A molecular probe design for tumor microenvironment response and its photodynamic enhanced treatment and a preparation method thereof

Abstract

The application belongs to the field of organic molecule fluorescent probe, and a small molecule fluorescent probe HX is rationally designed, and the integration of diagnosis and treatment is realized by monitoring the related physical properties of tumor microenvironment and the photodynamic therapy of tumor. The tumor microenvironment of solid tumor usually has the characteristics of low pH, high viscosity, low oxygen and the like, and it is of great significance to carry out real-time monitoring and effective inhibition on tumor tissue based on the characteristic parameters of tumor site. Herein, a near-infrared fluorescent probe HX is designed, the probe does not respond to the change of surrounding viscosity in a high pH environment, however, in a low pH microenvironment of tumor, the "Donor-π-Acceptor (D-π-A)" structure thereof is opened, and sensitive viscosity response characteristics are exhibited, so that the dynamic monitoring of the tumor microenvironment is realized. In addition, through structure optimization, it is also confirmed that the probe can be used as a photosensitizer with type I reaction for photodynamic therapy of tumor tissue under hypoxic conditions. With the help of a mouse tumor model in vivo, it is confirmed that the probe HX not only has the ability to identify tumor tissue and normal tissue, but also has excellent tumor inhibition effect and good biocompatibility under hypoxic conditions, thereby providing a new technology for the diagnosis and treatment integration design of tumor diseases.

Description

Technical Field

[0001] This invention belongs to the field of organic molecular fluorescent probes, and specifically discloses a molecular probe that can be used for in vivo tumor microenvironment response and photodynamic enhancement therapy, as well as its preparation method and application. Background Technology

[0002] The tumor microenvironment is typically characterized by low pH, high viscosity, and hypoxia, which are closely related to the development, metastasis, and spread of malignant tumors. Monitoring pH / viscosity changes in specific organelles is crucial for early cancer detection and undoubtedly improves treatment outcomes. Small molecule fluorescent probes have become powerful tools for dynamic and visual detection of tumor cell viscosity. Most viscosity-sensitive probes possess a "donor-π-acceptor (D-π-A)" framework, where molecular rotors promote the emergence of twisted intramolecular charge transfer (TICT) states, thereby enhancing their sensitivity to viscosity changes. However, these D-π-A probes are susceptible to the effects of intramolecular charge transfer (ICT), facing the challenge of false positive signals in complex cellular microenvironments, affecting the signal-to-background ratio (SBR). To address this issue, a "dual-locking" strategy, particularly fluorescent probe design based on multi-parameter responses related to the tumor microenvironment, is a highly effective solution, but such studies are currently rare. This patent designs and prepares a near-infrared fluorescent probe that is pH-locked and responds to tumor viscosity. The probe does not respond to changes in surrounding viscosity in a high pH environment, but in the low pH microenvironment of the tumor, its D-π-A structure is opened, exhibiting sensitive viscosity response characteristics, thereby realizing dynamic monitoring of the tumor microenvironment.

[0003] Furthermore, traditional photosensitizers utilize a type II photodynamic therapy (PDT) mechanism. These photosensitizers kill cancer cells by inducing the production of reactive oxygen species (ROS) under high O2 concentrations. However, the hypoxic microenvironment of solid tumors and the exacerbated O2 deficiency during PDT severely limit therapeutic efficacy. Developing novel type I photosensitizers can effectively address these issues. Type I photosensitizers react directly with substrates (such as water and oxygen) under light irradiation, generating superoxide radicals (O2-). •− It generates cationic or anionic free radicals such as hydroxyl radicals (•OH), thereby consuming less O2, which is suitable for the hypoxic environment deep in tumor tissue. Summary of the Invention

[0004] To address the above issues, this invention designs a dual-lock fluorescent probe molecule, HX, to monitor viscosity changes in a tumor microacid environment in real time. Through molecular structure regulation, it is demonstrated that the probe molecule possesses a type I PDT mechanism, enhancing the therapeutic effect on tumors and achieving the integration of diagnosis and treatment.

