A dual-mode responsive probe, and a preparation method and application thereof

Abstract

The application discloses a kind of dual-mode response probe and its preparation method and application, the probe is compound MB-BIO, it has tumor cell targeting and lysosome positioning ability, can realize tumor-specific imaging and photodynamic therapy under the stimulation of HClO or near infrared light.Experiments in vitro show that MB-BIO response is rapid (HClO response <5s, near infrared light response 10min), high sensitivity (HClO detection limit 15.6nM), excellent selectivity, while active oxygen can be efficiently produced.The probe described in the application can effectively distinguish cancer cells from normal cells, realize precise positioning of lysosome, and can kill cancer cells by light control powerful.The zebra fish and tumor-bearing mouse live fluorescence imaging experiment verifies the dual-mode activation characteristics of the probe for HClO and NIR, can realize high-contrast tumor imaging, and shows excellent photodynamic therapy effect at the level of live body, confirms its application potential in precise tumor diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a dual-mode response probe, its preparation method, and its application. Background Technology

[0002] Cancer remains a serious threat to human health, with the global mortality rate rising year by year. Current clinical cancer diagnosis mainly relies on imaging techniques such as computed tomography (CT) and positron emission tomography (PET), as well as histopathological biopsies. Traditional imaging can achieve gross localization and morphological assessment of tumors, but its sensitivity for detecting small lesions and early-stage tumors is insufficient; histopathological biopsies are invasive and difficult to use for dynamic monitoring of tumor progression and treatment response.

[0003] In terms of treatment, surgical resection, radiotherapy, and chemotherapy remain the mainstream methods, but they have problems such as high invasiveness, easy drug resistance, significant systemic toxicity, and poor targeting, so there is an urgent need to supplement treatment strategies. More importantly, the existing diagnostic and treatment models are often separated, lacking an integrated and intelligent diagnosis and treatment system, which makes it difficult to meet the urgent needs of personalized and efficient tumor treatment in the era of precision medicine.

[0004] In recent years, activated fluorescent probes have demonstrated great potential in precision tumor diagnosis and treatment due to their advantages such as real-time visualization, non-invasive detection, high sensitivity, high specificity, and integrated imaging-therapy, becoming an important strategy to overcome the limitations of traditional methods. The unique biological characteristics of the tumor microenvironment provide specific targets for the rational design of intelligent responsive probes. Among them, the abnormal enrichment of reactive oxygen species (ROS) is an important marker of malignant tumors: metabolic disorders in tumor cells lead to intracellular ROS levels that are approximately 10 times higher than in normal cells, providing a reliable basis for tumor-specific identification. Hypochlorous acid (HClO), as one of the major ROS, has a significantly higher concentration in tumor tissues than in normal tissues. It is not only a specific biomarker reflecting tumor progression and oxidative stress, but also participates in the processes of tumor cell proliferation, invasion, and drug resistance, making it a key target for the development of tumor-specific diagnostic and therapeutic probes.

[0005] Although various HClO-responsive probes have been developed for tumor detection, existing systems still face numerous technical bottlenecks: most probes are only used for detection and lack diagnostic-therapeutic integration capabilities; single response modes and single signal outputs can easily lead to high false negative rates and poor in vivo imaging results; some probes with diagnostic and therapeutic functions suffer from slow response dynamics, insufficient therapeutic effects, and poor targeting of cancer cells and subcellular organelles, resulting in low tumor enrichment efficiency and failing to meet the stringent requirements of integrated precision tumor diagnosis and treatment.

[0006] Therefore, developing fluorescent probes that can simultaneously target cancer cells and subcellular organelles, provide rapid dual-mode response, and integrate imaging and therapy is of great scientific significance for overcoming the aforementioned bottlenecks and achieving precise tumor monitoring.

[0007] Achieving precise targeting of tumor cells and subcellular organelles is a core strategy for improving probe enrichment efficiency, reducing systemic toxicity, and enhancing diagnostic and therapeutic specificity. Many cancer cells highly express biotin receptors on their surface, providing a feasible pathway for active targeting: modifying the probe backbone with biotin units can significantly enhance the passive enrichment and active uptake of probes in tumor tissues through receptor-mediated active targeting.

[0008] Furthermore, lysosomes, as key subcellular organelles within cancer cells, participate in metabolic and apoptosis regulation, containing a series of enzymes and secreted proteins related to apoptosis signals, invasion, and metastasis. Their internal acidic environment (pH 4.5–6.0) and high ROS (especially HClO) levels provide ideal triggering conditions for designing tumor-specific probes. Introducing basic targeting groups such as dimethylformyl chloride can achieve lysosome-specific enrichment, providing a simple chemical modification strategy for precision diagnosis and treatment at the subcellular level.

