Gold nano-mediated RNAi hydrogel biological agent for preventing and treating fall webworm and preparation method

Abstract

The invention discloses a gold nano-mediated RNAi hydrogel biological agent for preventing and treating fall webworms and a preparation method, and relates to the technical field of nano pesticides. According to the preparation method, a layer-by-layer method is adopted, siRNA, polyethyleneimine and sulfosuccinic acid dioctyl sodium salt are sequentially adsorbed on the surfaces of gold nanoparticles under the action of charges, and then the gold nanoparticles and high-esterification apple pectin are subjected to ultrasonic treatment to prepare the gold nanoparticle mediated RNAi hydrogel biological agent. The prepared gold nano-mediated RNAi hydrogel biological agent has good adhesive force, permeability and high spreadability on the surfaces of branches and leaves, and the retention rate, the conveying efficiency and the drug utilization rate of the gold nano-mediated RNAi biological pesticide on the branches and leaves can be improved. Meanwhile, the prepared gold nano-mediated RNAi hydrogel biological agent can silence a plurality of key genes of the fall webworm and can be used for preventing and treating the fall webworm.

Description

Technical Field

[0001] The present invention relates to the technical field of nanopesticides, and in particular to a gold nano-mediated RNAi hydrogel biopharmaceutical for preventing and controlling the gypsy moth and a preparation method thereof. Background Art

[0002] The cunea moth (Hyphantria cunea) is a common defoliator in my country's agriculture and forestry sectors. It is particularly destructive to forestry, often causing significant economic losses. Hyphantria cunea hosts a wide variety of host species, is widely distributed, has strong adaptability, and reproduces rapidly. Furthermore, its larvae spin silk and weave webs during feeding, making its control extremely difficult. Currently, chemical control is the primary method for controlling the cunea both domestically and internationally, offering high efficiency and low cost. Commonly used pesticides include chlorfenapyr 3, tebufenozide, abamectin emulsifiable concentrate, and cypermethrin. While these traditional pesticide formulations offer good insecticidal efficacy, they suffer from drawbacks such as easy decomposition, low specificity, environmental pollution, and low drug availability. Therefore, designing and developing pesticide formulations with strong stability, good permeability, environmental safety, and high specificity for controlling the cunea moth has become a current research priority.

[0003] RNA pesticides are hailed as the third revolution in the history of pesticides and are a type of exogenous interfering RNA preparation. Compared with traditional small-molecule chemical pesticides, RNA pesticides have the advantages of better targeting of pests, being non-toxic to humans and animals, and leaving no residue in the environment. RNA interference (RNAi) is a technology that interferes with or inhibits the expression of specific genes in target organisms. RNAi technology has excellent targeting, but in agricultural and forestry production applications, it has disadvantages such as low gene interference efficiency, low prevention effect, short duration of effect, and poor stability, which seriously restrict the application of RNA preparations in agricultural and forestry environments. When RNAi technology is used to control the gypsy moth, since the gypsy moth is a herbivorous pest with a strongly alkaline intestinal environment, the key to improving the efficiency of RNAi is to be able to bypass the hemolymph.

[0004] Therefore, it is particularly necessary to develop an RNAi hydrogel biopharmaceutical with high interference efficiency on the genes of the gypsy moth, which can quickly reach the target site to achieve excellent control effects when used to control the gypsy moth. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a gold nanoparticle-mediated RNAi hydrogel biopesticide for controlling the gypsy moth and its preparation method. The present invention utilizes a layer-by-layer method, utilizing charge interactions to sequentially adsorb siRNA, polyethyleneimine, and dioctyl sodium sulfosuccinate onto the surface of gold nanoparticles. The gold nanoparticle-mediated RNAi hydrogel biopesticide is then ultrasonically combined with highly esterified apple pectin to prepare the gold nanoparticle-mediated RNAi hydrogel biopesticide. The gold nanoparticle-mediated RNAi hydrogel biopesticide exhibits excellent adhesion, permeability, and high spreadability on the surfaces of branches and leaves, improving the retention rate, delivery efficiency, and drug utilization of the gold nanopesticide on branches and leaves. Furthermore, the gold nanoparticle-mediated RNAi hydrogel biopesticide produced by the present invention can silence multiple key genes in the gypsy moth, making it highly effective for controlling the gypsy moth. The present invention also effectively addresses issues such as poor siRNA stability, difficulty in cellular uptake, and low efficacy, achieving sustainable siRNA release.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing a gold nanoparticle-mediated RNAi hydrogel biopharmaceutical, comprising the following steps:

