WO2023104058A1 - Macrophage-based living cell drug-loading system, preparation method therefor and use thereof - Google Patents

Macrophage-based living cell drug-loading system, preparation method therefor and use thereof Download PDF

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WO2023104058A1
WO2023104058A1 PCT/CN2022/137029 CN2022137029W WO2023104058A1 WO 2023104058 A1 WO2023104058 A1 WO 2023104058A1 CN 2022137029 W CN2022137029 W CN 2022137029W WO 2023104058 A1 WO2023104058 A1 WO 2023104058A1
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drug
yeast
loaded
macrophages
macrophage
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Chinese (zh)
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蔡林涛
张保珍
郑明彬
潘宏
唐晓帆
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of drug carriers, and in particular relates to a macrophage-based living cell drug delivery system and its preparation method and application.
  • Macrophages are the most plastic cells in the hematopoietic system, present in all tissues and display enormous functional diversity. They play important roles in reproductive development, maintenance of homeostasis, tissue modification and immune regulation. Abnormal macrophages are closely related to many related diseases caused by inflammation, including tumors, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative diseases, arthritis, gastric ulcer, colitis, diabetes, etc. . It has become an important therapeutic target for inflammation-related diseases.
  • the macrophages in the body are extracted and loaded with drugs and reinfused, and the extracted macrophages need to be subjected to steps such as in vitro expansion, drug loading, and cell sorting, and the process is complicated; using targeted modification methods, antibodies, etc.
  • Modified on the surface of nanoparticles, intravenous or oral administration needs to overcome many physiological barriers, including mucus layer barrier, intestinal barrier, gastrointestinal emptying, blood viscosity, protein corona formation, etc., which seriously affect the absorption and efficacy of drugs .
  • micro-nano robots can actively penetrate physiological barriers and actively avoid obstacles due to their motion characteristics, which embodies important application value in the field of drug delivery. How to use micro-nano bio-robot technology to realize targeted drug loading or gene editing of macrophages to treat inflammation-related diseases is an urgent problem to be solved.
  • the living cell drug delivery system based on macrophages includes macrophages and drug-loaded yeast biomimetic micromotors inside the macrophages.
  • the drug-loaded yeast bionic micromotor has a self-driving function and can move actively. After reaching the environment of macrophages, it can be endocytized by macrophages, thus forming a living cell drug-loading system based on macrophages.
  • a living cell drug delivery system based on macrophages comprising living cells and drug-loaded yeast biomimetic micromotors inside the living cells, wherein the living cells are macrophages, and the drug-loaded yeast biomimetic micromotors are It includes yeast cell wall, drug-loaded nanoparticles inside the yeast cell wall and a compound enzyme structure half-surface modified on the yeast cell wall, and the compound enzyme structure is a Yin-Yang structure composed of glucose oxidase and catalase.
  • the purpose of designing a yin-yang structure is to make the driving force on the surface asymmetrical so that it can move.
  • Glucose oxidase oxidizes glucose to gluconic acid and hydrogen peroxide, and catalase further oxidizes hydrogen peroxide to water and oxygen.
  • catalase further oxidizes hydrogen peroxide to water and oxygen.
  • it can decompose toxic hydrogen peroxide, and on the other hand, the cascade reaction produces a greater driving force.
  • the main component of the yeast cell wall is ⁇ -(1,3)-D-glucan.
  • the macrophages are M1 or M2 macrophages.
  • the drug-loaded yeast bionic micromotor generates power by decomposing glucose, and the glucose concentration is greater than or equal to 8 mM.
  • the drug loaded on the drug-loaded nanoparticles is one or more of small molecule drugs, polypeptides, macromolecular protein drugs and gene drugs.
  • the present invention also provides a preparation method of the above macrophage-based living cell drug delivery system, comprising the following steps:
  • the drug-loaded yeast biomimetic micromotor is introduced into an environment with macrophages, and the macrophages endocytose the drug-loaded yeast biomimetic micromotor to form a living cell drug delivery system based on macrophages.
  • the "preparation of drug-loaded yeast biomimetic micromotor” specifically includes the following steps:
  • Yeast cells are extracted by acid-base method or enzyme digestion method
  • step (3) Spread the drug-loaded yeast cell wall microcapsules prepared in step (2) in a container, and then add an activator to perform surface activation to obtain surface-activated drug-loaded yeast cell walls;
  • step (3) Co-incubating glucose oxidase and catalase with the surface-activated drug-loaded yeast cell wall prepared in step (3), and after purification, the drug-loaded yeast biomimetic micromotor is obtained.
  • step (3) the container is a petri dish, and the container is coated with polylysine.
  • step (4) the mass ratio of catalase to glucose oxidase is 1:(2-5).
  • the present invention also provides the application of the above macrophage-based living cell drug delivery system in the preparation of drugs for targeted treatment of macrophage diseases.
  • macrophage diseases include but are not limited to the following: tumor, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative disease, arthritis, gastric ulcer, colitis, One or more of diabetes.
  • the drug-loaded yeast bionic micromotor is self-driven and can self-assemble with macrophages to form a living cell drug-loading system. It is safe, simple and convenient, and does not require complicated steps such as extraction of macrophages, cultivation, amplification, drug loading, and sorting. ;
  • Yeast bionic micro-nano motors can actively penetrate the intestinal barrier and other biological tissue barriers, greatly improving the drug delivery efficiency;
  • Yeast bionic micro-nano motors have specific macrophage targeting, and can treat diseases caused by macrophages more efficiently.
  • Fig. 1 is the structural schematic diagram of macrophage living cell drug
  • Fig. 2 is a schematic diagram of the preparation process of drug-loaded yeast biomimetic micromotor
  • Figure 3 is a fluorescent imaging diagram of glucose oxidase and catalase on the drug-loaded yeast biomimetic micromotor
  • Fig. 4 is the fluorescence imaging picture of macrophage living cell drug
  • Figure 5 is the distribution of drugs in the intestines of mice after oral administration of Free Cur, CP@Y and CP@Y-robot respectively;
  • Figure 6 is a comparison chart of the therapeutic effects of PBS, Free Cur, CP@Y and CP@Y-robot drugs on colitis in mice, in which Figure 6 (1) is the control group orally administered with PBS, and Figure 6 (2) Oral free curcumin group, Figure 6 (3) is oral administration of yeast microcapsules loaded with curcumin nanoparticles group, Figure 6 (4) is oral administration of curcumin nanoparticles loaded yeast bionic micromotor group.
  • the present invention provides a living cell drug delivery system based on macrophages, comprising living cells and drug-loaded yeast biomimetic micromotors inside the living cells, wherein the living cells are macrophages, and the drug-loading
  • the yeast biomimetic micromotor includes a yeast cell wall with a cystic structure, drug-loaded nanoparticles located inside the yeast cell wall, and a composite enzyme structure half-surface modified on the yeast cell wall.
  • the composite enzyme structure is composed of glucose oxidase and catalase Yin and Yang structure.
  • the yeast cell wall with a cystic structure can provide a good accommodation space for the drug-loaded nanoparticles, and has a regular cell wall surface, which is easy to carry out half-surface modification of biological enzymes.
  • the macrophages can be cultured in vitro, or naturally exist in vivo.
  • macrophages naturally existing in vivo which may be of human or mouse origin, etc., can be used.
