Nano-starch particle drug delivery system and its application in preventing and controlling aquatic animal diseases

Abstract

The present invention relates to the technical field of carrier drug delivery systems, and specifically to a nano starch granule drug delivery system and its application in preventing and treating aquatic animal diseases. The present invention provides a method for preparing a nano starch granule drug delivery system, comprising the following steps: S1. oxidizing starch with sodium periodate to obtain oxidized starch with aldehyde groups; S2. reacting cholesterol chloroformate with oxidized starch in molten imidazole to obtain cholesterol-imidazole modified amphoteric oxidized starch; S3. placing cholesterol-imidazole modified oxidized starch in a solution containing an antiviral drug, preparing starch nanoparticles encapsulating the antiviral drug, and obtaining a nano starch granule drug delivery system. The nano starch granule drug delivery system provided by the present invention can be used for preventing and treating aquatic animal diseases, and the preparation method of the drug delivery system of the present invention is simple, has low production cost, and is suitable for rapid large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of carrier drug delivery systems, and in particular to a nano starch particle delivery system and its application in preventing and treating aquatic animal diseases. Background Art

[0002] In recent years, the aquaculture industry has flourished, but the breeding of aquatic animals faces many challenges. Fish, shrimp, crab or shellfish are important economic aquaculture water bodies.

[0003] Largemouth bass ( Micropterus salmoides ), commonly known as California bass and black bass, has rapidly developed into a major economic aquaculture species due to its advantages such as fast growth, few diseases, delicious meat and high economic value. Micropterus salmoides Rhabdovirus disease is one of the main diseases that harm largemouth bass. The largemouth bass rhabdovirus ( M.Salmoides The MSRV strain belongs to the genus Vesiculovirus and is a linear negative-stranded single-stranded RNA virus. The main epidemic season of sea bass rhabdovirus disease is from April to May each year. The disease is contagious and causes huge economic losses to largemouth bass farming.

[0004] Nano drug delivery system refers to the use of pharmaceutical methods to prepare drug delivery systems, mainly in the form of nanoparticles, nanospheres and nanocapsules. Drugs can be dissolved or covalently bound inside or on the outer surface of nanoparticles, so that the drugs can be accurately delivered to the lesions through nanocarriers. Based on natural starch, two types of nano starch can be prepared: starch nanoparticles and starch nanocrystals. Starch nanoparticles (SNP), also known as nano-modified starch, are molecular aggregates with a size between 1 and 1000nm. Compared with starch nanocrystals, starch nanoparticles have a wider range of applications, especially in the delivery system. The advantages of starch nanoparticles are that they are natural in origin, high in safety, strong in adsorption capacity, small in particle size and easy to store. Therefore, they have broad application potential in the fields of pharmacy and adsorbents. For example, as a drug carrier, they can achieve sustained release of drugs and protective effects on drugs.

[0005] Isoimperatorin (ISO) is widely distributed in the plant kingdom, especially in Apiaceae Chinese medicines such as Angelica dahurica, Angelica dahurica, Peucedanum chinense, Notopterygium wilfordii, and Glehnia littoralis, and is its main pharmacologically effective component. According to existing reports, isoimperatorin has antibacterial and anti-inflammatory effects. However, there are relatively few research reports on isoimperatorin in the field of aquaculture.

[0006] Therefore, it is particularly important to prepare a nano-starch particle drug delivery system and apply it to the prevention and treatment of aquatic animal diseases. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a nano starch particle drug delivery system, which can be used to prevent and treat aquatic animal diseases. The drug delivery system of the present invention has a simple preparation method, low production cost, simple use method, and is suitable for rapid large-scale production.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a nano starch particle drug delivery system, the preparation method comprising the following steps:

[0010] S1. oxidizing starch using sodium periodate to obtain oxidized starch with aldehyde groups;

[0011] S2. reacting cholesterol chloroformate with oxidized starch in molten imidazole to obtain cholesterol-imidazole modified amphoteric oxidized starch;

[0012] S3. Place the cholesterol-imidazole modified amphoteric oxidized starch in a solution containing antiviral drugs to prepare starch nanoparticles encapsulating antiviral drugs, thereby obtaining a nano starch particle drug delivery system.

