Fluorescent probe as well as preparation method and application thereof in marking collagen

Abstract

The invention belongs to the technical field of fluorescent probe detection, and particularly relates to a fluorescent probe as well as a preparation method and application thereof in marking collagen. Specifically, a compound is designed based on carbazole and a triphenylamine group, and carboxyl is further introduced to adjust the labeling efficiency of a probe molecule to collagen; finally, the fluorescent probe molecule which has the characteristics of aggregation-induced emission (AIE), high fluorescence quantum yield, low cytotoxicity, good biocompatibility and capability of efficiently marking collagen is successfully prepared, and the fluorescent probe molecule can mark the collagen and research related physiological functions and properties of the collagen. A new method and a new tool are provided for future research of the fluorescence labeled protein, and a new molecular design concept is also provided for related scientific fields, so that the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence probe detection, and particularly relates to a fluorescence probe, a preparation method thereof, and an application thereof in labeling collagen. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Collagen is a highly structured protein and one of the most abundant proteins in the human body, accounting for approximately 30% of the total body protein. It exists in various parts of the body, including tissues such as skin, bones, tendons, ligaments, joints, eyes, blood vessels, and internal organs, and is a key substance for maintaining the structure and function of these tissues. Collagen not only plays an important physiological role in the living body, but also has wide applications in many fields such as medicine, skin care, and health care.

[0004] Currently, the research on collagen mainly stays in in vitro tests. By extracting and purifying collagen in vitro and using chemical crosslinking agents or enzymatic methods to study the crosslinking state of collagen and its impact on biological functions, it is a main way to observe and study the physiological functions of collagen. However, there are certain problems in studying the distribution, metabolism, and functions of collagen in vivo. In recent years, with the rise of the application of fluorescent materials in biomedicine, the fluorescent labeling protein technology has become a simple and effective molecular biology technology for observing protein structures, with advantages such as non-invasive, real-time, highly sensitive, multiplex analysis, and long-term tracking, which can help people deeply explore the dynamic behavior, spatial distribution, functional roles of proteins in vivo and in cells, as well as their interactions with other molecules. As a macromolecular protein, collagen can also be studied through the fluorescent labeling protein technology. However, traditional fluorescent labeling probes have problems such as slow labeling process, poor labeling effect, low fluorescence efficiency, and poor biocompatibility, which have become important factors hindering the use of the fluorescent labeling method to study collagen. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a fluorescence probe, a preparation method thereof, and an application thereof in labeling collagen. Specifically, the present invention designs a compound based on carbazole and a triphenylamine group with aggregation-induced emission (AIE) characteristics, and further introduces a long alkyl chain carboxyl group to regulate the labeling effect of the probe molecule on collagen. Finally, a fluorescence probe molecule with high fluorescence quantum yield, low cytotoxicity, good biocompatibility, and capable of efficiently labeling collagen is successfully prepared. Based on the above research results, the present invention is completed.

[0006] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, a compound is provided, and the structural formula of the compound is shown as Formula (I) or Formula (II):

[0008]

[0009] In the present invention, the compound shown in Formula (I) is named Ka12, and the compound with the structural formula shown in Formula (II) is named TPA12.

[0010] The compound may also include its pharmaceutically acceptable salts or esters or solvates, tautomers, mesomers, racemates, stereoisomers or metabolites.

[0011] In the second aspect of the present invention, a method for synthesizing the above compound is provided, and the method includes preparing the compound by respectively reacting derivatives of carbazole and triphenylamine with a pyridinium salt containing a long alkyl chain carboxyl group through the Knoevenage reaction.

[0012] In the third aspect of the present invention, an application of the above compound as a fluorescent probe or in the preparation of a fluorescent probe is provided.

[0013] In the fourth aspect of the present invention, a detection product is provided, and the detection product at least includes the above compound.

[0014] In the fifth aspect of the present invention, an application of the above compound or detection product in labeling collagen is provided.

[0015] In the sixth aspect of the present invention, a method for labeling collagen is provided, and the method includes: using the above compound or detection product to perform labeling detection on a test sample containing or suspected of containing collagen.

[0016] The beneficial technical effects of the above technical solution:

[0017] The above technical solution designs two fluorescent probes that can be used for fluorescently labeling collagen: Ka12 and TPA12. Fluorescent probes with high fluorescence quantum yields were constructed by selecting carbazole and triphenylamine fluorophores. In order to enable the probes to covalently bind to collagen, the above technical solution further introduced a carboxyl unit with a long alkyl chain to regulate the labeling effect of the probe molecules on collagen. The excellent performance of the precisely designed Ka12 and TPA12 probe molecules can meet the fluorescent labeling of collagen, with high quantum yields, low cytotoxicity, and good biocompatibility, and can label collagen to study its related physiological functions and properties. This provides new methods and tools for future research on fluorescently labeled proteins, and also provides new molecular design concepts for related scientific fields, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0019] Figure 1 : 1H NMR spectrum (A); 13C NMR spectrum (B); high-resolution mass spectrum (C) of probe Ka12.

