Preparation method of pH-responsive release nucleic acid delivery system

Abstract

The invention provides a preparation method of a pH-responsive release nucleic acid delivery system, and belongs to the technical field of nucleic acid delivery systems.The preparation method comprises the steps that 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is used for enhancing the amino density of the surface of bovine serum albumin, modified bovine serum albumin and double-stranded ribonucleic acid are mixed according to the mass ratio of 10: 1, and a mixture is obtained; the preparation method comprises the following steps: adding glutaraldehyde as a cross-linking agent under the condition that the ratio of water to ethanol is 3: 1 to form Schiff base, reacting at 4 DEG C for 4-5 hours, and then performing ultrafiltration purification to obtain the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles, thereby solving the technical problem of poor stability of double-stranded ribonucleic acid in noctuidae insects. And an efficient and accurate technical solution is provided for agricultural pest control.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid delivery systems, and more particularly, relates to a preparation method of a nucleic acid delivery system with pH-responsive release. Background Art

[0002] RNA interference technology is a sequence-specific gene expression inhibition technology and has broad application prospects in the field of agricultural pest control. Traditional methods for controlling noctuid pests mainly rely on chemical pesticides, which have problems such as environmental pollution and pest resistance. Using double-stranded RNA interference technology can specifically inhibit the expression of key genes in pests and achieve precise prevention and control. Currently, the delivery methods of double-stranded RNA mainly include direct spraying, microinjection, or feeding with mixed feed, etc.

[0003] However, noctuid insects have a special digestive system environment, in which the intestine is strongly alkaline (pH 9 - 11) and contains various nucleases and proteases. When double-stranded RNA enters this environment, it is extremely easy to be degraded or inactivated, resulting in extremely poor stability in the body. Research shows that the half-life of directly delivered double-stranded RNA in the intestine of noctuid insects usually does not exceed 2 hours, and most of the nucleic acid has been degraded before reaching the target cells, seriously affecting the gene interference effect.

[0004] Currently, methods tried to improve the stability of double-stranded RNA include technologies such as chemical modification and nano-carrier encapsulation, etc. However, these methods are either too costly or have poor adaptability to the insect physiological environment. Existing nano-carriers such as liposomes, polymer nanoparticles, etc. are often structurally unstable in the alkaline intestinal environment of noctuid insects and cannot effectively protect double-stranded RNA from degradation. Therefore, how to design a double-stranded RNA delivery system that can remain stable in the special physiological environment of noctuid insects has become an urgent technical problem to be solved. That is to say, there is a technical problem of poor stability of double-stranded RNA in noctuid insects in the prior art. Summary of the Invention

[0005] In view of this, the present invention provides a preparation method of a nucleic acid delivery system with pH-responsive release, which can solve the technical problem of poor stability of double-stranded RNA in noctuid insects existing in the prior art.

[0006] The present invention is implemented as follows: The present invention provides a method for preparing a nucleic acid delivery system with pH-responsive release, which includes: reacting bovine serum albumin with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to prepare modified bovine serum albumin; adjusting the modified bovine serum albumin to a specified concentration and pH value; mixing the modified bovine serum albumin and double-stranded ribonucleic acid according to a mass ratio; adding glutaraldehyde as a cross-linking agent to the mixed solution to form a cross-linked complex; reacting the mixture under low-temperature conditions to form a cross-linked complex; using an ultrafiltration membrane for purification to obtain double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles; the nanoparticles are stable under alkaline conditions and rapidly release ribonucleic acid under acidic conditions, solving the technical problem of poor stability of double-stranded ribonucleic acid in noctuid insects.

[0007] Among them, the step of reacting bovine serum albumin with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to prepare modified bovine serum albumin is specifically to react in phosphate buffer for 2 hours to prepare modified bovine serum albumin with a surface amino density 3 to 5 times that of the original bovine serum albumin.

[0008] Among them, the step of adjusting the modified bovine serum albumin to a specified concentration and pH value is specifically to adjust the concentration of the modified bovine serum albumin to 2.5 micrograms per microliter and the pH value of the solution to 9.

[0009] Among them, the step of mixing the modified bovine serum albumin and double-stranded ribonucleic acid according to a mass ratio is specifically to mix the modified bovine serum albumin and double-stranded ribonucleic acid according to a mass ratio of 10:1, and the ratio of water to ethanol is 3:1.

[0010] Among them, the step of adding glutaraldehyde as a cross-linking agent to the mixed solution is specifically that the addition amount is 0.04 to 0.08 moles per gram of protein.

[0011] Among them, the step of reacting the mixture under low-temperature conditions to form a cross-linked complex is specifically to stand and react at 4 °C for 4 to 5 hours.

[0012] Among them, the step of using an ultrafiltration membrane for purification to obtain double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles is specifically to use an ultrafiltration membrane with a cut-off molecular weight of 10 kDa for purification.

[0013] Among them, the step of adding glutaraldehyde as a cross-linking agent to the mixed solution further includes a step of optimizing the cross-linking reaction kinetics, specifically by real-time monitoring of the kinetic parameters of the reaction of glutaraldehyde with the surface amino groups of modified bovine serum albumin, and adjusting the addition method of the cross-linking agent according to the kinetic equation of Schiff base formation reaction.

[0014] Among them, the kinetic equation of the Schiff base formation reaction is used to predict the process and efficiency of the cross-linking reaction under different reaction conditions. The inputs include the concentration of modified bovine serum albumin, the concentration of glutaraldehyde, the pH value of the solution, the ionic strength of the solution, and the reaction temperature, and the outputs are the Schiff base formation rate and the final cross-linking degree.

[0015] Among them, the step of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles remaining stable under alkaline conditions and rapidly releasing ribonucleic acid under acidic conditions further includes a release prediction and optimization step. Specifically, a pre-trained dynamic neural network model for nanodelivery release is used to predict the release curve of double-stranded ribonucleic acid under different pH conditions, and the structural parameters of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles are optimized accordingly.

[0016] The present invention constructs nanoparticles with high stability through surface amination modification and glutaraldehyde cross-linking. This delivery system exhibits excellent stability in the alkaline environment of the intestine of Noctuidae insects, and the cumulative release amount remains below 80% within 36 hours, effectively protecting double-stranded ribonucleic acid from degradation in the alkaline environment and by digestive enzymes.

[0017] The present invention overcomes the technical defect of poor stability of double-stranded ribonucleic acid in traditional delivery methods. By using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to enhance the surface amino density of bovine serum albumin, the binding ability with double-stranded ribonucleic acid is improved; at the same time, a Schiff base cross-linked structure is formed using glutaraldehyde to construct a solid nucleic acid protection network, significantly enhancing the stability of double-stranded ribonucleic acid in the alkaline environment and prolonging its residence time in the body of Noctuidae insects, solving the technical problem of poor stability of double-stranded ribonucleic acid in the body of Noctuidae insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a flowchart of the method of the present invention; Figure 2 is the characterization of BSA·NPs treated with different cross-linking agents; Figure 3 is the characterization of dsRNA-BSA·NPs under different treatment conditions; Figure 4 is the release curves of dsRNA-GBSA·NPs and dsRNA-BSA·NPs; Figure 5 is the release curves of dsRNA-GBSA·NPs and dsRNA-BSA·NPs under different pH conditions; Figure 6 is the release curves of dsRNA-GBSA·NPs and dsRNA-BSA·NPs under different temperature conditions; Figure 7The RNA interference efficiencies of dsRNA, dsRNA-BSA·NPs and dsRNA-GBSA·NPs at different concentrations. Detailed implementation manners

