Dual-network hydrogel vaccine as well as preparation method and application thereof

Abstract

The invention provides double-network hydrogel. The double-network hydrogel is prepared by the following method: (1) preparing a Ga-Van compound; (2) GaCuVanamp, (3) And synthesizing the HACHO-BSA double-network hydrogel. The preparation method comprises the following steps: preparing GaCuVanamp; the HACHO-BSA double-network hydrogel can be used for capturing bacteria and adsorbing inflammatory factors, serum proteins and the like, so that GaCuVanamp is formed; the HACHO-BSA double-network hydrogel vaccine not only can provide long-term immunity to resist bacteria, but also can promote bone tissue regeneration, so that several key problems in osteomyelitis treatment are solved. A promising prospective hydrogel vaccine research strategy is provided for promoting clinical treatment and prevention of osteomyelitis.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a double network hydrogel vaccine and a preparation method and application thereof. Background Art

[0002] Bone infection, especially osteomyelitis, remains a major clinical challenge. Osteomyelitis is a serious chronic bone inflammatory disease mainly caused by Gram-positive Staphylococcus aureus (S. aureus), characterized by persistent infection, bone destruction and abscess formation. Traditional treatments include the use of systemic antibiotics, but their efficacy is limited by many factors, such as the difficulty of drug penetration into the site of infection, bacterial biofilm formation and the growing problem of antibiotic resistance. What's more, bone tissue healing problems and recurrent infections in patients with osteomyelitis remain major obstacles to clinical treatment, especially when the infection involves multidrug-resistant bacteria.

[0003] Recent advances in local drug delivery systems such as hydrogels and microspheres have shown promise, but most studies have focused on the antibacterial properties of topical materials, with limited attention paid to vaccine strategies that enhance the body's immune response and promote bone repair. Vaccination is considered one of the most effective means of preventing bacterial diseases. By activating the immune system and inducing the production of specific antibodies, vaccines can fundamentally prevent infection. However, existing vaccines have several limitations in practical applications, such as insufficient routes of administration, immunogenicity, and targeting, which prevent traditional vaccines from providing adequate protection. In addition, current vaccine research in the treatment of osteomyelitis is still in its early stages and has not yet been fully developed and applied.

[0004] Therefore, new therapeutic strategies that can simultaneously address infection and bone repair are urgently needed.

[0005] Technical content

[0006] The present invention first provides a method for preparing a double network hydrogel, comprising the following steps:

[0007] (1)Ga Van Preparation of complexes

[0008] Dissolve glycyrrhizic acid (Ga) in water to prepare a 1 mg / mL Ga aqueous solution; add vancomycin (Van); wherein the molar ratio of vancomycin to glycyrrhizic acid is 3-5:1;

[0009] NHS and EDC were dissolved in PBS buffer and added to the Ga and Van solution, stirred for 4 hours, the reaction temperature was maintained at 25°C, and the pH value was adjusted to 7.4; after the reaction, ethylene glycol was added to terminate the reaction; wherein the amount of NHS and EDC was 1.2 times the molar amount of Ga, respectively;

[0010] The reaction solution was transferred to a dialysis bag and dialyzed with PBS buffer for 24 hours to remove unreacted NHS, EDC and other small molecule impurities; after the dialysis was completed, the product was freeze-dried to obtain the Ga-Van complex.

[0011] (2)GaCu Van &HA CHO Synthesis of BSA double network hydrogel

[0012] Preheat deionized water to 60°C and weigh Ga Van and CuCl2 (the weight ratio of the two is 30-50:1), dissolved therein (the material-liquid ratio is 31-51 mg / mL), and continued to incubate at 60°C to promote the rapid dissolution of the components to form a precursor A solution;

[0013] Sodium periodate oxidizes hyaluronic acid HA CHO Dissolve in an aqueous solution containing 20% ​​BSA (forming a 10-30% by weight solution), and mix thoroughly at 60° C. to prepare a precursor B solution;

[0014] The precursor A solution and the precursor B solution were mixed in a volume ratio of 1-3:3-1 to prepare GaCu Van &HA CHO -BSA double network hydrogel.

[0015] The dialysis bag used was a regenerated fiber plastic film dialysis bag: flattened width (MD): 34 mm, molecular weight cut-off: (3500).

[0016] The present invention also provides GaCu prepared by the above method Van &HA CHO -BSA double network hydrogel.

[0017] The GaCu prepared by the present invention Van &HA CHO -BSA double network hydrogel is able to capture bacteria, adsorb inflammatory factors and serum proteins. Its properties are attributed to the physical capture effect caused by the hydrogel network structure and hydrogen bonding, as well as the chemical adsorption mediated by Schiff base reaction and coordination.

