Visible light absorbing cell membrane dye based on membrane-embedded conjugated oligoelectrolyte and preparation method and application thereof

Abstract

The present invention provides a visible light absorbing cell membrane dye based on a membrane-embedded conjugated oligoelectrolyte, comprising a conjugated hydrophobic backbone and polar hydrophilic side chains connected to both sides of the conjugated hydrophobic backbone, so that the cell membrane dye has amphiphilicity and can self-assemble with the lipid bilayer on the cell membrane; the conjugated hydrophobic backbone is a fluorescent group; the conjugated hydrophobic backbone is a D-A-D structure; the D-A-D structure with a conjugated backbone and two arms of hydrophilic side chains connected at both ends, constructed by the technical solution of the present invention, can not only adjust the absorption and emission wavelengths by changing the donor-acceptor unit, but also place the donor-acceptor part in the hydrophobic environment of the phospholipid membrane, reduce the collision of small solvent molecules with the hydrophilic part of the receptor, and improve the fluorescence quantum yield; the stability of the organic conjugated backbone can reduce the photobleaching of the probe, ensure the high stability of the probe molecule, and show a more uniform and stable cell membrane staining effect compared with commercial membrane dyes in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of organic small molecule fluorescent dyes, and in particular to a visible light absorbing cell membrane dye based on a membrane-embedded conjugated oligoelectrolyte, and a preparation method and application thereof. Background Art

[0002] As a crucial component of cellular structure, the cell membrane not only maintains a stable metabolic intracellular environment, regulates and selects substances entering and exiting the cell, but also serves as a site for intracellular and extracellular information exchange. Therefore, revealing the biological composition and function of the cell membrane is crucial for studying the molecular mechanisms underlying disease. However, the current model of the cell membrane remains the fluid mosaic model proposed by Singer et al. in 1972. This model fails to adequately explain many phenomena, such as multiprotein collaboration, leading to the proposal of improved models such as lipid rafts, but further refinement is still needed. Optical imaging, on the other hand, provides a convenient method for visualizing and quantifying multiple dynamic biological events at the molecular level. It typically possesses high spatiotemporal resolution and sensitivity, making it a primary method for in situ and real-time analysis of biological membrane structure under physiological conditions. While commercial membrane dyes, such as DiO, exemplified by the cyanine series of dyes, have been widely used, they are limited by poor water solubility, the need for organic solvents for solubilization, uneven staining, and poor photostability, making them unable to meet the stringent dye requirements imposed by emerging optical imaging technologies such as super-resolution imaging. Therefore, the development of a new generation of membrane structure imaging reagents with low toxicity, high sensitivity, high photostability, and high membrane embedding stability is an urgent need in the field of research on the fine structure and function of biological membranes.

[0003] Conjugated polyelectrolytes (CPEs) are usually composed of a lipophilic polymer conjugated main chain and an ionizable hydrophilic side chain. Due to their simultaneous ionization and molecular wire properties, they are widely used in water environment detection and biological detection, as well as biosensing and optoelectronic fields (Adv. Mater., 2008, 20, 3793-3810). Membrane-embedded conjugated oligoelectrolytes (MICOEs), as a type of conjugated polyelectrolyte, are a type of water-soluble optical small molecule with a special linear structure having a hydrophobic backbone in the middle and hydrophilic positively charged side chains at both ends. They are composed of a hydrophobic main chain with an electronically delocalized structure and suspended ionic functional groups. They can be orderly assembled with a bilayer phospholipid membrane and have excellent water solubility, photostability, tunable energy bands, and membrane-embedded fluorescence enhancement. They have a wide range of applications in membrane-related antibacterial, biocatalysis, fluorescence imaging, etc. (Chem. Soc. Rev., 2022, 51, 9917-9932).

