Targeted lysosome near-infrared fluorescent probe for photothermal therapy and preparation method of targeted lysosome near-infrared fluorescent probe

Abstract

The invention discloses a targeted lysosome near-infrared fluorescent probe for photothermal therapy and a preparation method thereof.The method comprises the steps that S1, nitrite and sulfuric acid are mixed, and nitrosyl sulfuric acid is obtained; s2, nitrosyl sulfuric acid is added into 2-amino-3-cyano-4-chloro-5-formyl thiophene, a solution A is obtained, phosphoric acid is added, and a diazotization solution is obtained; s3, dissolving the 1-N-R1-6-hydroxy-4-methyl-2-pyridone, so as to obtain a pyridone solution; r1 is one of hydrogen, C1-8 alkyl, hydroxyalkyl, alkoxy alkyl and phenyl; adding the diazotization solution into a pyridone solution to react, carrying out suction filtration, washing a filter cake, and recrystallizing to obtain an intermediate compound; and S4, adding the intermediate compound and 6-hydroxy-1, 3-R2 pyrimidine-2, 4 (1H, 3H)-diketone into acetic anhydride, carrying out heating reaction, cooling, filtering, washing and drying to obtain the near-infrared fluorescent probe. And R2 is one of hydrogen, C1-8 alkyl and phenyl.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to a targeted lysosomal near-infrared fluorescent probe for photothermal therapy and its preparation method. Background Technology

[0002] Cancer has long been considered one of the most intractable diseases. While traditional treatments such as surgical resection, radiation therapy, and chemotherapy have brought benefits to patients, these treatments have limitations such as high invasiveness, high dose requirements, and a high tendency for off-target effects, which can lead to unintended damage to healthy tissues (Mura, S., Adv. Drug Delivery Rev., 2012). 64 , 1394-1416; Li, Z., Nanomed., 2018, 13 , 2283-2300; Zheng, X.,Biomaterials, 2018, 185 , 133-141; Zhu, M., J. Am. Chem. Soc. 2021, 143 (7541-7552). Photoactivation technology provides a highly advantageous non-invasive tool for biological tumor imaging or treatment systems through photoactive triggering factors, and has become a promising alternative for the efficient eradication of tumors.

[0003] Photothermal therapy is a treatment modality that utilizes photothermal agents to achieve photothermal conversion at specific lesion sites, thereby generating heat to destroy tumor cells or pathogens. Due to its minimally invasive and high-precision characteristics, this treatment method has received widespread attention (Dash, SR, Curr. Nanosci., 2022). 18 , 31-47; Zou, L., Theranostics,2016, 6 , 762-772; Wu, L., Angew. Chem. Int. Ed., 2024, 63 , e202405937.).

[0004] Lysosomal polarity is an important biological parameter, and changes in lysosomal polarity can affect normal lysosomal functions, including degradation, nutrient sensing, and immune responses. Recent studies have shown that loss of lysosomal polarity is associated with apoptosis and cell death. In cancer cells, lysosomal polarity is lower than in normal cells, making it a potential biomarker for cancer diagnosis. To date, fluorescent probes capable of monitoring lysosomal polarity have primarily been designed based on intramolecular charge transfer mechanisms, and significant progress has been made in lysosomal-targeted fluorescence imaging. It is well known that photothermal effects are typically generated through nonradiative relaxation processes (Miao, XF, Adv. Mater., 2023, ). 35 , 23017; Zhang, W., Adv. Mater., 2024, 36 (2314021.). Typically, photothermal agents should satisfy a low fluorescence quantum yield to maximize the conversion of absorbed photons into heat. This is why most reported probes for detecting lysosomal polarity exhibit unsatisfactory photothermal performance, as their enhanced emission hinders non-radiative processes. Therefore, significantly improving the photothermal therapeutic efficiency of probes while maintaining fluorescence imaging capabilities is a significant challenge. Summary of the Invention

[0005] The purpose of this invention is to provide a near-infrared fluorescent probe for targeting lysosomes in photothermal therapy, aiming to solve the problems of unsatisfactory photothermal performance and low efficiency of photothermal therapy in existing probes for detecting lysosomal polarity.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a targeted lysosomal near-infrared fluorescent probe for photothermal therapy, the molecular structure of which is shown in formula (I): (I); In the formula, R1 is selected from hydrogen, C 1～8 R2 is selected from any one of alkyl, hydroxyalkyl, alkoxyalkyl, and phenyl; R2 is selected from hydrogen and C. 1～8 Any one of alkyl or phenyl.

