CN118005556A

CN118005556A - COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis and preparation method and application thereof

Info

Publication number: CN118005556A
Application number: CN202410155352.8A
Authority: CN
Inventors: 李海东; 曾爽; 王静云; 刘晓胜; 陈晨; 彭孝军
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2024-02-02
Filing date: 2024-02-02
Publication date: 2024-05-10

Abstract

The invention discloses a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis and a preparation method and application thereof, belonging to the technical fields of biological materials and biomedicine. The invention takes the cyanine dye as a fluorophore matrix, introduces the indomethacin group of the COX-2 inhibitor through an alkyl chain, designs and synthesizes the type I photosensitizer of COX-2 enzyme targeted on an endoplasmic reticulum, and the photosensitizer can effectively inhibit COX-2 and activate the scorching of tumor cells and can realize the inhibition effect on distant tumors.

Description

COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis and preparation method and application thereof

Technical Field

The invention belongs to the technical fields of biological materials and biomedicine, and in particular relates to a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis, and a preparation method and application thereof.

Background

Pyrodeath is programmed cell death caused by inflammatory corpuscles, and is characterized by the fact that cells are continuously swelled until cell membranes are broken, so that cell contents are released, a strong inflammatory reaction is caused, and the immune activation of an organism is obviously stimulated. Thus, in tumor therapy, focal death with immunostimulation is considered to be an excellent programmed cell death pathway, and has become a new wind mark for tumor immunotherapy. Compared with the traditional tumor treatment methods such as surgery, chemotherapy and the like, photodynamic therapy (Photodynamic therapy, PDT) has been attracting attention as a tumor treatment method which is small in wound and toxic and side effects and can be accurately controlled in time and space. In recent years, some researches have fully confirmed that photodynamic therapy can activate tumor cell pyro-death pathway and enhance immunogenic death, and is a novel potential photoactivation immunotherapy. However, most photosensitizers are oxygen-dependent type II photosensitizers at present, namely, the surrounding oxygen molecules are sensitized by the excited triplet photosensitizers to generate cytotoxicity singlet oxygen to kill cancer cells, and the singlet oxygen generation process of the type II photosensitizers is seriously blocked due to insufficient oxygen content (hypoxia) of tumor tissues, so that the damage to specific organelles in the tumor cells is reduced, and the activation of the apoptosis of the type II photosensitizers in deep tissue tumor PDT treatment is limited.

Furthermore, the immunosuppressive microenvironment inherent in tumor tissue is another major factor limiting the ability of tumor focal death to activate immunity. Cyclooxygenase 2 (COX-2) is an enzyme that catalyzes prostaglandin production, which is mainly overexpressed in inflammatory and neoplastic tissues, but hardly expressed in normal tissues, and is a potential target for cancer diagnosis and treatment. Transcription of COX-2 is regulated mainly by inflammatory factors and hypoxia-inducible factors (HIF-1. Alpha.), and its main downstream product PGE2 can significantly induce tumor environment to present immunosuppressive state, thereby facilitating survival and proliferation of cancer cells. Due to further consumption of oxygen during PDT, the tumor tissue is further hypoxized to stimulate over-expression of the intracellular COX-2 enzyme and promote prostaglandin E2 (PGE 2) production. And PGE2 can inhibit the activation of immune T cells and promote the differentiation of macrophages to M2 type with immunosuppressive function after being released into tumor microenvironment by cells. In addition, apoptosis, which is an inflammatory death mode, releases intracellular pro-inflammatory factors that further induce tumor cells to overexpress COX-2 enzymes, thereby attenuating the anti-tumor immune response elicited by apoptosis. Therefore, in order to realize effective anti-tumor immunity, a novel photosensitizer is urgently needed, can overcome the defect of hypoxia under the anoxic condition, efficiently activate tumor cell apoptosis, and has the function of inhibiting COX-2 enzyme, so that tumor immunosuppression microenvironment is remodeled, and dual combined amplification of PDT anti-tumor immune response effect is realized, which has important significance in reducing tumor recurrence and improving clinical application.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a COX-2 targeted inhibition photosensitizer capable of activating tumor cell pyrosis, a preparation method and application thereof, in particular to an anti-hypoxia I-type photosensitizer (Indo-Cy) which has the functions of remodelling immune inhibition microenvironment, efficiently activating tumor pyrosis and generating O ₂ ^-· and OH two active oxygen and a preparation method thereof. And further provides the application of the photosensitizer in the treatment of the living tumor of the mice, and the photosensitizer has the functions of activating the scorch of tumor cells and remodelling the immunosuppressive microenvironment, so that the photosensitizer not only has better PDT curative effect on in-situ tumors, but also activates the natural immunity of organisms and realizes the inhibition of distal tumors.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

In a first aspect, the invention provides a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis, wherein the structural general formula of the photosensitizer is shown in formula I:

In formula i, n=an integer of 2 to 8.

