Small-particle-size hyphantria cunea nuclear polyhedrosis virus nano-emulsion as well as preparation method and application thereof

Abstract

The invention discloses a small-particle-size hyphantria cunea nuclear polyhedrosis virus nano-emulsion as well as a preparation method and application thereof. Improvement is carried out on the basis of hyphantria cunea nuclear polyhedrosis virus, nano material preparation and Pickering emulsification technologies are combined, natural renewable polysaccharide polymer sodium alginate is used as a raw material, a sodium alginate derivative (CSAD) is obtained through cholesterol grafting modification, a stable Pickering emulsion is formed after the nuclear polyhedrosis virus is entrapped, and the stable Pickering emulsion is prepared. The novel dosage form has nanoscale particle diameter and is suitable for being used under low volume, the fast-acting property of the dosage form on the fall webworm is remarkably improved, the insecticidal activity is based on natural virus particles of the fall webworm, and the novel dosage form has specificity on target pests and is small in environmental influence. According to the method, the used materials are low in price, the operation process is simple and easy to standardize, and the nano Pickering emulsion has better persistence compared with a common aqueous solution, and is convenient to use and easy to popularize.

Description

Technical Field

[0001] The present invention belongs to the field of biological pesticides, and particularly relates to a small-particle-size nuclear polyhedrosis virus of Hyphantria cunea nanoemulsion, a preparation method thereof, and an application thereof. Background Art

[0003] Pickering emulsion is an emulsion that does not contain surfactants and is stabilized by tiny solid particles. It has the advantages of less foam, low toxicity, low irritation without surfactants, high stability and reproducibility, and overcomes the dependence of traditional emulsions on surfactants. At the same time, it is also a multiphase system. The solid particles at the aqueous phase, oil phase and interface act synergistically, and can encapsulate various active substances separately or simultaneously. The emulsion has strong coalescence stability, and the unique core-shell structure can also control the release of active ingredients, and also shows unique advantages in improving the permeability and diffusibility of active ingredients. Sodium alginate is the sodium salt of alginic acid, has the gel properties of alginic acid, has a certain responsiveness to pH value, temperature and salt concentration, is safe and non-toxic, and has good biocompatibility and biodegradability. By grafting cholesterol, a hydrophobic molecule, onto sodium alginate for modification, a new amphiphilic derivative CSAD is synthesized, which is low in cost, environmentally friendly, easy to biodegrade, good in stability, improves the amphiphilicity of the material, and improves the performance as a carrier material.

[0004] The nuclear polyhedrosis virus of Hyphantria cunea is an important insect pathogenic microorganism, which has the characteristics of strong specificity, high activity, no pollution to the environment, safety and non-toxicity, and is an environmentally friendly microbial biological control agent. Using insect viruses for control can reduce the use of highly toxic pesticides and plays an important role in pest control. However, directly using nuclear polyhedrosis virus for control is easily affected by seasons and climate, has a long insecticidal cycle, reduced activity, and limited effects. Summary of the Invention

[0005] The present invention provides a preparation method and an application of a small-particle-size nuclear polyhedrosis virus of Hyphantria cunea nano Pickering emulsion. Using sodium alginate as a raw material, sodium alginate derivative (CSAD) is obtained through cholesterol grafting modification, and then CSAD is used to activate and modify silicon dioxide SiO2 to optimize the interfacial adsorption ability. After encapsulating the nuclear polyhedrosis virus, a stable Pickering emulsion is formed, which improves the stability and release efficiency of the active ingredient, the nuclear polyhedrosis virus, in the alkaline environment of the midgut of Hyphantria cunea, and solves the problems in the prior art that directly using nuclear polyhedrosis virus for control is easily affected by the environment, has a long insecticidal cycle, and low activity.

