An ion -π + Applications of type organic fluorescent dyes

Abstract

This invention belongs to the field of biochemical materials technology, specifically relating to an ion-π + Applications of type-π organic fluorescent dyes. This invention utilizes ionic-π... + Type I organic fluorescent dyes exhibit weak fluorescence in solution but produce strong fluorescence upon aggregation, making them typical AIE-emitting molecules. This invention enhances the electron-donating ability of the Ar group, enabling the organic fluorescent dye to exhibit a significant Stokes shift in the biological environment. This results in pronounced AIE in low-concentration, unsuitable solvents, effectively generating type I ROS. At low concentrations, this leads to good photodynamic killing ability against cancer cells, enhancing anticancer activity and showing broad prospects for applications in antitumor drugs or non-therapeutic intracellular fluorescence imaging.

Description

Technical Field

[0001] This invention belongs to the field of biochemical materials technology, specifically relating to an ion-π + Applications of organic fluorescent dyes in the preparation of antitumor drugs, detection reagents, or in intracellular fluorescence imaging for non-therapeutic purposes. Background Technology

[0002] Since their first appearance in 1856, organic fluorescent dyes have become indispensable research tools in physics, chemistry, and life sciences. Throughout their development, researchers have consistently sought high sensitivity, good selectivity, controllable excitation and emission wavelengths, and easily modifiable functions in organic fluorescent dyes. However, the aggregation-induced quenching (ACQ) effect significantly limits the applications of traditional fluorescent molecules. Unlike ACQ, aggregation-induced emission (AIE) can produce highly efficient and stable light in aggregated or solid-state states.

[0003] Cancer has become one of the most serious threats to human health. Traditional treatments such as chemotherapy, surgery, and radiotherapy are ineffective and have significant side effects, causing immense physical and psychological suffering to patients. Summary of the Invention

[0004] The purpose of this invention is to provide an ion-π + The present invention utilizes ion-π organic fluorescent dyes for the application of antitumor drugs, detection reagents, or intracellular fluorescence imaging for non-therapeutic purposes. + Antitumor drugs prepared from organic fluorescent dyes have good antitumor effects and few toxic side effects.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides an ion-π + Applications of type-π organic fluorescent dyes, including the preparation of antitumor drugs, detection reagents, or non-therapeutic intracellular fluorescence imaging, wherein the ion-π + The structure of type I organic fluorescent dyes is shown in Formula I:

[0007]

[0008] In formula I, Ar is:

[0009] Preferably, the ion-π + The structure of type II organic fluorescent dyes is shown in Formula II:

[0010]

[0011] Preferably, the ion-π +The structure of type III organic fluorescent dyes is shown in Formula III:

[0012]

[0013] Preferably, the ion-π + The structure of type IV organic fluorescent dyes is shown in Formula IV:

[0014]

[0015] Preferably, the ion-π + The application of type-3 organic fluorescent dyes in intracellular fluorescence imaging for non-therapeutic purposes includes the following steps:

[0016] Cancer cells in an environment containing ions -π + After incubation with the culture medium containing the organic fluorescent dye, the sample was washed with PBS buffer and subjected to fluorescence imaging.

[0017] Preferably, the fluorescence imaging device is an inverted fluorescence microscopy system or a laser scanning confocal microscope; the fluorescence imaging is performed under an excitation wavelength of 375 nm.

[0018] Preferably, the cancer cells are inoculated and cultured before incubation, and then treated with a solution containing the ion -π. + Replace the original culture medium with the culture medium containing the type of organic fluorescent dye;

[0019] The containing ion-π + The π-ion in the culture medium of type organic fluorescent dyes + The effective concentration of the organic fluorescent dye should not be less than 5 μmol / L.

[0020] Preferably, the culture time is 24 hours.

[0021] Preferably, the incubation time is 30 minutes.

[0022] Preferably, the ion containing -π + The dye co-solvent in the culture medium for the organic fluorescent dye is DMSO; the concentration of the dye co-solvent is 0.25 wt%.

[0023] This invention provides an ion-π + Applications of type-π organic fluorescent dyes, including the preparation of antitumor drugs, detection reagents, or non-therapeutic intracellular fluorescence imaging, wherein the ion-π + The structure of the type-π organic fluorescent dye is shown in Formula I. The ionic-π dye used in this invention... + Antitumor drugs prepared from type-π organic fluorescent dyes exhibit good antitumor effects and low toxicity, while also possessing AIE properties, strong reactive oxygen species generation capacity, and live-cell imaging capabilities. This invention utilizes ion-π...+ Type I organic fluorescent dyes exhibit weak fluorescence in solution but produce strong fluorescence upon aggregation, making them typical AIE-emitting molecules. This invention enhances the electron-donating ability of the Ar group, enabling the organic fluorescent dye to exhibit a significant Stokes shift in the biological environment, thus demonstrating a pronounced AIE phenomenon in low-concentration unsuitable solvents and effectively generating type I ROS.