Preferably, in the general formula I, n is 4.

The invention takes the cyanine dye as a fluorophore matrix, introduces the indomethacin group of the COX-2 inhibitor through an alkyl chain, designs and synthesizes the type I photosensitizer of COX-2 enzyme targeted on an endoplasmic reticulum, and the photosensitizer can effectively inhibit COX-2 and activate the scorching of tumor cells and can realize the inhibition effect on distant tumors.

In a second aspect, the invention provides a method for preparing a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis, comprising the following steps:

S1, one of N-tert-butoxycarbonyl-1, 2-ethylenediamine, N-tert-butoxycarbonyl-1, 3-propylenediamine, N-tert-butoxycarbonyl-1, 4-butylenediamine, N-tert-butoxycarbonyl-1, 5-pentyldiamine, N-tert-butoxycarbonyl-1, 6-hexamethylenediamine, N-tert-butoxycarbonyl-1, 7-heptylenediamine or N-tert-butoxycarbonyl-1, 8-octylenediamine and indomethacin are subjected to amidation reaction to generate a compound Ind-1;

S2, performing ester hydrolysis reaction on the compound Indo-1 prepared in the step S1 and trifluoroacetic acid to generate a compound Indo-2;

s3, 2, 3-trimethyl-3H-benzo [ g ] indole and p-bromomethylbenzoic acid react through nucleophilic substitution to generate a compound S-1;

S4, performing aldol condensation reaction on the compound S-1 prepared in the step S3 and 3, 5-diiodo-4-hydroxybenzaldehyde to generate a compound Cy;

S5, performing amidation reaction on the compound Cy prepared in the step S4 and the compound Indo-2 prepared in the step S2 to generate the photosensitizer Indo-Cy;

Further, in the above technical scheme, in step S1, HATU and DIEA are used as catalysts; the molar ratio of indomethacin, HATU, DIEA and N-t-butyloxycarbonyl-1, 4-butanediamine is 1:1-2:1-2:1; the reaction condition is that the reaction is carried out for 12-14h at room temperature.

Further, in the above technical scheme, in step S2, the reaction condition is room temperature reaction for 12-14h.

Further, in the above technical scheme, in step S3, the molar ratio of 2, 3-trimethyl-3H-benzo [ g ] indole to p-bromomethylbenzoic acid is 1:1.1-1.2; the reaction condition is reflux for 12-14h at 90-95 ℃.

Further, in the above technical scheme, in step S4, piperidine is used as a catalyst; the mol ratio of the compound S-1 to the 3, 5-diiodo-4-hydroxy benzaldehyde is 1:1.2-1.5; the reaction condition is that the reaction is carried out for 12-14h at room temperature.

Further, in the technical scheme, the addition amount of the piperidine is 1-3 drops.

Further, in the above technical scheme, in step S5, HATU and DIEA are used as catalysts; the molar ratio of the compound Cy, HATU, DIEA to the compound Indo-2 is 1:1-2:1-2:1; the reaction condition is that the reaction is carried out for 12-14h at room temperature.

More specifically, the preparation method of the COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis comprises the following steps:

(1) Synthesis of Compound Indo-1: indometacin (1 eq.) was added to a round bottom flask containing anhydrous DMF solvent, HATU (2 eq.) and DIEA (2 eq.) were stirred at room temperature for 0.5h, then N-t-butoxycarbonyl-1, 4-butanediamine (1 eq.) was added and stirring was continued at room temperature for 12h. After the reaction is finished, the solvent is decompressed, distilled and separated and purified by column chromatography, and finally the white solid Indo-1 is obtained.

(2) Synthesis of Compound Indo-2: indo-1 was added to a round bottom flask containing anhydrous dichloromethane solvent and trifluoroacetic acid was added and stirred at room temperature for 12h. After the reaction, the white solid powder Indo-2 obtained after the solvent is decompressed and distilled is directly used for the next reaction without purification.

(3) Synthesis of Compound S-1 2, 3-trimethyl-3H-benzo [ g ] indole (1 eq.) and p-bromomethylbenzoic acid (1.1 eq.) were added separately to acetonitrile solution and refluxed at 90℃for 12H. After the reaction was completed, cooled to room temperature, the precipitate was filtered and washed 3 times with cold acetone.

(4) Synthesis of Compound Cy Compound S-1 (1 eq.) obtained in the above step and 3, 5-diiodo-4-hydroxybenzaldehyde (1.2 eq.) were added to ethanol solution, respectively, and 3 drops of piperidine were dropped to react at room temperature for 12 hours. After the reaction was completed, the precipitate was filtered, and the precipitate was washed 3 times with ice absolute ethanol.

