WO2021115072A1 - 一种巨噬细胞示踪荧光探针的制备方法及应用 - Google Patents

一种巨噬细胞示踪荧光探针的制备方法及应用 Download PDF

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WO2021115072A1
WO2021115072A1 PCT/CN2020/129547 CN2020129547W WO2021115072A1 WO 2021115072 A1 WO2021115072 A1 WO 2021115072A1 CN 2020129547 W CN2020129547 W CN 2020129547W WO 2021115072 A1 WO2021115072 A1 WO 2021115072A1
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macrophage
freeze
room temperature
fluorescent probe
dialysis
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French (fr)
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郑海荣
胡德红
罗新平
盛宗海
马腾
高笃阳
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof

Definitions

  • the invention relates to the field of material synthesis and application, in particular to a preparation method and application of a dextran nanoprobe targeted to macrophages.
  • Malignant tumors are a type of disease that seriously threatens the health of residents. According to the latest statistics, deaths from malignant tumors account for 23.91% of the total deaths in my country. In the past ten years, the incidence and deaths of malignant tumors have continued to increase. The medical expenses caused by tumors exceed 220 billion, and the prevention and control situation is severe. It is urgent to develop real-time, quantitative, and sensitive tumor monitoring probes. Direct targeting and monitoring of tumor cells is a common idea, and the tumor area is not only tumor cells, but also infiltrated with many immune cells that regulate tumor process, forming a tumor matrix environment, which can regulate tissue remodeling and angiogenesis, so it is not only It is important to monitor the division, apoptosis, resting, and molecular expression of tumor cells. It is also important to monitor the tumor matrix environment.
  • macrophages which are an important component of the innate immune process. They are distributed from monocytes in the bone marrow to different parts of the body and differentiate into resident macrophages in various tissues. It can usually play a positive role in removing foreign and harmful substances, but the cytokines secreted by tumor cells at the tumor site are taught to be harmful cells, forming tumor-associated macrophages (TAM), and promoting tumor growth, proliferation, invasion and metastasis , Immunosuppression, vascular and lymphangiogenesis, and establish a suitable stromal environment for tumor cells to survive, leading to poor prognosis for tumor patients. Therefore, targeting macrophages at the tumor site, developing tumor treatment strategies against macrophages, and developing corresponding The means of evaluation is very important.
  • TAM tumor-associated macrophages
  • TAM tumor area macrophages
  • methods such as depleting tumor area macrophages, inhibiting tumor area macrophage recruitment, and promoting TAM phenotype transformation.
  • preclinical and clinical studies have shown good tumor prognosis.
  • removing TAM in pancreatic cancer models can reduce tumor resistance to gemcitabine. Therefore, screening, monitoring, and evaluation of these methods are very important.
  • CT electronic computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission computed tomography
  • fluorescence imaging and so on.
  • CT is the most commonly used clinical diagnostic method, but it requires a large number of nanoparticles to trace macrophages, even at the molar level, and the high atomic number elements that need to be used are usually highly toxic. These two points limit the display of this method.
  • PET can achieve rapid and quantitative whole-body imaging, but it has high requirements for equipment and radiation. It can only be done 1-2 times a year, and it cannot provide accurate anatomical positioning.
  • MRI Magnetic resonance Imaging
  • Gadolinium (Gd) is a commonly used MRI contrast agent in medical treatment. It is the main object of MRI contrast agent research. However, due to its small molecular weight, there are problems such as short half-life. In summary, traditional imaging detection techniques have played an important role in tracing macrophages, but they all have some limitations.
  • fluorescence imaging Compared with the commonly used clinical imaging methods such as ultrasound, MRI, CT, PET, fluorescence imaging has the advantages of fast imaging speed, high sensitivity, no ionizing radiation, simple equipment, etc., and it can be used in early detection of cancer and guided surgical treatment during surgery. Playing an increasingly important role. Even if MRI, PET and other research are carried out, fluorescent tracing methods are needed to optimize the design and verify the targeting mechanism and effect at the cell level. In fact, most of the results of how macrophages and other cells dispose of nanoparticles are fluorescent imaging. Therefore, fluorescence imaging has unparalleled unique advantages in tracking macrophages to screen macrophage therapies.
  • Indocyanine green is a diagnostic reagent currently used in clinical practice. It has high safety and good imaging effect. In recent years, it has been found that in addition to the traditional near-infrared fluorescence imaging, it is in the near-infrared fluorescence.
  • the second zone (NIR-II, 1000-1700 nm) also has a good fluorescence imaging effect.
  • the second zone of near-infrared fluorescence has less tissue absorption and scattering and lower tissue autofluorescence characteristics in living tissues, which can greatly improve the tissue penetration depth and spatial resolution of fluorescence imaging, and greatly reduce the traditional fluorescence imaging of the first zone.
  • the limitations of low penetration depth and low spatial resolution have broad application prospects in biomedical imaging.
  • contrast agent delivery systems such as the modification of CD206 antibody or small molecule mannose to target the CD206 protein on the surface of macrophages (also known as Mannose receptor), etc.
  • antibody targeting has good specificity, but it is difficult to purify and expensive, and small molecule modification has the problem of being easily eliminated by kidney metabolism.
