WO2021104088A1 - 一种纳米颗粒载药系统及其制备方法和应用 - Google Patents

一种纳米颗粒载药系统及其制备方法和应用 Download PDF

Info

Publication number
WO2021104088A1
WO2021104088A1 PCT/CN2020/129167 CN2020129167W WO2021104088A1 WO 2021104088 A1 WO2021104088 A1 WO 2021104088A1 CN 2020129167 W CN2020129167 W CN 2020129167W WO 2021104088 A1 WO2021104088 A1 WO 2021104088A1
Authority
WO
WIPO (PCT)
Prior art keywords
macrophages
tumor
cancer
nanoparticles
drug
Prior art date
Application number
PCT/CN2020/129167
Other languages
English (en)
French (fr)
Inventor
蔡林涛
周海梅
刘兰兰
何华美
梁锐晶
Original Assignee
深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院 filed Critical 深圳先进技术研究院
Publication of WO2021104088A1 publication Critical patent/WO2021104088A1/zh

Links

Classifications

    • 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
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of nanomedicine, and specifically relates to a drug-carrying system with nano-particles loaded on the surface of macrophage cells, a preparation method thereof, and application in tumor immunotherapy.
  • Tumor immunotherapy is known as the fourth tumor treatment method following surgery, chemotherapy, and radiotherapy.
  • tumor immunotherapy is a treatment method that targets the human immune system instead of directly targeting tumors. By stimulating or mobilizing the body's immune system, it enhances the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells .
  • cellular immunotherapy has received widespread attention as the main tumor immunotherapy.
  • Cellular immunotherapy the full name of adoptive immune cell therapy, refers to the transportation of immune cells (specific and non-specific) with anti-tumor activity to tumor patients, which directly kills tumors or stimulates the body's immune response to kill tumor cells.
  • Macrophages are an important component of the body's innate immune response. They are a type of plastic and heterogeneous cell population. They maintain the homeostasis of normal tissues by removing abnormal cells and play an important role in the body's non-specific immune function. Macrophages can exert a wide range of anti-tumor effects through multiple pathways and multiple steps. Bacterial cell wall components and cytokines can activate macrophages. Activated macrophages can efficiently and specifically recognize and lyse tumor cells, including those tumor cells that are resistant to cytotoxic drugs, but damage normal cells. less.
  • Macrophages and tumor cells can secrete and release some cytotoxic substances (such as tumor necrosis factor, nitric oxide, serine proteases, lysosomal enzymes, reactive oxygen species, etc.) after direct contact with tumor cells for 1 to 3 days, which can cause the combined tumor cells to dissolve Or apoptosis, this process is slow and requires direct contact between cells.
  • Macrophages can also directly kill tumor cells through antibody-dependent cytotoxicity.
  • Activated macrophages can process and present tumor antigens, activate T cells, and stimulate the body to produce specific immune responses to tumor cells. Compared with T cells, the killing of tumor cells by macrophages has nothing to do with the immunogenicity, metastatic potential and drug sensitivity of tumor cells.
  • macrophages have tumor targeting properties and are widely used as tumor targeting vectors.
  • CN109893515 A published a patent titled "A macrophage drug-loaded microparticle preparation and its preparation method".
  • Molecular active ingredients, cell vesicles are derived from the apoptosis of macrophages modified by mannose. They believe that the drug-loaded microparticles provided by it are conducive to being highly enriched in tumor tissues and more easily taken up by M2 tumor-associated macrophages, improving the inverse polarization effect of small molecule drugs on M2 tumor-associated macrophages, and improving tumors
  • the microenvironment enhances the killing of tumor cells.
  • CN104771764 A published a patent titled "A macrophage targeting vector system and its preparation", pointing out that the macrophage targeting vector is mannosylated protamine, and the positively charged mannosylated protamine is charged with negatively charged nucleic acid formation A positively charged nanoparticle.
  • mannosylated protamine has nuclear localization function and macrophage targeting, which can improve the efficiency of gene transfection mediation of protamine in macrophages.
  • the above-mentioned prior art uses macrophage drug delivery, macrophage targeting system, etc., although it has a certain effect on the treatment of tumors, but these methods only use macrophages as carriers to deliver drugs to the tumor site. It does not use the function of macrophages, the natural immune cells themselves. This not only increases the preparation process and cost, but also wastes the role of macrophages.
  • Our research has found that macrophages can also exert their own unique immune function and tumor targeting while serving as a carrier to transport drugs. The two work together to improve the anti-tumor efficacy.
  • nanomaterials have obvious advantages in targeted drug delivery due to their unique physical and chemical properties and targeted modification.
  • the use of cells or cell-derived vesicles as drug carriers has attracted widespread attention. Therefore, the combination of nanomaterials and cellular immunotherapy may provide new ideas for tumor treatment.
  • the present invention discloses a nano drug delivery system with simple preparation method, mature technology, stable properties and good biocompatibility, which loads nanoparticles on the surface of macrophages and utilizes the immune function of macrophages. Regulating the function and the function of nano-particles to combine the treatment of tumors, has good application prospects in the fields of nano-biomedicine and tumor cell therapy.
  • An object of the present invention is to provide a nano drug delivery system, including macrophages and nanoparticles, the nanoparticles have loading, delivery and/or slow release performance; the nanoparticles include polymers, the polymers Selected from polyester, polyanhydride, polyorthoester, polyphosphazene, polyphosphate, polyhydroxy acid, polypropyl fumarate, polyamide, polyamino acid, polyacetal, polyether, polyurethane, polymethyl Acrylate, polyacrylate, polycyanoacrylate, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polyvalerolactone, poly(lactide-co-glycolide) ) (PLG), polylactic acid-glycolic acid (PLGA), polyglycolic acid-polylactic acid-polyethylene glycol (PLGA-PEG), poly(lactide-co-caprolactone) (PLC), poly(ethylene glycol) Any one or more of lactide-co-
