Nanocomposite for nervous necrosis virus, preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of prevention and control of aquatic animal diseases, and specifically relates to a nanocomposite for a nervous necrosis virus, a preparation method therefor, and the use thereof. Provided in the present invention is a nanocomposite consisting of poly(lactic-co-glycolic acid), polyethyleneimine and a siRNA. Also provided in the present invention are a preparation method for the nanocomposite and the use thereof in the preparation of a drug for improving the survival rate of fish eggs infected with a nervous necrosis virus. The present invention constructs a vector for a capsid protein of the nervous necrosis virus by means of simulating the nervous necrosis virus and expresses same in cells, and screens out a siRNA that can effectively inhibit the expression of the viral capsid protein, thus constructing the nanocomposite for the nervous necrosis virus. The nanocomposite can block NNVs at the embryonic phase (fertilized eggs), thereby improving the survival rate of fish fry, and further opening up new possibilities for the treatment of early stage diseases in fish fry. The method is innovative in the field of artificial breeding of aquatic animals and provides new ideas for virus prevention and control in the field of aquaculture.

Description

Nanocomposites targeting nerve necrosis virus, their preparation methods and applications Technical Field

[0001] This invention relates to the field of aquatic animal disease prevention and control technology, specifically to a nanocomposite targeting nerve necrosis virus, its preparation method, and its application. Background Technology

[0002] Although my country has made significant breakthroughs in the artificial breeding of marine fish, the number of species bred on a large scale is still limited. Furthermore, large-scale mortality of fry occurs in the early stages of artificial breeding, which is one of the main bottlenecks restricting the scale of marine fish farming and stock enhancement. One of the causes of early mass mortality in marine fish is the neuronecrosis virus (NNV), which was discovered in 1989 and first reported in 1990. Since then, over 100 species of marine fish have been reported worldwide to carry NNV, with more than 40 susceptible species, such as European sea bass (Dicentrarchus labrax), Asian sea bass (Lates calcarifer), various groupers (Ephinephelus spp.), yellowtail trevally (Pseudocaranx dentex), Senegal sole (Solea senegalensis), Atlantic flounder (Hippoglossus hippoglossus), Atlantic cod (Gadus morhua), and Pacific cod (Gadus macrocephalus) (Bandin I and Souto S., 2020). NNV mainly causes disease in larvae and juveniles, with a mortality rate of up to 100%. Currently, there are no effective methods, either domestically or internationally, to block NNV, which has been a bottleneck hindering marine fish breeding.

[0003] Studies have found that one of the main routes of NNV transmission is vertical transmission from parent to offspring. Both eggs and sperm can carry the virus. Before hatching, the embryo is protected by the egg membrane, making it an ideal time to block NNV, and this period is also convenient for manipulation. However, hatched larvae and juveniles are less resistant to disease in the water and are not suitable for medication. Therefore, blocking NNV from the embryonic stage can effectively avoid its impact on larvae and juveniles. Many drugs exist for blocking viruses, such as vaccines, small interfering RNA (siRNA), and interferons. Among these, RNA interference technology (RNAi) has received considerable attention for its application in the treatment of viral infectious diseases (Zhu et al., 2006). However, there is currently no effective method to deliver these drugs to fish embryos, necessitating the development of an effective and feasible drug delivery vector.

[0004] With the continuous innovation of new technologies in the medical field, delivery systems based on various materials such as proteins, liposomes, polymers, and inorganic materials have been applied to the research of high-efficiency siRNA transport and have made significant progress in tumor targeted diagnosis and treatment. However, there is relatively little research on siRNA nanodelivery technology in the aquaculture field. 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 nerve necrosis virus, its preparation method and application. The nanocomposite provided by the present invention can effectively improve the survival rate of fish eggs and can be effectively applied in the field of aquaculture.

[0006] This invention provides a nanocomposite whose raw materials are composed of polylactic acid-glycolic acid copolymer, polyethyleneimine, and siRNA, wherein:

[0007] The mass-to-volume ratio of the polylactic acid-glycolic acid copolymer to the 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 following: 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; a sense strand of the nucleotide sequence shown in SEQ ID NO:3 and an antisense strand of the nucleotide sequence shown in SEQ ID NO:4; and a sense strand of the nucleotide sequence shown in SEQ ID NO:5 and an 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 existing technologies, the polylactic acid-glycolic acid copolymer in the nanocomposite provided by this invention has good biocompatibility, is non-toxic, and has good encapsulation and film-forming properties. Its degradation products are lactic acid and glycolic acid, which are also byproducts of human metabolism. Therefore, it will not have toxic side effects when used in medicine and biomaterials. The siRNA can effectively inhibit neuronecrosis virus, and the polyethyleneimine can better form a complex with the siRNA, further increasing the loading capacity of the polylactic acid-glycolic acid copolymer. The components are closely coordinated, and the successfully constructed neuronecrosis virus nanocomposite can effectively enter the fish egg and block NNV during the embryonic stage, thereby improving the survival rate of fish fry and achieving more accurate technical effects.

