Intermediate aldehydes, photosensitizers and nanoplatforms and methods of making and uses thereof

Abstract

The application provides an intermediate aldehyde, a photosensitizer and a nano platform and a preparation method and application thereof, the intermediate aldehyde can be used for preparing the photosensitizer, the preparation method is simple, the obtained photosensitizer can more efficiently generate type I ROS (O2- and OH), under the crosslinking of Fe (III) and the natural polyphenol - rosemary acid (RA, containing active carboxylic acid and catechol structure) effectively form a hybrid nano platform, the nano platform shows a small particle size, is beneficial to the infiltration into a tumor cell membrane, and enhances the uptake efficiency, and has excellent stability in an aqueous solution and a physiological environment, and has a good anticancer effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to an intermediate aldehyde, a photosensitizer and a nano platform as well as a preparation method and application thereof. BACKGROUND

[0002] As an important tumor treatment method, photodynamic therapy (PDT) has taken a place in non-invasive tumor treatment technology. Photosensitizer (PS) is the core of PDT. When a certain wavelength of laser irradiates PS, reactive oxygen species (ROS) are generated in the presence of tissue oxygen. Currently known ROS mainly include singlet oxygen ( 1 O2), superoxide anion radical (·O2 - ) and hydroxyl radical (·OH). These ROS, especially ·O2 - and ·OH, as cytotoxic oxidants, react with biological macromolecules in tumor cells, destroy the normal structure and function of cells, and cause cell death. However, the currently widely studied PDT is mainly based on traditional organic photosensitizers such as phthalocyanine and porphyrin, and the generation of 1 O2 is dominant. Although it has been proved to have clinical efficacy, there are many inherent defects, including complex synthesis and purification, poor water solubility, light stability and penetration, aggregation-induced quenching and type I photochemical reaction (strong dependence on oxygen), and limited by insufficient oxygen supply (hypoxia) in solid tumors, resulting in low ROS generation efficiency.

[0003] In addition, endogenous oxidative resistance can also protect cancer cells from ROS-induced damage, which is like two shackles that seriously hinder the actual effect of PDT treatment. In order to overcome these problems, researchers have begun to develop strategies, such as using metal-based or metal-organic nanomaterials, such as manganese dioxide (MnO2) and MOFs to consume intracellular GSH or using oxygen supplement techniques, such as hyperbaric oxygen therapy and in situ oxygen generation to counteract hypoxia. Despite this, there is still an urgent need to create a more economical, easy-to-manufacture, low-toxicity and inherently degradable multifunctional nano platform to further optimize PDT treatment. Therefore, in PDT, improving the ROS generation efficiency and reducing the oxidative defense mechanism are the keys to improving the efficacy of PDT. SUMMARY

[0004] The technical problem to be solved by the present application is to provide an intermediate aldehyde.

[0005] Another technical problem to be solved by the present application is to provide a preparation method of the intermediate aldehyde.

[0006] Another technical problem to be solved by the present application is to provide an application of the intermediate aldehyde.

[0007] Another technical problem to be solved by the present application is to provide a photosensitizer obtained by using the intermediate aldehyde.

[0008] Another technical problem to be solved by the present application is to provide a preparation method of the photosensitizer.

[0009] Another technical problem to be solved by the present application is to provide an application of the photosensitizer.

[0010] Another technical problem to be solved by the present application is to provide a nano-platform containing the photosensitizer.

[0011] Another technical problem to be solved by the present application is to provide a preparation method of the nano-platform.

[0012] Another technical problem to be solved by the present application is to provide an application of the nano-platform.

[0013] To solve the above technical problems, the technical solution of the present application is:

[0014] An intermediate aldehyde (MCBT) has the following chemical structure:

[0015]

[0016] The preparation method of the intermediate aldehyde is to obtain the intermediate aldehyde MCBT through Buchwald-Hartwig amination reaction.

[0017] Preferably, the preparation method of the intermediate aldehyde has the following specific steps:

[0018] (1) Dissolve the mixture of MC, 7-bromo-2,1,3-benzothiadiazole-4-formaldehyde, cesium carbonate, Pd2(dba)3 and Ruphos in toluene, and reflux under N2 protection at 110℃;

[0019] (2) After the reaction is completed, cool the reaction to room temperature, add water and dichloromethane, and then stir vigorously, extract with a separatory funnel, collect the lower organic phase, and dry with anhydrous sodium sulfate;

[0020] (3) The dried crude product is purified by gradient elution on a silica gel column with petroleum ether / ethyl acetate as the eluent, and the pure product is obtained at a volume ratio of 8:1, and after drying, a red solid is obtained.

[0021] Preferably, the preparation method of the intermediate aldehyde, the MC is prepared by Suzuki-Miyaura coupling reaction.

[0022] Preferably, the preparation method of the intermediate aldehyde, the MC is prepared by the following method:

[0023] (1) A mixture of 3,6-dibromocarbazole, 4-methoxyphenylboronic acid, XPhos Pd G2 and potassium carbonate in a dioxane-water solution was stirred at 90°C for 2 hours under nitrogen protection;

[0024] (2) After the reaction was completed, it was cooled to room temperature, and water and ethyl acetate were added. Vigorous stirring was carried out during the extraction process;

[0025] (3) The organic layer was separated and dried with anhydrous sodium sulfate;

[0026] (4) The crude product was evaporated to dryness, purified by silica gel column chromatography with petroleum ether / ethyl acetate as the eluent, and finally obtained as a gray-white solid after rotary evaporation.

[0027] The application of the above-mentioned intermediate aldehyde in the preparation of a photosensitizer.

[0028] A photosensitizer (MCBTA) has the following chemical structural formula:

[0029]

[0030] The above-mentioned photosensitizer (MCBTA) is a purple-red solid, 1 H NMR (400 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.36 (s, 2H), 8.03 (d, J = 7.7 Hz, 2H), 7.95 (d, J = 7.7 Hz, 4H), 7.64 (dd, J = 18.7, 8.6 Hz, 2H), 7.35 (d, J = 2.5 Hz, 2H), 7.03 (d, J = 8.5 Hz, 4H), 5.00 (s, 2H), 3.88 (s, 6H). MALDI-TOF-MS: m / z calcd for C 38 H 26 N4O5S3, M 714.83, found M 714.050.

