A water-soluble type I photosensitizer, its preparation method and application

Abstract

This invention discloses a water-soluble type I photosensitizer, its preparation method, and its applications, belonging to the field of materials preparation technology. The invention uses the Suzuki reaction to prepare the intermediate product MTPABZCHO and synthesize PyC. Under alkaline conditions, MTPABZCHO and PyC dehydrate to generate MTPABZ-PyC, which is then further linked with PEG to obtain the water-soluble type I photosensitizer (PEG-MTPABZ-PyC) through a dehydration reaction. PEG-MTPABZ-PyC exhibits excellent water solubility in aqueous solution. Furthermore, PEG-MTPABZ-PyC can effectively generate type I ROS of O2·− and ·OH and type II ROS of 1O2, and shows good biocompatibility in the absence of white light irradiation. It also exhibits good PDT efficiency even under hypoxic conditions, demonstrating significant application potential in clinical tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of materials preparation technology, specifically relating to a water-soluble type I photosensitizer, its preparation method, and its application. Background Art

[0002] Photodynamic therapy (PDT) continues to attract increasing research attention due to its significant advantages, including being minimally invasive, highly selective, and less prone to drug resistance. Photosensitizers are a crucial component of PDT, and based on their energy transfer process from the triplet state to oxygen, they can be classified into Type II (primarily energy transfer) and Type I (primarily electron transfer). Typical tumor hypoxia caused by abnormal tumor cell proliferation, apoptosis, and tumor vascular malformations reduces the efficiency of reactive oxygen species (ROS) generation by photosensitizers (PSs) in the tumor, severely impacting the therapeutic efficacy of PDT. Therefore, there is a need to develop drugs that can achieve good therapeutic effects on hypoxic tumors under both normoxic and hypoxic conditions.

[0003] Photosensitizers (PSs) are generally classified into two categories: Type I, which generates superoxide radicals (O2·-) and hydroxyl radicals (·OH) primarily through electron transfer from the triplet state to oxygen; and Type II, which generates singlet oxygen (1O2) primarily through energy transfer. This difference in ROS generation mechanisms gives Type I PSs the advantage of low oxygen dependence. Therefore, Type I PSs, such as O2·- and ·OH generators, are considered promising candidates for treating hypoxic tumors. However, Type I PSs exhibit significant fluorescence self-absorption and photobleaching effects, which are detrimental to long-term fluorescence imaging of cells and tumors. Although more and more AIE-active Type I PSs have been developed in recent years, their hydrophobicity (due to their molecular structure composed of many aromatic rings) poses a significant obstacle to their practical application in the biomedical field. Currently reported Type I photosensitizers suffer from poor water solubility and short emission wavelengths, which limit their clinical application. Therefore, the pursuit of biocompatibility, stability, and water solubility of type I photosensitizers with aggregation-induced near-infrared fluorescence properties has enormous potential for clinical biomedical applications, and the development of water-soluble near-infrared type I photosensitizers is urgently needed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a water-soluble type I photosensitizer, its preparation method and application, so as to solve the technical problems of poor water solubility and poor PDT efficiency under hypoxic conditions.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for preparing a water-soluble type I photosensitizer, comprising the following steps:

[0006] S1. 4,7-Dibromo-2,1,3-benzothiadiazole, β-[4-[bis(4-methoxyphenyl)amino]phenyl]boric acid, and Pd catalyst were mixed in an organic solvent, followed by the addition of an alkaline solution. The mixture was reacted at 80-100℃ for 10-14 h to obtain the MTPABZCHO intermediate. The ratio of 4,7-dibromo-2,1,3-benzothiadiazole, β-[4-[bis(4-methoxyphenyl)amino]phenyl]boric acid, Pd catalyst, and alkaline solution was 30-50 mmol: 30-50 mmol: 1-3 mmol: 100-150 mL.