[0007] Specifically, to address the problems of unsatisfactory photothermal performance and low photothermal therapy efficiency of existing probes for detecting lysosomal polarity, this invention designs a near-infrared fluorescent probe for targeting lysosomes. This is a fluorescent probe based on hydrazone structure. A key feature of this probe is polarity-triggered azo-hydrazone conversion. The fluorescent hydrazone form (–NH–N=) can identify the low-polarity microenvironment of cancer cell lysosomes, while the azo form (–N=N–) generates a photothermal effect by enhancing nonradiative relaxation. The balanced coexistence of the hydrazone and azo forms endows the probe of this invention with dual functions of fluorescent imaging and photothermal therapy, enabling it to specifically target cancer cell lysosomes and simultaneously treat tumors.

[0008] Specifically, this invention relates to an improvement in fluorescent probe technology that integrates tumor diagnosis and treatment, particularly to a polar-activated near-infrared fluorescent probe based on an azo-hydrazone conversion strategy for lysosomal targeting and its preparation method, which can realize visualized diagnosis and non-invasive photothermal therapy of tumors.

[0009] This invention also provides a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy, the method comprising the following steps: Preparation of S1, nitrosylsulfuric acid: Nitrite is mixed with sulfuric acid and dissolved by stirring under heating or no heating conditions to obtain nitrite sulfuric acid; S2. Preparation of diazotization solution: Under ice bath conditions, the nitrosylsulfuric acid was added dropwise to 2-amino-3-cyano-4-chloro-5-formylthiophene to obtain solution A, and then phosphoric acid was added dropwise to carry out the reaction, and a diazotized solution was obtained after the reaction. S3. Preparation of intermediate compounds: Dissolve 1-N-R1-6-hydroxy-4-methyl-2-pyridone to obtain a pyridone solution; R1 is selected from hydrogen, C 1～8 Any one of alkyl, hydroxyalkyl, alkoxyalkyl, and phenyl; Under ice bath conditions, the diazotization solution was added dropwise to the pyridone solution to carry out the reaction. After the reaction, the mixture was filtered, the filter cake was washed, and the mixture was recrystallized to obtain the intermediate compound. S4. Preparation of near-infrared fluorescent probes targeting lysosomes: The intermediate compound and 6-hydroxy-1,3-R2-pyrimidine-2,4(1H,3H)-dione were added to acetic anhydride and heated to react. After the reaction, the mixture was cooled, filtered, washed, and dried to obtain a near-infrared fluorescent probe targeting lysosomes, the molecular structure of which is shown in formula (I); R2 is selected from hydrogen, C 1～8 Any one of alkyl or phenyl.

[0010] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: in step S1, sodium nitrite is selected as the nitrite, and concentrated sulfuric acid is used as the sulfuric acid.

[0011] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: the molar ratio of nitrite to sulfuric acid in step S1 is 1:(10-15).

[0012] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: the reaction temperature in step S2 is 0-5℃, and the reaction time is 20-30 minutes.

[0013] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: the volume ratio of nitrosylsulfuric acid to phosphoric acid in step S2 is 1:(1-1.5); the addition ratio of nitrosylsulfuric acid to 2-amino-3-cyano-4-chloro-5-formylthiophene is (2-3) mL:(0.9-1) g.

[0014] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: the solvent in step S3 is a mixed solvent of methanol and water with a volume ratio of (1-4):1; the reaction temperature is 0-5℃, and the reaction time is 1.5-2 hours.

[0015] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: in step S3, the mass-to-volume ratio of 1-N-R1-6-hydroxy-4-methyl-2-pyridone to the solvent is 0.03-0.05 g / mL; the mass ratio of 2-amino-3-cyano-4-chloro-5-formylthiophene to 1-N-R1-6-hydroxy-4-methyl-2-pyridone is (0.8-1):(1-1.1).