[0020] Figure 2 : 1H NMR spectrum (A); 13C NMR spectrum (B); high-resolution mass spectrum (C) of probe TPA12.

[0021] Figure 3 : UV-visible absorption spectrum and fluorescence emission spectrum of probe Ka12 (A); UV-visible absorption spectrum and fluorescence emission spectrum of probe TPA12 (B).

[0022] Figure 4 : Fluorescence emission spectra of probe Ka12 in solvents with different ratios of methanol:water (A); Fluorescence emission spectra of probe TPA12 in solvents with different ratios of methanol:water (B).

[0023] Figure 5 : Live cell permeability and imaging diagrams at different concentrations of the probe.

[0024] Figure 6 : Flow chart of the labeling of collagen by probe molecules Ka12 and TPA12 and electrophoresis experimental diagram.

[0025] Figure 7 : Absorption spectra of collagen, Ka12-labeled collagen, and Ka12 (A); Absorption spectra of collagen, TPA12-labeled collagen, and TPA12 (B).

[0026] Figure 8:Cytotoxicity of collagen to HACAT cells (A); cytotoxicity of collagen to HSF cell line (B); cytotoxicity of TPA12 to HACAT cells (C); cytotoxicity of TPA12 to HSF cell line (D).

[0027] Figure 9 :Cell scratch repair experiment of HACAT cell line (A); cell scratch repair experiment of HSF cell line (B).

[0028] Figure 10 :Gene expression of HACAT (A); gene expression of HSF (B); protein expression of HACAT and HSF (C).

[0029] Figure 11 :Preparation of photoaging models of HACAT and HSF cells, expression of JC-1 (A) and activity level of reactive oxygen species (B), with carbonyl cyanide m-chlorophenylhydrazone (CCCP) as the control group.

[0030] Figure 12 :Metabolic toxicity evaluation of fluorescently labeled collagen and fluorescent probe molecules in mice (heart, liver, spleen, lung, kidney).

[0031] Figure 13 :Toxicity evaluation of fluorescently labeled collagen and fluorescent probe molecules on mouse skin.

[0032] Figure 14 :Mouse skin absorption and metabolism imaging of fluorescently labeled collagen and fluorescent probe molecules. Detailed implementation manners

[0033] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present invention application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0035] As mentioned above, traditional fluorescent labeling probes have problems such as slow labeling process, poor labeling effect, low fluorescence efficiency, and poor biocompatibility, which have become an important problem hindering the use of fluorescent labeling method to study collagen.

[0036] Therefore, in order to achieve efficient labeling and high-brightness observation of collagen, there is an urgent need to develop some fluorescent probe molecules with high luminous efficiency and the ability to label collagen with high efficiency. Through long-term research, the inventors found that designing the fluorescent probe into a D-π-A structure can optimize its optical and electrical properties to improve its performance in various applications. The electron-donating group (D) and the electron-accepting group (A) are connected by a conjugated bridge (π). This structure can enhance the fluorescence intensity and quantum efficiency of the probe, enabling it to meet different application requirements. As a traditional fluorescent group, the carbazole group usually serves as an electron-donating group because the nitrogen atom in its structure can provide additional electron cloud density, thereby enhancing its electron conduction performance. When combined with other groups, it can enhance the electron supply ability. After absorbing photons, it can be excited to a high-energy state to achieve intermolecular electron transfer. When an electron transitions from the ground state to the excited state, the molecule can absorb light of a certain wavelength, and when the electron returns from the excited state to the ground state, photons (i.e., fluorescence) are emitted. Due to the rigid planar structure, high conjugation system, and the influence of the nitrogen atom of carbazole, the energy difference of its electron transition is appropriate, so it can effectively emit fluorescence. The triphenylamine group contains three benzene rings and one nitrogen atom. The benzene rings are conjugated to form a broad π electron cloud. This structure gives the triphenylamine molecule a long conjugation system, enabling it to absorb light of a longer wavelength and emit fluorescence of a longer wavelength. It can effectively avoid non-radiative relaxation, allowing excited-state electrons to lose less energy through other channels and more energy through fluorescence emission, resulting in a high fluorescence quantum yield. The triphenylamine molecule also has an aggregation-induced emission (AIE) effect, showing enhanced fluorescence in the aggregated state. This is mainly because when triphenylamine molecules aggregate into larger aggregates, the intermolecular interaction inhibits free rotation and vibration, thereby reducing non-radiative energy loss and leading to enhanced emission. Using the above two fluorescent groups, a fluorescent probe with a D-π-A structure and good luminescent properties can be designed. By introducing a long alkyl chain carboxyl unit that covalently binds to its characteristic amino group according to the structural characteristics of collagen, efficient labeling of collagen can be achieved.

[0037] In view of this, in a typical specific embodiment of the present invention, a compound is provided. The structural formula of the compound is shown in Formula (I) or Formula (II):

[0038]

[0039] In the present invention, the compound shown in Formula (I) is named Ka12, and the compound with the structural formula shown in Formula (II) is named TPA12.

