Water-soluble nuclear targeting probes with V-shaped structure and red fluorescence and their applications

Abstract

This invention belongs to the field of new materials and discloses a water-soluble nuclear targeting probe with a V-shaped structure and red fluorescence, as well as its applications. This probe is an aggregation-induced emission fluorescent probe material with both lipophilic and hydrophilic properties, possessing a general structural formula as shown in formula (I) or (II), where M is a hydrophobic aromatic ring and A is a hydrophilic nitrogen heterocyclic cation. Due to its unique structure, the aggregation-induced emission fluorescent probe material of this invention exhibits amphiphilicity and better universality for the physiological environment of cells. This probe can individually target and stain the cell nucleus without background fluorescence, enabling wash-free imaging.

Description

Technical Field

[0001] This invention belongs to the field of new materials, and more specifically, relates to a water-soluble cell nucleus-targeting probe with a V-shaped structure and red fluorescence and its application. The probe is an amphiphilic / water-soluble fluorescent probe based on aggregation-induced emission properties, and is particularly capable of targeting and locating cell nuclei in bioimaging. Background Technology

[0002] Cellular fluorescence imaging has become an indispensable technique in modern cell biology, enabling the fluorescence visualization of cells and their constituent structures, and is gradually becoming an effective tool for disease diagnosis and treatment. The cell nucleus is the largest and most important component of the cell; it is the center of cellular metabolism, the repository of genetic information, and the genetic control center. Genetic material, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are primarily stored in the nucleus. DNA damage is associated with a range of diseases, including neurodegenerative diseases, immunodeficiency, and cancer.

[0003] Common dyes used for nuclear imaging are lipophilic dyes. Due to their inherent hydrophobic properties, lipophilic dyes spontaneously aggregate under physiological conditions, resulting in strong background fluorescence during imaging, which is detrimental to image formation. Therefore, excess dye needs to be washed away with a buffer solution after staining to reduce background fluorescence and improve contrast. Summary of the Invention

[0004] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide a water-soluble nuclear targeting probe with a V-shaped structure and red fluorescence, along with its applications. This type of molecule is amphiphilic; its lipophilicity allows it to penetrate the cell phospholipid membrane and enter the cell, while its hydrophilicity allows it to dissolve in water. Due to non-radiative transitions, it does not fluoresce in aqueous solutions. The V-shaped structure allows the molecule to be trapped within the grooves of the DNA double helix. Because intramolecular movement is restricted, it emits strong fluorescence upon excitation, thus achieving wash-free labeling of the cell nucleus. The probe of this invention is particularly applicable to bioimaging, capable of individually targeting and staining the cell nucleus, providing clear imaging without the need for secondary rinsing.

[0005] To achieve the above objectives, according to one aspect of the present invention, a water-soluble nuclear targeting probe material with a V-shaped structure and red fluorescence is provided, characterized in that it has a general structural formula as shown in formula (I) or formula (II):

[0006]

[0007] Where M is a hydrophobic aromatic ring and A is a hydrophilic nitrogen heterocyclic cation;

[0008] The probe material is an aggregation-induced emission fluorescent probe material with both lipophilic and hydrophilic properties.

[0009] As a further preferred embodiment of the present invention, M is selected from benzene rings, naphthyl rings, anthracene rings, thiophene, thiazole, imidazole, indole, aniline, diphenylamine, triphenylamine, carbazole, and fluorene, with or without substituents.

[0010] As a further preferred embodiment of the present invention, M is selected from:

[0011]

[0012] R1 and R2 are hydrogen, fluorine, chlorine, bromine, iodine, nitro, cyano, carboxyl, hydroxyl, ester, alkyl, terminal amino alkyl, or terminal hydroxy alkyl.

[0013] As a further preferred embodiment of the present invention, A is selected from pyridine cations, quinoline cations, pyrimidine cations, and their derivative hydrophilic groups.