(5) Synthesis of the Compound Ind-Cy after adding Cy (1 eq.) and HATU (2 eq.) and DIEA (2 eq.) in a round bottom flask with anhydrous DMF solvent and stirring at room temperature for 0.5h, ind-2 (1 eq.) was added and stirring was continued at room temperature for 12h, the solvent was distilled off under reduced pressure and then purified by column chromatography to finally obtain Ind-Cy as a purple solid.

In a third aspect, the invention provides the application of a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis in preparing a medicine for photodynamic therapy of tumor and a medicine for enhancing tumor immunotherapy.

The beneficial effects of the invention are as follows:

1) The synthesis steps of the photosensitizer are few, the raw materials are economical, and the post-treatment process is relatively simple.

2) The photosensitizer synthesized by the invention can have maximum fluorescence emission at 620nm under 580nm laser irradiation, so that the photosensitizer can be incubated with a commercial organelle targeting fluorescent probe in cells to determine the organelle targeting positioning capability through a confocal laser fluorescence microscope. The co-localization coefficient of the photosensitizer with a commercial endoplasmic reticulum fluorescent probe in a 4T1 tumor cell is 0.91, which shows that the photosensitizer has excellent targeting performance on the endoplasmic reticulum.

3) The photosensitizer synthesized by the invention can effectively generate free radical active oxygen in solution or cells under 580nm laser irradiation, and can activate tumor cells to generate pyrodeath through damaging endoplasmic reticulum under hypoxia, thereby enhancing immunogenic death of the tumor cells.

4) The photosensitizer synthesized by the invention can specifically target COX-2 binding on the endoplasmic reticulum of tumor cells, inhibit the activity of COX-2 and PGE2 synthesis, thereby remodelling tumor immunosuppression microenvironment and greatly converting M2 type macrophages for promoting tumor growth into M1 type macrophages with tumor inhibiting function.

5) The synthesized photosensitizer of the invention can greatly promote organism immunity by the pyrosis and the inhibition of COX-2 in the PDT process, has obvious treatment effect on in-situ tumor in the treatment of mouse living tumor, and also has obvious inhibition on distal tumor which is not treated by PDT. Therefore, the photosensitizer can activate the cell coke death channel and inhibit the COX-2 activity in the cell through the accurate targeting treatment of the organelle in the tumor cell, so that photodynamic treatment and immunotherapy strategies are perfectly combined, and the photosensitizer has wide application prospect in the field of cancer treatment.

Drawings

FIG. 1 is an ultraviolet spectrum of a photosensitizer Indo-Cy.

FIG. 2 is a fluorescence spectrum of the photosensitizer Indo-Cy.

FIG. 3 is a fluorescence spectrum of the superoxide anion radical generating capacity of photosensitizer Ind-Cy containing dihydrorhodamine 123 (DHR 123, superoxide anion radical probe).

FIG. 4 is a fluorescence spectrum of the ability of a photosensitizer Indo-Cy containing hydroxyphenyl fluorescein (HPF, hydroxyl radical fluorescent probe) to generate hydroxyl radicals.

FIG. 5 is a fluorescence spectrum of the ability of a photosensitizer Ind-Cy containing 2',7' -dichlorofluorescein diacetate (DCFH-DA, active oxygen fluorescence probe) to generate active oxygen.

FIG. 6 is a graph showing the in vitro COX-2 enzyme activity inhibition assay of the photosensitizer Indo-Cy.

FIG. 7 is a graph of fluorescence imaging of subcellular organelle co-localization of the photosensitizer Indo-Cy in tumor cells.

FIG. 8 is a fluorescent imaging of the photosensitizer Indo-Cy in different tumor cells.

FIG. 9 is a morphological observation of the scorch of tumor cells activated by the photosensitizer Indo-Cy.

FIG. 10 is a graph showing the results of MTT cytotoxicity test on the photosensitizer Indo-Cy.

FIG. 11 is a graph showing the results of an experiment for promoting GSDMD protein cleavage after PDT of the photosensitizer Indo-Cy in tumor cells.

FIG. 12 is a graph showing the results of experiments for inhibiting intracellular PGE2 synthesis by the photosensitizer Indo-Cy.

Fig. 13 shows in situ tumor growth 14 days after photodynamic therapy in tumor-bearing mice.

Figure 14 shows distal tumor growth in tumor-bearing mice following photodynamic therapy of in situ tumors.

FIG. 15 is a graph of HE slice staining of heart, liver, spleen, lung, kidney, etc. tissues after treatment in each group of mice.

FIG. 16 is a nuclear magnetic hydrogen spectrum of the photosensitizer Indo-Cy.

FIG. 17 is a nuclear magnetic carbon spectrum of photosensitizer Indo-Cy.

FIG. 18 is a high resolution mass spectrum of the photosensitizer Indo-Cy.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings.