[0013] Preferably, the specific steps of step S1 are:

[0014] A 10.56% (w / v) sodium periodate solution was added dropwise to a 16% (w / v) starch solution and reacted at 37°C in the dark for 3 hours; ethanol was used for precipitation, sodium periodate was removed by dialysis, and oxidized starch with aldehyde groups was obtained by freeze-drying.

[0015] Preferably, the specific steps of step S2 are:

[0016] The oxidized starch is added into molten imidazole and stirred evenly; cholesterol chloroformate is added and stirred at 100° C. for reaction for 1 hour; unreacted imidazole and cholesterol chloroformate are removed by dialysis, and the mixture is freeze-dried to obtain cholesterol-imidazole modified amphoteric oxidized starch.

[0017] It should be noted that the oxidized starch should be added to the molten imidazole and mixed thoroughly before adding cholesterol chloroformate.

[0018] Preferably, the specific steps of step S3 are:

[0019] The amphoteric oxidized starch is dissolved in DMSO, and a solution containing antiviral drugs is added; the mixed solution is placed in a dialysis bag and dialyzed in a PBS solution for 3 days; the dialysate is centrifuged, washed, and freeze-dried to obtain a nano-starch particle drug-carrying system.

[0020] Preferably, the antiviral drug is an anti-MSRV drug.

[0021] More preferably, the antiviral drug is isoimperatorin.

[0022] In a second aspect, a nano starch particle drug delivery system is provided, which is prepared by the preparation method described in the present invention.

[0023] In a third aspect, a nano starch particle-loaded isoimperatorin system is provided, which is prepared by the preparation method of the present invention.

[0024] In a fourth aspect, there is provided an application of the nano starch particle drug delivery system or the nano starch particle isoimperatorin delivery system of the present invention in preventing and treating aquatic animal diseases, wherein the aquatic animal is fish, shrimp, crab or shellfish.

[0025] Preferably, the nano starch particle drug delivery system or the nano starch particle isoimperatorin delivery system is diluted during production and used to soak sick aquatic animals, or is directly diluted and mixed with water and then sprayed on aquatic animals to be treated; wherein the concentration of the antiviral drug is 8-40 mg / L, and the soaking time is 2-5 hours.

[0026] Preferably, the aquatic animal is largemouth bass.

[0027] It should be noted that the nano starch particle drug delivery system is applied by immersion. When the nano starch particle drug delivery system is applied by immersion, it is dissolved in clean water to form a solution. The concentration of the drug (such as isoimperatorin) in the solution is 8-40 mg / L, and the immersion time is 2-5 hours.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) The present invention confirmed through in vitro experiments that isoimperatorin has a certain inhibitory effect on largemouth bass rhabdovirus, so it was selected as the anti-disease model drug of the nano starch particle drug delivery system.

[0030] (2) The nano starch particle drug delivery system prepared by the present invention comprises amphiphilic cholesterol-imidazole nano starch particles and an antiviral drug encapsulated in the hydrophobic core of the amphiphilic cholesterol-imidazole nano starch particles. Furthermore, the nano starch particle drug delivery system prepared by the present invention comprises amphiphilic cholesterol-imidazole nano starch particles and an anti-largemouth bass rhabdovirus drug encapsulated in the hydrophobic core of the amphiphilic cholesterol-imidazole nano starch particles.

[0031] (3) The present invention utilizes nanoparticle carriers to encapsulate antiviral drugs. The nanoparticle carriers are amphiphilic and their hydrophobic cores can encapsulate hydrophobic drugs to solve the problem of immersion administration of drugs that are poorly soluble in water, while their hydrophilic surfaces can allow the drug delivery system to be fully dissolved in water. At the same time, the size of the nanoparticles and the size of the starch nanoparticle drug delivery system can improve the cellular uptake efficiency of the drug. The drug carrier can prevent the drug from being rapidly degraded in the external environment. The nanoparticles with negative charges on the surface have good blood compatibility and also reduce the toxicity of the drug delivery system to normal tissues.

