WO2022048470A1 - 介孔氧化硅纳米粒子控释系统、其制备方法及其应用 - Google Patents

介孔氧化硅纳米粒子控释系统、其制备方法及其应用 Download PDF

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WO2022048470A1
WO2022048470A1 PCT/CN2021/114083 CN2021114083W WO2022048470A1 WO 2022048470 A1 WO2022048470 A1 WO 2022048470A1 CN 2021114083 W CN2021114083 W CN 2021114083W WO 2022048470 A1 WO2022048470 A1 WO 2022048470A1
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mesoporous silica
group
functionalized
aryl
silica nanoparticles
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French (fr)
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朱维平
傅云
徐玉芳
钱旭红
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华东理工大学
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Priority to EP21863537.3A priority Critical patent/EP4209230A1/en
Priority to US18/043,594 priority patent/US20230310333A1/en
Publication of WO2022048470A1 publication Critical patent/WO2022048470A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

  • the invention belongs to the field of drug controlled release, and in particular relates to a mesoporous silica nanoparticle controlled release system, a preparation method thereof and its application in a microneedle patch.
  • porous materials are widely used in catalysts and adsorption carriers due to their large specific surface area and adsorption capacity. According to the pore size, porous materials can be divided into: microporous (Microporous), mesoporous (Mesoporous) and macroporous (Macroporous) materials.
  • the pore size of inorganic microporous materials is generally less than 2 nm, including tobermorite, activated carbon, zeolite, etc. The small pore size of microporous materials limits its catalysis and adsorption to organic macromolecules.
  • the pore size of macroporous materials is generally >50nm, including porous ceramics, cement, aerogel, etc., which are characterized by large pore size and wide distribution range. Materials with a pore size in between are called mesoporous materials. Mesoporous materials have the characteristics of extremely high specific surface area, regular and ordered pore structure, narrow pore size distribution, continuously adjustable pore size, and no physiological toxicity. It has good application prospects in the fields of burial and controlled release.
  • mesoporous silica materials in drug delivery has been widely studied.
  • the surface modification of mesoporous silica can realize the controlled release of the internal cargo, making it an ideal drug delivery carrier (Chem. Soc. Rev., 2012, 41(7): 2590-2605).
  • the mesoporous silica drug delivery platform is mainly composed of three parts: mesoporous silica, pore blocking agent, and sensitive response unit.
  • Surface-functionalized mesoporous silica with closed pore ports has the property of "zero premature release", which has attracted extensive attention of researchers and is known as an effective stimulus-responsive controlled release system.
  • Microneedle patch is a potential means of transdermal drug delivery.
  • the dissolving microneedle patch based on polysaccharide has the advantages of high drug delivery efficiency, high drug loading rate, convenient manufacturing process, and easy storage. Combining the microneedle patch with the insulin drug delivery system has broad application prospects.
  • the invention uses the mesoporous silica nanoparticles functionalized on the surface of the polyhydroxy compound as the carrier, and the nanoparticles functionalized on the surface of the phenylboronic acid matching the size of the mesopores as the pore blocking agent.
  • a controlled release system was designed and synthesized, which can be used for the controlled release of biological macromolecules such as insulin.
  • the present invention provides a mesoporous silica nanomaterial that can be used for controlled release
  • the mesoporous silica nanomaterial that can be used for controlled release comprises polyhydroxy compound-functionalized mesoporous silica nanomaterials Particles and Phenylboronic Acid Functionalized Nanoparticle Pore Blockers.
  • the phenylboronic acid group forms a linking group with the hydroxyl group of the polyhydroxy compound, thereby connecting the phenylboronic acid functionalized nanoparticle pore blocking agent and the polyhydroxy compound functionalized mesoporous dioxide Silicon nanoparticles.
  • the present invention can be used for controlled release mesoporous silica nanomaterials
  • the nanoparticle pore blocking agent is directly linked to the phenylboronic acid group or is linked to the phenylboronic acid group via a linker, consisting of The phenylboronic acid group reacts with the hydroxyl group of the polyhydroxy compound to form a linking group, and the polyhydroxy compound is then linked to the mesoporous silica nanoparticles via another linker.
  • the mesoporous silica nanoparticles have a particle size of 90-350 nm.
  • the mesoporous silica nanoparticles have a mesopore size of 5-20 nm.
  • the polyhydroxy compound is selected from monosaccharides and derivatives thereof, polysaccharides and derivatives thereof, and synthetic block copolymers, preferably from glucose and derivatives thereof.
  • the polyhydroxy compound is gluconic acid.
  • the polyol-functionalized mesoporous silica nanoparticles have the structure shown in Formula I below:
  • MSN refers to mesoporous silica nanoparticles
  • L 1 is a linker group; preferably, L 1 is a C 1-6 alkylene group, preferably a propylene group.
  • the size of the nanoparticle pore blocking agent matches the mesopore size of the mesoporous silica nanoparticles.
  • the size of the nanoparticle pore blocking agent is 3-25 nm.
  • the nanoparticle pore blocking agent is selected from the group consisting of metal oxide nanoparticles, metal sulfide nanoparticles, metal nanoparticles, quantum dots, block copolymers, natural polymers and biological macromolecules, Metal oxide nanoparticles are preferred, and zinc oxide nanoparticles are more preferred.
  • the phenylboronic acid-functionalized nanoparticle pore blocking agent has the structure shown in the following formula III or IV:
  • X refers to nanoparticle pore blocking agent
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from hydrogen, electron-withdrawing group substituent and electron-donating group substituent, preferably each independently selected from hydrogen, C 1 -6 alkyl, halogen, nitro, carboxyl and amino, more preferably each independently selected from hydrogen, C 1-6 alkyl and halogen
  • the relative positions of Z 1 , Z 2 , Z 3 , Z 4 and L 3 , L 4 and the phenylboronic acid group on the benzene ring are not limited, that is, L 3 or L 4 can be located in the phenyl boronic acid group The meta, para or ortho position of a group.
  • the phenylboronic acid-functionalized nanoparticle pore blocking agent is:
  • X refers to nanoparticle pore blocking agent.
  • the present invention also provides a method for preparing the mesoporous silica nanomaterial that can be used for controlled release according to any one of the present invention, characterized in that, the method comprises the following steps:
  • the mesoporous silica nanomaterials that can be used for controlled release further comprise functional active molecules.
  • the present invention also provides a functional active molecule nano-drug loading system, which comprises the mesoporous silica nanomaterials and functional active molecules that can be used for controlled release according to any embodiment of the present invention.
  • the size of the functionally active molecule is smaller than the mesopore size of the mesoporous silica nanoparticles.
  • the functionally active molecule comprises a diabetes therapeutic.
  • the diabetes therapeutic agent is insulin or a biologically active analog thereof.
  • the insulin or biologically active analog thereof is selected from the group consisting of human insulin, recombinant human insulin, insulin from a non-human animal, rapid-acting insulin, rapid-acting insulin analogs, intermediate-acting insulin, and long-acting insulin .
  • the functionally active molecule is loaded inside the mesoporous silica nanoparticles, eg, within the mesopores of the mesoporous silica nanoparticles.
  • the functionally active molecules include small molecule detection or diagnostic reagents, such as dyes, drugs, indicators, and the like.
  • the functionally active molecule is a functional quantum dot or nanoparticle having a size smaller than the mesopore size of the mesoporous silica nanoparticle.
  • the present invention also provides a method for preparing the functionally active molecule nano-drug-carrying system according to any embodiment of the present invention, the method comprising the following steps:
  • step (3) adding the pore blocking agent functionalized with phenylboronic acid to the reaction solution in step (2) for reaction, thereby obtaining the functional active molecule nano-drug-carrying system.
  • the present invention also provides a kit, which contains polyhydroxy compound-functionalized mesoporous silica nanoparticles and phenylboronic acid-functionalized nanoparticle pore blocking agent; preferably, the polyhydroxylated
  • the compound-functionalized mesoporous silica nanoparticles are as described in any embodiment of the present invention; preferably, the phenylboronic acid-functionalized nanoparticle pore blocking agent is as described in any of the embodiments of the present invention; optionally
  • the kit further contains functionally active molecules; preferably, the functionally active molecules are as described in any of the embodiments of the present invention; Silica nanoparticles, phenylboronic acid-functionalized nanoparticle pore blocking agents, and optional functional active molecules are stored in separate containers.
  • the present invention also provides a microneedle or microneedle array patch, the microneedle or microneedle array patch contains the mesoporous silica nanomaterial that can be used for controlled release according to any embodiment of the present invention or the present invention
  • the functionally active molecule nano-drug delivery system according to any one of the embodiments.
  • the present invention also provides the use of the mesoporous silica nanomaterial that can be used for controlled release according to any embodiment of the present invention in the preparation of a medicament for treating diabetes or controlling the blood sugar level of a patient.
  • the present invention also provides the use of the functionally active molecule nano-drug loading system according to any embodiment of the present invention in the preparation of a medicament for treating diabetes or controlling the blood sugar level of a patient, wherein the functionally active molecule comprises a diabetes therapeutic agent; preferably Preferably, the diabetes therapeutic agent is insulin or a biologically active analog thereof; preferably, the insulin or a biologically active analog thereof is selected from the group consisting of human insulin, recombinant human insulin, insulin from non-human animals, rapid-acting insulin, rapid-acting insulin Analogs, intermediate-acting insulin, and long-acting insulin.
  • the present invention also provides the mesoporous silica nanomaterial that can be used for controlled release according to any embodiment of the present invention or the functional active molecule nano-drug loading system according to any embodiment of the present invention, which is prepared for controlled release administration Use in medicines or diagnostic reagents or diagnostic kits.
  • FIG. 1 is a transmission electron microscope image of the nano-zinc oxide with a size of about 10 nm in Example 1.
  • Example 2 is an infrared spectrum diagram of ZnO-P1, mercaptophenylboronic acid, and nano-zinc oxide of Example 2.
  • FIG. 3 is a transmission electron microscope image of the mesoporous silica nanoparticles (M0-3) with a size of about 150 nm and uniform and ordered pores in Example 5.
  • M0-3 mesoporous silica nanoparticles
  • FIG. 4 is a transmission electron microscope image of the mesoporous silica nanoparticles (M1-1) loaded with fluorescein-labeled insulin and pore-blocking by zinc oxide of Example 8.
  • M1-1 mesoporous silica nanoparticles
  • Figure 5 shows the relationship between the release of FITC-insulin and the time of M1-1 of Example 29 in sugar solutions of different concentrations.
  • Figure 6 shows the relationship between the release of FITC-insulin and time of M2-1 of Example 30 in sugar solutions of different concentrations.
  • Figure 7 shows the relationship between the release of FITC-insulin and time of M3-1 of Example 31 in sugar solutions of different concentrations.
  • Figure 8 shows the relationship between the release of FITC-insulin and time of M4-1 of Example 32 in sugar solutions of different concentrations.
  • Example 9 is the cell viability of the cells of Example 33 cultured in the medium containing M0-3, M0-4 and M1-1.
  • Example 10 is a scanning electron microscope image of the SGRM microneedle patch of Example 35.
  • FIG. 11 is a scanning electron microscope image of the microneedles on the SGRM microneedle patch of Example 35.
  • FIG. 11 is a scanning electron microscope image of the microneedles on the SGRM microneedle patch of Example 35.
  • Figure 12 shows the changes in blood glucose concentration with time after the diabetic rats of Example 37 were injected with 1.5 mg M3-2, 2.0 mg M3-2 (4.1 IU), 1.0 mL PBS buffer solution and insulin (4.1 IU), respectively.
  • Figure 13 shows that the diabetic rats of Example 38 were injected with 2.0 mg M3-2 (4.1 IU) and insulin (4.1 IU), respectively, after 2.5 hours, and after the diabetic rats and normal rats were injected with glucose (1.5 g/kg) Changes in blood glucose concentration over time.
  • Figure 14 shows the changes in blood glucose concentration with time after injection of 2.0 mg M3-2 (4.1 IU) and insulin (4.1 IU) in the normal rats of Example 39, respectively.
  • Figure 15 shows the changes of blood glucose concentration with time in the diabetic mice of Example 40 after patching the blank patch, injecting insulin (12.0IU/kg) and patching the SGRM microneedle patch (12.0IU/kg) respectively.
  • Figure 16 shows that the diabetic mice of Example 41 were injected with insulin (12.0 IU/kg) and the patch SGRM microneedle patch (12.0 IU/kg), respectively. After 3 hours, the diabetic mice and normal mice were injected with glucose (1.5 g Changes in blood glucose concentration over time after /kg).
  • Fig. 17 shows the changes of blood glucose concentration with time in the normal mice of Example 42 after patching the SGRM microneedle patch (12.0 IU/kg) and injecting insulin (9.5 IU/kg) respectively.
  • FIG. 18 is a transmission electron microscope image of mesoporous silica nanoparticles with a size of about 170 nm.
  • FIG. 19 is a transmission electron microscope image of mesoporous silicon oxide nanoparticles with a size of about 300 nm.
  • halogen includes F, Cl, Br and I.
  • alkyl refers to a linear or branched monovalent saturated hydrocarbon group, usually containing 1-12 carbon atoms (C 1-12 alkyl), preferably 1-6 carbon atoms (C 1-6 alkyl).
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.
  • alkylene refers to a straight-chain or branched divalent saturated hydrocarbon group, usually containing 1-12 carbon atoms (C 1-12 alkylene), preferably containing 1-6 carbon atoms (C 1-6 alkylene) alkyl).
  • alkylene groups include, but are not limited to, methylene, ethylene, propylene, and the like.
  • alkenyl refers to a group having at least one double bond in a straight or branched chain, and the carbon chain length is usually 2-12 carbon atoms, preferably 2-6 carbon atoms.
  • Typical alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl and 2-butenyl.
  • alkynyl refers to a group having at least one triple bond in a straight or branched chain, and the carbon chain length is usually 2-12 carbon atoms, preferably 2-6 carbon atoms.
  • Typical alkynyl groups include ethynyl, 1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl and 2-butynyl.
  • alkoxy refers to "alkyl-O-", wherein the alkyl group may have 1-12 carbon atoms, preferably 1-6 carbon atoms.
  • electron withdrawing group substituents can be selected from nitro, cyano, halogen, carboxyl, alkynyl and alkenyl; electron donating group substituents can be selected from alkyl, secondary amine, primary amine, tertiary amine, hydroxyl and alkoxy.
  • reactive functional groups refer to functional groups that can undergo addition, condensation, cyclization, polymerization and other reactions with each other to be covalently linked, including but not limited to: amino group, hydroxyl group, carboxyl group, sulfonic acid group, mercapto group, alkenyl group , alkynyl, azide, tetrazine structure, halogen, hydrazine, epoxy, isocyanate, isothiocyanate, etc.
  • derivatives refer to more complex products derived from the substitution of hydrogen atoms or atomic groups in a compound by other atoms or atomic groups.
  • silicon oxide and silicon dioxide have the same meaning.
  • the mesoporous silica nanomaterials (herein referred to as controlled release nanomaterials) that can be used for controlled release of the present invention contain polyhydroxy compound-functionalized mesoporous silica nanoparticles and phenylboronic acid-functionalized nanoparticles to block pores agent.
  • the controlled release nanomaterials of the present invention consist of polyol functionalized mesoporous silica nanoparticles and phenylboronic acid functionalized nanoparticle pore blocking agents.
  • the phenylboronic acid-functionalized nanoparticle pore blocking agent can be connected to the polyhydroxy compound-functionalized mesoporous silica through the phenylboronic ester bond formed between the phenylboronic acid functional group and the polyhydroxy functional group. on the nanoparticles, thereby blocking the mesopores of the mesoporous silica nanoparticles.
  • the mesoporous silica nanoparticles can be various biocompatible or non-biocompatible mesoporous silica nanoparticles known in the art, preferably biocompatible mesoporous silica nanoparticles Nanoparticles. Its particle size is between 90-350nm and the pore size is between 5-20nm.
  • Mesoporous silica nanoparticles are usually prepared by a hydrothermal method, for example, using a cationic surfactant such as cetyltrimethylammonium bromide (CTAB) as a template, supplemented by a small molecular organic amine (or ammonia) and organic additives (such as n-hexane, etc.) to react with ethyl orthosilicate under appropriate conditions to form the mesoporous silica nanoparticles of the present invention.
  • a cationic surfactant such as cetyltrimethylammonium bromide (CTAB) as a template
  • CTAB cetyltrimethylammonium bromide
  • organic additives such as n-hexane, etc.
  • the mesoporous silica nanoparticles suitable for the present invention are the reaction product of a cationic surfactant and ethyl orthosilicate, supplemented with a small molecular organic amine (or ammonia) and an organic auxiliary, More preferred is the reaction product of CTAB with ethyl orthosilicate supplemented with ammonia and n-hexane.
  • CTAB, ethyl orthosilicate, ammonia water and n-hexane can be reacted in pure water at about 35°C for about 12 hours, and the mesoporous silica nanoparticles of the present invention can be obtained.
  • Methyl orthosilicate, propyl orthosilicate, etc. can be used to replace ethyl orthosilicate.
  • the particle size and pore size of the mesoporous silica nanoparticles can be controlled by controlling the reaction conditions and the like.
  • the pore size of the mesoporous silica nanoparticles of the present invention is preferably between 5 and 20 nm, for example, between 5 and 10 nm, between 10 and 20 nm, between 10 and 15 nm, and the like.