[0004] However, there are few studies on the use of conjugated polyelectrolytes as cell membrane dye reagents, especially for commercial membrane dyes such as DiO, which are represented by the cyanine series dyes. They are composed of a hydrophilic part on one side and a highly hydrophobic conjugated structure and alkyl chain on the other side. They are lipophilic membrane dyes. They gradually diffuse laterally into the hydrophobic area of ​​the cell membrane to stain the cell membrane. Therefore, they have problems such as poor water solubility and the need for organic solvents to assist in dissolution and imaging. Although other commercial membrane dyes such as FM 2-10 provide a larger hydrophilic polar group at one end, they are also based on the electrostatic adsorption of the hydrophilic group on one side and the hydrophilic part of one end of the cell membrane phospholipid bilayer and the insertion of the hydrophobic structure on the other side of the molecule into the hydrophobic end of the cell membrane phospholipid layer for staining. Such a structure may lead to problems such as uneven staining and poor photostability. Therefore, there is an urgent need to provide a commercial membrane dye with excellent stability and good water solubility.

[0005] Based on this, the present invention provides a conjugated polyelectrolyte molecule as a cell membrane dye reagent on the basis of the existing technology. Summary of the Invention

[0006] Based on the existing technology, the present invention provides a cell membrane dye based on membrane-embedded conjugated oligoelectrolytes, a preparation method and application thereof, and solves the technical problems of cell membrane dyes in the existing technology through a conjugated hydrophobic backbone structure with a DAD structure, thereby promoting the commercial application of membrane dye reagents based on membrane-embedded conjugated oligoelectrolytes.

[0007] To achieve the above objectives, the present invention provides a visible light absorbing cell membrane dye based on a membrane-embedded conjugated oligoelectrolyte, comprising a conjugated hydrophobic backbone and polar hydrophilic side chain arms connected to both sides of the conjugated hydrophobic backbone, so that the cell membrane dye has amphiphilicity and can self-assemble with the lipid bilayer on the cell membrane; the conjugated hydrophobic backbone is a fluorescent group; the conjugated hydrophobic backbone is a DAD structure; wherein D is a donor group, A is an acceptor group, and the acceptor group; the conjugated hydrophobic backbone adjusts the absorption and emission wavelengths by changing the donor-acceptor unit, so that the cell membrane dye can absorb visible light;

[0008] The wavelength of the visible light is 450-700 nm.

[0009] Preferably, the donor group in the conjugated hydrophobic backbone includes any one of a double bond, a benzene ring, furan or thiophene.

[0010] Preferably, the receptor groups in the conjugated hydrophobic backbone include

[0011] Preferably, the polar hydrophilic side chain in the polar hydrophilic side chain double arms comprises one of the following groups:

[0012] Wherein, n is the number of CH2 on the alkyl quaternary ammonium chain, and n=2 to 20.

[0013] Preferably, the cell membrane dye has a structure represented by any one of formula (I1), formula (I2) or formula (I3);

[0014] The structures of general formulas (I1) to (I3) are shown below:

[0015]

[0016] In general formula (I1), general formula (I2) and general formula (I3):

[0017] n is the number of CH2 on the alkyl quaternary ammonium chain, where n = 2-20;

[0018] D is a donor group, including any one of a double bond, a benzene ring, furan or thiophene;

[0019] A is an acceptor group, including Any of .

[0020] As one of the most preferred embodiments of the present invention, in general formula (I1), general formula (I2) and general formula (I3), n=8, D is thiophene, A is benzothiadiazole, and the compound represented by formula (I2) is TBT-DC8.

[0021] As another object, the present invention also provides a method for preparing the above-mentioned cell membrane dye based on membrane-embedded conjugated oligoelectrolyte, comprising the following steps:

[0022] S1. Coupling reaction;

[0023] The compound represented by formula (II) is mixed with the compounds represented by formula (III1), formula (III2) and formula (III3), respectively, and then a coupling reaction is carried out under the catalysis of tetrakistriphenylphosphine palladium to obtain the compounds represented by formula (IV1), formula (IV2) and formula (IV3), respectively;

[0024] S2. Quaternization reaction;

[0025] The compounds represented by formula (IV1), formula (IV2) and formula (IV3) are subjected to quaternization reaction under the action of trimethylamine to obtain compounds represented by formula (I1), formula (I2) or formula (I3); wherein the structure of formula (II) is as follows:

[0026] is a donor group, including any one of a double bond, a benzene ring, furan or thiophene;

[0027] A is an acceptor group, including Any of;

[0028] The structure of formula (III1) is shown below: Wherein, n=2 to 20;

[0029] The structure of formula (III2) is shown below: Wherein, n=2 to 20;

[0030] The structure of formula (III3) is shown below: Wherein, n=2 to 20;

[0031] The structural formula of formula (IV1) is shown below:

[0032] Wherein, n=2 to 20;

[0033] The structural formula of formula (IV2) is shown below:

[0034] Wherein, n=2 to 20;

[0035] The structural formula of formula (IV3) is shown below:

[0036] Among them, n=2~20.

