WO2019218594A1 - Nanoparticule phosphorescente hydrosoluble pour la détection d'acide hypochloreux à l'aide d'un procédé de rapport et procédé de préparation et application associés - Google Patents

Nanoparticule phosphorescente hydrosoluble pour la détection d'acide hypochloreux à l'aide d'un procédé de rapport et procédé de préparation et application associés Download PDF

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WO2019218594A1
WO2019218594A1 PCT/CN2018/111540 CN2018111540W WO2019218594A1 WO 2019218594 A1 WO2019218594 A1 WO 2019218594A1 CN 2018111540 W CN2018111540 W CN 2018111540W WO 2019218594 A1 WO2019218594 A1 WO 2019218594A1
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hypochlorous acid
water
nanoparticle
soluble
complex
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PCT/CN2018/111540
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English (en)
Chinese (zh)
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赵强
孟祥春
石玉祥
刘淑娟
陈泽晶
宋林娜
黄维
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南京邮电大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0033—Iridium compounds
    • 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
    • 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
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/185—Metal complexes of the platinum group, i.e. Os, Ir, Pt, Ru, Rh or Pd

Definitions

  • the invention belongs to the technical field of organic photoelectric functional materials, and particularly relates to a water-soluble phosphorescent nano particle which can be used for detecting hypochlorous acid by a ratio method, a preparation method thereof and an application of the nano particle to detect hypochlorous acid in the living body.
  • Active oxygen is a general term for a series of chemically active and oxidizing oxygen-containing substances produced by organisms.
  • Reactive oxygen contains both free radicals and some non-free radicals such as hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HClO), hydroxyl radicals (HO ⁇ ) and singlet oxygen ( 1 O 2 ).
  • Reactive oxygen species play an important role in biological systems.
  • HClO is a relatively common reactive oxygen species.
  • the endogenous HClO is mediated by myeloperoxidase (MPO).
  • MPO myeloperoxidase
  • ClO - has high reactivity, short life span and more physiological activity. The process is an important and powerful oxidant that exerts an anti-microbial effect under physiological conditions and protects the body. Studies have shown that HClO is also a natural adaptive immune adjuvant. However, under certain conditions, if the excess of HClO produced by the MPO catalytic reaction exceeds the defense reaction of the local antioxidant, it will cause oxidative stress and oxidative tissue damage.
  • Oxidative stress caused by excessive HClO has been shown to be associated with various diseases such as leukemia, nephritis, small vasculitis, tumors and atherosclerosis. Therefore, rapid, sensitive and real-time detection of hypochlorous acid has important physiological and pathological effects, which can provide reliable information for the pathogenesis, diagnosis and intervention of diseases.
  • Phosphorescent transition metal complexes such as Pt(II)-, Ir(III)-, Ru(II)-, Cu(I)-, Au(I)-, etc., have been used in the field of living cell imaging in recent years. To gradually attract people's attention.
  • the ruthenium complex exhibits special photoelectric properties in charge transfer and energy transfer between the metal center and the ligand, and has the advantages of high-efficiency triplet phosphorescence emission, long life, and large Stokes shift. It has not been found to be significantly toxic to cells and has great potential for application in cell biology imaging.
  • the present invention aims to provide a water-soluble phosphorescent nanoparticle capable of detecting hypochlorous acid by a ratio method and to disclose a preparation method thereof and related applications, and the ratio method can reduce interference factors and accurately and specifically detect hypochlorous acid.
  • the nanoparticle has excellent water solubility and biocompatibility, and has good application prospects in the fields of intracellular detection and living body detection.
  • a water-soluble phosphorescent nanoparticle for detecting hypochlorous acid by a ratio method characterized in that the structure is as follows:
  • C ⁇ N ligand in Ir(1-9) is any one of the following:
  • the N ⁇ N ligand in Ir(1-6) * is any one of the following:
  • water-soluble phosphorescent nanoparticles can be used for specific detection of hypochlorous acid by a ratio method.