[0031] The preparation method of the above-mentioned photosensitizer (MCBTA) is synthesized by the knoevenagel condensation reaction of the above-mentioned intermediate aldehyde (MCBT) and the precursor MC to obtain an AI Egen-MCBTA with a D-A structure.

[0032] A nano-platform containing the above-mentioned photosensitizer is MCBTA NPs, MCBTA-Fe NPs and / or MCBTA@RA-Fe NPs, wherein,

[0033] The MCBTA NPS is prepared by the following method: dissolving MCBTA and DSPE-PEG2000 in a DMSO solvent, ultrasonic mixing, and then quickly adding to deionized water, ultrasonic, filtration, and dialysis.

[0034] The MCBTA-Fe NPS is prepared by dissolving MCBTA and DSPE-PEG2000 in DMSO solvent, mixing under ultrasonic, then quickly adding into ferric chloride hexahydrate, ultrasonic, filtration, dialysis, and then obtaining;

[0035] The MCBTA@RA-Fe NPS is prepared by dissolving MCBTA, RA (rosmarinic acid) and DSPE-PEG2000 in DMSO solvent, mixing under ultrasonic, then quickly adding into ferric chloride hexahydrate, ultrasonic, filtration, dialysis, and then obtaining.

[0036] The preparation method of the above-mentioned nano platform is as follows:

[0037] (1) MCBTA NPS:

[0038] Dissolve 4 mg of MCBTA and 5 mg of DSPE-PEG2000 in DMSO (1 mL) solvent, ultrasonic treatment to form a mixed solution; then quickly add into deionized water (9 m L), the ultrasonic power is 45%; then, the prepared MCBTA NPs are ultrasonic treated for 3 minutes, filtered through a 220 nm filter membrane, and then transferred to a dialysis membrane with a molecular weight cut-off of 8000 to 14000; dialyze with deionized water for 24 hours to eliminate residual DMSO; the MCBTA NPs after ultrafiltration concentration are stored at 4-8℃;

[0039] (2) MCBTA-Fe NPS:

[0040] Dissolve 2 mg of MCBTA and 5 mg of DSPE-PEG2000 in DMSO (1 mL), ultrasonic treatment to form a mixed solution; then add 50 μL of ferric chloride hexahydrate (DMSO 30 mg·m L -1 ), ultrasonic for 2 minutes; then add the mixture into 9 m L of distilled water, ultrasonic at 45% power for 3 min; then filter the mixture using a 220 nm filter membrane, and transfer to a dialysis membrane with a molecular weight cut-off of 8000 to 14000; then immerse it in distilled water for 24 hours to eliminate DMSO, and finally the obtained MCBTA-Fe NPs are concentrated by ultrafiltration and stored at 4-8℃;

[0041] (3) MCBTA@RA-Fe NPS:

[0042] Dissolve 2 mg of MCBTA, 4 mg of RA and 5 mg of DSPE-PEG2000 in DMSO (1 mL) solvent, ultrasonic treatment to form a mixed solution; then add 50 μL of ferric chloride hexahydrate (DMSO 30 mg·m L-1 ), ultrasonic 2 min; the mixture was quickly dropped into deionized water (9 m L) and treated with ultrasonic for 3 min at 45% output power; then the mixture was filtered using 220 nm filter membrane and transferred into a dialysis membrane with a molecular weight cut-off of 8000-14000; then it was immersed in distilled water for 24 h to eliminate DMSO, and the final MCBTA@RA-Fe NPs were concentrated by ultrafiltration and stored at 4-8℃.

[0043] The above MCBTA, MCBTA-Fe and MCBTA@RA-Fe nano-platforms mainly showed the UV characteristic peaks of MCBTA, and characteristic UV absorption peaks appeared at 410 nm and 530 nm. The MCBTA and MCBTA-Fe nano-platforms presented maximum absorbance at 400 nm, and the MCBTA@RA-Fe slightly blue-shifted and presented maximum absorbance at 395 nm. At the same time, due to the UV absorption of RA-Fe in the wavelength range of 260-360 nm, MCBTA@RA-Fe had stronger and wider UV absorption than MCBTA-Fe in this range. The infrared experiment further proved that there was a characteristic peak of RA at 3200 cm -1 The X-ray photoelectron spectroscopy (XPS) was used to determine the existence of Fe 3+ ions in MCBTA-Fe NPs and MCBTA@RA-Fe NPs, and the characteristic signal at 710 eV corresponded to Fe 2p 3 / 2 ).

[0044] The above MCBTA NPs, MCBTA-Fe NPs and MCBTA@RA-Fe NPs showed relatively uniform shape and small particle size, with average particle sizes of 37.39, 61.38 and 70.07 nm, respectively, which was beneficial to their penetration into tumor cell membranes and enhancement of their uptake efficiency. The DLS stability study also showed that the above nano-platforms had excellent stability.

[0045] The above nano-platforms (MCBTA NPs, MCBTA-Fe NPs and / or MCBTA@RA-Fe NPs) are used in the preparation of anticancer drugs or anticancer diagnostic agents.

[0046] Beneficial effects:

[0047] The intermediate aldehyde can be used for preparing a photosensitizer, the preparation method is simple, the obtained photosensitizer can more efficiently generate type I ROS (·O2- and ·OH), effectively form a hybrid nano platform under the cross-linking of Fe(III) and natural polyphenol-rosemary acid (RA containing active carboxylic acid and catechol structure), the nano platform exhibits a small particle size, is beneficial to its penetration into a tumor cell membrane, and enhances its uptake efficiency, and has excellent stability in an aqueous solution and a physiological environment, and has a good anticancer effect.