[0018] The present invention also provides a GaCu Van &HA CHO -BSA double network hydrogel vaccine, which is composed of the above-mentioned GaCu Van &HA CHO -BSA double network hydrogel cross-linking MRSA / S. aureus formation;

[0019] Specifically, the GaCu Van &HACHO -BSA double network hydrogel vaccine is prepared by the following method:

[0020] Preheat deionized water to 60°C and weigh Ga Van and CuCl2 (the weight ratio of the two is 30-50:1), dissolved therein (the material-liquid ratio is 31-51 mg / mL), and continued to incubate at 60°C to promote the rapid dissolution of the components to form a precursor A solution;

[0021] Sodium periodate oxidizes hyaluronic acid HA CHO Dissolved in an aqueous solution containing 20% ​​BSA and 2×10^6 CFU / mL MRSA / S. aureus (forming a 10-30% weight ratio solution), and mixed thoroughly at 60° C. to prepare a precursor B solution;

[0022] The precursor A solution and the precursor B solution were mixed in a volume ratio of 1-3:3-1 to prepare GaCu Van &HA CHO -BSA double network hydrogel vaccine.

[0023] The present invention also provides a drug for preventing or treating osteomyelitis, comprising GaCu Van &HA CHO -BSA double network hydrogel and a pharmaceutically acceptable carrier.

[0024] The present invention also provides the GaCu Van &HA CHO -Application of BSA double network hydrogel, which can be used to prepare drugs for preventing or treating osteomyelitis or bone damage.

[0025] The innovative features and beneficial technical effects of the present invention are as follows:

[0026] The GaCu Van &HA CHO -BSA double network hydrogel and GaCu Van &HA CHO-BSA double network hydrogel vaccine, with glycyrrhizic acid (Ga) as the starting material, Ga is a natural herbal small molecule extracted from licorice, with a unique structure, including hydrophobic triterpenoid aglycones and hydrophilic diglucuronic acid units. Studies have shown that the self-assembly process of Ga hydrogel is significantly accelerated in the presence of Cu ions. In particular, copper ions play an important role in the repair of bone tissue, which further promotes bone repair by regulating the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSC) and promoting angiogenesis. In addition, Ga can also regulate the Wnt / β-catenin signaling pathway, which is essential for promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC), thereby effectively repairing bone defects.

[0027] Therefore, the GaCu Van &HA CHO -BSA dual-network hydrogel, by combining two different networks with complementary properties, not only enhances the mechanical properties of the hydrogel, but also has excellent sustained-release properties, making it an ideal drug delivery system and vaccine carrier.

[0028] The GaCu Van &HA CHO -BSA double network hydrogel, combined with a variety of physical-chemical cross-linked networks, the primary network is formed by the complexation of glycyrrhizic acid grafted with vancomycin (Van) and copper ions (GaCu Van ), while the secondary network is formed by modified oxidized hyaluronic acid (HA CHO ) and multiple amino groups of bovine serum albumin (BSA) through Schiff base reaction to form (HA CHO -BSA), the introduction of HA-based network endows the hydrogel vaccine with macrophage targeting ability.

[0029] In addition, the GaCu Van &HA CHO -The double network structure of BSA hydrogel enhances the mechanical properties and stability of the hydrogel, and can capture and adsorb a variety of pathogen-related molecules in situ, including bacterial components, bacterial antigens, inflammatory cytokines and serum proteins. This process benefits from the unique combination of hydrogen bonds, coordination interactions and Schiff base bond cross-linking. Furthermore, the hydrogel vaccine targets the immunogenic components it carries to macrophages and can also continuously release pathogen-related molecules. This mechanism enhances the activation of the immune system and effectively promotes the clearance of bacteria. More importantly, the hydrogel vaccine can also activate immune memory and effectively prevent the recurrence of osteomyelitis. At the same time, the Ga and Cu released by the hydrogel vaccine 2+ It has the effect of stimulating bone regeneration and is particularly suitable for treating bone defects associated with osteomyelitis.

[0030] In summary, GaCu Van &HA CHO -BSA double network hydrogel vaccine not only has antibacterial effect, but also has immunomodulatory function, providing a promising and comprehensive treatment plan for infection control, bone regeneration and recurrence prevention of osteomyelitis, and is an ideal choice for minimally invasive treatment of osteomyelitis bone defects. At the same time, the vaccine releases Ga, which can promote bone tissue repair and enhance immunomodulation in osteomyelitis. The role of Ga in promoting bone repair is not as good as that of Cu. 2+ The multifunctional hydrogel vaccine provides long-term immunity against bacteria and promotes bone tissue regeneration, thus solving several key problems in the treatment of osteomyelitis. It provides a promising forward-looking hydrogel vaccine research strategy for promoting the clinical treatment and prevention of osteomyelitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 ,Ga,Van and Ga Van FT-IR spectrum comparison

