A method for improving the antiviral efficacy of rainbow trout type i interferon alpha protein

Abstract

The application discloses a method for improving the antiviral efficacy of rainbow trout type I interferon alpha protein and belongs to the technical field of medical drugs. The method is that resistant starch and carboxymethyl chitosan are used as microencapsulation materials to perform microencapsulation treatment on the interferon alpha protein. A new combination mode of coating materials, resistant starch-carboxymethyl chitosan, is adopted, wherein the resistant starch has excellent enzyme resistance and colon targeting property, can prolong the half-life of the interferon protein in the body and has a long action time. The carboxymethyl chitosan has excellent water solubility and film forming property. After the two composite materials are used for coating, the highest encapsulation rate can reach 23.09%, the highest drug loading rate can reach 81.20%, and the effect is remarkable. The prepared oral interferon microcapsules by using the method are simple in use, have more obvious antiviral effect and have excellent application prospect, and a new idea is provided for microencapsulation coating.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for enhancing the antiviral efficacy of rainbow trout type I interferon α protein. Background Technology

[0002] Viral diseases have always been a significant problem in fish aquaculture and are one of the main constraints on the further expansion of aquaculture production. Fish viruses, especially RNA viruses, have a devastating economic impact on the aquaculture industry every year.

[0003] Fish exhibit strong immune responses against many viruses. Non-specific immunity is the host's primary defense against viral infection, and interferon (IFN) genes are involved in this process. These key antiviral cytokines induce the expression of numerous interferon-stimulated genes (ISGs), which possess various antiviral effects and regulatory functions. Therefore, extensive research on fish viruses and antiviral responses in commercially valuable species (such as salmonids) is of great significance. However, interferon proteins have short half-lives and are easily inactivated, making intraperitoneal injection inefficient and laborious compared to aquatic animals. Therefore, finding more effective ways to utilize interferon for antiviral treatment is of great importance. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, this invention provides a method for enhancing the antiviral efficacy of type I interferon α protein in rainbow trout. This invention designs a method for rapid antiviral action through oral administration mixed with feed. Two materials were selected for the coating: resistant starch and carboxymethyl chitosan. Resistant starch exhibits excellent resistance to enzymatic hydrolysis and colon-targeting properties, prolonging the half-life of interferon protein in vivo and resulting in a longer duration of action. Carboxymethyl chitosan possesses excellent water solubility and film-forming properties. The oral interferon microcapsules prepared using this method are easy to use, exhibit more pronounced antiviral effects, and have excellent application prospects.

[0005] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0006] A method for enhancing the antiviral efficacy of rainbow trout type I interferon α protein involves using resistant starch and carboxymethyl chitosan as microencapsulation materials to microencapsulate the interferon α protein.

[0007] Furthermore, resistant starch and carboxymethyl chitosan were used to microencapsulate interferon α protein via emulsification.

[0008] Furthermore, the specific process is as follows:

[0009] (1) After mixing the resistant starch solution with interferon α protein, it was added dropwise to liquid paraffin for emulsification;

[0010] (2) After emulsification, calcium chloride solution is added to calcify and form microspheres. Then, the precipitate is collected by centrifugation and washed 3 to 5 times.

[0011] (3) Add the precipitate obtained in step (2) to the carboxymethyl chitosan solution and stir for 10-20 min. Then, centrifuge to collect the precipitate, wash it 3-5 times, add mannitol and stir evenly, and finally freeze dry for storage.

[0012] Furthermore, the concentration of the resistant starch solution is 0.5% to 2.5%.

[0013] Furthermore, the concentration of the calcium chloride solution is 4-20%.

[0014] Furthermore, the concentration of the carboxymethyl chitosan solution is 0.4%–2.4%.

[0015] Furthermore, the W / O phase ratio during the preparation process is 1:9 to 5:5.

[0016] Furthermore, the resistant starch is RS3 resistant starch.

[0017] The microencapsulated interferon α protein was prepared by the above method.

[0018] The beneficial effects of this invention are:

[0019] (1) Using recombinant interferon protein as the core, the antiviral effect of interferon is more significant than that of drugs.

[0020] (2) This invention employs a novel combination of coating materials: resistant starch and carboxymethyl chitosan. Resistant starch exhibits excellent resistance to enzymatic hydrolysis and colon-targeting properties, prolonging the half-life of interferon protein in vivo and resulting in a longer duration of action. Carboxymethyl chitosan, on the other hand, possesses excellent water solubility and film-forming properties. The oral interferon microcapsules prepared using this method are easy to use, exhibit more pronounced antiviral effects, and have excellent application prospects, providing a new approach to microencapsulation coating.

[0021] (3) Microcapsule preparation is carried out by emulsification, which is simple and allows for large-scale preparation at one time, making it suitable for factory production.