[0037] Preferably, in S1, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III1) or formula (III2) or formula (III3) is 1:2 to 1:5, and the molar ratio of the compound represented by formula (II) to tetrakistriphenylphosphine palladium is 1:0.5 to 1:2.

[0038] Preferably, the reaction conditions of the catalytic reaction include reacting in tetrahydrofuran under nitrogen protection at 80-90° C. for 24-48 hours;

[0039] In S2, the molar ratio of the compound represented by formula (IV1) or formula (IV2) or formula (IV3) to trimethylamine is 1:100 to 1:200.

[0040] The reaction conditions of the coupling reaction include reacting in tetrahydrofuran and methanol at 40° C. for 12 h to 48 h.

[0041] The cell membrane dye is used as a membrane fluorescent marker or a visible light absorption fluorescent probe of the cell membrane dye in mammalian cell membrane for fluorescence imaging of the cell membrane.

[0042] The mammalian cells include but are not limited to suspension cells such as red blood cells and white blood cells, and adherent cells such as fibroblasts, lung cancer cells, breast cancer cells, and cervical cancer cells.

[0043] The beneficial technical effects obtained by the present invention are:

[0044] 1. Using the technical solution of the present invention, the membrane-embedded conjugated oligoelectrolyte series molecules provided by the present invention contain conjugated hydrophobic backbone fluorophores and polar hydrophilic side chains, are amphiphilic, can self-assemble with the lipid bilayer on the cell membrane, and exhibit good biocompatibility, and can be used as cell membrane-specific fluorescent probes for cell membrane imaging. Among them, the conjugated backbone constructed with the "DAD" structure and the double arms of the hydrophilic side chains at both ends of the molecules can not only adjust the absorption and emission wavelengths by changing the receptor unit to achieve visible light absorption of the cell membrane dye, but also make the receptor part in the hydrophobic environment of the phospholipid membrane, thereby reducing the collision of small solvent molecules in the environment with the hydrophilic part of the receptor, and ensuring a higher fluorescence quantum yield. The stability of the selected organic conjugated backbone can reduce the photobleaching of the probe, ensuring the high stability of this series of molecules.

[0045] 2. The membrane-embedded conjugated oligoelectrolyte membrane dye provided by the technical solution of the present invention can exhibit a more uniform and stable cell membrane staining effect compared with commercial dyes in the prior art, such as DiO, CellMask Green, FM 2-10, etc., and can be used as a new cell membrane dye for stable imaging on mammalian cell membranes, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the preparation mechanism of the compounds represented by formula I1, I2 and I3 of the present invention.

[0047] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the TBT-DC8 molecule in Example 1 of the present invention.

[0048] Figure 3 This is the carbon NMR spectrum of the TBT-DC8 molecule in Example 1 of the present invention.

[0049] Figure 4 The UV-visible absorption spectra of TBT-DC8 molecules in different solution systems in Example 2 of the present invention are shown.

[0050] Figure 5 2 is the fluorescence emission spectrum of the TBT-DC8 molecule in different solution systems in Example 2 of the present invention.

[0051] Figure 6 This is the dark toxicity of TBT-DC8 to L929 cells in Example 3 of the present invention.

[0052] Figure 7 This is a laser scanning confocal imaging diagram showing the changes in the incubation time of TBT-DC8 molecules in L929 cells in Example 4 of the present invention.

[0053] Figure 8The figure shows a comparison of the cell membrane staining effects of the TBT-DC8 molecule in Example 5 of the present invention and common commercial dyes.

[0054] Figure 9a and Figure 9b The figures are respectively a comparison of the photostabilization effects of the TBT-DC8 molecule in Example 6 of the present invention and a commercial membrane dye.

[0055] Figure 10 Flow cytometry results comparing the staining stability of the TBT-DC8 molecule and commercial membrane dyes in Example 7 of the present invention.

