WO2022105039A1 - 具有多级缓控释作用的载药微粒及其制备方法 - Google Patents

具有多级缓控释作用的载药微粒及其制备方法 Download PDF

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WO2022105039A1
WO2022105039A1 PCT/CN2021/000002 CN2021000002W WO2022105039A1 WO 2022105039 A1 WO2022105039 A1 WO 2022105039A1 CN 2021000002 W CN2021000002 W CN 2021000002W WO 2022105039 A1 WO2022105039 A1 WO 2022105039A1
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drug
hydroxyethyl methacrylate
microspheres
loaded
active factor
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French (fr)
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刘文婷
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西安组织工程与再生医学研究所
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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
    • A61K47/6927—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
    • A61K47/6931—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 the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2006—IL-1
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K47/52—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 inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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

Definitions

  • the invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a drug-carrying particle with multi-stage sustained and controlled release and a preparation method thereof.
  • microspheres as the carrier of drugs and entering the body in the form of microparticles will break the traditional drug delivery method and completely change the dosage form of drugs. Whether it is oral or intravenous administration, some of the same shortcomings of the drug can be improved to meet the increasing drug demand for clinical treatment. Compared with traditional medicines, this type of polymer carrier system can greatly reduce the number of times of taking medicines, shield the irritating smell of medicines, prolong the activity of medicines, control the dose of medicines released, and improve the efficacy of medicines, so it has obvious advantages over general pharmaceutical preparations. . It is a new direction in the development of pharmaceutical preparations.
  • Liposomes are a commonly used nanocarrier with a size of about 80300 nm. They are spherical and composed of phospholipids and steroids. They can be prepared spontaneously by dispersing lipids in an aqueous medium. Drugs can be encapsulated within liposomes and subsequently released from the drug by changing parameters such as pH, osmotic gradient, and surrounding environment. Different surface modifications also increased the half-life of liposomes. For example, the addition of polyethylene glycol (PEG) increases the half-life of liposomes by preventing phagosome recognition. Likewise, a polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate was also added. PEG-PE conjugates are nontoxic and can be used to specifically target nanocarriers to mitochondria.
  • the patent with the application number of CN201610010573.1 provides a preparation method of a co-loaded daunorubicin and gambogic acid CdTe quantum dot nano-drug-loading system, which realizes the synergy of reversing multidrug resistance drugs and chemotherapeutic drugs/nano-loading Controllable and sustained release of drugs, PH-sensitive targeting of tumors and other advantages are integrated, thereby reducing the amount of chemotherapy drugs used, reducing the toxic and side effects of chemotherapy and enhancing the efficacy of chemotherapy.
  • the patent with the application number CN201710071053.6 provides a preparation method of a nano-drug-loading system that co-loads chemical anti-tumor drugs and anti-Fas antibodies, which can respond to MMPs in the tumor microenvironment, programmatically, and continuously in tumor tissue.
  • Medium-release antibody and small molecule anti-tumor drugs enhance tumor targeting and anti-tumor effects of drugs, integrate nano-loaded drug controllable release, passive targeting, active targeting, enzyme responsiveness and immunotherapy-chemotherapy, etc. advantages in one.
  • the multi-drug loading system in the prior art can only meet the requirements of simultaneously loading multiple drugs, and some systems can achieve targeted drug delivery by connecting targeted molecules, but many drugs are required in the process of disease treatment.
  • the multi-drug carrier system in the prior art cannot meet the requirement of releasing drugs at different times by performing synergistic action at different times, so that the synergistic effect of multiple drugs cannot be maximized and the therapeutic effect is affected.
  • the purpose of the present invention is to provide a drug-loaded microparticle with multi-stage sustained and controlled release and a preparation method thereof in view of the problems existing in the prior art.
  • a drug-loaded particle with multi-stage slow-release and controlled-release effect wherein the drug-loaded particle is formed by combining drug microspheres and mesoporous silica nanoparticles; the drug microspheres are oil-in-water double emulsions Liquid microspheres, the drug microspheres contain a first active factor; the mesoporous silica nanoparticles are bound with a second active factor and a third active factor.
  • the first active factor is selected from B lymphocyte chemokine-1 or IL-17 antibody
  • the second active factor is selected from IL-1 or CCL3
  • the third active factor is selected from TNF- ⁇ or IFN- ⁇ .
  • the first active factor, the second active factor or the third active factor can select different active factors according to specific diseases, not only limited to the B lymphocyte chemokine-1, IL-17 antibody, IL listed here -1, CCL3, TNF- ⁇ or IFN- ⁇ , which are only used for illustration here in order to easily understand the concept of the present invention.
  • the drug microspheres are selected from B lymphocyte chemokine-1 microspheres or IL-17 antibody microspheres.
  • the structure of the drug microspheres is:
  • an outer layer consisting of an aqueous solution of polyvinyl alcohol
  • the oil layer is composed of a mixture of polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer and polylactic acid-glycolic acid copolymer;
  • the first active factor aqueous solution layer wrapped inside the oil layer.
  • the polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer is prepared by the following method:
  • the hydroxyethyl methacrylate-polylactic acid copolymer and acrylic acid are polymerized to form a polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer substance, having the structural formula shown in formula II:
  • the molar ratio of the hydroxyethyl methacrylate to L-lactide is 1:20.
  • the catalyst is selected from stannous isooctanoate; calculated on the assumption that the molar content of hydroxyethyl methacrylate is 1, the molar content of the stannous isooctanoate is 0.2.
  • the conditions of the polymerization reaction are: under nitrogen protection, heating until hydroxyethyl methacrylate and L-lactide are completely melted, and then heating at 140 degrees for 2 hours.
  • the initiator is selected from azobisisobutyronitrile.
  • the first organic medium is selected from dioxane.
  • the molar ratio of hydroxyethyl methacrylate-polylactic acid copolymer, acrylic acid and initiator is 5:50:1.
