MRNA vaccine delivery nanoparticles for nasal mucosa as well as preparation and application of mRNA vaccine delivery nanoparticles

Abstract

The invention discloses nano-particles for delivery of nasal mucosa mRNA vaccines as well as preparation and application of the nano-particles, and belongs to the technical field of biological medicines. The nano-particles are formed by quaternized chitosan, low molecular weight polyethyleneimine, aldehyde polyethylene glycol and mRNA (messenger ribonucleic acid) through self-assembly. QCS and PEI adsorb mRNA through electrostatic interaction, OPEG is cross-linked with PEI / QCS through a Schiff base bond, and pH response type nanoparticles are formed. The particles are stable at physiological pH and protect mRNA from being degraded; dissociation is carried out in an endosome acid environment, mRNA is rapidly released, and endosome escape is promoted. The QCS endows the particles with excellent mucous membrane adhesiveness and adjuvant effect. The invention also provides a preparation method of the particles and application of the particles in preparation of nasal mucosa mRNA vaccines. The vaccine can effectively induce mucous membrane sIgA and whole body IgG / IgA response, and is used for preventing viral infectious diseases and tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to mRNA vaccine delivery nanoparticles for nasal mucosa and their preparation and application. Background Technology

[0002] Mucosal tissues are the primary gateways for over 90% of pathogens entering the body. Mucosal vaccines can mimic natural infection pathways, simultaneously activating local mucosal immune responses and systemic immunity, effectively blocking viral replication and transmission in the early stages of infection, and demonstrating significant advantages in controlling highly infectious diseases. Messenger RNA (mRNA) vaccines have become a new platform for vaccine development due to their rapid development, good safety profile, and ability to induce strong cellular and humoral immunity. However, naked mRNA molecules are highly susceptible to degradation by nucleases in the environment and body fluids, and their negative charge makes them difficult to effectively cross cell membranes. Therefore, developing vector systems that can effectively protect mRNA and facilitate its intracellular delivery is crucial.

[0003] For mucosal delivery, the challenges are even more severe. Vaccine antigens must overcome mucosal barriers (such as the mucus layer and epithelial cells) and avoid being cleared in order to be taken up by antigen-presenting cells (APCs) and induce an effective mucosal immune response. Chitosan (CS), as a natural cationic polysaccharide, has good biocompatibility, degradability, and mucosal adhesion, and has been widely studied for mucosal delivery. However, its low solubility under physiological pH conditions and poor transfection efficiency with mRNA complexes limit its application. Polyethyleneimine (PEI) is another classic gene delivery vector. In particular, high molecular weight PEI (such as 25 kDa) has high transfection efficiency but high cytotoxicity; low molecular weight PEI (such as 1.8 kDa) has significantly reduced cytotoxicity and retains some nucleic acid compression and "proton sponge" effect to promote endosome escape, but its transfection efficiency is relatively low and the complex is unstable.

[0004] Therefore, there is an urgent need in this field to develop a novel delivery system that combines good biocompatibility, efficient mRNA loading and protection capabilities, strong mucosal adhesion, pH-responsive release characteristics, and efficient cell transfection capabilities for effective mucosal mRNA vaccination. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes mRNA vaccine delivery nanoparticles for nasal mucosa, their preparation, and their application, in order to achieve efficient delivery of mRNA vaccines to the nasal mucosa.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides mRNA vaccine delivery nanoparticles for nasal mucosa, said nanoparticles (PQOm) being formed by self-assembly of quaternized chitosan (QCS), polyethyleneimine (PEI), aldehyde-modified polyethylene glycol (OPEG) and mRNA.

