Viscosity-responsive cell membrane targeting fluorescent probe CMN and application thereof in photodynamic therapy

Abstract

The application discloses a viscosity-responsive cell membrane-targeting fluorescent probe CMN and application thereof in photodynamic therapy, wherein the structure of the fluorescent probe is shown in the following formula: The fluorescent probe in the application not only serves as a photosensitizer for generating active oxygen, but also can specifically target cell membranes (the positioning coefficient is 0.91) and has strong fluorescence opening in a high-viscosity environment. The fluorescent probe can be used for photodynamic therapy of cancer cells in a normoxic or anoxic state. Cell toxicity test shows that the fluorescent probe has good biocompatibility, and confocal fluorescence microscopic imaging experiment shows that the fluorescent probe has good cell membrane specificity for HepG2 cells. Experimental results show that the CMN can generate active oxygen and singlet oxygen under LED light source irradiation, can destroy the integrity of cell membranes, and has a photodynamic therapy effect.

Description

Technical Field

[0001] This invention relates to a viscosity-responsive cell membrane-targeting fluorescent probe (CMN) and its application in photodynamic therapy to achieve photodynamic therapy of cancer cells. Background Technology

[0002] The cell membrane, primarily composed of phospholipids, is an elastic, semi-permeable membrane that serves as a two-dimensional protective layer between living cells and their surroundings. The phospholipid bilayer forms the basic framework of the cell membrane. The cell membrane acts as a barrier preventing the free entry of extracellular substances, ensuring the relative stability of the intracellular environment and enabling the orderly conduct of various biochemical reactions. It has been shown to participate in various cellular processes and biological functions, such as cell adhesion, endocytosis, extracellular interactions, and selective osmosis, and is closely related to cell growth and death. On the other hand, abnormalities in the cell membrane state are often associated with cellular status and many diseases. In various tumor cells, due to the high viscosity of their cell membranes, the need for cell membrane viscosity imaging and monitoring is increasing. Therefore, using small-molecule fluorescent probes to monitor the state of the cell membrane is of great significance.

[0003] Photodynamic therapy (PDT) is an emerging technology for treating diseases such as cancer and tumors. It comprises three key elements: light, oxygen, and a photosensitizer. Under light irradiation, the photosensitizer interacts directly with the cell membrane, target tissues, and biomolecules through electron or proton transfer, generating free radicals or free radical ions. These free radicals further react with oxygen molecules to produce reactive oxygen species (ROS), a process known as type I PDT. In type II PDT, the photosensitizer in a triplet state can convert oxygen surrounding the tissue into highly oxidizing singlet oxygen (…). 1 In photodynamic therapy (PDT), both forms of photochemical reactions can occur. Reactive oxygen species (ROS) generated by PDT oxidize subcellular organelles and intracellular biomolecules, leading to cell damage and inducing tumor cell apoptosis or necrosis. PDT has attracted considerable attention due to its excellent local targeting ability and minimal side effects. Within a certain PDT dose, photosensitizing molecules selectively binding to the cell membrane cause cell necrosis first. This may be because locally generated ROS oxidizes unsaturated phospholipids such as cholesterol, leading to changes in membrane permeability, loss of membrane fluidity and integrity. During cell necrosis, cell morphology changes, followed by cell swelling, blistering, cell membrane rupture, and finally, the release of cell contents into the extracellular fluid, triggering an inflammatory response. Therefore, designing cell membrane photosensitizers for PDT, which can act as both ROS generators and near-infrared fluorescent probes specifically targeting the cell membrane, is crucial. Summary of the Invention

[0004] To address the above issues, this invention provides a viscosity-targeting cell membrane-targeting fluorescent probe (CMN) and its application in photodynamic therapy. The technical problem to be solved is to obtain an organic small molecule structure through molecular design that can respond to viscosity, specifically target the cell membrane, and generate reactive oxygen species under light irradiation. This structure possesses advantages such as good cell membrane specificity (localization coefficient of 0.91) and the ability to effectively generate reactive oxygen species and singlet oxygen. Cytotoxicity tests show that the probe of this invention has good cell compatibility.