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

[0012] The polylactic acid-glycolic acid copolymer has a molecular weight of 10,000 Da, and the polyethyleneimine has a molecular weight of 25,000 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 using the components in the above embodiments has the best loading capacity and the best particle size, resulting in the best effect when delivered into fish eggs, thus achieving the most accurate technical results.

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

[0017] Step 1: Mix the siRNA with the polyethyleneimine to form solution A, and mix the polylactic acid-glycolic acid copolymer with the organic phase solution to form solution B;

[0018] Step 2: Take solution A from step 1 and solution B and mix them using ultrasound;

[0019] Step 3: Take the ultrasonically mixed solution from Step 2 and ultrasonically mix it again with the external aqueous phase solution;

[0020] Step 4: Take the solution after ultrasonic mixing in step 3, filter and centrifuge to obtain the nanocomposite.

[0021] In some embodiments, in step 1, the solvent used for mixing solution A includes water.

[0022] Preferably, the concentration of polyethyleneimine in solution A is 5 vol% to 50 vol%.

[0023] More preferably, the concentration of polyethyleneimine in solution A is 10 vol%.

[0024] In some embodiments, in step 1, the organic phase solution includes chloroform and / or dimethylformamide.

[0025] Preferably, the organic phase solution is chloroform.

[0026] Preferably, in solution B, the concentration of the polylactic acid-glycolic acid copolymer is 1 to 10 mg / mL.

[0027] In some embodiments, the concentration of the polylactic acid-glycolic acid copolymer can 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 solution B, the concentration of the polylactic acid-glycolic acid copolymer is 5 mg / mL.

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

[0030] Preferably, the solute in the external aqueous phase solution is polyethylene glycol.

[0031] Preferably, the concentration of polyethylene glycol in the external aqueous phase solution 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 steps 2 and 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 solution A, solution B and the external aqueous phase solution is 1:5:15.

[0035] In some embodiments, in step 4, the filtration is performed using ultrafiltration, and the centrifugation is followed by washing with PBS to obtain the nanocomposite.

[0036] This invention provides the application of the nanocomposite described herein or the nanocomposite prepared by the method described herein in the preparation of drugs for treating neuronecrosis virus and / or improving the survival rate of fish eggs infected with neuronecrosis virus.

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

[0038] This invention provides a method for improving the survival rate of fish eggs infected with nerve necrosis virus, including bathing the fish eggs with the drug.

[0039] This invention provides a nanocomposite composed of polylactic acid-glycolic acid copolymer, polyethyleneimine, and siRNA. It also provides a method for preparing the nanocomposite and its application in the preparation of drugs that inhibit neuronecrosis virus (NNV) and / or improve the survival rate of fish eggs infected with NNV. This invention simulates NNV by constructing a vector for the NNV capsid protein and expressing it in cells; screening for siRNA that can effectively inhibit CP expression; and then constructing a nanocomposite for NNV to block NNV during the embryonic stage, thereby improving the survival rate of fish fry and opening up new possibilities for treating early-stage diseases in fish fry. This method is innovative in the field of artificial breeding of aquatic animals and provides new ideas for aquaculture. Attached Figure Description

[0040] [Corrected according to Rule 91, 24.09.2025] Figures 1-16 show EPC cells co-transfected with siRNA and pEGFP-CP at different transfection times (×100). Figure 1 shows 24h pEGFP-CP, Figure 2 shows 24h pEGFP-CP+NNV-18, Figure 3 shows 24h pEGFP-CP+NNV-144, Figure 4 shows 24h pEGFP-CP+NNV-720, Figure 5 shows 48h pEGFP-CP, Figure 6 shows 48h pEGFP-CP+NNV-18, Figure 7 shows 48h pEGFP-CP+NNV-144, Figure 8 shows 48h pEGFP-CP+NNV-720, Figure 9 shows 72h pEGFP-CP, Figure 10 shows 72h pEGFP-CP+NNV-18, and Figure 11 shows 72h pEGFP-CP+NNV-18. Figure 12 shows pEGFP-CP+NNV-144, Figure 13 shows 72h pEGFP-CP+NNV-720, Figure 14 shows 24h hpEGFP-empty vector, Figure 15 shows 48h hpEGFP-empty vector, Figure 16 shows 72h hpEGFP-empty vector, and Figure 17 shows 48h siRNA-Cy3.

[0041] [Corrected according to Rule 91 24.09.2025] Figure 17 shows the cell viability of EPC in different control groups. The asterisk (*) indicates that the difference between the group and the control group is significant (P<0.05), the ** indicates that the difference is extremely significant (P<0.01), and ns indicates that there is no significant difference.

[0042] [Corrected according to Rule 91 24.09.2025] Figure 18 shows the cell viability of EPC after co-transfection with siRNA and pEGFP-CP. The asterisk (*) indicates that the difference between this group and the control group is significant (P<0.05), and the ** indicates that the difference is extremely significant (P<0.01).