[0016] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: in step S4, the reaction temperature is 80–120°C and the reaction time is 1–3 hours.

[0017] Furthermore, a method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy: the molar ratio between the intermediate compound, 6-hydroxy-1,3-R2 pyrimidine-2,4(1H,3H)-dione and acetic anhydride in step S4 is 1:(1-2):(20-50).

[0018] The beneficial effects of this invention are: The near-infrared fluorescent probe for photothermal therapy of this invention achieves a high sensitivity response of the probe to lysosomal polarity through an azo-hydrazone conversion strategy, and has the integrated function of tumor fluorescence imaging and photothermal therapy.

[0019] This invention presents a near-infrared fluorescent probe for photothermal therapy targeting lysosomes. Addressing the challenge of existing lysosomal targeting probes failing to simultaneously achieve fluorescence imaging and photothermal therapy functions, this probe is designed based on azo-hydrazone conversion. This probe undergoes azo-hydrazone structural conversion in different polarity environments. Under low polarity conditions (such as cancer cell lysosomes), it is predominantly hydrazone in structure, producing strong fluorescence emission. In high polarity environments, it converts to an azo structure, resulting in reduced fluorescence. This achieves specific targeting of cancer cell lysosomes and highly efficient photothermal therapy.

[0020] The near-infrared fluorescent probe for targeting lysosomes prepared in this invention has absorption and emission in the near-infrared region, good biological tissue penetration ability, and has demonstrated excellent tumor diagnostic imaging and photothermal therapy effects in mouse models. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The 1H NMR spectrum of the targeted lysosomal near-infrared fluorescent probe of Example 1; Figure 2 The carbon NMR spectrum of the near-infrared fluorescent probe targeting lysosomes in Example 1; Figure 3 This is a high-resolution mass spectrum of the near-infrared fluorescent probe targeting lysosomes in Example 1; Figure 4 The absorption spectra of the targeted lysosomal near-infrared fluorescent probe of Example 1 in different polar solvents are shown. Figure 5 The fluorescence emission spectra of the targeted lysosomal near-infrared fluorescent probe of Example 1 in different polar solvents are shown. Figure 6 This is a graph evaluating the photothermal properties of the targeted lysosomal near-infrared fluorescent probe from Example 1. Figure 7 This is a cellular colocalization fluorescence image of the targeted lysosomal near-infrared fluorescent probe from Example 1; Figure 8 Fluorescence imaging and quantitative analysis of the targeted lysosomal near-infrared fluorescent probe in cancer cells and normal cells, as shown in Example 1; Figure 9 This is a graph showing the cytotoxicity and apoptosis assessment of the near-infrared fluorescent probe targeting lysosomes in Example 1. Figure 10The image shows fluorescence imaging of the targeted lysosomal near-infrared fluorescent probe of Example 1 in living organs and tumor sites. Figure 11 The image shows a photothermal image of the targeted lysosomal near-infrared fluorescent probe of Example 1 in tumor-bearing mice and a real-time record of temperature changes at the tumor site during light irradiation. Figure 12 This is an in vivo antitumor therapeutic effect evaluation diagram of the targeted lysosomal near-infrared fluorescent probe of Example 1. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Example 1

[0024] This embodiment 1 provides a targeted lysosomal near-infrared fluorescent probe for photothermal therapy, the molecular structure of which is shown in formula (Ⅰ): (I); In the formula, R1 is 3-isopropoxypropyl (one of the alkoxyalkyl groups); R2 is methyl (C 1～8 (One of the alkyl groups).