[0032] (4) In vivo experiments on largemouth bass showed that the nano-starch particle drug delivery system could increase the survival rate of infected bass by more than 20%, achieving significant anti-disease effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a preparation process of amphoteric oxidized starch.

[0034] Figure 2 This is the particle size distribution diagram of nano starch particles.

[0035] Figure 3 This is the particle size distribution diagram of the nano starch particle drug delivery system.

[0036] Figure 4 It is the drug release rate of nano starch particle drug delivery system under different pH environments.

[0037] Figure 5 The blood compatibility of nano starch particle drug delivery system.

[0038] Figure 6 This is the survival curve for largemouth bass.

[0039] Figure 7 This is a graph showing the viral load analysis in largemouth bass after MSRV infection. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0041] Example 1 Preparation of amphoteric oxidized starch

[0042] 1 Materials and methods

[0043] 1.1 Materials

[0044] 1.1.1 Test samples

[0045] Soluble starch, sodium periodate (NaIO4), anhydrous ethanol and cholesterol chloroformate (analytical grade) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; imidazole (analytical grade) was purchased from Chengdu Kelong Chemical Co., Ltd.

[0046] 1.1.2 Test equipment

[0047] The HH-4 digital constant temperature water bath was purchased from Shanghai Techeng Machinery Equipment Co., Ltd.; the H1650-W desktop high-speed microcentrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; the ALC-1100.2 electronic balance was purchased from Beijing Sartorius Instrument System Co., Ltd.; the Classic ultrapure water meter was purchased from Veolia Water Treatment Technology (Shanghai) Co., Ltd.; the F-101S magnetic stirrer was purchased from China Yingyu Yuhua Instrument System Co., Ltd.; and the SCIENTZ-10N vacuum freeze dryer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0048] 1.2 Test methods

[0049] 1.2.1 Preparation of oxidized starch

[0050] Starch was dissolved in deionized water to prepare a (16%, w / v) solution; sodium periodate was prepared into a (10.56%, w / v) solution; 200 mL of sodium periodate solution was added dropwise to 200 mL of starch solution at a ratio of 0.6 mol of sodium periodate per mol of anhydroglucose unit (AGU), and the mixed solution was placed at 37°C and stirred for 4 h in the dark. The reaction product was precipitated with 400 mL of anhydrous ethanol, and then the precipitate was collected in a dialysis bag (MWCO=3500 Da), dialyzed with deionized water for 3 days, and the water was changed every 4-6 hours. The dialyzed liquid was placed in a vacuum freeze dryer and freeze-dried to obtain oxidized starch powder.

[0051] 1.2.2 Oxidized starch modification

[0052] Melt 20 g of imidazole at 100°C, and slowly add 2 g of oxidized starch while stirring. Then, slowly add cholesterol chloroformate to the stirring oxidized starch system at a ratio of 0.01 mol of cholesterol chloroformate per mol of anhydroglucose unit (AGU), and stir at 100°C for 1 h. Collect the product in a dialysis bag (MWCO=3500 Da), dialyze it in deionized water for 3 days, change the water every 4 to 6 hours, and freeze-dry the dialyzed mixture in a vacuum freeze dryer to obtain modified oxidized starch powder (amphoteric oxidized starch). The preparation route of the amphoteric oxidized starch of the present invention is as follows: Figure 1 shown.

[0053] 2 Results and Analysis

[0054] Oxidized starch was successfully prepared by oxidizing starch with sodium periodate. The CC bond of the original glucose ring of oxidized starch was broken to form a dialdehyde structure, and an active aldehyde group was obtained, which was conducive to the next modification reaction. At the same time, the surface of the starch granules became rough, the structure collapsed, and the crystal characteristics were lost. This is because the reaction destroyed the crystal structure of the starch. At the same time, the presence of aldehyde groups caused cross-linking between molecular chains, making the particles easy to form clusters.

