Supramolecular hybrid hydrogel with pH responsiveness and preparation method of supramolecular hybrid hydrogel

Abstract

The invention relates to supramolecular hybrid hydrogel with pH responsiveness and a preparation method of the supramolecular hybrid hydrogel, the supramolecular hybrid hydrogel can load agricultural chemicals and can control release of the agricultural chemicals, so that the problem of environmental pollution caused by excessive use of pesticides and chemical fertilizers in agricultural production can be solved. Besides, by utilizing the pH response property of the hydrogel, the hydrogel loaded with the agricultural chemicals is applied to the saline-alkali soil, so that the soil environment can be improved, and the saline-alkali soil can be reused for agricultural cultivation.

Description

Technical Field

[0001] The invention relates to the technical field of hydrogels, and in particular to a supramolecular hybrid hydrogel with pH responsiveness and a preparation method thereof, a pesticide and a preparation method thereof. Background Art

[0002] Excessive use of fertilizers and pesticides has led to soil fertility degradation, reduced biodiversity, and even eutrophication of rivers and groundwater in some areas, leading to water pollution and other problems, which in turn make the soil unsuitable for cultivation. Therefore, in order to achieve the rational use of fertilizers and pesticides, maximize the benefits of chemical agricultural products, and reduce unnecessary pollution, it is necessary to develop a new type of environmentally responsive supramolecular hybrid hydrogel controlled release system to achieve this goal. Summary of the invention

[0003] The purpose of the present invention is to provide a supramolecular hybrid hydrogel with pH responsiveness. The hydrogel is a new type of environmentally responsive supramolecular hybrid hydrogel controlled-release system, which can solve the environmental pollution problem caused by excessive use of pesticides and fertilizers in agricultural production. In addition, by utilizing its pH responsiveness, it can be used on saline-alkali soil to improve the soil environment, which is conducive to the reuse of salinized soil for agricultural cultivation.

[0004] Another object of the present invention is to provide a method for preparing the supramolecular hybrid hydrogel.

[0005] Another object of the present invention is to provide a pesticide comprising the supramolecular hybrid hydrogel.

[0006] Another object of the present invention is to provide a method for preparing the pesticide.

[0007] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0008] In the first aspect, the present invention provides a supramolecular hybrid hydrogel with pH responsiveness, which includes laponite, a host and a guest; the host is chitosan modified with cyclodextrin; the guest includes one or more of group-modified adamantane, group-modified arylazopyrazole, and group-modified ferrocene, and the group is a quaternary ammonium group containing an ester group.

[0009] The supramolecular hybrid hydrogel of the invention comprises laponite, a host and a guest. The guest contains quaternary ammonium cations and can be combined with the laponite through electrostatic action, while the guest and the host can be combined through host-guest interaction to form a supramolecular hybrid hydrogel.

[0010] The guest of the present invention contains an ester group, which is pH-responsive and can be hydrolyzed under acidic or alkaline conditions, thereby causing a gel-sol phase transition. When the gel-sol phase transition occurs, the gel network of the supramolecular hybrid hydrogel loaded with agricultural chemicals is destroyed, thereby promoting the release of agricultural chemicals and achieving the purpose of better regulating plant growth. At the same time, the amino groups on the chitosan and the quaternary ammonium ions on the guest (such as a group-modified adamantane) react with the OH groups in the alkaline conditions. - The purpose of regulating saline-alkali soil is achieved by combining the hydrogel and hydrolyzing the ester bond under alkaline conditions. Therefore, by utilizing the pH response property of the hydrogel, the hydrogel loaded with agricultural chemicals is used on saline-alkali soil, which can improve the soil environment and facilitate the reuse of salinized soil for agricultural cultivation. At the same time, the hydrogel of the present invention has a controlled release effect, which can solve the environmental pollution problem caused by excessive use of pesticides and fertilizers in agricultural production.

[0011] In some embodiments of the present invention, the guest is a group-modified adamantane. Compared with azopyrazole, adamantane has the following advantages:

[0012] 1. It has photostability. Azopyrazole is easily decomposed by light. As a stereoester cycloalkane, adamantane has a simpler structure than azopyrazole and has relatively stable chemical properties.

[0013] 2. Adamantane can be synthesized in two steps from cyclopentadiene dimer, which is relatively simple. The synthesis process of azopyrazole is relatively complicated and has a lower purity.

[0014] The adamantane molecules and cyclodextrin-modified chitosan have better host-guest interactions and can be better combined together to form a stable hydrogel system together with laponite.

[0015] In some embodiments of the present invention, the group-modified adamantane has one of the following structures:

[0016]

[0017] In some embodiments of the present invention, the group-modified adamantane has the following structure: As can be seen from the structure, the guest molecule of the present invention contains two ester groups and two quaternary ammonium ions, the ester group has a pH response property, and the quaternary ammonium ion can increase the potential of the guest molecule, so that the guest and the hectorite can be well combined through electrostatic interaction. Compared with a guest with only one ester group and one quaternary ammonium ion, the use of the above guest is more conducive to forming a stable hydrogel network and improving the pH response property.

[0018] In some embodiments of the present invention, the cyclodextrin-modified chitosan has the following structure:

[0019]

[0020] Among them, n can be 150-170. If the n value is too small, the chitosan molecular chain may be too short, which is not conducive to the gelation of the gel and the formation of the internal structure of the hydrogel. The chitosan with a short molecular chain has no antibacterial effect. At the same time, the molecular chain with a small n value has few chemical active sites, which is not conducive to modification. If the n value is too large, it may affect the modification degree and modification rate of cyclodextrin, and the utilization rate of the modification site is low. Chitosan has a large number of amino groups, which can well control the controlled release of agricultural chemicals, reduce the use of pesticides, and at the same time can slow down plant fungal diseases and achieve plant protection.

[0021] In some specific embodiments, n may be 155-165, for example, 150, 155, 160, 165 or 170.

[0022] In some embodiments of the present invention, the mass ratio of hectorite, the host and the guest may be 50:(2.5-3.5):(0.8-1.6)

[0023] Controlling the mass ratio of the three substances is conducive to constructing a stable supramolecular hybrid hydrogel with a three-dimensional network structure. When the mass ratio is not within this range, it is difficult to form a supramolecular hybrid hydrogel with a three-dimensional network structure.

[0024] In some specific embodiments, the mass ratio of laponite, the host, and the guest can be 50:2.5:0.8, 50:2.5:1, 50:3:0.8, 50:3:1, 50:3.5:0.8, or 50:3.5:1.

[0025] In some embodiments of the present invention, the mass ratio of hectorite, the host and the guest is 50:(3-3.1):(0.8-0.9), preferably 50:3.05:0.85.

[0026] Optimizing the mass ratio of laponite, host and guest is beneficial to the gelation and good mechanical properties of the gel, while maximizing the internal three-dimensional network structure during gel formation, which can further increase the drug loading capacity and maintain the drug release time.

[0027] In a second aspect, the present invention provides a method for preparing the supramolecular hybrid hydrogel having pH responsiveness, comprising the following steps:

[0028] Mixing hectorite, a stripping agent, and water to obtain a hectorite solution;

[0029] mixing the guest with water to obtain a guest solution;

[0030] mixing a mixed solution of an organic acid and water with a main body to obtain a main body solution;

[0031] The laponite solution, the guest solution and the host solution are mixed and stirred until coagulation occurs, thereby obtaining the supramolecular hybrid hydrogel with pH responsiveness.

[0032] In some embodiments of the present invention, the release agent includes one or more of sodium polyacrylate, modified polycarboxylate, sodium polyepoxysuccinate, phosphonocarboxylic acid copolymer (POCA), and the like.