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

[0006] In a first aspect, the present invention provides a probe molecule with tumor microenvironment responsiveness and photodynamic therapy function, the probe molecule being denoted as HX, and its structure is shown in Formula I:

[0007] Formula I;

[0008] Under alkaline conditions, the hydroxyl group of the naphthalene ring in the probe molecule HX undergoes a 1,4-cycloaddition reaction with the spiro-benzofuran structure, forming a neutral six-membered ring structure that exhibits non-fluorescence due to photoinduced electron transfer. However, this structure is disrupted under acidic conditions, leading to the opening of the pyridine-like ring of the probe. At this stage, the probe possesses a D-π-A framework, thereby enhancing its sensitivity to viscosity changes. Therefore, HX possesses the ability to act as a pH switch and detect the viscosity of the tumor microenvironment.

[0009] Secondly, the preparation method of the probe molecule HX includes the following steps:

[0010] Step 1: Sodium metabisulfite, dimethylamine, and 2,7-dihydroxynaphthalene were added to water, and the mixture was stirred in a pressure-resistant flask and heated to 150°C. After reacting for 6 hours, the mixture was cooled and the pH was adjusted to 6 with 2 M hydrochloric acid. After the solid was completely precipitated, it was filtered and dried, and then purified by silica gel column chromatography to obtain the white crystalline compound 1-7-dimethylamino-2-naphthol.

[0011] Step 2: Under nitrogen protection, compound 1-7-dimethylamino-2-naphthol was added to an N,N-dimethylformamide solution. After 0.5 hours in an ice bath, phosphorus oxychloride was added, and stirring was continued for another 0.5 hours in an ice bath. The temperature was then raised to 50°C and reacted for 4 hours. After the reaction was completed, ice water was added, and the pH of the solution was adjusted to 8 using sodium bicarbonate in an ice bath. After the solid was completely precipitated, it was filtered, washed with pure water, and dried to finally obtain the yellow-green solid compound 2-6-(dimethylamino)-3-hydroxy-2-naphthal.

[0012] Step 3: Under nitrogen protection, 2,3,3-trimethyl-4,5-benzo-3H-indole was added to anhydrous acetonitrile. After adding iodoethane, the mixture was stirred and heated to 60°C for 6 hours. After the reaction was completed, anhydrous diethyl ether was added for recrystallization. The mixture was filtered and dried to obtain a gray-green solid compound, 3-3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-1-onium.

[0013] Step 4: Under nitrogen protection, compound 2-6-(dimethylamino)-3-hydroxy-2-naphthal and compound 3-3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-1-onium were dissolved in anhydrous ethanol and refluxed at 80°C for 8 hours. The mixture was then distilled under reduced pressure using a rotary evaporator and purified by silica gel column chromatography to obtain the final probe molecule HX.

[0014] Thirdly, the present invention provides the following uses for the probe molecule HX:

[0015] (1) Use in the preparation of reagents for responding to and diagnosing changes in pH and viscosity of the tumor microenvironment;

[0016] (2) Use in the preparation of drugs for photodynamic therapy of tumors.

[0017] In a preferred embodiment, the tumor is breast cancer.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention presents a simple synthetic method for designing a hemicyanine-based small-molecule fluorescent probe that responds to both pH and viscosity, two key physical properties of the tumor microenvironment, thus enhancing the reliability of the probe for tumor detection. Simultaneously, the probe molecule is a type I photosensitizer and releases ROS under hypoxic conditions, which is beneficial for photodynamic therapy of deep tumor tissues. Furthermore, based on the probe's own tumor tissue indication effect, phototherapy can be applied to lesions more precisely, thereby reducing biological damage.

[0020] The advantages include the following:

[0021] 1. The present invention provides a method for preparing and synthesizing the probe molecule HX for mouse tumor microenvironment response and photodynamic enhancement therapy. The synthesis route is simple, low-cost, and has high raw material utilization, making it suitable for industrial production.

[0022] 2. Viscosity and pH Response: HX, a small-molecule near-infrared fluorescent probe, exhibits viscosity-dependent changes. As the pH of the surrounding buffer solution decreases, HX is shown to be protonated, emitting weak fluorescence due to the TICT effect. However, in high-viscosity environments, the fluorescence intensity of HX at 670 nm significantly increases.