[0006] The present invention is realized through the following technical solutions:

[0007] A preparation method of a small-particle-size nuclear polyhedrosis virus of Hyphantria cunea nano Pickering emulsion, comprising the following steps:

[0008] 1) Preparation of cholesterol-grafted sodium alginate (CSAD): Using alginic acid and cholesterol dispersed in a solvent as raw materials, N,N'-dicyclohexylcarbodiimide (DCC, 90.0%) and 4-dimethylaminopyridine (DMAP, 99%) were added, and the reaction was stirred at room temperature. After purification, cholesterol-grafted sodium alginate (CSAD) was obtained;

[0009] 2) Preparation of cholesterol-grafted sodium alginate-modified silica (CSAD-SiO2): CSAD in step 1) was ultrasonically dispersed in water, nano-silica was added, and after stirring, shear treatment was carried out to obtain an emulsion. After centrifuging the emulsion, the precipitate was taken and freeze-dried to obtain the CSAD-SiO2 lyophilized powder;

[0010] 3) Preparation of nano-Pickering emulsion: The freeze-dried powder of the nucleopolyhedrovirus of Hyphantria cunea (HcNPV) was dissolved in deionized water to obtain a virus solution. The CSAD-SiO2 lyophilized powder in step 2) was added to paraffin oil and ultrasonically treated to obtain a dispersion; the dispersion was mixed with the virus solution and ultrasonically emulsified to obtain a nano-Pickering emulsion (CSAD-SiO2@HcNPV).

[0011] Preferably, in step 1), the mass ratio of alginic acid, cholesterol, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is (2.5 - 3.5):(2.5 - 3.4):(0.8 - 1.0):(0.05 - 0.15), and more preferably 3:3:0.9:0.1.

[0012] Preferably, in step 1), the M of the alginic acid w is 30000 - 300000; more preferably 112230.

[0013] Preferably, the specific steps of step 1) are: dissolving dry alginic acid in dimethyl sulfoxide to obtain solution A; dissolving cholesterol in chloroform and slowly adding it to solution A to obtain solution B; weighing N,N'-dicyclohexylcarbodiimide (DCC, 90.0%) and 4-dimethylaminopyridine (DMAP, 99%) respectively, dissolving them in dimethyl sulfoxide, and then dropping them into solution B, and stirring the reaction at room temperature for 24 h to obtain solution C; adding absolute ethanol to precipitate in solution C, centrifuging to remove the supernatant, and obtaining precipitate D by vacuum freezing; dissolving precipitate D in distilled water, adjusting the pH to 7.0 with sodium bicarbonate, standing and then centrifuging to take the supernatant E, loading it into a dialysis bag for dialysis, adding absolute ethanol to precipitate and drying to obtain CSAD. Further, the cut-off molecular weight of the dialysis bag is 2000 Da.

[0014] Preferably, in step 2), CSAD is ultrasonically dispersed in water at 1 mg / mL.

[0015] Preferably, in step 2), the mass ratio of CSAD to nano-silica is 1:(8 - 10). More preferably, it is 1:9 (20 mg: 180 mg).

[0016] Preferably, in step 2), nano-silica is added, and it is stirred at 1000 rpm for 18 min and sheared at 22000 rpm by a shear emulsifier for 4 min to obtain an emulsion.

[0017] Preferably, in step 3), the mass concentration of the freeze-dried powder of Hyphantria cunea nucleopolyhedrovirus in the virus solution is 0.5 mg / 5 g, and the mass concentration of the CSAD-SiO2 freeze-dried powder in the dispersion liquid is 0.1 g / 10 mL; the volume ratio of the dispersion liquid to the virus solution is 1:(0.3 - 0.8). More preferably, it is 1:0.5.

[0018] Preferably, in step 3), the conditions for ultrasonic emulsification are: put it into an ultrasonic cell disruptor and perform ultrasonic emulsification at an output frequency of 315 W for 14 min.

[0019] Preferably, in step 3), the preparation method of the freeze-dried powder of Hyphantria cunea nucleopolyhedrovirus is: take the dead larvae of Hyphantria cunea nucleopolyhedrovirus, grind them, filter, centrifuge at 500 r / min for 10 min, aspirate the supernatant, and then centrifuge at 6000 r / min for 30 min. Remove the supernatant, repeat several times, remove the supernatant, leave the precipitate, suspend the precipitate with double-distilled water, and purify it by sucrose density gradient centrifugation. Wash away the sucrose to obtain a white precipitate, that is, obtain the purified Hyphantria cunea nucleopolyhedrovirus precipitate (HcNPV), and remove the excess water by vacuum freezing to obtain the freeze-dried powder of Hyphantria cunea nucleopolyhedrovirus.