[0019] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0020] As Figure 1 shown, it is a flowchart of a preparation method of a nucleic acid delivery system with pH-responsive release provided by the present invention. This method includes the following steps: S01. React bovine serum albumin with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in phosphate buffer for 2 hours to prepare modified bovine serum albumin with a surface amino density 3 to 5 times that of the original bovine serum albumin; S02. Adjust the concentration of the modified bovine serum albumin to 2.5 micrograms per microliter and adjust the pH value of the solution to 9; S03. Mix the modified bovine serum albumin and double-stranded ribonucleic acid according to a mass ratio of 10:1, and the ratio of water to ethanol is 3:1; S04. Mix the modified bovine serum albumin and double-stranded ribonucleic acid according to a mass ratio of 10:1, and the ratio of water to ethanol is 3:1; S05. Let the mixture stand and react at 4 °C for 4 to 5 hours to form a cross-linked complex; S06. Purify using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to obtain double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles; S07. Store the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles at room temperature to ensure that the cumulative release amount remains below 80% within 36 hours; S08. The release rate of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles does not exceed 75% in 24 hours under alkaline conditions and is greater than 90% in 12 hours under acidic conditions; S09. Treat noctuid insects with the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles, and the RNA interference efficiency reaches more than 90%; Optionally, step S04 further includes a cross-linking reaction kinetics optimization step, specifically by monitoring in real time the reaction kinetic parameters of the formation of Schiff base between glutaraldehyde and the surface amino groups of the modified bovine serum albumin, and adjusting the addition method of the cross-linking agent according to the Schiff base formation reaction kinetic equation; Optionally, step S08 further includes a release prediction optimization step, specifically, a pre-trained dynamic neural network model for predicting the release curve of the double-stranded ribonucleic acid under different pH conditions is used, and based on this, the structural parameters of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles are optimized; Among them, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is a modifier used to increase the amino density on the surface of the bovine serum albumin and enhance the binding ability with the double-stranded ribonucleic acid; Among them, the modified bovine serum albumin refers to bovine serum albumin with an increased amino density on the surface after being treated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, which can bind more tightly to the double-stranded ribonucleic acid through electrostatic interaction; Among them, glutaraldehyde, as a cross-linking agent, can form Schiff base with the modified bovine serum albumin, so that the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles remain stable in an alkaline environment and are prone to breakage in an acidic environment, realizing pH-responsive release; Among them, the double-stranded ribonucleic acid refers to a double-stranded ribonucleic acid fragment with a length within 500 base pairs, which is used to specifically inhibit the expression of a target gene through the ribonucleic acid interference mechanism; Among them, the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles refer to a nano-level complex formed by cross-linking the double-stranded ribonucleic acid with the modified bovine serum albumin through glutaraldehyde, and the particle size is about 290 to 300 nanometers; Among them, the pH-responsive release refers to the property that the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles remain stable in the alkaline environment of the Noctuidae insect intestine, while rapidly releasing the double-stranded ribonucleic acid in the acidic environment after entering the cell; Among them, the ribonucleic acid interference efficiency refers to the degree of decrease in the expression level of the target gene caused by the double-stranded ribonucleic acid. Compared with directly using the double-stranded ribonucleic acid for treatment, the delivery system prepared by this method can increase the ribonucleic acid interference efficiency by more than 3 times; Among them, the kinetic equation for Schiff base formation is used to predict the process and efficiency of the cross-linking reaction under different reaction conditions. The inputs include the concentration of the modified bovine serum albumin, the concentration of glutaraldehyde, the pH value of the solution, the ionic strength of the solution, and the reaction temperature, and the outputs are the Schiff base formation rate and the final cross-linking degree; the optimal glutaraldehyde concentration and addition rate are determined through the kinetic equation for Schiff base formation to achieve the optimal cross-linking of the double-stranded ribonucleic acid and the modified bovine serum albumin, and improve the stability and delivery efficiency of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles; Among them, the solution ionic strength refers to the total concentration of ions in the solution, in moles per liter, which affects the cross-linking efficiency between the modified bovine serum albumin and the glutaraldehyde; Among them, the reaction temperature refers to the ambient temperature when the cross-linking reaction between the modified bovine serum albumin and the glutaraldehyde occurs, in degrees Celsius, which affects the reaction rate of Schiff base formation; Among them, the Schiff base formation rate refers to the number of Schiff bases formed between the surface amino groups of the modified bovine serum albumin and the glutaraldehyde per unit time, in moles per second, which is used to guide the optimization of the cross-linking agent addition method; Among them, the final cross-linking degree refers to the proportion of surface amino groups of the modified bovine serum albumin participating in the cross-linking reaction, expressed as a percentage, which is used to evaluate the stability of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles; Among them, the specific structure of the nano-drug delivery release dynamic neural network model is a hybrid neural network architecture combining a multi-layer perceptron and a long short-term memory network, including an input layer, three hidden layers and an output layer. Among them, the hidden layer uses an attention mechanism to model the structural changes of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles in different environments and the double-stranded ribonucleic acid release process. The number of heads in the attention mechanism is determined according to the particle size, zeta potential value and polydispersity index value of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles; Among them, the steps for establishing the training data set of the nano-drug delivery release dynamic neural network model specifically include collecting the release curve data of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles with different final cross-linking degrees, different particle sizes and different zeta potential values under various pH conditions, and simultaneously recording the corresponding reaction temperature, solution ionic strength and degradation state of the modified bovine serum albumin. Pairing the release curve data with the physicochemical property data of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles to form input-output sample pairs, and constructing a large-scale training set containing more than 10,000 groups of samples; Among them, the steps for training the nano-drug delivery release dynamic neural network model specifically include optimizing the hybrid neural network architecture using the batch gradient descent method, selecting the weighted sum of the root mean square error and the custom release curve shape loss as the loss function, introducing an early stopping strategy during the training process to avoid overfitting, and simultaneously using a learning rate decay strategy to improve the model convergence performance. Finally, a high-precision prediction effect with an average release curve prediction error less than 5% is achieved on the validation set; Among them, the zeta potential value refers to the electric potential on the surface of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles, in millivolts, which affects the stability of the double-stranded ribonucleic acid modified bovine serum albumin nanoparticles and the double-stranded ribonucleic acid release behavior; Among them, the polydispersity index value is a dimensionless parameter representing the uniformity of the particle size distribution of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles, ranging from 0 to 1. The smaller the value, the more uniform the particle size distribution, which affects the stability of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles; Among them, the degradation state of the modified bovine serum albumin refers to the structural integrity of the modified bovine serum albumin under different conditions, which is determined by protein electrophoresis or circular dichroism spectroscopy and is used to evaluate the stability of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles in different environments; Among them, the release curve data refers to the cumulative percentage of the release of the double-stranded ribonucleic acid by the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles over time under certain conditions, which is used as the basic data for establishing the release prediction optimization step; Among them, the custom release curve shape loss is a loss function that measures the difference in shape between the predicted release curve and the actual release curve, focusing on the change rate of the release of the double-stranded ribonucleic acid and the release characteristics at key time points, and is used to improve the prediction accuracy of the nano-drug delivery release dynamic neural network model for the release dynamics of the double-stranded ribonucleic acid.

[0021] The following is a detailed description of the specific implementation manners of the above steps.

[0022] The specific implementation manner of step S01 is to dissolve 1 gram of bovine serum albumin in 100 milliliters of phosphate buffer solution to prepare a bovine serum albumin solution with a concentration of 10 milligrams per milliliter, adjust the pH value of the solution to 7.4, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to make its concentration 1 milligram per milliliter, stir and react at room temperature for 2 hours, and then perform dialysis purification using a dialysis membrane with a molecular weight of 3500 daltons to remove the unreacted 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and other small molecule impurities, thereby obtaining modified bovine serum albumin with an increased surface amino density. This step is based on the principle of peptide bond condensation. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is used as a dehydrating condensing agent to activate the carboxyl groups on the surface of bovine serum albumin, promoting its reaction with amino compounds in the solution to form new amino bonds, thereby increasing the amino density on the surface of bovine serum albumin. Measured by the fluorescamine labeling method, the surface amino density of the modified bovine serum albumin is 4.2 times that of the original bovine serum albumin. The purpose of this step is to increase the positive charge density on the surface of bovine serum albumin, provide more binding sites for subsequent binding with negatively charged double-stranded ribonucleic acid, and enhance the electrostatic interaction.

[0023] The specific implementation of step S02 is to freeze-dry and preserve the modified bovine serum albumin obtained in step S01. When in use, weigh 12.5 mg of the modified bovine serum albumin, dissolve it in 5 mL of ultrapure water to prepare a modified bovine serum albumin solution with a concentration of 2.5 μg / μL. Then, gradually adjust the pH value of the solution to 9.0 ± 0.1 drop by drop using 0.1 mol / L sodium hydroxide solution, and monitor it in real time with a pH meter. Under this pH condition, the amino groups on the surface of the modified bovine serum albumin exist in an unprotonated state, which is conducive to forming stable electrostatic interactions with double-stranded ribonucleic acid. The precise control of the pH value adopts the buffer system stabilization method, and by adding an appropriate amount of borax-sodium hydroxide buffer system to the solution, the pH value of the solution is stabilized at 9.0. This step is based on the principle of the isoelectric point of proteins. Under the pH condition higher than the isoelectric point of the modified bovine serum albumin, the negatively charged groups on the protein surface increase, which has a certain impact on the spatial structure of the protein itself and is conducive to the subsequent binding with double-stranded ribonucleic acid. The purpose of step S02 is to optimize the physicochemical state of the modified bovine serum albumin and create conditions for the efficient loading of double-stranded ribonucleic acid.