[0025] The preparation method of the targeted lysosomal near-infrared fluorescent probe for photothermal therapy described in Example 1 above includes the following specific steps: Preparation of S1, nitrosylsulfuric acid: Mix 0.227 g (3.30 mmol) of sodium nitrite (NaNO2) with 2.5 mL of concentrated sulfuric acid (98% by mass) and stir rapidly until dissolved. If the dissolution is slow, heat slightly to dissolve it to obtain nitrosylsulfuric acid. S2. Preparation of diazotization solution: Under ice bath conditions (0–5 °C), the above-mentioned nitrosylsulfuric acid was added dropwise to 0.94 g (5.00 mmol) of 2-amino-3-cyano-4-chloro-5-formylthiophene in a three-necked flask to obtain a dark brown solution A. Then, 2.5 mL of phosphoric acid was added dropwise, and the mixture was stirred rapidly for 30 minutes to obtain a diazotized solution. S3. Preparation of intermediate compounds: 1.05 g (5.00 mmol) of N-(3-isopropoxypropyl)pyridine-2,6-dione was dissolved in 30.0 mL of solvent (a mixture of methanol and water in a volume ratio of 2:1) to obtain a pyridinone solution. Then, under ice bath stirring conditions (0–5 °C), the above diazotized solution was added dropwise to the pyridinone solution and reacted for 2 hours. After the reaction, the mixture was filtered under reduced pressure, and the filter cake was washed successively with water and ethanol. The crude product was purified by recrystallization from ethanol to obtain approximately 2.46 g of a red intermediate compound, with a yield of approximately 84.0%. S4. Preparation of near-infrared fluorescent probes targeting lysosomes: 2.00 mmol of the intermediate compound and 2.00 mmol of 6-hydroxy-1,3-dimethylpyrimidine-2,4(1H,3H)-dione were added to 5.00 mL (approximately 53.00 mmol) of acetic anhydride and heated to 90 °C for 2 hours. After the reaction, the mixture was cooled to room temperature, filtered, and the precipitate was washed with isopropanol and dried to obtain approximately 0.56 g of a purple targeting lysosome near-infrared fluorescent probe, with a yield of approximately 48.0%.

[0026] The reaction process for preparing the targeted lysosomal near-infrared fluorescent probe is as follows: test: (1) The near-infrared fluorescent probe targeting lysosomes prepared in Example 1 was subjected to NMR testing, and the results are as follows: Figure 1 As shown, Figure 1 The 1H NMR spectrum of the targeted lysosome near-infrared fluorescent probe prepared in Example 1 confirms that the final product, the targeted lysosome near-infrared fluorescent probe, was obtained. A characteristic proton resonance peak appears at δ = 15.54 ppm, which corresponds to the N–H protons that form hydrogen bonds with the carbonyl group, indicating that the targeted lysosome near-infrared fluorescent probe mainly exists in the hydrazone structure in CDCl3.

[0027] 1 ¹H NMR (400 MHz, CDCl₃, ppm): δ = 15.54 (s, 1H), 8.81 (s, 1H), 4.13(t, J = 6.7 Hz, 2H), 3.49 (m, 3H), 3.43 (s, 6H), 2.65 (s, 3H), 1.92 (m, J =6.4 Hz, 2H), 1.07 (d, J = 6.1 Hz, 6H). (2) The near-infrared fluorescent probe targeting lysosomes prepared in Example 1 was subjected to NMR spectroscopy, and the results are as follows: Figure 2 As shown, Figure 2 The image shows the carbon NMR spectrum of the near-infrared fluorescent probe targeting lysosomes prepared in Example 1. The carbon NMR spectrum further confirms that the final product, the near-infrared fluorescent probe targeting lysosomes, was obtained.

[0028] 13 C NMR (100 MHz, CDCl3, ppm): δ = 162.10, 161.65, 161.62, 161.07, 158.75, 156.74, 150.86, 142.50, 140.82, 128.29, 122.96, 113.06, 111.97, 110.31, 107.85, 97.71, 71.57, 65.96, 39.03, 29.11, 28.40, 27.90, 21.98, 16.70. (3) The near-infrared fluorescent probe targeting lysosomes prepared in Example 1 was subjected to NMR spectroscopy, and the results are as follows: Figure 3 As shown, Figure 3 The high-resolution mass spectrum of the near-infrared fluorescent probe targeting lysosomes prepared in Example 1 further confirms that the final product, the near-infrared fluorescent probe targeting lysosomes, was obtained.