  • the particle size of the mesoporous silica nanoparticles is preferably in the range of 90-350 nm, for example, it can be between 100-150 nm, 100-300 nm (as shown in FIG. 18 ), 200-300 nm (as shown in FIG. 19 ) )Wait.
  • the size of the mesopores corresponding to the particle size of 100-150nm can be between 5-10nm (as shown in Figure 3), or between 8-12nm; the size corresponding to 200-300nm It can be 5-10nm, or 10-20nm, etc.
  • the shape of the channel can be radial, ordered mesopore, random, etc.
  • the polyhydroxy compound-functionalized mesoporous silica nanoparticles refers to the reactive polyhydroxy functional groups on the surface of the mesoporous silica nanoparticles, such as 1,2-dihydroxy functional groups, the reactivity refers to the The polyhydroxy functional group is capable of reacting with the phenylboronic acid functional group.
  • Polyhydroxy compounds suitable for use in the present invention generally have the following structure: R 1 -R 2 , wherein R 1 is a group that can react with the linker molecule described below, including but not limited to amino and carboxyl groups, and R 2 is a straight Chain or branched C 2-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl, which alkyl, alkenyl and alkynyl are substituted with at least 2 hydroxy groups and have at least two adjacent C Atoms are each substituted with 1 hydroxyl group.
  • Exemplary polyhydroxy compounds include, but are not limited to, carbohydrate molecules such as monosaccharides and their derivatives, polysaccharides and their derivatives, and also include synthetic block copolymers containing more than 2 hydroxyl groups.
  • the polyhydroxy compounds used in the present invention are selected from the group consisting of monosaccharides and their derivatives, including but not limited to glucose and its derivatives, fructose and its derivatives, mannose and its derivatives, and galactose and its derivatives derivative.
  • the open-chain structures of these carbohydrate molecules are used.
  • derivatives refer to derivatives that retain more than 2 hydroxyl groups, especially sugar acids, that is, under the action of weak oxidants or enzymes, the aldehyde groups in the open-chain sugar molecules are oxidized to carboxyl groups. of an acid.
  • the polyhydroxy compound used in the present invention is selected from the group consisting of open-chain glucose and derivatives thereof, such as gluconic acid.
  • the polyol can be attached to the mesoporous silica nanoparticles through a linker molecule, which usually has both a group capable of reacting with the polyol and a group capable of reacting with the mesoporous silica nanoparticle.
  • linker molecules are typically molecules comprising trialkoxysilane moieties, eg, aminoalkyltrialkoxysilanes, wherein the group capable of reacting with the mesoporous silica nanoparticles is a trialkoxy group Silane group.
  • the connection between the polyhydroxy compound and the linker molecule can be an amide group (-NH-CO-).
  • the linker molecule is a silane coupling agent having a group capable of reacting with a polyhydroxy compound, such as 3-isocyanatopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, and the like .
  • the linker moiety is aminopropyltriethoxysilane or aminopropyltrimethoxysilane.
  • the linker molecule can be reacted with mesoporous silica nanoparticles first to obtain mesoporous silica nanoparticles with groups capable of reacting with polyhydroxy compounds, and then react with polyhydroxy compounds to obtain polyhydroxy compound-functionalized mesoporous Silica nanoparticles.
  • 3-aminopropyltrimethoxysilane is first reacted with gluconic acid to give N-(3-trimethoxysilylpropyl)glucamide, which is then reacted with N-(3- Trimethoxysilylpropyl) glucamide reacts with mesoporous silica to obtain polyhydroxy compound-functionalized mesoporous silica nanoparticles.
  • the polyol may also be a commercially available polyol having groups capable of reacting with mesoporous silica nanoparticles (eg, trialkoxysilane groups), such as N-(3-trimethoxysilane) propyl) glucamide, ie, in certain embodiments, the polyol can be directly attached to the mesoporous silica nanoparticles. In these embodiments, the polyol may also be considered to already contain a linker molecule.
  • groups capable of reacting with mesoporous silica nanoparticles eg, trialkoxysilane groups
  • N-(3-trimethoxysilane) propyl) glucamide ie
  • the polyol can be directly attached to the mesoporous silica nanoparticles.
  • the polyol may also be considered to already contain a linker molecule.
  • polyol-functionalized mesoporous silica nanoparticles have the structure shown in Formula I below:
  • MSN refers to mesoporous silica nanoparticles
  • L 1 is a linker group
  • the linker group L 1 is not particularly limited, as long as it is a group that can firmly connect the polyhydroxy compound and the mesoporous silica nanoparticles together. It means that the linking group will not be under the action of the competitive substrates of phenyl boronate (such as glucose, fructose and other monosaccharide compounds, oligosaccharides, polysaccharides and catechol and other compounds containing catechol structure) fracture occurs.
  • phenyl boronate such as glucose, fructose and other monosaccharide compounds, oligosaccharides, polysaccharides and catechol and other compounds containing catechol structure
  • the linker group L 1 can be regarded as part of the linker molecule described above that connects the mesoporous silica nanoparticles and the polyol, or part of the polyol having a group capable of reacting with the mesoporous silica nanoparticles , or a combination of a portion of the linker molecule and a portion of the polyol described above that connect the mesoporous silica nanoparticles and the polyol.
  • L 1 is a C 1-6 alkylene group, such as propylene (-CH 2 CH 2 CH 2 -).
  • the polyol-functionalized mesoporous silica nanoparticles are:
  • the nanoparticle pore blocking agent suitable for the present invention can be various nanoparticles matching the mesopore size of the mesoporous silica nanoparticles, including but not limited to metal oxide nanoparticles, metal sulfide nanoparticles, metal nanoparticles , quantum dots, block copolymers, natural polymers and biological macromolecules, such as zinc oxide nanoparticles, zinc sulfide nanoparticles, zinc sulfide-zinc oxide core-shell nanoparticles, manganese zinc sulfide nanoparticles, cadmium selenide, Ferric oxide, nano-gold, etc.
  • the nanoparticle pore blocking agents used in the present invention are metal oxide nanoparticles, such as zinc oxide nanoparticles.
  • the size of the nanoparticle pore blocking agent can match the mesopore size of the mesoporous silica nanoparticles, that is, the size of the nanoparticle pore blocking agent can be slightly smaller than, approximately equal to or slightly larger than the mesopore size of the mesoporous silica nanoparticles.
  • the size of the nanoparticle pore blocking agent may be between 3-25 nm, such as between 3-15 nm, between 8-25 nm, between 8-20 nm, and the like.
  • the nanoparticle pore blocking agent After the nanoparticle pore blocking agent is functionalized with phenylboronic acid, it can be connected to the mesoporous silica nanoparticles functionalized by polyhydroxy compound through the phenylboronic ester structure, so as to effectively block the mesoporous silica nanoparticles.
  • the mesoporous opening prevents the release of the internal cargo; at the same time, the nanoparticle pore blocking agent can be used as a contrast agent, a photothermal therapy agent or an additional source of some special elements in the organism.
  • the phenylboronic acid compound refers to a small molecule compound used for phenylboronic acid functionalization of the nanoparticle pore blocking agent, which has a phenylboronic acid group, and usually also has one or more other than the phenylboronic acid group. a reactive functional group. Phenylboronic acid groups are capable of reacting with polyhydroxy functional groups (eg, 1,2-dihydroxy functional groups) to form phenylboronic ester linkages.
  • polyhydroxy functional groups eg, 1,2-dihydroxy functional groups
  • the phenylboronic acid compound has a reactive functional group (eg, hydroxyl, amino, carboxyl, etc.) capable of reacting with a linker molecule, which can be attached to the pore-blocking agent nanoparticles through a linker molecule that simultaneously It has a group capable of reacting with a phenylboronic acid compound and a group capable of reacting with a nanoparticle pore blocking agent.
  • linker molecules are typically molecules comprising trialkoxysilane moieties, eg, aminoalkyltrialkoxysilanes, wherein the group capable of reacting with the pore blocker nanoparticles is a trialkoxysilane group.
  • the group capable of reacting with the phenylboronic acid compound can be, for example, a reactive functional group (eg, amino group, isocyanate group, etc.) as described herein.
  • a reactive functional group eg, amino group, isocyanate group, etc.
  • the connection between the phenylboronic acid compound and the linker molecule can be an amide group (-NH-CO-).
  • the linker molecule is 3-isocyanatopropyltrimethoxysilane or 3-aminopropyltriethoxysilane.
  • the phenylboronic acid compound has a group capable of reacting with the nanoparticle pore blocking agent (eg, a trialkoxysilane group or a mercapto group), and can be directly attached to the nanoparticle pore blocking agent.
  • the phenylboronic acid compound may be considered to already contain a linker molecule.
  • the phenylboronic acid compound having a group capable of reacting with the nanoparticle pore blocking agent may be the following phenylboronic acid compound having a mercapto group:
  • the phenylboronic acid compound is selected from the structure represented by the following formula (A):
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from H, electron withdrawing group substituent or electron donating group substituent, preferably each independently selected from hydrogen, C 1-6 alkyl, halogen, nitro, Carboxyl and amino, more preferably each independently selected from hydrogen, C1-6 alkyl, and halogen (eg, fluorine);
  • Y is a group that reacts with the linker molecule on the pore-blocking agent nanoparticles, selected from amino, hydroxyl, carboxyl, sulfonic acid, mercapto, alkenyl, alkynyl, azide, tetrazine structure, halogen, hydrazine, Epoxy group, isocyanate group and isothiocyanate group; preferably hydroxyl group, amino group, carboxyl group, sulfonic acid group or mercapto group.
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from hydrogen, C 1-6 alkyl and halogen;
  • the phenylboronic acid compound can be selected from the following structures:
  • the phenylboronic acid-functionalized nanoparticle pore-blocking agent refers to the reactive phenylboronic acid functional group on the surface of the nanoparticle pore-blocking agent, and the reactivity means that the phenylboronic acid functional group can interact with the polyhydroxy functional group reaction.
  • the phenylboronic acid-functionalized nanoparticle pore blocking agent can be obtained by directly reacting the nanoparticle pore blocking agent with a phenylboronic acid compound having a group capable of reacting with the nanoparticle pore blocking agent, or it can be obtained by the nanoparticle pore blocking agent and a linker.
  • Molecular reaction to obtain a nanoparticle pore blocking agent with surface functionalization that is, a group capable of reacting with phenyl boronate compound
  • phenyl boronate compound React with the linker molecule to obtain a molecule capable of reacting with the nanoparticle pore blocking agent, and then react with the nanoparticle pore blocking agent to obtain the molecule.
  • the phenylboronic acid compound having a group capable of reacting with the nanoparticle pore blocking agent may be a directly commercially available phenylboronic acid ester compound, or may be a compound obtained by reacting a phenylboronic acid ester compound with a linker molecule.
  • the phenylboronic acid-functionalized nanoparticle pore blocking agent has the structure shown in Formula III or IV below:
  • X refers to nanoparticle pore blocking agent
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from hydrogen, electron-withdrawing group substituent and electron-donating group substituent, preferably each independently selected from hydrogen, C 1 -6 alkyl, halogen, nitro, carboxyl and amino, more preferably each independently selected from hydrogen and halogen (eg fluorine);
  • L 3 and L 4 are not particularly limited, as long as they are groups that can firmly connect the polyhydroxy compound and the mesoporous silica nanoparticles together That is, the firmness means that the linking group will not be in the competitive substrate of phenylboronate (such as monosaccharides such as glucose and fructose, oligosaccharides, polysaccharides, and catechol-containing catechol structures). cleavage occurs under the action of the compound).
  • phenylboronate such as monosaccharides such as glucose and fructose, oligosaccharides, polysaccharides, and catechol-containing catechol structures.
  • the relative positions of Z 1 , Z 2 , Z 3 , Z 4 and L 3 , L 4 and the phenylboronic acid group on the benzene ring are not limited, that is, L 3 or L 4 can be located in the phenyl boronic acid group The meta, para or ortho position of a group.
  • the phenylboronic acid functionalized nanoparticle pore blocking agent is:
  • X refers to nanoparticle pore blocking agent.
  • the mesoporous silica nanomaterial that can be used for controlled release of the present invention can be prepared by the following steps:
  • the polyhydroxy compound-functionalized mesoporous silica nanoparticles and the phenylboronic acid-functionalized pore blocking agent can be prepared by the methods described herein, and the specific reaction conditions can be determined according to the actual reaction.
  • step (2) is carried out under conditions that allow the pore-blocking agent to block the mesopores.
  • step (2) can be reacted at a temperature lower than 40°C (eg room temperature), and the solvent used is not particularly limited, usually toluene, dimethylformamide, dimethylacetamide, dimethylmethylene can be used Sulfone (DMSO), dichloromethane, ethanol, methanol, water, one of various aqueous based buffers or mixtures thereof, eg the solvent may be a mixture of phosphate buffered saline (PBS buffer) and DMSO.
  • PBS buffer phosphate buffered saline
  • the substrates suitable for the present invention may be various substrates that can bind to phenylboronic acid, and the binding force thereof to phenylboronic acid is not less than that of the polyhydroxy compound that forms an ester with phenylboronic acid, so that the substrate can be
  • the phenylboronate bond can be broken to release the nanoparticle pore blocking agent, and finally open the mesoporous channel to release the internal cargo (such as various types of insulin, dyes, drugs, indicators or other biological macromolecules, etc.) .
  • the substrate used is biocompatible.
  • the substrate itself is carried by the human or animal body, such as naturally occurring in blood (such as glucose, catechols, etc.), or artificially administered (such as fructose, other types of monosaccharides, oligosaccharides, etc.) or polysaccharides, etc.).
  • the surface of the mesoporous silica nanoparticles and the nanoparticle pore blocking agent can be further modified to increase their biological safety, such as carrying a Layer PEG materials, pH-responsive medical polymer materials or other biodegradable materials.
  • the controlled release nanomaterials of the present invention may also contain a composition or mixture of other ingredients such as pharmaceutically acceptable carriers or excipients. Those skilled in the art can select appropriate pharmaceutically acceptable carriers or excipients according to the use of the controlled release nanomaterials of the present invention.
  • the controlled release nanomaterials of the present invention may also contain functional active molecules.
  • the controlled release nanomaterials of the present invention consist of polyol-functionalized mesoporous silica nanoparticles, phenylboronic acid-functionalized nanoparticle pore blocking agents, and functionally active molecules.
  • the controlled release nanomaterial containing polyhydroxy compound-functionalized mesoporous silica nanoparticles, phenylboronic acid-functionalized nanoparticle pore blocking agent and functional active molecules of the present invention can realize the loading and control of functional active molecules Therefore, the present invention is also referred to as functional active molecular nano-drug delivery system.
  • the functionally active molecule nano-drug loading system of the present invention refers to a product that can control the loading and controlled release of functionally active molecules (eg, insulin).
  • the functional active molecule nano-drug loading system of the present invention can be a simple product containing only mesoporous silica nanoparticles functionalized with polyhydroxy compounds, nano-particle pore blocking agents functionalized with phenylboronic acid and functional active molecules, It may also contain polyol-functionalized mesoporous silica nanoparticles, phenylboronic acid-functionalized nanoparticle pore-blocking agents and functionally active molecules and other ingredients such as pharmaceutically acceptable carriers or excipients.
  • a composition or mixture Those skilled in the art can select appropriate pharmaceutically acceptable carriers or excipients according to the use of the composition or mixture.
  • functionally active molecules that can be used in the present invention include functionally active molecules for various purposes, for example, biological macromolecules such as proteins and nucleic acids, small molecular compounds such as anti-tumor drugs and fluorescent probes, and nanoparticles smaller than mesoporous silica nanoparticles Various functional quantum dots or nanoparticles of mesopore size.
  • small molecules and small molecule compounds refer to various compounds that can enter the mesoporous channels of mesoporous carbon dioxide nanoparticles.
  • Proteins can be, for example, various enzymes and antibodies that can be used for therapeutic or diagnostic purposes.
  • Nucleic acids can be, for example, various functional nucleic acids that can be used in gene therapy, such as siRNA.
  • the functionally active molecules loaded by the controlled release nanomaterials and functionally active molecule nano-drug-carrying systems of the present invention can be a combination of one or more of the above-mentioned functionally active molecules for diagnosing or treating the same or different diseases or symptoms .
  • the size of the functionally active molecules is preferably smaller than the mesopore size of the mesoporous silica nanoparticles.
  • the functionally active molecules are loaded inside the mesoporous silica nanoparticles, eg, within the mesoporous channels of the mesoporous silica nanoparticles.
  • the functional active ingredient preferably contains a diabetes therapeutic agent.
  • the functionally active molecule may comprise only the diabetes therapeutic agent, or may consist of the diabetes therapeutic agent and other small molecule detection or diagnostic reagents (eg, dyes, drugs, indicators, etc.).
  • the diabetes therapeutic agent suitable for use in the present invention is preferably insulin or a biologically active analog thereof.
  • the insulin or its biologically active analog is selected from the group consisting of human insulin, recombinant human insulin, insulin from non-human animals, fast-acting insulin, fast-acting insulin analogs, intermediate-acting insulin and long-acting insulin.
  • Insulin suitable for use in the present invention includes, but is not limited to, monomeric insulin, dimers, hexamers or other aggregated forms of insulin, modified insulin, labeled insulin (eg, fluorescein labeled insulin) or other insulin substitutes.