[0040] The compounds Ka12 and TPA12 of the present invention are pyridinium salts containing carbazole and triphenylamine groups, which are designed by introducing carboxyl groups and long carbon chains to regulate the luminescence properties. On the one hand, carbazole with a rigid plane and high quantum yield and triphenylamine unit with aggregation-induced emission (AIE) characteristics are selected as fluorophores, providing a large conjugated unit, thereby achieving the high fluorescence characteristics of the probe. On the other hand, carboxyl groups are used as binding units to improve the performance of the probe for labeling collagen. Through the above structural optimization design, pyridinium salt fluorescent probes Ka12 and TPA12 based on carbazole and triphenylamine units are developed. In a cuvette, Ka12 emits bright yellow fluorescence in methanol, while TPA12 emits strong orange-red fluorescence in water. Both have high fluorescence quantum yields and show good labeling effects on collagen.

[0041] The compound may also include its pharmaceutically acceptable salts or esters or solvates, tautomers, mesomers, racemates, stereoisomers or metabolites.

[0042] As understood by those of ordinary skill in the art, the pharmaceutically acceptable salts include alkali metal salt forms of the above compounds (specific examples are sodium salts or potassium salts), or salts formed by the compound with inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid, and salts formed with organic acids, such as methanesulfonic acid, toluenesulfonic acid, citric acid, acetic acid or trifluoroacetic acid. The term "pharmaceutically acceptable" or its interchangeable term "medicinal" when describing "pharmaceutically acceptable salts" means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance with medicinal value, such as a salt formed as an intermediate during chiral resolution. Although this intermediate salt cannot be directly administered to the subject, it can play a role in obtaining the end product of the present invention.

[0043] In another specific embodiment of the present invention, a method for synthesizing the above compound is provided. The method includes preparing the compound by subjecting derivatives of carbazole and triphenylamine and pyridinium salts containing long-chain carboxyl groups to the Knoevenage reaction respectively.

[0044] Furthermore, the synthesis route of the compound is as follows:

[0045]

[0046] In another specific embodiment of the present invention, there is provided the use of the above compound as a fluorescent probe or in the preparation of a fluorescent probe. Through research, it has been found that the above compound Ka12 and TPA12 have large Stokes shift characteristics, reducing the decrease in fluorescence efficiency caused by energy transfer. In in vitro spectral tests, it was found that the absorption peak of Ka12 is at 436 nm, and the fluorescence emission peak after excitation with 436 nm is at 550 nm. The absorption peak of TPA12 is at 470 nm, and the fluorescence emission peak after excitation with 470 nm is at 575 nm.

[0047] In another specific embodiment of the present invention, there is provided a detection product, which at least includes the above compound.

[0048] Through research, the present invention has found that the above compound can efficiently label collagen as a fluorescent probe. After the probe molecule labels collagen, the absorption peak of Ka12 blue-shifts from 436 nm to 420 nm, and the absorption peak of TPA12 blue-shifts from 470 nm to 440 nm, indicating that after the fluorescent probe successfully binds to collagen, its own luminescence properties change. This shows that Ka12 and TPA12 can efficiently label collagen.

[0049] The detection product can be a detection kit, a detection device, and / or a detection equipment.

[0050] In another specific embodiment of the present invention, the product may further contain substances and components commonly used in detection kits, detection devices, and / or detection equipment. For example, in a detection kit, it may contain a solvent, and the solvent can be a phosphate buffer solution, physiological saline, etc. In detection devices and equipment, it may further contain a fluorescence spectrophotometer for detecting the emitted fluorescence signal, and specific limitations are not made here.

[0051] In another specific embodiment of the present invention, there is provided the use of the above compound or detection product in labeling collagen.

[0052] In another specific embodiment of the present invention, there is provided a method for labeling collagen, which includes: using the above compound or detection product to label and detect a test sample containing or suspected of containing collagen, so as to be used for studying its related physiological functions and properties. For example, through experiments, the present invention has proved that collagen has good biocompatibility, the function of accelerating cell growth and healing, and the function of blocking ultraviolet rays, etc.

[0053] Due to the advantages of good biocompatibility, permeability, and low toxicity of the fluorescent probe, it is particularly suitable for the detection of living samples. Therefore, the sample to be measured can be (living) biological cells, biological tissues, biological organs, or biological individuals. In a specific embodiment of the present invention, the biological cells can be human immortalized epidermal cells (HACAT) and human skin fibroblasts (HSF), the biological organs can be the heart, liver, spleen, lungs, kidneys, and skin, etc., the biological individuals can be humans or non-human animals, and the non-human animals can further be non-human mammals, such as mice, rats, guinea pigs, monkeys, chimpanzees, etc., which are not specifically limited herein.

