WO2020259489A1 - 一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体及制备方法 - Google Patents

一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体及制备方法 Download PDF

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WO2020259489A1
WO2020259489A1 PCT/CN2020/097678 CN2020097678W WO2020259489A1 WO 2020259489 A1 WO2020259489 A1 WO 2020259489A1 CN 2020097678 W CN2020097678 W CN 2020097678W WO 2020259489 A1 WO2020259489 A1 WO 2020259489A1
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silk fibroin
manganese dioxide
dioxide composite
core
shell structure
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French (fr)
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杨明英
王捷
陈玉银
陈玉平
范鑫
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Zhejiang University ZJU
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/501—Inorganic compounds
    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • 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 invention belongs to the field of biomedical materials, and specifically relates to a preparation method of a silk fibroin/manganese dioxide composite microsphere drug carrier with a core-shell structure.
  • MnO 2 Sol-shaped manganese dioxide nanoparticles have become an emerging material in the field of biomedicine due to their specific catalytic properties and good biocompatibility.
  • manganese dioxide nanoparticles can specifically react with hydrogen peroxide in the tumor microenvironment to catalyze its degradation to produce oxygen and water, thereby improving the hypoxic environment and lowering the hypoxia-inducible factors, which both weaken the tumor Tissue drug resistance also helps photosensitizers convert oxygen into singlet oxygen, and at the same time improves the efficiency of tumor chemotherapy and photodynamic therapy.
  • the rich H + and glutathione (GSH) in the tumor microenvironment can degrade MnO 2 to generate Mn 2+ enhanced MRI for tumor imaging and real-time monitoring.
  • Mn 2 can be quickly excreted by the kidneys, which has high biological safety. Therefore, MnO 2 nanoparticles have high application prospects in the field of tumor therapy.
  • the synthesis method of MnO 2 nanoparticles still has defects such as complicated process, harsh reaction conditions (high temperature, high pressure), uneven product size, and the colloidal stability of single MnO 2 nanoparticles circulating in the blood is uncontrollable. The rapid degradation hinders its clinical application in vivo.
  • Silk protein is synthesized in insects and forms silk protein fibers with good mechanical properties after spinning.
  • Silk protein is a natural biopolymer with excellent mechanical properties, biocompatibility and low immunogenicity. It is an ideal material in the field of tissue engineering.
  • silk protein is easier to form a micro-nano sphere structure for drug loading and transportation.
  • silk protein can be used as a biological template to regulate the synthesis of other inorganic nanoparticles and generate composite microspheres as drug carriers.
  • silk fibroin/hydroxyapatite composite microspheres can be prepared by a biomineralization method.
  • the use of silk protein to regulate the production of manganese dioxide optimize the performance of manganese dioxide nanoparticles, and improve their application value in the field of tumor therapy, so far, there is no relevant report.
  • the invention also discloses a core-shell structure silk protein/manganese dioxide composite microsphere product prepared by the above method.
  • the method of the present invention has simple process and mild reaction conditions.
  • the prepared silk protein/manganese dioxide composite microspheres have good dispersibility, high specific surface area, controllable drug release ability, and excellent singlet oxygen generation ability. Therefore, the problems existing in the prior art are fundamentally solved.
  • a preparation method of silk fibroin/manganese dioxide composite microsphere drug carrier with core-shell structure comprising:
  • step (1) the silk fibroin aqueous solution can be prepared by the following method:
  • the concentration of the silk fibroin aqueous solution is 0.1-30 mg/mL. More preferably, it is 2-20 mg/mL.
  • the water-soluble organic solvent is ethanol, isopropanol or acetone. It is further preferably isopropanol.
  • isopropanol is used, the microspheres obtained are more regular, smaller in particle size, and better in drug loading. According to actual needs, isopropanol can be used, or other reagents such as ethanol and acetone can be substituted according to the size of microspheres.
  • the volume of the water-soluble organic solvent added is 1/15-2/5 of the volume of the silk fibroin aqueous solution. More preferably, it is 1/10 to 2/5.
  • the water-soluble organic solvent is isopropanol, and the added volume is 1/10 to 2/10 of the volume of the silk fibroin aqueous solution.
  • the water-soluble organic solvent is ethanol
  • the volume of the water-soluble organic solvent added is 1/5 to 2/5 of the volume of the silk fibroin aqueous solution.
  • the freezing conditions are: the temperature is -10 ⁇ -90°C; the time is 10 ⁇ 20 hours; as a further preference: when the water-soluble organic solvent is isopropanol: the temperature is -70 ⁇ -90°C; when the water-soluble organic solvent is ethanol The temperature is -10 ⁇ -30°C.
  • KMnO 4 is the raw material for the synthesis of manganese dioxide, and polypropylene amine hydrochloride is the reducing agent and surface modifier. KMnO 4 is reduced to generate MnO 2 and the surface is modified with positive charges.
  • the molecular weight range of PAH is 10KDa -35KDa.
