SiRNA delivery system based on mPEG-PEI-c-SWCNTs as well as preparation method and application of siRNA delivery system

Abstract

The invention provides a siRNA delivery system based on mPEG-PEI-c-SWCNTs and a preparation method and application of the siRNA delivery system, carboxylated single-walled carbon nanotubes c-SWCNTs and polyethylene glycol grafted polyethyleneimine mPEG-PEI are combined through non-covalent modification to obtain mPEG-PEI-c-SWCNTs, siRNA is safely and effectively delivered to fertilized eggs of litopenaeus vannamei by optimizing the loading proportion of siRNA, silencing of a target gene is achieved, and the siRNA delivery system has the advantages that the siRNA delivery efficiency is improved, and the siRNA delivery efficiency is improved. The method has the advantages of high transfection efficiency and good safety, and is helpful for promoting the application of gene therapy means in the aspects of improving the disease resistance of the litopenaeus vannamei and cultivating healthy seed shrimps. Moreover, the preparation process of the siRNA delivery system based on mPEG-PEI-c-SWCNTs is mild, economical and time-saving, and the siRNA delivery system can be stably dispersed in seawater and is suitable for basic application of marine organisms.

Description

Technical Field

[0001] The invention belongs to the technical field of RNA interference, and relates to a siRNA delivery system based on mPEG-PEI-c-SWCNTs, and a preparation method and application thereof. Background Art

[0002] Litopenaeus vannamei, also known as Pacific white shrimp, has become one of the largest aquaculture species in the world due to its fast growth rate, high survival rate and strong adaptability. With the expansion of aquaculture scale and the extension of aquaculture years, diseases caused by various viral and bacterial pathogens in the aquaculture process have had a serious impact on the aquaculture economy. Common diseases mainly include white spot syndrome virus (WSSV), peach syndrome virus (TSV), yellow head virus (YHV) and infectious myonecrosis virus (IMNV).

[0003] Traditional disease management methods in shrimp farming rely mainly on antibiotics and chemical treatments. However, these methods also have some drawbacks, such as the overuse of antibiotics leading to the emergence of drug-resistant strains, which increases the difficulty of disease control. In addition, chemical treatments have adverse effects on the aquatic environment, affecting biodiversity and water quality safety. In addition, the residues of antibiotics and chemicals in shrimp products pose a hidden danger to consumer health and supervision, which in turn leads to economic losses in shrimp farming and increased antibiotic consumption.

[0004] Currently, more and more alternative strategies are being promoted, including RNA interference (RNAi) technology. This is a promising strategy for gene silencing using small interfering RNA (siRNA) to enhance disease resistance in aquatic species. siRNA achieves its function by degrading specific mRNA molecules, thereby preventing the translation of target genes. This technology can be used to downregulate genes related to immune response, metabolic pathways or pathogen susceptibility, potentially enhancing the health and resilience of farmed species. Although RNAi is currently considered a very promising strategy for shrimp viral disease control, the effective delivery of siRNA to target cells and tissues of shrimp remains a major challenge. Summary of the invention

[0005] Based on the above, the technical problem solved by the present invention is to provide a siRNA delivery system based on mPEG-PEI-c-SWCNTs and its preparation method and application. By characterizing the properties of nanomaterials, the loading ratio of siRNA is optimized to synthesize a nanocomposite with high transfection efficiency and low cytotoxicity, thereby safely and effectively delivering siRNA to the fertilized eggs of Penaeus vannamei.

[0006] The present invention provides a method for preparing a siRNA delivery system based on mPEG-PEI-c-SWCNTs, comprising:

[0007] Polyethylene glycol grafted polyethylene imine mPEG-PEI was added to the suspension of carboxylated single-walled carbon nanotubes c-SWCNTs. After stirring at room temperature for 24 hours, the excess mPEG-PEI was removed by ultrafiltration centrifugation, and the liquid in the filter was collected to obtain single-walled carbon nanotubes mPEG-PEI-c-SWCNTs non-covalently modified with polyethylene glycol grafted polyethylene imine.

[0008] Preferably, the mass ratio of the suspension of polyethylene glycol grafted polyethyleneimine mPEG-PEI to carboxylated single-walled carbon nanotubes c-SWCNTs is 2:1.