[0056] Figure 11 The figure shows the comparison of the staining stability of the TBT-DC8 molecule in Example 7 of the present invention and a commercial membrane dye when the cell number reaches the peak under flow cytometry testing. DETAILED DESCRIPTION

[0057] In view of the shortcomings of the prior art, the present invention proposes a membrane-embedded conjugated oligoelectrolyte molecule targeting the cell membrane and provides a method for preparing the same. The specific technical solutions of the present invention are described in detail below through specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. For the sake of concise description, it is impossible for this specification to provide a detailed description of all features of the actual implementation methods. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0058] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in this specification should be known to those skilled in the art of this application.

[0059] The present invention provides a visible light absorbing cell membrane dye based on a membrane-embedded conjugated oligoelectrolyte, comprising a conjugated hydrophobic backbone and polar hydrophilic side chain arms connected to both sides of the conjugated hydrophobic backbone, so that the cell membrane dye has amphiphilicity; the membrane-embedded conjugated oligoelectrolyte can self-assemble with the lipid bilayer on the cell membrane; wherein the conjugated hydrophobic backbone is a DAD structure; wherein D is a donor group and A is an acceptor group. The technical solution has a conjugated hydrophobic backbone (DAD structure) and hydrophilic side chain arms connected to both sides of the backbone. On the one hand, the overall structure improves the water solubility of the cell membrane dye, and can adjust the absorption and emission wavelengths by changing the donor unit and the acceptor unit to achieve visible light absorption, and the wavelength of visible light absorption is 450-700nm; on the other hand, when the molecule is embedded in the cell membrane, the donor and acceptor parts can be placed in the hydrophobic environment of the phospholipid membrane, thereby reducing the collision of small solvent molecules in the environment with the hydrophilic part of the acceptor, ensuring a higher fluorescence quantum yield. In addition, the hydrophilic groups at both ends of the molecule are electrostatically bonded to the hydrophilic ends on both sides of the cell membrane phospholipid bilayer, which can improve its stability in cell membrane staining, thereby solving the technical problems existing in the existing technology.

[0060] The visible light absorbing cell membrane dye based on the membrane-intercalated conjugated oligoelectrolyte has a structure represented by any one of formula (I1), formula (I2) or formula (I3);

[0061] The structures of formulas (I1) to (I3) are shown below:

[0062]

[0063]

[0064] In formula (I1), formula (I2) and formula (I3):

[0065] n is the number of CH2 on the alkyl quaternary ammonium chain, where n = 2-20;

[0066] D is a donor group, including any one of a double bond, a benzene ring, furan or thiophene;

[0067] A is an acceptor group, including Any of .

[0068] The method for preparing the cell membrane dye based on the membrane-embedded conjugated oligoelectrolyte comprises the following steps:

[0069] S1. Coupling reaction;

[0070] The compound represented by formula (II) is mixed with the compounds represented by formula (III1), formula (III2) and formula (III3), respectively, and then a coupling reaction is carried out under the catalysis of tetrakistriphenylphosphine palladium to obtain the compounds represented by formula (IV1), formula (IV2) and formula (IV3), respectively;

[0071] S2. Quaternization reaction;

[0072] The compounds represented by formula (IV1), formula (IV2) and formula (IV3) are subjected to quaternization reaction under the action of trimethylamine to obtain compounds represented by formula (I1), formula (I2) or formula (I3); wherein the structure of formula (II) is as follows:

[0073] is a donor group, including any one of a double bond, a benzene ring, furan or thiophene;

[0074] A is an acceptor group, including Any of;

[0075] The structure of formula (III1) is shown below: Wherein, n=2 to 20;

[0076] The structure of formula (III2) is shown below: Wherein, n=2 to 20;

[0077] The structure of formula (III3) is shown below: Wherein, n=2 to 20;

[0078] The structural formula of formula (IV1) is shown below:

[0079] Wherein, n=2 to 20;

[0080] The structural formula of formula (IV2) is shown below:

[0081] Wherein, n=2 to 20;

[0082] The structural formula of formula (IV3) is shown below:

[0083] Among them, n=2~20.

[0084] The synthesis method of the compound described in Formula II refers to Angew.Chem.Int.Ed., 2017, 56, 5031-5034. Eur.J.Org.Chem.2020, 348-361.; Dyes and Pigments, 2022, 197: 109884.; J.Mater.Chem.C, 2018, 6, 10456-10463.