[0055] Oxidized starch was conjugated with cholesterol chloroformate via an imidazole system to successfully prepare modified oxidized starch. Oxidized starch can be quickly dissolved in molten imidazole, thereby obtaining homogeneous reaction conditions. Imidazole reacts with the dialdehyde group of oxidized starch and connects to the oxidized starch, while cholesterol chloroformate covalently connects with the hydroxyl group of oxidized starch, thereby forming cholesterol-imidazole modified amphoteric oxidized starch.

[0056] Example 2 Self-assembly of Nanostarch Particles and Drug Encapsulation

[0057] 1 Materials and methods

[0058] 1.1 Materials

[0059] 1.1.1 Test samples

[0060] Dimethyl sulfoxide (chromatographic grade) used in the experiment was purchased from Chengdu Kelong Chemical Co., Ltd.; isoimperatorin (98%), potassium dihydrogen phosphate (analytical grade), and disodium hydrogen phosphate dodecahydrate (analytical grade) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0061] 1.1.2 Experimental equipment

[0062] ALC-1100.2 electronic balance was purchased from Beijing Sartorius Instrument Systems Co., Ltd.; Classic ultrapure water meter was purchased from Veolia Water Treatment Technology (Shanghai) Co., Ltd.; DF-101T constant temperature oil bath stirrer was purchased from Zhengzhou Haiqi Instrument Technology Co., Ltd.; H1650-W desktop high-speed microcentrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; SCIENTZ-10N vacuum freeze dryer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; Liquid Zeta particle size / potential analyzer was purchased from Anton Paar GmbH, Austria.

[0063] 1.2 Test methods

[0064] 40 mg of cholesterol-imidazole modified amphoteric oxidized starch was dissolved in 20 mL of DMSO and stirred at 100°C for 25 min; 4 mL of 0.1% (w / v) isoimperatorin was completely dissolved in DMSO and then added dropwise to the cooled cholesterol-imidazole modified amphoteric oxidized starch solution. After complete mixing, the mixed solution was placed in a dialysis bag (MWCO=3500 Da) and dialyzed in PBS buffer for 3 days, with the dialysate changed every 4 to 6 hours. After the dialysis was completed, the solution in the dialysis bag was filtered with a 0.45 μm microporous filter membrane, and the filtrate was centrifuged (10000 rpm, 10 min) to obtain starch nanoparticles encapsulated with isoimperatorin, that is, a nano starch particle drug delivery system, specifically a nano starch particle isoimperatorin delivery system.

[0065] The average particle size and polydispersity index of starch nanoparticles in aqueous solution were measured using a particle size / potential analyzer. Before testing, the solution was filtered through a 0.45 μm microporous filter membrane to remove aggregates.

[0066] The drug loading rate and encapsulation efficiency of starch nanoparticles were determined by spectrophotometry. A certain amount of isoimperatorin was dissolved in DMSO, its absorbance at a wavelength of 322 nm was measured, and a concentration-absorbance standard curve was drawn. 5 mg of starch nanoparticles encapsulated with isoimperatorin were weighed and placed in 5 mL of DMSO, stirred for 10 min to completely dissolve, and then its absorbance at 322 nm was measured. The drug loading rate of starch nanoparticles was calculated according to the standard curve, and the experiment was repeated three times. The encapsulation efficiency was calculated accordingly according to the drug loading rate. Among them, the calculation formulas for drug loading rate and encapsulation efficiency are as follows:

[0067] Drug loading rate = (mass of isoimperatorin in the isoimperatorin nanostarch particle system / isoimperatorin nanostarch particle system) × 100%

[0068] Encapsulation efficiency = (mass of isoimperatorin in the isoimperatorin nanostarch particle system / total mass of isoimperatorin input into the reaction) × 100%

