Arginine and hydroxybutyl co-modified chitosan nanodelivery carrier, preparation method and application thereof

Abstract

The present application relates to a kind of arginine and hydroxybutyl co-modified chitosan nano delivery carrier, the chemical structural formula of the delivery carrier is as follows general formula I: Wherein, m represents the molar number of hydroxybutyl graft in chitosan, n represents the molar number of arginine in chitosan or the substitution degree of arginine, k represents the molar number of deacetyl chitosan monomer not involved in reaction.The present application also relates to corresponding preparation method and application.Nucleic acid drug delivery efficiency can be improved, the cell immune regulation effect of drug-loaded nano particles is enhanced, and it is good in water-soluble, green and non-toxic.

Description

Technical Field

[0001] This invention belongs to the field of gene biotechnology, specifically relating to a chitosan nanocarrier co-modified with arginine and hydroxybutyl, its preparation method, and its application. Background Technology

[0002] Gene therapy involves identifying the relevant mutated or altered genes in diseased or mutated cells and using advanced gene-editing techniques to repair or rebuild the defective genetic material. Viral or non-viral vectors deliver and release the modified genes into target cells, modulating the genes to improve the patient's health.

[0003] Nucleic acid drugs, such as DNA and RNA, have the potential to treat a variety of diseases. Polyinosinic:polycytidylicacid (poly I:C) is a synthetic double-stranded RNA widely used as a vaccine adjuvant. It activates the body's immune system by mimicking viral RNA, particularly inducing a strong antiviral immune response via the Toll-like receptor 3 (TLR3) pathway. Poly I:C can enhance the immunogenicity of vaccines and improve their protective effect against pathogens. Furthermore, poly I:C has been studied for use in cancer vaccines, enhancing anti-tumor immune responses by activating the immune system. However, naked nucleic acids are easily degraded by nucleases in the external environment and are negatively charged biomolecules, making it difficult for them to cross biological membranes and enter cells to exert their effects. Therefore, suitable delivery carriers are needed to deliver them into cells to improve nucleic acid stability and cellular uptake efficiency.

[0004] Chitosan (CS) is a cationic polysaccharide found in nature, widely present in the exoskeletons of crustaceans. Due to its biocompatibility, degradability, and low toxicity, chitosan shows great potential in gene delivery. It offers many unique advantages as a gene carrier. First, chitosan exhibits excellent biocompatibility and degradability. It can be enzymatically degraded in vivo into non-toxic products without triggering an immune response. Due to the amino groups in its molecular structure, chitosan exhibits a positive charge under physiological conditions, allowing it to form stable complexes with negatively charged DNA or RNA molecules, thereby protecting nucleic acids from degradation and promoting their entry into cells. Chitosan also possesses good membrane permeability. Studies have shown that chitosan can enter cells via endocytosis mediated by receptors on the cell membrane (ZHOU H, WAN F, JIAN Y, et al. Chitosan / dsRNA polyplex nanoparticles advance environmental RNAinterference efficiency through activating clathrin-dependent endocytosis[J]. International Journal of Biological Macromolecules, 2023, 253:127021.). This property enables chitosan to efficiently deliver genetic material to target cells, thereby improving the efficacy of gene therapy. Furthermore, the application of chitosan in gene delivery also includes its tunable physicochemical properties. By changing the molecular weight, degree of deacetylation, and modifying groups of chitosan, its solubility, viscosity, and drug loading capacity can be controlled, thereby optimizing the performance of gene delivery systems.