[0024] The organic fluorescent dye used in this invention has ionic -π + The interaction forces can effectively suppress the π-π stacking of organic light-emitting molecules, inhibiting the non-radiative deactivation process by restricting the rotation of Ar groups, and increasing the luminescence of the molecules; simultaneously, the ion-π + The interaction between the organic fluorescent dye and the cell surface can increase the binding ability of organic fluorescent dyes. Furthermore, the presence of DA interaction in the molecular structure of organic fluorescent dyes is beneficial for the red shift of the absorption and emission wavelengths of organic fluorescent dyes, which increases the penetration depth into biological tissues, reduces the influence of cell autofluorescence in live cell imaging, improves the imaging ability of organic fluorescent dyes on cells, enables rapid imaging in cells, and has certain stability and resistance to photobleaching.

[0025] Cytotoxicity studies have demonstrated that the organic fluorescent dye of this invention can stably image 4T1 cells in a short time, exhibiting excellent resistance to photobleaching. The organic fluorescent dye of this invention also exhibits a certain photodynamic (PDT) killing ability against 4T1 cells with minimal toxicity to humans. In summary, the organic fluorescent dye of this invention can demonstrate good photodynamic (PDT) killing ability against cancer cells at low concentrations, improving anti-cancer activity with minimal toxicity, and shows broad prospects for application in antitumor drugs or non-therapeutic intracellular fluorescence imaging. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 To test the normalized absorption and fluorescence spectra of organic fluorescent dyes of formulas II, III, and IV in a 1% DMSO / water mixed solution in Example 1;

[0028] Figure 2 The fluorescence intensity graphs of organic fluorescent dyes of formulas II, III, and IV in mixed solutions with different water volume fractions in Test Example 1 are shown.

[0029] Figure 3The graph shows the relationship between the fluorescence value at 538 nm and the illumination time of the mixed aqueous solution of organic fluorescent dyes of formulas II, III, and IV and reactive oxygen species scavenger DCFH-DA in Test Example 2.

[0030] Figure 4 The graph shows the relationship between the absorbance of the mixed aqueous solutions of organic fluorescent dyes of formulas II, III, and IV and singlet oxygen scavenger ABDA in test example 3 at 320–420 nm and the illumination time.

[0031] Figure 5 The images shown are inverted fluorescence microscopy images of organic fluorescent dyes of formulas II, III, and IV in 4T1 cells in Example 1; wherein, Figures A to C are inverted fluorescence microscopy images of organic fluorescent dye of formula II, Figures D to F are inverted fluorescence microscopy images of organic fluorescent dye of formula III, and Figures G to I are inverted fluorescence microscopy images of organic fluorescent dye of formula IV.

[0032] Figure 6 In Example 2, at 10mW / cm -2 Photostability of Formula II organic fluorescent dye in 4T1 cells under white light irradiation for 5 minutes using inverted fluorescence microscopy.

[0033] Figure 7 In Example 2, at 10mW / cm -2 Photostability of Formula III organic fluorescent dye in 4T1 cells under white light irradiation for 5 minutes using inverted fluorescence microscopy.

[0034] Figure 8 The cell activity diagram of photodynamic killing of 4T1 cells by different concentrations of the formula II organic fluorescent dye in Example 3;

[0035] Figure 9 This is a cell activity diagram showing the photodynamic killing effect of different concentrations of formula III organic fluorescent dye on 4T1 cells in Example 3. Detailed Implementation

[0036] This invention provides an ion-π + Applications of type-π organic fluorescent dyes, including the preparation of antitumor drugs, detection reagents, or non-therapeutic intracellular fluorescence imaging, wherein the ion-π + The structure of type I organic fluorescent dyes is shown in Formula I:

[0037]

[0038] In formula I, Ar is:

[0039] In this invention, the ion-π +The preferred structure of the type II organic fluorescent dye is shown in Formula II:

[0040]

[0041] In this invention, the ion-π + The preferred structure of the type III organic fluorescent dye is shown in Formula III:

[0042]

[0043] In this invention, the ion-π + The preferred structure of the type organic fluorescent dye is shown in Formula IV:

[0044]

[0045] In this invention, the ion-π + The preferred method for preparing the type organic fluorescent dye is the synthesis method described in A facile accessto substituted cationic 12-azapyrene salts byrhodium(III)-catalyzed C–Hannulation of N-arylpyridinium salts (Boya Feng, Danyang Wan, Lipeng Yan, Vilas D. Kadam, Jingsong You and Ge Gao, RSCAdv, 2016.06).

