CN115444003A - Method for preparing high-drug-loading phoxim nano-structure lipid carrier based on micro-fluidic control - Google Patents
Method for preparing high-drug-loading phoxim nano-structure lipid carrier based on micro-fluidic control Download PDFInfo
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- CN115444003A CN115444003A CN202211039810.9A CN202211039810A CN115444003A CN 115444003 A CN115444003 A CN 115444003A CN 202211039810 A CN202211039810 A CN 202211039810A CN 115444003 A CN115444003 A CN 115444003A
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- phoxim
- lipid carrier
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- lipid
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Images
Classifications
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/14—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing aromatic radicals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/22—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients stabilising the active ingredients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/24—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients to enhance the sticking of the active ingredients
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- A—HUMAN NECESSITIES
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- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
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- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
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- Agronomy & Crop Science (AREA)
- Toxicology (AREA)
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- Dispersion Chemistry (AREA)
- Insects & Arthropods (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method for preparing a high-drug-loading phoxim nano-structure lipid carrier based on micro-fluidic control. The invention is beneficial to preparing the phoxim nanostructure lipid carrier which is environment-friendly, high in drug loading capacity, strong in leaf surface residue capacity, stable in preparation, stable in storage and good in control effect, and realizes the high-efficiency control of the field lepidoptera pests such as spodoptera litura and the like.
Description
Technical Field
The invention belongs to the field of plant protection, and particularly relates to a method for preparing a phoxim nano-structure lipid carrier with high drug loading based on microfluidics.
Background
Currently, the main control method of field pests is still chemical pesticide control (Journal of halocushion materials,2020, 385. Organophosphorus pesticide phoxim is a commonly used conventional pesticide, which targets acetylcholinesterase of pests, but decreases in effectiveness with increasing application rate (Journal of Applied immunology, 2021,145 (5): 440-448). Phoxim has a broad spectrum of toxicity against insects such as Lepidoptera, and a representative pest is Spodoptera litura (Ningxia agricultural science, 1982 (04): 40). 1-2 instar spodoptera litura larvae feed on a small amount of mesophyll, the food intake of larvae above 3 instar is significantly increased, and even the whole plant is damaged, and meanwhile, the resistance of older spodoptera litura larvae to insecticides is enhanced, and the larvae are difficult to kill in the field (Biological Control,2020, 150.
In order to effectively prevent and control prodenia litura and solve the problem that phoxim is insoluble in water, the conventional formula missible oil of patents and commercial medicines usually contains a large amount of organic solvents (CN 110999900A; CN1729781; CN 102845461A), and the organic solvents in the phoxim missible oil are not only difficult to degrade in the environment, but also increase the risk of pesticide storage and transportation. Therefore, the new formulation of phoxim should reduce the content of organic solvent or use degradable components while ensuring the foliar deposition ability and insecticidal ability of the pesticide.
The nano-structure lipid carrier can effectively improve the drug encapsulation efficiency, enhance the sustained release and transdermal capacity of the drug (Chinese traditional medicine journal, 2017,42 (13): 2473-2478), and is expected to realize the decrement and synergy of the pesticide. In the fields of medicine and cosmetics, nanostructured lipid carriers have been widely used due to their transdermal capabilities, and the application in the field of pesticides is still at a relatively primary stage. Most pesticides such as phoxim and the like are lipophilic, so that lipid can be used as a nano pesticide carrier material, and the phoxim is encapsulated by using a nano-structure lipid carrier, so that the adsorption capacity of pesticide leaves can be improved, and the insecticidal capacity is enhanced. Meanwhile, according to the principle of environmental friendliness, the surfactant in the structural lipid carrier raw material can be selected from natural nonionic surfactants or degradable anionic surfactants. In addition to the composition of the components, the preparation method also has an important influence on the quality of the nanostructured lipid carrier.
