Nanocomposite targeting neuronecrosis virus and preparation method and application thereof

Abstract

The present invention relates to the technical field of aquatic animal disease prevention and control, and specifically to a nanocomposite for neuronecrosis virus, a preparation method thereof and an application thereof. The present invention provides a nanocomposite composed of polylactic-glycolic acid copolymer, polyethyleneimine and siRNA, and the present invention also provides a preparation method of the nanocomposite and an application thereof in the preparation of a drug for improving the survival rate of fish eggs infected with neuronecrosis virus. The present invention simulates neuronecrosis virus, constructs a vector of neuronecrosis virus capsid protein, and expresses it in cells; screens out siRNA that can effectively inhibit the expression of viral capsid protein, and then constructs a nanocomposite of neuronecrosis virus, blocks NNV in the embryonic period (fertilized eggs), and then improves the survival rate of fry, further opening up new possibilities for treating early diseases of fry, and the method is innovative in the field of artificial breeding of aquatic animals, and provides a new idea for virus prevention and control in the field of aquaculture.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic animal disease prevention and control, and in particular to a nanocomposite targeting neuronecrosis virus and a preparation method and application thereof. Background Art

[0002] Although my country has made great breakthroughs in artificial breeding of marine fish, there are not many species that can be artificially bred on a large scale, and a large number of fry die in the early stages of artificial breeding, which is one of the main bottlenecks restricting the scale of marine fish breeding and proliferation and release. One of the causes of the early large-scale death of marine fish is the Nervousnecrosis virus (NNV), which was discovered in 1989 and first reported in 1990. Since then, more than 100 marine fish species have been reported to carry NNV around the world, and more than 40 species are susceptible, such as European seabass (Dicentrarchuslabrax), Asian seabass (Lates calcarifer), various groupers (Ephinephelus spp.), yellow-banded trevally (Pseudocaranx dentex), Senegal sole (Solea senegalensis), Atlantic halibut (Hippoglossus hippoglossus), Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), etc. (Bandin I and Souto S., 2020). NNV mainly occurs in larvae and juveniles, with a mortality rate of up to 100%. At present, there is no effective method to block NNV at home and abroad, which has always been a bottleneck that plagues marine fish breeding.

[0003] Studies have found that one of the main ways of NNV transmission is vertical transmission from parents to offspring. Both eggs and sperm may carry the virus. Before the fertilized eggs are hatched, the embryos are protected by the egg membrane and the operation is convenient, which is an ideal period to block NNV. However, the hatched fry have weak resistance in the water and are not suitable for medication. Therefore, blocking NNV from the embryo can effectively avoid the impact of NNV on fry. There are many drugs to block viruses, such as vaccines, small interfering RNA (siRNA), interferon, etc. Among them, the application of RNA interference technology (RNAi) in the treatment of viral infectious diseases has received great attention (Zhu Zuoyan et al., 2006), but there is currently no effective method to deliver these drugs to fish embryos. It is urgent to develop an effective and feasible drug delivery carrier.

[0004] With the continuous innovation of new technologies in the medical field, delivery systems based on various materials such as proteins, liposomes, polymers, inorganic materials, etc. have been applied to the research on high-efficiency transport of siRNA, and have made important progress in tumor targeted diagnosis and treatment. However, there is little research on siRNA nanodelivery technology in the field of aquaculture. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a nanocomposite for neuronecrosis virus and a preparation method and application thereof. The nanocomposite provided by the present invention can effectively improve the survival rate of fish eggs and can be effectively applied to the field of aquaculture.

[0006] The present invention provides a nanocomposite, the raw materials of which are composed of polylactic acid-glycolic acid copolymer, polyethyleneimine and siRNA, wherein:

[0007] The mass volume ratio of the polylactic acid-glycolic acid copolymer and polyethyleneimine is (5-50) mg: (0.05-1) mL, and each 100 mL of the polyethyleneimine contains 0.5-2.5 μmol of siRNA;

[0008] The siRNA comprises at least one of the sense strand of the nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 2, the sense strand of the nucleotide sequence shown in SEQ ID NO: 3 and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 4, and the sense strand of the nucleotide sequence shown in SEQ ID NO: 5 and the antisense strand of the nucleotide sequence shown in SEQ ID NO: 6;

[0009] The particle size of the nanocomposite is 100-200 nm.

[0010] Compared with the prior art, in the nanocomposite provided by the present invention, the polylactic acid-glycolic acid copolymer has good biocompatibility, is non-toxic, and has good capsule-forming and film-forming properties. Its degradation products are lactic acid and glycolic acid, which are also byproducts of human metabolic pathways. Therefore, when it is used in medicine and biomaterials, it will not have toxic side effects. The siRNA can effectively inhibit the neural necrosis virus. The polyethyleneimine can better form a complex with the siRNA, further increasing the loading amount of the polylactic acid-glycolic acid copolymer. The components cooperate closely, and the successfully constructed nanocomposite of the neural necrosis virus can effectively enter the fish eggs, block the NNV in the embryonic period, and then improve the survival rate of the fry, thereby obtaining a more accurate technical effect.