[0024] The specific implementation of step S03 is to dissolve double-stranded ribonucleic acid in nuclease-free ultrapure water to prepare a double-stranded ribonucleic acid solution with a concentration of 1 μg / μL. Take 1 mL of this solution and add it to 10 mL of the modified bovine serum albumin solution obtained in step S02 to make the mass ratio of double-stranded ribonucleic acid to modified bovine serum albumin 1:10. After mixing evenly, add 3.67 mL of absolute ethanol to make the volume ratio of water to ethanol 3:1, and gently stir at room temperature for 10 minutes to form a homogeneous mixture. This step is based on the principle of antisolvent precipitation and electrostatic self-assembly. First, the positively charged modified bovine serum albumin and the negatively charged double-stranded ribonucleic acid form a primary complex through electrostatic attraction. Then, by adding the organic solvent ethanol, the dielectric constant of the mixture is reduced, the electrostatic interaction is enhanced, and at the same time, the protein is partially dehydrated, resulting in the precipitation of the protein-nucleic acid complex to form nanoparticles. The volume ratio of water to ethanol is crucial for the formation of nanoparticles. When the ratio is lower than 2:1, the protein denatures too quickly and is prone to forming irregular aggregates; when the ratio is higher than 4:1, the precipitation efficiency decreases. The purpose of step S03 is to form a stable primary complex of double-stranded ribonucleic acid and modified bovine serum albumin by controlling the mass ratio of double-stranded ribonucleic acid to modified bovine serum albumin and the solvent composition.

[0025] The specific implementation of step S04 is to add a 25% glutaraldehyde solution drop by drop to the mixture obtained in step S03 at a ratio of 0.06 moles per gram of protein. The calculation method is as follows: first, determine that the total mass of modified bovine serum albumin in the mixture is 25 mg, and then calculate the number of moles of glutaraldehyde required as 25 mg × 0.06 moles per gram = 1.5 mmol. Considering that the density of the 25% glutaraldehyde solution is 1.06 g / mL and the molar mass is 100.12 g / mol, the volume of the 25% glutaraldehyde solution to be added is calculated as 0.056 mL. It is added drop by drop in 10 times using a micro syringe, and after each addition, it is gently stirred for 30 seconds to ensure uniform mixing. This step is based on the principle of cross-linking and curing. Glutaraldehyde, as a dialdehyde cross-linking agent, can undergo a nucleophilic addition reaction with the amino groups on the protein surface to form Schiff bases, connecting multiple protein molecules together to form a three-dimensional network structure, fixing double-stranded ribonucleic acid in the network, and improving the stability of the nanoparticles. The control of the glutaraldehyde addition amount is crucial for the performance of the nanoparticles. When the addition amount is less than 0.04 moles per gram of protein, insufficient cross-linking results in poor nanoparticle stability; when the addition amount is higher than 0.08 moles per gram of protein, over-cross-linking will cause a decrease in the surface potential of the nanoparticles, affecting the dispersibility. The purpose of step S04 is to form a stable complex of double-stranded ribonucleic acid and modified bovine serum albumin through glutaraldehyde cross-linking, laying a foundation for the subsequent selective release in different pH environments.

[0026] The specific implementation of step S05 is to transfer the mixture obtained in step S04 into a sealed container and place it in a refrigerator at 4 °C for static reaction for 4.5 hours. During this period, the container is gently turned over 1 to 2 times every 30 minutes to mix evenly, but not shaken violently to avoid forming bubbles and affecting the cross-linking uniformity. The cross-linking reaction proceeds according to the nucleophilic addition reaction mechanism between glutaraldehyde and protein amino groups. In the initial 30 minutes of the reaction, mainly single-point connections are formed, and then a network structure gradually forms. During the static reaction process, the temperature is controlled within the range of 4 ± 1 °C. The low temperature condition is beneficial to slowing down the self-polymerization reaction of glutaraldehyde and improving the selective reaction efficiency with protein amino groups. The reaction time is controlled within the range of 4 to 5 hours. When the reaction time is less than 4 hours, the cross-linking degree is insufficient; when it exceeds 5 hours, over-cross-linking begins to occur, manifested as an increase in the particle size of the nanoparticles and a decrease in the dispersibility. This step uses a dynamic cross-linking reaction monitoring technique. By taking a small amount of samples to measure the change in Zeta potential to understand the cross-linking process, when the Zeta potential reaches below -55 mV and the change does not exceed 1 mV within 30 minutes, it indicates that the cross-linking reaction is approaching completion. The purpose of step S05 is to form a stable Schiff base cross-linking structure of double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles by controlling the time and temperature of the cross-linking reaction, ensuring that the product has ideal physical and chemical properties.

[0027] The specific implementation of step S06 is to transfer the reaction product obtained in step S05 into an ultrafiltration device, and use a polyethersulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa. First, add an equal volume of phosphate buffer to dilute the sample, then centrifuge at a speed of 1500 revolutions per minute for 10 minutes at 4 degrees Celsius, discard the filtrate, and repeat the above process 2 times by adding an equal volume of phosphate buffer. For the third time, add nuclease-free ultrapure water to replace the phosphate buffer, and perform ultrafiltration purification 3 times in total. The ultrafiltration process is based on the principle of molecular sieving. By controlling the pore size of the membrane, substances smaller than the membrane pore size, such as unreacted glutaraldehyde, small molecule reaction products, and free double-stranded ribonucleic acid, pass through the membrane pores and are discharged, while the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles larger than the membrane pore size are retained above the membrane. The purified double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles are redispersed in nuclease-free ultrapure water to obtain the final product. The ultrafiltration technology used in this step has the advantages of high separation efficiency and less sample loss compared with the traditional dialysis method. The selection of the membrane molecular weight cut-off is based on the consideration of the molecular weight of double-stranded ribonucleic acid and the size of nanoparticles, ensuring that impurities can be effectively removed while the target product can be retained to the greatest extent. The purpose of step S06 is to remove unreacted substances and by-products in the reaction system through ultrafiltration purification and obtain high-purity double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles.

[0028] The specific implementation of step S07 is to aliquot the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles obtained in step S06 into sterile centrifuge tubes, 0.5 ml per tube, and store them under constant temperature and light protection conditions at 20 ± 2 degrees Celsius, and regularly sample to measure the release of double-stranded ribonucleic acid. The sampling method is to take 50 μl of the sample, centrifuge it at 12000 revolutions per minute for 5 minutes through an ultrafiltration tube with a molecular weight cut-off of 10 kDa, collect the filtrate, measure the concentration of double-stranded ribonucleic acid in the filtrate, and calculate the cumulative release rate. The measurement results show that under the storage condition of 20 degrees Celsius, the cumulative release amount of double-stranded ribonucleic acid from the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles is 23.5% at 12 hours, 42.7% at 24 hours, and 71.6% at 36 hours, all lower than the limit value of 80%, indicating that the prepared nanoparticles have good stability and sustained release characteristics. This step is based on the principle of drug release kinetics. By monitoring the release behavior of double-stranded ribonucleic acid under certain conditions, the stability and release characteristics of the nano-delivery system are evaluated. Controlling the cumulative release amount below 80% is a key parameter to ensure that the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles can maintain an adequate action time in the in vivo environment. The purpose of step S07 is to verify the stability and release behavior of the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles at room temperature and provide a basis for subsequent applications.

[0029] The specific implementation of step S08 is to separately take 1 mL of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticle solution obtained in step S06, place it in buffer solutions with a pH of 10.0 and a pH of 5.0, perform constant-temperature oscillation at 37 °C, with an oscillation speed of 100 revolutions per minute, and regularly sample to measure the release of double-stranded ribonucleic acid. The specific sampling time points are 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and the sampling method is the same as that in step S07. The measurement results show that under the alkaline condition with a pH of 10.0, the cumulative release rate of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles within 24 h is 71.84%, which is lower than the limit value of 75%; under the acidic condition with a pH of 5.0, the cumulative release rate within 12 h is 92.3%, which is higher than the limit value of 90%. This pH-responsive release behavior is based on the principle of the difference in the stability of Schiff base chemical bonds under different pH conditions. The Schiff base formed by glutaraldehyde and the protein amino group exists stably under alkaline conditions, while it is easily hydrolyzed and broken under acidic conditions, resulting in changes in the nanoparticle structure and accelerating the release of double-stranded ribonucleic acid. The purpose of step S08 is to verify the pH-responsive release characteristics of the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles, ensuring that they can remain stable in the alkaline environment of the Noctuidae insect intestine and rapidly release double-stranded ribonucleic acid in the acidic environment after entering the cells.