[0023] 3. Release of Type I ROS: In in vitro tests, the types of ROS produced after HX irradiation were identified using various commercial ROS dyes. It was found that HX mainly produces Type I ROS. This type of ROS has the advantage of being able to react even under hypoxic conditions, which has a unique advantage for the treatment of deep hypoxic environments of tumors.

[0024] 4. High biocompatibility: The present invention conducted a cell dark toxicity experiment on the probe molecule HX, and the results showed that HX was non-toxic to cells and organisms under no-light conditions and had biocompatibility.

[0025] 5. Imaging and Treatment of a Mouse Breast Cancer Model: In a mouse breast cancer model, HX was injected orally. In vivo imaging revealed a significant difference in fluorescence intensity between the breast cancer tissue and surrounding normal tissue. Furthermore, light irradiation effectively suppressed breast cancer in the mice, indicating that HX can be used as a photosensitizer for photodynamic therapy of tumors. Attached Figure Description

[0026] To make the above and / or other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described below:

[0027] Figure 1 Synthetic route and response mechanism of probe molecule HX;

[0028] Figure 2 These are (i) proton spectrum, (ii) carbon spectrum, and (iii) mass spectrometry data of probe molecule HX;

[0029] Figure 3 The fluorescence emission spectra of probe molecule HX in PBS solutions at different pH values ​​are (i) and (ii) the fluorescence intensity and pKa value at 670 nm.

[0030] Figure 4 The fluorescence emission spectra of the probe molecule HX in solvent systems with different ratios of water and glycerol are shown.

[0031] Figure 5 Selectivity testing of probe molecule HX against 17 interfering substances in physiological fluid;

[0032] Figure 6 These are (i) singlet oxygen of HX tested at different time intervals using DPBF. 1 (ii) O2) release content and (ii) HX and RB within 5 minutes 1 O2 release time-dependent;

[0033] Figure 7 These are (i) the superoxide anion radicals (O2) of HX and RB detected by DHR123 after 5 minutes of irradiation.•− (ii) Release H and (iii) O2 of HX and RB within 5 minutes •− Release time dependence;

[0034] Figure 8 The results are (i) the detection of ROS release of HX and RB after 6 minutes of DCFH irradiation and (ii) the time dependence of ROS release of HX and RB within 6 minutes.

[0035] Figure 9 The tests included (i) toxicity assays of 4T1 cells under HX-free illumination, and (ii) toxicity assays of 4T1 cells treated with HX under normoxic (21% O2) and hypoxic (1% O2) conditions. Illumination: 600 nm, 60 mW / cm². 2 10 min;

[0036] Figure 10 Fluorescence images of tumor and normal tissue sites in mice after HX injection at different time points;

[0037] Figure 11 The figures show: (a) a schematic diagram of the establishment and treatment of 4T1 breast tumor-bearing mice; (b) anatomical diagram of tumor tissue in each group of breast cancer mice after day 14 of treatment (n = 5); (c) tumor tissue weight in each group of mice in Figure b; (d) changes in tumor volume in each group of mice during treatment; (e) changes in body weight during the 14-day treatment period; (f) H&E, Ki67, and TUNEL staining of tumors on day 14 after diversified treatment. Scale bar: 50 μm; (g) serum ALT, AST, and BUN levels in each group of mice. Error bars (n = 3) represent the mean ± SD.

[0038] Figure 12 H&E staining of heart, liver, spleen, lung, and kidney tissues from different groups of mice. Scale bar: 50 μm. Detailed Implementation

[0039] The present invention will be described in detail below, specifically with reference to the accompanying drawings, to provide a clearer and more complete description of the embodiments of the invention, without limiting its scope. The embodiments described herein are only a part, not all, of the embodiments, and all are within the protection scope of the present invention.

[0040] Example 1: Preparation of probe molecule HX.