[0032] Figure 2 , HA and HA CHO FT-IR spectrum comparison

[0033] Figure 3 、GaCu Van &HA CHO -FT-IR (A) and XPS results (B) of BSA double network hydrogel

[0034] Figure 4 、GaCu Van &HA CHO -XPS results of BSA double network hydrogel

[0035] Figure 5 、GaCu Van &HA CHO -Typical SEM image of BSA double network hydrogel

[0036] Figure 6 、GaCu Van &HA CHO -Study on the plasticity, injectability and self-healing properties of BSA double network hydrogel

[0037] Figure 7 、GaCu Van &HA CHO -Schematic diagram of the formation and mechanism of BSA double network hydrogel

[0038] Figure 8 、GaCu Van &HA CHO Compression recovery properties of BSA double network hydrogels

[0039] Fig. 9 、GaCu Va n&HA CHO Representative compressive stress-strain curves (A) and corresponding compressive strengths (B) of BSA double network hydrogels with BSA concentrations of 10%, 20%, and 30%, respectively.

[0040] Fig.10 , Representative compressive stress-strain curves (A) and corresponding compressive strength (B) of GaCuVan&HACHO-BSA double network hydrogels or their vaccines with and without MRSA. Van &HA CHO - Representative compressive stress-strain curves of BSA double network hydrogel vaccine (C) and corresponding compressive strength (D)

[0041] Fig.11 , bacterial plate coating images (A) and CFU counts of MRSA and S. aureus in supernatants and hydrogels (B)

[0042] Fig.12 Representative MRSA bioluminescence images (A) and corresponding quantitative data (B) analyzed using IVIS at 1, 3, and 7 days after injection of different hydrogels

[0043] Fig.13 Representative standard plate coating photos (A) and CFU counts (B) of MRSA in the bone marrow of rats in each group on days 7, 14, and 28 of infection, including PBS, Ga Van 、GaCu Van 、GaCu Van &HA CHO -BSA hydrogel and Van were pretreated

[0044] Fig.14 、GaCu Van &HA CHO -BSA double network hydrogel promotes bone repair in vivo

[0045] Fig.15 、GaCu Van &HA CHO Heat map and functional classification of differentially expressed genes in the bone marrow of rats with ectopic osteomyelitis pretreated with BSA hydrogel and PBS

[0046] Fig.16 、GaCu Van &HA CHO GO enrichment analysis of upregulated DEGs in the BSA hydrogel group and PBS group

[0047] Fig.17 、GaCu Van &HA CHO -KEGG enrichment analysis of upregulated DEGs in the BSA hydrogel group and PBS group

[0048] Fig.18 , GSEA analysis was performed on the rat GO, KEGG, Reactome, and Wikipathway datasets, and the top 5 significantly enriched gene sets with absolute NES values ​​were selected by default for map display

[0049] Fig.19 , the interaction energy between each molecule and different complexes after molecular docking

[0050] Fig. 20 , Interaction analysis of the three-dimensional docking results of various complexes. A) Cu ions and Ga Van Molecular interaction model. Enlarged image: Cu ions and Ga Van The molecules form a coordination. B) HA CHO Molecules and GaCu Van Interaction mode of the complex, the black box represents GaCu Van O and N atoms in the molecule and HA CHO Multiple hydrogen bonds are formed between hydroxyl O atoms in the molecule. C)GaCu Van &HA CHO Visual analysis of the complex interaction after docking between the complex and BSA DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments and accompanying drawings are only used to explain and illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make corresponding changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0052] The sources of the main raw materials and equipment in the following examples are as follows, and the remaining reagents or raw materials are common commercial products:

[0053] Glycyrrhizic acid: Shanghai Yuanye Biotechnology Co., Ltd., B20417

[0054] Vancomycin: Sigma, 1404-93-9

[0055] N-hydroxysuccinimide (NHS): Sigma, 6066-82-6

[0056] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC): Sigma, 22572-40-3

[0057] Sodium periodate (NaIO4): Sigma, 7790-28-5

[0058] Hyaluronic acid (HA): RHAWN, 9076-32-7

[0059] LB broth: Haibo Biotechnology, HBKP0128

[0060] Regenerated fiber plastic film dialysis bag: flattened width (MD): 34mm, molecular weight cut-off: (3500), Titan Technology Exploration Platform, 02037431

[0061] 8-week-old male Sprague-Dawley rats: Vital River

[0062] MRSA is a bacterium cultured from clinical patients in Ruijin Hospital by conventional methods. The screening process of RJ0068 is shown in Table 1 below:

[0063] Table 1 Antibiotic resistance of microorganisms (S: susceptible; R: resistant)

[0064]

[0065]

[0066] Example 1GaCu Van &HA CHO -BSA double network hydrogel and GaCu Van &HA CHO - Preparation of BSA double network hydrogel vaccine