  • it may be an M1 macrophage or an M2 macrophage.
  • the main component of the yeast cell wall is ⁇ -(1,3)-D-glucan.
  • the drug-loaded yeast bionic micromotor generates power by decomposing glucose, and the glucose concentration is greater than or equal to 8 mM.
  • the drug loaded on the drug-loaded nanoparticles is one or more of small molecule drugs, polypeptides, macromolecular protein drugs and gene drugs.
  • the present invention also provides a preparation method of the above macrophage-based living cell drug delivery system, comprising the following steps:
  • the drug-loaded yeast biomimetic micromotor is introduced into an environment with macrophages, and the macrophages endocytose the drug-loaded yeast biomimetic micromotor to form a living cell drug delivery system based on macrophages.
  • the "preparation of drug-loaded yeast biomimetic micromotor” specifically includes the following steps:
  • Yeast cells are extracted by acid-base method or enzyme digestion method
  • step (3) Spread the drug-loaded yeast cell wall microcapsules prepared in step (2) in a container, and then add an activator to perform surface activation to obtain surface-activated drug-loaded yeast cell walls;
  • step (3) Co-incubating glucose oxidase and catalase with the surface-activated drug-loaded yeast cell wall prepared in step (3) to obtain the drug-loaded yeast biomimetic micromotor.
  • step (3) the container is a petri dish, and the container is coated with polylysine.
  • step (4) the mass ratio of catalase to glucose oxidase is 1:(2-5).
  • the following steps are further included: wrapping the drug-carrying yeast biomimetic micromotor with an acrylic resin casing to obtain an oral drug-carrying micromotor.
  • Medicinal yeast biomimetic micromotor Encapsulated by the casing, the drug-loaded yeast biomimetic micromotor can enter the target area such as the stomach and intestines.
  • the oral drug-loaded yeast biomimetic micromotor after the oral drug-loaded yeast biomimetic micromotor enters the body, it can form macrophage living cell drugs in the intestinal Peyer's junction, that is, form a macrophage-based living cell drug delivery system.
  • the present invention also provides the application of the above macrophage-based living cell drug delivery system in the preparation of drugs for targeted treatment of macrophage diseases.
  • macrophage diseases include but are not limited to the following: tumor, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative disease, arthritis, gastric ulcer, colitis, One or more of diabetes.
  • a preparation method for forming a living cell drug delivery system based on macrophages in vivo :
  • Yeast cell wall extraction Dissolve 500mg of Saccharomyces cerevisiae in 10mL of 1M NaOH solution, incubate at 80°C for 1h, then centrifuge at 3000rpm for 10min, discard the supernatant, wash twice with double distilled water, then add 10mL of 1M HCl , incubate at 60°C for 1 hour, then centrifuge at 3000rpm and wash twice with water, add 50mL isopropanol to wash four times, and wash twice with 50mL acetone, centrifuge to discard the supernatant, and dry to obtain empty negatively charged yeast cell walls.
  • Plating add 10 mg of the aqueous solution of drug-loaded yeast cell wall microcapsules prepared in (3) to a 6 cm petri dish. After standing still for 1-2 hours, absorb the supernatant, wash with double distilled water for 3 times, and put it in a 37°C oven to dry for later use.
  • Ligation Add 1-3 mg catalase and 3-9 mg glucose oxidase to (6), react at 4°C overnight, or incubate at room temperature for 4-6 hours.
  • macrophage living cell drug is formed in the intestinal Peyer's junction, that is, a macrophage-based living cell drug delivery system.
  • Fig. 1 is a schematic diagram of the structure of a macrophage living cell drug, wherein, 1 is a macrophage, and 2 is a drug-loaded yeast biomimetic micromotor.
  • Figure 2 is a schematic diagram of the preparation process of the drug-loaded yeast bionic micromotor. First, the yeast cell A is treated with acid and alkali to extract its cell wall, and then the drug-loaded nanoparticles are introduced into the yeast cell wall by electrostatic adsorption to form a drug-loaded yeast cell wall microcapsule C. .
  • the drug-loaded yeast cell wall microcapsules C were subjected to operation D, that is, tiled activation, and then ultrasonically dispersed (operation E), to obtain half-surface activated drug-loaded yeast cell wall microcapsules F.
  • the drug-loaded yeast cell wall microcapsule F was reacted with glucose oxidase G and catalase H to obtain yeast biomimetic micromotor I, which was wrapped with casing (operation J) to obtain an oral yeast biomimetic micromotor.
  • Figure 3 is the fluorescence imaging diagram of drug-loaded yeast biomimetic micromotor, in which Figure 3 (1) is the fluorescence imaging diagram of glucose oxidase, and Figure 3 (2) is the fluorescence imaging diagram of catalase, Fig. 3 (3) is the simultaneous fluorescence imaging of glucose oxidase and catalase. It can be seen that the half surface of the yeast cell wall microcapsule is modified with glucose oxidase and catalase.
  • Figure 4 is the fluorescence imaging image of macrophage living cell drug, in which Figure 4 (1) is the yeast biomimetic micromotor loaded with curcumin, Figure 4 (2) is the Dil-labeled macrophage, and Figure 4 (3) is DAPI-stained yeast cell nuclei, Figure 4 (4) is the simultaneous fluorescence imaging of curcumin-loaded yeast biomimetic micromotors, macrophages, and yeast cell nuclei.
  • Figure 5 shows the drug distribution diagram. After oral administration of Free Cur, CP@Y and CP@Y-robot, the mice were euthanized, and the intestinal tract was taken out for small animal imaging to observe the retention of curcumin in the intestinal tract.
  • Figure 5 (1) is the fluorescence imaging image after oral administration of Free Cur drug
  • Figure 5 (2) is the fluorescence imaging image after oral administration of CP@Y drug
  • Figure 5 (3) is the fluorescence imaging image after oral administration of CP@Y-robot drug Fluorescence image.
  • Figure 6 is a graph of the treatment effect after the 10th day. After injecting the inflammatory probe (L-012), small animal imaging was performed, the stronger the fluorescence, the more severe the inflammation. It can be seen from the results that the control group (see Figure 6 (1)) had strong fluorescence, indicating that the colitis model was successfully established.
  • the drug-loaded yeast bionic micromotor is self-driven, and can self-assemble with macrophages into a living cell drug-loading system to form a living cell drug in the body, which is safe, simple and convenient, and does not require complicated extraction, cultivation, and expansion of macrophages , drug loading, sorting and other steps;
  • Yeast bionic micro-nano motors can actively penetrate the intestinal barrier and other biological tissue barriers, greatly improving the drug delivery efficiency;
  • Yeast bionic micro-nano motors have specific macrophage targeting, and can treat diseases caused by macrophages more efficiently.

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Abstract

The present invention provides a macrophage-based living cell drug-loading system, comprising living cells and drug-loading yeast bionic micro-motors located inside the living cells, wherein the living cells are macrophages, and each drug-loading yeast bionic micro-motor comprises a yeast cell wall, drug-loaded nanoparticles located inside the yeast cell wall, and glucose oxidase and catalase half-surface modified on the yeast cell wall. In vivo self-assembled living cell drugs refer to that the drug-loaded yeast bionic micro-motors actively penetrate the intestinal wall after entering a human body, enter the lymphatic system in the intestine and are endocytosed by the macrophages therein, so that the drug-loaded yeast bionic micro-motors and the macrophages are self-assembled in vivo into macrophage living cell drugs, thereby achieving targeting transport of drugs.