[0069] The drug release curve of the nano starch particle-loaded isoimperatorin system was determined by dialysis. 400 μL of 10 mg / mL starch nanoparticle-isoimperatorin suspension was placed in a dialysis bag (MWCO=3500 Da), and the dialysis bag was immediately transferred to 10 mL of PBS solution (pH=5.5 and pH=7.4) with 0.5% Tween 80 added. The dialysis system was placed on an oscillator and shaken at 50 rpm and 37°C. After the expected time, 2 mL of PBS solution was taken out, its absorbance at 322 nm was measured, and 2 mL of PBS solution was added to the original dialysis solution. The cumulative release of isoimperatorin was calculated as follows:

[0070] Cumulative release of isoimperatorin (%) =

[0071] Among them, C n represents the concentration of isoimperatorin released from the PBS taken out at time n (μg / mL), M represents the mass of isoimperatorin in the isoimperatorin nanostarch particle-loaded system (μg); Cx represents the calculation starting from x=1 to x=n-1 (in this calculation method, 2 mL of PBS solution was taken out each time to measure the drug concentration, and the last time the drug concentration of 10 mL of PBS solution containing the dialysis bag was measured).

[0072] The release experiments were repeated three times.

[0073] The blood compatibility of starch nanoparticle-isoimperatorin drug delivery system was determined. 4 mL of fresh blood was collected from healthy largemouth bass and anticoagulated with 1 mL of anticoagulant citrate dextrose solution. The blood was diluted with an equivalent of 0.9% saline (5 mL). 300 μL of diluted blood was incubated with 3 mL of nano starch particle-loaded isoimperatorin system (1 mg / mL) at 37°C with slow stirring for 2 h. The mixture was centrifuged at 1200 rpm for 5 min, and the supernatant was taken and its absorbance at 545 nm was measured. Deionized water and 0.9% saline were used as positive and negative controls, respectively. The hemolysis rate (HR) of erythrocytes and nano starch particle-loaded isoimperatorin system was calculated using the absorbance of released hemoglobin according to the following formula:

[0074] HR(%)=(A 样本- A 阴性 / A 阳性- A 阴性 )×100%

[0075] Among them, A 阳性 , A 阴性 and A 样本 They represent the absorbance under the environments of deionized water, 0.9% saline and nano-starch particles loaded with isoimperatorin, respectively.

[0076] 2 Results and Analysis

[0077] Based on the dialysis method, the amphiphilic oxidized starch self-assembled into nanoparticles, with its hydrophobic cholesterol fragments aggregated in the core of the nanoparticles, while the hydrophilic oxidized starch and imidazole parts formed the shell of the nanoparticles. The hydrophobic drug isoimperatorin was encapsulated into its hydrophobic core during the self-assembly process of the nanoparticles, forming a nano starch particle-loaded isoimperatorin system.

[0078] The average particle size of the self-assembled starch nanoparticles without drug encapsulation was 251.1±5.3 nm, and the PDI was low, with good dispersibility; while the average particle size of the starch nanoparticles encapsulated with isoimperatorin was 382.6±7.6 nm, which was slightly larger, but still maintained good dispersibility. Figure 2 , Figure 3 shown.

[0079] Calculated by the above method, the drug loading rate of the nano starch granule drug delivery system of the present invention is 5.12±0.31%, and the drug encapsulation efficiency of the nano starch granule drug delivery system of the present invention is 21.63±2.03%. Therefore, it can be considered that the nano starch granule drug delivery system of the present invention can effectively load antiviral drugs.