[0011] In some embodiments, the mass volume ratio of the polylactic acid-co-glycolic acid and polyethyleneimine is 25 mg:0.1 mL, and each 100 mL of polyethyleneimine contains 1.25 μmol of siRNA, wherein:

[0012] The molecular weight of the polylactic acid-glycolic acid copolymer is 10000 Da, and the molecular weight of the polyethyleneimine is 25000 Da.

[0013] In some embodiments, the siRNA comprises a sense strand of the nucleotide sequence shown in SEQ ID NO:1 and an antisense strand of the nucleotide sequence shown in SEQ ID NO:2.

[0014] In some embodiments, the nanocomposite has a particle size of 160 nm.

[0015] Experiments show that the nanocomposite obtained by using the components in the above embodiments has the closest coordination between the components, the best loading capacity, the best particle size of the nanocomposite, and the best effect when delivered into fish eggs, thereby obtaining the most accurate technical effect.

[0016] The present invention also provides a method for preparing the nanocomposite, comprising the following steps:

[0017] The steps include:

[0018] Step 1, mixing the siRNA and the polyethyleneimine to form solution A, and mixing the polylactic acid-glycolic acid copolymer and the organic phase solution to form solution B;

[0019] Step 2, ultrasonically mixing the solution A and the solution B in step 1;

[0020] Step 3, taking the solution after ultrasonic mixing in step 2 and the external aqueous phase solution and ultrasonically mixing them again;

[0021] Step 4: Take the solution mixed by ultrasonic again in step 3, filter and centrifuge it to obtain the nanocomposite.

[0022] In some embodiments, in step 1, in step 1, the solvent used for mixing solution A includes water;

[0023] Preferably, the concentration of polyethyleneimine in the solution A is 5 vol% to 50 vol%;

[0024] More preferably, the concentration of the polyethyleneimine in the solution A is 10 vol %.

[0025] In some embodiments, in step 1, the organic phase solution comprises chloroform and / or dimethylformamide;

[0026] Preferably, the organic phase solution is chloroform;

[0027] Preferably, in the solution B, the concentration of the polylactic acid-co-glycolic acid copolymer is 1-10 mg / mL. In some embodiments, the concentration of the polylactic acid-co-glycolic acid copolymer may be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL;

[0028] More preferably, in the solution B, the concentration of the poly(lactic acid-co-glycolic acid) is 5 mg / mL.

[0029] In some embodiments, in step 3, the solute of the external aqueous phase solution includes polyethylene glycol and / or Tween 80;

[0030] Preferably, the solute of the external aqueous phase solution is polyethylene glycol;

[0031] Preferably, in the external aqueous phase solution, the concentration of polyethylene glycol is 1-20 mg / mL;

[0032] More preferably, the concentration of polyethylene glycol in the external aqueous phase solution is 10 mg / mL.

[0033] In some embodiments, in step 2 and step 3, the volume ratio of solution A, solution B and the external aqueous phase solution is (0.5-2) (1-10): (1-30);

[0034] More preferably, the volume ratio of the solution A, the solution B and the external aqueous phase solution is 1:5:15.

[0035] In some embodiments, in step 4, the filtration is ultrafiltration, and after the centrifugation, the process further comprises washing with PBS to obtain the nanocomplex.

[0036] The present invention provides the use of the nanocomposite or the nanocomposite prepared by the preparation method in preparing drugs for resisting neuronecrosis virus and / or improving the survival rate of fish eggs infected with neuronecrosis virus.

[0037] The invention provides a drug for resisting neuronecrosis virus and / or improving the survival rate of fish eggs infected with neuronecrosis virus, comprising the nanocomposite or the nanocomposite prepared by the preparation method.

[0038] The invention provides a method for improving the survival rate of fish eggs infected with neuronecrosis virus, comprising the step of bathing the fish eggs with the medicine.

