WO2024051809A1 - 一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法 - Google Patents

一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法 Download PDF

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WO2024051809A1
WO2024051809A1 PCT/CN2023/117689 CN2023117689W WO2024051809A1 WO 2024051809 A1 WO2024051809 A1 WO 2024051809A1 CN 2023117689 W CN2023117689 W CN 2023117689W WO 2024051809 A1 WO2024051809 A1 WO 2024051809A1
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nanocarrier
asp
targeting
targeted
drug
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PCT/CN2023/117689
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English (en)
French (fr)
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师冰洋
夏雪
郑蒙
刘洋
李爱杰
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河南大学
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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
    • A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels
    • 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
    • A01N25/08—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
    • 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
    • A01N25/08—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
    • A01N25/10—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the technical field of functional materials, and in particular to a targeted nanocarrier and its preparation method and application, and a targeted drug-loaded nanocarrier and its preparation method.
  • Plant cells have cell walls, which constitute the main barrier for the delivery of drugs such as exogenous biological macromolecules.
  • drugs such as exogenous biological macromolecules.
  • traditional gene guns have shortcomings such as causing target tissue damage and low gene expression levels.
  • the biggest challenge of Agrobacterium transformation method is narrow host selectivity and tissue specificity.
  • the callus generation required by these methods also limits the applicable plant species.
  • excessive application and high residues caused by extremely low utilization rates have put great pressure on production safety and the environment.
  • Nano-delivery carriers are widely used in the field of biomedicine and make important contributions to human health. Nano-delivery can greatly reduce the dosage of drugs and only target and aggregate in the required tissues, which can reduce drug costs, improve drug efficiency, and extend drug action time. , reduce toxicity and pollution, and can reduce the probability of drug resistance.
  • nano-delivery carriers used in plants. This is because in plant systems, the existence of cell walls forms a natural barrier, making it impossible for almost all nano-carriers to effectively penetrate the cell wall and carry out delivery in living tissues and cells. Delivery, the few studies on plant nanodelivery vectors also require external force assistance (such as leaf injection, magnetism, etc.) to achieve their purpose of crossing the cell wall and entering living plant tissues.
  • the purpose of this disclosure is to provide a targeted nanocarrier and its preparation method and application, a targeted drug-loaded nanocarrier and its preparation method.
  • the targeted nanocarrier provided by this disclosure can actively penetrate plant cell walls and cell membranes, and is suitable for treating Plants deliver drugs.
  • the present disclosure provides a targeting nanocarrier, including a nanocarrier and a targeting substance chemically bonded to the nanocarrier, wherein the nanocarrier is a nanoparticle formed of an organic polymer or an inorganic material, and the The target substance is aspartic acid or an aspartic acid derivative.
  • the targeting group provided by the aspartic acid includes a group with a structure shown in any one of formulas I to IV:
  • the particle size of the nanocarrier is 10 to 1000 nm.
  • the number average molecular weight of the organic high molecular polymer is 3 to 50 kDa.
  • the organic polymer includes a hydrophobic polymer and a hydrophilic linker covalently connected to the hydrophobic polymer, and the hydrophilic linker is chemically bonded to the target. connect.
  • the present disclosure provides a method for preparing targeted nanocarriers according to the above technical solution, which includes the following steps:
  • the targeting substance is chemically bonded and modified on the nanocarrier to obtain the targeting nanocarrier.
  • the present disclosure provides the targeted nanocarriers described in the above technical solutions or the targeted nanocarriers prepared by the preparation method described in the above technical solutions as living plant plants, tissues of living plant plants, organs of living plant plants, and cells of living plant plants. , the application of active targeting nanocarriers to explants cultured in vitro, callus cultured in vitro, plant tissues cultured in vitro or plant cells cultured in vitro.
  • the present disclosure provides the application of the targeted nanocarriers described in the above technical solutions or the targeted nanocarriers prepared by the preparation method described in the above technical solutions as active targeting nanodelivery carriers in living plants.
  • the present disclosure provides a targeted drug-loaded nanocarrier, including a targeted nanocarrier and a drug contained in the targeted nanocarrier.
  • the targeted nanocarrier is the targeted nanocarrier described in the above technical solution or the above-mentioned targeted nanocarrier.
  • Targeted nanocarrier prepared by the preparation method described in the technical solution.
  • the drug includes small molecule drugs or biological macromolecules, and the drug loading rate of the targeted drug-loaded nanocarrier is 1 to 99%.
  • the present disclosure provides a method for preparing targeted drug-loaded nanocarriers described in the above technical solutions, including the following steps:
  • the targeted nanocarrier, drug and solvent are mixed and encapsulated to obtain the targeted drug-loaded nanocarrier.
  • the present disclosure provides a targeting nanocarrier, including a nanocarrier and a targeting substance chemically bonded to the nanocarrier, wherein the nanocarrier is a nanoparticle formed of an organic polymer or an inorganic material, and the The target substance is aspartic acid or an aspartic acid derivative.
  • the targeted nanocarriers provided by the present disclosure can actively penetrate plant cell walls and cell membranes, are suitable for drug delivery to living plants or tissues, etc., can reduce drug dosage and cost, have a protective effect on loaded drugs, improve drug efficiency, and prolong drug effects. time, reduce toxicity and contamination, and reduce the probability of drug resistance.
  • Figure 1 is a flow chart for preparing Asp-NP according to the disclosure and a schematic diagram of the application of the targeted drug-loaded nanocarrier as a drought-resistant agent, taking ABA as an example;
  • Figure 2 is a nuclear magnetic resonance image of the Asp-PEG-PDPA whose targeting group is L-Asp prepared in Example 1;
  • Figure 3 is a transmission electron microscope image of L-Asp-NP in Example 1;
  • Figure 4 shows the Asp-NP@ABA obtained after loading ABA (specifically D-Asp-NP@ABA, A-Asp-NP@ABA, N-Asp-NP@ABA and L-Asp-NP@ABA) and those without Comparison of particle size and drug loading rate of NP@ABA targeted as a control;
  • Figure 5 shows the observation results of using a laser confocal microscope to track DiO at a depth of 20 ⁇ m after spraying Arabidopsis leaves with different treatments for 36 hours in Application Example 1;
  • Figure 6 is a statistical diagram of the depth of DiO brought into the leaf tissue by nanoparticles using a laser confocal microscope after spraying Arabidopsis leaves for 36 hours using different treatments in Application Example 1;
  • Figure 7 shows that in Application Example 1, different treatments were used to mix and co-culture isolated Arabidopsis mesophyll cell protoplasts for 4 hours. After replacing fresh MS (Murashige and Skoog) medium for 20 hours, laser confocal microscopy was used to track the DiO signal induced by nanoparticles. Comparison pictures brought into protoplasts;
  • Figure 8 shows the observation results of using a laser confocal microscope to track FITC at different depths after spraying Commelina leaves with different treatments for 6 hours in Application Example 1;
  • Figure 9 is a comparison chart of the germination rate of Arabidopsis thaliana seeds after using different treatments.
  • Figure 10 is a comparison chart of Asp-NP-FITC penetrating Arabidopsis root tissue to different depths at different time points;
  • Figure 11 is a comparison of Asp-NP-FITC penetrating into soybean root tissue at different depths after 4h and 6h;
  • Figure 12 is a comparison of Asp-NP-FITC penetrating corn root tissue to different depths after 4h and 6h;
  • Figure 13 is a comparison chart of the induction of leaf senescence and yellowing by using targeted nanocarriers absorbed by roots of Arabidopsis seedlings under different treatments in a hydroponic system;
  • Figure 14 is a comparison chart of application example 3 of using different treatments to extend the survival period of Arabidopsis seedlings under drought conditions after spraying them;
  • Figure 15 is a comparison of the survival rates of Arabidopsis thaliana seedlings after spraying with different treatments in Application Example 3;
  • Figure 16 is a scatter plot showing the percentage statistics of the extended survival period of Arabidopsis thaliana seedlings under drought conditions compared to the extended survival period of the MS treatment group after spraying Arabidopsis thaliana seedlings with different treatments in Application Example 3;
  • Figure 17 is a graph showing the minimum effective concentration results of Asp-NP@ABA measured using the survival cycle under drought conditions in Application Example 3 (using the results of ABA treatment as a reference);
  • Figure 18 is a comparison chart of the ABA content in the cytoplasm and apoplast of Arabidopsis leaves 24 hours after spraying with different treatments in Application Example 3;
  • Figure 19 is a comparison chart of application example 4 of using different treatments to extend the survival period of soybean seedlings under drought conditions after spraying them;
  • Figure 20 is a statistical diagram of the median survival value of soybean seedlings that are sprayed with different treatments to extend their survival period under drought conditions in Application Example 4;
  • Figure 21 is a comparison chart of application example 4 of using different treatments to extend the survival period of corn seedlings under drought conditions after spraying them;
  • Figure 22 is a comparative chart of the effects of drug-loaded products adding different proportions of non-targeting polymers (i.e. MeO-PEG-PDPA) on the germination rate of Arabidopsis thaliana seeds in Application Example 5;
  • non-targeting polymers i.e. MeO-PEG-PDPA
  • Figure 23 is a comparative chart of the effects of adding different proportions of non-targeting polymers (ie, MeO-PEG-PDPA) to drug-loaded products in Application Example 5 on extending the survival period of Arabidopsis thaliana under drought conditions.
  • non-targeting polymers ie, MeO-PEG-PDPA
  • the present disclosure provides a targeting nanocarrier, including a nanocarrier and a targeting substance chemically bonded to the nanocarrier, wherein the nanocarrier is a nanoparticle formed of an organic polymer or an inorganic material, and the The target substance is aspartic acid or an aspartic acid derivative.
  • the targeted nanocarriers provided by the present disclosure include nanocarriers, which are nanoparticles formed of organic polymers or inorganic materials.
  • the particle size of the nanocarrier can be selected from 10 to 1000 nm, and more preferably from 20 to 200 nm.
  • the number average molecular weight of the organic high molecular polymer can be selected from 3 to 50 kDa, and more preferably from 5 to 20 kDa.
  • the organic polymer may optionally include a hydrophobic polymer and a hydrophilic linker covalently linked to the hydrophobic polymer, and the hydrophilic linker and the targeting substance Chemical bonding links.
  • the hydrophobic polymer optionally includes any one of the substances involved in (1), (2) and (3) below;
  • PLGA Polylactic acid-co-glycolic acid
  • PLA polylactic acid
  • PLA polylactic acid
  • PLA polycaprolactone
  • PCL polycaprolactone
  • PCL polycarbonate
  • PMC PMC derivatives
  • glycolide, lactide, caprolactone and carbonate (2) One or more of glycolide, lactide, caprolactone and carbonate; a copolymer of at least two of glycolide, lactide, caprolactone and carbonate;
  • PU Polyurethane
  • PU derivatives polyether ether ketone (PEEK), PEEK derivatives, polymethyl methacrylate (PMMA), PMMA derivatives, polyvinyl alcohol (PVA), PVA derivatives, poly Ethylene (PE), PE derivatives, hydrophobic polyamino acid, hydrophobic polyamino acid derivatives; the hydrophobic polyamino acid can be polyphenylalanine.
  • hydrophilic linker optionally includes any one of the substances involved in (a) and (b) below:
  • PEG Polyethylene glycol
  • PEO polyethylene oxide
  • POEG poly(ethylene glycol) methacrylate
  • PMPC poly-2-methacryloyloxyethylphosphoryl
  • PCB polycarboxylic acid betaine
  • dextran dextran
  • hyaluronic acid polycarboxylic acid betaine
  • HPMA hyperbranched polyglycidyl ether
  • PPMA polyN-(2-hydroxypropane base) methacrylamide
  • PHEMA polyhydroxyethyl methacrylate
  • PAM polyvinylpyrrolidone
  • PVP polyacrylic acid
  • HPMA polymaleic anhydride
  • PEI Polyethylenimine
  • PEI derivatives pharmaceutically acceptable salts of PEI, poly N,N-dimethylaminoethyl methacrylate (PDMAEMA), PDMAEMA derivatives, PDMAEMA pharmaceutically acceptable salts salt, polylysine (PLL), PLL derivatives, PLL pharmaceutically acceptable salts, hydrophilic polyamino acids, hydrophilic polyamino acid derivatives, hydrophilic polyamino acid pharmaceutically acceptable salts; the hydrophilic The polyamino acid may be polyglutamic acid (PGu) or polyaspartic acid (PAsp).
  • PGu polyglutamic acid
  • PAsp polyaspartic acid
  • the morphology of the nanocarrier may be a micelle or a vesicle, which is not particularly limited in the present disclosure.