[0029] Mass spectrometry (ESI negative ion mode for [MH] - ): Calc. forC 25 H 23 ClN7O6S: 584.1119; found: 584.1117. (4) Polarity sensitivity test of the fluorescent probe prepared in Example 1: A stock solution of dimethyl sulfoxide (DMSO) containing a 2.0 mM fluorescent probe was prepared. Then, 30.0 μL of the stock solution was transferred to 3.0 mL volumetric flasks numbered ①, ②, ③, ④, ⑤, ⑥, and ⑦, respectively. The flasks were then diluted to volume with different polar solvents: 1,4-dioxane (1,4-Dioxane), chloroform (TCM), ethyl acetate (EA), dichloroform (DCM), methanol (MeOH), dimethyl sulfoxide (DMSO), and PBS buffer solution, respectively, to obtain a final test solution concentration of 2 × 10⁻⁶. -5 M, respectively test the absorption spectra of the fluorescent probe in solvents of different polarities (e.g. Figure 4 (as shown) and fluorescence emission spectra (as shown) Figure 5 (as shown) Depend on Figure 4It can be seen that in a weakly polar environment, the fluorescent probe prepared in Example 1 shows an absorption peak at ~505 nm, which is caused by the intramolecular π–π* electronic transition of the dominant hydrazone structure; when the solvent polarity increases from dielectric constant ε = 2.21 (1,4-dioxane) to ε = 46.45 (DMSO), the absorption peak at ~505 nm is significantly red-shifted to 657 nm in DMSO, which indicates that the fluorescent probe changes from a fluorescent hydrazone structure to a non-fluorescent deprotonated azo structure; Figure 5 The fluorescence intensity of the fluorescent probe decreased by nearly 43 times at 626 nm, further confirming this structural transformation. In addition, the absorption peaks of the fluorescent probe in MeOH and DMSO have similar characteristics to the absorption peaks in the low-energy bands of 1,4-dioxane, TCM and EA (marked by the dashed box in Figure 4), indicating that the fluorescent probe exhibits a coexistence of hydrazone and azo structures in weakly polar solvents.

[0030] (5) Photothermal performance test of the fluorescent probe prepared in Example 1: A 40 μM fluorescent probe solution was prepared using 1,4-dioxane, PBS, and DMSO. Using a blank PBS solution as a reference, the solution was irradiated for 5 minutes with a 635 nm laser (power density 1.33 W / cm²), and temperature changes were monitored in real-time using a thermal imager. The results are as follows: Figure 6 As shown in (a), the fluorescent probe exhibited only a mild heating effect in 1,4-dioxane, but a significant heating effect in DMSO. After 5 minutes of laser irradiation, the temperature rapidly rose to approximately 75°C. This result confirms that the dominant azo structure in DMSO endows the fluorescent probe with excellent photothermal efficiency. In PBS solution, the fluorescent probe reached ~50°C after 5 minutes of laser irradiation, indicating that the fluorescent probe coexists with both azo and hydrazone structures. This temperature is very suitable for photothermal therapy of tumors in vivo. Calculations of the photothermal conversion efficiency from the heating and natural cooling curves of the fluorescent probe samples showed that the photothermal conversion efficiency of the fluorescent probe in PBS was 40%. Figure 6 (b); Similarly, the PBS solution of the fluorescent probe was subjected to continuous laser irradiation to study different concentrations (e.g., Figure 6 (c) and laser power (e.g.) Figure 6 (d) Effect on photothermal performance: The results showed that the photothermal performance of the probe was concentration-dependent and laser power-dependent, and remained stable after three cycles, indicating that its superior azo structure has excellent photothermal performance and is suitable for photothermal therapy.

[0031] (6) Cell colocalization test of fluorescent probes prepared in Example 1: HeLa cells were loaded at a rate of 2 × 10 5 Inoculate at a density of cells / well in glass-bottomed petri dishes, incubate for 12 hours, then remove the culture medium and add 2×10⁻⁶ cells / well.-5 M fluorescent probe was incubated at 37°C for 2 hours; subsequently, these HeLa cells were co-incubated with the commercial tracking agent LysoTracker Green (200 nM) at 37°C for 30 minutes; then, the HeLa cells were imaged using a laser confocal scanning microscope, and the results are as follows. Figure 7 As shown, the red fluorescence of the fluorescent probe highly overlaps with the green fluorescence of LysoTrackerGreen, with an average Pearson colocalization coefficient as high as 0.97, indicating that the fluorescent probe has excellent lysosomal targeting ability.