[0014] As a further preferred embodiment of the present invention, A is selected from:

[0015]

[0016] Wherein, R3 is a water-soluble group, and R4 is hydrogen, fluorine, chlorine, bromine, iodine, nitro, cyano, carboxyl, hydroxyl, ester, alkyl, terminal aminoalkyl or terminal hydroxyalkyl.

[0017] Preferably, R3 is selected from:

[0018]

[0019] According to another aspect of the present invention, the present invention provides the application of the above-mentioned water-soluble nuclear targeting probe material with red fluorescence having a V-shaped structure in the preparation of nuclear dyes.

[0020] According to another aspect of the present invention, the present invention provides the application of the above-described water-soluble nuclear targeting probe material with a V-shaped structure and red fluorescence in the preparation of dyes for nuclear imaging.

[0021] As a further preferred embodiment of the present invention, the dye for nuclear imaging is capable of targeting and locating the cell nucleus.

[0022] As a further preferred embodiment of the present invention, the dye used for nuclear imaging does not require washing away excess dye with a buffer solution after staining, and can be directly used for subsequent imaging.

[0023] As a further preferred embodiment of the present invention, the concentration of the fluorescent probe in the dye is from 0.1 μmol / L to 2 mol / L, preferably from 5 μmol / L to 50 μmol / L.

[0024] Compared with existing technologies, the technical solutions conceived in this invention, due to the unique structure of the probe, possess excellent water solubility compared to other commercially available nuclear fluorescent probes. Simultaneously, they exhibit both lipophilic and hydrophilic properties (i.e., amphiphilic), providing greater versatility for the physiological environment of cells. Therefore, they offer many significant advantages over lipophilic compounds in fluorescent imaging for targeted nuclear staining. Using the fluorescent probes of this invention for cell staining allows for targeted staining of the cell nucleus (i.e., singular targeting and staining of the nucleus) without background fluorescence, enabling wash-free imaging. Furthermore, due to the unique molecular structure of the fluorescent probes, they possess excellent aggregation-induced emission properties, making them amphiphilic fluorescent probes based on aggregation-induced emission.

[0025] The fluorescent probes in this invention, with structures shown in Formulas (I) and (II), are V-shaped, red-fluorescent, water-soluble nuclear targeting probes. These probes are amphiphilic, completely soluble in aqueous solutions, and exhibit negligible fluorescence in the aqueous solution. They only emit strong fluorescence after binding to the target, enabling wash-free imaging (i.e., the probe material of this invention produces no background fluorescence during nuclear imaging, eliminating the need for washing away excess dye with a buffer solution after staining, as required by other existing staining methods; the imaging process is simple and rapid; for example, commercial nuclear dye DAPI requires washing 2-3 times with TBST, PBS, or physiological saline for 3-5 minutes each time during imaging). This provides unique advantages for long-term monitoring and tracking of dynamic cellular biological processes. Taking TPA-2OH and TPA-3OH in this invention as examples, their lipid-water partition coefficients (P) are -1.58 and -2.15, respectively, indicating strong water solubility. The fluorescent probe material of this invention is amphiphilic, making it more universally compatible with the physiological environment of cells. Commercially available nuclear dyes, such as DAPI, are highly irritating to humans and suffer from fluorescence quenching. However, the fluorescent probe material of this invention does not exhibit quenching with increasing probe solution concentration. When used in imaging, the concentration of the fluorescent probe material in the solution can range from 0.1 μmol / L to 2 mol / L, providing a wide applicable concentration range.

[0026] Specifically, the present invention can achieve the following beneficial effects:

[0027] (1) The V-shaped red fluorescent water-soluble nuclear targeting probe of the present invention has amphiphilic solubility properties and can be dissolved in both aqueous and oily environments. The application range of the fluorescent probe is wider and it has better universality.

[0028] (2) The V-shaped red fluorescent water-soluble nuclear targeting probe of the present invention can also be dissolved in the physiological environment of cells, and has better biocompatibility.

[0029] (3) The V-shaped red fluorescent water-soluble nuclear targeting probe in this invention has virtually no fluorescence in the buffer solution and no background fluorescence during imaging (the background fluorescence is extremely low). The probe exhibits far-red fluorescence emission when it is in the aggregated state, and has better signal-to-noise ratio and penetration.