  • the functional active molecule nano-drug loading system of the present invention can be prepared by the following steps:
  • step (2) dispersing the polyhydroxy compound-functionalized mesoporous silica nanoparticles obtained in step (1) in a solvent containing functionally active molecules, so that the mesoporous silica nanoparticles are loaded with the functionally active molecules;
  • step (3) adding the phenylboronic acid-functionalized nanoparticle pore-blocking agent to the reaction solution in step (2), and performing the reaction under conditions that allow the pore-blocking agent to block the mesopores, thereby obtaining the functional active molecule Nano drug delivery system.
  • Mesoporous silica nanoparticles can be prepared by a hydrothermal method.
  • An exemplary method for preparing mesoporous silica nanoparticles can be found in Example 3 of the present application.
  • Various mesoporous silica nanoparticles known in the art eg, commercially available can also be directly used.
  • the method for functionalizing the mesoporous silica nanoparticles may be well known in the art, and is illustrated in Example 4 of the present application.
  • Functionalization of the mesoporous silica nanoparticles can be performed by dispersing the mesoporous silica nanoparticles in a solvent (such as ethanol), reacting with a polyhydroxy compound, or reacting with a polyhydroxy compound and a linker molecule. reaction, or reaction with the linker molecule followed by the reaction with the polyol; the conditions for the above reaction may be known in the art.
  • a surfactant is present in the mesoporous structure of the mesoporous silica nanoparticles prepared by the hydrothermal method.
  • step (1) Also included: removing the surfactant after functionalizing the mesoporous silica nanoparticles with the polyol.
  • Methods for removing surfactants are well known in the art, and are illustrated in Example 5 of the present application.
  • the mesoporous silica nanoparticles can be dispersed in a solvent (such as ethanol), and a certain amount of salt solution (such as ethanol) is added. Aqueous solution of ammonium nitrate), reflux reaction.
  • the mesoporous silica nanoparticles functionalized with polyhydroxy compounds After obtaining the mesoporous silica nanoparticles functionalized with polyhydroxy compounds, they can be contacted with functional active molecules in a solvent, so that the functional active molecules can be loaded into the mesoporous silica nanoparticles; further, the loading can be carried out in a solvent.
  • the polyhydroxy compound-functionalized mesoporous silica nanoparticles with functional active molecules react with the phenylboronic acid-functionalized nanoparticle pore blocking agent, and the mesoporous openings are blocked by the mutual combination of phenylboronic acid and polyhydroxy compounds. .
  • steps (1) to (3) There are no special restrictions on the solvent used in steps (1) to (3), which can be determined according to the actual reaction, usually toluene, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and dichloromethane can be selected. , ethanol, methanol, water, one of various buffers based on aqueous solutions, or mixtures thereof.
  • the present invention has no special restrictions on the amount of reactants, reaction time, temperature, etc. in steps (1) to (3).
  • the skilled person can select appropriate reaction conditions according to the actual preparation situation. Exemplary preparation procedures can be found in the Examples section of this application.
  • the solvent used in step (2) is a phosphate buffer with a pH value of about 7.4, and the reaction is performed in the dark at a temperature lower than 40° C. (eg, room temperature).
  • the solvent used in step (3) is a mixture of phosphate buffer with a pH value of about 7.4 and DMSO, and the reaction is performed at a temperature lower than 40° C. (eg, room temperature).
  • the functional active molecule nano-drug loading system of the present invention can be evenly dispersed in the corresponding test system, and then fully contacted with the corresponding stimulus (substrate concentration), so that the mesopores are opened, and the internal load is the functional activity.
  • fluorescently labeled insulin can be used as a load molecule for characterization, but is not limited thereto.
  • the contact time and the amount of the controlled-release nanomaterials or functional active molecule nano-drug-carrying system used can be determined by the skilled person according to the actual situation, for example, the contact time and amount are determined according to factors such as the content of different substrates in the corresponding system.
  • the mesoporous silica nanoparticles and the test system can be separated by means of centrifugation, etc. Chromatography is used to detect the intensity of ultraviolet absorption in the system or the content of the functional active molecules in the released load, so as to determine the release rate and release amount of the loaded molecules.
  • the present invention can construct different controlled-release nanomaterials and functional active molecule nano-drug loading systems by adjusting the structure of the phenyl boronate ester, so as to realize the response to substrates in different concentration ranges.
  • the present invention selects a phenyl boronate structure that responds to the blood glucose concentration range, and constructs a mesoporous silica-based controlled release nanomaterial and functional active molecule nano-drug loading system, which can realize the change according to the blood glucose concentration.
  • Real-time controlled release of the cargo controlled release of insulin as shown in Figures 5-8).
  • the controlled-release nanomaterial or functional active molecule nano-drug loading system of the present invention can exist in the form of dry solid powder, or in the form of solution, or can be loaded on other materials or implantable devices, such as microneedles or microneedles Array patch (referred to as microneedle patch).
  • the present invention includes a kit comprising the polyhydroxy compound-functionalized mesoporous silica nanoparticles according to any embodiment of the present invention, the phenylboronic acid-functionalized Nanoparticle pore blocking agent and optional functional active molecule according to any embodiment of the present invention.
  • the polyhydroxy compound-functionalized mesoporous silica nanoparticles, the phenylboronic acid-functionalized nanoparticle pore blocking agent and the optional functional active molecules contained in the kit are stored in different containers respectively. middle.
  • the present invention includes a microneedle or microneedle array patch, which contains the mesoporous silica nanomaterial for controlled release described in any embodiment of the present invention or the functional active molecule described in any embodiment of the present invention Nano drug delivery system.
  • microneedles and microneedle array patches have meanings well known in the art.
  • microneedles refer to tiny needles with a diameter of less than 500 ⁇ m (the diameter of the needle tip is usually less than 20 ⁇ m) and a length of 100 to 1000 ⁇ m.
  • the microneedle array patch usually refers to a tiny patch with microneedles densely distributed on the substrate, and the density of the microneedles on the microneedle array patch is usually 100-10000/cm 2 .
  • the microneedle is attached to the skin, and the needle tip can penetrate into the stratum corneum to achieve intradermal controlled drug release.
  • the present invention also includes a method for controlled release, which comprises allowing the controlled release nanomaterial or functional active molecule nano-drug loading system of the present invention to fully contact with a corresponding substrate, so as to achieve the purpose of controlled release.
  • the present invention also includes the application of the controlled-release nanomaterials or functionally active molecule nano-drug loading systems of the present invention in the preparation of medicines or diagnostic reagents or diagnostic kits for controlled-release administration.
  • the drug for controlled release administration can be used to treat various diseases, and the diagnostic reagent or diagnostic kit can be used to diagnose various diseases, depending on the controlled release nanomaterials or functional active molecule nanomedicine loaded in the drug delivery system functionally active molecules.
  • the controlled-release nanomaterial or functionally active molecule nano-drug loading system of the present invention can be used to prepare a drug for treating diseases such as diabetes and its complications or for controlling the blood sugar level of a patient (as shown in Figure 7 ). Controlled release based on blood glucose concentration range).
  • the mesoporous silica-based controlled-release nanomaterials or functional active molecule nano-drug-carrying systems involved in the present invention can be applied to the living body in any suitable pharmaceutical preparation form, in any suitable route and dosage.
  • it in vivo application, it can be administered by oral, topical route, intravenous or intramuscular injection route, microneedle, microneedle patch, implant or exogenous pump form, and the like.
  • the controlled-release nanomaterials or nano-drug delivery systems of functional active molecules described in the present invention can achieve "zero premature release” of loaded molecules (eg, insulin) in the absence of a stimulus (substrate).
  • the controlled release nanomaterials or functional active molecule nano drug delivery systems corresponding to the same phenyl boronate structure described in the present invention have different response concentration ranges to different substrates.
  • the skilled person can select an appropriate amount according to the actual concentration range of the substrate and the desired content of the internal load, i.e., the functionally active molecule content.
  • the controlled-release nanomaterials or functional active molecule nano-drug loading systems corresponding to different phenyl boronate structures described in the present invention respond to the same substrate in different degrees, and the content of the released internal cargo is also different. Differently, in practical applications, the skilled person can choose the appropriate structure and dosage according to the actual concentration range of the substrate and the required content of the internal load.
  • the present invention also provides the application of the polyhydroxy compound of the present invention and the optional phenylboronic acid compound of the present invention in the preparation of controlled release nanomaterials or nanometer drug-carrying systems of functional active molecules.
  • CAB cetyltrimethylammonium bromide
  • 160mL deionized water and ammonia water 5mL, 70.8mmol
  • magnetically stir 35°C until clear.
  • 20 mL of n-hexane and 5 mL of ethyl orthosilicate in a 50 mL beaker stir evenly, and add dropwise to the above system with a constant pressure dropping funnel. Heating was stopped, suction filtered, the filter cake was washed three times with ethanol and deionized water, and dried in an oven at 100° C. for 8 hours to obtain 2.15 g of white powdery solid M0-1.
  • the reaction was stopped, filtered with suction, and the filter cake was washed three times with ethanol and deionized water, and then dried in an oven at 80 °C for 8 h to obtain 143 mg of white powdery solids, that is, mesoporous silica nanoparticles M0-3 functionalized with polyhydroxy compounds. .
  • the TEM image of M0-3 prepared in Example 5 is shown in FIG. 3 . It can be seen from Fig. 3 that the size of M0-3 is about 150 nm and has uniform and ordered pores.
  • 4-Aminobenzoic acid (0.905 g, 6.60 mmol) was added to a 50 mL round-bottomed flask, 10 mL of hydrochloric acid (5 M) was added, and the solution was magnetically stirred under ice bath conditions, and the solution was white and turbid.
  • Sodium nitrite (0.455 g, 6.60 mmol) was weighed and dissolved in 4 mL of ultrapure water, gradually added dropwise to the above reaction system with a constant pressure dropping funnel, and the solution was gradually turned clear and transparent while stirring in an ice bath.
  • reaction solution was poured into 200 mL of ice water, and a large amount of yellow flocs were precipitated. After standing for half an hour, suction filtration was performed, and the filter cake was washed with a large amount of ultrapure water. After drying in an infrared oven, it was directly used in the next reaction.
  • Example 12 The product in Example 12 (474 mg, 2.0 mmol) was added to a 100 mL round-bottomed flask, 25 mL of anhydrous methanol and 15 mL of tetrahydrofuran were added, and the mixture was stirred at room temperature, and the solution turned into a yellow suspension. Aqueous methylamine solution (25%-30%, wt) 8 mL was added. The solution gradually turned to a yellow clear liquid. After 16 h, sodium borohydride (378 mg, 10.0 mmol) was added portionwise to the above reaction system.
  • PBA1-1 (1.0 g, 3.759 mmol), 0.41 mL (5.639 mmol) of thionyl chloride and 5 mL of anhydrous DCM were added to a 50 mL round-bottomed flask, under argon protection, and stirred at 60 °C for 4 h. The reaction was stopped, the solvent was spin-dried, and 5 mL of anhydrous DCM was added for use.
  • the test results of Example 29 are shown in FIG. 5 . It can be seen from Figure 5 that the release rate of FITC-insulin in M1-1 increases with the increase of glucose concentration.
  • PBS buffer pH 7.4
  • Example 30 The test results of Example 30 are shown in FIG. 6 . It can be seen from Figure 6 that the release rate of FITC-insulin in M2-1 increases with the increase of glucose concentration, and the concentration of M2-1 in response to glucose is relatively close to the blood sugar range of diabetic patients.
  • PBS buffer pH 7.4
  • Example 31 The test results of Example 31 are shown in FIG. 7 . It can be seen from Figure 7 that the release rate of FITC-insulin in M3-1 increases with the increase of glucose concentration, and the concentration of M3-1 in response to glucose is relatively close to the blood sugar range of diabetic patients.
  • the test results of Example 32 are shown in FIG. 8 . It can be seen from FIG. 8 that the release rate of FITC-insulin in M4-1 increases with the increase of glucose concentration.
  • Chang Liver cells in good growth state were seeded into 96-well plates, and the cell density observed under the microscope was about 50%, and cultured in a cell incubator (37°C, 5% CO 2 ) for 8-12 hours to make them adhere to the wall.
  • the medium in the 96-well plate was discarded, 200 ⁇ L of the above-mentioned 5 nanoparticle suspensions of different concentrations were added to each well, and the wells were repeated 4 times, and cultured in a cell incubator (37° C., 5% CO 2 ) for 24 h.
  • MTT solution 5 mg/mL was added to each well, and the cells were incubated in a cell incubator (37° C., 5% CO 2 ) for 4 h.
  • Succinate dehydrogenase in the mitochondria of living cells can reduce MTT to blue-violet crystalline formazan and deposit in cells.
  • streptozotocin was 180 mg/kg (administered according to the specific body weight of the rats), and it was administered once.
  • STZ streptozotocin
  • Sodium citrate buffer solution with a concentration of 1% STZ (1g/100ml) was prepared, and the tail vein injection was completed within 30min to prevent inactivation. After the injection of STZ, they were given enough water and feed every day, and the fasting blood glucose was measured on the 1st, 7th, and 10th days, and the diabetes model was stable if it was greater than 16.65mmol/L.
  • FIGS. 10 and 11 Micrographs of the microneedle patch of the present invention are shown in FIGS. 10 and 11 .
  • C57bl/6 mice male, 4-6 weeks were selected and reared adaptively for two weeks, room temperature 24°C ⁇ 1°C, humidity 55%, 12h bright cycle (8:00-20:00), given sufficient water and feed. Fasting overnight for more than 12 hours before modeling, fasting but not water.
  • the dose of STZ was 180 mg/kg, one dose.
  • the sodium citrate buffer solution of STZ with a concentration of 1% was prepared, and the intraperitoneal injection was completed within 1 min to prevent inactivation. Fasting was continued for 2 h after STZ injection. After that, they were given enough water and feed every day.
  • the fasting blood glucose was measured on the 3rd, 5th, 7th, 10th and 21st days, and the diabetes model was stable if it was greater than 16.65mmol/L
  • Diabetic rats were taken and divided into 4 groups (4 rats in each group), and were subcutaneously injected into the neck with (1) 1.5 mg M3-2, (2) 2.0 mg M3-2 (4.1 IU), and (3) 1.0 mL PBS buffer. solution and (4) insulin (4.1 IU). Rat blood glucose was monitored for a period of time after injection. The experimental results are shown in Figure 12. At the same dose, the blood glucose of the rats injected with the insulin nano-drug loading system was 100-200 mg/dL for about 8 hours, while the blood glucose of the rats injected with pure insulin was 100-200 mg/dL.
  • the duration of 200mg/dL is only 4.5 hours, indicating that the insulin nano-drug loading system of the present invention can effectively release or not release insulin according to the blood sugar of rats, thereby increasing the effective duration of insulin and preventing it from being rapidly metabolized in the body. . Moreover, with the increase of the dosage of the nano-drug-loading system, no obvious symptoms of hypoglycemia appeared, indicating the high reliability and stability of the nano-drug-loading system.
  • Diabetic rat groups were subcutaneously injected with (1) 2.0 mg M3-2 (4.1 IU) and (2) insulin (4.1 IU) in the neck, respectively. 2.5h after injection.
  • Glucose 1.5g/kg was intraperitoneally injected into the diabetic group and the normal group, and the blood glucose was detected.
  • Figure 13 Compared with the normal group, the experimental group injected with the nano-drug-loading system of the present invention can simulate the function of normal pancreatic islets, release insulin when blood sugar rises, and then regulate the blood sugar of rats, while In the experimental group that was injected with insulin alone, after the injection of glucose, the blood sugar quickly returned to a higher blood sugar level.
  • the above results show that the insulin nano-drug loading system of the present invention has an obvious effect of regulating blood sugar.
  • mice Diabetic mice were taken, their backs were shaved and divided into 3 groups (4 mice in each group), respectively (1) blank patch, back patch, (2) subcutaneous injection of insulin (12.0IU/kg) in the neck, and ( 3) SGRM microneedle patch (12.0IU/kg), back patch.
  • the blood glucose of the mice was monitored for a period of time after injection or patch, and the experimental results are shown in Figure 15.
  • the SGRM microneedle patch of the present invention can effectively release or not release insulin according to the blood sugar of mice, thereby increasing the effective duration of insulin and preventing it from being rapidly metabolized in the body.
  • mice Two groups of diabetic mice and one group of normal mice were taken (4 mice in each group). The backs of diabetic mice were shaved, and (1) insulin (12.0 IU/kg) subcutaneously injected into the neck, and (2) SGRM microneedle patch (12.0 IU/kg) back patch, respectively. 3h after injection or patch. Glucose (1.5g/kg) was injected intraperitoneally in the diabetic group and the normal group, and the blood glucose was detected. The experimental results are shown in Figure 16. Experiments show that, compared with the normal group, the experimental group administered with the SGRM microneedle patch of the present invention can simulate the function of normal pancreatic islets, release insulin when blood sugar rises, and then regulate the blood sugar of mice, while the simple injection of insulin. In the experimental group, after the injection of glucose, the blood sugar quickly returned to a higher blood sugar level, indicating that the SGRM microneedle patch of the present invention has an obvious effect of regulating blood sugar.
  • mice Take normal mice, shave their backs, divide them into 2 groups (4 mice in each group), and receive (1) SGRM microneedle patch (12.0IU/kg) back patch, and (2) insulin (9.5IU/kg) ) subcutaneously in the neck, and blood sugar was measured.
  • the experimental results are shown in Figure 17.
  • the SGRM microneedle patch of the present invention does not release insulin, and there is no danger of hypoglycemia, indicating the "intelligence" and safety of the SGRM microneedle patch.

