Preparation method, product and application of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions

Abstract

The present invention discloses a preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions. The preparation steps are as follows: Prepare the cascade protein, which is a cascade protein of superoxide dismutase and catalase with a molecular weight of 80 kDa. Dissolve the cascade protein in deionized water to obtain a cascade protein solution; Configure the polycation into a polycation solution, and blend the polycation solution and the cascade protein solution in step 1) at a mass ratio of 1:1 to 5:1 to obtain a polycation cascade protein solution; Configure sodium hyaluronate into a sodium hyaluronate solution, and blend the polycation cascade protein solution in step 2) and the hyaluronic acid solution at a mass ratio of 10:1 to 120:1 to obtain a hyaluronic acid / polycation cascade protein preparation; The concentration of the cascade protein solution is 1 to 5 mg / mL, and the concentration of the polycation solution in step 2) is 1 to 5 mg / mL. The present invention is applicable to diseases caused by bacterial infections such as pneumonia.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical applications, and relates to a preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions. Background Art

[0002] Bacterial infectious diseases are increasingly harmful to human life and health. According to the statistics of the World Health Organization, by 2050, the number of deaths caused by bacterial infections each year may exceed 10 million, far exceeding the number of deaths from cancer. In addition, planktonic bacteria aggregate and adhere on the surface to form a dense and difficult-to-eradicate biofilm, which further increases the difficulty of treatment. At the same time, continuous bacterial stimulation causes cells to continuously produce reactive oxygen species (ROS), further activating immune cells and secreting high levels of cytokines. Although these immune cells and cytokines play a role in killing bacteria, they also make the cells in an oxidative stress state. The elevated inflammatory level increases the pain of patients and may also bring serious systemic sequelae: under the stimulation of inflammation, cells produce more ROS, which may ultimately cause a cytokine storm, multiple organ failure, and immune system collapse. The immune deficiency of the body further limits the effect of antibacterial treatment, resulting in a vicious cycle of "bacterial stimulation-oxidative stress-immune collapse". Therefore, it is necessary to develop efficient antibacterial and inflammation-regulating materials to solve the treatment problem of bacterial inflammatory diseases.

[0003] To overcome diseases caused by bacterial infections, various antibacterial materials have been widely developed. Among them, cationic polymers exhibit certain antibacterial effects because the positive charges they carry can bind to negatively charged bacterial membranes. Polymers with quaternary ammonium groups can disrupt the integrity of the bacterial membrane, causing the leakage of bacterial contents and resulting in bacterial death. At the same time, cationic polymers can bind to negatively charged or hydrophobic small molecule drugs and biomacromolecule drugs (nucleic acids, proteins, etc.), help them cross multiple biological barriers, protect them from degradation or clearance, and be delivered into cells to play their roles, showing promising prospects in the treatment of bacterial inflammatory diseases. Eliminating excessive ROS may be an important means to control the development of inflammation, and natural redox enzymes have broad application prospects in the fields of biotechnology, biomedicine, and pharmacy. The antioxidant system of organisms is defined as an oxidative defense system. It should be noted that the antioxidant defense system should not completely eliminate ROS, but maintain normal ROS levels so that they can perform appropriate functions. The complex antioxidant defense system includes endogenous antioxidant enzymes, mainly including SOD, CAT, and glutathione peroxidase (GPx), as well as small molecule scavengers, including vitamins, β-carotene, coenzyme Q, selenium, and zinc. Directly delivering antioxidant enzymes into cells is also one of the strategies to effectively relieve cellular oxidative damage. In addition to single-enzyme delivery systems, researchers are no longer satisfied with delivering only one antioxidant enzyme into cells at the same time. To obtain higher ROS scavenging efficiency, multi-enzyme co-delivery systems have gradually been proposed. Summary of the Invention

[0004] In view of this, the present invention provides a preparation method for a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions. The present invention specifically provides the following technical solutions:

[0005] 1. A preparation method for a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions, which forms a complex by electrostatic interaction between a positively charged polycation, a cascade protein, and a negatively charged hyaluronic acid. The specific steps are as follows:

[0006] 1) Prepare the cascade protein, and the cascade protein is a cascade protein expressed by a superoxide dismutase and catalase fusion plasmid;

[0007] 2) The polycation and the cascade protein are blended in solution at a mass ratio of 1:1 to 5:1 to obtain a polycation cascade protein;

[0008] 3) The polycation cascade protein and hyaluronic acid are blended in solution at a mass ratio of 10:1 to 120:1 to obtain a hyaluronic acid / polycation cascade protein preparation.

