WO2023056717A1 - Functional silk fibroin drug carrier and preparation method therefor - Google Patents

Functional silk fibroin drug carrier and preparation method therefor Download PDF

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WO2023056717A1
WO2023056717A1 PCT/CN2021/142944 CN2021142944W WO2023056717A1 WO 2023056717 A1 WO2023056717 A1 WO 2023056717A1 CN 2021142944 W CN2021142944 W CN 2021142944W WO 2023056717 A1 WO2023056717 A1 WO 2023056717A1
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drug
preparation
silk fibroin
solution
hours
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PCT/CN2021/142944
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French (fr)
Chinese (zh)
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王建南
罗元泽
郑昌懂
裔洪根
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苏州大学
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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
    • A61K47/51—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
    • 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
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a functional silk fibroin drug carrier and a preparation method thereof.
  • Cancer is one of the leading causes of death in the 21st century.
  • chemotherapy is the main means of fighting cancer.
  • chemotherapeutic drugs such as paclitaxel and doxorubicin
  • drug delivery systems have been ingeniously developed to release drugs effectively with less toxic side effects.
  • nanoparticles are widely used due to their small particle size, large specific surface area, large drug loading capacity, and easy phagocytosis by cells.
  • SF is a natural protein with good biocompatibility and biodegradability, non-toxic and no side effects, and is a biomaterial with broad application prospects. It has also received great attention as a drug carrier.
  • TK Thiketal
  • pH-responsive, magnetic-responsive, temperature-responsive, and light-responsive types were mainly disclosed regarding responsive SF materials.
  • a porous SF particle grafted with folic acid on the surface and loaded with doxorubicin the in vitro drug release shows pH-dependent release (Journal of Materials Science, 2019, 54(4):3319).
  • a doxorubicin-loaded magnetic SF nanoparticle can be used as a drug delivery system in magnetic field-guided (magnetic targeting) multidrug-resistant cancer chemotherapy (Advanced Materials, 2014, 26:7393).
  • a multifunctional drug delivery microcarrier combined with SF inverse opal scaffold and temperature-responsive poly(N-isopropylacrylamide) hydrogel the drug release of the microcarrier can be triggered by external temperature stimulation (Applied Materials Today, 2020, 19: UNSP 100540).
  • a photoresponsive CO-releasing nano drug-loaded particle based on SF exhibits improved CO release (Dalton Transactions, 2018, 47:10434).
  • the ROS-responsive materials are mainly ROS-responsive carriers containing sulfur, selenium, tellurium and unsaturated lipids.
  • a tellurium polymer in the presence of ultra-low concentrations of hydrogen peroxide, the polymer micelles can rapidly expand and evolve into irregular aggregates, which can be used as a potential therapeutic method for the combination of chemical and radiotherapy (Chemical Communications, 2015, 51:7069).
  • a near-infrared light-triggered liposome encapsulating a photosensitizer and tetrodotoxin provides on-demand adjustable local anesthesia (Proceedings of the National Academy of Sciences, 2015, 112:15719).
  • Another example is a TK/polymer nanocarrier coated with the photosensitizer chlorin e6 and the chemotherapeutic drug doxorubicin, which can trigger the rapid release of the chemotherapeutic drug doxorubicin under 660nm red light irradiation (Chemistry of Materials, 2018, 30 :517).
  • ROS-responsive drug-loaded nanomaterials There are some intellectual property rights in the research and development of ROS-responsive drug-loaded nanomaterials.
  • a nanoscale particle obtained by free radical polymerization of vinylpyridine and polyethylene glycol can be used as a transport carrier for photosensitizers (application number: 201611126310.3 ); a polycurcumin prodrug nanoparticle can be used to load anticancer drugs and cyanine molecules (application number: 201910191720.3); a surface-modified selective antagonist AMD3100 with dextran and lipoic acid as the substrate
  • the ROS-responsive amphiphilic material is used for the entrapment and specific release of drugs for the treatment of liver fibrosis (application number: 202010087783.7), etc.
  • the inventor once disclosed a research on a ROS-responsive SF drug-loaded delivery system, and showed that there is a certain ROS-responsive effect but not significant. Therefore, it is very important to develop a ROS-responsive SF drug-loaded nanoparticle. necessary.
  • the technical problem to be solved by the present invention is to provide a method for preparing a functional silk fibroin drug carrier.
  • the functional silk fibroin drug carrier provided by the present invention improves the accuracy and effectiveness of drug treatment at diseased tissue sites; At the same time, it has remarkable ROS response performance.
  • the invention provides a preparation method of a functional silk fibroin drug carrier, comprising:
  • the degumming in step A) is degumming with sodium carbonate or sodium bicarbonate; the dissolving is dissolving with lithium bromide; the molecular weight cut-off of the dialysis is 10-50kDa; the concentration of the silk fibroin solution is 10 ⁇ 200mg/mL.
  • step B) the preparation method of the ketal thiol with carboxyl groups at both ends is specifically:
  • the molar ratio of the 3-mercaptopropionic acid to anhydrous acetone is 1:1.5-2.5; the acidic condition is hydrochloric acid; the concentration of the hydrochloric acid is 7-8M; 8 hours; the freeze-drying specifically includes: pre-freezing at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
  • the mass ratio of the silk fibroin to the thioketal with carboxyl groups at both ends is 100:0.5-20; the dissolving solvent for the thioketal with carboxyl groups at both ends is water.
  • the cross-linking agent in step B) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; the addition of the cross-linking agent The amount is 2 to 2.5 times that of ketal thiols with carboxyl groups at both ends;
  • the activation reaction temperature is 4-6°C;
  • the reaction temperature is 20-30° C.; the reaction time is 10-60 minutes.
  • the concentration of absolute ethanol in step C) is 0.5-1.5M; the concentration of polyvinyl alcohol is 5-60 mg/mL, and the molecular weight of polyvinyl alcohol is 30,000-70,000;
  • the freezing is specifically: freezing at -20°C for 8-48 hours; the thawing is thawing at 20-30°C;
  • the centrifugal is 13000rpm centrifugal
  • the freeze-drying of the precipitate specifically includes: pre-freezing the precipitate at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
  • the invention provides a functional silk fibroin drug carrier, which is prepared by the preparation method described in any one of the above technical solutions.
  • the present invention provides the application of the functional silk fibroin drug carrier prepared by any one of the above preparation methods in loading anti-tumor/anti-inflammatory drugs.
  • the invention provides a drug-loaded nanoparticle, which includes a drug and a functional silk fibroin drug carrier prepared by the preparation method described in any one of the above technical solutions.
  • the present invention provides a method for preparing a functional silk fibroin drug carrier, comprising: A) degumming silkworm silk, dissolving, dialysis, filtering, and concentrating to obtain a silk fibroin solution; B) mixing two The ketal thiol with a carboxyl group is activated with a cross-linking agent, and then mixed with the SF solution to obtain a reacted solution; C) the reacted solution is added to the mixed system of absolute ethanol and polyvinyl alcohol to stir and freeze , thawing, centrifuging, and finally freeze-drying the precipitate.
  • the SF/TK nanoparticles of the present invention introduce more ROS-responsive molecules TK, and TK is covalently bonded to a large number of hydroxyl groups on the SF molecular chain with the dual-active functional group carboxyl at its end, and bridges between SF molecules.
  • TK is covalently bonded to a large number of hydroxyl groups on the SF molecular chain with the dual-active functional group carboxyl at its end, and bridges between SF molecules.
  • the C-S bond of the TK molecule in the SF/TK nanoparticle is broken to break the covalent bond between the SF molecules, and the internal molecules of the nanoparticle Inter-cracking, so that the drug in the nanoparticles is released quickly, and the directional killing of tumor and inflammatory cells is targeted.
  • the nanoparticles that enter the normal cells do not cleavage or have a small degree of cleavage. They only rely on osmotic release or release after degradation, and are metabolized. Prodrugs are rarely released very slowly, causing minimal damage to normal cells.
  • SF composed of 20 kinds of ⁇ -amino acids which are the same as the composition of the human body, is selected as the main body of the drug-loaded nanoparticles, which has no toxic and side effects after entering the human body, and is an ideal drug delivery carrier.
  • SF can adjust the crystalline structure of nanoparticles through preparation technology, thereby regulating the osmotic release and degradation release of drugs, and reducing the amount of drugs released in normal cells before the metabolism of nanoparticles is excreted. It is a drug delivery carrier for targeted treatment of inflammation and tumors. .
  • the invention provides a functional silk fibroin drug carrier and a preparation method thereof.
  • Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it.
  • all similar substitutions and modifications are obvious to those skilled in the art, and they all belong to the protection scope of the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • the invention provides a preparation method of a functional silk fibroin drug carrier, comprising:
  • the preparation method of a functional silk fibroin drug carrier provided by the invention firstly degummes silkworm silk.
  • Degumming described in the present invention is preferably to adopt sodium carbonate or sodium bicarbonate to carry out degumming; More preferably specifically:
  • the degummed silk is placed in an electric heating constant temperature drying oven, dried, and pulled until fluffy to obtain the degummed silkworm silk fiber.
  • the present invention does not limit the specific temperature of the drying, which may be 50-70°C.
  • the dissolving in the present invention is preferably carried out by lithium bromide; more preferably, it is as follows: weigh a certain amount of degummed silk fibroin fiber, cut it into pieces, put it into a lithium bromide solution, heat and stir at 60-70° C. for 1-2 hours to obtain silkworm SF solution.
  • the present invention does not limit the concentration of the lithium bromide, which is well known to those skilled in the art, and is preferably 9-10M.
  • the silkworm SF solution is poured into a dialysis bag and placed in a container filled with deionized water for dialysis; the dialysis molecular weight cut-off of the present invention is 10-50 kDa; more preferably 25-50 kDa.
  • the concentration of the silk fibroin solution in the present invention is preferably 10-200 mg/mL; more preferably 15-190 mg/mL; most preferably 20-80 mg/mL.
  • 3-mercaptopropionic acid and anhydrous acetone the molar ratio of 3-mercaptopropionic acid and anhydrous acetone is preferably 1:1.5-2.5; more preferably 1:2-2.5; and react under hydrochloric acid conditions;
  • the concentration of the hydrochloric acid is 7-8M; the reaction is specifically 20-30°C for 4-8 hours; more preferably 22-28°C for 4-8 hours; the precipitated crystals are washed, centrifuged, and freeze-dried , to obtain TK with carboxyl groups at both ends.
  • the centrifugation is preferably at 13000 rpm.
  • the freeze-drying specifically includes: pre-freezing at -20 to -80° C. for 2 to 6 hours and then freeze-drying for 8 to 16 hours.