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Abstract

本发明涉及介孔氧化硅纳米粒子控释系统、其制备方法及其应用，具体提供了可用于控制释放的介孔二氧化硅纳米材料，该可用于控制释放的介孔二氧化硅纳米材料包含经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸化合物功能化的纳米粒子堵孔剂。在底物浓度较低时，该纳米材料实现"零提前释放"；在底物浓度较高时，被堵孔剂封堵的介孔孔道被打开，活性成分得到释放。该纳米材料可以加载于用于治疗糖尿病或控制患者血糖水平的微针和微针阵列贴片中。

Description

介孔氧化硅纳米粒子控释系统、其制备方法及其应用 技术领域
本发明属于药物控释领域，具体涉及介孔氧化硅纳米粒子控释系统、其制备方法及其在微针贴片中的应用。
背景技术
无机多孔材料，因具有较大的比表面积和吸附容量，而被广泛应用于催化剂和吸附载体中。按照孔径大小，多孔材料可分为：微孔(Microporous)、介孔(Mesoporous)和大孔(Macroporous)材料。无机微孔材料的孔径一般＜2nm，包括硅钙石、活性炭、泡沸石等，微孔材料的小孔径限制了其对有机大分子的催化与吸附作用。大孔材料的孔径一般＞50nm，包括多孔陶瓷、水泥、气凝胶等，其特点是孔径尺寸大，但分布范围宽。孔径介于两者之间的称为介孔材料。介孔材料具有极高的比表面积、规则有序的孔道结构、狭窄的孔径分布、孔径大小连续可调、无生理毒性等特点，在酶、蛋白质等的固定和分离、生物芯片、药物的包埋和控释等领域具有很好的应用前景。
介孔氧化硅材料在药物传输方面的应用已获得广泛的研究。通过对介孔氧化硅进行表面修饰可以实现对内载物的控制释放，使其成为较为理想的药物运输载体(Chem.Soc.Rev.，2012，41(7)：2590-2605)。介孔氧化硅药物运输平台主要由介孔氧化硅、堵孔剂、敏感响应单元三部分组成。表面功能化的、孔端口被封闭的介孔氧化硅具有“零提前释放”的特性，引起了研究者的广泛关注，被称为有效的刺激响应控制释放系统。
生物大分子由于结构不稳定，其活性容易受到环境因素影响，如温度、pH等。因此，在生物大分子传输领域，如何保持生物大分子的活性，提高生物利用度与可控释放是当前研究的重点。利用介孔氧化硅介孔对生物大分子结构完整性的保护特点构建的生物大分子传输系统已有部分报道。但是，由于生物大 分子具有较大的尺寸，因此需要对应尺寸较大的介孔氧化硅来满足装载的要求，由此导致的介孔氧化硅的粒径会随孔径的增大而增加。然而，该类介孔氧化硅在生物体内应用时不易通过生物膜，且容易被网状内皮吞噬系统摄取，药物生物利用度低。
目前，尚未有利用介孔孔径≥5nm，而介孔氧化硅的尺寸小于350nm，且介孔间相对独立的介孔氧化硅运载系统的报道，特别是系统地针对生物大分子的运载系统的报道。
微针贴片是一种极具潜力的经皮给药的手段，基于聚多糖类的溶解型微针贴片具有给药效率高、载药率高、制作工艺方便、易存储等优点，将微针贴片与胰岛素载药系统相结合，具有广阔的应用前景。
发明内容
本发明以经多羟基化合物表面功能化的介孔氧化硅纳米粒子为载体，以与介孔尺寸相匹配的经苯基硼酸表面功能化的纳米粒子为堵孔剂，基于由多羟基化合物与苯基硼酸所形成的苯基硼酸酯与底物间的竞争性结合原理，设计并合成了一种控制释放系统，该系统能够用于生物大分子如胰岛素等的控制释放。
具体而言，本发明提供一种可用于控制释放的介孔二氧化硅纳米材料，所述可用于控制释放的介孔二氧化硅纳米材料包含经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的纳米粒子堵孔剂。所述苯基硼酸基团与所述多羟基化合物的羟基形成连接基团，从而连接所述经苯基硼酸功能化的纳米粒子堵孔剂与所述经多羟基化合物功能化的介孔二氧化硅纳米粒子。
在一个或多个实施方案中，本发明可用于控制释放的介孔二氧化硅纳米材料中，纳米粒子堵孔剂与苯基硼酸基团直接连接或经由接头与苯基硼酸基团连接，由该苯基硼酸基团与多羟基化合物的羟基反应形成连接基团，该多羟基化合物再经由另一接头与介孔二氧化硅纳米粒子连接。
在一个或多个实施方案中，所述介孔二氧化硅纳米粒子的粒径为90-350nm。
在一个或多个实施方案中，所述介孔二氧化硅纳米粒子的介孔尺寸为5-20nm。
在一个或多个实施方案中，所述多羟基化合物选自单糖及其衍生物、多糖及其衍生物以及合成嵌段共聚物，优选选自葡萄糖及其衍生物。
在一个或多个实施方案中，所述多羟基化合物为葡萄糖酸。
在一个或多个实施方案中，所述经多羟基化合物功能化的介孔二氧化硅纳米粒子具有如下式I所示的结构：
Figure PCTCN2021114083-appb-000001
其中，MSN指介孔二氧化硅纳米粒子；L 1为接头基团；优选地，L 1为C 1-6亚烷基，优选为亚丙基。
在一个或多个实施方案中，所述纳米粒子堵孔剂的尺寸与所述介孔二氧化硅纳米粒子的介孔尺寸相匹配。
在一个或多个实施方案中，所述纳米粒子堵孔剂的尺寸为3-25nm。
在一个或多个实施方案中，所述纳米粒子堵孔剂选自金属氧化物纳米粒子、金属硫化物纳米粒子、金属纳米粒子、量子点、嵌段共聚物、天然高分子和生物大分子，优选为金属氧化物纳米粒子，更优选为氧化锌纳米粒子。
在一个或多个实施方案中，所述经苯基硼酸功能化的纳米粒子堵孔剂具有如下式III或IV所示的结构：
Figure PCTCN2021114083-appb-000002
Figure PCTCN2021114083-appb-000003
其中，X指纳米粒子堵孔剂；Z 1、Z 2、Z 3和Z 4各自独立选自氢、吸电子基团取代基和供电子基团取代基，优选各自独立选自氢、C 1-6烷基、卤素、硝基、羧基和氨基，更优选各自独立选自氢、C 1-6烷基和卤素；L 3和L 4各自独立不存在或是接头基团，优选地，所述接头基团选自：C 1-6亚烷基、-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-、-(CH 2) n-NR’-COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-、-(CH 2) n-NR’-CO-、-(CH 2) n-CO-NR’-、-(CH 2) n-NR’-芳基-(CH 2) m-、-(CH 2) n-NR’-(CH 2) m-芳基-(CH 2) o-、-(CH 2) n-NR’-(CH 2) m-芳基-N＝N-芳基-(CH 2) o-和-C 2-6亚烯基-芳基-(CH 2) o-；其中，n、m、o和p各自独立为0-4的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代。
优选地，所述L 3和L 4各自独立不存在，或各自独立选自：-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-、-(CH 2) n-NR’-COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-和-酰胺基-C 1-6亚烷基-；其中，n、m、o和p各自独立为0-4的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代。
式III和IV中，Z 1、Z 2、Z 3、Z 4和L 3、L 4与苯基硼酸基团在苯环上的相对位置没有限制，即L 3或L 4可位于苯基硼酸基团的间位、对位或邻位。
在一个或多个实施方案中，所述经苯基硼酸功能化的纳米粒子堵孔剂为：
Figure PCTCN2021114083-appb-000004
Figure PCTCN2021114083-appb-000005
其中，X指纳米粒子堵孔剂。
本发明还提供一种制备本发明任一项所述的可用于控制释放的介孔二氧化硅纳米材料的方法，其特征在于，所述方法包括以下步骤：
(1)提供经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂；和
(2)将经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂分散在溶剂中，进行反应，从而得到所述可用于控制释放的介孔二氧化硅纳米材料。
在一个或多个实施方案中，所述可用于控制释放的介孔二氧化硅纳米材料还包含功能活性分子。
本发明还提供一种功能活性分子纳米载药体系，其包括本发明任一实施方案所述的可用于控制释放的介孔二氧化硅纳米材料和功能活性分子。
在一个或多个实施方案中，所述功能活性分子的尺寸小于所述介孔二氧化硅纳米粒子的介孔尺寸。
在一个或多个实施方案中，所述功能活性分子包含糖尿病治疗剂。
在一个或多个实施方案中，所述糖尿病治疗剂为胰岛素或其生物活性类似物。
在一个或多个实施方案中，所述胰岛素或其生物活性类似物选自人胰岛素、重组人胰岛素、来自非人类的动物的胰岛素、速效胰岛素、速效胰岛素类似物、中效胰岛素和长效胰岛素。
在一个或多个实施方案中，所述功能活性分子装载在所述介孔二氧化硅纳米粒子内部，例如装载在所述介孔二氧化硅纳米粒子的介孔内。
在一个或多个实施方案中，所述功能活性分子包括小分子检测或诊断试剂，如染料、药物、指示剂等。
在一个或多个实施方案中，所述功能活性分子为尺寸小于介孔二氧化硅纳米粒子介孔尺寸的功能性的量子点或纳米粒子。
本发明还提供一种制备本发明任一实施方案所述的功能活性分子纳米载药体系的方法，所述方法包括以下步骤：
(1)提供经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂；
(2)将经多羟基化合物功能化的介孔二氧化硅纳米粒子分散在含有功能活性分子的溶剂中，使所述介孔二氧化硅纳米粒子装载所述功能活性分子；和
(3)将经苯基硼酸功能化的堵孔剂加到步骤(2)的反应溶液中进行反应，从而得到所述功能活性分子纳米载药体系。
本发明还提供一种试剂盒，所述试剂盒含有经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的纳米粒子堵孔剂；优选地，所述经多羟基化合物功能化的介孔二氧化硅纳米粒子如本发明任一实施方案所述；优选地，所述经苯基硼酸功能化的纳米粒子堵孔剂如本发明任一实施方案所述；任 选地，所述试剂盒还含有功能活性分子；优选地，所述功能活性分子如本发明任一实施方案所述；优选地，所述试剂盒中含有的经多羟基化合物功能化的介孔二氧化硅纳米粒子、经苯基硼酸功能化的纳米粒子堵孔剂以及任选的功能活性分子分别保存在不同的容器中。
本发明还提供一种微针或微针阵列贴片，所述微针或微针阵列贴片含有本发明任一实施方案所述的可用于控制释放的介孔二氧化硅纳米材料或本发明任一实施方案所述的功能活性分子纳米载药体系。
本发明还提供本发明任一实施方案所述的可用于控制释放的介孔二氧化硅纳米材料在制备治疗糖尿病或控制患者血糖水平的药物中的用途。
本发明还提供本发明任一实施方案所述的功能活性分子纳米载药体系在制备用于治疗糖尿病或控制患者血糖水平的药物中的用途，其中，所述功能活性分子包含糖尿病治疗剂；优选地，所述糖尿病治疗剂为胰岛素或其生物活性类似物；优选地，所述胰岛素或其生物活性类似物选自人胰岛素、重组人胰岛素、来自非人类的动物的胰岛素、速效胰岛素、速效胰岛素类似物、中效胰岛素和长效胰岛素。
本发明还提供本发明任一实施方案所述的可用于控制释放的介孔二氧化硅纳米材料或本发明任一实施方案所述的功能活性分子纳米载药体系在制备用于控制释放给药的药物或诊断试剂或诊断试剂盒中的用途。
附图说明
图1为实施例1的尺寸在10nm左右的纳米氧化锌透射电镜图。
图2为实施例2的ZnO-P1、巯基苯硼酸、纳米氧化锌的红外光谱图。
图3为实施例5的尺寸在150nm左右，具有均匀有序孔道的介孔二氧化硅纳米粒子(M0-3)透射电镜图。
图4为实施例8的装载荧光素标记的胰岛素、氧化锌堵孔的介孔二氧化硅纳米粒子(M1-1)透射电镜图。
图5为实施例29的M1-1在不同浓度的糖溶液中，FITC-胰岛素的释放与时间的关系。
图6为实施例30的M2-1在不同浓度的糖溶液中，FITC-胰岛素的释放与时间的关系。
图7为实施例31的M3-1在不同浓度的糖溶液中，FITC-胰岛素的释放与时间的关系。
图8为实施例32的M4-1在不同浓度的糖溶液中，FITC-胰岛素的释放与时间的关系。
图9为实施例33的细胞在含M0-3、M0-4和M1-1的培养基中培养的细胞存活率。
图10为实施例35的SGRM微针贴片的扫面电镜图。
图11为实施例35的SGRM微针贴片上的微针的扫面电镜图。
图12为实施例37的糖尿病大鼠在分别注射1.5mg M3-2、2.0mg M3-2(4.1IU)、1.0mL PBS缓冲溶液和胰岛素(4.1IU)后血糖浓度随时间的变化。
图13为实施例38的糖尿病大鼠在分别注射2.0mg M3-2(4.1IU)和胰岛素(4.1IU)后，2.5h后，糖尿病大鼠和正常大鼠注射葡萄糖(1.5g/kg)后血糖浓度随时间的变化。
图14为实施例39的正常大鼠在分别注射2.0mg M3-2(4.1IU)和胰岛素(4.1IU)后血糖浓度随时间的变化。
图15为实施例40的糖尿病小鼠在分别贴片空白贴片、注射胰岛素(12.0IU/kg)和贴片SGRM微针贴片(12.0IU/kg)后血糖浓度随时间的变化。
图16为实施例41的糖尿病小鼠在分别注射胰岛素(12.0IU/kg)和贴片SGRM微针贴片(12.0IU/kg)，3h后，糖尿病小鼠和正常小鼠注射葡萄糖(1.5g/kg)后血糖浓度随时间的变化。
图17为实施例42的正常小鼠在分别贴片SGRM微针贴片(12.0IU/kg)和注射胰岛素(9.5IU/kg)后血糖浓度随时间的变化。
图18为尺寸在170nm左右的介孔氧化硅纳米粒子透射电镜图。
图19为尺寸在300nm左右的介孔氧化硅纳米粒子透射电镜图。
具体实施方式
为使本领域技术人员可了解本发明的特点及效果，以下谨就说明书及权利要求书中提及的术语及用语进行一般性的说明及定义。除非另有指明，否则文中使用的所有技术及科学上的字词，均为本领域技术人员对于本发明所了解的通常意义，当有冲突情形时，应以本说明书的定义为准。
本文描述和公开的理论或机制，无论是对或错，均不应以任何方式限制本发明的范围，即本发明内容可以在不为任何特定的理论或机制所限制的情况下实施。
本文所描述的数值范围应视为已涵盖且具体公开所有可能的次级范围及范围内的任何单独的数值。例如，“含有1至20个碳原子”将包括含有1至10个碳原子、含有2至10个碳原子、含有5个碳原子等。
本文中，为使描述简洁，未对各个实施方案、实施例或实例中的各个技术特征的所有可能的组合都进行描述。因此，只要这些技术特征的组合不存在矛盾，各个实施方案、实施例或实例中的各个技术特征可以进行任意的组合，所有可能的组合都应当认为是本说明书记载的范围。
本文中，卤素包括F、Cl、Br和I。
本文中，烷基是指直链或支链单价饱和烃基，通常含有1-12个碳原子(C 1-12烷基)，优选含有1-6个碳原子(C 1-6烷基)。烷基的例子包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基等。
本文中，亚烷基是指直连或支链二价饱和烃基，通常含有1-12个碳原子(C 1-12亚烷基)，优选含有1-6个碳原子(C 1-6亚烷基)。亚烷基的例子包括但不限于亚甲基、亚乙基、亚丙基等。
本文中，烯基指直链或支链的链中至少具有一个双键的基团，其碳链长度通常为2-12个碳原子，优选2-6个碳原子。典型的烯基包括乙烯基、1-丙烯基、2-丙烯基、2-甲基-1-丙烯基、1-丁烯基和2-丁烯基。亚烯基指直链或支链二价烯基，如亚乙烯基(-CH＝CH-)。
本文中，炔基指直链或支链的链中至少具有一个三键的基团，其碳链长度通常为2-12个碳原子，优选2-6个碳原子。典型的炔基包括乙炔基、1-丙炔基、1-甲基-2-丙炔基、2-丙炔基、1-丁炔基和2-丁炔基。
本文中，烷氧基指被“烷基-O-”，其中，烷基可以具有1-12个碳原子，优选1-6个碳原子。
本文中，吸电子基团取代基可选自硝基、氰基、卤素、羧基、炔基和烯基；供电子基团取代基可选自烷基、仲胺、伯胺、叔胺、羟基和烷氧基。