[0008] The nanoparticles adsorb and compress mRNA through electrostatic interactions between the amino groups of QCS and PEI and the phosphate backbone of mRNA. They are cross-linked through dynamic Schiff base bonds formed between the aldehyde groups of OPEG and the amino groups of QCS and PEI, thus forming structurally stable core-shell nanoparticles. The Schiff base bonds endow the nanoparticles with pH-responsive properties, making them stable under physiological pH conditions (~7.4), but breaking under the acidic pH conditions of endosomes / lysosomes (~5.0-6.5), causing the nanoparticles to dissociate and rapidly release mRNA, while triggering the "proton sponge" effect to promote endosome escape.

[0009] The polyethyleneimine is a low molecular weight PEI with a molecular weight of 1.8 kDa.

[0010] The quaternized chitosan has a molecular weight of 30 kDa.

[0011] The aldehyde-based polyethylene glycol has a molecular weight of 2 kDa.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned nanoparticles, comprising the following steps:

[0013] S1. Dissolve QCS and PEI separately in enzyme-free sterile water, then mix them to form a PQ solution;

[0014] S2. Add diluted mRNA solution to PQ solution and incubate at room temperature to form PQm complex;

[0015] S3. Add an OPEG solution dissolved in enzyme-free sterile water to the PQm complex, and incubate with shaking at room temperature to allow the aldehyde groups of OPEG to cross-link with the amino groups in PQm, forming the final nanoparticles PQOm.

[0016] Preferably, the mass concentration ratio of QCS to PEI in S1 is (0.8-1.5):1.

[0017] Preferably, the concentration of QCS in S1 is 2-4 mg / mL.

[0018] Preferably, the amount of mRNA added in S2 is such that the molar ratio (N / P) of nitrogen atoms (N) of the cationic polymers PEI and QCS to phosphate groups (P) of the mRNA is 40:1.

[0019] Preferably, the room temperature incubation time in S2 is 20-40 minutes.

[0020] Preferably, the concentration of OPEG in S3 is 2-4 mg / mL.

[0021] Preferably, the ratio of the amount of OPEG added in S3 to the total molar amount of amino groups in PEI and QCS is (0.8-1.5):1.

[0022] Preferably, the shaking incubation speed in S3 is 600-1000 rpm, and the time is 20-40 minutes.

[0023] Thirdly, the present invention provides the application of the above-mentioned nanoparticles in the preparation of preventive or therapeutic vaccines, which are administered via the nasal mucosa.

[0024] The beneficial effects of this invention are:

[0025] Compared with existing technologies, this method employs a unique dual-mechanism catalytic strategy, achieving significantly different regioselectivity simply by selectively adding or omitting the palladium catalyst, without requiring changes to ligands or additives. It is simple to operate and cost-effective. This method exhibits excellent regioselectivity for a variety of pyranoside substrates (including mannose, glucose, galactose, rhamnose, fucose, and arabinose), is applicable to both primary and secondary hydroxyl groups, and has broad substrate compatibility, suitable for various common protecting groups and sulfonyl chloride reagents, laying a solid foundation for subsequent derivatization. Furthermore, the obtained sulfonated products are key intermediates in the synthesis of saccharide drugs, natural products, and biological probes; therefore, this method has excellent industrial application potential and broad application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0027] Figure 1 The particle size distribution (A) and zeta potential (B) of the PQOm nanoparticles prepared in Example 1 are shown.

[0028] Figure 2 Transmission electron microscopy (TEM) images of PQOm nanoparticles under different pH conditions: A represents neutral conditions and B represents acidic conditions.

[0029] Figure 3 The images show the X-ray photoelectron spectroscopy (XPS) spectra of PQOm nanoparticles. A is the XPS C1s spectrum, and B is the XPS N1s spectrum.

[0030] Figure 4 The images show the 1H NMR spectra of the OPEG and PQO complex under different pH conditions.

[0031] Figure 5 This is an agarose gel electrophoresis image showing the effect of PQOm on mRNA loading.

[0032] Figure 6 This is a diagram showing the enzymatic protection effect of PQOm nanoparticles on mRNA.