[0030] (4) The aggregation-induced emission fluorescent probe described in this invention is used for targeted nuclear imaging. It is simple to operate, eliminates the need for secondary rinsing, and provides clear imaging, giving it unique advantages for long-term monitoring and tracking of cellular ecological processes.

[0031] In summary, the fluorescent probe of this invention is more universally applicable to the physiological environment of cells during nuclear imaging, can target and locate stained cell nuclei, and is simple to operate, produces clear images, has no background fluorescence, and does not require secondary washing (i.e., it can achieve wash-free imaging). Attached Figure Description

[0032] Figure 1 These are imaging images of HeLa cells stained alone and co-stained with compound TPA-2OH and nuclear dye DAPI, as shown in Example 1 of this invention. Figure 1 (a) in the image corresponds to the DAPI-stained image. Figure 1 Image (b) in the image corresponds to TPA-2OH staining alone. Figure 1 (c) in the image corresponds to the confocal image under co-staining.

[0033] Figure 2 This is a schematic diagram of the molecular structure of the fluorescent probe TPA-2OH in Example 1 of the present invention.

[0034] Figure 3 This is a schematic diagram of the molecular structure of the fluorescent probe TPA-3OH in Example 2 of the present invention.

[0035] Figure 4 This is a schematic diagram of the fluorescent probe molecule structure in Example 3 of the present invention.

[0036] Figure 5 This is a schematic diagram of the fluorescent probe molecule structure in Example 4 of the present invention.

[0037] Figure 6 This is a schematic diagram of the fluorescent probe molecule structure in Example 5 of the present invention.

[0038] Figure 7 This is a schematic diagram of the fluorescent probe molecule structure in Example 6 of the present invention.

[0039] Figure 8 The images shown are from Example 2 of this invention, showing HeLa cells stained individually and co-stained with compound TPA-3OH and the nuclear dye DAPI. Figure 8 (a) in the image corresponds to the DAPI-stained image. Figure 8 Image (b) in the image corresponds to TPA-3OH staining alone. Figure 8 (c) in the image corresponds to the confocal image under co-staining.

[0040] Figure 9 These are imaging images of HeLa cells stained individually and co-stained with the compound and nuclear dye DAPI in Example 3 of this invention. Figure 9 (a) in the image corresponds to the DAPI-stained image. Figure 9 Image (b) in this example corresponds to the image obtained by staining the compound alone in Example 3. Figure 9 (c) in the image corresponds to the confocal image under co-staining.

[0041] Figure 10 These are imaging images of HeLa cells stained individually and co-stained with the compound and nuclear dye DAPI, as shown in Example 4 of this invention. Figure 10 (a) in the image corresponds to the DAPI-stained image. Figure 10 Image (b) in this example corresponds to the image obtained by staining the compound alone in Example 4. Figure 10 (c) in the image corresponds to the confocal image under co-staining.

[0042] Figure 11 These are imaging images of HeLa cells stained individually and co-stained with the compound and nuclear dye DAPI, as shown in Example 5 of this invention. Figure 11 (a) in the image corresponds to the DAPI-stained image. Figure 11 Image (b) in the image corresponds to the image of the compound stained alone in Example 5. Figure 11 (c) in the image corresponds to the confocal image under co-staining.

[0043] Figure 12 These are imaging images of HeLa cells stained individually and co-stained with the compound and nuclear dye DAPI, as shown in Example 6 of this invention. Figure 12 (a) in the image corresponds to the DAPI-stained image. Figure 12 Image (b) in this example corresponds to the image obtained by staining the compound alone in Example 6. Figure 12 (c) in the image corresponds to the confocal image under co-staining.

[0044] Figure 13 This is a schematic diagram of the interaction between compound TPA-2OH and DNA in Example 1.

[0045] Figure 14 This is a schematic diagram of the interaction between compound TPA-3OH and DNA in Example 2.

