Preparation of nano biological agent for dual response-step-by-step delivery of small interfering RNA for pest control

Abstract

The invention discloses preparation of a nano biological agent for dual response-step-by-step delivery of small interfering RNA (Ribonucleic Acid) for preventing and treating diseases and insect pests, and relates to the technical field of pesticide preparations. The small interfering RNA is adsorbed on the surface of the gold nanoparticle through a layer-by-layer method and is embedded into the zinc-based organic metal framework nanoparticle, the small interfering RNA is controlled-released in a pH and enzyme dual-response manner, the gold nanoparticle loaded with the small interfering RNA is released through pH response decline of the zinc-based organic metal framework nanoparticle, the small interfering RNA is released under the action of intracellular enzyme, and a step-by-step release system is formed. The nano biological agent disclosed by the invention can be used for silencing key target genes of various diseases and insect pests and reducing the viability or lethal of the diseases and insect pests, and also can be used for adsorbing various small molecular pesticide raw medicines and realizing the effect of controlling the diseases and insect pests through co-carrying and co-delivery, so that the problem of RNAi (Ribonucleic Acid Interference) delivery is fundamentally solved, and a new thought is provided for the application of an RNAi technology.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide preparations, and in particular to the preparation of a nano-biological agent capable of dual response and step-by-step delivery of small interfering RNA for preventing and controlling pests and diseases. Background Art

[0002] RNA interference (RNAi) is a highly effective and specific gene silencing technique. It is triggered by double-stranded RNA and targets target genes. By inducing the specific degradation of homologous messenger RNA, it can reduce or shut down the expression of the relevant gene. This has been demonstrated in various insects to cause stunted development, lethality, or loss of vital functions. Compared with traditional pest control methods, RNAi is more specialized and targeted, providing not only sustained and effective pest control but also ecologically safe and friendly. The effective preventive and therapeutic applications of RNAi depend on the appropriate delivery of effective RNA. However, small interfering RNA (siRNA) is a large polyanionic molecule. Naked siRNA is unstable in tissue cells. Besides being immunogenic, it has difficulty escaping endosomal structures and is prone to off-target effects. To reach the cytoplasm where it is effective, it must traverse multiple biological barriers, ultimately resulting in low gene silencing efficiency. Furthermore, traditional delivery methods such as microinjection, oral administration, or immersion make it difficult to apply small interfering RNA in agricultural and forestry settings. To achieve effective delivery of double-stranded RNA, nano-delivery technologies have been developed and are widely used in agricultural and forestry pest control.

[0003] Currently, many nanomaterials are being used in nano-drug delivery systems due to their unique physicochemical properties, such as controllable size, low cytotoxicity, enhanced activity of the loaded components, and the ability to disrupt biomembrane barriers. The key to addressing RNAi technology is the development of efficient delivery systems. Nanoparticle-mediated RNAi technology can overcome specific biological barriers and induce functional gene silencing.

[0004] Therefore, a nanobiological agent is needed that can protect siRNA while having stable controlled release performance and long circulation ability in the body, thereby extending the duration of RNAi effect, improving the enrichment ability in target genes, and improving the prevention and control efficiency of pests such as agricultural and forestry diseases and insects. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a nanobiological agent for the dual-response, stepwise delivery of small interfering RNA for pest control. This nanobiological agent protects small interfering RNA (RNAi) while imparting stable controlled-release properties and long-term in vivo circulation, extending the duration of RNAi action and enhancing its ability to accumulate at target genes, thereby improving the effectiveness of controlling agricultural and forestry pests and diseases. This effectively addresses the problems of traditional pesticide formulations, such as low efficacy, poor controllability, inability to kill pests within tree trunks, and the tendency of pests to develop resistance.

[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 nanobiological agent capable of dual response and stepwise delivery of small interfering RNA, comprising the following steps:

[0008] (1) adding a sodium citrate solution to a boiling chloroauric acid solution to obtain a gold nanoparticle solution, then adding a polyethylene glycol solution to the gold nanoparticle solution to react, and centrifuging to obtain a polyethylene glycol-stabilized gold nanoparticle solution;

[0009] (2) adding the polyethyleneimine solution to the polyethylene glycol-stabilized gold nanoparticle solution, sonicating, centrifuging, and washing to obtain a first mixed solution, then adding the siRNA to the first mixed solution, sonicating, centrifuging, and washing to obtain a second mixed solution, and then adding the polyethyleneimine solution to the second mixed solution, sonicating, centrifuging, and washing to obtain the gold nanoparticle-loaded siRNA;

[0010] (3) The gold nanoparticle-loaded siRNA is dissolved in methanol to obtain a gold nanoparticle-loaded siRNA solution, and the zinc nitrate solution and the 2-methylimidazole solution are sequentially added to the gold nanoparticle-loaded siRNA solution. After the reaction, the mixture is centrifuged, and the precipitate is collected, washed, and dried to obtain a dual-response nanopharmaceutical for stepwise delivery of small interfering RNA.