[0009] Furthermore, the molecular weight of the cascading protein described in step 1) is 80 kDa, the concentration of the cascading protein solution described in step 1) is 1-5 mg / mL, and the concentration of the polycation solution described in step 2) is 1-5 mg / mL.

[0010] Furthermore, the polycation described in step 2) is chitosan substituted with guanidine or imidazole or quaternary ammonium salt.

[0011] Furthermore, the substitution degree of the chitosan substituted with guanidine or imidazole or quaternary ammonium salt described in step 2) is 5%-40%.

[0012] Furthermore, the particle size of the sodium hyaluronate described in step 3) is between 1-2 μm, the molecular weight of the sodium hyaluronate is 10-100 kDa, and the concentration of the sodium hyaluronate solution is 10-100 mg / mL.

[0013] Furthermore, for the preparation of the cascading protein, first design the functional sequence of the cascading protein and the prokaryotic expression vector, then express the cascading protein with competent cells, and obtain the cascading protein by purification with a nickel column.

[0014] Furthermore, the polycation and / or the cascading protein solution described in step 2) are blended at a mass ratio of 2.5:1.

[0015] Furthermore, the polycation / cascading protein solution and the sodium hyaluronate solution described in step 3) are blended at a mass ratio of 40:1.

[0016] 2. A sodium hyaluronate / polycation cascading protein preparation with antibacterial and anti-inflammatory functions prepared according to the above preparation method.

[0017] 3. Applications of the above sodium hyaluronate / polycation cascading protein preparation with antibacterial and anti-inflammatory functions in anti-Klebsiella pneumoniae and in reducing inflammatory factors in lung tissue.

[0018] The beneficial effects of the present invention are as follows: The present invention uses polycations and the cascade protein SOAT to complex into a polycation-cascade protein complex through electrostatic adsorption, and further hyaluronic acid and the polycation-cascade protein complex complex into a hyaluronic acid / polycation-cascade protein complex, as a hyaluronic acid / polycation-cascade protein preparation with antibacterial and anti-inflammatory dual functions. The cascade protein SOAT is constructed through techniques such as plasmid design and protein purification. SOAT has both catalase and superoxide dismutase activities, and regulates inflammation by scavenging various ROS. The preparation of the present invention can combine the pathological characteristics of bacterial pneumonia diseases, construct a fusion protein delivery system with size and charge regulated by pH based on hyaluronic acid and polycations, and through the method of aerosol inhalation, utilize the size and charge advantages to quickly penetrate mucus and reach deep tissues. The surface charge of the hyaluronic acid / polycation-cascade protein complex is negative, and the particle size is between 1-2 μm. It can penetrate the mucus barrier caused by bacterial infection. In a low-pH environment, hyaluronic acid falls off, releasing positively charged nanoparticles. On the one hand, it exerts antibacterial effects, and on the other hand, it delivers the cascade protein into cells, regulating intracellular reactive oxygen species (ROS) while antibacterial, reducing normal cell apoptosis, lowering the inflammation level, restoring the body's immunity, and restoring the efficacy of antibacterial drugs, solving the treatment problem of bacterial inflammatory diseases. The present invention is applicable to diseases caused by bacterial infections such as pneumonia. The preparation process of the present invention is simple, has good controllability, simple process conditions, low production cost, easily available raw materials, stable product performance, and the selected materials do not require chemical cross-linking agents and have good safety. Description of the Drawings

[0019] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings:

[0020] Figure 1 Schematic diagram of the composition of the hyaluronic acid / polycation-cascade protein preparation;

[0021] Figure 2 Dynamic light scattering average particle size and surface potential diagram of the hyaluronic acid / polycation-cascade protein preparation;

