Preparation method of polyol oxalate polymer drug-loaded sustained-release microspheres

Abstract

The present invention belongs to the field of pesticide preparation, and specifically relates to a method for preparing a polyol oxalate polymer drug-loaded sustained-release microsphere, comprising the following steps: 1) using polyol, oxalyl chloride, and an acid-binding agent to react to obtain a polyol oxalate polymer; 2) dissolving the polyol oxalate polymer and the pesticide agent in a volatile organic solvent as an oil phase; dissolving a surfactant in water as an aqueous phase; 3) adding the oil phase to the aqueous phase, performing cell crushing and dispersion after high-speed shear emulsification to obtain an O / W type emulsion; the O / W type emulsion is volatilized to remove the volatile organic solvent to obtain a polyol oxalate polymer drug-loaded sustained-release microsphere. The present invention can achieve the regulation of the release rate of the encapsulated drug, and because it can be gradually disintegrated in a weakly alkaline environment, it can effectively avoid the environmental pollution problem caused by microplastic residues.

Description

Technical Field

[0001] The invention belongs to the field of pesticide preparations, and in particular relates to a method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres. Background Art

[0002] Pesticides play a vital role in preventing and controlling crop diseases and ensuring food safety. However, the utilization rate of traditional pesticide formulations is low, and improper use can easily lead to a series of ecological and environmental safety issues; and the large amount of organic solvents, emulsifiers and surfactants contained in traditional pesticide formulations can cause certain harm to human health. The rapid development of nanotechnology in recent years is of great significance for the development of efficient and safe new pesticide formulations and the realization of sustainable agricultural development. It can solve the limitations of traditional pesticides in the use process; and can use nanomaterials and technologies to construct nano drug delivery systems to achieve slow and controlled release of pesticides, thereby reducing the loss of pesticides in the environment, improving the utilization rate of pesticides, and reducing pesticide residues and environmental pollution. At the same time, the occurrence of pests and diseases will be affected by environmental factors, such as soil pH, temperature, humidity and other factors. Introducing an appropriate "switch" in the carrier allows the pesticide to be released under a specific environment to achieve a more precise release effect and achieve "reduced amount and increased efficiency", which is a performance that traditional pesticide formulations do not have.

[0003] In the process of realizing pesticide loading, it is necessary to comprehensively consider the chemical properties of pesticides, the safety of materials, environmental friendliness, the applicability of preparation processes, and the cost and economy of materials. According to existing research results, materials such as mesoporous silica, metal organic framework compounds (MOFs), and bio-based polymers have been proven to be used for nano-scale pesticides and sustained and controlled release of active ingredients. For example, in patent CN115316382A, modified silica is used as a carrier for drug encapsulation, but due to the limited internal space capacity of silica, the drug loading amount is easily limited, and because most of its pores are open structures, the interaction between it and most drugs is weak, resulting in more serious drug leakage, thereby affecting the controlled release effect. The preparation process of the glyceric acid copper core nanospheres prepared in patent CN118000194A is relatively complicated and not suitable for large-scale production. In addition, copper is a heavy metal element, and improper handling can easily lead to potential environmental risks. Patent CN114794092A achieves the preparation of sustained-release pesticides through plant fiber gel. Although this method has certain advantages in using degradable natural polymer materials, the bio-based modification process steps are cumbersome, and its controlled-release performance is limited by the material itself, which cannot meet the preparation needs of most sustained-release pesticides.

[0004] At present, the commonly used sustained-release pesticide carriers on the market usually use artificially synthesized polymers such as polyurethane, polyurea, and polyester as pesticide encapsulation means. Although the polyurethane carrier described in patent CN115777705A has good light protection and controlled release performance, this type of artificial synthetic material degrades slowly in the natural environment and is prone to microplastic pollution, thereby affecting soil ecology. The degradable polyester capsules mentioned in patent CN108471741B have good encapsulation performance for water-soluble pesticides, but there is no detailed report on the encapsulation of fat-soluble pesticides; in addition, although the problem of difficult degradation is avoided, the large amount of organic solvents used in the preparation process will still cause potential damage to the environment, and the capsules prepared by this method are relatively large in size, all in the micron level, which limits the uniform dispersion of pesticide molecules during application.

[0005] CN 113603874 B discloses a method for preparing bio-based diol polyester. The raw material vanillyl alcohol is modified with chloroethanol. High temperature and high pressure are used in the polyester production process, which consumes a lot of energy and has a long process, which is not conducive to industrialized production. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres.

[0007] In order to solve the above problems, the present invention provides a method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres, comprising the following steps:

[0008] 1) Add polyol to organic solvent I, add oxalyl chloride dropwise at -20°C to 20°C with stirring, mix evenly after the addition is complete (stirring time is about 0.5 to 1 hour), then add acid binding agent, keep warm (-20°C to 20°C) and react for 8 to 24 hours, then separate the reaction product (remove reaction by-products) to obtain polyol oxalate polymer (ester polymer);

[0009] The mass ratio of the polyol, oxalyl chloride and acid binding agent is 1:1-2:2-3 (preferably 1:1.4-1.5:2.2-2.3);

[0010] illustrate:

[0011] The rate of the chain growth reaction can be controlled by regulating the reaction temperature, thereby achieving control of the degree of polymerization of the polymer to obtain the corresponding polyol oxalate polymer;

[0012] During the dropwise addition of oxalyl chloride, the system temperature is controlled to be ≤ (reaction temperature + 5°C); during the addition of the acid binding agent, the system temperature is controlled to be ≤ (reaction temperature + 5°C);

[0013] 2) dissolving the polyol oxalate polymer and the pesticide obtained in step 1) in a volatile organic solvent, and using the obtained mixed organic solution as the oil phase; the mass ratio of the polyol oxalate polymer to the pesticide is 1:1-10;

[0014] Dissolving a surfactant in water, and obtaining an aqueous surfactant solution as a water phase;

[0015] 3) Add the oil phase obtained in step 2) to the water phase obtained in step 2), perform cell crushing and dispersion after high-speed shear emulsification, and obtain an O / W type emulsion; the volume ratio of the oil phase to the liquid phase is 1:18-22;

[0016] The O / W emulsion is volatilized to remove the volatile organic solvent, thereby obtaining polyol oxalate polymer drug-loaded sustained-release microspheres (abbreviated as drug-loaded microspheres).