[0030] The specific implementation of step S09 is to prepare a series of different concentrations of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles obtained in step S06, including four concentration gradients of 500 ng / μL, 1000 ng / μL, 1500 ng / μL, and 2000 ng / μL. The third-instar larvae of Noctuidae pests are treated by the artificial feeding method, 5 μL is fed each time, and the feeding is continuous for 3 days. Then, the real-time fluorescence quantitative polymerase chain reaction method is used to measure the expression level of the target gene. Compared with the untreated control group, the RNA interference efficiency is calculated. The measurement results show that the RNA interference efficiency of the 500 ng / μL concentration treatment group is 45.7%, that of the 1000 ng / μL concentration treatment group is 68.3%, that of the 1500 ng / μL concentration treatment group is 82.6%, and that of the 2000 ng / μL concentration treatment group is 93.57%, achieving a high inhibition effect of over 90%. At the same time, compared with the control group treated directly with the same concentration of double-stranded ribonucleic acid, the RNA interference efficiency is increased by 3.2 times. This step is based on the RNA interference mechanism. By introducing double-stranded ribonucleic acid targeting specific genes, the RNA interference pathway is triggered to form an RNA-induced silencing complex, specifically degrade the messenger RNA of the target gene, and inhibit the expression of the target protein, thereby achieving the purpose of interfering with the physiological functions of Noctuidae insects. The purpose of step S09 is to verify the actual RNA interference effect of the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles on Noctuidae insects and confirm their application potential as biological pesticides for pest control. Among them, the target specific gene sequences include at least: Hsp90, CYP302A1, V_ATPase, acetylcholinesterase, Ecdyson Receptor, chitinase gene.

[0031] Optionally, step S04 also includes a cross-linking reaction kinetics optimization step. Specifically, the reaction kinetic parameters of Schiff base formed between glutaraldehyde and the surface amino group of modified bovine serum albumin are monitored in real time, and the cross-linking agent addition method is adjusted according to the Schiff base formation reaction kinetic equation, which is used to predict the progress and efficiency of the cross-linking reaction under different reaction conditions. The input includes the concentration of modified bovine serum albumin, the concentration of glutaraldehyde, the pH value of the solution, the ionic strength of the solution and the reaction temperature, and the output is the Schiff base formation rate and the final cross-linking degree. This optimization step uses a microfluidic injection system to add glutaraldehyde to the reaction system according to a preset rate gradient, and monitors the absorbance change at 450 nanometers by a real-time spectrophotometer, and adjusts the subsequent glutaraldehyde addition rate and interval time according to the absorbance change trend. The advantage of the cross-linking reaction kinetics optimization step is that it solves the problem of uneven reaction in the traditional one-time addition of the cross-linking agent method. The Schiff base formation reaction is a reversible reaction and is affected by a variety of environmental factors. By establishing a reaction kinetic equation, the influencing factors are quantified into specific parameters to achieve accurate prediction and control of the reaction process. The microfluidic injection system combined with real-time monitoring forms a closed-loop feedback control, which allows the cross-linking reaction to proceed evenly throughout the system, avoiding local over-cross-linking or under-cross-linking, and in principle ensuring the uniformity and stability of the nanoparticle structure. Compared with traditional methods, this optimization step transforms the cross-linking process from empirical operation to precise control, fundamentally improving the batch consistency and pH response accuracy of double-stranded RNA-modified bovine serum albumin nanoparticles.

[0032] Optionally, in step S08, a release prediction optimization step is further included. Specifically, a pre-trained dynamic neural network model for nanoparticle drug release is used to predict the release curves of double-stranded ribonucleic acid under different pH conditions, and the nanoparticle structure parameters are optimized accordingly. The specific structure of the dynamic neural network model for nanoparticle drug release is a hybrid neural network architecture combining a multi-layer perceptron and a long short-term memory network, including an input layer, three hidden layers, and an output layer. Among them, the hidden layer uses an attention mechanism to model the structural changes of double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles in different environments and the double-stranded ribonucleic acid release process. The number of heads in the attention mechanism is determined according to the particle size, zeta potential value, and polydispersity index value of the nanoparticles. This optimization step first prepares nanoparticle samples with different parameter combinations through orthogonal experimental design, measures the release curve data under various conditions, constructs a large-scale training dataset, then trains the dynamic neural network model for nanoparticle drug release, and finally realizes accurate prediction and reverse optimization design of the release behavior. The advantage of the release prediction optimization step is to achieve the transformation from experiment-oriented to mechanism-oriented, and capture the complex release laws of the nucleic acid delivery system through machine learning technology. The convolutional layer in the hybrid neural network architecture identifies local features in the release curve, the long short-term memory network layer captures the temporal dependence in the release process, and the attention mechanism adaptively allocates computing resources according to the nanoparticle characteristics. This model can not only predict the release behavior of known parameter combinations, but more importantly, through reverse design, it can deduce the optimal preparation parameters according to the desired release characteristics. In principle, this optimization step elevates the nanoparticle drug release behavior from qualitative description to quantitative prediction, providing a theoretical basis and technical support for designing different delivery systems for the digestive tract environments of different noctuid pests, and fundamentally improving the targeting and efficiency of RNA interference.

[0033] Specifically, the principle of the present invention is as follows: Based on the principles of protein chemical modification, molecular self-assembly, and chemical cross-linking, the present invention constructs a double-stranded ribonucleic acid delivery system with high stability in noctuid insects. The core technical principle can be analyzed from the following aspects:

[0034] First, bovine serum albumin is selected as the basic carrier material in the present invention, which has good biocompatibility and modifiability. Through treatment with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the carboxyl groups on the surface of bovine serum albumin are activated and react with amino compounds in the solution to form amide bonds, thereby increasing the amino density on the protein surface. This modification increases the surface amino density to 3 to 5 times the original level, significantly enhancing the electrostatic interaction between bovine serum albumin and negatively charged double-stranded ribonucleic acid, forming a tighter primary complex and providing the first barrier of stability.

[0035] Secondly, glutaraldehyde, as a bifunctional aldehyde cross-linking agent, can form Schiff base chemical bonds with the amino groups on the surface of modified bovine serum albumin. This cross-linking reaction is carried out under alkaline conditions with a pH value of 9, which is similar to the intestinal environment of Noctuidae insects, facilitating the formation of a stable three-dimensional network structure. By precisely controlling the amount of glutaraldehyde added (0.04 to 0.08 moles per gram of protein), a moderately cross-linked network is formed, which can firmly protect double-stranded ribonucleic acid without affecting its subsequent release in target cells. This cross-linked network structure provides a physical barrier for double-stranded ribonucleic acid, effectively blocking the contact with nucleases in the alkaline environment and significantly improving the stability of double-stranded ribonucleic acid.

[0036] Thirdly, the optimization of the water-to-ethanol ratio (3:1) in the present invention creates a solvent environment favorable for the formation of nanoparticles, promoting the ordered arrangement and close packing of protein molecules. At the same time, the low-temperature reaction condition at 4 °C slows down the cross-linking reaction rate, ensuring the formation of a uniform and dense nanostructure, and further improving the structural stability of the complex. Purification is carried out through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to remove free components and unstable small molecules, improving the homogeneity and stability of the final product.

[0037] Finally, the formed double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles have a particle size of about 290 to 300 nanometers and are positively charged on the surface. This structural characteristic makes them not easily disintegrate or aggregate in the alkaline intestinal environment of Noctuidae insects, maintaining a good dispersed state and structural integrity. Through multiple protection mechanisms, the present invention solves the technical problem of poor stability of double-stranded ribonucleic acid in Noctuidae insects and achieves an efficient gene interference effect.

[0038] A specific Example 1 of the present invention is provided below, and the specific implementation manners of each step in this Example 1 are described in detail as follows.

[0039] The specific implementation manner of step S01 is to dissolve 1 gram of bovine serum albumin in 100 milliliters of phosphate buffer to prepare a bovine serum albumin solution with a concentration of 10 milligrams per milliliter, adjust the pH value of the solution to 7.4, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to a concentration of 1 milligram per milliliter, stir and react at room temperature for 2 hours, and then carry out dialysis purification using a dialysis membrane with a molecular weight of 3500 Da to remove unreacted 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and other small molecule impurities, obtaining modified bovine serum albumin with an increased surface amino group density. This reaction is based on the principle of peptide bond condensation and follows the following reaction kinetic equation: ; In the formula, is the surface amino group concentration of bovine serum albumin, with the unit of millimole per liter; is the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, in millimoles per liter; is the concentration of carboxyl groups on the surface of bovine serum albumin, in millimoles per liter; is the concentration of hydrogen ions in the solution, in moles per liter; is the reaction rate constant, in liters per millimole per second, and takes the value of 0.35 under the condition of 25 degrees Celsius; is the reverse reaction rate constant, in liters per millimole per second, and takes the value of 0.02 under the condition of 25 degrees Celsius; is the reaction time, in seconds.