[0033] In some embodiments of the present invention, the preparation of the hectorite solution includes: suspending hectorite in water and stirring to obtain a hectorite suspension; adding a stripping agent to water and ultrasonically dissolving it to obtain a stripping agent solution; adding the stripping agent solution to the hectorite suspension and continuing to stir to obtain a hectorite solution. Hectorite is a layered material, and the addition of a stripping agent can strip the hectorite into negatively charged nanosheets, which is beneficial to improving the electrostatic interaction between the guest and the hectorite and stabilizing the structure of the supramolecular hybrid hydrogel.

[0034] In some embodiments of the present invention, the hectorite solution and the guest solution are first mixed, and then the host solution is added and mixed and stirred until coagulation occurs.

[0035] In some embodiments of the present invention, the organic acid includes one or more of acetic acid, oxalic acid, lactic acid, malic acid, and the like.

[0036] In some embodiments of the present invention, the object is (hereinafter referred to as adamantane diquaternary ammonium salt conjugate). The adamantane diquaternary ammonium salt conjugate can be prepared according to the method reported in the literature. For example, it can be prepared by a method comprising the following steps: reacting 5-hydroxydimethyl isophthalate and 1-adamantyl bromketone under alkaline conditions under heating to obtain 5-(2-(1-adamantyl)-2-oxoethoxy)dimethyl isophthalate; hydrolyzing 5-(2-(1-adamantyl)-2-oxoethoxy)dimethyl isophthalate to obtain 5-(2-(1-adamantyl)-2-oxoethoxy)isophthalic acid; reacting 5-(2-(1-adamantyl)-2-oxoethoxy)isophthalic acid with N-hydroxysuccinimide to obtain bis(2,5-dioxopyrrolidinyl)-5-(2-(1-adamantane)-2-oxoethoxy)isophthalic acid; The invention relates to a method for preparing a diquaternary adamantane diquaternary ammonium salt conjugate of the present invention. The method comprises reacting bis(2,5-dioxopyrrolidinyl)-5-(2-(1-adamantyl)-2-oxoethoxy)isophthalate with 3-dimethylaminopropylamine under alkaline conditions to obtain (5-(2-(1-adamantyl)-2-oxoethoxy)-1,3-phenylene)bis(((3-(dimethylamino)propyl)amino)oxy)methanone; and finally, reacting (5-(2-(1-adamantyl)-2-oxoethoxy)-1,3-phenylene)bis(((3-(dimethylamino)propyl)amino)oxy)methanone with ethyl bromoacetate to obtain a diquaternary adamantane diquaternary ammonium salt conjugate.

[0037] In some embodiments of the present invention, the main structural formula is as follows: Wherein, n is 150-170. The main body can be prepared according to the method reported in the literature. For example, it can be prepared by a method comprising the following steps: reacting β-cyclodextrin with p-toluenesulfonyl chloride to obtain mono-(6-p-toluenesulfonyl)-β-cyclodextrin; reacting mono-(6-p-toluenesulfonyl)-β-cyclodextrin with 4-hydroxybenzaldehyde to obtain 6-oxy-(4-formylphenyl)-β-cyclodextrin; reacting 6-oxy-(4-formylphenyl)-β-cyclodextrin with chitosan under alkaline conditions to obtain the main body.

[0038] In some embodiments of the present invention, ultrasound is used to mix the guest with water to obtain a guest solution.

[0039] In some embodiments of the present invention, a mixed solution of organic acid and water is mixed with a main body by ultrasound to obtain a main body solution.

[0040] In a third aspect, the present invention provides a pesticide, comprising the supramolecular hybrid hydrogel having pH responsiveness and agricultural chemicals loaded thereon.

[0041] In a fourth aspect, the present invention provides a method for preparing the above pesticide, comprising the following steps:

[0042] Mixing hectorite, a stripping agent, and water to obtain a hectorite solution;

[0043] mixing the guest with water to obtain a guest solution;

[0044] mixing a mixed solution of an organic acid and water with a main body to obtain a main body solution;

[0045] dissolving an agricultural chemical in a solvent to obtain an agricultural chemical solution;

[0046] The hectorite solution, the guest solution, the host solution and the agricultural chemical solution are mixed and stirred until coagulation occurs, thereby obtaining the pesticide.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention provides a supramolecular hybrid hydrogel with pH responsiveness, which can load agricultural chemicals and control the release of agricultural chemicals, thereby solving the environmental pollution problem caused by excessive use of pesticides and fertilizers in agricultural production. In addition, the guest of the present invention contains an ester group, which is pH responsive and can be hydrolyzed under acidic or alkaline conditions, thereby causing a gel-sol phase transition. When the gel-sol phase transition occurs, the gel network of the supramolecular hybrid hydrogel loaded with agricultural chemicals is destroyed, thereby promoting the release of agricultural chemicals and achieving the purpose of better regulating plant growth. At the same time, the amino groups on chitosan and the quaternary ammonium ions on the guest (such as group-modified adamantane) react with OH in alkaline conditions. - The purpose of regulating saline-alkali soil is achieved by combining the ester bond and hydrolyzing the ester bond under alkaline conditions. Therefore, by utilizing the pH-responsive properties of the hydrogel and applying the hydrogel loaded with agricultural chemicals on saline-alkali soil, the soil environment can be improved, which is conducive to reusing the salinized soil for agricultural cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 β-cyclodextrin modified chitosan 1 H NMR spectrum.

[0050] Figure 2 It is a diquaternary ammonium salt conjugate of adamantane 1 H NMR spectrum.

[0051] Figure 3 (a) Mixed solution after adding guest compounds but before adding host; (b) upright image of the gel after adding host compounds; (c) inverted image of the gel after adding host compounds; (d) scanning electron microscope image of the gel at 430 times magnification; (e) microscopic image of the gel after partial magnification; (f) microscopic image of the gel at 1000 times magnification.

[0052] Figure 4 Zeta potential diagram of the host-guest compounds and the supramolecular hybrid hydrogel prepared in Example 3.

[0053] Figure 5 Rheological property test results of the supramolecular hybrid hydrogel prepared in Example 3: (a) shear rate, viscosity and shear stress test results; (b) strain sweep test results; (c) frequency sweep test results; (d) continuous step strain test results; (e) temperature sweep test results.

[0054] Figure 6 This is a sustained release performance analysis diagram of the CSCD-ADA2+@Lp hydrogel drug delivery system prepared in Example 4.

[0055] Figure 7 These are the data for Chinese cabbage using the plant growth regulator IAA as a model.

[0056] Figure 8 These are the data for Chinese cabbage using the plant growth regulator NAA as a model.

[0057] Fig. 9 (a) Cumulative release curve of CSCD-ADA2+@Lp hydrogel loaded with NAA in carbonate buffer solution at pH = 9.00; Fig. 9 (b) Cumulative release curve of CSCD-ADA2+@Lp hydrogel loaded with NAA in 4 M NaBr aqueous solution.

[0058] Fig.10 (a) Photos of plants in each group under blank conditions (b) Plant height data of plants in each group (c) Stem length data of plants in each group (d) Wet weight data of plants in each group (e) Dry weight data of plants in each group.

[0059] Fig.11 (a) Photos of plants in each group under salt stress conditions (b) Plant height data of plants in each group (c) Stem length data of plants in each group (d) Wet weight data of plants in each group (e) Dry weight data of plants in each group.

[0060] Fig.12 (a) Photos of plants in each group under alkaline stress conditions (b) Plant height data of plants in each group (c) Stem length data of plants in each group (d) Wet weight data of plants in each group (e) Dry weight data of plants in each group.

[0061] Fig.13 The MDA concentration of each group of plants is shown.

[0062] Fig.14 These are photos of each treatment group co-cultured with Botrytis cinerea for 72 hours.

[0063] Fig.15 The inhibition rate of each treatment group after 72 h of co-culture with Botrytis cinerea is shown.

[0064] Fig.16 These are photos of each treatment group co-cultivated with pepper phytophthora for 72 hours.

[0065] Fig.17 The inhibition rate of each treatment group co-cultured with pepper phytophthora for 72 hours is shown.

