A chitosan composite nano-preparation prolonging the persistence period of citrus psylla RNA pesticide

Abstract

The application discloses a chitosan composite nano preparation for prolonging the persistence period of citrus psylla RNA pesticide, and a preparation method and application thereof. The preparation adopts a four-layer core-shell structure design of "core-inner layer-intermediate layer-outer layer", is composed of dsRNA for targeting key genes of citrus psylla and a composite nano carrier shell, and is formed by ion cross-linking and surface hydrophilic modification of modified chitosan, carboxymethyl starch, sodium tripolyphosphate, polyethylene glycol 2000 and sodium alginate. Through the synergistic effect of the composite carrier, the problems of easy degradation of naked dsRNA, poor weather resistance and short persistence period of conventional chitosan nano preparations are effectively solved, ultraviolet resistance, nuclease degradation resistance, rainwater erosion resistance and wood psylla midgut pH intelligent response release are realized. The field persistence period of the preparation can reach 15-20 days, the dsRNA encapsulation rate reaches 85%-92%, the dsRNA retention rate after rainwater erosion resistance reaches more than 75%, the number of pesticide application is significantly reduced, the labor cost is reduced, and the preparation is suitable for green prevention and control of citrus psylla in citrus orchards.

Description

Technical Field

[0001] This invention belongs to the field of biopesticides and nanodelivery formulation technology, specifically involving a chitosan composite nanoformulation for targeted control of citrus psyllids and long-acting sustained-release dsRNA. It is suitable for the control of citrus psyllids in citrus orchards and the integrated management of vector insects of citrus Huanglongbing (HLB). It falls under the category of RNA interference biopesticide carrier modification and processing technology. Background Technology

[0002] Citrus psyllids are the only natural vector for citrus Huanglongbing (HLB), and eradicating them is the core means to stop the spread of HLB. RNA interference (dsRNA) pesticides have become a hot topic in green pest control due to their advantages of targeted specificity, environmental safety, no residue, and low likelihood of inducing resistance. However, the field application of naked dsRNA pesticides has significant shortcomings:

[0003] In the natural environment, it is easily degraded by ultraviolet light and field nucleases. It becomes inactive 2-3 days after foliar spraying. Conventional formulations have an effective period of only 3-5 days, requiring frequent and multiple applications, which results in high labor costs.

[0004] Citrus psyllids have piercing-sucking mouthparts. Naked dsRNA is difficult to penetrate the insect's epidermis and midgut barrier and is hydrolyzed by RNase in the insect's midgut. As a result, the pests have low uptake efficiency and unstable control efficacy.

[0005] Conventional single chitosan nanoparticles have poor resistance to rain erosion. After being dissolved by rainwater, the carrier disintegrates and dsRNA leaks out, making it impossible to achieve long-term controlled release.

[0006] Existing modified chitosan carriers mostly employ single cross-linking modification, which only slightly improves stability and cannot simultaneously achieve the triple properties of resistance to UV degradation, resistance to rain washout, and pH-responsive release in the insect gut. This makes it difficult to solve the problem of long-lasting RNA pesticides in the hot and rainy environments of citrus-producing regions. Therefore, there is an urgent industrial need to develop composite nano-formulations that can significantly extend the effective period of dsRNA pesticides, improve environmental stress resistance, and achieve gut-targeted drug release. (Invention Content)

[0007] (I) Technical Problem Solved: Overcoming the shortcomings of existing naked dsRNA, such as easy degradation, poor weather resistance, short duration of action, easy leaching by rainwater, and low utilization rate by insects, this paper provides a chitosan composite nanocarrier-encapsulated composite nano-formulation targeting the key gene dsRNA of the citrus psyllid. This achieves UV resistance, resistance to nuclease degradation, resistance to rainwater erosion, and intelligent slow release at the midgut pH of the psyllid, significantly extending the duration of pesticide effectiveness in the field. (II) Technical Solution

[0008] Option 1: A chitosan composite nano-formulation for prolonging the residual effect of RNA pesticides on citrus psyllids

[0009] As shown in Figure 1, the formulation of the present invention adopts a four-layer core-shell structure design of "core-inner layer-middle layer-outer layer", which is composed of core active components and composite nanocarrier shell;