  • the main technical problem solved by the present invention is to provide a method for preparing a macrophage tracer probe with simple method, mild reaction conditions, good reproducibility, low toxicity and good biocompatibility and its application.
  • a technical solution adopted by the present invention is:
  • a preparation method of macrophage tracer fluorescent probe includes the following steps:
  • S140 Perform dialysis on the filtrate at room temperature, filter the membrane after the dialysis, pre-freeze at the first temperature, transfer to the second temperature to freeze, and then freeze-dry to obtain a freeze-dried powder;
  • the crosslinking agent is lysine.
  • the dextran is at least one carboxymethyl dextran with a molecular weight of 2-40 kD and a degree of substitution of carboxyl groups of 2%-10%.
  • the activating carboxyl reagent is at least one of EDC, DCC, CDI and DIC.
  • the tracer small molecule is at least one of a fluorescence imaging small molecule or an MRI imaging small molecule.
  • the small tracer molecule is at least one of COOH-ICG, CN-ICG and Gd-DOT.
  • the microfiltration membrane is a 0.22 ⁇ m filter membrane.
  • the room temperature dialysis is room temperature dialysis with a 10 kD dialysis bag using ultrapure water as a dialysis medium for 0.5-5 days.
  • said pre-freezing at the first temperature and then transferring to freezing at the second temperature, and then freeze-drying to obtain the freeze-dried powder pre-freezing in the refrigerator at -20 degrees for 2 h, and transferring to the refrigerator at-80 degrees for freezing For 24 hours, freeze-dry in a freeze dryer for 48 hours to obtain freeze-dried powder.
  • a technical solution adopted by the present invention is to apply the macrophage tracing fluorescent probe obtained by the method for preparing the macrophage tracing fluorescent probe in the targeting of macrophages.
  • the present invention synthesizes a macrophage-targeting dextran nanoprobe, which has better macrophage targeting ability and higher biological safety.
  • This probe chemically cross-links carboxymethyl dextran with one of the essential amino acids, lysine, to form uniform and stable dextran cross-linked nanoparticles, on which the indole cyanine is covalently bonded Green and other imaging small molecules have constructed nanoparticles with both macrophage targeting ability and imaging ability.
  • the invention helps to promote the research and development of macrophage tracing, and at the same time provides new theories and new methods for the early diagnosis and prognosis of tumors.
  • the preparation method of the present invention is simple, the reaction conditions are mild, the reproducibility is good, the toxicity is low, the biocompatibility is good, and it has a wide range of application prospects.
  • the experimental results prove that the probe prepared by the present invention is non-toxic to cells. And it has strong targeting of macrophages.
  • Fig. 1 is a schematic diagram of a synthetic route of a macrophage tracking probe according to an embodiment
  • Figure 2 shows the particle size and TEM characterization of the macrophage tracking fluorescent probe in Example 1, with a scale of 50 ⁇ m;
  • Figure 3 is a graph showing the effects of the same amount of probes prepared in Example 1 on the cell viability of three types of cells;
  • Figure 4 shows the near-infrared two-zone imaging when the same amount of probes prepared in Example 1 are added to different cells.
  • a preparation method of macrophage tracer fluorescent probe includes the following steps:
  • the dextran is at least one of carboxymethyl dextran having a molecular weight of 2-40 kD and a degree of substitution of carboxyl groups of 2%-10%.
  • the activating carboxyl reagent is at least one of EDC, DCC, CDI and DIC.
  • the buffer can be MES buffer or PBS buffer.
  • the crosslinking agent is lysine.
  • the microfiltration membrane is 0.22 ⁇ m filter membrane.
  • S140 Perform dialysis on the filtrate at room temperature, filter the membrane after the dialysis, pre-freeze at the first temperature, transfer to the second temperature to freeze, and then freeze-dry to obtain a freeze-dried powder;
  • the room temperature dialysis is to perform room temperature dialysis with a 10 kD dialysis bag using ultrapure water as a dialysis medium for 0.5-5 days.
  • pre-frozen at the first temperature and then transferred to the second temperature to freeze, and then freeze-dried to obtain the freeze-dried powder pre-frozen at -20 degrees refrigerator for 2 h, and transferred to-80 degrees refrigerator Freeze for 24 hours and freeze-dry in a freeze dryer for 48 hours to obtain freeze-dried powder.
  • the tracer small molecule is at least one of a fluorescence imaging small molecule or an MRI imaging small molecule.
  • the small tracer molecule is at least one of COOH-ICG, CN-ICG, and Gd-DOT.
  • the microfiltration membrane is 0.22 ⁇ m filter membrane.
  • Room temperature dialysis is to use ultrapure water as the dialysis medium to perform room temperature dialysis with a 10 kD dialysis bag for 0.5-5 days.
  • a method for preparing a macrophage tracking fluorescent probe includes the following steps:
  • step 3 Add the clear solution obtained in step 2 dropwise to (0.03-30 mL) pre-cooled absolute ethanol, and centrifuge (0.25-2.5 k ⁇ g, 3-30 min) Collect the white precipitate, re-dissolve the obtained white precipitate in water, and pass through a 0.22 ⁇ m filter membrane to obtain the filtrate.
