WO2023173824A1 - Hydrophobic methylene blue fluorescent dye, preparation method therefor and use thereof - Google Patents

Hydrophobic methylene blue fluorescent dye, preparation method therefor and use thereof Download PDF

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WO2023173824A1
WO2023173824A1 PCT/CN2022/137054 CN2022137054W WO2023173824A1 WO 2023173824 A1 WO2023173824 A1 WO 2023173824A1 CN 2022137054 W CN2022137054 W CN 2022137054W WO 2023173824 A1 WO2023173824 A1 WO 2023173824A1
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methylene blue
fluorescent dye
preparation
blue fluorescent
hydrophobic
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龚萍
马功成
刘中轲
蔡林涛
张鹏飞
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
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    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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    • 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
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    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
    • C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
    • C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
    • C07D279/18—[b, e]-condensed with two six-membered rings
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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Definitions

  • the invention relates to the field of material science, and in particular to a hydrophobic methylene blue fluorescent dye and its preparation method and application.
  • Photodynamic therapy is a treatment method based on the interaction of light, photosensitizer and tissue oxygen. Its mechanism of action is to use the photosensitizer to absorb photons under illumination of a specific wavelength, and the photosensitizer molecule changes from the singlet ground state to (S0) transitions to the singlet excited state (S1), and finally reaches the triplet excited state (T1), and in the process of decaying from the triplet excited state back to the ground state, it collides with tissue oxygen and transfers energy to generate reactive oxygen species (ROS). Inducing tumor cell necrosis and apoptosis to achieve the purpose of cancer treatment. Compared with traditional cancer treatments such as surgical resection, radiotherapy, and chemotherapy, PDT has the advantages of non-invasiveness, rapid treatment process, good cell killing effect, and repeatable operation, and has become a promising tumor treatment method.
  • ROS reactive oxygen species
  • Photosensitizers are different from ordinary chemotherapeutic drugs. They are a special chemical substance whose basic function is to transfer energy. It can absorb photons and be excited, and then quickly transfer the absorbed light energy to another patient. A group of molecules causes it to be excited to produce a photochemical reaction, while the photosensitizer itself does not participate in the chemical reaction and returns to its original state (ground state). As the core element of PDT, the main function of photosensitizer is to absorb light energy to generate ROS. The cytotoxic effect of ROS can cause cell apoptosis and eliminate tumors.
  • Methylene blue as a common dye-based photosensitizer, is often used in photodynamic antibacterial or antiviral treatments.
  • the currently commonly used new methylene blue photosensitizer is water-soluble. Although it is easy to dissolve, it has poor biocompatibility in mice, a short half-life, poor targeting, and difficulty in entering the cells.
  • the present invention proposes a hydrophobic methylene blue fluorescent dye.
  • the hydrophobic methylene blue fluorescent dye of the present invention has a structural formula shown in formula (I):
  • R is a saturated perfluoro chain and its isomers. "+" means carrying a positive charge.
  • saturated perfluoro chain and its isomers are selected from C 4 F 8 , C 5 F 11 , C 8 F 17 , C 9 F 19 , C 10 F 21 , C 11 F 23 , C 12 Any one of F 25 , C 15 F 31 .
  • the invention also provides a method for preparing the hydrophobic methylene blue fluorescent dye, which includes the following steps:
  • New methylene blue and perfluoroalkane are reacted at 80-150°C for 24-36 hours to generate perfluorodecane new methylene blue.
  • the structural formula of the new methylene blue is as shown in formula (II),
  • the mass ratio of the new methylene blue and the perfluoroalkane in step (1) is 1: (1.2 ⁇ 3), and the mass ratio of the anhydrous organic solvent to the new methylene blue is is (150 ⁇ 500): 1.
  • anhydrous organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.
  • step (2) the oil bath is heated to 80-150°C, 300-800 rpm, and the reaction is conducted in the dark for 24-36 hours.
  • drying time of the vacuum drying oven in step (3) is 24-36 hours.
  • methylene chloride is used for extraction in step (4).
  • the invention also provides the application of the hydrophobic methylene blue fluorescent dye in fluorescence imaging.
  • the invention also provides the application of the hydrophobic methylene blue fluorescent dye in photodynamic therapy.
  • the present invention synthesizes NMBF by connecting hydrophobic side chains to new methylene blue.
  • NMBF is very hydrophobic, so it can be wrapped into nanoparticles to enhance transportation and targeting functions.
  • folic acid, etc. can also be coupled to the nanoparticles.