  • the mesoporous silica nanoparticles are mesoporous silica nanoparticles co-functionalized with amino groups and thiols.
  • the ethylene oxide-polyethylene glycol co-modified drug microsphere-mesoporous silica nanoparticle connector particles are in distilled water, contacted with the third active factor, stirred, filtered, washed, and freeze-dried to obtain the drug loading particle.
  • the condensation reaction conditions are: under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide Stirring at room temperature, the mass ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 71:22.
  • amino and thiol co-functionalized mesoporous silica nanoparticles are prepared by the following method:
  • the mesoporous silica nanoparticles are dispersed in the first organic medium, and ultrasonically treated for 20 minutes to obtain a suspension; 3-trimethoxysilylpropanethiol and (3-aminopropyl)trimethoxysilane are added to the suspension The solution was stirred at room temperature for 12 hours, filtered, washed, and dried in a vacuum oven at 40 °C to obtain amino and thiol co-functionalized mesoporous silica nanoparticles;
  • the drug microspheres are prepared by the following method: dissolving 50 mg of polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer and 50 mg of polylactic acid-glycolic acid copolymer in the second organic medium, and then Add to 100 microliters of the aqueous solution containing 1 mg B lymphocyte chemokine-1, utilize super-production emulsification to form a water-in-oil emulsion, and gradually add the water-in-oil (w/o) emulsion to the polyvinyl alcohol solution, Oil-in-water (w/o/w) double emulsion was formed by stirring or ultrasonic treatment, the second organic medium was evaporated by stirring at room temperature, and the microspheres with embedded pores were collected by centrifugation. Lyophilized to obtain the drug microspheres.
  • the drug microspheres are prepared by the following method: 75 mg of polyacrylic acid-hydroxyethyl methacrylate-polylactic acid and 75 mg of polylactic acid-glycolic acid copolymer are dissolved in the second organic medium, and then added to 100 microliters of the aqueous solution containing 3 mg of IL-17 antibody was formed by ultrasonic emulsification to form a water-in-oil emulsion, the water-in-oil (w/o) emulsion was gradually added to the polyvinyl alcohol solution, and water was formed by stirring or ultrasonic treatment.
  • the polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer is prepared by the following method:
  • the hydroxyethyl methacrylate-polylactic acid copolymer and acrylic acid are polymerized to form a polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer substance; has the structural formula as shown in formula II:
  • the molar ratio of the hydroxyethyl methacrylate to L-lactide is 1:20.
  • the catalyst is selected from stannous isooctanoate; calculated on the assumption that the molar content of hydroxyethyl methacrylate is 1, the molar content of the stannous isooctanoate is 0.2.
  • the conditions of the polymerization reaction are: under nitrogen protection, heating until hydroxyethyl methacrylate and L-lactide are completely melted, and then heating at 140 degrees for 2 hours.
  • the initiator is selected from azobisisobutyronitrile.
  • the first organic medium is selected from ethanol.
  • the second organic medium is selected from dichloromethane.
  • the third organic medium is selected from dioxane.
  • the molar ratio of hydroxyethyl methacrylate-polylactic acid copolymer, acrylic acid and initiator is 5:50:1.
  • the first active factor is selected from B lymphocyte chemokine-1 or IL-17 antibody
  • the second active factor is selected from IL-1 or CCL3
  • the third active factor is selected from TNF- ⁇ or IFN- ⁇ .
  • the drug-loaded microparticles with multi-stage sustained and controlled release of the present invention have better stability, can be loaded with a variety of drugs or biological macromolecules, and can control different drugs and The administration time of biological macromolecules can achieve the requirement of precise control of drug release to achieve the maximum bioavailability of different drugs.
  • the drug-loaded microparticles with multi-stage slow-release and controlled-release effects of the present invention can be linked to different cytokines, so as to be targeted to the lesion site, and release different drugs only at the lesion site for different periods of time, so as to achieve the purpose of active targeted therapy and achieve the maximum The purpose of chemical drug availability and precise treatment regulation.
  • the intravenous drug delivery system of the present invention uses PLGA and acrylic acid-methacrylic acid hydroxy-lactide polymer, and has the advantages of low toxicity, prevention of hydrolysis of sensitive drugs, combined administration, and controlled drug release. Compared with liposomes, the combined structure intravenous drug delivery system of the present invention has better stability, can be loaded with a variety of drugs or biological macromolecules, and can control the administration time of different drugs and biological macromolecules. To achieve precise control of drug release to achieve the maximum bioavailability of different drugs.
  • the main components of the pharmaceutical microspheres of the present invention are LLA, hydroxyethyl methacrylate, stannous isooctanoate, AA monomer, and azobisisobutyronitrile, all of which are low-toxicity products and have high safety. Good, and can encapsulate a variety of drugs such as hydrophilic and lipophilic.
  • the silica nanoparticles are modified by EO-PEG-EO and PEGMEMA, and the targeted cytokines containing COOH- can be connected, and then the cytokines are adsorbed in the micropores of the silica nanoparticles by the absorption method,
  • the prepared nanoparticles can drive the nanoparticles to actively migrate and bind to the lesion area by linking with appropriate targeted cytokines, and then perform precise treatment of the disease by releasing the cytokine 2 adsorbed on the nanoparticles.
  • the invention connects the drug microspheres with the silica nanoparticles, which can achieve the purpose of co-loading a variety of drugs, and the release of the drugs on the microspheres and the nanoparticles in the body can meet the requirements of staged release.
  • Targeted cytokines can migrate to the lesion area.
  • the packaging, release sequence and release amount of the drug can be rationally designed according to the requirements of disease treatment, so as to achieve the maximum utilization of the drug.