[0009] In summary, integrating active tumor cell targeting, lysosomal subcellular targeting, and HClO-specific response represents the optimal design paradigm for constructing high-performance intelligent diagnostic and therapeutic probes. This approach is expected to improve lesion specificity and treatment precision, laying the foundation for targeted imaging and on-demand treatment.

[0010] In the field of cancer treatment, photodynamic therapy (PDT) is a non-invasive targeted therapy technology: photodynamic agents generate a large amount of reactive oxygen species (ROS) with strong oxidizing capabilities under specific wavelengths of light, selectively killing cancer cells. The effect of PDT is mainly based on three factors: photosensitizers (PS), light, and oxygen; the interaction of these three components leads to the production of ROS. PDT has advantages such as high specificity, low systemic toxicity, repeatability, and no induction of drug resistance, and has been gradually applied to the treatment and research of various malignant tumors.

[0011] Methylene blue (MB) is a high-performance near-infrared fluorophore and photosensitizer with high fluorescence quantum yield, good biocompatibility, easy molecular modification, and singlet oxygen (…). 1 Near-infrared chemiluminescence (MB) and other reactive oxygen species (ROS) have high quantum yields, making them potent photosensitizers for clinical photodynamic therapy. Simultaneously, MB possesses excellent photoacoustic (PA) imaging capabilities, allowing for the construction of photoacoustic probes with superior tissue penetration depth compared to traditional fluorescence imaging, making it suitable for high-resolution imaging of deep tumors. Therefore, MB is widely used in near-infrared fluorescence imaging, photoacoustic imaging, and photodynamic therapy. However, traditional MB formulations suffer from clinical bottlenecks such as low delivery efficiency, poor tumor targeting, ambiguous lesion localization, and separation of diagnostic and therapeutic functions, severely limiting the efficacy of photodynamic therapy.

[0012] Based on the aforementioned background and technological gaps, this application aims to rationally design and prepare a fluorescent probe MB-BIO that combines cancer cell targeting, lysosome specificity, and HClO / near-infrared dual-mode activation for the integration of tumor-specific imaging and efficient photodynamic therapy. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to provide a dual-mode response probe that integrates cancer cell-lysosome dual targeting, HClO / near-infrared dual-mode activated fluorescence imaging and photodynamic therapy, as well as its preparation method and application.

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0015] A dual-mode response probe, denoted as compound MB-BIO, has the structure shown below:

[0016] .

[0017] The preparation method of the above-mentioned dual-mode response probe includes the following steps:

[0018] 1) Take compound MB-Cl and 1,3-diamino-2-propanol in a first organic solvent, and carry out a substitution reaction with or without the addition of a basic substance to obtain compound MB-NH2-OH;

[0019] 2) Take compound MB-NH2-OH and (+)-biotin-N-hydroxysuccinimide ester (BNHS, CAS No.: 35013-72-0) in a second organic solvent, and carry out a substitution reaction with or without the addition of an alkaline substance to obtain compound MB-BIO-OH;

[0020] 3) Place the compound MB-BIO-OH in a third organic solvent, add an alkaline substance, and then add dimethylformyl chloride to react and obtain the crude product of the target compound;

[0021] , , .

[0022] In each step of the above preparation method, the addition of an alkaline substance is to make the system alkaline (pH value greater than 7). Higher yields can be obtained when the reaction is carried out under alkaline conditions. Specifically, in step 1), the preferred amount of alkaline substance added is to make the pH of the system 7.8~9.2; in step 2), the preferred amount of alkaline substance added is to make the pH of the system 8.0~8.5; and in step 3), the preferred amount of alkaline substance added is to make the pH of the system 8.0~9.5.

[0023] The alkaline substance can be a conventional organic base and / or inorganic base. Further, the organic base can be one or a combination of two or more selected from triethylamine (TEA), pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, potassium tert-butoxide, and sodium tert-butoxide, with triethylamine being preferred. The inorganic base is one or a combination of two or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, cesium hydroxide, and cesium carbonate, with sodium carbonate being preferred.

[0024] In steps 1) to 3) of the above preparation method, the reactions can be carried out under heating or non-heating conditions; that is, the reaction temperature can be room temperature or between room temperature and the boiling point of the organic solvent. The reaction is monitored by TLC until it is complete. Preferably, the reaction is carried out under heating conditions, more preferably at 30-50°C.

[0025] Furthermore, the reaction described in step 2) is carried out under protective atmosphere (inert atmosphere such as nitrogen or argon) to prevent interference from external humidity, thereby further improving the product yield.

