Molecular probe design for tumor microenvironment response and photodynamic enhancement therapy and preparation method thereof

Abstract

The invention belongs to the field of organic molecule fluorescent probes, and relates to a small molecule fluorescent probe HX which realizes the integration of diagnosis and treatment by monitoring related physical properties of a tumor microenvironment and photodynamic therapy of tumors. A tumor microenvironment of a solid tumor generally has the characteristics of low pH, high viscosity, low oxygen and the like, and has important significance on real-time monitoring and effective inhibition of tumor tissues on the basis of characteristic parameters of tumor parts. Therefore, a near-infrared fluorescent probe HX is designed, the probe does not respond to surrounding viscosity change in a high pH value environment, however, in a tumor low pH value microenvironment, a donor-pi-receptor (Donor-pi-acceptor, D-pi-A) structure of the probe is opened, and the sensitive viscosity response characteristic is shown, so that the dynamic monitoring of the tumor microenvironment is realized. Besides, through structural optimization, the probe is proved to be capable of serving as a photosensitizer with I-type reaction and used for tumor tissue photodynamic therapy under the hypoxic condition. By means of a mouse living tumor model, we prove that the probe HX not only has the capability of identifying tumor tissues and normal tissues, but also has an excellent tumor inhibition effect and good biocompatibility under the hypoxic condition, and a new technology is provided for integrated design of diagnosis and treatment of tumor diseases. # imgabs0 #

Description

Technical Field

[0001] The present 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 thereof, as well as a preparation method and application thereof. Background Art

[0002] The tumor microenvironment is often 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 detection of cancer and undoubtedly improves therapeutic efficacy. Small molecule fluorescent probes have become powerful tools for dynamic and visual detection of tumor cell viscosity. Most viscosity-sensitive probes have a "donor-π-acceptor" (D-π-A) framework, in which a molecular rotor promotes the emergence of twisted states during intramolecular charge transfer (TICT), thereby enhancing their sensitivity to viscosity changes. However, such D-π-A probes are susceptible to intramolecular charge transfer (ICT) effects, resulting in false positive signals in complex cellular microenvironments and poor imaging signal-to-background ratio (SBR). To address this issue, the design of fluorescent probes based on a "double-locking" strategy, particularly those responsive to multiple parameters relevant to the tumor microenvironment, is a highly effective solution, but few have been reported. This patent designs and prepares a pH-locked tumor viscosity-responsive near-infrared fluorescent probe. The probe does not respond to changes in surrounding viscosity in a high pH environment. However, in the low pH microenvironment of the tumor, its D-π-A structure is opened, showing sensitive viscosity response characteristics, thereby realizing dynamic monitoring of the tumor microenvironment.

[0003] In addition, traditional photosensitizers are type II photodynamic therapy (PDT) mechanisms, which kill cancer cells by generating reactive oxygen species (ROS) under high concentrations of O2 through light induction. However, the hypoxic microenvironment of solid tumors and the O2 shortage caused by the consumption of O2 during PDT seriously limit the therapeutic effect. The development of new type I photosensitizers can solve the above problems well. Type I photosensitizers react directly with substrates (such as water and oxygen) under light to produce superoxide radicals (O2 ·- ), hydroxyl radical (·OH) and other cationic or anionic free radicals, thereby consuming less O2 and is suitable for the hypoxic environment deep in tumor tissue. Summary of the Invention

[0004] To address the above issues, the present invention designed a dual-locked molecular fluorescent probe HX to monitor viscosity changes in the micro-acidic environment of tumors in real time. Through molecular structure regulation, it was demonstrated that the probe molecule has a type I PDT mechanism, enhancing the tumor treatment effect and realizing the integration of diagnosis and treatment.

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

[0006] In the first aspect, a molecular probe for in vivo tumor microenvironment response and photodynamic enhancement therapy thereof is provided, denoted as HX, and its molecular structure is shown below:

[0007]

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

[0009] In a second aspect, the preparation method of the molecular fluorescent probe HX comprises the following steps:

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

[0011] Step 2: Under nitrogen protection, compound 1 was added to N,N-dimethylformamide solution, and phosphorus oxychloride was added after ice bathing for 0.5 hours. Stirring was continued for 0.5 hours under ice bath, and the temperature was raised to 50 ° C and reacted for 4 hours. After the reaction was completed, ice water was added, and sodium bicarbonate was used to adjust the pH of the solution to 8 under ice bath. After the solid was completely precipitated, it was filtered, washed with pure water, and dried to finally obtain yellow-green solid compound 2.