[0017] In the present invention, the suitable volatilization temperature or the temperature for separating the organic solvent by rotary evaporation is 0°C to 60°C.

[0018] As an improvement of the preparation method of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention:

[0019] In step 2), in the oil phase, the solid-liquid ratio of the polyol oxalate polymer to the volatile organic solvent is 0.1 g: 0.8-1.2 ml; in the water phase, the mass concentration of the surfactant is 0.2-1.6% (preferably 0.4-0.6%);

[0020] In step 3), the volume ratio of the oil phase to the liquid phase is 1:18-22.

[0021] As a further improvement of the preparation method of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, in step 1):

[0022] The polyol is at least any one of the following: 1,2-ethylene glycol, 1,3-propylene glycol, 1,2,3-propanetriol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol;

[0023] The acid binding agent is at least one of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide, cesium carbonate, cesium hydroxide, triethylamine, N,N Diisopropylethylamine, pyridine.

[0024] As a further improvement of the preparation method of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, in step 1):

[0025] The organic solvent I is at least any one of the following: acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, dimethylacetamide, dichloromethane, dichloroethane, dimethyl sulfoxide;

[0026] The usage ratio of the organic solvent I to the polyol is 8-20 mL / 1g (preferably 8-10 mL / 1g).

[0027] As a further improvement of the method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, the surfactant in step 2) is at least any one of the following:

[0028] Hexadecyltrimethylammonium bromide, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, polyvinyl alcohol, sodium lauroylamphoacetate, sodium lignin sulfonate, phenylethylphenol polyoxyethylene ether, polyoxyethylene sorbitan monooleate, polyoxyethylene polyoxypropylene block polyether, alkylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, silicone.

[0029] As a further improvement of the method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, the volatile organic solvent in step 2) is at least any one of the following:

[0030] Tetrahydrofuran, 1,4-dioxane, dichloromethane, dichloroethane, cyclohexane, hexane, cyclohexanone, methyl tetradecanoate, methyl hexadecanoate, xylene.

[0031] As a further improvement of the method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, the pesticide in step 2) is a fat-soluble pesticide or a water-soluble pesticide.

[0032] Description: The pesticide of the present invention is an insecticide, a fungicide or a herbicide, and has the functions of UV resistance and alkali response to release the pesticide.

[0033] As a further improvement of the preparation method of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention:

[0034] The fat-soluble pesticide is any one of the following: avermectin, cypermethrin, fipronil, chlorpyrifos, trifloxystrobin, sclerotin, iprodione, carbendazim, indoxacarb, prochloraz, pyraclostrobin, metconazole, difenoconazole, fenthion, promethazine, and quizalofop-p-ethyl;

[0035] The water-soluble pesticide is any one of the following: thiamethoxam, metalaxyl, kasugamycin, and glyphosate.

[0036] As a further improvement of the preparation method of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, the separation treatment in step 1) includes extraction, liquid separation, drying, and rotary evaporation;

[0037] The extraction and separation are as follows: the reaction product is heated to room temperature (25-28°C), and a 3±0.5 mol / L HCl solution is added for stirring to dissolve the solids in the reaction product; then the water layer is separated by a separatory funnel; 40±5 ml of HCl solution is added for every 0.05 mol of oxalyl chloride;

[0038] The drying and rotary evaporation are as follows: the organic phase layer obtained by separation is recovered and dried with anhydrous sodium sulfate, and then the organic solvent I is removed (by a rotary evaporator) to obtain a polyol oxalate polymer.

[0039] As a further improvement of the preparation method of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention, in step 3):

[0040] The high-speed shear emulsification is an emulsification treatment at a rotation speed of 10000-20000 rpm for 1-50 minutes;

[0041] The cell disruption and dispersion is performed at a power of 100 W for 1 to 5 minutes;

[0042] The O / W emulsion is rapidly stirred at room temperature (the speed of rapid stirring is 1000~1500 rpm) to evaporate and remove the volatile organic solvent.

[0043] The polyol oxalate polymer drug-loaded sustained-release microspheres obtained by the method of the present invention have controllable molecular weight and adjustable sustained-release rate (mainly by adjusting the molecular weight of the polyol oxalate polymer); they are mainly nanoscale (1-100 nm), submicron scale (100-1000 nm), and can also be micron scale (1μm-50 μm).

[0044] During the invention process, the present invention fully considered the following problems to be solved:

[0045] Some of the existing patent literatures are polyester microcapsule drug delivery systems synthesized through multiple complex steps, and some are complex systems in which polyester is only used to load water-soluble pesticides. The particle sizes are mostly in the micron level, and the detailed controlled release or corresponding release function is unclear. No more ideal polyester drug delivery nano / micro systems have been reported.