[0043] [Correction 24.09.2025 according to Rule 91] Figure 19 shows the electron microscopy observation results of the nanocomposite in the filter membrane after secondary ultrasonication and ultrafiltration of the optimal ratio group in Example 2.

[0044] [Correction 24.09.2025 according to Rule 91] Figure 20 shows the fluorescence detection results of the nanocomposite in the filter membrane after secondary ultrasonication and ultrafiltration of the optimal ratio group in Example 2.

[0045] [Correction 24.09.2025 according to Rule 91] Figure 21 shows the effect of the nanocomposite in the filter membrane after secondary ultrasound and ultrafiltration of the optimal ratio group in Example 2 on grouper eggs, where Ctr is the control group; C1 is the concentration diluted 10 times; and C2 is the concentration diluted 1000 times.

[0046] [Corrected according to Rule 91, September 24, 2025] This invention provides nanocomposites targeting neuronecrosis viruses, their preparation methods, and applications. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] [Corrected according to Rule 91, 24.09.2025] It should be noted that, in this document, relational terms such as "first" and "second" are used only 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. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

[0048] [Corrected according to Article 91, September 24, 2025] Preliminary experiments have verified that, compared with other nanomaterials, the nanocomposite selected in this invention has the highest compatibility with fish fertilized eggs. Its particle size is suitable, preventing blockage of egg pores and channels, thus ensuring normal material exchange; it is chemically stable, does not release toxic substances, and reduces interference with the internal biochemical processes of the fish eggs; it is less likely to induce oxidative stress, reducing the production of reactive oxygen species and protecting intracellular biomolecules; it has no endocrine interference effect, maintaining normal hormone signal transduction and developmental processes in the fish eggs; and it has minimal impact on gene expression in the fish eggs, ensuring normal expression of development-related genes and promoting normal development and differentiation. Therefore, this invention uses polylactic acid-glycolic acid copolymer and polyethyleneimine-loaded siRNA to construct the nanocomposite. Preliminary experimental results were unsatisfactory, therefore, they are not included in this application.

[0049] [Corrected according to Article 91 24.09.2025] When siRNA and nanomaterials polylactic acid-glycolic acid copolymer and polyethyleneimine are used in the proportions selected in this invention to form nanocomposites, the components are closely matched, the loading is optimal, and the successfully constructed nanocomposites have the best particle size, which is more conducive to entering the fish eggs. They can block NNV during the embryonic stage, thereby improving the survival rate of fish fry.

[0050] [Revised according to Article 91, September 24, 2025] All test materials used in this invention are common commercially available products. The invention will be further described below with reference to embodiments.

[0051] [Corrected according to Rule 91, September 24, 2025] Example 1: Screening of siRNA interference sequences

[0052] [Corrected according to detailed rules 91, 24.09.2025] 1. Materials and Methods

[0053] [Corrected according to Rule 91 24.09.2025] (1) Simulation of virus synthesis

[0054] [Correction based on Rule 91 24.09.2025] RGNNV has a simple structure with only one structural protein, namely the capsid protein (CP). We inserted the gene encoding CP into the cell transfection vector pEGFP to lay the foundation for subsequent expression of the virus in cells. Detailed Implementation

[0055] [Corrected according to Rule 91 24.09.2025] (2) Design of siRNA interference sequences

[0056] [Correction based on Rule 91, September 24, 2025] To achieve effective silencing of the NNV CP gene, siRNA sequences were designed using online tools provided by Ambion (http: / / www.ambion.com) based on the existing neural necrosis virus (NNV) CP gene sequence in the laboratory. During the design process, it was ensured that the selected siRNA sequence was completely complementary to the target CP gene mRNA to achieve specific gene silencing. Online BLAST was used to ensure that the selected sequence had no homology with other genes besides this virus. Simultaneously, to verify the specificity of the siRNA, a negative control (siRNA-Cy3, siRNA labeled with the red fluorescent dye Cy3) was also designed in this experiment. All sequences were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0057] [Corrected according to Rule 91, 24.09.2025] Table 1 siRNA sequences

[0058] [Correction based on Rule 91, 24.09.2025] Note: Since the base U is not available among the characters that can be used in the nucleotides in the sequence listing, the base U in the RNA sequence in Table 1 above was changed to the base T when the sequence listing was created.

[0059] [Corrected according to Rule 91 24.09.2025] (3) Inhibitory effect of siRNA on intracellular CP

[0060] [Corrected according to Rule 91, 24.09.2025] Plasmid extraction via culture (endotoxin-free plasmid mini-prep kit):

[0061] [Corrected according to Rule 91 24.09.2025] (1) Prepare LB liquid culture medium according to the ratio, take out the Escherichia coli bacterial solution frozen at -20℃, thaw it, and then take 200μL into the culture medium. After sealing and labeling, put it into a shaker and set the temperature to 37℃ and the rotation speed to 200rpm.

[0062] [Correction based on Rule 91 24.09.2025] (2) Centrifuge the cultured bacteria (usually at 4°C, about 4000-6000g, for 5-10 minutes). Discard the supernatant and retain the bacterial precipitate.