[0039] The present invention provides a nanocomposite composed of polylactic acid-glycolic acid copolymer, polyethyleneimine and siRNA, and also provides a preparation method of the nanocomposite and a drug for preparing an anti-neural necrosis virus and / or improving the survival rate of fish eggs infected with the neuronecrosis virus. The present invention simulates the neuronecrosis virus, constructs a vector of the neuronecrosis virus capsid protein, and expresses it in cells; screens out siRNA that can effectively inhibit CP expression, and then constructs a nanocomposite of the neuronecrosis virus, blocks NNV in the embryonic period, and then improves the survival rate of fry, further opening up new possibilities for treating early diseases of fry. The method is innovative in the field of artificial breeding of aquatic animals and provides new ideas for the field of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Figure 3 EPC cells co-transfected with siRNA and pEGFP-CP at different transfection times (×100), where a is 24h EGFP-CP, b is 24h pEGFP-CP+NNV-18, c is 24h pEGFP-CP+NNV-144, d is 24h pEGFP-CP+NNV-720, e is 48h pEGFP-CP, f is 48h pEGFP-CP+NNV-18, g is 48h pEGFP-CP+NNV-144, h is 48h pEGFP-CP+NNV-720, i is 72h pEGFP-CP, j is 72h pEGFP-CP+NNV-18, k is 72h pEGFP-CP+NNV-144, l is 72h pEGFP-CP+NNV-720, and m is 24h pEGFP-empty vector, n is pEGFP-empty vector at 48 h, o is pEGFP-empty vector at 72 h, and p is siRNA-Cy3 at 48 h;

[0041] Figure 2 The cell survival rates of EPCs in different control groups are shown, where * indicates that the group is significantly different from the control group (P<0.05), ** indicates that the difference is extremely significant (P<0.01), and ns indicates that there is no significant difference;

[0042] Figure 3 The cell survival rate of EPC after co-transfection of siRNA and pEGFP-CP is shown, where the * indicates that the group is significantly different from the control group (P<0.05), and ** indicates an extremely significant difference (P<0.01);

[0043] Figure 4 Figure 2 shows the particle size analysis results of the secondary ultrasonic products of Group A1 in Example 2, wherein Figure A is a particle size-intensity relationship diagram, and Figure B is a particle size correlation coefficient;

[0044] Figure 5 Figure 2 shows the particle size analysis results of the secondary ultrasonic products of group A2 in Example 2, wherein Figure A is a particle size intensity relationship diagram, and Figure B is a particle size correlation coefficient;

[0045] Figure 6 Figure 2 shows the particle size analysis results of the secondary ultrasonic products of group A3 in Example 2, wherein Figure A is a particle size intensity relationship diagram, and Figure B is a particle size correlation coefficient;

[0046] Figure 7 Figure 2 shows the particle size analysis results of the secondary ultrasonic products of group A4 in Example 2, wherein Figure A is a particle size intensity relationship diagram, and Figure B is a particle size correlation coefficient;

[0047] Figure 8 Figure 2 shows the particle size and zeta potential analysis results of the secondary ultrasonic product of the best ratio group in Example 2, wherein Figure A is a particle size light intensity relationship diagram, Figure B is a particle size correlation coefficient, Figure C is a zeta potential voltage current, and Figure D is a zeta potential statistical diagram;

[0048] Fig. 9 The particle size and zeta potential analysis results of the filtrate after the secondary ultrasonic product and ultrafiltration of the best ratio group in Example 2 are shown, wherein Figure A is a particle size light intensity relationship diagram, Figure B is a particle size correlation coefficient, Figure C is a zeta potential voltage current, and Figure D is a zeta potential statistical diagram;

[0049] Fig.10 The results of particle size and zeta potential analysis of the nanocomposite in the membrane after ultrafiltration of the secondary ultrasonic product of the best ratio group in Example 2 are shown, wherein Figure A is a particle size-light intensity relationship diagram, Figure B is a particle size correlation coefficient, Figure C is a zeta potential voltage-current, and Figure D is a zeta potential statistical diagram;

[0050] Fig.11 The electron microscope observation results of the nanocomposite in the filter membrane after the second ultrasonic product and ultrafiltration of the best ratio group in Example 2 are shown;

[0051] Fig.12 The fluorescence detection results of the nanocomplex in the filter membrane after the secondary ultrasonic product and ultrafiltration of the best ratio group in Example 2 after washing with water are shown;

[0052] Fig.13 The fluorescence detection results of the nanocomplex in the filter membrane after the second ultrasonic product and ultrafiltration of the best ratio group in Example 2 after washing with PBS are shown;

[0053] Fig.14 The fluorescence detection results of the nanocomplex in the filter membrane after the second ultrasonic product and ultrafiltration of the best ratio group in Example 2 are shown;

[0054] Fig.15The effect of the nanocomposite in the filter membrane after ultrafiltration of the secondary ultrasonic product of the best ratio group in Example 2 on grouper eggs is shown, wherein Ctr is the control group; C1 is a 10-fold diluted concentration; and C2 is a 1000-fold diluted concentration. DETAILED DESCRIPTION