[0011] Preferably, in step (1), the mass fraction of the sodium citrate solution is 1%, the mass fraction of the chloroauric acid solution is 0.01%; and the volume ratio of the chloroauric acid solution to the trisodium citrate solution is 100:(1-5).

[0012] Preferably, in step (1), the chloroauric acid solution is heated to 155-160° C. to obtain a boiling chloroauric acid solution.

[0013] Preferably, in step (1), the molecular weight of polyethylene glycol in the polyethylene glycol solution is 2000-5000, and the concentration of the polyethylene glycol solution is 0.01-5 mg / mL.

[0014] Wherein, the polyethylene glycol solution is added to the gold nanoparticle solution for reaction. One end group of the polyethylene glycol is a thiol group, which can form an Au-S bond with the gold nanoparticles.

[0015] Preferably, in step (1), the volume ratio of the gold nanoparticle solution to the polyethylene glycol solution is 100:(2.5-10).

[0016] Preferably, in step (1), the reaction time is 0.5-24h.

[0017] Preferably, in step (1), the centrifugal speed is 14000-20000 rpm, the centrifugal temperature is 0°C-8°C, and the centrifugal time is 20 min-50 min.

[0018] Preferably, in step (2), the polyethyleneimine solution is prepared by mixing polyethyleneimine and enzyme-free sterile water, and the concentration of the polyethyleneimine solution is 0.1-2 mg / mL.

[0019] Preferably, in step (2), the ultrasonic treatment is ice bath ultrasonication, and the ultrasonication time is 10-240 min.

[0020] Preferably, in step (2), during the centrifugation process, the centrifugal speed is 14000-16000 rpm, the centrifugal temperature is 0°C-4°C, and the centrifugal time is 20 min-50 min.

[0021] Preferably, in step (2), the washing operation is: washing with enzyme-free sterile water for 2-3 times.

[0022] Preferably, in step (2), the concentration of siRNA is 0.01-2.0 μM.

[0023] Preferably, in step (2), the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution, the siRNA solution and the polyethyleneimine solution is (4-6):1:(4-6).

[0024] Preferably, in step (3), the zinc nitrate solution is prepared by mixing zinc nitrate hexahydrate and methanol, and the concentration of the zinc nitrate solution is 25 mM; the 2-methylimidazole solution is prepared by mixing 2-methylimidazole and methanol, and the concentration of the 2-methylimidazole solution is 25 mM.

[0025] Preferably, in step (3), the concentration of the gold nanoparticles loaded with siRNA is 2-4 μg / mL.

[0026] Preferably, in step (3), the volume ratio of the gold nanoparticle-loaded siRNA solution, the zinc nitrate solution, and the 2-methylimidazole solution is (0.001-1000):1:1.

[0027] Preferably, in step (3), the reaction time is 12-24 hours.

[0028] Preferably, in step (3), during the centrifugation process, the centrifugal speed is 14000-16000 rpm, the centrifugal temperature is 0°C-4°C, and the centrifugal time is 20 min-40 min.

[0029] The second aspect of the present invention provides a nanobiological agent for dual response and stepwise delivery of small interfering RNA prepared by the above preparation method.

[0030] The dual-responsive, step-by-step delivery nanobiological agent of the present invention features dual pH and enzyme responses and step-by-step delivery of small interfering RNA. Specifically, the dual-responsive, step-by-step delivery of small interfering RNA involves pH- and enzyme-dependent controlled release of small interfering RNA. The zinc-based organometallic framework nanobiological agent degrades and releases small interfering RNA-loaded gold nanoparticles in response to pH. The small interfering RNA is then released under the action of intracellular enzymes, thereby forming a step-by-step release system. This dual-responsive, step-by-step delivery nanobiological agent protects the small interfering RNA while providing it with stable controlled-release properties and long-term in vivo circulation, extending the duration of RNAi activity and improving its ability to enrich target genes.

[0031] Preferably, the pH-responsive environment includes an acidic environment, a neutral environment and an alkaline environment.

[0032] The third invention of the present invention provides a use of a nanobiological agent in any one of the following 1)-5):

[0033] 1) Silencing target genes of pests and diseases;

[0034] 2) Prevent and control pests on fruit trees;

[0035] 3) Prevent and control harmful mites on crops;

[0036] 4) Prevent and control plant nematodes.

[0037] Preferably, the target genes include acetylcholinesterase gene, chitin deacetylase gene, tyrosine hydroxylase gene, epidermal protein gene, follicle stimulating hormone gene, luteinizing gene, apoptosis gene, cytochrome P450 oxidase gene, and glutathione-S-transferase gene.

[0038] Preferably, the forest and fruit trees include pine trees, birch trees, poplar trees, chestnut trees, willow trees, ginkgo trees, banyan trees, sycamore trees, mulberry trees, locust trees, and fruit trees.

[0039] Preferably, the pests include Lepidoptera, Coleoptera, Orthoptera, and Homoptera.

[0040] Preferably, the harmful mites include Acarina and Acarina.