[0008] (1) adding a first polyethyleneimine solution to a polyethylene glycol-stabilized gold nanoparticle solution, performing ultrasonic and centrifugal purification, adding siRNA, performing ultrasonic and centrifugal purification, and then adding a second polyethyleneimine solution, performing ultrasonic and centrifugal purification to obtain a gold nanoparticle-mediated RNAi biopesticide;

[0009] (2) dispersing the gold nanopesticide mediated RNAi biopesticide in deionized water to obtain a gold nanopesticide solution; adding an aqueous solution of dioctyl sodium sulfosuccinate to the gold nanopesticide solution, and subjecting the solution to ultrasonic and centrifugal purification to obtain a gold nanopesticide mediated RNAi biopesticide;

[0010] (3) Adding a highly esterified apple pectin solution to the gold nano-mediated RNAi biopharmaceutical, and ultrasonicating to obtain a gold nano-mediated RNAi hydrogel biopharmaceutical.

[0011] Preferably, in step (1), the polyethylene glycol-stabilized gold nanoparticle solution is prepared by the following method:

[0012] After heating the chloroauric acid solution, trisodium citrate solution is added to react to obtain a gold nanoparticle solution; polyethylene glycol solution is added to the gold nanoparticle solution, stirred, and then centrifuged for purification to obtain a polyethylene glycol-stabilized gold nanoparticle solution.

[0013] Furthermore, the mass fraction of the chloroauric acid solution is 0.01%, the mass fraction of the trisodium citrate solution is 1%, the concentration of the polyethylene glycol solution is 0.5 mg / mL, and one end group of the polyethylene glycol in the polyethylene glycol solution is a thiol group with a molecular weight of 5000.

[0014] Furthermore, the heating temperature of the chloroauric acid solution is 155-163°C.

[0015] Furthermore, the volume ratio of the chloroauric acid solution, the trisodium citrate solution and the polyethylene glycol solution is 100:(2.8-3.2):(5-10).

[0016] Furthermore, the reaction time is 15-25 minutes and the stirring time is 0.5 hours to 24 hours.

[0017] Furthermore, the centrifugal speed is 10000 rpm-15000 rpm, the centrifugal temperature is 0°C-8°C, and the centrifugal time is 15 min-45 min.

[0018] Preferably, in step (1), the concentrations of the first polyethyleneimine solution and the second polyethyleneimine solution are both 0.1-2 mg / mL; and the solvent for preparing the first polyethyleneimine solution and the second polyethyleneimine solution is enzyme-free sterile water.

[0019] Preferably, in step (1), the ultrasound is performed in an ice bath, and the ultrasound time is 10 min to 240 min.

[0020] Preferably, in step (1), the concentration of the siRNA is (0.01-2) μM.

[0021] Preferably, in step (1), the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution to the first polyethyleneimine solution is 1:50-50:1; the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution to the second polyethyleneimine solution is 1:50-50:1; and the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution to siRNA is 1:100-100:1.

[0022] Preferably, in step (1), enzyme-free sterile water is used for the centrifugal purification; the centrifugal speed is 14000-16000 rpm, the centrifugal time is 30-50 min, and the centrifugal temperature is 2°C-6°C.

[0023] Preferably, in step (1), the siRNA is a small interfering RNA targeting the developmental genes, olfactory genes and / or reproductive genes of the cunea.

[0024] Preferably, in step (2), the concentration of the aqueous solution of dioctyl sodium sulfosuccinate is 10 mg / mL.