[0020] The present invention also protects the application of the nano Pickering emulsion (CSAD-SiO2@HcNPV) prepared by the above preparation method in controlling Hyphantria cunea.

[0021] The present invention also provides a method for controlling Hyphantria cunea, which is to use the nano Pickering emulsion prepared by the above method to kill the larvae of Hyphantria cunea. Preferably, the dosage is 330 - 350 PIBs per larva.

[0022] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0023] 1. The present invention is based on the improvement of the nucleopolyhedrovirus of Hyphantria cunea, combined with the preparation of nanomaterials and Pickering emulsion technology. Using the natural renewable polysaccharide polymer sodium alginate as the raw material, sodium alginate derivative (CSAD) is obtained by cholesterol graft modification, and the surface of nano-silica is modified by CSAD to form composite nanoparticles, obtaining modified silica nanoparticles (CSAD-SiO2), which improves stability and loading capacity. After encapsulating the active ingredient nucleopolyhedrovirus, a stable Pickering emulsion is formed. The new dosage form has a nanoscale particle diameter and is suitable for use at low volumes. The quick-acting property of the dosage form against Hyphantria cunea is significantly increased. The insecticidal activity is based on the natural virus particles of Hyphantria cunea, with specificity for target pests and little environmental impact.

[0024] 2. The nano-Pickering emulsion prepared by the method of the present invention prolongs the continuous action time of the virus and has better persistence than ordinary aqueous solutions; the materials used are inexpensive, the preparation conditions are mild, and the operation process is simple and easy to standardize; the raw materials used are safe and non-toxic, do not contain surfactants, and are easy to promote.

[0025] 3. Traditional Pickering emulsions are limited by the interfacial adsorption capacity of solid particles, and the encapsulated particle size is usually small, and only small molecule chemical pesticides can be loaded. However, in the method of the present invention, under high-speed shear conditions, CSAD successfully activates SiO2 to make the amphiphilic modification of the material more thorough, and it is easy to form a water-in-oil emulsion. Further, through ultrasonic emulsification technology, the macromolecule nucleopolyhedrovirus is successfully loaded, which not only expands the particle size of the encapsulated particles, but also has better stability. Description of the Drawings

[0026] Figure 1 It is the virulence results of the nano-Pickering emulsion CSAD-SiO2@HcNPV and the virus aqueous solution on the larvae of Hyphantria cunea in Example 2.

[0027] Figure 2 It is the statistical result of the particle diameter distribution of the nano-Pickering emulsion CSAD-SiO2@HcNPV and CSAD-SiO2 dispersed particles in Example 3.

[0028] Figure 3 It is the statistical result of the Zata potential of the nano-Pickering emulsion CSAD-SiO2@HcNPV, CSAD-SiO2 and SiO2 dispersed particles and the result in Example 4.

[0029] Figure 4 It is the FT-IR diagram of sodium alginate and CSAD in Example 5. Detailed Embodiments

[0030] In order to make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Example 1 Preparation of nano Pickering emulsion (CSAD-SiO2@HcNPV)

[0032] In September 2024, the nano Pickering emulsion (CSAD-SiO2@HcNPV) was prepared at the Institute of Zoology, Guangdong Academy of Sciences. The specific steps are as follows:

[0033] 1. Preparation of freeze-dried powder of Hyphantria cunea nucleopolyhedrovirus (HcNPV):

[0034] Take an appropriate amount of Hyphantria cunea nucleopolyhedrovirus-killed insect corpses, put them into a mortar and grind them, filter, centrifuge at 500 r / min for 10 min, suck the supernatant, and then centrifuge at 6000 r / min for 30 min. Remove the supernatant. Repeat several times to remove the supernatant and leave the precipitate. Suspend the precipitate with double-distilled water and purify it by sucrose density gradient centrifugation. After washing away the sucrose, a white precipitate is obtained, that is, the purified Hyphantria cunea nucleopolyhedrovirus precipitate (HcNPV) is obtained. Excess water is removed by vacuum freezing to obtain the freeze-dried powder of nucleopolyhedrovirus. Each 1 mg of virus freeze-dried powder contains 1×10 8 PIBs.