[0046] In response to the above problems, the inventor's research group conducted in-depth research and found that some diol basic raw materials with simple structures can be used to obtain polyester components with different molecular weights by a simple and quick esterification method by controlling the temperature during the polymerization reaction. Different pesticides are then added to make different drug delivery systems, which have rapid activity while taking into account a certain lasting effect, and are economical and environmentally friendly.

[0047] That is, in order to improve the utilization rate of pesticides in practical applications, the present invention has developed a method for preparing pesticide sustained-release drug-loaded microspheres with a simple process; by selecting suitable diols and oxalyl chloride as cheap raw materials, the preparation is used to construct pesticide nano-carriers and for encapsulation of pesticides. The technology of the present invention can achieve the regulation of the release rate of the drug encapsulated in the drug-loaded microspheres by regulating the preparation conditions of the carrier material, and has obvious technical advantages. In addition, since the material obtained by the present invention has the characteristics of weak alkaline stimulus response, it can be gradually disintegrated in a weak alkaline environment, which can effectively avoid the environmental pollution problem caused by microplastic residues.

[0048] Compared with the prior art, the present invention has the following technical advantages:

[0049] 1. The present invention uses commercially available polyols and oxalyl chloride as raw materials. The raw materials are cheap and readily available, the preparation process is simple, the reaction conditions are mild, and the pesticide can be used to load fat-soluble pesticides such as insecticides, fungicides or herbicides.

[0050] 2. The degree of polymerization of the polyol oxalate polymer can be adjusted by the reaction temperature, thereby achieving a rate-controlled release of the loaded pesticide, and realizing precise prevention and control with adjustable effective period for different crops and different diseases.

[0051] 3. The polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention can improve the photostability of the loaded drug in the environment and greatly extend the effective period of the pesticide.

[0052] 4. The drug loading rate of the polyol oxalate polymer drug-loaded sustained-release microspheres of the present invention is adjustable, and the maximum drug loading rate can reach about 83%.

[0053] 5. The polyol oxalate polymer involved in the present invention has the characteristics of weak alkaline stimulus response and can be gradually disintegrated in a weak alkaline environment, thereby avoiding the pollution problem caused by microplastic residues.

[0054] Conventional microcapsules or drug-carrying materials are polyurethane or polyurea, which have a strong chemical structure, are non-degradable, and are also the main source of "microplastics".

[0055] 6. The particle size of the present invention is nanometer or submicron, which helps the pesticide to be better dispersed and increases its dispersibility.

[0056] In summary, the present invention uses commercially available cheap polyols and oxalyl chloride as raw materials, based on a chain growth esterification reaction, and controls the molecular weight of the polymer by controlling the reaction temperature, thereby achieving a rate-controlled disintegration of the polymer material, and then realizing the controlled release of the encapsulated pesticide preparation. Specifically, polyols and oxalyl chloride are used as reactants, and polyester is obtained by acid binding with an acid binding agent, and then the polyol oxalate polymer and the pesticide agent are dissolved in an organic solvent to form an oil phase, and the oil phase is dispersed in the water phase to prepare an oil-in-water emulsion (O / W), and then the organic solvent in the emulsion is removed to finally form a polyol oxalate polymer drug-loaded microsphere loaded with pesticides. The polyol oxalate polymer drug-loaded controlled-release microspheres with controllable molecular weight and adjustable controlled-release rate not only release slowly, but also have the characteristics of weak alkaline stimulus response release, and have the function of avoiding ultraviolet light degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The infrared spectra of 1,4-butanediol and the polyol oxalate polymer (polyoxalate) prepared in Example 1.

[0058] Figure 2 Infrared spectra of metconazole and the ester polymer drug-loaded microspheres loaded with metconazole prepared in Examples 1, 2 and 3.

[0059] Figure 3 The release rate curves of metconazole-loaded ester polymer drug-loaded microspheres prepared in Examples 1, 2, and 3 under pH 9 conditions.

[0060] Figure 4 The anti-photolysis performance curve of metconazole and the ester polymer drug-loaded microspheres loaded with metconazole prepared in Example 1.

[0061] Figure 5 This is the SEM image of the ester polymer drug-loaded microspheres loaded with metconazole prepared in Example 1.

[0062] Figure 6 This is an evaluation of the effect of the polyol oxalate polymer prepared in Example 1 on the germination rate of wheat.

[0063] Figure 7 The effect of the polyol oxalate polymer prepared in Example 1 on the plant height and fresh weight of wheat seedlings was evaluated.

[0064] Figure 8 The natural degradation of the polyol oxalate polymers prepared in Examples 1 to 3 in a weakly alkaline water environment.

[0065] Fig. 9 This is the natural degradation of the polyol malonate polymer prepared in Comparative Example 1 in a weakly alkaline water environment. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0067] The transmission electron microscope used in the examples is a HT-7700 field emission transmission electron microscope (Hitachi, Japan); the thermal field emission scanning electron microscope (Zeiss G300); the liquid chromatograph is a Shimadzu LC-10AT; and the particle size analyzer is a Nano-ZS90 laser particle size analyzer.

[0068] The stirring speed of the reaction involved in the present invention is 200-400 rpm; the speed of rapid stirring is 1000-1500 rpm.