[0007] S2. Dissolve 4-methylpyridine and 11-bromoundecanoic acid in an organic solvent at a molar ratio of 4-6:5-7, stir the reaction at room temperature for 10-14 h, filter to obtain PyC intermediate;

[0008] S3. Dissolve MTPABZCHO intermediate and PyC intermediate in an organic solvent, then add alkaline solution and react at room temperature for 10-14 h to obtain MTPABZ-PyC intermediate; the ratio of MTPABZCHO intermediate, PyC intermediate and alkaline solution is 1-3 mmol: 1-3 mmol: 100-150 μL.

[0009] S4. Dissolve the MTPABZ-PyC intermediate, PEG and catalyst in an organic solvent at a molar ratio of 3-5:1-3:6-8, and react at 50-70℃ for 22-26 h to obtain a water-soluble type I photosensitizer (PEG-MTPABZ-PyC).

[0010] Based on the above technical solution, the present invention can be further improved as follows:

[0011] Furthermore, the ratio of 4,7-dibromo-2,1,3-benzothiadiazole, β-[4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid, Pd catalyst, and alkaline solution is 20 mmol: 20 mmol: 1 mmol: 60 mL.

[0012] Furthermore, the reaction temperature in S1 is 90℃, and the reaction time is 12h.

[0013] Furthermore, the organic solvent is tetrahydrofuran, toluene, or methanol.

[0014] Furthermore, the Pd catalyst is Pd(PPh3)4.

[0015] Furthermore, the alkaline solution is either a K2CO3 solution or a piperidine solution.

[0016] Furthermore, the concentration of the alkaline solution is 1.5-2.5 mol / L.

[0017] Furthermore, the catalyst in S4 is a mixture of carbodiimide and 4-dimethylaminopyridine in a molar ratio of 15-25:1-3.

[0018] The present invention also discloses a water-soluble type I photosensitizer prepared by the above preparation method.

[0019] This invention also discloses the application of water-soluble type I photosensitizers in the preparation of drugs for treating tumors.

[0020] The beneficial effects of this invention are as follows: This invention uses biocompatible macromolecular polyethylene glycol (PEG) to modify the hydrophobic AIE-active photosensitizer (MTPABZ-PyC), resulting in a water-soluble type I photosensitizer (PEG-MTPABZ-PyC) with bright near-infrared fluorescence, a large Stokes shift, and better photostability. PEG-MTPABZ-PyC exhibits excellent water solubility in aqueous solution (37 μg / mL). Furthermore, PEG-MTPABZ-PyC can effectively generate type I ROS such as O2·- and ·OH, and type II ROS such as 1O2. In vitro cell fluorescence imaging results show that the PEG-TPABZ-PyC probe can be rapidly phagocytosed by cells and exhibits bright red fluorescence, and can also effectively generate ROS such as 1O2, O2·-, and ·OH after white light irradiation. Cytotoxicity assessment results show that PEG-TPABZ-PyC has good biocompatibility with cells without white light irradiation, but most cancer cells are killed after irradiation with appropriate power of white light. Even under hypoxic conditions, PEG-MTPABZ-PyC exhibits good PDT efficiency and has significant application potential in clinical tumor treatment. Attached Figure Description

[0021] Figure 1 This is the synthetic route of the present invention;

[0022] Figure 2 The 1H NMR spectra of PEG, MTPAPZ-PyC, and PEG-MTPAPZ-PyC are shown.

[0023] Figure 3 The absorption and fluorescence spectra of MTPBZ-PyC and PEG-MTPBZ-PyC in tetrahydrofuran are shown.

[0024] Figure 4 The fluorescence spectrum of MTPBZ-PyC in a dimethyl sulfoxide / toluene mixed solution;

[0025] Figure 5 The UV absorption spectra of MTPATZ-PyC in different organic solvents;

[0026] Figure 6The fluorescence spectra of MTPATZ-PyC in different organic solvents;

[0027] Figure 7 This is a photograph of an aqueous solution of PEG MTPATZ-PyC.