  • Tumor-targeting groups or other disease-targeting groups facilitate the construction of treatment platforms.
  • Figure 1 is a schematic diagram of NMBF synthesis of the present invention
  • Figure 2 shows the dissolution and luminescence properties of NMBF of the present invention in water and DMSO;
  • Figure 3 is the ROS release detection of NMBF of the present invention.
  • Figure 4 is the ultraviolet absorption curve of NMBF of the present invention.
  • Figure 5 is a fluorescence luminescence image of NMBF of the present invention.
  • Figure 6 is the fluorescence luminescence curve of NMBF of the present invention.
  • Figure 7 is a fluorescence imaging picture of macrophages using NMBF of the present invention.
  • neomethylene blue and perfluoroalkyl iodide are reacted under conditions of 80-150°C for 24-36 hours to produce the neomethylene blue derivative perfluorodecane neomethylene blue NMBF of the invention.
  • perfluoroalkane is Iodide
  • the reaction is a substitution reaction, and the compound after the reaction does not contain iodide ions. It can be used as an AIE fluorescent dye for biological imaging, emitting strong fluorescence at 650nm, or using its AIE properties to achieve "Turn-on" and "Turn-off" of fluorescence.
  • NMBF can also be used as a photosensitizer for photodynamic therapy.
  • Example 1 Design and synthesis of the hydrophobic methylene blue fluorescent dye NMBF of the present invention
  • perfluoroalkyl iodide used in this embodiment is perfluorodecane iodide, and its chemical structural formula is as follows:
  • the reaction temperature may be 50-80°C.
  • the reaction temperature should be greater than 75°C, and as n increases, the reaction temperature should gradually increase.
  • the saturated perfluoro chain and its isomers have similar structures and properties, those skilled in the art can know that the side chain R groups are C 4 F 8 , C 5 F 11 , C 8 F 17 , C 9 F 19 , C 10 F 21 , C 11 F 23 , C 12 F 25 , and C 15 F 31 can all achieve the technical effects of the present invention.
  • the prepared NMBF precipitates in water (shown in circles) and has no fluorescence, because fluorescence quenching occurs when the dye is not dissolved.
  • NMBF has no fluorescence in water, proving that it is insoluble in water.
  • the strong luminescence in DMSO proves that NMBF is soluble in organic solvents.
  • step (3) Repeat step (3) 3 times for a total of 5 UV curves.
  • step (2) Reconfigure the solution according to step (2), and add DMSO-dissolved NMBF to a final concentration of 100-200 mM/L, keep the final concentration of DPBF consistent, and put it into a 1mL cuvette to measure the UV curve.
  • Figure 4 is the UV absorption curve of NMBF
  • Figure 5 is the luminescence picture of the NMBF solution under a fluorescence camera, indicating that it has luminescent properties.
  • Figure 6 shows the fluorescence emission curve of NMBF.
  • the excitation wavelength of fluorescence is 570nm, and the emission peak of fluorescence is 640nm.
  • the results are shown in Figure 7.
  • the red fluorescence is the fluorescence of NMBF, and the blue is the fluorescence of Hoechst.
  • the figures are all expressed in grayscale. It shows that the NMBF of the present invention can be used for fluorescence imaging.
  • the present invention discloses a hydrophobic methylene blue fluorescent dye and its preparation method and application.
  • the hydrophobic methylene blue fluorescent dye has the structural formula shown in formula (I), can effectively release ROS for photodynamic therapy or participate in subsequent reactions, and can emit strong fluorescence at 650nm for tumor imaging. It also has AIE properties and can Enhanced luminescence and strong hydrophobicity, it can be easily encapsulated into nanoparticles to enhance transport and targeting functions. Tumor-targeting groups such as folic acid or other disease-targeting groups can also be coupled to the nanoparticles. Conveniently build a treatment platform.

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Abstract

Disclosed in the present invention are a hydrophobic methylene blue fluorescent dye, and a preparation method therefor and the use thereof. The hydrophobic methylene blue fluorescent dye has a structural formula as shown in formula (I), can effectively release ROS for photodynamic therapy or participating in subsequent reactions, can emit strong fluorescence at 650 nm for tumor imaging, has AIE properties, can enhance the luminescence, and has a strong hydrophobicity, allowing for easy encapsulation in order to form a nanoparticle, and thereby enhancing the transporting and targeting functions.