[0054] The present invention will be further explained and illustrated below through examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0055] Example 1: Synthesis of Probe Ka12

[0056]

[0057] Carbazole (Compound 1, 11.67 g, 10 mmol) was dissolved in 250 mL of dichloromethane. After adding 2 mL of triethylamine, the mixture was stirred at room temperature for 20 min. N-Bromosuccinimide (567 mg, 10 mmol) was dissolved in 90 mL of dichloromethane and slowly added dropwise to the above solution. The mixture was stirred at room temperature overnight. After quenching with cold water, it was extracted with dichloromethane, washed, and the product was purified by column chromatography (dichloromethane: petroleum ether = 1:5) to obtain 3-bromo-9H-carbazole (Compound 2, 2.1 g, yield 85.4%). Compound 2 (2.46 g, 10 mmol) was dissolved in 50 mL of DMF. After adding 2 g of potassium carbonate, the mixture was stirred at room temperature for 1.5 h. After the solution turned light yellow, bromoethane (1.08 g, 10 mmol) was slowly added. After stirring at room temperature for 6 h, DMF was removed by vacuum distillation to obtain 3-bromo-9-ethylcarbazole (Compound 3, 2.7 g, yield 99%). Compound 3 (2.79 g, 10 mmol) was dissolved in a mixed solvent of 40 mL of DMF and triethylamine (volume ratio = 1:1). Under a nitrogen atmosphere, 2 g of triphenylphosphine and 150 mg of palladium acetate were added. After stirring at 90 °C for 48 h, the insoluble solid was removed by filtration, the DMF solvent was removed by vacuum distillation, and the solid residue was recrystallized from dichloromethane and n-hexane. After cooling at -20 °C, it was filtered to obtain (E)-3-(2-(4-pyridyl)vinyl)-9-ethylcarbazole (Compound 4, 1.9 g, yield 64%). Compound 4 (298 mg, 1 mmol) was dissolved in 15 mL of acetonitrile. 1-Bromododecanoic acid (279 mg, 1 mmol) was added thereto, and the mixture was stirred at 85 °C overnight. After cooling, it was filtered, and the yellow solid was obtained after washing three times with ether, namely: Ka12 (247 mg, yield 43%).

[0058] The experimental results are shown in Figure 1 。

[0059] 1 H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.91 (d, J = 6.7 Hz, 2H), 8.59 (d, J = 1.6 Hz, 1H), 8.32–8.13 (m, 4H), 7.90 (dd, J = 8.7, 1.7 Hz, 1H), 7.74 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.59–7.48 (m, 2H), 7.29 (t, J = 7.4 Hz, 1H), 4.58–4.40 (m, 4H), 2.18 (t, J = 7.3 Hz, 2H), 1.91 (q, J = 7.3 Hz, 2H), 1.47 (p, J = 7.2 Hz, 2H), 1.37–1.20 (m, 17H). 1313C NMR (101 MHz, DMSO-d6) δ 174.99, 153.94, 144.41, 143.11, 141.46, 140.64, 126.96, 126.77, 126.71, 123.56, 123.22, 122.61, 121.71, 120.98, 120.43, 120.18, 110.38, 110.22, 59.91, 37.74, 34.16, 30.98, 29.32, 29.24, 29.20, 29.02, 28.86, 25.91, 24.97, 14.25. HRMS: Calcd for: 554.3867; Found for: 554.3809.

[0060] Example 2: Synthesis of Probe TPA12

[0061]

[0062] Dissolve 4-bromotriphenylamine (Compound 5, 3.24 g, 10 mmol) in a mixed solvent of 40 mL DMF and NEt3 (volume ratio = 1:1). Under a nitrogen atmosphere, add 2 g of triphenylphosphine and 150 mg of palladium acetate. After stirring at 90 °C for 48 h, filter off the insoluble solids by suction, remove the DMF solvent by vacuum distillation. After the solid residue is dissolved in dichloromethane, it is purified by column chromatography (ethyl acetate: petroleum ether = 1:5), and distilled to obtain (E)-4-(2-(4-pyridyl)vinyl)-triphenylamine (Compound 6, 2.6 g, yield 75%). Dissolve Compound 6 (348 mg, 1 mmol) in 15 mL of acetonitrile, add 1-bromododecanoic acid (279 mg, 1 mmol) thereto, stir at 85 °C overnight, filter by suction after cooling, and wash three times with ether to obtain an orange-yellow solid, namely: TPA12 (225 mg, yield 41%).

[0063] The experimental results are shown in Figure 2 .