[0009] Preferably, the molecular weight of the polyethylene glycol used is Mn=5000Da, the molecular weight of the branched polyethyleneimine is Mn=25000Da; and / or the specifications of the carboxylated single-walled carbon nanotubes are diameter 4-5nm, length 0.5-1.5μm, and purity>90%.

[0010] Preferably, the ultrafiltration centrifugation comprises centrifugation at 800 rpm for 1 hour at room temperature using a 100,000 MWCO filter.

[0011] Preferably, the suspension of carboxylated single-walled carbon nanotubes c-SWCNTs is prepared by the following method:

[0012] Take carboxylated single-walled carbon nanotube c-SWCNTs powder and add deionized water. Perform ultrasonic treatment at a power of 300-600 W at room temperature for 30 minutes. After standing for 10 minutes, centrifuge at a maximum speed of 14000 rpm for 1 hour, collect all supernatant except the last 2-3 mL, and obtain a uniformly suspended c-SWCNTs solution.

[0013] Preferably, the preparation method further comprises: fully mixing the mPEG-PEI-c-SWCNTs solution with the siRNA, and incubating them together at room temperature for 30 minutes to obtain the mPEG-PEI-c-SWCNTs-siRNA nanocomplex.

[0014] More preferably, the concentration of the mPEG-PEI-c-SWCNTs solution is 5 μg / mL; and / or the mass ratio of the mPEG-PEI-c-SWCNTs solution to the siRNA is 4:1.

[0015] The present invention also provides a siRNA delivery system based on mPEG-PEI-c-SWCNTs, which is prepared by any of the above preparation methods.

[0016] The present invention further provides application of the siRNA delivery system in gene silencing of Litopenaeus vannamei.

[0017] Preferably, the concentration of the mPEG-PEI-c-SWCNTs solution is below 10 μg / mL.

[0018] The beneficial effects of the present invention are mainly reflected in:

[0019] The mPEG-PEI-c-SWCNTs-based siRNA delivery system prepared by the present invention successfully, safely and effectively delivers siRNA to the fertilized eggs of Penaeus vannamei, achieves silencing of the target gene, and has the advantages of high transfection efficiency and good safety. The establishment of this delivery system is conducive to the study of functional genes, and promotes the application of RNAi therapy in shrimp species, and helps to promote the application of gene therapy in improving the disease resistance of Penaeus vannamei and cultivating healthy shrimp species. In addition, the preparation process of the mPEG-PEI-c-SWCNTs-siRNA nanocomplex adopted by the present invention is mild, economical and time-saving, can be stably dispersed in seawater, and is suitable for basic applications in marine organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The morphology of the c-SWNT and mPEG-PEI-c-SWCNTs nanocomposite dispersion prepared in Example 1 after being placed at 30°C, pH 7.8, and salinity 30‰ for one week. Figure A is c-SWCNTs, and Figure B is mPEG-PEI-c-SWCNTs.

[0021] Figure 2 Scanning electron microscopy images of the mPEG-PEI-c-SWCNTs nanocomposite prepared in Example 1, Figure A is c-SWCNTs, and Figure B is mPEG-PEI-c-SWCNTs.

[0022] Figure 3 Fourier transform infrared spectrum of the mPEG-PEI-c-SWCNTs nanocomposite prepared in Example 1.

[0023] Figure 4 X-ray photoelectron spectrum of the mPEG-PEI-c-SWCNTs nanocomposite prepared in Example 1, Figure A is c-SWCNTs, and Figure B is mPEG-PEI-c-SWCNTs.

[0024] Figure 5 Particle size distribution diagram of c-SWCNTs and mPEG-PEI-c-SWCNTs nanocomposites prepared in Example 1 and their zeta potential measurement results.

[0025] Figure 6 Electrophoresis diagram of the complexes formed by the mPEG-PEI-c-SWCNTs nanocomplex prepared in Example 2 and siRNA at different mass ratios, the electrophoresis was performed at a 2% agarose concentration.

[0026] Figure 7 Evaluation of the protective ability of the mPEG-PEI-c-SWCNTs nanocomplex prepared in Example 2 on loaded siRNA.

[0027] Figure 8 The toxic effects of different concentrations of mPEG-PEI-c-SWCNTs nanocomposites prepared in the application example on the fertilized eggs of Penaeus vannamei.