  • FIG. 1 is a schematic structural diagram of drug-loaded microparticles with multi-stage sustained and controlled release in the embodiment of the present invention
  • Figure 2 is an electron microscope image of drug-loaded microparticles co-loaded with IL-17 antibody, CCL3, and IFN- ⁇ in the embodiment of the present invention
  • Fig. 3 is the ratio of IFN- ⁇ released over time in the drug-loaded microparticles with multi-stage sustained and controlled release effect in the embodiment of the present invention
  • Figure 4 shows the proportion of IL-17 antibody released over time in the drug-loaded microparticles with multi-stage sustained and controlled release in the example of the present invention.
  • B lymphocyte chemokine-1 was purchased from Saiye Bio, product number: HECXP-1301;
  • IL-17 antibody was purchased from Saiye Biology, product number: HETNP-0101;
  • IL-1 was purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd., product number: TL-109;
  • CCL3 was purchased from Beijing Biolab Technology Co., Ltd., product number: JN0292;
  • TNF- ⁇ or IFN- ⁇ was purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd., product number: TL-105;
  • L-lactide was purchased from Sigma-Aldrich Inc, product number: L09031;
  • M-Hydroxyethyl methacrylate was purchased from Sigma-Aldrich Inc, product number: B24260;
  • Hydroxyethyl methacrylate-polylactic acid, acrylic acid, azobisisobutyronitrile were purchased from Sigma Aldrich;
  • FIG. 1 is a schematic diagram of the structure of the drug-loaded microparticles with multi-stage sustained and controlled release in this embodiment; this embodiment provides a drug-loaded microparticle with multi-stage sustained and controlled release, the drug-loaded microparticles are composed of
  • the drug microspheres are combined with mesoporous silica nanoparticles; the drug microspheres are oil-in-water double emulsion microspheres, and the drug microspheres contain the first active factor; the mesoporous silica nanoparticles A second active factor and a third active factor are bound to the particles.
  • the first active factor is selected from B lymphocyte chemokine-1 or IL-17 antibody
  • the second active factor is selected from IL-1 or CCL3
  • the third active factor is selected from TNF- ⁇ or IFN- ⁇ .
  • the structure of the drug microspheres is:
  • an outer layer consisting of an aqueous solution of polyvinyl alcohol
  • the oil layer is composed of a mixture of acrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer and polylactic acid-glycolic acid copolymer;
  • the first active factor aqueous solution layer wrapped inside the oil layer.
  • the polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer is prepared by the following method:
  • the mesoporous silica nanoparticles are mesoporous silica nanoparticles co-functionalized with amino groups and thiols.
  • the present embodiment provides a preparation method of drug-loaded microparticles, comprising the following steps:
  • the amino and thiol co-functionalized mesoporous silica nanoparticles are prepared by the following methods:
  • the drug microspheres are prepared by dissolving 50 mg of polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer and 50 mg of polylactic acid-glycolic acid copolymer in a second organic medium , and then added to 100 microliters of an aqueous solution containing 1 mg of B-lymphocyte chemokine-1 to form a water-in-oil emulsion by ultrasonic emulsification, and the water-in-oil (w/o) emulsion was gradually added to the polyvinyl alcohol.
  • an oil-in-water (w/o/w) double emulsion is formed by stirring or ultrasonic treatment, the second organic medium is evaporated by stirring at room temperature, the microspheres with embedded pores are collected by centrifugation, and the obtained microspheres are treated with distilled water. After washing and lyophilization, the drug microspheres were obtained.
  • the polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer is prepared by the following method: hydroxyethyl methacrylate-polylactic acid (0.3 mmol, 0.771 g), acrylic acid (3mmol, 0.216g) and azobisisobutyronitrile (0.06mmol, 9.8mg) were added to 5ml of dioxane and stirred until dissolved, and the polymerization reaction was carried out at 70 degrees for 24 hours.
  • the hydroxyethyl methacrylate-polylactic acid is prepared by the following method:
  • the present embodiment provides a preparation method of drug-loaded microparticles, comprising the following steps:
  • Figure 2 shows the electron microscope image of the drug-loaded system co-loaded with IL-17 antibody, CCL3, and IFN- ⁇ ; The proportion of IFN- ⁇ released over time in the drug-loaded system loaded with IL-17 antibody, CCL3, IFN- ⁇ ;
  • Figure 4 shows the release of IL-17 antibody over time in co-loaded IL-17 antibody, CCL3, IFN- ⁇ Proportion.
  • the amino and thiol co-functionalized mesoporous silica nanoparticles are prepared by the following methods:
  • Disperse mesoporous silica nanoparticles in ethanol ultrasonically treat for 20 minutes to obtain a suspension; add 3-trimethoxysilylpropanethiol and (3-aminopropyl)trimethoxysilane to the suspension , stirred at room temperature for 12 hours, filtered, washed, and dried in a vacuum oven at 40 °C to obtain amino and thiol co-functionalized mesoporous silica nanoparticles.
  • the drug microspheres are prepared by the following method: 100 microliters of aqueous solution containing 3 mg of IL-17 antibody is added with 15% (mass fraction) polymer (75 mg of polyacrylic acid-hydroxyethyl methacrylate) Ester-polylactic acid and 75 mg of polylactic acid-co-glycolic acid) in dichloromethane were emulsified using a sonicator (15 W output for 20 s) on an ice bath to form a water-in-oil (w/o) emulsion.
  • the w/o emulsion was gradually added to 10 ml of 1% (mass fraction) polyvinyl alcohol solution, followed by stirring or ultrasonic treatment to form an oil-in-water (w/o/w) double emulsion.
  • the solution was stirred at room temperature for 3 h to evaporate the dichloromethane, and the microspheres with embedded pores were collected by centrifugation. The resulting microspheres were washed three times with distilled water and lyophilized.