[0067] (I)GaCu Van &HA CHO Preparation of -BSA double network hydrogel

[0068] (1) Preparation of Ga-Van composite

[0069] Dissolve 20 mg of glycyrrhizic acid (Ga) in 20 mL of water to prepare a 1 mg / mL Ga solution; similarly, dissolve 8.81 mg of vancomycin (Van) in a molar ratio of 4:1 with glycyrrhizic acid;

[0070] 32.7 mg N-hydroxysuccinimide (NHS) and 47.7 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in PBS buffer and slowly added to the Ga and Van solution. The reaction was stirred for 4 hours. The reaction temperature was maintained at 25 ° C and the pH value was adjusted to 7.4. After the reaction was completed, 30 mL of ethylene glycol was added to terminate the reaction; the reaction solution was transferred to a dialysis bag and dialyzed with PBS buffer for 24 hours to remove unreacted NHS, EDC and other small molecule impurities. After the dialysis was completed, the product was lyophilized to obtain the Ga-Van complex.

[0071] Sodium periodate oxidized hyaluronic acid HA CHO Preparation

[0072] Sodium periodate (NaIO4) aqueous solution (0.5M, 3 mL) was added dropwise to hyaluronic acid (HA) aqueous solution (10 mg / mL, 400 mL), and the mixture was stirred at 25°C and 600 rpm for 24 hours. The reaction was kept in the dark, and 3 mL of ethylene glycol was added to the solution to terminate the reaction.

[0073] Subsequently, the reaction solution was placed in a dialysis bag, and deionized water was replaced every morning and evening for 7 days to remove unreacted small molecules; finally, the dialysate was freeze-dried in a freeze dryer for 2 days to obtain oxidized hyaluronic acid (HA CHO ) and stored at -20°C;

[0074] (2)GaCu Van &HA CHO -BSA double network hydrogel synthesis

[0075] Preheat 10 mL of deionized water to 60 °C and weigh 400 mg of Ga Van and 10 mg CuCl2, dissolved therein, and continued to incubate at 60 °C to promote rapid dissolution of the components to form a precursor A solution;

[0076] 2g HA CHO Dissolve in 10 mL of a solution containing 20% ​​BSA and mix thoroughly at 60°C to prepare a precursor B solution;

[0077] The precursor A solution and the precursor B solution were quickly mixed in a volume ratio of 1:1 to prepare GaCu Van &HA CHO -BSA double network hydrogel.

[0078] (ii) GaCu Van &HA CHO -Preparation of BSA double network hydrogel vaccine:

[0079] Preheat 10 mL of deionized water to 60 °C and weigh 400 mg of Ga Van and 10 mg CuCl2, dissolved therein, and continued to incubate at 60 °C to promote rapid dissolution of the components to form a precursor A solution;

[0080] 2g HA CHO Dissolve in 10 mL of a solution containing 20% ​​BSA and 2×10^6 CFU / mL MRSA / S. aureus, and mix thoroughly at 60°C to prepare a precursor B solution;

[0081] The precursor A solution and the precursor B solution were quickly mixed in a volume ratio of 1:1 to prepare GaCu Van &HA CHO -BSA double network hydrogel vaccine.

[0082] Example 2: GaCu prepared in Example 1 Van &HA CHO - Characterization of BSA double network hydrogel and its vaccine:

[0083] (1) The chemical interactions between different hydrogels were analyzed by FT-IR (Nicolet 6700, USA) spectroscopy and X-ray photoelectron spectroscopy (XPS).

[0084] ① For Ga Van The synthesis of the Ga-based quartz crystal was carried out by using EDC and NHS to induce the condensation between the carboxyl group on Ga and the amino group on Van, and the process was verified by FT-IR spectra. Figure 1 , showing Ga Van The characteristic absorption peaks of Ga and Van appeared in the ions, including C=O stretching vibration, C–O bond vibration, and C–H stretching vibration, which further supports the conclusion that Ga Van successful synthesis.

[0085] ②To synthesize HA CHO -BSA, HA was oxidized by sodium periodate, and the effectiveness of the oxidation process was further verified by FT-IR. Figure 2 Compared with hyaluronic acid HA, HA CHO The FT-IR spectrum of HA showed a new C=O stretching vibration at 1735 cm-1, indicating that CHO Successful preparation.

[0086] ③ In order to verify the successful construction of the hydrogel, FT-IR analysis and X-ray photoelectron spectroscopy (XPS) analysis were performed. First, the FT-IR results ( Figure 3A) shows the characteristic absorption peaks of Ga and Van, including C=O stretching vibration and C–O bond vibration; however, in Cu 2+ After complexation, the absorption peaks of these functional groups shifted slightly, indicating that complexation did occur. CHO The characteristic absorption peaks (such as C–H stretching vibration, etc.) of GaCu were also verified. At the same time, the characteristic peak of C=O stretching vibration showed a slight red shift, which further illustrates the enhancement of hydrogen bonding in the co-assembled hydrogel. These results support the GaCu Van &HA CHO -Successful synthesis of BSA double network hydrogel.