[0064] 11H NMR (400 MHz, DMSO-d6) δ 12.01 (s, 1H), 8.89 (d, J = 6.8 Hz, 2H), 8.17 (d, J = 7.0 Hz, 2H), 7.97 (d, J = 16.2 Hz, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.42–7.35 (m, 4H), 7.32 (d, J = 16.2 Hz, 1H), 7.20–7.09 (m, 6H), 6.94 (d, J = 8.8 Hz, 2H), 4.46 (t, J = 7.3 Hz, 2H), 2.17 (t, J = 7.3 Hz, 2H), 1.88 (t, J = 7.2 Hz, 2H), 1.53–1.41 (m, 2H), 1.26 (d, J = 19.4 Hz, 14H). 13 13C NMR (101 MHz, DMSO-d6) δ 175.02, 153.67, 149.96, 146.67, 144.42, 141.23, 130.33, 130.18, 128.48, 125.88, 124.99, 123.69, 121.17, 120.94, 59.93, 34.21, 29.31, 29.21 (d, J = 1.8 Hz), 29.03, 28.84, 25.89, 24.99. HRMS: Cal for: 548.3397 Found for: 548.3347

[0065] Example 3: Absorption and Emission of Probe Ka12 and TPA12

[0066] Stock solutions of Ka12 and TPA12 at 10 μM were prepared with PBS. The UV-visible absorption spectrum of Ka12 in PBS solvent was obtained using a HITACH U-2910 ultraviolet spectrophotometer, and the fluorescence emission spectrum was obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp at 436 nm ( Figure 3 A) excitation to obtain the fluorescence emission spectrum of Ka12. The UV-visible absorption spectrum of TPA12 in PBS solvent was obtained using a HITACH U-2910 ultraviolet spectrophotometer, and the fluorescence emission spectrum was obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp at 470 nm ( Figure 3 B) excitation to obtain the fluorescence emission spectrum of TPA12.

[0067] The experimental results are shown in Figure 3 .

[0068] Example 4: Luminescence Property Test of Probe Ka12 and TPA12

[0069] Prepare 10 μM stock solutions of Ka12 and TPA12 with different ratios of methanol:water. The fluorescence emission spectra of Ka12 in solvents with different ratios of methanol:water were obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp at 436 nm ( Figure 4 A) excitation to obtain the fluorescence emission spectrum of Ka12. The fluorescence emission spectra of TPA12 in solvents with different ratios of methanol:water were obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp at 470 nm ( Figure 4 B) excitation to obtain the fluorescence emission spectrum of TPA12.

[0070] The experimental results are shown in Figure 4 .

[0071] From Figure 4 the results, it can be seen that the probe Ka12 has strong fluorescence emission in methanol, and as the proportion of water increases, the fluorescence intensity continuously decreases. The probe TPA12 has weak fluorescence in methanol, and as the proportion of water increases, the fluorescence intensity continuously increases, indicating that the probe molecule TPA12 has the property of aggregation-induced emission (AIE).

[0072] Example 5: To evaluate the penetration ability of the probe molecule in living cells and its ability to image living cells at different concentrations, first culture HACAT or HSF cells in an appropriate cell culture medium. After the cells reach 80-90% confluence, add probe solutions with different concentrations (0.1 μM, 1 μM, 5 μM, 10 μM), incubate for 1-2 hours and wash the cells to remove unbound probes. Then, image the cells using a confocal microscope, set appropriate laser wavelengths and detection channels, and record the fluorescence signal intensity of the probe. By analyzing the fluorescence intensity at different concentrations, evaluate the permeability and imaging ability of the probe in living cells.

[0073] The experimental results are shown in Figure 5 .

[0074] The research shows that the probe molecule has good penetration ability and imaging ability in living cells.

[0075] Example 6: The process of labeling collagen with probes Ka12 and TPA12 and the electrophoresis experiment

[0076] Take 1 mg of the fluorescent probe (dissolved in 10 mL of DMSO), and according to the molar ratio, the ratio of fluorescent dye: EDC: NHS = 1:2:2, add EDC and NHS, stir in the dark for 12 h for activation. According to the ratio of 0.01 mg of fluorescent dye per 1 mg of protein, add 100 mg of collagen, dissolve it with 15 mL of MES buffer solution (pH = 5.5), and stir and bind at 4 °C for 24 h. Activate the dialysis bag (cut-off molecular weight: 14,000 Da) with ultrapure water at 90 °C, take the combined mixed solution and place it in the dialysis bag, and dialyze it in an environment of MES buffer solution (pH = 5.5) until there is no fluorescence in the dialysis solution. The whole process is carried out in a dark environment. Take out the dialyzed mixed solution, freeze-dry it and store it for later use. After labeling, perform an electrophoresis experiment on the fluorescently labeled collagen, and it is obtained that the molecular weight of the fluorescently labeled collagen is about 70 KDa, and observe the luminescence of the fluorescently labeled collagen under fluorescence irradiation.

[0077] The experimental results are shown in Figure 6 .

[0078] From Figure 6 the results, it can be seen that: under fluorescence irradiation, the fluorescence of the fluorescently labeled collagen is observed, indicating that the fluorescent probe molecules are successfully labeled onto the collagen.

[0079] Example 7: Absorbance of Probe Ka12 and TPA12 Labeled Collagen

[0080] Prepare a stock solution (1 mM) of the probe-labeled collagen in DMSO. Add 20 μL of the stock solution to 2 mL of PBS solution to obtain a working solution of the probe-labeled collagen with a concentration of 10 μM. The ultraviolet-visible absorption spectra of the two probe-labeled collagens are obtained using a HITACH U-2910 ultraviolet spectrophotometer.

[0081] The experimental results are shown in Figure 7 .