  • the molar amount of potassium permanganate required for 1 g of silk fibroin is 5-25 mmol; the volume of polyacrylamine hydrochloride required for 1 g of silk fibroin is 10-250 ml.
  • the incubation conditions are: the temperature is 20-50°C, and the time is 1-10 hours. More preferably, it is 2-6 hours, and the temperature is 30-40 degreeC.
  • the silk fibroin is selected from one or more of Bombyx mori silk fibroin, wild silk fibroin, tussah silk fibroin, spider silk protein, and recombinant silk protein.
  • Step (2) After the reaction is completed, centrifugation and washing can obtain core-shell structure silk fibroin/manganese dioxide composite microspheres, the particle size of the microspheres is 100-500nm, the inner layer is the silk fibroin nanosphere layer, and the outer layer is The manganese dioxide layer has a rough surface structure and can be used as a vehicle for tumor drug therapy and photodynamic therapy.
  • a silk fibroin/manganese dioxide composite microsphere drug carrier with a core-shell structure is prepared by the preparation method described in any one of the above technical solutions.
  • the chemotherapeutic drugs may include hydrophilic anticancer drugs such as doxorubicin hydrochloride, etc., and can also be loaded with hydrophobic anticancer drugs paclitaxel, etc.
  • the drugs include doxorubicin hydrochloride and photosensitizer Ce6.
  • the drug loading method can be combined with silk protein for in-situ synthetic loading, or surface adsorption loading. The two methods can be used singly or simultaneously to increase the drug loading rate.
  • the silk fibroin/manganese dioxide composite microspheres of the present invention can catalyze H 2 O 2 in the tumor microenvironment due to the presence of MnO 2 to generate a large amount of O 2 and improve the photodynamic therapy efficiency of the tumor.
  • the silk fibroin/manganese dioxide composite microspheres are used as carriers to transport the photosensitizer to the tumor site. Under laser irradiation, the generated oxygen can be converted into singlet oxygen, thereby effectively killing tumor cells.
  • the invention discloses a preparation method of a silk fibroin/manganese dioxide composite microsphere drug carrier with a core-shell structure.
  • the present invention sequentially includes the following steps: sequentially degumming, dissolving, and concentrating the silk fibroin shells to obtain a silk fibroin solution; treating the silk fibroin solution with isopropanol and freezing and thawing at a low temperature to obtain silk fibroin Nanospheres; using silk fibroin nanospheres as a template to induce potassium permanganate (KMnO 4 ) to be reduced with the assistance of polyacrylamine hydrochloride (PAH), and generate MnO 2 in situ on the surface of the silk fibroin nanosphere template, Finally, the core-shell structure silk fibroin/manganese dioxide composite microspheres are obtained, which are used as drug carriers, and are loaded with anti-cancer drugs and photosensitizers to implement chemotherapy and
  • the process of the present invention is simple and easy to operate.
  • the obtained silk fibroin/manganese dioxide composite microspheres have rough surface.
  • the composite microspheres have excellent properties of combining silk fibroin and manganese dioxide.
  • the drug loading rate is high, it can respond to the controlled release of tumor microenvironment, degrade hydrogen peroxide in the tumor environment to generate oxygen, improve the efficiency of tumor chemistry and photodynamic therapy, and has a wide range of application prospects in the field of tumor therapy and medicine.
  • the present invention has the following outstanding advantages:
  • the silk protein template selected in the present invention is a natural biopolymer with a wide range of sources, providing good biosafety and effectively reducing preparation costs.
  • Silk fibroin can be loaded with both hydrophilic and hydrophobic anti-cancer drugs.
  • the rough surface of MnO 2 particles is also conducive to the loading of photosensitizers.
  • the silk fibroin/manganese dioxide composite microspheres can not only carry the drug by in-situ synthesis, but also can carry out the surface adsorption and loading by electrostatic attraction. The rate is higher.
  • Figure 2 is a cell viability diagram showing the effect of different concentrations of silk fibroin/manganese dioxide composite microsphere drug carrier on human fibroblasts in Example 1.
  • Figure 3 is the loading and release diagram of the silk fibroin/manganese dioxide composite microsphere drug carrier with the core-shell structure in Example 2 after loading the antitumor drug adriamycin hydrochloride and the photosensitizer Ce6.
  • Figure 4 is a graph showing the lethal efficiency of drug-loaded silk fibroin/manganese dioxide composite microspheres on human breast cancer MCF-7 cells in the presence of light and H 2 O 2 .
  • step (1) Adjust the concentration of silk fibroin in step (1) to 2 mg/mL (the solvent is water, the same in other embodiments), and place 10 mL in the reaction vessel. Slowly add 1 mL of isopropanol dropwise to the silk protein solution, and continue to stir for 30 minutes to make it uniform.
  • step (3) Place the mixed solution of step (2) in a -80°C refrigerator, freeze for 12 hours, and then allow it to thaw naturally at room temperature to obtain a milky white silk protein suspension.
