A sepiolite in-situ grown nanozyme, its preparation method and application

Abstract

The present invention relates to the technical field of nanomaterials for vaccines, and particularly relates to a sepiolite in-situ grown nanozyme, a preparation method thereof and an application thereof. The preparation method is to mix ferric chloride, ethylene glycol and sodium acetate to form a suspension, and then mix sepiolite with the suspension, and react at a temperature of 180-200 °C to obtain a black precipitate. After washing and drying, sepiolite in-situ grown nanozyme is obtained. The sepiolite nanozyme realizes the in-situ growth of iron-based nanozyme on the surface of sepiolite rod-like nanostructures under hydrothermal conditions, significantly enhancing the peroxidase activity. After Sep@IONzyme is connected with inactivated influenza virus, the complex forms an appropriate positive charge distribution, enhancing the adhesion ability of the antigen to nasal mucosa. Immunizing mice with it as a nasal drop vaccine significantly improves the levels of respiratory mucosal specific sIgA antibody and serum specific IgG antibody, and can completely protect against the lethal attack of influenza virus.

Description

Technical Field

[0001] The present invention relates to the technical field of nano - materials for vaccines, and particularly to a sepiolite - in - situ - grown nanozyme and its preparation method and application. Background Art

[0002] Influenza is a highly contagious zoonosis caused by the influenza virus. Currently, the prevention and control of influenza mainly rely on intramuscular injection of vaccines. Although the serum - circulating antibody titer is very high, it is often difficult to block virus excretion. The nasal cavity is one of the main entrances for influenza virus to enter the host. If good immune protection can be established in the nasal mucosa, it will prevent the invasion and spread of influenza virus. Due to the obstruction of the nasal mucosa, including mucus, cilia, and a dense epithelial barrier, the number of inactivated influenza vaccines that enter the submucosa and are taken up by antigen - presenting cells is very small. Therefore, the immune effect of a single inactivated influenza vaccine is often not ideal. The nasal - drip immunization of inactivated influenza vaccine combined with an adjuvant significantly improves the mucosal immune level of the host. Existing mucosal immune adjuvants include cholera toxin (CT), Escherichia coli heat - labile enterotoxin (LT), poly(I:C), and cytokines, etc. However, due to their safety and cost issues, their further clinical application is limited.

[0003] Iron - based nanozymes (Iron oxide nanozyme, IONzyme) are a class of inorganic nano - materials with enzyme - like activities, which can simulate various enzyme - like activities, such as peroxidase, catalase, etc., and have potential application prospects in the fields of anti - microorganism, tumor treatment, sewage purification, etc. However, the existing strategies are still limited in enhancing the enzyme - like activities of iron - based nanozymes. Sepiolite (Sep) is a magnesium - containing porous chain silicate mineral. The nano - structure pore diameter is unique, with a large specific surface area and pore volume, and strong adsorption. Due to its rich acid - base centers, it is helpful for the in - situ growth of other materials and can regulate catalytic performance by changing the structure. Sep is also a food additive recognized by national standards, and its safety can be guaranteed, and the cost is low. Therefore, it is expected to enhance peroxidase activity and convert the positive - charge distribution on the particle surface by in - situ growing IONzyme on the surface of Sep rod - like nano - structures under hydrothermal conditions, so as to achieve the purpose of being used as a mucosal adjuvant for influenza vaccines. So far, there has been no research report on sepiolite - in - situ - grown nanozyme (Sep@IONzyme). Summary of the Invention

[0004] The present invention aims to solve the technical problems existing in the prior art. For this purpose, the present invention provides a sepiolite - in - situ - grown nanozyme and its preparation method and application, aiming to provide new ideas for influenza prevention and control and improve the mucosal immune response level of the host.

[0005] For the above purposes, the present invention provides a preparation method of sepiolite in-situ grown nanozyme. The preparation method is to mix ferric chloride, ethylene glycol and sodium acetate to form a suspension, and then mix sepiolite with the suspension, react at a temperature of 180 - 200 °C to obtain a black precipitate, and then obtain sepiolite in-situ grown nanozyme after washing and drying.

[0006] Preferably, the ferric chloride is anhydrous ferric chloride, and the sodium acetate is sodium acetate trihydrate.

[0007] Preferably, the mass ratio of anhydrous ferric chloride, sodium acetate and sepiolite is 0.8 - 0.9:3.4 - 3.6:0.1 - 1.