[0022] Figure 3 Transmission electron microscopy images of the polycation-cascade protein and the hyaluronic acid / polycation-cascade protein preparation;

[0023] Figure 4 In vivo antibacterial activity diagram of the hyaluronic acid / polycation-cascade protein preparation;

[0024] Figure 5 In vivo anti-inflammatory activity diagram of the hyaluronic acid / polycation-cascade protein preparation;

[0025] Figure 6Hematology index chart of hyaluronic acid / polycation cascade protein preparation in vivo;

[0026] Figure 7 Blood biochemical index chart of hyaluronic acid / polycation cascade protein preparation in vivo;

[0027] Figure 8 Inflammatory factor level chart of lung tissue of hyaluronic acid / polycation cascade protein preparation in vivo. Detailed implementation mode

[0028] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0029] The preparation principle of the hyaluronic acid / polycation cascade protein preparation of the present invention is as Figure 1 shown. It can be seen from Figure 1 that polycation and cascade protein are complexed into polycation cascade protein complex through electrostatic interaction. Further, the positively charged polycation protein complex and the anionic polyelectrolyte hyaluronic acid are complexed into a negatively charged complex through electrostatic interaction, so as to obtain a protein preparation. The hyaluronic acid with a negatively charged outer shell enables the particles to penetrate the mucus barrier. In a low pH environment, the hyaluronic acid falls off to form a positively charged polycation protein complex. The polycation has bactericidal activity and at the same time enables the cascade protein to enter the cell, effectively maintaining the anti-inflammatory activity of the cascade protein.

[0030] Example 1

[0031] The preparation of hyaluronic acid / polycation cascade protein preparation is as follows:

[0032] 1) Preparation of polycation

[0033] Dissolve 700 mg of chitosan hydrochloride in 15 mL of deionized water to obtain a clear solution, add 2.1 g of cyanoguanidine, and react to obtain a reaction solution of chitosan derivative with a substitution degree of 20%. The reaction temperature is 120 °C and the reaction time is 4 h. Dialyze the reaction solution in deionized water, the dialysis bag specification is MWCO 7000, and freeze-dry to obtain a product as a pale yellow flocculent solid (GuChi).

[0034] 2) Design of cascade protein SOAT

[0035]

[0036] 3) Preparation of Cascade Protein SOAT

[0037] Transform the SOAT expression plasmid into BL21(DE3) competent cells. Subsequently, add the bacterial solution to LB bacterial culture medium containing 0.1% kanamycin and culture at a temperature of 37°C and a rotation speed of 220 rpm. Culture in a shaker until the OD600 is about 0.6, then add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM, and perform low-temperature induction at 16°C and continue to culture in the shaker for 24 h. Aliquot the bacterial solution into centrifuge tubes, centrifuge at 6000 rpm for 6 min at 4°C, pour off the supernatant, and resuspend the precipitate in sterile PBS. After ultrasonically disrupting the bacteria in an ice bath, the ultrasonic power is 120 W, the total ultrasonic time is 10 min, the ultrasound is on for 3 s and off for 7 s. Centrifuge at 8000 rpm for 6 min at 4°C to remove bacterial debris and collect the supernatant. Purify the supernatant on a nickel column, separate the protein using Tris-HCl buffer containing different concentrations of imidazole, and concentrate the eluted protein solution by ultrafiltration. Test the concentration of SOAT using a BCA kit and store SOAT in a -80°C refrigerator to avoid repeated freezing and thawing.

[0038] 4) Preparation of Polycationic Cascade Protein

[0039] Dissolve the polycation in deionized water and shake to obtain a polycation solution with a concentration of 1.25 mg / mL; dissolve the cascade protein in deionized water to obtain a cascade protein solution with a concentration of 1 mg / mL. Add the polycation solution and the cascade protein solution to a centrifuge tube at a mass ratio of 2.5:1, shake and mix well, then let it stand at a temperature of 25°C for 30 min to obtain a polycationic cascade protein complex nano-solution. The obtained nano-solution is a clear and transparent nano-solution, abbreviated as GuChi(SOAT).