  • step 4 Use ultrapure water as the dialysis medium for the filtrate obtained in step 3 to perform room temperature dialysis (0.5-5 days) with a 10 kD dialysis bag. After dialysis, pass through a 0.22 ⁇ m filter membrane and pre-freeze in a refrigerator at -20 degrees for 2 hours. Transfer to-80 degrees refrigerator and freeze for 24 hours, freeze-dry in a freeze dryer for 48 hours to obtain freeze-dried powder.
  • step 6 Use ultrapure water as the dialysis medium for the solution obtained in step 5 to perform room temperature dialysis (0.5-5 days) with a 10 kD dialysis bag to remove free contrast small molecules, and concentrate to (50-200 ⁇ L) with a 10 kD ultrafiltration tube A concentrated solution is obtained.
  • step 3 Add the clear solution obtained in step 2 dropwise to 30 mL of pre-cooled absolute ethanol, collect the white precipitate by centrifugation (2.5 k ⁇ g, 3 min), and re-dissolve the obtained white precipitate in water. 0.22 ⁇ m filter membrane.
  • step 5 Accurately weigh 0.05 g of the lyophilized powder obtained in step 4, add 0.003 g EDC and 0.001 g NHS, dissolve in 0.2 mL dimethyl sulfoxide, add 0.001 g carboxylated ICG, and react gently at room temperature, protected from light, for 7 hours.
  • step 5 The solution obtained in step 5 is dialyzed at room temperature with a 10 kD dialysis bag using ultrapure water as the dialysis medium (0.5 days) to remove free ICG, and concentrated to (150 ⁇ L) concentrated solution with a 10 kD ultrafiltration tube.
  • step 3 Add the clear solution obtained in step 2 dropwise to 6 mL of pre-cooled absolute ethanol, collect the white precipitate by centrifugation (2.5 k ⁇ g, 3 min), and re-dissolve the obtained white precipitate in water. 0.22 ⁇ m filter membrane.
  • step 5 Accurately weigh 0.05 g of the lyophilized powder obtained in step 4, add 0.003 g EDC and 0.001 g NHS, dissolve in 0.2 mL dimethyl sulfoxide, add 0.0005 g carboxylated ICG, and react gently at room temperature, protected from light, for 7 hours.
  • step 5 The solution obtained in step 5 is dialyzed at room temperature with a 10 kD dialysis bag using ultrapure water as the dialysis medium (0.5 days) to remove free ICG, and concentrated to (150 ⁇ L) concentrated solution with a 10 kD ultrafiltration tube.
  • step 3 Add the clear solution obtained in step 2 dropwise to 12 mL of pre-cooled absolute ethanol, centrifuge (2.5 k ⁇ g, 3 min) to collect the white precipitate, and redissolve the obtained white precipitate in water. 0.22 ⁇ m filter membrane.
  • step 5 Accurately weigh 0.05 g of the lyophilized powder obtained in step 4, add 0.003 g EDC and 0.001 g NHS, dissolve in 0.2 mL dimethyl sulfoxide, add 0.002 g carboxylated ICG, and react gently at room temperature, protected from light, for 7 hours.
  • step 5 The solution obtained in step 5 is dialyzed at room temperature with a 10 kD dialysis bag using ultrapure water as the dialysis medium (0.5 days) to remove free ICG, and concentrated to (150 ⁇ L) concentrated solution with a 10 kD ultrafiltration tube.
  • step 3 Add the clear solution obtained in step 2 dropwise to 12 mL of pre-cooled absolute ethanol, centrifuge (2.5 k ⁇ g, 3 min) to collect the white precipitate, and redissolve the obtained white precipitate in water. 0.22 ⁇ m filter membrane.
  • step 5 The solution obtained in step 5 is dialyzed at room temperature with a 10 kD dialysis bag using ultrapure water as the dialysis medium (0.5 days) to remove free gadoteric acid (DOTA), and concentrated to (150 ⁇ L) with a 10 kD ultrafiltration tube liquid.
  • DOTA free gadoteric acid
  • Pancreatic cancer cell lines SW1990, SW1990-mcherry-Luc and macrophage cell line RAW264.7 are preserved in our laboratory. These three cell lines are commonly used in tumor research and can be purchased on the market.
  • the dextran-ICG nanoparticles obtained in Example 1 were diluted with DMEM medium in equal proportions to an appropriate concentration, and the cytotoxicity of the probe on SW1990, SW1990-mcherry-Luc, and RAW264.7 was determined by the CCK-8 rapid colorimetric method. .
  • Example 6 Targeting effect of the probe on macrophages.
  • pancreatic cancer cell line SW1990 is preserved in our laboratory. This cell is a commonly used cell line and can be purchased on the market. Macrophages are extracted from mouse bone marrow. This extraction method is a relatively mature and simple cell extraction method.
  • the dextran probe obtained in Example 1 was diluted with DMEM medium or the like to an appropriate concentration, and added to the two kinds of cells respectively and incubated for 4 hours, and the near-infrared two-zone imaging of the cells (wavelength after 1000 nm) was observed with a confocal microscope.