[0005] For selective targeting of the cell membrane, a balance of positive charge and good hydrophilicity and hydrophobicity is crucial for its approach and retention on the cell membrane. This invention selects xanthracene as the probe backbone, uses the butyl group as the hydrophobic portion of the probe, anchors it to the cell membrane through hydrophobic interactions, and introduces the [(trimethylammonium)propyl]pyridinium salt group to target it to the cell membrane through electrostatic interactions, enabling the amphiphilic CMN to selectively locate on the cell membrane (localization coefficient of 0.91). Experimental results show that light irradiation can generate ROS and singlet oxygen, oxidizing substances in the cell membrane, disrupting membrane integrity, and exhibiting photodynamic therapeutic effects.

[0006] The cell membrane-targeting fluorescent probe CMN of this invention has the following structure:

[0007]

[0008] The method for preparing the cell membrane-targeting fluorescent probe CMN of the present invention includes the following steps:

[0009] Step 1: Mix 40 mL of N,N-dimethylformamide (DMF) with 100 mL of dichloromethane (DCM), add phosphorus tribromide dropwise at 0 °C under nitrogen protection, remove from the ice-water bath and add cyclopentanone dropwise to carry out the reaction; after about six hours of reaction, add the reaction solution dropwise to 200 mL of water to quench the reaction, then neutralize with sodium carbonate, extract using water and DCM system, dry the organic phase with anhydrous sodium sulfate, and evaporate the organic phase to obtain intermediate 1;

[0010] Step 2: Add 7.2g of intermediate 1, 4.4g of 2-hydroxy-4-methoxybenzaldehyde, and 14.3g of potassium carbonate to 30mL of DMF and react for 12h. Then extract with water and DCM system, dry and evaporate the organic phase, slurry the crude product (ethyl acetate: petroleum ether = 1:1, v / v), and filter to obtain intermediate 2.

[0011] Step 3: Dissolve 3.2g of intermediate 2 in DCM, under nitrogen protection, cool to 0℃ in an ice bath, slowly add boron tribromide (17.5mL), stir at 0℃ for 1h after the addition is complete, then move to room temperature and continue the reaction for 12h; after the reaction is complete, slowly add water to the reaction solution under an ice bath to quench the reaction, then add methanol (25mL) to dissolve it, concentrate the solvent under reduced pressure, a large amount of solid precipitates out, slowly add water to slurry, filter, dry to obtain intermediate 3;

[0012] Step 4: Dissolve 1g of intermediate 3 and excess iodobutane in acetonitrile, heat and stir in a sealed bottle for about 8 hours, evaporate the solvent, and purify by column chromatography (methanol: dichloromethane = 1:50, v / v) to obtain intermediate 4;

[0013] Step 5: Dissolve 1g of tetramethylpyridine and 2.8g of 3-bromopropyltrimethylammonium bromide in acetonitrile, heat and reflux in a sealed bottle for 3 hours to produce a white solid, filter, wash the filter cake with acetonitrile, and dry the filter cake to obtain intermediate 5;

[0014] Step 6: Dissolve 184 mg of intermediate 4 and 200 mg of intermediate 5 in ethanol, then add a catalytic amount of piperidine dropwise, heat and stir, monitor the reaction by TLC, and after the reaction is complete, evaporate the solvent and extract with water and DCM system, dry the organic phase with anhydrous sodium sulfate, evaporate the organic phase, purify with DCM and ethyl acetate system, precipitate a black solid, filter, and dry the filter cake to obtain the target product CMN.

[0015] The synthesis route is shown below:

[0016]

[0017] The present invention relates to the application of the viscosity-responsive cell membrane-targeting fluorescent probe CMN in photodynamic therapy.

[0018] The fluorescent probe CMN can respond to viscosity, specifically target the cell membrane (localization coefficient is 0.91), and generate reactive oxygen species and singlet oxygen under light conditions, thus exhibiting photodynamic therapy effects.

[0019] The experiment was conducted using 3W LED lights for illumination.