[0008] More preferably, the mass ratio of anhydrous ferric chloride, sodium acetate and sepiolite is 0.82:3.6:0.5.

[0009] The present invention also provides a sepiolite in-situ grown nanozyme prepared by the above preparation method.

[0010] As an alternative embodiment, the preparation method is as follows:

[0011] Add 40 - 50 mL of ethylene glycol into a beaker, and add anhydrous ferric chloride (0.8 - 0.9 g) while stirring on a magnetic stirrer. After the ferric chloride is completely dissolved, slowly add 3.4 - 3.6 g of sodium acetate trihydrate. After a stable suspension is formed, add 0.1 - 1 g of Sep according to the formula respectively, and use ultrasonic waves to fully mix the solution. After a stable suspension is formed, transfer the solution to a reaction kettle and react at a high temperature of 180 - 200 °C for 11 - 13 h. Take the reaction product, discard the black liquid in the supernatant, wash out the black precipitate at the bottom of the reaction kettle with anhydrous ethanol, and first wash the product 2 - 4 times with anhydrous ethanol by centrifugation, then wash it 2 - 4 times with water, and finally wash it 2 - 4 times with anhydrous ethanol again. Dry the washed product in an oven at 58 - 62 °C, and then obtain sepiolite in-situ grown nanozyme (Sep@IONzyme) and put it into a drying pot for standby.

[0012] The present invention also provides the application of the sepiolite in-situ grown nanozyme in influenza vaccines.

[0013] The preparation method of the influenza vaccine includes the following steps:

[0014] S1. Purify and inactivate the influenza virus;

[0015] S2. Mix the sepiolite in-situ grown nanozyme solution and the influenza virus diluent at a mass ratio of 18 - 22:1 to obtain the influenza vaccine.

[0016] Preferably, the concentration of the sepiolite in-situ growth nanozyme solution is 10 mg / mL, the concentration of the influenza virus dilution is 1 mg / mL, and the volume ratio of the sepiolite in-situ growth nanozyme solution to the influenza virus dilution is 2:1.

[0017] As an alternative embodiment, the influenza vaccine is an intranasal influenza vaccine.

[0018] Preferably, the sepiolite in-situ growth nanozyme is a mucosal adjuvant for intranasal influenza vaccine.

[0019] Advantages of the present invention:

[0020] 1. The sepiolite nanozyme (Sep@IONzyme) prepared in the present invention realizes the in-situ growth of iron-based nanozyme (IONzyme) on the surface of sepiolite (Sepiolite, Sep) rod-like nanostructures under hydrothermal conditions, significantly enhancing the peroxidase activity. After the connection of Sep@IONzyme with inactivated influenza virus, the complex forms an appropriate positive charge distribution, enhancing the adhesion ability of the antigen to nasal mucosa. Immunizing mice with it as an intranasal vaccine significantly improves the levels of respiratory mucosal specific sIgA antibody and serum specific IgG antibody, and can completely protect against the lethal attack of influenza virus.

[0021] 2. The sepiolite in-situ growth nanozyme-inactivated influenza virus complex vaccine provided by the present invention breaks through the bottleneck problems of poor antigen delivery effect and poor immune effect of existing mucosal vaccines, which is of great significance for the prevention and control of influenza. The sepiolite in-situ growth nanozyme can be prepared in large quantities, with low cost, good stability and good safety, so it has certain economic value and application prospects. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is the transmission electron micrograph of Sep@IONzyme of the present invention;

[0024] Figure 2 It is the peroxidase enzymatic kinetic curve of Sep@IONzyme of the present invention;

[0025] Figure 3Transmission electron microscopy image and Zeta potential image of the Sep@IONzyme-inactivated influenza virus complex of the present invention; wherein, A is the transmission electron microscopy image; B is the Zeta potential image;

[0026] Figure 4 Effect diagram of the Sep@IONzyme-inactivated influenza virus complex vaccine of the present invention on the adhesion of H1N1 WIV to nasal epithelium;

[0027] Figure 5 Schematic diagram of the safety evaluation of Sep@IONzyme of the present invention; wherein, A is the survival rate diagram; B is the body weight change diagram;

[0028] Figure 6 Schematic diagram of the detection of antibody levels after intranasal immunization of mice with the Sep@IONzyme-inactivated influenza virus complex vaccine of the present invention; wherein, A is the specific IgA antibody level in nasal lavage fluid; B is the specific IgA antibody level in tracheal lavage fluid; C is the specific IgA antibody level in lung lavage fluid; D is the specific IgG antibody level in serum; E is the HI antibody level in serum;

[0029] Figure 7 Schematic diagram of the survival rate, body weight change and viral load in the lungs of immunized mice after virus challenge; wherein, A is the mouse survival rate diagram; B is the mouse body weight change diagram; C is the viral load diagram in the mouse lungs. Detailed implementation manners

[0030] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0031] It should be noted that unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings understood by those of ordinary skill in the art to which the present disclosure belongs.