  • the mass ratio of silk fibroin to thioketal with carboxyl groups at both ends is preferably 100:0.5-20; more preferably 100:1-10; most preferably 100:1-8.
  • the dissolving solvent of the ketal thioketal having carboxyl groups at both ends is water, and the dissolving solvent is 0.1-0.5 mg/mL.
  • the dissolving solvent is 0.1-0.5 mg/mL.
  • it can be placed in a water bath at 4° C. and stirred for 20 minutes.
  • the activation reaction temperature of the present invention is 4 ⁇ 6 °C;
  • the cross-linking agent of the present invention is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; the addition amount of the cross-linking agent is two ends 2 to 2.5 times that of ketal thiols with carboxyl groups;
  • the reaction temperature is preferably 20-30°C; more preferably 22-28°C; the reaction time is preferably 10-60min; more preferably 15-55min.
  • the preferred method of the present invention is: slowly drop the cross-linking agent into the TK solution, and then react in a water bath at 4° C. to 6° C. for 10 to 15 minutes to activate the carboxyl group. Then slowly drop the activated TK into the SF solution, stir slowly on a magnetic stirrer, and react at room temperature to obtain a SF/TK composite solution.
  • the reacted solution is added into a mixed system of absolute ethanol and polyvinyl alcohol, stirred, frozen, thawed, centrifuged, and finally the precipitate is freeze-dried to obtain the obtained product.
  • the concentration of absolute ethanol in the present invention is preferably 0.5-1.5M; the concentration of polyvinyl alcohol is preferably 5-60 mg/mL, more preferably 10-50 mg/mL, and the molecular weight of polyvinyl alcohol is 3-70,000 ;
  • the freezing of the present invention is specifically preferably: freezing at -20°C for 8 to 48 hours; more preferably freezing at -20°C for 10 to 48 hours;
  • the thawing is thawing at 20°C to 30°C;
  • the centrifugal is 13000rpm centrifugal
  • the freeze-drying of the precipitate specifically includes: pre-freezing the precipitate at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
  • the present invention provides a functional silk fibroin drug carrier, which is prepared by the preparation method described in any one of the above technical solutions.
  • the present invention provides the application of the functional silk fibroin drug carrier prepared by any one of the above preparation methods in loading anti-tumor/anti-inflammatory drugs.
  • anti-tumor/anti-inflammatory drugs include but not limited to camptothecin.
  • the invention provides a drug-loaded nanoparticle, which includes a drug and a functional silk fibroin drug carrier prepared by the preparation method described in any one of the above technical solutions.
  • the concentration of absolute ethanol in the present invention is preferably 0.5-1.5M; the concentration of polyvinyl alcohol is preferably 5-60 mg/mL, more preferably 10-50 mg/mL, and the molecular weight of polyvinyl alcohol is 3-70,000 ;
  • the invention provides a preparation method of a functional silk fibroin drug carrier, comprising: A) degumming silkworm silk, dissolving, dialysis filtering and condensing to obtain a silk fibroin solution; B) ketone sulfurization with carboxyl groups at both ends Alcohol is activated with a cross-linking agent, and then mixed with SF solution to obtain a reacted solution; C) add the reacted solution to a mixed system of absolute ethanol and polyvinyl alcohol to stir, freeze, thaw, and centrifuge, and finally remove the precipitate The product is freeze-dried.
  • the SF/TK nanoparticles of the present invention introduce more ROS-responsive molecules TK, and TK is covalently bonded to a large number of hydroxyl groups on the SF molecular chain with the dual-active functional group carboxyl at its end, and bridges between SF molecules.
  • TK is covalently bonded to a large number of hydroxyl groups on the SF molecular chain with the dual-active functional group carboxyl at its end, and bridges between SF molecules.
  • the C-S bond of the TK molecule in the SF/TK nanoparticle is broken to break the covalent bond between the SF molecules, and the internal molecules of the nanoparticle Inter-cracking, so that the drug in the nanoparticles is released quickly, and the directional killing of tumor and inflammatory cells is targeted.
  • the nanoparticles that enter the normal cells do not cleavage or have a small degree of cleavage. They only rely on osmotic release or release after degradation, and are metabolized. Prodrugs are rarely released very slowly, causing minimal damage to normal cells.
  • SF composed of 20 kinds of ⁇ -amino acids which are the same as the composition of the human body, is selected as the main body of the drug-loaded nanoparticles, which has no toxic and side effects after entering the human body, and is an ideal drug delivery carrier.
  • SF can adjust the crystalline structure of nanoparticles through preparation technology, thereby regulating the osmotic release and degradation release of drugs, and reducing the amount of drugs released in normal cells before the metabolism of nanoparticles is excreted. It is a drug delivery carrier for targeted treatment of inflammation and tumors. .
  • SF molecules have hydrophilic and hydrophobic amphipathic segments, not only have positively charged amino groups and negatively charged carboxyl groups, but also have a large number of hydroxyl groups for modification.
  • the present invention proposes to utilize the most functional group hydroxyl groups contained on the SF molecules To prepare ROS-responsive drug-loaded SF/TK nanoparticles. Based on the chemical structural characteristics of SF molecules, various drugs with different surface properties can be loaded, so the SF smart ROS-responsive drug delivery carrier of the present invention has great application prospects for the treatment of most inflammatory and tumor diseases.
  • the drug-loading ability of the prepared pure SF nanoparticles loaded with camptothecin is equivalent to that of SF/TK nanoparticles.
  • the particle size test before and after oxygen cracking is carried out.
  • the particle size of SF drug-loaded nanoparticles increases first and then decreases slightly with the increase of 50mM KO solution treatment time.
  • the initial average particle size of SF drug-loaded nanoparticles is 325nm, as the time of oxygen treatment increases to 2 hours, the particle size of SF drug-loaded nanoparticles increases to 400nm due to water absorption, and decreases after 16 hours of oxygen treatment To 370nm, it is still larger than the particle size of the original particles, indicating that the SF drug-loaded nanoparticles have not undergone significant cracking.

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Abstract

Provided is a preparation method for a functional silk fibroin (SF) drug carrier, comprising: degumming, dissolving, dialyzing, filtering, and concentrating Bombyx mori silkworm silk to obtain an SF dissolving solution; using a cross-linking agent to activate ketone thioketal (TK) having carboxyl groups at two ends, and mixing TK with the SF dissolving solution for reaction to obtain a reacted solution; and adding the reacted solution into a mixed system of ethanol absolute and polyvinyl alcohol, stirring, freezing, unfreezing, centrifuging, and finally freeze-drying the precipitate to obtain the SF drug carrier. Provided is using most functional groups contained in SF molecules, i.e., hydroxyl groups to prepare ROS-responsive drug-loaded SF/TK nanoparticles. On the basis of chemical structural characteristics of the SF molecules, various drugs having different surface properties can be loaded, a series of SF drug carriers having an intelligent responsive delivery function are developed, and the accuracy and effectiveness of drug treatment at diseased tissue sites are improved.

Description

一种功能丝素蛋白药物载体及其制备方法A kind of functional silk fibroin drug carrier and its preparation method
本申请要求于2021年10月08日提交中国专利局、申请号为202111170433.8、发明名称为“一种功能丝素蛋白药物载体及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application with the application number 202111170433.8 and the invention title "a functional silk fibroin drug carrier and its preparation method" submitted to the China Patent Office on October 08, 2021, the entire contents of which are incorporated by reference incorporated in this application.
技术领域technical field
本发明涉及生物医药技术领域，尤其是涉及一种功能丝素蛋白药物载体及其制备方法。The invention relates to the technical field of biomedicine, in particular to a functional silk fibroin drug carrier and a preparation method thereof.
背景技术Background technique
癌症是二十一世纪主要的死亡原因之一，目前，化学疗法是抗癌的主要手段。然而，诸如紫杉醇和阿霉素等化学治疗药物，对肿瘤细胞缺乏特异性，在杀死肿瘤细胞的同时无差别地会杀死正常细胞。近来，巧妙开发了药物递送系统，以定向释放药物有效减少毒副作用。其中，纳米颗粒由于其粒径小，比表面积大，载药量大，容易被细胞吞噬而得到广泛应用。Cancer is one of the leading causes of death in the 21st century. Currently, chemotherapy is the main means of fighting cancer. However, chemotherapeutic drugs, such as paclitaxel and doxorubicin, lack specificity for tumor cells and kill normal cells indiscriminately while killing tumor cells. Recently, drug delivery systems have been ingeniously developed to release drugs effectively with less toxic side effects. Among them, nanoparticles are widely used due to their small particle size, large specific surface area, large drug loading capacity, and easy phagocytosis by cells.
与其他天然和合成聚合物相比，SF是一种具有良好生物相容性和生物可降解性的天然蛋白质，无毒无副作用，是一种具有广阔应用前景的生物材料。在作为药物载体的研究方面也受到了极大的关注。Compared with other natural and synthetic polymers, SF is a natural protein with good biocompatibility and biodegradability, non-toxic and no side effects, and is a biomaterial with broad application prospects. It has also received great attention as a drug carrier.
但包括SF在内的药物载体面临的临床问题是缺乏精准的靶向性或定位性。因此，赋予SF药物载体的智能响应功能，提升药物载体在炎症和肿瘤治疗中的定向性和高效性，具有重要的研究价值。酮缩硫醇(TK)是一种ROS响应性化合物，能在ROS环境下被氧化断键。ROS环境存在于大多数炎症组织和肿瘤细胞中，因此TK多聚物被关注用于炎症组织和肿瘤细胞中的释放给药治疗。However, the clinical problem faced by drug carriers including SF is the lack of precise targeting or localization. Therefore, it is of great research value to endow the SF drug carrier with an intelligent response function to improve the directionality and efficiency of the drug carrier in inflammation and tumor treatment. Thiketal (TK) is a ROS-responsive compound that can be oxidized to break bonds under ROS environment. The ROS environment exists in most inflammatory tissues and tumor cells, so TK polymers are concerned for release and drug delivery in inflammatory tissues and tumor cells.