[0040] Determined by the fluorescamine labeling method, the calculation formula for the surface amino group density of modified bovine serum albumin is: ; In the formula, is the surface amino group density of modified bovine serum albumin, in per square nanometer; is the fluorescence intensity value of modified bovine serum albumin; is the fluorescence intensity value of unmodified bovine serum albumin; is the surface amino group density of unmodified bovine serum albumin, with a value of 0.8 per square nanometer. Experimentally measured The value is 3.36 per square nanometer, meeting the requirement of 3 to 5 times that of the original bovine serum albumin. The purpose of this step is to increase the positive charge density on the surface of bovine serum albumin, provide more binding sites for subsequent binding with negatively charged double-stranded ribonucleic acid, and enhance the electrostatic interaction.

[0041] The specific implementation method of step S02 is to freeze-dry and store the modified bovine serum albumin obtained in step S01. When in use, weigh 12.5 mg of modified bovine serum albumin, dissolve it in 5 ml of ultrapure water to prepare a modified bovine serum albumin solution with a concentration of 2.5 μg / μl, and then gradually adjust the pH value of the solution to 9.0 ± 0.1 dropwise using 0.1 mol / L sodium hydroxide solution, and monitor it in real-time with a pH meter. Under this pH condition, the amino groups on the surface of modified bovine serum albumin exist in an unprotonated state, which is conducive to forming a stable electrostatic interaction with double-stranded ribonucleic acid. The precise control of the pH value adopts the buffer system stabilization method, and by adding an appropriate amount of borax-sodium hydroxide buffer system to the solution, the pH value of the solution is stabilized at 9.0. This step is based on the principle of the isoelectric point of proteins. Under the pH condition higher than the isoelectric point of modified bovine serum albumin, the negatively charged groups on the protein surface increase, which has a certain impact on the spatial structure of the protein itself and is conducive to subsequent binding with double-stranded ribonucleic acid.

[0042] The specific implementation of step S03 is to dissolve double-stranded ribonucleic acid in nuclease-free ultrapure water to prepare a double-stranded ribonucleic acid solution with a concentration of 1 microgram per microliter. Take 1 milliliter of this solution and add it to 10 milliliters of the modified bovine serum albumin solution obtained in step S02, so that the mass ratio of double-stranded ribonucleic acid to modified bovine serum albumin is 1:10. After mixing evenly, add 3.67 milliliters of absolute ethanol to make the volume ratio of water to ethanol 3:1, and gently stir at room temperature for 10 minutes to form a homogeneous mixture. In this process, the binding between double-stranded ribonucleic acid and modified bovine serum albumin follows the principle of electrostatic self-assembly, and its binding model is expressed as: ; In the formula, is the concentration of the double-stranded ribonucleic acid and modified bovine serum albumin complex, in milligrams per milliliter; is the concentration of modified bovine serum albumin, in milligrams per milliliter; is the concentration of double-stranded ribonucleic acid, in milligrams per milliliter; is the binding rate constant, in milliliters per milligram per second, and its value at room temperature is 0.28; is the dissociation rate constant, in reciprocal seconds, and its value at room temperature is 0.015; is the reaction time, in seconds.

[0043] After adding ethanol, the calculation formula for the change in the dielectric constant of the solution is: ; In the formula, is the dielectric constant of the mixed solution; is the dielectric constant of water, with a value of 78.5; is the dielectric constant of ethanol, with a value of 24.3; is the volume fraction of water, with a calculated value of 0.75; is the volume fraction of ethanol, with a calculated value of 0.25.

[0044] The decrease in the dielectric constant leads to an increase in the electrostatic interaction. The calculation formula for the enhancement coefficient is: ; In the formula, is the electrostatic interaction enhancement coefficient, and the calculated value is 1.42, indicating that the strength of the electrostatic interaction increases by 42% after adding ethanol. The purpose of this step is to form a stable primary complex of double-stranded ribonucleic acid and modified bovine serum albumin by controlling the mass ratio of double-stranded ribonucleic acid to modified bovine serum albumin and the solvent composition.

[0045] The specific implementation of step S04 is to dropwise add a 25% glutaraldehyde solution to the mixture obtained in step S03 at a ratio of 0.06 moles per gram of protein. The calculation method is as follows: first, determine that the total mass of the modified bovine serum albumin in the mixture is 25 mg, and then calculate the number of moles of glutaraldehyde required as 25 mg × 0.06 moles per gram = 1.5 mmol. Considering that the density of the 25% glutaraldehyde solution is 1.06 g / mL and the molar mass is 100.12 g / mol, the calculated volume of the 25% glutaraldehyde solution to be added is 0.056 mL. Use a micro syringe to add it drop by drop in 10 times, and gently stir for 30 seconds after each addition to ensure uniform mixing. The cross-linking reaction equation between glutaraldehyde and protein amino groups is: ; .

[0046] The cross-linking reaction kinetic equation is expressed as: ; In the formula, is the concentration of the formed Schiff base, in mmol / L; is the concentration of protein amino groups, in mmol / L; is the concentration of glutaraldehyde, in mmol / L; is the concentration of water molecules, in mol / L; is the forward reaction rate constant, in L / (mmol·s), and its value at 25 °C is 0.42; is the reverse reaction rate constant, in L² / (mol²·s), and its value at 25 °C is 0.03; is the activation energy, in J / mol, and its value is 45000; is the gas constant, in J / (mol·K), and its value is 8.314; is the absolute temperature, in K; is the reaction time, in s.

[0047] The formula for calculating the final cross-linking degree is: ; In the formula, is the cross-linking degree, expressed as a percentage; is the initial protein amino group concentration, in mmol / L. The purpose of this step is to form a stable complex of double-stranded ribonucleic acid and modified bovine serum albumin through glutaraldehyde cross-linking, laying a foundation for subsequent selective release in different pH environments.

[0048] The specific implementation of step S05 is to transfer the mixture obtained in step S04 into a sealed container and place it in a refrigerator at 4 °C for static reaction for 4.5 hours. During this period, the container is gently turned over 1 to 2 times every 30 minutes to mix evenly, but not shaken violently to avoid forming bubbles that affect the cross-linking uniformity. The cross-linking reaction proceeds according to the nucleophilic addition reaction mechanism between glutaraldehyde and protein amino groups. In the first 30 minutes of the reaction, mainly single-point connections are formed, and then a network structure is gradually formed. During the static reaction process, the temperature is controlled within the range of 4 ± 1 °C. The low temperature condition is conducive to slowing down the self-polymerization reaction of glutaraldehyde and improving the selective reaction efficiency with protein amino groups. The reaction process can be predicted by the following equation: ; In the formula, is the time when the concentration of the cross-linked complex, with the unit of milligram per milliliter; is the theoretical maximum concentration of the cross-linked complex, with the unit of milligram per milliliter; is the apparent cross-linking rate constant, with the unit of per hour, and takes the value of 0.75 under the condition of 4 °C; is the reaction time, with the unit of hour.

[0049] The relationship between the cross-linking degree and temperature can be expressed as: ; In the formula, is the cross-linking rate constant at temperature , with the unit of per hour; is the cross-linking rate constant at the reference temperature , with the unit of per hour; is the activation energy of the cross-linking reaction, with the unit of joule per mole, and takes the value of 50000; is the gas constant, with the unit of joule per mole per kelvin, and takes the value of 8.314; and are the actual temperature and the reference temperature respectively, with the unit of kelvin.

[0050] Through the dynamic cross-linking reaction monitoring technology, the change of Zeta potential can be measured in real time to understand the cross-linking process. When the Zeta potential reaches below -55 mV and the change does not exceed 1 mV within 30 minutes, it indicates that the cross-linking reaction is approaching completion. The purpose of this step is to control the time and temperature of the cross-linking reaction to make the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles form a stable Schiff base cross-linking structure, ensuring that the product has ideal physical and chemical properties.

[0051] The specific implementation of step S06 is to transfer the reaction product obtained in step S05 into an ultrafiltration device, and use a polyethersulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa. First, add an equal volume of phosphate buffer to dilute the sample, then centrifuge at a speed of 1500 revolutions per minute for 10 minutes at 4 °C, discard the filtrate, and repeat the above process 2 times by adding an equal volume of phosphate buffer. For the third time, add nuclease-free ultrapure water to replace the phosphate buffer, and perform ultrafiltration purification 3 times in total. The ultrafiltration efficiency can be expressed as: ; In the formula, is the ultrafiltration efficiency, expressed as a percentage; is the initial impurity concentration, with the unit of milligram per milliliter; is the final impurity concentration, with the unit of milligram per milliliter. After three times of ultrafiltration, the residual amount of glutaraldehyde is less than 0.001%, and the content of free double-stranded ribonucleic acid is less than 0.5%, meeting the purity requirements. The purpose of this step is to remove the unreacted substances and by-products in the reaction system through ultrafiltration purification to obtain high-purity double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles.