[0041] The synthetic route and response mechanism of the probe molecule HX are attached. Figure 1 As shown, the specific synthesis steps are as follows:

[0042] Step 1: Sodium metabisulfite (11.85 g, 1 eq), dimethylamine (7.50 mL, 2 eq), and 2,7-dihydroxynaphthalene (5 g, 1 eq) were added to water (60 mL). The mixture was stirred in a pressure-resistant flask and heated to 150 °C. After reacting for 6 hours, the mixture was cooled and the pH was adjusted to 6 with 2 mol / L hydrochloric acid. After the solid was completely precipitated, it was filtered and dried. Then, it was purified by silica gel column chromatography (developing solvent: petroleum ether: ethyl acetate = 20:1) to obtain the white crystalline compound 1-7-dimethylamino-2-naphthol.

[0043] Step 2: Under nitrogen protection, compound 1 (1.87 g, 1 eq) was added to a solution of N,N-dimethylformamide (4.11 mL, 1 eq), and after incubating in an ice bath for 0.5 hours, phosphorus oxychloride (2 mL, 1 eq) was added. After stirring in an ice bath for 0.5 hours, the reaction was carried out at 50°C for 4 hours. After the reaction was completed, ice water was added, and the pH of the solution was adjusted to 8 using sodium bicarbonate in an ice bath. After the solid was completely precipitated, it was filtered, washed with pure water, and dried to finally obtain the yellow-green solid compound 2-6-(dimethylamino)-3-hydroxy-2-naphthal.

[0044] Step 3: Under nitrogen protection, 2,3,3-trimethyl-4,5-benzo-3H-indole (2.09 g, 1 eq) was added to anhydrous acetonitrile (5 mL). After adding iodoethane (3.11 g, 2 eq), the mixture was stirred and heated to 60 °C for 6 hours. After the reaction was completed, anhydrous diethyl ether (60 mL) was added for recrystallization. The mixture was filtered and dried to obtain a gray-green solid compound 3-3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-1-onium.

[0045] Step 4: Under nitrogen protection, compound 2 (0.22 g, 1 eq) and compound 3 (0.24 g, 1 eq) were dissolved in 25 mL of anhydrous ethanol and refluxed at 80 °C for 8 hours. The mixture was then distilled off under reduced pressure using a rotary evaporator and purified by silica gel column chromatography (evolving solvent: dichloromethane:methanol = 20:1) to obtain probe molecule HX (0.11 g, yield 22.98%).

[0046] Example 2: Structural characterization of probe molecule HX

[0047] The probe molecule HX prepared in this invention was characterized and confirmed by proton NMR spectroscopy, carbon NMR spectroscopy, and high-resolution mass spectrometry (see appendix). Figure 2 HX: 1H NMR (400 MHz, DMSO-d6, δ): 10.19 (d, J = 6.6 Hz, 1H), 8.70 (dd, J= 15.6, 6.1 Hz, 1H), 8.42 – 8.35 (m, 1H), 8.20 (dt, J = 26.7, 7.6 Hz, 2H),8.08 – 8.01 (m, 1H), 7.90 (dd, J = 8.9, 6.6 Hz, 1H), 7.77 – 7.71 (m, 2H),7.69 – 7.62 (m, 1H), 7.55 (d, J = 4.1 Hz, 1H), 7.27 (dd, J = 9.0, 6.7 Hz,1H), 6.98 (dt, J = 13.5, 7.0 Hz, 2H), 4.60 – 4.51 (m, 2H), 3.09 (d, J = 6.5Hz, 6H), 2.05 (d, J = 6.4 Hz, 6H), 1.51 – 1.45 (m, 3H). 13 C NMR (200 MHz, DMSO-d6, δ): 181.46, 159.01, 157.60, 148.90, 139.09, 137.22, 136.48, 136.34,133.12, 131.73, 131.49,130.61, 128.83, 127.60, 126.99, 123.34, 123.29,116.31, 116.18, 115.71, 113.15, 108.97, 106.19, 53.23, 45.22, 41.92, 27.16,13.79. HRMS (ESI, m / z): [M] + Calculated for C 30 H 31 N2O + , 435.2431; found, 435.2409 (attached) Figure 2 ).