[0069] Example 1: A method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres, comprising the following steps:

[0070] (1) Preparation of polyol oxalate polymers

[0071] Weigh 4.5 g of 1,4-butanediol and add it to a three-necked flask, then add 40 ml of dichloromethane and mix well; place the three-necked flask in a cold trap at 20°C, and cool the mixture in the three-necked flask to 20°C while stirring. Then, add 6.4 g of oxalyl chloride dropwise under stirring, and control the temperature in the three-necked flask to ≤25°C during the addition. After the addition is completed, keep stirring at 20°C for 0.5 hours to ensure uniform mixing. Finally, slowly add 10.1 g of triethylamine, and control the temperature in the three-necked flask to ≤25°C during the slow addition. After the addition of triethylamine, keep the reaction at 20°C for 8 hours.

[0072] The reaction product is separated and treated (to remove the reaction by-products) as follows:

[0073] Extraction and separation: After the reaction is completed, heat the reaction mixture to room temperature (25-28°C), add 40 ml of 3M HCl solution into a three-necked flask, stir to dissolve the solids in the flask; then use a separatory funnel to separate the water layer;

[0074] Drying and rotary evaporation: The dichloromethane layer obtained by separation is recovered and dried with anhydrous sodium sulfate, and finally the solvent (ie, dichloromethane) is removed by a rotary evaporator to obtain a polyol oxalate polymer (ie, polyethylene oxalate, as a solid).

[0075] After gel chromatography analysis, the molecular weight of the polyol oxalate polymer was Mw = 724620 g / mol, Mn = 590689 g / mol, and the degree of polymerization (DP) = 4102.

[0076] The degree of polymerization is the number average of the monomer units in a polymer or oligomer; the degree of polymerization is equal to Mn / Mo, where Mn is the number average molecular weight and Mo = 144 (the molecular weight of the monomer unit).

[0077] Note: The main component of the solid in the above text is triethylamine hydrochloride (a reaction by-product) generated during the reaction. It has a very high viscosity and is difficult to filter directly. Therefore, the above method of dissolving first and then separating is adopted.

[0078] (2) Preparation of polyol oxalate polymer drug-loaded microspheres loaded with the fungicide metconazole

[0079] 100 mg of the polyol oxalate polymer prepared in step (1) above and 100 mg of metconazole were dissolved in 1 ml of dichloromethane to form the oil phase.

[0080] Take 20 ml of 0.4% mass concentration of nonylphenol polyoxyethylene ether (TX-10) aqueous solution as the aqueous phase.

[0081] The oil phase and the water phase were mixed, sheared and emulsified at 10,000 rpm for 1 minute using a high-speed shear emulsifier, and then further dispersed under a cell disruptor (the power of the cell disruptor was 100 W and the dispersion time was 5 minutes) to obtain a stable O / W emulsion.

[0082] The O / W emulsion was rapidly stirred at room temperature to volatilize and remove dichloromethane, thereby obtaining metconazole-loaded polyol oxalate polymer drug-loaded sustained-release microspheres (i.e., metconazole-loaded ester polymer drug-loaded microspheres, referred to as drug-loaded microspheres).

[0083] Figure 5 This is a scanning electron microscope image of the ester polymer drug-loaded microspheres loaded with metconazole prepared in Example 1. It can be seen that the obtained drug-loaded microspheres are regular spherical in shape, with a relatively smooth surface and an average size of less than 300 nm.

[0084] The particle size distribution of the polyester drug-loaded microspheres in aqueous suspension prepared in Example 1 was measured using a Malvern Zetasizer Nano ZS90. The average particle size was 231.08 nm, and the drug loading rate was 81.2%.

[0085] .

[0086] Example 2: A method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres, comprising the following steps:

[0087] (1) Preparation of polyol oxalate polymers

[0088] Weigh 4.5 g of 1,4-butanediol and add it to a three-necked flask, then add 40 ml of dichloromethane and mix well; place the three-necked flask in a cold trap at 0°C, and cool the mixture in the three-necked flask to 0°C while stirring; then add 6.4 g of oxalyl chloride dropwise under stirring conditions, and control the temperature in the three-necked flask to ≤5°C during the addition process. After the addition is completed, keep stirring at 0°C for 0.5 hours to ensure uniform mixing. Finally, slowly add 10.1 g of triethylamine, and control the temperature in the three-necked flask to ≤5°C during the slow addition process. After the addition of triethylamine, keep the reaction at 0°C for 8 hours.

[0089] The reaction product is separated (to remove the by-products), and the separation method is as follows:

[0090] After the reaction is completed, the reaction product is heated to room temperature, 40 ml of 3M HCl solution is added to a three-necked flask, and the solid in the flask is dissolved by stirring. Then, the water layer is separated by a separatory funnel, and the dichloromethane layer is recovered and dried with anhydrous sodium sulfate. Finally, the solvent is removed by a rotary evaporator to obtain a polyol oxalate polymer.

[0091] Gel chromatography analysis showed that the molecular weight of the polyol oxalate polymer was Mw = 561971 g / mol, Mn = 546619 g / mol, and DP = 3796.

[0092] (2) Preparation of polyol oxalate polymer loaded with fungicide metconazole nano / microspheres

[0093] The polyol oxalate polymer prepared in step (1) of the above embodiment 2 is used instead, and the rest is the same as step (2) of embodiment 1.

[0094] The polyol oxalate polymer drug-loaded sustained-release microspheres loaded with metconazole were obtained.

[0095] The particle size distribution of the drug-loaded microspheres prepared in Example 2 was measured using a Malvern Zetasizer Nano ZS90, and the average particle size was 254.37 nm, and the drug loading rate was 78.8%.