[0026] In the preparation method described in this invention, the terms "first organic solvent," "second organic solvent," and "third organic solvent" all refer to organic solvents. The terms "first," "second," and "first" are merely used to distinguish different organic solvents used in different steps of the preparation method. Specifically, the first organic solvent can be one or a combination of two or more selected from dichloromethane (DCM), acetonitrile, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), dichloroethane (DCE), and tetrahydrofuran (THF); the second organic solvent can be one or a combination of two or more selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone; and the third organic solvent can be one or a combination of two or more selected from dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, and tetrahydrofuran. The amount of organic solvent used in each step can be determined as needed, generally sufficient to fully dissolve the raw materials to be reacted in that step. Specifically, based on 1 mmol of compound MB-Cl / compound MB-NH2-OH / compound MB-BIO-OH, the total amount of the first organic solvent / second organic solvent / third organic solvent is usually 2~6 mL.

[0027] Steps 1) and 2) above produce crude products of compounds MB-NH2-OH and MB-BIO-OH, respectively. Preferably, the crude products of compounds MB-NH2-OH and MB-BIO-OH are purified before being used in subsequent steps.

[0028] Further, the crude MB-NH2-OH obtained in step 1) is purified by silica gel column chromatography. The eluent used in the chromatography is preferably a mixed solvent of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 30:1 to 50:1. For the purification of the crude MB-BIO-OH obtained in step 2), it is preferable to first extract with water and ethyl acetate (or saturated sodium chloride aqueous solution and ethyl acetate), collect the organic phase, concentrate it under reduced pressure, and then perform silica gel column chromatography; the eluent used in the chromatography can be a mixed solvent of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 30:1 to 50:1.

[0029] The crude product of compound MB-BlO obtained by the above method is further purified to improve the purity of the target compound. Specifically, conventional purification methods can be used to improve the purity of compound MB-BlO, such as silica gel column chromatography. The eluent used in the chromatography is preferably a mixed solvent of dichloromethane and methanol, with a preferred volume ratio of 40:1 to 60:1.

[0030] In the preparation method described in this invention, the compound MB-Cl involved can be synthesized with reference to existing literature (P. Wei, L. Liu, Y. Wen, etc., Release of Amino- or Carboxy-Containing Compounds Triggered by HOCl: Application for Imaging and Drug Design, Angewandte Chemie 58(14)(2019)4547~4551. or W. Zheng, J. Yang, Y. Shen, etc., The near-infrared fluorescent probes based on phenoxazine for the rapid detection of hypochlorous acid, Dyes and Pigments 179 (2020) 108404. or P. Wei, W. Yuan, F. Xue, etc., Deformylation reaction-based probe for in vivo imaging of HOCl, Chemicalscience 9(2)(2018) 495~501.).

[0031] Through experiments, the applicant discovered that the probe MB-BIO described in this invention exhibits rapid response (HClO response <5s, near-infrared light response 10min), high sensitivity (HClO detection limit 15.6 nmol / L), and excellent selectivity, while also efficiently generating reactive oxygen species. This probe can effectively distinguish between cancer cells and normal cells, achieve precise lysosomal localization, and powerfully kill cancer cells through photocontrolled imaging. In zebrafish in vivo imaging, MB-BIO demonstrated excellent HClO activation performance, confirming its application potential in precision tumor diagnosis and treatment.

[0032] Based on the above findings, the present invention includes the application of the above dual-mode probe in at least one of the following (a1) to (a4):

[0033] (a1) Preparation of drugs for photodynamic therapy;

[0034] (a2) Cellular or biological imaging not for diagnostic or therapeutic purposes;

[0035] (a3) Preparation of in vivo tumor imaging drugs;

[0036] (a4) Preparation of reactive oxygen species.

[0037] Furthermore, when imaging within cells or organisms, the probe MB-BIO is excited at 660 nm.

[0038] Furthermore, the present invention also includes a product comprising the aforementioned dual-mode response probe MB-BIO.

[0039] Compared with existing technologies, the probe MB-BIO described in this invention uses biotin as the active tumor targeting unit and basic groups as the lysosomal targeting unit to achieve precise cancer cell identification and efficient lysosomal enrichment. The urea bond acts as a dual-mode response switch for HClO and near-infrared light. High concentrations of HClO at the lesion or exogenous near-infrared light irradiation can trigger irreversible specific cleavage of the urea bond, releasing reduced MB and simultaneously activating the near-infrared fluorescence signal, achieving real-time high-contrast tumor imaging. The activated MB photosensitizer efficiently generates ROS (containing...) under 660 nm laser irradiation. 1 O2) precisely kills cancer cells and destroys lysosomal structures, amplifying the anti-tumor treatment effect, and realizing an intelligent probe that integrates dual targeting of cancer cells and lysosomes, HClO / near-infrared dual-mode activated fluorescence imaging and photodynamic therapy. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the mechanism of action of MB-BlO.

