrast, as... Figure 8 As shown, mice treated with nanoparticle solution on their skin surface (blank group) exhibited barely detectable photon intensities even with prolonged exposure time (10 min, 60 min, 240 min). This demonstrates that microneedles can effectively deliver antigens subcutaneously.

[0102] In addition, the vaginal lavage fluid antibody IgA was detected in the immunized mice, such as... Figure 9 As shown, the results indicate that compared to blank microneedles loaded only with HpaA antigen without adjuvant, nanoparticle microneedles loaded with both HpaA and atRA produced more secretory immunoglobulin A (sIgA), triggering significantly stronger mucosal immunity. Nanoparticle microneedles loaded with HpaA, atRA, and CpG produced the most sIgA, indicating that CpG played a synergistic role.

[0103] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing nanoparticle microneedles loaded with subunit vaccines, characterized in that, The preparation method includes the following steps: (I) Preparation of nanoparticles: (I-1) The antigen aqueous solution is mixed with an organic solution containing a polymeric carrier and an adjuvant, and then sonicated to obtain a primary emulsion; (I-2) The primary emulsion is added to a polyvinyl alcohol aqueous solution and homogenized to obtain a secondary emulsion; (I-3) Add polyvinyl alcohol aqueous solution to the emulsion again, stir and centrifuge to wash, and obtain nanoparticles loaded with antigen and adjuvant; The antigen is recombinant Helicobacter pylori protein HpaA; the polymeric carrier is polylactic acid-glycolic acid; and the adjuvant is all-trans retinoic acid. (II) Preparation of nanoparticle microneedles: (II-1) The nanoparticles are mixed with an aqueous solution of polyvinylpyrrolidone to obtain a mixture; (II-2) Add the mixture to the microneedle mold and centrifuge to fill the tip of the microneedle mold with the mixture; (II-3) Add an aqueous solution of polyvinylpyrrolidone without nanoparticles to the microneedle mold to form a microneedle backing layer; (II-4) Dry and demold the microneedle mold to obtain soluble nanoparticle microneedles loaded with subunit vaccine.

2. The method for preparing nanoparticle microneedles loaded with subunit vaccines according to claim 1, characterized in that, In step (I-1), the volume ratio of the antigen aqueous solution to the adjuvant organic solution is 1:3 to 1:10; the concentration of the antigen in the antigen aqueous solution is 0.1 to 5 mg / mL; the concentration of the adjuvant in the organic solution is 0.01 to 5 mg / mL, and the concentration of the polymer carrier is 5 to 40 mg / mL; the power of the ultrasonic treatment is 20 to 200 W, the working time in each ultrasonic cycle is 3 to 5 seconds, the interval time is 3 to 5 seconds, and the total ultrasonic time is 1 to 30 minutes.

3. The method for preparing nanoparticle microneedles loaded with subunit vaccines according to claim 1, characterized in that, In step (I-2), the concentration of the polyvinyl alcohol aqueous solution is 1%; the rotation speed of the homogenizer is 7400~7600 r / min, and the homogenization time is 14~16 min.

4. Nanoparticle microneedles for subunit-loaded vaccines obtained by the preparation method according to any one of claims 1 to 3.

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