[0052] The specific implementation of step S07 is to aliquot the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles obtained in step S06 into sterile centrifuge tubes, 0.5 ml per tube, and store them under constant temperature and light protection conditions at 20 ± 2 °C, and regularly sample to measure the release of double-stranded ribonucleic acid. The release kinetics conforms to the following equation: ; In the formula, is time is the amount of double-stranded ribonucleic acid released, with the unit of microgram; is the theoretically maximum amount of double-stranded ribonucleic acid that can be released, with the unit of microgram; is the release rate constant, with the unit of per hour power, and its value at 20 °C is 0.15; is the release exponent, reflecting the release mechanism. When is equal to 0.5, it represents the Fickian diffusion mechanism. When is equal to 1, it represents zero-order release. When is between 0.5 and 1, it represents non-Fickian diffusion. The experimentally measured value is 0.62, indicating that the release process is a non-Fickian diffusion mechanism.

[0053] The cumulative release rate calculation formula is: ; In the formula, is the cumulative release rate, expressed as a percentage; is time The amount of double-stranded ribonucleic acid released, in micrograms; is the total amount of double-stranded ribonucleic acid in the nanoparticles, in micrograms. The purpose of this step is to verify the stability and release behavior of the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles under normal temperature conditions, providing a basis for subsequent applications.

[0054] The specific implementation of step S08 is to separately take 1 milliliter of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticle solution obtained in step S06, place it in buffer solutions with a pH of 10.0 and a pH of 5.0, and perform constant-temperature oscillation at 37 degrees Celsius with an oscillation speed of 100 revolutions per minute. Samples are taken at regular intervals to measure the release of double-stranded ribonucleic acid. The pH-responsive release behavior is determined by the difference in the stability of Schiff bases at different pH values and can be described by the following kinetic equation: ; In the formula, is the concentration of double-stranded ribonucleic acid released into the external phase, in micrograms per milliliter; is the concentration of Schiff base bonds in the nanoparticles, in millimoles per liter; is the concentration of double-stranded ribonucleic acid inside the nanoparticles, in micrograms per milliliter; is the concentration of double-stranded ribonucleic acid in the external phase, in micrograms per milliliter; is the hydrolysis rate constant of the Schiff base, in per hour, which is a function of pH; is the diffusion rate constant, in per hour, with a value of 0.08.

[0055] The pH-dependent expression of is: ; In the formula, is the hydrolysis rate constant at pH 0, in per hour, with a value of 5.2; is the pH sensitivity coefficient, with a value of 0.45. This equation explains why the release rate is significantly higher under acidic conditions than under basic conditions. When the pH is 5.0, is calculated to be 0.32 per hour; when the pH is 10.0, is calculated to be 0.05 per hour, and the difference between the two is 6.4 times, which is consistent with the observed release behavior in the experiment. The purpose of this step is to verify the pH-responsive release characteristics of the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles, ensuring that they can remain stable in the alkaline environment of the Noctuidae insect intestine and rapidly release double-stranded ribonucleic acid in the acidic environment after entering the cell.

[0056] The specific implementation of step S09 is to prepare a series of different concentrations of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles obtained in step S06, including four concentration gradients of 500 ng / μL, 1000 ng / μL, 1500 ng / μL, and 2000 ng / μL. The third-instar larvae of Noctuidae pests are treated by the artificial feeding method, 5 μL is fed each time, and feeding is continued for 3 days. Then, the real-time fluorescence quantitative polymerase chain reaction method is used to measure the expression level of the target gene. Compared with the untreated control group, the ribonucleic acid interference efficiency is calculated. The formula for calculating the ribonucleic acid interference efficiency is: ; In the formula, is the ribonucleic acid interference efficiency, expressed as a percentage; is the amplification efficiency of the target gene; is the amplification efficiency of the reference gene; is the difference in Ct values of the target gene between the treatment group and the control group; is the difference in Ct values of the reference gene between the treatment group and the control group.

[0057] The relationship between the ribonucleic acid interference efficiency under different concentration treatments and the concentration of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles can be described by the Hill equation: ; In the formula, is the ribonucleic acid interference efficiency, expressed as a percentage; is the maximum possible ribonucleic acid interference efficiency, with a value of 100%; is the concentration of the double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles, in ng / μL; is the concentration required to achieve 50% interference efficiency, in ng / μL, with a fitted value of 850; is the Hill coefficient, reflecting the steepness of the concentration-effect curve, with a fitted value of 1.8. The purpose of this step is to verify the actual ribonucleic acid interference effect of the prepared double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles on Noctuidae insects and confirm its application potential as a biological pesticide for pest control.

[0058] Optionally, in step S04, there is also an optimization step for the cross-linking reaction kinetics. Specifically, by real-time monitoring the kinetic parameters of the reaction of glutaraldehyde with the surface amino groups of the modified bovine serum albumin, the addition method of the cross-linking agent is adjusted according to the kinetic equation of the Schiff base formation reaction. The kinetic equation of the Schiff base formation reaction is: ; In the formula, is the concentration of the Schiff base, in mmol / L; is the surface amino group concentration of bovine serum albumin, in millimoles per liter; is the glutaraldehyde concentration, in millimoles per liter; is the Schiff base formation reaction rate constant, in liters per millimole per second; is the Schiff base hydrolysis reaction rate constant, in reciprocal seconds; is affected by the pH value , the solution ionic strength and the reaction temperature The adjustment function is expressed as: ; In the formula, is the activation energy of the Schiff base hydrolysis reaction, in joules per mole, with a value of 50000; is the gas constant, in joules per mole per kelvin, with a value of 8.314; is the absolute temperature, in kelvin. The Schiff base formation rate The calculation formula is: ; In the formula, is the Schiff base formation rate, in millimoles per liter per second. By controlling the addition rate of glutaraldehyde, make remain within the optimal range to avoid local over-crosslinking and achieve a uniform crosslinked structure. The final crosslinking degree The calculation formula is: ; In the formula, is the crosslinking degree, expressed as a percentage; is the Schiff base concentration at the end of the reaction, in millimoles per liter; is the initial surface amino group concentration of the modified bovine serum albumin, in millimoles per liter.

[0059] Optionally, in step S08, there is also a release prediction optimization step, specifically using a pre-trained nano-drug delivery release dynamic neural network model to predict the release curve of double-stranded ribonucleic acid under different pH conditions and optimizing the nanoparticle structure parameters accordingly. The mathematical expression of the nano-drug delivery release dynamic neural network model is: ; In the formula, is the predicted release curve data, which is a dimensional matrix, is the number of time points, is the number of prediction parameters; is the input feature vector, including nanoparticle parameters and environmental conditions; is the set of neural network weight parameters; is the neural network function mapping. The input feature vector is defined as: ; In the formula, is the nanoparticle size, in nanometers; is the potential value, in millivolts; is the polydispersity index value, dimensionless; is the environmental pH value; is the environmental temperature, in degrees Celsius; is the solution ionic strength, in moles per liter; is the crosslinking degree, expressed as a percentage; is the predicted time point, in hours.

[0060] The number of heads in the attention mechanism The calculation formula is: ; In the formula, represents rounding down; is the nanoparticle size, in nanometers; is the potential value, in millivolts; is the polydispersity index value, dimensionless. The mathematical expression of the attention mechanism is: ; In the formula, , , are the query matrix, key matrix, and value matrix respectively, obtained by linear transformation of the input features; is the dimension of the key vector; is the normalization function to ensure that the sum of all weights is 1. The calculation formula of the multi-head attention mechanism is: ; ; In the formula, , , are the weight matrices of the query, key, and value of the th head respectively; is the weight matrix of the output; is the concatenation operation.

[0061] The loss function is designed as the weighted sum of the root mean square error and the custom release curve shape loss: ; In the formula, is the root mean square error, and its calculation formula is: ; is the loss of the custom release curve shape, and its calculation formula is: ; In the formula, is the actual release rate at the -th time point; is the release rate predicted by the model at the -th time point; is the number of time points; and are weight coefficients, with values of 0.7 and 0.3 respectively.

[0062] The early stopping strategy judgment condition is: ; In the formula, is the loss value of the validation set at the -th training epoch. The learning rate decay strategy is: ; In the formula, is the learning rate at the -th training epoch; is the initial learning rate, with a value of 0.001; is the decay coefficient, with a value of 0.8; is the decay period, with a value of 20. The core of this optimization step lies in using machine learning techniques to capture the complex release laws of double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles under different environmental conditions, establishing a high-precision prediction model, so as to achieve the precise design and optimization of the delivery system.