[0041] Figure 2The spectral characteristics of MB-BIO are shown below. (a) Absorption spectra of MB-BIO (10 μM) under different concentrations of HClO (0–11.25 μM); (b) Fluorescence spectra; (c) Linear relationship between fluorescence intensity at 685 nm and HClO concentration (0–10 μM); (d) Fluorescence intensity of MB-BIO (10 μM) at 685 nm with and without HClO (0, 10 μM) as a function of time; (e) Fluorescence intensity of MB-BIO (10 μM) under laser irradiation (660 nm, 0.5 W / cm²). 2 (f) Absorption spectrum under laser irradiation (660 nm, 0.5 W / cm²); (g) Fluorescence spectrum; 2 (h) Evaluation of photostability and photoswitching behavior of MB-BIO (10 μM) under different pH conditions; (i) Evaluation of ROS generation capacity of MB-BIO (10 μM) by DPBF (20 μg / mL); (j) Photoacoustic images and corresponding signal intensities of MB-BIO (0~8 mM) at different concentrations after the addition of HClO (1 mM), with photoacoustic signals acquired at 680 nm; (k) Selectivity of MB-BIO (10 μM) for various interfering substances. Excitation / emission wavelengths: 620 / 685 nm; slit width: 10 × 10 nm. Data are expressed as mean ± standard error (n=3).

[0042] Figure 3 shows the DFT calculation of MB-BIO and the molecular docking simulation with the biotin transporter of human cancer cells. (a) DFT calculation of the corresponding energy levels of the frontier molecular orbitals of MB-BIO and MB; (b) Molecular docking simulation of MB-BIO with the biotin transporter of human cancer cells.

[0043] Figure 4 shows the fluorescence images of MB-BIO in A549 cells. (a) Fluorescence imaging of A549 cells under different conditions: blank control group; incubation with MB-BIO (10 μM, 0.5 h) only; incubation with MB-BIO (10 μM, 0.5 h) after pretreatment with HClO (10 μM, 30 min); incubation with MB-BIO (10 μM, 0.5 h) after pretreatment with LPS and PMA (1 μg / mL each, 3 h); pretreatment with LPS and PMA (1 μg / mL each, 3 h) → treatment with ABH (200 μM, 1 h) → incubation with MB-BIO (10 μM, 0.5 h); (b) Mean fluorescence intensity of cells in (a); (c) Fluorescence imaging of different cell lines after incubation with MB-BIO (10 μM, 0.5 h); (d) Mean fluorescence intensity of different cells in (c). Scale bar: 20 µm. Error bars represent the mean ± standard deviation (n=3). Excitation wavelength 640 nm, emission wavelength 650~750 nm.

[0044] Figure 5 shows the results of photodynamic therapy using calcein-AM and propidium iodide double staining to differentiate between live and dead cells and assess the cellular level of MB-BIO. (a) Co-localization of MB-BIO with lysosomes in A549 cells. Representative fluorescence images of A549 cells co-stained with MB-BIO (10 μM, excitation wavelength 640 nm, emission wavelength 650-750 nm) and Lyso-Tracker Green (0.1 μM, excitation wavelength 488 nm, emission wavelength 500-540 nm); (b, c) HepG2 (b) and A549 (c) cells incubated with MB-BIO (10 μM) and irradiated with near-infrared laser (660 nm, 0.5 W / cm²). 2 (d) Intracellular ROS were detected using H2DCFDA (10 μM); (d) Photodynamic therapy based on MB-BIO (660 nm, 0.5 W / cm²) 2 Following this, calcein-AM (green) / PI (red) staining was performed on live and dead cells. Scale bar: 20 μm.

[0045] Figure 6 shows in vivo fluorescence imaging of MB-BIO in zebrafish. (a) Representative confocal fluorescence images of zebrafish under different conditions: incubation with MB-BIO (20 μM, 1 h) only; incubation with MB-BIO (20 μM, 1 h) after pretreatment with HClO (5 μM or 10 μM, 1 h); incubation with MB-BIO (20 μM, 1 h) after pretreatment with PMA (1 μg / mL) and LPS (1 μg / mL) for 3 h; incubation with MB-BIO (20 μM, 1 h) after pretreatment with PMA (1 μg / mL) and LPS (1 μg / mL) for 3 h, followed by treatment with ABH (200 μM, 1 h), and finally incubation with MB-BIO (20 μM, 1 h); (b) Quantitative analysis of the average fluorescence intensity in (a). Error bars represent mean ± standard deviation (n=3). Scale bar: 200 μm. Excitation wavelength: 640 nm, emission wavelength: 650~750 nm.

[0046] Figure 7 This study presents the fluorescence imaging and photodynamic therapy effects of MB-BIO in tumor-bearing mice. (a) Schematic diagram of the A549 tumor-bearing BALB / c mouse model construction process and photodynamic therapy experimental operation; (b) In vivo tumor fluorescence imaging images after intratumoral injection of MB-BIO (100 μL, 200 μM) followed by irradiation with a 660 nm near-infrared laser (0.8 W / cm²) for different durations; (c) Curve of body weight change in tumor-bearing mice during the 10-day treatment cycle; (d) Tumor growth curve of mice during the 10-day treatment process; (e) Representative photographs of tumors in each treatment group. The sample size for each group was n=3, and the excitation wavelength was λ. ex =600nm, emission wavelength λ em =680 nm.