Description

一种基于巨噬细胞的活细胞载药系统及其制备方法与应用A living cell drug delivery system based on macrophages and its preparation method and application 技术领域technical field
本发明属于药物载体技术领域，具体涉及一种基于巨噬细胞的活细胞载药系统及其制备方法与应用。The invention belongs to the technical field of drug carriers, and in particular relates to a macrophage-based living cell drug delivery system and its preparation method and application.
背景技术Background technique
巨噬细胞是造血系统中最具可塑性的细胞，存在于所有组织中并显示出巨大的功能多样性。它们在生殖发育、维持稳态、组织修饰和免疫调节中发挥重要作用。巨噬细胞异常与许多炎症引起的相关疾病，包括肿瘤、肥胖、动脉粥样硬化、骨质疏松、骨硬化病、肝炎、神经退行性疾病、关节炎、胃溃疡、结肠炎、糖尿病等密切相关。其已成为炎症相关疾病的重要治疗靶点。近几年，依赖于巨噬细胞在炎症部位的募集作用，研究者通过将小鼠体内巨噬细胞提取载药后，再重新输入体内，或利用葡聚糖、壳聚糖、甘露糖等靶向巨噬细胞的特性，将其修饰于药物载体表面，成功将氧化铁纳米粒、siRNA、和小分子药物递送至炎症病灶，同时在靶向巨噬细胞给药在炎症相关疾病的成像方面也表现出巨大的应用前景。Macrophages are the most plastic cells in the hematopoietic system, present in all tissues and display enormous functional diversity. They play important roles in reproductive development, maintenance of homeostasis, tissue modification and immune regulation. Abnormal macrophages are closely related to many related diseases caused by inflammation, including tumors, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative diseases, arthritis, gastric ulcer, colitis, diabetes, etc. . It has become an important therapeutic target for inflammation-related diseases. In recent years, relying on the recruitment of macrophages in inflammatory sites, researchers have extracted and loaded macrophages in mice, and then reinjected them into the body, or used glucan, chitosan, mannose and other target The characteristics of targeting macrophages were modified on the surface of drug carriers, and iron oxide nanoparticles, siRNA, and small molecule drugs were successfully delivered to inflammatory lesions. Show great application prospects.
现有技术中，将体内巨噬细胞提取载药并回输，需要对提取的巨噬细胞进行体外扩增、药物负载、细胞分选等步骤，工艺复杂；利用靶向修饰方法，将抗体等修饰于纳米粒表面，静脉给药或口服给药均需克服众多生理屏障，包括黏液层屏障、肠屏障、胃肠排空、血液粘滞性、蛋白冠形成等，严重影响药物的吸收和疗效。随着微纳技术的发展，微纳机器人因运动特性，可主动穿透生理屏障，主动规避障碍，其在药物递送领域体现了重要的应用价值。如何利用微纳生物机器人技术实现巨噬细胞靶向载药或基因编辑，从而治疗炎症相关疾病是亟待解决的难题。In the prior art, the macrophages in the body are extracted and loaded with drugs and reinfused, and the extracted macrophages need to be subjected to steps such as in vitro expansion, drug loading, and cell sorting, and the process is complicated; using targeted modification methods, antibodies, etc. Modified on the surface of nanoparticles, intravenous or oral administration needs to overcome many physiological barriers, including mucus layer barrier, intestinal barrier, gastrointestinal emptying, blood viscosity, protein corona formation, etc., which seriously affect the absorption and efficacy of drugs . With the development of micro-nano technology, micro-nano robots can actively penetrate physiological barriers and actively avoid obstacles due to their motion characteristics, which embodies important application value in the field of drug delivery. How to use micro-nano bio-robot technology to realize targeted drug loading or gene editing of macrophages to treat inflammation-related diseases is an urgent problem to be solved.
技术问题technical problem
为了克服上述现有技术的缺陷，本发明提出了一种基于巨噬细胞的活细胞载药系统及其制备方法和应用。所述基于巨噬细胞的活细胞载药系统包括巨噬细胞和位于所述巨噬细胞内部的载药酵母仿生微马达。其中，载药酵母仿生微马达具有自驱动功能，能够主动运动，到达巨噬细胞的所处环境后可被巨噬细胞内吞，从而形成基于巨噬细胞的活细胞载药系统。In order to overcome the above-mentioned defects of the prior art, the present invention proposes a macrophage-based living cell drug delivery system and its preparation method and application. The living cell drug delivery system based on macrophages includes macrophages and drug-loaded yeast biomimetic micromotors inside the macrophages. Among them, the drug-loaded yeast bionic micromotor has a self-driving function and can move actively. After reaching the environment of macrophages, it can be endocytized by macrophages, thus forming a living cell drug-loading system based on macrophages.
技术解决方案technical solution
一种基于巨噬细胞的活细胞载药系统，包括活细胞和位于所述活细胞内部的载药酵母仿生微马达，其中，所述活细胞为巨噬细胞，所述载药酵母仿生微马达包括酵母细胞壁、位于酵母细胞壁内部的载药纳米粒和半面修饰于所述酵母细胞壁的复合酶结构，所述复合酶结构为葡萄糖氧化酶和过氧化氢酶构成的阴阳结构。A living cell drug delivery system based on macrophages, comprising living cells and drug-loaded yeast biomimetic micromotors inside the living cells, wherein the living cells are macrophages, and the drug-loaded yeast biomimetic micromotors are It includes yeast cell wall, drug-loaded nanoparticles inside the yeast cell wall and a compound enzyme structure half-surface modified on the yeast cell wall, and the compound enzyme structure is a Yin-Yang structure composed of glucose oxidase and catalase.
设计成阴阳结构的目的在于使表面的推动力不对称，这样才能运动。葡萄糖氧化酶将葡萄糖氧化为葡萄糖酸和双氧水，过氧化氢酶进一步将双氧水氧化为水和氧气。一方面可以分解有毒的双氧水，另一方面级联反应，产生更大的推动力。The purpose of designing a yin-yang structure is to make the driving force on the surface asymmetrical so that it can move. Glucose oxidase oxidizes glucose to gluconic acid and hydrogen peroxide, and catalase further oxidizes hydrogen peroxide to water and oxygen. On the one hand, it can decompose toxic hydrogen peroxide, and on the other hand, the cascade reaction produces a greater driving force.
进一步地，所述酵母细胞壁主要成分为β-(1,3)-D-葡聚糖。Further, the main component of the yeast cell wall is β-(1,3)-D-glucan.
进一步地，所述巨噬细胞为M1型或M2型巨噬细胞。Further, the macrophages are M1 or M2 macrophages.
所述载药酵母仿生微马达通过分解葡萄糖产生动力，所述葡萄糖浓度大于等于8 mM。The drug-loaded yeast bionic micromotor generates power by decomposing glucose, and the glucose concentration is greater than or equal to 8 mM.
进一步地，所述载药纳米粒所载药物为小分子药物、多肽、大分子蛋白药物和基因药物中的一种或多种。Further, the drug loaded on the drug-loaded nanoparticles is one or more of small molecule drugs, polypeptides, macromolecular protein drugs and gene drugs.
