WO2021042451A1 - 一种基于靶向核仁素的双重载药系统及制备方法与应用 - Google Patents

一种基于靶向核仁素的双重载药系统及制备方法与应用 Download PDF

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WO2021042451A1
WO2021042451A1 PCT/CN2019/111776 CN2019111776W WO2021042451A1 WO 2021042451 A1 WO2021042451 A1 WO 2021042451A1 CN 2019111776 W CN2019111776 W CN 2019111776W WO 2021042451 A1 WO2021042451 A1 WO 2021042451A1
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delivery system
drug delivery
mesoporous silica
nucleolin
silica nanoparticles
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French (fr)
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王宗花
高晚霞
张怀荣
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青岛大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/54—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
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure belongs to the technical field of biomedicine, and relates to a dual drug-carrying system based on targeted nucleolin as well as a preparation method and application.
  • Tumor refers to a new organism formed by the proliferation of local tissue cells under the action of various tumor-causing factors. According to the cell characteristics of new organisms and the degree of harm to the body, tumors are divided into two categories: benign tumors and malignant tumors. Malignant tumors can be divided into carcinomas and sarcomas. Cancer refers to malignant tumors derived from epithelial tissues. Sarcoma is a malignant tumor that occurs in mesenchymal tissue, including fibrous connective tissue, fat, muscle, vessel, bone and cartilage tissue. Anti-tumor drugs are a class of drugs used to treat tumor diseases. Simply put, there are chemotherapeutic drugs and biological agents.
  • tumor targeted therapy can be divided into two categories, namely tumor cell targeted therapy and tumor blood vessel targeted therapy.
  • Tumor cell targeted therapy uses specific antigens or receptors on the surface of tumor cells as targets, while tumor vascular targeted therapy uses specific antigens or receptors on the surface of newly formed capillary endothelial cells in the tumor area.
  • the purpose of the present disclosure is to provide a dual drug delivery system based on targeted nucleolin and its preparation method and application.
  • the dual drug delivery system of the present disclosure uses nucleolin as a target to directly release tumor drugs to the periphery of the cell nucleus. Make tumor drugs directly act on the nucleus, thereby effectively killing tumor cells.
  • the present disclosure provides a dual drug delivery system based on targeted nucleolin, which is a nanoparticle-like structure, including mesoporous silica nanoparticles, tumor drugs, and AS1411 aptamers.
  • targeted nucleolin which is a nanoparticle-like structure, including mesoporous silica nanoparticles, tumor drugs, and AS1411 aptamers.
  • the AS1411 aptamer is encapsulated on the surface of the mesoporous silica nanoparticles.
  • Nucleolin is a type of high molecular weight (>200kD) glycoprotein, type I transmembrane protein. Under normal circumstances, it is mainly expressed in a variety of tissues and organs near the lumen or glandular surface of epithelial cells. It is expressed at the apex and distributed in polarity. . It is highly expressed in the nucleus of eukaryotic cells that grow exponentially, while a smaller amount is expressed on the cytoplasm and cell surface. Nucleolin plays an important role in the regulation of ribosomal biological transcription, chromatin remodeling and nucleolar organization. The present disclosure takes advantage of the abnormal expression of nucleolin in cancer cells, and selects nucleolin as a marker for tumor targeted therapy.
  • the drug-carrying system of the present disclosure has a nano-particle structure that enables the drug to directly enter the cell, and the AS1411 aptamer encapsulated on the surface of the mesoporous silica is specifically identified with the nucleolin in the cell.
  • the AS1411 aptamer is separated from the surface of the mesoporous silica, so that the tumor drug loaded in the mesopore is exposed and released.
  • the released tumor drug is located in the tumor cell and can directly act on the nucleus, which improves the accuracy of targeting and treatment. effect.
  • the present disclosure provides a method for preparing the above-mentioned dual drug-carrying system based on targeted nucleolin, which uses aminopropyltriethoxysilane to modify mesoporous silica nanoparticles.
  • the modified mesoporous silica nanoparticles of the drug are added to the solution containing the AS1411 aptamer, so that the AS1411 aptamer is attached to the surface of the modified mesoporous silica nanoparticles.
  • the present disclosure uses aminopropyltriethoxysilane to modify the mesoporous silica nanoparticles, so that the surface of the mesoporous silica nanoparticles is positively charged, so that the AS1411 aptamer can be attached to the mesoporous dioxide.
  • the surface of the silicon nano-particles can seal the mesopores of the mesoporous silica nano-particles and avoid the leakage of tumor drugs in the mesopores.
  • the present disclosure proves through experiments that when doxorubicin (DOX) and phorbol ester (PMA) are used in combination as tumor drugs, the drug has a better targeting effect.
  • DOX doxorubicin
  • PMA phorbol ester
  • the present disclosure provides an application of the above-mentioned dual drug delivery system based on targeted nucleolin in the preparation of anti-tumor drugs.
  • the present disclosure uses mesoporous silica nanoparticles as a drug carrier, uses AS1411 aptamer to target tumor cell marker nucleolin, and combines the combined therapy of PMA and DOX to construct MSN@PMA for tumor targeted therapy @DOX dual drug delivery system, experimental results show that the dual drug delivery system has a high-efficiency targeting effect on HeLa tumor cell nucleolin, and the dual drug delivery system of the present disclosure has the advantages of precise targeting, high therapeutic effect and the like.