[0066] Fig.18 These are photos of each treatment group co-cultured with wheat sheath blight for 72 hours.

[0067] Fig.19 The inhibition rate of each treatment group after 72 h of co-culture with wheat sheath blight is shown.

[0068] Fig. 20 The biocompatibility test data of the hydrogel prepared in Example 3 to L929 cells are shown.

[0069] Fig.21 The biocompatibility test data of the hydrogel prepared in Example 3 to 3T3 cells are shown.

[0070] Fig. 22 The images of the uncoagulated mixed solution prepared in Comparative Example 1 are shown ((a) upright image, (b) sideways image, (c) inverted image).

[0071] Fig.23 The images of the uncoagulated mixed solution prepared in Comparative Example 2 are shown ((a) upright image, (b) sideways image, (c) inverted image). DETAILED DESCRIPTION

[0072] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific 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.

[0073] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the instruments and equipment used in the following examples, etc., can all be purchased on the market or can be obtained by existing methods; the amounts of the experimental reagents, unless otherwise specified, are the amounts of reagents used in conventional experimental operations; the experimental methods, unless otherwise specified, are all conventional methods.

[0074] The cyclodextrin used in the experiment was natural β-cyclodextrin, purchased from Shanghai Titan Technology Co., Ltd., p-toluenesulfonyl chloride, adamantyl bromide ketone, and sodium polyacrylate were purchased from Shanghai McLean Co., Ltd., sodium hydroxide, sodium carbonate, sodium bicarbonate, and sodium chloride were purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., 4-hydroxybenzaldehyde, NN dimethylformamide, ammonia water, chitosan, 5-hydroxyisophthalic acid dimethyl ester, anhydrous potassium carbonate, 300 mesh silica gel, quartz sand, tetrahydrofuran, and N-hydroxysuccinimide were purchased from Shanghai Titan Technology Co., Ltd., potassium hydroxide was purchased from Tianjin Jinhui Taiya Chemical Reagent Co., Ltd., acetonitrile, glacial acetic acid, anhydrous ethanol, and dichloromethane were purchased from Tianjin Fuchen Chemical Reagent Co., Ltd., hydrochloric acid, acetone, methanol, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd., and n-propanol was purchased from Tianjin Xiens Biochemical Technology Co., Ltd. The water used in the experiment was purchased from Hangzhou Wahaha Group. All materials used in the experiment were analytically pure unless otherwise specified.

[0075] (1) Preparation of pH-responsive supramolecular hybrid hydrogels

[0076] Example 1: Synthesis of cyclodextrin-modified chitosan

[0077] (1) Synthesis of mono-(6-p-toluenesulfonyl)-β-cyclodextrin (6-OTs-β-CD)

[0078]

[0079] Weigh 210g of natural β-cyclodextrin and suspend it in 1500mL of double distilled water. Keep the temperature of the suspension at about 10°C with an ice-water bath, then add 60mL of 8.33mol / L NaOH aqueous solution to the suspension within 6 minutes to completely dissolve the cyclodextrin. Then slowly add 90mL of 1.77mol / L acetonitrile solution of p-toluenesulfonyl chloride to the cyclodextrin solution within 8 minutes to generate a white precipitate. Then stir at a constant temperature for 2.5h and filter to remove insoluble matter. Adjust the pH value of the solution to about 8 with 2M hydrochloric acid solution. Let it stand overnight at 4°C to precipitate a large amount of precipitate, and collect the precipitate after filtering. After 2-3 times of impurity removal by recrystallization from distilled water, vacuum drying at 70°C for 12h obtains the compound, which is a white powdery solid.

[0080] (2) Synthesis of 6-oxy-(4-formylphenyl)-β-cyclodextrin

[0081]

[0082] Weigh 0.37 g (3 mmol) of 4-hydroxybenzaldehyde and dissolve it in 10 mL of DMF, then add 0.4 g (3 mmol) of anhydrous K2CO3. Stir the mixture at room temperature in the dark for 2 h under nitrogen protection. Then weigh 1.9 g (1.5 mmol) of 6-OTs-β-CD prepared in step (1) and dissolve it in 20 mL of dry DMF, then add it dropwise to the DMF solution of 4-hydroxybenzaldehyde at a uniform rate. Heat the oil bath to 80°C and react in the dark for 24 h. Then evaporate the mixed solution under reduced pressure to obtain a solid, which is then dissolved in a small amount of hot water and precipitated with 200 mL of acetone. Purify the crude product by chromatography (the chromatography column is filled with 300 mesh silica gel, and the volume ratio of the mobile phase is n-propanol: water: ammonia water = 6:3:1). Recrystallize the purified product for further purification. After vacuum drying, pure 6-O-(4-formylphenyl)-β-cyclodextrin is obtained. The compound is a white powdery solid.

[0083] (3) Synthesis of cyclodextrin-modified chitosan

[0084] Where n is 161,

[0085] Weigh 0.2g of chitosan and dissolve it in 14mL of 0.2M acetic acid methanol solution, then titrate with 0.5M NaOH aqueous solution to adjust the pH value to about 5.0. Then add an equal amount of 6-O-(4-formylphenyl)-β-cyclodextrin to the system, and then react at 60℃ for 24h under nitrogen protection. The reaction product is precipitated in the reaction system with 0.5M NaOH aqueous solution. Then wash with water, ethanol: water with a volume ratio of 1:1, and anhydrous ethanol respectively, and obtain the product after vacuum drying. The product is a light yellow solid.

[0086] Figure 1 The characterization analysis of the nuclear magnetic resonance hydrogen spectrum of β-cyclodextrin modified chitosan (CSCD) is as follows: 1 HNMR (400 MHz, 1 mol / L DCl in D2O, ppm) δ9.29 (1H, -CH=N-), 7.44-7.46 (2H, H of phenyl 1), 6.73-6.75 (2H, H of phenyl), 4.58 (7H, C-1H of β-CD), 2.87-3.46 (80.26H, C-2, C-3, C-4, C-5, C-6H of β-CD and nucleopolysaccharide protons), 1.61 (38H, chitosan-C=OCH3).

[0087] Further analysis of H-NMR spectrum shows that the modification degree of chitosan by cyclodextrin units is 12.2% according to the number of protons of benzene ring and glucosamine units, that is, one cyclodextrin molecule is modified for every 8.2 sugar units on chitosan. According to the molecular weight of chitosan used, which is about 100,000, the number of sugar units on chitosan is calculated to be 620.35, and thus the number of cyclodextrins modified on the chitosan chain is 75.65, and the molecular weight of the main compound is calculated to be about 191137.028.

[0088] Example 2: Synthesis of adamantane diquaternary ammonium salt conjugate

[0089] (1) Synthesis of dimethyl 5-(2-(1-adamantyl)-2-oxoethoxy)isophthalate

[0090]

[0091] Dissolve 5-hydroxyisophthalic acid dimethyl ester (1.05 g, 5 mmol) and 1-adamantyl bromide (1.03 g, 4 mmol) in acetone (50 mL), add potassium carbonate (2.07 g, 15 mmol). Reflux at 60 ° C overnight. After cooling to room temperature, filter the solution and dry the filtrate under reduced pressure to remove the solvent. Purify by column chromatography (silica gel, pure dichloromethane as eluent) to obtain the compound as a white powder.

[0092] (2) Synthesis of 5-(2-(1-adamantyl)-2-oxoethoxy)isophthalic acid

[0093]

[0094] The product of step (1) (410 mg, 1.06 mmol) was dissolved in THF (50 mL), and then 10 mL of 10 wt% potassium hydroxide aqueous solution was added. Refluxed at 70 ° C for 12 h, cooled to room temperature, and the mixture was evaporated under reduced pressure to remove the solvent. The residue was redissolved in 40 mL of water, and 0.5 mol / L HCl solution was added to adjust the pH value of the solution to <1, and a large amount of precipitate appeared. After filtering the precipitate, it was rinsed with water several times until the pH value of the filtrate was adjusted to 7. The compound was finally obtained as a white solid.

