Preparation method and application of long-time cell membrane fluorescent probe for selectively recognizing cancer cells
By designing a rigid benzene ring-connected indole vinyl pyridine dimer fluorescent probe, the problem that fluorescent probes in the prior art cannot be anchored to the cell membrane for a long time is solved, and long-term labels for selective identification of cancer cells in simulated tumor microenvironment are achieved, and the potential for application in in vitro imaging and cancer treatment is achieved.
Patent Information
- Application Number
- CN202510414614.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-08-15
AI Technical Summary
Existing fluorescent probes can only achieve short-term cell membrane detection, cannot be anchored to the cell membrane for a long time, and it is difficult to selectively identify cancer cells in simulated tumor microenvironment.
A fluorescent probe of indole vinyl pyridine dimer (DIP) connected by a rigid benzene ring was designed to synthesize DIP dyes with bipyridine salts through Knoevenagel condensation reaction, and long-term cell membrane imaging was achieved using its ‘U’ configuration and double-anchored and double-charge properties.
This fluorescent probe can selectively identify cancer cells in simulated tumor microenvironment, achieve long-term cell membrane labeling, and prove through experiments that its fluorescence intensity difference is related to cell membrane polarity, and is suitable for further in vitro imaging and cancer treatment.
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Figure CN120483965A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent probes and relates to a preparation method and application of a long-term cell membrane fluorescent probe for selectively identifying cancer cells. Background Art
[0002] Cancer is a complex disease in which cancer cells lose their ability to control their growth and continue to divide, forming tumors. The transformation of normal cells into cancer cells is a complex process, manifested not only in disordered cell proliferation and division but also in differences in the cellular microenvironment. The cellular microenvironment is a crucial parameter of cells and a crucial basis for identifying cancer cells through changes in their microenvironment.
[0003] Among many parameters, changes in cell membrane polarity are a key component of the cancer cell microenvironment. Cancer cells have a greater polarity than normal cells. This microenvironment can be exploited to selectively identify cancer cells. Among the small molecule fluorescent probes currently used for specific imaging of cell membranes, some fluorescent dyes can produce different fluorescent responses to this change in polarity. However, in methods using fluorescent molecular probes to detect polarity, most probes can only achieve short-term detection. Fluorescent probes will leave the cell membrane over time, so it is necessary to develop long-lasting cell membrane fluorescent probes that can selectively identify cancer cells. The long-lasting cell membrane fluorescent probe we developed for selectively identifying cancer cells can selectively identify cancer cells in a simulated tumor microenvironment and remain anchored to the cell membrane for a long time. This material may be an ideal candidate dye for further in vitro imaging and cancer treatment. Summary of the Invention
[0004] The present invention addresses the aforementioned issues with existing probes by providing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells, as well as a method for preparing this DIP fluorescent probe. This probe selectively identifies cancer cells in a simulated tumor microenvironment, and experimentally demonstrates that this phenomenon is due to differences in cell membrane polarity, resulting in different fluorescence intensities when the probe molecule is anchored to the cell membrane. This material may be an ideal candidate dye for further in vitro imaging and cancer therapy.
[0005] The purpose of the present invention can be achieved through the following technical solutions:
[0006] A long-lasting cell membrane fluorescent probe for selectively identifying cancer cells. The fluorescent probe is an indole vinyl pyridine dimer connected by rigid benzene rings, and its structural formula is shown below:
[0007] Wherein, n=1-11.
[0008] The fluorescent probe is a DIP fluorescent probe.
[0009] The above-mentioned method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells comprises the following steps:
[0010] a.1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine)
[0011] b. Synthesis of alkyl substituted 1H-indole-2-carboxaldehyde derivatives;
[0012] c. Synthesis of alkyl-substituted indole vinyl salt derivatives linked by m-phenylpyridine, namely the target product DIP fluorescent probe.
[0013] The specific operation of step a is: adding 1,3-bis(bromomethyl)benzene to a solution of 4-methylpyridine in acetonitrile, and then refluxing the mixture. After the reaction is completed, white crystals precipitate on the wall of the round-bottom flask, and the precipitate is filtered and washed with acetone. The final product is a white powder after drying to obtain 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine).