[0082] From Figure 7 the results, it can be seen that: after fluorescence labeling, the absorption peak of Ka12 blue-shifts from 436 nm to 420 nm, and the absorption peak of TPA12 blue-shifts from 470 nm to 440 nm, indicating that Ka12 and TPA12 can fluorescently label collagen, and after the fluorescent probe binds to collagen, its own luminescence properties are affected by the structural change.

[0083] Example 8: Cytotoxicity Test of Collagen with Probe Molecules Ka12 and TPA12

[0084] Inoculate the HACAT and HSF cell lines into 96-well plates at a density of 5×10 per well 3Cells. After allowing the cells to adhere, replace the original medium and rinse the wells with PBS. The control group uses serum-free medium, and the experimental group uses serum-free medium containing the sample. After incubating for 48 hours, add 50 μL of MTT solution (5 mg / mL) to each well. After 2 hours, remove the MTT solution and add 150 μL of DMSO to each well. Then measure the absorbance at 490 nm using a Bio-Tek microplate reader. Each concentration is tested five times for analysis.

[0085] The experimental results are shown in Figure 8 .

[0086] From Figure 8 the experimental results, it can be seen that the survival rates of the cell lines incubated with different concentrations of collagen and the cell lines incubated with the probe molecule both exceed 80%. The results show that collagen, probe Ka12, and TPA12 all exhibit good biocompatibility.

[0087] Example 9: Cell scratch repair experiment of Ka12 and TPA12 labeled collagen

[0088] To study how collagen affects the migration of HACAT and HSF cells, an in vitro wound healing model was established. HACAT and HSF cells (1×10 6 cells / well) were seeded into 6-well plates and cultured until a confluent monolayer was formed. The monolayer cells were scratched with a sterile 200 μL pipette tip to form a wound, and then rinsed with PBS to remove cell debris. Then the samples were treated with collagen-containing medium (1 mg / mL) for further incubation. Scratch images were captured using a microscope, and the healing process was analyzed using ImageJ (×64) at 0 and 24 hours.

[0089] The experimental results are shown in Figure 9 .

[0090] From the experimental results, it can be seen that the blank group of cells without collagen incubation has the slowest healing rate, indicating that recombinant collagen can promote the migration and healing process of the cell line, and the effect is better than commercial collagen.

[0091] Example 10: Real-time quantitative polymerase chain reaction and Western blot experiments

[0092] To study the effects of HACAT and HSF cells on cell differentiation, the cells were cultured in a collagen-containing medium (1 mg / mL), and the medium was changed every 2 days for a total of 4 times. After 1 week, the cells were collected into different centrifuge tubes for RNA extraction. After lysing with Trizol reagent, they were incubated at room temperature for 8 minutes. The lysate was transferred to a new 2 mL microtube, incubated at 25 °C for 10 minutes, mixed with 200 μL of chloroform, and incubated again at 25 °C for 10 minutes. Then, the mixture was centrifuged at 12,000 rpm at 4 °C for 20 minutes. The supernatant was transferred to a new 1.5 mL microtube, mixed with isopropanol, and incubated at 25 °C for 10 minutes. Centrifuged at 12,000 rpm at 4 °C for 10 minutes, the supernatant was discarded, the precipitate was washed with 75% ethanol and centrifuged, and the final precipitate was dissolved in enzyme-free water. Complementary DNA (cDNA) synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed using a Bio-Rad system (USA). The RNA expression level of the β-actin gene was used as a reference to normalize the RNA expression levels of other genes. To study the protein expression levels of HACAT and HSF cells in cell differentiation, Western blot analysis was performed. First, the cells were lysed using ice-cold RIPA lysis buffer (containing protease and phosphatase inhibitors), and the supernatant was collected by centrifugation at 12,000 rpm at 4 °C. After measuring the protein concentration, an equal amount of protein sample was added to 5× SDS-PAGE loading buffer and heated at 100 °C for 5 minutes to completely denature the protein. After electrophoresis on a 10%-12% SDS-PAGE gel, the protein was transferred to a methanol-activated PVDF membrane, and the membrane was transferred using a wet transfer system at a current of 300 mA in an ice bath for 90 minutes. After membrane transfer, it was blocked with TBST buffer containing 5% non-fat milk powder at room temperature for 1 hour, and specific primary antibodies (such as those against Col I, TGF-β1, VIM, etc.) were added and incubated overnight at 4 °C. The next day, after washing the membrane with TBST, a horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing the membrane again, the protein signal was detected using ECL luminescence reagent, and the bands were recorded using a chemiluminescence imager. Finally, software such as ImageJ was used to analyze the gray values of the bands, and β-actin or GAPDH was used as an internal reference to normalize the expression level of the target protein, thereby verifying the consistency between gene expression and protein level.