[0028] Fig. 9 Evaluation of transfection efficiency of mPEG-PEI-c-SWCNTs-siRNA formed by the application example, wherein siRNA is labeled with -FAM fluorescence. Figure A is a microscopic observation of mPEG-PEI-c-SWCNTs-siRNA under bright field after successful transfection; Figure B is a microscopic observation of mPEG-PEI-c-SWCNTs-siRNA under fluorescence field after successful transfection; Figure C is a microscopic observation of mPEG-PEI-c-SWCNTs-siRNA under bright field after only transfection of siRNA; Figure D is a microscopic observation of mPEG-PEI-c-SWCNTs-siRNA under fluorescence field after only transfection of siRNA.

[0029] Fig.10 The silencing effects of the mPEG-PEI-c-SWCNTs formed in the application example were compared with those of three commercially available siRNA transfection reagents. DETAILED DESCRIPTION

[0030] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0031] Example 1

[0032] In this example, we will introduce in detail the design, preparation process and characterization methods of mPEG-PEI-c-SWCNTs nanocomposites.

[0033] 1. Preparation of Carboxylated Single-walled Carbon Nanotube Suspension

[0034] 10 mg of powdered c-SWCNTs (4-5 nm × 0.5-1.5 μm (D × L), 652490, Merck, Germany) was weighed and placed in a 50 mL conical tube in a chemical fume hood, and 30 mL of deionized water was added. Ultrasonic treatment was performed at room temperature for 30 minutes. Then, the mixture was allowed to stand at room temperature for 10 minutes, followed by centrifugation at a maximum speed of 14,000 rpm for 1 hour. All supernatants except the last 2-3 mL were collected to obtain a solution containing COOH-SWCNTs suspended alone, i.e., the c-SWCNTs solution.

[0035] 2. Carboxylated single-walled carbon nanotubes non-covalently modified with mPEG-PEI

[0036] 20 mg of mPEG-PEI (PEG Mn 5 kDa, branched PEI Mn 25 kDa, 900743, Sigma-Aldrich, USA) was added to the c-SWCNTs suspension at a mass ratio of 2:1. After stirring at room temperature for 24 h under a magnetic stirrer, the suspension was stirred for 24 h using Ultra 100 kDa-MWCO filter (UFC910024, Millipore Sigma, USA) was centrifuged at 800 rpm for 1 hour at room temperature to remove excess mPEG-PEI. Subsequently, the liquid in the filter was collected and ultrasonicated for another 1 hour to obtain an mPEG-PEI-c-SWCNTs solution.

[0037] 3. Determination of mPEG-PEI-c-SWCNTs concentration

[0038] Take 10 mL of the uniformly dispersed mPEG-PEI-c-SWCNTs solution, divide it into three groups, and weigh it after vacuum freeze drying. The mass difference of each group of samples can be regarded as the mass of each group of samples, and the average can be used to estimate the concentration of mPEG-PEI-c-SWCNTs in the sample. The concentration calculated from the mass difference before and after freeze drying is about 0.5 mg / mL.

[0039] 4. Stability evaluation of mPEG-PEI-c-SWCNTs

[0040] The c-SWCNTs and mPEG-PEI-c-SWCNTs dispersions with a concentration of 0.5 mg / mL were placed at 30°C, salinity 30‰, and pH = 7.8 for one week to observe the stability of the two nano-solutions. Figure 1As shown in Figure 1, the c-SWCNTs solution in Figure A aggregates, forms agglomerates, and settles, while the mPEG-PEI-c-SWCNTs solution in Figure B remains uniformly opaque black. This indicates that mPEG-PEI-c-SWCNTs significantly improves the dispersibility of SWCNTs in water and can remain suspended at room temperature for at least one week.

[0041] 5. Characterization of the physicochemical properties of mPEG-PEI-c-SWCNTs

[0042] Take 2-3 drops of uniformly dispersed mPEG-PEI-c-SWCNTs dispersion with a disposable dropper and drop them on a clean copper mesh microgrid, then use a xenon lamp to dry it to prepare a transmission electron microscopy (TEM) sample. The internal structure of the sample was observed under a field emission electron transmission microscope (Talos F200X, USA). The results are as follows: Figure 2 As shown in Figure B, the mPEG-PEI-c-SWCNTs in Figure A showed a significantly larger tube diameter and shorter tube length than the c-SWCNTs, indicating that mPEG-PEI was successfully wrapped on the SWCNTs. Next, the vacuum freeze-dried c-SWCNTs and mPEG-PEI-c-SWCNTs samples were taken and their infrared absorption characteristic spectra were tested using a Fourier transform infrared spectrometer (Frontier, USA). The results are shown in Figure 2. Figure 3 As shown. At about 3349cm -1 、2860cm -1 、1624cm -1 、1464cm -1 、1341cm -1 and 1104cm -1 The strong absorption peak at proves that c-SWCNTs were non-covalently modified with mPEG-PEI.

