CN110967326A - Near-infrared light-emitting binuclear ruthenium complex as tumor cell recognition and imaging reagent - Google Patents

Near-infrared light-emitting binuclear ruthenium complex as tumor cell recognition and imaging reagent Download PDF

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CN110967326A
CN110967326A CN201911274064.XA CN201911274064A CN110967326A CN 110967326 A CN110967326 A CN 110967326A CN 201911274064 A CN201911274064 A CN 201911274064A CN 110967326 A CN110967326 A CN 110967326A
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ruthenium complex
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王克志
张思琪
刘慧宇
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Beijing Normal University
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Abstract

The invention discloses application of a near-infrared luminescent binuclear ruthenium (II) complex as a tumor cell (HeLa) recognition probe and a lysosome positioning probe. The fluorescence intensity of the complex in tumor cells is obviously higher than that of normal cells (HEK), and the recognition of the tumor cells can be realized. In addition, after entering cells, the complex is targeted and positioned at tumor cell lysosomes, and is almost free of cytotoxicity under the condition of imaging concentration, so that long-time real-time fluorescence monitoring of the cell lysosomes can be realized. The complex has great development potential as a tumor cell imaging reagent.

Description

Near-infrared light-emitting binuclear ruthenium complex as tumor cell recognition and imaging reagent
Technical Field
The patent belongs to the field of fluorescent biomolecular probes, and relates to application of a binuclear ruthenium complex in the related fields of tumor cell identification, lysosome positioning fluorescent probes and the like.
Background
At present, living cell imaging and biomolecular tracing become research hotspots in the fields of biomedicine, material chemistry and the like. The laser scanning confocal technology has unique optical sectioning and three-dimensional reconstruction capability, can dynamically observe cell and molecular changes in real time, and has high resolution, so the laser scanning confocal fluorescence microscope has become one of the most advanced tools for fluorescence imaging and cell research (S.A. Stricker, Microsc.Res.Techniq.,1999,6, 356-369). Compared with the traditional organic small molecule fluorescent probe, the near infrared luminescent ruthenium complex has the advantages of high adjustable ground state and excited state property, large Stokes shift, difficult photobleaching, strong biological tissue penetrability and the like, and has very wide biological fluorescent imaging application prospect (F.Tang, X.Wang, C.Yao, RSC adv.,2016,6, 77745-77751; C.S.Burke, A.Byrne, T.E.Keyes, J.Am.chem.Soc.,2018,140, 6945-6955). Malignant tumors are the "first killer" leading to the death of residents, and early diagnosis of tumors is the key to cure the tumors. The tumor cell targeting molecules can be highly aggregated in tumor cells, so that the tumor detection sensitivity is improved, and therefore, the design and synthesis of tumor targeting bioluminescence probes becomes one of the current research hotspots. However, few near-infrared luminescent ruthenium complexes with high selectivity for tumor cells are currently reported (j.li, l.zeng, chem.commun.,2019,73, 10972-.
Lysosomes are membrane saccular organelles, have weakly acidic pH (4.5-5.5), and play an important role in cell membrane repair, apoptosis, autophagy, and intracellular substance transport and metabolism (M.Ebner, A.Philipp, H.Volker, biochem. Soc.T.,2019,4, 1173-ion 1185). The labeling and tracing of the lysosome by utilizing the specific fluorescent probe has important practical significance for deeply researching the physiological function of the lysosome and diagnosing and treating related diseases.
Disclosure of Invention
The binuclear ruthenium complex fluorescent probe used in the invention is [ (bpy)2Ru(HL1)Ru(H2L2)](ClO4)4Wherein the ligand and the binuclear complex [ (bpy)2Ru(HL1)Ru(H2L2)](ClO4)4The structural formula is as follows:
Figure BDA0002315045730000021
the invention aims to provide application of the binuclear ruthenium complex in a tumor recognition probe.
The invention also aims to provide application of the binuclear ruthenium complex as a lysosome fluorescent probe.
Compared with the existing living cell fluorescent probe, the fluorescent probe has the beneficial effects that:
(1) the binuclear ruthenium complex has good near-infrared phosphorescence emission performance, has high selectivity on tumor cells, can realize the identification of the tumor cells by using a fluorescence imaging technology, and has great development potential in the aspects of early diagnosis and adjuvant therapy of tumors.
(2) Specifically localized to tumor cell lysosomes. Compared with the traditional lysosome fluorescent probe, the photochemical probe has stable photochemical property, almost has no cytotoxicity under the imaging concentration, can be excited by visible light, can emit light in a near infrared region, has less damage to cells compared with the ultraviolet excited cell probe, and can realize long-time cell fluorescence real-time monitoring.
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FIG. 1 is a graph showing the cell survival rate of HeLa cells incubated with different concentrations of binuclear complex after 48 h.
FIG. 2 is a confocal image and relative fluorescence intensity comparison of the binuclear ruthenium (II) complex (40. mu.M) incubated with HeLa cells and HEK293 cells for 8h, respectively.