In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. It should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

EXAMPLE 1 preparation of photosensitizer Indo-Cy

(1) Synthesis of Compound Indo-1

Indometacin (100 mg,0.28 mmol), HATU (213 mg,0.56 mmol) and DIEA (103. Mu.L, 0.56 mmol) were added to a round bottom flask containing anhydrous DMF solvent, stirred at room temperature for 30 min, N-t-butoxycarbonyl-1, 4-butanediamine (52.7 mg,0.28 mmol) was added, and after stirring at room temperature for 12h the solvent was isolated by column chromatography after rotary evaporation under reduced pressure, eluent MeOH/CH ₂Cl₂ (v/v, 1/20) to give Ind-1 (110 mg, 74%) as a white solid.

(2) Synthesis of Compound Indo-2

Indo-1 (100 mg,0.19 mmol) was added to a round bottom flask containing anhydrous dichloromethane (10 mL) solvent, and trifluoroacetic acid (0.5 mL) was added and stirred at room temperature for 12h. After the completion of the reaction, the white solid powder Indo-2 (80 mg) obtained after the solvent was distilled under reduced pressure was used in the next reaction without purification.

(3) Synthesis of Compound S-1

2, 3-Trimethyl-3H-benzo [ g ] indole (1.06 g,5.08 mmol) and p-bromomethylbenzoic acid (1.21 g,20.6 mmol) were dissolved in 24mL acetonitrile and refluxed at 85℃for 12H. The reaction was checked by TLC plate, after completion of the reaction, the precipitate was filtered after cooling to room temperature, and washed 3 times with cold acetone, and dried to give compound S-1 in 40% yield.

(2) Synthesis of Compound Cy

Compound S-1 (200 mg,0.47 mmol) and 3, 5-diiodo-4-hydroxybenzaldehyde (264 mg,0.71 mmol) were dissolved in 10mL of an absolute ethanol solution, and 3 drops of piperidine were added dropwise. After the reaction is completed, the precipitate is filtered, washed with ice absolute ethanol for 3 times and dried to obtain the compound Cy with the yield of 51 percent.

(3) Synthesis of Compound Indo-Cy

Cy (100 mg,0.13 mmol), HATU (99 mg,0.26 mmol) and DIEA (48. Mu.L, 0.26 mmol) were added to a round bottom flask containing anhydrous DMF solvent, stirred at room temperature for 30 min, ind-2 (60 mg,0.14 mmol) was added, and after stirring at room temperature for 12h the solvent was evaporated under reduced pressure and purified by column chromatography eluting with MeOH/CH ₂Cl₂ (v/v, 1/20) to give finally Ind-Cy (80 mg, 52%) as a purple solid. The products Indo-Cy were subjected to nuclear magnetic hydrogen spectroscopy, nuclear magnetic carbon spectroscopy and high resolution mass spectrometry detection as shown in fig. 16-18:

¹H NMR(400MHz,CDCl₃,δ):8.71(s,1H),8.38(d,J＝5.1,2H),8.28(d,J＝8.7,1H),8.12(d,J＝14.8,1H),8.03(s,1H),8.01(s,1H),7.99(s,1H),7.80(d,J＝8.1,2H),7.68(s,1H),7.66(s,2H),7.64(s,1H),7.61(s,1H),7.58(d,J＝8.7,1H),7.52(t,J＝7.4,1H),7.39(d,J＝7.9,2H),7.08(s,1H),6.92(d,J＝5.7,1H),6.89(d,J＝11.0,1H),6.66(d,J＝9.0,1H),5.77(d,J＝13.0,2H),3.70(s,3H),3.46(s,2H),3.20(d,J＝5.8,2H),3.05(d,J＝5.7,2H),2.20(s,3H),2.01(s,6H),1.44(s,4H).

¹³C NMR(126MHz,DMSO-d6,δ)177.08,172.37,169.67,168.30,166.15,155.98,149.29,139.88,138.84,138.01,135.52,134.75,134.72,134.05,131.94,131.60,131.36,130.72,130.37,129.49,128.34,127.89,126.94,125.51,123.64,122.72,114.99,114.91,112.25,111.69,102.30,98.85,55.85,54.06,51.65,42.31,38.93,31.65,27.30,27.12,27.02,18.56(s),17.21(s),13.84(s),12.97.

HRMS (ESI) M/z: [ M-Br ] ⁺ calculated as C ₅₃H₄₈ClI₂N₄O₅, 1109.1398, detected as 1109.1418.

The results show that the target compound Indo-Cy was successfully obtained in this example.

Example 2 UV, fluorescence Spectrometry test of photosensitizer Indo-Cy

(1) Experimental method

The spectrum characterization comprises the following specific steps of dissolving a proper amount of compound end product Indo-Cy in dimethyl sulfoxide (DMSO) to prepare a test mother solution with the concentration of 1mM for standby. The test mother liquor was pipetted into a PE tube with a final volume of 3mL of DMSO, at a concentration of 10. Mu.M. The solution was transferred to a cuvette and the spectral properties of the photosensitizer Indo-Cy in the solvent were tested on an ultraviolet-visible spectrometer and a fluorescence spectrometer, respectively.