[0063] [Correction 24.09.2025 according to Rule 91] (3) Add the lysis buffer provided by the kit to the bacterial pellet, mix thoroughly to lyse the bacteria, and let stand at room temperature for a period of time to promote lysis.

[0064] [Correction 24.09.2025 according to Rule 91] (4) Add an equal volume of neutralization buffer, mix well and let stand at room temperature to neutralize the active ingredients in the lysis buffer.

[0065] [Correction 24.09.2025 according to Rule 91] (5) The mixture in the centrifuge tube is usually centrifuged at 4°C, at about 12,000-14,000 g for 10-15 min. Discard the supernatant and retain the precipitate containing plasmid DNA.

[0066] [Correction 24.09.2025 according to Rule 91] (6) Add washing buffer to the precipitate, mix well, and centrifuge to remove the washing buffer. Repeat the washing step at least once to ensure that all impurities such as proteins and RNA are removed.

[0067] [Correction 24.09.2025 according to Rule 91] (7) Add a certain volume of dissolving buffer to the final precipitate, usually sterile water or TE buffer. After mixing, place the tube at room temperature or 37°C to incubate and fully dissolve the plasmid DNA.

[0068] [Correction 24.09.2025 according to Rule 91] (8) Transfer the dissolved plasmid DNA to a new sterile tube and store it at -20°C or -80°C for use in subsequent experiments.

[0069] [Corrected according to Rule 91, 24.09.2025] Pre-culture of EPC cells:

[0070] [Correction 24.09.2025 according to Rule 91] (1) The activated EPC cells were cultured in M199 medium containing 20% ​​fetal bovine serum, and the growth status of the cells was judged based on the cell adhesion.

[0071] [Corrected according to Rule 91 24.09.2025] (2) After the cells have covered the bottom of the culture flask, remove the waste liquid in the clean bench, add trypsin for digestion, gently tap the flask to disperse the cells, and then use M199 medium containing 5% fetal bovine serum to blow and aspirate the flask wall, and decide whether to passage according to the cell density.

[0072] [Corrected according to Rule 91 24.09.2025] (3) During subculturing, after digestion, aspirate half of the liquid and transfer it to a new culture flask. Observe the cell growth status every 2 to 3 days and change the culture medium to ensure sufficient nutrients to promote cell growth.

[0073] [Corrected according to detailed rule 91, September 24, 2025] Experimental group design:

[0074] [Corrected according to Rule 91 24.09.2025] (1) (Blank) Control group 1: Cells without any treatment.

[0075] [Correction 24.09.2025 according to Rule 91] (Negative) Control group 2: Transfected only with pEGFP-empty vector plasmid, which is a blank vector plasmid with green fluorescence.

[0076] [Correction based on Rule 91 24.09.2025] (Positive) Control group 3: Only transfected with pEGFP-CP, which is a plasmid carrying the CP pathogenic gene and green fluorescence.

[0077] [Correction based on Rule 91 24.09.2025] (Negative) Control group 4: only transfected with siRNA-Cy3, which is siRNA labeled with the red fluorescent dye Cy3 and has no homology with the CP sequence.

[0078] [Corrected according to Rule 91 24.09.2025] Experimental group 1: pEGFP-CP and NNV-18 co-transfected.

[0079] [Corrected according to Rule 91 24.09.2025] Experimental group 2: pEGFP-CP and NNV-144 co-transfected.

[0080] [Corrected according to Rule 91 24.09.2025] Experimental group 3: pEGFP-CP and NNV-720 co-transfected.

[0081] [Corrected according to Rule 91, 24.09.2025] Cell transfection experiment:

[0082] [Corrected according to Rule 91, 24.09.2025] Taking a 24-well plate as an example, 0.5–2 × 10⁻⁶ mg / L of the virus should be inoculated into each well 18–24 hours before the transfection experiment. 5 One cell was added and cultured in 0.5 mL of cell culture medium.

[0083] [Correction 24.09.2025 based on Rule 91] The number of cells selected for initial seeding should be sufficient to achieve a cell density of 70% to 90% at transfection, and care should be taken to ensure that the cells are not overgrown or in a resting phase at transfection.

[0084] [Correction based on Rule 91 24.09.2025] Take 25 μL of Opti-MEM medium and add it into two 1.5 mL sterile EP tubes, and label them as No. 1 and No. 2 respectively.

[0085] [Corrected according to Rule 91 24.09.2025] Place the Lipofectamin 3000 transfection reagent at room temperature and mix gently before use; add 0.75 μL of Lipofectamin 3000 transfection reagent to tube 1 containing serum-free medium, mix by pipetting, and let stand at room temperature for 5 min.

[0086] [Corrected according to Rule 91, 24.09.2025] Add 1 μg of NNV plasmid / empty vector (calculate the required volume based on the desired amount) to tube 2, gently mix by pipetting, and then add 1 μL of P3000. TM Mix the reagent mixture gently by pipetting. Add 0.625 μL of the siRNA transfection solution, without adding P3000. TM Reagent.