[0014] Preferably, in step a, the usage ratio of 1,3-bis(bromomethyl)benzene, 4-methylpyridine and acetonitrile is: 38 mmol: 76 mmol: 50 mL.
[0015] Preferably, the specific operation of step b is: adding indole-2-carboxaldehyde to DMF in a two-necked flask, then adding a brominated alkyl chain, K2CO3 and KI to the above solution, heating to reflux, cooling to room temperature, filtering the resulting solution to remove the precipitate, pouring the filtrate into water, and then extracting with DCM, washing the organic layer with water 3 times (3×50 mL), and then drying with anhydrous Na2SO4, rotary evaporation to remove the solvent, and the crude product is purified by column chromatography (PE) to obtain a yellow liquid.
[0016] Preferably, in step b, the usage ratio of indole-2-carboxaldehyde, DMF, bromoalkyl chain, K2CO3, and KI is: 6.89 mmol: 30 mL: 13.78 mmol: 13.79 mmol: 6.89 mmol.
[0017] Preferably, the specific operation of step c is: stirring a mixture of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine), alkyl chain-substituted 1H-indole-3-carbaldehyde and pyrrolidine (5 drops) in ethanol overnight, evaporating the solvent under reduced pressure, and purifying the residue by alumina chromatography (DCM:MeOH=50:1) to obtain a yellow solid.
[0018] Preferably, in step c, the usage ratio of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine), alkyl chain substituted 1H-indole-3-carbaldehyde, pyrrolidine and ethanol is: 2.22mmol:4.45mmol:5drop:6.89mmol:30mL.
[0019] The present invention has the following beneficial effects: In this study, we designed a dual-anchored bipyridinium cationic plasma membrane fluorescent dye (DIP) linked to a rigid benzene ring. Not only can the DIP be opened in living cells after plasma membrane insertion, but it also exhibits high responsiveness and long-term cell membrane labeling, and can selectively identify cancer cells. This work provides a rational design strategy for long-term plasma membrane staining probes and opens up new avenues for cancer diagnosis. The DIP dye with a bipyridyl salt was cleverly designed and synthesized via a Knoevenagel condensation reaction between indolealdehyde and the intermediate 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridin-1-ium). Its "U"-shaped configuration, dual anchoring, and dual charge properties enable long-term cell membrane imaging. Furthermore, universal imaging experiments with normal and cancer cells demonstrate that this fluorescent probe is suitable for selectively identifying cancer cells in a simulated tumor microenvironment. Experiments demonstrate that this phenomenon is due to the different fluorescence intensities of the probe molecule anchored to the cell membrane, resulting from differences in cell membrane polarity. This material may be an ideal candidate dye for further in vitro imaging and cancer therapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 a is the photophysical properties of DIP. (A) UV-visible absorption spectrum of DIP aqueous solution (10.0 μM).
[0021] Figure 1 b is the fluorescence emission spectrum of DIP aqueous solution (10.0 μM).
[0022] Figure 1 c Fluorescence spectra of DIP (10.0 μM) in water before and after adding DOPC (5.0 mg / mL).
[0023] Figure 1 d is the fluorescence enhancement ratio of FM 1-43 and DIP in DOPC solution to that in water (5.0 mg / mL).
[0024] Figure 2 Confocal images of normal cells (293T and 16HBE) and cancer cells (HeLa, SY5Y, SW982) stained with DIP (20 μM). Scale bar = 10 μm.
[0025] Figure 3a is the fluorescence intensity distribution of HeLa cells (square) and 293T cells (circle) cultured in the same culture dish
[0026] Figure 3 b shows the fluorescence intensity of two cell lines stained with DIP. All fluorescence intensities of different cell lines were obtained using the cell image processing software ImageJ. Scale bar = 10 μm.
[0027] Figure 4 a Confocal cell imaging of HeLa cells at different cholesterol levels after staining with DIP.
[0028] Figure 4 b Fluorescence intensity on the cell membrane under different cholesterol levels. DETAILED DESCRIPTION
[0029] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.
[0030] Example 1:
[0031] The present invention provides a method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells. The structural formula of the fluorescent probe DIP is as follows:
[0032] At this time, n=5.