[0010] Core active ingredient (1): dsRNA targeting the chitin synthase gene (CHS1) / acetylcholinesterase gene (AChE) of citrus psyllid; the nucleotide sequence of the dsRNA is selected from either SEQ ID NO.1 or SEQ ID NO.2, and the molecular weight of the dsRNA is 400-800 bp;

[0011] Composite nanocarrier raw materials (mass ratio): 1.8-3.2 parts modified chitosan, 0.4-0.8 parts carboxymethyl starch, 0.15-0.3 parts sodium tripolyphosphate, 0.2-0.5 parts polyethylene glycol 2000 (PEG2000), and 0.2-0.6 parts sodium alginate;

[0012] The carrier is cross-linked with ion and hydrophilic surface modification to form core-shell structured nanospheres, in which dsRNA is completely encapsulated in the internal cavity of the nanoparticles; the inner cross-linking network (2) is formed by cross-linking modified chitosan and sodium tripolyphosphate through ion bonds, which is responsible for encapsulating dsRNA and providing pH-responsive release performance; the middle reinforcing layer (3) is formed by carboxymethyl starch and sodium alginate, which is responsible for enhancing the mechanical strength of the carrier and leaf adhesion; the outer hydrophilic modification layer (4) is formed by graft modification with PEG2000, which is responsible for improving the carrier's resistance to UV degradation and dispersion stability; the formulation is spherical (5) with an average particle size of 180-320 nm and a Zeta potential of +22-+35 mV.

[0013] Optimal proportions: 2.5 parts modified chitosan, 0.6 parts carboxymethyl starch, 0.22 parts sodium tripolyphosphate, 0.35 parts PEG2000, and 0.4 parts sodium alginate.

[0014] The modified chitosan is a product of oligochitosan lightly grafted with citric acid. The degree of deacetylation of the oligochitosan is ≥92%, the molecular weight is 3000-12000 Da, and the grafting degree is 15%-25%, which improves the ultraviolet shielding performance and pH response of the carrier.

[0015] Option 2: Preparation method of the above chitosan composite nano-formulation

[0016] As shown in Figure 7, the preparation method of the present invention includes the following steps:

[0017] S1. Preparation of modified chitosan aqueous solution: Dissolve modified chitosan in glacial acetic acid-sodium acetate buffer (pH=5.0~5.6) to prepare a chitosan stock solution with a mass fraction of 0.25%~0.4%; add carboxymethyl starch and PEG2000 in sequence, stir at low speed at room temperature for 30~50 min to obtain solution A;

[0018] S2. Preparation of crosslinking aid: Sodium tripolyphosphate and sodium alginate are dissolved in sterile pure water to prepare a 0.12% (w / w) crosslinking solution B.

[0019] S3, dsRNA dilution: Dissolve the targeting dsRNA in sterile DEPC water to prepare a dsRNA solution with a concentration of 1.2–1.8 μg / μL, and store at 4°C for later use;

[0020] S4. Nanoparticle self-assembly: Place solution A on a magnetic stirrer and control the speed at 600-800 rpm. Use a syringe to slowly add solution B dropwise at a rate of 0.5-1.0 mL / min. After the addition is complete, continue stirring for 15 min. Then add dsRNA solution dropwise at the same rate. After the addition is complete, continue stirring for 30 min to obtain a nanoparticle suspension.

[0021] S5. Solidification and purification: The suspension was solidified in a 4°C refrigerator for 2–4 h, and then dialyzed with ultrapure water for 24 h (molecular weight cutoff 8000–14000 Da). The dialysate was changed every 6 h to remove unencapsulated dsRNA and free cross-linking agent.

[0022] S6. Freeze-drying: Add 5%–8% mannitol by mass to the dialysis suspension as a freeze-drying protectant, pre-freeze at -40℃ for 4 h, and then freeze-dry under vacuum for 24 h to obtain a white powdered chitosan composite nano-formulation.

[0023] Option 3: Application of the above-mentioned chitosan composite nano-formulation in the control of citrus psyllids

[0024] The chitosan composite nano-formulation was diluted with water to a final dsRNA concentration of 50–200 μg / mL and applied to citrus plants via foliar spraying every 15–20 days. (III) Beneficial Effects

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] Significantly extended duration of action: The dense four-layered core-shell structure formed by the composite carrier can effectively shield against ultraviolet light and the degradation of dsRNA by nucleases, extending the field duration of action from 3-5 days for conventional formulations to 15-20 days, reducing the number of applications by 3-4 times per year, and significantly reducing labor costs.