[0040] 5) Preparation of Hyaluronic Acid / Polycationic Cascade Protein

[0041] Dissolve sodium hyaluronate (molecular weight 50000 g / mol) in water to obtain a clear hyaluronic acid solution (abbreviated as HA solution) with a concentration of 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above step to a centrifuge tube at a mass ratio of 10:1, shake and mix well, then let it stand at a temperature of 25°C for 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-1(SOAT).

[0042] Example 2

[0043] Steps 1) to 4) are the same as those in Example 1;

[0044] 5) Dissolve sodium hyaluronate (molecular weight: 50,000 g / mol) in water to obtain a clear and transparent hyaluronic acid solution (abbreviated as HA solution), with the solution concentration being 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above steps into a centrifuge tube at a mass ratio of 20:1. After shaking and mixing evenly, let it stand. The standing temperature is 25 °C and the standing time is 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-2(SOAT).

[0045] Example 3

[0046] Steps 1) to 4) are the same as those in Example 1;

[0047] 5) Dissolve sodium hyaluronate (molecular weight: 50,000 g / mol) in water to obtain a clear and transparent hyaluronic acid solution (abbreviated as HA solution), with the solution concentration being 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above steps into a centrifuge tube at a mass ratio of 40:1. After shaking and mixing evenly, let it stand. The standing temperature is 25 °C and the standing time is 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-3(SOAT).

[0048] Example 4

[0049] Steps 1) to 4) are the same as those in Example 1;

[0050] 5) Dissolve sodium hyaluronate (molecular weight: 50,000 g / mol) in water to obtain a clear and transparent hyaluronic acid solution (abbreviated as HA solution), with the solution concentration being 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above steps into a centrifuge tube at a mass ratio of 60:1. After shaking and mixing evenly, let it stand. The standing temperature is 25 °C and the standing time is 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-4(SOAT).

[0051] Example 5

[0052] Steps 1) to 4) are the same as those in Example 1;

[0053] 5) Dissolve sodium hyaluronate (molecular weight: 50,000 g / mol) in water to obtain a clear and transparent hyaluronic acid solution (abbreviated as HA solution), with the solution concentration being 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above steps into a centrifuge tube at a mass ratio of 80:1. After shaking and mixing evenly, let it stand. The standing temperature is 25 °C and the standing time is 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-5(SOAT).

[0054] Example 6

[0055] Steps 1) to 4) are the same as in Example 1;

[0056] 5) Dissolve sodium hyaluronate (molecular weight 50000 g / mol) in water to obtain a clear and transparent hyaluronic acid solution (abbreviated as HA solution), with the solution being 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above steps into a centrifuge tube at a mass ratio of 100:1. After shaking and mixing evenly, let it stand. The standing temperature is 25 °C and the standing time is 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-6(SOAT).

[0057] Example 7

[0058] Steps 1) to 4) are the same as in Example 1;

[0059] 5) Dissolve sodium hyaluronate (molecular weight 50000 g / mol) in water to obtain a clear and transparent hyaluronic acid solution (abbreviated as HA solution), with the solution being 50 mg / mL. Add the polycationic cascade protein solution and the hyaluronic acid solution from the above steps into a centrifuge tube at a mass ratio of 120:1. After shaking and mixing evenly, let it stand. The standing temperature is 25 °C and the standing time is 30 min to obtain a hyaluronic acid-polycationic cascade protein complex solution, abbreviated as HyGuChi-7(SOAT).

[0060] Test Example 1 Morphology and Particle Size Potential Characterization

[0061] 1) Determination of hydrodynamic particle size and surface potential

[0062] Dilute the polycationic cascade protein complexes prepared in Examples 1 - 6, take 1 mL and measure the hydrodynamic particle size and surface potential on a Malvern dynamic light scattering instrument. Dilute the hyaluronic acid / polycationic cascade protein complexes prepared in the examples, take 1 mL and measure the hydrodynamic particle size and potential on a Malvern dynamic light scattering instrument. Figure 2 It is the particle size potential diagram of the hyaluronic acid / polycationic cascade protein preparation. As can be seen from Figure 2 :

[0063] As the mass ratio of hyaluronic acid to GuChi(SOAT) increases (10:1 - 120:1), the particle size of the complex HyGuChi(SOAT) gradually increases, and the potential always remains above -30 mV. When w / w = 40, the particle size enters a plateau period, and the polycationic cascade protein complex is a negatively charged nanoparticle.