本发明还提供上述基于巨噬细胞的活细胞载药系统的制备方法，包括如下步骤：The present invention also provides a preparation method of the above macrophage-based living cell drug delivery system, comprising the following steps:
（1）制备载药酵母仿生微马达；(1) Preparation of drug-loaded yeast biomimetic micromotor;
（2）将载药酵母仿生微马达进入具有巨噬细胞的环境，巨噬细胞将所述载药酵母仿生微马达内吞，形成基于巨噬细胞的活细胞载药系统。(2) The drug-loaded yeast biomimetic micromotor is introduced into an environment with macrophages, and the macrophages endocytose the drug-loaded yeast biomimetic micromotor to form a living cell drug delivery system based on macrophages.
进一步地，所述“制备载药酵母仿生微马达”具体包括以下步骤：Further, the "preparation of drug-loaded yeast biomimetic micromotor" specifically includes the following steps:
（1）将酵母细胞利用酸碱法或酶消化法提取酵母细胞壁；(1) Yeast cells are extracted by acid-base method or enzyme digestion method;
（2）将载药纳米粒通过静电沉积吸附进入所述酵母细胞壁内，制得载药酵母细胞壁微囊；(2) Adsorbing the drug-loaded nanoparticles into the yeast cell wall through electrostatic deposition to prepare drug-loaded yeast cell wall microcapsules;
（3）将步骤（2）制备的载药酵母细胞壁微囊平铺在容器中，之后加入活化剂，进行表面活化，得到表面活化载药酵母细胞壁；(3) Spread the drug-loaded yeast cell wall microcapsules prepared in step (2) in a container, and then add an activator to perform surface activation to obtain surface-activated drug-loaded yeast cell walls;
（4）将葡萄糖氧化酶和过氧化氢酶与步骤（3）制备的表面活化载药酵母细胞壁共孵育，纯化后，得到载药酵母仿生微马达。(4) Co-incubating glucose oxidase and catalase with the surface-activated drug-loaded yeast cell wall prepared in step (3), and after purification, the drug-loaded yeast biomimetic micromotor is obtained.
进一步地，步骤（3）中，所述容器为平皿，所述容器经过多聚赖氨酸包被处理。Further, in step (3), the container is a petri dish, and the container is coated with polylysine.
进一步地，步骤（4）中，过氧化氢酶与葡萄糖氧化酶的质量比为1:（2-5）。Further, in step (4), the mass ratio of catalase to glucose oxidase is 1:(2-5).
本发明还提供上述基于巨噬细胞的活细胞载药系统在制备靶向治疗巨噬细胞疾病药物中的应用。The present invention also provides the application of the above macrophage-based living cell drug delivery system in the preparation of drugs for targeted treatment of macrophage diseases.
进一步地，所述巨噬细胞疾病包括但不限于以下几种：肿瘤、肥胖、动脉粥样硬化、骨质疏松、骨硬化病、肝炎、神经退行性疾病、关节炎、胃溃疡、结肠炎、糖尿病中的一种或多种。Further, the macrophage diseases include but are not limited to the following: tumor, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative disease, arthritis, gastric ulcer, colitis, One or more of diabetes.
有益效果Beneficial effect
1. 载药酵母仿生微马达具有自驱动，可以与巨噬细胞自组装成活细胞载药系统，安全简单方便，不需要复杂的提取巨噬细胞、培养、扩增、载药、分选等步骤；1. The drug-loaded yeast bionic micromotor is self-driven and can self-assemble with macrophages to form a living cell drug-loading system. It is safe, simple and convenient, and does not require complicated steps such as extraction of macrophages, cultivation, amplification, drug loading, and sorting. ;
2. 酵母仿生微纳马达具有主动穿透肠屏障等生物组织障碍的作用，大大提高了药物的给药效率；2. Yeast bionic micro-nano motors can actively penetrate the intestinal barrier and other biological tissue barriers, greatly improving the drug delivery efficiency;
3. 酵母仿生微纳马达具有特异的巨噬细胞靶向性，更加高效治疗由巨噬细胞引起的疾病。3. Yeast bionic micro-nano motors have specific macrophage targeting, and can treat diseases caused by macrophages more efficiently.
附图说明Description of drawings
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.
图1为巨噬细胞活细胞药物的结构示意图；Fig. 1 is the structural schematic diagram of macrophage living cell drug;
图2为载药酵母仿生微马达的制备过程示意图；Fig. 2 is a schematic diagram of the preparation process of drug-loaded yeast biomimetic micromotor;
图3为对载药酵母仿生微马达上的葡萄糖氧化酶及过氧化氢酶的荧光成像图；Figure 3 is a fluorescent imaging diagram of glucose oxidase and catalase on the drug-loaded yeast biomimetic micromotor;
图4为巨噬细胞活细胞药物的荧光成像图；Fig. 4 is the fluorescence imaging picture of macrophage living cell drug;
图5为分别口服Free Cur、CP@Y和CP@Y-robot药物后小鼠肠道内的药物分布图；Figure 5 is the distribution of drugs in the intestines of mice after oral administration of Free Cur, CP@Y and CP@Y-robot respectively;
图6为分别用PBS、Free Cur、CP@Y和CP@Y-robot药物对小鼠结肠炎的治疗效果对比图，其中，图6（1）为口服PBS的对照组，图6（2）为口服游离姜黄素组，图6（3）为口服载姜黄素纳米粒酵母微囊组，图6（4）为口服载姜黄素纳米粒酵母仿生微马达组。Figure 6 is a comparison chart of the therapeutic effects of PBS, Free Cur, CP@Y and CP@Y-robot drugs on colitis in mice, in which Figure 6 (1) is the control group orally administered with PBS, and Figure 6 (2) Oral free curcumin group, Figure 6 (3) is oral administration of yeast microcapsules loaded with curcumin nanoparticles group, Figure 6 (4) is oral administration of curcumin nanoparticles loaded yeast bionic micromotor group.
本发明的实施方式Embodiments of the present invention
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
本发明提供了一种基于巨噬细胞的活细胞载药系统，包括活细胞和位于所述活细胞内部的载药酵母仿生微马达，其中，所述活细胞为巨噬细胞，所述载药酵母仿生微马达包括囊状结构的酵母细胞壁、位于酵母细胞壁内部的载药纳米粒和半面修饰于所述酵母细胞壁的复合酶结构，所述复合酶结构为葡萄糖氧化酶和过氧化氢酶构成的阴阳结构。The present invention provides a living cell drug delivery system based on macrophages, comprising living cells and drug-loaded yeast biomimetic micromotors inside the living cells, wherein the living cells are macrophages, and the drug-loading The yeast biomimetic micromotor includes a yeast cell wall with a cystic structure, drug-loaded nanoparticles located inside the yeast cell wall, and a composite enzyme structure half-surface modified on the yeast cell wall. The composite enzyme structure is composed of glucose oxidase and catalase Yin and Yang structure.
在本发明中，采用囊状结构的酵母细胞壁，能够为载药纳米粒提供良好的容纳空间，并且具有规整的细胞壁表面，容易进行生物酶的半面修饰。In the present invention, the yeast cell wall with a cystic structure can provide a good accommodation space for the drug-loaded nanoparticles, and has a regular cell wall surface, which is easy to carry out half-surface modification of biological enzymes.