[0096] Example 3: A method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres, comprising the following steps:

[0097] (1) Preparation of polyol oxalate polymers

[0098] Weigh 4.5 g of 1,4-butanediol and add it to a three-necked flask, then add 40 ml of dichloromethane and mix well; place the three-necked flask in a -20°C cold trap and cool the mixture in the three-necked flask to -20°C while stirring. Then, add 6.4 g of oxalyl chloride dropwise under stirring conditions, and control the temperature in the three-necked flask to ≤-15°C during the addition process. After the addition is completed, keep stirring at -20°C for 0.5 hours to ensure uniform mixing. Finally, slowly add 10.1 g of triethylamine, and control the temperature in the three-necked flask to ≤-15°C during the slow addition process. After the addition of triethylamine, keep the reaction at -20°C for 8 hours.

[0099] The reaction product is separated (to remove the by-products), and the separation method is as follows:

[0100] After the reaction is completed, the reaction product is heated to room temperature, 40 ml of 3M HCl solution is added to a three-necked flask, and the solid in the flask is dissolved by stirring. Then, the water layer is separated by a separatory funnel, and the dichloromethane layer is recovered and dried with anhydrous sodium sulfate. Finally, the solvent is removed by a rotary evaporator to obtain a polyol oxalate polymer.

[0101] After gel chromatography analysis, the molecular weight of the polyol oxalate polymer was Mw = 424125 g / mol, Mn = 346188 g / mol, and DP = 2404.

[0102] (2) Preparation of polyol oxalate polymer drug-loaded microspheres loaded with the fungicide metconazole

[0103] The polyol oxalate polymer prepared in step (1) of the above Example 3 is used instead, and the rest is the same as step (2) of Example 1.

[0104] The polyol oxalate polymer drug-loaded sustained-release microspheres loaded with metconazole were obtained.

[0105] The particle size distribution of the drug-loaded microspheres prepared in Example 3 was measured using a Malvern Zetasizer Nano ZS90, and the average particle size was 227.09 nm, and the drug loading rate was 80.3%.

[0106] Example 4: With respect to Example 1, the following changes are made: the dispersion time of the cell disruptor in step (2) of Example 1 is changed from 5 minutes to 1 minute, and the rest is the same as Example 1.

[0107] The particle size distribution of the drug-loaded microspheres prepared in Example 4 was measured by Malvern Zetasizer Nano ZS90, and the average particle size was 560.20 nm, which was within the submicron particle size range. The drug loading rate was 78.2%.

[0108] Example 5. Compared with Example 1, the following changes are made: the cell crushing and dispersion is eliminated, that is, the dispersion time of the cell crusher in step (2) of Example 1 is changed from 5 minutes to 0 minutes, and the rest is the same as Example 1.

[0109] The particle size distribution of the drug-loaded microspheres prepared in Example 5 was measured by a laser particle size analyzer, and the average particle size was 5-20 μm, and the drug loading rate was 78.2%.

[0110] Example 6: The "metconazole" in step (2) of Example 1 is replaced with "photosensitive insecticide avermectin", and the dosage remains unchanged, still 100 mg, and the rest is the same as Example 1.

[0111] The polyol oxalate polymer drug-loaded sustained-release microspheres loaded with avermectin are obtained.

[0112] The particle size distribution of the drug-loaded microspheres prepared in Example 6 was measured using a Malvern Zetasizer Nano ZS90. The average particle size was 207.01 nm, and the drug loading rate of avermectin was 79.7%.

[0113] Experiment 1

[0114] In order to verify the successful modification of the polyol oxalate polymer, the polyol oxalate polymer obtained in Example 1, 1,4-butanediol oxalate polyester and the raw material 1,4-butanediol were characterized by infrared spectroscopy. Figure 1 The absorption peak caused by the stretching vibration of the hydroxyl group of the raw material 1,4-butanediol is at 3340 cm -1 The absorption intensity of 1,4-butanediol oxalate polyester at this position is significantly weakened, indicating that the alcohol hydroxyl group is continuously consumed during the synthesis process. -1 There are obvious stretching vibration absorption peaks from C=O in the ester bond on the left and right, indicating that the reaction has successfully synthesized the target polyol oxalate polymer (1,4-butanediol oxalate polyester).

[0115] In order to verify the successful preparation of metconazole-loaded polyol oxalate polymer drug-loaded sustained-release microspheres, infrared spectroscopy was used to characterize the metconazole-loaded ester polymer drug-loaded microspheres (hereinafter referred to as drug-loaded microspheres) prepared in Examples 1, 2, and 3. Figure 2 As shown in the infrared spectra of the three drug-loaded microspheres, the -1 There are obvious stretching vibration absorption peaks from C=O in the ester bond on the left and right, indicating that the microspheres contain polyol oxalate polymers. In addition, the stretching vibration of the C=N bond of the triazole ring structure in the metconazole molecular structure is at 678 cm -1The absorption peak is located at the left and right sides of the three drug-loaded microspheres. Since the polyol oxalate polymer itself has no obvious characteristic absorption peak at this location, the existence of the C=N bond stretching vibration absorption peak at this location can verify the successful loading of metconazole in the drug-loaded microspheres.