[0025] Preferably, in step (2), the volume ratio of the gold nanoparticle-mediated RNAi biopesticide solution to the dioctyl sodium sulfosuccinate solution is 1:1000-1000:1.

[0026] Preferably, in step (2), the ultrasound is performed in an ice bath, and the ultrasound time is 0.05-24 h.

[0027] Preferably, in step (3), the concentration of the highly esterified apple pectin solution is 1 mg / mL.

[0028] Preferably, in step (3), the volume ratio of the gold nanoparticle-mediated RNAi biopharmaceutical to the highly esterified apple pectin solution is 1:1000-1000:1.

[0029] Preferably, in step (3), the ultrasonic time is 0.01-1h.

[0030] The second aspect of the present invention provides a gold nanoparticle-mediated RNAi hydrogel biopharmaceutical prepared by the above preparation method.

[0031] The third aspect of the present invention provides the use of gold nanoparticle-mediated RNAi hydrogel biopharmaceuticals in the following (1) or (2):

[0032] (1) Prevent and control pests that eat branches and leaves on fruit trees;

[0033] (2) Prevent and control agricultural pests.

[0034] Preferably, the forest and fruit trees include but are not limited to pine trees, cypress trees, birch trees, poplar trees, chestnut trees, willow trees, fir trees, camphor trees, maple trees, ginkgo trees, kapok trees, banyan trees, sycamore trees, mulberry trees, locust trees, fruit trees, and ancient trees.

[0035] Preferably, the foliage-eating pests include the gypsy moth.

[0036] Beneficial effects of the present invention:

[0037] This invention, for the first time, utilizes a layer-by-layer method to adsorb siRNA onto the surface of gold nanoparticles using positively charged polyethyleneimine. Polyethyleneimine is then added to impart a positive charge to the surface of the gold nanoparticles, which is then used to adsorb the negatively charged sodium dioctyl sulfosuccinate. The resulting gold nanoparticles are then ultrasonically treated with highly esterified apple pectin to produce a gold nanoparticle-mediated RNAi hydrogel biopharmaceutical. The gold nanoparticle-mediated RNAi hydrogel biopharmaceutical produced via the layer-by-layer method has extremely small nanometer dimensions, allowing it to quickly penetrate the physical barriers of biomembranes and exhibit excellent dispersibility. The strong positive charge on the surface of the gold nanoparticles enhances cell membrane uptake, promotes transduction within various tissues on the surface and inside plants and the gypsy moth, shortens intracellular retention, accelerates intercellular transport, and reduces hemolymph clearance.

[0038] The present invention combines dioctyl sodium sulfosuccinate with highly esterified apple pectin to enhance adhesion, permeability, and high spreading properties on the surface of branches and leaves after spraying, thereby improving the retention rate, delivery efficiency, and drug utilization of gold nanoparticle-mediated RNAi biopesticides on branches and leaves. Furthermore, the combination of dioctyl sodium sulfosuccinate and highly esterified apple pectin synergistically improves the permeability and spreading properties of the gold nanoparticle-mediated RNAi hydrogel biopesticide.

[0039] This invention combines gold nanoparticles with siRNA molecules to create an efficient, simple, and safe nano-delivery system, enabling rapid transport and transfer within plants and pests, reaching their target sites and exerting their effects. This gold nanoparticle-mediated RNAi delivery system enhances the stability of siRNA in its application environment, improving its delivery efficiency within organisms and even at the cellular level. It facilitates early escape of siRNA from the endosomal-lysosomal system, reduces nuclease degradation, enables sustained release of siRNA to target sites, and increases cellular uptake, resulting in a more efficient RNAi effect.