[0080] The drug release curve of the nano starch particle-loaded isoimperatorin system was determined by the above dialysis method. The cumulative drug release in 66 hours reached 70% and 42% under pH = 5.5 and pH = 7.5 respectively. Figure 4 As shown. The results showed that the release of isoimperatorin at pH 5.5 was greater and faster than that at pH 7.5. The nanostructure and pH-triggered drug release properties of the nanostarch particle-loaded isoimperatorin system indicate that nanostarch particles have the potential to serve as an effective carrier for targeting and delivering drugs to acidic body fluid environments. The pH responsiveness of the nanostarch particle-loaded isoimperatorin system is provided by the imidazole group. The imidazole group has pH-sensitive properties and is often used to construct pH-sensitive nanocarriers. Studies have shown that the protonation of the imidazole group at acidic pH causes the nanostarch particle-loaded isoimperatorin system to swell and disintegrate more rapidly, thereby more efficiently triggering the release of encapsulated isoimperatorin, which indicates that the nanostarch particle drug delivery system can be further designed and optimized for pH environments in the future.

[0081] According to the blood compatibility determination method mentioned above, the hemolysis rate of the nano starch particle-loaded isoimperatorin system was finally measured to be 2.4%, which is lower than the international standard of 5%. This shows that the nano starch particle drug delivery system of the present invention has good blood compatibility and will not cause serious damage to red blood cells. Figure 5 shown.

[0082] Example 3 Evaluation of the antiviral effect of the nanostarch particle-loaded isoimperatorin system

[0083] 1 Materials and methods

[0084] 1.1 Materials

[0085] 1.1.1 Viruses and experimental animals

[0086] Largemouth bass rhabdovirus (MSRV) was isolated and preserved by the Fisheries Science Laboratory, College of Animal Science and Technology, Northwest Agriculture and Forestry University. California bass fry (body length: 4~6±0.5 cm) were purchased from Yongsheng Fishery Development Co., Ltd. In the experiment, 800 California bass fry were raised in two identical 500 L aquariums, each containing 300 L of fully aerated tap water. The water temperature was maintained at 26±0.5℃ by heating rods, and the dissolved oxygen in the water was maintained above 6 mg / L by aeration. The light / dark cycle within 24 hours was adjusted to 14 h:10 h by fluorescent lamps. The commercial feed (0.5 mm) of bass fry was fed once a day in the morning, and the feed was eaten within 10 min. The live test was carried out after 2 weeks of temporary rearing.

[0087] 1.1.2 Test reagents

[0088] The starch nanoparticle-loaded isoimperatorin system was synthesized by the Fisheries Science Laboratory of the College of Animal Science and Technology of Northwest Agriculture and Forestry University, with an isoimperatorin content of 5.12%; isoimperatorin (98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; reverse transcription kit and quantitative PCR kit were purchased from Bao Bioengineering (Dalian) Co., Ltd.; viral RNA extraction kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; HiScript III RT SuperMix for qPCR (+gDNA wiper) kit was purchased from Nanjing Vazyme Biotech Co., Ltd.; chloroform, isopropanol, and ethanol were purchased from Xi'an Chemical Reagent Factory; Trizol reagent was purchased from Invitrogen; and DNA Marker DL2000 was purchased from Dongsheng Biotechnology Co., Ltd. All reagents were analytical grade.

[0089] 1.1.3 Test equipment

[0090] ALC-1100.2 1% electronic balance was purchased from Beijing Sartorius Instrument System Co., Ltd.; KQ-500E ultrasonic cleaner; 5~10 μL and 20~200 μL pipettes; HH-4 digital constant temperature water bath was purchased from Shanghai Techeng Machinery Equipment Co., Ltd.; H1650-W desktop high-speed microcentrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; 1-15K high-speed refrigerated centrifuge was purchased from Sigma, USA; DYCZ-24DN mini dual vertical electrophoresis instrument and WD-9413A gel imaging analysis system were purchased from Beijing Liuyi Instrument Factory; Thermo Multiskan MK3 microplate reader was purchased from Thermo Fisher Scientific, USA; Bio-Rad PCR instrument was purchased from Bio-Rad, USA; automatic temperature control heating rod was purchased from Zhejiang Zhoushan Fishing Tackle Factory; 500L aquarium was purchased from Changzhou Hengzun Plastic Industry Co., Ltd.