(2) Experimental results

The ultraviolet spectrum result shows that the ultraviolet absorption band range of the Indo-Cy is 500-625nm in DMSO medium, and the ultraviolet absorption peaks are obvious at 550nm and 580 nm; in DMSO medium, the Indo-Cy has a fluorescence emission band range of 600-750nm under 580nm excitation, and has a significant fluorescence emission peak at 620 nm. This result indicates that the photosensitizer Indo-Cy has near infrared imaging potential (fig. 1 and 2).

Example 3 photosensitizer Indo-Cy in solution generating reactive oxygen species test

(1) Experimental method

The ability of the photosensitizer Indo-Cy to generate superoxide anion free radical, hydroxyl free radical and total active oxygen was detected with three probes of dihydrorhodamine 123 (DHR 123), hydroxyphenyl fluorescein (HPF) and 2, 7-dichlorofluorescein diacetate (DCFH-DA), respectively.

The DHR123 detection steps are: an aqueous solution containing a photosensitizer Indo-Cy (10. Mu.M) and DHR123 was prepared in a cuvette, and then the cuvette was irradiated with monochromatic light of 580nm wavelength (optical power: 10mW/cm ²), the fluorescence emission spectrum of the solution was measured every 10 seconds, and the presence or absence of the production of superoxide anion radical was determined by observing the change in fluorescence intensity of the DHR123 solution at 525 nm. The fluorescence excitation wavelength is 500nm, the emission range is 505-600nm, the excitation slit is 1.5nm, and the emission slit is 5nm. An aqueous DHR123 solution without photosensitizer and a PBS solution containing photosensitizer but no light were used as a negative control group.

And (3) detecting HPF: an aqueous solution containing a photosensitizer Indo-Cy (10. Mu.M) and HPF was prepared in a cuvette, and then the cuvette was irradiated with monochromatic light of 580nm wavelength (light power: 5mW/cm ²), the fluorescence emission spectrum of the solution was measured every 10 seconds, and the presence or absence of the generation of hydroxyl radicals was determined by observing the change in fluorescence intensity of the HPF solution at 525 nm. The fluorescence excitation wavelength is 491nm, the emission range is 492-600nm, the excitation slit is 2.5nm, and the emission slit is 5nm. An aqueous HPF solution without photosensitizer and a PBS solution containing photosensitizer but without light were used as a negative control group.

Detection step of DCFH-DA: DCFH-DA itself is non-fluorescent, but can become DCFH after hydrolysis. In the presence of active oxygen, DCFH is oxidized to generate fluorescent material DCF, which has obvious green fluorescence emission. Therefore, the ability of the photosensitizer to generate active oxygen can be qualitatively evaluated according to the intensity of the green fluorescence of DCF before and after illumination. First, a proper amount of DCFH-DA was dissolved in 0.5mL of ethanol solution at a final concentration of 1mM. This was added to 2mL of NaOH (1X 10 ^-2 M) solution and allowed to stand at room temperature for 30min to allow DCFH-DA to hydrolyze sufficiently to DCFH. A further 10mL of PBS (1X) was added to adjust the pH of the solution to 7.4. In the test, PBS solution containing photosensitizer (10 mu M) and DCFH is prepared in a cuvette, then the cuvette is irradiated under 580nm wavelength monochromatic light (light power: 5mW/cm ²), the fluorescence emission spectrum of the solution is measured every 15 seconds, and whether active oxygen is generated or not is judged by observing the change of fluorescence intensity of the DCFH solution at 525 nm. The fluorescence excitation wavelength is 488nm, the emission range is 490-600nm, and the excitation and emission slits are 2.5nm. As a negative control group, DCFH aqueous solution without photosensitizer and PBS solution containing photosensitizer but without light were used.

(2) Experimental results

As shown in FIGS. 3-5, in the solution of the photosensitizer Indo-Cy containing DHR123, the fluorescence intensity value at 525nm is increased gradually along with the illumination time, which shows that the photosensitizer Indo-Cy has the capability of efficiently generating superoxide anion free radicals. In the HPF-containing photosensitizer Indo-Cy solution, the fluorescence intensity value at 515nm is gradually increased along with the increase of illumination time, which shows that the photosensitizer Indo-Cy has high-efficiency capability of generating hydroxyl free radicals. In an aqueous solution containing only 2',7' -dichlorodihydrofluorescein diacetate (DCFH-DA, active oxygen fluorescent probe), the fluorescence intensity at 525nm hardly changed with illumination time. In the solution containing the photosensitizer Indo-Cy with the same concentration of DCFH, the fluorescence intensity at 525nm is gradually enhanced along with the increase of illumination time, and the increase amplitude is similar, so that the capability of the photosensitizer Indo-Cy for generating active oxygen is further verified.