  • the silk protein suspension was centrifuged to remove excess isopropanol, and washed with deionized water several times. Finally, the precipitate was resuspended in 5 mL deionized water and ultrasonically processed to obtain well-dispersed silk fibroin nanospheres.
  • step (3) Take 1 mL of the silk fibroin nanosphere suspension of step (3), adjust the reaction volume to 5 mL with deionized water, then add 2 mL of potassium permanganate aqueous solution (22 mmol/l), stir for a few minutes and add 0.2 mL Polyacrylamine hydrochloride (PAH), continue to stir for 30 minutes.
  • PHA Polyacrylamine hydrochloride
  • step (4) Incubate the reaction solution of step (4) at 37°C for 2 hours, then centrifuge at 8000 rpm for 10 minutes to remove the supernatant, resuspend the precipitate and wash, and freeze-dry to obtain 100-200 nm diameter silk fibroin
  • Figure 1 The morphology of the protein/manganese dioxide composite microspheres is shown in Figure 1.
  • the preparation method of the silk fibroin/manganese dioxide composite microsphere drug carrier of the core-shell structure in this embodiment sequentially includes the following steps:
  • step (3) Place the mixed solution of step (2) in a refrigerator at -20°C, freeze for 24 hours, and thawed naturally at room temperature to obtain a silk protein suspension.
  • the silk protein suspension was centrifuged, washed and ultrasonically processed to obtain silk protein microspheres with a diameter in the range of 200-500 nm, and resuspended with 10 mL of deionized water.
  • step (4) Incubate the reaction solution of step (4) at 37°C for 2 hours, then centrifuge at 8000 rpm for 10 minutes to remove the supernatant, resuspend the precipitate and wash, and freeze-dry to obtain silk fibroin with a diameter of 200-500 nm Protein/manganese dioxide composite microspheres.
  • the preparation method of the silk fibroin/manganese dioxide composite microsphere drug carrier of the core-shell structure in this embodiment sequentially includes the following steps:
  • step (2) Adjust the silk fibroin concentration in step (1) to 2mg/mL, take 10mL and place it in the reaction vessel, add 2mg of doxorubicin hydrochloride (DOX) to the silk fibroin solution, and ultrasonically disperse it with the silk
  • DOX doxorubicin hydrochloride
  • step (3) Place the mixed solution of step (2) in a -80°C refrigerator, freeze for 12 hours, and then allow it to thaw naturally at room temperature to obtain a silk protein suspension.
  • the silk protein suspension was centrifuged to remove excess isopropanol and doxorubicin hydrochloride, and washed with deionized water several times. Finally, the precipitate was resuspended in 5 mL of deionized water and subjected to ultrasonic treatment to obtain well-dispersed drug-loaded silk fibroin nanospheres.
  • step (3) Take 1 mL of the silk fibroin nanosphere suspension of step (3), adjust the reaction volume to 5 mL with deionized water, then add 2 mL of potassium permanganate aqueous solution (22 mmol/l), stir for a few minutes and add 0.2 mL Polyacrylamine hydrochloride (PAH), continue to stir for 30 minutes.
  • PHA Polyacrylamine hydrochloride
  • step (4) Incubate the reaction solution of step (4) at 37°C for 2 hours, then centrifuge at 8000 rpm and 10 min to remove the supernatant, resuspend the pellet and wash it, and freeze-dry to obtain a drug loading with a diameter of 100-200 nm Silk fibroin/manganese dioxide composite microspheres.
  • step (6) Disperse the drug-loaded silk fibroin/manganese dioxide composite microspheres of step (5) in 10 mL of absolute ethanol, add 1 mg of photosensitizer Ce6, sonicate to make the dispersion uniform, and incubate in the dark for 24 hours for drug adsorption . Subsequent centrifugation removes unbound drugs, and the drug loading can be calculated based on the absorbance of the supernatant.
  • the drug-loaded silk fibroin/manganese dioxide composite microspheres of step (6) were used for in vitro release experiments, and different pH conditions (see Figure 3) were set to simulate the acidic microenvironment of the tumor.
  • the silk fibroin/manganese dioxide composite microspheres can release drugs faster under acidic pH conditions, and the release amount is close to 80% in 24 hours, especially under pH 5.7 conditions, the release amount is close to 90% in 24 hours. This proves that the silk fibroin/manganese dioxide composite microspheres loaded with DOX and Ce6 can release drugs in a controlled manner in response to the tumor microenvironment.
  • the drug loading and release characteristics of silk fibroin/manganese dioxide composite microspheres are shown in Figure 3.
  • the preparation method of the silk fibroin/manganese dioxide composite microsphere drug carrier of the core-shell structure in this embodiment sequentially includes the following steps:
  • step (2) Adjust the concentration of silk fibroin in step (1) to 2 mg/mL, and place 10 mL in the reaction container. Slowly add 1 mL of isopropanol dropwise to the silk protein solution, and continue to stir for 30 minutes to make it uniform.