[0046] The ion-π used in this invention + The organic fluorescent dye molecule contains multiple freely rotating benzene rings. In solution, the energy absorbed by the molecule is mainly dissipated through the free rotation of the benzene rings, resulting in weak luminescence. In the aggregated state, the free rotation of the benzene rings is suppressed, and the energy absorbed by the molecule is mainly dissipated in the form of luminescence, thus enhancing the luminescence. This molecule has AIE properties.

[0047] In this invention, the ion-π + The application of type-3 organic fluorescent dyes in intracellular fluorescence imaging for non-therapeutic purposes preferably includes the following steps:

[0048] Cancer cells in an environment containing ions -π + After incubation with the culture medium containing the organic fluorescent dye, the sample was washed with PBS buffer and subjected to fluorescence imaging.

[0049] In this invention, the cancer cells are preferably cultured by inoculation before incubation, and then treated with a solution containing the ion -π. + The culture medium containing the type-π organic fluorescent dye replaces the original culture medium; the culture time is preferably 24 hours; the culture medium containing the ion-π+ The preferred volume of the culture medium for the type-π organic fluorescent dye is 1 mL; the culture medium containing the ion -π + The π-ion in the culture medium of type organic fluorescent dyes + The effective concentration of the type organic fluorescent dye is preferably not less than 5 μmol / L, more preferably 5–10 μmol / L; the dye containing the ion -π + The preferred dye co-solvent for the culture medium of the organic fluorescent dye is DMSO; the concentration of the dye co-solvent is preferably 0.25 wt%.

[0050] In this invention, the incubation time is preferably 30 minutes.

[0051] In this invention, the concentration of the PBS buffer (phosphate buffer) is preferably 5-10 mmol / L, more preferably 10 mmol / L.

[0052] In this invention, the fluorescence imaging device is an inverted fluorescence microscopy system or a laser scanning confocal microscope; the fluorescence imaging is performed under an excitation wavelength of 375 nm.

[0053] The present invention also provides the ion-π + Application of type-2 organic fluorescent dyes in the preparation of antitumor drugs.

[0054] In this invention, the ion-π + The concentration of the organic fluorescent dye in the antitumor drug is preferably 5-10 μmol / L, more preferably 6-9 μmol / L, and even more preferably 7-8 μmol / L; the dosage form of the antitumor drug is preferably injection, tablet or capsule.

[0055] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0056] Test Example 1

[0057] Absorption and emission spectra of organic fluorescent dyes: The dye was prepared into a 2 mmol / L DMSO stock solution. 20 μL of this stock solution was added to 2 mL of PBS solution. The absorption and fluorescence spectra of the dye were measured using a UV / Vis absorption spectrometer and a fluorescence spectrometer, respectively. The results are as follows: Figure 1 As shown.

[0058] according to Figure 1 It can be seen that as the electron donor (Ar) in the organic fluorescent dyes of Formula II, Formula III and Formula IV increases, the absorption and emission spectra of the dyes gradually red-shift. Longer wavelength absorption and emission are beneficial to increasing the tissue penetration depth and reducing the self-absorption of biological tissues.

[0059] Test Example 2

[0060] AIE property determination of organic fluorescent dyes: Dimethyl sulfoxide solutions (2 mmol / L) of organic fluorescent dyes of formulas II, III, and IV were added to mixed solutions of dimethyl sulfoxide (DMSO) in water with different volume fractions (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%) to obtain test solutions with a concentration of 20 μmol / L. The fluorescence intensity of organic fluorescent dyes of formulas II, III, and IV in the mixed solutions with different water volume fractions was measured at 475 nm, 475 nm, and 600 nm. The results are shown below. Figure 2 As shown.

[0061] according to Figure 2 It can be seen that the fluorescence intensity of the formula II organic fluorescent dye increased from 730 (water volume fraction of 0%) to 1600 (water volume fraction of 60%), and then decreased to 380 (water volume fraction of 99%); the fluorescence intensity of the formula III organic fluorescent dye increased from 280 (water volume fraction of 0%) to 880 (water volume fraction of 10%), then decreased slightly, and then continued to increase to 1000 (water volume fraction of 50%), and then decreased to 170 (water volume fraction of 99%); the fluorescence intensity of the formula IV organic fluorescent dye increased from 56... The fluorescence intensity of organic fluorescent dyes of formulas II, III, and IV gradually increased to 2000 (water volume fraction of 40%), and then decreased to 700 (water volume fraction of 99%). It can be seen that as the water volume fraction increases, water, as a poor solvent for dyes, causes dye aggregation to become more and more serious. The fluorescence intensity of organic fluorescent dyes of formulas II, III, and IV gradually increases. Later, due to the change in aggregation state, the fluorescence intensity begins to decrease. The fluorescence of dyes in the aggregated state is stronger than that in the solution state, indicating that organic fluorescent dyes of formulas II, III, and IV all have AIE characteristics.