[0033] Its synthetic route is as follows:
[0034]
[0035] The preparation method of the DIP fluorescent probe specifically includes the following steps:
[0036] a. Synthesis of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine): 1,3-Bis(bromomethyl)benzene (1.00 g, 38 mmol) was added to a solution of 4-methylpyridine (0.71 g, 76 mmol) in 50 mL of acetonitrile, and the mixture was refluxed for 8 hours. After the reaction, white crystals precipitated on the walls of the round-bottom flask. The precipitate was filtered and washed with acetone. The final product was dried to a white powder with an 80% yield.
[0037] Product NMR data: 1 H NMR (400MHz, D2O) δ (ppm): 8.69 (d, J=6.8Hz, 4H), 7.88 (d, J=6.5Hz, 4H), 7.61-7.51 (m, 4H), 5.77 (s, 4H), 2.65 (s, 6H). 13C NMR (101MHz, D2O) δ (ppm): δ 160.84, 143.24, 134.31, 130.64, 130.04, 129.05, 128.88, 63.07, 57.42, 21.37, 16.80.
[0038] b. Synthesis of 1-hexyl-1H-indole-2-carbaldehyde: In a 50 mL two-necked flask, indole-2-carbaldehyde (1.00 g, 6.89 mmol) was added to 30 mL of DMF. 1-bromohexane (2.28 g, 13.788 mmol), KCO (1.91 g, 13.79 mmol), and KI (1.14 g, 6.89 mmol) were then added to the solution. After cooling to room temperature, the resulting solution was filtered to remove the precipitate. The filtrate was poured into water and extracted with DCM. The organic layer was washed three times with water (3 x 50 mL) and then dried over anhydrous NaSO. The solvent was removed by rotary evaporation. The crude product was purified by column chromatography (PE) to give a yellow liquid in a 45% yield.
[0039] Product NMR data: 1 H NMR (400MHz, CDCl3) δ (ppm): 9.80 (s, 1H), 7.66 (d, J = 8.0Hz, 1H), 7.34-7.33 (m, 2H), 7.19 (s, 1 H), 7.13-7.06 (m, 1H), 4.53-4.42 (m, 2H), 1.75-1.65 (m, 2H), 1.28-1.21 (m, 6H), 0.80 (t, 3H). 13 C NMR (101MHz, CDCl3) δ12.98, 21.53, 25.48, 29.46, 30.49, 43.78, 109.70, 116.86, 119.79, 122.41, 124.95, 125.76, 134.34, 139.28, 181.57.
[0040] c. Synthesis of 1,1'-(1,3-phenylenebis(methylene)bis(4-(2-(1-hexyl-1H-indol-2-yl)vinyl)pyridine): A mixture of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridin-1-ium) (1.00 g, 2.22 mmol), 1-hexyl-1H-indole-3-carbaldehyde (1.02 g, 4.45 mmol), and pyrrolidine (5 drops) was stirred in 30 mL of ethanol overnight. The solvent was evaporated under reduced pressure. The residue was purified by alumina chromatography (DCM:MeOH = 50:1) to give a yellow solid in a 65% yield.
[0041] Product NMR data: 1H NMR (400MHz, DMSO-d6) δ (ppm): 9.21 (d, J = 6.5Hz, 4H), 8.44 (d, J = 6.8Hz, 3H), 8.24 (d, J = 15.9Hz, 2H), 8.14-8.01 (m, 1H), 7.92 (s, 1H), 7.74-7.29 (m, 1 0H), 7.27-7.19 (m, 3H), 7.07 (t, J=7.3Hz, 2H), 5.85 (s, 4H), 4.50 (t, J=7.0 Hz, 4H), 1.64 (q, J=7.0, 6.0Hz, 4H), 1.37-1.07 (m, 12H), 0.89-0.66 (t, 6H). 13 C NMR (101MHz, DMSO-d6) δ153.57, 144.73, 138.82, 136.37, 136.25, 130.33, 129.72, 129.71, 129.50, 127.78, 124.51, 124.16, 124.03, 121.65, 120.85, 111.05, 103.77, 61.99, 42.82, 31.35, 30.85, 26.24, 22.48, 14.27.