[0047] Figure 8 For MB-BIO 1 1H NMR spectrum (600 MHz, CDCl3).

[0048] Figure 9 For MB-BIO 13 C10 NMR spectrum (150 MHz, CDCl3).

[0049] Figure 10 This is a high-resolution mass spectrum of MB-BIO.

[0050] Figure 11 This is a high-resolution mass spectrum of the reaction product obtained after incubation of MB-BIO and HClO.

[0051] Figure 12 This is a high-resolution mass spectrum of the reaction products after near-infrared light irradiation of MB-BIO.

[0052] Figure 13 To evaluate the reactive oxygen species (ROS) generation performance of MB-BIO using 1,3-diphenylisobenzofuran (DPBF) as a detection indicator. (a) Positive control group: methylene blue (MB, 10 μM); (b) MB-BIO probe group (10 μM); DPBF-containing mixed solution at 660 nm (power density 0.5 W / cm²). 2 Changes in the ultraviolet-visible absorption spectrum under illumination.

[0053] Figure 14 This is a two-dimensional schematic diagram of the interaction between MB-BIO and biotin transporter.

[0054] Figure 15 The results show the cell viability of A549 cells and BEAS-2B cells after treatment with MB-BIO. (a) A549 cells; (b) BEAS-2B cells. Detailed Implementation

[0055] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0056] The compound MB-Cl involved in the following examples was prepared according to the following synthetic route:

[0057]

[0058] Preparation of compound MB-Cl: Methylene blue (1918.9 mg, 6.0 mmol) was dissolved in dichloromethane (50 mL) and stirred at room temperature under an inert nitrogen atmosphere to isolate it from oxygen. Then, a deionized aqueous solution (30 mL) of sodium carbonate (1260.0 mg, 18.0 mmol) and sodium dithionite (2089.3 mg, 12.0 mmol) was added. The resulting two-phase mixture was stirred at room temperature until the system turned distinctly yellow, indicating that methylene blue had been successfully reduced to its reduced state (compound 1). The mixture was cooled to 0 °C, and triethylamine (0.52 mL, 3.8 mmol) was added, followed by the slow dropwise addition of a dichloromethane solution (3.0 mL) of triphosgene (819.2 mg, 3.0 mmol). The reaction mixture was then heated to room temperature and stirred for 1 hour. After the reaction was complete, the mixture was quenched in ice water (100 mL) and extracted with dichloromethane (3 × 40 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (ethyl acetate / petroleum ether, v / v) to give compound MB-Cl. Yield: 1380.0 mg (3.98 mmol, 66.4%). 1 H NMR (600MHz, CDCl3) δ 7.39 (s, 2H), 6.70 (d, J=2.7Hz, 2H), 6.62 (dd, J=9.0, 2.8Hz, 2H), 2.95 (s, 12H). 13 C NMR (150MHz, CDCl3) δ 149.96, 149.39, 127.95, 126.65, 110.78, 110.28, 40.61. HRMS (ESI + C 17 H 18 ClN3OS [M+H] + Theoretical value: 348.0932, measured value: 348.0933.

[0059] Example 1: Preparation of compound MB-BIO

[0060]

[0061] 1) Preparation of compound MB-NH2-OH: 1,3-Diamino-2-propanol (0.2 mL) and triethylamine (0.2 mL) were placed in dichloromethane (5 mL), followed by the addition of MB-Cl (694 mg, 2.0 mmol). The reaction mixture was stirred at 40 °C for 3 hours. After the reaction was completed by thin-layer chromatography (TLC), the solvent was removed under reduced pressure to obtain the crude product MB-NH2-OH. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 40:1) to obtain compound MB-NH2-OH (light blue solid, 368 mg, 0.92 mmol, yield 46%). 1 H NMR (600MHz, CDCl3) δ 7.36–7.29 (m, 2H), 6.67 (d, J=3.5Hz, 2H), 6.60 (dd, J=7.5, 3.4Hz, 2H), 5.48 (q, J=4.7Hz, 1H), 3.63 (q, J=5.4, 4.9Hz, 1H), 3.33 ( dt, J=15.0, 4.6Hz, 1H), 3.28–3.12 (m, 6H), 2.90 (d, J=3.2Hz, 12H), 2.74 (dd, J=12.8, 4.2Hz, 1H), 2.62 (dd, J=13.0, 7.3Hz, 1H). 13 C NMR (150MHz, CDCl3) δ 157.37, 149.11, 134.30, 128.23, 127.27, 111.45, 111.01, 71.12, 44.59, 40.76. HRMS (ESI + C 20 H 28 N5O2S [M+H] + Theoretical value: 402.1958, measured value: 402.1975.