[0035] 2. Preparation of cholesterol-grafted sodium alginate (CSAD):

[0036] (1) After drying sodium alginic acid (CP, M w =112230) with calcium hydride, dissolve it in dimethyl sulfoxide (DMSO) at a ratio of 3 g:90 ml to obtain solution A;

[0037] (2) Dissolve 3 g of cholesterol in 10 ml of chloroform and slowly add it to solution A to obtain solution B;

[0038] (3) Weigh 0.9 g of N,N'-dicyclohexylcarbodiimide (DCC, 90.0%) and 0.1 g of 4-dimethylaminopyridine (DMAP, 99%) and dissolve them in 15 ml of DMSO, and then add them dropwise to solution B. Stir and react at room temperature for 24 h to obtain solution C;

[0039] (4) Add 4 times the volume of absolute ethanol to solution C for precipitation, centrifuge and separate, remove the supernatant, and obtain dry precipitate D by vacuum freezing;

[0040] (5) Dissolve precipitate D in 50 ml of distilled water, adjust the pH to 7.0 with sodium bicarbonate, let it stand and then centrifuge to obtain the supernatant E;

[0041] (6) Load the supernatant E into a dialysis bag with a molecular weight cut-off of 2000 Da for dialysis, add 4 times the volume of absolute ethanol to precipitate and dry it to obtain CSAD.

[0042] 3. Preparation of cholesterol-grafted sodium alginate modified silica (CSAD-SiO2):

[0043] (1) Take 20 mg of the prepared CSAD and dissolve it in 20 mL of water at 1 mg / mL, and ultrasonicate for 5 min to fully disperse it to obtain solution F;

[0044] (2) Add 180 mg of nano-silica to solution F (20 mL), stir at 1000 rpm for 18 min, and shear for 4 min at 22000 rpm in D gear of a shear emulsifier (LABGIC homogenizer / disperser, model: L-HO-M; 5-6 gears) to obtain emulsion G;

[0045] (3) Centrifuge emulsion G at 12000 rpm for 15 min, take the precipitate and freeze-dry it with a vacuum freeze dryer to obtain the CSAD-SiO2 lyophilized powder.

[0046] 4. Preparation of Pickering emulsion of Hyphantria cunea nuclear polyhedrosis virus (CSAD-SiO2@HcNPV):

[0047] (1) Dissolve 0.5 mg of the virus freeze-dried powder in 5 g of deionized water to obtain solution H;

[0048] (2) Add 0.1 g of the CSAD-SiO2 lyophilized powder to 10 mL of paraffin oil and ultrasonicate for 20 min to obtain dispersion I;

[0049] (3) Mix dispersion I with the virus solution H, with the volume ratio of the mixture being 2:1, put it into an ultrasonic cell disruptor, and ultrasonically emulsify for 14 min at an output frequency of 315 W to obtain the nano-Pickering emulsion (CSAD-SiO2@HcNPV).

[0050] Example 2 Determination of the insecticidal effect of the nano-Pickering emulsion (CSAD-SiO2@HcNPV)

[0051] In September 2024, the insecticidal effect of CSAD-SiO2@HcNPV (prepared in Example 1) was determined in the Biosafety Level 2 Laboratory (BSL-2) of the Institute of Zoology, Guangdong Academy of Sciences.

[0052] 1. Experimental materials:

[0053] 3rd instar larvae of Hyphantria cunea: Select 3rd instar larvae of Hyphantria cunea with consistent growth and development and basically the same individual size, and place them in a sterilized plastic box (specification: 7 cm × 7 cm × 11.2 cm) for 8 h of starvation treatment.

[0054] Mulberry leaves: Clean and wipe the mulberry leaves, and cut the leaves into pieces of 6 × 6 cm in size.

[0055] Nano Pickering emulsion: Prepared in Example 1.