[0005] Chitosan exhibits good solubility in acidic solutions but poor solubility in neutral and alkaline solutions, limiting its application in certain drug delivery systems. Numerous studies have focused on improving the water solubility of chitosan, such as CN116284498A, entitled "A Hydroxybutyl Chitosan and Its Application," which utilizes hydroxybutyl groups to replace the hydroxyl and amino groups in chitosan chains to prepare hydroxybutyl chitosan (HBC), which is then used as an emulsifier to stabilize oil-in-water emulsions for preparing delivery carriers of lipid-soluble substances. Because the substitution reaction partially occurs on the amino groups in chitosan, the positive charge density of HBC decreases, hindering the binding of negatively charged nucleic acid drugs via electrostatic interactions. Currently, chitosan delivery carriers are primarily used for protein or antitumor drug delivery, such as CN113318234A, entitled "An Arginine and Ursolic Acid Modified Chitosan Nanoparticle Drug Delivery Carrier and Its Preparation Method and Application," which modifies chitosan with ursolic acid and arginine to prepare an amphiphilic micelle solution with good membrane penetration and tumor drug resistance reversal. Whether water-soluble chitosan derivatives grafted with arginine are suitable for binding macromolecular nucleic acid drugs and serving as delivery carriers for the nucleic acid immunoadjuvant polyI:C has not been found in existing research or reports. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, this invention provides a chitosan nanocarrier co-modified with arginine and hydroxybutyl, its preparation method, and applications. This addresses the limitation of current chitosan delivery carriers, which are only soluble in acidic solutions and are primarily used for small-molecule nucleic acid drug delivery. This invention utilizes hydroxybutyl and arginine to graft chitosan, preparing a chitosan derivative with a broad solubility range, and then loads and delivers the macromolecular nucleic acid immunoadjuvant polyI:C. The chitosan derivative delivery carrier of this invention can protect nucleic acid drugs, enhance their immunomodulatory effects, and improve therapeutic efficiency.

[0007] To achieve the above objectives, a first aspect of the present invention provides a chitosan nanocarrier co-modified with arginine and hydroxybutyl, characterized in that the chemical structural formula of the delivery carrier is shown in the following general formula I:

[0008]

[0009]

[0010] Where m represents the number of moles of hydroxybutyl grafted onto chitosan, n represents the number of moles of arginine on chitosan or the degree of substitution of arginine, and k represents the number of moles of unreacted deacetylated chitosan monomers.

[0011] The chitosan nanocarrier co-modified with arginine and hydroxybutyl was prepared by substituting the active sites in the chitosan molecular chain with 1,2-epoxybutane and arginine.

[0012] Preferably, in the delivery carrier, the chitosan has a molecular weight of 1-300 kDa, a degree of deacetylation of 80%-95%, an arginine modification ratio of 1%-15%, and a hydroxybutyl modification ratio of 10%-80%.

[0013] A second aspect of the present invention provides a method for preparing the chitosan nanocarrier, the method comprising the following steps:

[0014] (1) Add an alkaline solution to chitosan, stir until homogeneous, and allow to swell at room temperature to prepare an alkaline chitosan solution;

[0015] (2) Isopropanol and ultrapure water were added to the chitosan alkalization solution, and the solution was swollen at room temperature. Then, 1,2-epoxybutane solution was added dropwise to react and hydroxybutyl chitosan was obtained.

[0016] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the hydroxybutyl chitosan solution, mix, then add arginine aqueous solution, adjust the pH value to the range of 4 to 7, and react to obtain chitosan co-modified with arginine and hydroxybutyl.

[0017] Preferably, in step (1), the alkaline solution is a NaOH solution, and the concentration of the chitosan alkalization solution is 50-200 mg / ml; the NaOH mass concentration is 50% NaOH, and the chitosan concentration is 150 mg / ml.

[0018] In step (2), the volume ratio of isopropanol to ultrapure water in the isopropanol aqueous solution is 0.5 to 2, the volume ratio of 1,2-epoxybutane solution to isopropanol is 0.5 to 3; the reaction temperature is 35℃ to 70℃; the volume ratio of isopropanol to ultrapure water is 1:1, the volume ratio of 1,2-epoxybutane to isopropanol is preferably 2:1; and the reaction temperature is 45℃.

[0019] In step (2), the reaction solution is neutralized, dialyzed, centrifuged to remove impurities, and freeze-dried to obtain hydroxybutyl chitosan; the reaction solution is neutralized with dilute hydrochloric acid, dialyzed with a dialysis bag with a molecular weight cutoff of 8K to 14K to remove small molecule impurities, centrifuged at 6000 rpm to remove insoluble impurities, and freeze-dried at -50℃ to obtain solid hydroxybutyl chitosan.

[0020] In step (3), the concentration of the arginine aqueous solution is 1–250 mg / ml; the concentration of the hydroxybutyl chitosan solution is 1–20 mg / ml; and the reaction time is 12–36 h. Alternatively, the concentration of the arginine aqueous solution is 40 mg / ml; the concentration of the hydroxybutyl chitosan solution is 20 mg / ml; and the reaction time at room temperature is 24 h.