  • Figure 1 is a schematic diagram of the application of the MSN@PMA@DOX dual drug delivery system prepared in Example 1 of the disclosure in tumor treatment;
  • Figure 2 is a transmission electron micrograph of the MSN@PMA@DOX dual drug delivery system prepared in Example 1 of the disclosure, A is MSN, and B is MSN@PMA@DOX dual drug delivery system;
  • Figure 3 is a bar graph of the cytotoxicity test results of the MSN@PMA@DOX dual drug delivery system prepared in Example 1 of the disclosure;
  • Figure 4 is a flow cytometric data structure diagram of the MSN@PMA@DOX dual drug delivery system prepared in Example 1 of the disclosure, stained with the Annexin-FITC/PI kit, A is the dual of LO-2 cells and MSN@PMA@DOX The drug delivery system acts for 24 hours, B is HeLa cells and MSN@PMA@DOX dual drug delivery system for 24 hours, C is HeLa cells and MSN@DOX single drug delivery system for 24 hours, D is HeLa cells and MSN@PMA single The drug-loading system acts for 24 hours;
  • Figure 5 is a laser confocal image of LO-2 cells and the MSN@PMA@DOX dual drug delivery system prepared in Example 1 of the present disclosure, stained with AnnexinV-EGFP/PI apoptosis kit for 24 hours, A is LO-2 cells Bright field image, B is LO-2 cytoplasmic staining image, C is LO-2 nuclear staining image, D is LO-2 cell superimposed image;
  • Figure 6 is a laser confocal image of HeLa tumor cells stained with AnnexinV-EGFP/PI apoptosis kit after 24 hours incubation with MSN@PMA@DOX dual drug delivery system prepared in Example 1 of the present disclosure
  • A is a bright field image of HeLa cells
  • B is a HeLa cytoplasmic staining image
  • C is a HeLa nuclear staining image
  • D is an overlay image of HeLa cells.
  • the present disclosure proposes a dual drug-carrying system based on targeted nucleolin as well as a preparation method and application.
  • the dual drug-carrying system enables tumor drugs to directly act on the nucleus, thereby effectively killing tumor cells.
  • a typical embodiment of the present disclosure provides a dual drug delivery system based on targeted nucleolin, which is a nanoparticle-like structure, including mesoporous silica nanoparticles, tumor drugs, and AS1411 aptamers.
  • Tumor drugs are loaded in the mesopores of mesoporous silica nanoparticles, and the AS1411 aptamer is encapsulated on the surface of the mesoporous silica nanoparticles.
  • Nucleolin is a type of high molecular weight (>200kD) glycoprotein, type I transmembrane protein. Under normal circumstances, it is mainly expressed in a variety of tissues and organs near the lumen or glandular surface of epithelial cells. It is expressed at the apex and distributed in polarity. . It is highly expressed in the nucleus of eukaryotic cells that grow exponentially, while a smaller amount is expressed on the cytoplasm and cell surface. Nucleolin plays an important role in the regulation of ribosomal biological transcription, chromatin remodeling and nucleolar organization. The present disclosure takes advantage of the abnormal expression of nucleolin in cancer cells, and selects nucleolin as a marker for tumor targeted therapy.
  • the drug-carrying system of the present disclosure has a nano-particle structure that enables the drug to directly enter the cell, and the AS1411 aptamer encapsulated on the surface of the mesoporous silica is specifically identified with the nucleolin in the cell.
  • the AS1411 aptamer is separated from the surface of the mesoporous silica, so that the tumor drug loaded in the mesopore is exposed and released.
  • the released tumor drug is located in the tumor cell and can directly act on the nucleus, which improves the accuracy of targeting and treatment. effect.
  • the tumor drugs are adriamycin and phorbol ester. Experiments show that when DOX and PMA are used in combination as tumor drugs, they can kill tumor cells more efficiently.
  • the particle size of the nano-particle-like structure is 170-190 nm.
  • Another embodiment of the present disclosure provides a method for preparing the above-mentioned dual drug delivery system based on targeted nucleolin, which uses aminopropyltriethoxysilane to modify mesoporous silica nanoparticles, Adding tumor drugs to the suspension of modified mesoporous silica nanoparticles, the tumor drugs diffuse into the mesopores of the modified mesoporous silica nanoparticles, so that the mesoporous silica nanoparticles are loaded with tumor drugs.
  • the modified mesoporous silica nanoparticles loaded with tumor drugs are added to the solution containing the AS1411 aptamer, so that the AS1411 aptamer is attached to the surface of the modified mesoporous silica nanoparticles.
  • the present disclosure uses aminopropyltriethoxysilane to modify the mesoporous silica nanoparticles, so that the surface of the mesoporous silica nanoparticles is positively charged, so that the AS1411 aptamer can be attached to the mesoporous dioxide.
  • the surface of the silicon nano-particles can seal the mesopores of the mesoporous silica nano-particles and avoid the leakage of tumor drugs in the mesopores.
  • the method for modifying the mesoporous silica nanoparticles is: adding aminopropyltriethoxysilane to the suspension of mesoporous silica nanoparticles, and heating to Reaction at 35 ⁇ 40°C.
  • reaction time is 5-7h.
  • the solvent in the suspension of mesoporous silica nanoparticles is ethanol.
  • reaction is followed by filtration, and the filter residue is washed with ethanol and dried.
  • the temperature at which the tumor drug is loaded is room temperature.
  • the room temperature mentioned in the present disclosure refers to the indoor ambient temperature, which is generally 15-30°C.
  • the modified mesoporous silica nanoparticles loaded with tumor drugs are added to the solution containing the AS1411 aptamer, and the reaction is carried out at the temperature of the human body.
  • the temperature of the human body is generally 36.2 to 37.2°C.
  • the reaction time is 0.5 to 1.5 hours.
  • the addition ratio of the modified mesoporous silica nanoparticles loaded with tumor drugs and the AS1411 aptamer is 0.5-1.5: 5 ⁇ 10 -10 , mg: mol.