[0020] The detection method is as follows:

[0021] A 2 mM mother liquor was prepared by dissolving CMN in DMSO. 15 μL of this mother liquor was then dissolved in 3 mL of solvents of different viscosities, and the UV and fluorescence spectra of CMN in different test solutions were obtained. The fluorescence spectra showed that CMN emitted more strongly at 650 nm in glycerol than in other solvents. Further analysis of CMN's response to viscosity was conducted by adjusting the ratio of glycerol to water to obtain mixed solutions of different viscosities. As the viscosity of the mixed solution increased, the fluorescence of CMN at 660 nm also increased, showing a good linear relationship. These results indicate that CMN has a good response to viscosity. To investigate CMN's ability to generate ROS, the commercially available fluorescent probe H2DCFDA was used to detect ROS. H2DCFDA itself does not emit light, but it emits fluorescence at 525 nm after binding ROS. Under the same irradiation conditions for different durations (0-30 min), changes in fluorescence up to 525 nm were observed in the group containing both CMN and H2DCFDA, while no significant fluorescence change was observed in the group containing only H2DCFDA or CMN, indicating that CMN can effectively generate ROS under light irradiation. The ability of CMN to generate singlet oxygen was investigated, analyzed from three perspectives: electron paramagnetic resonance (EPR), ultraviolet light, and fluorescence. 2,2,6,6-Tetramethyl-4-piperidinone hydrochloride (TEMP) was used as a singlet oxygen scavenger for EPR testing, revealing a singlet oxygen signal peak that increased with irradiation time (1 min, 5 min). 9,10-Anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) was used as a UV indicator for singlet oxygen. This water-soluble anthracene derivative reacts with singlet oxygen, photobleaching to generate the corresponding internal peroxide. This reaction was monitored by spectrophotometric recording of the decrease in optical density at 400 nm. With increasing illumination time (0-3 min), the characteristic absorption peak of ABDA gradually weakened, while the blank group showed no significant change in absorption signal. SOSG is a commercially available fluorescent probe for detecting singlet oxygen. It is non-luminescent and insensitive to other reactive oxygen species such as peroxides and hydroxyl radicals. Using SOSG as a fluorescent indicator for singlet oxygen, in the presence of both CMN and SOSG, with increasing illumination time (0-20 min), the intensity of the characteristic emission peak of SOSG at 525 nm gradually increased, while the fluorescence intensity of SOSG solution alone remained essentially unchanged. These results indicate that CMN can effectively generate singlet oxygen. Dark cytotoxicity experiments were conducted using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) method. Adding different concentrations of CMN to viable HepG2 cells and incubating them in the dark did not affect cell viability, indicating that CMN is suitable for cell imaging. After irradiation (20 min, 40 min), the cell viability decreased significantly, indicating that CMN has high phototoxicity to tumor cells under light and is suitable for PDT application.

[0022] To investigate the cell membrane targeting ability of CMN, co-incubation with a commercial cell membrane dye (Dio) yielded a co-localization coefficient of 0.91, indicating that the probe possesses good cell membrane targeting ability. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the cell membrane of living cells. However, when cells are in the late stage of apoptosis, it can cross the cell membrane and bind to the cell nucleus, exhibiting red fluorescence. Therefore, PI is commonly used to stain the nuclei of dead cells that have leaked from the cell membrane, and thus, PI reagent can be used to detect the effect of photodynamic therapy. After co-incubating the probe CMN with PI in live HepG2 cells for 30 min and then irradiating them with light (15 min), a significant enhancement of red fluorescence was observed in the cell nuclei.

[0023] The probe of this invention exhibits good viscosity response, cell membrane targeting ability (localization coefficient of 0.91), and biocompatibility. Detection of reactive oxygen species and singlet oxygen generated by light, along with confocal fluorescence microscopy, demonstrates that CMN possesses certain photodynamic therapeutic effects. Attached Figure Description

[0024] Figure 1 This is the response of CMN (10 μM) to viscosity. (a) UV absorption spectra of CMN (10 μM) in different solvents;

[0025] (b) Fluorescence emission spectra of CMN (10 μM) in different solvents; (c) Fluorescence emission spectra of CMN (10 μM) in glycerol / water at different ratios; (d) Linear relationship graph with the logarithm of different viscosity values ​​as the x-axis and the logarithm of the fluorescence intensity value at 655 nm as the y-axis.