[0055] The present invention provides a nanocomposite for neuronecrosis virus and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0056] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. However, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0057] Preliminary experiments have shown that compared with other nanomaterials, the nanocomposite selected by the present invention has the highest compatibility with fish fertilized eggs, and its particle size is suitable, so it will not block the pores and channels of fish eggs, and ensure normal material exchange; it has stable chemical properties, does not release toxic substances, and reduces interference with the internal biochemical process of fish eggs; it is not easy to cause oxidative stress, reduces the production of reactive oxygen species, and can protect intracellular biomacromolecules; it has no endocrine disrupting effect, and maintains the normal hormone signal transduction and development process of fish eggs; it has little effect on the gene expression of fish eggs, ensures the normal expression of development-related genes, and promotes normal development and differentiation. Therefore, the present invention uses polylactic acid-glycolic acid copolymer and polyethyleneimine loaded siRNA to construct nanocomposite. The preliminary experiment results are not good, so it is not provided in this application for the time being.

[0058] When the nanocomposite is composed of siRNA in the selected ratio of the present invention and the nanomaterials polylactic acid-glycolic acid copolymer and polyethyleneimine, the components cooperate closely with each other, the loading amount is optimal, and the particle size of the successfully constructed nanocomposite is optimal, which is more conducive to entering the inside of the fish eggs, and NNV can be blocked during the embryonic period, thereby improving the survival rate of the fry.

[0059] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0060] Example 1 Screening of siRNA interference sequences

[0061] 1 Materials and methods

[0062] (1) Synthesis of simulated viruses

[0063] RGNNV has a simple structure and has only one structural protein, namely the capsid protein (CP). We inserted the gene encoding CP into the cell transfection vector pEGFP to lay a good foundation for the subsequent simulation of the expression of the virus in cells.

[0064] (2) Design of siRNA interference sequence

[0065] In order to achieve effective silencing of the NNV CP gene, based on the existing neuronecrosis virus (NNV) CP gene sequence in the laboratory, the siRNA sequence was designed using the online tool provided by Ambion (http: / / www.ambion.com). During the design process, it was ensured that the selected siRNA sequence was completely complementary to the mRNA of the target CP gene to achieve specific gene silencing. The online BLAST was used to ensure that the selected sequence had no homology with other genes outside the virus. At the same time, in order to verify the specificity of siRNA, a negative control (siRNA-Cy3, siRNA labeled with the red fluorescent dye Cy3) was also designed in this experiment. All sequences were commissioned to Shanghai Jima Pharmaceutical Technology Co., Ltd. for synthesis.

[0066] Table 1 siRNA sequences

[0067]

[0068] Note: Since there is no base U among the characters that can be used for nucleotides in the sequence listing, when preparing the sequence listing, the base U in the RNA sequence in Table 1 above was changed to base T.

[0069] (3) Inhibitory effect of siRNA on intracellular CP

[0070] Plasmid extraction by shaking bacteria (endotoxin-free plasmid extraction kit):

[0071] (1) Prepare LB liquid culture medium according to the proportion, take out the E. coli culture liquid frozen at -20℃, thaw it, pipette 200μL into the culture medium, seal it, mark it and place it in a shaker, set the temperature to 37℃ and the speed to 200rpm.

[0072] (2) Centrifuge the cultured bacteria (usually at 4°C, about 4000-6000g, for 5-10 minutes). Discard the supernatant and retain the bacterial pellet.

[0073] (3) Add the lysis buffer provided by the kit to the bacterial pellet, mix thoroughly to lyse the bacteria, and let it stand at room temperature for a while to promote lysis.

[0074] (4) Add an equal volume of Neutralization Buffer, mix well, and let stand at room temperature to neutralize the active ingredients in the lysate.

[0075] (5) Centrifuge the mixture in the centrifuge tube, usually at 4°C, about 12,000-14,000g, for 10-15 minutes. Discard the supernatant and retain the pellet containing the plasmid DNA.

[0076] (6) Add wash buffer to the precipitate, mix well, and centrifuge to remove the wash buffer. Repeat the washing step at least once to ensure that all impurities such as proteins and RNA are removed.

[0077] (7) Add a certain volume of elution buffer, usually sterile water or TE buffer, to the final precipitate. After mixing, incubate the tube at room temperature or 37°C to fully dissolve the plasmid DNA.

[0078] (8) Transfer the dissolved plasmid DNA to a new sterile tube and store at -20°C or -80°C for subsequent experimental use.

[0079] Preliminary culture of EPC cells:

[0080] (1) The activated EPC cells were cultured in M199 medium containing 20% ​​fetal bovine serum, and the cell growth status was determined based on the cell adhesion.