[0040] The gold nanoparticle-mediated RNAi hydrogel biopharmaceutical prepared by this invention can silence multiple key genes of the gypsy moth, making it suitable for a variety of pest and disease control applications, including those on different tree species. Furthermore, the preparation method is environmentally friendly, non-toxic, and pollution-free, making it suitable for both single and combined control in a variety of scenarios, particularly large-scale control measures such as aerial spraying. The product boasts high yield, a simple preparation process, and minimal equipment requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : State diagram of a 1% mass fraction chloroauric acid solution and the prepared gold nanoparticle solution in Example 1;

[0042] Figure 2 : In Example 1, the high-magnification transmission electron microscopy image and X-ray energy spectrum image of the gold nanoparticle-mediated RNAi biopesticide prepared in step (2);

[0043] Figure 3 : In Experimental Example 1, the sustained release curve of the gold nanoparticle-mediated RNAi biopesticide;

[0044] Figure 4 : Fluorescence images of the gold nanoparticle-mediated fluorescently labeled RNAi hydrogel biopharmaceutical on the body wall and intestinal sections of the American white moth in Experimental Example 2;

[0045] Figure 5 : In Experimental Example 2, fluorescence distribution images of the gold nanoparticle-mediated fluorescently labeled RNAi hydrogel biopharmaceutical on willow leaves, sycamore leaves, and the web of the cuneiform moth;

[0046] Figure 6 : In Experimental Example 3, the effect of gold nanoparticle-mediated RNAi biopesticide on the acetylcholinesterase concentration of the American white moth. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0048] As described in the background technology section above, the gypsy moth (Hyphantria cunea) is one of the important agricultural and forestry leaf-feeding pests in my country and has caused serious damage to my country's agricultural and forestry ecosystems, especially garden plants. The gypsy moth has many host species, is widely distributed, has strong adaptability, and reproduces quickly. In addition, the larvae spin silk and weave webs during feeding activities, making it extremely difficult to prevent and control. Using RNA interference (RNAi) to target essential genes for silencing and induce toxic effects in target species without harming other organisms in the ecosystem is one of the most promising pest control strategies. Gold nanoparticle-mediated RNAi delivery systems can protect siRNA from the effects of nucleic acid degrading enzymes, extreme pH, etc., allowing siRNA to be continuously released to the target site and increasing cellular uptake to produce a more efficient RNAi effect.

[0049] Based on this, the present invention provides a gold nanoparticle-mediated RNAi hydrogel biopharmaceutical for controlling the gypsy moth and its preparation method. siRNA is first adsorbed onto the surface using a layer-by-layer method. The outermost layer is a highly positively charged polyethyleneimine, onto which the negatively charged sodium dioctyl sulfosuccinate is adsorbed, yielding the gold nanoparticle-mediated RNAi biopharmaceutical. The gold nanoparticle-mediated RNAi hydrogel biopharmaceutical is then ultrasonically treated with highly esterified apple pectin to obtain the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical. The gold nanoparticle-mediated RNAi hydrogel biopharmaceutical can silence multiple key genes in the gypsy moth, such as developmental, olfactory, and reproductive genes, achieving precise delivery to target cells in the moth.

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0052] Example 1: Preparation of gold nanoparticle-mediated RNAi hydrogel biopharmaceuticals

[0053] (1) 100 mL of 0.01% chloroauric acid solution was heated to 160°C, and 3 mL of 1% trisodium citrate solution was added and reacted for 20 min. The solution turned into a bright red clear liquid, which was then placed in ice water and cooled to room temperature to obtain a gold nanoparticle solution.

[0054] 7 mL of a 0.5 mg / mL polyethylene glycol solution containing a thiol end group and a molecular weight of 5000 was dropwise added to the gold nanoparticle solution. The mixture was stirred at room temperature for 2 hours, centrifuged at 14,000 rpm at 4°C for 30 minutes, and purified three times with deionized water to obtain a polyethylene glycol-stabilized gold nanoparticle solution. The resulting gold nanoparticles had a size of 13 nm and a yield of 90%.