[0042] Performance Characterization
[0043] The UV-visible absorption spectrum and fluorescence emission spectrum of the DIP fluorescent probe prepared in Example 1 were characterized. The test results of the UV-visible absorption spectrum of the DIP fluorescent probe in dimethyl sulfoxide (DMSO) solvent are as follows: Figure 1 As shown in a, the DIP fluorescent probe aqueous solution is yellow in sunlight, and the maximum absorption peak of the aqueous solution is located at 438nm. The test results of its fluorescence emission spectrum are shown in Figure 1 As shown in b, the emission spectrum of the DIP fluorescent probe was tested, and the maximum emission peak wavelength of the DIP fluorescent probe aqueous solution was 600 nm.
[0044] To evaluate the fluorescence response of DIP to cell membranes, we used stable micelles formed by DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) in water, which mimics the structure of cell membranes. In aqueous solution, the dye molecules show very weak fluorescence under UV irradiation. However, after the addition of DOPC, their fluorescence intensity increases significantly. This increase is accompanied by a blue shift of about 15-30 nm due to its inherent TICT properties. Figure 1 As shown in cd, after adding DOPC, the fluorescence intensity of the traditional commercial dye FM1-43 increased by 47.7 times, and the fluorescence enhancement of DIP reached an astonishing 140 times, so the probe can show a fluorescence turn-on process for cell membrane mimics.
[0045] The above characterization confirmed the fluorescence turn-on behavior of the cell membrane mimetic of the DIP fluorescent probe.
[0046] The present invention also provides a method for selectively identifying cancer cells in a simulated tumor microenvironment using this selective cancer cell identification fluorescent probe, and experiments have shown that this phenomenon is due to different cell membrane polarity leading to different fluorescence intensities of the probe molecules anchored on the cell membrane.
[0047] Application in selective recognition of cancer cells in simulated tumor microenvironment.
[0048] The following will provide a detailed description through specific application examples.
[0049] Application Example 1
[0050] Application of DIP fluorescent probe to selectively identify cancer cells in a simulated tumor microenvironment.
[0051] Specific implementation method: Human cervical cancer cell line HeLa and human embryonic kidney cell line HEK293T were routinely cultured in Dulbecco's Modified Eagle Medium (DMEM; GENOM, Zhejiang, China) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China) and 1% penicillin / streptomycin (Gibco, Gaithersburg, MD, USA) at 37°C and 5% CO2.
[0052] Human cervical cancer cell line HeLa and human embryonic kidney cell line HEK293T were incubated in the same culture dish with 20.0 μM DIPI for 5 min at 22°C. Fluorescence imaging experiments were performed on a Leica TCS SP5 laser confocal scanning microscope (Germany) with an excitation wavelength of 488 nm.
[0053] like Figure 2, as shown in Figure 3, which shows that various cancer cells and normal cells were cultured separately and stained with DIP for 5 minutes. The cells were then immediately observed under a confocal microscope. It was obvious that the imaging effect of these cells was poor, while the imaging of cancer cells was clear. Cancer cells and normal cells (HeLa cells and 293T cells) were also co-cultured in the same culture medium to simulate a real tumor environment. After 5 minutes of DIP staining, the cells were immediately observed under a confocal microscope. In the bright field, 293T cells with small nuclei and densely arranged pseudopodia and spindle-shaped HeLa cells without scattered pseudopodia were searched. The image shows that the yellow fluorescent signal on HeLa cells is significantly greater than that on 293T cells, reaching a level visible to the human eye. To further quantify the fluorescent signal, the fluorescence intensity signal contrast is more obvious under the grayscale image processed by ImageJ software. Quantification was performed using ImageJ software. The ratio of the fluorescent signal area on HeLa cells (cancer cells) to HeLa cells (normal cells) is 9.5 times. This shows that DIP dye can be used to selectively identify cancer cells and may be an ideal candidate dye for further in vitro imaging and cancer treatment.
[0054] Application Example 2
[0055] The DIP fluorescent probe was used to verify that the fluorescence intensity of the probe molecules anchored on the cell membrane was different due to the different polarity of the cell membrane.
[0056] The specific implementation method and instrument conditions are the same as those in Application Example 1.