  • the inorganic material optionally includes silicon, silicon oxide, iron, iron oxide, calcium, calcium oxide, or carbon nanomaterials.
  • the surface of the inorganic material contains an active group.
  • the active group can be directly chemically bonded to the target, or the active group can be connected through a hydrophilic linker.
  • the target substance can be chemically bonded to the hydrophilic linker first, and then on this basis, the hydrophilic linker can be chemically bonded to the active group on the surface of the inorganic material, or The active groups on the surface of the inorganic material can be chemically bonded to the hydrophilic linker first, and then the hydrophilic linker can be chemically bonded to the target substance through the hydrophilic linker.
  • the optional types of the hydrophilic linker may be consistent with the optional types of the above-mentioned hydrophilic linker, and will not be described again here.
  • the present disclosure does not specifically limit the specific type of the reactive group, as long as it can achieve chemical bonding with a hydrophilic linker or targeting substance.
  • the reactive group can be a hydroxyl group.
  • the present disclosure has no special limitations on the source of the inorganic material, and commercially available products well known to those skilled in the art can be used.
  • the targeting nanocarrier provided by the present disclosure includes a targeting substance, the targeting substance is chemically bonded to the nanocarrier, and the targeting substance is aspartic acid or an aspartic acid derivative.
  • the specific manner of chemical bonding between the target substance and the nanocarrier may be an ester group or an amide group, which is not particularly limited in the present disclosure.
  • the targeting group provided by aspartic acid in the targeting nanocarrier can optionally include a group with a structure shown in any one of formulas I to IV, which are recorded as D-Asp and L-Asp-A respectively. ,L-Asp-N,L-Asp:
  • the present disclosure provides a method for preparing targeted nanocarriers according to the above technical solution, which includes the following steps:
  • the targeting substance is chemically bonded and modified on the nanocarrier to obtain the targeting nanocarrier.
  • an appropriate method can be selected according to the specific types of nanocarriers and targeting substances to chemically bond the two to prepare targeting nanocarriers, which will be described in detail below.
  • the present disclosure can optionally chemically bond the nanocarrier and the target substance through a chemical reaction in the presence of a solvent to obtain the target substance.
  • Nanocarrier; the solvent and chemical reaction conditions are determined according to the types of inorganic materials and targeting substances, and this disclosure is not particularly limited.
  • the present disclosure can refer to the first case above to prepare targeted nanoparticles.
  • the carrier that is, first prepare the nanocarrier, and then chemically bond the nanocarrier and the target substance through a chemical reaction in the presence of a solvent to obtain the targeted nanocarrier; the conditions of the solvent and chemical reaction are based on organic polymers.
  • the types of molecular polymers and targeting substances are determined, and this disclosure is not particularly limited.
  • the present disclosure can also chemically bond the targeting substance to the monomer for preparing the organic polymer, and then realize the preparation of the targeting nanocarrier based on the monomer modified with the targeting substance;
  • the structure of the monomer modified with the targeting substance and the conditions for further preparing the targeting nanocarrier based on it are determined according to the type of the organic polymer and the targeting substance, and this disclosure is not particularly limited.
  • L-Asp is used as the targeting group
  • PDPA is the hydrophobic polymer
  • PEG is the hydrophilic linker to prepare the targeting nanocarrier (Asp-PEG-PDPA) as an example.
  • the preparation method of Asp-PEG-PDPA with L-Asp as the targeting group optionally includes the following steps:
  • compound S9 i.e., Asp-PEG-PDPA with L-Asp as the targeting group
  • compound S1, compound S6, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, 4-dimethylaminopyridine and the first organic solvent are mixed, and an amidation reaction is performed to obtain compound S7.
  • the molar ratio of compound S1, compound S6, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and 4-dimethylaminopyridine can be selected as 1: (0.2 ⁇ 3): ( 1 ⁇ 3): (1 ⁇ 3): (0.01 ⁇ 1), more optionally 1:1:1.2:1.2:0.1; the first organic solvent can be dichloromethane, and the present disclosure is There is no special limit on the amount of organic solvent used, as long as the reaction proceeds smoothly.
  • compound S1, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and 4-dimethylaminopyridine can be optionally dissolved in the first organic solvent to obtain a mixed material; compound S6 can be dissolved in the first organic solvent.
  • a compound S6 solution is obtained; the compound S6 solution is added to the mixture material at one time to perform amidation reaction.
  • the amidation reaction can be carried out at room temperature, and the amidation reaction time can be selected from 4 to 48 hours, and more preferably 24 hours; the amidation reaction can be carried out under nitrogen protection conditions. conduct.
  • the present disclosure optionally performs rotary evaporation of the obtained product system to remove the solvent, dissolves the obtained crude product in ethyl acetate, removes insoluble matter through filtration, and concentrates the filtrate to obtain compound S7.
  • the present disclosure mixes compound S7, compound S4, azobisisobutyronitrile and a second organic solvent, and performs RAFT polymerization to obtain compound S8.
  • the molar ratio of compound S4, compound S7 and azobisisobutyronitrile can be 25: (0.5 ⁇ 1.2): (0.01 ⁇ 0.5), and more preferably 25:1:0.1; so
  • the second organic solvent may be N,N-dimethylformamide.
  • the present disclosure has no special limit on the amount of the second organic solvent, as long as the reaction proceeds smoothly.
  • compound S4 and azobisisobutyronitrile can be dissolved in a second organic solvent, and compound S7 is added to the resulting mixed solution under nitrogen protection to perform RAFT polymerization.
  • the temperature of the RAFT polymerization reaction can be selected from 40 to 100°C, and more preferably 70°C; the time can be selected from 4 to 48 hours, and more preferably 24 hours; the RAFT polymerization reaction can be carried out in nitrogen. under protective conditions.
  • the present disclosure optionally cools the obtained product system to room temperature, and then places it in a dialysis bag for dialysis to obtain compound S8.
  • the dialysate used in the dialysis can be selected from absolute ethanol and high-purity water in order, and the time for dialysis using absolute ethanol and high-purity water can be independently selected from 6 to 72 hours. More optionally, it can be 24 hours; after the dialysis, the present disclosure can optionally take out the materials in the dialysis bag, and after freeze-drying, compound S8 is obtained as a pink amorphous solid.
  • the present disclosure mixes the compound S8, trifluoroacetic acid and a third organic solvent to perform a de-tert-butoxycarbonyl reaction to obtain compound S9.
  • the dosage ratio of the compound S8 to trifluoroacetic acid can be 1 mmol: (10-1000) mL, or more preferably 1 mmol: 100 mL; the third organic solvent can be dichloromethane. It is disclosed that there is no special limit on the amount of the third organic solvent, as long as the reaction proceeds smoothly.
  • the removal of tert-butoxycarbonyl reaction can be carried out at room temperature, and the time of the removal of tert-butoxycarbonyl reaction can be selected from 0.5 to 48 hours, more preferably 24 hours; the removal of tert-butoxycarbonyl reaction can be carried out at room temperature.
  • the carbonyl reaction can optionally be carried out under nitrogen protection.
  • the present disclosure can optionally rotary evaporate the obtained product system to remove the solvent and trifluoroacetic acid, dissolve the obtained crude product in ethyl acetate, and then place it in a dialysis bag for dialysis to obtain the compound S9.
  • the dialysate used in the dialysis can be anhydrous ethanol and high-purity water in that order, and the time for dialysis using anhydrous ethanol and high-purity water can be independently selected from 6 to 72 hours, and more preferably 24 hours; after the dialysis , the present disclosure can optionally take out the materials in the dialysis bag and freeze-dry them to obtain compound S9 as a pink amorphous solid.
  • D-Asp, L-Asp-A or L-Asp-N is used as the targeting group to prepare the corresponding targeting nanocarrier
  • PDPA is the hydrophobic polymer
  • PEG is the hydrophilic linker
  • the preparation method can be basically the same as the above-mentioned method of preparing targeting nanocarriers using L-Asp as the targeting group, and will not be described again here.
  • the present disclosure provides the targeted nanocarriers described in the above technical solutions or the targeted nanocarriers prepared by the preparation method described in the above technical solutions as living plant plants, tissues of living plant plants, organs of living plant plants, and cells of living plant plants. , the application of active targeting nanocarriers to explants cultured in vitro, callus cultured in vitro, plant tissues cultured in vitro or plant cells cultured in vitro.
  • the living plant plant optionally includes a monocot or a eudicot.
  • the monocotyledonous plants may optionally include plants of the order Asparagus, plants of the order Gramineales, plants of the order Commelodea or plants of the order Palmidae; the plants of the order Asparagus may optionally include plants of the family Orchidaceae; the plants of the order Gramineales may be selected.
  • the grass plants may include corn, rice, wheat, sorghum, bamboo or buckwheat; the Commelina plants may include Commelina plants; the Commelina plants may be optionally included Optional includes commeldera.
  • the eudicots include Asteraceae plants, Cucurbitales plants, Leguminosae plants, Solanaceae plants or Brassicaceae plants; the Asteraceae plants optionally include Asteraceae plants; the Cucurbitales plants
  • the leguminous plants may optionally include Cucurbitaceae plants; the leguminous plants may optionally include leguminous plants, and the leguminous plants may optionally include soybeans or peas;
  • the Solanaceae plants may optionally include Solanaceae plants, and the Solanaceae plants may optionally include Including tomatoes, peppers or potatoes; the cruciferous plants optionally include cruciferous plants, and the cruciferous plants optionally include Arabidopsis thaliana or rapeseed.
  • the organs of the living plant may optionally include leaves, seeds or roots; the plant cells cultured in vitro may optionally include protoplasts cultured in vitro, and the protoplasts cultured in vitro may specifically be leaves, embryos
  • the shaft or root tip is prepared.
  • the targeting nanocarrier can achieve active targeting and crossing of plant cell walls and cell membranes, forming a "Trojan horse" type of cell penetration and targeting. Using the targeted nanocarriers described in the present disclosure to deliver drugs to plants can reduce drug dosage and cost, have a protective effect on loaded drugs, improve drug efficiency, extend drug action time, reduce toxicity and contamination, and reduce the probability of drug resistance.
  • the targeting nanocarriers can be used alone or mixed with non-targeting modified nanocarriers.
  • the target nanocarriers are The mass of the targeted nanocarrier can be more than 1% of the total mass of the targeted nanocarrier and the non-targeted modified nanocarrier, specifically it can be 20 to 80%; the non-targeted modified nanocarrier described in the present disclosure specifically refers to a non-targeted modified nanocarrier. Any modified nanocarrier or nanocarrier modified with substances other than the targeting agents described in this disclosure.
  • the present disclosure provides a targeted drug-loaded nanocarrier, including a targeted nanocarrier and a drug contained in the targeted nanocarrier.
  • the targeted nanocarrier is the targeted nanocarrier described in the above technical solution or the above-mentioned targeted nanocarrier.
  • the drug loading rate of the targeted drug-loaded nanocarrier can be selected from 1 to 99%, and more preferably from 30 to 80%.
  • the drug can optionally include small molecule drugs or biological Macromolecules;
  • the biological macromolecules may optionally include nucleic acids, proteins, amino acids, polypeptides, sugar substances or lipid substances, and the nucleic acids may specifically be DNA or RNA;
  • the small molecule drugs may optionally include plant hormones, water retaining agents , growth-promoting drugs, anti-pest drugs, anti-freeze drugs, anti-heat drugs, anti-ultraviolet drugs, fluorescein, genetically modified drugs or isotope-labeled compounds.
  • the plant hormones optionally include auxin, gibberellin, cell Mitogens, ethylene, jasmonic acid, brassinosteroids, strigolactones, abscisic acid (ABA) or ABA analogs (such as Pyrabactin, Quinabactin, Opabacti, AM1, AMF1 ⁇ , AMF1 ⁇ , AMF2 ⁇ , AMF2 ⁇ , AMF4 or AMC1 ⁇ ).
  • ABA is taken as an example for explanation; ABA is a plant endogenous hormone. When plants are stressed by drought, salt, etc., ABA can close leaf stomata, reduce water transpiration, and activate downstream stress resistance signals.
  • the binding method between the drug and the targeted nanocarrier may include hydrophilic and hydrophobic forces, hydrogen bonds, electrostatic forces or chemical bonding.
  • the present disclosure provides a method for preparing targeted drug-loaded nanocarriers described in the above technical solutions, including the following steps:
  • the targeted nanocarrier, drug and solvent are mixed and encapsulated to obtain the targeted drug-loaded nanocarrier.
  • optional targeting nanocarriers and drugs are dissolved in organic solvents respectively, and the resulting targeting nanocarrier solution is mixed with the drug solution to obtain a mixed solution; the mixed solution is added dropwise to water for encapsulation treatment to obtain the targeted nanocarrier solution.