[0081] (2) When the cells have covered the bottom of the culture bottle, remove the waste liquid in the clean bench, add trypsin for digestion, tap the bottle to disperse the cells, and then use M199 culture medium containing 5% fetal bovine serum to blow and suck the bottle wall. Determine whether to subculture based on the cell density.

[0082] (3) When subculturing, half of the liquid after digestion is transferred to a new culture bottle. Observe the cell growth status every 2 to 3 days and replace the culture medium to ensure sufficient nutrients to promote cell growth.

[0083] Experimental group design:

[0084] (1) (Blank) Control group 1: cells without any treatment.

[0085] (Negative) Control group 2: Only pEGFP-empty vector plasmid was transfected, which is a blank vector plasmid with green fluorescence.

[0086] (Positive) Control group 3: Only pEGFP-CP was transfected, which is a plasmid carrying the CP pathogenic gene and green fluorescence.

[0087] (Negative) Control group 4: Only siRNA-Cy3 was transfected. siRNA-Cy3 is siRNA labeled with red fluorescent dye Cy3 and has no homology with CP sequence.

[0088] Experimental group 1: pEGFP-CP was co-transfected with NNV-18.

[0089] Experimental group 2: pEGFP-CP was co-transfected with NNV-144.

[0090] Experimental group 3: pEGFP-CP was co-transfected with NNV-720.

[0091] Cell transfection experiments:

[0092] Taking a 24-well plate as an example, 18 to 24 hours before the transfection experiment, 0.5 to 2 × 10 5 cells and added 0.5 mL of cell culture medium for culture.

[0093] The number of cells selected for initial inoculation should be such that the cell density reaches 70% to 90% during transfection, and care should be taken to ensure that the cells are not overgrown or in a dormant phase during transfection.

[0094] Take 25 μL of Opti-MEM culture medium and add it to two 1.5 mL sterile EP tubes, and mark them as No. 1 and No. 2 respectively.

[0095] Place Lipofectamin 3000 transfection reagent at room temperature and gently mix before use; add 0.75 μL of Lipofectamin 3000 transfection reagent to tube 1 containing serum-free culture medium, mix by pipetting, and place at room temperature for 5 minutes.

[0096] Add 1 μg of NNV plasmid / empty vector to tube 2 (calculate the required volume based on the required amount), pipette gently to mix, and then add 1 μL of P3000 TM Reagent, gently pipette to mix. For siRNA transfection, add 0.625 μL and do not add P3000 TM Reagent.

[0097] Add the transfection complex to the Lipofectamin 3000 mixture, that is, add 25 μL of the mixture in tube 2 to tube 1, mix well by pipetting, and let stand at room temperature for 10 to 15 minutes to allow DNA / siRNA and Lipofectamin 3000 transfection reagent to form a transfection complex.

[0098] Add 50 μL of the complex to the culture plate containing cells (for co-transfection, add plasmid complex and siRNA complex separately, 100 μL in total), tap and shake gently to disperse the cells evenly. Set up 3 wells for each group.

[0099] The culture plate was placed in a cell culture incubator at 25°C and cultured for 24 h, 48 h, and 72 h, respectively.

[0100] Cell observation and counting:

[0101] At different transfection times, the growth of cells in the culture plate was observed under an inverted microscope, and the fluorescence was recorded under a fluorescence microscope. The microscope was set to 100x magnification (10x objective × 10x eyepiece), and 5 to 8 fields of view were observed in each experimental well to ensure a comprehensive evaluation of the transfection effect.

[0102] The supernatant in the wells of the culture plate was collected into a new sterile EP tube, and each well in the culture plate was washed with PBS.

[0103] Add 200 μL of trypsin to each well, tap gently to digest for 2 minutes, add 300 μL of culture medium, pipette to mix, add to the EP tube containing the supernatant, add 500 μL of trypan blue solution, pipette to mix.

[0104] Pipette 25 μL of cell solution from the tube and add it to the counting chamber, and count using a cell counter.

[0105] Data Analysis:

[0106] GraphPad Prism v.8.0.2 was used to analyze the significance of data differences between groups and plot the data. All experiments were repeated three times, and two-way ANOVA was used for data comparison. *: P < 0.05; **: P < 0.01; ns: no significant difference.

[0107] 2. Results and Analysis

[0108] 2.1 Fluorescence observation of each group

[0109] At different transfection time points (24h, 48h and 72h), the positive control group (e.g. Figure 1In Figures a, e, and i, the number of green fluorescent cells is relatively large, with the highest number of fluorescent cells at 48h, and the successfully transfected cells are round. The experimental group co-transfected with pEGFP-CP and siRNA showed fewer green fluorescent signals, and they were all less than the positive control group at the corresponding time. Especially at the 48h transfection time point, the green fluorescent signal of the pEGFP-CP and siRNA co-transfection group was significantly reduced (e.g. Figure 1 (Figs. b, c, d, f, g, h, j, k and l).