[0041] Beneficial effects of the present invention:

[0042] The present invention first uses polyethylene glycol and chloroauric acid to prepare polyethylene glycol-stabilized gold nanoparticles. Small interfering RNA is then adsorbed onto the gold nanoparticle surface using a layer-by-layer method and embedded within zinc-based organometallic framework nanoparticles to produce a nanobiological agent. The resulting nanobiological agent exhibits dual-response characteristics, enabling step-by-step delivery of small interfering RNA.

[0043] The dual-responsive, step-by-step delivery nanopharmaceutical for small interfering RNA (siRNA) prepared by the present invention can achieve pH- and enzyme-responsive controlled release of siRNA. The zinc-based organometallic framework (MOF) nanoparticles release siRNA-loaded gold nanoparticles in response to pH degradation, and the siRNA is then released under the action of intracellular enzymes, forming a step-by-step release system. This protects the siRNA while imparting stable controlled-release properties and long-term in vivo circulation, extending the duration of the RNAi effect. Furthermore, by combining gold nanoparticles and zinc-based MOF nanoparticles to load siRNA, the present invention achieves a synergistic effect in increasing the duration of the nanopesticide's effectiveness.

[0044] The small interfering RNA nanopharmaceuticals prepared by this invention can silence key target genes of various pests and diseases, reducing their viability or even causing their lethality. Furthermore, by adsorbing multiple small molecule pesticide technicals, they can achieve co-loading and co-delivery, effectively and synergistically controlling pests and diseases. The preparation method of this invention is environmentally friendly and pollution-free, with high product yield, simple preparation process, and low equipment requirements. It is suitable for both single and combined control in various scenarios, and is particularly well-suited for large-scale control measures such as aerial spraying. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The zeta potential diagrams at various stages in the preparation process of the nanobiological preparation of the present invention are shown.

[0046] Figure 2 The figures are scanning electron microscope morphology images and transmission electron microscope morphology images of the nanobiopreparation prepared at different preparation times, wherein (a) is a scanning electron microscope image of the nanobiopreparation prepared in Example 2, (b) is a scanning electron microscope image of the nanobiopreparation prepared in Example 3, (c) is a scanning electron microscope image of the nanobiopreparation prepared in Example 4, (d) is a scanning electron microscope image of the nanobiopreparation prepared in Example 1, (e) is a transmission electron microscope morphology image of the nanobiopreparation prepared in Example 2, (f) is a transmission electron microscope morphology image of the nanobiopreparation prepared in Example 3, (g) is a transmission electron microscope morphology image of the nanobiopreparation prepared in Example 4, and (h) is a transmission electron microscope morphology image of the nanobiopreparation prepared in Example 1.

[0047] Figure 3 The element overlay image and element distribution image of the nanobiological agent prepared in Example 1.

[0048] Figure 4 This is a graph showing the expression of acetylcholinesterase after contact / stomach poisoning of nymphalidae larvae by the nanobiological agent prepared in Example 1.

[0049] Figure 5 :The diagram shows the silencing effect of different concentrations of nanobiological agents on acetylcholinesterase gene, chitin synthase gene and tyrosine hydroxylase gene.

[0050] Figure 6 This is a diagram showing the control effect of the pesticides in each treatment group on pine sawyer.

[0051] Figure 7 This is a diagram showing the inhibitory effect of the pesticides in each treatment group on citrus mites.

[0052] Figure 8 The diagram shows the control effect of the pesticides in each treatment group on nematodes. DETAILED DESCRIPTION

[0053] 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.

[0054] The "room temperature" in the present invention refers to a temperature of 10°C-30°C.

[0055] As introduced in the background technology section, RNA interference (RNAi) technology can reduce or shut down the expression of related genes by inducing the specific degradation of homologous messenger RNA. However, naked siRNA is unstable in tissue cells. In addition to being immunogenic, it is difficult to escape from endosomes and is prone to off-target. To reach the cytoplasm where it exerts its effect, it must pass through multiple biological barriers, which ultimately leads to low gene silencing efficiency. Nanomaterials are used in nano-delivery systems due to their unique physicochemical properties, such as controllable size, low cytotoxicity, enhanced activity of loaded components, and the ability to break through biological membrane barriers. Nanoparticle-mediated RNAi technology can overcome specific biological barriers to induce functional gene silencing.

[0056] Based on this, the present invention provides a dual-responsive nanobiological agent for pest control, featuring step-by-step delivery of small interfering RNA. Through a layer-by-layer approach, small interfering RNA is adsorbed onto the surface of gold nanoparticles and then embedded within zinc-based organometallic framework nanoparticles, resulting in a pH- and enzyme-responsive controlled release of the small interfering RNA. The zinc-based organometallic framework nanoparticles release the small interfering RNA-loaded gold nanoparticles in response to pH, and the small interfering RNA is then released under the action of intracellular enzymes, forming a step-by-step release system. This protects the small interfering RNA while providing it with stable controlled-release properties and long-term in vivo circulation, extending the duration of the RNAi effect and improving its enrichment in target genes. The small interfering RNA nanobiological agent of the present invention can silence key target genes of various pests and diseases, reduce the survival ability of pests and diseases or cause death. In addition, the biological agent can adsorb a variety of small molecule pesticide original drugs, achieve co-carrying, co-delivery, high efficiency, and synergistic control of pests and diseases, and is suitable for single and combined prevention and control in various scenarios, especially for large-scale prevention and control measures such as aerial spraying. It fundamentally solves the problem of RNAi delivery and provides new ideas for the application of RNAi technology.