[0021] Preferably, the molar ratio of chitosan, arginine, EDC·HCl, and NHS is 1–4:0.5–2:1–4:2–8. The preferred molar ratio of EDC·HCl to NHS is 2:1.

[0022] The above preparation process has the advantages of safe operation, simple process and low manufacturing cost. The degree of grafting of exogenous groups can be adjusted by changing the feed ratio, reaction time and reaction temperature, thereby changing the drug loading capacity and particle size of the carrier. The average particle size of the arginine-hydroxybutyl-chitosan nanoparticles formed is 100 to 600 nm.

[0023] A third aspect of the invention provides the application of the chitosan nanocarrier in the preparation of nucleic acid vaccines.

[0024] Preferably, the chitosan nanocarrier is used to deliver nucleic acid vaccine adjuvants.

[0025] Preferably, the nucleic acid vaccine adjuvant is a poly I:C solution.

[0026] This invention also provides a method for preparing a chitosan delivery carrier co-modified with arginine and hydroxybutyl to load a macromolecular nucleic acid immune adjuvant, comprising the following steps: uniformly mixing a positively charged aqueous solution of arginine-modified hydroxybutyl chitosan with a negatively charged synthetic polyI:C solution to homogenize.

[0027] Preferably, the arginine-modified hydroxybutyl chitosan is dissolved in DEPC water at a concentration of 0.1–5 mg / ml, more preferably 1 mg / ml; the poly I:C solution has a concentration of 0.1–2 mg / ml, more preferably 0.2 mg / ml; the poly I:C solution is added dropwise to the chitosan derivative aqueous solution, and the homogenization time is 0.5–1.5 h, more preferably 1 h.

[0028] The present invention also provides a nucleic acid immune adjuvant, which is a polyI:C solution loaded with the chitosan delivery carrier.

[0029] In application, a cationic delivery carrier solution is prepared, mixed with a double-stranded RNA analog polyI:C solution, incubated, and excess drug is removed to obtain arginine-hydroxybutyl chitosan drug-loaded particles. This invention demonstrates that the arginine-hydroxybutyl chitosan delivery carrier has a certain protective effect on nucleic acid drugs and enhances cellular immune responses.

[0030] This invention is the first to use arginine, 1,2-epoxybutane, and chitosan as raw materials to load and deliver the macromolecular nucleic acid vaccine adjuvant polyI:C, preparing a drug-loaded nanoparticle—arginine-hydroxybutyl-chitosan (Arg-HBC)—that effectively protects macromolecular double-stranded RNA, enhances cellular immune regulation, and improves therapeutic efficiency.

[0031] The chitosan nanoparticle delivery carrier co-modified with arginine and hydroxybutyl groups of the present invention and its preparation method are simple, environmentally friendly, and low in cost. The delivery carrier prepared by the present invention not only has excellent water solubility but also a high positive charge density, synergistically exerting an immunostimulatory effect with the loaded nucleic acid adjuvant polyI:C. Currently, in aquaculture and poultry farming, polyI:C combined with kanamycin (a stabilizer) is commonly used to form a PolyI:C-kanamycin (PICK) complex as an immunoadjuvant to enhance animals' immune defense against various pathogens. The use of antibiotics increases the risk of bacterial resistance and seriously impacts the environment. Kanamycin, an aminoglycoside antibiotic, is prohibited in aquaculture. To address these problems, the present invention uses chitosan derivatives to load antibiotic-free polyI:C to prepare drug-loaded nanoparticles to improve the delivery efficiency of polyI:C and solve the problem of antibiotic overuse. Furthermore, polyI:C is unstable in aqueous solution and is easily degraded by nucleases. Excessive polyI:C may trigger an overactive immune response, leading to excessive inflammation and adverse effects on animals. To enhance the immunomodulatory effect, this invention introduces arginine into water-soluble hydroxybutyl chitosan. Arginine's positive charge enhances the charge density of chitosan, binds to negatively charged nucleic acids, reduces the degradation rate of polyI:C, improves the loading efficiency of polyI:C, and achieves time-dependent drug release. Arginine also possesses certain immunomodulatory functions, synergistically enhancing the immunostimulatory effect of chitosan. The combined immunostimulatory effect of the delivered nanoparticles and polyI:C significantly enhances the cellular immune response. Attached Figure Description

[0032] Figure 1 A schematic diagram of the formation of Arg-HBC nanoparticles loaded with polyI:C in Example 1 of this invention.