[0048] The present application proves that MCBTA can more efficiently generate type I ROS (·O2- and ·OH) than the traditional commonly used photosensitizer chlorin e6 (Ce6) through active oxygen (ROS) identification experiments in an aqueous solution, that is, reduces the dependence on oxygen to play a type I photochemical process. In addition, based on the fact that MCBTA can effectively form a hybrid nano platform under the cross-linking of Fe(III) and natural polyphenol-rosemary acid (RA containing active carboxylic acid and catechol structure). Through characterization of the particle size, morphology, optical properties and the like of the nano particles, the experimental results prove that the corresponding MCBTA, MCBTA-Fe and MCBTA@RA-Fe nano platforms can be effectively constructed under the encapsulation of the amphiphilic polymer DSPE-PEG2000 which has high biological safety. The three nano platforms exhibit a small particle size, are beneficial to their penetration into a tumor cell membrane, and enhance their uptake efficiency. The particle size monitoring for ten consecutive days also shows that the above nano particles have excellent stability in an aqueous solution and a physiological environment.

[0049] The present application takes breast cancer 4T1 cells as a model, and studies the treatment effect of MCBTA, MCBTA-Fe and MCBTA@RA-Fe nano platforms in tumor cells through intracellular ROS generation experiments, toxicity detection, apoptosis experiments and mouse xenograft models. The results prove that MCBTA-mediated PDT can exert a good anticancer effect, and under the assistance of RA / Fe(III), the treatment effect of a single PDT nano platform can be further enhanced. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is a total synthesis route of AIEgen-MCBTA;

[0051] Figure 2 is 1 HNMR spectra of MC;

[0052] Figure 3 is a MALDI-TOF-MS spectrum of MC;

[0053] Figure 4 is 1HNMR spectra of MCBT;

[0054] Figure 5 MALD I-TOF-MS spectrum of MCBT;

[0055] Figure 6 are 1 HNMR spectra of MCBTA;

[0056] Figure 7 MALD I-TOF-MS spectrum of MCBTA;

[0057] Figure 8 are the qualitative and quantitative evaluation of the optical properties of MCBTA, MCBTA-Fe and MCBTA@RA-Fe three nano-platforms. Among them, (A) UV-VIS absorption spectra of MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs; (B) UV-VIS ultraviolet spectrum absorption standard curve of MCBTA (λmax 530 nm); (C) FT-IR spectra of RA, MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs; (D) PL spectra of MCBTA in DMSO-water mixed solution with different water fractions (λex: 410 nm); (E) Fe 2p XPS spectra of MCBTA-Fe, and (F) MCBTA@RA-Fe.

[0058] Figure 9 are the particle size distribution / zeta potential distribution / morphology characterization of MCBTA NPs, wherein (A) particle size distribution; (B) zeta potential distribution; (C) morphology characterization of MCBTA NPs (scale: 100 and 50 nm).

[0059] Figure 10 are the particle size distribution / zeta potential distribution / morphology characterization of MCBTA-Fe NPs, wherein (A) particle size distribution; (B) zeta potential distribution; (C) morphology characterization of MCBTA-Fe NPs (scale: 100 and 50 nm).

[0060] Figure 11 are the particle size distribution / zeta potential distribution / morphology characterization of MCBTA@RA-Fe NPs, wherein (A) particle size distribution; (B) zeta potential distribution; (C) morphology characterization of MCBTA@RA-Fe NPs (scale: 100 and 50 nm).

[0061] Figure 12Stability evaluation of MCBTA, MCBTA-Fe and MCBTA@RA-Fe three nano platforms. (A) in aqueous solution, (B) in cell culture medium containing 10% FBS at different time points.

[0062] Figure 13 Determination of ROS production type of MCBTA, MCBTA-Fe and MCBTA@RA-Fe three nano platforms in aqueous solution;

[0063] Figure 14 In vitro efficacy evaluation of MCBTA, MCBTA-Fe and MCBTA@RA-Fe three nano platforms, including (A) cytotoxicity evaluation, (B) apoptosis detection, (C) intracellular ROS generation after different treatments;

[0064] Figure 15 Anti-tumor effect of MCBTA, MCBTA-Fe and MCBTA@RA-Fe three nano platforms in 4T1 tumor-bearing nude mice model. Among them, (A) comparison of relative tumor volume changes among different treatment groups. (B) Changes in relative body weight after different treatment regimens. (C) Evaluation of tumor size in different treatment groups. DETAILED DESCRIPTION

[0065] In order to make those skilled in the art better understand the technical solutions of the present application, the technical solutions described in the present application will be further described in detail in combination with specific embodiments.

[0066] 4T1 (mouse breast cancer cells) involved in the examples were purchased from Punsai, China in December 2023, contact number 400-999-2100.

[0067] Example 1

[0068] 1. Test instruments and reagents

[0069] Experimental instruments: 400MHz liquid nuclear magnetic resonance spectrometer (Ascend 400, Germany Bruker), matrix assisted laser desorption ionization-time of flight mass spectrometer (Maldi-TOF, Germany Bruker).

[0070] Experimental reagents:

[0071] 3,6-dibromo-carbazole, 7-bromo-2,1,3-benzothiazole-4-carboxaldehyde, cesium carbonate, Pd2(dba)3, Ruphos, rhodanine-3-acetic acid and XPhos Pd G2 were purchased from Shanghai Biotechmed Co., Ltd. 4-methoxy-phenylboronic acid and sodium acetate were purchased from Tianjin Hensheng Biochemical Technology Co., Ltd. Dichloromethane, 1,4-dioxane, anhydrous sodium sulfate, petroleum ether, ethyl acetate, methanol, toluene were purchased from Tianjin Jiangtian Chemical Technology Co., Ltd. Deuterated chloroform and DMSO were purchased from Aldrich. Potassium carbonate was purchased from Tianjin Fengchen Chemical Reagent Technology Co., Ltd.