Description

一种疏水性亚甲蓝荧光染料及其制备方法和应用A hydrophobic methylene blue fluorescent dye and its preparation method and application 技术领域Technical field
本发明涉及材料科学领域，特别涉及一种疏水性亚甲蓝荧光染料及其制备方法和应用。The invention relates to the field of material science, and in particular to a hydrophobic methylene blue fluorescent dye and its preparation method and application.
背景技术Background technique
光动力治疗法（PDT）是一种以光、光敏剂和组织氧相互作用为基础的治疗方法，其作用机制是利用光敏剂在特定波长的光照下吸收光子，光敏剂分子从单重态基态(S0)跃迁到单重态激发态(S1)，最终到达三重激发态(T1)，并在从三重激发态衰变回基态的过程中与组织氧发生碰撞传递能量，产生活性氧（ROS），诱导肿瘤细胞坏死和凋亡，达到癌症治疗的目的。相较于手术切除、放疗和化疗等传统癌症治疗手段，PDT具有非侵入性、治疗过程迅速、细胞杀伤效果好、可重复操作等优点，已成为具有发展前景的肿瘤治疗方法。Photodynamic therapy (PDT) is a treatment method based on the interaction of light, photosensitizer and tissue oxygen. Its mechanism of action is to use the photosensitizer to absorb photons under illumination of a specific wavelength, and the photosensitizer molecule changes from the singlet ground state to (S0) transitions to the singlet excited state (S1), and finally reaches the triplet excited state (T1), and in the process of decaying from the triplet excited state back to the ground state, it collides with tissue oxygen and transfers energy to generate reactive oxygen species (ROS). Inducing tumor cell necrosis and apoptosis to achieve the purpose of cancer treatment. Compared with traditional cancer treatments such as surgical resection, radiotherapy, and chemotherapy, PDT has the advantages of non-invasiveness, rapid treatment process, good cell killing effect, and repeatable operation, and has become a promising tumor treatment method.
光敏剂（光动力治疗药物）与通常的化学治疗药不同，它是一种特殊的化学物质，其基本作用是传递能量，它能够吸收光子而被激发，又将吸收的光能迅速传递给另一组分的分子，使其被激发产生光化学反应，而光敏剂本身则不参与化学反应，回到原来的状态（基态）。光敏剂作为PDT的核心要素，其主要作用是吸收光能产生ROS，ROS的细胞毒性作用可以使细胞凋亡，达到消灭肿瘤的作用。Photosensitizers (photodynamic therapy drugs) are different from ordinary chemotherapeutic drugs. They are a special chemical substance whose basic function is to transfer energy. It can absorb photons and be excited, and then quickly transfer the absorbed light energy to another patient. A group of molecules causes it to be excited to produce a photochemical reaction, while the photosensitizer itself does not participate in the chemical reaction and returns to its original state (ground state). As the core element of PDT, the main function of photosensitizer is to absorb light energy to generate ROS. The cytotoxic effect of ROS can cause cell apoptosis and eliminate tumors.
亚甲蓝作为一种常见染料型光敏剂，常用于光动力抗菌或抗病毒治疗。目前常用的新亚甲蓝光敏剂水溶性，虽然方便溶解，但在小鼠体内生物相容性差，半衰期短，且靶向性差，难以入胞。Methylene blue, as a common dye-based photosensitizer, is often used in photodynamic antibacterial or antiviral treatments. The currently commonly used new methylene blue photosensitizer is water-soluble. Although it is easy to dissolve, it has poor biocompatibility in mice, a short half-life, poor targeting, and difficulty in entering the cells.
技术问题technical problem
针对现有技术中的缺陷，本发明提出了一种疏水性亚甲蓝荧光染料。In view of the defects in the prior art, the present invention proposes a hydrophobic methylene blue fluorescent dye.
技术解决方案Technical solutions
本发明的疏水性亚甲蓝荧光染料具有如式（I）所示的结构式：The hydrophobic methylene blue fluorescent dye of the present invention has a structural formula shown in formula (I):
（I）(I)
其中，R为饱和全氟链及其同分异构体。“＋”表示带一个正电荷。Among them, R is a saturated perfluoro chain and its isomers. "+" means carrying a positive charge.
进一步的，所述饱和全氟链及其同分异构体选自C 4F 8、C 5F 11、C 8F 17、C 9F 19、C 10F 21、C 11F 23、C 12F 25、C 15F 31中的任意一种。 Further, the saturated perfluoro chain and its isomers are selected from C 4 F 8 , C 5 F 11 , C 8 F 17 , C 9 F 19 , C 10 F 21 , C 11 F 23 , C 12 Any one of F 25 , C 15 F 31 .