[0032] (7) Fluorescence imaging test of the fluorescent probe prepared in Example 1 in cancer cells and normal cells: Cancer cells (HeLa and Panc1) and normal cells (HK2 and L929) were first cultured in DMEM or RPMI 1640 medium (containing 1% antibiotics and 10% fetal bovine serum) for 12 hours, and then 2 × 10⁻⁶ cells were added to the cells. -5 M fluorescent probe was incubated for 2 hours, and then cell images were captured using a Leica laser scanning confocal microscope, as shown below. Figure 8 As shown in (a), the average fluorescence intensity within the cells was quantitatively analyzed, as follows: Figure 8 As shown in (b), the average fluorescence intensity of cancer cells (HeLa, Panc1) is 2.0 times that of normal cells (HK2, L929), which indicates that the fluorescent probe can effectively distinguish cancer cells from normal cells by taking advantage of the lower polarity of cancer cell lysosomes.

[0033] (8) In vitro cytotoxicity and apoptosis test of the fluorescent probe prepared in Example 1: Cytotoxicity was determined using the MTT assay. HeLa cells were seeded at a density of 5000 cells per well in 96-well plates, with 100 μL of culture medium added to each well. The cells were incubated overnight at 37°C. Subsequently, the cells were treated with a 0–40 μM fluorescent probe for 120 minutes. After two washes, the cells were treated with a 635 nm laser (0.5 W / cm²) for 5 minutes each. Untreated cells served as a control. Cell viability was then assessed after another 24 hours of incubation. The results are shown below. Figure 9 As shown in (a), the fluorescent probe exhibits low cytotoxicity and a cell viability >90% under dark conditions. However, after irradiation with a 635nm laser, it induces dose-dependent apoptosis. Live and dead cell staining experiments were performed using Calcein-AM and propidium iodide (PI), and the results are as follows. Figure 9As shown in (b), HeLa cells showed significant cell death after treatment with the fluorescent probe combined with 635nm laser irradiation; however, no such phenomenon was observed in the group treated with the fluorescent probe alone or in the blank phosphate-buffered saline (PBS) control group; further flow cytometry experiments showed that cells treated with the fluorescent probe combined with laser underwent early apoptosis within a short period of time, such as Figure 9 (c).

[0034] (9) Fluorescence imaging tests of the fluorescent probes prepared in Example 1 on living organs and tumor sites: 1×10⁻⁶ mice were subcutaneously injected. 6 One HeLa cell, until the tumor grows to 50-70 mm. 3 Subsequently, a fluorescent probe (1.0 mg / kg, 125 μL) was injected via the tail vein; mice were sacrificed at different time points after injection, and their major organs and tumors were isolated. Fluorescence imaging was performed using an in vivo imaging system, such as... Figure 10 As shown, one hour after the probe was injected, the tumor site exhibited a significantly higher fluorescence signal than other organs, indicating that the fluorescent probe can specifically accumulate at the tumor site and has cancer targeting properties.

[0035] (10) Photothermal imaging test of the fluorescent probe prepared in Example 1 in tumor-bearing mice: 1×10⁻⁶ mice were subcutaneously injected. 6 One HeLa cell, until the tumor grows to 50-70 mm. 3 Subsequently, a fluorescent probe (1.0 mg / kg, 125 μL) was injected via the tail vein; one hour after injection, the tumor site was irradiated with a 635 nm laser (power density 0.5 W / cm²) for 5 minutes, and temperature changes were recorded in real time using a thermal imager. Figure 11 As shown in (a) and 11(b), the tumor surface temperature in the fluorescent probe + laser group rapidly increased from 32°C to 50°C within 5 minutes, while the PBS control group only showed a slight increase in temperature. This indicates that the fluorescent probe can still produce an effective photothermal effect in living tumors.