本文中，反应性官能团是指彼此之间能够发生加成、缩合、环化、聚合等反应从而共价连接的官能团，包括但不限于：氨基、羟基、羧基、磺酸基、巯基、烯基、炔基、叠氮基、四嗪类结构、卤素、肼、环氧基、异氰酸酯基、异硫氰酸酯基等。
本文中，衍生物是指一种化合物中的氢原子或原子团被其他原子或原子团取代而衍生的较复杂的产物。
本文中，氧化硅和二氧化硅具有相同的含义。
本发明的可用于控制释放的介孔二氧化硅纳米材料(本文简称控制释放纳米材料)含有经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的纳米粒子堵孔剂。在某些实施方案中，本发明的控制释放纳米材料由经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的纳米粒子堵孔剂组成。本发明中，经苯基硼酸功能化的纳米粒子堵孔剂可以通过苯基硼酸官能团与多羟基官能团之间形成的苯基硼酸酯键连接到经多羟基化合物功能化的介孔二氧化硅纳米粒子上，从而堵住介孔二氧化硅纳米粒子的介孔口。
本发明中，介孔氧化硅纳米粒子可以是本领域周知的各种具有生物相容性或不具有生物相容性的介孔氧化硅纳米粒子，优选为具有生物相容性的介孔氧化硅纳米粒子。其粒径在90-350nm之间，孔径在5-20nm之间。通常采用水热法制备介孔氧化硅纳米粒子，例如使用阳离子表面活性剂(例如十六烷基三甲基溴化铵(CTAB))作为模板剂，同时辅以小分子有机胺(或氨水)和有机助剂(如正己烷等)，使其在适当条件下与正硅酸乙酯反应，形成本发明的介孔氧化硅纳米粒子。因此，在优选实施例中，适用于本发明的介孔氧化硅纳米粒子是阳离子表面活性剂与正硅酸乙酯，同时辅以小分子有机胺(或氨水)和有机助剂的反应产物，更优选的是CTAB与正硅酸乙酯，同时辅以氨水和正己烷的反应产物。通常，CTAB、正硅酸乙酯、氨水和正己烷可在例如纯水中在 35℃左右反应12小时左右，可获得本发明的介孔氧化硅纳米粒子。可采用正硅酸甲酯、正硅酸丙酯等替换正硅酸乙酯。
可通过控制反应的条件等来控制介孔氧化硅纳米粒子的粒径和孔径。本发明的介孔氧化硅纳米粒子的孔径优选在5-20nm之间，例如可以在5-10nm之间、10-20nm之间、10-15nm之间等。介孔氧化硅纳米粒子的粒径优选在90-350nm范围内，例如可以在100-150nm之间、100-300nm之间(如图18所示)、200-300nm之间(如图19所示)等。孔径和粒径无对应的限制，例如100-150nm粒径对应的介孔尺寸可以在5-10nm之间(如图3所示)，也可以在8-12nm之间；200-300nm对应的尺寸可以为5-10nm，也可以为10-20nm等。孔道形状可以为辐射状、有序介孔、无规状等。
本文中，经多羟基化合物功能化的介孔氧化硅纳米粒子是指该介孔氧化硅纳米粒子表面具有反应性的多羟基官能团，例如1，2-二羟基官能团，所述反应性是指该多羟基官能团能够与苯基硼酸官能团反应。
适用于本发明的多羟基化合物通常具有如下结构：R 1-R 2，其中，R 1为可与下文所述的接头分子进行反应的基团，包括但不限于氨基和羧基，R 2为直链或支链C 2-12烷基、C 2-12烯基或C 2-12炔基，该烷基、烯基和炔基被至少2个羟基取代，且至少有两个相邻的C原子分别被1个羟基取代。示例性的多羟基化合物包括但不限于糖类分子，如单糖及其衍生物、多糖及其衍生物，也包括含2个羟基以上的合成嵌段共聚物。在某些实施方案中，本发明使用的多羟基化合物选自单糖及其衍生物，包括但不限于葡萄糖及其衍生物、果糖及其衍生物、甘露糖及其衍生物和半乳糖及其衍生物。通常，使用这些糖类分子的开链式结构。本文所述的“衍生物”指保留了2个以上羟基的衍生物，尤其包括糖酸，即在弱氧化剂或酶的作用下，开链式糖类分子中的醛基被氧化为羧基而成的一种酸。在某些实施方案中，本发明使用的多羟基化合物选自开链式葡萄糖及其衍生物，例如葡萄糖酸。
多羟基化合物可通过接头分子而连接到介孔氧化硅纳米粒子上，所述接头分子通常同时具有能够与多羟基化合物反应的基团和能够与介孔氧化硅纳米粒子反应的基团。举例而言，这类接头分子通常是包含三烷氧基硅烷部分的分 子，例如，氨基烷基三烷氧基硅烷，其中，能够与介孔氧化硅纳米粒子反应的基团是三烷氧基硅烷基团。通常，多羟基化合物与所述接头分子间的连接方式可以是酰胺基(-NH-CO-)。在某些实施方案中，所述接头分子为具有能够与多羟基化合物反应的基团的硅烷偶联剂，例如3-异氰酸酯基丙基三甲氧基硅烷、3-氨基丙基三甲氧基硅烷等。在一些优选的实施方案中，所述接头部分是氨基丙基三乙氧基硅烷或氨基丙基三甲氧基硅烷。可先使接头分子与介孔氧化硅纳米粒子反应，得到具有能够与多羟基化合物反应的基团的介孔氧化硅纳米粒子，再与多羟基化合物反应，得到经多羟基化合物功能化的介孔氧化硅纳米粒子。
也可先使接头分子与多羟基化合物反应，得到具有能够与介孔氧化硅纳米粒子反应的基团的多羟基化合物，再与介孔氧化硅纳米粒子反应，得到经多羟基化合物功能化的介孔氧化硅纳米粒子。例如，在某些实施方案中，3-氨基丙基三甲氧基硅烷先与葡萄糖酸反应，得到N-(3-三甲氧基甲硅烷基丙基)葡糖酰胺，再将N-(3-三甲氧基甲硅烷基丙基)葡糖酰胺与介孔氧化硅反应，得到经多羟基化合物功能化的介孔氧化硅纳米粒子。
多羟基化合物也可以是市售可得的具有能够与介孔氧化硅纳米粒子反应的基团(例如三烷氧基硅烷基团)的多羟基化合物，例如N-(3-三甲氧基甲硅烷基丙基)葡糖酰胺，即在某些实施方案中，多羟基化合物可直接连接到介孔氧化硅纳米粒子上。在这些实施方案中，多羟基化合物也可视为是已经包含了接头分子。
在某些实施方案中，经多羟基化合物功能化的介孔二氧化硅纳米粒子具有如下式I所示的结构：
Figure PCTCN2021114083-appb-000006
其中，MSN指介孔二氧化硅纳米粒子；L 1为接头基团。
本领域技术人员可以理解的是，本发明中，接头基团L 1没有特别限制，只要是能够牢固地将多羟基化合物与介孔氧化硅纳米粒子连接在一起的基团 即可，所述牢固是指连接基团不会在苯基硼酸酯的竞争性底物(如葡萄糖、果糖等单糖类化合物、寡糖、多糖以及儿茶酚等含邻苯二酚结构的化合物)的作用下发生断裂。接头基团L 1可视为上文所述的连接介孔氧化硅纳米粒子和多羟基化合物的接头分子的一部分，或具有能够与介孔氧化硅纳米粒子反应的基团的多羟基化合物的一部分，或上文所述的连接介孔氧化硅纳米粒子和多羟基化合物的接头分子的一部分与多羟基化合物的一部分的组合。
在某些实施方案中，L 1为C 1-6亚烷基，例如亚丙基(-CH 2CH 2CH 2-)。
在某些实施方案中，经多羟基化合物功能化的介孔二氧化硅纳米粒子为：
Figure PCTCN2021114083-appb-000007
适用于本发明的纳米粒子堵孔剂可以是各种与介孔氧化硅纳米粒子的介孔尺寸相匹配的纳米粒子，包括但不限于金属氧化物纳米粒子、金属硫化物纳米粒子、金属纳米粒子、量子点、嵌段共聚物、天然高分子和生物大分子，例如可以是氧化锌纳米粒子、硫化锌纳米粒子、硫化锌-氧化锌核壳纳米粒子、硫化锰锌纳米粒子、硒化镉、四氧化三铁、纳米金等。在某些实施方案中，本发明使用的纳米粒子堵孔剂为金属氧化物纳米粒子，例如氧化锌纳米粒子。
本领域技术人员可以理解的是，纳米粒子堵孔剂的尺寸与介孔氧化硅纳米粒子的介孔尺寸相匹配即可，即纳米粒子堵孔剂的尺寸可以稍小于、约等于或稍大于介孔氧化硅纳米粒子的介孔尺寸。例如，纳米粒子堵孔剂的尺寸可以在3-25nm之间，例如3-15nm之间、8-25nm之间、8-20nm之间等。
纳米粒子堵孔剂经过苯基硼酸功能化后，可通过苯基硼酸酯结构连接到经多羟基化合物官能化的介孔氧化硅纳米粒子上，从而有效地堵住介孔氧化硅纳米粒子的介孔口，阻止内载物的释放；同时该纳米粒子堵孔剂可作为造影剂、光热治疗剂或生物体某些特殊元素的额外来源。
本发明中，苯基硼酸化合物是指用于对纳米粒子堵孔剂进行苯基硼酸功能化的小分子化合物，具有苯基硼酸基团，通常还具有除苯基硼酸基团以外的一个或多个反应性官能团。苯基硼酸基团能够与多羟基官能团(例如1，2-二羟基官能团)反应形成苯基硼酸酯键。
在某些实施方案中，苯基硼酸化合物具有能够与接头分子反应的反应性官能团(例如羟基、氨基、羧基等)，可通过接头分子而连接到堵孔剂纳米粒子上，所述接头分子同时具有能够与苯基硼酸化合物反应的基团和能够与纳米粒子堵孔剂反应的基团。举例而言，这类接头分子通常是包含三烷氧基硅烷部分的分子，例如，氨基烷基三烷氧基硅烷，其中，能够与堵孔剂纳米粒子反应的基团是三烷氧基硅烷基团。所述能够与苯基硼酸化合物反应的基团例如可以是本文所述的反应性官能团(例如氨基、异氰酸酯基等)。通常，苯基硼酸化合物与所述接头分子间的连接方式可以是酰胺基(-NH-CO-)。在某些实施方案中，接头分子是3-异氰酸酯基丙基三甲氧基硅烷或3-氨丙基三乙氧基硅烷。
在某些实施方案中，苯基硼酸化合物具有能够与纳米粒子堵孔剂反应的基团(例如三烷氧基硅烷基团或巯基)，可以直接到纳米粒子堵孔剂上。在这类实施方案中，苯基硼酸化合物可视为已包含了接头分子。例如，具有能够与纳米粒子堵孔剂反应的基团的苯基硼酸化合物可以为如下的具有巯基的苯基硼酸化合物：
Figure PCTCN2021114083-appb-000008
在一些实施方案中，所述苯基硼酸化合物选自下式(A)所示的结构：
Figure PCTCN2021114083-appb-000009
式中，
Z 1，Z 2，Z 3和Z 4各自独立选自H、吸电子基团取代基或供电子基团取代基，优选各自独立选自氢、C 1-6烷基、卤素、硝基、羧基和氨基，更优选各自独立选自氢、C 1-6烷基和卤素(例如氟)；
L不存在，或是连接苯环和Y的接头基团；优选地，所述接头基团选自：C 1-6亚烷基、-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-、-(CH 2) n-NR’ -COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-、-(CH 2) n-NR’-CO-、-(CH 2) n-CO-NR’-、-(CH 2) n-NR’-芳基-(CH 2) m-、-(CH 2) n-NR’-(CH 2) m-芳基-(CH 2) o-、-(CH 2) n-NR’-(CH 2) m-芳基-N＝N-芳基-(CH 2) o-和-C 2-6亚烯基-芳基-(CH 2) o-；其中，n、m、o和p各自独立为0-4的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代；和
Y为与堵孔剂纳米粒子上的接头分子反应的基团，选自氨基、羟基、羧基、磺酸基、巯基、烯基、炔基、叠氮基、四嗪类结构、卤素、肼、环氧基、异氰酸酯基和异硫氰酸酯基；优选为羟基、氨基、羧基、磺酸基或巯基。
在一些优选的实施方案中，所述式A结构中，Z 1，Z 2，Z 3和Z 4各自独立选自氢、C 1-6烷基和卤素；L不存在，或是：C 1-6亚烷基、-(CH 2) n-NR’-苯基-N＝N-苯基-、-(CH 2) n-NR’-(CH 2) m-芳基-(CH 2) o-、或-C(O)-NH-C 1-6烷基-，其中，n、m和o各自独立为1-4的整数，R’为H或C 1-4烷基，所述苯基或芳基可任选地被1-3个选自C1-4烷基的取代基取代，所述芳基为苯基、萘基或蒽基。
在某些实施方案中，所述苯基硼酸化合物可以选自以下结构：
Figure PCTCN2021114083-appb-000010
Figure PCTCN2021114083-appb-000011
Figure PCTCN2021114083-appb-000012
以及
Figure PCTCN2021114083-appb-000013
本发明中，经苯基硼酸功能化的纳米粒子堵孔剂是指该纳米粒子堵孔剂表面具有反应性的苯基硼酸官能团，所述反应性是指该苯基硼酸官能团能够与多羟基官能团反应。
苯基硼酸功能化的纳米粒子堵孔剂可以由纳米粒子堵孔剂与具有能够与纳米粒子堵孔剂反应的基团的苯基硼酸化合物直接反应得到，也可以由纳米粒子堵孔剂与接头分子反应得到表面官能化(即具有能够与苯基硼酸酯化合物反应的基团)的纳米粒子堵孔剂，再与苯基硼酸酯化合物反应而得到，也可以由苯基硼酸酯化合物与接头分子反应得到能够与纳米粒子堵孔剂反应的分子，再与纳米粒子堵孔剂反应得到。
具有能够与纳米粒子堵孔剂反应的基团的苯基硼酸化合物可以是直接市售可得的苯基硼酸酯化合物，也可以是由苯基硼酸酯化合物与接头分子反应得到的化合物。
在某些实施方案中，经苯基硼酸功能化的纳米粒子堵孔剂具有如下式III或IV所示的结构：
Figure PCTCN2021114083-appb-000014
其中，X指纳米粒子堵孔剂；Z 1、Z 2、Z 3和Z 4各自独立选自氢、吸电子基团取代基和供电子基团取代基，优选各自独立选自氢、C 1-6烷基、卤素、硝基、羧基和氨基，更优选各自独立选自氢和卤素(例如氟)；L 3和L 4各自独立不存在或是接头基团；优选地，所述接头基团选自：C 1-6亚烷基、-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-、-(CH 2) n-NR’-COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-、-(CH 2) n-NR’-CO-、-(CH 2) n-CO-NR’-、-(CH 2) n-NR’-芳基-(CH 2) m-、-(CH 2) n-NR’-(CH 2) m-芳基-(CH 2) o-、-(CH 2) n-NR’-(CH 2) m-芳基-N＝N-芳基-(CH 2) o-和-C 2-6亚烯基-芳基-(CH 2) o-；其中，n、m、o和p各自独立为0-4的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代。
优选地，所述L 3和L 4各自独立不存在，或各自独立选自：-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-(如-(CH 2) n-NR’-CO-苯基-N＝N-苯基-NR’-(CH 2) o-)、 -(CH 2) n-NR’-COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-和-酰胺基-C 1-6亚烷基-；其中，n、m、o和p各自独立为0-4的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代。
本领域技术人员可以理解的是，本发明中，作为接头基团时，L 3、L 4没有特别限制，只要是能够牢固地将多羟基化合物与介孔氧化硅纳米粒子连接在一起的基团即可，所述牢固是指连接基团不会在苯基硼酸酯的竞争性底物(如葡萄糖、果糖等单糖类化合物、寡糖、多糖以及儿茶酚等含邻苯二酚结构的化合物)的作用下发生断裂。示例性的L 3和L 4可如前文式A中L的定义所述。
式III和IV中，Z 1、Z 2、Z 3、Z 4和L 3、L 4与苯基硼酸基团在苯环上的相对位置没有限制，即L 3或L 4可位于苯基硼酸基团的间位、对位或邻位。
在某些实施方案中，经苯基硼酸功能化的纳米粒子堵孔剂为：
Figure PCTCN2021114083-appb-000015
Figure PCTCN2021114083-appb-000016
其中，X指纳米粒子堵孔剂。
本发明的可用于控制释放的介孔氧化硅纳米材料可通过以下步骤制备：
(1)提供经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂；和
(2)将经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂分散在溶剂中，进行反应，从而得到所述可用于控制释放的介孔二氧化硅纳米材料。
经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂可以通过本文所述的方法制备得到，具体的反应条件可以根据实际反应确定。
步骤(2)的反应在允许堵孔剂将介孔堵上的条件下进行。例如，步骤(2)可以在低于40℃的温度(例如室温)下反应，所用的溶剂并无特殊限制，通常可选用甲苯、二甲基甲酰胺、二甲基乙酰胺、二甲基亚砜(DMSO)、二氯甲烷、乙醇、甲醇、水、基于水溶液的各种缓冲液中的一种或它们的混合物，例如溶剂可以是磷酸盐缓冲液(PBS缓冲液)和DMSO的混合物。