  • Drug-loaded nanoparticles The present disclosure has no special restrictions on the type of organic solvent used to prepare the targeted nanocarrier solution and the drug solution. It can be selected according to the type of targeted nanocarrier and drug.
  • Asp (including D-Asp , L-Asp-A, L-Asp-N or L-Asp) is the targeting group
  • PDPA is the hydrophobic polymer
  • PEG is the hydrophilic linker prepared targeting nanocarrier (Asp-PEG-PDPA)
  • the organic solvent used can be tetrahydrofuran; taking ABA as the carried drug as an example, the organic solvent used can be ethanol.
  • the concentrations of the targeting nanocarrier solution and the drug solution can be independently selected from 1 to 10 mg/mL, and more preferably 5 mg/mL.
  • the targeted nano The volume ratio of the carrier solution to the drug solution can be selected based on obtaining a targeted drug-loaded nanocarrier with a required drug loading amount, which is not specifically limited in this disclosure.
  • the volume ratio of the mixed solution to water can be selected from (0.2 to 0.5): 1, and more preferably from (0.3 to 0.4): 1; in this disclosure, the mixed solution can be added dropwise to In water, the volume of each drop can be selected to be 10 ⁇ L.
  • the targeted nanocarrier and the drug are self-assembled (for example, self-assembly can be performed under hydrophobic or charge adsorption forces) to obtain the targeted drug-loaded nanocarrier.
  • appropriate encapsulation treatment conditions can be selected based on the characteristics of the targeted nanocarriers and drugs. Specifically, the encapsulation treatment can be performed under stirring, ultrasound, electrical stimulation or heating conditions to improve efficiency. In this disclosure In the embodiment, taking Asp-PEG-PDPA as the targeted nanocarrier and ABA as the loaded drug, the encapsulation treatment can be carried out at room temperature and stirring conditions, and the encapsulation treatment time can be selected as 2 ⁇ 4h, optionally 3h.
  • the present disclosure can optionally centrifuge the resulting product system in a Millipore Amicon Ultra-4 5K centrifugal filtration device, take out the supernatant, and the targeted drug-loaded nanocarriers are dispersed in the supernatant.
  • the rotation speed of the centrifugal filtration can be selected as 3600 rpm, and the time can be selected as 16 minutes.
  • the targeted drug-loaded nanocarriers may be optionally used in a protected form of a targeted drug-loaded nanocarrier dispersion.
  • the targeted drug-loaded nanocarrier dispersion can be obtained by dispersing the targeted drug-loaded nanocarrier in a solvent; the solvent can be water and/or an organic solvent, and the organic solvent can be Including ethanol, dimethyl sulfoxide or tetrahydrofuran; the concentration of the targeted drug-loaded nanocarrier dispersion can be selected from 0.1 to 10 mg/mL.
  • the usage methods of the targeted drug-loaded nanocarrier dispersion include spraying, soaking, smearing or injection; taking the application example as an example, specifically, the targeted drug-loaded nanocarrier dispersion is used to soak seeds, soak The time can be selected from 1 to 168 hours; or the targeted drug-loaded nanocarrier dispersion can be sprayed on the plant leaves, and the dosage of the targeted drug-loaded nanocarrier dispersion can be selected from 10 to 10000 ⁇ L/cm 2 , or more Select 10 to 500 ⁇ L/cm 2 .
  • a targeting nanocarrier (Asp-PEG-PDPA) is prepared using Asp as the targeting group, PDPA as the hydrophobic polymer, and PEG as the hydrophilic linker.
  • ABA is used as the The loaded drug is self-assembled with Asp-PEG-PDPA to form a targeted drug-carrying nanocarrier (Asp-NP@ABA), which is then sprayed on plant leaves to improve its drought resistance.
  • the flow chart for preparing Asp-NP and the loading method Taking ABA as an example, the schematic diagram of the application of targeted drug-loaded nanocarriers as drought-resistant agents is shown in Figure 1.
  • the corresponding targeting nanocarriers self-assemble to form micelles Asp-
  • the NPs are respectively recorded as D-Asp-NP, A-Asp-NP, N-Asp-NP and L-Asp-NP.
  • Each targeted nanocarrier carries ABA and is self-assembled to form a targeted drug-loaded nanocarrier Asp-NP@ABA. They are recorded as D-Asp-NP@ABA, A-Asp-NP@ABA, N-Asp-NP@ABA and L-Asp-NP@ABA respectively.
  • compound S1 (4-cyano-4-(thiobenzoyl)pentanoic acid, 27.9 mg, 0.1 mmol, 1.0 equiv.) and dicyclohexylcarbodiimide (DCC, 24.8 mg, 0.12 mmol, 1.2 equiv.), 1-hydroxybenzotriazole (HOBt, 16.2 mg, 0.12 mmol, 1.2 equiv.) and 4-dimethylaminopyridine (DMAP, 1.2 mg, 0.01 mmol, 0.1 equiv.) ) was dissolved in dichloromethane (DCM, 1 mL), stirred for 5 min to obtain a mixture; compound S2 (Amino-PEG5000-OMe, 500 mg, 0.1 mmol, 1.0 equiv.) was dissolved in DCM (5 mL), and then mixed once Add to the mixture and stir for 24 hours at room temperature; remove the solvent DCM from the product system by rotary evaporation,
  • compound S8 was dissolved in a mixed solution of 5 mL DCM and 5 mL trifluoroacetic acid (TFA), and the resulting mixture was stirred and reacted at room temperature for 24 h; the solvent DCM and TFA were removed from the resulting product system by rotary evaporation.
  • the crude product was dissolved in EtOAc (5 mL), and then placed in a dialysis bag for purification. Specifically, absolute ethanol was used as the dialysate, dialyzed for 24 hours (the dialysate was changed 5 times during the period), and then the dialysate was replaced with high-purity water. , dialyzed for 24 hours (during which the dialysate was replaced 5 times), then the materials in the dialysis bag were taken out, and after freeze-drying, compound S9 was obtained as a pink amorphous solid.
  • TFA trifluoroacetic acid
  • Asp-PEG-PDPA with D-Asp, L-Asp-A and L-Asp-N as targeting groups were prepared respectively.
  • the Asp-PEG-PDPA powder prepared in Example 1 (targeting group is D-Asp, L-Asp-A, L-Asp-N or L-Asp) was prepared into micelle Asp-NP (specifically D- Asp-NP, A-Asp-NP, N-Asp-NP and L-Asp-NP) were then characterized by transmission electron microscopy. Specifically, Asp-PEG-PDPA powder (1 mg, 0.625 mmol) was dissolved in tetrahydrofuran (0.2 mL).
  • Figure 3 is a transmission electron microscope image of L-Asp-NP in Example 1 (scale bar is 50nm). It can be seen from Figure 3 that the Asp-NP nanoparticles are uniform in size, with a particle diameter of about 100nm.
  • the Asp-PEG-PDPA powder prepared in Example 1 (1 mg, 0.625 mmol; targeting group is D-Asp, L-Asp-A, L-Asp-N or L-Asp) was dissolved in tetrahydrofuran (0.2 mL) to obtain an Asp-PEG-PDPA solution; dissolve abscisic acid (ABA, 5 mg, 18.9 mmol) in ethanol (1 mL) to obtain an ABA solution; add 0.2 mL of the Asp-PEG-PDPA solution (containing 1 mg of Asp- PEG-PDPA) is mixed with a certain volume of ABA solution so that the mass of ABA is 40% of the total mass of Asp-PEG-PDPA and ABA to obtain a mixed solution;
  • the obtained product system is centrifugally filtered (3600rpm, 16min) in a Millipore Amicon Ultra-4 5K centrifugal filtration device, and the supernatant (specifically, the liquid in the inner tube of the centrifugal filtration device) is taken out, and the drug-loaded product (Denoted as Asp-NP@ABA, specifically D-Asp-NP@ABA, A-Asp-NP@ABA, N-Asp-NP@ABA and L-Asp-NP@ABA) were dispersed in the supernatant and stored at 4°C.
  • Asp-NP@ABA specifically D-Asp-NP@ABA, A-Asp-NP@ABA, N-Asp-NP@ABA and L-Asp-NP@ABA
  • the drug-loaded product was prepared according to the method of Example 2, except that Asp-PEG-PDPA powder was replaced with MeO-PEG-PDPA, and the final drug-loaded product was recorded as NP@ABA.
  • NP specifically MeO-PEG-PDPA prepared in Comparative Example 1
  • NP@ABA specifically MeO-PEG-PDPA prepared in Comparative Example 1
  • Asp-NP specifically D-Asp-NP, A-Asp-NP, N-Asp -NP and L-Asp-NP
  • Asp-NP@ABA specifically D-Asp-NP@ABA, A-Asp-NP@ABA, N-Asp-NP@ABA and L-Asp-NP@ABA
  • the particle size distribution and polydispersity coefficient are shown in Figure 4.
  • the particle size of the newly prepared L-Asp-NP@ABA is 135.5 ⁇ 4.2nm, and the PDI is 0.164 ⁇ 0.038 (after being placed for 12 months, the particle size of L-Asp-NP@ABA is 139.4 ⁇ 5.9nm , PDI is 0.15 ⁇ 0.03); the particle size of the newly prepared NP@ABA is 139.53 ⁇ 1.21nm, and the PDI is 0.156 ⁇ 0.012 (after being placed for 9 months, the particle size of NP@ABA is 136.6 ⁇ 13.1nm, and the PDI is 0.32 ⁇ 0.08).
  • the particle size of the newly prepared A-Asp-NP@ABA is 177.6 ⁇ 1.5nm and the PDI is 0.17 ⁇ 0.01; the particle size of the newly prepared D-Asp-NP@ABA is 173.9 ⁇ 3.8nm and the PDI is 0.12 ⁇ 0.02; The particle size of the newly prepared N-Asp-NP@ABA is 169.7 ⁇ 3.1nm and the PDI is 0.18 ⁇ 0.03.
  • the drug loading rates of Asp-NP@ABA and NP@ABA were measured, and the results are shown in Figure 4.
  • the drug loading rate of L-Asp-NP@ABA is 72.07 ⁇ 1.92%
  • the drug loading rate of A-Asp-NP@ABA is 65.3 ⁇ 1.4%
  • the drug loading rate of D-Asp-NP@ABA The drug loading rate of N-Asp-NP@ABA was 64.2 ⁇ 2.8%
  • the drug loading rate of N-Asp-NP@ABA was 69.1 ⁇ 1.0%
  • the drug loading rate of NP@ABA was 60.09 ⁇ 2.79%.
  • the Asp targeting group is L-Asp; when Asp targeting groups of other configurations are used, they will be labeled and explained.
  • Application Example 1 Asp-NP can efficiently penetrate plant tissue cell walls and cell membranes
  • Asp-NP@DiO was prepared according to the method of Example 2, except that ABA was replaced with 3,3′-dioctadecyloxycarbocyanine perchlorate (DiO; CAS: 34215-57-1 ).
  • NP@DiO was prepared according to the above method, except that Asp-PEG-PDPA was replaced by MEO-PEG-PDPA.
  • Asp-NP@DiO was dispersed in ultrapure water to obtain an Asp-NP@DiO dispersion with a concentration of 31.8 ⁇ M; the Asp-NP@DiO dispersion was sprayed on 14-day-old Arabidopsis leaves.
  • the strain is about 60 ⁇ L; after 36 hours, use a laser confocal microscope to track the penetration depth of the DiO fluorescence signal from the Arabidopsis leaf surface to the vertically downward mesophyll tissue based on its 3D layer scanning function, and set the MS, DiO and NP@DiO control group, the specific results are shown in Figure 5 (scale bar is 50 ⁇ m) and Figure 6.
  • Figure 5 shows the confocal microscopy observation results at 20 ⁇ m.
  • the Asp-NP@DiO dispersion was co-cultured with the isolated Arabidopsis thaliana mesophyll cell protoplasts for 4 hours; the fresh W5 medium was replaced and cultured for 20 hours, and then a laser confocal microscope was used to track the DiO fluorescence signal inward from the protoplast plasma membrane. Intracellular subcellular localization, and W5 buffer, DiO and NP@DiO control groups were set. The specific results are shown in Figure 7 (scale bar is 50 ⁇ m).
  • Fluorescein isothiocyanate is used to label Asp-NP to obtain Asp-NP-FITC, and the Asp-NP-FITC is dispersed in ultrapure water to obtain Asp-NP-FITC with a concentration of 12.5 ⁇ M. Dispersion; Spray the Asp-NP-FITC dispersion on the leaves of Commelina communis, each leaf is about 10 ⁇ L; after 6 hours, use a laser confocal microscope to track the FITC fluorescence signal in the duck based on its 3D layer scanning function. The depth of penetration from the surface of the plantweed blade to the vertically downward mesophyll tissue, and the MS group was set as a control. The specific results are shown in Figure 8 (scale bar is 50 ⁇ m).