[0043] The chemical bonds and elemental compositions of c-SWCNTs and mPEG-PEI-c-SWCNTs were determined using an X-ray photoelectron spectrometer (ESCALAB QXi, Thermo Fisher, USA), and the data analysis and peak fitting were performed using ThermoScientific Avantage software. Figure 4 As shown, c-SWCNTs only have C peak and O peak, while the appearance of N peak and the enhancement of O peak indicate that mPEG-PEI has been successfully modified on c-SWCNTs.

[0044] The particle size and surface potential of c-SWCNTs and mPEG-PEI-c-SWCNTs were measured using a nanoparticle size and Zeta potential instrument (Malvern Zetasizer Nano ZSE, UK). Figure 5 As shown in the figure, the particle size increased from 96nm to 275nm before and after modification, proving that mPEG-PEI was successfully modified on the surface of SWCNTs, resulting in an increase in the diameter of SWCNTs. Among them, the measured DPI was less than 0.2, indicating that the particle size distribution was narrow and the composition was relatively uniform.

[0045] Example 2

[0046] 1. Formation and optimization of mPEG-PEI-c-SWCNTs-siRNA nanocomplexes

[0047] The mPEG-PEI-c-SWCNTs solution with a concentration of 5 μg / mL after dilution 100 times was added dropwise to a certain amount (500 ng) of siRNA according to different mass ratios (1:1, 2:1, 3:1, 4:1, 5:1), and sterile seawater was added to make up the volume to 10 μL. The mixture was incubated at room temperature for 30 minutes to obtain PEI-c-SWCNTs-siRNA nanocomplexes with different mass ratios. Next, a 2% agarose gel was prepared, electrophoresed at a voltage of 110 V for 30 minutes, and analyzed by agarose gel retardation method. The electrophoresis diagram is shown in FIG. Figure 6 As shown. The results showed that the optimal mass ratio of mPEG-PEI-c-SWCNTs to fully load siRNA was 4:1. Therefore, this study selected mPEG-PEI-c-SWCNTs-siRNA complex (w / w% = 4:1) for subsequent cell transfection experiments.

[0048] 2. Analysis of the protective effect of mPEG-PEI-c-SWCNTs on siRNA

[0049] According to step 1 of this embodiment, after the mPEG-PEI-c-SWCNTs-siRNA nanocomplex was formed at the optimal ratio, sterile seawater was added to the complex to a total volume of 20 μL, and incubated at room temperature for 0 hours, 1 hour, 2 hours, and 6 hours. After incubation for different periods of time, 10 μL of a heparin solution with a concentration of 1 mg / mL was used for 10 minutes, and then electrophoresis analysis was performed in a 2% agarose gel. The final results are shown in FIG. Figure 7 As shown, even if the mPEG-PEI-c-SWCNTs-siRNA complex was incubated in seawater for 6 h, the heparin solution was still able to effectively displace the siRNA, indicating that the complex had good siRNA protection ability.

[0050] Application Examples

[0051] 1. Biotoxicity evaluation of mPEG-PEI-c-SWCNTs on fertilized eggs of Litopenaeus vannamei

[0052] In order to investigate the embryotoxicity of mPEG-PEI-c-SWCNTs on the embryos of Penaeus vannamei and its developmental effects, we chose to conduct an exposure experiment of mPEG-PEI-c-SWCNTs during the cleavage stage of fertilized eggs. The experiment set 7 concentration gradients of mPEG-PEI-c-SWCNTs: 0, 2, 5, 10, 20, 50, 100 μg / mL, and 7 time gradients: 0h, 12h, 24h, 36h, 48h, 60h, 72h, and the survival of embryos or larvae at different concentrations at each time point was counted. Three parallel experiments were set for each concentration. The experiment used a 6-well plate as an exposure container, and 2mL of native seawater and 100 normally developed fertilized eggs of Penaeus vannamei were added to each well, and incubated normally for 12 hours. After the set period, the embryos were filtered using a 70-mesh cell sieve and transferred to a new 6-well plate. While keeping the native seawater conditions unchanged, they were placed in a cell culture incubator at 30°C and continued to incubate for 32 hours. 100 embryos of the corresponding developmental period were taken as blank controls by a rubber-tipped dropper. No feeding was carried out during the experiment, and the temperature was maintained within the range of ±0.5°C. The number of hatched and surviving larvae was observed under an optical microscope, and the larval survival rate (%) was calculated as follows: number of surviving nauplii / total number of nauplii × 100. The experimental results were statistically analyzed and statistical graphs were prepared. The final results are shown in Figure 8 shown.