[0028] Figure 8 The absorption spectra of MTPABZ-PyC and PEG MTPABZ-PyC are shown.

[0029] Figure 9 The linear fitting results for the concentration and absorbance of PEG MTPBZ-PyC are shown.

[0030] Figure 10 Comparison of water solubility of MTPBZ-PyC and PEG MTPBz-PyC;

[0031] Figure 11 The fluorescence intensity of H2DCF-DA probe samples with added MTPABZ-PyC and PEG-MTPABZPy-PyC changes with illumination time.

[0032] Figure 12 The trend of UV absorption spectrum of sample with ABDA probe added to MTPABZ-PyC as a function of illumination time;

[0033] Figure 13 The trend of UV absorption spectrum of PEG-MTPABZ-PyC sample with ABDA probe added as a function of light exposure time;

[0034] Figure 14 The fluorescence intensity of samples with MTPABZ-PyC and PEG-MTPABZPy-PyC added to the DHE probe changes with illumination time.

[0035] Figure 15 The fluorescence intensity of HPF probes with MTPABZ-PyC and PEG-MTPABZPy-PyC as a function of illumination time is shown in the figure.

[0036] Figure 16 The fluorescence imaging effect of PEG-MTPABZ-PyC on MKN-45 cells;

[0037] Figure 17 Phototherapy efficacy of PEG-MTPABZ-PyC on MKN-45 cells;

[0038] Figure 18 Cell viability under hypoxic conditions;

[0039] Figure 19 The staining pattern of cancer cells after the addition of PEG-MTPABZ-PyC. Detailed Implementation

[0040] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0041] Example 1

[0042] A method for preparing a water-soluble type I photosensitizer, the synthetic route is as follows: Figure 1 As shown, the following steps are included:

[0043] S1. 4,7-Dibromo-2,1,3-benzothiadiazole (4 mmol, 1.12 g), β-[4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid (4 mmol, 1.4 g), and Pd(PPh3)4 (0.2 mmol, 231 mg) were added to a two-necked flask filled with nitrogen. Tetrahydrofuran (20 mL) and K2CO3 solution (2 M, 12 mL) were then added, and the mixture was reacted at 90 °C for 12 h. After the reaction was complete and the mixture was cooled, it was dichloroisocyanuric acid was used to treat the mixture. The mixture was extracted three times with methane (DCM) and ethyl acetate / petroleum ether (1:3, v:v) as eluent. The resulting red powder (978 mg) was obtained by column chromatography, which was the MTPABZCHO intermediate. The yield of the MTPABZCHO intermediate was 45%. The ¹H NMR (400 MHz, d-DMSO) δ = 10.05 (s, 1H), 8.19–7.99 (m, 8H), 7.06–6.83 (m, 10H), 3.72 (s, 6H).

[0044] S2. 4-Methylpyridine (5 mmol, 465 mg) and 11-bromoundecanoic acid (6 mmol, 1.59 g) were dissolved in toluene (20 mL), and the mixture was stirred at room temperature for 12 h. After filtration, a white precipitate was obtained. The precipitate was washed three times with ethyl acetate to obtain the PyC intermediate. The yield of the PyC intermediate was 87%. ¹H NMR (400 MHz, d-DMSO) δ = 11.95 (s, 1H), 8.95 (d, 2H), 7.99 (d, 2H), 4.52 (t, 2H), 2.62 (s, 3H), 2.22 (t, 2H), 1.89 (t, 2H), 1.47 (t, 2H), 1.23 (m, 12H).