  • Figure 8 shows the observation results of confocal microscopy at different depths. According to the observation of the FITC signal in Figure 8, it can be seen that Asp-NP can penetrate and bring FITC into the tissue depth close to 50 ⁇ m. Because the upper epidermis of Commelina communis leaves does not have stomata, this experiment confirmed that the targeting nanocarriers deliver the target molecules through the cell wall rather than through the stomata.
  • Application Example 2 Asp-NP@ABA can efficiently enter the plant through the seed coat and roots and deliver it
  • Place wild-type (WT) Arabidopsis seeds in a 24-well plate, 20 seeds per well; dilute Asp-NP@ABA (specifically D-Asp-NP@ABA) with 1/2MS (pH 6.7) liquid medium , A-Asp-NP@ABA, N-Asp-NP@ABA or L-Asp-NP@ABA), respectively, obtained Asp-NP@ABA dispersions with different targeting groups (based on ABA concentration, the concentration is 0.1 ⁇ M), add each Asp-NP@ABA dispersion (400 ⁇ L) to each well for incubation, set 6 wells for each concentration in duplicate, place in an incubator for germination (16h light/8h dark), and count every 12h Germination rate was measured continuously for 7 days.
  • MS, ABA, NP, Asp-NP specifically D-Asp-NP, A-Asp-NP, N-Asp-NP or L-Asp-NP
  • NP@ABA were set as controls.
  • Figure 9 is a comparison chart of the germination rate of Arabidopsis thaliana seeds after using different treatments. It can be seen from Figure 9 that Asp-NP@ABA with different targeting groups can enter the seed tissue more effectively than ABA, and is used by Asp-NP@ABA. - ABA delivered into seed tissue by NP can significantly delay seed germination.
  • Asp-NP-FITC Disperse Asp-NP-FITC in 1/2MS culture medium so that the concentration of Asp-NP-FITC is 12.5 ⁇ M to obtain a culture medium containing Asp-NP-FITC; then 7-day-old Arabidopsis thaliana The roots of the seedlings, the roots of the 4-day-old soybean seedlings and the roots of the 4-day-old corn seedlings were soaked in the medium containing Asp-NP-FITC, and the penetration of Asp-NP-FITC into the root tissue of each plant was observed at different time points. depth. At the same time, the MS group was set as a control.
  • Figure 10 is a comparison chart of Asp-NP-FITC penetrating into Arabidopsis root tissue at different depths at different time points (scale bar is 100 ⁇ m).
  • Figure 11 is a comparison of Asp-NP-FITC penetrating into soybean root tissue at different depths after 4h and 6h (scale bar is 100 ⁇ m).
  • Figure 12 is Comparison of Asp-NP-FITC penetrating into corn root tissue at different depths after 4h and 6h (scale bar is 100 ⁇ m). It can be seen from Figure 10, Figure 11 and Figure 12 respectively that Asp-NP-FITC can efficiently enter the root tissue of plants of different species through soaking.
  • Figure 13 is a statistical diagram showing the senescence and yellowing of leaves caused by ABA after absorption by the roots of Arabidopsis thaliana using different treatments in the hydroponic system.
  • Asp-NP@ABA with different targeting groups can more efficiently deliver ABA into plant tissues through roots. After ABA is delivered into cells, it can cause leaf senescence, showing There is etiolation, so a larger number of etiolated leaves shows that the corresponding Asp-NP has a higher efficiency in delivering ABA.
  • Asp-NP@ABA dispersion Disperse Asp-NP@ABA in ultrapure water to obtain Asp-NP@ABA dispersion.
  • concentration of ABA in the Asp-NP@ABA dispersion is 10 ⁇ M; Spray about 60 ⁇ L of the seedlings on the Arabidopsis leaves (3 weeks old), no longer water after spraying, and place them in an incubator to grow (8 hours of light/16 hours of darkness, the temperature in the light is 22°C, and the temperature in the dark is 19.8°C), record the plant growth status every day.
  • Figure 14 is a comparison chart of spraying Arabidopsis thaliana seedlings with different treatments to extend the survival period of the seedlings under drought conditions. From Figure 14, it can be seen that the Asp-NP@ABA treatment group still has better performance than other groups after 18 days of drought. vitality. This shows that each plant can extend its survival period after being treated with Asp-NP@ABA.
  • Figure 15 is a comparison chart of the survival rate of Arabidopsis thaliana seedlings after spraying with different treatments. Specifically, after spraying Arabidopsis thaliana seedlings with different treatments, they were rehydrated once on the 12th day.
  • Figure 16 is a scatter plot of percentages of spraying Arabidopsis thaliana seedlings under drought conditions with different treatments relative to the extended survival period of the MS treatment group (set up 4 parallel experiments, the Asp-NP used in this experiment @ABA is specifically D-Asp-NP@ABA, A-Asp-NP@ABA, N-Asp-NP@ABA or L-Asp-NP@ABA, and the Asp-NP used is specifically D-Asp-NP, A-Asp-NP, N-Asp-NP or L-Asp-NP), as shown in Figure 16, after plants were treated with Asp-NP@ABA with different targeting groups, the survival cycle was extended by an average of 57%. .
  • Figure 17 is the result of the lowest effective concentration of Asp-NP@ABA measured based on the survival cycle under drought conditions (using the results of ABA treatment as a reference). From Figure 17, it can be seen that under the same drought conditions, Asp-NP@ABA Abscisic acid concentration can be reduced to 100,000 to one million times that of ABA.
  • FIG. 18 is a comparison of the ABA content in the cytoplasm and apoplasm after spraying NP@ABA and Asp-NP@ABA on Arabidopsis leaves for 24 hours. It can be seen from Figure 18 that both NP@ABA and Asp-NP@ABA can effectively ABA is transported into the cell wall, causing ABA to be enriched in the extracellular area; however, the enrichment of ABA in protoplasts extracted from the same leaves is completely different.
  • Asp-NP@ABA Disperse Asp-NP@ABA in ultrapure water to obtain Asp-NP@ABA dispersion.
  • concentration of ABA in the Asp-NP@ABA dispersion is 10 ⁇ M; add the Asp-NP@ABA with the concentration of 10 ⁇ M.
  • Figure 19 is a comparison chart of using different treatments to extend the life cycle of soybeans under drought conditions after spraying soybeans. It can be seen from Figure 19 that the Asp-NP@ABA treatment group still has good vitality compared to other groups after 8 days of drought. This shows that each soybean plant can enhance drought resistance after being treated with Asp-NP@ABA.
  • Figure 20 is a statistical chart of the median values of spraying soybeans using different treatments to extend the life cycle of soybeans under drought conditions. It can be seen from Figures 19 and 20 that after soybean plants were treated with Asp-NP@ABA, the median survival period was extended by an average of 50%.
  • Asp-NP@ABA was dispersed in ultrapure water to obtain Asp-NP@ABA dispersion.
  • concentration of ABA in the Asp-NP@ABA dispersion was 50 ⁇ M; the Asp-NP with the concentration of 50 ⁇ M was @ABA dispersion was sprayed on corn leaves (10 weeks old) at an amount of about 7-8 mL per seedling. After spraying, no watering was performed, and the plants were placed outdoors for growth. The growth status of the plants was recorded every day.
  • Figure 21 is a comparison chart of using different treatments to extend the survival period of corn under drought conditions after spraying corn. As can be seen from Figure 21, the Asp-NP@ABA treatment group still has good vitality compared to the ABA group after 34 days of drought. This shows that each corn plant can enhance drought resistance after being treated with Asp-NP@ABA.
  • the Asp-PEG-PDPA powder prepared in Example 1 was mixed with a non-targeted polymer (ie, MeO-PEG-PDPA) to obtain a mixed powder.
  • the mass content of the Asp-PEG-PDPA powder in the mixed powder was 20 %, 40%, 60%, 80% or 100%; the mixed powder (1mg) was dissolved in tetrahydrofuran (0.2mL) to obtain a mixed solution; abscisic acid (ABA, 5mg, 18.9mmol) was dissolved in ethanol ( 1 mL) to obtain an ABA solution; mix 0.2 mL of the mixed solution with a certain volume of ABA solution to make ABA
  • the mass is 40% of the total mass of the mixed powder and ABA (i.e.
  • Asp-PEG-PDPA+MeO-PEG-PDPA+ABA to obtain an ABA-containing mixed solution; add ultrapure water (1mL) to the reaction bottle equipped with a rotor ), add the ABA-containing mixed solution drop by drop into the reaction bottle at a rate of 10 ⁇ L.
  • the targeted nanocarrier provided by the present disclosure has the potential to be widely used, especially its characteristics that provide excellent advantages in polar and extremely harsh environments, such as scientific expeditions and extraterrestrial exploration.
  • the targeted nanocarrier provided by the present disclosure includes a nanocarrier and a targeting substance chemically bonded to the nanocarrier, wherein the nanocarrier is a nanoparticle formed of an organic polymer or an inorganic material, and the targeting substance It is aspartic acid or aspartic acid derivatives.
  • the targeted nanocarriers provided by the present disclosure can actively penetrate plant cell walls and cell membranes, are suitable for drug delivery to living plants or tissues, etc., can reduce drug dosage and cost, have a protective effect on loaded drugs, improve drug efficiency, and prolong drug effects. time, reduce toxicity and pollution, reduce the probability of drug resistance, and have excellent industrial practicability.