[0087] Next, the XPS test results show ( Figure 3 B. Figure 4 ), in GaCu Van &HA CHO -Ga, Van, and Cu were identified in BSA double network hydrogel 2+ , HA CHO and BSA characteristic peaks (C 1s, O 1s, N 1s, Cu 2p, Cl2p); at the same time, the changes in N 1s and C1s spectra indicate the occurrence of Schiff base reaction, while the changes in O1s and N1s peaks reveal the further strengthening of hydrogen bonding. The above results fully prove that GaCu Van &HA CHO -BSA double network hydrogel was successfully synthesized. In summary, these results confirm the Van &HA CHO -Successful formation of BSA double network hydrogel vaccine.

[0088] (2) GaCu was characterized by SEM (JSM-7800F, JEOL). Van , HA CHO -BSA and GaCu Van &HA CHO -BSA hydrogel microstructure and morphology. GaCu Van , HA CHO -BSA and GaCu Van &HA CHO -Typical SEM images of BSA hydrogels show ( Figure 5 ), all three hydrogels showed porous microstructures and rough mesh features on the surface, further indicating that they have good bacterial adsorption capabilities.

[0089] (3)GaCu Van &HA CHO -Plasticity, injectability and self-healing properties of BSA double network hydrogels

[0090] In order to evaluate the plasticity of the hydrogel, the hydrogel was poured into a five-pointed star and a circular model, and after the gel was formed, it was taken out with tweezers to observe its shape change. The results showed ( Figure 6 A), the hydrogel was able to be injected into containers of different shapes and completely filled the star-shaped and circular grooves. However, when removed from the grooves, the hydrogel formed a complete block with uniform thickness, a finding that indicates the dual network structure of GaCu Van &HA CHO -BSA hydrogel has excellent shape adaptability and is therefore expected to effectively fill the complex irregular environment within the bone marrow cavity.

[0091] To evaluate the injectability of the hydrogels, the hydrogels were injected using a 1 mL syringe and injected into different letter markers as needed. Figure 6 B provides GaCu Van &HA CHO -BSA double network hydrogel has good injectability and will not cause needle clogging, which is crucial for minimally invasive treatment of osteomyelitis.

[0092] The self-healing properties of the hydrogel were investigated by pre-treating the GaCu Van &HA CHO -BSA double network hydrogels were dyed. Then, the two hydrogels were cut and left at room temperature for a period of time to promote self-healing. After repair, the self-healing efficiency of the hydrogels was evaluated, mainly based on the recovery of the mechanical strength of the repaired hydrogels and the changes in their visual appearance. The results showed ( Figure 6 C)GaCu Van &HA CHO -BSA double network hydrogel showed excellent self-healing ability. Van &HA CHO The -BSA double network hydrogel was cut in half and then the different colored hydrogel parts were rejoined. After 30 minutes of self-healing, no cracks were observed. These results indicate that the physicochemical double cross-linked network significantly improved the mechanical properties of the gel.

[0093] (4)GaCu Van &HA CHO -BSA double network hydrogel can not only recognize and capture surrounding bacteria ( Fig.11 ), and can also adsorb bacterial antigens, inflammatory factors and serum proteins through Schiff base reaction, hydrogen bonding and metal coordination ( Figure 7 ).

[0094] To investigate the limit of bacterial adsorption by hydrogels, we incubated the hydrogels with bacteria for 12 hours. This extended incubation time allowed the bacteria to grow to a plateau and for the bacteria to fully react with the hydrogels. It was observed that the bacterial concentration increased significantly with time, while the HA CHO -BSA hydrogel also increased the number of bacteria captured. This may be because long-term co-incubation caused the hydrogel to form more Schiff base bonds and hydrogen bonds with bacteria, and the gaps in the hydrogel were saturated with bacteria. Van and GaCu Van &HA CHO No bacteria were observed in the -BSA hydrogels, suggesting that the bacteria might be killed by the released Van after long-term incubation with the hydrogels.

[0095] (5)GaCu Van &HA CHO -Mechanical testing of BSA double network hydrogel

[0096] Cylindrical GaCu Van &HA CHO -BSA double network hydrogel samples (8 mm in diameter and 10 mm in height) were placed on the specimen platform of a universal material testing machine (Instron 4202, USA) equipped with a 200 N load cell and subjected to compression testing under the condition that the ultimate strain was fixed at 80%. Van &HA CHO -BSA double network hydrogel vaccine was added with 10%, 20% and 30% BSA solution, or MRSA / S. aureus, to form new hydrogel vaccine samples, and its compression properties were tested using a universal material testing machine.