[0033] Figure 7 The figure shows the effect of PQOm nanoparticles on the viability of DC2.4 cells.

[0034] Figure 8 This diagram illustrates how PQOm nanoparticles promote mRNA uptake in cells. In the diagram, A represents the flow cytometry analysis, and B represents the corresponding quantitative results.

[0035] Figure 9 This diagram illustrates how PQOm nanoparticles promote lysosomal escape of mRNA. In the diagram, A shows the lysosomal escape ability of PQOm and the control group analyzed by CLSM, and B shows the quantitative analysis of the Pearson correlation coefficient of red and green fluorescence colocalization in the lysosomal escape experiment.

[0036] Figure 10 The effect of PQOm nanoparticles on transfecting Fluc-mRNA in DC2.4 cells is shown, where A represents neutral conditions and B represents acidic conditions.

[0037] Figure 11 The images show flow cytometry analysis of PQOm nanoparticles transfected with eGFP-mRNA in DC2.4 cells. A represents the eGFP-mRNA expression efficiency as determined by CLSM, B is the quantitative fluorescence plot of eGFP-mRNA expression efficiency, and C is the flow cytometry result of eGFP expression. + A representative histogram of the proportion of DC2.4 cells, where D represents their eGFP. + Quantitative results of DC2.4 cells.

[0038] Figure 12 This is a schematic diagram illustrating the long-term adhesion ability of PQOm nanoparticles to mucosa after intranasal administration via nasal drops; where A is a representative IVIS image of nasal fluorescence signal at different time points (1, 2, 4, 6, 12 h and 2, 4, 6 d) after intranasal administration of 10 μg Cy5-mRNA PQOm nanoparticles in BALB / c mice, and B is the quantification of IVIS signal in the nasal cavity of mice in each administration group in Figure A.

[0039] Figure 13 The immunogenicity of PQOm nanoparticles as a nasal mucosal vaccine is shown in Figure AE, where A, E, and E represent the IgA antibody levels in nasal wash, saliva, BALF, vaginal wash, and plasma, respectively; Figure F shows the IgG antibody level in plasma. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0041] Example 1: Preparation and Characterization of PQOm Nanoparticles

[0042] S1. Weigh appropriate amounts of quaternized chitosan (QCS, 30kDa) and polyethyleneimine (PEI, 1.8kDa), dissolve them separately in enzyme-free sterile water to prepare a 3mg / mL QCS solution and an appropriate concentration of PEI solution (so that the mass ratio of PEI to QCS is 1:1.15). Mix the PEI solution and QCS solution, let stand at room temperature for 10 minutes, and obtain a clear PQ solution.

[0043] S2. Add the diluted mRNA solution to the PQ solution, mix gently, and incubate at room temperature for 30 minutes to allow the mRNA to fully complex with the cationic polymer through electrostatic interactions, forming the PQm complex. In this step, the N / P ratio should be controlled at 40:1.

[0044] S3. Weigh aldehyde-modified polyethylene glycol (OPEG, 2kDa), dissolve it in enzyme-free sterile water to prepare a 3mg / mL solution, add the OPEG solution to the above PQm complex, and react with shaking at 800rpm for 30 minutes at room temperature to allow the aldehyde group of OPEG to crosslink with the amino groups of PEI and QCS in PQm to form Schiff base bonds, finally obtaining a nanoparticle suspension, denoted as PQOm.

[0045] PEI+mRNA (Pm) and PEI+QCS+mRNA (PQm) samples were prepared using the same method. The morphology and size of the vectors were characterized by TEM. The hydration kinetic diameter, PDI, and zeta potential of the vectors were characterized by nanoparticle size and zeta potential analyzers.