[0072] 2. Test method

[0073] Synthesis of precursor compound MC

[0074] A mixture of 3,6-dibromo-carbazole (325.00 g / mol, 325 mg, 1 mmol, 1.0 eq.), 4-methoxy-phenylboronic acid (151.96 g / mol, 456 mg, 3 mmol, 3.0 eq.), XPhos Pd G2 (784.79 g / mol, 78 mg, 0.1 mmol, 0.1 eq.) and potassium carbonate (138.21 g / mol, 415 mg, 3.00 mmol, 3.0 eq.) in 5:1 dioxane-water solution was reacted under nitrogen protection at 90 °C for 2 hours. After the reaction was completed, it was cooled to room temperature, and water and ethyl acetate were added. Vigorous stirring was carried out during the extraction process. The organic layer was separated and dried over anhydrous sodium sulfate. The crude product was evaporated to dryness, purified by silica gel column with petroleum ether / ethyl acetate (1:1, v / v) as eluent, and finally dried by rotary evaporation to obtain off-white solid.

[0075] Synthesis of intermediate aldehyde MCBT

[0076] A mixture of MC (379.11 g / mol, 380 mg, 1 mmol, 1.0 eq.), 7-bromo-2,1,3-benzothiazole-4-carboxaldehyde (243.08 g / mol, 730 mg, 3 mmol, 3.0 eq.), cesium carbonate (325.82 g / mol, 815 mg, 2.5 mmol, 2.5 eq.), Pd2(dba)3 (1.02 g / mol, 2.5 mg, 0.0025 mmol, 0.0025 eq.), Ruphos (1.02 g / mol, 2.5 mg, 0.0025 mmol, 0.0025 eq.) and rhodanine-3-acetic acid (243.08 g / mol, 730 mg, 3 mmol, 3.0 eq.) in 5:1 dioxane-water solution was reacted under nitrogen protection at 90 °C for 2 hours. After the reaction was completed, it was cooled to room temperature, and water and ethyl acetate were added. Vigorous stirring was carried out during the extraction process. The organic layer was separated and dried over anhydrous sodium sulfate. The crude product was evaporated to dryness, purified by silica gel column with petroleum ether / ethyl acetate (1:1, v / v) as eluent, and finally dried by rotary evaporation to obtain off-white solid. 2(A mixture of dba)3 (511.05 g / mol, 26 mg, 0.05 mmol, 0.05 eq.) and Ruphos (466.64 g / mol, 47 mg, 0.1 mmol, 0.1 eq.) was dissolved in toluene and heated under reflux at 110 °C for 8 hours under nitrogen protection. After the reaction was complete, the reaction mixture was cooled to room temperature, and water and dichloromethane were added. After vigorous stirring, the mixture was extracted using a separatory funnel. The lower organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by gradient elution using a silica gel column with petroleum ether / ethyl acetate as the eluent at a volume ratio of 8:1 to obtain the pure product, which was then evaporated to dryness to give a red solid.

[0077] Synthesis of target product MCBTA

[0078] A mixture of intermediate aldehyde MCBT (541.106 g / mol, 270 mg, 0.5 mmol, 1.0 eq.), rhodanine-3-acetic acid (191.23 g / mol, 115 mg, 0.6 mmol, 1.2 eq.), and sodium acetate (82.03 g / mol, 82 mg, 1 mmol, 2.0 eq.) was dissolved in acetic acid and heated under reflux at 100 °C for 3 hours under nitrogen protection. The reaction mixture was then cooled to room temperature, and water and dichloromethane were added with vigorous stirring. Extraction was performed using a separatory funnel, and the lower organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by gradient elution using a silica gel column with dichloromethane / methanol as the eluent at a volume ratio of 20:1 to obtain a pure product, which was then evaporated to dryness to give a purple-red solid.

[0079] 3. Test Results

[0080] like Figure 1 The synthesis was carried out according to the steps shown, and the compound synthesized in each step was obtained through... 1 The characterization was verified by H NMR and MALDI-TOF-MS.

[0081] The precursor compound MC was synthesized in 81% yield. 1 H NMR (400MHz, Ch l oroform-d) δ8.28 (s, 2H), 7.64 (dd, J = 7.8, 4.4Hz, 6H), 7.47 (d, J = 8.4Hz, 2H), 7.03 (d, J = 8.2Hz, 4H), 3.88 (s, 6H). (See Figure 2 )MALDI-TOF-MS:m / z ca l cd for C 26 H 21 NO2, M 379.46, found M 379.107. (See) Figure 3 )

[0082] The intermediate aldehyde MCBT was synthesized in 89% yield, 1 H NMR (400 MHz, Chloroform-d) δ 10.88 (s, 1H), 8.48 (d, J = 7.6 Hz, 1H), 8.36 (d, J = 1.8 Hz, 2H), 8.07 (d, J = 7.5 Hz, 1H), 7.70 - 7.62 (m, 4H), 7.61 (dd, J = 8.6, 1.8 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 7.08 - 6.95 (m, 4H), 3.89 (s, 6H).(See Figure 4 ) MALDI-TOF-MS: m / z calcd for C 33 H 23 N3O3S, M 541.63, found M 541.106.(See Figure 5 )

[0083] The target product MCBTA was synthesized in 63% yield, 1 H NMR (400 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.36 (s, 2H), 8.03 (d, J = 7.7 Hz, 2H), 7.95 (d, J = 7.7 Hz, 4H), 7.64 (dd, J = 18.7, 8.6 Hz, 2H), 7.35 (d, J = 2.5 Hz, 2H), 7.03 (d, J = 8.5 Hz, 4H), 5.00 (s, 2H), 3.88 (s, 6H).(See Figure 6 ) MALDI-TOF-MS: m / z calcd for C 38 H 26 N4O5S3, M 714.83, found M 714.050.(See Figure 7 )

[0084] Example 2

[0085] 1. Experimental instruments and reagents

[0086] Experimental instruments: Nanoparticle size analyzer (Zetasizer Nano ZS, Malvern, UK), Field emission transmission electron microscope (Tecnai G2 F20, Philips, Netherlands), Ultraviolet visible spectrophotometer (CARY 60, Agilent, China), Infrared spectrometer (TENSOR 27, Bruker, Germany).

[0087] Experimental reagents: Ferric chloride hexahydrate and rosmarinic acid were purchased from Tianjin Hisun biochemical technology Co., Ltd., High-sugar medium DMEM (cell culture medium) was purchased from Gibco company.