  • the polyacrylic acid-hydroxyethyl methacrylate-polylactic acid copolymer is prepared by the following method: hydroxyethyl methacrylate-polylactic acid (0.3 mmol, 0.771 g), acrylic acid (3mmol, 0.216g) and azobisisobutyronitrile (0.06mmol, 9.8mg) were added to 5ml of dioxane and stirred until dissolved, and the polymerization was carried out at 70 degrees for 24 hours. The original product after polymerization was treated with chloroform It was purified by repeated precipitation with methanol 3 times and finally dried under vacuum for 40 hours.
  • the hydroxyethyl methacrylate-polylactic acid is prepared by the following method:

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Abstract

具有多级缓控释作用的载药微粒，所述载药微粒由药物微球和介孔二氧化硅纳米粒子结合而成；所述药物微球为水包油双乳化液微球，所述药物微球中含有第一活性因子；所述介孔二氧化硅纳米粒子上结合有第二活性因子和第三活性因子。载药微粒与脂质体相比，具有较好的稳定性，可负载多种药物或生物大分子，且可实现控制不同药物及生物大分子的给药时间，达到精确控制药物释放，以实现不同药物最大生物利用度的要求。

Description

具有多级缓控释作用的载药微粒及其制备方法 技术领域
本发明属于药物制剂技术领域，具体涉及一种具有多级缓控释作用的载药微粒及其制备方法。
背景技术
将微球作为药物的载体，以微颗粒的形式进入体内，这将打破传统的药物给药方式，彻底改变药物的剂型。无论是口服，还是静脉给药，都能改进药物的一些同有缺点，以满足临床治疗日益增加的用药需求。与传统的药剂相比，这类高分子载体系统可大大减少服药次数，屏蔽药物的刺激性气味、延长药物的活性、控制药物释放剂量、提高药物疗效，因此比一般药物制剂具有明显的优越性。是药物制剂发展的一个新方向。
脂质体是一种常用的纳米载体，大小约为80300nm。它们是球形的，由磷脂和类固醇组成。它们可以通过将脂质分散在水性介质中自发制备。药物可被包封在脂质体内，随后通过改变pH、渗透梯度和周围环境等参数从药物中释放出来。不同的表面修饰也提高了脂质体的半衰期。例如，加入聚乙二醇(PEG)通过阻止吞噬体的识别来增加脂质体的半衰期。同样，也加入了聚乙二醇-磷脂酰乙醇胺(PEG-PE)缀合物。PEG-PE缀合物无毒，可用于将纳米载体特异性靶向线粒体。
申请号为CN201610010573.1的专利提供了一种共载柔红霉素和藤黄酸CdTe量子点纳米载药体系的制备方法，实现了集逆转多药耐药药物与化疗药物协同作用/纳米载药可控缓释，PH敏感靶向肿瘤等多种优势为一体，从而减少化疗药物使用量，降低化疗毒副作用增强化疗疗效。申请号为CN201710071053.6的专利提供了一种共载化学抗肿瘤药物和anti-Fas抗体的纳米载药体系的制备方法，实现与肿瘤微环境中MMPs响应，程序性，持续性地在肿瘤组织中释放抗体及小分子抗肿瘤药物，增强药物的肿瘤靶向性及抗肿瘤效果，集纳米载药可控释放，被动靶向，主动靶向，酶响应性与免疫治疗-化学药物治疗等多种优势于一体。但是现有技术的多药载药系统仅能做到可同时负载多种药物的要求，部分系统可通过连接靶向分子起到靶向给药的目的，但在疾病治疗过程中很多药物是需要分不同时间进行协同作用，现有技术的多药载体系统无法达到不同时间释放药物的要求，从而无法使多种药物协同作用最大化，影响治疗效果。
发明内容
本发明的目的在于，针对现有技术存在的问题，提供一种具有多级缓控释作用的载药微粒及其制备方法。
本发明的目的是通过以下技术方案来实现的：
一方面，提供一种具有多级缓控释作用的载药微粒，所述载药微粒由药物微球和介孔二氧化硅纳米粒子结合而成；所述药物微球为水包油双乳化液微球，所述药物微球中含有第一活性因子；所述介孔二氧化硅纳米粒子上结合有第二活性因子和第三活性因子。
优选地，所述第一活性因子选自B淋巴细胞趋化因子-1或IL-17抗体，所述第二活性因子选自IL-1或CCL3，所述第三活性因子选自TNF-α或IFN-γ。应当理解，第一活性因子、第二活性因子或第三活性因子可以根据具体的疾病选择不同的活性因子，不仅仅限于此处列举的B淋巴细胞趋化因子-1、IL-17抗体、IL-1、CCL3、TNF-α或IFN-γ，此处仅用于举例说明，以便容易理解本发明的构思。
优选地，所述药物微球选自B淋巴细胞趋化因子-1微球或IL-17抗体微球。
优选地，所述药物微球的结构为：
由聚乙烯醇水溶液构成的外层；
包裹于所述外层内部的油层，所述油层由聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物和聚乳酸-羟基乙酸共聚物的混合物组成；
包裹于所述油层内部的第一活性因子水溶液层。