[0097] The experimental results show that GaCu Van &HA CHO -BSA double network hydrogel will not show obvious cracking during compression and can return to its original shape after the external force is removed ( Figure 8 ). When the W / W ratio of BSA increases from 10% to 30%, GaCu Van &HA CHO -BSA double network hydrogel's maximum compressive strain also increased accordingly, reaching up to 280.80 kPa ( Fig. 9 ). This shows that the content of BSA is crucial to the overall mechanical properties of the hydrogel, and the increase of BSA content helps to enhance the mechanical properties of the hydrogel.

[0098] In addition, this embodiment also detects the Van &HA CHO -BSA double network hydrogel was supplemented with 107 Compression performance after CFU / mLMRSA and S. aureus, i.e., GaCu Van &HA CHO -BSA hydrogel vaccine compression performance test. The results show that ( Fig.10 ), GaCu after adding MRSA Van &HA CHO -BSA hydrogel vaccine compression strength increased from 52.662±13.894kPa to 89.536±14.7kPa, and after mixing with S. aureus, the compression strength increased from 52.522±17.149kPa to 134.097±20.915kPa. This shows that the artificially introduced bacteria are Van &HA CHO After the effective adsorption and capture of the GaCu-BSA double network hydrogel, the cross-linking degree increased, thus significantly improving its mechanical properties. Van &HA CHO -BSA double network hydrogel is able to capture bacteria, adsorb inflammatory factors and serum proteins. Its properties are attributed to the physical capture effect caused by the hydrogel network structure and hydrogen bonding, as well as the chemical adsorption mediated by Schiff base reaction and coordination.

[0099] Example 3GaCu Van &HA CHO -BSA double network hydrogel vaccine against osteomyelitis in rats (I) Establishment of rat osteomyelitis model and in situ GaCu Van &HA CHO -BSA hydrogel vaccination

[0100] The rat osteomyelitis model was established by inoculating 6-week-old male Sprague-Dawley rats (200-250 g) with 1×10^6 CFU of MRSA into the tibial medullary cavity. Prior to inoculation, the rats were anesthetized with 3% pentobarbital and the tibial area was disinfected with iodine to prevent contamination. The tibia was exposed through a small incision, and 200 μL of MRSA bacterial suspension was injected directly into the medullary cavity using a sterile syringe. After inoculation, the surgical area was disinfected and sutured, and the rats were placed under standard laboratory conditions to recover. The animals were monitored for signs of infection, such as redness, swelling, and limited movement at the injection site.

[0101] For in situ hydrogel vaccination, one day after MRSA inoculation, different hydrogels (Ga Van 、GaCu Van and GaCu Van &HA CHO-BSA group) was carefully injected into the bone marrow cavity of the rat tibia. Each group of rats received 200 μL of hydrogel once. After injection, the rats were closely observed for adverse reactions, changes in vital signs (weight and body temperature), and changes in blood counts.

[0102] (ii) The MRSA bioluminescent signal in the medullary cavity of rats with infectious osteomyelitis was recorded using the IVIS system (PerkinElmer), and the severity of infection was evaluated on days 1, 3, and 7. In addition, medullary bone marrow was sampled from rats infected for 7, 14, and 28 days. 0.1 mL of the bone marrow dilution was inoculated onto LB culture plates, which were cultured at 37°C for 24 hours, and the colonies were recorded and CFU counts were performed.

[0103] Experimental results ( Fig.12 ): On the first day of the experiment, obvious bioluminescence was observed at the injection site (box) of the tibia of rats in each group, and there was no significant difference in luminescence intensity, indicating that the tibial osteomyelitis model was successfully established. Van &HA CHO The MRSA bioluminescence in the -BSA group was no longer detectable, while bioluminescence was still detectable in the remaining groups, indicating that in situ vaccination with the hydrogel vaccine was able to quickly control the bacterial infection, while the other groups maintained a persistent infection state.

[0104] (III) Tibia samples were collected from rats at 7, 14, and 28 days after MRSA infection. After the epiphysis was removed at both ends and the infected bone marrow fluid was flushed out with PBS, it was plated on a standard plate and CFU was counted. No bacterial colony growth was observed in the bone marrow fluid of the sham group, while a large number of colonies grew in the PBS group, with a CFU count of up to 1×10 6 CFU / mL. Compared with the PBS group, Van and Ga Van and GaCu Van There was no significant change in the CFU counts of the GaCu Van &HA CHO -BSA CFU counts were significantly reduced. More significantly, on day 7, GaCu Van &HA CHO - The number of bacteria in the BSA group had decreased by 5 log units, and on days 14 and 28, there was almost no bacterial growth in the GaCu group. Van &HA CHO - No bacterial survival was observed in the BSA group, while the remaining groups still had obvious bacterial infection ( Fig.13 ).