本发明还提供所述的疏水性亚甲蓝荧光染料的制备方法，包括如下步骤：The invention also provides a method for preparing the hydrophobic methylene blue fluorescent dye, which includes the following steps:
将新亚甲蓝与全氟烷在80-150℃条件下反应24-36小时生成全氟癸烷新亚甲蓝，所述新亚甲蓝的结构式如式（II）所示，New methylene blue and perfluoroalkane are reacted at 80-150°C for 24-36 hours to generate perfluorodecane new methylene blue. The structural formula of the new methylene blue is as shown in formula (II),
（II）(II)
所述全氟癸烷新亚甲蓝结构式如式（I）所示。The structural formula of the perfluorodecane neomethylene blue is shown in formula (I).
进一步的，具体包括如下步骤：Further, the specific steps include:
（1）将新亚甲蓝与全氟烷碘装入容器，随后加入无水有机溶剂，1-2滴三乙胺；容器内充入氮气；(1) Put new methylene blue and perfluoroalkyl iodide into a container, then add anhydrous organic solvent and 1-2 drops of triethylamine; fill the container with nitrogen;
（2）在油浴锅加热，避光反应；(2) Heat in an oil bath and avoid light reaction;
（3）抽滤，除去多余固体；将抽滤得到的溶液旋转蒸发干燥，干燥后的固体放真空干燥箱继续干燥；(3) Suction filtration to remove excess solids; rotary evaporate the solution obtained by suction filtration to dryness, and place the dried solids in a vacuum drying oven to continue drying;
（4）干燥后的固体用水完全溶解，随后萃取，并旋转蒸发干燥；(4) The dried solid is completely dissolved in water, then extracted, and dried by rotary evaporation;
（5）干燥后的固体用水清洗去除水溶性杂志，收集剩下固体，真空干燥后得到本发明的疏水性亚甲蓝荧光染料。(5) Wash the dried solid with water to remove water-soluble impurities, collect the remaining solid, and obtain the hydrophobic methylene blue fluorescent dye of the present invention after vacuum drying.
进一步的，所述步骤（1）中所述新亚甲蓝和所述全氟烷的质量比为1：（1.2~3），所述无水有机溶剂与所述新亚甲蓝的质量比为（150~500）：1。Further, the mass ratio of the new methylene blue and the perfluoroalkane in step (1) is 1: (1.2~3), and the mass ratio of the anhydrous organic solvent to the new methylene blue is is (150~500): 1.
进一步的，所述无水有机溶剂为N,N-二甲基甲酰胺或二甲基亚砜。Further, the anhydrous organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.
进一步的，所述步骤（2）中油浴锅加热至80-150℃，300~800rpm，避光反应24-36 h。Further, in step (2), the oil bath is heated to 80-150°C, 300-800 rpm, and the reaction is conducted in the dark for 24-36 hours.
进一步的，所述步骤（3）中真空干燥箱干燥时间为24-36小时。Further, the drying time of the vacuum drying oven in step (3) is 24-36 hours.
进一步的，所述步骤（4）中使用二氯甲烷萃取。Further, methylene chloride is used for extraction in step (4).
本发明还提供所述的疏水性亚甲蓝荧光染料在荧光成像中的应用。The invention also provides the application of the hydrophobic methylene blue fluorescent dye in fluorescence imaging.
本发明还提供所述的疏水性亚甲蓝荧光染料在光动力治疗中的应用。The invention also provides the application of the hydrophobic methylene blue fluorescent dye in photodynamic therapy.
有益效果beneficial effects
综上，与现有技术相比，本发明达到了以下技术效果：In summary, compared with the prior art, the present invention achieves the following technical effects:
本发明通过在新亚甲蓝上连接疏水性侧链合成NMBF，NMBF疏水性很强，因此可以将其包裹成纳米颗粒，增强运输与靶向功能，同时也可以在纳米颗粒上耦连叶酸等肿瘤靶向基团或其他疾病靶向基团，方便构建治疗平台。The present invention synthesizes NMBF by connecting hydrophobic side chains to new methylene blue. NMBF is very hydrophobic, so it can be wrapped into nanoparticles to enhance transportation and targeting functions. At the same time, folic acid, etc. can also be coupled to the nanoparticles. Tumor-targeting groups or other disease-targeting groups facilitate the construction of treatment platforms.
附图说明Description of the drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，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相关的附图。In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.