[0046] Figure 15 The images show the UV-Vis absorption spectra of compounds TPA-2OH and TPA-3OH; among them, Figure 15 (a) in the figure corresponds to TPA-2OH, and the curves from bottom to top correspond to TPA-2OH concentrations of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM and 35 μM respectively; Figure 15 (b) in the figure corresponds to TPA-3OH, and the curves from bottom to top correspond to TPA-3OH concentrations of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM and 35 μM respectively.

[0047] Figure 16 The fluorescence spectra of compounds TPA-2OH and TPA-3OH in relation to changes in DNA content are shown; among them, Figure 16 (a) in the figure corresponds to TPA-2OH, and the curves from bottom to top correspond to DNA concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL, respectively. Figure 16 (b) in the figure corresponds to TPA-3OH, and the curves from bottom to top correspond to DNA concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL respectively. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0049] When the fluorescent probe of this invention is applied to targeted nuclear fluorescence imaging, the solution containing the fluorescent probe can stain the cells. The fluorescent probe can target and stain the cell nucleus without the need for secondary rinsing, and can form nuclear fluorescence imaging with high clarity, contrast and signal-to-noise ratio.

[0050] The solution of the fluorescent probe described in this invention is a solution obtained by dissolving the fluorescent probe in a solvent. In some embodiments, the solvent is deionized water or an organic solvent.

[0051] In a preferred embodiment, the solution of the fluorescent probe is an aqueous solution of the fluorescent probe.

[0052] In some embodiments, the concentration of the fluorescent probe in the fluorescent probe solution is from 0.1 μmol / L to 2 mol / L, preferably from 5 μmol / L to 50 μmol / L.

[0053] The following are specific examples:

[0054] Example 1

[0055] A structure such as Figure 2 The fluorescent probe shown is abbreviated as TPA-2OH. HeLa cells were stained with TPA-2OH and the nuclear dye DAPI at a concentration of 2 μM and 5 μM, respectively, and fluorescence imaging was performed using confocal microscopy.

[0056] Through the imaging image Figure 1 As shown, both the red channel of TPA-2OH and the blue channel of the nuclear dye DAPI show obvious elliptical patches. When the two channel images are superimposed, the red and blue parts overlap and turn into purple, proving that TPA-2OH and the nuclear dye DAPI target the same cellular structure, namely the cell nucleus.

[0057] Example 2

[0058] One structure is as follows Figure 3 The fluorescent probe shown is abbreviated as TPA-3OH. HeLa cells were stained with TPA-3OH and the nuclear dye DAPI at a concentration of 2 μM and 5 μM, respectively, and fluorescence imaging was performed using a confocal microscope.

[0059] Through imaging Figure 8 As shown, both the red channel of TPA-3OH and the blue channel of the nuclear dye DAPI show obvious elliptical patches. When the two channel images are superimposed, the red and blue parts overlap and turn into purple, proving that TPA-3OH and the nuclear dye DAPI target the same cellular structure, namely the cell nucleus.

[0060] Example 3

[0061] One structure is as follows Figure 4 The fluorescent probe shown was used to stained HeLa cells with the nuclear dye DAPI at a concentration of 2 μM and 5 μM, and fluorescence imaging was performed using a confocal microscope.

[0062] Through imaging Figure 9As shown, both the red channel of the probe and the blue channel of the nuclear dye DAPI show obvious elliptical patches. When the two channel images are superimposed, the red and blue parts overlap and turn into purple, proving that the probe molecule and the nuclear dye DAPI target the same cellular structure, namely the cell nucleus.

[0063] Example 4

[0064] One structure is as follows Figure 5 The fluorescent probe shown was used to stained HeLa cells with the nuclear dye DAPI at a concentration of 2 μM and 5 μM, and fluorescence imaging was performed using a confocal microscope.

[0065] Through imaging Figure 10 As shown, both the red channel of the probe and the blue channel of the nuclear dye DAPI show obvious elliptical patches. When the two channel images are superimposed, the red and blue parts overlap and turn into purple, proving that the probe molecule and the nuclear dye DAPI target the same cellular structure, namely the cell nucleus.