[0032] Description of the test materials used in the present invention

[0033] Sep, anhydrous ferric chloride, ethylene glycol, 3,3',5,5'-tetramethylbenzidine (TMB), and cholera toxin B subunit (CTB) were all purchased from Sigma. Sodium acetate trihydrate and 30% H2O2 solution were both purchased from Aladdin. HRP-labeled anti-mouse IgG antibody was purchased from Santa Cruz Biotechnology. HRP-labeled anti-mouse IgA antibody was purchased from Southern Biotech. Anhydrous ethanol was purchased from Sinopharm Group. Influenza virus A / Puerto Rico / 8 / 34 (H1N1, PR8) was preserved by the Key Laboratory of Veterinary Infectious Diseases of Ministry of Agriculture and Rural Affairs (College of Veterinary Medicine, Yangzhou University).

[0034] Example 1

[0035] Preparation of sepiolite in-situ grown nanozyme

[0036] Add 40 mL of ethylene glycol to a beaker, and while stirring on a magnetic stirrer, add 0.82 g of anhydrous ferric chloride. After the ferric chloride is completely dissolved, slowly add 3.6 g of sodium acetate trihydrate. After a stable suspension is formed, add 0.1 g, 0.5 g, and 1 g of Sep respectively according to the formula. Use ultrasound to fully mix the solution. After a stable suspension is formed, transfer the solution to a reaction kettle and react at 200 °C for 12 h. Take the reaction product, discard the black liquid in the supernatant, rinse the black precipitate at the bottom of the reaction kettle with anhydrous ethanol, and first wash the product 3 times with anhydrous ethanol by centrifugation, then wash it 3 times with water, and finally wash it 3 times with anhydrous ethanol again. After drying the washed product in an oven at 60 °C, the sepiolite in-situ grown nanozyme (Sep@IONzyme) is obtained and placed in a drying pan for standby.

[0037] Morphological observation of sepiolite in-situ grown nanozyme

[0038] Take 50 μg / mL Sep@IONzyme and observe the morphological structure through a transmission electron microscope (TEM). As Figure 1As shown, Sep@IONzyme is composed of a combination of spherical and rod-like structures, and IONzyme can grow on the surface of rod-shaped Sep. In the formulation with 0.1 g of Sep added, the in-situ growth of IONzyme is the most dense, and there is a situation where it does not grow on the rod-shaped Sep; in the formulation with 0.5 g of Sep added, the in-situ growth of IONzyme is more, and it all grows on the rod-shaped Sep; however, in the formulation with 1 g of Sep added, the in-situ growth of IONzyme is relatively sparse, and many rod-shaped Sep do not show the growth of IONzyme. Therefore, from the morphological observation, the optimal formulation for preparing in-situ growth nanozyme is the one with 0.5 g of Sep.

[0039] Determination of the Peroxidase Activity of In-Situ Growth Nanozyme on Sepiolite

[0040] Dissolve three different ratios of Sep@IONzyme in 1 mL of sodium acetate buffer solution with a pH of 4.5, then add 4 μL of 3,3',5,5'-tetramethyl benzidine (3,3',5,5'-Tetramethyl benzidine, TMB), and add different amounts of 30% H2O2 solution (0, 0.375, 0.75, 1.5, 3, 6, 12, 24 μL) respectively. Use an enzyme-labeled instrument to detect the absorbance at 652 nm (300 s, 37 °C). The peroxidase activities of the three different ratios of Sep@IONzyme are all higher than that of the single IONzyme, indicating that after IONzyme is loaded on Sep, its peroxidase activity is significantly enhanced. Loading IONzyme on the surface of Sep makes IONzyme fully dispersed, significantly increasing the contact area between IONzyme and the substrate. ( Figure 2 ) Among the three different ratios, the peroxidase activities of the formulations with 0.5 g and 1 g of Sep added are significantly higher than that of the formulation with 0.1 g of Sep added. Combining the results of transmission electron microscopy and peroxidase-like activity, the formulation with more, uniform in-situ growth of IONzyme and strong peroxidase-like activity (adding 0.5 g of Sep) is selected for the subsequent experiments of the present invention.