[0045] S3. MTPABZCHO intermediate (1 mmol, 543 mg) and PyC intermediate (1 mmol, 357 mg) were dissolved in a mixed solution of tetrahydrofuran (10 mL) and methanol (10 mL), followed by the addition of piperidine (60 μL) and reaction at room temperature for 12 h. After the reaction was completed, the solvent was removed by rotary evaporation, and a small amount of dichloromethane was added to dissolve the product. The product was then precipitated three times in low-polarity hexane to obtain a purple powder (600 mg), which is the MTPABZ-PyC intermediate. The yield of the MTPABZ-PyC intermediate was 78%. ¹H NMR (400 MHz, d-DMSO) δ = 1H NMR (500MHz, d-DMSO) δ = 9.02 (s, 2H), 8.16-7.91 (m, 12H), 7.13-6.88 (m, 10H), 4.52 (s, 2H), 3.76 (s, 6H), 2.04 (d, 4H), 1.44-1.23 (m, 16H);

[0046] S4, mix MTPABZ-PyC intermediate (0.2 mmol, 154 mg) and PEG (PEG 1000 0.1 mmol (200 mg) and carbodiimide (0.3 mmol, 61 mg) were dissolved in anhydrous dichloromethane (10 mL) under nitrogen atmosphere. Then, 4-dimethylaminopyridine (DMAP) (0.03 mmol, 4 mg) dissolved in dichloromethane (2 mL) was injected into the above reaction system, and the reaction was carried out at 60 °C for 24 h. After the reaction was completed, the solution was concentrated and poured into cooled diethyl ether, and a purple powder was precipitated. The powder was repeatedly precipitated with diethyl ether to obtain the purple product, a water-soluble type I photosensitizer (PEG-MTPABZ-PyC).

[0047] Example 2

[0048] A method for preparing a water-soluble type I photosensitizer, the synthetic route is as follows: Figure 1 As shown, the following steps are included:

[0049] S1. 4,7-Dibromo-2,1,3-benzothiadiazole (3 mmol), β-[4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid (3 mmol) and Pd(PPh3)4 (0.1 mmol) were added to a two-necked flask filled with nitrogen atmosphere, followed by the addition of tetrahydrofuran (20 mL) and K2CO3 solution (2 M, 10 mL). The mixture was reacted at 80 °C for 14 h. After the reaction was completed and the mixture was cooled, it was extracted three times with dichloromethane (DCM) using ethyl acetate / petroleum ether (1:3, v:v) as eluent. The resulting red powder, the MTPABZCHO intermediate, was obtained by column chromatography.

[0050] S2. Dissolve 4-methylpyridine (4 mmol) and 11-bromoundecanoic acid (7 mmol) in toluene (20 mL), stir the mixture at room temperature for 10 h, filter to obtain a white precipitate, wash the precipitate three times with ethyl acetate to obtain the PyC intermediate;

[0051] S3. Dissolve MTPABZCHO intermediate (2 mmol) and PyC intermediate (1 mmol) in a mixed solution of tetrahydrofuran (10 mL) and methanol (10 mL), then add piperidine (60 μL) and react at room temperature for 14 h. After the reaction is complete, remove the solvent with a rotary evaporator, add a small amount of dichloromethane to dissolve the product, and precipitate it three times in low-polarity hexane to obtain a purple powder (600 mg), which is the MTPABZ-PyC intermediate.

[0052] S4, add MTPABZ-PyC intermediate (0.25 mmol), PEG (PEG) 1000 0.15 mmol) and carbodiimide (0.3 mmol) were dissolved in anhydrous dichloromethane (10 mL) under nitrogen atmosphere. Then, 0.03 mmol of 4-dimethylaminopyridine (DMAP) dissolved in dichloromethane (2 mL) was injected into the above reaction system, and the reaction was carried out at 50 °C for 26 h. After the reaction was completed, the solution was concentrated and poured into cooled diethyl ether, and a purple powder was precipitated. The powder was repeatedly precipitated with diethyl ether to obtain the purple product, a water-soluble type I photosensitizer (PEG-MTPABZ-PyC).