[0087] [Correction 24.09.2025 according to Rule 91] Add the transfection complex to the Lipofectamin 3000 mixture, that is, add 25 μL of the mixture in tube 2 to tube 1, mix by pipetting, and let stand at room temperature for 10-15 min to allow the DNA / siRNA to form a transfection complex with the Lipofectamin 3000 transfection reagent.

[0088] [Corrected according to Rule 91, 24.09.2025] Add 50 μL of the complex to a culture plate containing cells (for co-transfection, add 100 μL of plasmid complex and siRNA complex separately), and gently shake to disperse the cells evenly. Set up 3 wells for each group.

[0089] [Corrected according to Rule 91 24.09.2025] The culture plates were placed in a cell culture incubator at 25°C and cultured for 24h, 48h, and 72h respectively.

[0090] [Corrected according to Rule 91, 24.09.2025] Cell observation and counting:

[0091] [Corrected according to Rule 91, 24.09.2025] Cell growth in culture plates was observed under an inverted microscope at different transfection times, and fluorescence was recorded by photographing under a fluorescence microscope. The microscope was set to 100x magnification (10x objective lens × 10x eyepiece), and 5 to 8 fields of view were observed in each well to ensure a comprehensive evaluation of the transfection effect.

[0092] [Correction 24.09.2025 according to Rule 91] Collect the supernatant from the wells of the culture plate into a new sterile EP tube and wash each well of the culture plate with PBS.

[0093] [Corrected according to Rule 91 24.09.2025] Add 200 μL of trypsin to each well, gently tap to digest for 2 min, add 300 μL of culture medium, mix by pipetting, add to an EP tube containing supernatant, add 500 μL of phenol blue solution, and mix by pipetting.

[0094] [Correction 24.09.2025 according to Rule 91] Draw 25 μL of cell fluid from the tube and add it to the counting chamber, and count the cells using a cell counter.

[0095] [Corrected according to detailed rule 91, September 24, 2025] Data analysis:

[0096] [Corrected according to Rule 91, 24.09.2025] GraphPad Prism v.8.0.2 was used to analyze and plot the significance of differences between groups. All trials were set to three replicates, and two-way ANOVA analysis was used for data comparison. *: P < 0.05; **: P < 0.01; ns: no significant difference.

[0097] [Corrected according to detailed rule 91, September 24, 2025] 2. Results and Analysis

[0098] [Corrected according to detailed rule 91, 24.09.2025] 2.1 Fluorescence observation of each group

[0099] [Corrected according to Rule 91, September 24, 2025] At different transfection time points (24h, 48h, and 72h), the positive control group transfected only with pEGFP-CP (Figures 1, 5, and 9) showed a higher amount of green fluorescence, with the highest fluorescence count at 48h, and the successfully transfected cells were round. The experimental groups co-transfected with pEGFP-CP and siRNA showed less green fluorescence signal, and all were less than the positive control group at the corresponding time points. Particularly at the 48h transfection time point, the green fluorescence signal in the pEGFP-CP and siRNA co-transfected group was significantly reduced (Figures 2, 3, 4, 6, 7, 8, 10, 11, and 12).

[0100] [Correction based on Rule 91, 24.09.2025] In addition, the amount of green fluorescence in the pEGFP-empty vector group gradually increased over time, and the cells appeared as irregular green shapes under a microscope (Figures 13, 14 and 15). Red fluorescence could also be observed in siRNA-Cy3 (as shown in Figure 16).

[0101] [Corrected according to detailed rule 91, 24.09.2025] 3.2 EPC Counting Status

[0102] [Corrected according to Rule 91, September 24, 2025] The cell viability of EPC cells in different control groups is shown in Figure 17. The cell viability of the pEGFP-empty vector group and the siRNA-Cy3 group at 48 h were 84.55%, 71.69%, and 74.69%, respectively. At 72 h, the viability 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 showed a highly significant difference compared to the cell group at 48 h and a significant difference at 72 h. The cell viability of EPC cells after co-transfection with siRNA and pEGFP-CP is shown in Figure 18. After co-transfection with NNV-18, NNV-144, and NNV-720 and pEGFP-CP for 48 h and 72 h, the cell viability of EPC cells significantly increased, indicating that these three pairs of siRNAs all inhibited CP expression and had relatively low cytotoxicity. With a transfection time of 48 h, the cell viability of the pEGFP-CP group was approximately 27%, while the cell viability of the pEGFP-CP+NNV-18, pEGFP-CP+NNV-144, and pEGFP-CP+NNV-720 groups were 61%, 59%, and 62%, respectively. The differences from the pEGFP-CP control group were extremely significant (P<0.01), while there were no significant differences among the three groups. At a transfection time of 72 h, the cell viability 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, all of which were increased compared to 48 h. Among them, the pEGFP-CP+NNV-18 group was significantly different from the pEGFP-CP control group (P<0.01), and the pEGFP-CP+NNV-144 and pEGFP-CP+NNV-720 groups were significantly different from the pEGFP-CP control group (P<0.05). However, there were no significant differences among the three groups.