[0105] 4. Micro-CT imaging and histopathological examination of bone defects in osteomyelitis

[0106] In the presence of different hydrogels (Ga Van 、GaCu Van and GaCu Van &HA CHO -BSA group) 4 weeks and 8 weeks after the rats were killed and the tibia was collected. The bone specimens were fixed in 4% paraformaldehyde for 24 hours and then scanned using a Micro-CT system (Skyscan 1172, Aartselaar, Belgium) with a scanning resolution of 15μm voxel size. The acquired three-dimensional images were reconstructed and analyzed to evaluate bone mineral density (BMD), bone volume / volume ratio (BV / TV), trabecular number (Tb.N) and trabecular spacing (Tb.Sp). These parameters were quantitatively analyzed by Micro-CT software (CTAn, Bruker) to evaluate the degree of bone repair and bone integration.

[0107] Micro-CT reconstruction of the damaged tibia shows ( Fig.14 A, B), the bone defect (in the dotted box) in the Sham group was significantly smaller at 8 weeks than at 4 weeks, which is consistent with the bone defect healing speed of normal rats. Compared with the Sham group, the bone defects of the tibia of rats in the PBS and Van groups were not effectively repaired at either 4 or 8 weeks, and this area was consistent with the drilling site during osteomyelitis modeling. This result shows that when bacterial infection is not controlled, the repair of bone defects cannot be resolved. Van 、GaCu Van and GaCu Van &HA CHO The bone repair effect of the -BSA group was significantly better than that of the PBS group. At 4 weeks, the bone defects of the three groups of rats had been significantly improved, and at 8 weeks, they were almost completely repaired. Van and GaCu Van &HA CHO -The repair effect of BSA group is better than that of Ga Van The results showed that the Ga released by the hydrogel vaccine can significantly promote the bone regeneration disorder caused by osteomyelitis in vivo, and this effect is further enhanced by the addition of Cu. This conclusion is also further verified by data such as bone tissue volume / total tissue volume ratio (BV / TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Fig.14 C, D).

[0108] (V) In vivo systemic immune response studies

[0109] Fourteen days after infection, bone marrow tissues of rats were collected for RNA-seq analysis to investigate changes in gene expression associated with osteogenesis and immune response. Total RNA was extracted from rat bone marrow using the ABclonal mRNA-seq Lib Prep Kit (Invitrogen, USA), and the A260 / A280 absorbance ratio of RNA samples was measured using Nanodrop ND-2000 (Thermo Scientific, USA). The RIN value of RNA was determined using Agilent Bioanalyzer 4150 (Agilent Technologies, California, USA).

[0110] (ABclonal, China) instructions for preparing PE libraries. Finally, sequencing was performed using the Illumina Novaseq 6000 / MGISEQ-T7 sequencing platform.

[0111] The results show that Fig.15 ), in Sham, PBS and GaCu Van &HA CHO A total of 184 differentially expressed genes (DEGs) related to hydrogel vaccine were identified in the bone marrow of rats in the -BSA group (Fold Change ≥ 2 and q < 0.05). Gene ontology (GO) database analysis revealed significant changes in differentially expressed genes in specific biological processes (BP), molecular functions (MF), or cellular components (CC). Fig.16 Compared with G2(PBS), G6(GaCu Van &HA CHO The expression of genes related to immune response, response to bacteria, immune system process, MyD88-independent Toll-like receptor signaling pathway regulation, response to exogenous biological stimulation, pattern recognition receptor signaling pathway regulation, CXCR chemokine receptor binding and chemokine activity (such as Irf7, Dhx58, Ifit3, Cxcl2) was significantly upregulated in the GaCu-BSA group (rectangular box), further confirming that the GaCu Van &HA CHO -The role of BSA hydrogel vaccine in activating immune response at the molecular level.

[0112] In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that after hydrogel vaccine treatment, the expression levels of genes involved in RIG-I-like receptor signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, bacterial invasion of epithelial cells, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, and TNF signaling pathway were upregulated (rectangular boxes) (see Fig.17 ). Encouragingly, gene set enrichment analysis (GSEA) also revealed multiple changes in the immune response activated by hydrogel vaccination ( Fig.18), including upregulation of genes in cytokine and inflammatory response pathways (e.g., CXCL2, CSF3, CXCL3, IFNb1, IL2, IL6, IL11, CSF2, IL12b, TBX21, XCL1, CCL3, CCL4, IL12b, IL18), while downregulation of genes related to negative regulation of cytokine production in immune response, negative regulation of production of immune response molecular mediators, and interleukin 10 signaling pathway (e.g., IFNb1, ARG1, RT1-M6-2, CD96, IL10, FOXP3, Zpbp2, CCL22).