在本发明之前，关于响应型SF材料主要公开了pH响应型、磁响应型、温度响应型和光响应型等。如一种表面接枝叶酸并载有阿霉素的多孔SF颗粒，体外药物释放显示pH依赖性释放(Journal of Materials Science,2019,54(4):3319)。一种载有阿霉素的磁性SF纳米粒子，可用于磁场指导下(磁性靶向)的多药耐药性癌症化学疗法中的药物递送系统(Advanced Materials,2014,26:7393)。一种结合SF反蛋白石支架和温度 响应型聚(N-异丙基丙烯酰胺)水凝胶的多功能药物递送微载体，通过外部温度刺激可以触发微载体的药物释放(Applied Materials Today,2020,19:UNSP 100540)。一种基于SF的光响应型一氧化碳释放纳米载药颗粒，表现出改善的一氧化碳释放(Dalton Transactions,2018,47:10434)。Prior to the present invention, pH-responsive, magnetic-responsive, temperature-responsive, and light-responsive types were mainly disclosed regarding responsive SF materials. For example, a porous SF particle grafted with folic acid on the surface and loaded with doxorubicin, the in vitro drug release shows pH-dependent release (Journal of Materials Science, 2019, 54(4):3319). A doxorubicin-loaded magnetic SF nanoparticle can be used as a drug delivery system in magnetic field-guided (magnetic targeting) multidrug-resistant cancer chemotherapy (Advanced Materials, 2014, 26:7393). A multifunctional drug delivery microcarrier combined with SF inverse opal scaffold and temperature-responsive poly(N-isopropylacrylamide) hydrogel, the drug release of the microcarrier can be triggered by external temperature stimulation (Applied Materials Today, 2020, 19: UNSP 100540). A photoresponsive CO-releasing nano drug-loaded particle based on SF exhibits improved CO release (Dalton Transactions, 2018, 47:10434).
关于ROS响应型的材料主要是含硫、硒、碲和不饱和脂质ROS响应载体等。一种二羟基烷基硒化物与二异氰酸酯缩合聚合、并终止于聚乙二醇单甲基醚的含单硒化物的两亲嵌段共聚物，在氧化环境中发生了结构分解和负载药物的受控释放(Polymer Chemistry,2010,1:1609)。一种碲聚合物，在超低浓度过氧化氢存在下，聚合物胶束可以迅速膨胀并演变成不规则的聚集体，可以作为化学和放射治疗相结合的潜在治疗手段(Chemical Communications,2015,51:7069)。一种封装光敏剂和河豚毒素的近红外光触发脂质体，可提供按需可调的局部麻醉(Proceedings of the National Academy of Sciences,2015,112:15719)。又如一种包覆光敏剂二氢卟酚e6和化疗药物阿霉素的TK/聚合物纳米载体，在660nm红光照射下可触发化疗药物阿霉素的快速释放(Chemistry of Materials,2018,30:517)。ROS响应型载药纳米材料的研发已有一些知识产权，如一种以乙烯基吡啶和聚乙二醇为单体进行自由基聚合得到的纳米级粒子可以作为光敏剂的传输载体(申请号：201611126310.3)；一种聚姜黄素前药型纳米粒子可以用于负载抗癌药物和花箐分子(申请号：201910191720.3)；一种表面修饰选择性拮抗剂AMD3100且以葡聚糖和硫辛酸为基材的ROS响应型两亲性材料，用于肝纤维化治疗药物的包载和特异性释药(申请号：202010087783.7)等。The ROS-responsive materials are mainly ROS-responsive carriers containing sulfur, selenium, tellurium and unsaturated lipids. A monoselenide-containing amphiphilic block copolymer of dihydroxyalkyl selenide and diisocyanate condensation polymerization, terminated in polyethylene glycol monomethyl ether, undergoes structural decomposition and drug-loaded block copolymers in an oxidative environment Controlled release (Polymer Chemistry, 2010, 1:1609). A tellurium polymer, in the presence of ultra-low concentrations of hydrogen peroxide, the polymer micelles can rapidly expand and evolve into irregular aggregates, which can be used as a potential therapeutic method for the combination of chemical and radiotherapy (Chemical Communications, 2015, 51:7069). A near-infrared light-triggered liposome encapsulating a photosensitizer and tetrodotoxin provides on-demand adjustable local anesthesia (Proceedings of the National Academy of Sciences, 2015, 112:15719). Another example is a TK/polymer nanocarrier coated with the photosensitizer chlorin e6 and the chemotherapeutic drug doxorubicin, which can trigger the rapid release of the chemotherapeutic drug doxorubicin under 660nm red light irradiation (Chemistry of Materials, 2018, 30 :517). There are some intellectual property rights in the research and development of ROS-responsive drug-loaded nanomaterials. For example, a nanoscale particle obtained by free radical polymerization of vinylpyridine and polyethylene glycol can be used as a transport carrier for photosensitizers (application number: 201611126310.3 ); a polycurcumin prodrug nanoparticle can be used to load anticancer drugs and cyanine molecules (application number: 201910191720.3); a surface-modified selective antagonist AMD3100 with dextran and lipoic acid as the substrate The ROS-responsive amphiphilic material is used for the entrapment and specific release of drugs for the treatment of liver fibrosis (application number: 202010087783.7), etc.
本发明人曾经公开了一种ROS响应型的SF载药递送系统的探索研究，并显示有一定的ROS响应性效果但不显著，因此，开发一种ROS高效响应的SF载药纳米颗粒是非常必要的。The inventor once disclosed a research on a ROS-responsive SF drug-loaded delivery system, and showed that there is a certain ROS-responsive effect but not significant. Therefore, it is very important to develop a ROS-responsive SF drug-loaded nanoparticle. necessary.
发明内容Contents of the invention
有鉴于此，本发明要解决的技术问题在于提供一种功能丝素蛋白药物载体的制备方法，本发明提供的功能丝素蛋白药物载体提升了药物在病变组织部位治疗的精确性和有效性；同时具有显著的ROS响应性能。In view of this, the technical problem to be solved by the present invention is to provide a method for preparing a functional silk fibroin drug carrier. The functional silk fibroin drug carrier provided by the present invention improves the accuracy and effectiveness of drug treatment at diseased tissue sites; At the same time, it has remarkable ROS response performance.
本发明提供了一种功能丝素蛋白药物载体的制备方法，包括：The invention provides a preparation method of a functional silk fibroin drug carrier, comprising:
A)将家蚕蚕丝脱胶、溶解、透析、过滤、浓缩得到丝素蛋白溶解液；A) degumming, dissolving, dialysis, filtering and concentrating silkworm silk to obtain a silk fibroin solution;
B)将两端带有羧基的酮缩硫醇用交联剂活化，再与SF溶解液混合反应，得到反应后的溶液；B) activating the thioketal ketal with carboxyl groups at both ends with a crosslinking agent, and then mixing and reacting with the SF solution to obtain a reacted solution;
C)将反应后的溶液加入无水乙醇和聚乙烯醇的混合体系搅拌、冷冻、解冻、离心，最后将沉淀物冷冻干燥即得。C) adding the reacted solution into a mixed system of absolute ethanol and polyvinyl alcohol, stirring, freezing, thawing, centrifuging, and finally freeze-drying the precipitate to obtain the product.
优选的，步骤A)所述脱胶为采用碳酸钠或碳酸氢钠进行脱胶；所述溶解为采用溴化锂进行溶解；所述透析的截留分子量为10～50kDa；所述丝素蛋白溶解液的浓度为10～200mg/mL。Preferably, the degumming in step A) is degumming with sodium carbonate or sodium bicarbonate; the dissolving is dissolving with lithium bromide; the molecular weight cut-off of the dialysis is 10-50kDa; the concentration of the silk fibroin solution is 10～200mg/mL.
优选的，步骤B)所述两端带有羧基的酮缩硫醇的制备方法具体为：Preferably, step B) the preparation method of the ketal thiol with carboxyl groups at both ends is specifically:
3-巯基丙酸和无水丙酮的混合物在酸性条件下反应，制备的晶体经洗涤、离心、冻干获得两端带有羧基的TK；The mixture of 3-mercaptopropionic acid and anhydrous acetone reacts under acidic conditions, and the prepared crystals are washed, centrifuged, and freeze-dried to obtain TK with carboxyl groups at both ends;
所述3-巯基丙酸和无水丙酮的摩尔比为1：1.5～2.5；所述酸性条件为盐酸；所述盐酸的浓度为7～8M；所述反应具体为20～30℃反应4～8小时；所述冻干具体为：-20～-80℃预冷冻2～6小时后冻干8～16小时。The molar ratio of the 3-mercaptopropionic acid to anhydrous acetone is 1:1.5-2.5; the acidic condition is hydrochloric acid; the concentration of the hydrochloric acid is 7-8M; 8 hours; the freeze-drying specifically includes: pre-freezing at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
优选的，步骤B)所述丝素蛋白和两端带有羧基的酮缩硫醇质量比为100:0.5～20；所述两端带有羧基的酮缩硫醇的溶解溶剂为水。Preferably, in step B), the mass ratio of the silk fibroin to the thioketal with carboxyl groups at both ends is 100:0.5-20; the dissolving solvent for the thioketal with carboxyl groups at both ends is water.
优选的，步骤B)所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶；所述交联剂的添加量为两端带有羧基的酮缩硫醇的2～2.5倍；Preferably, the cross-linking agent in step B) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; the addition of the cross-linking agent The amount is 2 to 2.5 times that of ketal thiols with carboxyl groups at both ends;
所述活化反应温度为4～6℃；The activation reaction temperature is 4-6°C;
所述反应温度为20～30℃；所述反应时间为10～60min。The reaction temperature is 20-30° C.; the reaction time is 10-60 minutes.
优选的，步骤C)所述无水乙醇的浓度为0.5～1.5M；所述聚乙烯醇的浓度为5～60mg/mL，聚乙烯醇的分子量为3～7万；Preferably, the concentration of absolute ethanol in step C) is 0.5-1.5M; the concentration of polyvinyl alcohol is 5-60 mg/mL, and the molecular weight of polyvinyl alcohol is 30,000-70,000;
所述冷冻具体为：-20℃冷冻8～48h；所述解冻为20～30℃解冻；The freezing is specifically: freezing at -20°C for 8-48 hours; the thawing is thawing at 20-30°C;
所述离心为13000rpm离心；The centrifugal is 13000rpm centrifugal;
所述沉淀物冷冻干燥具体为：将沉淀物-20～-80℃预冷冻2～6h后冻干8～16h。The freeze-drying of the precipitate specifically includes: pre-freezing the precipitate at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
本发明提供了一种功能丝素蛋白药物载体，由上述技术方案任意一项 所述的制备方法制备得到。The invention provides a functional silk fibroin drug carrier, which is prepared by the preparation method described in any one of the above technical solutions.
本发明提供了上述任意一项所述的制备方法制备得到的功能丝素蛋白药物载体在担载抗肿瘤/抗炎药物中的应用。The present invention provides the application of the functional silk fibroin drug carrier prepared by any one of the above preparation methods in loading anti-tumor/anti-inflammatory drugs.
