A fluorescent probe for detecting cell membrane integrity using polarity response ratio and its application
By developing a polar response ratio-type fluorescent probe ANVP-CA, which produces red fluorescence after cell membrane damage, solves the problem of the fluorescence wavelength of existing fluorescence probes, and achieves more accurate cell membrane integrity detection and apoptosis assessment.
Patent Information
- Application Number
- CN202411534577.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2044-10-31
AI Technical Summary
When detecting cell membrane integrity, the fluorescence wavelength remains unchanged and is easily disturbed by factors such as uneven staining and changing incident light intensity, making it difficult to provide a more accurate signal.
A polar response ratio-type fluorescent probe ANVP-CA was developed, which was able to relocate into the cytoplasm after cell membrane damage and produce significant red fluorescence, which was visually evaluated by calculating the ratio of green to red fluorescence.
Through the use of ANVP-CA probe, changes in cell membrane integrity can be detected more accurately, interference is reduced, detection sensitivity is improved, and is suitable for applications such as evaluating cell membrane integrity and detecting cell apoptosis.
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Figure CN119080688B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for detecting cell membrane integrity using a polarity response ratio type and an application thereof. Background Art
[0002] The cell membrane is composed of a phospholipid bilayer, which is decorated with sugars and proteins on the outside to form a barrier to protect the cell. As a lipophilic barrier, the phospholipid bilayer works together with channel proteins to enable the cell membrane to selectively control the entry and exit of substances, thereby maintaining the stability of the cytoplasmic environment. Once the integrity of the cell membrane is damaged, various substances may flow in and out non-selectively, which will destroy the stability of the intracellular environment and even cause cell death. Cell membrane damage is common under both physiological and pathological conditions. For example, under external stimuli such as mechanical force, cracks will appear in the skeletal muscle cell membrane; under high sugar conditions, the osmotic pressure inside and outside the cell will be unbalanced, increasing the mechanical pressure of the cell membrane. Excessive sugar can also bind to membrane proteins and lipids through non-enzymatic glycosylation reactions, thereby reducing the stability of the membrane and causing cell dysfunction or death. The destruction of cell membrane integrity is also an important feature for evaluating late cell apoptosis. Therefore, evaluating the integrity of the cell membrane is an important task to gain a deep understanding of the physiological and pathological changes of the body. However, subtle changes in cell membrane integrity are often difficult to observe directly using traditional optical microscopes or even electron microscopes.
[0003] Currently, the commonly used method to evaluate cell membrane integrity is to detect the uptake of membrane-impermeable substances by cells. For example, Bowman et al. (The Journal of Membrane Biology, 2010, 236: 15-26) used the tantalum ion (Tl + ) to detect cell membrane integrity. If the cells are resistant to Tl + Positive uptake indicates that the cell membrane is damaged. Trypan blue is a commercially available colorimetric dye that can mark the nuclei of cells with damaged cell membranes in blue. Compared with traditional methods, fluorescence imaging technology has the advantages of high sensitivity, simple operation, and in situ real-time monitoring of cell membrane integrity. Therefore, in the past few decades, domestic and foreign scholars have developed a variety of fluorescent probes for assessing cell membrane integrity. Zou (Materials Today, 2022, 61: 117-128) developed a fluorescent probe designed to visualize and assess changes in cell membrane damage in real time. The probe targets the cell membrane in normal cells and migrates to the nucleus after the membrane integrity is damaged. However, existing fluorescent probes usually emit only in a single emission channel when detecting cell membrane integrity, and the fluorescence wavelength remains unchanged after membrane damage. This makes the emission signals of these cells susceptible to interference from multiple factors such as uneven staining and changes in incident light intensity. Summary of the invention
[0004] In view of the technical problem that the fluorescence wavelength of existing fluorescent probes remains unchanged after membrane damage, the present invention proposes a fluorescent probe for detecting cell membrane integrity using a polarity response ratio and its application. The probe can relocate to the cytoplasm after cell membrane damage and produce significant red fluorescence, thereby providing a more accurate fluorescence signal.
[0005] In the first aspect, the present invention provides a fluorescent probe for detecting cell membrane integrity using a polarity response ratio, named ANVP-CA, and having a chemical structural formula of:
[0006] .
[0007] Furthermore, the preparation method of the fluorescent probe ANVP-CA comprises the following steps:
[0008] 6-bromonaphthalene-2-amine reacts with iodomethane to obtain compound 1, compound 1 reacts with 4-vinylpyridine to obtain compound 2 through Heck coupling reaction, and compound 2 reacts with bromoacetic acid to obtain the fluorescent probe ANVP-CA;
[0009] The synthetic route is as follows:
[0010] .