[0033] Figure 2 This is a schematic diagram of the particle size distribution of Arg-HBC nanoparticles loaded with polyI:C in Example 1 of the present invention.

[0034] Figure 3 The infrared spectrum of Arg-HBC in Example 2.

[0035] Figure 4 The NMR spectrum of Arg-HBC in Example 2 is shown.

[0036] Figure 5 This is a nucleic acid gel electrophoresis image of Arg-HBC detecting the polyI:C loading capacity in Example 2.

[0037] Figure 6 The results of the toxicity experiment on L929 cells by Arg-HBC nanoparticles loaded with polyI:C in Example 3 are shown.

[0038] Figure 7 A and Figure 7 B represents the synergistic stimulatory effect of Arg-HBC nanoparticles loaded with polyI:C on RAW264.7 immune cells in Example 4. Detailed Implementation

[0039] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0040] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0041] Arginine and hydroxybutyl-modified chitosan exhibits good water solubility, and the aqueous solution of the chitosan derivative falls within the physiological pH range. Similar to the chitosan backbone, it possesses the unique advantages of chitosan while overcoming the limitation of chitosan's solubility only in acidic solutions, thus better meeting the solubility requirements during delivery. Using arginine and hydroxybutyl-modified chitosan as a delivery carrier for polyI:C effectively protects polyI:C from nuclease degradation and improves its stability in vivo. Simultaneously, the positive charge of the arginine and hydroxybutyl-modified chitosan can interact with the negative charge of polyI:C to form a stable complex, promoting cellular uptake of polyI:C, thereby enhancing the immunomodulatory effects of polyI:C and improving its antiviral and antitumor therapeutic efficacy, potentially playing an important role in gene therapy and vaccine development.

[0042] The arginine and hydroxybutyl-modified chitosan synthesized in this invention exhibits good water solubility within the physiological pH range. Its effective loading and delivery of polyI:C compensates for chitosan's limitation of being only soluble in acidic solutions, making it more suitable for solubility requirements during delivery. The introduction of arginine significantly enhances chitosan's ability to bind nucleic acid molecules. The cationic polymer chains entangle with nucleic acid molecules through electrostatic interactions, forming a stable complex that effectively protects polyI:C from nuclease degradation and improves its stability. Simultaneously, this positively charged complex not only increases the loading rate of nucleic acid drugs but also readily adsorbs onto negatively charged cell membranes, entering cells via endocytosis. This promotes cellular uptake and enhances the immunomodulatory effects of polyI:C, improving its antiviral and antitumor therapeutic efficacy, and holds promise for playing a significant role in gene therapy and vaccine development.

[0043] Example 1

[0044] Preparation of Arg-HBC nanoparticles loaded with polyI:C

[0045]

[0046] Weigh 3g of chitosan and add it to 20ml of 50% NaOH. Stir well and react at room temperature for 24 hours. Filter to remove excess alkali, and wash thoroughly three times with ultrapure water to obtain alkalized chitosan. Disperse the alkalized chitosan evenly in 20ml of isopropanol and 20ml of ultrapure water, and stir at room temperature until fully swollen. Add 80ml of 1,2-epoxybutane dropwise to the chitosan solution and react at 45℃ for 24 hours. The reaction was terminated by neutralization with dilute hydrochloric acid, followed by dialysis, centrifugation, and freeze-drying of the supernatant to obtain hydroxybutyl chitosan (1); 1g of hydroxybutyl chitosan was weighed and dissolved in 50ml of water, 1.5g of EDC·HCl and 0.5g of NHS were added, and the mixture was stirred evenly. 2g of arginine was added, and the pH was adjusted to 5.0. The mixture was reacted at room temperature for 24h, followed by dialysis, centrifugation, and freeze-drying of the supernatant to obtain Arg-HBC; 10mg of Arg-HBC was dissolved in 10ml of water and vortexed to obtain Arg-HBC nanosolution (2).