[0053] Example 3

[0054] A method for preparing a water-soluble type I photosensitizer, the synthetic route is as follows: Figure 1 As shown, the following steps are included:

[0055] S1. 4,7-Dibromo-2,1,3-benzothiadiazole (5 mmol), β-[4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid (5 mmol) and Pd(PPh3)4 (0.3 mmol) were added to a two-necked flask filled with nitrogen atmosphere, followed by the addition of tetrahydrofuran (20 mL) and K2CO3 solution (2 M, 12 mL). The mixture was reacted at 100 °C for 10 h. After the reaction was completed and the mixture was cooled, it was extracted three times with dichloromethane (DCM) using ethyl acetate / petroleum ether (1:3, v:v) as eluent. The resulting red powder (978 mg) was obtained by column chromatography, which is the intermediate MTPABZCHO.

[0056] S2. Dissolve 4-methylpyridine (6 mmol) and 11-bromoundecanoic acid (5 mmol) in toluene (20 mL), stir the mixture at room temperature for 10 h, filter to obtain a white precipitate, wash the precipitate three times with ethyl acetate to obtain the PyC intermediate;

[0057] S3. Dissolve MTPABZCHO intermediate (1 mmol) and PyC intermediate (3 mmol) in a mixed solution of tetrahydrofuran (10 mL) and methanol (10 mL), then add piperidine (60 μL) and react at room temperature for 10 h. After the reaction is complete, remove the solvent with a rotary evaporator, add a small amount of dichloromethane to dissolve the product, and precipitate it three times in low-polarity hexane to obtain a purple powder (600 mg), which is the MTPABZ-PyC intermediate.

[0058] S4, add MTPABZ-PyC intermediate (0.15 mmol), PEG (PEG) 1000 0.05 mmol) and carbodiimide (0.4 mmol) were dissolved in anhydrous dichloromethane (10 mL) under nitrogen atmosphere. Then, 0.03 mmol of 4-dimethylaminopyridine (DMAP) dissolved in dichloromethane (2 mL) was injected into the above reaction system, and the reaction was carried out at 70 °C for 22 h. After the reaction was completed, the solution was concentrated and poured into cooled diethyl ether, and a purple powder was precipitated. The powder was repeatedly precipitated with diethyl ether to obtain the purple product, a water-soluble type I photosensitizer (PEG-MTPABZ-PyC).

[0059] Comparative Example

[0060] The difference between this comparative example and Example 1 is that step S4 is omitted, while the remaining steps are the same as in Example 1, to obtain MTPABZ-PyC.

[0061] The following experiments were conducted using MTPABZ-PyC prepared in the comparative example and PEG-MTPABZ-PyC prepared in Example 1 as examples.

[0062] Experimental Example 1: Structural Characterization

[0063] To confirm the successful preparation of the water-soluble type I photosensitizer PEG-MTPABZ-PyC, the 1H NMR spectra of PEG, MTPABZ-PyC (comparative example), and PEG-MTPABZ-PyC (Example 1) were compared. The results are as follows: Figure 2 As shown, commercial PEG 1000 The chemical shift of PEG-MTPABZ-PyC is 3.51 ppm. MTPABZ-PyC shows some special chemical shifts, such as the chemical shift at 9.02 ppm, which is attributed to the proton hydrogen (c-position) of pyridine, while the chemical shifts at 3.76 ppm and 4.52 ppm belong to the methoxy (b-position) and methylene (d-position), respectively. The 1H NMR spectrum of PEG-MTPABZ-PyC shows the special chemical shifts of both PEG and MTPABZ-PyC, which proves that the target product has been successfully prepared.