[0055] (2) Adding an equal volume of 1 mg / mL polyethyleneimine solution to the polyethylene glycol-stabilized gold nanoparticle solution, ultrasonicating the solution on ice for 40 minutes, centrifuging the solution at 4°C and 14,000 rpm for 30 minutes, and purifying the solution three times with enzyme-free sterile water to remove excess polyethyleneimine; then adding 1 μM siRNA, ice bathing the solution in the dark for 40 minutes to allow the negatively charged siRNA to be adsorbed by the strongly positively charged polyethyleneimine, centrifuging the solution at 4°C and 14,000 rpm for 30 minutes, and purifying the solution three times with enzyme-free sterile water to remove unbound siRNA; then adding an equal volume of 1 mg / mL polyethyleneimine solution again to allow the surface of the gold nanoparticle-mediated RNAi biopesticide to be positively charged, centrifuging the solution at 4°C and 14,000 rpm for 30 minutes, and purifying the solution three times with deionized water to remove unbound polyethyleneimine to obtain the gold nanoparticle-mediated RNAi biopesticide;

[0056] Among them, the siRNA targets the acetylcholinesterase gene of the gypsy moth, and its nucleotide sequence is as follows:

[0057] 5'-CUCGAAGCAUGUCGUUUAATT-3'

[0058] 5'-UUAAACGACAUGCUUCGAGTT-3'.

[0059] The encapsulation efficiency of siRNA in nano-mediated RNAi biopesticide was 86%;

[0060] The encapsulation efficiency calculation formula is:

[0061]

[0062] (3) dispersing the gold nanopesticide mediated RNAi biopesticide in deionized water to obtain a gold nanopesticide solution; adding 100 μL of a 10 mg / mL aqueous solution of dioctyl sodium sulfosuccinate to the gold nanopesticide solution, ultrasonicating the mixture in an ice bath for 30 minutes, and centrifuging and purifying the mixture to obtain a gold nanopesticide mediated RNAi biopesticide;

[0063] (4) Add 100 μL of 1 mg / mL highly esterified apple pectin solution to the gold nanoparticle-mediated RNAi biopharmaceutical and ultrasonicate for 10 minutes to obtain the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical.

[0064] Test Example 1: Sustained Release Performance

[0065] (1) Determination of pH in the intestine of gypsy moth larvae:

[0066] After dissecting the larvae of the American white moth, the intestine was picked out, fully ground and centrifuged, and the pH of the upper liquid was measured with a pH meter. This was repeated 5 times and the average pH was taken to be 8.2.

[0067] (2) Determination of sustained-release performance:

[0068] 50 mL of the gold nanopesticide-mediated RNAi biopesticide prepared in step (2) of Example 1 was added to a PBS phosphate buffer solution with a pH of 8.2, and the mixture was placed in a constant temperature shaker at 5°C and 200 rpm in the dark. Samples were taken at different time points (20 h, 40 h, 60 h, 80 h, 100 h, 120 h, and 160 h), with a sampling volume of 4 mL each time.

[0069] After centrifuging the sample, retain the supernatant and use an ultra-micro UV spectrophotometer to measure the concentration of siRNA in the supernatant and calculate the cumulative release rate of siRNA. Set up three groups of repetitions and draw the siRNA release curve based on the cumulative release rate of siRNA. The results are as follows: Figure 3 shown.

[0070] The cumulative release rate of siRNA = (amount of siRNA released / gold nanopesticide-mediated RNAi biopesticide) × 100%.

[0071] As can be seen from Table 3, the siRNA release rate shows a trend of first increasing rapidly, then decreasing, and finally reaching saturation. The entire sustained-release process reaches equilibrium in about 120 hours, and the siRNA release amount is close to 80% at 60 hours. This shows that the gold nanoparticle-mediated RNAi biopesticide prepared by the present invention has a good siRNA sustained-release effect.

[0072] Experimental Example 2: Biodistribution of gold nanoparticle-mediated RNAi hydrogel biopharmaceuticals on the bodies of the gypsy moth, willow leaves, sycamore leaves, and net curtains

[0073] (1) Cy3-labeled siRNA was used to prepare gold nanoparticle-mediated fluorescently labeled RNAi hydrogel biopharmaceutical according to the method in Example 1.