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Abstract

本公开提供了一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法，属于功能材料技术领域。本公开提供的靶向纳米载体包括纳米载体以及化学键合在所述纳米载体上的靶向物，其中，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子，所述靶向物为天冬氨酸或天冬氨酸衍生物。本公开提供的靶向纳米载体能够主动穿透植物细胞壁和细胞膜，适用于对活体植物或组织等进行药物递送，能够降低用药剂量和成本、对搭载药物具有保护作用并提高药物效率、延长药物作用时间，降低毒性和污染性，降低耐药概率。

Description

一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法
相关申请的交叉引用
本公开要求于2022年09月08日提交中国专利局的申请号为202211094750.0、名称为“一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及功能材料技术领域，尤其涉及一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法。
背景技术
科学家们利用植物生物技术进行高产和抗逆作物筛选，改良药物生物合成以及发展可持续农业等。然而，即使经过数十年的发展，生物技术在植物科学乃至农业生产上的应用仍然面临各种各样的问题与挑战。植物细胞具有细胞壁，构成了外源生物大分子等药物递送的主要屏障。在植物遗传转化进行分子育种优化过程中，传统的基因枪具有造成目标组织损伤且基因表达水平低等不足，而农杆菌转化法最大的挑战是狭窄的宿主选择性和组织特异性。同时，这些方法所需的愈伤组织生成也限制了可应用的植物物种。而在传统的物理和化学农药及肥料应用方面，极低的利用率造成的过量施用及高残留则对生产安全和环境造成了巨大的压力。
纳米递送载体在生物医药领域被广泛应用，对人类健康有重要贡献，纳米递送能极大的减少药物用量，仅在需要的组织靶向聚集，能够降低用药成本、提高药物效率、延长药物作用时间、降低毒性和污染性，而且能够降低耐药概率。然而，当前用于植物的纳米递送载体极少，这是因为在植物体系中，细胞壁的存在形成了天然的屏障，使得几乎所有纳米载体都无法有效的穿透细胞壁进而在活体组织和细胞中进行递送，仅有的几例关于植物纳米递送载体的研究也需要外力辅助(如叶片注射、磁力等)实现其跨越细胞壁进入活体植物组织的目的。而针对植物生物技术尤其是农业生产中的应用往往需要庞大的样本量，即便在实验室中的操作应用也需要数百个样本，因而任何需要外力辅助的方法都较为繁琐并且效率低下。因此，开发能够主动穿透植物细胞壁并且进一步穿透细胞膜的纳米递送载体，同时不需要借助外力并且不引起组织损伤，能够轻易的施用于大量植物，用于对植物递送药物，是目前亟需解决的技术问题。
发明内容
本公开的目的在于提供一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法，本公开提供的靶向纳米载体能够主动穿透植物细胞壁和细胞膜，适用于对植物递送药物。
为了实现上述发明目的，本公开提供以下技术方案：
本公开提供了一种靶向纳米载体，包括纳米载体以及化学键合在所述纳米载体上的靶向物，其中，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子，所述靶向物为天冬氨酸或天冬氨酸衍生物。
可选地，所述天冬氨酸提供的靶向基团包括式I～IV任一所示结构的基团：
可选地，所述纳米载体的粒径为10～1000nm。
可选地，所述有机高分子聚合物的数均分子量为3～50kDa。
可选地，所述有机高分子聚合物包括疏水性聚合物以及与所述疏水性聚合物共价连接的亲水性连接物，所述亲水性连接物与所述靶向物经化学键合连接。
本公开提供了上述技术方案所述靶向纳米载体的制备方法，包括以下步骤：
在溶剂存在条件下，在纳米载体上经化学键合修饰靶向物，得到靶向纳米载体。
本公开提供了上述技术方案所述靶向纳米载体或上述技术方案所述制备方法制备得到的靶向纳米载体作为活体植物植株、活体植物植株的组织、活体植物植株的器官、活体植物植株的细胞、体外培养的外植体、体外培养的愈伤组织、体外培养的植物组织或体外培养的植物细胞的主动靶向纳米载体的应用。
本公开提供了上述技术方案所述靶向纳米载体或上述技术方案所述制备方法制备得到的靶向纳米载体作为活体植物主动靶向纳米递送载体的应用。
本公开提供了一种靶向载药纳米载体，包括靶向纳米载体以及包载在所述靶向纳米载体中的药物，所述靶向纳米载体为上述技术方案所述靶向纳米载体或上述技术方案所述制备方法制备得到的靶向纳米载体。
可选地，所述药物包括小分子药物或生物大分子，所述靶向载药纳米载体的载药率为1～99％。
本公开提供了上述技术方案所述靶向载药纳米载体的制备方法，包括以下步骤：
将靶向纳米载体、药物与溶剂混合，进行包载处理，得到靶向载药纳米载体。
本公开提供了一种靶向纳米载体，包括纳米载体以及化学键合在所述纳米载体上的靶向物，其中，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子，所述靶向物为天冬氨酸或天冬氨酸衍生物。本公开提供的靶向纳米载体能够主动穿透植物细胞壁和细胞膜，适用于对活体植物或组织等进行药物递送，能够降低用药剂量和成本、对搭载药物具有保护作用并提高药物效率、延长药物作用时间，降低毒性和污染性，降低耐药概率。
附图说明
图1为本公开制备Asp-NP的流程图以及以搭载ABA为例所得靶向载药纳米载体作为抗旱剂的应用示意图；
图2为实施例1制备的靶向基团为L-Asp的Asp-PEG-PDPA的核磁共振图；
图3为实施例1中L-Asp-NP的透射电镜图；
图4为搭载ABA后所得Asp-NP@ABA(具体为D-Asp-NP@ABA、A-Asp-NP@ABA、N-Asp-NP@ABA和L-Asp-NP@ABA)和不具有靶向作为对照的NP@ABA的粒径和载药率对比图；
图5为应用例1中采用不同处理喷洒拟南芥叶片36h后，使用激光共聚焦显微镜在20μm的深度时追踪DiO的观察结果图；
图6为应用例1中采用不同处理喷洒拟南芥叶片36h后，使用激光共聚焦显微镜追踪DiO被纳米粒子带入穿透叶片组织的深度统计图；
图7为应用例1中采用不同处理与分离的拟南芥叶肉细胞原生质体混合共培养4h，更换新鲜MS(Murashige and Skoog)培养基培养20h后，使用激光共聚焦显微镜追踪DiO信号被纳米粒子带入原生质体的对比图；
图8为应用例1中采用不同处理喷洒鸭跖草叶片6h后，使用激光共聚焦显微镜在不同深度追踪FITC的观察结果图；
图9为采用不同处理后拟南芥种子发芽率对比图；
图10为Asp-NP-FITC在不同时间点穿透拟南芥根部组织不同深度的对比图；
图11为Asp-NP-FITC在4h和6h后穿透大豆根部组织不同深度的对比图；
图12为Asp-NP-FITC在4h和6h后穿透玉米根部组织不同深度的对比图；
图13为在水培系统中采用不同处理拟南芥小苗根吸收靶向纳米载体对叶片衰老黄化诱导的对比图；
图14为应用例3中采用不同处理喷洒拟南芥小苗后在干旱条件下延长小苗生存周期的对比图；
图15为应用例3中采用不同处理喷洒拟南芥小苗后的生存率对比图；
图16为应用例3中采用不同处理喷洒拟南芥小苗后在干旱条件下延长小苗的生存周期相对于MS处理组延长的生存周期统计的百分比散点图；
图17为应用例3中以干旱条件下的生存周期为衡量标准测定的Asp-NP@ABA最低有效浓度结果图(以ABA处理的结果作为参照)；
图18为应用例3中采用不同处理喷洒拟南芥叶片24h后细胞质内和质外体内的ABA含量对比图；
图19为应用例4中采用不同处理喷洒大豆小苗后在干旱条件下延长小苗生存周期的对比图；
图20为应用例4中采用不同处理喷洒大豆小苗后在干旱条件下延长小苗生存周期的中位生存值的统计图；
图21为应用例4中采用不同处理喷洒玉米小苗后在干旱条件下延长小苗生存周期的对比图；
图22为应用例5中添加不同比例非靶向的聚合物(即MeO-PEG-PDPA)的载药产物对于拟南芥种子发芽率的影响对比图；
图23为应用例5中添加不同比例非靶向的聚合物(即MeO-PEG-PDPA)的载药产物对于拟南芥在干旱条件下的生存周期延长的影响对比图。
具体实施方式
本公开提供了一种靶向纳米载体，包括纳米载体以及化学键合在所述纳米载体上的靶向物，其中，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子，所述靶向物为天冬氨酸或天冬氨酸衍生物。
本公开提供的靶向纳米载体包括纳米载体，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子。在本公开中，所述纳米载体的粒径可选为10～1000nm，更可选为20～200nm。
在本公开中，所述有机高分子聚合物的数均分子量可选为3～50kDa，更可选为5～20kDa。在本公开中，所述有机高分子聚合物可选包括疏水性聚合物以及与所述疏水性聚合物共价连接的亲水性连接物，所述亲水性连接物与所述靶向物化学键合链接。
在本公开中，所述疏水性聚合物可选包括以下(1)、(2)和(3)中涉及的物质中的任一种；
(1)聚乳酸-羟基乙酸共聚物(PLGA)、PLGA衍生物、聚乳酸(PLA)、PLA衍生物、聚己内酯(PCL)、PCL衍生物、聚碳酸酯(PMC)、PMC衍生物；
(2)乙交酯、丙交酯、己内酯和碳酸酯中的一种或几种；乙交酯、丙交酯、己内酯和碳酸酯中至少两种形成的共聚物；
(3)聚氨酯(PU)、PU衍生物、聚醚醚酮(PEEK)、PEEK衍生物、聚甲基丙烯酸甲酯(PMMA)、PMMA衍生物、聚乙烯醇(PVA)、PVA衍生物、聚乙烯(PE)、PE衍生物、疏水聚氨基酸、疏水聚氨基酸衍生物；所述疏水聚氨基酸可选为聚苯丙氨酸。
在本公开中，所述亲水性连接物可选包括以下(a)和(b)中涉及的物质中的任一种：
(a)聚乙二醇(PEG)、聚环氧乙烷(PEO)、聚(乙二醇)甲基丙烯酸酯(POEG)、聚2-甲基丙烯酰氧基乙基磷酰(PMPC)、聚羧酸甜菜碱(PCB)、葡聚糖、葡聚糖、透明质酸、壳聚糖、β-环糊精、超支化聚缩水甘油醚(HPG)、聚N-(2-羟丙基)甲基丙烯酰胺(PHPMA)、聚甲基丙烯酸羟乙酯(PHEMA)、聚丙烯酰胺(PAM)、聚乙烯吡咯烷酮(PVP)、聚丙烯酸(PAA)、聚马来酸酐(HPMA)、聚季胺盐；
(b)聚乙烯亚胺(PEI)、PEI衍生物、PEI药学上可接受的盐、聚甲基丙烯酸N,N-二甲基氨基乙酯(PDMAEMA)、PDMAEMA衍生物、PDMAEMA药学上可接受的盐、聚赖氨酸(PLL)、PLL衍生物、PLL药学上可接受的盐、亲水聚氨基酸、亲水聚氨基酸衍生物、亲水聚氨基酸药学上可接受的盐；所述亲水聚氨基酸可选为聚谷氨酸(PGu)或聚天冬氨酸(PAsp)。
在本公开中，当所述纳米载体为有机高分子聚合物形成的纳米粒子时，所述纳米载体的形貌具体可以为胶束或囊泡，本公开对此没有特殊限定。
在本公开中，所述无机材料可选包括硅、硅氧化物、铁、铁氧化物、钙、钙氧化物或碳纳米材料。在本公开中，具体的，所述无机材料表面含有活性基团，本公开可选直接通过所述活性基团与靶向物化学键合连接，或者通过亲水性连接物将所述活性基团与靶向物化学键合连接，即可以先将靶向物与亲水性连接物化学键合，然后在此基础上再通过所述亲水性连接物与无机材料表面的活性基团化学键合，也可以先将无机材料表面的活性基团与亲水性连接物化学键合，然后在此基础上再通过所述亲水性连接物与靶向物化学键合。在本公开中，所述亲水性连接物的可选种类可选与上述亲水性连接物的可选种类一致，在此不再赘述。本公开对所述活性基团的具体种类没有特殊限定，能够实现与亲水性连接物或靶向物进行化学键合即可，具体的，所述活性基团可以为羟基。本公开对所述无机材料的来源没有特殊限定，采用本领域技术人员熟知的市售商品即可。
本公开提供的靶向纳米载体包括靶向物，所述靶向物化学键合在所述纳米载体上，所述靶向物为天冬氨酸或天冬氨酸衍生物。在本公开中，所述靶向物与纳米载体化学键合的具体方式可以为酯基，也可以为酰胺基，本公开对此没有特殊限定。在本公开中，所述靶向纳米载体中天冬氨酸提供的靶向基团可选包括式I～IV任一所示结构的基团，分别记为D-Asp、L-Asp-A、L-Asp-N、L-Asp：