[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 at 60°C for 6 hours. After the reaction, anhydrous ether was added for recrystallization, and the mixture was filtered and dried to obtain a gray-green solid compound 3.

[0013] Step 4: Under nitrogen protection, compound 2 and compound 3 were dissolved in anhydrous ethanol and refluxed at 80°C for 8 hours. The mixture was subjected to reduced pressure distillation of the ethanol solvent using a rotary evaporator and purified by silica gel column chromatography to obtain the final probe HX.

[0014] In a third aspect, a use of an HX fluorescent probe is provided.

[0015] Specifically, HX can respond to the physical properties of the tumor microenvironment, such as viscosity and pH.

[0016] Specifically, HX can effectively generate ROS under light excitation and effectively generate type I ROS under hypoxic conditions to perform photodynamic therapy on tumor cells.

[0017] Specifically, HX can respond to the tumor microenvironment of mice and distinguish the boundaries of tumors under in situ injection. It can effectively perform photodynamic therapy on mouse breast cancer models and has good biosafety.

[0018] The beneficial effects of the present invention are:

[0019] This invention designs a simple hemicyanine small-molecule fluorescent probe that responds to two major physical properties of the tumor microenvironment: pH and viscosity. This enhances the probe's reliability in tumor detection. Furthermore, the probe molecule is a Type I photosensitizer and releases ROS under hypoxic conditions, facilitating photodynamic therapy of deep-lying tumor tissue. Furthermore, the probe's excellent tumor tissue-indicating properties enable more precise phototherapy of lesions, thereby minimizing biological damage.

[0020] The advantages include the following:

[0021] 1. The present invention provides a method for preparing and synthesizing a molecular probe HX for detecting tumor microenvironmental responses in mice and enhancing their photodynamic therapy. The method has a simple synthesis route, low cost, high raw material utilization, and is suitable for industrial production.

[0022] 2. Excellent viscosity and pH responsiveness: HX, a small molecule near-infrared fluorescent probe that changes viscosity, was shown to be protonated as the pH of the surrounding buffer decreased, emitting weak fluorescence due to the TICT effect. In a high-viscosity environment, the fluorescence intensity of HX at 670 nm increased significantly.

[0023] 3. Good release of type I ROS: In in vitro tests, various ROS commercial dyes were used to identify the types of ROS produced after HX irradiation. It was found that HX mainly produces type I ROS. This type of ROS has the advantage of being able to react under hypoxic conditions and has unique advantages for treating deep hypoxic environments of tumors.

[0024] 4. High biological safety: The present invention conducted a dark cell toxicity experiment on the fluorescent probe molecules. The results showed that the fluorescent probe molecules were non-toxic to cells and organisms under non-illumination conditions and had good biocompatibility.

[0025] 5. Imaging and Treatment of Mouse Breast Cancer Models: HX was injected orally into a mouse model of breast cancer. In vivo imaging revealed a significant difference in fluorescence intensity between breast cancer tissue and surrounding normal tissue. Furthermore, after illumination, breast cancer in the mice was effectively suppressed, demonstrating that HX can serve as an excellent photosensitizer for photodynamic therapy of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the above and / or other objects, features, advantages and embodiments of the present invention more apparent, the accompanying drawings are described as follows:

[0027] Figure 1 The synthetic route and response mechanism of molecular fluorescent probes;

[0028] Figure 2 They are (i) hydrogen spectrum, (ii) carbon spectrum, and (iii) mass spectrum data of the molecular fluorescent probe HX;

[0029] Figure 3 The fluorescence emission spectra of probe HX in PBS solutions with different pH values ​​and the fluorescence intensity at 670 nm and its pKa value are shown respectively.