[0062] 2) Preparation of compound MB-BIO-OH: (+)-Biotin-N-hydroxysuccinimide ester (BNHS, 341 mg, 1.0 mmol) was dissolved in N,N-dimethylformamide (3 mL), triethylamine (0.14 mL, 1.0 mmol) was added, followed by compound MB-NH2-OH (360 mg, 0.9 mmol). The reaction mixture was heated to 50 °C and stirred for 8 hours under a nitrogen inert atmosphere. After the reaction was completed by TLC monitoring, saturated sodium chloride aqueous solution was added, and the mixture was extracted with ethyl acetate to remove DMF. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude MB-BIO-OH. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 40:1) to obtain compound MB-BIO-OH (light blue solid, 282 mg, 0.7 mmol, yield 45%). 1H NMR (600MHz, CDCl3) characteristic signals: δ 7.78 (d, J=8.0 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.29 (d, J=8.8 Hz, 2H), 7.22 (t, J=8.8 Hz, 2H), 6.84 (d, J=28.6 Hz, 1H), 6.65 (s, 2H), 6.58 (d, J=9.0 Hz, 1H), 6.10 (d, J=15.1 Hz, 1H), 5.59 (d, J=7.7 Hz, 1H). Hz, 1H), 4.38–4.35 (m, 1H), 4.20 (s, 1H), 3.72 (s, 1H), 3.27–3.24 (m, 2H), 3.08–3.04 (m, 5H), 2.89 (s, 12H), 2.78 (d, J=12.0 Hz, 1H), 2.61 (d, J=12.8Hz, 1H), 2.14 (t, J=7.9Hz, 2H), 1.68–1.53 (m, 6H). 13 150MHz, CDCl3 NMR (150MHz) characteristic signals: δ 174.90, 164.49, 157.36, 149.08, 143.57, 134.28, 128.15, 127.68, 127.24, 124.54, 123.77, 118.71, 111.38, 110.93, 70.59, 61.74, 60.32, 55.85, 44.41, 43.04, 40.73, 40.59, 35.89, 28.32, 28.07, 25.78. HRMS (ESI) + C 50 H42N7O4S2 + [M+H] + Theoretical value: 628.2734, measured value: 628.2759.

[0063] 3) Preparation of compound MB-BIO: Compound MB-BIO-OH (250 mg, 0.4 mmol) was dissolved in acetonitrile (5 mL), triethylamine (0.1 mL, 0.72 mmol) was added, and then dimethylformyl chloride (0.2 mL, 1.86 mmol) was slowly added dropwise. The reaction solution was refluxed at 50 °C and stirred for 4 h. After the reaction was completed, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol, v / v 50:1) to obtain compound MB-BIO (light blue solid, 134 mg, 0.2 mmol, yield 48%). 1H NMR (600MHz, CDCl3) characteristic signals: δ 7.30 (dt, J = 8.7, 3.9Hz, 2H), 7.11–7.04 (m, 1H), 6.91 (t, J = 6.3Hz, 1H), 6.69 (t, J = 3.1Hz, 1H), 6.61 (ddd, J = 9.5, 7.2, 2.8 Hz, 1H), 6.36 (s, 1H), 5.48 (q, J = 6.3Hz, 1H), 5.15 (dt, J = 8.3, 4.2 Hz, 1H), 4.64 (d, J = 14.2Hz, 1H), 4.15 (dddd, J = 10.5, 7.6, 5.1, 1.9 Hz, 1H), 3.68 (ddd,J = 34.3, 11.2, 5.2 Hz, 2H), 3.44 (ddt, J = 9.2, 6.5, 4.2Hz, 1H), 3.34 (dq, J= 14.4, 5.7Hz, 1H), 3.29–3.22 (m, 1H), 3.15 (s, 3H), 3.05 (s, 3H), 2.93 (d, J= 3.9Hz, 12H), 2.87 (ddd, J = 13.1, 4.5, 2.5Hz, 1H), 2.70 (d, J = 13.3 Hz,1H), 1.93 (s, 4H), 1.74–1.66 (m, 2H), 1.59 (J = 7.3Hz, 2H) Figure 8 ). 13 150MHz, CDCl3 NMR (150MHz) characteristic signals: δ 174.76, 174.60, 157.95, 157.78, 157.62, 154.74, 154.70, 149.82, 149.20, 134.52, 128.09, 127.26, 111.38, 111.35, 111.06, 111.04, 71.05, 70.88, 70.66, 63.38, 58.42, 58.30, 55.51, 55.15, 45.90, 44.19, 43.94, 42.73, 42.53, 40.77, 40.32, 38.90, 38.52, 35.59, 35.50, 29.82, 28.05, 25.51 Figure 9 Melting point: 185–195 ℃. HRMS (ESI) + ):C 33 H47 N8O5S2 + [M+H] + Theoretical value: 699.3105; measured value: 699.3143. Figure 10 ).