所述巨噬细胞可以是体外培养，或是活体内自然存在。在本发明中，尤其可应用活体内自然存在的巨噬细胞，可为人源或鼠源等。具体可为M1型巨噬细胞或M2型巨噬细胞。The macrophages can be cultured in vitro, or naturally exist in vivo. In the present invention, in particular, macrophages naturally existing in vivo, which may be of human or mouse origin, etc., can be used. Specifically, it may be an M1 macrophage or an M2 macrophage.
进一步地，所述酵母细胞壁主要成分为β-(1,3)-D-葡聚糖。Further, the main component of the yeast cell wall is β-(1,3)-D-glucan.
所述载药酵母仿生微马达通过分解葡萄糖产生动力，所述葡萄糖浓度大于等于8 mM。The drug-loaded yeast bionic micromotor generates power by decomposing glucose, and the glucose concentration is greater than or equal to 8 mM.
在一些具体实施例中，载药纳米粒所载药物为小分子药物、多肽、大分子蛋白药物和基因药物中的一种或多种。In some specific embodiments, the drug loaded on the drug-loaded nanoparticles is one or more of small molecule drugs, polypeptides, macromolecular protein drugs and gene drugs.
本发明还提供上述基于巨噬细胞的活细胞载药系统的制备方法，包括如下步骤：The present invention also provides a preparation method of the above macrophage-based living cell drug delivery system, comprising the following steps:
（1）制备载药酵母仿生微马达；(1) Preparation of drug-loaded yeast biomimetic micromotor;
（2）将载药酵母仿生微马达进入具有巨噬细胞的环境，巨噬细胞将所述载药酵母仿生微马达内吞，形成基于巨噬细胞的活细胞载药系统。(2) The drug-loaded yeast biomimetic micromotor is introduced into an environment with macrophages, and the macrophages endocytose the drug-loaded yeast biomimetic micromotor to form a living cell drug delivery system based on macrophages.
进一步地，所述“制备载药酵母仿生微马达”具体包括以下步骤：Further, the "preparation of drug-loaded yeast biomimetic micromotor" specifically includes the following steps:
（1）将酵母细胞利用酸碱法或酶消化法提取酵母细胞壁；(1) Yeast cells are extracted by acid-base method or enzyme digestion method;
（2）将载药纳米粒通过静电沉积吸附进入所述酵母细胞壁内，制得载药酵母细胞壁微囊；(2) Adsorbing the drug-loaded nanoparticles into the yeast cell wall through electrostatic deposition to prepare drug-loaded yeast cell wall microcapsules;
（3）将步骤（2）制备的载药酵母细胞壁微囊平铺在容器中，之后加入活化剂，进行表面活化，得到表面活化载药酵母细胞壁；(3) Spread the drug-loaded yeast cell wall microcapsules prepared in step (2) in a container, and then add an activator to perform surface activation to obtain surface-activated drug-loaded yeast cell walls;
（4）将葡萄糖氧化酶和过氧化氢酶与步骤（3）制备的表面活化载药酵母细胞壁共孵育，得到载药酵母仿生微马达。(4) Co-incubating glucose oxidase and catalase with the surface-activated drug-loaded yeast cell wall prepared in step (3) to obtain the drug-loaded yeast biomimetic micromotor.
进一步地，步骤（3）中，所述容器为平皿，所述容器经过多聚赖氨酸包被处理。Further, in step (3), the container is a petri dish, and the container is coated with polylysine.
进一步地，步骤（4）中，过氧化氢酶与葡萄糖氧化酶的质量比为1:（2-5）。Further, in step (4), the mass ratio of catalase to glucose oxidase is 1:(2-5).
在本发明中，当所述基于巨噬细胞的活细胞载药系统是在活体内自组装时，还包括以下步骤：用丙烯酸树脂肠衣包裹所述载药酵母仿生微马达，得到可口服的载药酵母仿生微马达。通过肠衣包裹，可以使载药酵母仿生微马达能够进入肠胃等目标区域。In the present invention, when the macrophage-based living cell drug-carrying system is self-assembled in vivo, the following steps are further included: wrapping the drug-carrying yeast biomimetic micromotor with an acrylic resin casing to obtain an oral drug-carrying micromotor. Medicinal yeast biomimetic micromotor. Encapsulated by the casing, the drug-loaded yeast biomimetic micromotor can enter the target area such as the stomach and intestines.
具体的，所述可口服的载药酵母仿生微马达进入体内后，可在肠道派尔式结内形成巨噬细胞活细胞药物，即形成基于巨噬细胞的活细胞载药系统。Specifically, after the oral drug-loaded yeast biomimetic micromotor enters the body, it can form macrophage living cell drugs in the intestinal Peyer's junction, that is, form a macrophage-based living cell drug delivery system.
本发明还提供上述基于巨噬细胞的活细胞载药系统在制备靶向治疗巨噬细胞疾病药物中的应用。The present invention also provides the application of the above macrophage-based living cell drug delivery system in the preparation of drugs for targeted treatment of macrophage diseases.
进一步地，所述巨噬细胞疾病包括但不限于以下几种：肿瘤、肥胖、动脉粥样硬化、骨质疏松、骨硬化病、肝炎、神经退行性疾病、关节炎、胃溃疡、结肠炎、糖尿病中的一种或多种。Further, the macrophage diseases include but are not limited to the following: tumor, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative disease, arthritis, gastric ulcer, colitis, One or more of diabetes.
实施例Example
一种在活体内形成基于巨噬细胞的活细胞载药系统的制备方法：A preparation method for forming a living cell drug delivery system based on macrophages in vivo:
（1）提取酵母细胞壁：将500mg酿酒酵母溶解于10mL浓度为1M的NaOH溶液中，于80℃孵育1h，之后3000rpm离心10min，弃去上清，双蒸水洗涤2次后，加入10mL1M的HCl，60℃孵育1h，之后3000rpm离心水洗2次，再加入50mL异丙醇洗涤4次，用50mL丙酮洗涤2次，离心弃上清，干燥后得到空的带负电荷的酵母细胞壁。(1) Yeast cell wall extraction: Dissolve 500mg of Saccharomyces cerevisiae in 10mL of 1M NaOH solution, incubate at 80°C for 1h, then centrifuge at 3000rpm for 10min, discard the supernatant, wash twice with double distilled water, then add 10mL of 1M HCl , incubate at 60°C for 1 hour, then centrifuge at 3000rpm and wash twice with water, add 50mL isopropanol to wash four times, and wash twice with 50mL acetone, centrifuge to discard the supernatant, and dry to obtain empty negatively charged yeast cell walls.
（2）制备负载姜黄素的阳离子纳米粒：将10mg姜黄素和5mg分子量为2 kDa的枝化聚乙烯亚胺溶于二甲基亚砜中，在水中透析，得到载姜黄素的阳离子纳米粒。(2) Preparation of cationic nanoparticles loaded with curcumin: 10 mg of curcumin and 5 mg of branched polyethyleneimine with a molecular weight of 2 kDa were dissolved in dimethyl sulfoxide and dialyzed in water to obtain cationic nanoparticles loaded with curcumin .