  • step (3) Place the mixed solution of step (2) in a refrigerator at -80°C, freeze for 12 hours, and then allow it to thaw naturally at room temperature, centrifuge to remove excess isopropanol, and wash with deionized water several times. Finally, it was resuspended in 5 mL deionized water and sonicated to obtain well-dispersed silk fibroin nanospheres.
  • step (3) Take 1 mL of the silk fibroin nanosphere suspension of step (3), adjust the reaction volume to 10 mL with deionized water, then add 5 mL of potassium permanganate aqueous solution (22 mmol/l), stir for a few minutes and add 0.2 mL Polyacrylamine hydrochloride (PAH), continue to stir for 30 minutes.
  • PHA Polyacrylamine hydrochloride
  • the method of calculating the drug concentration in Figure 4 is: disperse the calculated drug-loaded nanoparticles in the cell culture medium, and then divide the calculated drug amount by the volume of the medium (ml) to obtain the drug concentration ), the experiment is divided into three groups: (1) drug-loaded silk fibroin/manganese dioxide composite microspheres + hydrogen peroxide, adding about 10 ⁇ l of hydrogen peroxide; (2) drug-loaded silk fibroin/manganese dioxide composite microspheres + Laser treatment; (3) Drug-loaded silk fibroin/manganese dioxide composite microspheres + hydrogen peroxide + laser treatment.
  • the experimental results show that the silk fibroin/manganese dioxide composite microspheres prepared by the present invention perform dual drug loading.
  • the dual effects of chemotherapy and photodynamic therapy can effectively kill tumor cells and can be used as a good tumor therapy drug carrier.
  • the cell lethal experiment is shown in Figure 4 (the abscissa is the concentration of DOX or Ce6 in the test sample; the ordinate is human breast cancer MCF-7 cell activity).

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Abstract

一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，包括：(1)向丝素蛋白水溶液中加入水溶性有机溶剂，经过冷冻-自然解冻后，去除溶剂，得到丝素蛋白纳米微球；(2)将得到的丝素蛋白纳米微球与高锰酸钾、聚丙烯胺盐酸盐混合均匀，孵育完成，去除溶剂，得到里层为丝素蛋白外层为二氧化锰的核壳结构的丝素蛋白/二氧化锰复合微球药物载体。该工艺简单，操作简便，获得的丝素蛋白/二氧化锰复合微球表面粗糙，结合了丝素蛋白和二氧化锰的优良性能，药物装载率高，能够响应肿瘤微环境可控释放，降解肿瘤环境中的过氧化氢生成氧气，提高肿瘤化学和光动力治疗效率，在肿瘤治疗和药物领域有广泛的应用前景。

