阅读说明：本技术 一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途 (Cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material and preparation method and application thereof ) 是由 田玉鹏 杜文丽 王飞 郭辉 张琼 马文 吴杰颖 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途,其中杂化材料简记为Eu(TTA)-(4)P,结构式如下所示：本发明铕配合物/噻吩吡啶盐杂化材料Eu(TTA)-(4)P具有低的细胞毒性,在不同波段可以分别靶向脂滴和线粒体,通过不同窗口采集信息Eu(TTA)-(4)P可以很好地跟踪细胞中的脂滴与线粒体,这种双靶向的荧光探针在疾病诊断方面具有重要的应用价值。(The invention discloses a cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material and a preparation method and application thereof, wherein the hybrid material is abbreviated as Eu (TTA) 4 P, the structural formula is as follows: the invention relates to a europium complex/thiophene pyridine salt hybrid material Eu (TTA) 4 P has low cytotoxicity, can target lipid droplets and mitochondria respectively at different wave bands, and collects information Eu (TTA) through different windows 4 P can well track lipid droplets and mitochondria in cells, and the dual targeting is realizedThe fluorescent probe has important application value in disease diagnosis.)

1. A cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material is characterized in that the structural formula is as follows:

2. a method for preparing the cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material of claim 1, which is characterized by comprising the following steps:

placing HTTA and triethylamine in a round-bottom flask, adding methanol, stirring and dispersing uniformly at normal temperature, and dropwise adding dissolved Eu (NO)₃)₃·6H₂A methanol solution of O to obtain a clear solution; dissolving compound P⁺Slowly adding a methanol solution of diphenylamine thiophene vinyl methyl pyridine salt into the clear solution, carrying out reflux reaction for 12 hours, concentrating under reduced pressure to obtain an oily state, adding a small amount of ethanol to dissolve, standing to separate out a solid, carrying out suction filtration, washing with methanol and drying to obtain a hybrid material Eu (TTA)₄P；

The synthetic route is as follows:

3. use of the europium complex/thiophene pyridinium hybrid material of claim 1, wherein: the europium complex/thiophene pyridinium hybrid material is used for preparing a double-target biological probe material; the double-targeting biological probe material can respectively target lipid droplets and mitochondria at different wave bands.

Technical Field

The invention relates to a cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material and a preparation method and application thereof. The hybrid material has a multi-photon effect, and can respectively target lipid droplets and mitochondria at different wave bands.

Background

The multi-photon absorption material is a luminescent material with low energy excitation and high energy emission, has the advantages of strong penetrating power to cells and biological tissues, small light damage, weak autofluorescence interference and the like, and has important application value in the field of biomedicine. The fluorescent probe can well track the change of lipid drops and mitochondria in cells, and has a certain application prospect in the aspect of clinical disease diagnosis.

Lipid droplets are the primary storage site for intracellular neutral lipids. Recent studies have shown that lipid droplets are not a simple energy reservoir within a cell, but rather a complex, motile, dynamically changing, multifunctional organelle. Lipid droplets are able to move along the cytoskeleton and interact with other organelles, possibly playing important roles in lipid metabolism and storage, membrane transport, protein degradation, and signal transduction processes. In addition, studies have shown that various metabolic diseases, such as obesity, fatty liver, cardiovascular diseases and diabetes, and neutral lipid storage diseases, are often accompanied by abnormal lipid storage. Therefore, biological studies on lipid droplets are increasingly receiving attention.

Mitochondria are the "energy factory" of cells, which are organelles encapsulated by two membranes, an important site for aerobic respiration of cells. Mitochondria not only supply energy to cells, but also participate in a plurality of cell metabolism, such as cell information transmission, immunoreaction, biosynthesis, cell differentiation, apoptosis and the like, and have certain regulation and control functions on the aspects of cell cycle, cell growth and the like. Therefore, by detecting the state of the cell mitochondria, the diagnosis and treatment of body diseases can be accurately carried out, and the research on mitochondrial fluorescent probes with high efficiency and low toxicity is subject to the squint of researchers.

Most of the currently commonly used commercial nuclear dyes DAPI, Hoechst and the like are organic small molecules, are mainly limited to single-photon fluorescence imaging, and are not favorable for long-time tracking along with the defects of poor water solubility, high cytotoxicity, high photodamage, poor light stability and the like, so that the application and action mechanism research of the dyes in organisms is limited. In addition, the stokes shift between the emission energy and excitation energy is small, resulting in interference from autofluorescence of the endogenous fluorophores. Therefore, the development of the biological-friendly multi-photon fluorescent probe with good light stability and long-wave excitation has important scientific significance and application prospect.