[0095] (3) Synthesis of bis(2,5-dioxopyrrolidinyl)-5-(2-(1-adamantyl)-2-oxoethoxy)isophthalate

[0096]

[0097] The product of step (3) (716.8 mg, 2 mmol), NHS (N-hydroxysuccinimide) (460.4 mg, 4 mmol), and DCC (824.7 mg, 4 mmol) were dissolved in THF (30 mL) and stirred at room temperature for 24 h to obtain a large amount of white precipitate. The mixture was filtered and the filtrate was evaporated under reduced pressure to remove the solvent. The residue was purified by column chromatography (silica gel, dichloromethane / methanol = 60:1 as eluent) to obtain the compound as a white solid.

[0098] (4) Synthesis of (5-(2-(1-adamantyl)-2-oxoethoxy)-1,3-phenylene)bis(((3-(dimethylamino)propyl)amino)oxy)ketone

[0099]

[0100] The product of step (3) (1.15 g, 2.08 mmol) and 3-dimethylaminopropylamine (1.02 g, 10 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (1 mL) was added. After stirring at room temperature for 24 h, the mixture was washed with 3×100 mL of clean water, and the organic layer was dried over anhydrous Na2SO4. The solvent was removed under reduced pressure to obtain a yellow solid compound.

[0101] (5) Synthesis of diquaternary ammonium salt conjugate of adamantane

[0102]

[0103] The product of step (4) (526.7 mg, 1 mmol) was dissolved in acetonitrile (15 mL), ethyl bromoacetate (277 μL, 2.5 mmol) was added, stirred at room temperature for 24 h, and the solvent was evaporated under reduced pressure. After that, ether (50 ml) was added to the residue, and a white precipitate appeared immediately. The solution was centrifuged to remove the solvent and washed twice with ether (2×50 mL). The diquaternary ammonium salt of adamantane conjugate (ADA2+) was a light yellow solid.

[0104] Figure 2 It is a diquaternary ammonium salt conjugate of adamantane 1 H NMR spectrum. The specific characterization is as follows: 1H NMR (400MHz, D2O, TMS): δ1.15-1.19 (t, 6H, J=7.2Hz, methyl), 1.66-1.77 (m, 6H, adamantane), 1.88 (m, 6H, adamantane), 2.02 (s, 3H, adamantane), 2.12 (m, 4H, -CH2), 3.27 (s, 12H, H of methyl), 3.41-3.71 (m, 8H, ethyl and -CH2), 4.14-4.15 (m, 4H, -CH2), 4.30 (s, 4H, -CH2 of ethyl acetate), 5.19 (s, 2H, -CH2), 7.43 (s, 2H, phenyl), 7.77ppm (s, 1H, phenyl). The characteristic peaks of phenyl and adamantane can be seen from the NMR spectrum, indicating that the guest compound adamantane diquaternary ammonium salt was successfully synthesized.

[0105] Example 3: Construction of supramolecular hybrid hydrogel with pH responsiveness

[0106] Weigh 50 mg of hectorite and suspend it in 2 mL of ultrapure water and stir for 10 minutes to make the solution clear and transparent. Then weigh 1.7 mg of sodium polyacrylate and fully dissolve it in 200 microliters of ultrapure water by ultrasonication, then add it to the hectorite suspension and continue stirring for 10 minutes. Then weigh 0.85 mg of adamantane diquaternary ammonium salt conjugate and dissolve it in 100 microliters of ultrapure water, add it to the above solution after ultrasonic dissolution and stir for 10 minutes. Next, weigh 3.05 mg of chitosan-modified cyclodextrin and dissolve it in 100 microliters of 1% acetic acid aqueous solution, and ultrasonically dissolve it until no solid is visible, then add it to the above mixed solution and stir until it forms a coagulation.

[0107] Example 4: Preparation of supramolecular hybrid hydrogel loaded with naphthyl acetic acid

[0108] Weigh 50 mg of laponite and suspend it in 2 mL of ultrapure water and stir for 10 min to make the solution clear and transparent. Then weigh 1.7 mg of sodium polyacrylate and dissolve it in 200 microliters of ultrapure water by ultrasound. After fully dissolving, add it to the laponite suspension and stir for 10 min. Then weigh 2.4 mg of naphthaleneacetic acid (NAA) and dissolve it in 10 microliters of DMSO. After fully dissolving, add it to the stripped laponite suspension and stir. Then weigh 0.85 mg of adamantane diquaternary ammonium salt conjugate and dissolve it in 100 microliters of ultrapure water. After ultrasonic dissolution, add it to the above solution and stir for 10 min. Next, weigh 3.05 mg of chitosan-modified cyclodextrin and dissolve it in 100 microliters of 1% acetic acid aqueous solution, and ultrasonically dissolve it until no solid is visible, then add it to the above mixed solution and stir until it is coagulated, thereby preparing a supramolecular hybrid hydrogel loaded with naphthaleneacetic acid.

[0109] Example 5: Preparation of supramolecular hybrid hydrogel loaded with indoleacetic acid

[0110] Weigh 50 mg of laponite and suspend it in 2 mL of ultrapure water and stir for 10 minutes to make the solution clear and transparent. Then weigh 1.7 mg of sodium polyacrylate and dissolve it in 200 microliters of ultrapure water by ultrasound. After fully dissolved, add it to the laponite suspension and stir for 10 minutes. Then weigh 2.4 mg of indoleacetic acid (IAA) and dissolve it in 10 microliters of DMSO. After fully dissolved, add it to the peeled laponite suspension and stir. Then weigh 0.85 mg of adamantane diquaternary ammonium salt conjugate and dissolve it in 100 microliters of ultrapure water. After ultrasonic dissolution, add it to the above solution and stir for 10 minutes. Next, weigh 3.05 mg of chitosan-modified cyclodextrin and dissolve it in 100 microliters of 1% acetic acid aqueous solution, and ultrasonically dissolve it until no solid is visible, then add it to the above mixed solution and stir until it is coagulated, thereby preparing a supramolecular hybrid hydrogel loaded with indoleacetic acid.

[0111] Comparative Example 1

[0112] Weigh 50 mg of hectorite and suspend it in 2 mL of ultrapure water and stir for 10 minutes to make the solution clear and transparent. Then weigh 1.7 mg of sodium polyacrylate and dissolve it in 200 microliters of ultrapure water by ultrasonication, then add it to the hectorite suspension and continue stirring for 10 minutes. Then weigh 1.7 mg of adamantane diquaternary ammonium salt conjugate and dissolve it in 200 microliters of ultrapure water and add it to the above solution and stir for 10 minutes. Next, weigh 3.05 mg of chitosan-modified cyclodextrin and dissolve it in 100 microliters of 1% acetic acid solution. After ultrasonic dissolution, add it dropwise to the above solution and stir for 10 minutes. No coagulation occurs, as shown in FIG. Fig. 22 shown.

[0113] Comparative Example 2

[0114] Weigh 50 mg of hectorite and suspend it in 2 mL of ultrapure water and stir for 10 minutes to make the solution clear and transparent. Then weigh 1.7 mg of sodium polyacrylate and dissolve it in 200 microliters of ultrapure water by ultrasonication, then add it to the hectorite suspension and continue stirring for 10 minutes, then weigh 0.75 mg of adamantane diquaternary ammonium salt conjugate and dissolve it in 100 microliters of ultrapure water and stir for 10 minutes, then weigh 3.05 mg of chitosan modified cyclodextrin and dissolve it in 100 microliters of 1% acetic acid solution, and then add it dropwise into the above solution after ultrasonic dissolution and stir for 10 minutes, no coagulation, such as Fig.23 shown.