[0063] In this embodiment, through systematic parameter optimization and reaction control, double-stranded ribonucleic acid-modified bovine serum albumin nanoparticles with pH responsiveness were successfully prepared. These nanoparticles can remain stable in the alkaline environment (pH about 10) of the intestine of Noctuidae insects and rapidly release double-stranded ribonucleic acid in the acidic environment (pH about 5) after entering the cells, achieving an efficient ribonucleic acid interference effect. By adopting two optional steps of optimizing the cross-linking reaction kinetics and the release prediction, the quality stability and performance predictability of the nanoparticles were further improved, laying a foundation for the industrial application of the nucleic acid delivery system.

[0064] To better understand and implement the present invention, Example 2 of a specific application scenario of the present invention is provided below: Taking Helicoverpa armigera as the research object, a double-stranded ribonucleic acid delivery system targeting its cholesterol synthase (HaCS) gene is designed. The cholesterol synthase of Helicoverpa armigera plays a key role in its growth and development. Silencing this gene can significantly inhibit the growth, development, and reproductive ability of Helicoverpa armigera. First, the gene fragment of the cholesterol synthase of Helicoverpa armigera is amplified by polymerase chain reaction, with a length of 487 base pairs. The amplification product is transcribed in vitro to obtain double-stranded ribonucleic acid (dsRNA-HaCS). Bovine serum albumin (BSA) is selected as the carrier, and a pH-responsive delivery system is prepared by carbodiimide modification and glutaraldehyde cross-linking. The specific preparation process is as follows:

[0065] Weigh 2 grams of bovine serum albumin and dissolve it in 200 milliliters of phosphate buffer to prepare a solution with a concentration of 10 milligrams per milliliter, and adjust the pH of the solution to 7.4. Add 4 grams of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to make its concentration 20 milligrams per milliliter, and stir and react at 25 °C for 2 hours. Dialyze using a dialysis membrane with a molecular weight cut-off of 3500 daltons to remove unreacted EDC and other small molecular substances, and obtain modified bovine serum albumin (cBSA). The surface amino density is measured by the fluorescamine labeling method, and the results are shown in Table 1: Table 1 Comparison of surface amino density before and after modification of bovine serum albumin

[0066] Select the cBSA reacted for 2 hours for subsequent experiments and store it frozen and dried. When in use, weigh 25 milligrams of cBSA, dissolve it in 10 milliliters of ultrapure water to prepare a solution with a concentration of 2.5 micrograms per microliter, and adjust the pH to 9.0 with sodium hydroxide solution. Dissolve the prepared dsRNA-HaCS in nuclease-free ultrapure water to prepare a solution with a concentration of 1 microgram per microliter. Take 2.5 milliliters of the dsRNA-HaCS solution and add it to 25 milliliters of the cBSA solution so that the mass ratio of dsRNA-HaCS to cBSA is 1:10. After mixing evenly, slowly add 9.17 milliliters of absolute ethanol to make the volume ratio of water to ethanol 3:1. Stir gently at room temperature for 15 minutes to form the primary complex of dsRNA-HaCS and cBSA.

[0067] As Figure 2As shown, according to the kinetic equation of Schiff base formation reaction, the optimal dosage of the crosslinking agent was calculated. Considering that the total amount of protein in the system was 62.5 mg, 3.75 mmol of glutaraldehyde was required according to the ratio of 0.06 mol per gram of protein. Using a 25% glutaraldehyde solution (density 1.06 g / mL, molar mass 100.12 g / mol), the required volume was calculated to be 0.141 mL. A microfluidic injection system was used to gradually add the glutaraldehyde solution at a rate of 0.05 mL per minute, and the change in absorbance at 450 nm was monitored in real time using a UV-visible spectrophotometer. The relationship between the change in absorbance and the degree of crosslinking is shown in Table 2: Table 2 Relationship between the change in absorbance at 450 nm and the degree of crosslinking during the addition of glutaraldehyde

[0068] As Figure 3 shown, when the change rate of absorbance decreased to 30% of the initial value, the intermittent injection method of adding 0.01 mL every 10 minutes was adopted until the absorbance was stable, and a total of 0.141 mL of glutaraldehyde solution was added. The mixture was transferred to a sealed container and allowed to stand and react at 4°C for 5 hours, during which the container was gently inverted 2 times every 30 minutes to mix evenly. After the crosslinking reaction was completed, purification was carried out through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. An equal volume of phosphate buffer was added to dilute the sample, and it was centrifuged at 1500 rpm at 4°C for 10 minutes, and the filtrate was discarded, repeating 3 times. Finally, the purified dsRNA-HaCS-GBSA·NPs were dispersed in nuclease-free ultrapure water, and their physicochemical properties were measured. The results are shown in Table 3: Table 3 Physicochemical property parameters of dsRNA-HaCS-GBSA·NPs

[0069] To verify the release characteristics of the prepared dsRNA-HaCS-GBSA·NPs under different pH conditions, an in vitro release experiment was carried out. 1 mL of dsRNA-HaCS-GBSA·NPs solution was taken and placed in buffers with pH 10.0 and pH 5.0, and incubated at 37°C with constant shaking at a shaking speed of 100 rpm. Samples were taken at regular intervals to measure the release of double-stranded ribonucleic acid. The specific release curves are shown in Table 4: Table 4 Cumulative release rate (%) of dsRNA-HaCS-GBSA·NPs under different pH conditions

[0070] By applying the trained nano-drug delivery dynamic neural network model, the release behavior of dsRNA-HaCS-GBSA·NPs with different degrees of crosslinking in the body environment of Helicoverpa armigera was predicted. The model input parameters included nanoparticle size (293.6 nm), Zeta potential (-57.8 mV), polydispersity index (0.115), degree of crosslinking (87.4%), intestinal pH (10.2), and intracellular pH in midgut cells (5.3). The prediction results showed that the release rate of dsRNA-HaCS-GBSA·NPs in the intestinal environment of Helicoverpa armigera was 27.3% within 24 hours, while the release rate reached 89.6% within 12 hours after entering the midgut cells, meeting the design requirements.

[0071] As Figure 4 shown, the RNA interference effect of dsRNA-HaCS-GBSA·NPs on Helicoverpa armigera was evaluated by the artificial feeding method. The third-instar Helicoverpa armigera larvae were divided into five groups, namely the control group (fed only artificial diet), the dsRNA control group (directly added dsRNA-HaCS), the BSA·NPs control group (uncrosslinked dsRNA-HaCS-BSA·NPs), the low-dose treatment group (1000 ng / μL dsRNA-HaCS-GBSA·NPs), and the high-dose treatment group (2000 ng / μL dsRNA-HaCS-GBSA·NPs). There were 30 larvae in each group, and they were continuously fed for 3 days, with each larva fed 5 μL per day. After 7 days of treatment, the expression level of the cholesterol synthase gene was measured by real-time fluorescence quantitative polymerase chain reaction, and the growth and development of the larvae were observed. The results are shown in Table 5: Table 5 Effects of different treatments on the expression of cholesterol synthase gene and the growth and development of Helicoverpa armigera larvae

[0072] Traditional pesticide control of Helicoverpa armigera mainly uses chemical insecticides spraying, such as pyrethroids, organophosphorus compounds, etc. Although these methods can quickly reduce the pest population density, there are problems such as rapid development of pest resistance, serious environmental pollution to the ecosystem, and great harm to non-target organisms. Direct use of double-stranded ribonucleic acid as a biopesticide has high specificity and environmental friendliness, but it faces problems such as easy degradation of ribonucleic acid, low delivery efficiency, and high cost in practical applications. The previously developed ribonucleic acid delivery carriers mainly include cationic liposomes, polymer nanoparticles, etc. Although these carriers can protect ribonucleic acid to a certain extent, the delivery efficiency is not high, and most of them lack environmental stimulus responsiveness and are difficult to achieve targeted release.

[0073] As Figure 7As shown, in addition to the cholesterol synthase (HaCS) gene used in the above embodiments, the pH-responsive double-stranded ribonucleic acid delivery system of the present invention can also be applied to target a variety of key functional genes to achieve efficient prevention and control of Noctuidae insects. The following are the application schemes for several important target genes:

[0074] Heat shock protein 90 (Hsp90) is a molecular chaperone protein that plays an important role in the growth, development, and environmental adaptation of insects. For the Hsp90 gene of Noctuidae insects, a double-stranded ribonucleic acid fragment with a length of 450-500 base pairs can be designed to specifically target its conserved region. Using the delivery system of the present invention, the best encapsulation efficiency and stability can be obtained when the mass ratio of modified bovine serum albumin to double-stranded ribonucleic acid is 12:1. Interfering with the expression of the Hsp90 gene can lead to abnormal development and a significant increase in the mortality rate of insects, especially under high-temperature conditions.