[0026] Figure 2 This represents the ability of CMN (10 μM) to generate reactive oxygen species (ROS) after illumination for different durations. (a) Changes in fluorescence emission spectra of H2DCFDA (5 μM) in the presence of CMN (10 μM) over time (0-30 min); (b) Changes in fluorescence emission spectra of H2DCFDA (5 μM) over time (0-30 min); (c) Changes in fluorescence emission spectra of CMN (10 μM) over time (0-30 min); (d) Linear relationship between illumination time and fluorescence intensity (I525 nm) of different groups.

[0027] Figure 3 The EPR signal of singlet oxygen generated by CMN (10 μM) under different light exposure times using TEMP as a trapping agent.

[0028] Figure 4The UV absorption spectra of singlet oxygen generated by CMN (10 μM) under different illumination times using ABDA as an indicator are shown. (a) Change of UV absorption spectrum of ABDA (50 μM) over time (0-3 min); (b) Change of UV absorption spectrum of ABDA (50 μM) and CMN (10 μM) over time (0-3 min).

[0029] Figure 5 The fluorescence emission spectra of singlet oxygen generated by CMN (10 μM) under different illumination times using SOSG as an indicator are shown. (a) Fluorescence emission spectra of SOSG (5 μM) and CMN (10 μM) coexisting with time (0-20 min); (b) Fluorescence emission spectra of SOSG (5 μM) with time (0-20 min).

[0030] Figure 6 The graph shows the survival rates of HepG2 cells with different concentrations of CMN under light-free (0 min) and light-bearing (20, 40 min) conditions.

[0031] Figure 7 This is a confocal fluorescence image of the cell membrane of HepG2 cells simultaneously co-stained with CMN (10 μM) and a 0.5 μM commercial cell membrane probe (Dio).

[0032] Figure 8 This is a confocal cell image of HepG2 cells containing CMN (10 μM) and SOSG (2.5 μM) under light-free (0 min) and light-bearing (15 min) conditions, respectively.

[0033] Figure 9 This is a confocal cell image of CMN (10 μM) and PI (3 μM) in HepG2 cells under light (15 min) conditions. Detailed Implementation

[0034] The present invention will be further illustrated by the following examples.

[0035] Example 1: Synthesis of CMN

[0036] 184 mg of intermediate 4 and 200 mg of intermediate 5 were dissolved in ethanol, and a catalytic amount of piperidine was added dropwise. The mixture was heated and stirred, and the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to dryness, and the mixture was extracted with water and DCM. The organic phase was dried with anhydrous sodium sulfate and evaporated to dryness. The mixture was then purified by slurrying with DCM and ethyl acetate, resulting in the precipitation of a black solid. The solid was filtered, and the filter cake was dried to obtain the product CMN.

[0037] 1H NMR (400MHz, DMSO-d6) δ8.77(d,J=6.6Hz,2H),8.06(d,J=6.4Hz,2H),7.89(d,J=15.5Hz,1H),7.16 (d,J=8.5Hz,1H),6.74(d,J=2.5Hz,1H),6.69(dd,J=8.5,2.4Hz,1H),6.65(s,1H),6.48(d,J=15.5 Hz,1H),4.47(t,J=7.4Hz,2H),3.97(t,J=6.6Hz,2H),3.41(t,J=8.1Hz,2H),3.08(s,9H),2.81-2. 59(m,4H),2.43-2.29(m,2H),1.70-1.63(m,2H),1.40(q,J=7.6Hz,2H),0.90(t,J=7.4Hz,3H).13C NMR(101MHz,DMSO-d6)δ160.19,153.87,153.29,143.94,138.00,132.85,128.08,122.86,120.11,119.68,1 16.35,116.24,111.88,102.08,68.26,62.36,56.39,53.02,31.13,25.99,24.70,24.47,19.21,14.22.FT-MS m / z:[M-2Br - ] 2+ :C 29 H 38 N2O2 2+ calcd.,223.146; found,223.145.