[0041] Preparation and Charge Determination of In-Situ Growth Nanozyme-Inactivated Influenza Virus Complex on Sepiolite

[0042] Purify influenza virus by sucrose gradient density centrifugation. Dilute 37% formaldehyde with sterile PBS at a ratio of 1:50, and the volume ratio of the formaldehyde dilution to the virus dilution is 7:43. Mix well and inactivate for 24 h. Dilute the purified virus with PBS to 1 mg / mL, prepare 10 mg / mL Sep@IONzyme, and add it to a 1.5 mL centrifuge tube at a volume ratio of virus dilution to Sep@IONzyme solution of 1:2. Rotate and mix for 1 h at a speed of 300 rpm to prepare the Sep@IONzyme combined inactivated influenza vaccine complex. It was found by transmission electron microscopy that virus particles were bound to the surface of the Sep@IONzyme rod-shaped structure ( Figure 3 A). Use a Zeta sizer analyzer to detect the Zeta potential of the Sep@IONzyme-inactivated influenza virus complex. As shown in Figure 3 B, the overall complex shows a positive charge, between 0.1 and 0.3 mV. A certain range of positive charge levels is beneficial for adhesion to the mucosa.

[0043] Evaluation of the ability of sepiolite in-situ grown nanozyme-inactivated influenza virus complex to adhere to mouse nasal mucosa epithelium

[0044] Divide 6-8-week-old C57BL / 6 mice into 5 groups: PBS group, influenza vaccine alone group (DyLight 633-H1N1WIV), Sep combined with influenza vaccine group (Sep+DyLight 633-H1N1 WIV), IONzyme combined with influenza vaccine group (IONzyme+DyLight 633-H1N1 WIV), Sep@IONzyme combined inactivated influenza virus complex vaccine group (Sep@IONzyme+DyLight 633-H1N1 WIV). According to the above grouping, instill the mice (20 μL / mouse), euthanize the mice at 1 h, 6 h, and 12 h after instillation respectively, dissect the nasal cavities of the mice, fix them in 4% paraformaldehyde for 24 h, and then decalcify them in the decalcifying solution for 7 d. After embedding the nasal cavity with OCT, section it with a thickness of 8-10 μm, wash the sections twice with PBS, stain with DAPI in the dark for 5 min, and observe under confocal microscopy after washing with PBS. As shown in Figure 4 , a large amount of virus adhered to the nasal epithelium (indicated by the white arrow) in the Sep@IONzyme combined inactivated influenza virus complex vaccine group 1 h after instillation, and there was no adhesion in other groups; 6 h and 12 h after instillation, virus still adhered to the nasal mucosa in the complex group, indicating that Sep@IONzyme significantly prolonged the nasal adhesion time of inactivated influenza virus up to 12 h.

[0045] Example 2

[0046] Safety Test of Nanzyme In-situ Grown on Sepiolite

[0047] C57BL / 6 mice aged 6 - 8 weeks were divided into 7 groups (10 mice / group): PBS group, Sep (0.25 mg / kg) group, Sep (5 mg / kg) group, IONzyme (0.25 mg / kg) group, IONzyme (5 mg / kg) group, Sep@IONzyme (0.25 mg / kg) group, and Sep@IONzyme (5 mg / kg) group. Each mouse was instilled with 20 μL of the corresponding material by nasal drip according to the above grouping, and the body weight changes and survival status of the mice were monitored for 15 days. As Figure 5 shown, compared with the mice in the PBS group, the survival rates of the mice in the IONzyme, Sep, and Sep@IONzyme groups were 100% and their body weights did not decrease significantly, indicating that Sep@IONzyme has high safety in mice.

[0048] Mucosal and Systemic Immune Response Levels in Mice Immunized by Nasal Drip with Nanzyme In-situ Grown on Sepiolite-Inactivated Influenza Virus Complex