[0064] 2) Characterization by transmission electron microscope

[0065] Drop 20 μL of the hyaluronic acid-polycation cascade protein complex onto a 200-mesh copper grid. After air-drying, collect images under a transmission electron microscope at 80 kV to observe the morphology of the polyion complex. Characterize the hyaluronic acid-polycation cascade protein complex by transmission electron microscopy (TEM), and collect images at a scale of 2 μm. Figure 3 This is the TEM image of the hyaluronic acid / polycation cascade protein preparation. As can be seen from Figure 3 it, the particle size of the hyaluronic acid / polycation cascade protein complex is about 1.5 μm, and the shape is spherical.

[0066] Test Example 2 In vivo antibacterial activity test

[0067] Use a mouse model of acute lung injury infected with Klebsiella pneumoniae to detect in vivo antibacterial and anti-inflammatory properties. The steps are as follows:

[0068] 1) Model establishment: Take 25 female Balb / c mice, weighing about 20 g, and randomly divide them into 5 groups: normal control group, model control group, GuChi (SOAT) administration group, HyGuChi-3 (bovine serum albumin (BSA)) administration group, and HyGuChi-3 (SOAT) administration group, with 5 mice in each group. On the 3rd day and 1st day before modeling, intraperitoneally inject the immunosuppressant cyclophosphamide to reduce the immune response of the mice and facilitate the construction of a bacterial ALI model. Subsequently, except for the normal control group, the mice in the other groups are injected with 1×10 8 CFU / mL of Klebsiella pneumoniae into the trachea by atomization to construct an ALI model infected with Klebsiella pneumoniae. 2) Administration: 4 h after infection, perform the first treatment, and inject sterile PBS, GuChi (SOAT), HyGuChi-3 (BSA), and HyGuChi-3 (SOAT) into the trachea of the mice by atomization respectively. Within 32 h, a total of six administrations are carried out, with a dose of 3.5 μg / kg. At 48 h after infection, weigh the mice, lavage the lungs of the mice with 1 mL of sterile PBS to obtain bronchoalveolar lavage fluid, and take out the lung tissue.

[0069] 3) Bacterial load in lung tissue and bronchoalveolar lavage fluid

[0070] Dilute the lung tissue of the mice with sterile PBS at a ratio of 1:9, homogenize it with a tissue homogenizer, centrifuge at 2000 rpm for 5 min at 4 °C to remove tissue and cell debris, and take the supernatant. Dilute the bronchoalveolar lavage fluid and the supernatant of the lung tissue homogenate, use a spiral inoculator to evenly spread the bacterial solution on an agar culture dish, and culture it in a 37 °C bacterial incubator until obvious single colonies appear, and take pictures and count the culture dishes.

[0071] 4) Results of bacterial load in lung tissue and bronchoalveolar lavage fluid

[0072] The antibacterial activity of HyGuChi-3(SOAT) against Klebsiella pneumoniae pulmonary infection in mice was evaluated. Figure 4 For the bacterial loads in the bronchoalveolar lavage fluid (BALF) and lung tissue homogenate of mice after treatment with HyGuChi-3(SOAT) for acute lung injury mice induced by Klebsiella pneumoniae infection, Figure 4 it can be seen from

[0073] There were almost no colonies in the BALF and lung tissue of normal mice, but there were a large number of Klebsiella pneumoniae in the BALF and lung tissue of mice in the bacterial infection group, which proved that the ALI model induced by Klebsiella pneumoniae was successfully constructed. The number of colonies in the BALF and lung tissue of mice after treatment with GuChi(SOAT) hardly decreased, and the average survival rate of bacteria hardly decreased (about 95% in BALF and about 102% in lung tissue). Due to the nano-size and positive charge of GuChi(SOAT), it is difficult to reach the deep part of the lung and penetrate the mucus layer of the lung during inhalation administration, and it cannot enter the lesion site of bacterial infection, so the antibacterial effect cannot be achieved. However, HyGuChi-3(SOAT) of the present invention showed obvious antibacterial effects, and the average survival rates of bacteria in the BALF and lung tissue of mice were only about 1% and 25% respectively.