  • water-soluble phosphorescent nanoparticles can be used in the field of cell sensing and in vivo imaging sensing.
  • water soluble phosphorescent nanoparticles can be used to establish a model of in vivo inflammation.
  • the invention has the beneficial effects that the present invention prepares a water-soluble phosphorescent nanoparticle by solving the problems of poor biocompatibility, poor water solubility, short life span and high toxicity of the small molecule hypochlorous acid probe in the prior art.
  • the nanoparticle binding ratio method realizes self-calibration, and can specifically detect the change of hypochlorous acid content; has a long emission life, combines time-resolved technology to eliminate interference of background fluorescent signal, and improves detection signal-to-noise ratio; meanwhile, the nanoparticle has Good water solubility and biocompatibility, can detect intracellular hypochlorous acid; low toxicity, less damage to biological samples, can achieve detection of hypochlorous acid in the living field.
  • Example 1 is a responsive ultraviolet absorption spectrum of ruthenium complex Ir1 to hypochlorite in Example 4 of the present invention
  • Figure 2 is a responsive ultraviolet absorption spectrum of ruthenium complex Ir1* to hypochlorite in Example 4 of the present invention
  • Figure 3 is the embodiment Ir1-ClO 5 * Ir1 embodiment of the present invention, hypochlorous acid in response - emission spectrum;
  • Example 6 is a statistical diagram showing ion exchange experiment results of Ir1 and Ir1* in Example 6 of the present invention
  • Example 7 is a TEM test chart of phosphorescent water-soluble nanoparticle Ir NPs in Example 7 of the present invention.
  • Example 6 is a DLS test chart of phosphorescent water-soluble nanoparticle Ir NPs in Example 8 of the present invention.
  • Figure 7 is an absorption spectrum diagram of the complexes Ir1, Ir1* and the nanoparticles Ir NPs in Example 9 of the present invention.
  • Figure 8 is a graph showing the titration spectrum of phosphorescent water-soluble nanoparticle Ir NPs in Example 10 of the present invention.
  • Figure 9 is a test chart showing the relationship between the ratio of two emission peaks (I 600 /I 680 ) in the titration emission spectrum of the phosphorescent water-soluble nanoparticle Ir NPs according to the concentration of NaClO in the tenth embodiment of the present invention
  • Figure 10 is a graph showing the experimental results of MTT cytotoxicity of phosphorescent water-soluble nanoparticle Ir NPs in Example 11 of the present invention.
  • Figure 11 is a cell confocal imaging map of phosphorescent water-soluble nanoparticle Ir NPs in Example 12 of the present invention.
  • the chemical reagents used in the present invention are all commercially available.
  • the instruments used include:
  • UV spectrometer UV-3600 UV-VIS-NIR, Shimadzu
  • the probe complex Ir1 (1.0 mg) and the reference complex Ir1* (0.9 mg) were dissolved in a certain amount of tetrahydrofuran (2.0 mL), and a solution of 10.0 mg of phospholipid polyethylene glycol in PBS (10.0 mL) was added. Mix quickly and sonicate for 2.0 min. Then, the mixture was purged with a nitrogen balloon to tetrahydrofuran, and finally centrifuged in an ultrafiltration centrifuge tube to obtain an orange-red emulsion product, which was lyophilized to obtain an orange-red solid, that is, a nanoparticle Ir NPs.
  • the ruthenium complex Ir1, Ir1* used in the present invention has a spectral test concentration of 10 ⁇ M, and the test solvent is a PBS solution mixed with 1% DMSO.
  • Figure 1 is a UV absorption spectrum of the probe complex Ir1 after adding different concentrations of hypochlorite.
  • Example 5 Responsive emission spectroscopy of hypobromite complexes Ir1 and Ir1*
  • the ruthenium complex Ir1, Ir1* used in the present invention has a spectral test concentration of 10 ⁇ M, and the test solvent is a PBS solution mixed with 1% DMSO.
  • the hypochlorite was 5 times the equivalent concentration, the response time was 1 minute, the remaining ions were 20 times the equivalent concentration, and the response time was 5 minutes.
  • the results are shown in Fig. 4.
  • the intensity of the emission peak of the reference complex Ir1* at 680 nm is almost constant, and the emission of the probe complex Ir1 at 680 nm is obtained.
  • the peak is specifically illuminated by hypochlorite. Therefore, Ir1* can be used as a reference and imaged with the Ir1 construction ratio method for specific detection of changes in hypochlorite.