[0030] Figure 4 The fluorescence emission spectra of probe HX in solvent systems with different ratios of water and glycerol;

[0031] Figure 5 To test the selectivity of probe HX against 17 related interfering substances in physiological fluids;

[0032] Figure 6 They are (i) the singlet oxygen of HX tested by DPBF at different time intervals ( 1 O2) release content and (ii) HX and RB within 5 minutes 1 O2 release time dependence;

[0033] Figure 7 (i) The superoxide anion radicals (O2 ·- ) release of H and (ii) O2 from HX and RB within 5 min ·- Release time dependency;

[0034] Figure 8These are (i) DCFH detection of ROS release from HX and RB after 6 min of irradiation and (ii) time dependence of ROS release from HX and RB within 6 min;

[0035] Figure 9 The toxicity tests were (i) HX toxicity test on 4T1 cells without illumination, and (ii) HX toxicity test on 4T1 cells treated with illumination under normoxic (21% O2) and hypoxic (1% O2) conditions. Illumination: 600 nm, 60 mW / cm -2 , 10min; Figure 10 Fluorescence images of tumor tissue and normal tissue sites in mice at different times after HX injection;

[0036] Figure 11 (a) Schematic diagram of the establishment and treatment of 4T1 breast tumor-bearing mice; (b) Tumor tissue anatomy of each group of breast cancer mice after 14 days of treatment (n = 5); (c) Tumor tissue weight of each group of mice in Figure b; (d) Changes in tumor volume of 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) ALT, AST, and BUN levels in the serum of each group of mice were measured. Error bars (n = 3) represent mean ± SD.

[0037] Figure 12 H&E staining of heart, liver, spleen, lung, and kidney tissues from different groups of mice. Scale bar: 50 μm. DETAILED DESCRIPTION

[0038] The present invention is described in detail below, specifically with reference to the accompanying drawings of the present invention, to provide a clearer and more complete description of the embodiments of the present invention, without limiting the contents thereof. The embodiments described in the present invention are only a part, not all, of the embodiments, which are all within the scope of protection of the present invention.

[0039] Example 1 Preparation of probe HX.

[0040] The synthesis route and response mechanism of probe HX are shown in the attached Figure 1 As shown, the synthesis steps are as follows:

[0041] 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), and the mixture was stirred and heated to 150°C in a pressure bottle. After reacting for 6 hours, it 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, and then purified by silica gel column chromatography (developing solvent: petroleum ether: ethyl acetate = 20:1) to obtain white crystalline compound 1 (4.03 g, yield 71.83%).

[0042] 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 phosphorus oxychloride (2 mL, 1 eq) was added after ice bathing for 0.5 hours. After stirring for 0.5 hours under ice bath, the mixture was reacted at 50°C for 4 hours. After the reaction was completed, ice water was added, and sodium bicarbonate was used to adjust the pH of the solution to 8 under ice bath. After the solid was completely precipitated, it was filtered, washed with pure water, and dried to finally obtain a yellow-green solid compound 2 (1.02 g, yield 47.44%).

[0043] 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 at 60°C for 6 hours. After the reaction, anhydrous ether (60 mL) was added for recrystallization. The mixture was filtered and dried to obtain a gray-green solid compound 3 (1.97 g, yield 84.36%).

[0044] 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 subjected to reduced pressure distillation of the ethanol solvent using a rotary evaporator and purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 20:1) to obtain the probe molecule HX (0.11 g, yield 22.98%).

[0045] Example 2 Structural Characterization of Probe HX

[0046] The probe HX prepared by the present invention was characterized and confirmed by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high resolution mass spectrum (see Appendix Figure 2 ). HX: 1H NMR (400MHz, DMSO-d6, δ): 10.19 (d, J=6.6Hz, 1H), 8.70 (dd, J=15.6, 6.1Hz, 1H), 8.42–8.35 (m, 1H),8.20(dt,J=26.7,7.6Hz,2H),8.08–8.01(m,1H),7.90(dd,J=8.9,6.6Hz,1H),7.77–7.71(m ,2H),7.69–7.62(m,1H),7.55(d,J=4.1Hz,1H),7.27(dd,J=9.0,6.7Hz,1H),6.98(dt,J=13.5, 7.0Hz,2H),4.60–4.51(m,2H),3.09(d,J=6.5Hz,6H),2.05(d,J=6.4Hz,6H),1.51–1.45(m,3H). 13 C NMR(200MHz,DMSO-d6,δ):181.46,159.01,157.60,148.90,139.09,137.22,136.48,136.34,133.12,131.73,131.49,130.61,128.83,12 7.60,126.99,123.34,123.29,116.31,116.18,115.71,113.15,108.97,106.19,53.23,45.22,41.92,27.16,13.79.HRMS(ESI,m / z):[M] + calculated for C 30 H 31 N2O + ,435.2431;found,435.2409(attached Figure 2 ).