[0056] Aqueous solution of Hyphantria cunea nucleopolyhedrovirus (as a control): Dissolve 0.5 mg of virus freeze-dried powder in water to obtain a pure virus aqueous solution with a concentration of about 1×10 7 PIBs / mL. The insecticidal rate is 100% at this concentration. By diluting to a low concentration, the rapid efficacy of the emulsion and the aqueous solution is compared.

[0057] 2. Experimental method (feeding method):

[0058] Apply the aqueous solution of Hyphantria cunea nucleopolyhedrovirus or nano Pickering emulsion containing the same virulence of 1×10 4 PIBs on the leaves. (Take 30 larvae of Hyphantria cunea as a group, with the same virulence, and each group is fed a total of 1×10 4 PIBs of nano Pickering emulsion or virus aqueous solution). After drying, put them into the plastic box containing the larvae, and transfer the test insects to a constant temperature incubator at a temperature of 25 ± 1°C, a relative humidity of 75 ± 5%, and a photoperiod of L:D = 14:10 for observation. After 24 h of feeding, replace with non-toxic fresh mulberry leaves. Repeat the experiment 3 times. Observe and record the number of live insects, dead insects, and the disease-infected situation every 24 h (for 8 consecutive days) after inoculation until all the insects die or pupate.

[0059] 3. Experimental results

[0060] Figure 1 It is a comparison of the mortality rate of Hyphantria cunea larvae within 8 days between the nano Pickering emulsion and the aqueous solution of polyhedrovirus. The ordinate is the total newly added dead insect number in 3 repeated experiments on the 2nd, 4th, 6th, and 8th days. First of all, the death of larvae can be observed 2 days after inoculation with CSAD-SiO2@HcNPV prepared in the present invention. From the observation of the dead larvae to 8 days, regression is performed with the total number of newly added dead larvae every 2 days. Taking the slope to represent the rapid efficacy of the medicament, it can be seen that the slope of the regression line of CSAD-SiO2@HcNPV is 1.35, compared with 0.75 of the virus aqueous solution, and the rapid efficacy against Hyphantria cunea larvae is increased by 80.0% in the first week of feeding the poison.

[0061] Particle size distribution test of Example 3

[0062] The particle size distribution of the CSAD-SiO2 prepared in step 3 of Example 1 and the nano Pickering emulsion CSAD-SiO2@HcNPV prepared in step 4 was characterized.

[0063] As can be seen from Figure 2 the particle size of the CSAD-SiO2 prepared in the present invention is less than 500 nm. After loading the nuclear polyhedrosis virus, the particle size of the CSAD-SiO2@HcNPV particles reaches nearly 600 nm. While the material morphology of the present invention reaches the nanoscale, it encapsulates virus particles of a larger size, and at the same time, the particle size distribution is relatively uniform.

[0064] Example 4 ζ-potential characterization

[0065] The ζ-potential of SiO2, the CSAD-SiO2 prepared in step 3 of Example 1, and the nano Pickering emulsion CSAD-SiO2@HcNPV prepared in step 4 was characterized.

[0066] As can be seen from Figure 3 Table 1, after loading the virus, the ζ-potential of the particle system of the CSAD-SiO2@HcNPV emulsion prepared in the present invention is higher than that of the reported similar technologies, reaching nearly -40 mV, indicating that the virus has been successfully encapsulated into the aqueous phase in the emulsion system. Moreover, in the process of encapsulating particles with a larger particle size by the technical process of the present invention, a more stable emulsion system can be formed.

[0067] Table 1

[0068]

[0069] Example 5 FT-IR characterization

[0070] The FT-IR of sodium alginate (Alg) and cholesterol-grafted sodium alginate (CSAD) prepared in step 2 of Example 1 was characterized.

[0071] As can be seen from Figure 4 the newly emerged absorption peak at 1737 cm -1 in the spectrum of CSAD relative to Alg is the stretching vibration peak of in the ester group. At the same time, the absorption peak at 2851 cm -1 is the C-H stretching vibration absorption peak of methylene. Since there is no methylene in the molecular structure of sodium alginate itself, it can be proved that the carboxyl group of sodium alginate has an esterification reaction with the hydroxyl group of cholesterol.