[0113] Example 4 Binding energy of hydrogel vaccine components and molecular mechanism of gelation

[0114] In order to study GaCu Van &HA CHO -BSA hydrogel components, in turn, bind to Cu 2+ , Ga Van Molecules and HA CHO First, Ga Van With Cu 2+ The molecular docking predicted binding energy between them was 0.998 kcal / mol; subsequently, HA CHO With GaCu Van The molecular docking predicted binding energy is 3.816 kcal / mol. Van &HA CHO The molecular docking binding energy between the complex and BSA is 8.854 kcal / mol.

[0115] In order to study GaCu Van &HA CHO -BSA hydrogel gelation mechanism, and visualized the complex after molecular docking. The first visualization analysis results showed ( Fig. 20 ), Cu 2+ Combined with Ga Van In a cavity of HA, it forms a coordination effect with the four surrounding O atoms, thus promoting its stable binding. CHO With GaCu Van Visual analysis of the complex showed that HA CHO The hydroxyl O atoms of GaCu Van Five groups of hydrogen bonds are formed between the O atoms of HA CHO The hydroxyl groups of O also react with GaCu Van A group of hydrogen bonds are formed between the N atoms, which further promotes the stable combination of the ternary complex. Van &HA CHOThe results of docking of the complex with BSA showed that GaCu Van &HA CHO A large number of hydrogen bonds are formed between the complex and BSA, which promotes its binding to the cavity above the protein. 2+ In addition to Ga Van In addition to forming four groups of coordination between them, it also forms two groups of coordination with the Asp135 amino acid side chain on BSA. Van and HA CHO It contains a large number of hydrogen bond acceptors and donors, so a total of 26 groups of hydrogen bonds are formed between the ternary complex molecule and the protein, further promoting the stable binding of the hydrogel.

Claims

1. A method for preparing a double network hydrogel, characterized in that The steps include: (1)Ga Van Preparation of complexes Dissolve glycyrrhizic acid Ga in water to prepare a 1 mg / mL Ga aqueous solution; add vancomycin Van; wherein the molar ratio of vancomycin to glycyrrhizic acid is 3-5:1; NHS and EDC were dissolved in PBS buffer and added to the Ga and Van solution, stirred for 4 hours, the reaction temperature was maintained at 25°C, and the pH value was adjusted to 7.4; after the reaction, ethylene glycol was added to terminate the reaction; wherein the amount of NHS and EDC was 1.2 times the molar amount of Ga, respectively; The reaction solution was transferred to a dialysis bag and dialyzed with PBS buffer for 24 hours to remove unreacted NHS, EDC and other small molecule impurities; after the dialysis was completed, the product was lyophilized to obtain a Ga-Van complex; (2)GaCu Van &HA CHO Synthesis of BSA double network hydrogel Preheat deionized water to 60°C and weigh Ga Van and CuCl2, the weight ratio of which is 30-50:1, are dissolved in water, the solid-liquid ratio is 31-51 mg / mL, and the mixture is incubated at 60°C to promote the rapid dissolution of the components, thereby forming a precursor A solution; Sodium periodate oxidizes hyaluronic acid HA CHO Dissolved in an aqueous solution containing 20% ​​BSA to form a 10-30% by weight solution, and mixed thoroughly at 60° C. to prepare a precursor B solution; The precursor A solution and the precursor B solution were mixed in a volume ratio of 1-3:3-1 to prepare GaCu Van &HA CHO -BSA double network hydrogel.

2. GaCu prepared by the method of claim 1 Van &HA CHO -BSA double network hydrogel.

3. A GaCu Van &HA CHO -BSA double network hydrogel vaccine, which is GaCu according to claim 2 Van &HA CHO -BSA double network hydrogels were formed by cross-linking MRSA or S. aureus.

4. GaCu according to claim 3 Van &HA CHO -BSA double network hydrogel vaccine is prepared by the following method: Preheat deionized water to 60°C and weigh Ga Van and CuCl2, the weight ratio of which is 30-50:1, dissolved therein, The solid-liquid ratio was 31-51 mg / mL, and the incubation was continued at 60°C to promote the rapid dissolution of the components to form a precursor A solution; Sodium periodate oxidizes hyaluronic acid HA CHO Dissolved in an aqueous solution containing 20% ​​BSA and 2×10^6 CFU / mL MRSA / S. aureus, and mixed thoroughly at 60°C to prepare a precursor B solution; The precursor A solution and the precursor B solution were mixed in a volume ratio of 1-3:3-1 to prepare GaCu Van &HA CHO -BSA double network hydrogel vaccine.

5. A drug for preventing or treating osteomyelitis, characterized in that Containing the GaCu according to claim 2 Van &HA CHO -BSA double network hydrogel and a pharmaceutically acceptable carrier.

6. GaCu according to claim 2 Van &HA CHO -Application of BSA double network hydrogel, which can be used to prepare drugs for preventing or treating osteomyelitis or bone damage.

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