[0047] Example 3 In vitro spectroscopic performance test of probe HX

[0048] The in vitro test reaction system used a 384-well microtiter plate, with a total test volume of 100 μL per well. The test solution contained 99.80% PBS buffer (100 mM, pH 7.4) and 0.20% DMSO. The final concentration of the probe HX in the test solution was 100 μM, and the test temperature was 37°C. The specific experimental results are as follows:

[0049] (1) In vitro pH response test: The mother solution probe 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 sample was added to a 384-well plate, and its fluorescence emission at an excitation wavelength of 590 nm was measured (see Appendix). Figure 3 ).

[0050] (2) In vitro viscosity response test: The probe 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 ELISA plate, and its fluorescence emission at an excitation wavelength of 590 nm was measured (see Appendix). Figure 4 ).

[0051] (3) In vitro selectivity test: The mother solution probe is dissolved in various interfering substances (Na + Mg 2+ 、Al 3+ , K + , Ca 2+ 、Cu 2+ 、Cu + 、Fe 3+ 、Fe 2+ 、Cl - 、SO4 2- , Alanine, Lysine, Arginine, Proline, Histidine, Bovine serum albumin (BSA), Glycerol solution), the final concentration of the relevant interfering substances was 100 μM, the final sample was incubated at 37 ° C for 30 minutes, 100 μL of sample was added to each 384 ELISA plate well, and the fluorescence emission was measured in an ELISA instrument (attached Figure 5 ).

[0052] Experimental data showed that probe HX exhibited minimal fluorescence changes at different pH values, with fluorescence increasing as the pH decreased, demonstrating a D-π-A structure. Based on the fluorescence changes at different pH values, the probe's pKa value was calculated to be 7.10. Below the pKa value, the probe exhibited good viscosity responsiveness, with fluorescence significantly increasing with increasing viscosity (increased glycerol ratio). Furthermore, in the presence of interfering substance 17, probe HX exhibited fluorescence enhancement only in response to changes in viscosity, demonstrating good response selectivity.

[0053] Example 4 In vitro ROS release ability test of probe HX

[0054] The reaction system for in vitro testing is as follows: a 96-well ELISA plate is used, the total volume of the test liquid in each well is 100 μL, the final concentration of the probe HX in the test solution is 50 μM, the test temperature is 37°C, the wavelength of the xenon lamp light source is 600 nm, and the power is 0.06 W / cm 2 At the same time, a reference group, Rose Bengal (RB), was set up to compare the ROS release capacity of HX. The specific experimental results are as follows:

[0055] (1) In vitro singlet oxygen ( 1 O2) release test: 1,3-diphenylisobenzofuran (DPBF) was added to a sample solution (EtOH:H2O=1:1) containing HX (50μM) and RB (50μM) respectively, so that the absorbance at 410nm was close to 1.0. After irradiation with 600nm wavelength light for different time intervals, the absorption spectrum of DPBF was measured and the absorption intensity values ​​under different irradiation time were recorded (see Appendix). Figure 6 The control group was subjected to the same experiment using the same procedure.

[0056] (2) In vitro superoxide anion free radicals (O2 ·- ) Release test: Dihydrorhodamine 123 (DHR123) was added to PBS sample solution (10 mM, pH = 7.0) containing HX and RB (50 μM), and DHR123 was used as O2 ·- After the mixed solution was irradiated with a 600nm xenon lamp for different time intervals, the fluorescence spectrum of DHR123 under 515nm excitation was measured and the fluorescence emission intensity at 535nm was recorded (see Appendix Figure 7 ).