[0103] [Corrected according to Rule 91, 24.09.2025] The above results show that the simulated virus can cause a high lethality rate to cells, while the cell survival rate 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 showed the best effect and was used for the subsequent preparation of nanocomposites.

[0104] [Corrected according to Rule 91, September 24, 2025] Example 2: Preparation of PLGA-siRNA Nanocomposite

[0105] [Corrected according to Rule 91, September 24, 2025] 1. Synthesis of PLGA-siRNA nanocomposite

[0106] [Corrected according to Rule 91 24.09.2025] (1) Organic phase: PLGA with a molecular weight of 10000 was selected. 5 mg of PLGA was dissolved in different volumes of organic solvent (chloroform or DMF) and the optimal ratio was screened to form an organic phase.

[0107] [Corrected according to Rule 91 24.09.2025] (2) Inner aqueous phase: PEI (polyethyleneimine) with a molecular weight of 25000 was selected. PEI is dissolved in water, and the optimal ratio was screened. siRNA was dissolved in PEI aqueous solution at 4℃ to form an inner aqueous phase.

[0108] [Corrected according to Rule 91 24.09.2025] (3) External aqueous phase: Dissolve PEG or Tween80 in water, select the optimal ratio, and form an external aqueous phase;

[0109] [Corrected according to Rule 91 24.09.2025] (4) Mix the organic phase and the internal aqueous phase in proportion and perform the first ultrasonic mixing using an ultrasonic instrument.

[0110] [Corrected according to Rule 91 24.09.2025] (5) Add the sonicated solution to the prepared external aqueous phase in proportion, and sonicate a second time.

[0111] [Corrected according to Rule 91 24.09.2025] (6) After processing, take a certain amount and centrifuge to obtain the supernatant. Add the supernatant to an ultrafiltration tube and centrifuge. The spherical complex encapsulating siRNA is filtered onto the filter membrane. Then, use PBS or water to suspend the nanocomplex on the filter membrane in PBS solution or aqueous solution and store it at low temperature in the dark.

[0112] [Correction 24.09.2025 according to Rule 91] (7) Use a nano-laser particle size analyzer to detect the particle size of the secondary ultrasonic product and measure the zeta potential, and use an electron microscope to observe the final product.

[0113] [Corrected according to Rule 91, 24.09.2025] In the above steps, the PEI concentration, PLGA concentration, the volume ratio of polyethyleneimine to siRNA, the organic phase solution, the main solute and its concentration in the outer aqueous phase, and the volume ratio of the inner aqueous phase, organic phase solution, and outer aqueous phase solution are prepared according to the groups in Table 2. After modification according to the above synthesis, the particle size of the secondary ultrasonic product is detected. The optimal reagent is determined by comparing the particle size distribution and dilution factor. For the dilution factor test, 2 mL of the PLGA-siRNA nanocomposite prepared in groups A1 to A4 of Table 2 is taken and logarithmically diluted with deionized water. The optimal prepared product is obtained by maintaining the maximum stable ratio of the final product.

[0114] [Corrected according to detailed rule 91, September 24, 2025] Table 2: Different Grouping Situations

[0115] [Corrected according to Rule 91, 24.09.2025] 2. Encapsulation efficiency of nanocomposites

[0116] [Correction based on Rule 91, 24.09.2025] After synthesis, the nanocomposite was centrifuged in a special way and then washed with distilled water and PBS respectively. The washing products (suspended matter) were detected by nanoflow cytometer to compare the encapsulation rate of siRNA in the two methods.

[0117] [Corrected according to Rule 91 24.09.2025] 3. Effect of nanocomposite on hatching of fertilized eggs: The nanocomposite was suspended in PBS and the fertilized grouper eggs were given a bath. The groups included: Ctr: control group, C1: 10-fold dilution, C2: 1000-fold dilution. The hatching rate was observed and calculated.

[0118] [Corrected according to detailed rule 91, 24.09.2025] 4. Results and Analysis

[0119] [Corrected according to detailed rule 91, 24.09.2025] 4.1 Effect of different groupings on particle size

[0120] [Corrected according to Rule 91, 24.09.2025] Group A1 chloroform-PEG:

[0121] [Correction based on Rule 91, 24.09.2025] After functionalizing the organic phase chloroform with the external aqueous phase PEG, the particle size of the secondary ultrasonic product was found to be 156.5 nm, and the light intensity distribution was 200 nm.

[0122] [Corrected according to Rule 91, 24.09.2025] Group A2 Chloroform-Tween 80:

[0123] [Correction based on Rule 91, 24.09.2025] After functionalizing the organic phase chloroform with the external aqueous phase Tween 80, the particle size of the secondary ultrasonic product was found to be 146 nm, and the light intensity distribution was 200 nm.