  • Example 2 2. Add the SW1990 cells in logarithmic growth phase and the extracted macrophages into an eight-well chamber at 1 ⁇ 5*10 4 cells/well, and culture them overnight until they adhere to the wall, and then use the appropriate concentration of the obtained in Example 1 respectively. Incubate the dextran probe in DMEM medium at 37°C for 4 hours, aspirate the probes that are not bound to the cells, and wash the cells 3 times with PBS to remove the unbound dextran probes, and observe the near-infrared two of the cells with a confocal microscope Area imaging situation (wavelength after 1000 nm).

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Abstract

本发明涉及一种巨噬细胞示踪荧光探针的制备方法及其应用，本探针以人体必需氨基酸之一赖氨酸对羧甲基葡聚糖进行化学交联，形成均一、稳定的葡聚糖交联纳米颗粒，在此纳米颗粒上以共价键连接吲哚菁绿等成像小分子，构建了兼具巨噬细胞靶向能力和成像能力的纳米颗粒。本发明制备的探针具有较好的巨噬细胞靶向能力和较高的生物安全性。

Description

一种巨噬细胞示踪荧光探针的制备方法及应用 技术领域
本发明涉及材料合成及应用领域，具体而言，涉及一种靶向巨噬细胞的葡聚糖纳米探针的制备方法及其应用。
 背景技术
恶性肿瘤是严重威胁居民健康的一类疾病，根据最新的统计数据显示，恶性肿瘤死亡占我国居民总死亡人数的23.91％，且近十几年来恶性肿瘤的发病死亡均呈持续上升态势,每年恶性肿瘤所致的医疗花费超过2200亿，防控形势严峻，发展实时、定量、灵敏的肿瘤监测探针十分迫切。直接靶向并监测肿瘤细胞是一种常见的思路，而肿瘤区域不仅仅只有肿瘤细胞，也浸润着很多调控肿瘤进程的免疫细胞，构成肿瘤基质环境，可调控组织重塑和血管生成，因此不仅监测肿瘤细胞的分裂、凋亡、静息和分子表达等很重要，监测肿瘤基质环境同样很重要。
其中，肿瘤基质环境中占据数量最多的调控细胞是巨噬细胞，其为先天性免疫过程的重要组分，由骨髓中单核细胞分布至全身各处分化为各组织中的驻留巨噬细胞，通常可以发挥清除外来、有害物质等积极作用，但在肿瘤部位却被肿瘤细胞分泌的细胞因子教育为有害细胞，形成肿瘤相关巨噬细胞（TAM），促进肿瘤的生长、增殖、侵袭、转移、免疫抑制、血管及淋巴管生成，为肿瘤细胞建立起适宜生存的基质环境，从而导致肿瘤患者的恶劣预后，因而靶向肿瘤部位巨噬细胞、发展针对巨噬细胞的肿瘤治疗策略并发展相应的评估手段十分重要。
当今靶向TAM以干预肿瘤的手段越来越多，且正在快速推进至临床应用，主要包括消耗肿瘤区域巨噬细胞、抑制肿瘤区域巨噬细胞招募、促进TAM表型转化等方法，有些手段在临床前和临床研究中都表现出了很好的肿瘤预后，如通过清除胰腺癌模型中的TAM可降低肿瘤对吉西他滨的耐药性等，故筛选、监测、评估这些手段十分重要。
这迫切需要示踪肿瘤相关巨噬细胞的成像手段及对应成像造影剂。当前示踪肿瘤相关巨噬细胞的方式有电子计算机 X 线断层扫描（CT）、磁共振成像（MRI）、正电子发射计算机断层显像（PET）和荧光成像等等。CT 是现在临床最常用的一种诊断手段，但是需要很大量纳米颗粒才能示踪巨噬细胞，甚至摩尔水平，而且需要用到的高原子序数元素通常毒性高，这两点限制了该手段示踪巨噬细胞的临床应用。PET能够实现快速定量全身成像，但对仪器要求较高，有辐射，一年只能做1~2次，且不能提供精确的解剖学定位。MRI 是一种临床上常用的成像手段，无电离辐射，亚毫米分辨率，能清楚显示肿瘤区域和血管的关系，对肿瘤手术可切除性的判断具有重要作用，但 MRI 的空间分辨率较差，对早期肿瘤的诊断作用有限，钆（Gd）为医疗中常用的一种MRI造影剂，是MRI显影剂研究的主要对象，但因分子量小，存在半衰期短等问题。综上所述，传统影像学检测技术对于巨噬细胞的示踪发挥了重要作用，但均存在一些局限性。与临床常用的超声、MRI、CT、PET 等影像方法相比，荧光成像具有成像速度快、灵敏度高、无电离辐射、仪器设备简单等优点，在癌症的早期检测和术中引导手术治疗等方面发挥着越来越重要的作用。即使开展MRI、PET等研究，也需要荧光示踪手段来进行优化设计、细胞层面验证靶向机制和效果，事实上现今大多数关于巨噬细胞及其他细胞如何处置纳米粒的结果都是荧光成像得到的，因此荧光成像在示踪巨噬细胞从而筛选巨噬细胞疗法有着无可比拟的独特优势。