  • Example 7 TEM test of phosphorescent water-soluble nanoparticle Ir NPs
  • the phosphorescent water-soluble nanoparticle Ir NPs was dissolved in ethanol, dropped on a copper mesh, and subjected to TEM test after being naturally volatilized. As a result, as shown in Fig. 5, the nanoparticles were regular in shape, uniform in distribution, and all of them were circular, and the particle radius was about 105 nm.
  • Example 8 DLS test of phosphorescent water-soluble nanoparticle Ir NPs
  • the phosphorescent water-soluble nanoparticle Ir NPs was dissolved in ultrapure water, and the bubbles were removed by ultrasonication, and the DLS test was performed. As a result, as shown in Fig. 6, the nanoparticles were concentrated and had a hydration kinetic radius of about 125 nm.
  • Example 9 Absorption spectroscopy test of complex Ir1, Ir1* and nanoparticle Ir NPs
  • the ruthenium complexes Ir1 and Ir1* used in the present invention have a spectral concentration of 10 ⁇ M
  • the test solvent is a PBS solution mixed with 1% DMSO
  • the nanoparticles Ir NPs is 1 mg/mL
  • the test solvent is a PBS solution.
  • the ultraviolet absorption spectrum of the nanoparticle Ir NPs contains characteristic absorption peaks of the ruthenium complexes Ir1 and Ir1*.
  • Example 11 MTT cytotoxicity assay of phosphorescent water-soluble nanoparticle Ir NPs
  • the digested cells were seeded in a 96-well plate at a seeding density of 10 4 cells/well per well, and culture was continued for 24 hours at 37 ° C under 5% CO 2 . After the old culture solution was aspirated, the cells were further cultured for 24 hours with cell culture solutions of different concentrations of Ir NPs (10, 50, 100, 200, 300 ⁇ g/mL). The culture was terminated by adding 10 ⁇ L of MTT (5 mg/mL) to each well and continuing the culture for 4 hours. The culture solution was aspirated, 150 ⁇ L of DMSO was added to each well, and the shaker was shaken for 10 minutes, and then the OD570 was tested using a microplate reader.
  • Fig. 10 The results of the MTT cytotoxicity experiment are shown in Fig. 10. It can be seen from the figure that when the concentration of the complex is 10 to 300 ⁇ g/mL, the cell survival rate after 24 hours of culture is greater than 90%, which proves that the nanoparticles have a lower concentration. Cytotoxicity can be used for cell imaging.
  • Example 12 Confocal imaging experiment of phosphorescent water-soluble nanoparticle Ir NPs
  • the cell confocal imaging experiment of the nanoparticles Ir NPs the experimental results are shown in Figure 11, using a concentration of 10 ⁇ g / mL.
  • the specific procedure was to incubate HeLa cells in a 37 ° C incubator for 24 hours, then incubate HeLa cells with nanoparticle Ir NPs for 1 hour at 37 ° C, and then incubate with different concentrations of hypochlorous acid culture. Confocal testing was performed at the end of the incubation. The test results are shown in Fig. 11. The luminescence of the green channel increases with the increase of sodium hypochlorite concentration, while the luminescence of the red channel does not change significantly.
  • the ratio of I 600nm /I 680nm increases with the increase of sodium hypochlorite. It is therefore possible to monitor changes in intracellular sodium hypochlorite by monitoring the luminescence and the ratio of the two. It is indicated that the phosphorescent water-soluble nanoparticle Ir NPs can specifically detect intracellular hypochlorous acid by ratiometric method combined with cell life imaging.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Optics & Photonics (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract

L'invention concerne une nanoparticule phosphorescente hydrosoluble pour la détection d'acide hypochloreux à l'aide d'un procédé de rapport, constituée de complexes d'iridium Ir(1‑9) et Ir(1‑6)* et de phospholipide-polyéthylène glycol. L'Ir(1‑9) peut répondre spécifiquement à l'acide hypochloreux, et l'Ir(1‑6)* est utilisé en tant que complexe de référence et ne répond pas à l'acide hypochloreux. L'invention concerne également un procédé de préparation d'une nanoparticule phosphorescente hydrosoluble.
PCT/CN2018/111540 2018-05-17 2018-10-24 Nanoparticule phosphorescente hydrosoluble pour la détection d'acide hypochloreux à l'aide d'un procédé de rapport et procédé de préparation et application associés WO2019218594A1 (fr)

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CN108535233B (zh) * 2018-05-17 2020-07-17 南京邮电大学 一种用于比率法检测次氯酸的水溶性磷光纳米粒子及其制备方法与应用
CN111233754B (zh) * 2020-01-19 2023-06-02 广东省生物工程研究所（广州甘蔗糖业研究所） 基于铂配合物的磷光探针及其在次氯酸检测中的应用

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