优选地，所述聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物由以下方法制备而成：
(1)在催化剂催化作用下，使甲基丙烯酸羟乙基酯与L-丙交酯在聚合反应条件下发生聚合反应，生成甲基丙烯酸羟乙基酯-聚乳酸共聚物，具有如式I所示的结构式：
Figure PCTCN2021000002-appb-000001
(2)在引发剂参与下，在第三有机介质中，使甲基丙烯酸羟乙基酯-聚乳酸共聚物与丙烯酸发生聚合反应，生成聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物，具有如式II所示的结构式：
Figure PCTCN2021000002-appb-000002
优选地，所述甲基丙烯酸羟乙基酯与L-丙交酯的摩尔比为1∶20。
优选地，所述催化剂选自异辛酸亚锡；以甲基丙烯酸羟乙基酯的摩尔含量为1计算，所述异辛酸亚锡的摩尔含量为0.2。
优选地，步骤(1)中，所述聚合反应的条件为：在氮气保护下，加热至甲基丙烯酸羟乙基酯与L-丙交酯完全融化，然后在140度加热2小时。
优选地，所述引发剂选自偶氮二异丁腈。
优选地，所述第一有机介质选自二氧六环。
优选地，甲基丙烯酸羟乙基酯-聚乳酸共聚物、丙烯酸和引发剂的摩尔比为5∶50∶1。
优选地，所述介孔二氧化硅纳米粒子为经过氨基和硫醇共功能化的介孔二氧化硅纳米粒子。
另一方面，提供上述的载药微粒的制备方法，包含以下步骤：
(1)在缩合反应条件下，使介孔二氧化硅纳米粒子与药物微球在蒸馏水中接触，获得药物微球-介孔二氧化硅纳米粒子连接体；所述介孔二氧化硅纳米粒子为氨基和硫醇共功能化介孔二氧化硅纳米粒子；
(2)将药物微球-介孔二氧化硅纳米粒子连接体分散于含聚(乙二醇)二甲基丙烯酸酯和2，2-二甲氧基-2-苯乙酮的蒸馏水中，在紫外线照射下搅拌30分钟，然后用蒸馏水清洗形成聚乙二醇改性的药物微球-介孔二氧化硅纳米粒子连接体；
(3)将所述聚乙二醇改性的药物微球-介孔二氧化硅纳米粒子连接体在含有环氧乙烷-聚乙二醇的蒸馏水中于室温下搅拌12小时；过滤、冷冻干燥获得所述聚乙二醇/环氧乙烷-聚乙二醇共修饰的药物微球-介孔二氧化硅纳米粒子连接体颗粒；
(4)将所述聚乙二醇/环氧乙烷-聚乙二醇共修饰的药物微球-介孔二氧化硅纳米粒子连接体颗粒在蒸馏水中与第二活性因子接触，获得结合第二活性因子的聚乙二醇/环氧乙烷-聚乙二醇共修饰的药物微球-介孔二氧化硅纳米粒子连接体颗粒；将所述结合第二活性因子的聚乙二醇/环氧乙烷-聚乙二醇共修饰的药物微球-介孔二氧化硅纳米粒子连接体颗粒在蒸馏水中，与第三活性因子接触，搅拌、过滤、洗涤、冷冻干燥得到所述载药微粒。
优选地，步骤(1)中，所述缩合反应条件为：在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺作用下室温搅拌，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的质量比为71∶22。
优选地，所述氨基和硫醇共功能化介孔二氧化硅纳米粒子由以下方法制备而成：
将介孔二氧化硅纳米粒子分散于第一有机介质中，超声处理20分钟，获得悬浮液；将3-三甲氧基硅丙硫醇和(3-氨基丙基)三甲氧基硅烷加入所述悬浮液中，室温搅拌12小时，过滤，洗涤后，在40℃的真空烘箱中干燥，获得氨基和硫醇共功能化介孔二氧化硅纳米粒子；
优选地，所述药物微球由以下方法制备而成：将50mg聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物和50mg聚乳酸-羟基乙酸共聚物溶解于第二有机介质中，然后加入到100微升含有1mg B淋巴细胞趋化因子-1的水溶液中，利用超产乳化形成形成油包水乳液，将所述油包水(w/o)乳液逐渐加入到聚乙烯醇溶液中，经搅拌或超声处理形成水包油(w/o/w)双乳化液，在室温下搅拌蒸发掉第二有机介质，离心收集具有包埋孔的微球，得到的微球用蒸馏水清洗后并冻干，获得所述药物微球。
优选地，所述药物微球由以下方法制备而成：将75mg聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸和75mg聚乳酸-羟基乙酸共聚物溶解于第二有机介质中，然后加入到100微升含有3mg IL-17抗体的水溶液，利用超声乳化形成形成油包水乳液，将所述油包水(w/o)乳液逐渐加入到聚乙烯醇溶液中，经搅拌或超声处理形成水包油(w/o/w)双乳化液，在室温下搅拌蒸发掉第二有机介质，离心收集具有包埋孔的微球，得到的微球用蒸馏水清洗后并冻干，获得所述药物微球。
优选地，所述聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物由以下方法制备而成：
(1)在催化剂催化作用下，使甲基丙烯酸羟乙基酯与L-丙交酯在聚合反应条件下发生聚合反应，生成甲基丙烯酸羟乙基酯-聚乳酸共聚物；具有如式I所示的结构式：
Figure PCTCN2021000002-appb-000003
(2)在引发剂参与下，在第三有机介质中，使甲基丙烯酸羟乙基酯-聚乳酸共聚物与丙烯酸发生聚合反应，生成聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物；具有如式II所示的结构式：
Figure PCTCN2021000002-appb-000004
优选地，所述甲基丙烯酸羟乙基酯与L-丙交酯的摩尔比为1∶20。
优选地，所述催化剂选自异辛酸亚锡；以甲基丙烯酸羟乙基酯的摩尔含量为1计算，所述异辛酸亚锡的摩尔含量为0.2。