吲哚菁绿（ICG）是当前已用于临床的一种诊断试剂，安全性较高，成像效果较好，且近几年来研究发现其除了传统近红外荧光一区成像外，在近红外荧光二区(NIR-II, 1000-1700 nm)也具有较好的荧光成像效果。近红外荧光二区在活体组织中具有更少的组织吸收和散射以及更低组织自发荧光特性,可以大大提高荧光成像的组织穿透深度和空间分辨率，大大减弱了传统荧光一区成像组织穿透深度低和空间分辨率低的局限性，在生物医学影像中具有广阔的应用前景，看得深、看得清，成像时器官轮廓清晰，有助于实时清晰观测各器官的颗粒分布情况。尤其在胰腺癌活体成像方面，肝脏和胰腺位置靠得很近，传统荧光成像很难区分清楚，二区荧光成像有着绝对的优势。然而吲哚菁绿存在光学稳定性差等缺陷，通过化学修饰等手段增强其稳定性是利用其进行巨噬细胞荧光成像的难点和关键问题。
有了荧光成像、MRI成像等合适的成像手段和造影剂，如何使造影剂特异性靶向肿瘤相关巨噬细胞便成了示踪肿瘤相关巨噬细胞的关键问题。当前靶向肿瘤相关巨噬细胞的策略包括在造影剂递送系统表面修饰巨噬细胞特异性配体、抗体等，如修饰CD206抗体或小分子甘露糖以靶向巨噬细胞表面CD206蛋白（又称甘露糖受体）等，抗体靶向具有很好的特异性，但纯化困难、费用昂贵，小分子修饰存在易被肾脏代谢清除的问题。
有鉴于此，有必要提出一种新的方法，以解决上述问题。
 技术解决方案
本发明主要解决的技术问题是提供一种方法简便、反应条件温和、重现性好、低毒、生物相容性好的巨噬细胞示踪探针制备方法和其应用。为解决上述技术问题，本发明采用的一个技术方案是：
一种巨噬细胞示踪荧光探针的制备方法，包括以下步骤：
S110、将羧甲基化葡聚糖、NHS和sulfo-NHS中的一个、和活化羧基试剂置于缓冲液中，室温搅拌反应得到活化好的羧甲基化葡聚糖溶液；
S120、将交联剂溶解在缓冲液中，加入活化好的羧甲基化葡聚糖溶液，室温搅拌反应后得到澄清溶液；
S130、将澄清溶液滴加至预冷的无水乙醇中去，离心得到沉淀，将得到的沉淀复溶于水，过微滤膜得到过滤液；
S140、将过滤液进行室温透析，透析结束后过滤膜，于第一温度下预冻后转移至第二温度下冷冻, 再冷冻干燥得到冻干粉；
S150、将活化羧基试剂和NHS溶于二甲基亚砜中，加入示踪小分子，示踪小分子活化后加之冻干粉的水溶液中，避光室温反应得到反应液；
S160、将反应液中的游离造影小分子去除后浓缩得到浓缩液；
S170、将丁二酸酐溶于二甲基亚砜中，加入催化量三乙胺与所述浓缩液避光室温反应，室温透析后过微滤膜，得到巨噬细胞示踪荧光探针。
在其中一个实施例，所述交联剂为赖氨酸。
在其中一个实施例，所述葡聚糖为分子量为2~40 kD且羧基取代度为2%~10%的羧甲基葡聚糖的至少一种。
在其中一个实施例，所述的活化羧基试剂为EDC、DCC、CDI和DIC中至少一种。
在其中一个实施例，所述示踪小分子为荧光成像小分子或MRI成像小分子中的至少一种。
在其中一个实施例，所述示踪小分子为COOH-ICG、CN-ICG和Gd-DOT中的至少一种。
在其中一个实施例，所述微滤膜为0.22μm滤膜。
在其中一个实施例，所述室温透析为以超纯水为透析介质用10 kD透析袋进行室温透析0.5-5天。
在其中一个实施例，所述于第一温度下预冻后转移至第二温度下冷冻, 再冷冻干燥得到冻干粉为：于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
为解决上述技术问题，本发明采用的一个技术方案是：将上述巨噬细胞示踪荧光探针的制备方法得到的巨噬细胞示踪荧光探针在巨噬细胞的靶向性中的应用。
本发明的有益效果是：相对于现有技术，本发明合成了一种靶向巨噬细胞的葡聚糖纳米探针，具有较好的巨噬细胞靶向能力和较高的生物安全性，本探针以人体必需氨基酸之一赖氨酸对羧甲基葡聚糖进行化学交联，形成均一、稳定的葡聚糖交联纳米颗粒，在此纳米颗粒上以共价键连接吲哚菁绿等成像小分子，构建了兼具巨噬细胞靶向能力和成像能力的纳米颗粒。本发明有助于推动巨噬细胞示踪的研究与发展，同时为肿瘤的早期诊断和预后提供新理论和新方法。此外，本发明的制备方法简便、反应条件温和、重现性好、低毒、生物相容性好，具有广泛的应用前景，经实验结果证明，通过本发明制备的探针对细胞无毒性，且对巨噬细胞的靶向性强。
 附图说明
图1是一实施方式的巨噬细胞示踪探针的合成路线示意图；
图2为实施例1巨噬细胞示踪荧光探针的粒径和TEM表征，比例尺为50 μm；
图3为实施例1制备的等量探针分别对三种细胞的细胞活性影响图；
图4为实施例1制备的等量探针分别加入不同细胞中时的近红外二区成像情况。
 本发明的实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。
一种巨噬细胞示踪荧光探针的制备方法，包括以下步骤：
S110、将羧甲基化葡聚糖、NHS和sulfo-NHS中的一个、和活化羧基试剂置于缓冲液中，室温搅拌反应得到活化好的羧甲基化葡聚糖溶液。
具体地，在一实施方式中，葡聚糖为分子量为2~40 kD且羧基取代度为2%~10%的羧甲基葡聚糖的至少一种。活化羧基试剂为EDC、DCC、CDI和DIC中至少一种。缓冲液可以为MES缓冲液或PBS缓冲液。
S120、将交联剂溶解在缓冲液中，加入至活化好的羧甲基化葡聚糖溶液中，室温搅拌反应后得到澄清溶液。
具体地，在一实施方式中，交联剂为赖氨酸。