[0027] Excellent resistance to rain washout: The synergistic effect of carboxymethyl starch and sodium alginate enhances the adhesion of nanoparticles to the surface of citrus leaves. After simulated rain washout (rainfall of 10 mm / h, washout for 30 min), the dsRNA retention rate on the leaf surface is still above 75%, which is much higher than the 32% of single chitosan nanoparticles.

[0028] Psyllid midgut pH-responsive smart release: Modified chitosan rapidly swells and disintegrates in an acidic environment (psyllid midgut pH=5.0~6.0), releasing the encapsulated dsRNA, while remaining stable in a neutral environment (leaf surface pH=6.5~7.5), achieving targeted drug release and improving dsRNA utilization.

[0029] High encapsulation efficiency and drug loading: The nano-formulation dsRNA prepared by this invention has an encapsulation efficiency of 85% to 92% and a drug loading of 12% to 18%, which is significantly higher than the existing technology level.

[0030] Environmentally friendly: All carrier raw materials are natural biodegradable polymer materials, which are safe for non-target organisms, leave no residue, and meet the requirements of green agricultural development. Attached Figure Description

[0031] To provide a more detailed description of the technical solutions of the embodiments of this invention, the accompanying drawings used in the following description of the embodiments are briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on such drawings without creative effort.

[0032] Figure 1. Schematic diagram of the core-shell structure of the chitosan composite nanoformulation of the present invention.

[0033] Figure 2. Transmission electron microscopy (TEM) image of the chitosan composite nanoparticles prepared in Example 2.

[0034] Figure 3. Particle size distribution and Zeta potential of the chitosan composite nanoparticles prepared in Example 2.

[0035] Figure 4. Agarose gel electrophoresis images of the UV degradation resistance of different samples

[0036] Figure 5. Bar chart comparing the rain erosion resistance of different samples

[0037] Figure 6. Comparison curves of the field efficacy duration of different samples

[0038] Figure 7. Flowchart of the preparation process of the chitosan composite nano-formulation of the present invention Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0040] Example 1

[0041] 10 g of oligochitosan (95% degree of deacetylation, molecular weight 5000 Da) was added to 100 mL of 0.5 mol / L citric acid solution and stirred in a 60℃ water bath for 3 h. After the reaction was completed, the pH was adjusted to 7.0 with 1 mol / L NaOH solution, and then dialyzed with ultrapure water for 48 h (molecular weight cutoff 3500 Da), with the dialysate being replaced every 12 h. Finally, the mixture was freeze-dried under vacuum to obtain citric acid grafted modified chitosan with a grafting degree of 21%.

[0042] Example 2

[0043] S1. Weigh 2.5 g of the modified chitosan prepared in Example 1, dissolve it in 1000 mL of glacial acetic acid-sodium acetate buffer (pH=5.2) to prepare a chitosan stock solution with a mass fraction of 0.25%; add 0.6 g of carboxymethyl starch and 0.35 g of PEG2000 in sequence, stir at low speed at room temperature for 40 min to obtain solution A;

[0044] S2. Weigh 0.22 g sodium tripolyphosphate and 0.4 g sodium alginate, dissolve them in 500 mL sterile pure water, and prepare a cross-linking solution B with a mass fraction of 0.12%.

[0045] S3. Dissolve the dsRNA (SEQ ID NO.1, molecular weight 520 bp) targeting the CHS1 gene of citrus psyllid in sterile DEPC water to prepare a dsRNA solution with a concentration of 1.5 μg / μL.

[0046] S4. Place solution A on a magnetic stirrer and control the speed at 700 rpm. Use a syringe to slowly add solution B dropwise at a rate of 0.8 mL / min. After the addition is complete, continue stirring for 15 min. Then add 100 mL of dsRNA solution dropwise at the same rate. After the addition is complete, continue stirring for 30 min to obtain a nanoparticle suspension.

[0047] S5. Place the suspension in a 4°C refrigerator to solidify for 3 hours, then dialyze with ultrapure water for 24 hours (molecular weight cutoff 10000 Da), changing the dialysate every 6 hours.