The applicant has conducted the following literature search on the subject matter of the present application:

1. xueshu.baidu.com net search results: (2021/6/29)

2. Chinese knowledge network retrieval results:

the first retrieval method comprises the following steps:

the discourse-cell double-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material: there is no relevant literature.

The preparation method of the discourse-cell double-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material comprises the following steps: there is no relevant literature.

And a second retrieval mode:

full text-cell dual-targeting multiphoton absorption europium complex/thiophene pyridinium hybrid material: item 2. Are all independent of the preparation method of the cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material

The preparation method of the full-text-cell double-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material comprises the following steps: item 2. Is irrelevant to the preparation method of the europium complex/thiophene pyridinium hybrid material with cell double-targeting multi-photon absorption.

Disclosure of Invention

The invention aims to provide a cell dual-target multi-photon absorption europium complex/thiophene pyridinium hybrid material and a preparation method and application thereof. Rare earth ion Eu^IIIThe thiophene pyridinium has excellent optical properties of good color rendering property, large Stokes shift, strong photobleaching resistance, long fluorescence life and the like, and the thiophene pyridinium has good nonlinear optical property, can be assembled to prepare the thiophene pyridinium/europium complex hybrid material with the advantages of the thiophene pyridinium and the europium complex, and is expected to become a novel double-targeting biological probe material.

The invention relates to a cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material, which is abbreviated as Eu (TTA)₄P, the structural formula is as follows:

the invention relates to a method for synthesizing a cell dual-targeting multi-photon absorption europium complex/thiophene pyridinium hybrid material, which comprises the following steps:

0.3996g (1.8mmol) of HTTA and 0.4mL (2.7mmol) of triethylamine were placed in a 100mL round-bottom flask, 15mL of methanol was added, stirring was performed at normal temperature for 5min, and 15mL of a solution in which 0.2007g (0.45mmol) of Eu (NO) was dissolved was added dropwise₃)₃·6H₂A methanol solution of O to obtain a clear solution; 15mL of dissolved 0.225mmol of Compound P⁺Slowly adding methanol solution of (diphenylamine thiophene vinyl methyl pyridinium) into the clear solution, carrying out reflux reaction for 12h, concentrating under reduced pressure to obtain oily substance, adding a small amount of ethanol for dissolving, standing overnight, precipitating a solid, carrying out suction filtration, washing with methanol and drying to obtain a complex Eu (TTA)₄And P. Yield 0.6504g, yield: 86.21 percent.

The compound P⁺Is diphenylamine thiophene vinyl methyl pyridine salt.

The synthetic route is as follows:

the invention relates to a europium complex/thiophene pyridine salt hybrid material Eu (TTA)₄The application of P is used for preparing the dual-target biological probe material. The double-targeting biological probe material can respectively target lipid droplets and mitochondria at different wave bands.

The invention has the beneficial effects that:

the invention enables Eu (TTA) to be prepared through the assembly of pyridinium cations₄The P probe has specific targeting to mitochondria. Eu (TTA)₄The P has the strongest two-photon fluorescence signal at the near infrared 880nm wavelength and the strongest three-photon fluorescence signal at the intermediate infrared 1250nm wavelength, and is a multi-photon fluorescent material with excellent nonlinear optical properties. As shown in fig. 1 and 2.

Eu (TTA) of the invention₄The P raw material is easy to obtain and simple to synthesize. The comprehensive property is superior to that of commercial lipid drops and mitochondrial dyes, and a fluorescent probe with similar function does not exist, so that the method has stronger commercial value.

Eu (TTA) of the invention₄P has low cytotoxicity, collects information Eu (TTA) through different windows₄P can well track lipid drops and mitochondria in cells, and the double-targeting fluorescent probe has important application value in the aspect of disease diagnosis.

Drawings

FIG. 1(a) is the hybrid material Eu (TTA)₄Cross-sectional view of effective two-photon absorption of P in DMSO solvent. Description of the drawings: eu (TTA)₄The optimum excitation wavelength of the two-photon fluorescence spectrum of P is 880nm, and the maximum absorption cross section is 212 GM. FIG. 1(b) shows Eu (TTA)₄Two-photon verification plot of P. Description of the drawings: output power (I)_out) And input power (I)_in) The slope of the linear relationship is 1.948, approximately 2, Eu (TTA)₄P has a two-photon fluorescence effect.