[0011] In a second aspect, the present invention also provides an application of a fluorescent probe ANVP-CA in cell imaging visualization.
[0012] Furthermore, the fluorescent probe ANVP-CA was used to observe cell apoptosis induced by ultraviolet irradiation, oxidative stress, toxins and / or anti-tumor drugs in a ratiometric manner.
[0013] Furthermore, the fluorescent probe ANVP-CA was used to measure the ratio of cell membrane damage in the late stage of apoptosis.
[0014] Furthermore, the fluorescent probe ANVP-CA was used to evaluate the cytotoxicity of formaldehyde and screen antidotes.
[0015] The beneficial effects of the present invention are:
[0016] The probe of the present invention has a polar response and high hydrophilicity, which enables it to act as a lipophilic barrier without penetrating the cell membrane. In normal cells, the probe can target the cell membrane and emit green fluorescence; while in cells with damaged cell membranes, the probe cannot enter the cell and target the cell membrane, and only produces red fluorescence. Therefore, changes in cell membrane integrity can be visually evaluated by calculating the ratio of green to red fluorescence. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0018] Figure 1 It is the fluorescent probe ANVP-CA 1 HNMR spectrum.
[0019] Figure 2 This is the membrane model construction diagram of the fluorescent probe ANVP-CA, where Figure 2 (A) is the cell imaging image of the fluorescent probe ANVP-CA of two GUVs. Figure 2 (B) is a bar graph showing the fluorescence intensity ratio of the red channel to the green channel in the two GUVs.
[0020] Figure 3 This is the localization map of the fluorescent probe ANVP-CA on the cell membrane in the cell, where Figure 3 (A) is the cell imaging image of the fluorescent probe ANVP-CA. Figure 3 (B) is a comparison of the fluorescence intensity of the cell membrane and cells.
[0021] Figure 4 This is an image of damaged cells using the fluorescent probe ANVP-CA to detect cell membrane integrity. Figure 4 (A) is the imaging image of fluorescent probe ANVP-CA cells after treatment with paraformaldehyde. Figure 4 (B) Histogram of the fluorescence intensity ratio of the red channel to the green channel in living cells and dead cells.
[0022] Figure 5 This is a fluorescent probe ANVP-CA to visualize apoptotic cells. Figure 5 (A) is the cell imaging of the fluorescent probe ANVP-CA at different 1 mM hydrogen peroxide treatment times. Figure 5 (B) Figure 5 (A) Enlarged view of the boxed area. Figure 5 (C) is the imaging image of apoptosis induced by the fluorescent probe ANVP-CA cells. Figure 5 (D) is a bar graph showing the fluorescence intensity ratio of the red channel to the green channel in live cells and apoptotic cells treated with hydrogen peroxide and induced by colchicine, rotenone, and paclitaxel.
[0023] Figure 6 These are cell imaging images of the fluorescent probe ANVP-CA treated with different ultraviolet irradiation times.
[0024] Figure 7 Cell imaging diagram of the toxicity and detoxification evaluation of formaldehyde using the fluorescent probe ANVP-CA. Figure 7 (A) is the imaging image of fluorescent probe ANVP-CA cells treated with different concentrations of formaldehyde. Figure 7 (B) is the chemical reaction formula of formaldehyde, sodium sulfite and thiol compounds. Figure 7 (C) is Na 2 SO 3 Cell imaging of the fluorescent probe ANVP-CA in the experiment of removing formaldehyde cytotoxicity using DTT and Cys. DETAILED DESCRIPTION
[0025] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
[0026] Example 1 Preparation of fluorescent probe ANVP-CA
[0027] 6-Bronaphthalene-2-amine (3.026 g, 13.6 mmol), iodomethane (9.576 g, 67.5 mmol) and potassium carbonate (1.870 g, 13.5 mmol) were added to 10 mL of anhydrous acetonitrile and stirred for 15 minutes to mix evenly. The mixture was then heated to reflux for 24 hours to complete the reaction. After the reaction, the system was cooled to room temperature and a solid was finally precipitated. The resulting product was separated by vacuum filtration. The solid product was purified by column chromatography (petroleum ether: ethyl acetate = 50: 1) to obtain 2.542 g of a white solid, i.e., compound 1, with a yield of 74.7%.
[0028] 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.96 (d, J = 1.8 Hz, 1H), 7.73 (d, J = 9.1 Hz,1H), 7.63 (d, J = 8.8 Hz, 1H), 7.43 (dd, J = 8.8, 2.0 Hz, 1H), 7.27 (dd, J = 9.1,2.5 Hz, 1H), 6.95 (d, J= 2.2 Hz, 1H), 3.01 (s, 6H).