图1为本发明的      NMBF合成示意图；Figure 1 is a schematic diagram of NMBF synthesis of the present invention;
图2为本发明的NMBF在水中和DMSO中的溶解和发光性质；Figure 2 shows the dissolution and luminescence properties of NMBF of the present invention in water and DMSO;
图3为本发明的NMBF的ROS释放检测；Figure 3 is the ROS release detection of NMBF of the present invention;
图4为本发明的NMBF的紫外吸收曲线；Figure 4 is the ultraviolet absorption curve of NMBF of the present invention;
图5为本发明的NMBF的荧光发光图像；Figure 5 is a fluorescence luminescence image of NMBF of the present invention;
图6为本发明的NMBF的荧光发光曲线；Figure 6 is the fluorescence luminescence curve of NMBF of the present invention;
图7为使用本发明的NMBF的巨噬细胞的荧光成像图片。Figure 7 is a fluorescence imaging picture of macrophages using NMBF of the present invention.
本发明的实施方式Embodiments of the invention
为了使本技术领域的人员更好地理解本发明方案，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，所描述的实施例仅仅是本发明一部分的实施例，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的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.
本发明将新亚甲蓝与全氟烷碘在80-150℃条件下反应24-36小时生成本发明的新亚甲蓝衍生物全氟癸烷新亚甲蓝NMBF，反应前为全氟烷碘化物，反应是取代反应，反应后的化合物中不包含碘离子。它可以作为AIE荧光染料进行生物成像，在650nm处发出强烈荧光，或者利用其AIE性质实现荧光的“Turn-on”与“Turn-off”。NMBF同时也可以作为光敏剂进行光动力治疗。In the present invention, neomethylene blue and perfluoroalkyl iodide are reacted under conditions of 80-150°C for 24-36 hours to produce the neomethylene blue derivative perfluorodecane neomethylene blue NMBF of the invention. Before the reaction, perfluoroalkane is Iodide, the reaction is a substitution reaction, and the compound after the reaction does not contain iodide ions. It can be used as an AIE fluorescent dye for biological imaging, emitting strong fluorescence at 650nm, or using its AIE properties to achieve "Turn-on" and "Turn-off" of fluorescence. NMBF can also be used as a photosensitizer for photodynamic therapy.
实施例 1 本发明的疏水性亚甲蓝荧光染料NMBF的设计合成 Example 1 Design and synthesis of the hydrophobic methylene blue fluorescent dye NMBF of the present invention
具体步骤如下：Specific steps are as follows:
（1）将40-70mg新亚甲蓝与80mg全氟烷碘装入50mL圆底烧瓶，随后加入8-10mL DMF，1-2滴三乙胺。在圆底烧瓶口接三通阀，三通阀一端接充满氮气的气球。(1) Put 40-70 mg of new methylene blue and 80 mg of perfluoroalkyl iodide into a 50 mL round-bottomed flask, then add 8-10 mL of DMF and 1-2 drops of triethylamine. Connect a three-way valve to the mouth of the round-bottomed flask, and connect one end of the three-way valve to a balloon filled with nitrogen.
本实施例使用的全氟烷碘是全氟癸烷碘，其化学结构式如下：The perfluoroalkyl iodide used in this embodiment is perfluorodecane iodide, and its chemical structural formula is as follows:
； ;
当2＜n＜5时，反应温度可以为50-80℃。当n＞5时，反应温度应大于75℃，且随着n的增加反应温度应该逐渐提高。When 2<n<5, the reaction temperature may be 50-80°C. When n>5, the reaction temperature should be greater than 75°C, and as n increases, the reaction temperature should gradually increase.
因为饱和全氟链及其同分异构体具有相似的结构和性质，本领域技术人员可以知晓侧链R基为C 4F 8、C 5F 11、C 8F 17、C 9F 19、C 10F 21、C 11F 23、C 12F 25、C 15F 31都可以实现本发明的技术效果。 Because the saturated perfluoro chain and its isomers have similar structures and properties, those skilled in the art can know that the side chain R groups are C 4 F 8 , C 5 F 11 , C 8 F 17 , C 9 F 19 , C 10 F 21 , C 11 F 23 , C 12 F 25 , and C 15 F 31 can all achieve the technical effects of the present invention.
（2）在油浴锅加热至80-90℃，500rpm，避光反应24h。(2) Heat in an oil bath to 80-90°C, 500 rpm, and react in the dark for 24 hours.
（3）抽滤，除去多余固体。将抽滤得到的溶液旋蒸，旋干后的固体放真空干燥箱干燥24h。(3) Suction filtration to remove excess solids. The solution obtained by suction filtration was rotary evaporated, and the spin-dried solid was dried in a vacuum drying oven for 24 hours.