[0110] In addition, the number of green fluorescent cells in the pEGFP-empty vector group gradually increased over time, and green irregular cells (e.g. Figure 1 In Figures m, n, and o, siRNA-Cy3 can also be observed with red fluorescence (e.g. Figure 1 (as shown in Figure p).

[0111] 3.2 EPC Counting

[0112] The cell survival rates of EPCs in different control groups are as follows Figure 2 As shown, the cell survival rates of the pEGFP-empty vector group and the siRNA-Cy3 group at 48h were 84.55%, 71.69%, and 74.69%, respectively, and at 72h the survival rates were 86.60%, 81.24%, and 82.23%, respectively. There was no significant difference between the pEGFP-empty vector group and the siRNA-Cy3 group and the cell control group. The pEGFP-CP positive control group was significantly different from the cell group at 48h, and the difference was significant at 72h. The cell survival rate of EPC after co-transfection of siRNA and pEGFP-CP is shown in Figure 2. Figure 3As shown in the figure, after co-transfection of NNV-18, NNV-144, NNV-720 and pEGFP-CP for 48h and 72h, the cell survival rate of EPC was significantly increased, indicating that these three pairs of siRNAs all showed an inhibitory effect on the expression of CP and had a small toxic effect on cells. When the transfection time was 48h, the cell survival rate of the pEGFP-CP group was about 27%, and the cell survival rates of the pEGFP-CP+NNV-18, pEGFP-CP+NNV-144, and pEGFP-CP+NNV-720 groups were 61%, 59%, and 62%, respectively, which were significantly different from the pEGFP-CP control group (P<0.01), while there were no significant differences among the three groups. When the transfection time was 72h, the cell survival rates of the pEGFP-CP control group, pEGFP-CP+NNV-18, pEGFP-CP+NNV-144, and pEGFP-CP+NNV-720 groups were 55%, 86%, 81%, and 73%, respectively, which were all increased compared with 48h. Among them, the pEGFP-CP+NNV-18 group was significantly different from the pEGFP-CP control group (P<0.01), the pEGFP-CP+NNV-144, pEGFP-CP+NNV-720 group was significantly different from the pEGFP-CP control group (P<0.05), and there was no significant difference among the pEGFP-CP+NNV-18, pEGFP-CP+NNV-144, and pEGFP-CP+NNV-720 groups.

[0113] The above results show that the simulated virus can cause a high lethality to cells, while the survival rate of cells can be significantly improved after adding siRNA, indicating that these siRNAs can effectively inhibit the virus. Among them, the siRNA in the NNV-18 group has the best effect and is used for the subsequent preparation of nanocomplexes.

[0114] Example 2 Preparation of PLGA-siRNA Nanocomplex

[0115] 1. Synthesis of PLGA-siRNA Nanocomplexes

[0116] (1) Organic phase: The molecular weight of PLGA was selected as 10,000, and 5 mg of PLGA was dissolved in different volumes of organic solvents (chloroform or DMF) to screen the optimal ratio to form an organic phase;

[0117] (2) Inner aqueous phase: PEI (polyethyleneimine) with a molecular weight of 25,000 was selected and dissolved in water to screen the optimal ratio; siRNA was dissolved in the PEI aqueous solution at 4°C to form an inner aqueous phase;

[0118] (3) External aqueous phase: Dissolve PEG or Tween80 in water and select the optimal ratio to form the external aqueous phase;

[0119] (4) The organic phase and the inner aqueous phase are mixed in proportion and ultrasonically mixed for the first time using an ultrasonic device.

[0120] (5) Add the sonicated solution into the prepared external aqueous phase in proportion and sonicate for the second time.

[0121] (6) After treatment, take a certain amount of supernatant by centrifugation. The supernatant is added to an ultrafiltration tube and centrifuged. The spherical complexes encapsulating siRNA are filtered onto the filter membrane. Then, PBS or water is used to suspend the nanocomplexes on the filter membrane in a PBS solution or aqueous solution and stored at low temperature and away from light.

[0122] (7) The particle size of the secondary ultrasonic product was detected using a nano-laser particle size analyzer, and the zeta potential was measured. The final product was observed using an electron microscope.

[0123] In the above steps, the PEI concentration, PLGA concentration, the molar mass ratio of the volume of polyethyleneimine to siRNA, the organic phase solution, the main solute of the external aqueous phase and its concentration, and the volume ratio of the internal aqueous phase, the organic phase solution and the external aqueous phase solution are grouped and prepared according to Table 2. After the above synthesis is operated and modified, the particle size of the secondary ultrasonic product is detected, and the best reagent is compared by particle size distribution and dilution multiple. The dilution multiple test takes 2 mL of the PLGA-siRNA nanocomplex prepared in groups A1 to A4 in Table 2, logarithmically dilutes it with deionized water, and the best preparation product is obtained by maintaining the maximum stable ratio of the final product.