The preparation method of the nano-structure lipid carrier comprises a high-energy emulsification method and a low-energy emulsification method (Nanoscale, 2021,13 (7): 4051-4059). The structural lipid carrier prepared by the high-energy emulsification method has smaller particle size and good stability, but the method has complex equipment and high energy consumption (Colloids and Surfaces A: physical and Engineering enterprises, 2020, 601. The phase inversion method is commonly used in the low energy emulsification method, but the preparation process usually adopts dropwise mixing of oil phase and water phase, so the controllability and stability of the method are poor (International journal of pharmaceutical, 2014,477 (1-2): 208-217). The microfluidic method is a preparation method of a highly controllable nano-structure lipid carrier, and can realize high product quality, high heat transfer efficiency, automatic preparation, good batch processing repeatability and less external pollution (Nanoscale, 2021,13,19352-19366, 2019,11 (19): 9410-9421. The method can control the preparation of nanostructured lipid carriers by adjusting the chip shape, composition of the liquid in the channels and flow rate (Langmuir, 2018,34 (13): 3961-3970 chemical Engineering science,2021, 235. The nano-structured lipid carrier prepared by micro-fluidic is mainly used for loading medical drugs, is not used for preparing pesticides, can be used for preparing a phoxim nano-structured lipid carrier with high drug loading through micro-fluidic, reduces the use of organic solvents in a formula, and is very important for efficient green prevention and treatment of lepidoptera insect pests such as prodenia litura and the like.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for preparing a phoxim nano-structure lipid carrier with high drug loading based on micro-fluidic. By optimizing the formula and the process in the preparation process, the environment friendliness of the phoxim pesticide is improved, the water accounts for more than 50%, and the raw materials are safe and degradable; the nano phoxim prepared in multiple batches has the drug loading rate of more than 98 percent, and the preparation stability is obviously improved; the leaf surface retention rate of the prepared phoxim nano-structure lipid carrier reaches more than 62 percent, the semi-lethal concentration of 72 hours is as low as 3.16mg/L, the cold storage stability reaches more than 95 percent, the heat storage stability reaches more than 59 percent, the phoxim nano-structure lipid carrier is superior to the original medicine and the commercial medicine, the control effect of phoxim is enhanced, and the high-quality and high-efficiency preparation of phoxim nano-pesticide is realized.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for preparing a phoxim nano-structure lipid carrier with high drug loading based on micro-fluidic comprises the following steps:
step 1, respectively preparing a water phase and an oil phase, wherein the water phase is formed by mixing a nonionic surfactant and water, and the oil phase is formed by mixing solid lipid, liquid lipid, phoxim, an anionic surfactant and a cosurfactant;
step 2, in a micro-fluidic system, injecting a water phase and an oil phase into a side channel or a main channel of a connection chip from a micro-injection pump 1 and a micro-injection pump 2 respectively, controlling the flow rate, mixing micro-fluid at the channel connection part of a heating platform, introducing a product into an ice-water bath for cooling, and filtering through a filter membrane to form a phoxim nano-structure lipid carrier;
wherein, the mass fraction of the water phase is 60-80%, and the mass fraction of the oil phase is 20-40%.
The improvement is that the mass fraction of the nonionic surfactant in the water phase is 70-90%, and the mass fraction of the nonionic surfactant in the water phase is 10-30%.
The improvement is that the mass fraction of the solid lipid in the oil phase is 1-10%, the mass fraction of the liquid lipid is 10-30%, the mass fraction of the phoxim is 30-60%, the mass fraction of the anionic surfactant is 10-30%, and the mass fraction of the cosurfactant is 10-30%.
The improvement is that the phoxim has a purity of 60-99%, the solid lipid is at least one of stearic acid, beeswax, monoglyceride, lecithin or cholesterol, the liquid lipid is at least one of oleic acid, palmitic acid, castor oil, isopropyl myristate, glyceryl caprylate caprate or isopropyl palmitate, the anionic surfactant is at least one of laureth sulfosuccinate disodium salt (MES), lauryl sulfonated succinate disodium monoester (DLS), cocoyl monoethanolamide sulfosuccinate Disodium Monoester (DMSS) or lauramidopropyl hydroxysultaine (LHSB-35), the nonionic surfactant is at least one of soyasaponin, theasaponin, tribulus terrestris saponin, ginsenoside, or sea cucumber saponin, and the cosurfactant is at least one of ethanol, n-propanol, isopropanol, n-butanol or isobutanol.
The improvement is that the connecting chip of the microfluidic system is a T-shaped, Y-shaped, cross-shaped O/W-shaped or W/O-shaped chip.
The improvement is that the flow rates of a micro-injection pump 1 and a micro-injection pump 2 in the micro-fluidic system are respectively 20-40 mu L/min and 5-15 mu L/min, the temperature of the joint of a heating platform channel is 60-120 ℃, the cooling time in an ice water bath is 5-60min, and the aperture of a filter membrane is 0.1-1 mu m.
As an improvement, the drug loading rate of the phoxim nano-structure lipid carrier reaches more than 98 percent.