[0091] 1.2 Test methods

[0092] The nano starch particle-loaded isoimperatorin system and isoimperatorin prepared in the embodiment of the present invention were dissolved in deionized water to drug concentrations of 8.0 mg / L, 16.0 mg / L, and 32.0 mg / L, respectively (the concentration of the drug-loaded system was calculated based on the effective drug concentration). 720 healthy sea bass fry were randomly collected from the aquarium, 20 in a group, and divided into nine groups, with 4 replicates in each group (3 groups were sampled and 1 group was observed for survival rate). Seven groups of sea bass fry were injected with the ventral fin base of each fry, and each sea bass fry was injected with 20 μL 6.125×10 2 TCID 50 mL -1 The remaining two groups of sea bass fry were injected with 20 μL of PBS in the same way as the blank group and the negative control group, and then placed in a new aquarium of 10 L, with the water temperature controlled at 26±0.5℃ and other rearing conditions unchanged, waiting for virus infection for 24 h. After 24 h, the infected sea bass fry were immersed in starch nanoparticles loaded with isoimperatorin system and isoimperatorin for 4 h. The specific scheme is shown in Table 1.

[0093] Table 1 In vivo experimental protocol

[0094]

[0095] Tissue sampling was performed on the 3rd, 6th, and 9th days after immersion in the drug / drug delivery system. Three fish were randomly selected from each group. The fry were first anesthetized with 0.01% benzocaine. After dissection, liver, spleen, and kidney tissue samples were collected. RNA extraction reagent (Trizol) was added and fully ground. Total RNA was extracted from liver, spleen, and kidney tissues according to the instructions of the kit. The quality and concentration of the extracted RNA were determined by agarose gel electrophoresis. The RNA concentration was adjusted to 1.0 μg / μL using nuclease-free water, and cDNA was synthesized according to the instructions of the kit. The synthesized cDNA product was used for qRT-PCR to detect the content of MSRV. The qRT-PCR reaction system was 10 μL, including 1 μL cDNA template, 5 μL ChamQ SYBR qPCR Master Mix (Vazyme Biotech), 3 μL nuclease-free water, and 0.5 μL of upstream and downstream primers. The reaction program was preheating at 95°C for 30 s, followed by 40 cycles (95°C for 5 s, 60°C for 30 s, and 72°C for 30 s). The primer pairs used for real-time PCR analysis of gene expression are shown in Table 2.

[0096] Table 2 Primer pairs used for real-time PCR analysis of gene expression

[0097]

[0098] 2 Results and Analysis

[0099] The results of the sea bass toxicity experiment showed that the starch nanoparticle-loaded isoimperatorin system had no significant toxicity to sea bass fry at an immersion administration concentration of 16 mg / L. When the concentration of isoimperatorin was 16 mg / L, the inhibition rate of the drug-loaded system treatment group on MSRV was significantly improved compared with the inhibition rate of the isoimperatorin treatment group. When the concentration of isoimperatorin reached 32 mg / L, the inhibition effect on MSRV was not significantly improved compared with 16 mg / L, but sea bass began to die at a concentration of 43 mg / L, so 16 mg / L was a better administration concentration.

[0100] At the same time, the embodiment of the present invention explores the survival rate of seabass after infection with MSRV under different treatment conditions, and the results are shown in Table 3 and Figure 6 shown.

[0101] Depend on Figure 6 It can be seen that when isoimperatorin was not used for treatment, the survival rate of seabass within 14 days after infection with MSRV was only 10%; when the treatment concentration of isoimperatorin was 16 mg / kg, the survival rate of seabass within 14 days after infection with MSRV reached 45%; when 16 mg / L starch nanoparticle-loaded isoimperatorin system was used for treatment, the survival rate of seabass within 14 days after infection with MSRV increased to 65%.

[0102] As shown in Table 3, compared with the survival rates of 45% and 55% in the isoimperatorin immersion group at 16 mg / L and 32 mg / L, respectively, the survival rates of the isoimperatorin immersion group loaded with starch nanoparticles reached 65% and 70% at 16 mg / L and 32 mg / L, respectively, with a relative survival rate increase of 15% to 20%, proving that the drug delivery system can improve the anti-disease effect of the drug.