[0085] The compound represented by formula (II) is mixed with the compound represented by formula (III1) or formula (III2) or formula (III3) and tetrakistriphenylphosphine palladium in a molar ratio of 1:2.5:0.5, and reacted in tetrahydrofuran protected by nitrogen at 80-90°C for 24 hours.

[0086] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0087] Example 1

[0088] Synthesis of TBT-DC8

[0089] Chemical reaction flow chart Figure 1 As shown, the specific reaction step conditions are as follows:

[0090] 1) Synthesis of compound 2-8 represented by formula III2

[0091] 1,8-dibromooctane (14 g, 52.9 mmol), 5-bromo-1,3-benzenediol (2 g, 10.58 mmol) and anhydrous K CO (4.5 g, 31.7 mmol) were dissolved in acetone (60 mL) and deoxygenated with nitrogen bubbling for 30 minutes. The reaction mixture was then heated to 90° C. and stirred for 12 hours. The residual K CO was removed by filtration, and the filtrate was collected and the solvent was removed by rotary evaporation. The mixed product was separated by silica gel column chromatography to give 2-8 (3.4 g, 56.02%) as a white solid.

[0092] The structural confirmation data of the compound represented by Formula III2 by hydrogen and carbon nuclear magnetic resonance spectra are as follows:

[0093] 1H NMR (600MHz, Chloroform-d): δ6.63 (d, J=2.1Hz, 2H), 6.36 (t, J=2.2Hz, 1H), 3.90 (t, J=6.5Hz , 4H), 3.41 (t, J=6.8Hz, 4H), 1.86 (p, J=7.0Hz, 4H), 1.75 (p, J=6.8Hz, 4H), 1.48-1.32 (m, 16H); 13 C NMR (100MHz, Chloroform-d): δ 160.74, 122.86, 110.24, 100.62, 68.22, 34.02, 32.78, 29.14, 29.08, 28.69, 28.09, 25.91.

[0094] The high-resolution mass spectrometric structure confirmation data of the compound represented by Formula III2 are as follows:

[0095] HRMS (APCI), m / z: calcd for C 22 H 35 Br3O2 + [M] + ,570.0167;found,570.0222;

[0096] From the above, it can be seen that the synthesized structural formula is consistent with the characterization results.

[0097] 2) Synthesis of compound 5-8 represented by formula IV2 (coupling reaction)

[0098] In a glove box, 4,7-bis(5-trimethyltinthienyl-2-)-2,1,3-benzothiadiazole (0.1 g, 0.15 mmol), compound 2-8 (0.22 g, 0.39 mmol), tetrakistriphenylphosphine palladium (0.09 g, 0.08 mmol), and anhydrous tetrahydrofuran (5 mL) were added to a microwave tube. The tube was sealed and the reactants were stirred at 90°C for 24 hours. After the reaction, the mixture was evaporated to remove the solvent and eluted by silica gel column chromatography to obtain compound 5-8 (0.1 g, yield: 48.3%).

[0099] The structural confirmation data of the compound represented by Formula IV2 by hydrogen and carbon nuclear magnetic resonance spectra are as follows:

[0100] 1H NMR (600MHz, Chloroform-d): δ8.11 (d, J=3.8Hz, 2H), 7.89 (s, 2H), 7.40 (d, J=3.8Hz, 2H), 6.84 (d, J=2.2Hz, 4H), 6.43 (d, J= 2.3Hz, 2H), 4.01 (t, J=6.5Hz, 8H), 3.42 (t, J=6.8Hz, 8H), 1.88 (p, J=7.0Hz, 8H), 1.81 (p, J=6.8Hz, 8H), 1.52-1.33 (m, 32H); 13 CNMR (151MHz, Chloroform-d): δ160.69, 145.73, 138.64, 135.83, 128.49, 125.84, 125 .40, 124.38, 104.73, 100.94, 68.14, 33.99, 32.82, 29.26, 29.21, 28.71, 28.12, 26.01;

[0101] The high-resolution mass spectrometric structure confirmation data of the compound represented by Formula IV2 are as follows:

[0102] HRMS (APCI), m / z: calcd for C 58 H 76 Br4N2O4S3+[M] + , 1280.1660; found, 1280.1800; From the above, it can be seen that the structural formula of the compound represented by formula IV2 is consistent with the characterization results.