  • the particle size of the mesoporous silica nanoparticles is 170-190 nm.
  • the third embodiment of the present disclosure provides an application of the above-mentioned dual drug delivery system based on targeted nucleolin in the preparation of antineoplastic drugs.
  • the anti-tumor is anti-cervical cancer.
  • Cytotoxicity kit (CCK-8) was purchased from DOJINDO Institute of Chemistry.
  • the Annexin-FITC/PI apoptosis detection kit was purchased from Unitech Biosciences, and the Annexin-EGFP/PI apoptosis detection kit was purchased from Shanghai Yeasen Biotechnology Co., Ltd. This experiment uses ultrapure water (18.25M ⁇ cm, 24°C), and other reagents are of analytical grade.
  • the CCK-8 determination was performed on a microplate reader (EP0CH2). Apoptosis images were performed on a confocal laser microscope (Nikon, C2plus) and a flow cytometer (cytoFLEX, A00-1-1102).
  • DNA was purchased from Shanghai Shenggong Biological Engineering Co., Ltd., and the sequence is as follows:
  • CTABr n-hexadecyltrimethylammonium bromide (0.052g) was dissolved in 25mL of ultrapure water. Then, 1 mL of NaOH solution (0.36M) was added to the solution, and the mixture was heated to 95°C. Under continuous stirring, 2.0 mL TEOS (Ethyl Orthosilicate) was added dropwise, and the reaction mixture was stirred at 95°C for 3 hours. The mixture was filtered and centrifuged with ultrapure water and ethanol. Finally, the white precipitate was dried at 60°C and calcined at 550°C for 5 hours to obtain MSN.
  • NaOH solution 0.36M
  • MSN mesoporous silica nanoparticles
  • APTES 1 mL aminopropyltriethoxysilane
  • the mixture was centrifuged to obtain the MSN@PMA@DOX dual drug delivery system coated with AS1411 aptamer, which was centrifuged twice with ultrapure water and resuspended in PBS. Finally, the obtained MSN@PMA@DOX dual drug delivery system was stored at 4°C and protected from light.
  • the MSN@PMA and MSN@DOX single drug delivery systems were synthesized by the same method steps, and stored at 4°C in the dark.
  • the mesoporous silica is filled with anticancer drugs PMA and DOX, and then encapsulated with AS1411 aptamer to obtain the MSN@PMA@DOX dual drug delivery system.
  • PMA and DOX anticancer drugs
  • AS1411 aptamer of the dual drug delivery system binds to the nucleolin of HeLa cells and falls off the surface of the mesoporous silicon.
  • the dual drug delivery system releases PMA and DOX, anticancer drugs A large number of accumulations around the nucleus of HeLa cells, which in turn leads to cell apoptosis.
  • the CCK-8 assay was used.
  • HeLa tumor cells (10 ⁇ L, 2.0 ⁇ 10 6 cells mL -1 ) were incubated with 90 ⁇ L of medium.
  • the experimental group consisted of HeLa tumor cells incubated with 6 ⁇ L MSN@PMA@DOX dual drug delivery system for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours and 24 hours, respectively.
  • the apoptosis rate was measured by the Annexin-FITC/PI apoptosis kit, and flow cytometry was performed according to the instructions.
  • Cell test In short, 2 ⁇ 10 5 cells were collected after incubation, stained with 2 ⁇ L Annexin-FITC for 5 minutes in the dark, and 5 ⁇ L PI for 10 minutes, and then analyzed by flow cytometry. Neither Annexin-FITC nor PI can stain live cells. Early apoptotic cells are negative for PI but positive for Annexin-FITC, while late apoptotic cells are positive for both PI and Annexin-FITC.
  • the apoptosis rate was determined by the apoptosis kit Annexin-EGFP/PI, and analyzed by laser confocal microscope imaging. In brief, 1 ⁇ 10 4 cells were incubated, stained with 0.5 ⁇ L Annexin-EGFP for 5 minutes in the dark, stained with 0.5 ⁇ L PI for 10 minutes, and then analyzed by confocal microscope. Living cells cannot be stained by Annexin-EGFP and PI. Early apoptotic cells are negative for PI but positive for Annexin-EGFP, and late apoptotic cells stain both PI and Annexin-EGFP.
  • the cell culture is as follows:
  • MSN The transmission electron microscope of MSN and MSN@PMA@DOX dual drug delivery system is shown in Figure 2.
  • MSN is uniform in size, with an average diameter of about 180nm, bright pore size, and hollow and porous structure.
  • Figure 2B the hole of MSN is darkened, indicating that the MSN@PMA@DOX dual drug delivery system was successfully synthesized.
  • Nucleolin does not exist in LO-2 cells as ordinary somatic cells, so the MSN@PMA@DOX dual drug delivery system is non-toxic to LO-2 cells.
  • the nucleolin in HeLa cells can be targeted to bind to the AS1411 aptamer of the dual drug delivery system to release PMA and DOX in the dual drug delivery system, the dual drug delivery system can target and efficiently kill HeLa cells.
  • FIG. 4A shows LO-2 cells incubated with MSN@PMA@DOX dual drug delivery system, showing that 2.50% of LO-2 cells are in the apoptotic stage.
  • Figure 4B is HeLa tumor cells incubated with MSN@PMA@DOX dual drug delivery system, showing 12.35% of HeLa tumor cells are in the apoptotic stage,
  • Figure 4C is HeLa tumor cells incubated with MSN@DOX single drug delivery system, showing 3.45 % Of HeLa tumor cells are in the apoptotic stage.