  • the functional group of the nanoparticle is a maleamide bond
  • the nanoparticle is a nanoparticle encapsulating the maleamide bond synthesized by an ultrasonic method.
  • the functional group of the macrophage is a sulfhydryl group, and the macrophage is treated with a thiol reducing agent before coupling with the nanoparticle; preferably, the thiol reducing agent is TCEP.
  • the nano drug delivery system further includes an anti-tumor drug.
  • the anti-tumor drugs include anti-tumor broad-spectrum drugs and/or anti-tumor targeted drugs.
  • the anti-tumor broad-spectrum drug is selected from any one or more of camptothecin drugs, doxorubicin drugs, paclitaxel drugs or platinum drugs.
  • the anti-tumor targeted drug is selected from the group consisting of zebutinib, nilotinib, imatinib, vermodil, verofenib, temsirolimus, sunitinib, and ceritin Ni, regorafenib, afatinib, trametinib, pranatinib, bortezomib, pazopanib, axitinib, romidepsin, everolimus, ibrutinib , Levatinib, Darafenib, Crizotinib, Carfilzomib, Ostinib, Cabotinib, Carbitinib, Gefitinib, Vorinostat, Vandetanib , Alectinib, denosumab, sondeji, sorafenib, bosutinib, belisstat, olaparib, aflibercept
  • the composition further includes a polypeptide substance, and the polypeptide includes an antigen or an antibody.
  • the antibody is selected from the group consisting of adalimumab, cetuximab, ibrituximab, trastuzumab, nivolumab, darrilimumab ramucirumab, and navastin Bevacizumab, pembrolizumab, pembrolizumab, ofatumumab, Bonatumumab, bevacizumab, panitumumab, maybezumab, bentuximab , Denutuximab, Tositumomab, Errotuzumab, Trastuzumab, or Rituximab.
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Any one or more of pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.
  • Another object of the present invention is to provide a drug containing the nano drug delivery system.
  • the drug is an anti-tumor drug.
  • the drug is administered by injection.
  • the injection administration includes any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymph node injection, intratumor injection or subfoot injection.
  • the medicine also includes medically or pharmaceutically acceptable auxiliary substances and/or excipients.
  • Another object of the present invention is to provide an application of the nano drug-carrying system or the drug in the preparation of anti-tumor drugs.
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Any one or more of pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.
  • Another object of the present invention is to provide a method for preparing the nano drug delivery system, which includes the following steps:
  • step (3) Transfer the nanoparticles obtained in step (1) and the macrophages processed in step (3) into a 15 mL centrifuge tube, continue to add phosphate buffer to a total volume of 4 mL, and then place them in a 37 °C centrifuge tube. Shaker to mix, and finally centrifuge the mixed liquid, that is, successfully load the nanoparticles on the surface of macrophages.
  • the preparation method of the step (1) is to synthesize the nanoparticles encapsulating the maleamide bond by an ultrasonic method.
  • the number of macrophages moved into the 1.5 mL centrifuge tube in the step (3) is 1 ⁇ 10 5 -1 ⁇ 10 8 , preferably 1-5 ⁇ 10 6 , more preferably 2 ⁇ 10 6 pieces.
  • the concentration of the TCEP solution in the step (3) is 1 mM.
  • the incubation time in the step (3) is 20 minutes.
  • the condition of the shaking table mixing in the step (4) is 120 revolutions for 20 minutes.
  • the condition of the centrifugation in the step (4) is 1000 revolutions for 4 minutes.
  • the present invention provides a method for preparing a nano drug-carrying system that loads nanoparticles on the surface of macrophages.
  • the technical problem to be solved is to solve the problem that small drug molecules in the prior art cannot be effectively enriched and targeted at tumor sites. problem.
  • the characteristics of the nano drug-carrying system prepared by the present invention are: 1 The macrophages used to load the nanoparticles must be treated with TCEP to expose the sulfhydryl groups on the cell surface; 2 The core-shell structured nanoparticles are surrounded by horses.
  • the amide bond is used to bind to the sulfhydryl groups on the surface of macrophages; 3Nanoparticles are loaded on the surface of macrophages instead of entering the cell; 4Macrophages loaded with nanoparticles have good biocompatibility and biological compatibility. Degradability can reduce the toxicity of drugs to the body; 5 Macrophages loaded with nanoparticles are pH-responsive and can achieve controlled release of drugs in response; 6 Macrophages loaded with nanoparticles are targeted to the tumor site and interact with the tumor. Direct contact can not only activate and release the drugs encapsulated in nanoparticles in situ, but also exert the immune regulation of macrophages and improve the anti-tumor efficacy.
  • Figure 1 is a scanning electron microscope image of macrophages loaded with nanoparticles.
  • Figure 2 is a laser confocal image of macrophages loaded with nanoparticles.
  • Figure 3 shows the in vitro killing effect of macrophages loaded with nanoparticles on the surface of cancer cells.
  • BMDM stands for macrophages
  • MPIP stands for macrophages loaded with nanoparticles.
  • the solution of macrophages loaded with nanoparticles on the surface was diluted 10 times, and then the samples were prepared according to the scanning electron microscope sample preparation method, and then the samples were observed under the scanning electron microscope. It was found that the nanoparticles were loaded on the surface of macrophages, as shown in Figure 1.
  • the breast tumor cells (4T1) were seeded in 96-well plates (10 4 cells / well), respectively, the number of macrophages containing 10 4 individual surface macrophages or macrophages loaded nanoparticles was added thereto, Let them incubate for 24 hours. Then the survival rate of breast cancer cells was detected by a killing kit, and it was found that macrophages loaded with nanoparticles on the surface have a much stronger killing effect on tumors than macrophages alone, as shown in Figure 3 (BMDM stands for macrophages, MPIP Represents macrophages loaded with nanoparticles).
  • BMDM stands for macrophages
  • MPIP Represents macrophages loaded with nanoparticles