（3）制备负载药物的酵母细胞壁微囊：将100mg空的带负电的酵母细胞壁溶解于0.1M碳酸氢纳缓冲液中，37℃孵育30min，之后加入载10mg载药阳离子纳米粒，37℃继续孵育24h，阳离子载药纳米粒吸附并沉积进入酵母细胞壁内，离心水洗4次，干燥即得载药酵母细胞壁微囊。(3) Preparation of drug-loaded yeast cell wall microcapsules: Dissolve 100mg of empty negatively charged yeast cell wall in 0.1M sodium bicarbonate buffer, incubate at 37°C for 30min, then add 10mg of drug-loaded cationic nanoparticles, and continue at 37°C After incubation for 24 hours, the cationic drug-loaded nanoparticles were adsorbed and deposited into the yeast cell wall, centrifuged and washed 4 times, and dried to obtain drug-loaded yeast cell wall microcapsules.
（4）制备多聚赖氨酸包被的平皿：将0.1mg/ml-0.025mg/ml多聚赖氨酸加入平皿中，37℃孵育2h或室温孵育过夜，然后吸取液体，表面晾干，即得多聚赖氨酸包被的平皿。(4) Prepare poly-lysine-coated plates: add 0.1mg/ml-0.025mg/ml poly-lysine to the plate, incubate at 37°C for 2 hours or overnight at room temperature, then absorb the liquid and dry the surface. That is, poly-lysine-coated plates.
（5）铺板：将10mg（3）中制得的载药酵母细胞壁微囊水溶液加入到6cm培养皿中。静置1-2h后，吸取上清，双蒸水清洗3遍后，放入37℃烘箱中烘干备用。(5) Plating: add 10 mg of the aqueous solution of drug-loaded yeast cell wall microcapsules prepared in (3) to a 6 cm petri dish. After standing still for 1-2 hours, absorb the supernatant, wash with double distilled water for 3 times, and put it in a 37°C oven to dry for later use.
（6）活化：将羰基二咪唑溶于四氢呋喃中，配置0.1-1M溶液，并将其加入到（5）平皿中，活化2-3小时，之后水洗3次。(6) Activation: Dissolve carbonyldiimidazole in tetrahydrofuran, prepare a 0.1-1M solution, and add it to (5) plate, activate for 2-3 hours, and then wash 3 times with water.
（7）连接：将1-3mg过氧化氢酶和3-9mg葡萄糖氧化酶加入到（6）中，4℃反应过夜，或室温孵育4-6小时。(7) Ligation: Add 1-3 mg catalase and 3-9 mg glucose oxidase to (6), react at 4°C overnight, or incubate at room temperature for 4-6 hours.
（8）将（7）制备的酵母微纳马达前体进行水洗3次，之后10kHz超声3-6min，收集溶液离心，沉淀即为酵母仿生微马达。(8) Wash the yeast micro-nanomotor precursor prepared in (7) with water three times, then sonicate at 10 kHz for 3-6 minutes, collect the solution and centrifuge, and precipitate to become the yeast biomimetic micromotor.
（9）将1-3mg丙烯酸树脂肠衣与10mg载药酵母仿生微马达放入磁力搅拌器中搅拌6-12小时，即得可口服的载药酵母仿生微马达；(9) Put 1-3mg of acrylic resin casing and 10mg of drug-loaded yeast bionic micromotor into a magnetic stirrer and stir for 6-12 hours to obtain a drug-loaded yeast bionic micromotor that can be taken orally;
（10）每次口服1-5mg载药酵母仿生微马达后，在肠道派尔式结内形成巨噬细胞活细胞药物，即基于巨噬细胞的活细胞载药系统。(10) After each oral administration of 1-5 mg drug-loaded yeast biomimetic micromotor, macrophage living cell drug is formed in the intestinal Peyer's junction, that is, a macrophage-based living cell drug delivery system.
实验例Experimental example
将3%的葡萄糖硫酸钠（DSS）加入到小鼠饮用水中，连续饮用9天。以制备小鼠结肠炎模型。从第三天开始分为四组：对照组（口服PBS）、游离姜黄素组（Free Cur）、载姜黄素纳米粒酵母微囊组（CP@Y）和载姜黄素纳米粒酵母仿生微马达组（CP@Y-robot）。每隔两天口服以上三种药物（姜黄素当量为10μg/g）。第10天腹腔注射炎症检测探针（L-012）利用小动物成像观察结肠炎的治疗效果。Add 3% dextrose sodium sulfate (DSS) into the drinking water of the mice for 9 days. To prepare a mouse colitis model. From the third day, they were divided into four groups: control group (oral PBS), free curcumin group (Free Cur), curcumin nanoparticle yeast microcapsule group (CP@Y) and curcumin nanoparticle yeast bionic micromotor group (CP@Y-robot). The above three drugs (curcumin equivalent 10 μg/g) were taken orally every two days. On day 10, the inflammation detection probe (L-012) was intraperitoneally injected to observe the therapeutic effect of colitis using small animal imaging.
图1为巨噬细胞活细胞药物的结构示意图，其中，1为巨噬细胞，2为载药酵母仿生微马达。图2为载药酵母仿生微马达制备过程示意图，先将酵母细胞A进行酸碱处理，提取其细胞壁，然后将载药纳米粒通过静电吸附引入到酵母细胞壁中，形成载药酵母细胞壁微囊C。接着将载药酵母细胞壁微囊C进行操作D，即平铺活化，然后超声分散（操作E），得到半面活化的载药酵母细胞壁微囊F。将载药酵母细胞壁微囊F与葡萄糖氧化酶G和过氧化氢酶H进行反应，得到酵母仿生微马达I，包裹上肠衣（操作J），得到可口服的酵母仿生微马达。Fig. 1 is a schematic diagram of the structure of a macrophage living cell drug, wherein, 1 is a macrophage, and 2 is a drug-loaded yeast biomimetic micromotor. Figure 2 is a schematic diagram of the preparation process of the drug-loaded yeast bionic micromotor. First, the yeast cell A is treated with acid and alkali to extract its cell wall, and then the drug-loaded nanoparticles are introduced into the yeast cell wall by electrostatic adsorption to form a drug-loaded yeast cell wall microcapsule C. . Then, the drug-loaded yeast cell wall microcapsules C were subjected to operation D, that is, tiled activation, and then ultrasonically dispersed (operation E), to obtain half-surface activated drug-loaded yeast cell wall microcapsules F. The drug-loaded yeast cell wall microcapsule F was reacted with glucose oxidase G and catalase H to obtain yeast biomimetic micromotor I, which was wrapped with casing (operation J) to obtain an oral yeast biomimetic micromotor.
图3为载药酵母仿生微马达的荧光成像图，其中，图3（1）为对葡萄糖氧化酶进行的荧光成像图，图3（2）为对过氧化氢酶进行的荧光成像图，图3（3）为对葡萄糖氧化酶和过氧化氢酶同时进行的荧光成像图。可以看出，酵母细胞壁微囊半面修饰有葡萄糖氧化酶和过氧化氢酶。Figure 3 is the fluorescence imaging diagram of drug-loaded yeast biomimetic micromotor, in which Figure 3 (1) is the fluorescence imaging diagram of glucose oxidase, and Figure 3 (2) is the fluorescence imaging diagram of catalase, Fig. 3 (3) is the simultaneous fluorescence imaging of glucose oxidase and catalase. It can be seen that the half surface of the yeast cell wall microcapsule is modified with glucose oxidase and catalase.