[0088] 2. Test method

[0089] (1) Preparation of MCBTA NPs

[0090] 4 mg of MCBTA and 5 mg of DSPE-PEG2000 were dissolved in DMSO (1 mL) solvent and ultrasonicated to form a mixed solution. Then it was quickly added to deionized water (9 mL) with an ultrasonic power of 45%. Subsequently, the prepared MCBTA NPs were ultrasonicated for 3 min, filtered through a 220 nm filter membrane, and then transferred to a dialysis membrane with a molecular weight cut-off of 8000 to 14000. Dialysis was performed with deionized water for 24 h to eliminate residual DMSO. The MCBTA NPs after ultrafiltration concentration were stored at 4-8°C.

[0091] (2) Preparation of MCBTA-Fe NPs

[0092] 2 mg of MCBTA and 5 mg of DSPE-PEG2000 were dissolved in DMSO (1 mL) and ultrasonicated to form a mixed solution. Then 50 μL of iron trichloride hexahydrate (DMSO 30 mg·mL-1) was added and ultrasonicated for 2 min. The mixture was then added to 9 mL of distilled water and ultrasonicated for 3 min at a power of 45%. The mixture was then filtered using a 220 nm filter membrane and transferred to a dialysis membrane with a molecular weight cut-off of 8000 to 14000. Subsequently, it was immersed in distilled water for 24 h to eliminate DMSO, and the final MCBTA-Fe NPs were concentrated by ultrafiltration and stored at 4-8°C.

[0093] (3) Preparation of MCBTA@RA-Fe NPs

[0094] 2 mg of MCBTA, 4 mg of RA, and 5 mg of DSPE-PEG2000 were dissolved in DMSO (1 mL) solvent and ultrasonicated to form a mixed solution. Then 50 μL of iron trichloride hexahydrate (DMSO 30 mg·mL-1) was added and ultrasonicated for 2 min. The mixture was quickly dropped into deionized water (9 mL) and ultrasonicated for 3 min at a power of 45%. The mixture was then filtered using a 220 nm filter membrane and transferred to a dialysis membrane with a molecular weight cut-off of 8000 to 14000. Subsequently, it was immersed in distilled water for 24 h to eliminate DMSO, and the final MCBTA@RA-Fe NPs were concentrated by ultrafiltration and stored at 4-8°C.

[0095] The particle size, zeta potential of MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs were measured by nanoparticle size analyzer and the stability of the above three nano-platforms in aqueous solution and 10% FBS-containing cell culture medium (simulating cell physiological relief) at different time points was monitored. The morphological characteristics of the nano-platforms were observed by field emission transmission electron microscopy. Further quantitative and qualitative analysis of MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs were carried out by UV-visible spectrophotometer, infrared spectrometer, microplate reader and XPS.

[0096] 3. Test results

[0097] The UV absorption spectra of MCBTA, MCBTA-Fe and MCBTA@RA-Fe are shown in Figure 8 A. MCBTA-Fe and MCBTA@RA-Fe NPs mainly show the UV characteristic peaks of MCBTA. It is worth noting that MCBTA shows maximum absorbance at 410 nm and 530 nm wavelengths. Considering that RA has weak absorption near 410 nm, the peak of MCBTA at 530 nm is selected as the reference for constructing the standard curve for quantitative evaluation of the three nano-platforms Figure 8 B). MCBTA and MCBTA-Fe NPs both have maximum absorbance at 410 nm. From the UV data graph, it can be observed that MCBTA and MCBTA-Fe have maximum absorbance at 400 nm, while the maximum of MCBTA@RA-Fe is blue-shifted to 395 nm. At the same time, due to the UV absorption of RA-Fe in the wavelength range of 260-360 nm, MCBTA@RA-Fe has stronger and wider UV absorption than MCBTA-Fe in this range.

[0098] Considering that the overlapping peaks in the UV data are not very intuitive to prove the presence of RA in MCBTA@RA-Fe, the characteristic peaks of MCBTA and RA in MCBTA@RA-Fe NPs can be clearly observed by FT-IR spectral analysis Figure 8 C), thereby confirming the effective loading of MCBTA and RA in MCBTA@RA-Fe nanocomposites.

[0099] The effect of different water fractions (fw) Figure 8AI E phenomenon under D). Weak fluorescence signal was observed in anhydrous pure DMSO. With the increase of water content, the emission intensity gradually increased. The fluorescence emission peak appeared at 50% water content (fw 50%). After that, due to the change of morphology and size of the nano-aggregates, the emission intensity decreased with the further increase of fw. This behavior confirmed the AI E properties of MCBTA and its applicability in fluorescence imaging applications.

[0100] In order to analyze the hybrid nano-platform in depth, X-ray photoelectron spectroscopy (XPS) was used to determine the presence of Fe 3+ in MCBTA-Fe NPs and MCBTA@RA-Fe NPs. 3 / 2 As shown in Figure 8 E and Figure 8 F, the characteristic signal at 710 eV corresponds to Fe 2p

[0101] By encapsulating MCBTA, MCBTA-Fe and MCBTA@RA-Fe nano-platforms in a biocompatible amphiphilic polymer DSPE-PEG2000, the nano-platforms can be effectively constructed. The results of nano-particle size analyzer analysis show that the average particle size of MCBTA NPs Figure 9 A) is 37.39 nm, the average particle size of MCBTA-Fe NPs Figure 10 A) is 61.38 nm, and the average particle size of MCBTA@RA-Fe NPs Figure 11 A) is 70.07 nm. The three nano-platforms show tiny particle size, which is conducive to their penetration into tumor cell membranes and enhances their uptake efficiency. Due to the wrapping of DSPE-PEG2000, the zeta potentials of the three nano-platforms are about 0 mV Figure 9 B, Figure 10 B, Figure 11 B). TEM analysis was used to further explore their morphological characteristics, which confirmed that these nano-platforms have relatively uniform shapes. However, due to different aggregation modes, the morphologies of the nanoparticles are different Figure 9 C, Figure 10 C, Figure 11 C). The stability of the three nanoparticles in water Figure 12 A) and cell culture medium containing 10% FBS Figure 12 B) at different time intervals was studied using a nano-particle size analyzer. The research results show that the material has excellent stability, and even after 9 days, the particle size change is very small.