[0048] Example 3: In vitro spectroscopic performance testing of probe molecule HX

[0049] The reaction system for the in vitro test was as follows: a 384-well microplate was used, with a total test volume of 100 μL per well. The solution contained 99.80% PBS buffer (100 mM, pH = 7.4) and 0.20% DMSO. The final concentration of the probe molecule HX in the test solution was 100 μM, and the test temperature was 37℃. The specific experimental results are as follows:

[0050] (1) In vitro pH response test: The mother liquor probe molecule HX was dissolved in PBS buffer solutions of different pH values ​​(4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.4, 8.0, 8.5, 9.0, 10.0, 11.0, 12.0). 100 μL of the sample was added to a 384-well plate, and its fluorescence emission at an excitation wavelength of 590 nm was measured (see attached). Figure 3 ).

[0051] (2) In vitro viscosity response test: The probe molecule HX with a final concentration of 100 μM was added to a mixed solution of ultrapure water and glycerol at different glycerol ratios (0%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%). 100 μL of the solution was transferred to a 384-well microplate, and its fluorescence emission at an excitation wavelength of 590 nm was measured (see attached figure). Figure 4 ).

[0052] (3) In vitro selectivity test: The mother liquor probe is dissolved in various different interfering substances (Na) + Mg 2+ Al 3+ K + Ca 2+ Cu 2+ Cu + Fe 3+ Fe 2+ Cl - SO4 2- The solution contained alanine, lysine, arginine, proline, histidine, bovine serum albumin (BSA), and glycerol. The final concentration of the interfering substances was 100 μM. After incubating the final sample at 37°C for 30 min, 100 μL of sample was added to each well of a 384-plate microplate, and the fluorescence emission was measured using a microplate reader. (See attached image) Figure 5 ).

[0053] Experimental data show that the probe molecule HX exhibits small fluorescence changes at different pH values, with fluorescence increasing as pH decreases, revealing a D-π-A structure. Based on the fluorescence changes at different pH values, the pKa value of this probe was calculated to be 7.10. Below the pKa value, the probe exhibits viscosity responsiveness; with increasing viscosity (increased glycerol content), the probe fluorescence significantly increases. Furthermore, in the presence of the 17 interfering substances, the probe molecule HX only shows fluorescence enhancement in response to viscosity changes, indicating that the probe has response selectivity.

[0054] Example 4: In vitro ROS release capacity test of probe molecule HX

[0055] The in vitro testing reaction system was as follows: a 96-well microplate was used, with a total test liquid volume of 100 μL per well, a final concentration of probe molecule HX in the test solution of 50 μM, a test temperature of 37℃, a xenon lamp light source wavelength of 600 nm, and a power of 0.06 W / cm². 2 Meanwhile, a control group, Rose Bengal (RB), was set up to compare its ROS release capacity with that of HX. The specific experimental results are as follows:

[0056] (1) In vitro singlet oxygen ( 1 O2 Release Test: 1,3-Diphenylisobenzofuran (DPBF) was added to sample solutions containing HX (50 μM) and RB (50 μM) (EtOH∶H2O = 1∶1), respectively, to make its absorbance at 410 nm close to 1.0. After irradiation with light at a wavelength of 600 nm for different time intervals, the absorption spectrum of DPBF was measured and the absorption intensity values ​​at different irradiation times were recorded (see attached diagram). Figure 6 The control group underwent the same procedure.

[0057] (2) In vitro superoxide anion free radicals (O2) •− Release test: Dihydrorhodamine 123 (DHR123) was added to PBS sample solutions containing HX and RB (50 μM) (10 mM, pH = 7.0), respectively. DHR123 was used as O2 release agent. •− Indicator. After irradiating the mixed solution with a 600 nm xenon lamp at different time intervals, the fluorescence spectrum of DHR123 under 515 nm excitation was measured, and the fluorescence emission intensity at 535 nm was recorded (see attached). Figure 7 ).

[0058] (3) In vitro total ROS release test: DCFH-DA solution (20 μM) was added to PBS sample solutions containing HX (50 μM) and RB (50 μM) respectively (10 mM, pH = 7.0). The samples were irradiated with a 600 nm xenon lamp at different time intervals. Then, the fluorescence emission spectrum of DCFH under 485 nm excitation was measured using a fluorescence spectrophotometer and the fluorescence intensity at 525 nm was recorded (see attached). Figure 8 Furthermore, the DCFH was tested under the same experimental conditions.