优选地，步骤(1)中，所述聚合反应的条件为：在氮气保护下，加热至甲基丙烯酸羟乙基酯与L-丙交酯完全融化，然后在140度加热2小时。
优选地，所述引发剂选自偶氮二异丁腈。
优选地，所述第一有机介质选自乙醇。
优选地，所述第二有机介质选自二氯甲烷。
优选地，所述第三有机介质选自二氧六环。
优选地，甲基丙烯酸羟乙基酯-聚乳酸共聚物、丙烯酸和引发剂的摩尔比为5∶50∶1。
优选地，所述第一活性因子选自B淋巴细胞趋化因子-1或IL-17抗体，所述第二活性因子选自IL-1或CCL3，所述第三活性因子选自TNF-α或IFN-γ。
相对于现有技术，本发明的有益效果在于；
本发明的具有多级缓控释作用的载药微粒与现有技术中的脂质体相比，具有较好的稳定性，可负载多种药物或生物大分子，且可实现控制不同药物及生物大分子的给药时间，达到精确控制药物释放，以实现不同药物最大生物利用度的要求。
本发明的具有多级缓控释作用的载药微粒可通过连接不同的细胞因子，从而靶向到病灶部位，并只在病灶部位分时段释放不同药物，实现主动靶向治疗的目的，达到最大化药物利用度及精准治疗调控的目的。
本发明静脉给药系统，以PLGA与丙烯酸-甲基丙烯酸羟-丙交酯聚合体，具有毒性低，防止敏感药物的水解，可组合式给药，控制药物释放等优点。本发明的组合式结构静脉给药系统与脂质体相比，具有较好的稳定性，可负载多种药物或生物大分子，且可实现控制不同药物及生物大分子的给药时间，可达到精确控制药物释放，以实现不同药物最大生物利用度的要求。
本发明的药物微球的主要成分LLA、甲基丙烯酸羟乙基酯、异辛酸亚锡、AA单体、偶氮二异丁腈，均为低毒性产品安全性高，所形成微球密封性好，且可包裹亲水亲油等多种药物。
本发明利用EO-PEG-EO和PEGMEMA对二氧化硅纳米粒子进行了改性，可连接含有COOH -的靶向细胞因子，然后通过吸收法吸附细胞因子在二氧化硅纳米粒子的微孔中，其所制备的纳米粒子可通过连接合适的靶向细胞因子驱使纳米粒子向病灶区域主动迁移并结合，然后通过释放纳米粒子上吸附的细胞因子2对疾病进行精准治疗。
本发明将药物微球与二氧化硅纳米粒子进行连接，可达到多种药物共同负载的目的，而且微球与纳米粒子上的药物在体内的释放可实现分阶段释放的要求，纳米粒子上设计靶向细胞因子，可迁移病灶区域，其在此设计下，可根据疾病治疗的要求，合理设计药物的包装，释放顺序及释放量，达到药物的最大利用度。
附图说明
图1为本发明实施例中具有多级缓控释作用的载药微粒的结构示意图；
图2为本发明实施例中共载IL-17抗体，CCL3，IFN-γ的载药微粒的电镜图；
图3为本发明实施例中具有多级缓控释作用的载药微粒中IFN-γ随时间释放的比例；
图4为本发明实施例中具有多级缓控释作用的载药微粒中IL-17抗体随时间释放的比例。
具体实施例
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
试剂说明：
B淋巴细胞趋化因子-1采购自赛业生物，产品编号：HECXP-1301；
IL-17抗体采购自赛业生物，产品编号：HETNP-0101；
IL-1采购自北京同立海源生物科技有限公司，产品编号：TL-109；
CCL3采购自北京百奥莱博科技有限公司，产品编号：JN0292；
TNF-α或IFN-γ采购自北京同立海源生物科技有限公司，产品编号：TL-105；
L-丙交酯采购自Sigma-Aldrich Inc，产品编号：L09031；
M-甲基丙烯酸羟乙基酯采购自Sigma-Aldrich Inc，产品编号：B24260；
N-异辛酸亚锡采购自Sigma-Aldrich Inc，产品编号：B23612；
甲基丙烯酸羟乙基酯-聚乳酸、丙烯酸、偶氮二异丁腈采购自Sigma Aldrich；
其他未特别说明来源的试剂均采购自Sigma Aldrich。
实施例1
参考图1，图1为本实施例中具有多级缓控释作用的载药微粒的结构示意图；本实施例提供一种具有多级缓控释作用的载药微粒，所述载药微粒由药物微球和介孔二氧化硅纳米粒子结合而成；所述药物微球为水包油双乳化液微球，所述药物微球中含有第一活性因子；所述介孔二氧化硅纳米粒子上结合有第二活性因子和第三活性因子。
在一些实施例中，所述第一活性因子选自B淋巴细胞趋化因子-1或IL-17抗体，所述第二活性因子选自IL-1或CCL3，所述第三活性因子选自TNF-α或IFN-γ。
在一些实施例中，所述药物微球的结构为：
由聚乙烯醇水溶液构成的外层；
包裹于所述外层内部的油层，所述油层由丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物和聚乳酸-羟基乙酸共聚物的混合物组成；
包裹于所述油层内部的第一活性因子水溶液层。
在一些实施例中，所述聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物由以下方法制备而成：
取L-丙交酯(40mmol，5.760g)、甲基丙烯酸羟乙基酯(2mmol，0.26g)和异辛酸亚锡(0.4mmol，0.162g)在50ml圆底烧瓶中搅拌并加氮净化，获得混合物，存氮气保护下，将混合物加热至120度，使其完全熔化后，在140度下聚合2小时，将聚合产物溶于20毫升氯仿，在100毫升冷甲醇中沉淀，然后真空干燥，获得甲基丙烯酸羟乙基酯-聚乳酸；
取甲基丙烯酸羟乙基酯-聚乳酸(0.3mmol，0.771g)，丙烯酸(3mmol，0.216g)和偶氮二异丁腈(0.06mmol，9.8mg)添加到二氧六环(5ml)中并搅拌直到溶解，聚合反应于70度下持续24小时，聚合后的原产品经氯仿至甲醇反复沉淀3次，真空干燥40小时，获得聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物。