[0038] Example 2: CMN's response to viscosity

[0039] The CMN of this invention was dissolved in DMSO to prepare a 2 mM stock solution. 15 μL of this stock solution was then dissolved in 3 mL of solvents of different viscosities to obtain the UV and fluorescence spectra of CMN in different test solutions. CMN showed the highest absorbance at 560 nm in glycerol. Figure 1 a). Using 560 nm as the excitation wavelength, CMN exhibits stronger fluorescence emission in glycerol than in other solvents at 650 nm. Figure 1 b). Different ratios of glycerol / water systems were selected as test solutions of varying viscosities. As the system viscosity increased, the fluorescence intensity of CMN at 650 nm gradually increased. Figure 1 c). A scatter plot was created with the logarithm of viscosity as the x-axis and the fluorescence intensity at 650 nm as the y-axis, and the logarithm of the fluorescence intensity at 650 nm as the y-axis. A good linear relationship was found between the two, with a correlation coefficient of 0.99. Figure 1 d).

[0040] Example 3: The ability of CMN to generate reactive oxygen species (ROS) after exposure to light for different durations.

[0041] ROS were detected using the commercially available fluorescent probe H2DCFDA. With increasing illumination time (0-30 min), the fluorescence intensity of H2DCFDA (5 μM) at 525 nm significantly increased in the presence of CMN (10 μM). Figure 2 a, Figure 2 d). However, the fluorescence intensity at 525 nm did not change significantly when H2DCFDA (5 μM) or CMN (10 μM) were present alone. Figure 2 a, Figure 2 b, Figure 2 d). The above results indicate that the probe CMN can effectively generate ROS under illumination.

[0042] Example 4: The ability of CMN to generate singlet oxygen after exposure to light for different durations

[0043] Using TEMP as a singlet oxygen scavenger, EPR signal peaks of singlet oxygen were generated in CMN (10 μM) at different illumination times (1 min, 5 min), and the peak intensity increased with increasing illumination time. Figure 3 ABDA was used as a UV indicator for singlet oxygen. With increasing illumination time (0-3 min), the characteristic UV absorption peak of ABDA (50 μM) alone showed no significant change. Figure 4 a) When ABDA (50 μM) and CMN (10 μM) coexist, the UV characteristic absorption peak gradually decreases. Figure 4 b). Using SOSG as a fluorescent indicator of singlet oxygen, the intensity of the characteristic emission peak at 525 nm gradually increased with increasing illumination time (0-20 min) under the coexistence of SOSG (5 μM) and CMN (10 μM). Figure 5 a). However, when SOSG (5 μM) is present alone, the intensity of the characteristic emission peak at 525 nm shows no significant change. Figure 5 b) The above results indicate that the probe CMN can effectively generate singlet oxygen under illumination.

[0044] Example 5: Cytotoxicity Test

[0045] Before applying the probe CMN for cell imaging, its toxicity needs to be tested using the MTT assay. HepG2 cells were cultured with 0 μM, 10 μM, 20 μM, and 30 μM CMN, respectively. It was found that in the absence of light (0 min), cell viability remained above 90%. However, after 20 min of light exposure, cell viability decreased significantly with increasing probe concentration, and further decreased when the light exposure time was increased to 40 min. Figure 6 Therefore, the probe CMN showed low toxicity to HepG2 cells in the absence of light (0 min), making it suitable for biological applications; while it showed high toxicity to HepG2 cells under light (20 min, 40 min), making it suitable for PDT applications.

[0046] Example 6: Cell localization test

[0047] To investigate the cell membrane targeting properties of CMN, co-localization studies were conducted in HepG2 cells using a commercial cell membrane dye (Dio, 0.5 μM). The results showed good overlap between the fluorescence images of the red channel of CMN (λem = 650 nm, λex = 560 nm) and Dio (λem = 500-540 nm, λex = 488 nm), and the Pearson co-localization coefficient between CMN and Dio was calculated to be 0.91. Figure 7 The results showed that CMN can be well localized within the cell membrane.