[0116] Experiment 2: Alkali response sustained release effect of ester polymer drug-loaded microspheres loaded with metconazole (hereinafter referred to as drug-loaded microspheres) obtained in Examples 1 to 3:

[0117] The drug-loaded microspheres obtained in Examples 1 to 3 were first freeze-dried at -40°C for 12 hours to obtain dried drug-loaded microspheres; and then the following operations were performed respectively:

[0118] Accurately weigh 0.05 g of dried drug-loaded microspheres into a dialysis bag with a molecular weight cutoff of 1000, add 3 mL of PBS buffer (pH 9) containing 0.1% Tween 80 into the dialysis bag, then place the dialysis bag into a brown dialysis bottle containing 100 mL of PBS buffer (pH 9) containing 0.1% Tween 80, and place the brown dialysis bottle in a constant temperature shaker at 150 rpm and 25 °C for continuous oscillation or stirring; sample 1 mL of solution from the outside of the dialysis bag at regular intervals and add 1 mL of fresh PBS buffer. After filtering the sample through a water-based syringe filter head, the concentration of metconazole was determined by high performance liquid chromatography, with time as the horizontal axis and the cumulative release percentage of metconazole as the vertical axis. Repeat three times and take the average value to draw the release curve of metconazole.

[0119] HPLC determination of metconazole content: the analytical column was an Agilent ZORBAX SB-C18 column (4.6×250mm, 5.0μm), the mobile phase was V(methanol):V(water)=80:20; the flow rate was 1.0 mL / min; the detector was an ultraviolet detector, the detection wavelength was 220 nm, the injection volume was 20 μL, the column temperature was 40 ℃, and the standard curve method was used for quantification.

[0120] That is, the weak alkaline response release performance of the three drug-loaded microspheres obtained in Examples 1, 2, and 3 was measured, and the results obtained are as follows: Figure 3 As shown. Figure 3 It can be seen that the release rates of the drug-loaded microspheres loaded with metconazole prepared under different conditions are significantly different. The release rate order is: Example 1 < Example 2 < Example 3. The drug-loaded microspheres prepared under lower temperature conditions show faster drug release rates, which makes the ability of the drug-loaded particles to release pesticides controllable and slow release.

[0121] The results showed that the drug release rate was related to the molecular weight of the polyester carrier, and the drug release rate of the polyol oxalate polymer with a large molecular weight was slower after drug loading.

[0122] Correspondingly, Examples 4 to 6 also exhibit corresponding sustained-release effects.

[0123] As common sense, the pesticide metconazole itself has no sustained-release effect.

[0124] Experiment 3

[0125] The ester polymer drug-loaded microspheres loaded with metconazole prepared in Example 1 were recorded as MEZ CS1, and the in vitro inhibitory effects of metconazole original drug and ester polymer drug-loaded microspheres loaded with metconazole on Fusarium graminearum were determined by the mycelial growth rate method. 1.232 grams of ester polymer drug-loaded microspheres loaded with metconazole were added to 1L of 0.1% Tween 80 aqueous solution to prepare a 1000ppm suspension as a mother liquor, and the mother liquor was drawn into a potato dextrose agar medium under a clean bench environment and shaken evenly to prepare a drug-containing medium, with concentrations of 0.0625 ppm, 0.125 ppm, 0.25 ppm, 0.5 ppm, 0.75 ppm, and 1 ppm, respectively. Then, a bacterial cake of Fusarium graminearum with a diameter of 0.8 cm was placed on the surface of the drug-containing medium, and the inhibition rate was statistically calculated after 4 days of growth. A 1000 ppm suspension of metconazole technical drug MEZ TC (the technical drug was dissolved in a small amount of acetone and then added dropwise to a 0.1% by mass Tween 80 aqueous solution) was prepared to prepare a drug-containing culture medium of equal concentration as a control, and the growth inhibitory effect on F. graminearum was determined; the results are shown in Table 1.

[0126] .

[0127] Table 1. Antibacterial effect

[0128]

[0129] *Not this invention.

[0130] The results shown in Table 1 show that the ester polymer drug-loaded microspheres loaded with metconazole exhibited an in vitro antibacterial efficiency comparable to that of metconazole, confirming that the ester polymer drug-loaded microspheres loaded with metconazole of the present invention have a rapid antibacterial ability.

[0131] Experiment 4

[0132] The ester polymer drug-loaded microspheres loaded with metconazole prepared in Examples 1 to 3 (respectively denoted as MEZ CS1, MEZ CS2, and MEZ CS3) were diluted to 100 mg / L with a 0.1% mass percentage Tween 80 aqueous solution. Compared with 100 mg / L metconazole original drug (MEZ TC), the control effect on wheat stem base rot was tested under artificial greenhouse conditions to verify the control effectiveness of the drug-loaded microspheres of the present invention.

[0133] First, experimental soil containing metconazole was prepared. Specifically, soil sterilized at 120°C was treated with MEZ CS1, MEZ CS2, MEZ CS3, and MEZ TC to achieve a metconazole effective active ingredient concentration of 20 mg / kg in the soil. The soil was then left to stand in the dark for 14 days before wheat sowing.

[0134] Prepare the infected wheat seeds and healthy germinating wheat seeds for the experiment. For the infected wheat seeds, soak the wheat seeds disinfected with 2% sodium hypochlorite aqueous solution in sterile water for 24 hours, transfer them to a conical flask, and sterilize them under high pressure at 120°C. The sterilized seeds are inoculated with Pseudomonas graminearum and cultured until the mycelium completely covers the conical flask, and used as infected wheat seeds. For healthy germinating wheat seeds, soak the wheat seeds disinfected with 2% sodium hypochlorite aqueous solution in sterile water for 48 hours for germination.

[0135] Ten healthy germinated wheat seeds and ten infected wheat seeds were evenly sown in the soil treated with MEZ CS1, MEZ CS2, MEZ CS3 and MEZ TC. The soil without metconazole was used as the blank control group (CK). The rest of the cultivation method was the same as usual. The incidence rate, disease index and inhibitory effect on wheat stem rot were calculated on the 14th day after sowing. The results are shown in Table 2.