[0029] Compound 1 (1.502 g, 6.0 mmol), 4-vinylpyridine (0.94 g, 9.0 mmol), palladium acetate (0.13 g, 0.6 mmol), tri-o-tolylphosphine (1.096 g, 3.6 mmol) and potassium carbonate (0.135 g, 0.6 mmol) were added to 10 mL of DMF and stirred for 15 minutes under a nitrogen atmosphere to allow uniform mixing. The mixture was then heated to reflux for 10 hours to complete the reaction. The solvent was then evaporated under reduced pressure and the crude product was purified by column chromatography (CH 2 Cl 2 :MeOH=100:1), to obtain 1.206 g of yellow solid, namely compound 2, with a yield of 73.5%.
[0030] 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.53 (d, J = 5.9 Hz, 2H), 7.88 (s, 1H), 7.75(t, J = 8.3 Hz, 2H), 7.68 (d, J = 8.6 Hz, 1H), 7.62 (d, J = 16.4 Hz, 1H), 7.56 (d, J = 5.9 Hz, 2H), 7.26 – 7.19 (m, 2H), 6.95 (d, J = 2.2 Hz, 1H), 3.03 (s, 6H).
[0031] Compound 2 (0.652 g, 2.4 mmol) and 2-bromoacetic acid (0.583 g, 4.7 mmol) were added to 10 mL of anhydrous ethanol in a flask and stirred for 15 min. The mixture was then heated to reflux for 12 h to complete the reaction. The solvent was evaporated under reduced pressure and the product was analyzed by column chromatography (CH 2 Cl 2 :MeOH=20:1) to obtain 0.681 g of a red solid compound, namely ANVP-CA, with a yield of 68.7%.
[0032] 1 H NMR (600 MHz, Methanol- d 4) δ 8.56 (s, 1H), 8.55 (s, 1H), 8.05 (s, 1H), 8.04 (s, 1H), 7.98 (d, J = 16.1 Hz, 1H), 7.94 (s, 1H), 7.76 (s, 1H), 7.75(s, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 16.1 Hz, 1H), 7.23 (dd, J = 9.1,2.5 Hz, 1H), 6.95 (d, J = 2.1 Hz, 1H), 5.02 (s, 2H), 3.10 (s, 6H).
[0033] Example 2 Cellular Imaging of Fluorescent Probe ANVP-CA
[0034] (1) Cell culture
[0035] HeLa cells were purchased from Wuhan Pronocell Life Science Co., Ltd. and cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum. The live HeLa cells were suspended and diluted, inoculated in a culture dish at a cell concentration of 10,000 cells / mL, and cultured for 24 hours.
[0036] (2) Membrane model construction
[0037] Since cell membranes and organelle membranes have different membrane phases, the fluorescent probe ANVP-CA has the potential to distinguish cell membranes from organelle membranes. To demonstrate that the fluorescent probe ANVP-CA can distinguish different membrane phases, lipid-dense giant unilamellar vesicles (DPPC GUVs) were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (CL) as the main raw materials, and giant unilamellar vesicles with loosely packed lipids (DOPC GUVs) were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the two model membranes were visualized using the fluorescent probe ANVP-CA.
[0038] The results are as follows Figure 2 (A) As shown, the fluorescent probe ANVP-CA is able to distinguish two GUVs with different emission colors, which shows very weak green emission and strong red emission in the DOPC GUV with loosely packed lipids, and strong green emission and weak red fluorescence in the DPPC GUV with tightly packed lipids. The different emission colors should be attributed to the different polarities of the two GUVs. The emission color difference can be clearly seen in the merged image and the ratio image. Figure 2 (B) The red to green emission intensity ratios of different GUVs were calculated, with DOPC GUV showing a large ratio, while DPPC GUV showed a small ratio, further demonstrating that the fluorescent probe ANVP-CA can distinguish the two different membranes in a ratiometric manner.
[0039] (3) Positioning of the intact cell membrane in the cell
[0040] The fluorescent probe ANVP-CA was added to normal living cells and cells with damaged cell membranes for incubation to evaluate its role in detecting membrane permeability and cell localization. Figure 3 As shown in (A), Figure 3 The white line in (A) represents a scale bar of 20 μm in length, and the same applies below. The emission of ANVP-CA is mainly distributed on the cell membrane, and the emission signal in the cytoplasm is very weak. ANVP-CA exhibits poor membrane permeability. The emission intensity of cells stained with ANVP-CA is plotted along the yellow line, as shown in Figure 2. Figure 3 As shown in (B), ANVP-CA exhibits strong fluorescence in the cell membrane and weak emission in the cell. This is because ANVP-CA is modified with a hydrophilic chain (carboxyl group), which enhances the hydration shell of the probe and reduces membrane permeability.