[0102] Example 3

[0103] 1. Test instruments and reagents

[0104] Test instruments:

[0105] Full-automatic multifunctional microplate detection platform (FlexStation 3, China Molecular Devices).

[0106] Test reagents:

[0107] 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA, CAS 307554-62-7, Macklin), hydroxyphenyl fluorescein (HPF, CAS 359010-69-8, Shanghai Mokang Biotechnology Co., Ltd.), dihydro rhodamine 123 (DHR 123, CAS 109244-58-8, Tianjin Xinsisheng Biotechnology Co., Ltd.), chlorin e6 (Ce6, CAS 19660-77-6, Macklin), D-PBS (Biosharp).

[0108] 2. Test method

[0109] 1 The amount of O2 generation was quantitatively analyzed with ABDA probe as an indicator. ABDA (final concentration: 10 μM) was mixed with Ce6, MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs (MCBTA final concentration was 1 μM) respectively, and irradiated under white light with an intensity of 100 mW cm -2 The absorbance decrease value at 380 nm relative to the initial value was recorded to represent the decomposition rate of ABDA, thereby characterizing the generation rate of O2. 1

[0110] The amount of ·OH generation was measured with hydroxyphenyl fluorescein (HPF) as an indicator. HPF (final concentration: 5 μM) was mixed with Ce6, MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs (MCBTA final concentration was 1 μM) respectively, and irradiated under white light with an intensity of 100 mW cm -2 Under 490 nm excitation, the fluorescence intensity increase value at 515 nm relative to the initial value was recorded to represent the generation rate of ·OH.

[0111] The amount of ·O2 - generation was measured with dihydro rhodamine 123 (DHR 123) as an indicator. DHR 123 (final concentration: 5 μM) was mixed with Ce6, MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs (MCBTA final concentration was 1 μM) respectively, and irradiated under white light with an intensity of 100 mW cm -2 ​Under white light illumination, and with excitation at 490 nm, the increase in fluorescence intensity at 525 nm relative to the initial value was recorded to represent the fluorescence intensity at ·O2. - The generation rate.

[0112] 3. Test Results

[0113] To identify the specific ROS generated by MCBTA and to evaluate the ROS generation capabilities of three nanoplatforms compared to the commonly used PS-Ce6, ABDA, HPF, and DHR123 were used as probes, and their ROS generation was measured under white light irradiation (100 mW cm⁻¹). -2 When generated 1 O2, ·OH and ·O2 - The ability.

[0114] like Figure 13 As shown, the relative fluorescence emission intensities of MCBTA, MCBTA-Fe, and MCBTA@RA-Fe NPs mixed with HPF or DHR123 are much stronger than those of Ce6, indicating that type I ROS (·OH and ·O2) are significantly stronger. - The efficient generation of ROS was significantly improved. Conversely, under the same conditions, ABDA exhibited no significant change in relative UV absorption when bound to the three nanoplatforms. This evidence suggests that MCBTA functions effectively to address the challenge of tumor hypoxia through a typical type I PDT mechanism. Although there may be differences in ROS generation rates between different nanoplatforms due to the different aggregation states of MCBTA, they do not hinder the generation of ROS in aqueous solution, which can be further confirmed by subsequent intracellular ROS.

[0115] Example 4

[0116] 1. Experimental instruments and reagents

[0117] Experimental instruments: Fully automated multi-functional microplate detection platform (Flex Statistics 3, Molecular Devices, China), BD analytical sorting flow cytometer (FACSAria III FACSVerse, BD, USA), and ultra-high resolution confocal microscope (SP8, Leica, Germany).

[0118] Experimental reagents: Trypsin-EDTA solution was purchased from Beijing Solarbio Science & Technology Co., Ltd. DPBS and CCK8 were purchased from White Shark (China). Annexin V-FITC apoptosis detection kit and DCFH-DA were purchased from Shanghai Beyotime. High-glucose culture medium DMEM was purchased from Gibco. Fetal bovine serum was purchased from Biologic Industries.

[0119] 2. Test methods

[0120] (1) Intracellular ROS generation detection

[0121] Intracellular ROS generation was monitored using DCFH-DA as a probe. First, 4T1 cells were seeded in culture dishes and allowed to adhere for 12 h. Then, the cells were treated with MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs at each concentration of 16 μΜ relative to MCBTA for 8 h. Then, the cells were washed with DPBS for 3 times and DCFH-DA (10 μΜ) was added for incubation at 37 °C for 30 min. For the light group, the cells were exposed to white light (100 mW cm -2 ) for 3 min, and imaged by CLSM after incubation for 10 min. The excitation wavelength used was 488 nm. For these nano-platforms, the emission filter range was set between 600-850 nm, while for DCFH-DA, the emission filter range was set between 500-570 nm.

[0122] (2) Cytotoxicity evaluation

[0123] In the cytotoxicity evaluation, 4T1 cells in the logarithmic growth phase were seeded in 96-well plates and incubated at 37 °C, 5% CO2 for 12 h. Different concentrations (reference to the concentration of MCBTA) of nano-platforms were divided into 6 groups, with 4 repeats for each concentration. The MCBTA (Dark) group, MCBTA-Fe (Dark) group and MCBTA@RA-Fe (Dark) group were incubated in the dark for 24 h. At the same time, the MCBTA (Laser) group, MCBTA-Fe (Laser) group and MCBTA@RA-Fe (Laser) group were irradiated with white light (100 mW cm -2 ) for 3 min after 8 h, and then the medium was discarded after incubation for 16 h in the incubator. Next, 100 μL of fresh cell culture medium (high-sugar DMEM medium) was added to each well, including 10% CCK8, to ensure that no bubbles were formed. The 96-well plate was further incubated in the cell incubator, and the absorbance was measured at 450 nm using a microplate reader after 2 h to determine the cell viability.