[0136] .

[0137] Table 2. Control effect

[0138]

[0139] *Not this invention.

[0140] The results shown in Table 2 indicate that the ester polymer drug-loaded microspheres loaded with metconazole can achieve long-term control of wheat stem base rot through controlled-release technology, and the control efficacy is increased by about 1.5 times compared with the original drug, effectively improving the utilization rate of metconazole during use.

[0141] Experiment 5: Anti-photolysis performance of metconazole-loaded ester polymer drug-loaded microspheres:

[0142] Weigh a certain mass of the ester polymer drug-loaded microspheres loaded with metconazole and metconazole original drug obtained in Example 1, and dilute them to 100 mL (active ingredient concentration is 500 ug / mL) with a 0.1% by mass Tween 80 aqueous solution. Pipette 1 mL of the above suspension into a 5 mL centrifuge tube and place it in an ultraviolet degradation box equipped with two ultraviolet lamps with a power of 6W and an emission wavelength of 254 nm (degradation box volume 25 cm×25 cm×25 cm). Take out the centrifuge tube at regular intervals, add 3 mL of acetonitrile and mix evenly, then filter the sample through a 0.45 μm organic syringe filter, and test the remaining amount of metconazole using high performance liquid chromatography. Repeat the experiment three times and take the average value. The results are as follows: Figure 4 As shown, compared with the original drug of metconazole, the ester polymer drug-loaded microspheres loaded with metconazole released the drug significantly slower under light conditions.

[0143] That is, in order to verify that the ester polymer drug-loaded microspheres loaded with metconazole have the ability to improve the photodegradation resistance of the original drug, the photolysis performance of the drug-loaded microspheres prepared in Example 1 was evaluated. Figure 4 As shown in the figure, the active ingredient of metconazole original drug was quickly consumed under 254 nm wavelength ultraviolet light, and only 11% remained after 24 hours. However, the active ingredient of ester polymer drug-loaded microspheres loaded with metconazole was still as high as 63% after 24 hours of ultraviolet light irradiation, which has good photostability, so it has higher sustained performance in actual application scenarios.

[0144] Experiment 6: Determination of insecticidal biological activity

[0145] The avermectin-loaded ester polymer drug-loaded microspheres prepared in Example 6 and the avermectin original drug were respectively prepared into 1000 ppm mother solutions and irradiated under ultraviolet light (254 nm, 6W), and the distance between the sample and the light source was 15 cm. Samples were taken every 0.5 h, and the samples were diluted 100 times. The diluted solution was used to treat the cabbage slices by immersion method and dried in the air. Subsequently, 10 third-instar larvae of diamondback moth were placed in a culture dish, and a treated cabbage leaf was placed in it. The experiment was carried out under artificial conditions of 25°C, 85% relative humidity and 16:8 hours (light / dark cycle). The bioavailability of avermectin-loaded ester polymer drug-loaded microspheres (AVM CS) and avermectin original drug (AVM TC) was tested. The evaluation range was 0-100% activity. 100% activity means that the tested diamondback moth is completely dead, and 0% activity means that all the tested diamondback moths survive. The results are shown in Table 3.

[0146] Table 3. Bioavailability

[0147]

[0148] *Not this invention.

[0149] The results shown in Table 3 indicate that the unencapsulated avermectin has poor photostability and its insecticidal activity decreases rapidly with increasing irradiation time, while the encapsulated avermectin-loaded ester polymer drug-loaded microspheres have excellent stability and its insecticidal activity can be maintained for a long time.

[0150] Experiment 7: To study the toxicity of polyester to wheat seeds, a polyol oxalate polymer suspension obtained in Example 1 was prepared with a 0.1% Tween 80 aqueous solution at concentrations of 50, 100, 250, 500, and 1000 μg / mL. The wheat seeds were disinfected with a 2% sodium hypochlorite solution, then thoroughly washed with sterile water and transferred to a culture dish (10 wheat seeds per dish), and 5 mL of the polyester suspension prepared above was added. Clean water without polyester was used as a blank control. The germination rate of the wheat seeds was observed and counted on the third day. Each experimental group was repeated three times and the average value was taken.

[0151] A polyester suspension with a concentration of 1000 μg / mL was prepared with a 0.1% Tween 80 aqueous solution. Wheat seedlings grown for 7 days were used as the test objects (10 wheat plants per pot), and 20 mL of the polyester suspension was used to irrigate the wheat seedlings, and clear water without polyester was used as a blank control. On the 7th day after treatment, the plant height and fresh weight data of the treated wheat in the two groups were observed and counted, and each experimental group was repeated three times to take the average value.

[0152] Figure 6 and Figure 7 This is the result of the biosafety evaluation of the polyol oxalate polymer prepared in Example 1 on wheat. From the results, it can be seen that the polyol oxalate polymer has no effect on the germination of wheat. In addition, during the growth and development of wheat, the polyol oxalate polymer will not affect the growth of wheat. The plant height and fresh weight of the treated wheat are not significantly different from those of the blank group. Therefore, the results show that the polyol oxalate polymer of the present invention has good safety during use.

[0153] Experiment 8: Degradation under alkaline conditions

[0154] 50 mg of each of the polyol oxalate polymers obtained in Examples 1 to 3 were pressed into thin slices with a diameter of 1 cm and a thickness of 1 mm by a tablet press. The thin slices and 50 mL of a 0.1 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution (pH 9) were placed in a 50 mL beaker and allowed to stand at room temperature; every 24 hours, the buffer solution was removed with a plastic dropper, and then 50 mL of a fresh 0.1 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution (pH 9) was added, and the degradation of the polyol oxalate polymer was observed within 21 days.