（4）干燥后的固体用水完全溶解，随后用二氯甲烷萃取，并旋蒸旋干。(4) The dried solid is completely dissolved in water, then extracted with dichloromethane, and evaporated to dryness.
（5）旋干后的固体用水清洗去除水溶性杂志，收集剩下固体，真空干燥后得到产物。(5) The spin-dried solid is washed with water to remove water-soluble impurities, the remaining solid is collected, and the product is obtained after vacuum drying.
图2所示，制备得到的NMBF在水中沉淀（圈内所示），且没有荧光，因为染料不溶解时会发生荧光淬灭，NMBF在水中无荧光，证明其不溶于水。而在DMSO中发光强烈，证明NMBF溶于有机溶剂。As shown in Figure 2, the prepared NMBF precipitates in water (shown in circles) and has no fluorescence, because fluorescence quenching occurs when the dye is not dissolved. NMBF has no fluorescence in water, proving that it is insoluble in water. The strong luminescence in DMSO proves that NMBF is soluble in organic solvents.
实施例 2  利用DPBF（1,3-二苯基异苯并呋喃）检测ROS释放 Example 2 Using DPBF (1,3-diphenylisobenzofuran) to detect ROS release
具体步骤如下：Specific steps are as follows:
（1）将紫外分光光度计用超纯水校准基线。(1) Calibrate the baseline of the UV spectrophotometer with ultrapure water.
（2）将1-2 mg DPBF溶于DMSO，随后吸取8-10μL加入1mL超纯水，混匀后加入1mL比色皿，并测试其紫外曲线。(2) Dissolve 1-2 mg DPBF in DMSO, then add 8-10 μL to 1 mL of ultrapure water, mix well, add 1 mL cuvette, and test its UV curve.
（3）将比色皿取出，用功率5W的660nm激光器照射4-5min，再次测试其紫外曲线。(3) Take out the cuvette, irradiate it with a 660nm laser with a power of 5W for 4-5 minutes, and test its UV curve again.
（4）再重复第（3）步3次，共计5条紫外曲线。(4) Repeat step (3) 3 times for a total of 5 UV curves.
（5）取每条紫外曲线在415nm处的数值，按时间顺序做折线图。(5) Take the value of each UV curve at 415nm and make a line chart in chronological order.
（6）按步骤（2）重新配置溶液，并加入DMSO溶解的的NMBF使其终浓度100-200 mM/L，并保持DPBF终浓度一致，装入1mL比色皿测紫外曲线。(6) Reconfigure the solution according to step (2), and add DMSO-dissolved NMBF to a final concentration of 100-200 mM/L, keep the final concentration of DPBF consistent, and put it into a 1mL cuvette to measure the UV curve.
（7）重复（3）（4）（5）步，比较加入NMBF前后曲线变化。(7) Repeat steps (3) (4) (5) and compare the changes in the curve before and after adding NMBF.
图3的结果说明NMBF在激光照射下能够释放ROS，是一种优秀的光敏剂。The results in Figure 3 illustrate that NMBF can release ROS under laser irradiation and is an excellent photosensitizer.
实施例 3紫外分光光度计测试NMBF的紫外吸收曲线以及NMBF溶液荧光图片 Example 3 UV absorption curve of NMBF tested by UV spectrophotometer and fluorescence picture of NMBF solution
具体步骤如下：Specific steps are as follows:
（1）将NMBF溶于DMSO，浓度为100-200mM/L，加入1mL比色皿。(1) Dissolve NMBF in DMSO at a concentration of 100-200mM/L, and add it to a 1mL cuvette.
（2）用紫外光谱仪测试其在200-700nm范围内的紫外吸收曲线。(2) Use a UV spectrometer to test its UV absorption curve in the range of 200-700nm.
（3）取NMBF的DMSO溶液，浓度100-1000μg/mL共1mL于1.5mL试管中。(3) Take 1 mL of DMSO solution of NMBF with a concentration of 100-1000 μg/mL in a 1.5 mL test tube.
（4）用580nm激光器照射下，用荧光成像相机拍摄的此时NMBF溶液的发光状态。(4) The luminescence state of the NMBF solution was captured with a fluorescence imaging camera under irradiation with a 580nm laser.
图4为NMBF的紫外吸收曲线，图5为荧光相机下NMBF溶液的发光图片，表明其具有发光性质。Figure 4 is the UV absorption curve of NMBF, and Figure 5 is the luminescence picture of the NMBF solution under a fluorescence camera, indicating that it has luminescent properties.