[0103] 3) Synthesis of the compound TBT-DC8 represented by formula I2 (quaternization)

[0104] First, compound 5-8 (0.03 g, 0.02 mmol) was dissolved in 5 mL of anhydrous tetrahydrofuran in a microwave tube, followed by the addition of 1 mL of a 2 M solution of trimethylamine in tetrahydrofuran. The mixture was then stirred at 40°C for 24 hours. The crude product was then evaporated to remove the solvent, and 1 mL of a 2 M solution of trimethylamine in methanol and 5 mL of ultra-dry methanol were added, followed by further stirring at 40°C for 24 hours. The mixture was evaporated to remove the solvent and redissolved in pure water. The mixture was then filtered through a 0.22 μm hydrophilic PVDF syringe filter and lyophilized to obtain TBT-DC8 (0.03 g, 100% yield) as a red solid.

[0105] See Figure 2 , is the H NMR spectrum of TBT-DC8, and the structural confirmation data are as follows:

[0106] 1H NMR (600MHz, DMSO-d6): δ8.20 (d, J=3.6Hz, 4H), 7.74 (d, J=3.8Hz, 2H), 6.88 (s, 4H), 6.49 (s, 2H), 4.04 (q, J=9 .5, 8.0Hz, 8H), 3.31-3.28 (m, 8H), 3.05 (d, J=3.0Hz, 36H), 1.72 (dq, J=40.0, 7.5Hz, 16H), 1.46-1.29 (m, 32H);

[0107] See Figure 3 This is the carbon NMR spectrum of TBT-DC8.

[0108] 13 C NMR (151MHz, DMSO-d6): δ160.92, 152.11, 138.17, 135.71, 129.07, 126.11, 125.74, 10 4.42, 101.27, 68.16, 65.77, 52.62, 40.54, 29.18, 29.07, 28.97, 26.23, 25.95, 22.51.;

[0109] Mass spectrometry data: HRMS (ESI), m / z: calculated for C 70 H 112 N6O4S3 4+ [M-4Br] 4+ , 299.1971; found, 299.1976;

[0110] From the above, it can be seen that the synthesis of TBT-DC8 is consistent with the results of NMR and mass spectrometry.

[0111] Example 2

[0112] Fluorescence performance test of compound TBT-DC8 represented by formula I2

[0113] TBT-DC8 was accurately weighed and dissolved in pure water to prepare a 1 mM aqueous stock solution. This stock solution was then diluted with DMSO, methanol, pure water, and 1× PBS to a 10 μM test solution. Simultaneously, a 100 μM TBT-DC8 liposome stock solution was prepared using dimyristoylphosphatidylglycerol sodium salt (DMPG) and cholesterol (molar ratio 1.7:1) via thin film hydration. This was further diluted with pure water to a 10 μM test solution. Subsequently, the absorption spectra of TBT-DC8 in various solution systems were measured at 350-800 nm using a UV-visible spectrophotometer.

[0114] The results are as follows Figure 4As shown, the absorption peak of the compound TBT-DC8 in different solution systems is around 500nm. Compared with pure water and 1x PBS buffer, TBT-DC8 has a larger molar extinction coefficient in the organic solvents DMSO and methanol. The molar extinction coefficient of TBT-DC8 in liposome solution is between that of pure water and 1x PBS. Next, fluorescence spectra of TBT-DC8 in different solution systems were measured under 500nm excitation using a fluorescence spectrometer.

[0115] like Figure 5 As shown, TBT-DC8 exhibits strong emission intensity in DMSO and methanol, with an emission peak around 630nm. However, as the polarity increases, the fluorescence intensity of TBT-DC8 in pure water and 1×PBS drops sharply, indicating that the fluorescence emission of TBT-DC8 is sensitive to the polarity of the solvent. Furthermore, after TBT-DC8 molecules are embedded in liposomes from aqueous solution, the fluorescence intensity is greatly enhanced, indicating that TBT-DC8 possesses membrane-embedded fluorescence enhancement properties, implying its potential as a membrane imaging probe.