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种制备方法简单、工艺成熟，性质稳定、生物相容性好的纳米载药系统，该纳米载药系统是将纳米颗粒通过马来酰胺键偶联到巨噬细胞表面，通过巨噬细胞本身的免疫调节功能和纳米颗粒的功能来联合治疗肿瘤，提高抗肿瘤疗效，解决了现有技术中存在的药物小分子无法在肿瘤部位有效富集和靶向的问题，该纳米载药系统在纳米医学领域及肿瘤治疗领域具有重要的应用前景。

Description

一种纳米颗粒载药系统及其制备方法和应用 技术领域
本发明属于纳米医药领域，具体涉及一种巨噬细胞细胞表面负载有纳米颗粒的载药系统及其制备方法和在肿瘤免疫治疗中的应用。
 背景技术
肿瘤免疫疗法被称为继手术，化疗，放疗后的第四种肿瘤治疗方法。与传统治疗方式不同，肿瘤免疫疗法是一种针对人体免疫系统而非直接针对肿瘤的治疗方法，通过激发或调动机体的免疫系统，增强肿瘤微环境的抗肿瘤免疫力，从而控制和杀伤肿瘤细胞。近年来，细胞免疫疗法作为主要的肿瘤免疫疗法被广泛关注。细胞免疫疗法，其全称为过继性免疫细胞疗法，是指向肿瘤患者运输具有抗肿瘤活性的免疫细胞（特异性和非特异性的），直接杀伤肿瘤或激发机体的免疫应答杀伤肿瘤细胞。
巨噬细胞是机体固有免疫反应的重要组分，是一类具有可塑性、异质性的细胞群体，通过清除异常细胞保持正常组织的稳态，在机体的非特异免疫功能中发挥重要作用。巨噬细胞可通过多途径、多步骤发挥广泛抗瘤作用。细菌细胞壁成分和细胞因子可以活化巨噬细胞，活化的巨噬细胞可以高效、专一地识别并裂解肿瘤细胞，包括那些对细胞毒性药物有抗性的肿瘤细胞，但对正常细胞的损伤却很少。巨噬细胞和肿瘤细胞直接接触 1～3天后可分泌释放一些细胞毒性物质(如肿瘤坏死因子、一氧化氮、丝氨酸蛋白酶、溶酶体酶、活性氧等)，可导致所结合的肿瘤细胞溶解或调亡，此过程缓慢并且需要细胞之间直接接触。巨噬细胞也可以通过抗体依赖的细胞毒性作用直接杀伤肿瘤细胞。活化的巨噬细胞可以处理和呈递肿瘤抗原、激活 T 细胞并刺激机体对肿瘤细胞产生特异性免疫应答。与 T 细胞相比，巨噬细胞杀伤肿瘤细胞与肿瘤细胞的免疫原性、转移潜能和对药物的敏感性无关。因此，于绝大多数肿瘤细胞，尤其是那些肿瘤抗原易发生变异的转移肿瘤细胞，在体内特异性 T 细胞难以发挥效应时，其对活化的巨噬细胞的杀伤作用却极少出现抗性。除此之外，巨噬细胞具有肿瘤靶向性，作为肿瘤靶向载体被广泛应用。
CN109893515 A公布了名称为“一种巨噬细胞载药微颗粒制剂及其制备方法”的专利，该专利指出巨噬细胞载药微颗粒制剂包括细胞囊泡和包裹在细胞囊泡内的药物小分子有效成分，细胞囊泡源自于甘露糖修饰的巨噬细胞凋亡所释放。他们认为其提供的载药微颗粒有利于在肿瘤组织高度富集、及更易被M2型肿瘤相关巨噬细胞摄取，提高小分子药物对M2型肿瘤相关巨噬细胞的逆极化效果，改善肿瘤微环境，增强对肿瘤细胞的杀伤。CN104771764 A公布了名称为“一种巨噬细胞靶向载体系统及其制备”的专利，指出巨噬细胞靶向载体为甘露糖化精蛋白，正电性的甘露糖化精蛋白荷载负电性的核酸形成一种荷正电的纳米粒子。与非病毒基因载体精蛋白相比，甘露糖化精蛋白具有核定位功能和巨噬细胞靶向性，能够提高精蛋白在巨噬细胞中的基因转染介导效率。
上述现有技术通过巨噬细胞载药、巨噬细胞靶向系统等方式，虽然对于肿瘤的治疗有一定的作用，但是这些方式仅仅是把巨噬细胞作为载体将药物递送到肿瘤部位发挥作用，并没有利用巨噬细胞这一天然免疫细胞本身的功能。这不仅增加了制剂制备的工艺和成本，还浪费了巨噬细胞的作用。我们的研究发现，巨噬细胞在作为载体运输药物的同时还可以发挥自身特有的免疫功能和肿瘤靶向性，两者共同发挥作用，从而提高了抗肿瘤疗效。
近年来，纳米材料因其独特的理化特性和靶向修饰性等，在靶向药物输送方面具有明显优势。相对于人工合成的纳米药物载体可能具有的生物相容性差等问题，利用细胞或细胞来源的囊泡用作药物载体引起广泛关注。因此，通过联合纳米材料与细胞免疫疗法，可能为肿瘤治疗提供新的思路。