  • Figure 4D shows HeLa tumor cells incubated with the MSN@PMA single drug delivery system. It shows that 2.23% of HeLa tumor cells are in the apoptotic stage. Flow cytometry shows that MSN@PMA@ The DOX dual drug delivery system can target and effectively kill tumor cells without damaging ordinary somatic cells, and the therapeutic effect on tumor cells is significantly higher than that of the single drug delivery system.
  • FIG. 5 is a confocal laser image of LO-2 cells incubated with dual drug delivery system for 24 hours, where Figure 5A is a bright field image of LO-2 cells; Figure 5B is a monochrome image of the cytoplasm of LO-2 cells without staining Phenomenon; Figure 5C is a single-color image of the nucleus of LO-2 cells without staining; Figure 5D is an overlay image of LO-2 cells.
  • FIG. 6 is a laser confocal image of HeLa tumor cells after 24 hours of incubation with the dual drug delivery system.
  • Figure 6A is a bright field image of HeLa tumor cells;
  • Figure 6B is a monochrome image of the cytoplasm of HeLa tumor cells with green cytoplasm.
  • Figure 6C is a single-color image of the nucleus of HeLa tumor cells, cells with red nuclei are late apoptotic cells
  • Figure 6D is an overlay image of HeLa tumor cells, early apoptotic HeLa tumor cells against Annexin-EGFP Staining, HeLa tumor cells with late apoptosis are double-stained for Annexin-EGFP and PI.
  • the reason is that the nucleolin in HeLa cells targets the AS1411 aptamer of the dual drug delivery system, and the dual drug delivery system releases PMA and DOX, leading to cell apoptosis.
  • the results show that the MSN@PMA@DOX dual drug delivery system can target HeLa tumor cells without harm to ordinary somatic cells.
  • the present disclosure uses MSN as a drug carrier, uses AS1411 aptamer to target tumor cell marker nucleolin, and combines PMA and DOX combined therapy to construct MSN@PMA@DOX dual drug delivery system for tumor targeted therapy .
  • the experimental results show that the dual drug-carrying system has a high-efficiency targeting effect on HeLa tumor cell nucleolin, and the present disclosure has the advantages of simple experimental operation, precise targeting, high-efficiency treatment effect and the like.

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Abstract