[0115] (2) Characterization and analysis of the supramolecular hybrid hydrogel (CSCD-ADA2+@Lp) of Example 3

[0116] like Figure 3As shown in (a), when the guest compound ADA2+ is added to the lithium laponite solution and stirred, the solution begins to become viscous and shows signs of coagulation, but it always maintains fluidity. This just proves that the positively charged guest compound ADA2+ is intercalated between Lp and stabilizes the negatively charged lithium laponite nanosheets through electrostatic interactions. Figure 3 (b) with Figure 3 (c) are the upright and inverted images of CSCD-ADA2+@Lp hydrogel. With the addition of the main compound CSCD, the solution gradually lost its fluidity and formed a supramolecular hybrid hydrogel. Figure 3 (d) Figure 3 (e) with Figure 3 (f) are all scanning electron microscope images of the gel. Through the SEM images of the gel micro-surface, we observed the nano-sheet structure of lithium laponite. At the same time, we observed the three-dimensional network structure formed by the main compound and the guest compound attached to the surface of the lithium laponite. Therefore, this structure can effectively load pesticides.

[0117] (3) Zeta potential analysis of host-guest compounds and supramolecular hybrid hydrogels

[0118] By performing Zeta potential analysis on the host and guest compounds and the hydrogel of Example 3, it was determined that the potential of the adamantane diquaternary ammonium salt conjugate was +23.02 mV, and the potential of the hectorite was -37.67 mV. Figure 4 As shown. Therefore, it can be proved that the hectorite and the diquaternary ammonium salt of adamantane conjugate are combined through electrostatic interaction. At the same time, the potentials of chitosan and cyclodextrin-modified chitosan are both positive, which also proves the presence of a large number of amino groups on chitosan.

[0119] (4) Rheological properties analysis of supramolecular hybrid hydrogels

[0120] The mechanical properties of the CSCD-ADA2+@Lp hydrogel of Example 3 were studied by rheological testing. Figure 5 (a) shows that as the shear rate increases, the hydrogel exhibits a decrease in viscosity and shear stress, and when the shear rate is roughly between 10 and 100 s- 1 The shear stress variation should be less than 0.1~10s- 1 In addition, the strain sweep test of the hydrogel is as follows Figure 5 As shown in (b), when the amplitude scan of the gel was fixed at ω = 6.28 rad / s, the hydrogel underwent a gel-sol phase transition at the critical strain region γ = 84.87%, indicating that the gel network was destroyed. Figure 5(c) shows the curves of the storage modulus (G') and loss modulus (G") of the hydrogel as a function of frequency. In the test range, the storage modulus of the hydrogel is much larger than its loss modulus, which indicates that the hydrogel has formed a relatively stable structure. Figure 5 (d) means that when γ =100%, ω=6.28rad / s, the storage modulus of the hydrogel decreased, and the hydrogel underwent a phase transition from gel to sol. However, when the amplitude was reduced, i.e., γ=0.1%, ω=6.28rad / s, the hydrogel returned to the gel state 95s after the phase transition. Figure 5 (e) is a curve that tests the effect of temperature change on the hydrogel. The figure shows the thermal stability of the hydrogel and the rheological properties at 70°C. When the gel is heated from room temperature to 70°C, its storage modulus is always greater than the loss modulus. Even when the temperature is maintained at 70°C, there is no phase transition between gel and sol, which shows that the CSCD-ADA2+@Lp hydrogel has a very high thermal stability. The above rheological test results show that the hybrid hydrogel formed by the electrostatic combination of CSCD-ADA2+ supramolecular complex and lithium laponite (Lp) nanoclay is environmentally friendly and has good mechanical strength and stability.

[0121] (5) Analysis of sustained release performance of CSCD-ADA2+@Lp hydrogel drug delivery system

[0122] Naphthaleneacetic acid (NAA) was used as a drug model, and the detailed preparation process was as described in Example 4.

[0123] Calculated according to the formula

[0124] Load rate (%) = 100 × (C0V0-C1V1) / m

[0125] Encapsulation efficiency (%) = 100 × (C0V0-C1V1) / C0V0

[0126] Where C0 and C1 are the concentrations of NAA in the solution before and after loading; V0 is the volume of the solution before loading, V1 is the volume of the solution after loading; and m is the mass of the dry hydrogel after freeze-drying. The concentration of NAA in the solution was determined by UV-visible spectroscopy.

[0127] Finally, it was calculated that the loading rate of CSCD-ADA2+@Lp hydrogel for NAA was 3.67% and the encapsulation efficiency was 100%.

[0128] Similarly, according to the above formula, the loading rate of IAA of CSCD-ADA2+@Lp hydrogel in Example 5 was calculated to be 3.67%, and the encapsulation efficiency was 100%.

[0129] Subsequently, the sustained-release behavior of the hydrogel in pure water was tested. Specifically, the hydrogel prepared in Example 4 was freeze-dried and ground at -70.4°C, and 20 mg of the dry gel was weighed and loaded into a dialysis bag of appropriate size (molecular weight cutoff of 500-1000Da). Dialyze in ultrapure water for 18 hours respectively, and extract the dialysate at intervals. Using the standard curve method, the absorbance of the drug in the dialysate was measured by a double-beam UV-visible spectrophotometer, and then the concentration of the drug was calculated and the amount of drug released was analyzed. The controlled-release performance of the hydrogel was thus evaluated. The sustained-release results are shown in FIG. Figure 6 As shown in the figure, the cumulative release rate of NAA is evaluated by the calculation method of Lambert-Beer's law, and according to the results shown by the curve in the figure, a stable release level is reached at 100 minutes, and it has been maintained between 25% and 30%. This shows that the supramolecular hybrid hydrogel has good drug sustained release performance, which can greatly prolong the efficacy of pesticides, improve the utilization rate of pesticides, reduce the use of pesticides, and reduce environmental pollution.

[0130] (6) Drug release experiment of assembled gel on plants

[0131] After verifying the sustained release behavior of the hydrogel by the above experiment (5), the ability of CSCD-ADA2+@Lp hydrogel to regulate plant growth was further studied using hydrogels loaded with indoleacetic acid (IAA) (Example 5) and naphthaleneacetic acid (NAA) (Example 4). We conducted a simulation experiment using cabbage as a plant model, and selected plant seeds of uniform size were immersed in a 5% NaClO aqueous solution for ten minutes for disinfection and activated by water bathing at 45°C for 30 minutes. The seeds were cultured under uniform conditions of 26°C and 60% humidity, and except for the blank control group, each group was added with 1 mL of drug (i.e., freeze-dried hydrogel (the hydrogel of Example 3 was freeze-dried and ground at -70.4°C, 24.52 mg was weighed and suspended in 50 mL of water), IAA aqueous solution (drug concentration was 0.02 mg / mL), NAA aqueous solution (drug concentration was 0.02 mg / mL), IAA-loaded hydrogel of Example 5 (the hydrogel of Example 5 was freeze-dried and ground at -70.4°C, 24.17 mg was weighed and suspended in 50 mL of water) or NAA-loaded hydrogel of Example 4 (the hydrogel of Example 4 was freeze-dried and ground at -70.4°C, 24.17 mg was weighed and suspended in 50 mL of water)) on the 2nd, 4th and 6th days of culture, and water was added on the 1st, 3rd, 5th and 7th days, and water was added once a day in the culture period to the control group. The plant height, stem length, dry weight and wet weight data were measured after 7 days of cultivation to analyze the plant body to prove the regulatory effect of the gel on the plant.

[0132] Figure 7The effect of the IAA-loaded CSCD-ADA2+@Lp hydrogel of Example 5 on the development of Chinese cabbage is shown. Figure 7 (a) shows the representative pictures of the blank group and each experimental group. It can be seen from the figure that both IAA and CSCD-ADA2+@Lp hydrogels have the effect of promoting plant growth. Figure 7 (b) also shows that the growth of the CSCD-ADA2+@Lp hydrogel group is greater than that of the pure plant growth regulator IAA group, so it can be inferred that the hydrogel plays a key role in promoting plant growth. Then, the wet weight and dry weight of each group of plants after 7 days of cultivation were analyzed. Figure 7 (c) The wet weight data of the plants showed that the mass of the hydrogel group and the hydrogel group loaded with IAA was much greater than that of the blank group and the IAA group. The plants in each group were then dried to obtain the dry weight data. Figure 7 (d) It was found that the mass of plants cultivated in the hydrogel group was greater than that of plants cultivated in the blank group, and the mass of plants cultivated in the hydrogel loaded with IAA was also higher than that of plants cultivated with IAA alone.