本发明提供了一种载药纳米粒子，包括药物和上述技术方案任意一项所述的制备方法制备得到的功能丝素蛋白药物载体。The invention provides a drug-loaded nanoparticle, which includes a drug and a functional silk fibroin drug carrier prepared by the preparation method described in any one of the above technical solutions.
与现有技术相比，本发明提供了一种功能丝素蛋白药物载体的制备方法，包括：A)将家蚕蚕丝脱胶、溶解、透析、过滤、浓缩得到丝素蛋白溶解液；B)将两端带有羧基的酮缩硫醇用交联剂活化，再与SF溶解液混合反应，得到反应后的溶液；C)将反应后的溶液加入无水乙醇和聚乙烯醇的混合体系搅拌、冷冻、解冻、离心，最后将沉淀物冷冻干燥即得。本发明SF/TK纳米粒子中引入了较多的具有ROS响应的分子TK，TK以其端部的双活性官能团羧基分别与SF分子链上大量的羟基共价结合，桥接于SF分子之间。在肿瘤和炎症病变治疗中，经肿瘤或炎症细胞吞噬后因胞中高浓度氧的穿透作用，SF/TK纳米粒子中TK分子的C-S键断裂使SF分子间共价键断裂，纳米粒子内部分子间裂解，从而纳米粒子中的药物迅速释放出来，定向杀死肿瘤和炎症细胞，同时进入正常细胞中的纳米粒子其不裂解或裂解程度很小，仅依赖于渗透释放或降解后释放，被代谢前药物释放很少很慢，对正常细胞的杀伤极小。另外，选用的与人体组成相同的20种α-氨基酸组成的SF作为载药纳米粒子主体，进入人体后无毒副作用，是一种理想的药物递送载体。SF可通过制备技术调节纳米粒子的结晶结构，从而调控药物的渗透释放与降解释放，降低纳米粒子代谢排出前释放于正常细胞中的药物量，是一种符合定向治疗炎症和肿瘤的药物递送载体。Compared with the prior art, the present invention provides a method for preparing a functional silk fibroin drug carrier, comprising: A) degumming silkworm silk, dissolving, dialysis, filtering, and concentrating to obtain a silk fibroin solution; B) mixing two The ketal thiol with a carboxyl group is activated with a cross-linking agent, and then mixed with the SF solution to obtain a reacted solution; C) the reacted solution is added to the mixed system of absolute ethanol and polyvinyl alcohol to stir and freeze , thawing, centrifuging, and finally freeze-drying the precipitate. The SF/TK nanoparticles of the present invention introduce more ROS-responsive molecules TK, and TK is covalently bonded to a large number of hydroxyl groups on the SF molecular chain with the dual-active functional group carboxyl at its end, and bridges between SF molecules. In the treatment of tumors and inflammatory lesions, after being phagocytized by tumor or inflammatory cells, due to the penetration of high concentration of oxygen in the cells, the C-S bond of the TK molecule in the SF/TK nanoparticle is broken to break the covalent bond between the SF molecules, and the internal molecules of the nanoparticle Inter-cracking, so that the drug in the nanoparticles is released quickly, and the directional killing of tumor and inflammatory cells is targeted. At the same time, the nanoparticles that enter the normal cells do not cleavage or have a small degree of cleavage. They only rely on osmotic release or release after degradation, and are metabolized. Prodrugs are rarely released very slowly, causing minimal damage to normal cells. In addition, SF composed of 20 kinds of α-amino acids, which are the same as the composition of the human body, is selected as the main body of the drug-loaded nanoparticles, which has no toxic and side effects after entering the human body, and is an ideal drug delivery carrier. SF can adjust the crystalline structure of nanoparticles through preparation technology, thereby regulating the osmotic release and degradation release of drugs, and reducing the amount of drugs released in normal cells before the metabolism of nanoparticles is excreted. It is a drug delivery carrier for targeted treatment of inflammation and tumors. .
具体实施方式Detailed ways
本发明提供了一种功能丝素蛋白药物载体及其制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都属于本发明保护的范围。本发明的方法及应用已经通过较佳实施例进行了描述， 相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention provides a functional silk fibroin drug carrier and a preparation method thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they all belong to the protection scope of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
本发明提供了一种功能丝素蛋白药物载体的制备方法，包括：The invention provides a preparation method of a functional silk fibroin drug carrier, comprising:
A)将家蚕蚕丝脱胶、溶解、透析、过滤、浓缩得到丝素蛋白溶解液；A) degumming, dissolving, dialysis, filtering and concentrating silkworm silk to obtain a silk fibroin solution;
B)将两端带有羧基的酮缩硫醇用交联剂活化，再与SF溶解液混合反应，得到反应后的溶液；B) activating the thioketal ketal with carboxyl groups at both ends with a crosslinking agent, and then mixing and reacting with the SF solution to obtain a reacted solution;
C)将反应后的溶液加入无水乙醇和聚乙烯醇的混合体系搅拌、冷冻、解冻、离心，最后将沉淀物冷冻干燥即得。C) adding the reacted solution into a mixed system of absolute ethanol and polyvinyl alcohol, stirring, freezing, thawing, centrifuging, and finally freeze-drying the precipitate to obtain the product.
本发明提供的一种功能丝素蛋白药物载体的制备方法首先将将家蚕蚕丝脱胶。The preparation method of a functional silk fibroin drug carrier provided by the invention firstly degummes silkworm silk.
本发明所述脱胶优选为采用碳酸钠或碳酸氢钠进行脱胶；更优选具体为：Degumming described in the present invention is preferably to adopt sodium carbonate or sodium bicarbonate to carry out degumming; More preferably specifically:
称取一定量的生丝，按浴比1:50(w:v)量取去离子水，往冷水中加入Na 2CO 3或碳酸氢钠搅拌均匀，待水煮沸后加入生丝脱胶，取出搓洗，放入锅中再次煮沸脱胶，重复2～3次。 Weigh a certain amount of raw silk, measure deionized water according to the bath ratio of 1:50 (w:v), add Na 2 CO 3 or sodium bicarbonate into the cold water and stir evenly, add raw silk to degumming after the water is boiled, take it out and scrub, Put it in the pot and boil again to degumming, repeat 2-3 times.
将脱胶后的丝置于电热恒温干燥箱中，烘干，扯至蓬松，得到脱胶后的家蚕丝素纤维。本发明对于所述烘干的具体温度不进行限定，可以为50～70℃。The degummed silk is placed in an electric heating constant temperature drying oven, dried, and pulled until fluffy to obtain the degummed silkworm silk fiber. The present invention does not limit the specific temperature of the drying, which may be 50-70°C.
脱胶后为溶解。本发明所述溶解优选为采用溴化锂进行溶解；更优选具体为：称取一定量的脱胶丝素纤维，剪碎放入溴化锂溶液中60～70℃加热搅拌1～2h小时得到家蚕SF溶解液。本发明对于所述溴化锂的浓度不进行限定，本领域技术人员熟知的即可，优选为9～10M。Dissolution after degumming. The dissolving in the present invention is preferably carried out by lithium bromide; more preferably, it is as follows: weigh a certain amount of degummed silk fibroin fiber, cut it into pieces, put it into a lithium bromide solution, heat and stir at 60-70° C. for 1-2 hours to obtain silkworm SF solution. The present invention does not limit the concentration of the lithium bromide, which is well known to those skilled in the art, and is preferably 9-10M.
溶解后为透析、过滤、浓缩得到丝素蛋白溶解液。After dissolving, it is dialyzed, filtered and concentrated to obtain silk fibroin solution.
将家蚕SF溶解液灌注于透析袋中置于盛有去离子水的容器内透析；本发明所述透析的截留分子量为10～50kDa；更优选为25～50kDa。The silkworm SF solution is poured into a dialysis bag and placed in a container filled with deionized water for dialysis; the dialysis molecular weight cut-off of the present invention is 10-50 kDa; more preferably 25-50 kDa.
透析后过滤、通风橱内旋转蒸发浓缩。After dialysis, filter and concentrate by rotary evaporation in a fume hood.
本发明所述丝素蛋白溶解液的浓度优选为10～200mg/mL；更优选为15～190mg/mL；最优选为20～80mg/mL。The concentration of the silk fibroin solution in the present invention is preferably 10-200 mg/mL; more preferably 15-190 mg/mL; most preferably 20-80 mg/mL.
将两端带有羧基的酮缩硫醇用交联剂活化，再与SF溶解液混合反 应；本发明所述两端带有羧基的酮缩硫醇(两端带有羧基的TK)的制备方法具体为：Activate the thioketal ketal with carboxyl groups at both ends with a cross-linking agent, and then mix and react with the SF solution; the preparation of the thioketal ketal with carboxyl groups at both ends (TK with carboxyl groups at both ends) of the present invention The method is specifically:
3-巯基丙酸和无水丙酮的混合物在酸性条件下反应，制备的晶体经洗涤、离心、冻干获得两端带有羧基的TK；The mixture of 3-mercaptopropionic acid and anhydrous acetone reacts under acidic conditions, and the prepared crystals are washed, centrifuged, and freeze-dried to obtain TK with carboxyl groups at both ends;
将3-巯基丙酸和无水丙酮混合，所述3-巯基丙酸和无水丙酮的摩尔比优选为1：1.5～2.5；更优选为1：2～2.5；并在盐酸条件下反应；所述盐酸的浓度为7～8M；反应具体为20～30℃反应4～8小时；更优选为22～28℃反应4～8小时；将反应沉析出来的晶体经洗涤、离心、冻干，获得两端带有羧基的TK。所述离心优选为13000rpm。所述冻干具体为：-20～-80℃预冷冻2～6小时后冻干8～16小时。Mix 3-mercaptopropionic acid and anhydrous acetone, the molar ratio of 3-mercaptopropionic acid and anhydrous acetone is preferably 1:1.5-2.5; more preferably 1:2-2.5; and react under hydrochloric acid conditions; The concentration of the hydrochloric acid is 7-8M; the reaction is specifically 20-30°C for 4-8 hours; more preferably 22-28°C for 4-8 hours; the precipitated crystals are washed, centrifuged, and freeze-dried , to obtain TK with carboxyl groups at both ends. The centrifugation is preferably at 13000 rpm. The freeze-drying specifically includes: pre-freezing at -20 to -80° C. for 2 to 6 hours and then freeze-drying for 8 to 16 hours.
按照本发明所述丝素蛋白和两端带有羧基的酮缩硫醇质量比为优选100:0.5～20；更优选为100:1～10；最优选为100:1～8。According to the present invention, the mass ratio of silk fibroin to thioketal with carboxyl groups at both ends is preferably 100:0.5-20; more preferably 100:1-10; most preferably 100:1-8.