实施例 4荧光光谱仪证实NMBF在650nm处有较强荧光 Example 4 Fluorescence spectrometer confirms that NMBF has strong fluorescence at 650nm
具体步骤如下：Specific steps are as follows:
（1）将NMBF溶于DMSO，浓度为100-200mM/L，加入1mL比色皿。(1) Dissolve NMBF in DMSO at a concentration of 100-200mM/L, and add it to a 1mL cuvette.
（2）用紫外光谱仪测试其吸收峰在570-620nm。(2) Use a UV spectrometer to test the absorption peak at 570-620nm.
（3）用570nm激发发现其在650nm有较强的荧光。(3) Using 570nm excitation, it was found that it has strong fluorescence at 650nm.
图6为NMBF的荧光发射曲线，荧光的激发波长为570nm，荧光的发射峰值为640nm。Figure 6 shows the fluorescence emission curve of NMBF. The excitation wavelength of fluorescence is 570nm, and the emission peak of fluorescence is 640nm.
实施例 5  NMBF在细胞中的荧光成像 Example 5 Fluorescence imaging of NMBF in cells
具体步骤如下：Specific steps are as follows:
（1）25mL培养瓶培养巨噬细胞，并传代至8孔共聚焦培养板。(1) Cultivate macrophages in a 25mL culture bottle and passage them into an 8-well confocal culture plate.
（2）将NMBF溶于DMSO，浓度1-2mg/mL。(2) Dissolve NMBF in DMSO at a concentration of 1-2 mg/mL.
（3）待共聚焦培养板中细胞数量长到3-5w时，将（2）中溶液按体积比1:100加入共聚焦培养板，使其在培养基中浓度为10-20μg/mL，共同孵育20min。(3) When the number of cells in the confocal culture plate reaches 3-5w, add the solution in (2) to the confocal culture plate at a volume ratio of 1:100 so that its concentration in the culture medium is 10-20 μg/mL. Incubate together for 20 minutes.
（4）按说明书进行Hoechst（靶向细胞核的荧光染料）染色.(4) Perform Hoechst (fluorescent dye targeting the nucleus) staining according to the instructions.
（5）将培养板利用共聚焦显微镜成像，通道设置为580nm激发，进行共聚焦成像。(5) Image the culture plate using a confocal microscope, set the channel to 580nm excitation, and perform confocal imaging.
结果见图7，红色荧光为NMBF的荧光，蓝色为Hoechst的荧光，图总均以灰度表示。说明本发明的NMBF可以用于荧光成像。The results are shown in Figure 7. The red fluorescence is the fluorescence of NMBF, and the blue is the fluorescence of Hoechst. The figures are all expressed in grayscale. It shows that the NMBF of the present invention can be used for fluorescence imaging.
综合以上实施例，本发明公开了一种疏水性亚甲蓝荧光染料及其制备方法和应用。所述疏水性亚甲蓝荧光染料具有式（I）所示的结构式，可以有效释放ROS进行光动力治疗或参与后续反应，并在650nm处能发出强烈荧光进行肿瘤成像，同时具有AIE性质，可以增强发光，而且疏水性很强，可以方便地被包载形成纳米颗粒，增强运输与靶向功能，同时也可以在纳米颗粒上耦连叶酸等肿瘤靶向基团或其他疾病靶向基团，方便构建治疗平台。Based on the above embodiments, the present invention discloses a hydrophobic methylene blue fluorescent dye and its preparation method and application. The hydrophobic methylene blue fluorescent dye has the structural formula shown in formula (I), can effectively release ROS for photodynamic therapy or participate in subsequent reactions, and can emit strong fluorescence at 650nm for tumor imaging. It also has AIE properties and can Enhanced luminescence and strong hydrophobicity, it can be easily encapsulated into nanoparticles to enhance transport and targeting functions. Tumor-targeting groups such as folic acid or other disease-targeting groups can also be coupled to the nanoparticles. Conveniently build a treatment platform.
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 are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

  1. 一种疏水性亚甲蓝荧光染料，其特征在于，所述疏水性亚甲蓝荧光染料具有如式（I）所示的结构式： A hydrophobic methylene blue fluorescent dye, characterized in that the hydrophobic methylene blue fluorescent dye has a structural formula as shown in formula (I):
    （I）(I)
    其中，R为饱和全氟链及其同分异构体。Among them, R is a saturated perfluoro chain and its isomers.