图4为巨噬细胞活细胞药物的荧光成像图，其中，图4（1）为负载姜黄素的酵母仿生微马达，图4（2）为Dil标记的巨噬细胞，图4（3）为DAPI染色的酵母细胞细胞核，图4（4）为同时对负载姜黄素的酵母仿生微马达、巨噬细胞以及酵母细胞细胞核进行荧光成像。Figure 4 is the fluorescence imaging image of macrophage living cell drug, in which Figure 4 (1) is the yeast biomimetic micromotor loaded with curcumin, Figure 4 (2) is the Dil-labeled macrophage, and Figure 4 (3) is DAPI-stained yeast cell nuclei, Figure 4 (4) is the simultaneous fluorescence imaging of curcumin-loaded yeast biomimetic micromotors, macrophages, and yeast cell nuclei.
图5为药物分布图，口服Free Cur、CP@Y和CP@Y-robot药物后，小鼠安乐死，取出肠道进行小动物成像观察姜黄素在肠道的保留情况结果。其中，图5（1）为口服Free Cur药物后的荧光成像图，图5（2）为口服CP@Y药物后的荧光成像图，图5（3）为口服CP@Y-robot药物后的荧光成像图。从结果可见，Free Cur组无可见荧光，说明几乎没有姜黄素保留在肠道内，CP@Y组有可见荧光，说明有姜黄素保留在肠道内，CP@Y-robot组有非常强的荧光，说明酵母机器人主动穿透肠粘液层并克服了胃肠的排空，显著增加了姜黄素保留，大大提高了给药效率。Figure 5 shows the drug distribution diagram. After oral administration of Free Cur, CP@Y and CP@Y-robot, the mice were euthanized, and the intestinal tract was taken out for small animal imaging to observe the retention of curcumin in the intestinal tract. Among them, Figure 5 (1) is the fluorescence imaging image after oral administration of Free Cur drug, Figure 5 (2) is the fluorescence imaging image after oral administration of CP@Y drug, and Figure 5 (3) is the fluorescence imaging image after oral administration of CP@Y-robot drug Fluorescence image. It can be seen from the results that there is no visible fluorescence in the Free Cur group, indicating that almost no curcumin is retained in the intestine, there is visible fluorescence in the CP@Y group, indicating that curcumin is retained in the intestine, and the CP@Y-robot group has very strong fluorescence, It shows that the yeast robot actively penetrates the intestinal mucus layer and overcomes the emptying of the gastrointestinal tract, significantly increases the retention of curcumin, and greatly improves the efficiency of drug administration.
图6为第10天后的治疗效果图。当注射炎症探针后（L-012）,进行小动物成像，荧光越强，炎症越严重。从结果可见，对照组（参见图6（1））有强烈的荧光，说明结肠炎模型造模成功，当口服Free Cur后（参见图6（2）），并没有降低荧光强度，说明Free Cur很少保留在肠道内，几乎对结肠炎无治疗效果；当口服CP@Y后（参见图6（3）），荧光强度有一定的降低，说明炎症减轻，CP@Y对结肠炎有一定的治疗效果；当口服CP@Y-robot后（参见图6（4）），腹腔部位有微弱的荧光，说明仅有微弱的炎症反应，CP@Y-robot大大增加了药物的治疗效果。因为小鼠一直在口服DSS，炎症刺激一直在，所以CP@Y-robot并不能完全治愈结肠炎。Figure 6 is a graph of the treatment effect after the 10th day. After injecting the inflammatory probe (L-012), small animal imaging was performed, the stronger the fluorescence, the more severe the inflammation. It can be seen from the results that the control group (see Figure 6 (1)) had strong fluorescence, indicating that the colitis model was successfully established. After oral administration of Free Cur (see Figure 6 (2)), the fluorescence intensity did not decrease, indicating that Free Cur It is rarely retained in the intestinal tract and has almost no therapeutic effect on colitis; after oral administration of CP@Y (see Figure 6 (3)), the fluorescence intensity decreases to a certain extent, indicating that inflammation is reduced, and CP@Y has a certain effect on colitis Therapeutic effect; after oral administration of CP@Y-robot (see Figure 6 (4)), there is weak fluorescence in the abdominal cavity, indicating that there is only a weak inflammatory response, and CP@Y-robot greatly increases the therapeutic effect of the drug. Because the mice have been taking DSS orally, the inflammatory stimulus is always there, so CP@Y-robot cannot completely cure the colitis.
本发明的有益效果包括以下几个方面：The beneficial effects of the present invention include the following aspects:
1. 载药酵母仿生微马达具有自驱动，可以与巨噬细胞自组装成活细胞载药系统在体自主装形成活细胞药物，安全简单方便，不需要复杂的提取巨噬细胞、培养、扩增、载药、分选等步骤；1. The drug-loaded yeast bionic micromotor is self-driven, and can self-assemble with macrophages into a living cell drug-loading system to form a living cell drug in the body, which is safe, simple and convenient, and does not require complicated extraction, cultivation, and expansion of macrophages , drug loading, sorting and other steps;
2. 酵母仿生微纳马达具有主动穿透肠屏障等生物组织障碍的作用，大大提高了药物的给药效率；2. Yeast bionic micro-nano motors can actively penetrate the intestinal barrier and other biological tissue barriers, greatly improving the drug delivery efficiency;
3. 酵母仿生微纳马达具有特异的巨噬细胞靶向性，更加高效治疗由巨噬细胞引起的疾病。3. Yeast bionic micro-nano motors have specific macrophage targeting, and can treat diseases caused by macrophages more efficiently.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (10)

  1. 一种基于巨噬细胞的活细胞载药系统，其特征在于，包括活细胞和位于所述活细胞内部的载药酵母仿生微马达；其中，所述活细胞为巨噬细胞，所述载药酵母仿生微马达包括酵母细胞壁、位于酵母细胞壁内部的载药纳米粒和半面修饰于所述酵母细胞壁的复合酶结构，所述复合酶结构为葡萄糖氧化酶和过氧化氢酶构成的阴阳结构。A living cell drug delivery system based on macrophages, characterized in that it includes living cells and drug-loaded yeast biomimetic micromotors located inside the living cells; wherein the living cells are macrophages, and the drug-loaded The yeast biomimetic micromotor comprises yeast cell walls, drug-loaded nanoparticles inside the yeast cell walls, and a composite enzyme structure half-surface modified on the yeast cell walls, and the composite enzyme structure is a yin-yang structure composed of glucose oxidase and catalase.
  2. 根据权利要求1所述的基于巨噬细胞的活细胞载药系统，其特征在于，所述酵母细胞壁主要成分为β-(1,3)-D-葡聚糖。The living cell drug delivery system based on macrophages according to claim 1, wherein the main component of the yeast cell wall is β-(1,3)-D-glucan.
  3. 根据权利要求1所述的基于巨噬细胞的活细胞载药系统，其特征在于，所述巨噬细胞为M1型或M2型巨噬细胞。The living cell drug delivery system based on macrophages according to claim 1, wherein the macrophages are M1 or M2 macrophages.
  4. 根据权利要求1所述的基于巨噬细胞的活细胞载药系统，其特征在于，所述载药纳米粒所载药物为小分子药物、多肽、大分子蛋白药物和基因药物中的一种或多种。The living cell drug delivery system based on macrophages according to claim 1, wherein the drug loaded on the drug-loaded nanoparticle is one or more of small molecule drugs, polypeptides, macromolecular protein drugs, and gene drugs. Various.