[0064] Absorption and fluorescence spectra were used to characterize the optical properties of MTPABZ-PyC and PEG-MTPABZ-PyC. The absorption and fluorescence spectra of MTPABZ-PyC and PEG-MTPABZ-PyC in tetrahydrofuran are shown below. Figure 3 As shown, the MTPABZ-PyC small molecule exhibits three absorption peaks at 320 nm, 383 nm, and 479 nm, which are attributed to position and charge transfer transitions. When the excitation wavelength is set to 479 nm, MTPABZ-PyC emits deep red fluorescence, with a maximum emission wavelength of 660 nm. For PEG-MTPABZ-PyC, its maximum absorption and fluorescence wavelengths are the same as those of the MTPABZ-PyC small molecule, indicating that the optical properties mainly originate from the MTPABZ-PyC core.

[0065] The fluorescence spectrum of MTPBZ-PyC in a dimethyl sulfoxide / toluene mixed solution is as follows: Figure 4 As shown, when MTPABZ-PyC is completely dissolved in dimethyl sulfoxide (DMSO) solvent, the fluorescence of MTPABZ-PyC is very weak. Low-polarity toluene acts as a poor solvent for the high-polarity MTPABZ-PyC, causing it to aggregate in DMSO / toluene mixed solvents with high toluene content. Therefore, with increasing toluene content, a significant increase in the fluorescence intensity of MTPABZ-PyC can be observed, indicating that MTPABZ-PyC possesses AIE properties. These AIE properties are attributed to the introduction of triphenylamine (TPA) as an electron donor.

[0066] like Figure 5 and Figure 6 As shown, the absorption spectra of MTPABZ-PyC in various organic solvents are similar, indicating that the ground-state optical properties are insensitive to environmental polarity. However, when the solvent polarity increases, the maximum fluorescence peak red-shifts, indicating that the MTPABZ-PyC molecule has a significant ICT effect. The significant ICT effect of the MTPABZ-PyC molecule is due to the inclusion of typical electron donor and acceptor units in its molecular structure, thus exhibiting a typical intramolecular charge transfer (ICT) effect, which is beneficial for accelerating inter-system channel crossings to generate ROS.

[0067] Experiment Example 2: Water Solubility Test

[0068] Actual sample of aqueous solution of PEG MTPATZ-PyC Figure 7 As shown, PEG MTPATZ-PyC can be uniformly dissolved in water without obvious precipitation, indicating that PEG MTPATZ-PyC has good water solubility.

[0069] The absorption spectra of MTPABZ-PyC and PEG MTPABZ-PyC are as follows: Figure 8 As shown, the maximum absorption wavelength of MTPABZ-PyC in the DMSO / H2O mixture is 482 nm, and the maximum emission wavelength peak is 660 nm. For PEG-MTPABZ-PyC in aqueous solution, its maximum absorption wavelength is the same as that of MTPABZ-PyC, indicating that the optical properties are mainly attributed to the MTPABZ-PyC core; however, the fluorescence peak red-shifts to the near-infrared fluorescence region, and the maximum emission wavelength is 670 nm, which is attributed to the solvation effect.

[0070] The solubility of PEG-MTPABZ-PyC in water was further characterized by studying the linear fit between concentration and absorbance. The results are as follows: Figure 9 As shown, based on the fitting formula y=0.034+0.016x, the saturation concentration of PEG-MTPABZ-PyC in water was calculated to be 37μg / mL by measuring the absorbance of the saturated PEG-MTPABZ-PyC solution.

[0071] from Figure 10 Enhanced water solubility of PEG-MTPABZ-PyC can also be observed. MTPABZ-PyC can dissolve in ethyl acetate organic solvent, but PEG-MTPABZ-PyC mainly dissolves in water, while the ethyl acetate layer is clear and transparent.

[0072] Experiment Example 3: Efficiency and Types of ROS

[0073] The types and efficiencies of ROS were identified by using a series of specific ROS probes. To ensure data accuracy, each MTPABZ-PyC and PEG-MTPABZ-PyC was tested three times using different ROS probes.