[0048] S6. Add 6% mannitol by mass to the dialysis suspension as a freeze-drying protectant, pre-freeze at -40℃ for 4 h, and then freeze-dry under vacuum for 24 h to obtain a white powdered chitosan composite nano-formulation.

[0049] Formulation characterization results:

[0050] As shown in Figure 2, transmission electron microscopy revealed that the nanoparticles were uniformly spherical, well dispersed, and without obvious aggregation. High-magnification electron microscopy clearly showed the core-shell layered structure, with the core being darker (dsRNA enrichment area) and the shell being lighter (composite carrier layer).

[0051] As shown in Figure 3, dynamic light scattering detection showed that the particle size of the formulation exhibited a single-peak distribution, with a PDI value of 0.128 and an average particle size of 245 nm; the Zeta potential also exhibited a single-peak distribution with an average potential of +28.6 mV.

[0052] According to ultraviolet spectrophotometry, the dsRNA encapsulation efficiency of this formulation was 89.2%, and the drug loading was 15.3%.

[0053] Example 3

[0054] Except for replacing the dsRNA with a dsRNA targeting the AChE gene of the citrus psyllid (SEQ ID NO.2, molecular weight 480 bp), the remaining steps are the same as in Example 2.

[0055] The formulation was tested and found to have an average particle size of 238 nm, a zeta potential of +27.9 mV, a dsRNA encapsulation efficiency of 88.7%, and a drug loading of 14.8%.

[0056] Comparative Example 1

[0057] Without adding carboxymethyl starch, PEG2000, and sodium alginate, the remaining steps are the same as in Example 2.

[0058] The formulation was tested and found to have an average particle size of 195 nm, a zeta potential of +32.1 mV, a dsRNA encapsulation efficiency of 72.5%, and a drug loading of 9.6%.

[0059] Comparative Example 2

[0060] Commercially available uncoated dsRNA formulations were used, and the dsRNA concentration was the same as in Example 2.

[0061] Experimental Example 1

[0062] The samples from Example 2, Comparative Example 1, and Comparative Example 2 were diluted to a dsRNA concentration of 100 μg / mL and irradiated under a UV lamp (30 W, 30 cm distance) for different times. The integrity of the dsRNA was detected by agarose gel electrophoresis, and the results are shown in Figure 4.

[0063] The results showed that the naked dsRNA in Comparative Example 2 was completely degraded after 2 h of UV irradiation (lane 2); the single chitosan nanoparticle formulation in Comparative Example 1 showed a dsRNA degradation rate of 65% after 8 h of UV irradiation (lane 4); while the composite nanoparticle formulation in Example 2 showed a dsRNA degradation rate of only 18% after 24 h of UV irradiation (lane 6), demonstrating excellent resistance to UV degradation.

[0064] Experiment Example 2

[0065] The samples from Example 2 and Comparative Example 1 were diluted to a dsRNA concentration of 100 μg / mL and sprayed evenly onto the surface of citrus leaves. After air drying, they were washed using an artificial rainfall simulation device (rainfall rate 10 mm / h, washing for 30 min). After washing, the nanoparticles remaining on the leaf surface were collected, dsRNA was extracted and its content was determined, and the retention rate was calculated. The results are shown in Figure 5.

[0066] The results showed that the single chitosan nanoparticle formulation in Comparative Example 1 had a dsRNA retention rate of only 31.8% after being washed away by rainwater; while the composite nanoparticle formulation in Example 2 had a dsRNA retention rate of 76.3%, and its resistance to rainwater erosion was significantly improved.

[0067] Experimental Example 3

[0068] The experiment was conducted in a citrus orchard in Nankang District, Ganzhou City, Jiangxi Province, using navel orange trees of uniform growth as the experimental subjects. Samples from Example 2, Comparative Example 1, and Comparative Example 2 were diluted to a final dsRNA concentration of 100 μg / mL and applied as foliar sprays, with each treatment repeated three times. The population of citrus psyllids was regularly monitored after application, and the population reduction rate was calculated. The results are shown in Figure 6.