其中，所述两端带有羧基的酮缩硫醇的溶解溶剂为水，配置成0.1～0.5mg/mL，优选可以为：置于4℃水浴中搅拌20分钟。Wherein, the dissolving solvent of the ketal thioketal having carboxyl groups at both ends is water, and the dissolving solvent is 0.1-0.5 mg/mL. Preferably, it can be placed in a water bath at 4° C. and stirred for 20 minutes.
本发明所述活化反应温度为4～6℃；The activation reaction temperature of the present invention is 4～6 ℃;
本发明所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶；所述交联剂的添加量为两端带有羧基的酮缩硫醇的2～2.5倍；The cross-linking agent of the present invention is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; the addition amount of the cross-linking agent is two ends 2 to 2.5 times that of ketal thiols with carboxyl groups;
所述反应温度优选为20～30℃；更优选为22～28℃；所述反应时间优选为10～60min；更优选为15～55min。The reaction temperature is preferably 20-30°C; more preferably 22-28°C; the reaction time is preferably 10-60min; more preferably 15-55min.
本发明优选为：向TK溶液里慢慢滴加交联剂，随后在4℃～6℃水浴中反应10～15分钟，以活化羧基。而后将活化后的TK缓慢滴加于SF溶解液中，在磁力搅拌器上慢速搅拌，室温反应得到SF/TK复合溶液。The preferred method of the present invention is: slowly drop the cross-linking agent into the TK solution, and then react in a water bath at 4° C. to 6° C. for 10 to 15 minutes to activate the carboxyl group. Then slowly drop the activated TK into the SF solution, stir slowly on a magnetic stirrer, and react at room temperature to obtain a SF/TK composite solution.
将反应后的溶液加入无水乙醇和聚乙烯醇的混合体系搅拌、冷冻、解冻、离心，最后将沉淀物冷冻干燥即得。The reacted solution is added into a mixed system of absolute ethanol and polyvinyl alcohol, stirred, frozen, thawed, centrifuged, and finally the precipitate is freeze-dried to obtain the obtained product.
本发明所述无水乙醇的浓度优选为0.5～1.5M；所述聚乙烯醇的浓度优选为5～60mg/mL，更优选为10～50mg/mL，聚乙烯醇的分子量为3～7万；The concentration of absolute ethanol in the present invention is preferably 0.5-1.5M; the concentration of polyvinyl alcohol is preferably 5-60 mg/mL, more preferably 10-50 mg/mL, and the molecular weight of polyvinyl alcohol is 3-70,000 ;
本发明所述冷冻具体优选为：-20℃冷冻8～48h；更优选为-20℃冷冻 10～48h；The freezing of the present invention is specifically preferably: freezing at -20°C for 8 to 48 hours; more preferably freezing at -20°C for 10 to 48 hours;
所述解冻为20℃～30℃解冻；The thawing is thawing at 20°C to 30°C;
所述离心为13000rpm离心；The centrifugal is 13000rpm centrifugal;
所述沉淀物冷冻干燥具体为：将沉淀物-20～-80℃预冷冻2～6h后冻干8～16h。The freeze-drying of the precipitate specifically includes: pre-freezing the precipitate at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
本发明提供了一种功能丝素蛋白药物载体，由上述技术方案任意一项所述的制备方法制备得到。The present invention provides a functional silk fibroin drug carrier, which is prepared by the preparation method described in any one of the above technical solutions.
本发明对于上述制备方法已经有了清楚的描述，在此不再赘述。The present invention has already clearly described the above-mentioned preparation method, and will not repeat them here.
本发明提供了上述任意一项所述的制备方法制备得到的功能丝素蛋白药物载体在担载抗肿瘤/抗炎药物中的应用。The present invention provides the application of the functional silk fibroin drug carrier prepared by any one of the above preparation methods in loading anti-tumor/anti-inflammatory drugs.
其中，抗肿瘤/抗炎药物包括但不限于喜树碱。Among them, anti-tumor/anti-inflammatory drugs include but not limited to camptothecin.
本发明提供了一种载药纳米粒子，包括药物和上述技术方案任意一项所述的制备方法制备得到的功能丝素蛋白药物载体。The invention provides a drug-loaded nanoparticle, which includes a drug and a functional silk fibroin drug carrier prepared by the preparation method described in any one of the above technical solutions.
当药物存在时，在制备载药纳米粒子时，最后步骤C)将变为：When the drug is present, when preparing the drug-loaded nanoparticles, the final step C) will become:
在搅拌条件下向上述SF/TK复合溶液中加入抗肿瘤药物喜树碱，再加入无水乙醇和聚乙烯醇的混合体系搅拌混合均匀，冷冻。室温下溶解、洗涤、离心，将离心管底端的沉淀物冷冻干燥，得到载药SF/TK纳米粒子。本发明所述无水乙醇的浓度优选为0.5～1.5M；所述聚乙烯醇的浓度优选为5～60mg/mL，更优选为10～50mg/mL，聚乙烯醇的分子量为3～7万；Add the antineoplastic drug camptothecin to the above SF/TK composite solution under stirring condition, then add the mixed system of absolute ethanol and polyvinyl alcohol, stir and mix evenly, and freeze. Dissolving, washing and centrifuging at room temperature, and freeze-drying the precipitate at the bottom of the centrifuge tube to obtain drug-loaded SF/TK nanoparticles. The concentration of absolute ethanol in the present invention is preferably 0.5-1.5M; the concentration of polyvinyl alcohol is preferably 5-60 mg/mL, more preferably 10-50 mg/mL, and the molecular weight of polyvinyl alcohol is 3-70,000 ;
本发明提供了一种功能丝素蛋白药物载体的制备方法，包括：A)将家蚕蚕丝脱胶、溶解、透析过滤凝缩得到丝素蛋白溶解液；B)将两端带有羧基的酮缩硫醇用交联剂活化，再与SF溶解液混合反应，得到反应后的溶液；C)将反应后的溶液加入无水乙醇和聚乙烯醇的混合体系搅拌、冷冻、解冻、离心，最后将沉淀物冷冻干燥即得。本发明SF/TK纳米粒子中引入了较多的具有ROS响应的分子TK，TK以其端部的双活性官能团羧基分别与SF分子链上大量的羟基共价结合，桥接于SF分子之间。在肿瘤和炎症病变治疗中，经肿瘤或炎症细胞吞噬后因胞中高浓度氧的穿透作用，SF/TK纳米粒子中TK分子的C-S键断裂使SF分子间共价键断 裂，纳米粒子内部分子间裂解，从而纳米粒子中的药物迅速释放出来，定向杀死肿瘤和炎症细胞，同时进入正常细胞中的纳米粒子其不裂解或裂解程度很小，仅依赖于渗透释放或降解后释放，被代谢前药物释放很少很慢，对正常细胞的杀伤极小。另外，选用的与人体组成相同的20种α-氨基酸组成的SF作为载药纳米粒子主体，进入人体后无毒副作用，是一种理想的药物递送载体。SF可通过制备技术调节纳米粒子的结晶结构，从而调控药物的渗透释放与降解释放，降低纳米粒子代谢排出前释放于正常细胞中的药物量，是一种符合定向治疗炎症和肿瘤的药物递送载体。The invention provides a preparation method of a functional silk fibroin drug carrier, comprising: A) degumming silkworm silk, dissolving, dialysis filtering and condensing to obtain a silk fibroin solution; B) ketone sulfurization with carboxyl groups at both ends Alcohol is activated with a cross-linking agent, and then mixed with SF solution to obtain a reacted solution; C) add the reacted solution to a mixed system of absolute ethanol and polyvinyl alcohol to stir, freeze, thaw, and centrifuge, and finally remove the precipitate The product is freeze-dried. The SF/TK nanoparticles of the present invention introduce more ROS-responsive molecules TK, and TK is covalently bonded to a large number of hydroxyl groups on the SF molecular chain with the dual-active functional group carboxyl at its end, and bridges between SF molecules. In the treatment of tumors and inflammatory lesions, after being phagocytized by tumor or inflammatory cells, due to the penetration of high concentration of oxygen in the cells, the C-S bond of the TK molecule in the SF/TK nanoparticle is broken to break the covalent bond between the SF molecules, and the internal molecules of the nanoparticle Inter-cracking, so that the drug in the nanoparticles is released quickly, and the directional killing of tumor and inflammatory cells is targeted. At the same time, the nanoparticles that enter the normal cells do not cleavage or have a small degree of cleavage. They only rely on osmotic release or release after degradation, and are metabolized. Prodrugs are rarely released very slowly, causing minimal damage to normal cells. In addition, SF composed of 20 kinds of α-amino acids, which are the same as the composition of the human body, is selected as the main body of the drug-loaded nanoparticles, which has no toxic and side effects after entering the human body, and is an ideal drug delivery carrier. SF can adjust the crystalline structure of nanoparticles through preparation technology, thereby regulating the osmotic release and degradation release of drugs, and reducing the amount of drugs released in normal cells before the metabolism of nanoparticles is excreted. It is a drug delivery carrier for targeted treatment of inflammation and tumors. .
再有，SF分子具有亲水和疏水的两性链段，不仅具有正电荷的氨基和负电荷的羧基，还具有大量的羟基用于改性，本发明提出利用SF分子上含有的最多的官能团羟基来制备ROS响应的载药SF/TK纳米粒子。基于SF分子的化学结构特征，可以加载各种不同表面性质的药物，所以本发明的SF智能ROS响应药物递送载体对于大多数炎症和肿瘤疾病的治疗具有很大的应用前景。Furthermore, SF molecules have hydrophilic and hydrophobic amphipathic segments, not only have positively charged amino groups and negatively charged carboxyl groups, but also have a large number of hydroxyl groups for modification. The present invention proposes to utilize the most functional group hydroxyl groups contained on the SF molecules To prepare ROS-responsive drug-loaded SF/TK nanoparticles. Based on the chemical structural characteristics of SF molecules, various drugs with different surface properties can be loaded, so the SF smart ROS-responsive drug delivery carrier of the present invention has great application prospects for the treatment of most inflammatory and tumor diseases.
为了进一步说明本发明，以下结合实施例对本发明提供的一种功能丝素蛋白药物载体及其制备方法进行详细描述。In order to further illustrate the present invention, a functional silk fibroin drug carrier provided by the present invention and its preparation method are described in detail below in conjunction with the examples.