[0046] Characterization results as follows Figure 1 As shown, PEI and QCS form unstable nanoparticles solely through electrostatic interactions with mRNA, while the addition of OPEG crosslinking agent results in more stable nanoparticles. Under acidic conditions, the Schiff base bonds in the nanoparticles partially break, leading to an increase in particle size and PDI. OPEG crosslinks some of the amino groups in PEI and QCS, resulting in a decrease in surface potential. Under acidic conditions, the crosslinked amino groups break, and the surface potential increases from +32.97 mV to +58.10 mV.

[0047] The morphology and size of PQOm were characterized by TEM, and the results are as follows: Figure 2As shown in Figure A, PQOm forms uniform spherical nanoparticles, such as Figure 2 As shown in Figure B, the nanoparticles disintegrate under acidic conditions, confirming their acid-responsiveness.

[0048] X-ray photoelectron spectroscopy (XPS) was used to analyze the intermolecular interactions of different nanoparticles. The characteristic binding energy spectra of carbon (C1s) and nitrogen (N1s) elements in PQOm nanoparticles were measured, and Schiff base structural characteristics were analyzed by high-resolution peak fitting. Figure 3 As shown in Figure A, the peaks at 284.8 eV, 285.8 eV, and 287.5 eV correspond to C / C=C, CN / CO, and C=N bonds, respectively, indicating that the aldehyde group of OPEG combines with the amino group to form a Schiff base. Furthermore, as... Figure 3 As shown in B, the peak at 398.7 eV in the N1s energy spectrum of PQOm belongs to pyridine nitrogen (C=N), corresponding to the Schiff base bond, and the characteristic peak at 401.8 eV corresponds to quaternary ammonium nitrogen, which originates from the quaternary ammonium group in QCS.

[0049] At the same time, using 1 The acid responsiveness of nanoparticles was characterized by the presence or absence of aldehyde groups under neutral and acidic conditions determined by ¹H NMR. The acidity of the deuterated solvent was adjusted by adding DCl to D₂O. The results are as follows: Figure 4 As shown, the aldehyde peak at 9.62 ppm in PQO nanoparticles disappears, indicating the formation of Schiff base bonds. The aldehyde peak reappears under acidic conditions, confirming the acid responsiveness of the nanoparticles.

[0050] Example 2: Loading and Enzyme Protection Assay of PQOm Nanoparticles on mRNA

[0051] The mRNA loading capacity of P, PQ, PQO (pH 7.4), and PQO (pH 6.8) was determined using an agarose gel retardation assay. Mixtures of different component-mRNA were prepared, incubated at room temperature for 30 min, and then 2.5 μL of 5×RNA loading buffer was added to obtain the loading mixture. The mixture was added to the wells of a prepared 1% agarose gel containing Gel-Red, and electrophoresis was performed at 80 V for 30 min using 1×TAE as the electrophoresis buffer. The mRNA bands were detected using a Tanon 1600 gel imaging system.

[0052] PQOm nanoparticles were prepared, and their ability to protect mRNA against enzymes was determined using an agarose gel retardation assay. 75 ng of RNase A was added to a solution of free mRNA and PQOm (1 μg mRNA), and the mixture was incubated at 37°C for 10 min. An RNase inhibitor was then added, and the mixture was incubated at 37°C for 15 min to inhibit enzyme activity. Finally, heparin solution was added, and the mixture was incubated at 50°C for 4 h to displace the mRNA from the system. Six sample groups (mRNA, PQOm, PQOm + heparin, mRNA + RNase A, PQOm + RNase A + heparin, and PQOm + RNase A) were mixed with RNA loading buffer and added to the wells of an agarose gel. Electrophoresis was performed at 80 V for 30 min, and the integrity of the mRNA bands was detected using a Tanon 1600 gel imaging system.

[0053] The results are as follows Figure 5 As shown, the PQOm group has a strong loading capacity for mRNA. For example... Figure 6 As shown, the mRNA displaced by PQOm nanoparticles remains structurally intact under enzymatic degradation, indicating that the nanoparticles can protect mRNA from enzymatic degradation.