[0124] [Corrected according to Rule 91, 24.09.2025] Group A3 DMF-PEG:

[0125] [Correction 24.09.2025 according to Rule 91] After functionalizing the organic phase DMF with the external aqueous phase PEG, the particle size of the secondary ultrasonic product was found to be 189.2 nm, and the light intensity distribution was 200 nm.

[0126] [Corrected according to Rule 91, 24.09.2025] Group A4 DMF-Tween 80:

[0127] [Correction based on Rule 91, 24.09.2025] After functionalizing the organic phase DMF with the external aqueous phase Tween 80, the particle size of the secondary ultrasonic product was found to be 79.53 nm, and the light intensity distribution showed a double peak.

[0128] [Corrected according to Rule 91 24.09.2025] Comparison results: In the relationship between particle size and light intensity, A1, A2, and A3 are all single peaks with peak values ​​concentrated around 200nm, while A4 is a double peak, indicating that the nanoparticle structure formed is not simple.

[0129] [Corrected according to Rule 91, 24.09.2025] 4.2 Stability of nanoparticles after logarithmic dilution of four formulations

[0130] [Corrected according to Rule 91, 24.09.2025] The prepared A1-A4 group nanoparticles were diluted by 2... 0 ~2 9 The ratio was increased by 10 times, and the maximum proportion of the final product that remained stable was investigated. The results are shown in Table 3 below.

[0131] [Corrected according to Detailed Rules 91, 24.09.2025] Table 3

[0132] [Corrected according to Rule 91, 24.09.2025] Results show that, by comparing chloroform and DMF, the nanostructures formed when using chloroform as the oil phase are single (unimodal), and when diluted to 2... -4 When the particle size exceeds 300 nm, the goal of this experiment is to form smaller particles so that the nanoparticles can more easily enter the fertilized egg; and when DMF is used as the oil phase and diluted to 2... -1 Since the particle size exceeds 300 nm, chloroform was chosen as the component of the oil phase. When using PS80 as the external aqueous phase, dilution 2... -1The nanoparticle size exceeds 300 nm, and the nanoparticle structure formed using Tween 80 is non-uniform (bimodal). However, when PEG is chosen as the external aqueous phase, the resulting nanoparticles have a size less than 300 nm and a uniform structure. Therefore, PEG was chosen as the external aqueous phase. Thus, chloroform-PEG was selected as the optimal reagent for both the organic and external aqueous phases.

[0133] [Corrected according to detailed rule 91, 24.09.2025] 4.3 Material parameters under optimal proportions

[0134] [Corrected according to Rule 91 24.09.2025] After obtaining the optimal reagents based on the above results and combining them with the optimal ratio, the volume ratio of the inner aqueous phase, organic phase solution and the outer aqueous phase solution is 1:5:15. After modifying the operation according to the operation steps of PLGA-siRNA nanocomposite synthesis, the particle size of the secondary ultrasonic product is detected and the zeta potential is measured. The product after thorough washing and drying is observed using an electron microscope.

[0135] [Corrected according to Rule 91 24.09.2025] (1) Results of the particle size and zeta potential of the secondary ultrasonic solution: The nanoparticle size of the secondary ultrasonic solution obtained under the optimal reagent and ratio is 109.7 nm, the light intensity distribution is a single peak, and the zeta potential is 30.91 mV.

[0136] [Corrected according to Rule 91 24.09.2025] (2) Results of ultrafiltration centrifugation solution particle size and zeta potential: The nanoparticle size of the ultrafiltration filtrate obtained under the optimal reagent and ratio was 15.78 nm, the light intensity distribution was a single peak, and the zeta potential was 5.075 mV.

[0137] [Corrected according to Rule 91 24.09.2025] (3) Electron microscopic observation of filter membrane washing solution particle size, zeta potential and final dried product: The nanoparticle size of the ultrafiltration filter membrane washing solution obtained under the optimal reagent and ratio was 106.7 nm, the light intensity distribution was single peak, and the zeta potential was 32.82 mV. After drying, the filter membrane washing solution was observed under an electron microscope, as shown in Figure 19. Obvious spherical particles appeared under a 100 nm scale bar. The washing effect of distilled water and PBS was compared. The results showed that the nanoparticles were more stable and the particle size was more concentrated after washing with PBS, around 160 nm. This is basically consistent with the structure of the nanoparticle size analyzer and electron microscope. Different instruments will have certain deviations in measuring particle size, which is normal.

[0138] [Corrected according to Rule 91 24.09.2025] (4) The encapsulation rate of siRNA by nanomaterials was detected by flow cytometry after the nanocomposite was suspended in pure water and PBS. The results are shown in Figure 20. It was found that the encapsulation rate of siRNA in PBS was more stable and higher than that in water.

[0139] [Corrected according to Rule 91, 24.09.2025] (5) Effect of nanocomposite on the hatching of grouper fertilized eggs: The nanocomposite was suspended in PBS and used as the maternal source. It was diluted 10 times and 1000 times to a final concentration and then used to bathe grouper fertilized eggs. The bathing time was 1-2 hours. The results are shown in Figure 21. 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 after hatching were 66% and 70%, respectively, while the control group was 60%. The results showed that the survival rate of the bathing group was higher than that of the experimental group. Based on the above results, the nanocomposite has no toxic effect on grouper fertilized eggs and can improve their survival rate.