[0133] Figure 8 The effect of CSCD-ADA2+@Lp hydrogel loaded with NAA on the development of Chinese cabbage is shown in Example 4. Figure 8 (a)- Figure 8 (d) It can be seen that the effect of NAA on the development of Chinese cabbage is almost the same as that of IAA, but because NAA has the effect of promoting root development, the dry weight and wet weight of NAA are slightly greater than those of IAA.

[0134] Therefore, it can be concluded that CSCD-ADA2+@Lp hydrogel not only has very good biocompatibility, but also can promote plant growth. When used with plant growth regulators such as IAA, it can also enhance the accumulation of organic matter in plants and promote plant growth, thereby achieving an increase in agricultural income while reducing the use of pesticides.

[0135] (7) Drug release experiments of assembled hydrogels under salt stress and alkali stress

[0136] In order to continue to prove that the assembled hydrogel can not only achieve the sustained release and regulation of pesticides under neutral conditions, but also can be used on salinized or alkaline land to achieve the release of drugs while regulating saline-alkali land, and theoretically achieve the effect of 1+1>2, the following experiment was conducted: The sustained release behavior of the hydrogel was verified according to the method described in the above experiment (5), except that the ultrapure water was replaced by 4 mol / L NaBr solution and pH = 9 alkaline salt (mixed solution of sodium carbonate and sodium bicarbonate) solution.

[0137] In pure water ( Figure 6 ) compared to Example 4, the CSCD-ADA2+@Lp hydrogel loaded with NAA under alkaline conditions ( Fig. 9 (a)) and under salt conditions ( Fig. 9 (b)) has a much higher release rate, and during the release period, the hydrogel system has been in a stable growth trend, which shows that the structure of the hydrogel can be changed under saline-alkali conditions, causing the gel to undergo a gel-sol phase transition, so that the drug release rate has always shown a high level of release concentration. Therefore, the supramolecular hybrid hydrogel can regulate plant growth under salt and alkaline conditions, while utilizing the amino groups on chitosan and the quaternary ammonium ions on the diquaternary ammonium salt of adamantane conjugate to react with OH in alkaline conditions. - Combined to achieve the purpose of regulating saline-alkali soil.

[0138] (8) Plant growth regulation experiment of CSCD-ADA2+@Lp hydrogel under saline-alkali conditions

[0139] In order to explore the effect of hydrogel on plants under salt stress and alkali stress, experiments were carried out under three conditions: salt stress (mixed solution A of 75 mM NaCl aqueous solution and 75 mM Na2SO4 aqueous solution, volume ratio of 1:1), alkali stress (mixed solution B of 75 mM NaHCO3 aqueous solution and 75 mM Na2CO3 aqueous solution, volume ratio of 1:1) and blank condition, and NAA was used as the loaded simulated drug (i.e., the hydrogel loaded with NAA prepared in Example 4). For each condition, four different conditions were designed, namely blank group, NAA group, hydrogel group and hydrogel group loaded with NAA. Specifically, wheat was used as the cultivation object in this experiment. The pre-sterilization, activation and seedling raising work were the same as the method of the above experiment (6). After the seeds were germinated for 2 days (germination conditions: 18°C, humidity: 70%), wheat seedlings with similar growth were selected and cultured with complete Hoagland culture medium (manufacturer: Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) for 14 days (cultivation conditions: light conditions: 25°C, humidity 70%, light duration: 14h, dark conditions: 15°C, humidity 70%, dark duration: 10h), and wheat seedlings with similar growth were selected again for the salt-alkali stress experiment. The selected wheat seedlings were divided into groups of 20 and cultured in paper cups in 30 mL of the corresponding solution (i.e., blank under pure water conditions (i.e., pure water), NAA (i.e., pure water added with NAA at a concentration of 0.02 mg / mL), the hydrogel of Example 3 (i.e., pure water added with the hydrogel of Example 3 at a concentration of 0.4708 mg / mL, the concentration calculation here is based on the dosage concentration of NAA and the dosage and concentration of the NAA-loaded hydrogel of Example 4, and then the dosage and concentration of the hydrogel of Example 3 equivalent to that of Example 4 are calculated.), the NAA-loaded hydrogel of Example 4 (i.e., pure water added with the NAA-loaded hydrogel of Example 4 at a concentration of 0.4908 mg / mL, the concentration here is based on the dosage concentration of NAA of 0.02 mg / mL (C NAA =0.02 mg / mL) to calculate the dosage of NAA, and then calculate the required gel mass of Example 4, and then calculate the required gel concentration of Example 4); blank (i.e., mixed solution A), NAA (i.e., NAA was added to the mixed solution A at a concentration of 0.02 mg / mL), hydrogel of Example 3 (i.e., the hydrogel of Example 3 was added to the mixed solution A at a concentration of 0.4708 mg / mL, and the concentration calculation here is based on the dosage concentration of NAA and the dosage and concentration of the NAA-loaded hydrogel of Example 4, and then the dosage and concentration of the hydrogel of Example 3 equivalent to that of Example 4 were calculated), and the NAA-loaded hydrogel of Example 4 (i.e., the NAA-loaded hydrogel of Example 4 was added to the mixed solution A at a concentration of 0.4908 mg / mL, and the concentration here is based on the dosage concentration of NAA of 0.02 mg / mL (CNAA =0.02 mg / mL) to calculate the dosage of NAA, and then calculate the required gel mass of Example 4, and then calculate the required gel concentration of Example 4); blank (i.e., mixed solution B), NAA (i.e., NAA was added to mixed solution B at a concentration of 0.02 mg / mL), hydrogel of Example 3 (i.e., the hydrogel of Example 3 was added to mixed solution B at a concentration of 0.4708 mg / mL, and the concentration calculation here is based on the dosage concentration of NAA and the dosage and concentration of the NAA-loaded hydrogel of Example 4, and then the dosage and concentration of the hydrogel of Example 3 equivalent to that of Example 4 were calculated), and hydrogel loaded with NAA of Example 4 (i.e., the hydrogel loaded with NAA of Example 4 was added to mixed solution B at a concentration of 0.4908 mg / mL, and the concentration here is based on the dosage concentration of NAA of 0.02 mg / mL (C NAA =0.02mg / mL) to calculate the dosage of NAA, and then calculate the required gel mass of Example 4, and then calculate the required gel concentration of Example 4), a total of 12 solutions) were continued to be cultivated for 14 days and then taken out. The plant height, stem length, wet weight and dry weight of the plant were analyzed, and the malondialdehyde concentration was detected by MDA kit (Beijing Solebow Biotechnology Co., Ltd., item number: BC0025). After being fixed at 105°C for 2h, the plant was digested after drying at 80°C for 12h (0.1g of the plant drying sample was weighed, ground and crushed, and placed in a digestion tube, and then 5mL of concentrated nitric acid was added for heating and boiling, and slowly evaporated to the minimum volume. When the digestion was nearly dry, 20mL of 2% dilute nitric acid was added after cooling slightly, and the volume was fixed to 50mL after filtering), and the potassium and sodium ion concentrations were determined by inductively coupled plasma mass spectrometry ICP-OES.

[0140] The experimental results under blank conditions, salt stress conditions, and alkaline stress conditions are as follows: Fig.10 , 11 and 12.