在一些实施例中，所述介孔二氧化硅纳米粒子为经过氨基和硫醇共功能化的介孔二氧化硅纳米粒子。
实施例2
本实施例提供一种载药微粒的制备方法，包含以下步骤：
(1)取71毫克EDC.HCl和22毫克N-羟基琥珀酰亚胺，加入3ml蒸馏水中，然后再加入20mg氨基和硫醇共功能化介孔二氧化硅纳米粒子和1mgB淋巴细胞趋化因子-1微球，获得悬浮液，将悬浮液在室温下搅拌24小时，然后用蒸馏水清洗，收集微球后，冷冻干燥后获得B淋巴细胞趋化因子-1微球-二氧化硅纳米粒子连接体；
(2)将100mgB淋巴细胞趋化因子-1微球-介孔二氧化硅纳米粒子连接体分散于20ml含1毫升聚(乙二醇)二甲基丙烯酸酯和30毫克2，2-二甲氧基-2-苯乙酮的蒸馏水中，获得悬浮液，将悬浮液在365nm的紫外线照射下搅拌30分钟，然后用蒸馏水清洗三次形成聚乙二醇改性的B淋巴细胞趋化因子-1微球-介孔二氧化硅纳米粒子连接体；
(3)将所述聚乙二醇改性的B淋巴细胞趋化因子-1微球-介孔二氧化硅纳米粒子连接体在含有800微升环氧乙烷-聚乙二醇的20毫升蒸馏水中重新分散，室温下搅拌12小时，过滤、冷冻干燥获得聚乙二醇/环氧乙烷-聚乙二醇共修饰的B淋巴细胞趋化因子-1微球-介孔二氧化硅纳米粒子连接体颗粒；
(4)将制备好的聚乙二醇/环氧乙烷-聚乙二醇共修饰的B淋巴细胞趋化因子-1微球-介孔二氧化硅纳米粒子连接体颗粒分散于20ml含有IL-1的蒸馏水，在室温下搅拌12小时；用3次蒸馏水洗涤去除未结合的IL-1，将其分散在蒸馏水中，然后通过吸收法增加TNF-α；将混合物在室温下搅拌24小时，所得固体进行过滤，用蒸馏水清洗，然后冷冻干燥得到共载B淋巴细胞趋化因子-1，IL-1，TNF-α的载药微球。
在一些实施例中，所述氨基和硫醇共功能化介孔二氧化硅纳米粒子由以下方法制备而成：
将介孔二氧化硅纳米粒子分散于乙醇中中，超声处理20分钟，获得悬浮液；将3-三甲氧基硅丙硫醇和(3-氨基丙基)三甲氧基硅烷加入所述悬浮液中，室温搅拌12小时，过滤，洗涤后，在40℃的真空烘箱中干燥，获得氨基和硫醇共功能化介孔二氧化硅纳米粒子；
在一些实施例中，所述药物微球由以下方法制备而成：将50mg聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物和50mg聚乳酸-羟基乙酸共聚物溶解于第二有机介质中，然后加入到100微升含有1mg B淋巴细胞趋化因子-1的水溶液中，利用超声乳化形成形成油包水乳液，将所述油包水(w/o)乳液逐渐加入到聚乙烯醇溶液中，经搅拌或超声处理形成水包油(w/o/w)双乳化液，在室温下搅拌蒸发掉第二有机介质，离心收集具有包埋孔的微球，得到的微球用蒸馏水清洗后并冻干，获得所述药物微球。
在一些实施例中，所述聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物由以下方法制备而成：将甲基丙烯酸羟乙基酯-聚乳酸(0.3mmol，0.771g)，丙烯酸(3mmol，0.216g)和偶氮二异丁腈(0.06mmol，9.8mg)添加到5ml二氧六环中并搅拌直到溶解，于70度下聚合反应24小时，聚合后的原产品经氯仿至甲醇反复沉淀3次纯化，并最后真空干燥40小时，获得聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物，具有如式II所示的结构式：
Figure PCTCN2021000002-appb-000005
在一些实施例中，所述甲基丙烯酸羟乙基酯-聚乳酸由以下方法制备而成：
取L-丙交酯(40mmol，5.760g)、甲基丙烯酸羟乙基酯(2mmol，0.26g)和异辛酸亚锡(0.4mmol，0.162g)在50ml圆底烧瓶中搅拌并加氮净化，在氮气保护下，将混合物加热至120度，使其完全熔化，在140度下聚合反应2小时，将聚合产物溶于20毫升氯仿，在100毫升冷甲醇中沉淀，然后真空干燥，获得甲基丙烯酸羟乙基酯-聚乳酸，具有如式I所示的结构式：
Figure PCTCN2021000002-appb-000006
实施例3
本实施例提供一种的载药微粒的制备方法，包含以下步骤：
(1)取71毫克EDC.HCl和22毫克N-羟基琥珀酰亚胺，加入3ml蒸馏水中，然后再加入20mg氨基和硫醇共功能化介孔二氧化硅纳米粒子和1mg IL-17抗体微球，获得悬浮液，将悬浮液在室温下搅拌24小时，然后用蒸馏水清洗，收集微球后，冷冻干燥后获得IL-17抗体微球-二氧化硅纳米粒子连接体；
(2)将100mgIL-17抗体微球-介孔二氧化硅纳米粒子连接体分散于20ml含1毫升聚(乙二醇)二甲基丙烯酸酯和30毫克2，2-二甲氧基-2-苯乙酮的蒸馏水中，获得悬浮液，将悬浮液在365nm的紫外线照射下搅拌30分钟，然后用蒸馏水清洗三次形成聚乙二醇改性的IL-17抗体微球-介孔二氧化硅纳米粒子连接体；
(3)将所述聚乙二醇改性的IL-17抗体微球-介孔二氧化硅纳米粒子连接体在含有800微升环氧乙烷-聚乙二醇的20毫升蒸馏水中重新分散，室温下搅拌12小时，过滤、冷冻干燥获得聚乙二醇/环氧乙烷-聚乙二醇共修饰的IL-17抗体微球-介孔二氧化硅纳米粒子连接体颗粒；