[0074] (2) Spray the gold nanoparticle-mediated fluorescently labeled RNAi hydrogel biopharmaceutical onto the body wall of the gypsy moth larvae. After 30 minutes, absorb the excess agent with absorbent paper and wash the larvae body wall slowly with clean water several times to wash away the agent remaining on the surface. The treated larvae were placed in a fluorescent stereoscope to observe the fluorescence distribution. The larvae were then cross-sectioned and stained to prepare tissue sections. The fluorescence distribution in the tissue was observed under a fluorescent stereoscope. The results are as follows: Figure 4 shown.

[0075] (3) Spray the gold nanoparticle-mediated fluorescently labeled RNAi hydrogel biopharmaceutical on the surface of willow leaves, sycamore leaves, and net curtains. After 30 minutes, rinse the biopharmaceuticals on the willow leaves and sycamore leaves with a slow flow of water. The fluorescence distribution of the leaves and net curtains was observed in a small animal live imaging device. The results are as follows: Figure 5 shown.

[0076] After being excited by green light, Cy3 emits red fluorescence. Figure 4 The original and fluorescent images of the gypsy moth larvae and cross-sections of the gypsy moth tissues after staining show that there is bright red fluorescence in the gypsy moth larvae and cross-sections of the gypsy moth tissues. This shows that the gold nanoparticle-mediated fluorescently labeled RNAi biopesticide can enter the body tissues through the body wall of the gypsy moth. Figure 5 It can be seen from the original images and fluorescence distribution images of the willow leaves, sycamore trees and net curtains that after being washed with water to eliminate the interference of surface agent residues on the fluorescence image, they still have a strong fluorescence distribution. This shows that the gold nano-mediated fluorescence-labeled RNAi biological pesticide can enter the interior of the leaves through the leaf surface, and the fluorescence on the net curtain gradually diffuses from the outer layer to the inner layer, indicating that the gold nano-mediated fluorescence-labeled RNAi hydrogel biological agent has good permeability.

[0077] Experimental Example 3: Genetic Interference Efficiency of Gold Nanoparticle-Mediated RNAi Biopesticide on the Third-Instar Larvae of the Hyphantria cuneiformis

[0078] The third-instar larvae of the gypsy moth with uniform size and good growth were selected and starved for 24 hours before the experiment.

[0079] This experimental example set up four experimental groups (C1-C4 groups), a simple naked siRNA group and a control group (CK).

[0080] The details are as follows:

[0081] The reagents used in groups C1-C4 were as follows: the gold nanoparticle-mediated RNAi biopesticide prepared in Example 1 was diluted to 6 ng / μL, 30 ng / μL, 15 ng / μL, and 5 ng / μL;

[0082] Naked siRNA group: Naked siRNA preparation was prepared, and the siRNA content in the siRNA preparation corresponded to that in group C1;

[0083] Control group: clean water was used as the medicine.

[0084] Each treatment group was set up with 3 replicates, and each replicate had 20 test insects.

[0085] Stomach poisoning test treatment: Take 10 mL of each test group of drugs and mix them evenly with fresh artificial feed of the American white moth, and feed them to the American white moth. The moth was fed with the drug-containing artificial feed throughout the experiment.

[0086] Contact toxicity test treatment: the larvae of the American white moth were completely immersed in the pesticides of each experimental group for 10-15 seconds, then removed and the excess pesticides were wiped off. During the experiment, the larvae were fed with fresh drug-free artificial feed.

[0087] After the treatment of the stomach poison test and the contact toxicity test, the surviving larvae were picked out at 12 hours and 24 hours, and the test insect samples were quickly frozen with liquid nitrogen. The acetylcholinesterase content of each sample was measured using an acetylcholinesterase content assay kit. The results are as follows: Figure 6 shown.