[0074] (1) The total ROS of MTPABZ-PyC and PEG-MTPABZ-PyC were characterized using the H2DCF-DA probe. The results are as follows: Figure 11 As shown, the fluorescence intensity of the H2DCF-DA probe significantly increased under frequent white light irradiation, indicating that MTPABZ-PyC and PEG-MTPABZ-PyC can effectively generate ROS, and from Figure 11 It can be seen that the trend of MTPABZ-PyC is higher than that of PEG-MTPABZ-PyC, that is, MTPABZ-PyC generates total ROS more efficiently than PEG-MTPABZ-PyC, which is attributed to the higher effective concentration of PEG-MTPABZ-PyC.

[0075] (2) Specific ABDA probes were used to demonstrate whether MTPABZ-PyC and PEG-MTPABZ-PyC produce type II ROS of 1O2. The results are as follows: Figure 12 and Figure 13 As shown, the absorption intensity of ABDA decreased significantly, indicating that it effectively generated 1O2.

[0076] (3) Type I ROS containing O2·- and ·OH were further measured using DHE and HPF probes. The results are as follows: Figure 14 As shown in the figure, the fluorescence intensity of the DHE probe is significantly increased, and the experimental group of PEG-MTPABZ-PyC shows greater fluorescence enhancement than MTPABZ-PyC. This indicates that both MTPABZ-PyC and PEG-MTPABZ-PyC can generate O2·- after white light irradiation, and PEG-MTPABZ-PyC is more efficient in generating O2·- than MTPABZ-PyC.

[0077] Furthermore, the results from the HPF probe also indicated that PEG-MTPABZ-PyC was more efficient than MTPABZ-PyC in generating ·OH. Figure 15 This is because the concentration of active ingredients in PEG-MTPABZ-PyC is lower than that in MTPABZ-PyC. Therefore, the PEGylation of PEG-MTPABZ-PyC not only improves hydrophilicity but also accelerates the generation of type I ROS.

[0078] Experiment Example 4 Fluorescence Imaging

[0079] Water-soluble photosensitizers can easily enter cells and are expected to exhibit good fluorescence imaging and therapeutic effects. Therefore, this experimental example evaluated the cellular fluorescence imaging and PDT effects of PEG-MTPABZ-PyC.

[0080] The prerequisite for PDT efficacy is that the photosensitizer can enter cells; therefore, the first step was to assess whether the water-soluble PEG-MTPABZ-PyC could enter cells. Fluorescence imaging of PEG-MTPABZ-PyC was used to evaluate its effectiveness, and the results are as follows: Figure 16 As shown, after incubating human gastric cancer cells (MKN-45 cells) with PEG-MTPABZ-PyC for 0.5 h, obvious red fluorescence was observed, indicating that PEG-MTPABZ-PyC can be rapidly absorbed by cells. Extending the cell culture time resulted in more cells displaying bright red fluorescence, indicating that more PEG-MTPABZ-PyC had entered the cells, which is beneficial for enhancing the PDT effect.

[0081] Phototherapy efficacy of PEG-MTPABZ-PyC on MKN-45 cells is as follows: Figure 17As shown, cell viability results indicate that the PEG-MTPABZ-PyC aqueous solution is almost non-toxic, as most cells remained viable even at an incubation concentration of 250 μg / mL. However, when cells absorbed different concentrations of PEG-MTPABZ-PyC and were irradiated with white light at a concentration of 250 μg / mL, cancer cells were effectively killed, reflecting better PDT efficiency.

[0082] Experimental Example 5: PDT Efficiency under Hypoxic Environment

[0083] Since reactive oxygen species (ROS) can effectively overcome the effects of hypoxia, this experimental case further evaluated cell viability under hypoxic conditions. The results are as follows: Figure 18 As shown, when the incubation concentration of PEG-MTPABZ-PyC is high (62.5 μg / mL, 125 μg / mL and 250 μg / mL), cell viability decreases after light irradiation, which strongly demonstrates that PDT is effective even under hypoxic conditions.