 技术问题
针对以上问题，本发明公开了一种制备方法简单、工艺成熟，性质稳定、生物相容性好的纳米载药系统，把纳米颗粒负载到巨噬细胞表面，并通过利用巨噬细胞本身的免疫调节功能和纳米颗粒的功能来联合治疗肿瘤，在纳米生物医学和肿瘤细胞治疗等领域具有良好的应用前景。
 技术解决方案
本发明的一个目的在于提供一种纳米载药系统，包括巨噬细胞和纳米颗粒，所述纳米颗粒具备装载、递送和/或缓释的性能；所述纳米颗粒包括聚合物，所述聚合物选自聚酯、聚酸酐、聚原酸酯、聚磷腈、聚磷酸酯、聚羟基酸、聚丙基富马酸酯、聚酰胺、聚氨基酸、聚缩醛、聚醚、聚氨酯、聚甲基丙烯酸酯、聚丙烯酸酯、聚氰基丙烯酸酯、聚乳酸(PLA)、聚乙醇酸(PGA)、聚己内酯(PCL)、聚戊内酯、聚(丙交酯-共-乙交酯)(PLG)、聚乳酸－羟基乙酸（PLGA）、聚羟基乙酸－聚乳酸－聚乙二醇（PLGA－PEG）、聚(丙交酯-共-己内酯)(PLC)、聚(乙交酯-共-己内酯)(PGC)、聚己内酯－聚乙二醇（PCL－PEG）中任一种或多种；所述纳米颗粒和/或巨噬细胞具有或被改变以具有一个或多个官能团，使所述纳米颗粒负载到所述巨噬细胞的表面。
优选地，所述纳米颗粒的官能团是马来酰胺键，所述纳米颗粒是通过超声的方法合成包裹马来酰胺键的纳米颗粒。
优选地，所述巨噬细胞的官能团是巯基，在与所述纳米颗粒偶联前所述巨噬细胞经过硫醇类还原剂处理；优选地，所述硫醇类还原剂为TCEP。
优选地，所述纳米载药系统还包括抗肿瘤药物。
优选地，所述抗肿瘤药物包括抗肿瘤广谱药物和/或抗肿瘤靶向药物。
优选地，所述抗肿瘤广谱药物选自喜树碱类药物、阿霉素类药物、紫杉醇类药物或铂类药物中任一种或多种。
优选地，所述抗肿瘤靶向药物选自泽布替尼、尼罗替尼、伊马替尼、维莫德吉、维罗非尼、替西罗莫司、舒尼替尼、赛立替尼、瑞格非尼、阿法替尼、曲美替尼、普钠替尼、硼替佐米、帕唑帕尼、阿西替尼、罗米地辛、依维莫司、依鲁替尼、乐伐替尼、达拉菲尼、克唑替尼、卡非佐米、奥斯替尼、卡博替尼、卡比替尼、吉非替尼、伏立诺他、凡德他尼、艾乐替尼、狄诺塞麦、索尼德吉、索拉非尼、博舒替尼、贝利司他、奥拉帕尼、阿柏西普、拉帕替尼、达沙替尼、帕博西尼、帕比司他或厄洛替尼中任一种或多种。
优选地，所述组合物还包括多肽类物质，所述多肽包括抗原或抗体。
优选地，所述抗体选自阿达木单抗、西妥昔单抗、替伊莫单抗、曲妥珠单抗、纳武单抗、达雷木单抗雷莫芦单抗、耐昔妥珠单抗、派姆单抗、派姆单抗、奥法木单抗、博纳吐单抗、贝伐珠单抗、帕尼单抗、奥宾尤妥珠单抗、本妥昔单抗、地努图希单抗、托西莫单抗、埃罗妥珠单抗、曲妥珠单抗或利妥昔单抗中任一种或多种。
优选地，所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤中任一种或多种。
本发明的另一目的在于提供含有所述纳米载药系统的药物。
优选地，所述药物为抗肿瘤药物。
优选地，所述药物通过注射给药。
优选地，所述注射给药包括皮下注射、肌肉注射、腹腔注射、静脉注射、淋巴结内注射、瘤内注射或足下注射中任一种或多种。
优选地，所述药物还包括医学或药学上可接受的辅助物质和/或赋型剂。
本发明的另一目的在于提供一种所述纳米载药系统或所述药物在制备抗肿瘤药物中的应用。
优选地，所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤中任一种或多种。
本发明的另一目的在于提供一种制备所述纳米载药系统的方法，包括如下步骤：
（1）制备包裹马来酰胺键的所述纳米颗粒；
（2）从脊髓处取出并诱导和培养原代巨噬细胞；
（3）待巨噬细胞生长状态良好时，巨噬细胞计数，并移入1.5mL离心管里，向其中加入用磷酸缓冲液稀释的TCEP溶液，在37℃，5%CO 2培养箱中共孵育，期间每隔10分钟上下颠倒一次离心管后，用磷酸缓冲液洗涤数次；