Has the beneficial effects that:
compared with the prior art, the method for preparing the phoxim nano-structure lipid carrier with high drug loading based on micro-fluidic has the advantages that by optimizing the formula and the process in the preparation process, the water accounts for more than 50%, and the raw materials are safe and degradable; the drug loading rate of the nano phoxim prepared in multiple batches reaches more than 98%, and the preparation stability is obviously improved; the leaf surface retention rate of the prepared phoxim nano-structure lipid carrier reaches more than 62%, the semi-lethal concentration of 72 hours is as low as 3.16mg/L, the cold storage stability reaches more than 95%, and the heat storage stability reaches more than 59%, which are all superior to original drugs and commercial drugs, the environment friendliness, the drug loading rate and the preparation stability of phoxim pesticides are improved, the control effect of phoxim is enhanced, and the high-quality and high-efficiency preparation of the phoxim nano-pesticides is realized. Through the establishment of the technology, a new product and a new process are provided for the field of pesticides, and an important reference basis is provided for realizing the reduction and the increase of the pesticides in the field.
Drawings
FIG. 1 is a schematic view of a microfluidic chip;
FIG. 2 is a schematic diagram of a nanostructured lipid carrier phoxim prepared in example 1 of the present invention;
FIG. 3 is a schematic representation of a dry structured lipid carrier by transmission electron microscopy;
FIG. 4 is an X-ray diffraction characterization of nanostructured lipid carriers and nano-phoxim;
FIG. 5 is an infrared characterization of the bulk drug, nanostructured lipid vehicle and nano-phoxim;
FIG. 6 is a Raman spectrum characterization of the nanostructured lipid carrier and the nano-phoxim;
fig. 7 is a schematic view of a microfluidic system used in embodiment 1 of the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
This example illustrates the microfluidic preparation of a high drug-loaded phoxim nanostructured lipid carrier.
Example 1-1
The oil phase formula comprises solid lipid (monoglyceride), liquid lipid (oleic acid), and anionic surfactant (MES), and the water phase formula comprises water and nonionic surfactant (theasaponin). Setting the mass ratio of different lipids (monoglyceride and oleic acid) -surfactants (MES and tea saponin) in a two-phase system as 1. Then the oil phase was additionally added with a total biphasic mass fraction of 10% co-surfactant n-propanol and 2% phoxim as shown in table 1.
Wherein, the purity of monoglyceride is 90%, the purity of phoxim is 99%, the purity of MES is 30%, the purity of tea saponin is 95%, and the rest components are analytically pure.
In the microfluidic system, the flow rates of the micro-syringe pumps 1 and 2 were controlled to 22 to 27. Mu.L/min and 6 to 13. Mu.L/min, respectively (Environmental Science and Pollution Research, 2022. And respectively injecting the water phase and the oil phase into a side channel and a main channel of the T-shaped connection chip, and mixing microfluid at the channel connection position of which the heating platform temperature is 85 ℃. And then introducing the product into an ice-water bath for cooling for 30min, and filtering through a 0.22 mu m filter membrane to form the nanostructured lipid carrier phoxim.
100 μ L of the nano-pesticide was mixed with 600 μ L of n-hexane and inverted 10 times to extract the unencapsulated phoxim into n-hexane. Subsequently, 100. Mu.L of the extract was aspirated into a 2mL centrifuge tube, the extract was dried and supplemented with 400. Mu.L of methanol, and C of phoxim was determined 1 And (4) concentration. On the other hand, 100. Mu.L of the nano-pesticide was mixed with 500. Mu.L of methanol, left to stand for 30 minutes until the loaded phoxim was dissolved, and the total phoxim concentration in the mixture was determined to be C 2 。
The Encapsulation Efficiency (EE) of the nanostructured lipid carrier to phoxim was calculated as follows: EE = (C) 2 -C 1 )/C 2 X 100%. Wherein EE is the encapsulation efficiency, C 1 Is the unencapsulated phoxim concentration, C 2 The total phoxim concentration is obtained to obtain the drug loading rate.
TABLE 1 Effect of lipid-surfactant ratio on encapsulation efficiency
As can be seen from table 1, as the ratio of the lipid surfactant increases, the encapsulation efficiency of phoxim decreases; when the ratio is 1. In order to ensure the encapsulation efficiency of phoxim and reduce the dosage of the surfactant, the ratio of the lipid-surfactant is determined to be 1.