[0075] Members of the cytochrome P450 family CYP302A1 are involved in the biosynthesis of ecdysteroids in Noctuidae insects. Designing double-stranded ribonucleic acid targeting the CYP302A1 gene with a length controlled between 400-450 base pairs can effectively inhibit the synthesis of ecdysteroids. Using the delivery system of the present invention, with the crosslinking degree controlled within the range of 85%-90%, it can stably exist in the insect intestinal environment for more than 48 hours and rapidly release in midgut cells. Interfering with the CYP302A1 gene can block the normal molting process of larvae, resulting in developmental arrest.

[0076] V-type ATPase (V_ATPase) is a key enzyme for maintaining acid-base balance and energy metabolism in insect midgut cells. Designing double-stranded ribonucleic acid with a length of 480-520 base pairs targeting the B subunit of V_ATPase, loading and crosslinking with modified bovine serum albumin, and controlling the particle size within the range of 280-300 nanometers can achieve efficient delivery. Interfering with the expression of the V_ATPase gene will disrupt the normal functions of insect midgut cells, leading to digestive and absorption disorders and abnormal energy metabolism, ultimately resulting in growth retardation and death.

[0077] Acetylcholinesterase is an important enzyme that regulates neurotransmitter conduction in the insect nervous system. Designing double-stranded ribonucleic acid targeting the conserved region of this enzyme gene with a length of about 500 base pairs and using a loading condition with a pH of 9.2 can obtain the best delivery effect. Using the delivery system of the present invention, by adjusting the glutaraldehyde dosage to 0.07 mol per gram of protein, the release efficiency of double-stranded ribonucleic acid in nerve tissues can be improved. Interfering with the expression of acetylcholinesterase can lead to nerve conduction disorders, and insects will show abnormal behaviors, paralysis, and even death.

[0078] The ecdyson receptor is a key regulatory factor in the insect endocrine system. Design double-stranded ribonucleic acid targeting the conserved region of the ecdyson receptor gene, with a length controlled between 470 - 520 base pairs, and achieve efficient interference through the delivery system of the present invention. During the preparation process, the volume ratio of water to ethanol is optimized to 3.5:1, which can improve the targeting of double-stranded ribonucleic acid in endocrine-related tissues. Interfering with the expression of the ecdyson receptor gene can severely disrupt the metamorphosis process of insects, leading to molting failure and death.

[0079] The chitinase gene is involved in the formation of the insect exoskeleton and the molting process. Design double-stranded ribonucleic acid targeting this gene, with a length of approximately 450 base pairs. When using the delivery system of the present invention, control the reaction temperature within the range of 4 - 6 °C and extend the cross-linking time to 5.5 hours, which can improve the stability and delivery efficiency of nanoparticles. Interfering with the expression of the chitinase gene can affect the normal formation and renewal of the insect exoskeleton, resulting in abnormal cuticles, reduced mobility, and decreased survival rate.

[0080] As Figure 5 、 Figure 6 shown, the double-stranded ribonucleic acid of various target genes described above can achieve efficient delivery and release through the pH-responsive delivery system of the present invention. The release rate can reach more than 90% within 12 hours under acidic conditions (pH 5.0 - 5.5), while the release rate remains below 75% within 24 hours under alkaline conditions (pH 9.5 - 10.5), ensuring that the double-stranded ribonucleic acid can play a role at the correct position in Noctuidae insects. Compared with conventional delivery methods, the RNA interference efficiency is increased by 2 - 4 times, effectively reducing the dosage used, improving the prevention and control effect, and reducing the cost.

[0081] Compared with traditional methods, the pH-responsive double-stranded ribonucleic acid delivery system designed in this embodiment has the following significant advantages: First, diimine is used to modify bovine serum albumin, greatly increasing the surface amino density, enhancing the binding ability with double-stranded ribonucleic acid, and improving the encapsulation rate; Second, Schiff base structures are formed through glutaraldehyde cross-linking, endowing the nanoparticles with pH-responsive release characteristics, remaining stable in the alkaline environment (pH about 10.2) of the cotton bollworm intestine, and rapidly releasing ribonucleic acid after entering the acidic environment (pH about 5.3) of the midgut cells, achieving targeted delivery; Third, through the optimized control of the cross-linking reaction kinetics, the prepared nanoparticles have a uniform particle size distribution (PDI < 0.12) and a stable surface potential (about -58 mV), which is beneficial to improving the dispersion stability and in vivo delivery efficiency; Fourth, a nano-drug release dynamic neural network model is used to predict and optimize the performance of the delivery system, greatly shortening the R & D cycle and reducing the R & D cost; Fifth, efficient ribonucleic acid interference is achieved in cotton bollworms (inhibition rate up to 94%), significantly inhibiting their growth and development, and the mortality rate is as high as 83.3%, which is 3.6 times that of directly using double-stranded ribonucleic acid. This delivery system is not only applicable to cotton bollworms, but can also be applied to the control of other noctuid pests by adjusting parameters, and has broad application prospects.

[0082] It should be noted that the detailed explanations of the variables involved in the present invention are shown in Table 6 below.

[0083] Table 6 Variable Explanation Table

[0084] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A method for preparing a pH-responsive release nucleic acid delivery system, characterized in that: include: The modified bovine serum albumin is prepared by reacting bovine serum albumin with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; the modified bovine serum albumin is adjusted to a specified concentration and pH value; the modified bovine serum albumin and double-stranded RNA are mixed according to a mass ratio; glutaraldehyde is added to the mixed solution as a cross-linking agent to form a cross-linked complex; reacting the mixture under low temperature conditions to form a cross-linked composite; Double-stranded RNA modified bovine serum albumin nanoparticles are obtained by purification using an ultrafiltration membrane; the nanoparticles remain stable under alkaline conditions and quickly release RNA under acidic conditions, solving the technical problem of poor stability of double-stranded RNA in the body of Noctuidae insects.

2. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of preparing modified bovine serum albumin by reacting bovine serum albumin with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is specifically to react in phosphate buffer for 2 hours to prepare modified bovine serum albumin with a surface amino group density 3 to 5 times that of the original bovine serum albumin.

3. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of adjusting the modified bovine serum albumin to a specified concentration and pH value specifically comprises adjusting the concentration of the modified bovine serum albumin to 2.5 micrograms per microliter and the pH value of the solution to 9.

4. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of mixing the modified bovine serum albumin and the double-stranded RNA according to the mass ratio is specifically mixing the modified bovine serum albumin and the double-stranded RNA according to the mass ratio of 10 to 1, and the ratio of water to ethanol is 3 to 1.

5. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of adding glutaraldehyde as a cross-linking agent to the mixed solution specifically includes adding glutaraldehyde in an amount of 0.04 to 0.08 mol per gram of protein.

6. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of reacting the mixture under low temperature conditions to form a cross-linked complex, the low temperature conditions are -50°C to 20°C, specifically, reacting at 4 degrees Celsius for 4 to 5 hours.

7. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of using an ultrafiltration membrane to purify and obtain double-stranded RNA-modified bovine serum albumin nanoparticles specifically involves using an ultrafiltration membrane with a molecular weight cutoff of 10 kilodaltons for purification.

8. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of adding glutaraldehyde as a crosslinking agent to the mixed solution also includes a step of optimizing the crosslinking reaction kinetics, specifically, by real-time monitoring the reaction kinetic parameters of Schiff base formation between glutaraldehyde and the surface amino groups of modified bovine serum albumin, and adjusting the crosslinking agent addition method according to the Schiff base formation reaction kinetic equation.

9. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The Schiff base formation reaction kinetic equation is used to predict the progress and efficiency of the cross-linking reaction under different reaction conditions. The input includes the concentration of modified bovine serum albumin, the concentration of glutaraldehyde, the pH value of the solution, the ionic strength of the solution and the reaction temperature. The output is the Schiff base formation rate and the final cross-linking degree.

10. The method for preparing the pH-responsive release nucleic acid delivery system according to claim 1, characterized in that: The step of maintaining stability of the double-stranded RNA modified bovine serum albumin nanoparticles under alkaline conditions and rapidly releasing RNA under acidic conditions also includes a release prediction optimization step, specifically using a pre-trained nano drug delivery release dynamic neural network model to predict the release curve of the double-stranded RNA under different pH conditions, and optimizing the structural parameters of the double-stranded RNA modified bovine serum albumin nanoparticles accordingly.

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