[0036] (11) In vivo antitumor therapy test of the fluorescent probe prepared in Example 1: When the tumor volume in HeLa tumor-bearing mice reached approximately 80 mm... 3 Mice were randomly divided into four groups: fluorescent probe + laser group, fluorescent probe group, PBS + laser group, and PBS group. Drug administration was administered via tail vein injection combined with laser treatment. Tumor volume and mouse weight were monitored regularly. After treatment, tumor tissue was collected for pathological analysis. Results are as follows: Figure 12 As shown, only the fluorescent probe + laser combination effectively inhibited tumor growth, achieving a tumor inhibition rate of 80% (e.g., Figure 12 (a) and 12(b)) and the mice's weight remained stable ( Figure 12 (c) H&E and TUNEL staining showed that this group of tumor cells exhibited extensive cell death and apoptosis. Figure 12 (d) indicates that fluorescent probe-mediated photothermal therapy has excellent anti-tumor effects in vivo. Example 2

[0037] This embodiment 2 provides a targeted lysosomal near-infrared fluorescent probe for photothermal therapy. The molecular structure of the targeted lysosomal near-infrared fluorescent probe is shown in formula (Ⅰ): (I); In the formula, R1 is methyl; R2 is phenyl.

[0038] The difference between the preparation method of the targeted lysosomal near-infrared fluorescent probe in Example 2 and the preparation method in Example 1 is that the raw materials containing R1 (methyl) and R2 (phenyl) groups are different; in step S1 of Example 2, the molar ratio of nitrite to sulfuric acid is 1:10; in step S2, the volume ratio of nitrosylsulfuric acid to phosphoric acid is 1:1.5, and the reaction time is 25 minutes; in step S3, the mass-volume ratio of 1-N-R1-6-hydroxy-4-methyl-2-pyridone to solvent is 0.05 g / mL, the mass ratio of 2-amino-3-cyano-4-chloro-5-formylthiophene to 1-N-R1-6-hydroxy-4-methyl-2-pyridone is 0.8:1, and the reaction time is 1.5 hours; in step S4, the reaction temperature is 100℃, the reaction time is 1.5 hours, and the molar ratio between the intermediate compound, 6-hydroxy-1,3-R2 pyrimidine-2,4(1H,3H)-dione, and acetic anhydride is 1:2:50. Example 3

[0039] This embodiment 3 provides a targeted lysosomal near-infrared fluorescent probe for photothermal therapy. The molecular structure of the targeted lysosomal near-infrared fluorescent probe is shown in formula (Ⅰ). (I); In the formula, R1 is phenyl; R2 is phenyl.

[0040] The preparation method of the targeted lysosomal near-infrared fluorescent probe in Example 3 differs from that in Example 1 in that the raw materials containing R1 (phenyl) and R2 (phenyl) groups are different. In step S1 of Example 2, the molar ratio of nitrite to sulfuric acid is 1:15; in step S2, the volume ratio of nitrosylsulfuric acid to phosphoric acid is 1:1.2, and the reaction time is 20 minutes; in step S3, the mass-volume ratio of 1-N-R1-6-hydroxy-4-methyl-2-pyridone to solvent is 0.04 g / mL, the mass ratio of 2-amino-3-cyano-4-chloro-5-formylthiophene to 1-N-R1-6-hydroxy-4-methyl-2-pyridone is 1:1.1, and the reaction time is 1.5 hours; in step S4, the reaction temperature is 80℃, the reaction time is 3 hours, and the molar ratio between the intermediate compound, 6-hydroxy-1,3-R2 pyrimidine-2,4(1H,3H)-dione, and acetic anhydride is 1:1.5:40.

[0041] This invention provides a near-infrared fluorescent probe for targeting lysosomes based on a hydrazone structure. The probe has a polarity-triggered azo-hydrazone switching structure. The fluorescent hydrazone form can identify the low-polarity microenvironment of cancer cell lysosomes, while the azo form generates a photothermal effect by enhancing the non-radiative relaxation effect. The balanced coexistence of the hydrazone and azo forms endows the probe of this invention with dual functions of fluorescence imaging and photothermal therapy, enabling it to specifically target cancer cell lysosomes and simultaneously treat tumors.