一种基于靶向核仁素的双重载药系统及制备方法与应用，该系统为纳米颗粒状结构，包括介孔二氧化硅纳米粒子、肿瘤药物和AS1411适配体，所述肿瘤药物负载在介孔二氧化硅纳米粒子的介孔内，所述AS1411适配体封装在介孔二氧化硅纳米粒子表面。该系统以介孔二氧化硅纳米粒子作为药物载体，利用AS1411适配体可以靶向肿瘤细胞的标志物核仁素，结合PMA和DOX的联合治疗，构建了针对肿瘤靶向治疗的MSN@PMA@DOX双重载药系统，实验结果表明，该双重载药系统对HeLa肿瘤细胞核仁素有高效的靶向作用，并且具有靶向精确、治疗效果高效等优点。

Description

一种基于靶向核仁素的双重载药系统及制备方法与应用 技术领域
本公开属于生物医药技术领域，涉及一种基于靶向核仁素的双重载药系统及制备方法与应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然构成现有技术。
肿瘤是指机体在各种致瘤因子作用下，局部组织细胞增生所形成的新生物。根据新生物的细胞特性及对机体的危害性程度，又将肿瘤分为良性肿瘤和恶性肿瘤两大类。恶性肿瘤可分为癌和肉瘤，癌是指来源于上皮组织的恶性肿瘤。肉瘤是指间叶组织，包括纤维结缔组织、脂肪、肌肉、脉管、骨和软骨组织等，发生的恶性肿瘤。抗肿瘤药物为治疗肿瘤疾病的一类药物。简单说来有化疗药物、生物制剂。近年来，分子肿瘤学、分子药理学的发展使肿瘤本质正在逐步阐明；大规模快速筛选、组合化学、基因工程等先进技术的发明和应用加速了药物开发进程；抗肿瘤药物的研究与开发已进入一个崭新的时代。目前，国际上临床常见的抗肿瘤药物约80余种，大致可分为以下6类：细胞毒类药物、激素类药物、生物反应调节剂、单克隆抗体药物、其他类药物、辅助药。但是，临床使用的抗肿瘤药物通常存在选择性不高等缺陷，即在杀伤肿瘤细胞的同时对机体正常细胞，特别是增殖旺盛的细胞具有损害作用。这些缺陷成为限制抗肿瘤药物用量、阻碍抗肿瘤药物疗效发挥的主要问题。
随着社会和科技的发展，癌症治疗观念正在发生根本性的改变，即由经验科学向循证医学、由细胞攻击模式向靶向性治疗模式转变。根据靶向部位的不同，又可以将肿瘤靶向治疗分为二大类，即肿瘤细胞靶向治疗和肿瘤血管靶向治疗。肿瘤细胞靶向治疗是利用肿瘤细胞表面的特异性抗原或受体作为靶向，而肿瘤血管靶向治疗则是利用肿瘤区域新生毛细血管内皮细胞表面的特异性抗原或受体起作用。
发明内容
本公开的目的是提供一种基于靶向核仁素的双重载药系统及制备方法与应用，本公开的双重载药系统以核仁素作为靶向，将肿瘤药物直接释放至细胞核周围，能够使肿瘤药物直接作用于细胞核，从而有效杀死肿瘤细胞。
为了实现上述目的，本公开的技术方案为：
第一方面，本公开提供了一种基于靶向核仁素的双重载药系统，为纳米颗粒状结构， 包括介孔二氧化硅纳米粒子、肿瘤药物和AS1411适配体，所述肿瘤药物负载在介孔二氧化硅纳米粒子的介孔内，所述AS1411适配体封装在介孔二氧化硅纳米粒子表面。
核仁素是一类高分子量(>200kD)糖蛋白，Ⅰ型跨膜蛋白，正常情况下主要表达于多种组织、器官中上皮细胞近管腔或腺腔面，呈顶端表达，极性分布。在指数生长的真核细胞细胞核中高度表达，而较小量在细胞质和细胞表面表达。核仁素在核糖体生物转录调节，染色质重塑和核仁组织中起重要作用。本公开利用核仁素在癌细胞中表达异常的特点，选择核仁素作为肿瘤靶向治疗的标志物。
本公开的载药系统为纳米颗粒状结构能够使得药物直接进入细胞内，并通过封装在介孔二氧化硅表面的AS1411适配体与细胞内的核仁素进行特异性识别，特异性识别后，AS1411适配体脱离介孔二氧化硅表面，从而使得负载在介孔内的肿瘤药物裸露，进而释放，释放的肿瘤药物位于肿瘤细胞内，能够直接作用于细胞核，提高了靶向精确、治疗效果。
通过实验表明，当采用阿霉素(DOX)和佛波酯(PMA)联合使用作为肿瘤药物时，能够更加高效的杀死肿瘤细胞。
第二方面，本公开提供了一种上述基于靶向核仁素的双重载药系统的制备方法，采用氨丙基三乙氧基硅烷对介孔二氧化硅纳米粒子进行改性，向改性后介孔二氧化硅纳米粒子的悬浮液中添加肿瘤药物，肿瘤药物扩散至改性后介孔二氧化硅纳米粒子的介孔中，使介孔二氧化硅纳米粒子负载肿瘤药物，将负载肿瘤药物的改性后介孔二氧化硅纳米粒子加入至含有AS1411适配体的溶液中，使AS1411适配体附着在改性后介孔二氧化硅纳米粒子的表面。
本公开采用氨丙基三乙氧基硅烷对介孔二氧化硅纳米粒子进行改性，使得介孔二氧化硅纳米粒子表面带有正电荷，从而能够将AS1411适配体附着在介孔二氧化硅纳米粒子的表面，从而实现封闭介孔二氧化硅纳米粒子的介孔，避免介孔中肿瘤药物泄漏。
本公开通过试验证明，当以阿霉素(DOX)和佛波酯(PMA)联合使用作为肿瘤药物，其药物的靶向效果更好。
第三方面，本公开提供了一种上述基于靶向核仁素的双重载药系统在制备抗肿瘤药物中的应用。
本公开的有益效果为：
本公开以介孔二氧化硅纳米粒子作为药物载体，利用AS1411适配体可以靶向肿瘤细胞的标志物核仁素，结合PMA和DOX的联合治疗，构建了针对肿瘤靶向治疗的MSN@PMA@DOX双重载药系统，实验结果表明，该双重载药系统对HeLa肿瘤细胞核仁素有高效的靶向作用，并且本公开的双重载药系统具有靶向精确、治疗效果高效等优点。
附图说明
构成本公开的一部分的说明书附图用来提供对本公开的进一步理解，本公开的示意性实施例及其说明用于解释本公开，并不构成对本公开的不当限定。
图1为本公开实施例1制备的MSN@PMA@DOX双重载药系统在肿瘤治疗中应用的示意图；
图2为本公开实施例1制备的MSN@PMA@DOX双重载药系统透射电镜照片，A为MSN，B为MSN@PMA@DOX双重载药系统；
图3为本公开实施例1制备的MSN@PMA@DOX双重载药系统细胞毒性实验结果柱状图；