[0124] Table 2 Different groups

[0125]

[0126] 2. Encapsulation efficiency of nanocomposites

[0127] After the synthesis of the nanocomplex, the nanocomplex was specially centrifuged and washed with distilled water and PBS respectively. The washed products (suspended matter) were detected using a nanoflow cytometer to compare the siRNA encapsulation rates of the two.

[0128] 3. Effect of nanocomplex on the hatching of fertilized eggs

[0129] The nanocomplex was suspended in PBS and used for bathing grouper fertilized eggs, wherein the groups included: Ctr: control group, C1: diluted 10 times the concentration, C2: diluted 1000 times the concentration, and the hatching rate was observed and calculated.

[0130] 4 Results and analysis

[0131] 4.1 Effect of different groups on particle size

[0132] Group A1: Chloroform-PEG:

[0133] After the organic phase chloroform and the external aqueous phase PEG were functionalized and synthesized, the particle size of the secondary ultrasonic product was 156.5nm, and the light intensity distribution was 200nm. Figure 4 .

[0134] Group A2 Chloroform-Tween 80:

[0135] The particle size of the secondary ultrasonic product was 146 nm after functionalization of the organic phase chloroform and the external aqueous phase Tween 80. The light intensity distribution was 200 nm. Figure 5 .

[0136] A3 group DMF-PEG:

[0137] After the organic phase DMF and the external aqueous phase PEG were functionalized and synthesized, the particle size of the secondary ultrasonic product was 189.2nm, and the light intensity distribution was 200nm. Figure 6 .

[0138] A4 group DMF-Tween 80:

[0139] After the organic phase DMF and the external aqueous phase Tween 80 were functionalized and synthesized, the particle size of the secondary ultrasonic product was 79.53nm, and the light intensity distribution showed a double peak. Figure 7 .

[0140] Comparison results: In the particle size and light intensity relationship diagram, A1, A2, and A3 are all single-peaked with the peak concentrated around 200 nm, and A4 is a double peak, indicating that the nanoparticle structure it forms is not single.

[0141] 4.2 Stability of nanoparticles after logarithmic dilution of four formulations

[0142] The prepared nanoparticles of groups A1 to A4 were diluted 2 0 ~2 -9 times, and explored the maximum proportion of the final product that remained stable. The results are shown in Table 3 below.

[0143] Table 3

[0144]

[0145] The results show that by comparing the two reagents chloroform and DMF, the nanostructure formed when chloroform is used as the oil phase is single (single peak) and the nanostructure formed when diluted to 2 -4 When the particle size exceeds 300nm, the goal of this experiment is to form smaller particles so that the nanoparticles can enter the fertilized egg more easily; when DMF is used as the oil phase and diluted to 2 -1When the particle size exceeds 300nm, chloroform is selected as the component of the oil phase. When PS80 is used as the external aqueous phase, dilution 2 -1 The nanoparticle size is more than 300nm, and the nanoparticle structure formed by Tween80 is not uniform (double peak); when PEG is selected as the external aqueous phase, the nanoparticle size is less than 300nm and the structure is uniform, so PEG is selected as the external aqueous phase. Therefore, chloroform-PEG is selected as the best reagent for the organic phase and the external aqueous phase.

[0146] 4.3 Material parameters under optimal ratio

[0147] After obtaining the optimal reagent based on the above results and combining it with the optimal ratio, the volume ratio of the inner aqueous phase, the organic phase solution and the outer aqueous phase solution was 1:5:15. After the operation was modified according to the operation steps of the synthesis of the PLGA-siRNA nanocomplex, the particle size of the secondary ultrasonic product was detected and the zeta potential was measured, and the product after thorough washing and drying was observed using an electron microscope.

[0148] (1) Secondary ultrasonic solution particle size and zeta potential results

[0149] The nanoparticle size of the secondary ultrasonic solution obtained under the optimal reagent and ratio is 109.7nm, and the light intensity distribution is a single peak. Figure 8 In (A), the zeta potential is 30.91 mV.

[0150] (2) Ultrafiltration centrifugal solution particle size and zeta potential results

[0151] The nanoparticle size of the secondary ultrasonic solution ultrafiltration filtrate obtained under the optimal reagent and ratio is 15.78nm, and the light intensity distribution is a single peak. Fig. 9 In (A), the zeta potential is 5.075 mV.