[0062] Test Example 3

[0063] ROS generation rate determination: The ROS generation capacity of organic fluorescent dyes of formulas II, III, and IV was determined using the ROS scavenger 2',7'-dichlorofluorescein diacetate (DCFH-DA).

[0064] Preparation method: First, weigh 2.44 mg of DCFH-DA solid powder and dissolve it in 5 mL of anhydrous ethanol to obtain an anhydrous ethanol solution of DCFH-DA with a concentration of 1 mmol / L. Note that it should be stored at low temperature and protected from light. Take 0.5 mL of the above DCFH-DA solution and mix it evenly in 2 mL of NaOH solution (0.01 mol / L). Store it at low temperature and protected from light for 30 min to fully activate DCFH-DA. Add the activated DCFH-DA / NaOH mixed solution to 10 mL of PBS buffer solution to obtain a test solution of 40 μmol / L DCFH-DA.

[0065] Test conditions: 5 μL of organic fluorescent dye solutions of formulas II, III, and IV (2 mmol / L) were added to 2 mL of PBS solution, respectively. Then, 20 μL of DCFH-DA solution was added to each solution under dark conditions. Fluorescence spectra from 490 nm to 600 nm were measured using a fluorescence spectrometer. First, the fluorescence spectrum of the mixed solution under no light (0 min) was measured, and then the fluorescence spectrum of the mixed solution under light was measured at the same time interval. The results at 538 nm are shown below. Figure 3 As shown.

[0066] according to Figure 3 It can be seen that organic fluorescent dyes of formulas II, III, and IV can all effectively generate ROS under light irradiation, and the rate of ROS generation of organic fluorescent dyes of formulas II, III, and IV follows the order of formula IV > formula III > formula II from fast to slow. This is because organic fluorescent dye of formula IV introduces a triphenylamine group, which greatly enhances the intramolecular DA interaction.

[0067] Test Example 4

[0068] 1 O2 Generation Capacity Test: To further determine whether the ROS generated by organic fluorescent dyes of formulas II, III, and IV is type I or type II, we used 9,10-anthrayl-bis(methylene)dimalonic acid (ABDA) as a scavenging agent to test the type II ROS generated by organic fluorescent dyes of formulas II, III, and IV (i.e., type I or type II). 1 The capabilities of O2 were tested respectively:

[0069] Organic fluorescent dyes, when exposed to light, will produce... 1 O2, 1 O2 reacts with ABDA, causing photodegradation. During this process, the absorbance in the ultraviolet absorption spectrum at 350–420 nm gradually decreases with increasing light exposure time. Without... 1 The production of O2 does not change the absorption value, as shown in the following figure. Figure 4 As shown.

[0070] according to Figure 4 It is known that under the same illumination conditions, the absorption spectra of organic fluorescent dyes of formulas II, III, and IV do not decrease significantly. Therefore, this invention believes that the ROS generated by organic fluorescent dyes of formulas II, III, and IV are not type II ROS, but type I ROS, namely superoxide anion radicals and hydroxyl radicals, etc.

[0071] Example 1

[0072] Study of organic fluorescent dyes in live cell imaging: 4T1 cells were seeded in glass dishes and cultured for 24 h. The original culture medium was then replaced with 1 mL of fresh culture medium containing organic fluorescent dyes of formula II, formula III, and formula IV, respectively. After incubation for 1 h, the cells were washed with PBS buffer and then imaged using an inverted fluorescence microscopy system.

[0073] After incubating 4T1 cells in a medium containing 5 μmol / L Formula II organic fluorescent dye (0.25% DMSO by volume) for 30 min, the cells were observed using an excitation wavelength of 375 nm. The test results are as follows: Figure 5 As shown.