本公开提供了上述技术方案所述靶向纳米载体的制备方法，包括以下步骤：
在溶剂存在条件下，在纳米载体上经化学键合修饰靶向物，得到靶向纳米载体。
本公开可选根据纳米载体以及靶向物的具体种类选择合适的方法将二者化学键合制备靶向纳米载体，下面进行详细说明。
第一种情况，当所述纳米载体为无机材料形成的纳米粒子时，本公开可选在溶剂存在条件下，将所述纳米载体与靶向物通过化学反应将二者化学键合，得到靶向纳米载体；所述溶剂以及化学反应的条件根据无机材料以及靶向物的种类确定，本公开对此没有特殊限定。
第二种情况，当所述纳米载体为有机高分子聚合物形成的纳米粒子时，根据所述有机高分子聚合物以及靶向物的性质，本公开可以参照上述第一种情况制备靶向纳米载体，即首先制备纳米载体，然后在溶剂存在条件下，将所述纳米载体与靶向物通过化学反应将二者化学键合，得到靶向纳米载体；所述溶剂以及化学反应的条件根据有机高分子聚合物以及靶向物的种类确定，本公开对此没有特殊限定。
第三种情况，当所述纳米载体为有机高分子聚合物形成的纳米粒子时，根据所述有机高分子聚合物 以及靶向物的性质，本公开还可以将靶向物化学键合于制备有机高分子聚合物的单体上，然后在此修饰有靶向物的单体基础上实现靶向纳米载体的制备；所述修饰有靶向物的单体的结构以及在此基础上进一步制备靶向纳米载体的条件根据有机高分子聚合物以及靶向物的种类确定，本公开对此没有特殊限定。
在本公开的实施例中，以L-Asp为靶向基团、PDPA为疏水性聚合物、PEG为亲水性连接物制备靶向纳米载体(Asp-PEG-PDPA)为例进行说明。在本公开中，以L-Asp为靶向基团的Asp-PEG-PDPA的制备方法可选包括以下步骤：
将化合物S1、化合物S6、二环己基碳二亚胺、1-羟基苯并三唑、4-二甲氨基吡啶与第一有机溶剂混合，进行酰胺化反应，得到化合物S7；
将所述化合物S7、化合物S4、偶氮二异丁腈与第二有机溶剂混合，进行可逆加成-断裂链转移(RAFT)聚合反应，得到化合物S8；
将所述化合物S8、三氟乙酸与第三有机溶剂混合，进行脱叔丁氧羰基(Boc)反应，得到化合物S9(即以L-Asp为靶向基团的Asp-PEG-PDPA)；
所述化合物S1、化合物S6、化合物S7、化合物S4、化合物S8以及化合物S9的结构式如下所示：
本公开将化合物S1、化合物S6、二环己基碳二亚胺、1-羟基苯并三唑、4-二甲氨基吡啶与第一有机溶剂混合，进行酰胺化反应，得到化合物S7。在本公开中，所述化合物S1、化合物S6、二环己基碳二亚胺、1-羟基苯并三唑与4-二甲氨基吡啶的摩尔比可选为1：(0.2～3)：(1～3)：(1～3)：(0.01～1)，更可选为1：1：1.2：1.2：0.1；所述第一有机溶剂可选为二氯甲烷，本公开对所述第一有机溶剂的用量没有特殊限定，保证反应顺利进行即可。本公开可选将化合物S1、二环己基碳二亚胺、1-羟基苯并三唑和4-二甲氨基吡啶溶解于第一有机溶剂中，得到混合物料；将化合物S6溶解于第一有机溶剂中，得到化合物S6溶液；将所述化合物S6溶液一次性加到所述混合物料中进行酰胺化反应。在本公开中，所述酰胺化反应可选在室温条件下进行，所述酰胺化反应的时间可选为4～48h，更可选为24h；所述酰胺化反应可选在氮气保护条件下进行。所述酰胺化反应后，本公开可选将所得产物体系进行旋蒸以除去溶剂，将所得粗产物溶解于乙酸乙酯中，经过滤除去不溶物，将滤液浓缩得到化合物S7。
得到化合物S7后，本公开将所述化合物S7、化合物S4、偶氮二异丁腈与第二有机溶剂混合，进行RAFT聚合反应，得到化合物S8。在本公开中，所述化合物S4、化合物S7与偶氮二异丁腈的摩尔比可选为25：(0.5～1.2)：(0.01～0.5)，更可选为25：1：0.1；所述第二有机溶剂可选为N,N-二甲基甲酰胺，本公开对所述第二有机溶剂的用量没有特殊限定，保证反应顺利进行即可。本公开可选将化合物S4与偶氮二异丁腈溶解于第二有机溶剂中，在氮气保护下，向所得混合液中加入化合物S7，进行RAFT聚合反应。在本公开中，所述RAFT聚合反应的温度可选为40～100℃，更可选为70℃；时间可选为4～48h，更可选为24h；所述RAFT聚合反应可选在氮气保护条件下进行。所述RAFT聚合反应后，本公开可选将所得产物体系冷却至室温，然后置于透析袋中进行透析，得到化合物S8。在本公开中，所述透析采用的透析液可选依次为无水乙醇与高纯水，采用无水乙醇与高纯水透析的时间独立地可选为6～72h， 更可选为24h；所述透析后，本公开可选将透析袋中物料取出，冷冻干燥后，得到化合物S8，为粉红色非晶状固体。
得到化合物S8后，本公开将所述化合物S8、三氟乙酸与第三有机溶剂混合，进行脱叔丁氧羰基反应，得到化合物S9。在本公开中，所述化合物S8与三氟乙酸的用量比可选为1mmol：(10～1000)mL，更可选为1mmol：100mL；所述第三有机溶剂可选为二氯甲烷，本公开对所述第三有机溶剂的用量没有特殊限定，保证反应顺利进行即可。在本公开中，所述脱叔丁氧羰基反应可选在室温条件下进行，所述脱叔丁氧羰基反应的时间可选为0.5～48h，更可选为24h；所述脱叔丁氧羰基反应可选在氮气保护条件下进行。所述脱叔丁氧羰基反应后，本公开可选将所得产物体系进行旋蒸除去溶剂和三氟乙酸，将所得粗产物溶解于乙酸乙酯中，然后置于透析袋中进行透析，得到化合物S9。在本公开中，所述透析采用的透析液可选依次为无水乙醇与高纯水，采用无水乙醇与高纯水透析的时间独立地可选为6～72h，更可选为24h；所述透析后，本公开可选将透析袋中物料取出，冷冻干燥后，得到化合物S9，为粉红色非晶状固体。
在本公开中，当以D-Asp、L-Asp-A或L-Asp-N为靶向基团制备相应的靶向纳米载体(PDPA为疏水性聚合物，PEG为亲水性连接物)时，采用本领域常规化学合成方法即可，具体的，其制备方法可以与上述以L-Asp为靶向基团制备靶向纳米载体的方法基本相同，在此不再赘述。
本公开提供了上述技术方案所述靶向纳米载体或上述技术方案所述制备方法制备得到的靶向纳米载体作为活体植物植株、活体植物植株的组织、活体植物植株的器官、活体植物植株的细胞、体外培养的外植体(explant)、体外培养的愈伤组织、体外培养的植物组织或体外培养的植物细胞的主动靶向纳米载体的应用。在本公开中，所述活体植物植株可选包括单子叶植物或真双子叶植物。在本公开中，所述单子叶植物可选包括天门冬目植物、禾本目植物、鸭跖草目植物或棕榈目植物；所述天门冬目植物可选包括兰科植物；所述禾本目植物可选包括禾本科植物；所述禾本科植物可选包括玉米、水稻、小麦、高粱、竹子或荞麦；所述鸭跖草目植物可选包括鸭跖草科植物；所述鸭跖草科植物可选包括鸭跖草。在本公开中，所述真双子叶植物包括菊目植物、葫芦目植物、豆目植物、茄目植物或十字花目植物；所述菊目植物可选包括菊科植物；所述葫芦目植物可选包括葫芦科植物；所述豆目植物可选包括豆科植物，所述豆科植物可选包括大豆或豌豆；所述茄目植物可选包括茄科植物，所述茄科植物可选包括番茄、辣椒或马铃薯；所述十字花目植物可选包括十字花科植物，所述十字花科植物可选包括拟南芥或油菜。在本公开中，所述活体植物植株的器官可选包括叶片、种子或根；所述体外培养的植物细胞可选包括体外培养的原生质体，所述体外培养的原生质体具体可以为叶片、胚轴或根尖制备得到。在本公开中，所述靶向纳米载体能够实现植物细胞壁及细胞膜的主动靶向和穿越，形成“特洛伊木马”式的细胞穿透和靶向。将本公开所述靶向纳米载体用于对植物递送药物，能够降低用药剂量和成本、对搭载药物具有保护作用并提高药物效率、延长药物作用时间，降低毒性和污染性，降低耐药概率。
在本公开中，所述靶向纳米载体可以单独使用，也可以与非靶向修饰的纳米载体混合使用，当所述靶向纳米载体与非靶向修饰的纳米载体混合使用时，所述靶向纳米载体的质量可选为靶向纳米载体与非靶向修饰的纳米载体总质量的1％以上，具体可以为20～80％；本公开所述非靶向修饰的纳米载体具体是指没有任何修饰的纳米载体或者采用除本公开所述靶向物以外的物质修饰的纳米载体。
本公开提供了一种靶向载药纳米载体，包括靶向纳米载体以及包载在所述靶向纳米载体中的药物，所述靶向纳米载体为上述技术方案所述靶向纳米载体或上述技术方案所述制备方法制备得到的靶向纳米载体。在本公开中，所述靶向载药纳米载体的载药率可选为1～99％，更可选为30～80％，在本公开中，所述药物可选包括小分子药物或生物大分子；所述生物大分子可选包括核酸、蛋白质、氨基酸、多肽、糖类物质或脂类物质，所述核酸具体可以为DNA或RNA；所述小分子药物可选包括植物激素、保水剂、促生长药物、抗虫害药物、抗冻药物、抗热药物、抗紫外线药物、荧光素、转基因药物或同位素标记的化合物，所述植物激素可选包括生长素(auxin)、赤霉素、细胞分裂素、乙烯、茉莉酸、油菜甾醇、独脚金内酯、脱落酸(ABA)或ABA类似物(如Pyrabactin、Quinabactin、Opabacti、AM1、AMF1α、AMF1β、AMF2α、AMF2β、AMF4或AMC1β)。在本公开的实施例中，具体以ABA为例进行说明；ABA是一种植物内源激素，当植物受到干旱、盐等胁迫时，ABA能够关闭叶片气孔，减少水分蒸腾，激活下游抗逆信号，从而达到植物抗旱的效果，但由于造价昂贵并且在体外及其不稳定，难以用于农业生产；通过采用本公开提供的靶向纳米载体包载ABA，能够有效增强植物抗旱效果，大大减少ABA用量。
在本公开中，所述药物与靶向纳米载体的结合方式可选包括亲疏水作用力、氢键、静电作用力或化学键结合。
本公开提供了上述技术方案所述靶向载药纳米载体的制备方法，包括以下步骤：
将靶向纳米载体、药物与溶剂混合，进行包载处理，得到靶向载药纳米载体。
本公开可选靶向纳米载体与药物分别溶解于有机溶剂中，将所得靶向纳米载体溶液与药物溶液混合，得到混合溶液；将所述混合溶液滴加至水中进行包载处理，得到靶向载药纳米颗粒。本公开对制备所述靶向纳米载体溶液与药物溶液采用的有机溶剂种类没有特殊限定，根据靶向纳米载体与药物种类选择即可；在本公开的实施例中，以Asp(包括D-Asp、L-Asp-A、L-Asp-N或L-Asp)为靶向基团、PDPA为疏水性聚合物、PEG为亲水性连接物制备的靶向纳米载体(Asp-PEG-PDPA)为例，所用有机溶剂可选为四氢呋喃；以ABA为所搭载药物为例，所用有机溶剂可选为乙醇。在本公开中，所述靶向纳米载体溶液与药物溶液的浓度独立地可选为1～10mg/mL，更可选为5mg/mL。在本公开中，所述靶向纳米 载体溶液与药物溶液的体积比可选以得到所需载药量的靶向载药纳米载体为基准，本公开对此不作特殊限定。在本公开中，所述混合溶液与水的体积比可选为(0.2～0.5)：1，更可选为(0.3～0.4)：1；本公开可选将所述混合溶液逐滴加入至水中，每滴的体积可选为10μL。
在本公开中，所述包载处理过程中，靶向纳米载体与药物进行自组装(例如可以在亲疏水或电荷吸附等作用力下进行自组装)，得到靶向载药纳米载体。本公开可选根据靶向纳米载体与药物的特性选择合适的包载处理条件，具体的，所述包载处理可以在搅拌、超声、电刺激或加热条件下进行，以提高效率，在本公开的实施例中，以Asp-PEG-PDPA为靶向纳米载体、ABA为所搭载药物为例，所述包载处理可选在室温、搅拌条件下进行，所述包载处理的时间可选为2～4h，更可选为3h。
所述包载处理后，本公开可选将所得产物体系于Millipore Amicon Ultra-4 5K离心过滤装置中离心过滤，取出上清液，靶向载药纳米载体即分散于所述上清液中，置于4℃保存；所述离心过滤的转速可选为3600rpm，时间可选为16min。
本公开对所述靶向载药纳米载体的使用方法没有特殊限定，采用本领域技术人员熟知的方法即可。在本公开中，所述靶向载药纳米载体可选以靶向载药纳米载体分散液的保护形式使用。在本公开中，所述靶向载药纳米载体分散液可选是将靶向载药纳米载体分散于溶剂中得到；所述溶剂可选为水和/或有机溶剂，所述有机溶剂可选包括乙醇、二甲基亚砜或四氢呋喃；所述靶向载药纳米载体分散液的浓度可选为0.1～10mg/mL。在本公开中，所述靶向载药纳米载体分散液的使用方式可选包括喷洒、浸泡、涂抹或注射；以应用例为例，具体是采用靶向载药纳米载体分散液浸泡种子，浸泡时间可选为1～168h；或者将所述靶向载药纳米载体分散液喷洒于植物叶片上，所述靶向载药纳米载体分散液的用量可选为10～10000μL/cm2，更可选为10～500μL/cm2。