[0141] pass Fig.10 (a) Comparison of the photos shows that the growth of the NAA gel group is better than that of the other three groups, and the growth of the pure hydrogel group and the pure NAA group is also better than that of the blank group. Fig.10 (b) Fig.10 (c) Fig.10 (d) and Fig.10(e) The results show that the other three groups showed very effective results compared with the blank in terms of plant height, stem length, wet weight and dry weight. However, considering the four groups of data comprehensively, the CSCD-ADA2+@Lp hydrogel loaded with NAA has a relatively good effect on promoting plant growth. While ensuring the water content of the plant, it also increases the accumulation of organic matter in the plant.

[0142] pass Fig.11 and Fig.10 By contrast, at high concentrations of Na + The growth of the blank samples grown under the conditions is obviously not as good as that of the blank samples grown under the blank conditions, and the growth of the hydrogel group under salt stress is worse than that under the blank conditions. Therefore, it can be proved that the CSCD-ADA2+@Lp hydrogel loaded with NAA has excellent salt regulation ability. In addition, compared with the group with simple application of NAA, the plant height, stem length and wet weight data of the hydrogel loaded with NAA have certain advantages. And the analysis of the data error found that the growth of the hydrogel group loaded with NAA is more uniform, proving that the hydrogel loaded with NAA can effectively achieve the controlled release of pesticides and stabilize the growth of plants.

[0143] pass Fig.12 (a) It can be observed that the growth of hydrogel loaded with NAA is better than that of the other three groups, and Fig.12 This conclusion can also be drawn through data comparison in (b)(c)(d)(e). Not only that, the wheat seedlings cultivated with hydrogels loaded with NAA grew more evenly, with errors much smaller than those in the NAA group. This shows that after the hydrogel encapsulates free fertilizers or pesticides, it can promote drug release under the stimulation of saline-alkali conditions. At the same time, by utilizing this property, the hydrogel can be used on soils that have become salinized due to excessive fertilization to improve the soil, which can increase the content of organic matter in the plant and achieve the effect of "1+1>2".

[0144] When plants are subjected to adverse stress such as salt and alkaline environments, a large number of superoxide free radicals will be produced, causing membrane lipids to undergo peroxidation and produce malondialdehyde (MDA). Excessive accumulation of MDA can cause cross-linking and polymerization of plant macromolecules such as proteins and nucleic acids, thereby causing changes in the structure and function of cell membranes. Therefore, the peroxidation of membrane lipids is an important sign of damage to plant cell membranes, and the peroxidation level of plant cell membranes is generally characterized by measuring the content of MDA.

[0145] After the wheat was subjected to stress culture as in the above experiment (8), 0.1 g of fresh leaf tissue was taken from each group and added with 1 mL of MDA extract for grinding. The supernatant was extracted by centrifugation at 8459 r / min (8000 g) for 10 min. The supernatant of each group was then cooled in an ice-water bath after being water-bathed at 100°C for 60 min and centrifuged at 9458 r / min (10000 g) for 10 min. 200 μ The supernatant of L was placed in a micro-cuvette and the absorbance at 532nm and 600nm was measured using a UV-visible spectrophotometer. And the following was calculated:

[0146] ΔA 532 =A 532测定 -A 532空白

[0147] ΔA 600 =A 600测定 -A 600空白

[0148] ΔA=ΔA 532 -ΔA 600

[0149] Thus, the MDA content (unit: nmol / g) is calculated:

[0150] MDA content (nmol / g·mass) = 32.258 × ΔA ÷ W × F

[0151] Among them, A 532测定 is the UV absorbance of each sample at 532 nm, A 532空白 is the UV absorbance of the blank sample at 532 nm, ΔA 532 It is the difference between the UV absorbance of each sample at 532nm and the UV absorbance of the blank sample at 532nm. 600测定 is the UV absorbance of each sample at 600 nm, A 600空白 is the UV absorbance of the blank sample at 600 nm, ΔA 600 The difference between the UV absorbance of each sample at 600nm and the UV absorbance of the blank sample at 600nm. W is the sample mass, i.e. 0.1g. F is the dilution factor, because the sample has not been diluted, so F = 1.

[0152] After MDA extraction and calculation of the MDA concentration, we get Fig.13The results show that under blank conditions (the first 4 groups of data in the figure) and salt stress conditions (the middle 4 groups of data in the figure), the NAA group is more effective than the gel group in reducing MDA. This may be because NAA, as a plant growth regulator, regulates the metabolism of wheat organically, and NAA can promote the growth of plant roots, which plays a very important role in protecting the roots of wheat seedlings. However, the gel group also played a certain role in reducing the MDA of plants. The NAA group, gel group and gel group loaded with NAA under blank conditions were analyzed, and it was found that the MDA content of the gel group loaded with NAA was much lower than that of the simple gel group, but still higher than that of the NAA group. This shows that the CSCD-ADA2+@Lp hydrogel loaded with NAA can achieve a slow release of the loaded drug in water to achieve the effect of regulating plant growth.

[0153] However, different results were obtained from the plant growth regulation behavior experiment under alkaline stress conditions. The results showed that the MDA concentration of the CSCD-ADA2+@Lp hydrogel group loaded with NAA was the lowest under alkaline stress, and the effect was more obvious under pure water conditions and salt stress conditions. This shows that under alkaline stress conditions, due to the pH stimulus response, the ester group on the diquaternary ammonium chain of the adamantane diquaternary ammonium salt conjugate (amphoteric compound) can be hydrolyzed to form zwitterions, so that the hydrogel undergoes a gel-sol phase transition, which promotes the release of NAA. At the same time, the CSCD-ADA2+@Lp hydrogel also reduces the impact of alkaline stress on plant growth and alleviates the peroxidation reaction in the plant body, achieving the effect of "1+1>2".

[0154] (9) Antibacterial experiment of CSCD-ADA2+@Lp hydrogel

[0155] The antibacterial concentrations of three CSCDs, 0.02 mg / mL, 0.03 mg / mL, and 0.04 mg / mL, were designed. At the same time, according to the antibacterial concentration of chitosan-modified cyclodextrin, the antibacterial concentration of the corresponding supramolecular hybrid hydrogel and the antibacterial concentration of the guest molecule were calculated. The three plant fungi, Botrytis cinerea, Phytophthora capsici, and Rhizoctonia solani, were used as models. The required subject of Example 1, the guest of Example 2, and the assembled hydrogel of Example 3 were weighed and loaded into a 50 mL centrifuge tube and mixed into 45 mL of potato dextrose agar (PDA), and then poured into three sterile culture dishes while hot, and 15 mL of culture medium was poured into each culture dish. After irradiating with ultraviolet light for 30 minutes, wait for 2 to 3 hours to ensure that the culture medium solidifies and no foreign bacterial colonies are generated, and then inoculate the bacteria to ensure that the diameter of each strain is 4 mm. After inoculation, the culture was continued in a biochemical constant temperature incubator for 72 hours. The antibacterial activity of the host, guest and assembled hydrogel was evaluated by measuring the diameter of the bacterial circle colonies, thereby verifying the antibacterial properties of the synthesized host, guest and assembled hydrogel.

[0156] The antibacterial activity images of each treatment group against Botrytis cinerea from 0 to 72 hours are shown in the figure. Fig.14 As shown. It was found that the main compound CSCD had a certain inhibitory effect on Botrytis cinerea, and it was found that the antibacterial rate of the assembled CSCD-ADA2+@Lp hydrogel could almost achieve a complete disinfecting effect on plant fungi. Fig.15 (and Fig.14 Dosage corresponding) It can be seen that after 72h of co-culture, when the data of the obtained inhibition rate were analyzed, it was found that when the mass of CSCD used was 0.3mg, 0.45mg and 0.6mg, the corresponding inhibition rates of the three groups were 35.49%, 56.00% and 63.93% respectively, so when the concentration of CSCD decreased, its inhibition rate also showed a downward trend. When the mass of the guest compound ADA2+ was 0.084mg, 0.13mg and 0.17mg, the corresponding inhibition rates were 9.38%, 7.43% and 4.26% respectively. Although the three groups showed a corresponding downward trend with the increase of the concentration of the compound ADA2+, the difference in change was not large, and it was found that the guest compound had almost no antibacterial effect. Surprisingly, the assembly gel showed very high antibacterial performance at different concentrations, and the inhibition rates at high, medium and low concentrations were close to 100%. It is very effective in inhibiting Botrytis cinerea.