Description

一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体及制备方法 技术领域
本发明属于生物医药材料领域，具体是涉及一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法。
背景技术
溶胶状二氧化锰(MnO 2)纳米粒子因其特定的催化特性和良好的生物相容性，成为生物医学领域的新兴材料。在肿瘤治疗领域，二氧化锰纳米粒子能特异性的与肿瘤微环境中的过氧化氢反应，催化其降解生成氧气和水，从而改善低氧的环境，下调缺氧诱导因子，既减弱了肿瘤组织耐药性，又有助于光敏剂将氧气转换成单线态氧，同时提高肿瘤的化学治疗和光动力治疗效率。肿瘤微环境中富含的H +和谷胱甘肽(GSH)能降解MnO 2，生成Mn 2+增强核磁共振成像，用于肿瘤成像和实时监测。此外，Mn 2能够被肾脏快速排出体外，具有很高的生物安全性。因此，MnO 2纳米粒子在肿瘤治疗领域具有很高的应用前景。然而，目前为止，MnO 2纳米粒子的合成方法仍存在过程复杂、反应条件苛刻(高温、高压)、产物尺寸不均一等缺陷，并且单一的MnO 2纳米粒子血液中循环的胶体稳定性不可控，降解迅速，阻碍其体内临床应用。
丝蛋白是在昆虫体内合成，吐丝后形成具有良好力学性能的丝蛋白纤维。丝蛋白是天然的生物高分子聚合物，具有优良的力学性能、生物相容性及低免疫原性，是组织工程领域的理想材料。此外，蚕丝蛋白因其多肽链结构的特殊性，较易形成微纳米球结构，用于药物的装载和运送。另一方面，蚕丝蛋白能够作为生物模板调控其他无机纳米粒子的合成，生成复合型微球用作药物载体。目前，已有专利文献报道通过生物矿化法能够制备丝素蛋白/羟基磷灰石的复合微球。然而，利用蚕丝蛋白调控二氧化锰的生成，优化二氧化锰纳米粒子的性能，提高其在肿瘤治疗领域的应用价值，目前为止，还没有相关报道。
发明内容
为了克服现有MnO 2纳米粒子合成技术复杂、反应条件苛刻，以及单纯MnO 2纳米粒子性质不稳定等缺陷，本发明提供了一种以蚕丝蛋白微球为模板，通过自组装制备核壳结构的丝蛋白/二氧化锰复合微球的方法。
本发明同时公开了一种由上述方法制备得到的核壳结构的丝蛋白/二氧化锰复合微球产品。
本发明的方法不仅过程简单，反应条件温和，制备的丝蛋白/二氧化锰复合微球具有良好的分散性，较高的比表面积，可控的药物释放能力，优异的单线态氧产生能力，因此从根本上解决了现有技术存在的问题。
一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，包括：
(1)向丝素蛋白水溶液中加入水溶性有机溶剂，经过冷冻-自然解冻后，去除溶剂，得到丝素蛋白纳米微球；
(2)将得到的丝素蛋白纳米微球与高锰酸钾、聚丙烯胺盐酸盐在水中混合均匀，孵育完成，去除溶剂，得到里层为丝素蛋白外层为二氧化锰的核壳结构的丝素蛋白/二氧化锰复合微球药物载体。
步骤(1)中，所述丝素蛋白水溶液可以有下述方法制备得到：
选取家蚕茧壳或者其他丝素蛋白原材料，通过脱胶、溶解、透析、浓缩等步骤，获得丝素蛋白水溶液。
作为优选，丝素蛋白水溶液的浓度为0.1-30mg/mL。进一步优选为2～20mg/mL。
作为优选，水溶性有机溶剂为乙醇、异丙醇或者丙酮。进一步优选为异丙醇，采用异丙醇时得到的微球更加规则，粒径更小，载药性更好。可根据实际需要，可用异丙醇，也可根据微球尺寸需求用乙醇、丙酮等其他试剂替代。
作为优选，水溶性有机溶剂加入的体积为丝素蛋白水溶液体积的1/15～2/5。进一步优选为1/10～2/5。作为优选，水溶性有机溶剂为异丙醇，其加入的体积为丝素蛋白水溶液体积的1/10～2/10。作为优选，水溶性有 机溶剂为乙醇时，水溶性有机溶剂加入的体积为丝素蛋白水溶液体积的1/5～2/5。
冷冻条件为：温度为-10～-90℃；时间为10～20小时；作为进一步优选：水溶性有机溶剂为异丙醇时：温度为-70～-90℃；水溶性有机溶剂为乙醇时温，度为-10～-30℃。
步骤(1)经过冷冻-自然解冻后，获得丝蛋白悬液。将丝蛋白悬液进行离心、洗涤超声处理，经过步骤(1)后，可以得到100-600nm直径的丝素蛋白/二氧化锰复合微球。
本发明中，KMnO 4为合成二氧化锰的原料，聚丙烯胺盐酸盐为还原剂和表面修饰剂，还原KMnO 4生成MnO 2，并对其进行表面正电荷修饰，PAH的分子量范围为10KDa-35KDa。
作为优选，1g丝素蛋白需要高锰酸钾的摩尔量为5～25mmol；1g丝素蛋白需要的聚丙烯胺盐酸盐的体积为10～250ml。
作为优选，孵育条件为：温度为20～50℃，时间为1～10小时。进一步优选为2～6小时，温度为30～40℃。
作为优选，所述丝素蛋白选自家蚕丝素蛋白、野蚕丝素蛋白、柞蚕丝素蛋白、蜘蛛丝蛋白、重组丝蛋白中的一种或多种。