Claims

1. A preparation method of a small-particle-size nucleopolyhedrovirus of Hyphantria cunea Pickering nanoemulsion, characterized in that, It includes the following steps: 1) Using alginic acid and cholesterol dispersed in a solvent as raw materials, adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, stirring and reacting at room temperature, and obtaining cholesterol-grafted sodium alginate (CSAD) after purification; 2) Ultrasonically dispersing the CSAD in step 1) in water, adding nano-silica, stirring and then subjecting to shear treatment to obtain an emulsion, centrifuging the emulsion and taking the precipitate, and obtaining the CSAD-SiO2 lyophilized powder after freeze-drying; 3) Dissolving the freeze-dried powder of the nuclear polyhedrosis virus of Hyphantria cunea in deionized water to obtain a virus solution, adding the CSAD-SiO2 lyophilized powder in step 2) into paraffin oil and performing ultrasonic treatment to obtain a dispersion; mixing the dispersion with the virus solution and obtaining a nano Pickering emulsion through ultrasonic emulsification.

2. The method according to claim 1, wherein In step 1), the mass ratio of alginic acid, cholesterol, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is (2.5 - 3.5):(2.5 - 3.4):(0.8 - 1.0):(0.05 - 0.15).

3. The method according to claim 1, characterized in that, The specific steps of step 1) are: dissolving dry alginic acid in dimethyl sulfoxide to obtain solution A; dissolving cholesterol in chloroform and slowly adding it to solution A to obtain solution B; respectively weighing N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, dissolving them in dimethyl sulfoxide, and then dropping them into solution B, stirring and reacting at room temperature for 24 h to obtain solution C; adding anhydrous ethanol to precipitate in solution C, centrifuging to remove the supernatant, and obtaining precipitate D through vacuum freezing; dissolving precipitate D in distilled water, adjusting the pH to 7.0 with sodium bicarbonate, standing and then centrifuging to take the supernatant E, loading it into a dialysis bag for dialysis, adding anhydrous ethanol to precipitate and drying to prepare CSAD.

4. The method according to claim 1, wherein In step 2), CSAD is ultrasonically dispersed in water at 1 mg / mL; the mass ratio of CSAD to nano-silica is 1:(8 - 10).

5. The method according to claim 1, wherein In step 2), adding nano-silica, stirring at 1000 rpm for 18 min, and subjecting to shear treatment with a shear emulsifier at 22000 rpm for 4 min to obtain an emulsion.

6. The method according to claim 1, wherein In step 3), the mass concentration of the freeze-dried powder of the nuclear polyhedrosis virus of Hyphantria cunea in the virus solution is 0.1 mg / g, and the mass concentration of the CSAD-SiO2 lyophilized powder in the dispersion is 0.01 g / mL; the volume ratio of the dispersion to the virus solution for mixing is 1:(0.3 - 0.8).

7. The method according to claim 1, wherein In step 3), the conditions for ultrasonic emulsification are: in an ultrasonic cell disruptor, performing ultrasonic emulsification at an output frequency of 315 W for 14 min.

8. The method according to claim 1, wherein In step 3), the preparation method of the freeze-dried powder of the nuclear polyhedrosis virus of Hyphantria cunea is: taking the dead insects killed by the nuclear polyhedrosis virus of Hyphantria cunea, grinding them, filtering and then centrifuging, sucking the supernatant and centrifuging again to remove the supernatant, repeating 3 - 5 times, then removing the supernatant and leaving the precipitate, suspending the precipitate with double-distilled water, performing purification by sucrose density gradient centrifugation, washing away the sucrose, and obtaining the freeze-dried powder of the nuclear polyhedrosis virus of Hyphantria cunea through vacuum freezing.

9. Application of the nano Pickering emulsion prepared by the method according to any one of claims 1 - 8 in controlling Hyphantria cunea.

10. A control method for Hyphantria cunea, characterized in that, The nano Pickering emulsion prepared by the method described in any one of claims 1-8 is used for killing the larvae of Hyphantria cunea.

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