[0069] The results show:

[0070] Comparative Example 2 (naked dsRNA): The insect population reduction rate was 62% 3 days after application, dropped to 28% after 5 days, and had almost no effect after 7 days;

[0071] Comparative Example 1 (single chitosan nano-formulation): The insect population reduction rate was 75% 7 days after application, dropped to 41% after 10 days, and had almost no control effect after 14 days;

[0072] Example 2 (composite nano-formulation): The insect population reduction rate was 92% 7 days after application, still reached 83% 14 days, and decreased to 56% 20 days, with a field effective period of 15-20 days.

Claims

1. A chitosan composite nano-formulation for prolonging the residual effect of RNA pesticides against citrus psyllids, characterized in that, It adopts a four-layer core-shell structure consisting of an inner core active component and a composite nanocarrier shell. Core active ingredient: dsRNA targeting the chitin synthase gene or acetylcholinesterase gene of citrus psyllid; Composite nanocarrier raw materials (mass ratio): modified chitosan 1.8-3.2 parts, carboxymethyl starch 0.4-0.8 parts, sodium tripolyphosphate 0.15-0.3 parts, polyethylene glycol 2000 0.2-0.5 parts, sodium alginate 0.2-0.6 parts; The carrier is cross-linked with ions and modified with hydrophilic surface to form core-shell structured nanospheres, in which dsRNA is encapsulated in the internal cavity of the nanoparticles; the average particle size of the formulation is 180–320 nm, and the Zeta potential is +22–+35 mV.

2. The chitosan composite nano-formulation according to claim 1, characterized in that, The dsRNA nucleotide sequence is selected from either SEQ ID NO.1 or SEQ ID NO.2, and the dsRNA molecular weight is 400–800 bp.

3. The chitosan composite nano-formulation according to claim 1, characterized in that, The preferred mass ratio of the composite nanocarrier raw materials is as follows: 2.5 parts modified chitosan, 0.6 parts carboxymethyl starch, 0.22 parts sodium tripolyphosphate, 0.35 parts polyethylene glycol 2000, and 0.4 parts sodium alginate.

4. The chitosan composite nano-formulation according to claim 1, characterized in that, The modified chitosan is a product of oligochitosan that has been lightly grafted with citric acid. The degree of deacetylation of the oligochitosan is ≥92%, the molecular weight is 3000-12000 Da, and the grafting degree is 15%-25%.

5. A method for preparing a chitosan composite nanoparticle formulation as described in any one of claims 1-4, characterized in that, It includes the following steps: S1. Preparation of modified chitosan aqueous solution: Dissolve modified chitosan in glacial acetic acid-sodium acetate buffer (pH=5.0~5.6) to prepare a chitosan stock solution with a mass fraction of 0.25%~0.4%; add carboxymethyl starch and polyethylene glycol 2000 in sequence, stir at low speed at room temperature for 30~50 min to obtain solution A; S2. Preparation of crosslinking aid: Sodium tripolyphosphate and sodium alginate are dissolved in sterile pure water to prepare a 0.12% (w / w) crosslinking solution B. S3, dsRNA dilution: Dissolve the targeting dsRNA in sterile DEPC water to prepare a dsRNA solution with a concentration of 1.2–1.8 μg / μL, and store at 4°C for later use; S4. Nanoparticle self-assembly: Place solution A on a magnetic stirrer and control the speed at 600-800 rpm. Use a syringe to slowly add solution B dropwise at a rate of 0.5-1.0 mL / min. After the addition is complete, continue stirring for 15 min. Then add dsRNA solution dropwise at the same rate. After the addition is complete, continue stirring for 30 min to obtain a nanoparticle suspension. S5. Solidification and purification: The suspension was solidified in a 4°C refrigerator for 2-4 hours, and then dialyzed with ultrapure water for 24 hours. The molecular weight cutoff was 8000-14000 Da. The dialysate was changed every 6 hours. S6. Freeze-drying: Add 5%–8% mannitol by mass to the dialysis suspension as a freeze-drying protectant, pre-freeze at -40℃ for 4 h, and then freeze-dry under vacuum for 24 h to obtain a white powdered chitosan composite nano-formulation.

6. The application of the chitosan composite nano-formulation as described in any one of claims 1-4 in the control of citrus psyllids, characterized in that, Dilute the chitosan composite nano-formulation with water to a final dsRNA concentration of 50–200 μg / mL, and apply it to citrus plants by foliar spraying once every 15–20 days.

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