（4）将步骤（1）所获得的纳米颗粒和步骤（3）处理后的巨噬细胞移入15 mL离心管，继续滴加磷酸缓冲液至总体积为4mL，然后将其放入37℃的摇床混合，最后将混合液体离心，即成功把纳米颗粒负载在巨噬细胞表面。
优选地，所述步骤（1）的制备方法是通过超声的方法合成包裹马来酰胺键和的纳米颗粒。
优选地，所述步骤（3）中移入1.5mL离心管里的所述巨噬细胞的数量为1×10 5-1×10 8个，优选为1-5×10 6个，更佳地为2×10 6个。
优选地，所述步骤（3）中所述TCEP溶液的浓度为1mM。
优选地，所述步骤（3）中所述孵育的时间为20 分钟。
优选地，所述步骤（4）中所述摇床混合的条件为120转20分钟。
优选地，所述步骤（4）中所述离心的条件为1000转4分钟。
 有益效果
本发明提供一种在巨噬细胞表面负载纳米颗粒的纳米载药系统的制备方法，所要解决的技术问题就是，解决现有技术中存在的药物小分子无法在肿瘤部位有效富集和靶向的问题。本发明制备的纳米载药系统的特征为：①用于负载纳米颗粒的巨噬细胞必须是经TCEP处理过的，目的是使细胞表面的巯基暴露出来；②核壳结构的纳米颗粒外围有马来酰胺键用于和巨噬细胞表面的巯基结合；③纳米颗粒是负载在巨噬细胞表面的，而不是进入细胞内部；④负载纳米颗粒的巨噬细胞具有较好的生物相容性、生物可降解性，可降低药物对机体的毒性；⑤负载纳米颗粒的巨噬细胞具有pH响应性，能实现药物的响应控制释放；⑥负载纳米颗粒的巨噬细胞靶向到肿瘤部位后，与肿瘤直接接触不仅能原位激活释放出包裹在纳米颗粒中的药物，而且可以发挥巨噬细胞的免疫调节，提高抗肿瘤疗效。
 附图说明
图1为负载纳米颗粒的巨噬细胞的扫描电镜图像。
图2为负载纳米颗粒的巨噬细胞的激光共聚焦图像。
图3为表面负载纳米颗粒的巨噬细胞对癌细胞的体外杀伤效果检测，BMDM代表巨噬细胞，MPIP代表负载纳米颗粒的巨噬细胞。
 本发明的实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1  表面负载纳米颗粒的巨噬细胞的制备
①通过超声的方法合成包裹马来酰胺键的纳米颗粒；
②在小鼠脊髓处取出并诱导原代巨噬细胞；
③待巨噬细胞生长状态良好时，计数2×10 6左右个巨噬细胞在1.5mL离心管里，向其中加入1mM TCEP溶液（用磷酸缓冲液稀释），在37℃，5%CO 2培养箱中共孵育20分钟，期间每隔10分钟上下颠倒一次离心管，后用磷酸缓冲液洗涤3-4次；
④将纳米颗粒和处理后的巨噬细胞移入15 mL离心管，继续滴加磷酸缓冲液至总体积为4mL，然后将其放入37℃的摇床内，120转20分钟，最后将混合液体放入1000转，离心4分钟，即成功把颗粒负载在巨噬细胞表面了。
实施例2  表面负载纳米颗粒的巨噬细胞的性能及表征
1. 表面负载纳米颗粒的巨噬细胞的扫描电镜图像
首先将表面负载纳米颗粒的巨噬细胞的溶液稀释10倍后按照扫描电镜制样方法制样，然后在扫描电镜下观察。结果发现纳米颗粒负载在巨噬细胞表面，如图1所示。
2. 表面负载纳米颗粒的巨噬细胞的激光共聚焦图像
首先将表面负载纳米颗粒的巨噬细胞的溶液稀释100倍后滴加在共聚焦小皿里，后将共聚焦小皿置于激光共聚焦显微镜下观察。结果显示纳米颗粒负载在巨噬细胞表面，如图2所示。
3. 表面负载纳米颗粒的巨噬细胞对癌细胞的体外杀伤效果检测
将乳腺癌瘤细胞（4T1）接种在96孔板中（10 4个/孔），分别将含有巨噬细胞数量为10 4个的单独巨噬细胞或表面负载纳米颗粒的巨噬细胞加入其中，让它们共孵育24小时。然后通过杀伤试剂盒检测乳腺癌细胞的存活率，结果发现表面负载纳米颗粒的巨噬细胞对肿瘤的杀伤作用比单独的巨噬细胞强很多，如图3所示（BMDM代表巨噬细胞，MPIP代表负载纳米颗粒的巨噬细胞）。
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。