[0057] To verify how the DIP fluorescent probe selectively recognizes cancer cells, we used methyl-β-cyclodextrin (Mβ-CD) to extract cholesterol from cell membranes, which can regulate the polarity of cell membranes. Figure 4 As shown, HeLa cells were extracted with Mβ-CD to remove cholesterol from the cell membrane and then stained with DIP. The fluorescence on the cell membrane was only 1 / 3.7 of that in the blank control. This indicates that changes in cell membrane polarity do affect the dye's staining of normal and cancer cells.
[0058] Application Examples 1-2 show that the DIP fluorescent probe can utilize the different cell membrane polarity between cancer cells and normal cells to cause different fluorescence intensities of the probe molecules anchored on the cell membrane, and can be used to selectively identify cancer cells in a simulated tumor microenvironment.
[0059] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.
[0060] It should be understood that in the claims and description of the present invention, all "including..." should be understood as open-ended, that is, its meaning is equivalent to "at least containing...", and should not be understood as closed-ended, that is, its meaning should not be understood as "only including...".
[0061] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.
Claims
1. A long-lasting cell membrane fluorescent probe for selectively identifying cancer cells, characterized by: The fluorescent probe is an indole vinyl pyridine dimer connected by rigid benzene rings, and its structural formula is shown below: Wherein, n=1-11.
2. In the above-mentioned long-term cell membrane fluorescent probe for selectively identifying cancer cells, it is characterized in that: The fluorescent probe is a DIP fluorescent probe.
3. A method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claims 1 and 2, characterized in that: It includes the following steps: a. Synthesis of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine); b. Synthesis of alkyl-substituted 1H-indole-2-carboxaldehyde derivatives; c. Synthesis of alkyl-substituted indole vinyl salt derivatives linked by m-phenylpyridine, namely the target product DIP fluorescent probe.
4. The method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claim 3, characterized in that: The specific operation of step a is: adding 1,3-bis(bromomethyl)benzene to a solution of 4-methylpyridine in acetonitrile, and then refluxing the mixture. After the reaction is completed, white crystals precipitate on the wall of the round-bottom flask, and the precipitate is filtered and washed with acetone. The final product is a white powder after drying, and 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine) is obtained.
5. The method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claim 4, characterized in that: In step a, the usage ratio of 1,3-bis(bromomethyl)benzene, 4-methylpyridine, and acetonitrile is: 38 mmol:76 mmol:50 mL.
6. The method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claim 3, characterized in that: The specific operation of step b is as follows: indole-2-carboxaldehyde is added to DMF in a two-necked flask, and then a brominated alkyl chain, K2CO3 and KI are added to the above solution, heated to reflux, cooled to room temperature, and the resulting solution is filtered to remove the precipitate. The filtrate is poured into water and then extracted with DCM. The organic layer is washed with water three times (3×50 mL), and then dried over anhydrous Na2SO4. The solvent is removed by rotary evaporation, and the crude product is purified by column chromatography (PE) to obtain a yellow liquid.
7. The method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claim 6, characterized in that: In the step b, the usage ratio of indole-2-carboxaldehyde, DMF, alkyl bromide chain, K2CO3, and KI is: 6.89 mmol: 30 mL: 13.78 mmol: 13.79 mmol: 6.89 mmol.
8. The method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claim 3, characterized in that: The specific operation of step c is: stirring a mixture of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine), alkyl chain-substituted 1H-indole-3-carbaldehyde and pyrrolidine (5 drops) in ethanol overnight, evaporating the solvent under reduced pressure, and purifying the residue by alumina chromatography (DCM:MeOH=50:1) to obtain a yellow solid.
9. The method for preparing a long-lasting cell membrane fluorescent probe for selectively identifying cancer cells according to claim 8, characterized in that: In step c, the usage ratio of 1,1'-(1,3-phenylenebis(methylene))bis(4-methylpyridine), alkyl chain substituted 1H-indole-3-carbaldehyde, pyrrolidine and ethanol is: 2.22 mmol: 4.45 mmol: 5 drops: 6.89 mmol: 30 mL.
10. Use of the long-lasting cell membrane fluorescent probe for selectively identifying cancer cells as claimed in claim 1 or 2 in plasma membrane imaging.