[0057] 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.

[0058] 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.

[0059] In the present invention, the nucleotide sequence of the acetylcholinesterase gene siRNA is:

[0060] 5'-CUCGAAGCAUGUCGUUUAATT-3'

[0061] 5'-UUAAACGACAUGCUUCGAGTT-3'.

[0062] Example 1: Preparation of Nanobiological Agents Loaded with Dual-Response-Gradual Delivery of Small Interfering RNAs Interfering with Acetylcholinesterase Genes of Hyphantria cuneiformis

[0063] (1) A 0.01% chloroauric acid solution was heated to boiling at 160° C., 7.5 mL of a 1% sodium citrate solution was added to the boiling chloroauric acid solution and mixed for 20 minutes to obtain a bright red solution, which was then placed in ice water and cooled to room temperature to obtain a gold nanoparticle solution. The gold nanoparticles in the gold nanoparticle solution had a size of 13 nm.

[0064] 15 mL of a 0.5 mg / mL polyethylene glycol solution was added to 150 mL of a gold nanoparticle solution and reacted at room temperature for 3 h. After the reaction was completed, the mixture was centrifuged at 4°C and 17,000 rpm for 35 min and purified three times to obtain a polyethylene glycol-stabilized gold nanoparticle solution, wherein the molecular weight of the polyethylene glycol in the polyethylene glycol solution was 2,000.

[0065] (2) Polyethyleneimine and enzyme-free sterile water were mixed to prepare a polyethyleneimine solution with a concentration of 1 mg / mL, and the polyethyleneimine solution was added to the polyethylene glycol-stabilized gold nanoparticle solution. After ultrasonic treatment in an ice bath for 30 minutes, the mixture was centrifuged at 15,000 rpm and 2°C for 35 minutes. The precipitate after centrifugation was then washed three times with enzyme-free sterile water to remove excess polyethyleneimine, thereby obtaining a first mixed solution.

[0066] 0.5 μM nymphal acetylcholinesterase siRNA was mixed with the first mixed solution, and then sonicated in an ice bath in the dark for 40 minutes. The mixture was centrifuged at 15,000 rpm and 2°C for 35 minutes, and the precipitate was washed three times with enzyme-free sterile water to remove unbound siRNA, thereby obtaining a second mixed solution.

[0067] The second mixed solution was then mixed with 10 mL of polyethyleneimine solution, and the mixture was ultrasonicated on ice for 30 minutes. The mixture was then centrifuged at 15,000 rpm and 2°C for 35 minutes. The precipitate was then washed three times with enzyme-free sterile water to remove unbound polyethyleneimine, thereby obtaining gold nanoparticle-loaded siRNA. The volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution, siRNA solution, and polyethyleneimine solution was 1:1:1.

[0068] (3) The gold nanoparticles loaded with siRNA and methanol were mixed in a mass ratio of 1:1 to obtain a gold nanoparticles loaded with siRNA solution; zinc nitrate hexahydrate and methanol were mixed to obtain a zinc nitrate solution with a concentration of 25 mM; 2-methylimidazole and methanol were mixed to obtain a 2-methylimidazole solution with a concentration of 25 mM; the zinc nitrate solution and the 2-methylimidazole solution were added to the gold nanoparticles loaded with siRNA solution in a volume ratio of 10:1:1, and the mixture was allowed to stand for 15 minutes at room temperature, centrifuged at 14000 rpm and 4°C for 30 minutes, the precipitate after centrifugation was collected and washed and purified with methanol three times, then transferred to the aqueous phase, and freeze-dried to obtain a nanoparticle biopharmaceutical that loaded with the dual response-stepwise delivery of small interfering RNA that interfered with the acetylcholinesterase gene of the American white moth.

[0069] Example 2: Preparation of Nanobiological Agents Loaded with Dual Response-Gradual Delivery of Small Interfering RNAs Interfering with Acetylcholinesterase Genes of Hyphantria cuneiformis

[0070] (1) A 0.01% chloroauric acid solution was heated to boiling at 160° C. 10 mL of a 1% sodium citrate solution (250 mL) was added to the boiling chloroauric acid solution and mixed for 20 min to obtain a red solution. The red solution was placed in ice water and cooled to room temperature to obtain a gold nanoparticle solution.

[0071] 3.75 mL of 0.01 mg / mL polyethylene glycol solution was added to 150 mL of gold nanoparticle solution and reacted at room temperature for 0.5 h. After the reaction was completed, the mixture was centrifuged at 0°C and 14,000 rpm for 20 min and purified three times to obtain a polyethylene glycol-stabilized gold nanoparticle solution, wherein the molecular weight of the polyethylene glycol in the polyethylene glycol solution was 3,500;

[0072] (2) Polyethyleneimine and enzyme-free sterile water were mixed to prepare a polyethyleneimine solution with a concentration of 0.1 mg / mL, and the polyethyleneimine solution was added to the polyethylene glycol-stabilized gold nanoparticle solution. After ultrasonic treatment in an ice bath for 10 minutes, the mixture was centrifuged at 14,000 rpm and 0°C for 20 minutes. The precipitate after centrifugation was then washed three times with enzyme-free sterile water to remove excess polyethyleneimine, thereby obtaining a first mixed solution.