图4为本公开实施例1制备的MSN@PMA@DOX双重载药系统用Annexin-FITC/PI试剂盒染色的流式细胞实验数据结构图，A为LO-2细胞与MSN@PMA@DOX双重载药系统作用24小时，B为HeLa细胞与MSN@PMA@DOX双重载药系统作用24小时，C为HeLa细胞与MSN@DOX单载药系统作用24小时，D为HeLa细胞与MSN@PMA单载药系统作用24小时；
图5为LO-2细胞与本公开实施例1制备的MSN@PMA@DOX双重载药系统孵育24小时用AnnexinV-EGFP/PI凋亡试剂盒染色的激光共聚焦图像，A为LO-2细胞明场图，B为LO-2细胞质染色图，C为LO-2细胞核染色图，D为LO-2细胞叠加图；
图6为HeLa肿瘤细胞与本公开实施例1制备的MSN@PMA@DOX双重载药系统孵育24小时用AnnexinV-EGFP/PI凋亡试剂盒染色的激光共聚焦图像，A为HeLa细胞明场图，B为HeLa细胞质染色图，C为HeLa细胞核染色图，D为HeLa细胞叠加图。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本公开提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本公开所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本公开的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本公开提出了一种基于靶向核仁素的双重载药系统及制备方法与应用。该双重载药系统能够使肿瘤药物直接作用于细胞核，从而有效杀死肿瘤细胞。
本公开的一种典型实施方式，提供了一种基于靶向核仁素的双重载药系统，为纳米颗粒状结构，包括介孔二氧化硅纳米粒子、肿瘤药物和AS1411适配体，所述肿瘤药物负载在介孔二氧化硅纳米粒子的介孔内，所述AS1411适配体封装在介孔二氧化硅纳米粒子表面。
核仁素是一类高分子量(>200kD)糖蛋白，Ⅰ型跨膜蛋白，正常情况下主要表达于多种组织、器官中上皮细胞近管腔或腺腔面，呈顶端表达，极性分布。在指数生长的真核细胞细胞核中高度表达，而较小量在细胞质和细胞表面表达。核仁素在核糖体生物转录调节，染色质重塑和核仁组织中起重要作用。本公开利用核仁素在癌细胞中表达异常的特点，选择核仁素作为肿瘤靶向治疗的标志物。
本公开的载药系统为纳米颗粒状结构能够使得药物直接进入细胞内，并通过封装在介孔二氧化硅表面的AS1411适配体与细胞内的核仁素进行特异性识别，特异性识别后，AS1411适配体脱离介孔二氧化硅表面，从而使得负载在介孔内的肿瘤药物裸露，进而释放，释放的肿瘤药物位于肿瘤细胞内，能够直接作用于细胞核，提高了靶向精确、治疗效果。
该实施方式的一种或多种实施例中，肿瘤药物为阿霉素和佛波酯。通过实验表明，当采用DOX和PMA联合使用作为肿瘤药物时，能够更加高效的杀死肿瘤细胞。
该实施方式的一种或多种实施例中，纳米颗粒状结构的粒径为170～190nm。
本公开的另一种实施方式，提供了一种上述基于靶向核仁素的双重载药系统的制备方法，采用氨丙基三乙氧基硅烷对介孔二氧化硅纳米粒子进行改性，向改性后介孔二氧化硅纳米粒子的悬浮液中添加肿瘤药物，肿瘤药物扩散至改性后介孔二氧化硅纳米粒子的介孔中，使介孔二氧化硅纳米粒子负载肿瘤药物，将负载肿瘤药物的改性后介孔二氧化硅纳米粒子加入至含有AS1411适配体的溶液中，使AS1411适配体附着在改性后介孔二氧化硅纳米粒子的表面。
本公开采用氨丙基三乙氧基硅烷对介孔二氧化硅纳米粒子进行改性，使得介孔二氧化硅纳米粒子表面带有正电荷，从而能够将AS1411适配体附着在介孔二氧化硅纳米粒子的表面，从而实现封闭介孔二氧化硅纳米粒子的介孔，避免介孔中肿瘤药物泄漏。
该实施方式的一种或多种实施例中，介孔二氧化硅纳米粒子改性的方法为：向介孔二氧化硅纳米粒子的悬浮液中添加氨丙基三乙氧基硅烷，加热至35～40℃反应。
该系列实施例中，反应时间为5～7h。
该系列实施例中，介孔二氧化硅纳米粒子的悬浮液中的溶剂为乙醇。
该系列实施例中，反应后过滤，滤渣采用乙醇洗涤后干燥。
该实施方式的一种或多种实施例中，负载肿瘤药物的温度为室温。本公开所述的室温是指室内环境温度，一般为15～30℃。
该实施方式的一种或多种实施例中，将负载肿瘤药物的改性后介孔二氧化硅纳米粒子加入至含有AS1411适配体的溶液中，在人体温度下进行反应。所述人体温度一般为36.2～37.2℃。
该系列实施例中，反应时间为0.5～1.5h。
该实施方式的一种或多种实施例中，负载肿瘤药物的改性后介孔二氧化硅纳米粒子与AS1411适配体的加入比例为0.5～1.5：5×10 -10，mg：mol。
该实施方式的一种或多种实施例中，介孔二氧化硅纳米粒子的粒径为170～190nm。
本公开的第三种实施方式，提供了一种上述基于靶向核仁素的双重载药系统在制备抗肿瘤药物中的应用。
该实施方式的一种或多种实施例中，所述抗肿瘤为抗宫颈癌。
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例详细说明本公开的技术方案。
仪器与试剂
PMA、DOX购买于美国Sigma公司。细胞毒性试剂盒(CCK-8)购买于DOJINDO化学研究所。Annexin-FITC/PI凋亡检测试剂盒购买于联科生物公司，Annexin-EGFP/PI凋亡检测试剂盒购买于上海Yeasen生物科技有限公司。本实验采用超纯水(18.25MΩcm，24℃)，其他试剂均为分析纯。CCK-8测定在酶标仪(EP0CH2)上进行。细胞凋亡图像在激光共聚焦显微镜(Nikon，C2plus)和流式细胞仪(cytoFLEX，A00-1-1102)上进行。DNA购买于上海生工生物工程股份有限公司，序列如下：
Figure PCTCN2019111776-appb-000001