Examples 1 to 2
The preparation method comprises the following steps of setting an oil phase formula as solid lipid (monoglyceride), liquid lipid (oleic acid) and an anionic surfactant (MES), selecting a water phase formula as water and a nonionic surfactant (theasaponin), selecting a mass ratio of 1. Then the oil phase was additionally added with a total biphasic mass fraction of 10% co-surfactant n-propanol and 2% phoxim as shown in table 2.
The component purity, microfluidic system preparation parameters, encapsulation efficiency test methods were as above, as shown in table 2.
TABLE 2 Effect of solid-liquid lipid ratio on encapsulation efficiency
As can be seen from Table 2, the encapsulation efficiency increases with decreasing solid-liquid lipid ratio, with a maximum of 99.35. + -. 0.04% at 1. However, when the ratio is reduced to 1. Therefore, the solid-liquid lipid ratio is preferably 1.
Examples 1 to 3
The method comprises the following steps of setting an oil phase formula as solid lipid (monoglyceride), liquid lipid (oleic acid) and an anionic surfactant (MES), setting a water phase formula as water and a nonionic surfactant (tea saponin), selecting the mass ratio of the lipid (monoglyceride and oleic acid) -surfactant (MES and tea saponin) in a two-phase system from 1. Then the oil phase was additionally added with a total biphasic mass fraction of 10% co-surfactant n-propanol and 2% phoxim as shown in table 3.
The component purity, microfluidic system preparation parameters, encapsulation efficiency test methods were as above, as shown in table 3.
TABLE 3 Effect of anionic-nonionic surfactant ratio on encapsulation efficiency
As can be seen from table 3, the increase in the ratio of anionic-nonionic surfactant increased the encapsulation efficiency of phoxim, which was close to 100%, and due to the cost of theasaponin, the preferred ratio was determined to be 1.
Examples 1 to 4
Setting an oil phase formula as solid lipid (monoglyceride), liquid lipid (oleic acid) and an anionic surfactant (MES), a water phase formula as water and a nonionic surfactant (tea saponin), selecting the mass ratio of the lipid (monoglyceride and oleic acid) -the surfactant (MES and tea saponin) of 1. Then, the oil phase was additionally added with co-surfactant n-propanol in an amount of 10% of the total biphasic mass fraction, and phoxim in different mass fractions of 2%, 4%, 6%, 8%, 10%, respectively, as shown in table 4.
The component purity, microfluidic system preparation parameters, encapsulation efficiency test methods were as above, as shown in table 4.
TABLE 4 Effect of Phoxim addition on encapsulation efficiency
As can be seen from Table 4, when the addition amount of phoxim is increased from 2% to 4%, the encapsulation efficiency of phoxim is remarkably reduced from 98.94 + -0.11% to 97.52 + -0.58%; however, the encapsulation efficiency increased significantly to 99.02 ± 0.10% after the addition of 10% phoxim. Meanwhile, in the system, the lipid content in the nano-structured lipid carrier is 6%, and the addition amount of the phoxim exceeding 10% exceeds the loading capacity of the system, so that the addition amount of the phoxim is determined to be 10% preferably.
Examples 1 to 5
The preparation method comprises the following steps of setting an oil phase formula as solid lipid (monoglyceride), liquid lipid (oleic acid) and an anionic surfactant (MES), selecting a water phase formula as water and a nonionic surfactant (theasaponin), selecting the mass ratio of lipid (monoglyceride and oleic acid) -surfactant (MES and theasaponin) of 1. Then, 10% of phoxim in total two-phase mass fraction is additionally added into the oil phase, and the addition of the cosurfactant n-propanol in different mass fractions is respectively 0%,5%,10%,15% and 20%, as shown in table 5.
The component purity, microfluidic system preparation parameters, encapsulation efficiency test methods were as above, as shown in table 5.
TABLE 5 Effect of cosurfactant n-propanol addition on encapsulation efficiency
As can be seen from table 5, the encapsulation efficiency of the nanostructured lipid vehicle decreased with the increase of the addition amount of the cosurfactant n-propanol, the maximum encapsulation efficiency of the nanostructured lipid vehicle was 99.02 ± 0.03% without the cosurfactant, and the encapsulation efficiency was significantly decreased to 87.26 ± 0.30% and 73.38 ± 2.74% when the addition amount of the cosurfactant was 15% and 20%, respectively. However, when no co-surfactant is added, it is found that insoluble matter is present in the oil phase in the experiment, and therefore, it is preferable to select 5% of the co-surfactant to be added.
Example 2
This example illustrates the stability of the microfluidic preparation process of a high drug-loaded phoxim nanostructured lipid carrier.