[0022] (4) The preparation of this microcapsule solves the dilemma of aquatic animals being unable to receive timely medication when threatened by pathogens, and provides a new method for aquatic animal farming. Attached Figure Description

[0023] Figure 1 A schematic diagram under a microscope of the recombinant type I interferon α microcapsules of rainbow trout prepared in this application;

[0024] Figure 2 A schematic diagram illustrating the in vitro antiviral activity verification of recombinant type I interferon α in rainbow trout prepared in this application;

[0025] Figure 3 A schematic diagram showing the drug loading and encapsulation efficiency of the rainbow trout recombinant type I interferon α microcapsules prepared for this application using different concentrations of resistant starch;

[0026] Figure 4 A schematic diagram showing the drug loading and encapsulation efficiency of the rainbow trout recombinant type I interferon α microcapsules prepared for this application using different water-oil phase ratios;

[0027] Figure 5 A schematic diagram showing the drug loading rate and encapsulation efficiency of the rainbow trout recombinant type I interferon α microcapsules prepared for this application using different concentrations of CaCl2;

[0028] Figure 6 A schematic diagram showing the drug loading and encapsulation efficiency of the rainbow trout recombinant type I interferon α microcapsules prepared for this application using different concentrations of carboxymethyl chitosan;

[0029] Figure 7 Schematic diagram of the in vitro antiviral activity of the rainbow trout recombinant type I interferon α microcapsules prepared for this application after being coated with single materials and composite materials;

[0030] Figure 8 Schematic diagram of the in vivo antiviral activity of the rainbow trout recombinant type I interferon α microcapsules prepared for this application after being coated with a single material and a composite material. Detailed Implementation

[0031] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0032] Example 1: Study on the antiviral function of recombinant interferon

[0033] EPC cells were seeded into 96-well cell culture plates. After a monolayer of cells was formed, renatured protein rtrIFNa was added and incubated for 12 hours, with PBS used as a control. The culture medium was then removed, the cells were washed once with PBS, and 3.24 × 10⁶ cells were seeded. 4 TCID 50100 μL of IHNV virus solution (at a dose of / mL) was incubated at 16°C for 1 h, followed by the addition of 100 μL of M199 medium (containing 1% carboxymethyl cellulose) with 2% FBS, and incubation continued at 25°C. Five days after infection, the supernatant was discarded, the cells were washed once with PBS, and diluted CCK-8 reagent was added. Cell viability was measured at 480 nm. Figure 2 As shown.

[0034] Example 2: Single-factor experiment

[0035] Factors influencing protein microsphere formation were investigated by varying the concentrations of carboxymethyl chitosan, w / o phase ratio, CaCl2 concentration, and resistant starch concentration. Using different levels of these four factors, the effects of encapsulation efficiency (EE) and drug-loaded rate (DR) on the EE and DR of the microsphere vaccine were examined. Figure 3-6 .

[0036] The concentrations of resistant starch were set to 0.5%, 1%, 1.5%, 2%, and 2.5%, respectively; the W / O ratio was 5:5; the CaCl2 concentration was 8%; and the carboxymethyl chitosan concentration was 0.8%. Microcapsules were prepared under different conditions to investigate the effect of carboxymethyl chitosan solution concentration on the EE and DR of the microcapsules.

[0037] The W / O phase ratios were set to 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively. The resistant starch concentration was set according to the optimal conditions in the previous step. Microcapsules were prepared under the conditions of 8% CaCl2 concentration and 0.8% carboxymethyl chitosan concentration. The effects of different W / O phase ratios on the EE and DR of the microcapsules were investigated.

[0038] The CaCl2 concentrations were set to 4%, 6%, 8%, 10%, 12%, 14%, 16%, and 20%, respectively. The resistant starch concentration and W / O ratio were set according to the above optimal conditions. The carboxymethyl chitosan concentration was 0.8%. Microcapsules were prepared under these conditions, and the effects of different CaCl2 concentrations on the EE and DR of the microcapsules were investigated.

[0039] The concentrations of carboxymethyl chitosan were set to 0.4%, 0.8%, 1.0%, 1.4%, 1.8%, 2.0%, and 2.0%, respectively. The concentrations of resistant starch, W / O phase ratio, and CaCl2 were set according to the above optimal conditions. The effects of different resistant starch concentrations on the EE and DR of the microcapsules were investigated.

[0040] like Figures 3-6 As shown, after processing with the technical solution of this application, the material has excellent encapsulation efficiency and drug loading rate.

[0041] Example 3: Response Surface Methodology Detection

[0042] Based on the analysis of single-factor experimental results, and using Design-Expert 8.0.6 software, with encapsulation efficiency (Y) as the response value, a Box-Behnken experimental design was selected. A four-factor, three-level response surface optimization experiment was designed as shown in Table 1 to optimize the microcapsule preparation process. The prepared microspheres were placed in PBS solution and smeared under a microscope for microscopic observation and analysis. The preparation conditions were optimized based on the morphological observation, encapsulation efficiency, and drug loading rate of the prepared samples.