[0140] [Corrected according to Rule 91, 24.09.2025] The above are merely preferred embodiments of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0141] [Corrected according to detailed rule 91 on 24.09.2025]

[0142] [Corrected according to detailed rule 91 on 24.09.2025]

[0143] [Corrected according to detailed rule 91 on 24.09.2025]

[0144] [Corrected according to detailed rule 91 on 24.09.2025]

[0145] [Corrected according to detailed rule 91 on 24.09.2025]

[0146] [Corrected according to detailed rule 91 on 24.09.2025]

[0147] [Corrected according to detailed rule 91 on 24.09.2025]

[0148] [Corrected according to detailed rule 91 on 24.09.2025]

Claims

1. Nanocomposite, characterized in that, Its raw materials consist of polylactic acid-glycolic acid copolymer, polyethyleneimine, and siRNA, wherein: The mass-to-volume ratio of the polylactic acid-glycolic acid copolymer to the polyethyleneimine is (5-50) mg:(0.05-1) mL, and each 100 mL of the polyethyleneimine contains 0.5-2.5 μmol of siRNA; The siRNA comprises at least one of the following: 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; a sense strand of the nucleotide sequence shown in SEQ ID NO:3 and an antisense strand of the nucleotide sequence shown in SEQ ID NO:4; and a sense strand of the nucleotide sequence shown in SEQ ID NO:5 and an antisense strand of the nucleotide sequence shown in SEQ ID NO:6; the nanocomposite has a particle size of 100–200 nm.

2. The nanocomposite of claim 1, wherein, The mass-to-volume ratio of the polylactic acid-glycolic acid copolymer to polyethyleneimine is 25 mg. 0.1 mL, each 100 mL of polyethyleneimine contains 1.25 μmol of siRNA, wherein: the molecular weight of the polylactic acid-glycolic acid copolymer is 10000 Da, and the molecular weight of the polyethyleneimine is 25000 Da.

3. The nanocomposite of claim 1, wherein, 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; the nanocomposite has a particle size of 160 nm.

4. Process for the preparation of the nanocomposite according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step 1: Mix the siRNA with the polyethyleneimine to form solution A, and mix the polylactic acid-glycolic acid copolymer with the organic phase solution to form solution B; Step 2: Take solution A from step 1 and solution B and mix them using ultrasound; Step 3: Take the ultrasonically mixed solution from Step 2 and ultrasonically mix it again with the external aqueous phase solution; Step 4: Take the solution after ultrasonic mixing in step 3, filter and centrifuge to obtain the nanocomposite.

5. The preparation method according to claim 4, characterized in that, In step 1, the solvent used for mixing solution A includes water, and the organic phase solution includes chloroform and / or dimethylformamide; The concentration of polyethyleneimine in solution A is 5 vol% to 50 vol%, and the concentration of polylactic acid-glycolic acid copolymer in solution B is 1 to 10 mg / mL.

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

7. The preparation method according to claim 4, characterized in that, In step 3, the solute in the external aqueous phase solution includes polyethylene glycol and / or Tween 80.

8. The preparation method according to claim 4, characterized in that, The solute in the external aqueous phase solution is polyethylene glycol.

9. The preparation method according to claim 4, characterized in that, The concentration of polyethylene glycol in the external aqueous phase solution is 1–20 mg / mL.

10. The method of claim 4, wherein, The concentration of polyethylene glycol in the external aqueous phase solution is 10 mg / mL.

11. The preparation method according to claim 4, characterized in that, In steps 2 and 3, the volume ratio of solution A, solution B, and the external aqueous phase solution is (0.5-2)(1-10):(1-30).

12. The method of claim 4, wherein, In steps 2 and 3, the volume ratio of solution A, solution B, and the external aqueous phase solution is 1:5:

15.

13. The preparation method according to claim 4, characterized in that, In step 4, the filtration is performed using ultrafiltration, and the centrifugation is followed by washing with PBS to obtain the nanocomposite.

14. The use of the nanocomposite according to any one of claims 1 to 3 or the nanocomposite prepared by the preparation method according to any one of claims 4 to 13 in the preparation of drugs for anti-neural necrosis virus and / or improving the survival rate of fish eggs infected with neuronecrosis virus.

15. A medicament against neuroparalytic viruses and / or to increase the survival rate of fish eggs infected with neuroparalytic viruses, characterized in that it comprises a compound of formula (I) as defined in any one of claims 1 to 14. The nanocomposite includes the nanocomposite according to any one of claims 1 to 3 or the nanocomposite prepared by the preparation method according to any one of claims 4 to 13.

16. A method of increasing the survival rate of eggs infected with a nervous necrosis virus, characterized by, This includes using the drug described in claim 15 to bathe fish eggs.

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