[0103] The present invention also uses real-time quantitative PCR to detect the MSRV viral load of sea bass liver, spleen and kidney on the 3rd, 6th and 9th day after infection with MSRV. The results are as follows: Figure 7 As shown, Figure 7It was proved that isoimperatorin itself has a significant inhibitory effect on MSRV, which will reach a peak on the 3rd day of administration and then gradually weaken. The use of the nano starch particle drug delivery system enhanced its inhibitory effect on the 3rd and 6th days of the liver, the 3rd day of the spleen, and the 3rd, 6th and 9th days of the kidney, and the enhancement effect was particularly significant on the 3rd day. After treatment with the starch nanoparticle-loaded isoimperatorin system, the infection rate of sea bass can be reduced relatively directly by administration, and the survival rate of diseased sea bass can be increased. The above results show that the starch nanoparticle drug delivery system can more efficiently deliver hydrophobic drugs to diseased tissues in a short time to improve the therapeutic effect of the drug.

[0104] Table 3 Survival results of sea bass fry

[0105]

[0106] In summary, the present invention prepares an amphoteric nano starch particle capable of encapsulating antiviral drugs (for example, isoimperatorin). The starch nano particle drug delivery system can greatly improve the solubility of hydrophobic drugs in water, and penetrate the body surface of aquatic animals such as fish, shrimp, crab, and shellfish in the water to enter the cells and tissues of individuals, inhibit or kill and eliminate the corresponding viruses in these aquatic animals, and has a wide range of application space and practical value in aquatic animal breeding.

[0107] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0108] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. Application of a nano starch particle drug delivery system in the preparation of a drug for preventing and treating rhabdovirus infection in aquatic animals, characterized in that: The aquatic animals are fish, shrimp, crab or shellfish; The preparation method of the nano starch particle drug delivery system comprises the following steps: S1. oxidizing starch using sodium periodate to obtain oxidized starch with aldehyde groups; S2. reacting cholesterol chloroformate with oxidized starch in molten imidazole to obtain cholesterol-imidazole modified amphoteric oxidized starch; S3. placing the cholesterol-imidazole modified amphoteric oxidized starch in a solution containing an antiviral drug to prepare starch nanoparticles encapsulating the antiviral drug, thereby obtaining a nano starch particle drug delivery system; The antiviral drug is isoimperatorin.

2. The use according to claim 1, characterized in that: The specific steps of step S1 are: A 10.56% (w / v) sodium periodate solution was added dropwise to a 16% (w / v) starch solution, and the mixture was reacted at 37° C. in the dark for 3 hours. The solution was precipitated with ethanol, the sodium periodate was removed by dialysis, and the solution was freeze-dried to obtain oxidized starch with aldehyde groups.

3. The use according to claim 1, characterized in that: The specific steps of step S2 are: The oxidized starch is added into molten imidazole and stirred evenly; cholesterol chloroformate is added and stirred at 100° C. for reaction for 1 hour; unreacted imidazole and cholesterol chloroformate are removed by dialysis, and the mixture is freeze-dried to obtain cholesterol-imidazole modified amphoteric oxidized starch.

4. The use according to claim 1, characterized in that: The specific steps of step S3 are: The amphoteric oxidized starch is dissolved in DMSO, and a solution containing antiviral drugs is added; the mixed solution is placed in a dialysis bag and dialyzed in a PBS solution for 3 days; the dialysate is centrifuged, washed, and freeze-dried to obtain a nano-starch particle drug-carrying system.

5. The use according to claim 1, characterized in that: The nano starch particle drug delivery system is diluted during production and used to soak sick aquatic animals, or is directly diluted and mixed with water and then sprayed on aquatic animals to be treated; wherein the concentration of the antiviral drug is 8-40 mg / L, and the soaking time is 2-5 hours.

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