[0041] (4) Detection of damage to cell membrane integrity
[0042] Living cells were fixed with paraformaldehyde and healthy and dead cells were observed using the fluorescent probe ANVP-CA. Figure 4 As shown in (A). ANVP-CA only stains the cell membrane of living cells with intact membranes and shows strong green emission and weak red fluorescence. In dead cells with damaged cell membranes, the probe is located inside the cell and targets the organelle membranes, producing strong emission in both the green and red channels. The different emission colors in living and dead cells can be clearly visualized in merged images and ratio images. The polarity of the cell membrane is similar to that of the DPPC GUV, while the organelle membrane is similar to that of the DOPC GUV. Since the polarity of the organelle membrane is higher than that of the cell membrane, the emission spectrum of dead cells is obviously red-shifted. Figure 4 As shown in (B), the red to green emission intensity ratio in live and dead cells was quantified. The intensity ratio of ANVP-CA in dead cells was more than 4 times that in live cells, indicating that ANVP-CA can detect damage to cell membrane integrity in a ratiometric manner with high sensitivity.
[0043] (5) Visualization of cell apoptosis
[0044] During apoptosis, especially in the late stage of apoptosis, the integrity of the cell membrane will be damaged. 2 O 2Live cells were treated for different time periods to obtain early and late apoptotic cells, and these cells were co-incubated with propidium iodide (PI) and Annexin V-FITC to detect changes in the apoptotic cycle. Figure 5 As shown in (A) and 5 (B), H 2 O 2 After 2 hours of treatment, Annexin V-FITC emitted strong green fluorescence and weak red fluorescence on the cell membrane, indicating that the cells were in the early stage of apoptosis; 2 O 2 After 6 hours of treatment, the cells showed strong fluorescence in both the green and red channels, indicating that the cells had entered the late stage of apoptosis.
[0045] Then, we use H 2 O 2 Cells treated for 2 hours and 6 hours were stained with ANVP-CA to verify its ability to detect cell apoptosis. The control normal group cells only showed strong green emission and weak red fluorescence on the cell membrane, indicating that the cell membrane was intact. Figure 5 (C) As shown, H 2 O 2 After 2 hours of treatment, the cell membrane emission was stronger and the cytoplasm emission was weaker. The fusion image and ratio image showed that the cytoplasm emission was red-shifted compared with the cell membrane, indicating that the cells were in the early stage of apoptosis and the cell membrane was only slightly damaged. 2 O 2 After 6 hours of treatment, the cell membrane fluorescence weakened, the intracellular fluorescence increased, and the cytoplasmic fluorescence was significantly red-shifted compared to the cell membrane, indicating that the integrity of the cell membrane has been completely damaged in late apoptosis. Figure 5 (D) The red to green intensity ratio was also quantified and the results showed a significant increase in the ratio during late apoptosis.
[0046] These results suggest that ANVP-CA can be used as a ratiometric probe to detect the late stages of cell apoptosis.
[0047] (6) Inducing cell apoptosis
[0048] Colchicine (Colc) and paclitaxel (Ptxl) are anti-tumor drugs, while rotenone (Rot) is a toxin that inhibits respiration. HeLa cells were treated with the above three reagents and then visualized with ANVP-CA to examine the apoptotic stage of the cells. ANVP-CA stained the cytoplasm of some cells incubated with the three reagents, and the emission of ANVP-CA was significantly red-shifted compared to healthy cells in the control group. In addition, the quantitative red to green intensity ratio also showed a significant increase in cells treated with the three drugs (see Figure 5(D)). These results indicate that colchicine, paclitaxel, and rotenone can effectively induce late apoptosis of tumor cells.
[0049] Living cells were treated with ultraviolet (UV) irradiation to induce apoptosis and visualized with the fluorescent probe ANVP-CA. Figure 6 As shown, living cells irradiated with UV light for 5 minutes showed strong emission on the cell membrane and weak fluorescence in the cytoplasm, and the cytoplasmic emission was red-shifted compared with the cell membrane, indicating that the cells were in the early stage of apoptosis; after 10 minutes of UV light irradiation, strong fluorescence emission appeared in the cell cytoplasm, and compared with healthy cells, the fluorescence emission was significantly red-shifted, indicating that the cells were in the late stage of apoptosis. Therefore, it can be determined based on the color change of the fluorescent probe ANVP-CA that 10 minutes of ultraviolet irradiation can effectively induce late apoptosis of tumor cells.