在本公开的实施例中，以Asp为靶向基团、PDPA为疏水性聚合物、PEG为亲水性连接物制备靶向纳米载体(Asp-PEG-PDPA)，在此基础上以ABA为所搭载药物，其与Asp-PEG-PDPA经自组装形成靶向载药纳米载体(Asp-NP@ABA)，然后喷洒于植物叶片可以提高其抗旱能力，制备Asp-NP的流程图以及以搭载ABA为例所得靶向载药纳米载体作为抗旱剂的应用示意图具体如图1所示。在本公开中，当所述靶向基团Asp分别为D-Asp、L-Asp-A、L-Asp-N或L-Asp时，对应的各靶向纳米载体自组装形成胶束Asp-NP分别记为D-Asp-NP、A-Asp-NP、N-Asp-NP和L-Asp-NP，各靶向纳米载体搭载ABA经自组装形成靶向载药纳米载体Asp-NP@ABA分别记为D-Asp-NP@ABA、A-Asp-NP@ABA、N-Asp-NP@ABA和L-Asp-NP@ABA。
下面将结合本公开中的实施例，对本公开中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
对比例1
制备MeO-PEG-PDPA(化合物S5)的反应式如下所示：
MeO-PEG-CPADN(化合物S3)的合成：
在氮气保护和室温条件下，将化合物S1(4-氰基-4-(硫代苯甲酰)戊酸，27.9mg，0.1mmol，1.0equiv.)、二环己基碳二亚胺(DCC，24.8mg，0.12mmol，1.2equiv.)、1-羟基苯并三唑(HOBt，16.2mg，0.12mmol，1.2equiv.)和4-二甲氨基吡啶(DMAP，1.2mg，0.01mmol，0.1equiv.)溶解在二氯甲烷(DCM，1mL)中，搅拌5min，得到混合物料；将化合物S2(Amino-PEG5000-OMe，500mg，0.1mmol，1.0equiv.)溶解在DCM(5mL)中，然后一次性加到所述混合物料中，在室温条件下搅拌反应24h；将所得产物体系通过旋蒸除去溶剂DCM，将所得粗产物溶解于EtOAc(5mL)中，经过滤除去不溶物，将滤液浓缩得到化合物S3，无需进一步纯化将其投入下一步反应中。
MeO-PEG-PDPA(化合物S5)的合成：
将化合物S4(533.3mg，2.5mmol，25equiv.)和偶氮二异丁腈(AIBN，1.6mg，0.01mmol，0.1equiv.)溶解在N,N-二甲基甲酰胺(DMF，5mL)中，在氮气保护下，向所得混合液中加入上一步产物化合物S3，然后在70℃条件下搅拌反应24h；反应结束后，将所得产物体系冷却至室温，然后置于透析袋中进行纯化，具体是先以无水乙醇作为透析液，透析24h，再把透析液换成高纯水，透析24h，然后将透析袋中物料取出，冷冻干燥后，得到化合物S5，即MeO-PEG-PDPA(具体为MeO-5kPEG-4kPDPA)， 为粉红色非晶状固体。
实施例1
制备以L-Asp为靶向基团的Asp-PEG-PDPA(化合物S9)的反应式如下所示：
在氮气保护和室温条件下，将化合物S1(27.9mg，0.1mmol，1.0equiv.)、DCC(24.8mg，0.12mmol，1.2equiv.)、1-羟基苯并三唑(HOBt，16.2mg，0.12mmol，1.2equiv.)和4-二甲氨基吡啶(DMAP，1.2mg，0.01mmol，0.1equiv.)溶解在二氯甲烷(DCM，1mL)中，搅拌5min，得到混合物料；将化合物S6(Amino-PEG5000-Boc-Asp-OtBu，500mg，0.1mmol，1.0equiv.)溶解在DCM(5mL)中，然后一次性加到所述混合物料中，在室温条件下搅拌反应24h；将所得产物体系通过旋蒸除去溶剂DCM，将所得粗产物溶解于乙酸乙酯(EtOAc，5mL)中，经过滤除去不溶物，将滤液浓缩得到化合物S7，无需进一步纯化将其投入下一步反应中；
将化合物S4(2-(二异丙基氨基)甲基丙烯酸乙酯，533.3mg，2.5mmol，25equiv.)和AIBN(1.6mg，0.01mmol，0.1equiv.)溶解在DMF(5mL)中，在氮气保护下，向所得混合液中加入化合物S7，然后在70℃条件下搅拌反应24h；反应结束后，将所得产物体系冷却至室温，然后置于透析袋中进行纯化，具体是先以无水乙醇作为透析液，透析24h，再把透析液换成高纯水，透析24h，然后将透析袋中物料取出，冷冻干燥后，得到化合物S8，为粉红色非晶状固体；
在氮气保护下，将化合物S8溶解在5mL DCM和5mL三氟乙酸(TFA)的混合溶液中，将所得混合物料在室温条件下搅拌反应24h；将所得产物体系通过旋蒸除去溶剂DCM和TFA，将所得粗产物溶解于EtOAc(5mL)中，然后置于透析袋中进行纯化，具体是先以无水乙醇作为透析液，透析24h(期间更换5次透析液)，再把透析液换成高纯水，透析24h(期间更换5次透析液)，然后将透析袋中物料取出，冷冻干燥后，得到化合物S9，为粉红色非晶状固体。
参照上述方法，分别制备以D-Asp、L-Asp-A和L-Asp-N为靶向基团的Asp-PEG-PDPA。
将实施例1制备的化合物S9进行核磁表征，结果如图2所示，由图2可确定实施例1制备的产物为Asp-PEG-PDPA。
将实施例1制备的Asp-PEG-PDPA粉末(靶向基团为D-Asp、L-Asp-A、L-Asp-N或L-Asp)制备成胶束Asp-NP(具体为D-Asp-NP、A-Asp-NP、N-Asp-NP和L-Asp-NP)后进行透射电镜表征，具体是将Asp-PEG-PDPA粉末(1mg，0.625mmol)溶解在四氢呋喃(0.2mL)中，得到Asp-PEG-PDPA溶液；在装有转子的反应瓶内加入超纯水(1mL)，将所述Asp-PEG-PDPA溶液以每10μL为一滴逐滴加入到反应瓶中，滴加结束后，室温条件下搅拌3h，全程在通风橱内操作；之后将所得体系于Millipore Amicon Ultra-4 5K离心过滤装置中离心过滤(3600rpm，16min)，取出上清液(具体为离心过滤装置的内管中的液体)，Asp-NP即分散于所述上清液中，置于4℃保存。
图3为实施例1中L-Asp-NP的透射电镜图(比例尺为50nm)，由图3可知，Asp-NP纳米粒子大小均一，粒径在100nm左右。
实施例2
将实施例1制备的Asp-PEG-PDPA粉末(1mg，0.625mmol；靶向基团为D-Asp、L-Asp-A、L-Asp-N或L-Asp)溶解在四氢呋喃(0.2mL)中，得到Asp-PEG-PDPA溶液；将脱落酸(ABA，5mg，18.9mmol)溶解在乙醇(1mL)中，得到ABA溶液；将0.2mL所述Asp-PEG-PDPA溶液(含1mg的Asp-PEG-PDPA)与一定体积ABA溶液混合，使ABA质量为Asp-PEG-PDPA和ABA总质量的40％，得到混合溶液；
在装有转子的反应瓶内加入超纯水(1mL)，将所述混合溶液以每10μL为一滴逐滴加入到反应瓶中，滴加结束后，室温条件下搅拌3h，全程在通风橱内操作；反应结束后，将所得产物体系于Millipore Amicon Ultra-4 5K离心过滤装置中离心过滤(3600rpm，16min)，取出上清液(具体为离心过滤装置的内管中的液体)，载药产物(记为Asp-NP@ABA，具体为D-Asp-NP@ABA、A-Asp-NP@ABA、 N-Asp-NP@ABA和L-Asp-NP@ABA)即分散于所述上清液中，置于4℃保存。
对比例2
按照实施例2的方法制备载药产物，不同之处仅在于将Asp-PEG-PDPA粉末替换为MeO-PEG-PDPA，最终所得载药产物记为NP@ABA。
采用动态光散射仪(DLS)检测NP(具体为对比例1制备的MeO-PEG-PDPA)、NP@ABA、Asp-NP(具体为D-Asp-NP、A-Asp-NP、N-Asp-NP和L-Asp-NP)和Asp-NP@ABA(具体为D-Asp-NP@ABA、A-Asp-NP@ABA、N-Asp-NP@ABA和L-Asp-NP@ABA)的粒径分布以及多分散系数，结果如图4所示。由图4可知，新制备L-Asp-NP@ABA的粒径为135.5±4.2nm，PDI为0.164±0.038(放置12个月后，L-Asp-NP@ABA的粒径为139.4±5.9nm，PDI为0.15±0.03)；新制备NP@ABA的粒径为139.53±1.21nm，PDI为0.156±0.012(放置9个月后，NP@ABA的粒径为136.6±13.1nm，PDI为0.32±0.08)。新制备的A-Asp-NP@ABA的粒径为177.6±1.5nm，PDI为0.17±0.01；新制备的D-Asp-NP@ABA的粒径为173.9±3.8nm，PDI为0.12±0.02；新制备的N-Asp-NP@ABA的粒径为169.7±3.1nm，PDI为0.18±0.03。
对Asp-NP@ABA和NP@ABA的载药率进行测定，结果如图4所示。由图4可知，L-Asp-NP@ABA的载药率为72.07±1.92％，A-Asp-NP@ABA的载药率为65.3±1.4％，D-Asp-NP@ABA的载药率为64.2±2.8％，N-Asp-NP@ABA的载药率为69.1±1.0％；NP@ABA的载药率为60.09±2.79％。
以下应用例中，若无特殊说明，所述的Asp靶向基团均为L-Asp；当采用其它构型的Asp靶向基团时均进行标注说明。
应用例1 Asp-NP能够高效穿透植物组织细胞壁和细胞膜
按照实施例2的方法制备Asp-NP@DiO，不同之处仅在于将ABA替换成3,3′-双十八烷基氧碳花菁高氯酸盐(DiO；CAS：34215-57-1)。
按照上述方法制备NP@DiO，不同之处仅在于将Asp-PEG-PDPA替换为MEO-PEG-PDPA。
将Asp-NP@DiO分散于超纯水中，得到浓度为31.8μM的Asp-NP@DiO分散液；将所述Asp-NP@DiO分散液喷洒在14天大的拟南芥叶片上，每株约为60μL；在36h后，使用激光共聚焦显微镜，基于其3D层扫功能追踪DiO荧光信号在拟南芥叶片表面到垂直往下的叶肉组织中的穿透深度，并设置MS、DiO以及NP@DiO对照组，具体结果见图5(标尺为50μm)和图6。图5展示了在20μm时的共聚焦显微镜的观察结果。根据图5中对DiO信号观察可以看出，在20μm这一深度时，只有Asp-NP能够穿透并将DiO带入这个组织深度。同时，通过叠加图可以看出，Asp-NP@DiO组有很强的叶绿体(红色颗粒)和DiO信号的共定位，说明Asp-NP@DiO很有可能已经穿过了细胞壁和细胞膜到达了胞内。图6中每一个点代表一次重复试验，总共采用了4株拟南芥。由图6可知，Asp-NP@DiO在喷洒后的叶片中组织穿透深度最大，这说明Asp-NP在喷洒后短时间内具有较强的组织穿透能力。
将所述Asp-NP@DiO分散液与分离的拟南芥叶肉细胞原生质体共培养4h；更换新鲜W5培养基培养20h，之后使用激光共聚焦显微镜，追踪DiO荧光信号在原生质体质膜往内到胞内的亚细胞定位，并设置W5缓冲液、DiO以及NP@DiO对照组，具体结果见图7(标尺为50μm)。
采用异硫氰酸荧光素(Fluorescein isothiocyanate；FITC)标记Asp-NP得到Asp-NP-FITC，将所述Asp-NP-FITC分散于超纯水中，得到浓度为12.5μM的Asp-NP-FITC分散液；将所述Asp-NP-FITC分散液喷洒在鸭跖草叶片上，每片叶约为10μL；在6h后，使用激光共聚焦显微镜，基于其3D层扫功能追踪FITC荧光信号在鸭跖草叶片表面到垂直往下的叶肉组织中的穿透深度，同时设置MS组作为对照，具体结果见图8(标尺为50μm)。图8展示了在不同深度时的共聚焦显微镜的观察结果。根据图8中对FITC信号观察可以看出，Asp-NP能够穿透并将FITC带入接近50μm这个组织深度。因为鸭跖草的叶片上表皮不具有气孔，此实验确认了靶向纳米载体是穿过细胞壁而不是通过气孔进行目标分子的递送。
综合图5、图6、图7和图8的结果，进一步证明了本公开制备的Asp-NP能够高效穿透不同植物的组织、细胞壁和细胞膜，把目标分子递送进入活体植物组织和细胞内。
应用例2 Asp-NP@ABA能够高效通过种皮和根部进入植物并进行递送