[0047] Different masses of polyI:C (1 mg, 2 mg, 5 mg, 10 mg) were weighed according to Table 1 and dissolved in 1 ml of DEPC water. 10 mg of Arg-HBC was weighed and dissolved in 10 ml of DEPC water. 100 μl of polyI:C of different concentrations was slowly added dropwise to 100 μl of chitosan derivative solution. The mixture was stirred at high speed in an emulsifying homogenizer for 10 minutes, followed by magnetic stirring at 800-1000 rpm for 1 hour. The two components self-assembled through intermolecular electrostatic interactions to form Arg-HBC nanoparticles loaded with polyI:C. A schematic diagram of the preparation is shown below. Figure 1 As shown.

[0048] The particle size, PDI, and zeta potential of the prepared polyI:C-loaded Arg-HBC nanoparticles were measured, and the results are shown in Table 1. Figure 2 As shown.

[0049] Table 1

[0050]

[0051] From Table 1 and Figure 2 It can be seen that when 100 μl of polyI:C (0.1 mg / ml) and 100 μl of Arg-HBC (1 mg / ml) are mixed, the prepared mixed nanoparticles have the smallest particle size, high zeta potential, and good dispersion index. As the polyI:C ratio increases to 1.0 mg / ml, the particle size of the drug-loaded particles increases to about 552.9 nm.

[0052] Example 2

[0053] Fourier transform infrared spectroscopy analysis

[0054] The Arg-HBC solid prepared in Example 1 was detected using a Nicolet 2000 Fourier Transform Infrared Spectrometer (Nicolet Corporation, USA). The detection method was as follows: 1–2 mg of the above sample was mixed with 200 mg of KBr particles, thoroughly ground, compressed into a tablet, and scanned in the infrared spectrometer within a scanning range of 4000 cm⁻¹ to 400 cm⁻¹. The infrared spectrum was recorded, and the results are as follows. Figure 3 As shown.

[0055] Nuclear magnetic resonance 1 H-NMR

[0056] The Arg-HBC lyophilized sample prepared above was dissolved in deuterated water, irradiated at a frequency of 400 MHz, and its spectrum was recorded at 20 °C using a Bruker (AVANCEⅢ) AV-400 nuclear magnetic resonance spectrometer (Bruker GmbH, Germany). The results are as follows: Figure 4 As shown.

[0057] Nucleic acid gel electrophoresis

[0058] Take 5 μl of the polyI:C-Arg-HBC nanoparticles prepared in Example 1, spot it into the sample well, and observe the gel electrophoresis results after 150V for 15 min. Figure 5 As shown.

[0059] Example 3

[0060] CCK-8 assay was used to detect the effect of polyI:C-loaded Arg-HBC nanoparticles on L929 cell viability.

[0061] The CCK-8 assay involved seeding cultured cells into 96-well plates and incubating them at 37°C with 5% CO2 for 24 hours. The culture medium was discarded, and then an appropriate amount of complete culture medium was added to each well. The control group received complete culture medium, while the experimental groups received Arg-HBC nanoparticles containing different concentrations of polyI:C. After 24 and 48 hours of incubation, the culture medium was aspirated from each well, and CCK-8 solution was added. The plates were incubated at 37°C for 1 hour, and after shaking, the OD value of each well was measured at 450 nm using a microplate reader. The blank group contained only complete culture medium. The average OD value of each well was used to calculate cell viability using the following formula:

[0062] Cell viability (%) = [(Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance)] × 100%

[0063] The results are as follows Figure 6 As shown, compared with the control group, there was no statistically significant difference in the proliferation inhibition rate of Arg-HBC nanoparticles loaded with different concentrations of polyI:C on L929 cells.

[0064] Example 4:

[0065] Stimulating effect of polyI:C-loaded Arg-HBC nanoparticles on immune cells

[0066] Take RAW 264.7 cells (mouse mononuclear macrophage leukemia cells) in the logarithmic growth phase, and divide them into 2 × 10⁻⁶ cells per well. 5 Cells were seeded in 24-well plates with 500 μl of cell suspension per well. After 12 h of culture, the culture medium was aspirated. A negative control group (without drug) and a Lipofectamine control group were included. TM The study included a positive control group (3000 cells), a group containing only polyI:C, a group containing polyI:C and pre-modified chitosan, and an experimental group with an Arg-HBC mass ratio of 5:1, with three replicates per group. 100 μl of working solution was added to each well, followed by 400 μl of complete culture medium to a final volume of 500 μl. Cells were incubated at 37°C with 5% CO2 for 6 h and 12 h. Cells were collected at different time points, and total RNA was extracted, reverse transcribed, and analyzed using real-time quantitative PCR to detect changes in cellular inflammatory factor levels.