实施例1Example 1
(1)称取一定量的生丝，按浴比1:50(w:v)量取去离子水，往冷水中加入Na 2CO 3(wt 0.06％)搅拌均匀，待水煮沸后加入生丝脱胶，30分钟后取出搓洗，放入锅中再次煮沸脱胶，重复3次。将脱胶后的丝置于电热恒温干燥箱中，60℃烘干，扯至蓬松，得到脱胶后的家蚕丝素纤维。 (1) Weigh a certain amount of raw silk, measure deionized water according to the bath ratio of 1:50 (w:v), add Na 2 CO 3 (wt 0.06%) into the cold water and stir evenly, after the water is boiled, add raw silk to degumming , After 30 minutes, take it out and scrub it, put it in a pot and boil it again for degumming, repeat 3 times. The degummed silk is placed in an electric heating constant temperature drying oven, dried at 60° C., and pulled until fluffy to obtain the degummed silkworm silk fibroin fiber.
(2)称取一定量的脱胶丝素纤维，剪碎放入9.3M的溴化锂溶液中65±2℃加热搅拌1小时得到家蚕SF溶解液。将家蚕SF溶解液灌注于透析袋中置于盛有去离子水的容器内透析得到纯化后的家蚕SF水溶液，调整透析后的SF水溶液浓度为10～200mg/mL。(2) Weigh a certain amount of degummed silk fibroin fiber, cut it into pieces, put it into a 9.3 M lithium bromide solution, heat and stir at 65±2° C. for 1 hour to obtain silkworm SF solution. The silkworm SF solution was poured into a dialysis bag, placed in a container filled with deionized water and dialyzed to obtain a purified silkworm SF aqueous solution, and the concentration of the dialyzed SF aqueous solution was adjusted to 10-200 mg/mL.
(3)称取一定量3-巯基丙酸和无水丙酮混合，加入浓盐酸，室温下密封搅拌6小时。将混合溶液置于冰浴中，急速冷却发生淬灭反应，直到全部变成白色晶体沉淀为止。将晶体过滤并洗涤三次。最后，离心洗涤，倒掉上清液，将沉淀物在-80℃冰箱冻4小时后，冷冻干燥机干燥12小 时，获得ROS可裂解的两端带有羧基的TK。(3) Weigh a certain amount of 3-mercaptopropionic acid and anhydrous acetone to mix, add concentrated hydrochloric acid, and stir at room temperature for 6 hours. The mixed solution was placed in an ice bath, and the reaction was quenched by rapid cooling until all white crystals were precipitated. The crystals were filtered and washed three times. Finally, wash by centrifugation, discard the supernatant, freeze the precipitate at -80°C for 4 hours, and dry it in a freeze dryer for 12 hours to obtain TK with carboxyl groups at both ends that can be cleaved by ROS.
(4)将制得的TK溶于去离子水配置成0.1～0.5mg/mL，向TK溶液里慢慢滴加摩尔比是TK的2～2.5倍的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶，随后在4℃水浴中反应10分钟，以活化羧基。按SF：TK质量比为100:(1～5)将活化后的TK缓慢滴加于SF水溶液中，在磁力搅拌器上慢速搅拌，室温下反应1小时，得到SF/TK复合溶液。(4) Dissolve the prepared TK in deionized water to make 0.1-0.5 mg/mL, and slowly add 1-(3-dimethylaminopropyl group whose molar ratio is 2-2.5 times that of TK) dropwise into the TK solution. )-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, followed by reaction in a water bath at 4° C. for 10 minutes to activate the carboxyl group. According to the SF:TK mass ratio of 100:(1~5), the activated TK was slowly added dropwise to the SF aqueous solution, stirred slowly on a magnetic stirrer, and reacted at room temperature for 1 hour to obtain the SF/TK composite solution.
(5)在搅拌条件下向上述SF/TK复合溶液中加入终浓度0.1～1mg/mL抗肿瘤药物喜树碱，再按体积比加入无水乙醇(V SF/TK:V 乙醇＝5:1)，然后加入2％聚乙烯醇溶液，体积V 聚乙烯醇＝2(V SF/TK+V 乙醇)，搅拌混合均匀，放置于-20℃冰箱冷冻48小时后取出。室温下溶解、洗涤、离心，将离心管底端的沉淀物-80℃冷冻3小时，然后冷冻干燥12小时，得到载药SF/TK纳米粒子。 (5) Add antineoplastic drug camptothecin at a final concentration of 0.1 to 1 mg/mL to the above-mentioned SF/TK composite solution under stirring conditions, and then add absolute ethanol by volume (V SF/TK :V ethanol =5:1 ), then add 2% polyvinyl alcohol solution, volume V polyvinyl alcohol = 2 (V SF/TK + V ethanol ), stir and mix evenly, place it in a -20°C refrigerator for 48 hours and take it out. Dissolving, washing and centrifuging at room temperature, freezing the precipitate at the bottom of the centrifuge tube at -80°C for 3 hours, and then freeze-drying for 12 hours to obtain drug-loaded SF/TK nanoparticles.
(6)将制备得到的包载喜树碱的纳米粒子漂洗后进行载药能力的测试，检测365nm处的喜树碱的吸光度值，经过计算SF/TK载药纳米粒子的载药率和包封率分别为1.5～7.5％和40～70％。(6) After rinsing the prepared nanoparticles loaded with camptothecin, test the drug-loading ability, detect the absorbance value of camptothecin at 365nm, and calculate the drug-loaded rate and the package ratio of the SF/TK drug-loaded nanoparticles. The sealing rates were 1.5-7.5% and 40-70% respectively.
实施例2Example 2
(1)称取一定量的生丝，按浴比1:50(w:v)量取去离子水，往冷水中加入Na 2CO 3(wt 0.06％)搅拌均匀，待水煮沸后加入生丝脱胶，30分钟后取出搓洗，放入锅中再次煮沸脱胶，重复3次。将脱胶后的丝置于电热恒温干燥箱中，60℃烘干，扯至蓬松，得到脱胶后的家蚕丝素纤维。 (1) Weigh a certain amount of raw silk, measure deionized water according to the bath ratio of 1:50 (w:v), add Na 2 CO 3 (wt 0.06%) into the cold water and stir evenly, after the water is boiled, add raw silk to degumming , After 30 minutes, take it out and scrub it, put it in a pot and boil it again for degumming, repeat 3 times. The degummed silk is placed in an electric heating constant temperature drying oven, dried at 60° C., and pulled until fluffy to obtain the degummed silkworm silk fibroin fiber.
(2)称取一定量的脱胶丝素纤维，剪碎放入9.3M的溴化锂溶液中65±2℃加热搅拌1小时得到家蚕SF溶解液。将家蚕SF溶解液灌注于透析袋中置于盛有去离子水的容器内透析得到纯化后的家蚕SF水溶液，调整透析后的SF水溶液浓度为10～200mg/mL。(2) Weigh a certain amount of degummed silk fibroin fiber, cut it into pieces, put it into a 9.3 M lithium bromide solution, heat and stir at 65±2° C. for 1 hour to obtain silkworm SF solution. The silkworm SF solution was poured into a dialysis bag, placed in a container filled with deionized water and dialyzed to obtain a purified silkworm SF aqueous solution, and the concentration of the dialyzed SF aqueous solution was adjusted to 10-200 mg/mL.
(3)称取一定量3-巯基丙酸和无水丙酮混合，加入浓盐酸，室温下密封搅拌6小时。将混合溶液置于冰浴中，急速冷却发生淬灭反应，直到全部变成白色晶体沉淀为止。将晶体过滤并洗涤三次。最后，离心洗涤， 倒掉上清液，将沉淀物在-80℃冰箱冻4小时后，冷冻干燥机干燥12小时，获得ROS可裂解的两端带有羧基的TK。(3) Weigh a certain amount of 3-mercaptopropionic acid and anhydrous acetone to mix, add concentrated hydrochloric acid, and stir at room temperature for 6 hours. The mixed solution was placed in an ice bath, and the reaction was quenched by rapid cooling until all white crystals were precipitated. The crystals were filtered and washed three times. Finally, centrifuge and wash, discard the supernatant, freeze the precipitate at -80°C for 4 hours, and then dry it with a freeze dryer for 12 hours to obtain TK with carboxyl groups at both ends that can be cleaved by ROS.
(4)将制得的TK溶于去离子水配置成0.1～0.5mg/mL，向TK溶液里慢慢滴加摩尔比是TK的2～2.5倍的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶，随后在4℃水浴中反应10分钟，以活化羧基。按SF：TK质量比为100:3将活化后的TK缓慢滴加于SF水溶液中，在磁力搅拌器上慢速搅拌，室温下反应1小时，得到SF/TK复合溶液。(4) Dissolve the prepared TK in deionized water to make 0.1-0.5 mg/mL, and slowly add 1-(3-dimethylaminopropyl group whose molar ratio is 2-2.5 times that of TK) dropwise into the TK solution. )-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, followed by reaction in a water bath at 4° C. for 10 minutes to activate the carboxyl group. According to the SF: TK mass ratio of 100:3, the activated TK was slowly added dropwise to the SF aqueous solution, stirred slowly on a magnetic stirrer, and reacted at room temperature for 1 hour to obtain the SF/TK composite solution.
(5)在搅拌条件下向上述SF/TK复合溶液中加入终浓度0.5mg/mL抗肿瘤药物喜树碱，再按体积比加入无水乙醇(V SF/TK:V 乙醇＝5:1)，然后加入2％聚乙烯醇溶液，体积V 聚乙烯醇＝2(V SF/TK+V 乙醇)，搅拌混合均匀，放置于-20℃冰箱冷冻48小时后取出。室温下溶解、洗涤、离心，将离心管底端的沉淀物-80℃冷冻3小时，然后冷冻干燥12小时，得到载药SF/TK纳米粒子。 (5) Add the antineoplastic drug camptothecin with a final concentration of 0.5 mg/mL to the above-mentioned SF/TK composite solution under stirring conditions, and then add absolute ethanol by volume ratio (V SF/TK :V ethanol =5:1) , and then add 2% polyvinyl alcohol solution, volume V polyvinyl alcohol = 2 (V SF/TK + V ethanol ), stir and mix evenly, place it in a -20°C refrigerator for 48 hours and take it out. Dissolve, wash, and centrifuge at room temperature, freeze the precipitate at the bottom of the centrifuge tube at -80°C for 3 hours, and then freeze-dry for 12 hours to obtain drug-loaded SF/TK nanoparticles.