(4)将制备好的聚乙二醇/环氧乙烷-聚乙二醇共修饰的IL-17抗体微球-介孔二氧化硅纳米粒子连接体颗粒分散于20ml含有CCL3的蒸馏水，在室温下搅拌12小时；用3次蒸馏水洗涤去除未结合的CCL3，将其分散在蒸馏水中，然后通过吸收法增加IFN-γ；将混合物在室温下搅拌24小时，所得固体进行过滤，用蒸馏水清洗，然后冷冻干燥得到共载IL-17抗体，CCL3，IFN-γ的载药微球，图2为共载IL-17抗体，CCL3，IFN-γ的载药系统电镜图；图3显示了共载IL-17抗体，CCL3，IFN-γ的载药系统中IFN-γ随时间释放的比例；图4显示了共载IL-17抗体，CCL3，IFN-γ中IL-17抗体随时间释放的比例。
在一些实施例中，所述氨基和硫醇共功能化介孔二氧化硅纳米粒子由以下方法制备而成：
将介孔二氧化硅纳米粒子分散于乙醇中中，超声处理20分钟，获得悬浮液；将3-三甲氧基硅丙硫醇和(3-氨基丙基)三甲氧基硅烷加入所述悬浮液中，室温搅拌12小时，过滤，洗涤后，在40℃的真空烘箱中干燥，获得氨基和硫醇共功能化介孔二氧化硅纳米粒子。
在一些实施例中，所述药物微球由以下方法制备而成：100微升含有3mg IL-17抗体的水溶液加入含有15％(质量分数)聚合物(75mg聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸和75mg聚乳酸-羟基乙酸共聚物)二氯甲烷中在冰浴上利用超声波仪(15W的输出功率持续20s)进行乳化形成油包水(w/o)乳液。将w/o乳液逐渐加入10ml 1％(质量分数)聚乙烯醇溶液经搅拌或超声处理形成水包油(w/o/w)双乳化液。将溶液在室温下搅拌3h以蒸发二氯甲烷，离心收集具有包埋孔的微球。得到的微球用蒸馏水清洗三次并冻干。
在一些实施例中，所述聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物由以下方法制备而成：将甲基丙烯酸羟乙基酯-聚乳酸(0.3mmol，0.771g)，丙烯酸(3mmol，0.216g)和偶氮二异丁腈(0.06mmol，9.8mg)添加到5ml二氧六环中并搅拌直到溶解，于70度下聚合反应24小时，，聚合后的原产品经氯仿至甲醇反复沉淀3次纯化，并最后真空干燥40小时。
在一些实施例中，所述甲基丙烯酸羟乙基酯-聚乳酸由以下方法制备而成：
取L-丙交酯(40mmol，5.760g)、甲基丙烯酸羟乙基酯(2mmol，0.26g)和异辛酸亚锡(0.4mmol，0.162g)在50ml圆底烧瓶中搅拌并加氮净化，在氮气保护下，将混合物加热至120度，使其完全熔化，在140度下聚合反应2小时，将聚合产物溶于20毫升氯仿，在100毫升冷甲醇中沉淀，然后真空干燥，获得甲基丙烯酸羟乙基酯-聚乳酸。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何在本申请揭露的技术范围内的变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 具有多级缓控释作用的载药微粒，其特征在于，所述载药微粒由药物微球和介孔二氧化硅纳米粒子结合而成；所述药物微球为水包油双乳化液微球，所述药物微球中含有第一活性因子；所述介孔二氧化硅纳米粒子上结合有第二活性因子和第三活性因子。
  2. 根据权利要求1所述的载药微粒，其特征在于，所述第一活性因子选自B淋巴细胞趋化因子-1或IL-17抗体，所述第二活性因子选自IL-1或CCL3，所述第三活性因子选自TNF-α或IFN-γ。
  3. 根据权利要求1所述的载药微粒，其特征在于，所述药物微球选自B淋巴细胞趋化因子-1微球或IL-17抗体微球。
  4. 根据权利要求1所述的载药微粒，其特征在于，所述药物微球的结构为：
    由聚乙烯醇水溶液构成的外层；
    包裹于所述外层内部的油层，所述油层由丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物和聚乳酸-羟基乙酸共聚物的混合物组成；
    包裹于所述油层内部的第一活性因子水溶液层。
  5. 根据权利要求3所述的载药微粒，其特征在于，所述丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物由以下方法制备而成：
    (1)在催化剂催化作用下，使甲基丙烯酸羟乙基酯与L-丙交酯在聚合反应条件下发生聚合反应，生成甲基丙烯酸羟乙基酯-聚乳酸共聚物，具有如式I所示的结构式：
    Figure PCTCN2021000002-appb-100001
    (2)在引发剂参与下，在第三有机介质中，使甲基丙烯酸羟乙基酯-聚乳酸共聚物与丙烯酸发生聚合反应，生成聚丙烯酸-甲基丙烯酸羟乙基酯-聚乳酸共聚物，具有如式II所示的结构式：
    Figure PCTCN2021000002-appb-100002
  6. 根据权利要求5所述的载药微粒，其特征在于，所述甲基丙烯酸羟乙基酯与L-丙交酯的摩尔比为1∶20；甲基丙烯酸羟乙基酯-聚乳酸共聚物、丙烯酸和引发剂的摩尔比为5∶50∶1。
  7. 根据权利要求5所述的载药微粒，其特征在于，所述催化剂选自异辛酸亚锡；以甲基丙烯酸羟乙基酯的摩尔含量为1计算，所述异辛酸亚锡的摩尔含量为0.2。
  8. 根据权利要求5所述的载药微粒，其特征在于，步骤(1)中，所述聚合反应的条件为：在氮气保护下，加热至甲基丙烯酸羟乙基酯与L-丙交酯完全融化，然后在140度加热2小时。
  9. 根据权利要求5所述的载药微粒，其特征在于，所述引发剂选自偶氮二异丁腈；所述第一有机介质选自二氧六环。
  10. 根据权利要求5所述的载药微粒，其特征在于，所述介孔二氧化硅纳米粒子为经过氨基和硫醇共功能化的介孔二氧化硅纳米粒子。
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