  2. 根据权利要求1所述的疏水性亚甲蓝荧光染料，其特征在于，所述饱和全氟链及其同分异构体选自C 4F 8、C 5F 11、C 8F 17、C 9F 19、C 10F 21、C 11F 23、C 12F 25、C 15F 31中的任意一种。 The hydrophobic methylene blue fluorescent dye according to claim 1, characterized in that the saturated perfluoro chain and its isomers are selected from C 4 F 8 , C 5 F 11 , C 8 F 17 , C Any one of 9 F 19 , C 10 F 21 , C 11 F 23 , C 12 F 25 , and C 15 F 31 .
  3. 权利要求1的疏水性亚甲蓝荧光染料的制备方法，其特征在于，包括如下步骤： The preparation method of hydrophobic methylene blue fluorescent dye according to claim 1, characterized in that it includes the following steps:
    将新亚甲蓝与全氟烷碘在80-150℃条件下反应24-36小时生成全氟癸烷新亚甲蓝，所述新亚甲蓝的结构式如式（II）所示，Neomethylene blue and perfluoroalkyl iodide are reacted at 80-150°C for 24-36 hours to generate perfluorodecane neomethylene blue. The structural formula of the neomethylene blue is as shown in formula (II),
    （II）(II)
    所述全氟癸烷新亚甲蓝结构式如式（I）所示。The structural formula of the perfluorodecane neomethylene blue is shown in formula (I).
  4. 根据权利要求3所述的制备方法，其特征在于，具体包括如下步骤： The preparation method according to claim 3, characterized in that it specifically includes the following steps:
    （1）将新亚甲蓝与全氟烷碘装入容器，随后加入无水有机溶剂，1-2滴三乙胺；容器内充入氮气；(1) Put new methylene blue and perfluoroalkyl iodide into a container, then add anhydrous organic solvent and 1-2 drops of triethylamine; fill the container with nitrogen;
    （2）在油浴锅加热，避光反应；(2) Heat in an oil bath and avoid light reaction;
    （3）抽滤，除去多余固体；将抽滤得到的溶液旋转蒸发干燥，干燥后的固体放真空干燥箱继续干燥；(3) Suction filtration to remove excess solids; rotary evaporate the solution obtained by suction filtration to dryness, and place the dried solids in a vacuum drying oven to continue drying;
    （4）干燥后的固体用水完全溶解，随后萃取，并旋转蒸发干燥；(4) The dried solid is completely dissolved in water, then extracted, and dried by rotary evaporation;
    （5）干燥后的固体用水清洗去除水溶性杂志，收集剩下固体，真空干燥后得到本发明的疏水性亚甲蓝荧光染料。(5) Wash the dried solid with water to remove water-soluble impurities, collect the remaining solid, and obtain the hydrophobic methylene blue fluorescent dye of the present invention after vacuum drying.
  5. 根据权利要求4所述的制备方法，其特征在于，所述步骤（1）中所述新亚甲蓝和所述全氟烷的质量比为1：（1.2~3），所述无水有机溶剂与所述新亚甲蓝的质量比为（150~500）：1。 The preparation method according to claim 4, characterized in that the mass ratio of the new methylene blue and the perfluoroalkane in the step (1) is 1: (1.2~3), and the anhydrous organic The mass ratio of the solvent to the new methylene blue is (150~500):1.
  6. 根据权利要求4所述的制备方法，其特征在于，所述无水有机溶剂为N,N-二甲基甲酰胺或二甲基亚砜。 The preparation method according to claim 4, characterized in that the anhydrous organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.
  7. 根据权利要求4所述的制备方法，其特征在于，所述步骤（2）中油浴锅加热至80-150℃，300~800 rpm，避光反应24-36 h。 The preparation method according to claim 4, characterized in that in the step (2), the oil bath is heated to 80-150°C, 300-800 rpm, and the reaction is conducted in the dark for 24-36 hours.
  8. 根据权利要求4所述的制备方法，其特征在于，所述步骤（4）中使用二氯甲烷萃取。 The preparation method according to claim 4, characterized in that dichloromethane is used for extraction in step (4).
  9. 权利要求1~2任一项所述的疏水性亚甲蓝荧光染料在荧光成像中的应用。 Application of the hydrophobic methylene blue fluorescent dye described in any one of claims 1 to 2 in fluorescence imaging.
  10. 权利要求1~2任一项所述的疏水性亚甲蓝荧光染料在光动力治疗中的应用。 Application of the hydrophobic methylene blue fluorescent dye described in any one of claims 1 to 2 in photodynamic therapy.
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