[0157] The antibacterial activity images of each treatment group against pepper phytophthora from 0 to 72h are shown in the figure Fig.16As shown. The conclusions drawn by comparing each group with the blank were almost the same as those of Botrytis cinerea mentioned above. The inhibitory effect of the guest compound on pepper antifungal fungi was also not obvious, but the antibacterial performance of the host compound on pepper phytophthora was better than that of Botrytis cinerea at the same concentration. Fig.17 (and Fig.16 It can also be seen from the dosage correspondence that the main compound can almost achieve an antibacterial rate of more than 90%, and the antibacterial performance of the assembled hydrogel is better than that of the simple main compound. The above shows that CSCD-ADA2+@Lp hydrogel can also play an extremely effective antibacterial role in inhibiting fungi in peppers.

[0158] The results of applying drugs to wheat blast fungus are as follows Fig.18 As shown in the figure, the wheat leaf rust was cultured for 72 hours, and the results were similar to those of Botrytis cinerea and Phytophthora capsici. From the culture medium doped with 0.3 mg of the main compound, it was found that there was some mycelial growth at 72 hours of cultivation, but the growth of the bacterial circle was very slow. The culture medium doped with the guest compound also shows that the guest compound has almost no antibacterial effect. Fig.19 (and Fig.18 The comparison of the antibacterial rates of each group can also be seen in the corresponding dosage).

[0159] From the above three groups of antibacterial experiments, it can be seen that the antibacterial activity of CSCD-ADA2+@Lp hydrogel is more effective than that of pure host compounds and pure guest compounds. In the culture medium doped with 5.47mg of CSCD-ADA2+@Lp hydrogel (equivalent to 0.3mg of CSCD), it can be found that the antibacterial rate of pure CSCD does not reach 100%, while in the culture medium doped with CSCD-ADA2+@Lp hydrogel, the antibacterial effect can be almost 100% at a lower concentration. At the same time, the three groups of antibacterial experiments can also show that the supramolecular hybrid hydrogel has a good antibacterial effect on most plant fungi, verifying that CSCD-ADA2+@Lp hydrogel has a wide range of adaptability and can be applied to plants as a pesticide carrier, thereby effectively treating large-scale fungal infections caused by plant fungi, while also slowing down the use of pesticides and reducing pollution to land and water resources.

[0160] (10) CSCD-ADA2+@Lp hydrogel biocompatibility experiment

[0161] Experiment (9) proved that the hydrogel has a strong antibacterial effect. In order to further prove that the hydrogel only has an inhibitory effect on bacteria or small fungi and does not have a large toxic effect on most living organisms, we designed a biocompatibility experiment. L929 cells (mouse fibroblasts) were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum). When the cell activity was relatively stable, L929 cells were inoculated in a 96-well plate (cell density: 5×10 4 cells / mL, 100μL per well) after culturing for 24h, the extracellular density of CSCD-ADA2+@Lp hydrogel (prepared by Example 3) was designed to be 5mg / mL, 10mg / mL, 15mg / mL and 20mg / mL, and then 5μL of hydrogel suspension was added to each corresponding well for co-culture for 24h. After that, CCK-8 solution was added for cell staining and continued to be cultured for 4h. The absorbance at 450nm was read by an ELISA instrument, and the cell survival rate was calculated, and the biocompatibility of CSCD-ADA2+@Lp hydrogel at different concentrations was evaluated.

[0162] Depend on Fig. 20 It can be seen that the effects of the four different concentrations of CSCD-ADA2+@Lp hydrogel on L929 cells were not obvious, and the cell survival rates were all above 90%, which is sufficient to prove that the supramolecular hybrid hydrogel has almost no toxicity to L929 cells.

[0163] Using the same experimental method as above, 3T3 cells were cultured in α-MEM medium containing 10% FBS, and the effect of CSCD-ADA2+@Lp hydrogel on the survival rate of 3T3 cells was observed, and almost the same conclusion as L929 was obtained. After 24 hours of co-culture with CSCD-ADA2+@Lp hydrogel, 3T3 cells still maintained nearly 100% cell activity, which was sufficient to prove that CSCD-ADA2+@Lp hydrogel had no effect on 3T3 cells. It also indirectly proved that CSCD-ADA2+@Lp hydrogel would not affect the ecology of the place where it was used.

[0164] In summary, the present invention has developed a green, environmentally friendly supramolecular hybrid hydrogel drug-carrying controlled release system, in which hectorite and guest compounds are combined through electrostatic action, and host-guest compounds are combined through hydrophilic-hydrophobic interaction, thereby achieving supramolecular self-assembly. Due to the presence of a large number of positively charged amino groups on the chitosan chain, while controlling the release of pesticides and fertilizers, plant fungal diseases are slowed down, the amount of pesticides used is reduced, and the effect of plant protection is achieved.

[0165] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A supramolecular hybrid hydrogel with pH responsiveness, characterized in that: The invention comprises laponite, a host and a guest; the host is chitosan modified with cyclodextrin; the guest comprises one or more of adamantane modified with a group, arylazopyrazole modified with a group, and ferrocene modified with a group, and the group is a quaternary ammonium group containing an ester group.

2. The pH-responsive supramolecular hybrid hydrogel according to claim 1, characterized in that: The guest is adamantane modified with a group.

3. The pH-responsive supramolecular hybrid hydrogel according to claim 2, characterized in that: The group-modified adamantane has one of the following structures: Preferably, the group-modified adamantane has the following structure:

4. The pH-responsive supramolecular hybrid hydrogel according to any one of claims 1 to 3, characterized in that: The cyclodextrin-modified chitosan has the following structure: Among them, n is 150-170.

5. The pH-responsive supramolecular hybrid hydrogel according to any one of claims 1 to 3, characterized in that: The mass ratio of hectorite, the host and the guest is 50:(2.5-3.5):(0.8-1.6).

6. The pH-responsive supramolecular hybrid hydrogel according to claim 5, characterized in that: The mass ratio of hectorite, the host and the guest is 50:(3-3.1):(0.8-0.9), preferably 50:3.05:0.

85.

7. The method for preparing the supramolecular hybrid hydrogel with pH responsiveness according to any one of claims 1 to 6, characterized in that: The following steps are involved: Mixing hectorite, a stripping agent, and water to obtain a hectorite solution; mixing the guest with water to obtain a guest solution; mixing a mixed solution of an organic acid and water with a main body to obtain a main body solution; The laponite solution, the guest solution and the host solution are mixed and stirred until coagulation occurs, thereby obtaining the supramolecular hybrid hydrogel with pH responsiveness.

8. The preparation method according to claim 7, characterized in that: The stripping agent includes one or more of sodium polyacrylate, modified polycarboxylate, sodium polyepoxysuccinate, and phosphonocarboxylic acid copolymer; The organic acid includes one or more of acetic acid, oxalic acid, lactic acid and malic acid.

9. A pesticide, characterized in that: The invention comprises the supramolecular hybrid hydrogel with pH responsiveness according to any one of claims 1 to 6 and the agricultural chemicals loaded thereon.

10. The method for preparing the pesticide according to claim 9, characterized in that: The following steps are involved: Mixing hectorite, a stripping agent, and water to obtain a hectorite solution; mixing the guest with water to obtain a guest solution; mixing a mixed solution of an organic acid and water with a main body to obtain a main body solution; dissolving an agricultural chemical in a solvent to obtain an agricultural chemical solution; The hectorite solution, the guest solution, the host solution and the agricultural chemical solution are mixed and stirred until coagulation occurs, thereby obtaining the pesticide.

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