[0073] 0.01 μM nymphaloid acetylcholinesterase siRNA was mixed with the first mixed solution, and then sonicated in an ice bath in the dark for 40 minutes. The mixture was centrifuged at 14,000 rpm and 0°C for 20 minutes, and the precipitate was washed three times with enzyme-free sterile water to remove unbound siRNA, thereby obtaining a second mixed solution.

[0074] The second mixed solution and the polyethyleneimine solution were then mixed, ultrasonicated on ice for 40 minutes, and centrifuged at 14,000 rpm and 0°C for 20 minutes. The precipitate was then washed three times with enzyme-free sterile water to remove unbound polyethyleneimine, thereby obtaining the gold nanoparticle-loaded siRNA.

[0075] The volume ratio of polyethylene glycol-stabilized gold nanoparticle solution, siRNA solution, and polyethyleneimine solution was 1:1:1;

[0076] (3) The gold nanoparticles loaded with siRNA and methanol were mixed at a mass ratio of 1:5 to obtain a gold nanoparticles loaded with siRNA solution; zinc nitrate hexahydrate and methanol were mixed to obtain a zinc nitrate solution with a concentration of 25 mM; 2-methylimidazole and methanol were mixed to obtain a 2-methylimidazole solution with a concentration of 25 mM; the zinc nitrate solution and the 2-methylimidazole solution were added to the gold nanoparticles loaded with siRNA solution at a volume ratio of 0.01:1:1, and the mixture was allowed to stand for 30 minutes at room temperature, centrifuged at 14000 rpm and 0°C for 20 minutes, the precipitate after centrifugation was collected and washed and purified with methanol for 3 times, then transferred to the aqueous phase, and freeze-dried to obtain a nanobiological agent loaded with a dual response-stepwise delivery of small interfering RNA that interferes with the acetylcholinesterase gene of the American white moth.

[0077] Example 3: Preparation of Nanobiological Agents Loaded with Dual Response-Gradual Delivery of Small Interfering RNAs that Interfere with Acetylcholinesterase Genes of Hyphantria cuneiformis

[0078] (1) A 0.01% chloroauric acid solution was heated to boiling at 160° C. 20 mL of a 1% sodium citrate solution was added to the boiling chloroauric acid solution and mixed for 20 min to obtain a red solution. The red solution was placed in ice water and cooled to room temperature to obtain a gold nanoparticle solution.

[0079] 7.5 mL of a 5 mg / mL polyethylene glycol solution was added to 150 mL of a gold nanoparticle solution and reacted at room temperature for 24 h. After the reaction was completed, the mixture was centrifuged at 8°C and 20,000 rpm for 50 min and purified three times to obtain a polyethylene glycol-stabilized gold nanoparticle solution, wherein the molecular weight of the polyethylene glycol in the polyethylene glycol solution was 5,000;

[0080] (2) Polyethyleneimine and enzyme-free sterile water were mixed to prepare a polyethyleneimine solution with a concentration of 2 mg / mL, and the polyethyleneimine solution was added to the polyethylene glycol-stabilized gold nanoparticle solution. After ultrasonic treatment in an ice bath for 240 min, the mixture was centrifuged at 16,000 rpm and 4°C for 50 min. The precipitate after centrifugation was washed three times with enzyme-free sterile water to remove excess polyethyleneimine, thereby obtaining a first mixed solution.

[0081] 2.0 μM nymphal acetylcholinesterase siRNA was mixed with the first mixed solution, and then sonicated in an ice bath in the dark for 240 min. The mixture was centrifuged at 16,000 rpm and 4°C for 50 min, and the precipitate was washed three times with enzyme-free sterile water to remove unbound siRNA, thereby obtaining a second mixed solution.

[0082] The second mixed solution and the polyethyleneimine solution were then mixed, ultrasonicated on ice for 40 minutes, and centrifuged at 16,000 rpm and 4°C for 50 minutes. The precipitate was then washed three times with enzyme-free sterile water to remove unbound polyethyleneimine, thereby obtaining the gold nanoparticle-loaded siRNA.