[0116] Example 3

[0117] Dark toxicity test of compound TBT-DC8 represented by formula I2 in L929 cells

[0118] L929 cells were plated at 3 × 10 3 Cells were seeded at a density of 100 cells / mL in a 96-well plate and allowed to adhere overnight. The original culture medium was then discarded and various concentrations of TBT-DC8 were added. The cells were incubated in the dark at room temperature for 30 minutes. After each drug was aspirated, the cells were replaced with fresh culture medium and incubated in a 37°C incubator for another 48 hours. Cell proliferation was assessed using MTT. 10 μL of MTT (5 mg / mL in PBS) was added to each well and incubated at 37°C for 4 hours. The original culture medium was then removed, and 150 μL of DMSO was added to dissolve the purple crystals. The absorbance at 570 nm was measured using a microplate reader.

[0119] The results are as follows Figure 6 As shown in the results, TBT-DC8 has good biocompatibility and safety and can be used in biological imaging.

[0120] Example 4

[0121] Fluorescence distribution of the compound TBT-DC8 represented by formula I2 in L929 cells over time:

[0122] TBT-DC8 was prepared into 1 mM 1× PBS stock solution. Before cell imaging, 7×10 4L929 cells were seeded at a density of 10 cells / mL in a 35 mm confocal dish and allowed to adhere for 24 hours at 37°C. TBT-DC8 was diluted to 3 μmol / L and incubated for 5 minutes. After that, the culture medium was removed and the cells were washed twice with DMEM basal medium. Images were taken at different time intervals using a laser confocal microscope.

[0123] like Figure 7 As shown, TBT-DC8 can specifically target the cell membrane for imaging within a short period of time, with low signal-to-noise ratio and uniform distribution on the membrane. As time increases, only a very small amount of TBT-DC8 enters the cytoplasm, demonstrating its ability to stably image the cell membrane.

[0124] Example 5

[0125] Comparison of membrane imaging effects of the compound TBT-DC8 represented by formula I2 and common commercial dyes:

[0126] Commercial membrane dyes 2-10 (Thermo Fisher Scientific), DiO (Shanghai Biyuntian Biotechnology Co., Ltd.) was prepared as a 1mM DMSO stock solution and diluted to 3μmol / L cell staining working solution, and the commercial membrane dye CellMaskGreen (Thermo Fisher Scientific) was diluted to 3μg / mL cell staining working solution. Before cell imaging, 7×10 4 L929 cells were seeded into 35 mm confocal dish at a density of 100 cells / mL and allowed to adhere to the wall in a 37°C incubator for 24 h. After incubating the cells with 2-10 cell staining working solutions for 5 minutes, the culture medium was removed and the cells were washed twice with DMEM basal medium, and confocal images were taken.

[0127] like Figure 8 As shown, under the same incubation conditions, TBT-DC8 is more sensitive than CellMask Green, DiO, 2-10 These commercial membrane dyes stained the cell membrane more uniformly and clearly, which proved that TBT-DC8 can achieve efficient, stable and specific imaging of the cell membrane.

[0128] Example 6

[0129] This example provides a photostability test of the compound TBT-DC8 molecule represented by Formula I2, and compares it with commercial membrane dyes DiO and CellMask Green.

[0130] The test method includes: diluting 6% rabbit red blood cells to 3×10 6The red blood cells were washed twice by centrifugation at 2000 rpm for 3 min and then transferred to a confocal dish.

[0131] Washed rabbit erythrocytes were incubated with 3 μM TBT-DC8, 3 μM commercial membrane dye DiO, and 3 μg / mL CellMask Green at 37°C for 10 minutes. After incubation, the cells were centrifuged at 2000 rpm for 3 minutes, washed once, and then imaged under a confocal fluorescence microscope. During imaging, the laser intensity of the confocal fluorescence microscope was set to 4% and all other parameters were kept constant. The confocal dish was imaged every 30 seconds under continuous laser irradiation for approximately 6.5 minutes ( Figure 9a ). The fluorescence intensity (I) of the confocal images taken at different time periods was counted and divided by the initial fluorescence intensity (I0) for normalization. The results were compared. Figure 9b .

[0132] like Figure 9a and Figure 9b As shown, TBT-DC8 exhibits a slower fluorescence decay under continuous laser irradiation than the commercial membrane dye DiO, and its fluorescence decay trend is close to that of CellMask Green. These results demonstrate that TBT-DC8 has excellent photostability, and its photobleaching resistance meets the benchmark for use as a commercial membrane dye.