[0048] Example 7: Real-time detection of PDT-induced singlet oxygen production in HepG2 cells by CMN

[0049] Four groups of experiments were conducted. The first group of cells was incubated with SOSG (2.5 μM) in the dark for 30 min. The second group was incubated with the same solution as the first group, but with additional light exposure (15 min). The third group of cells was incubated with both CMN (10 μM) and SOSG (2.5 μM) in the dark for 30 min. The fourth group was incubated with the same solution as the third group, but with additional light exposure (15 min). Compared to the first three groups, the fourth group showed a significant enhancement in green fluorescence. Figure 8 Experimental results show that CMN can effectively generate singlet oxygen in cells under light irradiation. The large amount of reactive oxygen species, including singlet oxygen, within the cells induces apoptosis and necrosis, laying the foundation for photodynamic therapy.

[0050] Example 8: Photodynamic Therapy Effect of CMN in HepG2 Cells

[0051] After incubating cells with CMN (10 μM) and PI (3 μM) in the dark for 30 min, followed by illumination (15 min), a significant red fluorescence was observed on the cell nuclei compared to the group treated with only PI (3 μM). Figure 9The experimental results showed that cell membrane leakage occurred, PI entered the cell and bound to the cell nucleus to produce red fluorescence, thus indicating that CMN has a certain photodynamic therapy effect.

Claims

1. A viscosity-responsive cell membrane-targeting fluorescent probe CMN, characterized in that... Its structure is as follows: 。 2. The method for preparing the viscosity-responsive cell membrane-targeting fluorescent probe CMN according to claim 1, characterized in that... Includes the following steps: Step 1: N,N-dimethylformamide was mixed with dichloromethane, and phosphorus tribromide was added dropwise under nitrogen protection and an ice-water bath. After removing the mixture from the ice-water bath, cyclopentanone was added dropwise to carry out the reaction. After the reaction was completed, the reaction solution was added dropwise to water to quench the reaction, and then neutralized with sodium carbonate. The mixture was extracted using water and a DCM system. The organic phase was dried with anhydrous sodium sulfate, and the organic phase was evaporated to dryness to obtain intermediate 1. Step 2: Add intermediate 1, 2-hydroxy-4-methoxybenzaldehyde and potassium carbonate to DMF for reaction, then extract with water and DCM system, dry and evaporate the organic phase, slurry the crude product and filter to obtain intermediate 2. Step 3: Dissolve intermediate 2 in DCM, under nitrogen protection, cool to 0°C in an ice bath, add boron tribromide dropwise, stir at 0°C for 1 hour after the addition is complete, then move to room temperature to continue the reaction; after the reaction is complete, add water dropwise to the reaction solution under an ice bath to quench the reaction, then add methanol to dissolve it, concentrate the solvent under reduced pressure, the solid precipitates out, add water to make a slurry, filter, and dry to obtain intermediate 3; Step 4: Dissolve intermediate 3 and excess iodobutane in acetonitrile, heat and stir the reaction in a sealed bottle, evaporate the solvent, and purify by column chromatography to obtain intermediate 4; Step 5: Dissolve tetramethylpyridine and 3-bromopropyltrimethylammonium bromide in acetonitrile, heat and reflux in a sealed bottle for 3 hours to produce a white solid, filter, wash the filter cake with acetonitrile, and dry the filter cake to obtain intermediate 5; Step 6: Dissolve intermediates 4 and 5 in ethanol, then add a catalytic amount of piperidine, heat and stir, monitor the reaction by TLC, and after the reaction is complete, evaporate the solvent and extract with water and DCM system, dry the organic phase with anhydrous sodium sulfate, evaporate the organic phase, purify with DCM and ethyl acetate system, precipitate black solid, filter, dry the filter cake to obtain the target product CMN. The synthesis route is shown below: 。 3. The use of the fluorescent probe CMN according to claim 1 in the preparation of photodynamic therapy drug formulations.

4. The application according to claim 3, characterized in that: The fluorescent probe CMN is used to prepare photosensitizers that target cell membranes.

5. The application according to claim 4, characterized in that: The fluorescent probe CMN can respond to viscosity, specifically target the cell membrane, and generate reactive oxygen species and singlet oxygen under light conditions.

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