[0074] Test Example 3 In vivo anti-inflammatory activity test

[0075] 1) Levels of inflammatory factors in lung tissue and bronchoalveolar lavage fluid

[0076] For the bronchoalveolar lavage fluid and lung tissue homogenate of mice, the levels of TNF-α and IL-6 pro-inflammatory cytokines were detected respectively using ELISA kits. High levels of pro-inflammatory cytokines represent severe pulmonary inflammation.

[0077] 2) Results of levels of inflammatory factors in lung tissue and bronchoalveolar lavage fluid

[0078] The anti-inflammatory activity of HyGuChi-3(SOAT) against Klebsiella pneumoniae pulmonary infection in mice was evaluated. Figure 5 It is a statistical chart of the levels of TNF-α and IL-6 pro-inflammatory cytokines in each group of mice with acute lung injury induced by Klebsiella pneumoniae infection after treatment.

[0079] From Figure 5 it can be seen that:

[0080] The levels of inflammatory factors in the normal control group of mice were low, while there was little difference in the inflammatory factors between the GuChi(SOAT)-administered group of mice and the PBS group, and there was almost no therapeutic effect on the inflammation in the lungs of mice. This was mainly because GuChi(SOAT) was difficult to reach the infected tissues in the lungs. In contrast, in the bronchoalveolar lavage fluid and lung tissue homogenate, the therapeutic effects of the HyGuChi-3(SOAT) group of mice in the present invention were obvious, the levels of inflammatory factors were significantly down-regulated, and were close to the levels of the normal group of mice. The results showed that the HyGuChi(SOAT) of the present invention had a good therapeutic effect on the inflammation of lung tissue and could reduce the lung inflammation to the normal level. This effect mainly came from the effective clearance of SOAT at the infected sites in the lung tissue.

[0081] Test Example 4 In vivo Compatibility Test

[0082] 1) Safety test model

[0083] Take 25 female Balb / c mice, weighing about 20 g, and randomly divide them into 5 groups with 5 mice in each group. Inhale different doses of HyGuChi-3(SOAT) and GuChi(SOAT) by aerosol administration, and the doses are 0.35, 3.5, and 35 μg / kg SOAT respectively. The Blank group injected with sterile PBS is set as the control for normal mice. At 48 h, weigh the mice in each group, take out the blood of the mice, lavage the lungs of the mice with 1 mL of sterile PBS to obtain bronchoalveolar lavage fluid, and take out the lung tissue.

[0084] 2) Blood routine indexes of mice

[0085] For the whole blood of mice, take out 100 μL, add anticoagulant ethylenediaminetetraacetic acid, conduct a blood routine test, and count the number of white blood cells, red blood cells, platelets, percentage of neutrophils, percentage of lymphocytes, and percentage of monocytes in the mice.

[0086] 3) Results of blood routine indexes of mice

[0087] The effects of HyGuChi-3(SOAT) and GuChi(SOAT) on the blood routine indexes of normal mice were evaluated. Figure 6 For the statistical chart of the main blood routine parameters of each group of mice, from Figure 6 it can be seen that:

[0088] Compared with the normal mice in the normal group, HyGuChi-3(SOAT) and GuChi(SOAT) at the above three doses did not cause obvious changes in the blood cell count, indicating that no blood diseases were caused.

[0089] 4) Blood biochemical indexes of mice

[0090] Whole blood was placed in 1.5 mL centrifuge tubes and allowed to stand at room temperature for 1 h. Then, it was centrifuged at 3000×g for 30 min at 4 °C. The supernatant was transferred to a new centrifuge tube and stored in a -80 °C refrigerator. Blood biochemical indices were detected, and the levels of aspartate aminotransferase (ALT), alanine aminotransferase (AST), total protein (TP), creatinine (CREA), urea nitrogen (UREA), and uric acid (UA) in mice were statistically analyzed to evaluate the liver and kidney functions of each group of mice.