FIG. 3 confocal images of a Co-culture model of incubated HeLa-HEK293 cells with dinuclear ruthenium (II) complex (40. mu.M) (HeLa: HEK ═ 1:1) after 8 h.
FIG. 4 is an image of confocal cells after HeLa cells incubated with binuclear ruthenium (II) complexes for 8h and co-stained with LysoTracker Green, MitoTracker Green and Hoechst 33342.
Detailed Description
Example 1: cell culture and cytotoxicity assay of binuclear ruthenium (II) complexes
CO of human cervical carcinoma HeLa cell at 37 DEG C2Cultured in an incubator (relative humidity 95%, CO)2 Content 5%), the cell culture medium is DMEM medium containing 10% fetal calf serum and 1% double antibody.
The MTT method detects the influence of the complex on the cell survival rate. Taking tumor cells in logarithmic growth phase, and obtaining the concentration of the cells at 1 × 104Wells were seeded in 96-well plates at 100 μ L per well, blank wells were filled with cell-free medium, and peripheral wells were filled with sterile PBS. IncubatorCulturing for 24h, discarding culture medium in each well, adding 100 μ L of binuclear ruthenium complex prepared from fresh culture medium with different concentrations, setting 5-6 multiple wells, and adding fresh culture medium without ruthenium complex to control group. After 48h incubation, 10. mu.L of 5mg/mL MTT solution was added to each well and incubation was continued for 4 h. Finally, 150 μ L of DMSO lysate is added to each well, after shaking for 15min, the OD value of each well at 490nm is detected by a microplate reader, and the blank control wells are zeroed. Cell viability was calculated according to the following formula:
Figure BDA0002315045730000031
the results of the detection are shown in FIG. 1. The concentration of the complex corresponding to 50% of inhibition rate and the IC50 value of more than 100 mu M are obtained, which shows that the binuclear ruthenium complex has lower cytotoxicity. At the imaging concentration of 40 mu M, the cell survival rate is over 80 percent, almost no cytotoxicity exists, and the influence on the cell viability is small.
Example 2: binuclear ruthenium (II) complex for recognizing tumor cells
At a cell density of 1X 104And (2) inoculating a HeLa cell and a HEK293 cell of a human embryonic kidney of a human cervical cancer into a confocal cuvette with the diameter of 20mm, culturing in an incubator for 24h, removing the old culture solution, replacing with a fresh culture medium containing 40 mu M binuclear ruthenium complex, continuously culturing for 8h, washing with a phosphate buffer solution with the pH value of 7.4 for three times, exciting with a Nikon A1R laser confocal fluorescence microscope at 562nm, and imaging with a red channel with the wavelength of 640-720 nm. The results shown in fig. 2 indicate that the binuclear ruthenium complex can be effectively taken up by tumor HeLa cells, while the human normal HEK293 cells have lower uptake rate, and the tumor cells are 13 times of the normal cells under the same experimental conditions with the fluorescence intensity. To further determine the target recognition ability of the ruthenium complex on tumor cells, a HeLa-HEK293 cell co-culture model was constructed (HeLa: HEK ═ 1:1) (fig. 3). According to the confocal imaging result, irregular oval cells with larger volume indicated by green arrows are tumor HeLa cells, slender cells indicated by white arrows are normal HEK293 cells, and the red fluorescence intensity in the HeLa cells is obviously higher than that of the HEK293 cells.
Example 3: subcellular localization of dinuclear ruthenium complexes in HeLa cells
HeLa cells were cultured at 1X 104The cell density of each well is inoculated in a 20mm confocal dish, and after 24h of culture, fresh culture medium containing 40 mu M binuclear ruthenium complex is added and incubated for 8h in an incubator. The culture medium in the dish was discarded, washed 2 times with phosphate buffer at pH 7.4, added lyso-tracker Green at 200nM, mito-tracker Green at 200nM and Hoechst33342 at 5 μ g/mL, stained for 30 minutes, washed 3 times with phosphate buffer, and observed for subcellular localization of the dinuclear ruthenium complex using Nikon A1R confocal laser fluorescence microscope. Binuclear ruthenium (II) complexes (lambda)ex＝562nm，λem640-ex＝488nm，λem＝505-530nm)，Hoechst33342(λex＝405nm，λem＝430-460nm)。
The imaging results (fig. 4) show that the red fluorescence of the binuclear ruthenium complex overlaps with the Green fluorescence of the lysosomal dye LysoTracker Green, presenting a distinct yellow color with a Pearson coefficient of 0.91, while there is almost no overlap with the Green fluorescence of the mitochondrial dye mitotracker Green and the blue fluorescence of the nuclear dye Hoechst 33342. From this, it was found that the dinuclear ruthenium (II) complex can be selectively localized in the lysosome of HeLa cells.

Claims (2)

1. An application of a binuclear ruthenium complex as a tumor cell targeted HeLa imaging reagent, wherein the binuclear ruthenium complex is [ (H)2L2)Ru(HL1)Ru(bpy)2](ClO4)4The structural formula is shown as follows:
Figure RE-FDA0002387992700000011
2. the use of the dinuclear ruthenium complex according to claim 1 as a lysosome localization probe for HeLa cells.
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CN116284147A (en) * 2023-02-20 2023-06-23 青岛大学 Fluorescent probe for selectively detecting hNQO1 with high expression in tumor cells and application thereof

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CN116284147A (en) * 2023-02-20 2023-06-23 青岛大学 Fluorescent probe for selectively detecting hNQO1 with high expression in tumor cells and application thereof
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