Preparing by a micro-fluidic method:
two-phase formula (components in parts by mass): 74.8% of water phase and 25.2% of oil phase;
the water phase formula (the components in parts by mass): 79.55% of water and 20.45% of tea saponin;
an oil phase formula (the components in parts by mass): 39.68% of phoxim, 4.05% of monoglyceride, 16.19% of oleic acid, 20.24% of MES and 19.84% of n-propanol.
The purity of the components and the preparation parameters of the microfluidic system are the same as those in example 1.
Preparing by a phase change method: the oil and water phases of the above formulation were placed in a test tube and heated at 85 ℃ in an oil bath magnetic stirrer at 200 rpm. The aqueous phase was then added all dropwise to the oil phase and stirred for 30 minutes. Finally, the mixture was subjected to ice-water bath for 30 minutes and passed through a 0.22 μm filter as a phase inversion sample.
The encapsulation efficiency of the nano pesticide prepared by the two methods is respectively measured, and the encapsulation efficiency testing method is the same as that of the example 1.
And calculating the standard error value of the encapsulation efficiency of the nano pesticide prepared for 10 times, and judging the batch stability of the two preparation methods.
TABLE 6 encapsulation efficiency of 10 batches of nano-pesticides prepared by microfluidic and phase transition methods
As shown in Table 6, the encapsulation efficiencies of 10 batches of nano-pesticides prepared by the microfluidic and phase-change methods were calculated, and the average values were 98.67 + -0.47% and 46.01 + -12.46%, respectively; the standard error of the encapsulation efficiency of the nano pesticide prepared by the micro-fluidic method is far smaller than that of the nano pesticide prepared by the phase inversion method, which shows that the nano pesticide prepared by the method has higher batch stability.
Example 3
This example illustrates the comparison of the compatibility of green high drug-loaded nano phoxim pesticide with other forms of phoxim for foliar application.
Phoxim nanostructured lipid vehicles were prepared as described in example 2 using microfluidic methods.
Selecting a technical sprayed with phoxim, a commercial formula drug of the phoxim and a lipid carrier with a phoxim nano structure for comparison, wherein the preparation method of the technical sprayed with the phoxim comprises the following steps: mixing 100 mu g of phoxim with the purity of 99 percent with 2mL of acetone to form a standard solution, and diluting the standard solution with water according to the required concentration; the content of phoxim in the commercial formulation of phoxim was 40% and purchased from eastern agricultural chemical plant (taixing, china).
The diluted concentration of the three dosage forms is 100mg/L phoxim, the diameter of potted mulberry seedlings is 80 +/-20 mm, the height of the potted mulberry seedlings is 20-30 cm, and the plants are used for retention and anti-scouring tests. Then punching fresh folium Mori with puncher (diameter 3 cm), weighing m 1 . Then, the perforated leaf disks were immersed in the aqueous pesticide solution at the same concentration as above for 15s, taken out with tweezers, allowed to stand for 30s, and weighed to m 2 . All three formulations were tested, 10 replicates each. The formula for calculating the leaf surface retention of the pesticide is as follows: r = (m) 2 -m 1 ) S, where R is the retention, m 1 Is the mass of the untreated leaf discs, m 2 Is the mass of the treated leaf disk, and S is the area of the leaf disk.
Spraying 1ml of insecticide diluent on the mulberry leaves, and naturally drying for 2h. Pure water was added dropwise to the mulberry leaves at a rate of 10 ml/min for 8 minutes to simulate rain wash. The leaves were punched, chopped and collected in a centrifuge tube containing 5 ml of methanol. The tube was sonicated at 200W for 10min, with working and rest times of 5s. Thereafter, the tube was centrifuged at 12000rpm for 3min, and 1mL of the supernatant was aspirated and mixed with an equal amount of methanol as a sample. And finally, measuring the concentration of phoxim in the sample, calculating the residual rate, and calculating the residual rate according to the percentage of the concentration of the time point to the concentration of the flushing time of 0 min.
TABLE 7 foliar affinities of Phoxim-sprayed bulk drug, phoxim commercial formulation, and Phoxim nanostructured lipid vehicle
As shown in table 7, the leaf retention of commercial formulations of phoxim was significantly lower than that of phoxim sprayed technical and phoxim nanostructured lipid carriers. The leaves are detained, so that the phoxim nanostructure lipid carrier is easier to be ingested by pests, and the waste caused by rain wash is reduced.