[0155] Figure 8 The degradation of the polyol oxalate polymers of Examples 1 to 3 under weak alkaline conditions (pH 9) within 21 days. From the results, it can be seen that the polyol oxalate polymers of Examples 1 to 3 can be gradually degraded, and the pollution caused by the material remaining in the natural environment can be avoided. Note: The black part represents the bottom of the beaker, and the gray part represents the polyol oxalate polymer.

[0156] Comparative Example 1: The 6.4 g (0.05 mol) oxalyl chloride in step (1) of Example 1 was replaced with the same molar amount of malonyl chloride (7.0 g, 0.05 mol), and the rest was the same as in Example 1 to obtain a polyol malonate polymer.

[0157] The obtained product was tested according to the above experiment 8, and the results were as follows Fig. 9 As shown: Polyol malonate polymers cannot achieve natural degradation under alkaline conditions.

[0158] Comparative Example 2: 6.4 g (0.05 mol) of oxalyl chloride in step (1) of Example 1 was replaced with the same molar amount of oxalic acid (0.05 mol), and the rest was the same as Example 1. As a result, the target polyol oxalate polymer could not be synthesized.

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres, characterized in that The following steps are involved: 1) Add polyol to organic solvent I, add oxalyl chloride dropwise at -20°C to 20°C with stirring, mix evenly after the addition is complete, then add acid binding agent, keep warm and react for 8 to 24 hours, and then separate the reaction product to obtain a polyol oxalate polymer; The mass ratio of the polyol, oxalyl chloride and acid binding agent is 1:1~2:2~3; The polyol is at least any one of the following: 1,2-ethylene glycol, 1,3-propylene glycol, 1,2,3-propanetriol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol; The acid binding agent is at least any one of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide, cesium carbonate, cesium hydroxide, triethylamine, N,N-diisopropylethylamine, pyridine; 2) dissolving the polyol oxalate polymer and the pesticide obtained in step 1) in a volatile organic solvent, and using the obtained mixed organic solution as the oil phase; the mass ratio of the polyol oxalate polymer to the pesticide is 1:1-10; Dissolving a surfactant in water, and obtaining an aqueous surfactant solution as a water phase; 3) adding the oil phase obtained in step 2) to the water phase obtained in step 2), performing cell crushing and dispersion after high-speed shear emulsification to obtain an O / W emulsion; The O / W type emulsion is volatilized to remove the volatile organic solvent, thereby obtaining polyol oxalate polymer drug-loaded sustained-release microspheres.

2. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 1, characterized in that: In step 2), in the oil phase, the solid-liquid ratio of the polyol oxalate polymer to the volatile organic solvent is 0.1 g: 0.8-1.2 ml; in the water phase, the mass concentration of the surfactant is 0.2-1.6%; In step 3), the volume ratio of the oil phase to the liquid phase is 1:18-22.

3. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 2, characterized in that In step 1): The organic solvent I is at least any one of the following: acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, dimethylacetamide, dichloromethane, dichloroethane, dimethyl sulfoxide; The usage ratio of the organic solvent I to the polyol is 8-20 mL / 1 g.

4. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to any one of claims 1 to 3, characterized in that The surfactant in step 2) is at least any one of the following: Hexadecyltrimethylammonium bromide, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, polyvinyl alcohol, sodium lauroylamphoacetate, sodium lignin sulfonate, phenylethylphenol polyoxyethylene ether, polyoxyethylene sorbitan monooleate, polyoxyethylene polyoxypropylene block polyether, alkylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, silicone.

5. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 4, characterized in that The volatile organic solvent in step 2) is at least one of the following: Tetrahydrofuran, 1,4-dioxane, dichloromethane, dichloroethane, cyclohexane, hexane, cyclohexanone, xylene.

6. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 5, characterized in that: The pesticide in step 2) is a fat-soluble pesticide or a water-soluble pesticide.

7. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 6, characterized in that: The fat-soluble pesticide is any one of the following: avermectin, cypermethrin, fipronil, chlorpyrifos, trifloxystrobin, sclerotin, iprodione, carbendazim, indoxacarb, prochloraz, pyraclostrobin, metconazole, difenoconazole, fenthion, promethazine, and quizalofop-p-ethyl; The water-soluble pesticide is any one of the following: thiamethoxam, metalaxyl, kasugamycin, and glyphosate.

8. The method for preparing the polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 7, characterized in that The separation treatment in step 1) includes extraction, liquid separation, drying, and rotary evaporation; The extraction and separation are as follows: the reaction product is heated to room temperature, and a 3±0.5 mol / L HCl solution is added to dissolve the solids by stirring; then the water layer is separated by a separatory funnel; 40±5 ml of HCl solution is added for every 0.05 mol of oxalyl chloride; Drying and rotary evaporation: The organic phase obtained by separation is recovered and dried with anhydrous sodium sulfate, and then the organic solvent I is removed to obtain a polyol oxalate polymer.

9. The method for preparing polyol oxalate polymer drug-loaded sustained-release microspheres according to claim 8, characterized in that In step 3): The high-speed shear emulsification is an emulsification treatment at a rotation speed of 10000-20000 rpm for 1-50 minutes; The cell disruption and dispersion is performed at a power of 100 W for 1 to 5 minutes; The O / W emulsion is rapidly stirred at room temperature to evaporate and remove the volatile organic solvent.

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