FIG. 2(a) is the hybrid material Eu (TTA)₄P different wave band excitation lower Eu (TTA)₄Fluorescence spectrum of P. Description of the drawings: fluorescence excited at 1250nm bandThe light is strongest. FIG. 2(b) is the complex Eu (TTA)₄P effective three-photon absorption cross section in DMSO solvent, wherein the inset is complex Eu (TTA) excited at optimal wavelength band 1250nm in DMSO₄P luminescence pattern. Description of the drawings: eu (TTA)₄The optimal excitation wavelength of the three-photon fluorescence spectrum of the P is 1250nm, the maximum absorption cross section of the three photons is 19.2GM, and the hybrid material has stronger red light emission. FIG. 2(c) shows Eu (TTA)₄P three-photon verification plot. Description of the drawings: output power (I)_out) And input power (I)_in) The slope of the linear relationship is 2.987, about 3, Eu (TTA)₄P has a three-photon fluorescence effect.

FIG. 3 is the hybrid material Eu (TTA)₄Cytotoxicity test pattern of P. Description of the drawings: eu (TTA)₄P has low cytotoxicity when Eu (TTA)₄When the concentration of P reaches 25 mu M, the cell survival rate is still higher than 60 percent.

FIG. 4 shows Eu (TTA)₄Confocal microscopy of P in HepG2 and HeLa cells. The blue signal is the signal for collecting 420-489nm window, the red is the signal for 600-650nm window, and the green is the signal for lipid droplet quotient staining. Wherein the signal collected by the 420-489nm window is completely overlapped with the lipid droplet quotient stain signal, and shows light blue in the superimposed channel map (Merge), but is not overlapped with the red signal collected by the 600-650nm window, which shows that: probe Eu (TTA)₄P can well distinguish lipid droplets from mitochondria in cells, and can well track lipid droplets from mitochondria in cells by collecting information through different windows. The same phenomenon was also confirmed in the cervical cancer cell HeLa.

Detailed Description

Example 1: hybrid materials Eu (TTA)₄Synthesis of P

0.3996g (1.8mmol) of HTTA and 0.4mL (2.7mmol) of triethylamine were placed in a 100mL round-bottom flask, 15mL of methanol was added, stirring was performed at normal temperature for 5min, and 15mL of a solution in which 0.2007g (0.45mmol) of Eu (NO) was dissolved was added dropwise₃)₃·6H₂A methanol solution of O to obtain a clear solution; 15mL of dissolved 0.225mmol of Compound P⁺Slowly adding methanol solution of (diphenylamine thiophene vinyl methyl pyridinium) into the clear solution, refluxing for 12h, and concentrating under reduced pressure to obtain oilDissolving with a little ethanol, standing overnight, separating out solid, filtering, washing with methanol, and drying to obtain hybrid material Eu (TTA)₄And P. Yield 0.6504g, yield: 86.21 percent.

¹HNMR(CD₃CN,400MHz,ppm)δ9.62(d,J＝6.6Hz,2H),8.53(d,J＝6.7Hz,2H),8.24(d,J＝15.7Hz,1H),7.45(t,J＝7.9Hz,4H),7.29(ddd,J＝23.4,12.9,4.5Hz,12H),6.85(d,J＝15.7Hz,1H),6.75(s,4H),6.69–6.56(m,4H),6.46(d,J＝4.1Hz,1H),5.13(s,3H),4.02(s,3H)；¹³C-NMR(CD₃CN,100MHz,ppm)):δ157.09,146.45,144.01,140.95,137.87,133.41,129.84,125.95,121.97,49.81,39.98,33.91；ESI-MS m/z in positive ion mode:369.52,found:369.67,in negative ion mode:1037.81[Eu(TTA)₄]^-。

Example 2: biological investigation of target molecules

1. In 96-well plates with 0, 5, 10, 15, 20 and 25 μ M Eu (TTA)₄P incubation of HepG2 cells for 24 hours, the survival rate of the cells observed at different concentrations is higher than 60%, indicating that Eu (TTA)₄P has lower dark toxicity.

2. For hybrid material Eu (TTA)₄P confocal cell visualization experiments were performed. Cells were cultured in HepG2 for 30min at a concentration of 1. mu.M. Mitochondrial and lipid droplet commercial staining were selected for co-localization experiments. The blue signal is the signal for collecting 420-489nm window, the red is the signal for 600-650nm window, and the green is the signal for lipid droplet quotient staining. Wherein the signal collected by the 420-489nm window is completely overlapped with the lipid droplet quotient stain signal, showing a light blue color in the superimposed channel map, and is completely not overlapped with the red signal collected by the 600-650nm window, indicating that the probe Eu (TTA)₄P can well distinguish lipid droplets from mitochondria in cells, and the change of the lipid droplets and the mitochondria in the cells can be well tracked by collecting information through different windows, and the same phenomenon is also proved in a cervical cancer cell HeLa.