[0088] As can be seen from Table 6, using the same agent and the same treatment time, the acetylcholinesterase concentration of the stomach poison treatment is lower than that of the contact poison treatment, indicating that the stomach poison has a better killing effect. Under the same treatment time, the lower the concentration of the gold nano-mediated RNAi biological pesticide, the higher the acetylcholinesterase concentration. When the same agent is used, the acetylcholinesterase concentration gradually decreases as the treatment time increases. Compared with the naked siRNA group, the acetylcholinesterase concentration of the gold nano-mediated RNAi biological pesticide (C1 group) with the same siRNA content is much lower than that of the naked siRNA group. At the same time, the acetylcholinesterase concentration of the C2-C4 groups, which have lower siRNA content than the naked siRNA group, is also lower than that of the naked siRNA group. This shows that the gold nano-mediated RNAi biological pesticide has a good insecticidal effect and can also achieve the effect of reducing the amount and increasing the efficiency.

[0089] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a gold nanoparticle-mediated RNAi hydrogel biopharmaceutical, characterized in that: The following steps are involved: (1) adding a first polyethyleneimine solution to a polyethylene glycol-stabilized gold nanoparticle solution, performing ultrasonic and centrifugal purification, adding siRNA, performing ultrasonic and centrifugal purification, and then adding a second polyethyleneimine solution, performing ultrasonic and centrifugal purification to obtain a gold nanoparticle-mediated RNAi biopesticide; (2) dispersing the gold nanopesticide mediated RNAi biopesticide in deionized water to obtain a gold nanopesticide solution; adding an aqueous solution of dioctyl sodium sulfosuccinate to the gold nanopesticide solution, and subjecting the solution to ultrasonic and centrifugal purification to obtain a gold nanopesticide mediated RNAi biopesticide; (3) Adding a highly esterified apple pectin solution to the gold nano-mediated RNAi biopharmaceutical, and ultrasonicating to obtain a gold nano-mediated RNAi hydrogel biopharmaceutical.

2. The method for preparing the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 1, wherein: In step (1), the polyethylene glycol-stabilized gold nanoparticle solution is prepared by the following method: After heating the chloroauric acid solution, a trisodium citrate solution is added to react to obtain a gold nanoparticle solution; a polyethylene glycol solution is added to the gold nanoparticle solution, stirred, and then centrifuged and purified to obtain a polyethylene glycol-stabilized gold nanoparticle solution; The volume ratio of the chloroauric acid solution, the trisodium citrate solution and the polyethylene glycol solution is 100:(2.8-3.2):(5-10); the heating temperature of the chloroauric acid solution is 155-163° C., the reaction time is 15-25 minutes, and the stirring time is 0.5 hours to 24 hours.

3. The method for preparing the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 1, wherein: In step (2), the concentrations of the first polyethyleneimine solution and the second polyethyleneimine solution are both 0.1-2 mg / mL; the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution to the first polyethyleneimine solution is 1:50-50:1; and the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution to the second polyethyleneimine solution is 1:50-50:

1.

4. The method for preparing the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 1, wherein: In step (2), the concentration of siRNA is (0.01-2) μM, and the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution to siRNA is 1:100-100:

1.

5. The method for preparing the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 1, wherein: In step (2), the siRNA is a small interfering RNA targeting the developmental genes, olfactory genes and / or reproductive genes of the cuneiform moth.

6. The method for preparing the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 1, wherein: In step (3), the concentration of the dioctyl sodium salt of sulfosuccinate aqueous solution is 10 mg / mL; the volume ratio of the gold nanopesticide solution to the dioctyl sodium salt of sulfosuccinate solution is 1:1000-1000:1; and the ultrasonic time is 0.05-24 h.

7. The method for preparing the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 1, wherein: In step (4), the concentration of the high-esterified apple pectin solution is 1 mg / mL; the volume ratio of the gold nanoparticle-mediated RNAi biopharmaceutical to the high-esterified apple pectin solution is 1:1000-1000:1; and the ultrasonication time is 0.01-1 h.

8. The gold nanoparticle-mediated RNAi hydrogel biopharmaceutical prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the gold nanoparticle-mediated RNAi hydrogel biopharmaceutical according to claim 8 in controlling pests that feed on branches and leaves of fruit trees or in controlling agricultural pests.

10. The use according to claim 9, characterized in that The foliage-eating pests include the gypsy moth.

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