Example 4
This example illustrates the comparison of the toxicity of green high drug-loaded nano phoxim pesticide and other forms of phoxim against prodenia litura.
Technical grade phoxim spray, commercial formulations of phoxim, phoxim nanostructured lipid vehicles and leaf discs were obtained as described in example 3.
Bioassay adopts a leaf soaking method to evaluate the toxicity of different phoxim formulas and theasaponin to prodenia litura. The dilution concentration of the treatment is 1-50g/L of tea saponin, 10-20mg/L of original pesticide sprayed by phoxim, 10-50mg/L of commercial phoxim and 2.5-12.5mg/L of phoxim nano-structure lipid carrier. Discs of mulberry leaves (diameter 3 cm) were soaked in the dilution for 30s and then dried.
The larvae are artificially fed in a breeding chamber with temperature of 25 + -1 deg.C, relative humidity of 60-70% and light/dark cycle of 16/8 h. Larvae from day 3 of 3 instars were selected for pesticide toxicity determination. Leaf discs and 24h starved larvae were then placed in 35mm plastic petri dishes with agar, and other conditions were the same. Calculating the larval mortality rate after 24h, 48h and 72h of treatment, and calculating the semi-lethal concentration LC of phoxim to prodenia litura in different formulas through a probit regression model 50 。
TABLE 8 toxicity of Phoxim-sprayed bulk drugs, phoxim commercial formulations, and Phoxim nanostructured lipid vehicles
Such as a watchThe three formulations shown in fig. 8 are a probit model for prodenia litura within 72 h. At 24h, original pesticide sprayed by phoxim, commercial formula drug of phoxim and LC using phoxim as standard in phoxim nano-structure lipid carrier 50 20.76, 78.29 and 6.39mg/L respectively, LC at 72h 50 Continue to drop to 19.90, 70.67 and 3.16mg/L. Under the semi-lethal concentration, the dosage of the phoxim nano-structure lipid carrier is 15.9-30.8 percent of the original pesticide sprayed by phoxim and 4.5-8.2 percent of the commercial formula of the phoxim. Meanwhile, in the embodiment, the spodoptera litura larvae treated by 10-50 g/L of theasaponin and 0-10 mg/L of the nanostructured lipid carrier (the preparation method is the same as that of the phoxim nanostructured lipid carrier, except that phoxim is removed) do not die after 24 hours, which indicates that the toxicity of the phoxim nanostructured lipid carrier is not influenced by the carrier.
Example 5
This example illustrates the comparison of the cold-heat storage performance of a green high drug-loaded nano phoxim pesticide with other formulations of phoxim.
Firstly diluting original pesticide, commercial pesticide and nano pesticide of phoxim to 100mg/L concentration of phoxim, and pouring 10mL of diluent into a test tube. In the cold storage experiment, 200 mu L of sample solution is sampled firstly, the initial phoxim concentration is detected by using liquid chromatography, and then the concentration of the sample phoxim is determined after the rest diluent is placed in a refrigerator for cold storage for 15 days at 0 ℃, namely the rest phoxim concentration. The heat storage experiment is basically the same as that of the sample, and the sample is placed in a water bath kettle at 54 ℃ for heat storage for 15 days. And calculating the residual rate of the phoxim of the sample after cold storage and hot storage treatment, wherein the residual rate is the percentage of the residual concentration of the phoxim in the original concentration.
TABLE 9 Cold storage and Heat storage Capacity of Phoxim-sprayed base pesticides, phoxim commercial formulations and Phoxim nanostructured lipid Carriers
As shown in Table 9, the residual rates of the three formulations of the phoxim after the pesticide is stored for 14 days in a cold mode are 52.21 +/-18.21, 25.84 +/-4.60 and 95.23 +/-3.90 respectively, and the residual rate of the phoxim nano-drug in the cold mode is obviously higher than that of the other two formulations.
As shown in Table 9, the residual rates of the three formulations after the pesticide is stored for 14 days are respectively 19.12 +/-1.36, 27.61 +/-8.95 and 59.30 +/-16.35 percent, and the residual rate of the phoxim nano-drug after the pesticide is stored for 14 days is obviously higher than that of the other two formulations.
The cold storage and heat storage stability of the pesticide is an evaluation index for analyzing whether the pesticide can be decomposed in storage, the cold storage stability of the phoxim nano-drug is higher, the heat storage stability of the phoxim nano-drug can be effectively improved by replacing a carrier or a concentrated component, and the cold storage and heat storage stability provides a foundation for long-term storage and practical application of the phoxim nano-drug.