[0042] This invention provides a near-infrared fluorescent probe targeting lysosomes. Through an azo-hydrazone conversion strategy, the probe achieves a high sensitivity response to lysosomal polarity and integrates tumor fluorescence imaging and photothermal therapy.

[0043] The above-described preferred embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of the invention. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A targeted lysosomal near-infrared fluorescent probe for photothermal therapy, characterized in that, The molecular structure of this lysosomal near-infrared fluorescent probe is shown in formula (Ⅰ): (I); In the formula, R1 is selected from hydrogen, C 1～8 R2 is selected from any one of alkyl, hydroxyalkyl, alkoxyalkyl, and phenyl; R2 is selected from hydrogen and C. 1～8 Any one of alkyl or phenyl.

2. The method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 1, characterized in that, The method includes the following steps: Preparation of S1, nitrosylsulfuric acid: Nitrite is mixed with sulfuric acid and dissolved by stirring under heating or no heating conditions to obtain nitrite sulfuric acid; S2. Preparation of diazotization solution: Under ice bath conditions, the nitrosylsulfuric acid was added dropwise to 2-amino-3-cyano-4-chloro-5-formylthiophene to obtain solution A, and then phosphoric acid was added dropwise to carry out the reaction, and a diazotized solution was obtained after the reaction. S3. Preparation of intermediate compounds: Dissolve 1-N-R1-6-hydroxy-4-methyl-2-pyridone to obtain a pyridone solution; R1 is selected from hydrogen, C 1～8 Any one of alkyl, hydroxyalkyl, alkoxyalkyl, and phenyl; Under ice bath conditions, the diazotization solution was added dropwise to the pyridone solution to carry out the reaction. After the reaction, the mixture was filtered, the filter cake was washed, and the mixture was recrystallized to obtain the intermediate compound. S4. Preparation of near-infrared fluorescent probes targeting lysosomes: The intermediate compound and 6-hydroxy-1,3-R2-pyrimidine-2,4(1H,3H)-dione were added to acetic anhydride and heated to react. After the reaction, the mixture was cooled, filtered, washed, and dried to obtain a near-infrared fluorescent probe targeting lysosomes, the molecular structure of which is shown in formula (I); R2 is selected from hydrogen, C 1～8 Any one of alkyl or phenyl.

3. The method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2, characterized in that, In step S1, sodium nitrite is used as the nitrite and concentrated sulfuric acid is used as the sulfuric acid.

4. A method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2 or 3, characterized in that, The molar ratio of nitrite to sulfuric acid in step S1 is 1:(10-15).

5. The method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2, characterized in that, The reaction temperature in step S2 is 0–5°C, and the reaction time is 20–30 minutes.

6. A method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2 or 5, characterized in that, In step S2, the volume ratio of nitrosylsulfuric acid to phosphoric acid is 1:(1-1.5); the addition ratio of nitrosylsulfuric acid to 2-amino-3-cyano-4-chloro-5-formylthiophene is (2-3) mL:(0.9-1) g.

7. The method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2, characterized in that, The solvent used in step S3 is a mixture of methanol and water with a volume ratio of (1-4):1; the reaction temperature is 0-5℃ and the reaction time is 1.5-2 hours.

8. A method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2 or 7, characterized in that, In step S3, the mass-to-volume ratio of 1-N-R1-6-hydroxy-4-methyl-2-pyridone to the solvent is 0.03–0.05 g / mL; The mass ratio of 2-amino-3-cyano-4-chloro-5-carboxythiophene to 1-N-R1-6-hydroxy-4-methyl-2-pyridone is (0.8-1):(1-1.1).

9. A method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2, characterized in that, In step S4, the reaction temperature is 80–120°C and the reaction time is 1–3 hours.

10. A method for preparing a targeted lysosomal near-infrared fluorescent probe for photothermal therapy according to claim 2 or 9, characterized in that, The molar ratio of the intermediate compound, 6-hydroxy-1,3-R2 pyrimidine-2,4(1H,3H)-dione, and acetic anhydride in step S4 is 1:(1-2):(20-50).

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