介孔二氧化硅纳米粒子(MSN)的合成：
将CTABr(正十六烷基三甲基溴化铵)(0.052g)溶解在25mL超纯水中。后向溶液中加入1mL NaOH溶液(0.36M)，并将混合物加热至95℃。在连续搅拌下，逐滴加入2.0mL TEOS(正硅酸乙酯)，将反应混合物在95℃下搅拌3小时。过滤混合物，并用超纯水和乙醇离心。最后，将白色沉淀物在60℃下干燥，并在550℃下煅烧5小时，得到MSN。
实施例1
将1.0000g介孔二氧化硅纳米粒子(MSN)悬浮在100mL无水乙醇中，并加入过量的氨丙基三乙氧基硅烷(APTES 1mL)。将混合物在36℃下连续搅拌6小时，过滤，用乙醇洗涤，在60℃下干燥，得到APTES-MSN。将1.0000g APTES-MSN粉末分散至1mL超纯水中获得悬浮液。然后在100μL悬浮液中加入PMA(0.05mL，5μg mL -1)和DOX(0.05mL，0.25mg mL -1)并在室温下搅拌过夜，用超纯水离心，真空干燥，得到白色粉末。将1mg白色粉末分散在1mL PBS(1mg mL -1)中并与AS1411适配体(50μL，10μM)混合，在37℃下反应1小时。然后，离心混合物，获得AS1411适配体包裹的MSN@PMA@DOX双重载药系统，用 超纯水离心两次并重悬于PBS中。最后，将获得的MSN@PMA@DOX双重载药系统在4℃避光存储。
以同样的方法步骤分别合成MSN@PMA和MSN@DOX单载药系统，在4℃避光存储。
本公开的原理如图1所示，首先在介孔二氧化硅中填充抗癌药物PMA和DOX，后用AS1411适配体封装，得到MSN@PMA@DOX双重载药系统。将双重载药系统与HeLa细胞孵育，双重载药系统的AS1411适配体与HeLa细胞的核仁素靶向结合，从介孔硅表面脱落从而双重载药系统释放出PMA和DOX，抗癌药物在HeLa细胞核周围大量聚集，进而导致细胞凋亡。
对获得的MSN@PMA@DOX双重载药系统进行实验如下：
细胞毒性实验：
为了评估MSN@PMA@DOX双重载药系统对细胞的毒性，采用CCK-8测定法。在96孔板的孔中，将HeLa肿瘤细胞(10μL，2.0×10 6个细胞mL -1)与90μL培养基一起孵育。为了显示MSN@PMA@DOX双重载药系统的生物相容性，我们设置了对照实验。实验组为HeLa肿瘤细胞与6μL MSN@PMA@DOX双重载药系统分别孵育2小时、4小时、6小时、8小时、10小时、12小时和24小时。对照组是HeLa细胞与6μL MSN和LO-2细胞与6μL MSN@PMA@DOX双重载药系统在相同时间梯度下孵育。然后，在每个孔中加入10μL CCK-8(5mg mL -1)并在37℃细胞培养箱中孵育1小时。最后，使用酶标仪测量每个孔在450nm处的吸光度。根据下式：细胞活力(％)＝[(A test-A blank)/(A control-A blank)]×100％。计算细胞的存活率。
流式细胞试验：
将HeLa肿瘤细胞或LO-2细胞与MSN@PMA@DOX双重载药系统一起孵育24小时后，通过膜联蛋白Annexin-FITC/PI凋亡试剂盒测定细胞凋亡率，按照的说明书进行流式细胞试验。简而言之，孵育后收集2×10 5个细胞，在黑暗中用2μL Annexin-FITC染色5分钟，用5μL PI染色10分钟，然后通过流式细胞仪分析。Annexin-FITC和PI均不能染色活细胞。早期凋亡的细胞对PI是阴性但对Annexin-FITC是阳性的，而晚期凋亡细胞对PI和Annexin-FITC都呈阳性。
激光共聚焦显微镜图像：
将HeLa肿瘤细胞或LO-2细胞与MSN@PMA@DOX双重载药系统一起孵育24小时后，通过凋亡试剂盒Annexin-EGFP/PI测定细胞凋亡率，并通过激光共聚焦显微镜成像分析。简言之，孵育1×10 4个细胞，在黑暗中用0.5μL Annexin-EGFP染色5分钟，用0.5μL PI染色10分钟，然后通过共聚焦显微镜分析。活细胞均不能被Annexin-EGFP和PI染色。早期凋亡的细 胞对PI是阴性但对Annexin-EGFP是阳性的，晚期凋亡细胞对PI和Annexin-EGFP都染色。
其中，细胞培养如下：
将HeLa肿瘤细胞和LO-2细胞在含有1％链霉素和青霉素，10％胎牛血清(FBS)的DMEM培养基，5％CO 2和95％空气，湿润的，37℃恒温箱中孵育。通过胰蛋白酶(0.1％，m/v)消化指数生长期的细胞1分钟，然后从细胞培养基中分离。用无菌磷酸盐缓冲溶液(PBS，10mM，pH 7.4)以800rpm离心3分钟洗涤三次，收集细胞。然后将细胞重新悬浮在10mL DMEM中。
表征结果：
MSN与MSN@PMA@DOX双重载药系统的透射电镜如图2所示，图2A中，MSN尺寸均匀，平均直径约为180nm，孔径明亮，结构呈中空多孔状。而图2B中，MSN的孔变暗，说明MSN@PMA@DOX双重载药系统成功合成。
采用CCK-8测定法评估MSN@PMA@DOX双重载药系统对细胞的生物相容性。将细胞与双重载药系统在设定的时间梯度下孵育，后与CCK-8试剂作用并在酶标仪中测量每个孔在450nm处的吸光度。根据下式：细胞活力(％)＝[(A test-A blank)/(A control-A blank)]×100％，获得细胞存活率。从图3中可以看出，HeLa细胞与MSN@PMA@DOX双重载药系统作用后，随着时间的不断增加，细胞的存活率不断减小，作用24小时后，细胞的存活率仅为46％。HeLa细胞与MSN双重载药系统孵育后存活率超过90％，LO-2细胞与MSN@PMA@DOX双重载药系统孵育后存活率超过90％。实验结果表明，MSN对LO-2细胞和HeLa细胞均无生物毒性，是一种优良的药物载体。LO-2细胞中作为普通体细胞不存在核仁素，因此MSN@PMA@DOX双重载药系统对LO-2细胞无毒性。但是，由于HeLa细胞中的核仁素可以与双重载药系统的AS1411适配体靶向结合，释放双重载药系统中的PMA和DOX，所以该双重载药系统可靶向并高效的杀死HeLa细胞。