(6)将制备得到的包载喜树碱的SF/TK纳米粒子漂洗后进行氧裂解前后的粒径测试，SF/TK载药纳米粒子的粒径随着50mM的KO 2溶液处理时间的增加呈递减趋势，处理16小时后，从最初的426nm减小到了257nm，即SF/TK纳米粒子中的TK遇到氧环境，化学键断裂释放，从内部破坏了纳米粒子，再次冻干后发生了严重收缩，及裂解导致纳米粒子破碎，说明该SF/TK载药纳米粒子可以在氧环境下发生裂解。 (6) After rinsing the prepared SF/TK nanoparticles loaded with camptothecin, carry out the particle size test before and after oxygen cracking, the particle size of SF/TK drug-loaded nanoparticles increases with the treatment time of 50mM KO solution It showed a decreasing trend. After 16 hours of treatment, it decreased from the initial 426nm to 257nm, that is, the TK in the SF/TK nanoparticles encountered an oxygen environment, and the chemical bonds were broken and released, destroying the nanoparticles from the inside. Shrinkage, and cleavage lead to the fragmentation of nanoparticles, indicating that the SF/TK drug-loaded nanoparticles can be cleaved in an oxygen environment.
(7)将制备得到的包载喜树碱的SF/TK纳米粒子在氧处理环境中观察其药物释放行为，在氧环境中，SF/TK载药纳米粒子释药较快，尤其是前12小时，随后释放有所平缓。氧处理8小时，SF/TK载药纳米粒子的累计释药率达到47％左右，48小时后，SF/TK载药纳米粒子的累积释药率约为72％，显著高于对比例的释药率。SF/TK载药纳米粒子在非氧环境中48小时药物释放量远小于其在氧环境中的释放量。即SF/TK纳米粒子在氧环境中因TK响应断裂，使得SF/TK纳米粒子能快速释放药物，具有显著的ROS响应性能。(7) Observe the drug release behavior of the prepared SF/TK nanoparticles loaded with camptothecin in the oxygen treatment environment. In the oxygen environment, the SF/TK drug-loaded nanoparticles released faster, especially the first 12 hours, and then the release leveled off. After oxygen treatment for 8 hours, the cumulative drug release rate of SF/TK drug-loaded nanoparticles reached about 47%. After 48 hours, the cumulative drug release rate of SF/TK drug-loaded nanoparticles was about 72%, which was significantly higher than that of the comparative example. drug rate. The drug release amount of SF/TK drug-loaded nanoparticles in non-oxygen environment for 48 hours is much smaller than that in oxygen environment. That is, the SF/TK nanoparticles are fractured due to the TK response in an oxygen environment, so that the SF/TK nanoparticles can release drugs rapidly and have significant ROS responsive properties.
实施例3Example 3
(1)称取一定量的生丝，按浴比1:50(w:v)量取去离子水，往冷水中加入Na 2CO 3(wt 0.06％)搅拌均匀，待水煮沸后加入生丝脱胶，30分钟后取出搓洗，放入锅中再次煮沸脱胶，重复3次。将脱胶后的丝置于电热恒温干燥箱中，60℃烘干，扯至蓬松，得到脱胶后的家蚕丝素纤维。 (1) Weigh a certain amount of raw silk, measure deionized water according to the bath ratio of 1:50 (w:v), add Na 2 CO 3 (wt 0.06%) into the cold water and stir evenly, after the water is boiled, add raw silk to degumming , After 30 minutes, take it out and scrub it, put it in a pot and boil it again for degumming, repeat 3 times. The degummed silk is placed in an electric heating constant temperature drying oven, dried at 60° C., and pulled until fluffy to obtain the degummed silkworm silk fibroin fiber.
(2)称取一定量的脱胶丝素纤维，剪碎放入9.3M的溴化锂溶液中65±2℃加热搅拌1小时得到家蚕SF溶解液。将家蚕SF溶解液灌注于透析袋中置于盛有去离子水的容器内透析得到纯化后的家蚕SF水溶液，调整透析后的SF水溶液浓度为10～200mg/mL。(2) Weigh a certain amount of degummed silk fibroin fiber, cut it into pieces, put it into a 9.3 M lithium bromide solution, heat and stir at 65±2° C. for 1 hour to obtain silkworm SF solution. The silkworm SF solution was poured into a dialysis bag, placed in a container filled with deionized water and dialyzed to obtain a purified silkworm SF aqueous solution, and the concentration of the dialyzed SF aqueous solution was adjusted to 10-200 mg/mL.
(3)称取一定量3-巯基丙酸和无水丙酮混合，加入浓盐酸，室温下密封搅拌6小时。将混合溶液置于冰浴中，急速冷却发生淬灭反应，直到全部变成白色晶体沉淀为止。将晶体过滤并洗涤三次。最后，离心洗涤，倒掉上清液，将沉淀物在-80℃冰箱冻4小时后，冷冻干燥机干燥12小时，获得ROS可裂解的两端带有羧基的TK。(3) Weigh a certain amount of 3-mercaptopropionic acid and anhydrous acetone to mix, add concentrated hydrochloric acid, and stir at room temperature for 6 hours. The mixed solution was placed in an ice bath, and the reaction was quenched by rapid cooling until all white crystals were precipitated. The crystals were filtered and washed three times. Finally, wash by centrifugation, discard the supernatant, freeze the precipitate at -80°C for 4 hours, and dry it in a freeze dryer for 12 hours to obtain TK with carboxyl groups at both ends that can be cleaved by ROS.
(4)将制得的TK溶于去离子水配置成0.1～0.5mg/mL，向TK溶液里慢慢滴加摩尔比是TK的2～2.5倍的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶，随后在4℃水浴中反应10分钟，以活化羧基。按SF：TK质量比为100:5将活化后的TK缓慢滴加于SF水溶液中，在磁力搅拌器上慢速搅拌，室温下反应1小时，得到SF/TK复合溶液。(4) Dissolve the prepared TK in deionized water to make 0.1-0.5 mg/mL, and slowly add 1-(3-dimethylaminopropyl group whose molar ratio is 2-2.5 times that of TK) dropwise into the TK solution. )-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, followed by reaction in a water bath at 4° C. for 10 minutes to activate the carboxyl group. According to the SF:TK mass ratio of 100:5, the activated TK was slowly added dropwise to the SF aqueous solution, stirred slowly on a magnetic stirrer, and reacted at room temperature for 1 hour to obtain the SF/TK composite solution.
(5)在搅拌条件下向上述SF/TK复合溶液中加入终浓度0.5mg/mL抗肿瘤药物喜树碱，再按体积比加入无水乙醇(V SF/TK:V 乙醇＝5:1)，然后加入2％聚乙烯醇溶液，体积V 聚乙烯醇＝2(V SF/TK+V 乙醇)，搅拌混合均匀，放置于-20℃冰箱冷冻48小时后取出。室温下溶解、洗涤、离心，将离心管底端的沉淀物-80℃冷冻3小时，然后冷冻干燥12小时，得到载药SF/TK纳米粒子。 (5) Add the antineoplastic drug camptothecin with a final concentration of 0.5 mg/mL to the above-mentioned SF/TK composite solution under stirring conditions, and then add absolute ethanol by volume ratio (V SF/TK :V ethanol =5:1) , and then add 2% polyvinyl alcohol solution, volume V polyvinyl alcohol = 2 (V SF/TK + V ethanol ), stir and mix evenly, place it in a -20°C refrigerator for 48 hours and take it out. Dissolve, wash, and centrifuge at room temperature, freeze the precipitate at the bottom of the centrifuge tube at -80°C for 3 hours, and then freeze-dry for 12 hours to obtain drug-loaded SF/TK nanoparticles.
(6)将制备得到的包载喜树碱的SF/TK纳米粒子漂洗后进行氧裂解前后的粒径测试，SF/TK载药纳米粒子的粒径随着50mM的KO 2溶液处理时间的增加呈递减趋势，处理16小时后，从最初的532nm减小到了 315nm，即SF/TK纳米粒子中的TK遇到氧环境，化学键断裂释放，从内部破坏了纳米粒子，再次冻干后发生了严重收缩，及裂解导致纳米粒子破碎，说明该SF/TK载药纳米粒子可以在氧环境下发生裂解。 (6) After rinsing the prepared SF/TK nanoparticles loaded with camptothecin, carry out the particle size test before and after oxygen cracking, the particle size of SF/TK drug-loaded nanoparticles increases with the treatment time of 50mM KO solution It showed a decreasing trend. After 16 hours of treatment, it decreased from the initial 532nm to 315nm, that is, the TK in the SF/TK nanoparticles encountered an oxygen environment, and the chemical bonds were broken and released, destroying the nanoparticles from the inside. Shrinkage, and cleavage lead to the fragmentation of nanoparticles, indicating that the SF/TK drug-loaded nanoparticles can be cleaved in an oxygen environment.
(7)将制备得到的包载喜树碱的SF/TK纳米粒子在氧处理环境中观察其药物释放行为，在氧环境中，SF/TK载药纳米粒子释药较快，尤其是前12小时，随后释放有所平缓。氧处理8小时，SF/TK载药纳米粒子的累计释药率达到59％左右，48小时后，SF/TK载药纳米粒子的累积释药率约为81％，显著高于对比例的释药率。SF/TK载药纳米粒子在非氧环境中48小时药物释放量远小于其在氧环境中的释放量。即SF/TK纳米粒子在氧环境中因TK响应断裂，使得SF/TK纳米粒子能快速释放药物，具有显著的ROS响应性能。(7) Observe the drug release behavior of the prepared SF/TK nanoparticles loaded with camptothecin in the oxygen treatment environment. In the oxygen environment, the SF/TK drug-loaded nanoparticles released faster, especially the first 12 hours, and then the release leveled off. After oxygen treatment for 8 hours, the cumulative drug release rate of SF/TK drug-loaded nanoparticles reached about 59%. After 48 hours, the cumulative drug release rate of SF/TK drug-loaded nanoparticles was about 81%, which was significantly higher than that of the comparative example. drug rate. The drug release amount of SF/TK drug-loaded nanoparticles in non-oxygen environment for 48 hours is much smaller than that in oxygen environment. That is, the SF/TK nanoparticles are fractured due to the TK response in an oxygen environment, so that the SF/TK nanoparticles can release drugs rapidly and have significant ROS responsive properties.
对比例1Comparative example 1
(1)称取一定量的生丝，按浴比1:50(w:v)量取去离子水，往冷水中加入Na 2CO 3(wt 0.06％)搅拌均匀，待水煮沸后加入生丝脱胶，30分钟后取出搓洗，放入锅中再次煮沸脱胶，重复3次。将脱胶后的丝置于电热恒温干燥箱中，60℃烘干，扯至蓬松，得到脱胶后的家蚕丝素纤维。 (1) Weigh a certain amount of raw silk, measure deionized water according to the bath ratio of 1:50 (w:v), add Na 2 CO 3 (wt 0.06%) into the cold water and stir evenly, after the water is boiled, add raw silk to degumming , After 30 minutes, take it out and scrub it, put it in a pot and boil it again for degumming, repeat 3 times. The degummed silk is placed in an electric heating constant temperature drying oven, dried at 60° C., and pulled until fluffy to obtain the degummed silkworm silk fibroin fiber.