  5. 一种根据权利要求1-4任一项所述的基于巨噬细胞的活细胞载药系统的制备方法，其特征在于，包括如下步骤：A method for preparing a macrophage-based living cell drug delivery system according to any one of claims 1-4, characterized in that it comprises the steps of:
    （1）制备载药酵母仿生微马达；(1) Preparation of drug-loaded yeast biomimetic micromotor;
    （2）将载药酵母仿生微马达进入具有巨噬细胞的环境，巨噬细胞将所述载药酵母仿生微马达内吞，形成基于巨噬细胞的活细胞载药系统。(2) The drug-loaded yeast biomimetic micromotor is introduced into an environment with macrophages, and the macrophages endocytose the drug-loaded yeast biomimetic micromotor to form a living cell drug delivery system based on macrophages.
  6. 根据权利要求5所述的基于巨噬细胞的活细胞载药系统的制备方法，其特征在于，所述“制备载药酵母仿生微马达”具体包括以下步骤：The method for preparing a macrophage-based living cell drug-carrying system according to claim 5, wherein said "preparing drug-carrying yeast biomimetic micromotor" specifically comprises the following steps:
    （1）将酵母细胞利用酸碱法或酶消化法提取酵母细胞壁；(1) Yeast cells are extracted by acid-base method or enzyme digestion method;
    （2）将载药纳米粒通过静电沉积吸附进入所述酵母细胞壁内，制得载药酵母细胞壁微囊；(2) Adsorbing the drug-loaded nanoparticles into the yeast cell wall through electrostatic deposition to prepare drug-loaded yeast cell wall microcapsules;
    （3）将步骤（2）制备的载药酵母细胞壁微囊平铺在容器中，之后加入活化剂，进行表面活化，得到表面活化载药酵母细胞壁；(3) Spread the drug-loaded yeast cell wall microcapsules prepared in step (2) in a container, and then add an activator to perform surface activation to obtain surface-activated drug-loaded yeast cell walls;
    （4）将葡萄糖氧化酶和过氧化氢酶与步骤（3）制备的表面活化载药酵母细胞壁共孵育，得到载药酵母仿生微马达。 (4) Co-incubating glucose oxidase and catalase with the surface-activated drug-loaded yeast cell wall prepared in step (3) to obtain the drug-loaded yeast biomimetic micromotor.
  7. 根据权利要求6所述的基于巨噬细胞的活细胞载药系统的制备方法，其特征在于，步骤（3）中，所述容器为平皿，所述容器经过多聚赖氨酸包被处理。The method for preparing a macrophage-based living cell drug delivery system according to claim 6, characterized in that, in step (3), the container is a plate, and the container is coated with polylysine.
  8. 根据权利要求6所述的基于巨噬细胞的活细胞载药系统的制备方法，其特征在于，步骤（4）中，过氧化氢酶与葡萄糖氧化酶的质量比为1:（2-5）。The preparation method of a macrophage-based living cell drug delivery system according to claim 6, characterized in that, in step (4), the mass ratio of catalase to glucose oxidase is 1: (2-5) .
  9. 根据权利要求1-4任一项所述的基于巨噬细胞的活细胞载药系统在制备靶向治疗巨噬细胞疾病药物中的应用。Application of the macrophage-based living cell drug delivery system according to any one of claims 1-4 in the preparation of drugs for targeted treatment of macrophage diseases.
  10. 根据权利要求9所述的应用，其特征在于，所述巨噬细胞疾病包括但不限于：肿瘤、肥胖、动脉粥样硬化、骨质疏松、骨硬化病、肝炎、神经退行性疾病、关节炎、胃溃疡、结肠炎、糖尿病中的一种或多种。The application according to claim 9, wherein the macrophage diseases include but not limited to: tumors, obesity, atherosclerosis, osteoporosis, osteosclerosis, hepatitis, neurodegenerative diseases, arthritis , one or more of gastric ulcer, colitis, diabetes.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103120795A (en) * 2011-10-27 2013-05-29 四川大学 Preparation targeting macrophages
CN105168180A (en) * 2015-09-30 2015-12-23 中国人民解放军第三军医大学 Oral targeted carrier system based on yeast capsules and preparation method of oral targeted carrier system
WO2020115124A1 (en) * 2018-12-05 2020-06-11 Fundació Institut De Bioenginyeria De Catalunya Functionalized enzyme-powered nanomotors
CN114425043A (en) * 2021-12-08 2022-05-03 深圳先进技术研究院 Live cell drug delivery system based on macrophages and preparation method and application thereof
CN114425088A (en) * 2021-12-08 2022-05-03 深圳先进技术研究院 Yeast bionic immune micro-nano biological robot and preparation method and application thereof
CN114425089A (en) * 2021-12-08 2022-05-03 深圳先进技术研究院 Yeast bionic micro-nano biological robot and preparation method thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102431966B (en) * 2011-12-27 2014-10-29 复旦大学 Tubular multi-pore micron motor and preparation method and application thereof
CN102556935B (en) * 2012-03-06 2014-10-08 哈尔滨工业大学 Artificial hollow micro-nano motor and preparation method thereof
WO2018129390A1 (en) * 2017-01-06 2018-07-12 The Regents Of The University Of California Micromotors and nanomotors for gastrointestinal diagnosis and treatment applications
CN110478247B (en) * 2019-08-27 2021-07-06 哈尔滨工业大学 Micro-nano motor capsule and preparation method thereof
CN111521545A (en) * 2020-05-29 2020-08-11 中山大学 Completely biocompatible cell micromotor assembly method and application
CN111632038B (en) * 2020-06-05 2022-02-11 南京师范大学 Platelet drug-loaded micro-nano motor and preparation method and application thereof
CN112755195A (en) * 2021-01-21 2021-05-07 南方医科大学 Preparation method and application of hydrogen power micro motor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103120795A (en) * 2011-10-27 2013-05-29 四川大学 Preparation targeting macrophages
CN105168180A (en) * 2015-09-30 2015-12-23 中国人民解放军第三军医大学 Oral targeted carrier system based on yeast capsules and preparation method of oral targeted carrier system
WO2020115124A1 (en) * 2018-12-05 2020-06-11 Fundació Institut De Bioenginyeria De Catalunya Functionalized enzyme-powered nanomotors
CN114425043A (en) * 2021-12-08 2022-05-03 深圳先进技术研究院 Live cell drug delivery system based on macrophages and preparation method and application thereof
CN114425088A (en) * 2021-12-08 2022-05-03 深圳先进技术研究院 Yeast bionic immune micro-nano biological robot and preparation method and application thereof
CN114425089A (en) * 2021-12-08 2022-05-03 深圳先进技术研究院 Yeast bionic micro-nano biological robot and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG MAOJIE, ZHANG PING, ZHAO RUIBIN, YANG YALAN, WANG WEI, CHU LIANGYIN: "Recent progress on controllable fabrication of bubble-propelled functional micromotors", ZHONGGUO KEXUE. HUAXUE - SCIENTIA SINICA CHIMICA, ZHONGGUO KEXUE ZAZHISHE, CN, vol. 49, no. 6, 1 June 2019 (2019-06-01), CN , pages 861 - 876, XP093069324, ISSN: 1674-7224, DOI: 10.1360/N032018-00206 *

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