S130、将澄清溶液滴加至预冷的无水乙醇中去，离心得到沉淀，将得到的沉淀复溶于水，过微滤膜得到过滤液；
具体地，在一实施方式中，微滤膜为0.22 μm滤膜。
S140、将过滤液进行室温透析，透析结束后过滤膜，于第一温度下预冻后转移至第二温度下冷冻, 再冷冻干燥得到冻干粉；
具体地，在一实施方式中，室温透析为以超纯水为透析介质用10 kD透析袋进行室温透析0.5-5天。
具体地，在一实施方式中，于第一温度下预冻后转移至第二温度下冷冻, 再冷冻干燥得到冻干粉为：于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
S150、将活化羧基试剂和NHS溶于二甲基亚砜中，加入示踪小分子，示踪小分子活化后加之冻干粉的水溶液中，避光室温反应得到反应液；
具体地，在一实施方式中，示踪小分子为荧光成像小分子或MRI成像小分子中的至少一种。具体地，示踪小分子为COOH-ICG、CN-ICG和Gd-DOT中的至少一种。
S160、将反应液中的离造影小分子去除后浓缩得到浓缩液；
S170、将丁二酸酐溶于二甲基亚砜中，加入催化量三乙胺与所述浓缩液避光室温反应，室温透析后过微滤膜，得到巨噬细胞示踪荧光探针。
具体地，在一实施方式中，微滤膜为0.22 μm滤膜。室温透析为以超纯水为透析介质用10 kD透析袋进行室温透析0.5-5天。
具体地，一种巨噬细胞示踪荧光探针的制备方法，包括以下步骤：
1、准确称取（0.00055-0.55g）羧甲基化葡聚糖，加入（0.0024-2.4 g）EDC活化羧基试剂和（0.0004572-0.4572 mg）NHS，溶于（0.0062-6.2 mL）MES缓冲液（50mM，pH 6.0-6.5）中，室温温和搅拌反应（0.25-48 h）。
2、准确称取交联剂 L-赖氨酸（0.0004-0.4 g），用（0.0007-0.7 mL）MES缓冲液（50mM，pH 6.0-6.5）溶解，加之步骤1活化好的羧甲基化葡聚糖溶液中，室温温和搅拌反应（5-48 h）。
3、将步骤2得到的澄清溶液逐滴滴加至（0.03-30 mL）预冷的无水乙醇中去，离心（0.25-2.5 k ×g，3-30 min）收集白色沉淀，将得到的白色沉淀复溶于水，过0.22 μm滤膜得到过滤液。
4、将步骤3得到的过滤液以超纯水为透析介质用10 kD透析袋进行室温透析（0.5-5天），透析结束后过0.22 μm滤膜，于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
5、准确称取（0.00002-0.02 g）步骤4得到的冻干粉，加入（0.000003-0.003 g）EDC和（0.000001-0.001 g）NHS，溶于（0.0002-0.2 mL）二甲基亚砜中，加入（0.000001-0.001 g）羧基化ICG或钆特酸（Gd-DOTA）示踪小分子，避光室温温和反应（1-48 h）。
6、将步骤5得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（0.5-5天），除去游离造影小分子，用10 kD超滤管浓缩至（50-200 μL）得到浓缩液。
7、称取（0.00015-0.015 g）丁二酸酐，溶于（0.0001-0.1 mL）二甲基亚砜中，加入催化量三乙胺（0.00001-0.01 μL）与浓缩液避光室温温和反应（0.5-48 h），以超纯水为透析介质用10 kD透析袋进行室温透析（0.5-5天），过0.22 μm滤膜，得到探针。
实施例1：
1、准确称取0.55 g羧甲基化葡聚糖，加入2.4 g EDC和0.4572 g NHS，溶于6.2 mL MES缓冲液（50mM，pH 6.0-6.5）中，室温温和搅拌反应10分钟。
2、准确称取 L-赖氨酸0.4 g，用0.7 mL MES缓冲液（50mM，pH 6.0-6.5）溶解，加入步骤1活化好的羧甲基化葡聚糖溶液中去，室温温和搅拌反应5 h。
3、将步骤2得到的澄清溶液逐滴滴加至30 mL预冷的无水乙醇中去，离心（2.5 k ×g，3 min）收集白色沉淀，将得到的白色沉淀复溶于水，过0.22 μm滤膜。
4、将步骤3得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（3天），透析结束后过0.22 μm滤膜，于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
5、准确称取0.05 g步骤4得到的冻干粉，加入0.003 g EDC和0.001 g NHS，溶于0.2 mL二甲基亚砜中，加入0.001 g羧基化ICG，避光室温温和反应7 h。
6、将步骤5得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），除去游离ICG，用10 kD超滤管浓缩至（150 μL）浓缩液。
7、称取0.015 g丁二酸酐，溶于0.1 mL二甲基亚砜中，加入催化量三乙胺4 μL，与浓缩液避光室温温和反应18 h，以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），过0.22 μm滤膜，得到最终探针溶液。