将野生型(WT)拟南芥种子置于24孔板中，每孔20颗；以1/2MS(pH＝6.7)液体培养基稀释Asp-NP@ABA(具体为D-Asp-NP@ABA、A-Asp-NP@ABA、N-Asp-NP@ABA或L-Asp-NP@ABA)，分别得到具有不同靶向基团的Asp-NP@ABA分散液(以ABA浓度计，浓度为0.1μM)，在每孔中加入每种Asp-NP@ABA分散液(400μL)进行孵育，每个浓度设置6个孔重复，置于培养箱中萌发(16h光照/8h黑暗)，每12h统计发芽率，连续统计7天。同时设置MS、ABA、NP、Asp-NP(具体为D-Asp-NP、A-Asp-NP、N-Asp-NP或L-Asp-NP)以及NP@ABA作为对照。
图9为采用不同处理后拟南芥种子发芽率对比图，由图9可知，具有不同靶向基团的Asp-NP@ABA相较于ABA均能够更有效的进入种子组织内部，而被Asp-NP递送进种子组织内部的ABA能够更显著延缓种子萌发。
将Asp-NP-FITC分散于1/2MS培养基中，使所述Asp-NP-FITC的浓度为12.5μM，得到含有Asp-NP-FITC的培养基；然后分别将7天大的拟南芥小苗根部、4天大的大豆小苗根部以及4天大的玉米小苗根部浸泡于所述含有Asp-NP-FITC的培养基中，在不同时间点观察Asp-NP-FITC穿透各植物根部组织的深度。同时设置MS组作为对照。
图10为Asp-NP-FITC在不同时间点穿透拟南芥根部组织不同深度的对比图(标尺为100μm)。图11为Asp-NP-FITC在4h和6h后穿透大豆根部组织不同深度的对比图(标尺为100μm)。图12为 Asp-NP-FITC在4h和6h后穿透玉米根部组织不同深度的对比图(标尺为100μm)。由图10、图11和图12分别能看出，Asp-NP-FITC通过浸泡的方式能够高效地进入不同物种植物的根部组织。
将10天大的拟南芥小苗置于水培系统中，仅根部浸泡于1/2MS(pH＝6.7)液体培养基稀释的Asp-NP@ABA分散液(所用Asp-NP@ABA具体为D-Asp-NP@ABA、A-Asp-NP@ABA、N-Asp-NP@ABA或L-Asp-NP@ABA；以ABA浓度计，浓度为2.5μM)。每株小苗的浸泡体积为1.8mL，每次实验设置10株重复，置于培养箱中萌发(16h光照/8h黑暗)，每12h统计叶片衰老黄化的数量，连续统计2天。同时设置MS、ABA、NP、Asp-NP(具体为D-Asp-NP、A-Asp-NP、N-Asp-NP或L-Asp-NP)以及NP@ABA作为对照。
图13为水培系统中采用不同处理后拟南芥的根部吸收后ABA引起的叶片衰老黄化的数量统计图。由图13可知，相较于其他处理，具有不同靶向基团的Asp-NP@ABA能够更高效地把ABA经由根部递送进入植物组织内部，ABA被递送进细胞后，能够引起叶片衰老，显示出黄化，因此较多的黄化叶片数量显示出相应的Asp-NP递送ABA的效率更高。
应用例3 Asp-NP@ABA赋予模式植物抗旱能力
将Asp-NP@ABA分散于超纯水中，得到Asp-NP@ABA分散液，所述Asp-NP@ABA分散液中ABA的浓度为10μM；将所述Asp-NP@ABA分散液以每棵苗约60μL的量喷洒于拟南芥叶片(3周龄)上，喷洒后不再浇水，置于培养箱中生长(8h光照/16h黑暗，光照时温度为22℃，黑暗时温度为19.8℃)，每天记录植物生长状况。
图14为采用不同处理喷洒拟南芥小苗后在干旱条件下延长小苗生存周期的对比图，由图14可知，Asp-NP@ABA处理组在干旱18日后相较于其他组相比仍然有良好的生命力。说明各个植株被Asp-NP@ABA处理后能够延长生存周期。图15为采用不同处理喷洒拟南芥小苗后的生存率对比图，具体是采用不同处理喷洒拟南芥小苗后开始至第12天复水一次，即前11天均保持干旱，在第13天检测生存率，进一步验证Asp-NP@ABA对于植株抗旱的准确性。由图15可知，Asp-NP@ABA处理组的拟南芥植株生存率为100％，而其他处理组生存率为0％。图16为采用不同处理喷洒拟南芥小苗后在干旱条件下延长小苗的生存周期相对于MS处理组延长的生存周期的百分比散点图(设置4次平行试验，本试验所采用的Asp-NP@ABA具体为D-Asp-NP@ABA、A-Asp-NP@ABA、N-Asp-NP@ABA或L-Asp-NP@ABA，所采用的Asp-NP具体为D-Asp-NP、A-Asp-NP、N-Asp-NP或L-Asp-NP)，由图16可知，植物植株被具有不同靶向基团的Asp-NP@ABA处理后，生存周期被延长了平均57％。图17为以干旱条件下的生存周期为衡量标准测定的Asp-NP@ABA最低有效浓度结果图(以ABA处理的结果作为参照)，由图17可知，相同干旱条件下，Asp-NP@ABA可以将脱落酸浓度降低至ABA的10万倍到百万倍。
进一步研究发现，拟南芥叶片表面喷洒Asp-NP@ABA后，使用高效液相色谱质谱联用技术在原生质体和叶片的细胞外基质(即细胞壁和细胞间隙)中明显检测到ABA的富集。图18为拟南芥叶片喷洒NP@ABA以及Asp-NP@ABA 24h后细胞质内和质外体内的ABA含量对比图，由图18可知，NP@ABA以及Asp-NP@ABA均能够有效的将ABA运输到细胞壁中，使ABA在细胞外区域富集；而在同样的叶片中提取的原生质体中ABA的富集情况所有不同，喷洒Asp-NP@ABA时原生质体中ABA富集较多，这说明当同样量的ABA被纳米载体载入细胞壁后，Asp的靶向修饰才能高效的跨越细胞膜进入细胞质内。
应用例4 Asp-NP@ABA赋予真双子叶植物和单子叶植物抗旱能力
将Asp-NP@ABA分散于超纯水中，得到Asp-NP@ABA分散液，所述Asp-NP@ABA分散液中ABA的浓度为10μM；将所述浓度为10μM的Asp-NP@ABA分散液以每棵苗约600～800μL的量喷洒于大豆叶片(5周龄)上，喷洒后不再浇水，置于培养箱中生长(8h光照/16h黑暗，光照时温度为22℃，黑暗时温度为19.8℃)，每天记录植物生长状况。
图19为采用不同处理喷洒大豆后在干旱条件下延长大豆生存周期的对比图，由图19可知，Asp-NP@ABA处理组在干旱8日后相较于其他组相比仍然有良好的生命力。说明各个大豆植株被Asp-NP@ABA处理后能够增强抗旱能力。图20为采用不同处理喷洒大豆后在干旱条件下延长大豆的生存周期的中位值统计图。由图19和图20可知，大豆植株被Asp-NP@ABA处理后，生存周期中位值被延长了平均50％。
同时，将Asp-NP@ABA分散于超纯水中，得到Asp-NP@ABA分散液，所述Asp-NP@ABA分散液中ABA的浓度为50μM；将所述浓度为50μM的Asp-NP@ABA分散液以每棵苗约7-8mL的量喷洒于玉米叶片(10周龄)上，喷洒后不再浇水，置于室外生长，每天记录植物生长状况。
图21为采用不同处理喷洒玉米后在干旱条件下延长玉米生存周期的对比图，由图21可知，Asp-NP@ABA处理组在干旱34日后相较于ABA组相比仍然有良好的生命力。说明各个玉米植株被Asp-NP@ABA处理后能够增强抗旱能力。
应用例5
将实施例1制备的Asp-PEG-PDPA粉末与非靶向的聚合物(即MeO-PEG-PDPA)混合，得到混合粉末，所述混合粉末中Asp-PEG-PDPA粉末的质量含量分别为20％、40％、60％、80％或100％；将所述混合粉末(1mg)溶解在四氢呋喃(0.2mL)中，得到混合溶液；将脱落酸(ABA，5mg，18.9mmol)溶解在乙醇(1mL)中，得到ABA溶液；将0.2mL所述混合溶液与一定体积ABA溶液混合，使ABA 质量为所述混合粉末与ABA(即Asp-PEG-PDPA+MeO-PEG-PDPA+ABA)总质量的40％，得到含ABA混合溶液；在装有转子的反应瓶内加入超纯水(1mL)，将所述含ABA混合溶液以每10μL为一滴逐滴加入到反应瓶中，滴加结束后，室温条件下搅拌3h，全程在通风橱内操作；反应结束后，将所得产物体系于Millipore Amicon Ultra-4 5K离心过滤装置中离心过滤(3600rpm，16min)，取出上清液(具体为离心过滤装置的内管中的液体)，载药产物即分散于所述上清液中，置于4℃保存。
按照应用例2和应用例3的方法分别对所述载药产物进行性能测试，结果如图22和图23所示。由图22和图23可知，在同一ABA浓度条件下，添加不同比例非靶向的聚合物(即MeO-PEG-PDPA)的载药产物对于种子萌发的抑制效果以及对拟南芥在干旱条件下的生存周期的延长效果没有统计学差异。
由以上应用例可知，本公开提供的纳米递送载体至少具有以下有益效果：
1)操作便捷，载药后可以直接分散于溶剂中喷洒在多种不同的植物表面，也可以采用浸泡、涂抹或注射方式；2)应用范围广，对被装载的药物种类具有高度兼容性，具有较高的载药率，并且可以用于不同的植物、品种和组织器官；3)递送效率极高，在应用例3条件下可以将脱落酸浓度降低10万倍到百万倍；4)作用时间久，在应用例3中干旱条件下的植物生存周期延长57％，生存率为100％，而其他处理组生存率为0％；5)性质稳定，12个月后粒径仍稳定不变；6)成本可控，技术路线简单，可大规模生产。
因此，本公开提供的靶向纳米载体具有广泛应用的潜力，尤其是其特性使得在极地、极端恶劣环境下具有极佳的优势，如科考拓荒、外星探索。
以上所述仅是本公开的可选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本公开原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本公开的保护范围。
工业实用性
本公开提供的靶向纳米载体包括纳米载体以及化学键合在所述纳米载体上的靶向物，其中，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子，所述靶向物为天冬氨酸或天冬氨酸衍生物。本公开提供的靶向纳米载体能够主动穿透植物细胞壁和细胞膜，适用于对活体植物或组织等进行药物递送，能够降低用药剂量和成本、对搭载药物具有保护作用并提高药物效率、延长药物作用时间，降低毒性和污染性，降低耐药概率，具有优异的工业实用性。

Claims (10)

  1. 一种靶向纳米载体，包括纳米载体以及化学键合在所述纳米载体上的靶向物，其中，所述纳米载体为有机高分子聚合物或无机材料形成的纳米粒子，所述靶向物为天冬氨酸或天冬氨酸衍生物。
  2. 根据权利要求1所述的靶向纳米载体，其特征在于，所述天冬氨酸提供的靶向基团包括式I～IV任一所示结构的基团：
  3. 根据权利要求1所述的靶向纳米载体，其特征在于，所述纳米载体的粒径为10～1000nm。
  4. 根据权利要求1或3所述的靶向纳米载体，其特征在于，所述有机高分子聚合物的数均分子量为3～50kDa。
  5. 根据权利要求1所述的靶向纳米载体，其特征在于，所述有机高分子聚合物包括疏水性聚合物以及与所述疏水性聚合物共价连接的亲水性连接物，所述亲水性连接物与所述靶向物经化学键合连接。
  6. 权利要求1～5任一项所述靶向纳米载体的制备方法，包括以下步骤：
    在溶剂存在条件下，在纳米载体上经化学键合修饰靶向物，得到靶向纳米载体。
  7. 权利要求1～5任一项所述靶向纳米载体或权利要求6所述制备方法制备得到的靶向纳米载体作为活体植物植株、活体植物植株的组织、活体植物植株的器官、活体植物植株的细胞、体外培养的外植体、体外培养的愈伤组织、体外培养的植物组织或体外培养的植物细胞的主动靶向纳米载体的应用。
  8. 一种靶向载药纳米载体，包括靶向纳米载体以及包载在所述靶向纳米载体中的药物，所述靶向纳米载体为权利要求1～5任一项所述靶向纳米载体或权利要求6所述制备方法制备得到的靶向纳米载体。
  9. 根据权利要求8所述的靶向载药纳米载体，其特征在于，所述药物包括小分子药物或生物大分子，所述靶向载药纳米载体的载药率为1～99％。
  10. 权利要求8或9所述靶向载药纳米载体的制备方法，包括以下步骤：
    将靶向纳米载体、药物与溶剂混合，进行包载处理，得到靶向载药纳米载体
PCT/CN2023/117689 2022-09-08 2023-09-08 一种靶向纳米载体及其制备方法和应用、靶向载药纳米载体及其制备方法 WO2024051809A1 (zh)

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