[0064] Example 2: Preparation of compound MB-BIO

[0065] Repeat Example 1, except that in step 3), N,N-dimethylformamide is used instead of acetonitrile, and the reaction is carried out at 25°C until the reaction is complete.

[0066] Finally, 112 mg of a light blue solid was obtained, with a yield of 40%. Characterization by 1H NMR, 1C NMR, and high-resolution mass spectrometry confirmed it to be the compound MB-BIO.

[0067] Example 3: Preparation of compound MB-BIO

[0068] Repeat Example 1, except that triethylamine is not added in steps 1) and 2).

[0069] Finally, 35 mg of a light blue solid was obtained, with a yield of 18%. Characterization by 1H NMR, 1C NMR, and high-resolution mass spectrometry confirmed it to be the compound MB-BIO.

[0070] Example 4: Preparation of compound MB-BIO

[0071] Repeat Example 1, except that:

[0072] In step 1), tetrahydrofuran is used instead of dichloromethane, and sodium carbonate is used instead of triethylamine to adjust to the appropriate alkalinity;

[0073] In step 2), dimethyl sulfoxide is used instead of N,N-dimethylformamide, and pyridine is used instead of triethylamine to adjust to the appropriate alkalinity;

[0074] In step 3), dichloromethane is used instead of acetonitrile, and sodium hydroxide is used instead of triethylamine to adjust to the appropriate alkalinity.

[0075] Finally, 58 mg of a light blue solid was obtained, with a yield of 21%. Characterization by 1H NMR, 1C NMR, and high-resolution mass spectrometry confirmed it to be the compound MB-BIO.

[0076] Example 5: Preparation of compound MB-BIO

[0077] Example 1 was repeated, except that the reactions in each step were carried out at 25°C.

[0078] Finally, 106 mg of a light blue solid was obtained, with a yield of 38%. Characterization by 1H NMR, 1C NMR, and high-resolution mass spectrometry confirmed it to be the compound MB-BIO.

[0079] Experimental Example: Detection Limit and Imaging Capability of the Dual-Mode Response Probe MB-BIO of the Present Invention

[0080] 1. Experimental Section

[0081] 1.1 Instruments and Materials

[0082] All chemical reagents were purchased from commercial suppliers and used directly upon receipt without further purification. (NMR 1H NMR spectroscopy) 1 H NMR and carbon spectroscopy 13 C10 NMR was performed using a Bruker 600MHz NMR spectrometer at 25 °C, with tetramethylsilane (TMS) as the internal standard and deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) as the deuterated solvent. NMR peak abbreviations are: br s (broad singlet), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet). Mass spectrometry data were acquired using a Thermo Fisher Scientific LTQ FT Ultra ultra-high resolution liquid chromatography-mass spectrometry system. UV-Vis absorption and fluorescence spectra were measured at room temperature using an Agilent Cary 3500 UV spectrophotometer and a CaryEclipse fluorescence spectrophotometer, respectively. High performance liquid chromatography (HPLC) was performed using an Agilent 1260 Infinity II quaternary gradient liquid chromatography system. Photoacoustic (PA) imaging was performed using the Vevo® LAZR photoacoustic imaging system. The photodynamic therapy (PDT) light source was a 660nm LASER660-3W laser from Tianjin Sente Optics, China.

[0083] Biological testing instruments: Cytotoxicity experiments were performed using the Swiss Tecan Infinite 200 Pro multi-functional microplate reader; cell and zebrafish fluorescence imaging was performed using the Olympus Fluoview FV3000 multiphoton laser confocal microscope; zebrafish embryos were cultured in a GZX-250E light incubator; and mouse in vivo fluorescence imaging was performed using the Chinese AniView600 multi-mode small animal in vivo imaging system.

[0084] MB-BIO probe spectroscopy in solution: A precise 1 mM MB-BIO stock solution was prepared using dimethyl sulfoxide (DMSO) as the solvent and stored at 4°C in the dark. For testing, 20 L of the stock solution was taken and diluted to 2 mL with phosphate-buffered saline (PBS) containing 1% DMSO at pH 7.4. 1 mL of the diluted probe solution was added to a standard 1 cm path length quartz cuvette for subsequent UV and fluorescence spectroscopy detection.

[0085] Limit of Detection (LOD) Determination: The formula for calculating the probe detection limit is: LOD = 3σ / k. Where σ is the standard deviation of the fluorescence signal in the blank PBS solution, and the number of parallel tests for the blank is n = 5; k is the slope of the linear fitting curve of fluorescence intensity at 685 nm and HClO concentration.