实施例2：
1、准确称取0.11 g羧甲基化葡聚糖，加入0.48g EDC和0.09144 g NHS，溶于1.24 mL MES缓冲液（50mM，pH 6.0-6.5）中，室温温和搅拌反应10分钟。
2、准确称取 L-赖氨酸0.08 g，用0.14 mL MES缓冲液（50mM，pH 6.0-6.5）溶解，加入步骤1活化好的羧甲基化葡聚糖溶液中去，室温温和搅拌反应5 h。
3、将步骤2得到的澄清溶液逐滴滴加至6 mL预冷的无水乙醇中去，离心（2.5 k ×g，3 min）收集白色沉淀，将得到的白色沉淀复溶于水，过0.22 μm滤膜。
4、将步骤3得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（3天），透析结束后过0.22 μm滤膜，于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
5、准确称取0.05 g步骤4得到的冻干粉，加入0.003 g EDC和0.001 g NHS，溶于0.2 mL二甲基亚砜中，加入0.0005 g羧基化ICG，避光室温温和反应7 h。
6、将步骤5得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），除去游离ICG，用10 kD超滤管浓缩至（150 μL）浓缩液。
7、称取0.015 g丁二酸酐，溶于0.1 mL二甲基亚砜中，加入催化量三乙胺4 μL，与浓缩液避光室温温和反应18 h，以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），过0.22 μm滤膜，得到最终探针溶液。
实施例3：
1、准确称取0.22 g羧甲基化葡聚糖，加入0.96g EDC和0.18288 g NHS，溶于2.48 mL MES缓冲液（50mM，pH 6.0-6.5）中，室温温和搅拌反应10分钟。
2、准确称取 L-赖氨酸0.16 g，用0.28 mL MES缓冲液（50mM，pH 6.0-6.5）溶解，加入步骤1活化好的羧甲基化葡聚糖溶液中去，室温温和搅拌反应5 h。
3、将步骤2得到的澄清溶液逐滴滴加至12 mL预冷的无水乙醇中去，离心（2.5 k ×g，3 min）收集白色沉淀，将得到的白色沉淀复溶于水，过0.22 μm滤膜。
4、将步骤3得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（3天），透析结束后过0.22 μm滤膜，于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
5、准确称取0.05 g步骤4得到的冻干粉，加入0.003 g EDC和0.001 g NHS，溶于0.2 mL二甲基亚砜中，加入0.002 g羧基化ICG，避光室温温和反应7 h。
6、将步骤5得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），除去游离ICG，用10 kD超滤管浓缩至（150 μL）浓缩液。
7、称取0.015 g丁二酸酐，溶于0.1 mL二甲基亚砜中，加入催化量三乙胺4 μL，与浓缩液避光室温温和反应18 h，以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），过0.22 μm滤膜，得到最终探针溶液。
实施例4：
1、准确称取0.22 g羧甲基化葡聚糖，加入0.96g EDC和0.18288 g NHS，溶于2.48 mL MES缓冲液（50mM，pH 6.0-6.5）中，室温温和搅拌反应10分钟。
2、准确称取 L-赖氨酸0.16 g，用0.28 mL MES缓冲液（50mM，pH 6.0-6.5）溶解，加入步骤1活化好的羧甲基化葡聚糖溶液中去，室温温和搅拌反应5 h。
3、将步骤2得到的澄清溶液逐滴滴加至12 mL预冷的无水乙醇中去，离心（2.5 k ×g，3 min）收集白色沉淀，将得到的白色沉淀复溶于水，过0.22 μm滤膜。
4、将步骤3得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（3天），透析结束后过0.22 μm滤膜，于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
5、准确称取0.05 g步骤4得到的冻干粉，加入0.003 g EDC和0.001 g NHS，溶于0.2 mL二甲基亚砜中，加入0.002 g钆特酸（DOTA），室温温和反应7 h。
6、将步骤5得到的溶液以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），除去游离钆特酸（DOTA），用10 kD超滤管浓缩至（150 μL）浓缩液。
7、称取0.015 g丁二酸酐，溶于0.1 mL二甲基亚砜中，加入催化量三乙胺4 μL，与浓缩液避光室温温和反应18 h，以超纯水为透析介质用10 kD透析袋进行室温透析（0.5天），过0.22 μm滤膜，得到最终探针溶液。
实施例5：探针对细胞无毒性实验
比较葡聚糖-ICG纳米探针对两株胰腺癌肿瘤细胞SW1990、SW1990-mcherry-Luc和巨噬细胞RAW264.7的细胞毒性作用，证明探针的生物相容性。