HeLa肿瘤细胞的凋亡通过流式细胞仪定量表达。将细胞与MSN@PMA@DOX双重载药系统一起孵育24小时，并用Annexin-FITC/PI试剂盒进行染色。图4A是用MSN@PMA@DOX双重载药系统孵育的LO-2细胞，显示2.50％的LO-2细胞处于凋亡阶段。图4B是用MSN@PMA@DOX双重载药系统孵育的HeLa肿瘤细胞，显示12.35％的HeLa肿瘤细胞处于凋亡阶段，图4C是用MSN@DOX单载药系统孵育的HeLa肿瘤细胞，显示3.45％的HeLa肿瘤细胞处于凋亡阶段，图4D是用MSN@PMA单载药系统孵育的HeLa肿瘤细胞，显示2.23％的HeLa肿瘤细胞处于凋亡阶段，通过流式细胞实验表明，MSN@PMA@DOX双重载药系统可以靶向并有效的杀死肿瘤细胞而对普通体细胞无损伤作用，且对肿瘤细胞的治疗效果明显高于单载药系统。
细胞激光共聚焦实验进一步研究了MSN@PMA@DOX双重载药系统诱导HeLa肿瘤细胞凋亡的过程。将细胞与MSN@PMA@DOX双重载药系统一起孵育24小时，并用Annexin-EGFP/PI试剂盒进行染色。图5是LO-2细胞与双重载药系统孵育24小时之后的激光共聚焦图像，其中图5A是LO-2细胞的明场图像；图5B是LO-2细胞的细胞质单色图，没有染色现象；图5C是LO-2细胞的细胞核单色图，无染色现象；图5D是LO-2细胞的叠加图。结果表明MSN@PMA@DOX双重载药系统对LO-2细胞无损伤作用。原因是LO-2细胞中无核仁素，双重载药系统在细胞中无法释放药物，因此对细胞无损伤。图6是HeLa肿瘤细胞与双重载药系统孵育24小时之后的激光共聚焦图像，其中图6A是HeLa肿瘤细胞的明场图像；图6B是HeLa肿瘤细胞的细胞质单色图，细胞质为绿色的细胞为早期凋亡细胞；图6C是HeLa肿瘤细胞的细胞核单色图，细胞核呈红色的细胞为晚期凋亡细胞；图6D是HeLa肿瘤细胞的叠加图，早期凋亡的HeLa肿瘤细胞对Annexin-EGFP染色，晚期凋亡的HeLa肿瘤细胞对Annexin-EGFP和PI双染色。原因是HeLa细胞中的核仁素靶向结合双重载药系统的AS1411适配体，双重载药系统释放出PMA和DOX，导致细胞凋亡。结果表明MSN@PMA@DOX双重载药系统可靶向治疗HeLa肿瘤细胞，而对普通体细胞无损害。
结论：
本公开以MSN作为药物载体，利用AS1411适配体可以靶向肿瘤细胞的标志物核仁素，结合PMA和DOX的联合治疗，构建了针对肿瘤靶向治疗的MSN@PMA@DOX双重载药系统。实验结果表明，该双重载药系统对HeLa肿瘤细胞核仁素有高效的靶向作用，并且本公开具有实验操作简便、靶向精确、治疗效果高效等优点。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
Figure PCTCN2019111776-appb-000002

Claims (10)

  1. 一种基于靶向核仁素的双重载药系统，其特征是，为纳米颗粒状结构，包括介孔二氧化硅纳米粒子、肿瘤药物和AS1411适配体，所述肿瘤药物负载在介孔二氧化硅纳米粒子的介孔内，所述AS1411适配体封装在介孔二氧化硅纳米粒子表面。
  2. 如权利要求1所述的基于靶向核仁素的双重载药系统，其特征是，肿瘤药物为阿霉素和佛波酯。
  3. 如权利要求1所述的基于靶向核仁素的双重载药系统，其特征是，纳米颗粒状结构的粒径为170～190nm。
  4. 一种权利要求1～3任一所述的基于靶向核仁素的双重载药系统的制备方法，其特征是，采用氨丙基三乙氧基硅烷对介孔二氧化硅纳米粒子进行改性，向改性后介孔二氧化硅纳米粒子的悬浮液中添加肿瘤药物，肿瘤药物扩散至改性后介孔二氧化硅纳米粒子的介孔中，使介孔二氧化硅纳米粒子负载肿瘤药物，将负载肿瘤药物的改性后介孔二氧化硅纳米粒子加入至含有AS1411适配体的溶液中，使AS1411适配体附着在改性后介孔二氧化硅纳米粒子的表面。
  5. 如权利要求4所述的基于靶向核仁素的双重载药系统的制备方法，其特征是，介孔二氧化硅纳米粒子改性的方法为：向介孔二氧化硅纳米粒子的悬浮液中添加氨丙基三乙氧基硅烷，加热至35～40℃反应；
    优选的，反应时间为5～7h；
    优选的，介孔二氧化硅纳米粒子的悬浮液中的溶剂为乙醇；
    优选的，反应后过滤，滤渣采用乙醇洗涤后干燥。
  6. 如权利要求4所述的基于靶向核仁素的双重载药系统的制备方法，其特征是，负载肿瘤药物的温度为室温。
  7. 如权利要求4所述的基于靶向核仁素的双重载药系统的制备方法，其特征是，将负载肿瘤药物的改性后介孔二氧化硅纳米粒子加入至含有AS1411适配体的溶液中，在人体温度下进行反应；
    优选的，反应时间为0.5～1.5h。
  8. 如权利要求4所述的基于靶向核仁素的双重载药系统的制备方法，其特征是，负载肿瘤药物的改性后介孔二氧化硅纳米粒子与AS1411适配体的加入比例为0.5～1.5：5×10 -10，mg：mol。
  9. 如权利要求4所述的基于靶向核仁素的双重载药系统的制备方法，其特征是，介孔二氧化硅纳米粒子的粒径为170～190nm。
  10. 一种权利要求1～3任一所述的基于靶向核仁素的双重载药系统在制备抗肿瘤药物中 的应用。
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