Example 6
This example illustrates the relevant characterization of green high-drug-loading nano phoxim pesticide and other formulations of phoxim.
The structure schematic diagram of the nanostructured lipid carrier phoxim prepared by the invention is shown in figure 2.
(1) Characterization of particle size and potential
The particle size and Zeta potential of the phoxim nanostructured lipid carrier, the phoxim sprayed technical and the phoxim commercial formulation are measured by a multi-angle particle size analyzer. The three dosage forms are diluted by 10 times to 10mL by adding water, and the working temperature of an instrument is 25 ℃.
TABLE 10 particle size and Zeta potential of Phoxim-sprayed bulk drug, phoxim commercial formulation, and Phoxim nanostructured lipid vehicle
The particle diameters and Zeta potentials of the three formulations are shown in Table 10. The particle sizes of the three dosage forms are 768.58 +/-27.47, 362.78 +/-1.29 and 120.08 +/-0.65 nm respectively, and the particle size of the phoxim nano-structure lipid carrier is greatly reduced. The Zeta potential of the three dosage forms is-0.77 +/-0.32, 0.61 +/-0.24 and-39.61 +/-1.33 mV respectively, and the absolute value of the potential of the phoxim nano-structure lipid carrier is larger and more stable.
(2) Characterization of particle morphology
The particle morphology is shown in figure 3, the particle morphology of the phoxim nanostructured lipid carrier is characterized by a Transmission Electron Microscope (TEM), the TEM sample of the phoxim nanostructured lipid carrier is prepared by diluting the carrier by 10 times with water, sucking 1 drop of diluent by a 10 mu L pipette, dripping the diluent on a copper net covered with a carbon film, standing for 5min, naturally drying the copper net after sucking the redundant liquid by using filter paper, continuously dripping 2% phosphotungstic acid on the dried copper net for negative dyeing for 1min, and then sucking the redundant liquid by using the filter paper and drying the copper net to serve as a TEM detection sample.
(3) Crystal form and chemical bond characterization
The samples used in the experiment are all powder, and the treatment method comprises the following steps: respectively adding 5 times of n-hexane in volume into the blank nanostructure lipid carrier and the phoxim nanostructure lipid carrier, reversing and mixing, then discarding the n-hexane, repeatedly cleaning for 3 times, volatilizing residual n-hexane in the sample, and freeze-drying for 48 hours to obtain powdery blank nanostructure lipid carrier and phoxim nanostructure lipid carrier samples, wherein the powdery blank nanostructure lipid carrier and the phoxim nanostructure lipid carrier samples are also used for infrared and Raman characterization experiments. Detecting the components and crystal forms of the blank nanostructure lipid carrier and the phoxim nanostructure lipid carrier by an X-ray diffraction instrument, wherein the scanning speed is 2 degrees/min, and the scanning angle is 5-90 degrees.
X-ray diffraction characterization As shown in figure 4, X-ray diffraction XRD results of the blank nanostructured lipid carrier and the phoxim nanostructured lipid carrier comprise monoclinic phase of glycerol monostearate GMS (JCPDS file number 08-0590) and oleic acid (JCPDS file number 37-1811), which indicates that the blank NLC contains GMS and oleic acid, and the phoxim-loaded P-NLC also contains the two lipids, thus proving that the P-NLC is the nanostructured lipid carrier. The broad impurity peaks indicate that the crystal forms of the blank NLC and the P-NLC are quasi-amorphous.
Whether the phoxim is successfully encapsulated into the blank nanostructure lipid carrier and the chemical bond change of the phoxim nanostructure lipid carrier component are determined by Fourier transform infrared spectroscopy FTIR. The phoxim raw medicine used in the infrared spectrum experiment does not contain acetone, and the blank nanostructure lipid carrier and the phoxim nanostructure lipid carrier treatment method are the same.
Fourier infrared spectrum characterization is shown in figure 5, and a phoxim-related absorption peak also appears in the phoxim nano-structured lipid carrier, so that the phoxim is proved to be successfully loaded into the nano-structured lipid carrier.
The Raman spectrum is used for verifying the change of chemical bonds of the lipid carrier with the phoxim nano structure. The blank nanostructure lipid carrier and the phoxim nanostructure lipid carrier are processed by the same method.