Claims (20)

  1. 一种纳米载药系统，包括巨噬细胞和纳米颗粒，所述纳米颗粒具备装载、递送和/或缓释的性能；所述纳米颗粒包括聚合物，所述聚合物选自聚酯、聚酸酐、聚原酸酯、聚磷腈、聚磷酸酯、聚羟基酸、聚丙基富马酸酯、聚酰胺、聚氨基酸、聚缩醛、聚醚、聚氨酯、聚甲基丙烯酸酯、聚丙烯酸酯、聚氰基丙烯酸酯、聚乳酸(PLA)、聚乙醇酸(PGA)、聚己内酯(PCL)、聚戊内酯、聚(丙交酯-共-乙交酯)(PLG)、聚乳酸－羟基乙酸（PLGA）、聚羟基乙酸－聚乳酸－聚乙二醇（PLGA－PEG）、聚(丙交酯-共-己内酯)(PLC)、聚(乙交酯-共-己内酯)(PGC)、聚己内酯－聚乙二醇（PCL－PEG）中任一种或多种；所述纳米颗粒和/或巨噬细胞具有或被改变以具有一个或多个官能团，使所述纳米颗粒负载到所述巨噬细胞的表面。
  2. 根据权利要求1所述的纳米载药系统，所述纳米颗粒的官能团是马来酰胺键，所述纳米颗粒是通过超声的方法合成包裹马来酰胺键的纳米颗粒。
  3. 根据权利要求1所述的纳米载药系统，所述巨噬细胞的官能团是巯基，在与所述纳米颗粒偶联前所述巨噬细胞经过硫醇类还原剂处理；优选地，所述硫醇类还原剂为TCEP。
  4. 根据权利要求1所述的纳米载药系统，所述纳米颗粒还包括抗肿瘤药物和/或多肽类物质；所述抗肿瘤药物包括抗肿瘤广谱药物和/或抗肿瘤靶向药物；所述多肽包括抗原或抗体。
  5. 根据权利要求4所述的纳米载药系统，所述抗肿瘤广谱药物选自喜树碱类药物、阿霉素类药物、紫杉醇类药物或铂类药物中任一种或多种。
  6. 根据权利要求4所述的纳米载药系统，所述抗肿瘤靶向药物选自泽布替尼、尼罗替尼、伊马替尼、维莫德吉、维罗非尼、替西罗莫司、舒尼替尼、赛立替尼、瑞格非尼、阿法替尼、曲美替尼、普钠替尼、硼替佐米、帕唑帕尼、阿西替尼、罗米地辛、依维莫司、依鲁替尼、乐伐替尼、达拉菲尼、克唑替尼、卡非佐米、奥斯替尼、卡博替尼、卡比替尼、吉非替尼、伏立诺他、凡德他尼、艾乐替尼、狄诺塞麦、索尼德吉、索拉非尼、博舒替尼、贝利司他、奥拉帕尼、阿柏西普、拉帕替尼、达沙替尼、帕博西尼、帕比司他或厄洛替尼中任一种或多种。
  7. 根据权利要求4所述的纳米载药系统，所述抗体选自阿达木单抗、西妥昔单抗、替伊莫单抗、曲妥珠单抗、纳武单抗、达雷木单抗雷莫芦单抗、耐昔妥珠单抗、派姆单抗、派姆单抗、奥法木单抗、博纳吐单抗、贝伐珠单抗、帕尼单抗、奥宾尤妥珠单抗、本妥昔单抗、地努图希单抗、托西莫单抗、埃罗妥珠单抗、曲妥珠单抗或利妥昔单抗中任一种或多种。
  8. 一种含有根据权利要求1-7任一项所述纳米载药系统的药物，所述药物为抗肿瘤药物。
  9. 根据权利要求8所述的药物，是通过注射给药。
  10. 根据权利要求9所述的药物，所述注射给药包括皮下注射、肌肉注射、腹腔注射、静脉注射、淋巴结内注射、瘤内注射或足下注射中任一种或多种。
  11. 根据权利要求8-10任一项所述的药物，还包括医学或药学上可接受的辅助物质和/或赋型剂。
  12. 一种权利要求1-7任一项所述纳米载药系统或权利要求8-11任一项所述药物在制备抗肿瘤药物中的应用。
  13. 根据权利要求4-7任一项所述的纳米载药系统或权利要求8-11任一项所述药物或权利要求12所述的应用，所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤中任一种或多种。
  14. 一种制备纳米载药系统的方法，包括如下步骤：
    （1）制备权利要求1-7任一项所述的纳米颗粒；
    （2）从脊髓处取出并诱导和培养原代巨噬细胞；
    （3）待巨噬细胞生长状态良好时，巨噬细胞计数，并移入1.5mL离心管里，向其中加入用磷酸缓冲液稀释的TCEP溶液，在37℃，5%CO 2培养箱中共孵育，期间每隔10分钟上下颠倒一次离心管后，用磷酸缓冲液洗涤数次；
    （4）将步骤（1）所获得的纳米颗粒和步骤（3）处理后的巨噬细胞移入15 mL离心管，继续滴加磷酸缓冲液至总体积为4mL，然后将其放入37℃的摇床混合，最后将混合液体离心，即成功把纳米颗粒负载在巨噬细胞表面。
  15. 根据权利要求14所述的方法，所述步骤（1）的制备方法是通过超声的方法合成包裹马来酰胺键和的纳米颗粒。
  16. 根据权利要求14所述的方法，所述步骤（3）中移入1.5mL离心管里的所述巨噬细胞的数量为1×10 5-1×10 8个，优选为1-5×10 6个，更佳地为2×10 6个。
  17. 根据权利要求14所述的方法，所述步骤（3）中所述TCEP溶液的浓度为1mM。
  18. 根据权利要求14所述的方法，所述步骤（3）中所述孵育的时间为20 分钟。
  19. 根据权利要求14所述的方法，所述步骤（4）中所述摇床混合的条件为120转，20分钟。
  20. 根据权利要求14所述的方法，所述步骤（4）中所述离心的条件为1000转，4分钟。
PCT/CN2020/129167 2019-11-27 2020-11-16 一种纳米颗粒载药系统及其制备方法和应用 WO2021104088A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911182894.XA CN110859817A (zh) 2019-11-27 2019-11-27 一种纳米颗粒载药系统及其制备方法和应用
CN201911182894.X 2019-11-27