[0093] The results are shown in Figure 10 。

[0094] Type I collagen (Colα(I)), as an important component of the extracellular matrix, plays a key role in wound healing and tissue strength. The study evaluated the effect of collagen on promoting the differentiation of Colα(I), TGF-β1, and VIM in cells by RT-qPCR method. The experiment found that when the collagen concentration was 1 mg / mL, the mRNA levels related to the differentiation of HACAT and HSF cells were significantly increased. Under the NewCoII culture conditions, the expressions of Colα(I) and TGF-β1 in HACAT and HSF cells increased respectively, and at the same time, the expression of vimentin also increased. When comparing the effects of Col I and NewCoII, it was found that the treatment with Col I increased the expression of vimentin in HACAT cells, which was significantly higher than that of NewCoII. Although there was no significant difference in enhancing the expression of TGF-β1 between the two, both Col I and NewCoII significantly promoted the differentiation of TGF-β1, thereby increasing the synthesis and deposition of Colα(I). In addition, both treatments up-regulated the expression of VIM, further verifying its key role in promoting cell stability and supporting wound healing.

[0095] Example 11: Testing of JC-1 and reactive oxygen species in the photoaging model of HACAT cells

[0096] After HACAT and HSF cells were cultured in a culture dish until they reached about 90% confluence, they were transferred to a 6-well plate at a density of 1×10 6 cells per well, grouped, modeled, and treated according to the protocol described in the section "Collagen, Circular Dichroism" for 24 hours. After removing the culture medium, 1 mL of DCFH-DA (10 μmol / L) was added and diluted to a serum-free medium solution at a ratio of 1:1000. The cells were incubated at 37 °C and 5% CO2 for 20 min, and gently mixed every 5 min to ensure full contact between the probe and the cells. Subsequently, the cells were washed three times with serum-free medium and then three times with PBS. Finally, 1 mL of PBS was added, and the level of reactive oxygen species (ROS) was evaluated by fluorescence microscopy imaging. After HACAT cells were cultured in a culture dish until they reached about 90% confluence, they were transferred to a 6-well plate at a density of 1×10 6Transfer the density of the cells to a 6-well plate. According to the experimental grouping, establish the model and process it for 24 hours according to the protocol described in the "Collagen, Circular Dichroism" section. JC-1 detection dye treatment: After removing the medium, add 1 mL of JC-1 dye working solution (serum-free medium solution diluted to 10 μg / mL). The cells are incubated at 37 °C and 5% CO2 for 20 minutes, and gently mixed every 5 minutes during this period to ensure full contact of the dye with the cells. Cell washing: Gently wash the cells three times with serum-free medium to remove the unbound JC-1 dye, and then wash the cells three times with PBS. Fluorescence microscopy imaging: Observe and evaluate the fluorescence signal of JC-1 dye in the cells under the microscope. Under normal mitochondrial membrane potential, JC-1 exists in an aggregated form and emits red fluorescence; while when the mitochondrial membrane potential is lost, JC-1 exists in a monomeric form and emits green fluorescence. The change in mitochondrial membrane potential can be evaluated by the red / green fluorescence ratio. ROS damage assessment: Compare the changes in the red / green fluorescence ratio of different experimental groups, and analyze the level of cellular ROS damage in combination with the results of fluorescence microscopy imaging. This experimental design combines the sensitivity of JC-1 dye to mitochondrial membrane potential and the intuitive observation of fluorescence microscopy, and can effectively evaluate the ROS damage and mitochondrial function changes under different treatment conditions.

[0097] The experimental results are shown in Figure 11 .

[0098] Studies have shown that the intracellular reactive oxygen species level of HACAT cells exposed to UVB irradiation at 15 mJ / cm 2 is significantly higher than that of unirradiated HACAT cells. When these UVB-irradiated cells are cultured in a medium containing type I collagen or NewCoII, their reactive oxygen species level is significantly lower than that of the untreated group, with a significance level of P less than 0.01. This indicates that type I collagen and NewCoII have the effect of repairing UVB-induced photoaging at the cellular level. Further comparison of the treatment effects of type I collagen and NewCoII found that there was no significant difference between them in reducing the reactive oxygen species level, indicating that they are equally effective in reducing UVB-induced cell damage. This further confirms the repair effect of type I collagen and NewCoII on UVB-induced photoaging at the cellular level. Similarly, there was no significant difference in the effect of the two treatment methods on reducing the reactive oxygen species level, further indicating their equivalence in repairing UVB-induced reactive oxygen species damage.

[0099] Example 12: Metabolic Toxicity Evaluation of Fluorescently Labeled Collagen and Fluorescent Probe Molecules in Mice

[0100] To evaluate the toxic damage caused by the metabolism of fluorescently labeled collagen and fluorescent probe molecules in mice, five groups of healthy mice were depilated at the same position, and then skin damage was created on the smooth skin using medical tape to establish a mouse damage model. One group was used as the blank group without any treatment. Two probe molecules and their labeled collagens were each in a group, resulting in a total of five groups of data. Each time, the fluorescently labeled collagen and the probe solution were used to damage and apply to the mouse skin, once every 12 h for a total of 7 days. Subsequently, the hearts, livers, spleens, lungs, and kidneys of the five groups of mice were taken for HE staining sections and observed.