[0053] As the concentration of mPEG-PEI-c-SWCNTs increased, the successful hatching rate of fertilized eggs gradually decreased. At a concentration of 100μg / mL, the proportion of fertilized eggs that failed to hatch was as high as 20%, while concentrations below 50μg / mL had no significant effect on the hatching rate of fertilized eggs. As the incubation time of nauplii increased, the increase in the concentration of mPEG-PEI-c-SWCNTs led to a gradual decrease in the survival rate of the larvae. Within 36 hours, except for the slow swimming and abnormal conditions of the larvae at a concentration of 100μg / mL, the concentrations had no obvious toxic effects on the development of the larvae, and the survival rate of the larvae below 50μg / mL was higher than 50%. Within 48 hours, the survival rate of the larvae was only 27% at a concentration of 100μg / mL, while the survival rate below 20μg / mL was still higher than 50%. Within 60 hours, the survival rate of larvae at a concentration of 100 μg / mL dropped to 11%, the survival rate below 50 μg / mL was 32%, and the survival rate below 10 μg / mL was higher than 50%. Within 72 hours, except for the control group with a survival rate higher than 50%, the larvae at other concentrations all showed a large-scale death, and the survival rate of larvae at a concentration of 100 μg / mL was 0%.

[0054] Therefore, the mPEG-PEI-c-SWCNTs concentration below 10 μg / mL has little toxic effect on larval development and can be regarded as a safe concentration.

[0055] 2. Evaluation of transfection effect of mPEG-PEI-c-SWCNTs-siRNA

[0056] The siRNA-FAM fluorescent label used in this experiment can show green fluorescence in the cytoplasm. According to the optimal loading ratio determined in Example 2, siRNA and mPEG-PEI-c-SWCNTs were incubated at room temperature for 30 minutes respectively to form a transfection complex solution. Subsequently, a transfection experiment was carried out on fertilized eggs in the cleavage stage, and 3 parallel experiments were set up. The experiment used a 6-well plate as an exposure container, and 2mL of native seawater and 100 freshly laid fertilized eggs of Penaeus vannamei were added to each well. After the start of the experiment, a disposable rubber-tipped dropper was used to gently blow air every hour to make the water body float slightly to prevent the fertilized eggs from sinking to the bottom and agglomerating. After 5 hours, a fully automatic inverted fluorescence microscope (Leica, DMi8, Germany) was used for observation, and the final results are as follows Fig. 9 Green fluorescence was observed after transfection, indicating that mPEG-PEI-c-SWCNTs could successfully deliver siRNA into the fertilized eggs of Penaeus vannamei.

[0057] 3. Evaluation of the silencing effect of mPEG-g-PEI-c-SWCNTs-siRNA

[0058] The specific siRNA for single insulin-like growth factor-binding domain protein-2 (LvIGFBP-2, XM_027379073.1) of Litopenaeus vannamei was designed using Thermofisher's BLOCK-iT RNAi Designer (https: / / rnaidesigner.thermofisher.com / rnaiexpress / ). The qRT-PCR primers were designed using Primer Premier 5.0 software. The siRNA and its qRT-PCR amplification primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and a FAM fluorescent label was added to the 5' end of the siRNA.

