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 PDFInfo
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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
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- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 title claims abstract description 160
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 129
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- 108090000623 proteins and genes Proteins 0.000 claims description 29
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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Images
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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
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- 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
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- 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
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- 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
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- 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
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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
Description
Claims (13)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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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