[0057] (3) In vitro total ROS release test: DCFH-DA solution (20 μM) was added to PBS sample solution (10 mM, pH = 7.0) containing HX (50 μM) and RB (50 μM). The samples were irradiated with a 600 nm xenon lamp for different time intervals. The fluorescence emission spectrum of DCFH under 485 nm excitation was then measured using a fluorescence spectrophotometer and the fluorescence intensity at 525 nm was recorded (see Appendix). Figure 8 ). In addition, DCFH was tested under the same experimental conditions.

[0058] The experimental results showed that compared with the RB reference group, the probe HX 1 The release ability of the probe on O2 is low, showing low type II photosensitizer properties. ·-The probe has obvious advantages in the release of type I ROS and has significant type I photosensitizer characteristics, indicating that the probe can have excellent ROS release ability and good photodynamic therapy effect under hypoxic conditions, and is more suitable for the deep treatment of solid tumors.

[0059] Example 4: Phototoxicity of probe HX in cells

[0060] In this part, 4T1 cells were cultured in RPMI-1640 at 37°C and 5% CO2.

[0061] (1) Dark toxicity test of the probe: cells (4T1) were plated at 1×10 5 The ratio of cells was placed in a 96-well cell culture plate. Then, after incubation at 37°C in a normoxia (21% O2) atmosphere for 24 hours, different concentrations of HX (0.1% DMSO) were added to the 4T1 cells and incubated for another 24 hours in the absence of light. After washing the cells with PBS, fresh culture medium (100 μL) and Cell Counting Kit-8 (CCK-8, 10 μL) were added to each well and incubated at 37°C for 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader, and the cell viability (see Appendix) was calculated. Figure 9 i).

[0062] (2) Phototoxicity test of the probe: cells (4T1) were plated at 1×10 5 Then, after incubation at 37°C for 24 hours under normoxic (21% O2) and hypoxic (1% O2) conditions, different concentrations of HX (0.1% DMSO) were added to 4T1 cells at a power density of 60 mW / cm 2 After irradiation under a 600nm xenon lamp for 10 minutes, the cells were cultured for 24 hours. After washing with PBS, fresh culture medium (100μL) and Cell Counting Kit-8 (CCK-8, 10μL) were added to each well and incubated at 37°C for 2 hours. Finally, the absorbance at 450nm was measured using a microplate reader, and the cell viability (appended) was calculated. Figure 9 ii).

[0063] The experimental results showed that the probe HX showed extremely low dark toxicity in the concentration range of 0-50 μM and had good biosafety. After illumination, HX showed moderate toxicity under normoxic conditions (IC 50 =45.37 μM), and showed significant phototoxicity enhancement under hypoxic conditions (IC 50 =28.37 μM), showing the characteristics of a typical type I photosensitizer. Therefore, probe HX has good photodynamic therapy effect and biosafety, and is suitable for the treatment of deep tumor hypoxia.

[0064] Example 5 Fluorescence imaging of probe HX in a breast cancer mouse model

[0065] All animal experiments involved in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (8th edition, 2011) and approved by the Institutional Animal Care and Use Committee of Nanjing Tech University (approval number: LL-20210310-01).

[0066] 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. After 10 days of in vivo imaging, 100 μL of HX solution (200 μM, 0.4% DMSO, 1% Tween 80) was injected into the tumor site (Tumor) and the same amount of probe solution was injected into the normal tissue adjacent to the tumor (Normal). The imaging performance of the tumor tissue was then observed under an in vivo imaging device (see Appendix). Figure 10 The experimental results showed that the probe HX can distinguish between tumor tissue and normal tissue, and can achieve good enrichment within 0.5 hours, confirming that the probe can monitor living tumor tissue in real time.