The Raman spectrum characterization is shown in figure 6, the nano-structured lipid carrier has no obvious characteristic peak, and the phoxim nano-structured lipid carrier contains the characteristic peak. The relative characteristic peaks of P-NLC in the Raman spectrum indicate that phoxim has been successfully loaded into NLC.
In conclusion, by optimizing the formula and the process in the preparation process, the water accounts for more than 50%, and the raw materials are safe and degradable; the drug loading rate of the nano phoxim prepared in multiple batches reaches more than 98%, and the preparation stability is obviously improved; the leaf surface retention rate of the prepared phoxim nano-structure lipid carrier reaches more than 62%, the semi-lethal concentration of 72 hours is as low as 3.16mg/L, the cold storage stability reaches more than 95%, and the heat storage stability reaches more than 59%, which are all superior to original drugs and commercial drugs, the environment friendliness, the drug loading rate and the preparation stability of phoxim pesticides are improved, the control effect of phoxim is enhanced, and the high-quality and high-efficiency preparation of the phoxim nano-pesticides is realized. Through the establishment of the technology, a new product and a new process are provided for the field of pesticides, and an important reference basis is provided for realizing the reduction and the increase of the pesticides in the field.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (7)
1. A method for preparing a phoxim nano-structure lipid carrier with high drug loading based on micro-fluidic is characterized by comprising the following steps: step 1, respectively preparing a water phase and an oil phase, wherein the water phase is formed by mixing a nonionic surfactant and water, and the oil phase is formed by mixing solid lipid, liquid lipid, phoxim, an anionic surfactant and a cosurfactant;
step 2, in a micro-fluidic system, injecting a water phase and an oil phase into a side channel or a main channel of a connection chip from a micro-injection pump 1 and a micro-injection pump 2 respectively, controlling the flow rate, mixing micro-fluid at the channel connection part of a heating platform, introducing a product into an ice-water bath for cooling, and filtering through a filter membrane to form a phoxim nano-structure lipid carrier;
wherein, the mass fraction of the water phase is 60-80%, and the mass fraction of the oil phase is 20-40%.
2. The microfluidic-based method for preparing the high drug loading phoxim nanostructured lipid carrier according to claim 1, wherein the mass fraction of the nonionic surfactant in the aqueous phase is 70-90%, and the mass fraction of the nonionic surfactant in the aqueous phase is 10-30%.
3. The microfluidic-based method for preparing the high drug loading phoxim nanostructured lipid carrier according to claim 1, wherein the mass fraction of the solid lipid in the oil phase is 1-10%, the mass fraction of the liquid lipid is 10-30%, the mass fraction of phoxim is 30-60%, the mass fraction of the anionic surfactant is 10-30%, and the mass fraction of the cosurfactant is 10-30%.
4. The method for preparing a high drug-loading phoxim nanostructured lipid carrier based on microfluidics according to claim 1, wherein the phoxim has a purity of 60-99%, the solid lipid is at least one of stearic acid, beeswax, monoglyceride, lecithin or cholesterol, the liquid lipid is at least one of oleic acid, palmitic acid, castor oil, isopropyl myristate, caprylic capric acid glyceride or isopropyl palmitate, the anionic surfactant is at least one of laureth sulfosuccinate disodium salt (MES), lauryl sulfosuccinate disodium monoester (DLS), coco monoethanolamide sulfosuccinate Disodium Monoester (DMSS) or laureth hydroxypropyl hydroxysultaine (LHSB-35), the nonionic surfactant is at least one of soyasaponin, theasaponin, tribulus saponin, ginsenoside, or sea cucumber saponin, and the co-surfactant is at least one of ethanol, n-propanol, isopropanol, n-butanol, or isobutanol.
5. The method for preparing the phoxim nanostructure lipid carrier with high drug loading based on microfluidics according to claim 1, wherein the connection chip of the microfluidic system is a T-shaped, Y-shaped, cross-shaped O/W-shaped or W/O-shaped chip.
6. The method for preparing the phoxim nanostructured lipid carrier with high drug loading based on microfluidics according to claim 1, wherein the flow rates of the micro-injection pump 1 and the micro-injection pump 2 in the microfluidic system are 20-40 μ L/min and 5-15 μ L/min respectively, the temperature of the connection part of the heating platform channel is 60-120 ℃, the cooling time in the ice-water bath is 5-60min, and the aperture of the filter membrane is 0.1-1 μm.
7. The method for preparing the high-drug-loading phoxim nano-structure lipid carrier based on the microfluidic control of claim 1, wherein the drug loading rate of the phoxim nano-structure lipid carrier is more than 98%.
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