[0133] Example 7

[0134] This example provides a comparison of the staining stability of the compound TBT-DC8 molecule represented by Formula I2 and the commercial membrane dye CellMask Green:

[0135] 6% rabbit red blood cells were diluted with 1× PBS to 3×10 7 / mL, and wash the red blood cells twice by centrifugation at 2000 rpm for 3 minutes. Then take a tube of diluted red blood cells and add the prepared 3μM TBT-DC8 1×PBS solution and stain it at room temperature for 5 minutes. The stained red blood cells were washed 3 times by centrifugation at 2000 rpm for 3 minutes, and then mixed with unstained red blood cells in equal proportions every half an hour, and detected by flow cytometry. The stained and unstained cells were cultured at 37°C during the test. The CellMask Green test concentration was 3μg / mL and was tested in the same way. Figure 10 As shown, the flow cytometer will detect the strong fluorescence signal peak of the treated group stained with TBT-DC8 or CellMask Green molecules and the weak fluorescence signal peak of the untreated group each time the sample is loaded and scanned. Among them, the detection channel of the cells stained with CellMaskGreen in the flow cytometer is the FITC-H channel (see Figure 10 B in Figure 1); the detection channel for cells stained with TBT-DC8 was the PerCP-Cy 5.5-A channel (see Figure 10 Figure A in the figure).

[0136] Further based on Figure 10 The relationship between the fluorescence intensity at the peak of cell number in each time period and its change over time Figure 11 ,in, Figure 11 Figure A is a trend chart showing the change of fluorescence intensity of unstained red blood cells over time. Figure 11 Figure B is a trend chart showing the fluorescence intensity of stained red blood cells changing with time. Figure 10 and Figure 11 As shown, TBT-DC8 has excellent staining stability. Within 1.5 hours, the fluorescence intensity of TBT-DC8-stained erythrocytes after mixing was essentially the same, indicating that TBT-DC8 molecules were not released from the cell membrane into the solution. The fluorescence intensity of erythrocytes not previously stained with TBT-DC8 also remained essentially unchanged, indicating that these erythrocytes were not labeled with TBT-DC8 during this period after mixing, thus verifying that TBT-DC8 on erythrocytes stained with TBT-DC8 did not diffuse into the solution. In contrast, CellMask Green exhibited poor staining stability. After mixing, the fluorescence intensity of pre-stained erythrocytes gradually decreased within 1.5 hours, indicating that CellMask Green, which had already been stained on the erythrocytes, gradually diffused into the solution, resulting in a decrease in fluorescence intensity. Meanwhile, the fluorescence intensity of unstained erythrocytes gradually increased within 1.5 hours, indicating that the erythrocytes were gradually stained by CellMask Green that had diffused into the solution, resulting in an increase in fluorescence intensity. The results showed that TBT-DC8 has a more stable cell membrane staining ability than the new generation commercial membrane dye CellMask Green, and is not easily released into the solution after staining the cell membrane.

[0137] Based on the above analysis, the cell membrane dye based on the membrane-embedded conjugated oligoelectrolyte provided by the present invention, as a visible light absorption fluorescent probe, has significant advantages in photostability and staining performance compared with the same visible light commercial membrane dyes in the prior art, and exhibits a more uniform and stable cell membrane staining effect. As a new cell membrane dye for stable imaging on mammalian cell membranes, it has great application prospects.

[0138] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any changes, modifications, substitutions, integrations, or parameter changes to these embodiments, which fall within the spirit and principles of the present invention and achieve the same functionality through conventional substitutions, without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A visible light absorbing cell membrane dye based on a membrane-embedded conjugated oligoelectrolyte, characterized in that: The structural formula is shown below: 。 2. Use of the visible light absorbing cell membrane dye according to claim 1 in the preparation of fluorescent imaging agents for mammalian cell membranes.

3. The use according to claim 2, characterized in that The mammalian cells are selected from suspension cells and / or adherent cells.

4. The use according to claim 3, characterized in that The suspended cells are selected from red blood cells and / or white blood cells.

5. The use according to claim 3, characterized in that The adherent cells are selected from fibroblasts, lung cancer cells, breast cancer cells, and cervical cancer cells.

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