[0084] The live / dead cell co-staining assay directly demonstrated the effectiveness of PDT; green fluorescence indicated cell viability, while red fluorescence indicated cell death. The results are as follows: Figure 19 As shown, when cells were exposed to light or not, the green fluorescence emitted by most cells indicated that the treated cells were viable. However, in the experimental group where cells were incubated with PEG-MTPABZ-PyC and exposed to light, prominent red fluorescence indicated that the cells were dead. Comparing the two groups, the experimental group showed more red fluorescence, indicating that PEG-MTPABZ-PyC has significant PDT efficiency against cancer cells.

[0085] The above experimental results show that PEG-MTPABZ-PyC exhibits good biocompatibility, can rapidly enter cells, and effectively kills cancer cells after light irradiation; even under hypoxic conditions, PEG-MTPABZ-PyC also shows good PDT efficiency, demonstrating that PEG-MTPABZ-PyC has important application potential in clinical tumor treatment.

Claims

1. A method for preparing a water-soluble type I photosensitizer, characterized in that, Includes the following steps: S1. 4,7-Dibromo-2,1,3-benzothiadiazole, β-[4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid, and Pd catalyst are mixed in tetrahydrofuran, followed by the addition of an alkaline solution. The mixture is reacted at 80-100℃ for 10-14 h to obtain the MTPABZCHO intermediate. The ratio of 4,7-dibromo-2,1,3-benzothiadiazole, β-[4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid, Pd catalyst, and alkaline solution is 30-50 mmol: 30-50 mmol: 1-3 mmol: 100-150 mL. S2. Dissolve 4-methylpyridine and 11-bromoundecanoic acid in toluene at a molar ratio of 4-6:5-7, stir and react at room temperature for 10-14 h, filter to obtain PyC intermediate; S3. Dissolve MTPABZCHO intermediate and PyC intermediate in a solution of tetrahydrofuran and methanol in a 1:1 volume ratio, then add alkali solution and react at room temperature for 10-14 h to obtain MTPABZ-PyC intermediate; the ratio of MTPABZCHO intermediate, PyC intermediate and alkali solution is 1-3 mmol: 1-3 mmol: 100-150 μL. S4. Dissolve the MTPABZ-PyC intermediate, PEG, and carbodiimide in dichloromethane, then inject 4-dimethylaminopyridine dissolved in dichloromethane into the above reaction system, and react at 50-70℃ for 22-26 h to obtain a water-soluble type I photosensitizer; the molar ratio of the MTPABZ-PyC intermediate, PEG, carbodiimide, and 4-dimethylaminopyridine is 0.15-0.25:0.05-0.15:0.3-0.4:0.

03.

2. The method for preparing the water-soluble type I photosensitizer according to claim 1, characterized in that, The ratio of 4,7-dibromo-2,1,3-benzothiadiazole, β-[4-[bis(4-methoxyphenyl)amino]phenyl]boric acid, Pd catalyst, and alkaline solution is 20 mmol: 20 mmol: 1 mmol: 60 mL.

3. The method for preparing the water-soluble type I photosensitizer according to claim 1, characterized in that, The reaction temperature in S1 is 90℃, and the reaction time is 12h.

4. The method for preparing the water-soluble type I photosensitizer according to claim 1, characterized in that, The Pd catalyst is Pd(PPh3)4.

5. The method for preparing the water-soluble type I photosensitizer according to claim 1 or 2, characterized in that, The alkaline solution is a K2CO3 solution or a piperidine solution.

6. The method for preparing the water-soluble type I photosensitizer according to claim 5, characterized in that, The concentration of the alkaline solution is 1.5-2.5 mol / L.

7. A water-soluble type I photosensitizer, characterized in that, It is prepared by any one of the preparation methods of claims 1-6.

8. The use of the water-soluble type I photosensitizer according to claim 7 in the preparation of a drug for treating tumors.

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