(2)称取一定量的脱胶丝素纤维，剪碎放入9.3M的溴化锂溶液中65±2℃加热搅拌1小时得到家蚕SF溶解液。将家蚕SF溶解液灌注于透析袋中置于盛有去离子水的容器内透析得到纯化后的家蚕SF水溶液，调整透析后的SF水溶液浓度为10～200mg/mL。(2) Weigh a certain amount of degummed silk fibroin fiber, cut it into pieces, put it into a 9.3 M lithium bromide solution, heat and stir at 65±2° C. for 1 hour to obtain silkworm SF solution. The silkworm SF solution was poured into a dialysis bag, placed in a container filled with deionized water and dialyzed to obtain a purified silkworm SF aqueous solution, and the concentration of the dialyzed SF aqueous solution was adjusted to 10-200 mg/mL.
(3)与上述实施例同比例向SF水溶液中加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶，随后在4℃水浴中反应10分钟，再在室温下搅拌反应1小时。向上述SF反应液中加入终浓度0.5mg/mL抗肿瘤药物喜树碱，再按体积比加入无水乙醇(V SF:V 乙醇＝5:1)，然后加入2％聚乙烯醇溶液，体积V 聚乙烯醇＝2(V SF+V 乙醇)，搅拌混合均匀，放置于-20℃冰箱冷冻48小时后取出。室温下溶解、洗涤、离心，将离心管底端的沉淀物-80℃冷冻3小时，然后冷冻干燥12小时，得到载药SF纳米粒子。 (3) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to the SF aqueous solution in the same proportion as in the above example, and then in a water bath at 4°C The reaction was carried out for 10 minutes, and the reaction was stirred at room temperature for 1 hour. Add final concentration 0.5mg/mL antineoplastic drug camptothecin to above-mentioned SF reaction liquid, then add absolute ethanol (V SF :V ethanol =5:1) by volume ratio, then add 2% polyvinyl alcohol solution, volume V polyvinyl alcohol = 2 (V SF + V ethanol ), stir and mix evenly, place in -20°C refrigerator for 48 hours and take out. Dissolving, washing and centrifuging at room temperature, freezing the precipitate at the bottom of the centrifuge tube at -80°C for 3 hours, and then freeze-drying for 12 hours to obtain drug-loaded SF nanoparticles.
(4)制备得到的包载喜树碱的纯SF纳米粒子的载药能力与SF/TK纳米粒子相当。将制备得到的包载喜树碱的SF纳米粒子漂洗后进行氧裂解前后的粒径测试，SF载药纳米粒子的粒径随着50mM的KO 2溶液处理时间的增加呈先增加后稍有减小的趋势，SF载药纳米粒子的初始平均粒径为325nm，随着氧处理的时间增加到2小时，SF载药纳米粒子的粒径因吸水溶胀增加到400nm，氧处理16小时后减小到370nm，仍然大于原始颗粒的粒径，说明SF载药纳米粒子没有发生明显的裂解。 (4) The drug-loading ability of the prepared pure SF nanoparticles loaded with camptothecin is equivalent to that of SF/TK nanoparticles. After rinsing the prepared SF nanoparticles loaded with camptothecin, the particle size test before and after oxygen cracking is carried out. The particle size of SF drug-loaded nanoparticles increases first and then decreases slightly with the increase of 50mM KO solution treatment time. Small trend, the initial average particle size of SF drug-loaded nanoparticles is 325nm, as the time of oxygen treatment increases to 2 hours, the particle size of SF drug-loaded nanoparticles increases to 400nm due to water absorption, and decreases after 16 hours of oxygen treatment To 370nm, it is still larger than the particle size of the original particles, indicating that the SF drug-loaded nanoparticles have not undergone significant cracking.
(5)将制备得到的包载喜树碱的SF纳米粒子在氧处理环境中观察其药物释放行为，在氧环境中，SF载药纳米粒子释药较缓慢，48小时后，SF载药纳米粒子的累积释药率小于40％。SF载药纳米粒子在非氧环境中48小时药物释放量接近于其在氧环境中的释放量，说明纯SF载药纳米粒子没有明显的ROS响应性能。(5) Observe the drug release behavior of the prepared SF nanoparticles loaded with camptothecin in the oxygen treatment environment. In the oxygen environment, the drug release of the SF drug-loaded nanoparticles is slow. The cumulative drug release rate of the particles is less than 40%. The drug release of SF drug-loaded nanoparticles in non-oxygen environment for 48 hours was close to that in oxygen environment, indicating that pure SF drug-loaded nanoparticles had no obvious ROS response performance.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (9)

  1. 一种功能丝素蛋白药物载体的制备方法，其特征在于，包括：A method for preparing a functional silk fibroin drug carrier, characterized in that it comprises:
    A)将家蚕蚕丝脱胶、溶解、透析、过滤、浓缩得到丝素蛋白溶解液；A) degumming, dissolving, dialysis, filtering and concentrating silkworm silk to obtain a silk fibroin solution;
    B)将两端带有羧基的酮缩硫醇用交联剂活化，再与SF溶解液混合反应，得到反应后的溶液；B) activating the thioketal ketal with carboxyl groups at both ends with a crosslinking agent, and then mixing and reacting with the SF solution to obtain a reacted solution;
    C)将反应后的溶液加入无水乙醇和聚乙烯醇的混合体系搅拌、冷冻、解冻、离心，最后将沉淀物冷冻干燥即得。C) adding the reacted solution into a mixed system of absolute ethanol and polyvinyl alcohol, stirring, freezing, thawing, centrifuging, and finally freeze-drying the precipitate to obtain the product.
  2. 根据权利要求1所述的制备方法，其特征在于，步骤A)所述脱胶为采用碳酸钠或碳酸氢钠进行脱胶；所述溶解为采用溴化锂进行溶解；所述透析的截留分子量为10～50kDa；所述丝素蛋白溶解液的浓度为10～200mg/mL。The preparation method according to claim 1, characterized in that, the degumming in step A) is degumming by using sodium carbonate or sodium bicarbonate; the dissolving is dissolving by using lithium bromide; the molecular weight cut-off of the dialysis is 10～50kDa ; The concentration of the silk fibroin solution is 10-200 mg/mL.
  3. 根据权利要求1所述的制备方法，其特征在于，步骤B)所述两端带有羧基的酮缩硫醇的制备方法具体为：preparation method according to claim 1, is characterized in that, step B) the preparation method of the thioketal with carboxyl at both ends is specifically:
    3-巯基丙酸和无水丙酮的混合物在酸性条件下反应，制备的晶体经洗涤、离心、冻干获得两端带有羧基的TK；The mixture of 3-mercaptopropionic acid and anhydrous acetone reacts under acidic conditions, and the prepared crystals are washed, centrifuged, and freeze-dried to obtain TK with carboxyl groups at both ends;
    所述3-巯基丙酸和无水丙酮的摩尔比为1：1.5～2.5；所述酸性条件为盐酸；所述盐酸的浓度为7～8M；所述反应具体为20～30℃反应4～8小时；所述冻干具体为：-20～-80℃预冷冻2～6小时后冻干8～16小时。The molar ratio of the 3-mercaptopropionic acid to anhydrous acetone is 1:1.5-2.5; the acidic condition is hydrochloric acid; the concentration of the hydrochloric acid is 7-8M; 8 hours; the freeze-drying specifically includes: pre-freezing at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
  4. 根据权利要求1所述的制备方法，其特征在于，步骤B)所述丝素蛋白和两端带有羧基的酮缩硫醇质量比为100:0.5～20；所述两端带有羧基的酮缩硫醇的溶解溶剂为水。The preparation method according to claim 1, characterized in that, in step B), the mass ratio of the silk fibroin to the thioketal with carboxyl groups at both ends is 100:0.5-20; the silk fibroin with carboxyl groups at both ends The dissolving solvent of thioketal is water.
  5. 根据权利要求1所述的制备方法，其特征在于，步骤B)所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶；所述交联剂的添加量为两端带有羧基的酮缩硫醇的2～2.5倍；The preparation method according to claim 1, characterized in that the crosslinking agent in step B) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-bis methylaminopyridine; the added amount of the crosslinking agent is 2 to 2.5 times that of the ketal ketal with carboxyl groups at both ends;
    所述活化反应温度为4～6℃；The activation reaction temperature is 4-6°C;
    所述反应温度为20～30℃，所述反应时间为10～60min。The reaction temperature is 20-30° C., and the reaction time is 10-60 minutes.
  6. 根据权利要求1所述的制备方法，其特征在于，步骤C)所述无水乙醇的浓度为0.5～1.5M；所述聚乙烯醇的浓度为5～60mg/mL，聚乙烯 醇的分子量为3～7万；The preparation method according to claim 1, characterized in that, in step C), the concentration of the dehydrated alcohol is 0.5-1.5M; the concentration of the polyvinyl alcohol is 5-60mg/mL, and the molecular weight of the polyvinyl alcohol is 30,000 to 70,000;
    所述冷冻具体为：-20℃冷冻8～48h；所述解冻为20～30℃解冻；The freezing is specifically: freezing at -20°C for 8-48 hours; the thawing is thawing at 20-30°C;
    所述离心为13000rpm离心；The centrifugal is 13000rpm centrifugal;
    所述沉淀物冷冻干燥具体为：将沉淀物-20～-80℃预冷冻2～6h后冻干8～16h。The freeze-drying of the precipitate specifically includes: pre-freezing the precipitate at -20 to -80° C. for 2 to 6 hours, and then freeze-drying for 8 to 16 hours.
  7. 一种功能丝素蛋白药物载体，其特征在于，由权利要求1～6任意一项所述的制备方法制备得到。A functional silk fibroin drug carrier, characterized in that it is prepared by the preparation method described in any one of claims 1-6.
  8. 权利要求1～6任意一项所述的制备方法制备得到的功能丝素蛋白药物载体在担载抗肿瘤/抗炎药物中的应用。Application of the functional silk fibroin drug carrier prepared by the preparation method described in any one of claims 1 to 6 in loading anti-tumor/anti-inflammatory drugs.
  9. 一种载药纳米粒子，包括药物和权利要求1～6任意一项所述的制备方法制备得到的功能丝素蛋白药物载体。A drug-loaded nanoparticle, comprising a drug and a functional silk fibroin drug carrier prepared by the preparation method described in any one of claims 1-6.
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