[0054] Example 3: Cytotoxicity assay of PQOm nanoparticles

[0055] DC2.4 cells were seeded at a density of 1×10⁴ cells / well in 96-well plates. After 24 h, the original culture medium was discarded, and 100 μL of complete culture medium containing different groups of nanoparticles was added to each well. After 24 h of incubation, cell viability was measured using a CCK8 assay at 450 nm, and cell survival rate was calculated for each group. Results are as follows: Figure 7 As shown, the cell viability of the PQOm group was above 80%, indicating that it has high safety.

[0056] Example 4 Intracellular uptake and lysosomal escape

[0057] To determine the uptake efficiency of PQOm by cells, DC2.4 cells were plated and cultured for 24 h. The original culture medium was discarded, and the cells were washed twice with PBS. PQOm nanoparticles encapsulating Cy5-mRNA were added to each well, and the cells were incubated for 0, 0.25, 0.5, 1, 2, 4, and 6 h, respectively. After incubation, the drug-containing culture medium was discarded, and the cells were washed three times with PBS. After trypsin digestion, the cells were harvested, centrifuged to remove the supernatant, resuspended in 1% FBS-PBS, and passed through a 70 μm sieve. The proportion of Cy5+ DC2.4 cells was detected by flow cytometry, and the uptake efficiency of nanoparticles was evaluated by the percentage of positive cells. Results are as follows: Figure 8 Cells take up nanoparticles quickly, with an uptake rate of 90% after 2 hours and 100% after 6 hours.

[0058] The lysosomal escape ability of different nanoparticles was determined. DC2.4 cells were seeded in confocal dishes and cultured for 24 h. The original culture medium was discarded, and nanoparticles encapsulating Cy5-mRNA were added to each well. After incubation for 24 h, the supernatant was discarded, and the cells were washed three times with PBS. Lysosomes and cell nuclei were stained with LysoTrackerRed and DAPI dyes sequentially according to the reagent instructions. The colocalization of LysoTrackerRed and Cy5 fluorescence was observed using CLSM to assess the lysosomal escape of the nanoparticles. The results are as follows: Figure 9 The red and green fluorescence of PQOm and PQOm (pH 6.8) groups did not show colocalization, indicating that they have a strong lysosomal escape ability.

[0059] Example 5: Transfection of mRNA by PQOm nanoparticles

[0060] Different nanoparticles encapsulating eGFP-mRNA or Fluc-mRNA were transfected into DC2.4 cells to evaluate the cell transfection efficiency. DC2.4 cells (1×10⁵ cells / mL) were seeded and incubated overnight. eGFP-mRNA encapsulated in nanoparticles was then cultured in serum-free RPMI 1640 for 6 hours, followed by incubation with serum for 18 hours. DC2.4 cells were transfected, and the proportion of eGFP+ DC2.4 cells was detected by flow cytometry. mRNA expression was observed using CLSM, and the transfection capacity was evaluated by comparing fluorescence intensities between different groups. Fluc-mRNA encapsulated in nanoparticles was transfected into DC2.4 cells. After cell lysis, fluorescein substrate was added to the cell lysate, and RLU was measured. Protein concentration was also measured using the BCA method. Transfection efficiency was expressed as RLU / mg. Subsequently, flow cytometry was used to quantitatively analyze fluorescent protein expression. Fluc-mRNA expression levels were expressed as RLU / mg.

[0061] The results are as follows Figure 10-11 As shown, PQOm nanoparticles can effectively transfect eGFP-mRNA and Fluc-mRNA into DC2.4 cells.