适用于本发明的底物可以是能与苯基硼酸结合的各种底物，且其与苯基硼酸的结合力不小于与苯基硼酸形成酯的多羟基化合物的结合力，以使底物可以将苯基硼酸酯键断开，从而释放纳米粒子堵孔剂，最终打开介孔孔道，释放出内载物(如各种类型胰岛素、染料、药物、指示剂或其他生物大分子等)。
优选的是，所使用的底物是具有生物相容性的。例如，所述底物本身就是人体或动物体自身所携带的，例如血液中天然存在的(如葡萄糖、儿茶酚类等)，或者人为给予的(例如果糖、其他各类单糖、寡糖或多糖等)。
因此，为了使本发明的控制释放纳米材料具有生物相容性，可对介孔氧化硅纳米粒子和纳米粒子堵孔剂表面做进一步修饰，以增加其生物安全性，如在其外表面包载一层PEG材料、pH响应医用高分子材料或其他生物可降解材料。
本发明的控制释放纳米材料还可以含有其他成分例如药学上可接受的载体或赋形剂的一种组合物或混合物。本领域技术人员能根据本发明的控制释放纳米材料的用途来选用适当的药学上可接受的载体或赋形剂。
本发明的控制释放纳米材料除含有上述经多羟基化合物功能化的介孔二氧化硅纳米粒子和上述经苯基硼酸功能化的纳米粒子堵孔剂以外，还可含有功能活性分子。在某些实施方案中，本发明的控制释放纳米材料由经多羟基化合物功能化的介孔二氧化硅纳米粒子、经苯基硼酸功能化的纳米粒子堵孔剂和功能活性分子组成。
本发明的含有经多羟基化合物功能化的介孔二氧化硅纳米粒子、经苯基硼酸功能化的纳米粒子堵孔剂和功能活性分子的控制释放纳米材料，能够实现功能活性分子的装载和控制释放，因此，本发明又称之为功能活性分子纳米载药体系。
应理解，本发明的功能活性分子纳米载药体系是指能够控制功能活性分子(例如胰岛素)的装载和控制释放的一种产品。本发明的功能活性分子纳米载药体系可以是简单的仅含有经多羟基化合物功能化的介孔二氧化硅纳米粒子、经苯基硼酸功能化的纳米粒子堵孔剂和功能活性分子的产品，也可以是含有经多羟基化合物功能化的介孔二氧化硅纳米粒子、经苯基硼酸功能化的纳米粒子堵孔剂和功能活性分子和其他成分例如药学上可接受的载体或赋形剂的一种组合物或混合物。本领域技术人员能根据该组合物或混合物的用途来选用适当的药学上可接受的载体或赋形剂。
本发明的控制释放纳米材料和功能活性分子纳米载药体系可用于控制功能活性成分的释放。因此，可用于本发明的功能活性分子包括各种用途的功能活性分子，例如，蛋白和核酸等生物大分子，抗肿瘤药物和荧光探针等小分子化合物，以及尺寸小于介孔氧化硅纳米粒子的介孔尺寸的各种功能性量子点或纳米粒子。本文中，小分子、小分子化合物是指各种可以进入介孔二氧化挂纳 米粒子介孔孔道的化合物。蛋白例如可以是各种可用于治疗或诊断用途的酶和抗体。核酸例如可以是可用于基因治疗的各种功能性核酸，如siRNA。本发明的控制释放纳米材料和功能活性分子纳米载药体系所负载的功能活性分子可以是上述功能活性分子中的一种或多种的组合，以用于诊断或治疗相同或不同的疾病或症状。
功能活性分子的尺寸优选小于介孔二氧化硅纳米粒子的介孔尺寸。优选地，功能活性分子装载在介孔二氧化硅纳米粒子内部，例如装载在介孔二氧化硅纳米粒子的介孔孔道内。
本发明中，功能活性成分优选包含糖尿病治疗剂。在某些实施方案中，功能活性分子可以仅包含糖尿病治疗剂，也可以由糖尿病治疗剂和其他小分子检测或诊断试剂(如染料、药物、指示剂等)组成。适用于本发明的糖尿病治疗剂优选为胰岛素或其生物活性类似物。优选地，胰岛素或其生物活性类似物选自人胰岛素、重组人胰岛素、来自非人类的动物的胰岛素、速效胰岛素、速效胰岛素类似物、中效胰岛素和长效胰岛素。适用于本发明的胰岛素包括但不限于胰岛素单体，胰岛素的二聚体、六聚体或其其他聚集形式，改性胰岛素，标记胰岛素(例如荧光素标记的胰岛素)或其他胰岛素替代物。
本发明的功能活性分子纳米载药体系可通过以下步骤制备：
(1)用多羟基化合物对介孔氧化硅纳米粒子进行功能化得到具有中空孔道的经多羟基化合物功能化的介孔氧化硅纳米粒子；
(2)将步骤(1)所得的经多羟基化合物功能化的介孔氧化硅纳米粒子分散在含有功能活性分子的溶剂中，使所述介孔氧化硅纳米粒子装载所述功能活性分子；和
(3)将经苯基硼酸功能化的纳米粒子堵孔剂加到步骤(2)的反应溶液中，在允许堵孔剂将介孔堵上的条件下进行反应，从而得到所述功能活性分子纳米载药体系。
可采用水热法制备介孔氧化硅纳米粒子。示范性的制备介孔氧化硅纳米粒子的方法可参见本申请的实施例3。也可直接使用现有技术已知的(如市售的)各种介孔氧化硅纳米粒子。
对介孔氧化硅纳米粒子进行功能化的方法可以是本领域周知的，在本申请实施例4中予以举例说明。对介孔氧化硅纳米粒子进行功能化可以是将介孔氧化硅纳米粒子分散在溶剂(例如乙醇)中，与多羟基化合物进行反应，或与多羟基化合物和接头分子反应后所形成的化合物进行反应，或先与接头分子反应，再与多羟基化合物反应；上述反应的条件可以是本领域已知的。
在某些实施方案中，水热法制备的介孔氧化硅纳米粒子的介孔结构中存在表面活性剂，为了去除表面活性剂，以便后续在介孔结构中装载功能活性分子，步骤(1)还包括：在用多羟基化合物对介孔氧化硅纳米粒子进行功能化之后，去除表面活性剂。去除表面活性剂的方法是本领域周知的，在本申请实施例5中予以举例说明，例如可以是将介孔氧化硅纳米粒子分散在溶剂(例如乙醇)中，加入一定量的盐溶液(例如硝酸铵的水溶液)，回流反应。
获得经多羟基化合物功能化的介孔氧化硅纳米粒子之后，可在溶剂中使其与功能活性分子接触，使功能活性分子装载到介孔氧化硅纳米粒子中；进一步地可在溶剂中使装载了功能活性分子的经多羟基化合物功能化的介孔氧化硅纳米粒子与经苯基硼酸功能化纳米粒子堵孔剂反应，通过苯基硼酸与多羟基化合物的相互结合而将介孔口堵上。
对步骤(1)到(3)中所用的溶剂并无特殊限制，可根据实际反应确定，通常可选用甲苯、二甲基甲酰胺、二甲基乙酰胺、二甲基亚砜、二氯甲烷、乙醇、甲醇、水、基于水溶液的各种缓冲液中的一种或它们的混合物。同样地，本发明对步骤(1)到(3)的反应物的用量、反应时间、温度等并没有特殊的限制。技术人员可根据实际的制备情况选择适当的反应条件。示范性的制备过程可见本申请实施例部分。在一个具体实施例中，步骤(2)所用的溶剂为pH值约为7.4的磷酸盐缓冲液，在低于40℃的温度(例如室温)下避光反应。在一个具体实施例中，步骤(3)所用的溶剂为pH值约为7.4的磷酸盐缓冲液和DMSO的混合物，在低于40℃的温度(例如室温)下反应。
可采用不同的方法测试本发明的控制释放纳米材料或功能活性分子纳米载药体系的性能。例如，可将本发明的功能活性分子纳米载药体系均分分散于相应的测试体系中，然后与相应的刺激源(底物浓度)经充分接触，使介孔打 开，内载物即功能活性分子释放。根据本发明，可用荧光标记的胰岛素作为负载分子用于表征，但不仅限于此。
接触时间以及所使用的控制释放纳米材料或功能活性分子纳米载药体系的量可由技术人员根据实际情况而定，例如根据不同底物在相应体系中的含量等因素而确定接触时间和用量。
在使刺激源与本发明的控制释放纳米材料或功能活性分子纳米载药体系接触一段时间后，可用离心等手段分离介孔氧化硅纳米粒子和测试体系，然后通过紫外分光光度计或高效液相色谱来检测体系中的紫外吸收强度或释放内载物即功能活性分子的含量，从而确定负载分子的释放速率和释放量。
本发明可通过调节苯基硼酸酯的结构构建不同的控制释放纳米材料和功能活性分子纳米载药体系，以实现对不同浓度范围底物的响应。在某些实施方案中，本发明选用针对血糖浓度范围响应的苯基硼酸酯结构，构建基于介孔氧化硅的控制释放纳米材料和功能活性分子纳米载药体系，可以实现根据血糖浓度变化而实时控制释放内载物(如图5-8所示胰岛素的控制释放)。
因此，基于本发明中不同结构苯基硼酸酯对应的控制释放纳米材料和功能活性分子纳米载药体系，在实际应用中，可以使用其中的一种，也可以两种或两种以上同时使用，以实现在不同阶段对不同底物浓度的响应，最终达到控制内载物的释放速度和释放量。两种或两种以上同时使用时，其使用比例根据实际条件可进行调节。
本发明的控制释放纳米材料或功能活性分子纳米载药体系可以是以干燥固体粉末的形式存在，也可以溶液的形式存在，也可以装载于其他材料或植入式器件，例如微针或微针阵列贴片(简称微针贴片)。
本发明包括一种试剂盒，其含有本发明任一实施方案所述的经多羟基化合物功能化的介孔二氧化硅纳米粒子、本发明任一实施方案所述的经苯基硼酸功能化的纳米粒子堵孔剂和任选的本发明任一实施方案所述的功能活性分子。优选地，所述试剂盒中含有的经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的纳米粒子堵孔剂以及任选的功能活性分子分别保存在不同的容器中。
本发明包括一种微针或微针阵列贴片，其含有本发明任一实施方案所述的可用于控制释放的介孔二氧化硅纳米材料或本发明任一实施方案所述的功能活性分子纳米载药体系。
本发明中，微针、微针阵列贴片具有本领域周知的含义。通常，微针是指直径小于500μm(针尖直径通常小于20μm)、长度为100～1000μm的微小针具。微针阵列贴片通常是指基底上密布有微针的微小贴片，微针阵列贴片上的微针密度通常为100-10000根/cm 2。微针贴于皮肤，针尖能够刺入角质层，实现皮内控释药物释放。
本发明也包括一种控制释放的方法，所述方法包括允许本发明的控制释放纳米材料或功能活性分子纳米载药体系与相应的底物充分接触，从而达到控制释放的目的。
本发明也包括本发明的控制释放纳米材料或功能活性分子纳米载药体系在制备用于控制释放给药的药物或诊断试剂或诊断试剂盒中的应用。该用于控制释放给药的药物可用于治疗各种疾病，该诊断试剂或诊断试剂盒可用于诊断各种疾病，这取决于所述控制释放纳米材料或功能活性分子纳米载药体系中所负载的功能活性分子。
例如，当负载糖尿病治疗剂如胰岛素时，本发明的控制释放纳米材料或功能活性分子纳米载药体系可用于制备治疗糖尿病及其并发症等疾病或控制患者血糖水平的药物(如图7所示基于血糖浓度范围的控制释放)。
本发明所涉及的基于介孔氧化硅的控制释放纳米材料或功能活性分子纳米载药体系可以通过任何合适的药物制剂形式，以任何合适的途径和剂量应用到生物体内。例如，在生物体内应用时，可以通过口服、局部给药途径、静脉或肌肉注射给药途径、微针、微针贴片、植入剂或外源性泵形式等。
在某些实施方案中，本发明所述的控制释放纳米材料或功能活性分子纳米载药体系在没有刺激源(底物)存在时，可达到负载分子(如胰岛素)的“零提前释放”。
在某些实施方案中，本发明所述的同一苯基硼酸酯结构对应的控制释放纳米材料或功能活性分子纳米载药体系对不同底物具有不同的响应浓度范围。在 实际应用中，技术人员可根据底物的实际浓度范围和所需的内载物即功能活性分子含量选择合适的用量。
在某些实施方案中，本发明所述的不同苯基硼酸酯结构对应的控制释放纳米材料或功能活性分子纳米载药体系对同一底物响应程度不同，所释放的内载物的含量也不同，在实际应用中，技术人员可根据底物的实际浓度范围和所需的内载物含量选择合适的结构与用量。
本发明还提供本发明所述的多羟基化合物和任选的本发明所述的苯基硼酸化合物在制备控制释放纳米材料或功能活性分子纳米载药体系中的应用。
下文将以具体实施例的方式描述本发明，其目的在于更好地理解本发明的内容。应理解，这些实施例仅仅是阐述性的，而非限制性的。实施例中所使用的试剂，除非另有说明，否则都是从市场上常规购得。其用法和用量都可根据常规的用法和用量使用。实施例中所采用的实验方法、实验条件和检测方法、检测条件，除非另有说明，否则采用本领域常规的方法、条件。
实施例1
Figure PCTCN2021114083-appb-000017
在500mL三口圆底烧瓶中加入3.95g乙酸锌二水合物和100mL乙醇，机械搅拌下加热至70℃。待反应液澄清透明后，称取2.10g KOH溶于90mL乙醇中，滴加至上述体系，半小时内滴完，继续机械搅拌(600rpm)，维持70℃反应3h。3h后，停止加热，停止机械搅拌，取反应液于12000rpm离心10min，弃上清液，沉淀物以乙醇洗涤3次，冷冻干燥得1.2g白色固体(纳米氧化锌ZnO)。实施例1制备得到的纳米氧化锌的投射电镜图如图1所示。由图1可见，纳米氧化锌的尺寸在160nm左右。
实施例2
Figure PCTCN2021114083-appb-000018
在250mL单口圆底烧瓶中加入189mg 4-巯基苯硼酸和50mL超纯水，室温磁力搅拌。将200mg纳米氧化锌ZnO于乙醇中超声分散，逐滴加入上述体系中，继续室温磁力搅拌30min。停止反应，取反应液于12000rpm离心10min，弃上清液，沉淀物以乙醇洗涤3次，冷冻干燥得180mg黄色固体，即经苯基硼酸功能化的纳米氧化锌ZnO(ZnO-P1)。ZnO-P1、4-巯基苯硼酸和纳米氧化锌的红外光谱图如图2所示。由图2可知，ZnO-P1中，4-巯基苯硼酸已修饰到纳米氧化锌的表面。
实施例3
Figure PCTCN2021114083-appb-000019
在500mL单口圆底烧瓶中加入十六烷基三甲基溴化铵(CTAB)(1.00g，2.7mmol)、160mL去离子水和氨水(5mL，70.8mmol)，35℃下磁力搅拌至澄清。量取20mL正己烷和5mL正硅酸乙酯于50mL烧杯中，搅拌均匀，用恒压滴液漏斗滴加至上述体系中，30min内滴完，滴加完毕后在35℃下反应12h。停止加热，抽滤，滤饼用乙醇和去离子水洗涤三次后，于100℃烘箱中干燥8h，得2.15g白色粉末状固体M0-1。
实施例4
Figure PCTCN2021114083-appb-000020
在500mL单口圆底烧瓶中加入1.00g研磨成细粉的介孔氧化硅纳米粒子M0-1，加入100mL无水乙醇，超声，待其分散均匀后，加入1mL N-(3-三甲氧基甲硅烷基丙基)葡糖酰胺，在氩气保护下室温磁力搅拌15h。停止反应，抽滤，滤饼用乙醇和去离子水洗涤三次后，于80℃烘箱中干燥8h，得0.917g白色粉末状固体M0-2。
实施例5
Figure PCTCN2021114083-appb-000021
在100mL单口圆底烧瓶中加入200mg研磨成细粉的介孔氧化硅纳米粒子M0-2和47.5mL无水乙醇，超声使其分散。称取500mg硝酸铵溶于2.5mL超纯水中，加入上述体系，回流反应24h。停止反应，抽滤，滤饼用乙醇和去离子水洗涤三次后，于80℃烘箱中干燥8h，得143mg白色粉末状固体，即经多羟基化合物功能化的介孔氧化硅纳米粒子M0-3。实施例5制备得到的M0-3的透射电镜图如图3所示。由图3可见，M0-3的尺寸在150nm左右，具有均匀有序的孔道。
实施例6
Figure PCTCN2021114083-appb-000022
称取0.84g NaHCO 3溶于100mL超纯水中，配置成0.1M NaHCO 3溶液；称取1.06g Na 2CO 3溶于100mL超纯水中，配置成0.1M Na 2CO 3溶液；以体积 比为1∶9的比例分别量取0.1M Na 2CO 3溶液和0.1M NaHCO 3溶液，配置成pH＝9.0的Na 2CO 3缓冲液。
称取200mg胰岛素溶于50mL 0.1M Na 2CO 3缓冲液中，然后称取异硫氰基荧光素(FITC)(2.5mg，6.4μmol)溶于2.5mL DMSO中，滴加至上述胰岛素溶液中，室温避光磁力搅拌2h。反应结束后，将反应液转移至透析袋(MWCO＝1000D)中，透析48h，每6h更换透析液。最后经冷冻干燥得188mg黄色固体，即荧光素标记的胰岛素(FITC-Insulin)，冷藏待用。
实施例7
Figure PCTCN2021114083-appb-000023
称取10mg M1-3于塑料离心管中，加入2mL PBS缓冲液(pH 7.4)，超声使其分散。称取5mg荧光素标记的胰岛素(FITC-Insulin)，加入1mL PBS(pH 7.4)缓冲液溶解后，加至上述体系中，室温避光磁力搅拌24h，得到装载了荧光素标记的胰岛素的介孔二氧化硅纳米粒子M0-4的混合液。
实施例8
Figure PCTCN2021114083-appb-000024
称取10mg ZnO-P1于塑料离心管中，加入2mL PBS缓冲液(pH 7.4)和200μL DMSO，超声使其分散后，加至实施例7的M0-4的混合液中，继续室温避光磁力搅拌24h。然后，取反应液离心(10000rpm，10min)，弃上清液， 沉淀物用PBS洗涤三次后，冷藏待用，得到装载了荧光素标记的胰岛素、纳米氧化锌堵孔的介孔二氧化硅纳米粒子M1-1。实施例8制备得到的M1-1的透射电镜图如图4所示。由图4可见，M1-1中，介孔二氧化硅纳米粒子表面的介孔口已被纳米氧化锌堵住。