Example 4 in vitro COX-2 enzyme Activity inhibition assay of photosensitizer Indo-Cy

(1) Experimental method

The experiment adopts an cyclooxygenase-2 (COX-2) inhibitor screening kit (Biyun) to evaluate the activity efficiency of the experimental medicine for inhibiting COX-2, and the experimental steps are described in the specification of the kit.

(2) Experimental results

Indometacin is used as a positive control inhibitor, and the inhibition rate of COX-2 enzyme activity can be as high as 99% at the concentration of 1 mu M. The inhibition rate of the photosensitizer Indo-Cy to COX-2 activity at 5 mu M can reach 70%. Cy molecules without indomethacin Xin Guan functional group structure do not have enzyme inhibition ability. The above results indicate that Indo-Cy is effective in inhibiting COX-2 enzyme activity (FIG. 6).

Example 5 photosensitizing agent Indo-Cy co-localized fluorescence imaging of subcellular organelles in tumor cells

Human cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) were cultured in a DMEM medium containing 10% inactivated Fetal Bovine Serum (FBS), 100U/mL streptomycin, 100U/mL penicillin, and placed in an incubator at 37℃under 5% CO ₂ and 95% humidity. Culture flask HeLa cells were grown to about 90% cell density, and subculture was noted.

Mouse breast cancer cells (4T 1) were cultured in an incubator containing 10% inactivated Fetal Bovine Serum (FBS), 100U/mL streptomycin, 100U/mL penicillin in RPMI-1640 medium, and 5% CO ₂, 95% humidity, and 37 ℃. Culture flask 4T1 cells had a cell density as high as about 90%, and subculture was noted.

(1) Experimental method

4T1 cells were selected as subjects. Cells were first incubated with photosensitizer Indo-Cy (10. Mu.M) for 15min, then different organelle-targeted commercial dye molecules (Mito-TRACKER GREEN, lyso-TRACKER GREEN, ER-TRACKER GREEN, hoechst 33342) were added for an additional 15min incubation. Then washed 3 times with PBS, 2mL of fresh medium was added, and finally imaged under laser confocal (FV 3000, olympus). After the fluorescent images are acquired, image J software is used for superposition or regional fluorescent intensity change comparison.

(2) Experimental results

After the photosensitizer Indo-Cy and different commercial organelle targeting dyes are respectively incubated with 4T1 cells for imaging, the co-localization coefficient of the Indo-Cy and the commercial mitochondrial localization dye is 0.31, the co-localization coefficient of the commercial lysosome localization dye and the commercial endoplasmic reticulum localization dye is 0.24, the co-localization coefficient of the commercial endoplasmic reticulum localization dye and the co-localization coefficient of the commercial nucleus localization dye is-0.04. The above results indicate that the photosensitizer Indo-Cy is precisely targeted to the endoplasmic reticulum of the cell (fig. 7).

Example 6 fluorescent imaging of the photosensitizer Indo-Cy in different tumor cells with active oxygen

Intracellular active oxygen detection was accomplished using DCFH-DA. DCFH-DA is a membrane permeable non-fluorescent dye that when it enters the intracellular environment, is converted to the non-fluorescent derivative DCFH by hydrolysis of intracellular acetate, and the polar structure cannot normally permeate the cell membrane and is therefore trapped inside the cell. Under the action of active oxygen, DCFH is oxidatively dehydrogenated and converted into strong green fluorescence DCF, so that the generation level of the active oxygen can be judged according to the green fluorescence signal intensity in cells.

(1) Experimental method

100. Mu.L of HeLa cell suspension (1X 10 ⁵/mL) was placed in a copoly Jiao Min containing 2mL of fresh medium, incubated at 37℃in 5% CO ₂ for 24h to logarithmic phase, then different organelle-targeted photosensitizers (5. Mu.M) were added and incubated with DCFH-DA (10. Mu.M) for 30min, the medium was discarded, and the excess photosensitizers and DCFH-DA dye were removed by washing 3 times with serum-free DMEM medium. After adding 2mL of fresh medium and irradiating with monochromatic light of a specific wavelength for 5min, the green fluorescence signal in the cells was observed under a confocal microscope. DCF excitation wavelength: 488nm, fluorescence emission reception wavelength: 500-530nm. MCF-7 and 4T1 cells were operated as above.

(2) Experimental results

In different tumor cells, the generation of active oxygen after illumination is detected by using an active oxygen probe DCFH-DA. After the Indo-Cy is taken up by tumor cells, the tumor cells are irradiated by a 580nm light source for 5min (40 mW/cm ²) and then are placed for confocal fluorescence microscopy imaging, so that strong green fluorescence can be observed in the cells, and a large amount of active oxygen is generated in the cells. Whereas under the same conditions, only relatively weak green fluorescence was observed in tumor cells ingested by incubation with no light sensitizer (fig. 8).