[0124] (3) Apoptosis detection

[0125] The anti-tumor effect of the nano-platform on 4T1 tumor cells was further verified based on the apoptosis experiment. 4T1 cells were seeded in 6-well plates and incubated at 37 °C, 5% CO2 in a cell incubator for 12 h. Then, MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs (MCBTA: 16 μΜ) were co-cultured for 8 h. The control group and the dark group continued to incubate. In contrast, the light group was irradiated with white light (100 mW cm -2Cells were irradiated. Subsequently, they were digested with trypsin and transferred to centrifuge tubes. After centrifugation at 1000 rpm for 5 minutes, the tubes were washed twice with DPBS. Annexin V-FITC / PI staining solution was added, and the cells were incubated in the dark for 15 minutes before being transferred to an ice box. Apoptosis was assessed using flow cytometry within 1 hour.

[0126] 3. Test Results

[0127] First, DCFH-DA was used as a molecular probe tool, with green fluorescence ( Figure 14 A) Labeling assessment of ROS generation in 4T1 cells. The study found that all three nanoplatforms (MCBTA: 16 μM) exhibited significant green fluorescence under white light irradiation. In contrast, the light-shielded group showed weak green fluorescence. This observation is consistent with the principle that ROS is generated only under light irradiation. Red fluorescence was observed from AI E-MCBTA, and differences in fluorescence intensity were observed between different groups, even at the same concentration. These differences may stem from different absorption rates or different aggregation modes. However, meta-analysis revealed that MCBTA@RA-Fe may also generate ROS due to RA, acting synergistically with AI E, exhibiting the strongest ROS generation capacity under laser irradiation, exceeding the individual effects of MCBTA and MCBTA-Fe.

[0128] To verify the anticancer potential of the MCBTA@RA-Fe nanocomposite, 4T1 tumor cells were used as the efficacy evaluation model. Figure 14 As shown in Figure B, cytotoxicity was assessed using the CCK8 assay. After 24 hours of incubation in the dark, all three nanoplatforms showed negligible cytotoxicity, indicating high biocompatibility. Due to MCBTA-mediated PDT, all three nanoplatforms exhibited high mortality after light exposure. The MCBTA@RA-Fe NPs (Laser) group showed the strongest cytotoxicity, attributed to the synergistic effect of RA / Fe(III). However, at selected low concentrations, the difference between the MCBTA and MCBTA-Fe NP groups may not be very significant, merely indicating a toxicity trend that will become more apparent in subsequent in vivo studies. Apoptosis analysis further corroborated the CCK8 results. Figure 14 C). Under laser irradiation, the apoptosis rates of the MCBTA, MCBTA-Fe, and MCBTA@RA-Fe NPs (Laser) groups were 27.92%, 35.90%, and 38.71%, respectively.

[0129] In summary, the results verified by intracellular ROS generation, cytotoxicity evaluation and apoptosis analysis indicated that all three nano-platforms effectively promoted MCBTA-based PDT under laser irradiation. Notably, MCBTA@RA-Fe nanocomposites exhibited the highest cytotoxicity effect and enhanced cancer treatment effect due to the assistance of natural nano-adjuvant RA / Fe(III).

[0130] Example 5 Anti-cancer effects of MCBTA, MCBTA-Fe and MCBTA@RA-Fe NPs in vivo in 4T1 (mouse breast cancer cell) tumor-bearing mice

[0131] 1. Test animals

[0132] SPF grade BALB / C nude mice (male) were purchased from Beijing Sbiopharm Co., Ltd.

[0133] 2. Test method

[0134] 4T1 cells were injected subcutaneously into the right dorsal skin of male BALB / C (nu / nu) nude mice to the middle and lower part of the armpit in complete cell culture medium. When the tumor volume exceeded 50 mm 3 , the mice were randomly divided into 7 groups (4 in each group): (1) Con, (2) MCBTA NPs (Dark), (3) MCBTA-Fe NPs (Dark), (4) MCBTA@RA-Fe NPs (Dark), (5) MCBTA NPs (Laser), (6) MCBTA-Fe NPs (Laser), (7) MCBTA@RA-Fe NPs (Laser) (20 μL of MCBTA at a concentration of 1.0 mg / mL was administered). The light irradiation group was irradiated with white light (300 mW cm -2 ) for 5 minutes continuously 8 hours after intratumoral injection, once every other day, for 10 consecutive days. During the entire treatment process, the tumor size (length and width) and the body weight of the mice were recorded before each administration. The tumor volume was calculated according to the formula: volume = length x width 2 / 2 (mm 3 ). The mice were sacrificed on the 11th day.

[0135] 3. Test results

[0136] In vivo tumor growth inhibition was performed for 10 consecutive days to evaluate the anti-cancer effect Figure 14 A) and body weight change monitoring Figure 14B) To assess drug safety. Tumor size continued to increase in both the control and TNTP NPs (Dark) groups, and body weight showed no significant changes, indicating that only TNTP NPs exhibited negligible dark toxicity. Conversely, treatment with TNTP-Ra NPs under dark conditions significantly inhibited tumor growth because the released Ra exerted a CHT effect to suppress tumor cell growth. For the TNTP NPs group under laser irradiation, tumor growth was also greatly inhibited due to PDT. The best tumor-suppressing effect was observed with TNTP-Ra NPs under laser irradiation, with almost complete inhibition of tumor growth and minimal impact on body weight changes, indicating good biocompatibility of the nanomedicine.

[0137] To evaluate the in vivo anticancer efficacy and differences of the nanoplatform, mice implanted with 4T1 tumors were divided into 7 groups, with 4 mice in each group. Tumor size was monitored every 2 days, and the relative tumor volume (N / A) was calculated. Figure 15 A) To evaluate the anticancer efficacy of different treatment regimens. On day 10 of the experiment, tumor tissue was removed to allow for more direct observation of tumor size. Figure 15 C). Experimental results showed that the MCBTA(Dark) group exhibited the least dark toxicity compared to the Con group. Tumor growth rates in the MCBTA-Fe(Dark) and MCBTA@RA-Fe(Dark) groups were moderately inhibited due to the hybridization of Fe and RA / Fe. A similar trend was observed in the light-exposed groups. Notably, the MCBTA@RA-Fe(Laser) group showed the most significant inhibitory effect on tumor growth, superior to the MCBTA(Laser) and MCBTA-Fe(Laser) groups, thus highlighting the enhancing role of RA / Fe(III) as an adjuvant in nanoparticle formulation in enhancing the anticancer effect of PDT. To assess the safety of the nanoplatform after administration, mouse body weight was measured every other day, such as... Figure 15 As shown in B. The body weight of all groups showed a relatively stable trend, indicating that there were no adverse reactions related to drug administration.