步骤(2)反应完成后，离心、洗涤后可以得到核壳结构的丝素蛋白/二氧化锰复合微球，微球粒径为100-500nm，里层为丝素纳米球层，外层为二氧化锰层，具有粗糙的表面结构，可作为肿瘤药物治疗和光动力治疗载体。
一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体，由上述任一项技术方案所述的制备方法制备得到。
所述化疗药物既可以包括亲水性抗癌药物如盐酸阿霉素等，又可以装载疏水性抗癌药物紫杉醇等，作为优选，所述药物包括盐酸阿霉素、光敏剂Ce6。药物装载方式可以通过与丝蛋白混合进行原位合成装载，也可以通过表面吸附装载，两种方法可以单一或同时使用，以提高药物装载率。
药物装载时，作为优选，在上述任一项技术方案中，所述的步骤(1)中加入药物，或者在最终得到的丝素蛋白/二氧化锰复合微球药物载体后对 药物进行加载。
本发明所述的丝素蛋白/二氧化锰复合微球，由于MnO 2的存在能够催化肿瘤微环境中的H 2O 2，生成大量O 2，提高肿瘤的光动力治疗效率。丝素蛋白/二氧化锰复合微球作为载体将光敏剂运送至肿瘤部位，在激光照射下，能够将产生的氧气转换成单线态氧，从而有效杀死肿瘤细胞。
本发明公开了一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法。目前还没有一种工艺简单、绿色环保、高效安全的方法制备二氧化锰纳米颗粒。本发明依次包括如下步骤：将家蚕茧壳依次经过脱胶、溶解、浓缩处理，获得丝素蛋白溶液；将丝素蛋白溶液用异丙醇进行处理，并在低温下进行冻融，获得丝素蛋白纳米球；以丝素蛋白纳米球为模板，诱导高锰酸钾(KMnO 4)在聚丙烯胺盐酸盐(PAH)辅助下还原，并在丝素蛋白纳米球模板表面原位生成MnO 2，最终获得核壳结构丝素蛋白/二氧化锰复合微球，作为药物载体，同时装载抗癌药物和光敏剂对肿瘤实施化疗和光动力双重治疗。本发明工艺简单，操作简便，获得的丝素蛋白/二氧化锰复合微球表面粗糙，与传统的丝素微球药物载体相比，复合微球结合了丝素蛋白和二氧化锰的优良性能，药物装载率高，能够响应肿瘤微环境可控释放，降解肿瘤环境中的过氧化氢生成氧气，提高肿瘤化学和光动力治疗效率，在肿瘤治疗和药物领域有广泛的应用前景。
具体讲，本发明与现有技术相比具有以下突出优点：
(1)本发明以丝素蛋白纳米球为模板，通过自组装的方式，构建丝素蛋白/二氧化锰复合微球药物载体，与常用的制备二氧化锰纳米粒子的方法相比，本方法工艺简单，反应条件温和，无副产物产生，对环境无污染，适合工厂化大批量生产。
(2)有别于其他以有机化学试剂作为模板进行的调控，本发明选取的丝蛋白模板，为天然的生物高分子，来源广泛，提供良好生物安全性的同时也有效降低了制备成本。
(3)装载药物的多样性，丝素蛋白既能装载亲水性抗癌药物又能装载疏水性抗癌药物，粗糙的MnO 2粒子表面也有利于光敏剂的装载。
(4)有别于传统药物载体单一的载药方式，丝素蛋白/二氧化锰复合 微球既能以原位合成的方式进行载药，也能通过静电引力进行表面吸附载药，药物装载率更高。
(5)能够响应肿瘤微环境进行药物的可控释放，外壳的MnO 2层在肿瘤微环境中的谷胱甘肽和H +存在下降解释放药物，丝素蛋白在肿瘤环境微酸的pH条件下结构发生变化，实现药物的突释。
附图说明
图1为实施例1中核壳结构的丝素蛋白/二氧化锰复合微球药物载体的扫描电镜图。
图2为实施例1中不同浓度的丝素蛋白/二氧化锰复合微球药物载体对人成纤维细胞影响的细胞活力图。
图3为实施例2中核壳结构的丝素蛋白/二氧化锰复合微球药物载体装载抗肿瘤药物盐酸阿霉素和光敏剂Ce6后的装载和释放图。
图4为载药的丝素蛋白/二氧化锰复合微球在光照和H 2O 2存在下对人乳腺癌MCF-7细胞的致死效率图。
具体实施方式
下面通过实施例对本发明做进一步的详细说明，以下实施例是对本发明的解释而本发明并不局限于以下实施例。
本发明的实施例如下：
实施例1
本实施例中核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法依次包括如下步骤：
(1)取家蚕茧壳适量，洗净晾干后采用现有的方法进行脱胶处理。随后，将脱胶丝纤维依次经过现有的溶解、透析、浓缩等步骤，获得家蚕丝素蛋白水溶液；
(2)将步骤(1)中的丝素蛋白浓度调整为2mg/mL(溶剂为水，其它实施例同)，取10mL置于反应容器。向丝蛋白溶液中缓慢滴加1mL异丙醇，持续搅拌30min使其混合均匀。
(3)将步骤(2)的混合溶液放置于-80℃冰箱，冷冻12h，然后使其 室温自然解冻，获得乳白色丝蛋白悬浊液。将丝蛋白悬浊液进行离心处理去除多余异丙醇，并用去离子水进行多次洗涤。最后将沉淀用5mL去离子水重悬并进行超声处理获得分散良好的丝素蛋白纳米球。
(4)取步骤(3)的丝素蛋白纳米球悬液1mL，用去离子水调整其反应体积为5mL，随后加入高锰酸钾水溶液2mL(22mmol/l)，搅拌数分钟后加入0.2mL聚丙烯胺盐酸盐(PAH)，继续搅拌30min。