Publications (1)

Publication Number Publication Date
WO2021104088A1 true WO2021104088A1 (zh) 2021-06-03

Family

ID=69656099

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/129167 WO2021104088A1 (zh) 2019-11-27 2020-11-16 一种纳米颗粒载药系统及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN110859817A (zh)
WO (1) WO2021104088A1 (zh)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110859817A (zh) * 2019-11-27 2020-03-06 深圳先进技术研究院 一种纳米颗粒载药系统及其制备方法和应用
CN111925448B (zh) * 2020-08-03 2022-06-21 山东大学 在体生成car-巨噬细胞的制备方法及肿瘤免疫治疗中的应用
CN112535678A (zh) * 2020-12-28 2021-03-23 烟台大学 连接曲妥珠单抗的美登素纳米粒组合物
CN112675313A (zh) * 2020-12-28 2021-04-20 烟台大学 连接曲妥珠单抗片段的美登素纳米粒组合物
CN112870194B (zh) * 2021-01-06 2022-06-21 广州医科大学附属肿瘤医院 治疗肝癌的组合物及其应用
CN112807289B (zh) * 2021-02-09 2022-12-06 中国医学科学院生物医学工程研究所 基于纳米颗粒的活细胞表面改造方法及其使用的纳米颗粒
CN113651959B (zh) * 2021-07-14 2024-05-07 中山大学 一种基于氨基酸-羟基酸共聚物的纳米载药体系及其制备方法和应用
CN116236461A (zh) * 2021-12-08 2023-06-09 深圳先进技术研究院 一种氧化还原型纳米颗粒和活细胞载体及其应用
CN114533888B (zh) * 2022-02-21 2024-06-11 同济大学 一种以细胞为载体的仿生载药系统及其制备方法和应用
CN114712486B (zh) * 2022-04-08 2023-12-12 南方医科大学 一种环五肽纳米制剂及其制备方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105796510A (zh) * 2016-02-19 2016-07-27 泰山医学院 肿瘤靶向的药剂及其制备方法和应用
US20170266317A1 (en) * 2016-03-15 2017-09-21 Massachusetts Institute Of Technology Synthetically Functionalized Living Cells for Targeted Drug Delivery
WO2017180820A1 (en) * 2016-04-15 2017-10-19 The Penn State Research Foundation Compositions and methods for targeted delivery of therapeutic and/or diagnostic agents
WO2019139892A1 (en) * 2018-01-12 2019-07-18 President And Fellows Of Harvard College Compositions and methods relating to macrophages and/or monocytes with adhered particles
CN110859817A (zh) * 2019-11-27 2020-03-06 深圳先进技术研究院 一种纳米颗粒载药系统及其制备方法和应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104771764B (zh) * 2015-04-14 2018-09-18 中国药科大学 一种巨噬细胞靶向载体系统及其制备
JP7455510B2 (ja) * 2016-04-29 2024-03-26 メモリアル スローン－ケタリング キャンサー センター 悪性脳腫瘍における標的化粒子の浸透、分布および応答のための組成物及び方法
CN108210482B (zh) * 2016-12-09 2020-08-07 中国科学院深圳先进技术研究院 一种载miRNA复合纳米颗粒及其制备方法和应用
CN109953972A (zh) * 2017-12-14 2019-07-02 复旦大学 基于巨噬细胞膜包被的乳腺癌靶向纳米粒及其制备方法
CN108837154B (zh) * 2018-08-31 2021-05-07 中国药科大学 一种细胞膜仿生脂蛋白靶向纳米递药系统的制备及应用
CN109893515B (zh) * 2019-02-26 2021-10-29 华中科技大学 一种巨噬细胞载药微颗粒制剂及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105796510A (zh) * 2016-02-19 2016-07-27 泰山医学院 肿瘤靶向的药剂及其制备方法和应用
US20170266317A1 (en) * 2016-03-15 2017-09-21 Massachusetts Institute Of Technology Synthetically Functionalized Living Cells for Targeted Drug Delivery
WO2017180820A1 (en) * 2016-04-15 2017-10-19 The Penn State Research Foundation Compositions and methods for targeted delivery of therapeutic and/or diagnostic agents
WO2019139892A1 (en) * 2018-01-12 2019-07-18 President And Fellows Of Harvard College Compositions and methods relating to macrophages and/or monocytes with adhered particles
CN110859817A (zh) * 2019-11-27 2020-03-06 深圳先进技术研究院 一种纳米颗粒载药系统及其制备方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DOSHI NISHIT ET AL.,: "Cell-Based Drug Delivery Devices Using Phagocytosis-Resistant Backpacks,", ADVANCED HEALTHCARE MATERIALS,, vol. 23, 1 March 2011 (2011-03-01), XP055815458 *
GUO LING, ZHANG YE, WEI RUNXIU, WANG CUIFENG, FENG MIN: "Lipopolysaccharide-anchored macrophages hijack tumor microtube networks for selective drug transport and augmentation of antitumor effects in orthotopic lung cancer", THERANOSTICS, IVYSPRING INTERNATIONAL PUBLISHER, AU, vol. 9, no. 23, 1 January 2019 (2019-01-01), AU, pages 6936 - 6948, XP055815448, ISSN: 1838-7640, DOI: 10.7150/thno.37380 *
HOLDEN CHRISTOPHER A, YUAN QUAN, YEUDALL W ANDREW, LEBMAN DEBORAH A, YANG HU: "Surface engineering of macrophages with nanoparticles to generate a cell-nanoparticle hybrid vehicle for hypoxia-targeted drug delivery", INTERNATIONAL JOURNAL OF NANOMEDICINE, DOVE MEDICAL PRESS LTD., AUCKLAND, NZ, vol. 5, 2 February 2010 (2010-02-02), AUCKLAND, NZ, pages 25 - 36, XP055815180, ISSN: 1176-9114 *
LUCÍA MARTÍNEZ-JOTHAR ET AL.: "Insights into maleimide-thiol conjugation chemistry: Conditions for efficient surface functionalization of nanoparticles for receptor targeting,", JOURNAL OF CONTROLLED RELEASE,, vol. 282, 9 March 2018 (2018-03-09), XP085408440, DOI: 10.1016/j.jconrel.2018.03.002 *