[0043] Table 1 Factors and Levels in Response Surface Analysis

[0044]

[0045] Example 4: Preparation of microcapsules by emulsification method

[0046] Based on the optimal concentration and dosage determined in the aforementioned examples, the resistant starch solution was thoroughly mixed with recombinant IFNα protein. The mixture was then slowly added dropwise to a certain amount of food-grade liquid paraffin (containing emulsifier Span-80 and magnesium stearate), and stirred at 1800 rpm until fully emulsified. CaCl2 solution was then added to the emulsified resistant starch protein to calcify and form microspheres. The precipitate was collected by centrifugation at 8000 rpm for 5 min, washed three times with 0.01 mol / L sodium acetate (pH = 4.0) washing solution, and then mixed with carboxymethyl chitosan (prepared with sodium acetate at pH = 5) at 1800 rpm for 10 min. The precipitate was collected by centrifugation at 8000 rpm for 5 min, washed three times with the same washing solution, resuspended in the same buffer solution, and then an equal volume of 2% mannitol was added and stirred until homogeneous. The mixture was then lyophilized and stored. The morphology of the microcapsules was observed under a microscope. Figure 1 .

[0047] Example 5: Comparison of the protective rates of recombinant IFNα protein microcapsules and mono-material IFNα microcapsules against viral infection in vitro.

[0048] Resistant starch protein microcapsules, carboxymethyl chitosan protein microcapsules, and resistant starch-carboxymethyl chitosan protein microcapsules were prepared. These microcapsules were ultrasonically filtered in an ice box and then filtered through a 0.22 μm filter to obtain an IFNα suspension. EPC cells were seeded into 96-well cell culture plates. After the cells reached a monolayer, the refolded protein RTRIFNa suspensions from the ultrasonically disrupted resistant starch group, carboxymethyl chitosan group, and composite material group were added and incubated for 12 h, with PBS as a control. The culture medium was removed, the cells were washed once with PBS, and then seeded with 3.24 × 10⁶ cells / well. 4100 μL of IPNV virus solution at a TCID50 / mL dose was incubated at 16°C for 1 h, followed by the addition of 100 μL of M199 medium (containing 1% carboxymethyl cellulose) with 2% FBS, and incubation continued at 15°C. Five days after infection, the supernatant was discarded, the cells were washed once with PBS, and diluted CCK-8 reagent was added. Cell viability was measured at 480 nm. Figure 7 As shown.

[0049] according to Figure 7 The test results show that the cells are well protected after treatment using the technical solution of this application.

[0050] Example 6: Evaluation of the clinical therapeutic effect of recombinant IFNα protein microcapsules on antiviral infected fish

[0051] One hundred rainbow trout were randomly divided into five groups (recombinant IFNa protein IP group, resistant starch microcapsule gavage group, carboxymethyl chitosan microcapsule gavage group, composite material microcapsule gavage group, and control group), with 20 fish in each group. The recombinant IFNa protein IP group received an intraperitoneal injection of approximately 100 μg / fish of rtrIFNa. The recombinant IFNa protein microcapsule feeding group received approximately 100 μg / fish of microcapsules via gavage. The control group received no treatment. Twenty-four hours after immunization, all experimental fish were intraperitoneally injected with 200 μL of IHNV virus solution. The fish were observed for a total of two weeks, and the survival rate was calculated. Figure 8 As shown.

[0052] according to Figure 8 The test results show that rainbow trout treated with the technical solution of this application can maintain a higher survival rate for a longer period of time.

[0053] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing oral interferon microcapsules that enhance the antiviral efficacy of rainbow trout type I interferon α protein, characterized in that, Resistant starch and carboxymethyl chitosan were used as microencapsulation materials to microencapsulate interferon α protein; the specific process is as follows: (1) After mixing the resistant starch solution with interferon α protein, it was added dropwise to liquid paraffin for emulsification; (2) After emulsification, calcium chloride solution is added to calcify and form microspheres. Then, the precipitate is collected by centrifugation and washed 3 to 5 times. (3) Add the precipitate obtained in step (2) to the carboxymethyl chitosan solution and stir for 10-20 min. Then, centrifuge to collect the precipitate, wash it 3-5 times, add mannitol and stir evenly, and finally freeze dry for storage.

2. The preparation method according to claim 1, characterized in that, The concentration of the resistant starch solution is 0.5-2.5%.

3. The preparation method according to claim 1, characterized in that, The concentration of calcium chloride solution is 4-20%.

4. The preparation method according to claim 1, characterized in that, The concentration of the carboxymethyl chitosan solution is 0.4-2.4%.

5. The preparation method according to claim 1, characterized in that, The W / O phase ratio during the preparation process is 1:9 to 5:

5.

6. The preparation method according to claim 1, characterized in that, The resistant starch is RS3 resistant starch.

7. Oral interferon microcapsules that enhance the antiviral efficacy of rainbow trout type I interferon α protein, prepared by the preparation method according to any one of claims 1 to 6.

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