(5)将步骤(4)的反应溶液置于37℃条件下孵育2h，随后进行8000rpm，10min离心，去除上清液，沉淀进行重悬洗涤，冷冻干燥后可获得100-200nm直径的丝素蛋白/二氧化锰复合微球，微球的扫描电镜形貌如图1所示。
(6)人成纤维细胞生物相容性实验表明，制备获得的核壳结构丝素蛋白/二氧化锰复合微球具有良好的生物相容性，成纤维细胞活力测试如图2所示(横坐标表示复合微球与细胞共培养处理的浓度，0μg/ml表示没有没有加入丝素蛋白/二氧化锰复合微球纳米颗粒；纵坐标表示细胞存活比例；NPs是指丝素蛋白/二氧化锰复合微球纳米颗粒)。
从图2可知，加入本发明得到的丝素蛋白/二氧化锰复合微球纳米颗粒后，与没有添加丝素蛋白/二氧化锰复合微球纳米颗粒的对照样进行对比，人成纤维细胞存活率均在70％以上，特别是在丝素蛋白/二氧化锰复合微球纳米颗粒含量为20～50μg/ml时，人成纤维细胞存活率比对照样的存活率更高，进一步说明，本发明制备获得的核壳结构丝素蛋白/二氧化锰复合微球没有毒性，具有广阔的应用前景。
实施例2
本实施例中核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法依次包括如下步骤：
(1)取家蚕茧壳适量，洗净晾干后进行脱胶处理。随后，将脱胶丝纤维依次经过溶解、透析、浓缩等步骤，获得家蚕丝素蛋白水溶液；
(2)调整步骤(1)中的丝素蛋白浓度为2％，取5mL置于反应容器，加入无水乙醇2mL搅拌均匀。
(3)将步骤(2)的混合溶液置于-20℃冰箱，冷冻处理24h，室温自 然解冻，获得丝蛋白悬液。将丝蛋白悬液进行离心、洗涤超声处理，获得直径为200-500nm范围的丝蛋白微球，并用10mL去离子水进行重悬。
(4)取步骤(3)的丝素蛋白微球球悬液1mL，用去离子水调整其反应体积为10mL，随后加入高锰酸钾水溶液4mL(22mmol/l)，搅拌数分钟后加入0.4mL聚丙烯胺盐酸盐(PAH)，继续搅拌30min。
(5)将步骤(4)的反应溶液置于37℃条件下孵育2h，随后进行8000rpm，10min离心，去除上清液，沉淀进行重悬洗涤，冷冻干燥后可获得200-500nm直径的丝素蛋白/二氧化锰复合微球。
实施例3(原位载药)
本实施例中核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法依次包括如下步骤：
(1)取家蚕茧壳适量，洗净晾干后进行脱胶处理。随后，将脱胶丝纤维依次经过溶解、透析、浓缩等步骤，获得家蚕丝素蛋白水溶液；
(2)将步骤(1)中的丝素蛋白浓度调整为2mg/mL，取10mL置于反应容器，向丝素蛋白溶液中加入盐酸阿霉素(DOX)2mg，并超声分散使其与丝素蛋白混合均匀，随后向反应溶液中缓慢滴加1mL异丙醇，持续搅拌30min使其混合均匀。
(3)将步骤(2)的混合溶液放置于-80℃冰箱，冷冻12h，然后使其室温自然解冻，获得丝蛋白悬浊液。将丝蛋白悬浊液进行离心处理去除多余异丙醇和盐酸阿霉素，并用去离子水进行多次洗涤。最后将沉淀用5mL去离子水重悬并进行超声处理获得分散良好的载药丝素蛋白纳米球。
(4)取步骤(3)的丝素蛋白纳米球悬液1mL，用去离子水调整其反应体积为5mL，随后加入高锰酸钾水溶液2mL(22mmol/l)，搅拌数分钟后加入0.2mL聚丙烯胺盐酸盐(PAH)，继续搅拌30min。
(5)将步骤(4)的反应溶液置于37℃条件下孵育2h，随后进行8000rpm，10min离心，去除上清液，沉淀进行重悬洗涤，冷冻干燥后可获得100-200nm直径的载药丝素蛋白/二氧化锰复合微球。
(6)将步骤(5)的载药丝素蛋白/二氧化锰复合微球分散于10mL无水乙醇，加入1mg光敏剂Ce6，超声使其分散均匀，置于暗处孵育24h， 进行药物吸附。随后离心去除未结合药物，并根据上清液吸光度值可计算药物装载量。
(7)将步骤(6)的载药丝素蛋白/二氧化锰复合微球用于体外释放实验，设置不同的pH条件(见图3)以模拟肿瘤酸性微环境。丝素蛋白/二氧化锰复合微球在pH酸性条件下能更快的释放药物，24h释放量接近80％，特别是在pH5.7条件下，24h释放量接近90％。由此证明，装载DOX和Ce6的丝素蛋白/二氧化锰复合微球能够响应肿瘤微环境可控释放药物。丝素蛋白/二氧化锰复合微球药物装载和释放特性如图3所示。
实施例4
本实施例中核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法依次包括如下步骤：
(1)取家蚕茧壳适量，洗净晾干后进行脱胶处理。随后，将脱胶丝纤维依次经过溶解、透析、浓缩等步骤，获得家蚕丝素蛋白水溶液；
(2)将步骤(1)中的丝素蛋白浓度调整为2mg/mL，取10mL置于反应容器。向丝蛋白溶液中缓慢滴加1mL异丙醇，持续搅拌30min使其混合均匀。
(3)将步骤(2)的混合溶液放置于-80℃冰箱，冷冻12h，然后使其室温自然解冻，离心处理去除多余异丙醇，并用去离子水进行多次洗涤。最后用5mL去离子水重悬并进行超声处理获得分散良好的丝素蛋白纳米球。
(4)取步骤(3)的丝素蛋白纳米球悬液1mL，用去离子水调整其反应体积为10mL，随后加入高锰酸钾水溶液5mL(22mmol/l)，搅拌数分钟后加入0.2mL聚丙烯胺盐酸盐(PAH)，继续搅拌30min。