[0066] Example 5

[0067] One structure is as follows Figure 6 The fluorescent probe shown was used to stained HeLa cells with the nuclear dye DAPI at a concentration of 2 μM and 5 μM, and fluorescence imaging was performed using a confocal microscope.

[0068] Through imaging Figure 11 As shown, both the red channel of the probe and the blue channel of the nuclear dye DAPI show obvious elliptical patches. When the two channel images are superimposed, the red and blue parts overlap and turn into purple, proving that the probe molecule and the nuclear dye DAPI target the same cellular structure, namely the cell nucleus.

[0069] Example 6

[0070] One structure is as follows Figure 7 The fluorescent probe shown was used to stained HeLa cells with the nuclear dye DAPI at a concentration of 2 μM and 5 μM, and fluorescence imaging was performed using a confocal microscope.

[0071] Through imaging Figure 12As shown, both the red channel of the probe and the blue channel of the nuclear dye DAPI show obvious elliptical patches. When the two channel images are superimposed, the red and blue parts overlap and turn into purple, proving that the probe molecule and the nuclear dye DAPI target the same cellular structure, namely the cell nucleus.

[0072] Results Analysis

[0073] The lipid-water partition coefficients (P) of TPA-2OH and TPA-3OH were measured to be -1.58 and -2.15, respectively, as shown in Table 1, indicating that they have strong water solubility.

[0074] Table 1. Lipid-water partition coefficients of compounds TPA-2OH and TPA-3OH

[0075]

[0076] As can be seen from Examples 1, 2, 3, 4, 5, and 6, the general structural formula is: and Amphiphilic aggregation-induced emission fluorescent probe materials can target and locate cell nuclei, and obtain clear, high-contrast fluorescence images by confocal microscopy without the need for secondary rinsing.

[0077] Depend on Figures 1 to 12 It can be seen that the fluorescence imaging after co-staining Hale cells with the fluorescent molecular probes synthesized in Examples 1, 2, 3, and 4 and the nuclear dye DAPI shows that the amphiphilic aggregation-induced emission fluorescent probe materials synthesized in Examples 1, 2, 3, 4, 5, and 6, together with the nuclear dye DAPI, have the ability to specifically target and stain mitochondria. The molecule exhibits amphiphilic solubility and maintains the same excellent properties in various environments. Furthermore, since the physiological environment of cells is closer to an aqueous environment, the amphiphilic / water-soluble fluorescent probe molecule has better solubility in the environment, thus exhibiting no background fluorescence and eliminating the need for secondary rinsing.

[0078] The three amphiphilic aggregation-induced emission fluorescent probe materials synthesized in Examples 1, 2, 3, 4, 5, and 6 exhibit superior solubility properties and higher imaging performance (clarity, penetration, contrast, and signal-to-noise ratio) compared to commercially available nuclear dyes such as DAPI. Furthermore, they hold promise for in-situ observation of cell imaging processes and for long-term tracking of cell states.

[0079] The water-soluble nuclear targeting probes with red fluorescence and V-shaped structure in this invention can be prepared by comprehensively designing based on the specific chemical structural formulas of these probes and referring to the reaction types known in the prior art, which will not be elaborated here.

[0080] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a water-soluble nuclear targeting probe material with a V-shaped structure and red fluorescence in the preparation of wash-free nuclear dyes for nuclear imaging, characterized in that, The probe material has a structural formula as shown in any one of the following equations: The wash-free nuclear dye used for nuclear imaging does not require washing away excess dye with a buffer solution after staining, allowing for direct imaging.

2. The application as described in claim 1, characterized in that, The dye used for nuclear imaging can target and locate the cell nucleus.

3. The application as described in claim 1, characterized in that, The concentration of the fluorescent probe in the dye is from 0.1 μmol / L to 2 mol / L.

4. The application as described in claim 3, characterized in that, The concentration of the fluorescent probe in the dye is from 5 μmol / L to 50 μmol / L.

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