Example 7 morphological observations of the photosensitizing agent Indo-Cy activated tumor cell apoptosis

(1) Experimental method

100. Mu.L of 4T1 cell suspension (1X 10 ⁵/mL) was placed in a copoly Jiao Min containing 2mL of fresh medium, incubated at 37℃in 5% CO ₂ for 24h to logarithmic phase, then incubated for 30min with different organelle-targeted photosensitizers (5. Mu.M), the medium was discarded and the excess photosensitizers were removed by washing 3 times with serum-free medium. 2mL of fresh medium is added, after irradiation of light with 580nm wavelength monochromatic light for 10min, the culture medium is placed in an incubator for incubation for 1h, and then the culture medium is placed under a confocal microscope for imaging. MCF-7 and HeLa cells were operated as above.

(2) Experimental results

Compared with normal cells, tumor cells developed cell swelling and significant membrane bubbling after PDT treatment with different organelle-targeted photosensitizers, which were characteristic of pyrosis (fig. 9).

EXAMPLE 8 MTT cytotoxicity assay of photosensitizer Indo-Cy

(1) Experimental method

4T1 cells were seeded at a density of 1X10 ⁵ cells per well in 96-well cell culture plates and incubated for 24 hours at 37℃under normoxic conditions (21% O ₂). After further culturing for 12 hours in normoxic (21% O ₂) or hypoxic (2% O ₂) conditions, different concentrations of Indo-Cy were added to incubate the cells for 1 hour under normoxic (21% O ₂) or hypoxic (2% O ₂) conditions. After the cells were subsequently washed with 100. Mu.L of fresh medium, the cells were placed under a 580nm LED lamp at a power density of 40mW/cm ² for 5 minutes, and then all cells were grown in a normoxic (21% O ₂) environment for an additional 24 hours. Finally, fresh medium (100. Mu.L) and MTT (10. Mu.L, 5 mg/mL) were added to each well, and the cells were cultured at 37℃for 4 hours. Cell viability was calculated by measuring absorbance at 570nm using a Bio-Rad microplate reader.

(2) Experimental results

The photosensitizer Indo-Cy was not significantly dark toxic to cells in the tested concentration range, but was significantly phototoxic to cells either in normoxic or hypoxic conditions after 10min (40 mW/cm ²) of irradiation with a 580nm light source (fig. 10).

Example 9 photosensitizer Indo-Cy after PDT in tumor cells, GSDMD protein cleavage experiment GSDMD is an executive protein of cell apoptosis, activated Caspase 1 can cleave the junction region of the N-terminal and C-terminal domains of GSDMD protein, release the N-terminal domain with membrane perforation activity on membrane phospholipid, and further destroy cell membranes to initiate cell apoptosis, thus GSDMD protein N-terminal high expression is a direct proof of the occurrence of apoptosis.

(1) Experimental method

4T1 cells are planted on a cell culture plate at a certain cell density, cultured for 24 hours at 5% CO ₂ and 37 ℃, then changed into a culture medium containing 5 mu M of different photosensitizers, incubated for 1 hour, then changed back to a normal culture medium, irradiated for 10 minutes (40 mW/cm ²) by a 580nm light source, and after 2 hours, 4T1 cells treated with Indo-Cy under light or darkness are collected and subjected to Western Blot method to detect protein expression.

(2) Experimental results

The expression of the cell GSDMD and the cleavage product GSDMD-N end thereof after PDT treatment of the 4T1 cells by the three photosensitizers is detected by a Western Blot method, and the expression of the GSDMD-N end is directly proved to cause cell apoptosis. The GSDMD protein expression in the Indo-Cy light group was decreased and the GSDMD-N expression level was significantly increased compared to 4T1 cells not subjected to light treatment and indomethacin (Indo) treatment; while the other groups hardly express GSDMD-N terminus. It was shown that the photosensitizer Indo-Cy promotes GSDMD protein cleavage to GSDMD-N-terminal protein and initiates apoptosis after PDT treatment of 4T1 cells (FIG. 11).

Example 10 experiment for inhibition of intracellular PGE2 Synthesis by photosensitizer Indo-Cy

PGE2 is a major downstream product of cyclooxygenase 2 (COX-2) that can significantly induce the tumor environment to assume an immunosuppressive state, thereby favoring survival and proliferation of cancer cells. Thus, detecting PGE2 levels is an important indicator for evaluating intracellular COX-2 activity and for evaluating reverse tumor immunosuppressive microenvironment.

(1) Experimental method

4T1 cells were seeded into 6-well plates at a density of 1X 10 ⁶ cells/well and cultured at 37 ℃. After complete growth on the plates, 4T1 cells were treated with 50. Mu.M indomethacin (Indo), cy or Indo-Cy for 24 hours. The supernatants were collected and assayed for PGE2 levels in the cell supernatants using the Elisa kit.