[0059] We selected mPEG-PEI-c-SWCNTs (5 μg / mL) and siRNA (10 nM) at a mass ratio (w / w) of 4:1 and incubated them at room temperature for 30 minutes to form an optimal ratio of mPEG-PEI-c-SWCNTs-siLvIGFBP-2 complex. According to the manufacturer's instructions, siRNA was incubated with four transfection reagents: mPEG-PEI-c-SWCNTs, (101000028, Polyplus, USA), RNAiMAX (13778150, ThermoFisher, USA) and small nucleic acid transfection reagent (abs60252, absin, China) were incubated at room temperature for 30 minutes, 10 minutes, 5 minutes and 20 minutes to form a transfection complex solution. Subsequently, the fertilized eggs in the cleavage stage were exposed to the experiment, and three parallel experiments were set up at the same time. 6-well plates were used as exposure containers, and 2 mL of native seawater and 100 freshly laid fertilized eggs of Penaeus vannamei were added to each well. Cells or larvae were collected 36 hours after transfection. Total RNA was extracted according to the manufacturer's protocol (DP451, Tiangen, China), and 100 ng of the extracted total RNA was reverse transcribed according to the manufacturer's protocol (KR116, Tiangen, China). The obtained cDNA was diluted 10 times as a template, and the β-actin gene was used as an internal reference gene for real-time quantitative PCR amplification according to the manufacturer's protocol (FP205, Tiangen, China). The PCR amplification conditions were: 95°C, 15 min, [95°C, 10 sec; 60°C, 30 sec, 40 cycles]. The results of real-time quantitative PCR were analyzed by 2 –△△Ct The results were analyzed by GraphPad Prism 8.0 software for multiple comparisons and significant difference analysis, and graphs were drawn. The silencing efficiency of the target gene was recorded, and GraphPad Prism 8.0 software was used to record and produce statistical graphs for each experiment. The final results are shown in Figure 2. Fig.10 As shown in Figure 2, the use of mPEG-PEI-c-SWCNTs to deliver siRNA resulted in the highest target gene silencing efficiency, reaching 77.92%; while in the other reagent groups, The silencing efficiency of RNAiMAX was 56.59%. The silencing efficiency was low, only 28.08%. Therefore, mPEG-PEI-c-SWCNTs can successfully mediate siRNA silencing in vivo, which has broad development prospects in the field of siRNA delivery to Litopenaeus vannamei.

[0060] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a siRNA delivery system based on mPEG-PEI-c-SWCNTs, comprising: Polyethylene glycol grafted polyethylene imine mPEG-PEI was added to the suspension of carboxylated single-walled carbon nanotubes c-SWCNTs. After stirring at room temperature for 24 hours, the excess mPEG-PEI was removed by ultrafiltration centrifugation, and the liquid in the filter was collected to obtain single-walled carbon nanotubes mPEG-PEI-c-SWCNTs non-covalently modified with polyethylene glycol grafted polyethylene imine.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the suspension of polyethylene glycol grafted polyethyleneimine mPEG-PEI to carboxylated single-walled carbon nanotubes c-SWCNTs is 2:

1.

3. The preparation method according to claim 1, characterized in that: The molecular weight of the polyethylene glycol used is Mn=5000Da, the molecular weight of the branched polyethyleneimine is Mn=25000Da; and / or the specifications of the carboxylated single-walled carbon nanotubes are 4-5nm in diameter, 0.5-1.5μm in length, and purity>90%.

4. The preparation method according to claim 1, characterized in that: The ultrafiltration centrifugation involved centrifugation at 800 rpm for 1 hour at room temperature using a 100,000 MWCO filter.

5. The preparation method according to claim 1, characterized in that: The suspension of carboxylated single-walled carbon nanotubes c-SWCNTs is prepared by the following method: Take carboxylated single-walled carbon nanotube c-SWCNTs powder and add deionized water. Perform ultrasonic treatment at a power of 300-600 W at room temperature for 30 minutes. After standing for 10 minutes, centrifuge at a maximum speed of 14000 rpm for 1 hour, collect all supernatant except the last 2-3 mL, and obtain a uniformly suspended c-SWCNTs solution.

6. The preparation method according to claim 1, characterized in that: Also includes: The mPEG-PEI-c-SWCNTs solution was fully mixed with siRNA and incubated together at room temperature for 30 minutes to obtain the mPEG-PEI-c-SWCNTs-siRNA nanocomplex.

7. The preparation method according to claim 6, characterized in that: The concentration of the mPEG-PEI-c-SWCNTs solution is 5 μg / mL; and / or the mass ratio of the mPEG-PEI-c-SWCNTs solution to the siRNA is 4:

1.

8. An mPEG-PEI-c-SWCNTs-based siRNA delivery system, prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the siRNA delivery system according to claim 8 in gene silencing of Litopenaeus vannamei.

10. The use according to claim 9, characterized in that: The concentration of the mPEG-PEI-c-SWCNTs solution is below 10 μg / mL.

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