[0083] The volume ratio of polyethylene glycol-stabilized gold nanoparticle solution, siRNA solution, and polyethyleneimine solution was 1:1:3;

[0084] (3) The gold nanoparticles loaded with siRNA and methanol were mixed in a mass ratio of 1:3 to obtain a gold nanoparticles loaded with siRNA solution; zinc nitrate hexahydrate and methanol were mixed to obtain a zinc nitrate solution with a concentration of 25 mM; 2-methylimidazole and methanol were mixed to obtain a 2-methylimidazole solution with a concentration of 25 mM; the zinc nitrate solution and the 2-methylimidazole solution were added to the gold nanoparticles loaded with siRNA solution in a volume ratio of 1000:1:1, and the mixture was allowed to stand for 1 hour at room temperature, centrifuged at 16000 rpm and 4°C for 50 minutes, the precipitate after centrifugation was collected and washed and purified with methanol for 3 times, then transferred to the aqueous phase and freeze-dried to obtain a nanobiological agent loaded with a dual response-stepwise delivery of small interfering RNA that interferes with the acetylcholinesterase gene of the American white moth.

[0085] Example 4: Preparation of Nanobiological Agents Loaded with Dual Response-Gradual Delivery of Small Interfering RNAs Interfering with Acetylcholinesterase Genes of Hyphantria cuneiformis

[0086] (1) A 0.01% chloroauric acid solution was heated to boiling at 160° C., 7.5 mL of a 1% sodium citrate solution was added to the boiling chloroauric acid solution and mixed for 20 minutes to obtain a bright red solution, which was then placed in ice water and cooled to room temperature to obtain a gold nanoparticle solution. The gold nanoparticles in the gold nanoparticle solution had a size of 13 nm.

[0087] 15 mL of a 0.5 mg / mL polyethylene glycol solution was added to 150 mL of a gold nanoparticle solution and reacted at room temperature for 3 h. After the reaction was completed, the mixture was centrifuged at 4°C and 17,000 rpm for 35 min and purified three times to obtain a polyethylene glycol-stabilized gold nanoparticle solution, wherein the molecular weight of the polyethylene glycol in the polyethylene glycol solution was 2,000.

[0088] (2) Polyethyleneimine and enzyme-free sterile water were mixed to prepare a polyethyleneimine solution with a concentration of 1 mg / mL, and the polyethyleneimine solution was added to the polyethylene glycol-stabilized gold nanoparticle solution. After ultrasonic treatment in an ice bath for 30 minutes, the mixture was centrifuged at 15,000 rpm and 2°C for 35 minutes. The precipitate after centrifugation was then washed three times with enzyme-free sterile water to remove excess polyethyleneimine, thereby obtaining a first mixed solution.

[0089] 0.5 μM nymphal acetylcholinesterase siRNA was mixed with the first mixed solution, and then sonicated in an ice bath in the dark for 40 minutes. The mixture was centrifuged at 15,000 rpm and 2°C for 35 minutes, and the precipitate was washed three times with enzyme-free sterile water to remove unbound siRNA, thereby obtaining a second mixed solution.

[0090] The second mixed solution was then mixed with 10 mL of polyethyleneimine solution, and then ultrasonicated on ice for 30 minutes. The mixture was then centrifuged at 15,000 rpm and 2°C for 35 minutes. The precipitate was then washed three times with enzyme-free sterile water to remove unbound polyethyleneimine, thereby obtaining the gold nanoparticle-loaded siRNA.

[0091] The volume ratio of ethylene glycol-stabilized gold nanoparticle solution, siRNA solution, and polyethyleneimine solution was 1:1:5;

[0092] (3) The gold nanoparticles loaded with siRNA and methanol were mixed at a mass ratio of 11:5 to obtain a gold nanoparticles loaded with siRNA solution; zinc nitrate hexahydrate and methanol were mixed to obtain a zinc nitrate solution with a concentration of 25 mM; 2-methylimidazole and methanol were mixed to obtain a 2-methylimidazole solution with a concentration of 25 mM; the zinc nitrate solution and the 2-methylimidazole solution were added to the gold nanoparticles loaded with siRNA solution at a volume ratio of 100:1:1, and the mixture was allowed to stand at room temperature for 24 hours, and centrifuged at 14000 rpm and 4°C for 30 minutes. The precipitate after centrifugation was collected and washed and purified with methanol three times, and then transferred to the aqueous phase and freeze-dried to obtain a nanobiological agent loaded with a dual response-stepwise delivery of small interfering RNA that interferes with the acetylcholinesterase gene of the American white moth.

[0093] Test Example 1:

[0094] Zeta potential analysis was performed on the materials at each stage of the preparation process of the nanobiological agent in Example 1. The results are as follows: Figure 1 The nanobiological preparations prepared in Examples 1-4 were subjected to scanning electron microscopy and transmission electron microscopy analysis, and the results were as follows: Figure 2 The element superposition and element distribution analysis of the nanobiological agent prepared in Example 1 were performed, and the results were as follows: Figure 3 shown.

[0095] pass Figure 1 From the Zata potential analysis diagram, it can be seen that after RNA encapsulation, the gold nanoparticle surface has a strong positive charge. Figure 2 The preparation time is the time for the MOF to combine with the gold nanoparticles in step (2). Figure 2 It can be seen that as the preparation time increases, the size of the nanocarrier gradually increases. Figure 3 It can be seen that there is P element on the gold nanoparticles, and P element is a characteristic element of RNA. This shows that RNA is successfully encapsulated on the surface of gold nanoparticles.