(2) Experimental results

After significant inhibition of intracellular COX-2 activity by indomethacin (Indo) and Indo-Cy, the PGE2 level of tumor cell supernatants was significantly reduced compared to the normal and Cy drug-treated groups, indicating that Indo-Cy was able to significantly inhibit COX-2 activity in the cells, with the ability to remodel the tumor immunosuppressive microenvironment (FIG. 12).

Example 11 therapeutic application of different organelle-targeting photosensitizers for in situ tumor and distal tumor in tumor-bearing mice

(1) Experimental method

Female BALB/c mice are selected as living body treatment models, strictly follow relevant regulations of animal welfare and animal ethics of experimental animals in China, and are approved by the animal ethics examination committee of university of great company. In situ tumor preparation: a suspension of 4T1 tumor cell lines was injected with 2X 10 ⁶ cells in front of the right chest of 8 week female BALB/c mice and related experiments were performed when tumors grew to a volume of 100mm ³. Preparation of distal tumor: the 4T1 tumor cell line suspension was injected at 1×10 ⁵ cells in the front left chest of mice 7 days after in situ tumor seeding. Photodynamic therapy test: mice with tumor bodies growing to a certain volume are randomly divided into different treatment groups, 4-6 of each group are injected with the photosensitizer, and after the photosensitizer is injected for 2 hours, the tumor part is irradiated with a 580nm light source (0.1W/cm ²) for 20 minutes for the tumor part, and the treatment is continued for two days. And measuring the volumes of the in-situ tumor and the distal tumor by using a caliper every two days, and examining the photodynamic treatment effect of the photosensitizer on the tumor in the body. On day 14, mice were euthanized to obtain tissues such as tumors, hearts, livers, spleens, lungs, kidneys, etc., and H & E staining was used to analyze the systemic toxicity of photosensitizers to different organs, i.e., biosafety assessment.

(2) Experimental results

Tumor volume growth was rapid in mice of the control group injected with PBS alone, and non-illuminated groups injected with Indo and Indo-Cy had a tendency to slow down tumor growth, whereas tumor volume was significantly inhibited in mice of the Indo-Cy combined photodynamic therapy group. For the far-end tumor, the combination photodynamic therapy group of Indo-Cy also has the optimal far-end tumor inhibition effect, which shows that the tumor scorch is initiated and the COX-2 activity of tumor cells is inhibited to activate the natural immunity of the organism more obviously, thereby realizing the immunotherapy of the far-end tumor. HE section staining was performed on heart, liver, spleen, lung, kidney, etc. tissues after treatment of each group of mice, and no significant abnormalities were found in the tissue sections of all groups, indicating that the photosensitizer Indo-Cy according to the present invention has a better biosafety during treatment (fig. 13, 14, and 15).

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis is characterized in that the structural general formula of the photosensitizer is shown in formula I:

In formula i, n=an integer of 2 to 8.

2. The COX-2 targeted inhibitory photosensitizer capable of activating tumor cell apoptosis according to claim 1, wherein n is 4 in formula i.

3. A preparation method of a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis is characterized by comprising the following steps:

4. the method for preparing a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis according to claim 3, wherein in step S1, HATU and DIEA are used as catalysts; the molar ratio of indomethacin, HATU, DIEA and N-t-butyloxycarbonyl-1, 4-butanediamine is 1:1-2:1-2:1; the reaction condition is that the reaction is carried out for 12-14h at room temperature.

5. The method for preparing a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis according to claim 3, wherein in step S2, the reaction condition is room temperature reaction for 12-14h.

6. The method of preparing a COX-2 targeted inhibitory photosensitizer capable of activating tumor cell apoptosis according to claim 3, wherein in step S3, the molar ratio of 2, 3-trimethyl-3H-benzo [ g ] indole to p-bromomethylbenzoic acid is 1:1.1-1.2; the reaction condition is reflux for 12-14h at 90-95 ℃.

7. The method for preparing a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis according to claim 3, wherein in step S4, piperidine is used as a catalyst; the mol ratio of the compound S-1 to the 3, 5-diiodo-4-hydroxy benzaldehyde is 1:1.2-1.5; the reaction condition is that the reaction is carried out for 12-14h at room temperature.

8. The method for preparing a COX-2 targeted inhibitory photosensitizer capable of activating tumor cell apoptosis according to claim 3, wherein in step S5, HATU and DIEA are used as catalysts; the molar ratio of the compound Cy, HATU, DIEA to the compound Indo-2 is 1:1-2:1-2:1; the reaction condition is that the reaction is carried out for 12-14h at room temperature.

9. The use of a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis as claimed in claim 1 or 2 or a COX-2 targeted inhibition photosensitizer capable of activating tumor cell apoptosis as prepared by the preparation method as claimed in any one of claims 3 to 8 in the preparation of medicaments for photodynamic therapy of tumors and medicaments for enhancing tumor immunotherapy.