[0074] according to Figure 5 As shown in Figures A to C, the organic fluorescent dye of formula II can enter cells and emit strong green fluorescence, with good overlap between the bright field and the fluorescence field; similarly, the organic fluorescent dye of formula III also exhibits the same properties as the organic fluorescent dye of formula II, such as... Figure 5 As shown in Figures D to F; however, for formula IV organic fluorescent dyes, no green fluorescence was observed after the compound entered the cells in a culture medium containing 0.25% DMSO. Therefore, we increased the concentration of DMSO to 5% and extended the incubation time to 60 min, and green fluorescence was observed, as shown in Figures D to F. Figure 5 Figures G through I. The above results indicate that organic fluorescent dyes of formulas II, III, and IV can all be used for fluorescence imaging of 4T1 cells.

[0075] Example 2

[0076] Stability of organic fluorescent dyes in cell imaging: 4T1 cells were seeded in glass dishes and cultured for 24 h. The original culture medium was then replaced with 1 mL of fresh medium containing organic fluorescent dyes of formulas II, III, and IV, respectively. After incubation for 1 h, the cells were washed with PBS buffer and then imaged using an inverted fluorescence microscopy system. The cells were placed under white light (10 mW / cm²). -2 The compound was continuously irradiated for 1 min, 2 min, 3 min, 4 min, and 5 min, and then its photostability within the cells was observed. The results are as follows: Figure 6 and Figure 7 As shown.

[0077] according to Figure 6 and Figure 7 It can be seen that the fluorescence intensity of organic fluorescent dyes of formula II and formula III remained basically stable after 5 minutes of white light irradiation, proving that organic fluorescent dyes of formula II and formula III have excellent photostability and resistance to photobleaching.

[0078] Example 3

[0079] Photodynamic killing ability of organic fluorescent dyes against 4T1 cells: 10 μL of dye was applied to each well of a 96-well plate. 4 Four T1 cells were seeded at a density of 10 cells / day and incubated for 24 hours. The DMEM culture medium was then removed, and the cells were incubated for another 20 hours in DMEM medium containing different concentrations of organic fluorescent dyes of formulas II and III (0 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, and 10 μmol / L). The light-treated group was placed under white light (10 mW / cm²). -2 Irradiate the cells under light for 5 min, then incubate for another 4 h. Simultaneously, under the same experimental conditions, perform a dark cytotoxicity study on cells incubated with the unirradiated compound. After incubation, remove the cell culture medium and add 100 μL of fresh DMEM medium containing 10 μL of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) (5 mg / mL) solution to each well, and wait another 4 h. After removing the MTT solution, add 100 μL of DMSO to each well, and record the absorbance at 490 nm using a microplate reader. The results are as follows: Figures 8-9 As shown, relative cell viability is calculated using the following formula:

[0080] Cell viability (%) = (OD) 样本 -OD 背景 ) / (OD 对照 -OD 背景 )×100%.

[0081] according to Figure 8 It can be seen that when the concentration of the formula II organic fluorescent dye is 10 μmol / L in the dark, the cell activity is slightly reduced, indicating that the formula II organic fluorescent dye has low dark toxicity to 4T1 cells; under white light irradiation for 5 min, the cell survival rate is only 37%, indicating that the formula II organic fluorescent dye can kill cancer cells through the type I ROS generated by photodynamic action.

[0082] according to Figure 9It can be seen that under dark conditions, cell survival rate is basically unaffected, indicating that the formula III organic fluorescent dye is essentially non-toxic to cells and has high safety. However, under light, cell survival rate decreases to 28%, which shows that the formula III organic fluorescent dye can also kill cancer cells through the type I ROS generated by photodynamic action.

[0083] In addition, according to Figures 8-9 The cytotoxicity test results showed that the cell survival rate of both formula II and formula III organic fluorescent dyes was higher than 70% under dark conditions, indicating that formula II and formula III organic fluorescent dyes have low toxicity to cells. The ion-π provided by this invention... + Type I organic fluorescent dyes can be used for intracellular fluorescence imaging for non-therapeutic purposes.

[0084] As can be seen from the above embodiments, the ion-π provided by the present invention + Type I organic fluorescent dyes can be used for intracellular fluorescence imaging of antitumor drugs, detection reagents, or non-therapeutic purposes, with good application results.

[0085] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An ion -π + The application of type-π organic fluorescent dyes, namely, in the preparation of antitumor drugs, wherein the ion-π + The structure of type I organic fluorescent dyes is shown in Formula I: In formula I, Ar is:

2. Use according to claim 1, characterized in that, The ion-π + The structure of the organic fluorescent dye of Formula II is shown below:

3. Use according to claim 1, characterized in that, The ion-π + The structure of the organic fluorescent dye of Formula III is shown below:

4. Use according to claim 1, characterized in that, The ion-π + The structure of the organic fluorescent dye of Formula IV is shown below:

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