实施例9
Figure PCTCN2021114083-appb-000025
在25mL圆底烧瓶中加入2-甲酰基苯硼酸(1.000g，6.66mmol)和间甲苯胺(0.314g，6.66mmol)，加入甲醇10mL，室温搅拌20min，冰浴冷却至0℃。称取硼氢化钠(0.302g，8.00mmol)分批次逐渐加入到上述反应体系中。2小时后停止反应，加入饱和氯化铵水溶液淬灭反应，调节pH至中性，旋蒸除去甲醇。乙酸乙酯(50mL×3)萃取，合并有机相，无水硫酸钠干燥，旋蒸除去溶剂，柱层析分离(EA/PE＝1/5(v/v))得微黄色油状液体(0.701g，收率43.7％)。 1H NMR(400MHz，DMSO-d 6)：δ(ppm)9.29(s，1H)，7.86(d，J＝7.2Hz，1H)，7.43-7.38(m，4H)，7.39-7.30(m，1H)，7.16(t，J＝8.0Hz，1H)，6.72(d，J＝7.6Hz，1H)，4.51(s，2H)，2.30(s，3H)。
实施例10
Figure PCTCN2021114083-appb-000026
在50mL圆底烧瓶中加入4-氨基苯甲酸(0.905g，6.60mmol)，加入10mL盐酸(5M)，冰浴条件下磁力搅拌，溶液呈白色浑浊状。称取亚硝酸钠(0.455g，6.60mmol)溶于4mL超纯水，用恒压滴液漏斗逐渐滴加至上述反应体系中，保持冰浴搅拌，溶液逐渐变为澄清透明。另取实施例9中的产物(1.061g，4.40mmol)于250mL圆底烧瓶中，加入10mL甲醇，10mL HCl溶液(1M)溶解，将该溶液逐滴加入到含重氮盐的澄清溶液中，溶液逐渐变为暗红色浑浊液，滴加完毕后加入40mL NaHCO 3饱和溶液，1h后停止反应。抽滤，滤饼用大量超纯水洗涤，柱层析分离(CH 2Cl 2/MeOH＝50/1(v/v))得暗红色固体P2(0.864g，收率50.5％)。 1H NMR(400MHz，DMSO-d6)：δ(ppm)8.13-8.04(m，2H)，7.92(d，J＝8.0Hz，1H)，7.82(d，J＝8.0Hz，2H)，7.72-7.57(m，2H)，7.48(d，J＝4.0Hz，1H)，7.35-7.24(m，2H)，6.68(s，1H)，4.65(s，2H)，2.74(s，1H)，2.56(s，2H)。
实施例11
Figure PCTCN2021114083-appb-000027
在250mL圆底烧瓶中加入蒽醌(2.08g，10mmol)和氢化钠(1.2g，30mmol，60％)，然后加入二甲基亚砜60mL，室温避光磁力搅拌。称取三甲基碘化锍(6.12g，30mmol)，溶于40mL二甲基亚砜中，于恒压滴液漏斗逐渐滴加入 上述避光反应体系中。滴加完毕后室温避光反应2h后停止反应。将反应溶液倒入200mL冰水中，析出大量黄色絮状物，静置半小时后抽滤，滤饼用大量超纯水洗涤。红外烘箱干燥后，直接用于下一步反应。
实施例12
Figure PCTCN2021114083-appb-000028
在250mL圆底烧瓶中加入实施例11中的产物和溴化锂(4.00g，46.1mmol)，加入乙腈150mL，加热至回流。16h后停止反应，溶液呈黄色透明。将该热溶液放置到-40℃冰箱中重结晶。3h后溶液中析出大量黄色针状固体。抽滤，滤饼用大量超纯水洗涤，红外烘箱干燥得黄色晶状固体(1.417g)，两步总收率60％。 1H NMR(400MHz，DMSO-d 6)：δ(ppm)11.46(s，1H)，8.95(d，J＝8.8Hz，2H)，8.61(d，J＝8.4Hz，2H)，7.75-7.65(m，4H)，5.58(t，J＝5.2Hz，1H)，5.47(d，J＝5.2Hz，2H)。
实施例13
Figure PCTCN2021114083-appb-000029
在100mL圆底烧瓶中加入实施例12中的产物(474mg，2.0mmol)，加入无水甲醇25mL和四氢呋喃15mL，室温搅拌，溶液呈黄色悬浊状。加入甲胺水溶液(25％-30％，wt)8mL。溶液逐渐变为黄色澄清液。16h后向上述反应体系中分批加入硼氢化钠(378mg，10.0mmol)。1h后加入30mL饱和氯化铵水溶液淬灭反应，旋除溶剂，以乙酸乙酯(50mL×5)萃取，合并有机相，无水硫酸钠干燥。旋蒸除去有机溶剂。柱层析分离(CH 2Cl 2/MeOH＝50/1(v/v))得淡黄色粉末状固体(0.319g，收率63.5％)。 1H NMR(400MHz，DMSO-d 6)： δ(ppm)8.50-8.43(m，4H)，7.57-7.54(m，4H)，5.44(s，2H)，4.57(s，2H)，3.27(s，3H)。
实施例14
Figure PCTCN2021114083-appb-000030
在50mL两口烧瓶中加入实施例13中的产物(122mg，0.485mmol)，2-甲酰基苯硼酸(73mg，0.485mmol)，加入甲醇8mL，室温密闭磁力搅拌，溶液呈淡黄色澄清状。45h后冰浴条件下分批加入硼氢化钠(92mg，2.425mmol)。1h加入饱和氯化铵水溶液淬灭反应，旋蒸除去溶剂，以二氯甲烷(50mL×5)萃取，合并有机相，无水硫酸钠干燥。旋除有机溶剂，柱层析分离(CH 2Cl 2/MeOH＝50/1(v/v))得淡黄色固体P3(90mg，收率48.4％)。 1H NMR(400MHz，CD 3OD)：δ(ppm)8.55(d，J＝8.0Hz，2H)，8.22(d，J＝8.0Hz，2H)，7.72(d，J＝8.0Hz，1H)，7.62-7.53(m，4H)，7.41-7.36(m，2H)，7.30(t，J＝8.0Hz，1H)，5.61(s，2H)，5.06(s，2H)，4.38(s，2H)，2.41(s，3H)。
实施例15
Figure PCTCN2021114083-appb-000031
在500mL三口圆底烧瓶中加入3.95g乙酸锌二水合物和100mL乙醇，机械搅拌下加热至70℃。待反应液澄清透明后，称取2.10g KOH溶于90mL乙醇中，滴加至上述体系，半小时内滴完，继续机械搅拌(600rpm)，维持70℃反应3h。3h后，量取5mL3-氨丙基三乙氧基硅烷(APTES)滴加至上述体系，继续机械搅拌1h。1h后，停止加热、机械搅拌，取反应液于12000rpm离 心10min，弃上清液，沉淀物以乙醇洗涤3次，冷冻干燥得1.2g白色固体ZnO-NH 2。
实施例16
Figure PCTCN2021114083-appb-000032
在100mL单口圆底烧瓶中加入P2(10mg，0.0257mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐EDCl(7.4mg，0.0385mmol)、N-羟基琥珀酰亚胺NHS(4.4mg，0.0385mmol)和30mL超纯水，并用稀NaOH调pH至7.0，室温磁力搅拌4h。将300mg ZnO-NH 2于超纯水中超声分散，加入上述体系中，继续室温磁力搅拌4h。4h后，停止反应，反应液于12000rpm离心10min，弃上清液，沉淀物以乙醇洗涤3次，冷冻干燥得280mg红色固体ZnO-P2(经苯基硼酸功能化的纳米氧化锌)。
实施例17
Figure PCTCN2021114083-appb-000033
在50mL两口圆底烧瓶中加入P3(12.2mg，0.0317mmol)、(3-异氰基丙基)三甲氧基硅烷(15.7mg，0.0634mmol)、10mL无水四氢呋喃和2-3滴三乙胺，氩气保护下回流24h。24h后，旋蒸除去溶剂，得黄色油状物，将该油状物溶于10mL无水DMF，待用。称取300mg ZnO，研磨成细粉，并于40mL无水DMF中超声分散均匀，加入上述油状物的DMF溶液，120℃下磁力搅拌30min。30min后，停止加热、搅拌，冷却至室温，反应液于12000rpm离心10min，弃上清液，沉淀物以乙醇洗涤3次，冷冻干燥得274mg灰白色固体ZnO-P3(经苯基硼酸功能化的纳米氧化锌)。
实施例18
Figure PCTCN2021114083-appb-000034
称取10mg M0-3于塑料离心管中，加入2mL PBS缓冲液(pH 7.4)，超声使其分散。称取5mg胰岛素，加1mL PBS缓冲液(pH 7.4)溶解，加至上述体系中，室温避光磁力搅拌24h，得到装载了胰岛素(未被荧光素标记)的介孔二氧化硅纳米粒子M0-5的混合液。
实施例19
Figure PCTCN2021114083-appb-000035
称取10mg ZnO-P2于塑料离心管中，加2mL PBS缓冲液(pH 7.4)和200μL DMSO，超声使其分散。加至实施例7的M0-4混合液中，继续室温避光磁力搅拌24h。然后取反应液离心(10000rpm，10min)，弃上清液，沉淀物用PBS洗涤三次后，冷藏待用，得到装载了荧光素标记的胰岛素、纳米氧化锌堵孔的介孔二氧化硅纳米粒子M2-1。
实施例20
Figure PCTCN2021114083-appb-000036
称取10mg ZnO-P2于塑料离心管中，加2mL PBS缓冲液(pH 7.4)和200μL DMSO，超声使其分散。加至实施例18的M0-5的混合液中，继续室温避光磁力搅拌24h。然后取反应液离心(10000rpm，10min)，弃上清液，沉淀物用PBS洗涤三次后，冷藏待用，得到装载了胰岛素(未被荧光素标记)、纳米氧化锌堵孔的介孔二氧化硅纳米粒子M2-2。
实施例21
Figure PCTCN2021114083-appb-000037
称取10mg ZnO-P3于塑料离心管中，加2mL PBS缓冲液(pH 7.4)和200μL DMSO，超声使其分散。加至上述M0-4混合液中，继续室温避光磁力搅拌24h。然后取反应液离心(10000rpm，10min)，弃上清液，沉淀物用PBS洗涤三次后，冷藏待用，得到装载了荧光素标记的胰岛素、纳米氧化锌堵孔的介孔二氧化硅纳米粒子M3-1。
实施例22
Figure PCTCN2021114083-appb-000038
称取10mg ZnO-P3于塑料离心管中，加2mL PBS缓冲液(pH 7.4)和200μL DMSO，超声使其分散。加至上述M0-5混合液中，继续室温避光磁力搅拌24h。然后取反应液离心(10000rpm，10min)，弃上清液，沉淀物用PBS洗 涤三次后，冷藏待用，得到装载了胰岛素(未被荧光素标记)、纳米氧化锌堵孔的介孔二氧化硅纳米粒子M3-2。
实施例23
Figure PCTCN2021114083-appb-000039
在100mL圆底烧瓶中加入3-羧基-4氟苯硼酸(5.0g)、频哪醇(4.8g)和DMF 20mL，室温磁力搅拌15min后加入无水硫酸镁(4.9g)，反应过夜。停止反应，EA萃取，旋干，所得固体柱层析分离(DCM)，得到白色固体4.5g PBA1-1。 1H NMR(400MHz，DMSO)δ7.87(t，J＝7.4Hz，1H)，7.56(d，J＝7.6Hz，1H)，7.42(d，J＝11.0Hz，1H)，1.31(s，12H)。
实施例24
Figure PCTCN2021114083-appb-000040
在50mL圆底烧瓶中加入PBA1-1(1.0g，3.759mmol)、二氯亚砜0.41mL(5.639mmol)和无水DCM 5mL，氩气保护，60℃回流搅拌4h。停止反应，旋干溶剂，加入5mL无水DCM待用。在另一50mL双口圆底烧瓶中加入胱胺二盐酸盐380mg(1.6915mmol)、三乙胺1.56mL和无水DCM 10mL，氩气保护，室温磁力搅拌15min。在冰浴条件下，将含有酰氯的DCM溶液缓慢加入上述体系，继续反应6h。停止反应，旋干溶剂，EA萃取，饱和食盐水洗3遍，旋干有机溶剂，得黄色固体650mg PBA1-2，产率54％。 1H NMR(400MHz，DMSO)δ8.54(s，2H)，7.63(t，J＝7.3Hz，2H)，7.53(d，J＝7.6Hz，2H)，7.40(d，J＝10.6Hz，2H)，3.56(dd，J＝12.7，6.4Hz，4H)，2.93(t，J＝6.8Hz，4H)，1.31(s，24H)。
实施例25
Figure PCTCN2021114083-appb-000041
在25mL圆底烧瓶中加入PBA1-2(1.27g)、NaOH(5.08g)、超纯水63.5mL和甲醇32.0mL，室温磁力搅拌48h。停止反应，旋干甲醇溶剂。用0.1M HCl酸化剩余溶液，析出大量白色固体，抽滤，所得固体柱红外烘干，柱层析分离(DCM/MeOH＝20∶1)，得到白色固体PBA1-3(560mg，产率86％)。 1H NMR(400MHz，DMSO)δ8.55(s，2H)，7.67(dd，J＝10.0，1.5Hz，2H)，7.60-7.47(m，4H)，3.55(dd，J＝12.7，6.4Hz，4H)，2.92(t，J＝6.8Hz，4H)。
实施例26
Figure PCTCN2021114083-appb-000042
在25mL圆底烧瓶中加入PBA1-3(100mg)、二硫苏糖醇(37mg)、三乙胺0.5mL和无水DMF 5mL，氩气保护，室温磁力搅拌3h，柱层析分离(DCM/MeOH＝20∶1)，得到白色固体P4(10mg，产率20％)。 1H NMR(400MHz，DMSO)δ8.54(s，1H)，7.67(dd，J＝10.0，1.5Hz，1H)，7.57(t，J＝7.9Hz，1H)，7.51(dd，J＝8.3，1.6Hz，1H)，3.55(dd，J＝12.7，6.4Hz，2H)，2.92(t，J＝6.8Hz，2H)，2.50(s，2H)。
实施例27
Figure PCTCN2021114083-appb-000043
在250mL单口圆底烧瓶中加入263mg P4和50mL DMF，室温磁力搅拌。将200mg纳米氧化锌ZnO于乙醇中超声分散，逐滴加入上述体系中，继续室温磁力搅拌30min。停止反应，取反应液于12000rpm离心10min，弃上清液，沉淀物以乙醇洗涤3次，冷冻干燥得170mg黄色固体ZnO-P4(经苯基硼酸功能化的纳米氧化锌)。
实施例28
Figure PCTCN2021114083-appb-000044
称取10mg ZnO-P4于塑料离心管中，加2mL PBS缓冲液(pH 7.4)和200μL DMSO，超声使其分散。加至上述M0-4混合液中，继续室温避光磁力搅拌24h。然后取反应液离心(10000rpm，10min)，弃上清液，沉淀物用PBS洗涤三次后，冷藏待用，得到装载了荧光素标记的胰岛素、纳米氧化锌堵孔的介孔二氧化硅纳米粒子M4-1。
实施例29
取装载完毕的M1-1于7mL塑料离心管中，加入PBS缓冲液(pH 7.4)洗涤表面吸附的胰岛素。洗涤完全后，加入4mL PBS缓冲液(pH 7.4)，37℃磁力搅拌，测定上清液荧光强度，待上清液荧光强度不变后，加入一定浓度的葡萄糖(0mM，25mM，50mM，100mM，300mM，500mM)，一定时间间隔后，离心，取3mL上清液，测定其荧光强度(Ex＝488nm，Em＝520nm)， 测试完成后将上清液转移回原测试体系。实施例29的测试结果如图5所示。由图5可知，M1-1中的FITC-胰岛素的释放速率随葡萄糖浓度的增大而增大。
实施例30
取装载完毕的M2-1于7mL塑料离心管中，加入PBS缓冲液(pH 7.4)洗涤表面吸附的胰岛素。洗涤完全后，加入4mL PBS缓冲液(pH 7.4)，37℃磁力搅拌，测定上清液荧光强度，待上清液荧光强度不变后，加入一定浓度的葡萄糖(0mM，25mM，50mM，100mM)，一定时间间隔后，离心，取3mL上清液，测定其荧光强度(Ex＝488nm，Em＝520nm)，测试完成后将上清液转移回原测试体系。实施例30的测试结果如图6所示。由图6可知，M2-1中的FITC-胰岛素的释放速率随葡萄糖浓度的增大而增大，且M2-1对葡萄糖响应的浓度比较接近糖尿病患者的血糖范围。
实施例31