[0101] The experimental results are shown in Figure 12 。

[0102] It can be seen from the experimental results that there were no obvious abnormalities in the hearts, livers, spleens, lungs, and kidneys of the five groups of mice, indicating that the metabolic process of fluorescently labeled collagen and fluorescent probe molecules in mice did not cause toxic damage to the organs of the mice themselves.

[0103] Example 13: Toxicity Evaluation of Fluorescently Labeled Collagen and Fluorescent Probe Molecules on Mouse Skin

[0104] To evaluate the toxic damage caused by fluorescently labeled collagen and fluorescent probe molecules to mouse skin during the absorption and metabolism processes in mouse skin, five groups of healthy mice were depilated at the same position, and then skin damage was created on the smooth skin using medical tape to establish a mouse damage model. One group was used as the blank group without any treatment. Two probe molecules and their labeled collagens were each in a group, resulting in a total of five groups of data. Each time, the fluorescently labeled collagen and the probe solution were used to damage and apply to the mouse skin for a total of 7 days, once every 12 h. Subsequently, the skin at the application sites of the five groups of mice was taken for HE staining sections and Masson staining sections and observed.

[0105] The experimental results are shown in Figure 13 。

[0106] It can be seen from the experimental results that there were no obvious abnormalities in the skin of the five groups of mice, indicating that the absorption and metabolism processes of fluorescently labeled collagen and fluorescent probe molecules in mouse skin did not cause toxic damage to the skin of the mice.

[0107] Example 14: Evaluation of Mouse Skin Absorption and Metabolism of Fluorescently Labeled Collagen and Fluorescent Probe Molecules

[0108] To evaluate the degree of absorption and metabolism of fluorescently labeled collagen and fluorescent probe molecules by mouse skin, after depilating five groups of healthy white mice at the same position, skin damage was created on the smooth skin using medical tape to establish a mouse damage model, and one group was used as the blank group without any treatment. Two probe molecules and their labeled collagens were each in a group, resulting in a total of five groups of data. Each time, the fluorescently labeled collagen and the probe solution were used to damage and apply to the mouse skin, once every 12 h for a total of 7 days. Subsequently, the skin at the application sites of the five groups of mice was taken for sectioning and imaged and observed under a fluorescence confocal microscope.

[0109] The experimental results are shown in Figure 14 .

[0110] From the experimental results, it can be seen that no obvious fluorescence or background fluorescence was observed in the skin sections of the mice in the blank group. Fluorescence was observed in the skin sections of the experimental group mice treated with the fluorescently labeled protein and probe molecules. The fluorescence intensity of the skin sections of the experimental group mice with fluorescently labeled collagen was relatively low and mainly distributed in the skin surface layer. The fluorescence intensity of the skin sections of the experimental group mice with the fluorescent probe was high and well distributed in all skin layers. This indicates that both probe molecules have good biocompatibility, can be permeated, absorbed, and metabolized by the skin, while the fluorescently labeled collagen is difficult to penetrate into the skin and enters the skin through absorption and metabolism.

[0111] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of them. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. Although the specific implementation manners of the present invention have been described above, it does not limit the protection scope of the present invention. Those skilled in the art should understand that based on the technical solutions of the present invention, various modifications or deformations that can be made without creative efforts by those skilled in the art are still within the protection scope of the present invention.

Claims

1. A compound, characterized in that The compound has a structural formula as shown in formula (I) or formula (II):

2. The compound according to claim 1, characterized in that The compound may also include a pharmaceutically acceptable salt or ester or solvate, tautomer, meso-racemate, racemate, stereoisomer or metabolite thereof.

3. A method for synthesizing the compound according to claim 1 or 2, characterized in that: The method comprises the steps of respectively reacting derivatives of carbazole and triphenylamine with a pyridinium salt containing a long-chain carboxyl group through a Knoevenage reaction to prepare the compound.

4. The method according to claim 3, characterized in that The synthetic route of the compound is as follows:

5. Use of the compound according to claim 1 or 2 as a fluorescent probe or in the preparation of a fluorescent probe.

6. A detection product, characterized in that: The test product at least comprises the compound according to claim 1 or 2.

7. The detection product according to claim 6, characterized in that: The detection product is a detection kit, a detection device and / or a detection equipment; Furthermore, the product also contains substances and components commonly used in detection kits, detection devices and / or detection equipment; further, the detection kit contains a solvent, and the solvent is further a phosphate buffer solution or physiological saline; the detection device or detection equipment contains a fluorescence spectrophotometer.

8. Use of the compound according to any one of claims 1 to 2 or the detection product according to any one of claims 6 to 7 in labeling collagen.

9. A method for labeling collagen, characterized in that: The method comprises: using the compound according to claim 1 or 2 or the detection product according to any one of claims 6 to 7 to perform labeling detection on a sample to be tested that contains or is suspected of containing collagen.

10. The method according to claim 9, characterized in that The sample to be tested is a biological cell, biological tissue, biological organ or biological individual.

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