[0096] Test Example 2:

[0097] The contact and stomach toxicity of the nanobiological agent prepared in Example 1 to the larvae of the American white moth and the new expression levels of the acetylcholinesterase gene, chitin synthase gene and tyrosine hydroxylase gene were measured. The results are as follows: Figure 4 As shown in Table 1-Table 2. The specific steps are:

[0098] (1) Select the third-instar larvae of the American moth with uniform size and good growth as the test insects and starve them for 24 hours.

[0099] (2) The nanobiological agent prepared in Example 1 was mixed with deionized water to obtain 60 μg / mL, 30 μg / mL, and 15 μg / mL nanobiological agent solutions, respectively. Two treatment groups, one containing the vehicle alone and one containing naked siRNA, and a water control group (CK) were also set up. Each treatment was replicated three times, with 20 test insects in each replicate.

[0100] 1. Stomach poison test: 10 mL of each agent prepared in step (1) was mixed evenly with fresh artificial diet for the cuneiform moth and fed to starved cuneiform moths. The medicated artificial diet was continuously fed during the experiment. Feeding was carried out at 25°C and 55% RH. The number of dead insects after 12 h, 24 h, 48 h, and 74 h of feeding was counted, and the adjusted mortality rate was calculated. The results are shown in Table 1.

[0101] 2. Contact Killing Experiment: The larvae of the cuneiform moth were completely immersed in the agent prepared in step (1) for 15 seconds. The excess agent was removed and wiped off. During the experiment, the larvae were fed a fresh, drug-free artificial diet at 25°C and 55% RH. The number of dead larvae was counted and the corrected mortality rate was calculated 12, 24, 48, and 72 hours after the agent treatment. The results are shown in Table 2.

[0102] The formula for calculating the adjusted mortality rate is: adjusted mortality rate (%) = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate)] × 100.

[0103] 3. Relative expression of acetylcholinesterase gene: The insects that survived the stomach poison test and the contact poison test were selected, rapidly frozen in liquid nitrogen, and ground. The total RNA of the samples was extracted using the Trizol method, reverse transcribed into cDNA, and primers were added. The expression of acetylcholinesterase in each treatment group was obtained by fluorescence quantitative PCR. The results are shown in the figure below. Figure 4 shown.

[0104] Table 1 Corrected mortality of nymphalid moth larvae after stomach poisoning

[0105]

[0106] As can be seen from Table 1, the insecticidal effect of the nanobiological agent with dual response and stepwise delivery of small interfering RNA prepared by the present invention is better than that of naked siRNA alone, and the carrier is almost non-toxic. Figure 4 It can be seen that the gene interference efficiency of the dual-response nanobiological agent with stepwise delivery of small interfering RNA is better than that of naked siRNA alone, and the carrier has almost no effect on the expression of the acetylcholinesterase gene. This shows that the nanobiological agent prepared by the present invention has a good insecticidal effect.

[0107] Table 2 Corrected mortality of nymphalid moth larvae after contact treatment As can be seen from Table 2, the insecticidal effect of the nanobiological agent with dual response and step-by-step delivery of small interfering RNA prepared by the present invention is better than that of naked siRNA alone, and the carrier is almost non-toxic.

[0108] Depend on Figure 4 It can be seen that the expression level of acetylcholinesterase in the dual-response-stepwise delivery of small interfering RNA nanopharmaceutical prepared by the present invention is significantly lower than that in the other treatment groups. This shows that the nanopharmaceutical prepared by the present invention can silence the acetylcholinesterase gene of the American cunea.

[0109] Test Example 3:

[0110] The nanopesticide prepared in Example 1 was mixed with deionized water and prepared into four different concentrations: 0.1 μg / μL, 0.5 μg / μL, 1.0 μg / μL, and 3.0 μg / μL. The nanopesticide solutions of different concentrations were sprayed onto the surface of diamondback moth. After 72 hours of treatment, the relative expression levels of acetylcholine gene, chitin synthase gene, and tyrosine hydroxylase gene were determined by fluorescence quantitative analysis. The results are as follows: Figure 5 shown.

[0111] Depend on Figure 5 It can be seen that the nanobiological agent prepared by the present invention has a good silencing effect on the acetylcholinesterase gene, chitin synthase gene, and tyrosine hydroxylase gene. Among them, the 3.0μg / μL nanobiological agent solution has the best silencing effect on the acetylcholinesterase gene, chitin synthase gene, and tyrosine hydroxylase gene.

[0112] Test Example 4:

[0113] The control effect of the nanobiological agent prepared by the present invention on Monochamus alternatus, Panonychus citri and nematodes was investigated. The details are as follows:

[0114] 1. The nanobiological agent and dsRNA prepared in Example 1 were prepared into solutions with concentrations of 0.5 μg / μL, 1.0 μg / μL, 2.0 μg / μL, and 3.0 μg / μL, respectively. A water control group was also set up. The different concentrations of the agent were sprayed on the surface of four-week-old Monochamus alternatus larvae for contact killing. The same volume of water was sprayed as a control. The results are shown in Figure 2. Figure 6 shown.