[0067] Example 6 Photodynamic therapy of a breast cancer mouse model using probe HX

[0068] BALB / c mice (4 weeks old, female) were selected and injected orally with 5×10 6 4T1 cells were used to establish an orthotopic breast cancer mouse model. When the tumor volume reached 62.50 mm 3 Tumor-bearing mice were randomly divided into three groups (n=5): control group (PBS), probe group (HX), and probe light therapy group (HX+L). The PBS group received no probe injection and the tumor site was irradiated with a 600 nm xenon lamp for 15 min (optical density 0.18 W / cm2) on the first, second, and fourth days, respectively. 2 ); HX group received 100 μL of HX solution (200 μM, 0.4% DMSO, 1% Tween 80) injected into the tumor site on the first, second, and fourth days; HX+L group received probe injection on the first, second, and fourth days and then irradiated with a 600 nm xenon lamp for 15 min (optical density 0.18 W / cm 2 The tumor volume calculation formula is: V = 1 / 2 (a × b2). Where V is the tumor volume of the mouse, a is the longest diameter of the tumor area, and b is the diameter of the tumor area perpendicular to a. The mouse treatment cycle is 14 days, during which the weight and tumor volume of the mouse are recorded. At the end of the treatment, the various vital signs of the mouse are tested (see Appendix). Figure 11 ).

[0069] Experimental results demonstrated that, under illumination, the HX probe significantly inhibited tumor growth, demonstrating excellent photodynamic therapy efficacy. During treatment, the mice's vital signs remained within normal ranges, and no abnormal expression was observed in tissue sections. This demonstrates HX's excellent photodynamic therapy capabilities and high biosafety, making it suitable for integrated diagnosis and treatment applications.

[0070] The materials, methods, and examples described herein are intended to be illustrative only and not restrictive. Those skilled in the art may refer to the disclosure herein and make appropriate substitutions and / or modifications to the process parameters. However, it is particularly important to note that all such substitutions and / or modifications will be apparent to those skilled in the art and will not depart from the spirit of the present invention or exceed the scope of the appended claims. Therefore, such substitutions and / or modifications are considered to be included herein.

Claims

1. A molecular probe design and preparation method for tumor microenvironment response and photodynamic enhancement therapy, characterized in that: The probe includes a core fluorophore composed of 2-dimethylamino-7-hydroxynaphthalene, which can respond to changes in the mouse tumor microenvironment and has a photodynamic therapy effect on mouse tumors. Its structural formula is as follows:

2. The method for preparing a molecular probe for tumor microenvironment response and photodynamic enhancement therapy thereof according to claim 1, characterized in that: The preparation scheme is as follows: Step 1: Sodium metabisulfite, dimethylamine, and 2,7-dihydroxynaphthalene were added to water, and the mixture was stirred and heated to 150°C in a pressure bottle. After reacting for 6 hours, it was cooled and the pH was adjusted to 6 with 2M hydrochloric acid. After the solid was completely precipitated, it was filtered and dried, and then purified using silica gel column chromatography to obtain white crystalline compound 1. Step 2: Under nitrogen protection, compound 1 was added to N,N-dimethylformamide solution, and phosphorus oxychloride was added after ice bath for 0.5 hours. After stirring for 0.5 hours under ice bath, the mixture was reacted at 50°C for 4 hours. After the reaction was completed, ice water was added, and sodium bicarbonate was used to adjust the pH of the solution to 8 under ice bath. After the solid was completely precipitated, it was filtered, washed with pure water, and dried to finally obtain 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 at 60°C for 6 hours. After the reaction, anhydrous ether was added for recrystallization, and the mixture was filtered and dried to obtain a gray-green solid compound 3. Step 4: Under nitrogen protection, compound 2 and compound 3 were dissolved in anhydrous ethanol and refluxed at 80°C for 8 hours. The mixture was subjected to reduced pressure distillation of the ethanol solvent using a rotary evaporator and purified by silica gel column chromatography to obtain the probe molecule HX.

3. The use of the molecular probe for tumor microenvironment response and photodynamic enhancement therapy thereof according to claim 1, characterized in that: It can respond to changes in pH and viscosity of the tumor microenvironment and perform photodynamic therapy for tumor diseases.

4. The use of the molecular fluorescent probe according to claim 1, characterized in that: It responds to the tumor microenvironment of breast cancer mouse models and can better distinguish the boundaries between tumor and normal tissue.

5. The use of the molecular fluorescent probe according to claim 1, characterized in that: During photodynamic therapy of breast cancer mouse models, it can effectively inhibit tumor growth and has high biosafety.

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