[0091] 5) Results of blood biochemical indices in mice

[0092] The effects of HyGuChi-3(SOAT) and GuChi(SOAT) on the biochemical indices of normal mice were evaluated. Figure 7 It is a statistical chart of the main blood biochemical parameters of each group of mice. As can be seen from Figure 7 it that HyGuChi-3(SOAT) and GuChi(SOAT) had no effect on blood biochemical indices and did not cause damage to liver and kidney functions.

[0093] 6) Levels of inflammatory factors in mouse lung tissue

[0094] The lung tissue was weighed, and a mixed solution of tissue lysate and sterile PBS was added at a ratio of 1:9. The lung tissue was thoroughly homogenized using a tissue homogenizer. Then, it was centrifuged at 2000 rpm for 10 min at 4 °C to remove tissue and cell debris. The supernatant was taken and stored in a -80 °C refrigerator for ELISA detection of inflammatory factors.

[0095] 7) Results of levels of inflammatory factors in mouse lung tissue and bronchoalveolar lavage fluid

[0096] The effects of HyGuChi-3(SOAT) and GuChi(SOAT) on the inflammatory factors in the lung tissue of normal mice were evaluated. Figure 8 It is a statistical chart of the pro-inflammatory cytokines TNF-α and IL-6 in each group of mice. As can be seen from Figure 8 it that:

[0097] For the lung tissue homogenate, different doses of HyGuChi-3(SOAT) and GuChi(SOAT) did not cause a significant increase in the levels of inflammatory factors, indicating that no obvious lung tissue inflammatory reaction was induced.

[0098] In summary, based on the results of the blood routine indices, blood biochemical indices, and levels of inflammatory factors in lung tissue of mice, the hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions of the present invention can be safely used in mice.

[0099] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in terms of form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions, characterized in that, A positively charged polycation, a tandem protein, and negatively charged hyaluronic acid are formed into a complex through electrostatic interaction. The specific steps are as follows: 1) Prepare the tandem protein, where the tandem protein is the tandem protein expressed by the fusion plasmid of superoxide dismutase and catalase; the sequence of the tandem protein is SEQ ID No.1; 2) Blend the polycation solution and the tandem protein in step 1) in a solution form at a mass ratio of 1:1 to 5:1 to obtain a polycation-tandem protein; the polycation in step 2) is chitosan substituted with guanidine or imidazole or quaternary ammonium salt; 3) Blend the polycation-tandem protein in step 2) and hyaluronic acid in a solution form at a mass ratio of 10:1 to 120:1 to obtain a hyaluronic acid / polycation-tandem protein preparation.

2. The preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, The molecular weight of the tandem protein in step 1) is 80 kDa, the concentration of the tandem protein solution in step 1) is 1 - 5 mg / mL, and the concentration of the polycation solution in step 2) is 1 - 5 mg / mL.

3. The preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, The degree of substitution of the chitosan substituted with guanidine or imidazole or quaternary ammonium salt in step 2) is 5% - 40%.

4. The preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, The preparation of the tandem protein in step 1) first designs the functional sequence of the tandem protein, constructs the sequences of superoxide dismutase and catalase into a prokaryotic expression vector, then expresses the tandem protein using competent cells, and obtains the tandem protein through nickel column purification.

5. The preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, The polycation and / or the tandem protein in step 2) are blended at a mass ratio of 2.5:

1.

6. The preparation method of a hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, The polycation / tandem protein and hyaluronic acid in step 3) are blended at a mass ratio of 40:

1.

7. A hyaluronic acid / polycation cascade protein preparation with antibacterial and anti-inflammatory functions prepared by the preparation method according to any one of claims 1-6, characterized in that, The particle size is 1 - 2 μm.

8. Use of a hyaluronic acid / polycation-tandem protein preparation with antibacterial and anti-inflammatory functions according to claim 7 in the preparation of a drug against Klebsiella pneumoniae or in the preparation of a drug for reducing the inflammatory factors TNF-α and IL-6 in lung tissue.

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