取装载完毕的M3-1于7mL塑料离心管中，加入PBS缓冲液(pH 7.4)洗涤表面吸附的胰岛素。洗涤完全后，加入4mL PBS缓冲液(pH 7.4)，37℃磁力搅拌，测定上清液荧光强度，待上清液荧光强度不变后，加入一定浓度的葡萄糖(0mM，10mM，25mM，50mM)，一定时间间隔后，离心，取3mL上清液，测定其荧光强度(Ex＝488nm，Em＝520nm)，测试完成后将上清液转移回原测试体系。实施例31的测试结果如图7所示。由图7可知，M3-1中的FITC-胰岛素的释放速率随葡萄糖浓度的增大而增大，且M3-1对葡萄糖响应的浓度比较接近糖尿病患者的血糖范围。
实施例32
取装载完毕的M4-1于7mL塑料离心管中，加入PBS缓冲液(pH 7.4)洗涤表面吸附的胰岛素。洗涤完全后，加入4mL PBS缓冲液(pH 7.4)，37℃磁力搅拌，测定上清液荧光强度，待上清液荧光强度不变后，加入一定浓度的葡萄糖(0mM，25mM，50mM)，一定时间间隔后，离心，取3mL上清液， 测定其荧光强度(Ex＝488nm，Em＝520nm)，测试完成后将上清液转移回原测试体系。实施例32的测试结果如图8所示。由图8可知，M4-1中的FITC-胰岛素的释放速率随葡萄糖浓度的增大而增大。
实施例33
将生长状态良好的Chang Liver细胞接种于96孔板中，显微镜下观察的细胞密度为50％左右，在细胞培养箱(37℃，5％CO 2)中培养8-12h使其贴壁生长。
次日，分别称取1.0mg的M0-3、M0-4和M1-1，加2mL DMEM培养基超声分散配成500μg/mL的母液，移取0.8mL母液至1.2mL DMEM培养基中，配制成200μg/mL的溶液；移取1.0mL上述溶液至1.0mL DMEM培养基中，配制成100μg/mL的溶液，以此类推逐渐稀释，得到200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL 5个不同浓度的悬浮液。弃去96孔板内的培养基，每孔分别加入200μL上述5个不同浓度纳米粒子的悬浮液，重复4个复孔，在细胞培养箱(37℃，5％CO 2)中培养24h。
24h后，每孔加入20μL MTT溶液(5mg/mL)，继续在细胞培养箱(37℃，5％CO 2)中培养4h。活细胞线粒体中的琥珀酸脱氢酶可以将MTT还原为蓝紫色结晶甲瓒并沉积在细胞中。
4h后，弃去96孔板内的培养基，每孔分别加入200μL DMSO溶解甲瓒，在酶标仪中测定吸光度(492nm，570nm，630nm)，计算细胞存活率，结果如图9所示。
实施例34
选择4-6周的wistar雄性大鼠，饲料饲养两周时间。链脲佐菌素(STZ)给药量为180mg/kg(按照大鼠具体体重给药)，一次给药。按鼠重称取STZ，放于灭菌瓶，外用锡箔纸包好，将柠檬酸钠缓冲溶液及装STZ的瓶子在冰浴条件下带进动物房。配置浓度为1％的STZ(1g/100ml)的柠檬酸钠缓冲溶液，30min内尾静脉注射完，防止失活。注射STZ后，每日给予充足饮水、饲料， 第1天、第7天、第10天测空腹血糖，大于16.65mmol/L为稳定的糖尿病模型。
实施例35
将1.5mg M3-2加入0.5mL超纯水中，超声使其分散均匀，加入0.25g透明质酸(HA)，室温搅拌至均匀体系，得到M3-2的HA水溶液，用作微针(Microneedles，MNs)的浇筑材料。将1g HA加入1mL超纯水中，室温搅拌至澄清透明，得到HA空白溶液，用作MNs的基底材料。取50μL M3-2的HA水溶液，添加到模具表面，将磨具置于50毫升康宁管中，离心机(TDZ5-WS，上海湘仪离心机仪器有限公司，中国)4390×g离心5min；使用移液枪吸走磨具表面的溶液，4390×g离心5min；取出磨具，干燥器干燥24h，得到本发明的微针贴片(SGRM微针贴片，SGRM patch)。取1mL HA空白溶液(1g HA/1mL H 2O)置于模具表面，4390×g离心5min；取出磨具，干燥器干燥24h，得到空白微针贴片(Blank patch)。本发明的微针贴片的显微照片如图10和图11所示。
实施例36
选取C57bl/6小鼠(雄性，4-6周)，适应性饲养两周，室温24℃±1℃，湿度55％，12h明亮循环(8：00-20：00)，给予充足的水和饲料。造模前禁食过夜12h以上，禁食不禁水。STZ给药量为180mg/kg，一次给药。按鼠重称取STZ，放于灭菌瓶，外用锡箔纸包好，将柠檬酸钠缓冲溶液及装STZ的瓶子在冰浴条件下带进动物房。配置浓度为1％的STZ的柠檬酸钠缓冲溶液，1min内腹腔注射完，防止失活。注射STZ后，继续禁食2h。之后每日给予充足饮水、饲料。第3、5、7、10、21天测空腹血糖，大于16.65mmol/L为稳定的糖尿病模型。
实施例37
取糖尿病大鼠，分为4组(每组4只)，分别颈部皮下注射(1)1.5mg M3-2、(2)2.0mg M3-2(4.1IU)、(3)1.0mL PBS缓冲溶液和(4)胰岛素(4.1IU)。在注射后的一段时间内对大鼠血糖进行监测。实验结果如图12所示，在相同的剂量下，注射了胰岛素纳米载药体系的大鼠血糖在100-200mg/dL的持续时间约为8h，而注射了纯胰岛素的大鼠血糖在100-200mg/dL的持续时间仅为4.5小时，表明本发明的胰岛素纳米载药体系可以有效地根据大鼠血糖释放或不释放胰岛素，从而增加了胰岛素的有效时长，使其在体内不被快速得代谢。并且，随着纳米载药体系剂量的升高，其没有出现明显的低血糖症状，表明纳米载药体系高的可靠性和稳定性。
实施例38
取糖尿病大鼠2组，正常大鼠1组(每组4只)。糖尿病大鼠组分别颈部皮下注射(1)2.0mg M3-2(4.1IU)和(2)胰岛素(4.1IU)。注射2.5h后。糖尿病组和正常组大鼠腹腔注射葡萄糖(1.5g/kg)，并对血糖进行检测。实验结果如图13所示，注射了本发明的纳米载药体系的实验组与正常组相比，其可以模拟正常胰岛的功能，在血糖升高时释放胰岛素，进而调控大鼠的血糖，而单纯注射胰岛素的实验组，在注射葡萄糖后，血糖迅速回升至较高血糖。上述结果表明本发明的胰岛素纳米载药体系有着明显的调控血糖的效果。
实施例39
取正常大鼠，分为2组(每组4只)，分别颈部皮下注射(1)2.0mg M3-2(4.1IU)和(2)胰岛素(4.1IU)，并对血糖进行检测。实验结果如图14所示。实验表明，在正常血糖的情况下，本发明的胰岛素纳米载药体系并不释放胰岛素，不存在低血糖的危险，表明纳米载药体系的“智能性”和安全性。
实施例40
取糖尿病小鼠，背部剃毛，分为3组(每组4只)，分别进行(1)空白贴片，背部贴片、(2)胰岛素(12.0IU/kg)颈部皮下注射，和(3)SGRM微针 贴片(12.0IU/kg)，背部贴片。在注射或贴片后的一段时间内对小鼠血糖进行监测，实验结果如图15所示。实验表明，本发明的SGRM微针贴片可以有效地根据小鼠血糖释放或不释放胰岛素，从而增加了胰岛素的有效时长，使其在体内不被快速地代谢。
实施例41
取糖尿病小鼠2组，正常小鼠1组(每组4只)。糖尿病小鼠背部剃毛，分别进行(1)胰岛素(12.0IU/kg)颈部皮下注射，和(2)SGRM微针贴片(12.0IU/kg)背部贴片。注射或贴片3h后。糖尿病组和正常组小鼠腹腔注射葡萄糖(1.5g/kg)，并对血糖进行检测。实验结果如图16所示。实验表明，施用了本发明的SGRM微针贴片的实验组与正常组相比，其可以模拟正常胰岛的功能，在血糖升高时释放胰岛素，进而调控小鼠的血糖，而单纯注射胰岛素的实验组，在注射葡萄糖后，血糖迅速回升至较高血糖，说明本发明的SGRM微针贴片有着明显的调控血糖的效果。
实施例42
取正常小鼠，背部剃毛，分为2组(每组4只)，分别进行(1)SGRM微针贴片(12.0IU/kg)背部贴片，和(2)胰岛素(9.5IU/kg)颈部皮下注射，并对血糖进行检测。实验结果如图17所示。实验表明，在正常血糖的情况下，本发明的SGRM微针贴片并不释放胰岛素，不存在低血糖的危险，表明SGRM微针贴片的“智能性”和安全性。
虽然以具体实施例的方式描述了本发明，但应理解，本发明的范围并不限于上述具体实施方式。在不偏离本发明精神和范围的情况下，本领域技术人员可对本发明做出各种修改和变动，这些修改和变动都在本发明的范围之内。

Claims (10)

  1. 一种可用于控制释放的介孔二氧化硅纳米材料，其特征在于，所述可用于控制释放的介孔二氧化硅纳米材料包含经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸化合物功能化的纳米粒子堵孔剂。
  2. 如权利要求1所述的可用于控制释放的介孔二氧化硅纳米材料，其特征在于，
    (1)所述多羟基化合物具有R 1-R 2所示的结构，其中，R 1为氨基或羧基，R 2为直链或支链C 2-12烷基、C 2-12烯基或C 2-12炔基，且该烷基、烯基和炔基被至少2个羟基取代，且至少有两个相邻的C原子分别被1个羟基取代；优选地，所述多羟基化合物为单糖及其保留了至少两个相邻的羟基的衍生物、多糖及其保留了至少两个相邻的羟基的衍生物、含2个羟基以上且至少有两个相邻的羟基的合成嵌段共聚物；更优选地，所述多羟基化合物选自开链式葡萄糖及其衍生物，例如葡萄糖酸；和/或
    (2)所述苯基硼酸化合物选自下式(A)所示的结构：
    Figure PCTCN2021114083-appb-100001
    式中，
    Z 1，Z 2，Z 3和Z 4各自独立选自H、吸电子基团取代基或供电子基团取代基，优选各自独立选自氢、C 1-6烷基、卤素、硝基、羧基和氨基，更优选各自独立选自氢、C 1-6烷基和卤素；
    L不存在，或是连接苯环和Y的接头基团，优选选自：C 1-6亚烷基、-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-、-(CH 2) n-NR’-COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-、-(CH 2) n-NR’-CO-、-(CH 2) n-CO-NR’-、-(CH 2) n-NR’-芳基-(CH 2) m-、-(CH 2) n-NR’-(CH 2) m-芳基-(CH 2) o-、-(CH 2) n-NR’-(CH 2) m-芳基-N＝N-芳基-(CH 2) o-和-C 2-6亚烯基-芳基-(CH 2) o-；其中，n、m、o和p各自独立为0-4 的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代；和
    Y选自氨基、羟基、羧基、磺酸基、巯基、烯基、炔基、叠氮基、四嗪类结构、卤素、肼、环氧基、异氰酸酯基和异硫氰酸酯基；优选为羟基、氨基、羧基或巯基；和/或
    (3)所述纳米粒子堵孔剂的尺寸与所述介孔二氧化硅纳米粒子的介孔尺寸相匹配；优选地，所述纳米粒子堵孔剂选自金属氧化物纳米粒子、金属硫化物纳米粒子、金属纳米粒子、量子点、嵌段共聚物、天然高分子和生物大分子，优选为金属氧化物纳米粒子，例如氧化锌纳米粒子。
  3. 如权利要求1所述的可用于控制释放的介孔二氧化硅纳米材料，其特征在于，所述经多羟基化合物功能化的介孔二氧化硅纳米粒子中，所述介孔二氧化硅纳米粒子表面连接有氨基烷基三烷氧基硅烷，所述多羟基化合物通过酰胺键与所述氨基烷基三烷氧基硅烷连接；
    优选地，所述经多羟基化合物功能化的介孔二氧化硅纳米粒子具有如下式I所示的结构：
    Figure PCTCN2021114083-appb-100002
    其中，MSN指介孔二氧化硅纳米粒子；L 1为接头基团；优选地，L 1为C 1-6亚烷基。
  4. 如权利要求1所述的可用于控制释放的介孔二氧化硅纳米材料，其特征在于，所述经苯基硼酸功能化的纳米粒子堵孔剂具有如下式III或IV所示的结构：
    Figure PCTCN2021114083-appb-100003
    其中，X指纳米粒子堵孔剂；Z 1、Z 2、Z 3和Z 4各自独立选自氢、吸电子基团取代基和供电子基团取代基，优选各自独立选自氢、C 1-6烷基、卤素、硝基、羧基和氨基，更优选各自独立选自氢、C 1-6烷基和卤素；L 3和L 4各自独立不存在或是接头基团，优选地，所述接头基团选自：C 1-6亚烷基、-(CH 2) n-NR’-CO-芳基-N＝N-芳基-NR’-(CH 2) o-、-(CH 2) n-NR’-COO-(CH 2) m-芳基-(CH 2) o-NR’-(CH 2) p-、-(CH 2) n-NR’-CO-、-(CH 2) n-CO-NR’-、-(CH 2) n-NR’-芳基-(CH 2) m-、-(CH 2) n-NR’-(CH 2) m-芳基-(CH 2) o-、-(CH 2) n-NR’-(CH 2) m-芳基-N＝N-芳基-(CH 2) o-和-C 2-6亚烯基-芳基-(CH 2) o-；其中，n、m、o和p各自独立为0-4的整数；R’为H或C 1-4烷基；所述芳基选自苯基、萘基、蒽基和菲基，且可任选地被1-3个选自卤素、C 1-4烷基和C 1-4烷氧基的取代基取代；
    优选地，所述经苯基硼酸功能化的纳米粒子堵孔剂为：
    Figure PCTCN2021114083-appb-100004
    Figure PCTCN2021114083-appb-100005
    其中，X指纳米粒子堵孔剂。
  5. 一种制备权利要求1-4中任一项所述的可用于控制释放的介孔二氧化硅纳米材料的方法，其特征在于，所述方法包括以下步骤：
    (1)提供经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂；和
    (2)将经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂分散在溶剂中，进行反应，从而得到所述可用于控制释放的介孔二氧化硅纳米材料。
  6. 一种功能活性分子纳米载药体系，其特征在于，所述功能活性分子纳米载药体系包含权利要求1-4中任一项所述的可用于控制释放的介孔二氧化硅纳米材料和功能活性分子；优选地，所述功能活性分子的尺寸小于所述介孔二 氧化硅纳米粒子的介孔尺寸；优选地，所述功能活性分子装载在所述介孔二氧化硅纳米粒子内部；优选地，所述功能活性分子包含糖尿病治疗剂；优选地，所述糖尿病治疗剂为各类胰岛素或其生物活性类似物；优选地，所述胰岛素或其生物活性类似物选自人胰岛素、重组人胰岛素、来自非人类的动物的胰岛素、速效胰岛素、速效胰岛素类似物、中效胰岛素和长效胰岛素。
  7. 一种制备权利要求6所述的功能活性分子纳米载药体系的方法，其特征在于，所述方法包括以下步骤：
    (1)提供经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的堵孔剂；
    (2)将经多羟基化合物功能化的介孔二氧化硅纳米粒子分散在含有功能活性分子的溶剂中，使所述介孔二氧化硅纳米粒子装载所述功能活性分子；和
    (3)将经苯基硼酸功能化的堵孔剂加到步骤(2)的反应溶液中进行反应，从而得到所述功能活性分子纳米载药体系。
  8. 一种试剂盒，其特征在于，所述试剂盒含有经多羟基化合物功能化的介孔二氧化硅纳米粒子和经苯基硼酸功能化的纳米粒子堵孔剂；优选地，所述经多羟基化合物功能化的介孔二氧化硅纳米粒子如权利要求2或3所述；优选地，所述经苯基硼酸功能化的纳米粒子堵孔剂如权利要求2或4所述；任选地，所述试剂盒还含有如权利要求6所述的功能活性分子；优选地，所述试剂盒中含有的经多羟基化合物功能化的介孔二氧化硅纳米粒子、经苯基硼酸功能化的纳米粒子堵孔剂以及任选的功能活性分子分别保存在不同的容器中。
  9. 一种微针或微针阵列贴片，其特征在于，所述微针或微针阵列贴片含有权利要求1-4中任一项所述的可用于控制释放的介孔二氧化硅纳米材料或权利要求6所述的功能活性分子纳米载药体系。
  10. 权利要求1-4中任一项所述的可用于控制释放的介孔二氧化硅纳米材料在制备用于治疗糖尿病或控制患者血糖水平的药物或试剂盒中的用途；或
    权利要求6所述的功能活性分子纳米载药体系在制备用于治疗糖尿病或控制患者血糖水平的药物或试剂盒中的用途，其中，所述功能活性分子包含糖尿病治疗剂；或
    权利要求1-4中任一项所述的可用于控制释放的介孔二氧化硅纳米材料或权利要求6所述的功能活性分子纳米载药体系在制备用于控制释放给药的药物或诊断试剂或诊断试剂盒中的用途。
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