[0067] PolyI:C, as an immune adjuvant, can bind to Toll-like receptor 3 (TLR3) on the surface of macrophages, triggering an inflammatory response. It can also stimulate the production of type I interferon, thereby exerting an antiviral effect. Figure 7 A and Figure 7As shown in Figure B, compared with the positive control, Arg-HBC nanoparticles loaded with polyI:C significantly increased the stimulation of inflammatory factors such as IFN-β and CXCL10 in RAW264.7 cells, with statistically significant differences (P < 0.05). The modified drug-loaded Arg-HBC nanoparticles exhibited a stronger stimulating effect on immune cells compared to the unmodified cationic polymer CS. This indicates that Arg-HBC drug-loaded nanoparticles have the ability to deliver polyI:C intracellularly and stimulate inflammation.

[0068] In summary, this invention provides a novel drug-loaded nanoparticle that improves drug delivery efficiency and enhances cellular immune regulation. It exhibits good water solubility, is green and non-toxic, and is inexpensive. Cytotoxicity experiments and real-time quantitative PCR experiments demonstrate that the drug delivery carrier of this invention better promotes intracellular delivery and effectively stimulates the upregulation of inflammatory cytokine expression, achieving superior therapeutic effects.

[0069] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. A chitosan nanocarrier co-modified with arginine and hydroxybutyl, characterized in that, The chemical structural formula of the delivery carrier is shown in General Formula I below: Where m represents the number of moles of hydroxybutyl grafted onto chitosan, n represents the number of moles of arginine on chitosan or the degree of substitution of arginine, and k represents the number of moles of unreacted deacetylated chitosan monomers.

2. The chitosan nanocarrier co-modified with arginine and hydroxybutyl according to claim 1, characterized in that, In the delivery carrier, the chitosan has a molecular weight of 1–300 kDa, a degree of deacetylation of 80%–95%, an arginine modification ratio of 1%–15%, and a hydroxybutyl modification ratio of 10%–80%.

3. The method for preparing the chitosan nanocarrier according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Add an alkaline solution to chitosan, stir until homogeneous, and allow to swell at room temperature to prepare an alkaline chitosan solution; (2) Isopropanol and ultrapure water were added to the chitosan alkalization solution, and the solution was swollen at room temperature. Then, 1,2-epoxybutane solution was added dropwise to react and hydroxybutyl chitosan was obtained. (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the hydroxybutyl chitosan solution, mix, then add arginine aqueous solution, adjust the pH value to the range of 4 to 7, and react to obtain chitosan co-modified with arginine and hydroxybutyl.

4. The preparation method according to claim 3, characterized in that, In step (1), the alkaline solution is a NaOH solution, and the concentration of the chitosan alkalization solution is 50-200 mg / ml; In step (2), the volume ratio of isopropanol to ultrapure water is 0.5 to 2, the volume ratio of 1,2-epoxybutane solution to isopropanol is 0.5 to 3, and the reaction temperature is 35℃ to 70℃. In step (2), the reaction solution is neutralized, dialyzed, centrifuged to remove impurities, and freeze-dried to obtain hydroxybutyl chitosan; In step (3), the concentration of the arginine aqueous solution is 1-250 mg / ml; the concentration of the hydroxybutyl chitosan solution is 1-20 mg / ml; and the reaction time is 12-36 h.

5. The preparation method according to claim 3, characterized in that, The molar ratio of chitosan, arginine, EDC·HCl, and NHS is 1–4:0.5–2:1–4:2–8.

6. The application of the chitosan nanodelivery carrier according to claim 1 or 2 in the preparation of nucleic acid vaccines.

7. The application according to claim 6, characterized in that, The chitosan nanocarrier described herein is used to deliver adjuvants for nucleic acid vaccines.

8. The application according to claim 7, characterized in that, The adjuvant for the nucleic acid vaccine is poly I:C.

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