[0138] The experimental results of the above embodiments demonstrate that the photosensitizer in this application can generate type I ROS (·O2) more efficiently than the traditional photosensitizer Chloroline6 (Ce6). -and ·OH), that is, to reduce the dependence on oxygen to play type I photochemical process. At the same time, under the encapsulation of the higher biological safety of amphiphilic polymer DSPE-PEG2000, the corresponding MCBTA, MCBTA-Fe and MCBTA@RA-Fe nano-platforms can be effectively constructed. With breast cancer 4T1 cells as a model, the therapeutic effect of MCBTA, MCBTA-Fe and MCBTA@RA-Fe nano-platforms in tumor cells was confirmed by in vitro and in vivo efficacy evaluation. The results confirmed that MCBTA-mediated PDT can exert good anticancer effect, and with the help of RA / Fe(Ⅲ), the therapeutic effect of single PDT nano-platform can be further enhanced. In addition, the hybrid nano-platform (MCBTA@RA-Fe NPs) based on metal ion cross-linking driven as a safe and stable new nano-preparation can also provide new insights and references for the development of multifunctional PDT nano-platform.

[0139] In summary, the type I AI E photosensitizer (MCBTA) is designed and synthesized in the application, which mediates the low oxygen-dependent type I PDT process. At the same time, RA / Fe(Ⅲ) as a building block of nano-preparation, aiming at the problem of tumor oxidative resistance, can realize the assistance to the single photosensitizer nano-platform and enhance the actual effect of PDT in the application of anti-cancer. In addition, the AI E molecule can effectively form a multifunctional hybrid nano-platform under the cross-linking of Fe(Ⅲ) and natural polyphenol-rosemary acid (RA, containing active carboxylic acid, catechol structure). The hybrid nano-platform (MCBTA@RA-Fe NPs) based on metal ion cross-linking driven as a safe and stable new nano-preparation can also provide new insights and references for the development of multifunctional PDT nano-platform.

[0140] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and the technical improvements and refinements made by those skilled in the art based on the technical solutions of the present application are considered to be within the protection scope of the present application.

Claims

1. An intermediate aldehyde, characterized by: has the following chemical structure formula: 。 2. Process for the preparation of the intermediate aldehyde according to claim 1, characterized in that: The intermediate aldehyde is obtained by Buchwald-Hartwig amination reaction with MC and 7-bromo-2, 1, 3-benzothiadiazole-4-formaldehyde as reactants, the MC has the following chemical structure formula: 。 3. The method of claim 2, wherein: The specific steps are as follows: (1) A mixture of MC, 7-bromo-2, 1, 3-benzothiadiazole-4-formaldehyde, cesium carbonate, Pd2(dba)3 and Ruphos is dissolved in toluene, and refluxed at 110°C under N2 protection; (2) After the reaction is completed, the reaction is cooled to room temperature, water and dichloromethane are added, and after stirring, extraction is carried out with a separatory funnel, the lower organic phase is collected, and dried with anhydrous sodium sulfate; (3) The dried crude product is purified by gradient elution on a silica gel column with petroleum ether / ethyl acetate as the eluent, and the pure product is obtained at a volume ratio of 8:1, and after drying, a red solid is obtained.

4. The method of claim 3, wherein: The MC is prepared by Suzuki-Miyaura coupling reaction with 3, 6-dibromocarbazole and 4-methoxyphenylboronic acid as reactants.

5. The method of claim 4, wherein: The MC is prepared by the following method: (1) A mixture of 3, 6-dibromocarbazole, 4-methoxyphenylboronic acid, XPhos Pd G2 and potassium carbonate is dissolved in a dioxane-water solution, and reacted at 90°C under nitrogen protection for 2 hours; (2) After the reaction is completed, the reaction is cooled to room temperature, water and ethyl acetate are added, and the mixture is stirred vigorously during the extraction process; (3) The organic layer is separated and dried with anhydrous sodium sulfate; (4) The crude product is evaporated to dryness, purified by silica gel column with petroleum ether / ethyl acetate as the eluent, and finally dried to obtain a gray-white solid.

6. Use of the intermediate aldehyde of claim 1 in the preparation of a photosensitizer having the following chemical structure formula: 。 7. A photosensitizer characterized by: has the following chemical structure formula: 。 8. A method of preparing the photosensitizer of claim 7, characterized by: The intermediate aldehyde of claim 1 is subjected to knoevenagel condensation reaction with rhodamine-3-acetic acid.

9. A nano-platform comprising the photosensitizer of claim 7, wherein: MCBTA NPs, MCBTA-FeNPs and / or MCBTA@RA-Fe NPs, wherein the RA is rosmarinic acid, and the photosensitizer has the following chemical structure formula: The MCBTA NPs are prepared by the following method: the photosensitizer and DSPE-PEG2000 are dissolved in DMSO solvent, ultrasonically mixed, and then quickly added to deionized water, ultrasonically mixed, filtered, and dialyzed to obtain the product; The MCBTA-Fe NPs are prepared by the following method: the photosensitizer and DSPE-PEG2000 are dissolved in DMSO solvent, ultrasonically mixed, and then quickly added to iron trichloride hexahydrate, ultrasonically mixed, filtered, and dialyzed to obtain the product; The MCBTA@RA-Fe NPs are prepared by the following method: the photosensitizer, RA and DSPE-PEG2000 are dissolved in DMSO solvent, ultrasonically mixed, and then quickly added to iron trichloride hexahydrate, ultrasonically mixed, filtered, and dialyzed to obtain the product.

10. Use of the nano-platform of claim 9 in the preparation of an anti-breast cancer drug.

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