[0049] C57BL / 6 mice aged 6 - 8 weeks were divided into 6 groups (20 mice / group): PBS group, single influenza vaccine group (H1N1 WIV), Sep combined with influenza vaccine group (Sep + H1N1 WIV), IONzyme combined with influenza vaccine group (IONzyme + H1N1 WIV), Sep@IONzyme combined with inactivated influenza virus complex group (Sep@IONzyme + H1N1 WIV), and cholera toxin B subunit (CTB) combined with inactivated influenza vaccine group (CTB + H1N1 WIV). According to the above grouping, the mice were immunized by nasal drip (20 μL / mouse) at 0 and 14 days respectively. 28 days after the first immunization, the mice were euthanized, and blood was collected from the mice by eye bleeding to prepare serum, which was stored at -70 °C. Nasal cavities (0.5 mL), tracheas (0.2 mL), and lung lavage fluids (0.5 mL) (washed with PBS) of the mice were taken and stored at -70 °C. The levels of specific IgA antibodies in the lavage fluids and specific IgG antibodies in the serum were detected by indirect enzyme-linked immunosorbent assay (ELISA). The levels of antibodies neutralizing the virus in the serum were detected by hemagglutination inhibition test. As Figure 6 shown in A - C, 28 days after the first immunization, the specific IgA antibodies against influenza virus in the nasal cavities, tracheas, and lung lavage fluids of the mice in the complex vaccine group were significantly higher than those in the single influenza vaccine group (P < 0.01); compared with the single influenza vaccine group, the levels of specific IgG antibodies against influenza virus in the serum of the complex vaccine group ( Figure 6 D), HI antibody levels (Figure 6 E) Significantly increased (P < 0.01). Therefore, after intranasal immunization of mice with the complex vaccine, the levels of respiratory mucosal and systemic immune responses in mice can be significantly enhanced.

[0050] Virus challenge protection after intranasal immunization of mice with the sepiolite in-situ grown nanozyme-inactivated influenza virus complex vaccine

[0051] Twenty-eight days after the first immunization, 15 immunized mice per group were taken, and challenged by intranasal instillation with 106 EID50 / 50 μL of the virus (50 μL), and the body weight changes and survival rates of the mice were monitored for 15 days. On the 5th day after virus challenge, 5 mice per group were sacrificed, and the lungs were aseptically isolated, added with 0.4 mL of PBS containing antibiotics, homogenized with a tissue homogenizer, centrifuged at 8000 rpm for 10 min, the supernatant was taken, the total RNA in the lungs was extracted, and the viral load in the lungs was measured by fluorescence quantitative PCR. As Figure 7 Shown in A and B, the survival rate of the group immunized with the inactivated virus vaccine alone was 50%, while the survival rate of the mice in the complex vaccine group was 100%, with a low body weight loss rate, and it could provide complete protection. As Figure 7 Shown in C, compared with the group immunized with the inactivated virus vaccine alone, the viral load in the lungs of the mice in the complex vaccine group was significantly reduced (P < 0.01). In summary, after intranasal immunization of mice with the Sep@IONzyme-inactivated influenza virus complex vaccine, it can provide 100% complete protection against lethal influenza virus challenge.

[0052] Those of ordinary skill in the art should understand that the discussion of any above embodiments is only exemplary, and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, and they are not provided in detail for the sake of brevity.

[0053] The embodiments of the present invention are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of sepiolite in-situ grown nanozyme, characterized in that, The preparation method is to mix ferric chloride, ethylene glycol and sodium acetate to form a suspension, and then mix sepiolite with the suspension, react at a temperature of 180 - 200 °C to obtain a black precipitate, and then obtain sepiolite in-situ grown nanozyme after washing and drying; the ferric chloride is anhydrous ferric chloride, and the sodium acetate is sodium acetate trihydrate; the mass ratio of the anhydrous ferric chloride, sodium acetate and sepiolite is 0.82: 3.6:0.

5.

2. A sepiolite in-situ growth nanozyme, characterized in that, Prepared by the preparation method described in claim 1.

3. Use of the sepiolite in-situ grown nanozyme described in claim 2 in the preparation of influenza vaccines.

4. The application according to claim 3, characterized in that The preparation method of the influenza vaccine includes the following steps: S1. Purify and inactivate the influenza virus; S2. Mix the sepiolite in-situ grown nanozyme solution and the influenza virus diluent at a mass ratio of 18 - 22:1 to obtain the influenza vaccine.

5. The application according to claim 4, wherein The concentration of the sepiolite in-situ grown nanozyme solution is 10 mg / mL, the concentration of the influenza virus diluent is 1 mg / mL, and the volume ratio of the sepiolite in-situ grown nanozyme solution to the influenza virus diluent is 2:

1.

6. The application according to claim 3, characterized in that, The influenza vaccine is an influenza nasal drop vaccine.

7. The application according to claim 6, characterized in that The sepiolite in-situ grown nanozyme is a mucosal adjuvant for the influenza nasal drop vaccine.

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