1、胰腺癌细胞株SW1990、SW1990-mcherry-Luc以及巨噬细胞株RAW264.7由本实验室保存，该三株细胞皆属于肿瘤研究中常用细胞株，均能通过市场购买获得。将实施例1所得葡聚糖-ICG纳米颗粒用DMEM培养基等比例稀释成适当浓度，采用CCK-8快速比色法测定探针对SW1990、SW1990-mcherry-Luc、RAW264.7的细胞毒性作用。
2、将对数生长期的SW1990、SW1990-mcherry-Luc、RAW264.7细胞以1~5*10 4个/孔加入九十六孔板中，过夜培养至贴壁，然后分别用含有相应浓度实施例1所得葡聚糖-ICG的DMEM培养基培养24h，每孔加入10 ul CCK-8，37℃，5% CO2及饱和湿度的培养箱中孵育1h，450nm酶标仪检测。
结果如图3所示，从图3中可以看到实施例1所得探针对细胞无明显杀伤作用，证明探针对细胞无毒。
实施例6：探针对巨噬细胞的靶向作用。
1、胰腺癌细胞株SW1990由本实验室保存，该细胞为常用细胞株，能通过市场购买获得，巨噬细胞由小鼠骨髓中提取得到，该提取方法为较为成熟、简单的细胞提取方法。将实施例1所得葡聚糖探针用DMEM培养基等稀释成适当浓度，分别加至两种细胞中孵育4h，用共聚焦显微镜观测细胞的近红外二区成像情况（1000 nm以后波段）。
2、将对数生长期的SW1990细胞和提取得到的巨噬细胞分别以1~5*10 4个/孔加入八孔小室中，过夜培养至贴壁，然后分别用适当浓度的实施例1所得葡聚糖探针DMEM培养基于37℃培养4h，吸去未与细胞结合的探针，并用PBS洗涤细胞3遍以除去未结合的葡聚糖探针，用共聚焦显微镜观测细胞的近红外二区成像情况（1000 nm以后波段）。
结果如图4所示，从图4中可以看到在加入等量葡聚糖探针后，明显巨噬细胞成像亮于胰腺癌细胞SW1990，说明巨噬细胞吞噬探针多于胰腺癌细胞，可证明探针对于巨噬细胞的靶向性。
以上仅为本发明的实施方式，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (10)

  1. 一种巨噬细胞示踪荧光探针的制备方法，其特征在于，包括以下步骤：
    S110、将羧甲基化葡聚糖、NHS或sulfo-NHS中的一个、和活化羧基试剂置于缓冲液中，室温搅拌反应得到活化好的羧甲基化葡聚糖溶液；
    S120、将交联剂溶解在缓冲液中，加入活化好的羧甲基化葡聚糖溶液，室温搅拌反应后得到澄清溶液；
    S130、将澄清溶液滴加至预冷的无水乙醇中去，离心得到沉淀，将得到的沉淀复溶于水，过微滤膜得到过滤液；
    S140、将过滤液进行室温透析，透析结束后过滤膜，于第一温度下预冻后转移至第二温度下冷冻, 再冷冻干燥得到冻干粉；
    S150、将活化羧基试剂、NHS溶于二甲基亚砜中，加入示踪小分子，示踪小分子活化后加之冻干粉的水溶液中，避光室温反应得到反应液；
    S160、将反应液中的游离造影小分子去除后浓缩得到浓缩液；
    S170、将丁二酸酐溶于二甲基亚砜中，加入催化量三乙胺与所述浓缩液避光室温反应，室温透析后过微滤膜，得到巨噬细胞示踪荧光探针。
  2. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述交联剂为赖氨酸。
  3. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述葡聚糖为分子量为2~40 kD且羧基取代度为2%~10%的羧甲基葡聚糖的至少一种。
  4. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述的活化羧基试剂为EDC、DCC、CDI和DIC中至少一种。
  5. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述示踪小分子为荧光成像小分子或MRI成像小分子中的至少一种。
  6. 根据权利要求5所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述示踪小分子为COOH-ICG、CN-ICG和Gd-DOT中的至少一种。
  7. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述微滤膜为0.22μm滤膜。
  8. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述室温透析为以超纯水为透析介质用10 kD透析袋进行室温透析0.5-5天。
  9. 根据权利要求1所述巨噬细胞示踪荧光探针的制备方法，其特征在于，所述于第一温度下预冻后转移至第二温度下冷冻, 再冷冻干燥得到冻干粉为：于- 20 度冰箱预冻2 h, 转移至- 80度冰箱冷冻24 h, 冷冻干燥机中冷冻干燥48 h, 得到冻干粉。
  10. 根据权利要求1-9任意一项权利要求所述巨噬细胞示踪荧光探针的制备方法得到的巨噬细胞示踪荧光探针在巨噬细胞的靶向性中的应用。
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