[0059] Experimental results show that, compared with the RB reference group, the probe molecule HX in 1 The probe exhibits low release capacity on O2, displaying inferior type II photosensitizer properties. However, the probe shows good release capacity on O2. •− It has a significant advantage in releasing type I ROS and exhibits remarkable type I photosensitizer characteristics, indicating that the probe can release ROS and provide photodynamic therapy effects under hypoxic conditions, making it more suitable for deep treatment of solid tumors.

[0060] Example 4: Phototoxicity of probe molecule HX in cells

[0061] This section describes the cell culture of 4T1 cells using RPMI-1640 at 37°C and 5% CO2.

[0062] (1) Dark toxicity test of the probe: Cells (4T1) were placed in a well at a density of 1 × 10⁻⁶ cells / well. 5 Cells were cultured in 96-well plates at a specific ratio. After incubation at 37°C in a normal aerobic (21% O2) atmosphere for 24 hours, different concentrations of HX (0.1% DMSO) were added to each 4T1 cell, and incubation continued for another 24 hours under darkness. After washing the cells with PBS, 100 μL of fresh culture medium and 10 μL of Cell Counting Kit-8 (CCK-8, 10 μL) were added to each well, and the cells were incubated at 37°C for 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated (see attached image). Figure 9 i).

[0063] (2) Phototoxicity test of the probe: Cells (4T1) were placed in a well at a density of 1 × 10⁻⁶ cells / well. 5 The cells were placed in 96-well cell culture plates at a ratio of 1:1. Then, after incubation at 37°C under normoxic (21% O2) and hypoxic (1% O2) conditions for 24 hours, different concentrations of HX (0.1% DMSO) were added to the 4T1 cells at a power density of 60 mW / cm². 2Cells were irradiated under a 600 nm xenon lamp for 10 min and then cultured for 24 h. After washing with PBS, 100 μL of fresh culture medium and 10 μL of Cell Counting Kit-8 (CCK-8, 10 μL) were added to each well, and the cells were incubated at 37 °C for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated (see attached image). Figure 9 ii).

[0064] Experimental results showed that the probe molecule HX exhibited extremely low dark toxicity in the concentration range of 0–50 μM, indicating that HX has low cytotoxicity. After light exposure, HX showed moderate toxicity (IC50) under normoxic conditions. 50 = 45.37 μM), exhibiting significantly enhanced phototoxicity (IC50) under hypoxic conditions. 50 = 28.37 μM), exhibiting typical characteristics of a type I photosensitizer. Therefore, the probe molecule HX possesses photodynamic therapeutic efficacy and biocompatibility, and is suitable for treatment under deep tumor hypoxia conditions.

[0065] Example 5: Fluorescence Imaging Study of Probe Molecule HX in Tumor-Bearing Animal Models

[0066] All animal experiments involved in this study were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (8th edition, 2011), and all animals were approved by the Animal Management and Use Committee of Nanjing University of Technology (Approval No.: LL-20210310-01).

[0067] BALB / c mice (4 weeks old, female) were selected and injected orally with 5×10 6 A mouse model of orthotopic breast cancer was established using 4T1 cells, and in vivo imaging was performed after 10 days of feeding. 100 μL of HX solution (200 μM, 0.4% DMSO, 1% Tween 80) was injected orally into the tumor site, while the same amount of probe solution was injected into adjacent normal tissue. The imaging performance of the tumor tissue was then observed using an in vivo imaging system (see attached image). Figure 10 Experimental results show that the probe molecule HX can distinguish between tumor tissue and normal tissue, and can achieve enrichment in 0.5 h, confirming that the probe can monitor in vivo tumor tissue in real time.