Also Published As

Publication number Publication date
CN110859817A (zh) 2020-03-06

Similar Documents

Publication Publication Date Title
WO2021104088A1 (zh) 一种纳米颗粒载药系统及其制备方法和应用
Irvine et al. Enhancing cancer immunotherapy with nanomedicine
Li et al. Tailoring materials for modulation of macrophage fate
Liang et al. Recent advances in engineered materials for immunotherapy‐involved combination cancer therapy
Han et al. Biomaterial-assisted biotherapy: A brief review of biomaterials used in drug delivery, vaccine development, gene therapy, and stem cell therapy
US9468687B2 (en) Immuno gene therapy for treatment of cancer and hyperproliferative diseases
Gu et al. Nano-delivery systems focused on tumor microenvironment regulation and biomimetic strategies for treatment of breast cancer metastasis
Xie et al. Immunoengineering with biomaterials for enhanced cancer immunotherapy
Sharma et al. Recent advances of metal-based nanoparticles in nucleic acid delivery for therapeutic applications
Chen et al. Nanomaterials: small particles show huge possibilities for cancer immunotherapy
Dai et al. Nanomedicines modulating myeloid-derived suppressor cells for improving cancer immunotherapy
WO2021098621A1 (zh) 一种包载免疫佐剂纳米颗粒及应用
Mohaghegh et al. Injectable hydrogels for personalized cancer immunotherapies
CN107708668A (zh) 用作治疗性疫苗的纳米粒子
Han et al. Biomimetic nano-drug delivery system: an emerging platform for promoting tumor treatment
Deng et al. Ferritin in cancer therapy: A pleiotropic tumoraffin nanocage‐based transport
CN114887070A (zh) 一种脾脏靶向的纳米药物
Lu et al. Research progress in nano-drug delivery systems based on the characteristics of the liver cancer microenvironment
Shao et al. KERS‐Inspired Nanostructured Mineral Coatings Boost IFN‐γ mRNA Therapeutic Index for Antitumor Immunotherapy
Chen et al. Injectable thermo-sensitive hydrogel enhances anti-tumor potency of engineered Lactococcus lactis by activating dendritic cells and effective memory T cells
WO2023109704A1 (zh) 一种抗体合成菌-纳米刺激剂杂合体及其抗肿瘤应用
CN111281981A (zh) 泊洛沙姆和/或泊洛沙胺与peg脂质组合的复合物纳米粒
Yu et al. Combined application of nanotechnology and multiple therapies with tumor immune checkpoints
Liu et al. Extracellular Vesicles:“Stealth Transport Aircrafts” for Drugs
CN115137748B (zh) 介孔二氧化硅-氧化铈-miR129复合材料及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20892624

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20892624

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 11/01/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20892624

Country of ref document: EP

Kind code of ref document: A1