[0050] In summary, ANVP-CA can be used as a probe for detecting the rate of late cell apoptosis in biomedical research.
[0051] (7) Toxicity evaluation and detoxification evaluation of formaldehyde
[0052] The fluorescent probe ANVP-CA was used to evaluate the cytotoxicity of formaldehyde. Figure 7 As shown in (A). ANVP-CA stains the cell membrane of healthy cells in the control group, producing strong green emission and weak red emission. With the addition of formaldehyde, ANVP-CA stably relocalizes to the cytoplasm, and a significant red shift in emission can be clearly observed in the fused image and the ratio image. The relocalization and red shift of ANVP-CA were clearly detected in cells treated with 1mM formaldehyde for 2h, indicating that the cell membrane can be significantly damaged by formaldehyde at a concentration of 1mM. In addition, in almost all cells treated with 3mM formaldehyde for 2h, ANVP-CA showed strong emission in the cytoplasm, indicating that the cell membrane was completely damaged, indicating that the cells were in the late stage of apoptosis. Therefore, based on the visualization of ANVP-CA, it can be concluded that millimolar levels of formaldehyde can rapidly cause cell death.
[0053] Formaldehyde can react chemically with sodium sulfite and thiol compounds, as shown in the reaction formula: Figure 7 As shown in (B), sodium sulfite, cysteine (Cys), and dithiothreitol (DTT) have the potential to act as detoxifiers to counteract the toxicity of formaldehyde. To verify the detoxification effect of the three substances on formaldehyde, formaldehyde was mixed with these three compounds, and then cells were treated with them and stained with ANVP-CA to verify whether they could reduce the cytotoxicity of formaldehyde.
[0054] like Figure 7 (C) As shown, formaldehyde (2 mM) and Na 2 SO 3Cells treated with / DTT (2mM and 4mM) showed strong emission in the cytoplasm and very weak emission on the cell membrane, which was obviously red-shifted compared with normal cells. 2 SO 3 and DTT cannot be used to eliminate the cytotoxicity of formaldehyde.
[0055] Living cells were treated with different amounts of Cys (2mM and 4mM) and formaldehyde (2mM). Figure 7 (C) It can be seen that the effect of 2 mM Cys on formaldehyde toxicity is negligible. Under this condition, ANVP-CA is mainly distributed in the cytoplasm. Figure 7 Compared with the control group in (A), the fluorescence red shift indicates cell membrane damage and cell apoptosis; when the dosage of Cys is increased to 4mM, most cell membranes show strong fluorescence after ANVP-CA loading, and weak fluorescence in the cytoplasm, indicating that the cell membrane is intact. Therefore, adding 4mM Cys can significantly reduce the cytotoxicity of formaldehyde. According to the ANVP-CA visualization analysis, it can be concluded that Na 2 SO 3 DTT itself has obvious cytotoxicity and cannot counteract the toxicity of formaldehyde, while Cys is a potential antidote to reduce the toxicity of formaldehyde.
[0056] In conclusion, ANVP-CA can be used as a suitable tool to evaluate formaldehyde cytotoxicity and screen antidotes.
[0057] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.
Claims
1. A fluorescent probe for detecting cell membrane integrity using a polarity response ratio, characterized in that: The chemical structure is: 。 2. The fluorescent probe according to claim 1, characterized in that The preparation method comprises the following steps: 6-bromonaphthalene-2-amine reacts with iodomethane to obtain compound 1, compound 1 reacts with 4-vinylpyridine to obtain compound 2 through Heck coupling reaction, and compound 2 reacts with bromoacetic acid to form a salt to obtain the compound; The synthetic route is as follows: 。 3. A use of the fluorescent probe as claimed in claim 1 in cell imaging visualization, characterized in that: The applications are for non-disease diagnosis and treatment purposes.
4. The use according to claim 3, characterized in that The fluorescent probe ANVP-CA is used to observe cell apoptosis induced by ultraviolet irradiation, oxidative stress, toxins and / or anti-tumor drugs in a ratiometric manner, and the application is for non-disease diagnosis and treatment purposes.
5. The use according to claim 3, characterized in that The fluorescent probe ANVP-CA is used to measure the ratio of cell membrane damage in the late stage of apoptosis, and the application is for non-disease diagnosis and treatment purposes.
6. The use according to claim 3, characterized in that The fluorescent probe ANVP-CA was used to evaluate the cytotoxicity of formaldehyde and screen antidotes for non-disease diagnosis and treatment purposes.
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