[0062] Example 6: Determination of the mucosal adhesion ability of PQOm nanoparticles

[0063] Cy5-mRNA was encapsulated in PQO and its control group. The nanoparticles were administered via nasal drops to mice. In vivo imaging was used to characterize the mucosal adhesion ability of the nanoparticles by detecting the fluorescence intensity at the nasal mucosa administration site at different time points after administration. Mice were shaved two days prior to administration. PQOm nanoparticles encapsulating Cy5-mRNA were prepared and administered via nasal drops (10 μg / 40 μL / mouse), with 20 μL administered to each nostril of BALB / c mice. The fluorescence intensity at the nasal mucosa administration site was measured using in vivo imaging at 1, 2, 4, 8, and 12 hours and 2, 4, and 6 days after administration. The fluorescence intensity at the nasal mucosa administration site was also measured using ex vivo imaging of the mouse palate. The mucosal adhesion ability of the PQOm nanoparticles was evaluated based on the fluorescence intensity.

[0064] The results are as follows Figure 12 As shown, the average fluorescence intensity at the nasal mucosa administration site gradually decreased 1 hour after administration, but still showed a certain fluorescence intensity 6 days after administration.

[0065] Example 7: Evaluation of the efficacy of PQOm nanoparticles as a mucosal vaccine

[0066] A mucosal vaccine prophylaxis model was established, using OVA-mRNA as the model antigen mRNA to evaluate the immunizing effect of the PQOm mucosal vaccine. OVA-mRNA was encapsulated in PQOm and its control group, and BALB / c mice were administered the vaccine three times via nasal mucosa, with each administration 14 days apart. Mice were anesthetized and administered the vaccine in a supine position, at a dose of 10 μg / 40 μL / mouse, 20 μL / nostril, with an interval of 30-60 seconds between each administration. The mice were kept in the anesthetized supine position for at least 5 minutes after administration to promote drug absorption and prevent outflow. The vaccine's immune activation effect was measured 14 days after the last administration.

[0067] Mouse body fluids were collected, and the titers of IgG and sIgA antibodies were measured to analyze the level of humoral immune response induced by the vaccine. Blood was collected from the mouse orbital cavity and purified to lithium heparin for blood collection. The plasma was collected at 6000 rpm for 8 min at 4°C, and the supernatant was collected and aliquoted and stored at -80°C. After anesthetizing the mice, vaginal wash was collected and irrigated with 75 μL of sterile PBS (containing 5 μL of 25× protease inhibitor), 25 μL per wash, 3 times. Each 25 μL was pipetted 3-5 times, and the supernatant was collected after centrifugation at 4°C for 10 min at 12000g and stored at -80°C. After anesthetizing the mice, saliva was collected by dripping 30 μL of sterile PBS (containing 5 μL of 25× protease inhibitor) between the cheek and gums. The saliva was pipetted 3-5 times, and each cheek was washed once. After centrifugation at 4°C for 10 min at 12000g, the supernatant was collected and stored at -80°C. Mice were anesthetized and euthanized. The skin and tissue around the neck were cut open to expose the trachea. A small hole was cut in the trachea, and an irrigation needle was inserted towards the mouse's head. The needle was brought close to the pharynx, and the syringe was injected to allow the irrigation fluid to flow out through the nostrils. The nasal irrigation fluid was collected. The irrigation needle was then inserted towards the mouse's lungs, and the fluid was repeatedly blown to collect the alveolar lavage fluid (BALF). The BALF was centrifuged at 12000g at 4℃ for 10 min, and the supernatant was collected and stored at -80℃. The IgG antibody titer of the plasma samples was determined using the following protocol: High-affinity 96-well plates were pre-coated with OVA protein (2 μg / 100 μL / well) overnight at 4℃. The plates were washed three times with PBST and blocked with 3% BSA (w / v) at 37℃ for 1 h (100 μL / well). After washing, 100 μL / well of plasma diluted 50-fold with 1% BSA was added, and the plates were blocked at 37℃ for 1.5 h. After washing the plate, add 100 μL / well of alkaline phosphatase-labeled goat anti-mouse IgG antibody (1:3000 dilution) and incubate at room temperature for 1 h. After washing the plate, add 100 μL of pNPP substrate to each well and incubate at room temperature for 15 min. Finally, add 100 μL of pNPP stop solution and measure the absorbance at 405 nm using a microplate reader. For all the above body fluid samples, determine the IgA antibody titer using the following protocol: coat 96-well plates using the same method, block with BSA, dilute the body fluid 5-fold, and block at 37°C for 1.5 h. After washing the plate, add 100 μL / well of alkaline phosphatase-labeled goat anti-mouse IgA antibody (1:1000 dilution) and incubate at room temperature for 1.5 h.