[0068] Example 6: Photodynamic Study of Probe Molecule HX in Tumor-Bearing Animal Models

[0069] BALB / c mice (4 weeks old, female) were selected and injected orally with 5×10 6 An orthotopic breast cancer mouse model was established using 4T1 cells. When the tumor volume reached 62.50 mm... 3In this study, tumor-bearing mice were randomly divided into three groups (n = 5): control group (PBS), probe group (HX), and probe phototherapy group (HX + L). The PBS group received no probe injection and was irradiated with a 600 nm xenon lamp for 15 min on days 1, 2, and 4 (optical density 0.18 W / cm²). 2 The HX group received an in-situ injection of 100 μL of HX solution (200 μM, 0.4% DMSO, 1% Tween 80) at the tumor site only on days 1, 2, and 4. The HX+L group received probe injection on days 1, 2, and 4, followed by irradiation with a 600 nm xenon lamp for 15 min (optical density 0.18 W / cm²). 2 The tumor volume is calculated using the formula: V = 1 / 2(a×b²). Where V is the mouse tumor volume, a is the longest diameter of the tumor region, and b is the diameter of the tumor region perpendicular to a. The treatment period for mice was 14 days. During this period, the mice's weight and tumor volume were recorded. After the treatment, various vital signs of the mice were tested (see attached). Figure 11 ).

[0070] Experimental results showed that under light irradiation, the probe molecule HX could significantly inhibit tumor growth, demonstrating photodynamic therapy efficacy. During treatment, the vital signs of the mice remained within the normal range, and no abnormal expression was observed in any tissue sections, indicating that HX possesses photodynamic therapy capabilities and biocompatibility, making it suitable for future integrated diagnostic and therapeutic applications.

[0071] The materials, methods, and embodiments described herein are exemplary and not restrictive. Those skilled in the art can appropriately substitute and / or modify process parameters based on the content of this document; however, it should be particularly noted that all such substitutions and / or modifications are obvious to those skilled in the art and do not depart from the spirit of the invention or exceed the scope defined by the appended claims. Therefore, they are all considered to be included in the present invention.

Claims

1. A probe molecule HX with tumor microenvironment responsiveness and photodynamic therapy function, characterized in that, The structural formula of the probe is shown in Formula I: Equation I.

2. A method for preparing the probe molecule HX as described in claim 1, characterized in that, Includes the following steps: Step 1: Sodium metabisulfite, dimethylamine, and 2,7-dihydroxynaphthalene were added to water. The mixture was stirred in a pressure-resistant flask and heated to 150°C. After reacting for 6 hours, the mixture was cooled and the pH was adjusted to 6 with 2 M hydrochloric acid. After the solid completely precipitated, it was filtered and dried, and then purified by silica gel column chromatography to obtain white crystalline compound 1. ; Step 2: Under nitrogen protection, compound 1 was added to an N,N-dimethylformamide solution, and after an ice bath for 0.5 hours, phosphorus oxychloride was added. The mixture was stirred for another 0.5 hours under an ice bath, and then reacted at 50°C for 4 hours. After the reaction was complete, ice water was added, and the pH of the solution was adjusted to 8 using sodium bicarbonate under an ice bath. After the solid completely precipitated, it was filtered, washed with pure water, and dried to obtain a yellow-green solid, compound 2. ; Step 3: Under nitrogen protection, 2,3,3-trimethyl-4,5-benzo-3H-indole was added to anhydrous acetonitrile. After adding iodoethane, the mixture was stirred and heated to 60°C for 6 hours. After the reaction was completed, anhydrous diethyl ether was added for recrystallization. The mixture was filtered and dried to obtain a gray-green solid compound 3. ; Step 4: Under nitrogen protection, compounds 2 and 3 were dissolved in anhydrous ethanol and refluxed at 80°C for 8 hours. The mixture was then distilled under reduced pressure using a rotary evaporator to obtain the probe molecule HX by silica gel column chromatography.

3. The use of the probe molecule HX as described in claim 1 in the preparation of a drug for diagnosing changes in pH and viscosity of the tumor microenvironment.

4. Use of the probe molecule HX as described in claim 1 in the preparation of a medicament for photodynamic therapy of tumors.

5. The use as described in claim 3 or 4, characterized in that, The tumor in question is a tumor from a mouse breast cancer model.

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