[0152] (3) Electron microscopy observation of the particle size and zeta potential of the membrane washing liquid and the final dried product

[0153] The nanoparticle size of the secondary ultrasonic solution ultrafiltration membrane washing liquid obtained under the optimal reagent and ratio is 106.7nm, and the light intensity distribution is a single peak. Fig.10 The zeta potential of (A) is 32.82 mV. After the filter membrane rinse solution is dried, it is observed under an electron microscope. Fig.11 , obvious spherical particles appear at a scale of 100 nm.

[0154] The effect of washing with distilled water and PBS is compared. Fig.12 and 13As shown in the figure, after washing with PBS, the nanoparticles are more stable and the particle size is more concentrated, around 160nm. This is basically consistent with the structure of the nanoparticle size analyzer and electron microscope. There will be a certain deviation in the particle size measured by different instruments, which is normal.

[0155] (4) Nanomaterial encapsulation efficiency of siRNA

[0156] After the nanocomplex was suspended in pure water and PBS, flow cytometry fluorescence detection was performed. The results are as follows: Fig.14 As shown, it was found that the encapsulation rate of siRNA in PBS was more stable and higher than that in water.

[0157] (5) Effects of nanocomposites on the hatching of grouper fertilized eggs

[0158] The nanocomplex was suspended in PBS as the mother source and diluted to 10 times and 1000 times the final concentration for bathing grouper fertilized eggs for 1-2 hours. Fig.15 As shown in the figure, the hatching rates were 92% and 94% respectively, while the control group was 88%, indicating that the material had no toxic effect on grouper fertilized eggs. The survival rates within a period of time after hatching were 66% and 70% respectively, while the control group was 60%. The results showed that the survival rate of the medicated bath group was higher than that of the experimental group. Based on the above results, the nanocomposite had no toxic effect on grouper fertilized eggs and could improve their survival rate.

[0159] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanocomposite, characterized in that The raw materials are composed of polylactic acid-glycolic acid copolymer, polyethyleneimine and siRNA, wherein: The mass volume ratio of the polylactic acid-glycolic acid copolymer and polyethyleneimine is 25 mg:0.1 mL, and each 100 mL of polyethyleneimine contains 1.25 μmol of siRNA; The molecular weight of the polylactic acid-glycolic acid copolymer is 10000Da, and the molecular weight of the polyethyleneimine is 25000Da; The sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 2; The particle size of the nanocomposite is 160 nm.

2. The method for preparing the nanocomposite according to claim 1, characterized in that: The steps include: Step 1, mixing the siRNA and the polyethyleneimine to form solution A, and mixing the polylactic acid-glycolic acid copolymer and the organic phase solution to form solution B; Step 2, ultrasonically mixing the solution A and the solution B in step 1; Step 3, taking the solution after ultrasonic mixing in step 2 and the external aqueous phase solution and ultrasonically mixing them again; Step 4: Take the solution after ultrasonic mixing again in step 3, filter and centrifuge it to obtain the nanocomposite.

3. The preparation method according to claim 2, characterized in that: In the step 1, the solvent used for mixing the solution A includes water, and the organic phase solution includes chloroform and / or dimethylformamide; The concentration of the polyethyleneimine in the solution A is 5 vol% to 50 vol%, and the concentration of the polylactic acid-glycolic acid copolymer in the solution B is 1 to 10 mg / mL.

4. The preparation method according to claim 3, characterized in that: The organic phase solution is chloroform; The concentration of polyethyleneimine in the solution A is 10 vol %, and the concentration of polylactic acid-glycolic acid copolymer in the solution B is 5 mg / mL.

5. The preparation method according to claim 2, characterized in that: In step 3, the solute of the external aqueous phase solution includes polyethylene glycol and / or Tween 80.

6. The preparation method according to claim 5, characterized in that: The solute of the external aqueous phase solution is polyethylene glycol, and the concentration of the polyethylene glycol in the external aqueous phase solution is 1-20 mg / mL.

7. The preparation method according to claim 2, characterized in that: In step 2 and step 3, the volume ratio of solution A, solution B and the external aqueous phase solution is (0.5-2) (1-10): (1-30).

8. The preparation method according to claim 2, characterized in that: In the step 4, the filtration is ultrafiltration, and after the centrifugation, PBS is used for washing to obtain the nanocomplex.

9. Use of the nanocomposite according to claim 1 or the nanocomposite prepared by the preparation method according to any one of claims 2 to 8 in preparing drugs for resisting neurological necrosis virus and / or improving the survival rate of fish eggs infected with neurological necrosis virus.

10. A drug for resisting neurological necrosis virus and / or improving the survival rate of fish eggs infected with neurological necrosis virus, characterized in that: The invention comprises the nanocomposite according to claim 1 or the nanocomposite prepared by the preparation method according to any one of claims 2 to 8.

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