[0068] After washing the plate, add 100 μL / well of alkaline phosphatase-labeled donkey anti-sheep IgG antibody (1:3000 dilution) and incubate at room temperature for 1 h. After washing the plate again, add substrate for color development for 30 min, then stop the reaction and measure the absorbance at 405 nm using a microplate reader.

[0069] The results are as follows Figure 13AE represents the IgA antibody level in nasal wash, saliva, BALF, vaginal wash, and plasma, while F represents the IgG antibody level. The PQOm group significantly increased the sIgA antibody level in the above-mentioned mucosal washes and significantly increased the IgA and IgG antibody titers in the blood.

[0070] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. mRNA vaccine delivery nanoparticles for nasal mucosa, characterized in that, The nanoparticles are formed by the self-assembly of quaternized chitosan, polyethyleneimine, aldehyde-modified polyethylene glycol, and mRNA. The polyethyleneimine and quaternized chitosan adsorb the mRNA through electrostatic interaction, and the aldehyde-modified polyethylene glycol crosslinks the complex of polyethyleneimine, quaternized chitosan, and mRNA by forming Schiff base bonds with the amino groups of the polyethyleneimine and / or quaternized chitosan through its aldehyde groups.

2. The mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 1, characterized in that, The molecular weight of the polyethyleneimine is 1.8 kDa.

3. The mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 2, characterized in that, The quaternized chitosan has a molecular weight of 30 kDa.

4. The mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 3, characterized in that, The aldehyde-based polyethylene glycol has a molecular weight of 2 kDa.

5. A method for preparing mRNA vaccine delivery nanoparticles for nasal mucosa, characterized in that, Includes the following steps: S1. Dissolve quaternized chitosan and polyethyleneimine separately in enzyme-free sterile water, and mix them to form a PQ solution; S2. Add diluted mRNA solution to PQ solution and incubate at room temperature to form PQm complex; S3. Add an aldehyde-modified polyethylene glycol solution dissolved in enzyme-free sterile water to the PQm composite, and incubate with shaking at room temperature to allow the aldehyde groups of the aldehyde-modified polyethylene glycol to crosslink with the amino groups in PQm, forming the final nanoparticles PQOm.

6. The method for preparing mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 5, characterized in that, The mass concentration ratio of quaternized chitosan to polyethyleneimine in S1 is (0.8-1.5):1; the concentration of quaternized chitosan in S1 is 2-4 mg / mL.

7. The method for preparing mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 6, characterized in that, The amount of mRNA added in S2 is such that the molar ratio of nitrogen atoms in the cationic polymer polyethyleneimine and quaternized chitosan to phosphate groups in the mRNA is 40:

1.

8. The method for preparing mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 7, characterized in that, The incubation time at room temperature in S2 is 20-40 minutes.

9. The method for preparing mRNA vaccine delivery nanoparticles for nasal mucosa according to claim 8, characterized in that, The ratio of the amount of aldehyde-modified polyethylene glycol added in S3 to the total amino molar amount of polyethyleneimine and quaternized chitosan is (0.8-1.5):1; the concentration of aldehyde-modified polyethylene glycol in S3 is 2-4 mg / mL; the shaking incubation speed in S3 is 600-1000 rpm, and the time is 20-40 minutes.

10. The use of mRNA vaccine delivery nanoparticles for nasal mucosa in the preparation of preventive or therapeutic vaccines administered via the nasal mucosa.

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