(5)将步骤(4)的反应溶液置于37℃条件下孵育2h，随后进行8000rpm，10min离心，去除上清液，沉淀进行重悬洗涤，冷冻干燥后可获得200nm直径的丝素蛋白/二氧化锰复合微球，微球表面为粗糙凸起颗粒。
(6)对步骤(5)的丝素蛋白/二氧化锰复合微球进行载药处理，采用分步吸附的方式进行载药以提高载药量。由于PAH赋予载药微球正电荷 表面，先进行负电荷的Ce6装载，按照图4所需浓度将微球分散于乙醇后与Ce6共培养24h，随后离心处理后，分散于水溶液进行DOX的装载，得到载药的丝素蛋白/二氧化锰复合微球。
(7)将载药的丝素蛋白/二氧化锰复合微球与人乳腺癌MCF-7细胞进行共培养(培养基为商业化的高糖DMEM培养基，实验在96孔培养板进行，培养液总体积为100μl，图4中药物浓度计算方法为：将计算好的载药纳米颗粒分散在细胞培养基中，然后用计算得到的所载药物量除以培养基体积(ml)得到药物浓度)，实验分为三组：(1)载药的丝素蛋白/二氧化锰复合微球+双氧水，双氧水加入约为10μl；(2)载药的丝素蛋白/二氧化锰复合微球+激光处理；(3)载药的丝素蛋白/二氧化锰复合微球+双氧水+激光处理，实验结果显示，本发明制备的丝素蛋白/二氧化锰复合微球进行双重载药后，发挥化学治疗和光动力治疗双重效果，能有效杀死肿瘤细胞，能够作为良好的肿瘤治疗药物载体，细胞致死实验如图4所示(横坐标为检测样品中DOX或Ce6的浓度；纵坐标为人乳腺癌MCF-7细胞的活性)。
最后，还需要注意的是，以上列举的仅是本发明的具体实施例子。显然，本发明不限于以上实施例子，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (13)

  1. 一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，包括：
    (1)向丝素蛋白水溶液中加入水溶性有机溶剂，经过冷冻-自然解冻后，去除溶剂，得到丝素蛋白纳米微球；
    (2)将得到的丝素蛋白纳米微球与高锰酸钾、聚丙烯胺盐酸盐混合均匀，孵育完成，去除溶剂，得到里层为丝素蛋白外层为二氧化锰的核壳结构的丝素蛋白/二氧化锰复合微球药物载体。
  2. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，丝素蛋白水溶液的浓度为0.1-30mg/mL。
  3. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，丝素蛋白水溶液的浓度为2～20mg/mL。
  4. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，水溶性有机溶剂为乙醇、异丙醇或者丙酮。
  5. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，水溶性有机溶剂异丙醇。
  6. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，水溶性有机溶剂加入的体积为丝素蛋白水溶液体积的1/15～2/5。
  7. 根据权利要求5或6所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，水溶性有机溶剂加入的体积为丝素蛋白水溶液体积的1/10～2/5。
  8. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，1g丝素蛋白需要高锰酸钾的摩尔量为5～25mmol；1g丝素蛋白需要的聚丙烯胺盐酸盐的体积为10～250ml。
  9. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，孵育条件为：温度为20～50℃，时间 为1～10小时。
  10. 根据权利要求1所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体的制备方法，其特征在于，所述丝素蛋白选自家蚕丝素蛋白、野蚕丝素蛋白、柞蚕丝素蛋白、蜘蛛丝蛋白、重组丝蛋白中的一种或多种。
  11. 一种核壳结构的丝素蛋白/二氧化锰复合微球药物载体，其特征在于，由权利要求1～10任一项所述的制备方法制备得到。
  12. 根据权利要求11所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体，其特征在于，在权利要求1～10任一项所述的步骤(1)中加入药物，或者在最终得到的丝素蛋白/二氧化锰复合微球药物载体后对药物进行加载。
  13. 根据权利要求12所述的核壳结构的丝素蛋白/二氧化锰复合微球药物载体，其特征在于，所述药物包括盐酸阿霉素、紫杉醇、光敏剂Ce6。
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