[0115] 2. The nanobiological agent and dsRNA prepared in Example 1 were prepared into solutions with concentrations of 0.2 μg / μL, 0.8 μg / μL, 1.6 μg / μL, and 3.2 μg / μL, respectively. A water control group was also set up. The different concentrations of the agent were sprayed on the surface of citrus larvae for contact killing. The same volume of water was sprayed as a control. The results are shown in Figure 2. Figure 7 shown.

[0116] 3. The nanobiological agent and dsRNA prepared in Example 1 were prepared into solutions with concentrations of 0.1 μg / μL, 0.5 μg / μL, 1.0 μg / μL, and 2.0 μg / μL, respectively. A water control group was also set up. The different concentrations of the agent were sprayed on the surface of the nematodes for contact killing, and the same volume of water was sprayed as a control. The results are shown in Figure 2. Figure 8 shown.

[0117] Depend on Figure 6-Figure 8 It can be seen that compared with the control group and the dsRNA group, the nanobiological agent prepared by the present invention has a good control effect on Monochamus alternatus, Panonychus citri and nematodes.

[0118] 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 nanobiological agent for dual response and stepwise delivery of small interfering RNA, characterized in that: The following steps are involved: (1) adding a sodium citrate solution to a boiling chloroauric acid solution to obtain a gold nanoparticle solution, then adding a polyethylene glycol solution to the gold nanoparticle solution to react, and centrifuging to obtain a polyethylene glycol-stabilized gold nanoparticle solution; (2) adding the polyethyleneimine solution to the polyethylene glycol-stabilized gold nanoparticle solution, ultrasonically, centrifugally, and washing to obtain a first mixed solution, then adding the siRNA to the first mixed solution, ultrasonically, centrifugally, and washing to obtain a second mixed solution, then adding the polyethyleneimine solution to the second mixed solution, ultrasonically, centrifugally, and washing to obtain the gold nanoparticle-loaded siRNA; (3) The gold nanoparticle-loaded siRNA is dissolved in a methanol solution to obtain a gold nanoparticle-loaded siRNA solution, and a zinc nitrate solution and a 2-methylimidazole solution are sequentially added to the gold nanoparticle-loaded siRNA solution. After the reaction, the solution is centrifuged, and the precipitate after centrifugation is collected, washed, and dried to obtain a dual-response nanopharmaceutical for step-by-step delivery of small interfering RNA.

2. The preparation method according to claim 1, wherein In step (1), the mass fraction of the sodium citrate solution is 1%, the mass fraction of the chloroauric acid solution is 0.01%; and the volume ratio of the chloroauric acid solution to the trisodium citrate solution is 100:(1-5).

3. The preparation method according to claim 1, wherein In step (1), the molecular weight of polyethylene glycol in the polyethylene glycol solution is 2000-5000, the concentration of the polyethylene glycol solution is 0.01-5 mg / mL; and the volume ratio of the gold nanoparticle solution to the polyethylene glycol solution is 100:(2.5-10).

4. The preparation method according to claim 1, wherein In step (1), the reaction time is 0.5-24h; the centrifugal speed is 14000-20000rpm, the centrifugal temperature is 0℃-8℃, and the centrifugal time is 20min-50min.

5. The preparation method according to claim 1, wherein In step (2), the concentration of siRNA is 0.01-2.0 μM; the volume ratio of the polyethylene glycol-stabilized gold nanoparticle solution, the siRNA solution and the polyethyleneimine solution is (4-6):1:(4-6).

6. The preparation method according to claim 1, wherein In step (3), the volume ratio of the gold nanoparticle-loaded siRNA solution, the zinc nitrate solution, and the 2-methylimidazole solution is (0.001-1000):1:1; the reaction time is 12-24 hours; during the centrifugation process, the centrifugal speed is 14000-16000 rpm, the centrifugal temperature is 0°C-4°C, and the centrifugal time is 20 min-40 min.

7. A nanobiological agent for dual response and stepwise delivery of small interfering RNA prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the dual-response nanobiological agent for stepwise delivery of small interfering RNA according to claim 7 in any one of the following 1) to 5): 1) Silencing target genes of pests and diseases; 2) Prevent and control pests on fruit trees; 3) Prevent and control harmful mites on crops; 4) Prevent and control plant nematodes.

9. The use according to claim 8, characterized in that The target genes include acetylcholinesterase gene, chitin deacetylase gene, tyrosine hydroxylase gene, epidermal protein gene, follicle-stimulating hormone gene, luteinizing gene, apoptosis gene, cytochrome P450 oxidase gene, and glutathione-S-transferase gene; the small molecule pesticide technical includes avermectin, emamectin benzoate, pyrethrin, matrine, azadirachtin, spirotetramat, rotenone, sophora flavescens, spirodiclofen, imidacloprid, fluopyram, kresoxim-methyl, and kresoxim-methyl.

10. The use according to claim 8, characterized in that The pests include Lepidoptera, Coleoptera, Orthoptera, and Homoptera; the harmful mites include Acarina and Acarina; and the plant pathogenic microorganisms include fungi, bacteria, and actinomycetes.

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