[0086] Evaluation of MB-BIO singlet oxygen generation capacity: 10 μM MB-BIO and 40 μM HClO were mixed thoroughly in PBS and incubated at 37 °C for 1 min. Singlet oxygen indicator DPBF was added to a final concentration of 20 μg / mL, and the mixture was irradiated with a 660 nm laser for 0–20 min, with continuous UV absorption spectra throughout. The level of reactive oxygen species generation after probe irradiation was quantitatively evaluated by monitoring the attenuation of the characteristic absorption peak intensity of DPBF at 410 nm.

[0087] Density functional theory (DFT) calculations: Molecular structure calculations were performed using density functional theory, with B3LYP as the functional and 6-31G(d) as the basis set. All calculations were performed in the Gaussian 16 software package. Full geometry optimization was performed on the probe molecule to obtain a stable optimal conformation. The molecular frontier orbital distribution was visualized using Gaussian View software.

[0088] Molecular docking simulation: Based on the UniProt ID corresponding to the human biotin transporter, the raw protein crystal structure was downloaded from the RCSB PDB database. The raw protein structure was imported into Discovery Studio 2019 for preprocessing: removal of water of crystallization, addition of hydrogen atoms, atomic charge distribution, and completion of missing amino acid residues and side chains. After optimization, the protein was exported as a PDB format file. The probe MB-BIO molecular structure was downloaded from the PubChem database, and energy minimization optimization was performed in Discovery Studio 2019, saving it as a PDB ligand file. Molecular docking simulation was conducted using AutoDock Vina 1.2.6; before docking, AutoDock4 was used to convert the protein and ligand PDB files to PDBQT format. The optimal binding conformation was visualized and quantitatively analyzed using PyMOL 3.1 and Discovery Studio 2019.

[0089] Cell culture and CCK-8 cytotoxicity assay: Four cell lines, HepG2, THLE-2, A549, and BEAS-2B, were used in this experiment. All cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C and 5% CO2 saturated humidity. Before imaging experiments, cells were seeded in confocal culture dishes and cultured for 24 hours. Cells were then washed three times with PBS before the experiments.

[0090] Cytotoxicity was assessed using the CCK-8 assay. Cells were seeded in 96-well plates and cultured for 24 h. The medium was then replaced with fresh medium containing different concentrations of MB-BIO (0, 10, 25, 50, 100 μM), with a PBS system containing 10% DMSO used as a solvent control. After 24 h of incubation, 10 μL of CCK-8 assay solution was added to each well, and the plates were incubated at 37°C in the dark for 4 h. Absorbance at 450 nm was measured using a Tecan Infinite200 Pro microplate reader. Cell viability was calculated as follows: Cell viability (%) = (Absorbance of drug-treated group / Absorbance of blank control group) × 100%.

[0091] Intracellular fluorescence confocal imaging: Each cell line was seeded and cultured adherently in confocal culture dishes for 12 h; all cell incubation operations were performed at 37℃ and 5%... Completed under saturated humidity conditions.

[0092] Selective imaging of tumor / normal cells: HepG2, THLE-2, A549, and BEAS-2B cell lines were incubated with 10 μM MB-BIO for 30 min under identical culture conditions to verify the probe's ability to target and image tumor cells. After incubation with all drugs, cells were washed three times with PBS to remove unbound free probes; 500 μL of preheated PBS was added to each culture dish to maintain cell viability and morphology. Cell fluorescence images were acquired using an Olympus FV3000 multiphoton laser confocal microscope; the MB-BIO excitation wavelength was 640 nm, and the fluorescence signal acquisition range was 650–750 nm; ImageJ software was used to quantitatively analyze the fluorescence intensity in the images.

[0093] Lysosomal colocalization assay: HepG2 cells were used at a concentration of 6 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / dish in confocal dishes and cultured for 12 h. Cells were simultaneously incubated with MB-BIO and the commercial lysosomal fluorescent probe Lyso-Tracker Green for 1 h. After incubation, the culture medium was discarded, cells were washed three times with PBS, and 500 μL of PBS was added before imaging. Confocal parameters were set as follows: Lyso-Tracker Green: excitation wavelength 488 nm, emission wavelength 500–540 nm; MB-BIO: excitation wavelength 640 nm, emission wavelength 650–750 nm. At least three independent biological replicates were set up for each experiment, and ImageJ was used for image colocalization analysis.

[0094] Detection of intracellular reactive oxygen species (ROS) generation: HepG2 and A549 cells were subjected to a growth rate of 6 × 10⁻⁶. 4Cells were seeded per well in confocal microscopy dishes and cultured overnight. The next day, the culture medium was discarded, cells were gently washed with PBS, and fresh culture medium containing 10 μM MB-BIO was added. The cells were incubated at 37°C for 1 hour. Then, the intracellular ROS fluorescent indicator H2DCFDA (final concentration 10 μM) was added, and the cells were incubated in the dark for 5 minutes. Unadsorbed indicator was removed by washing with PBS. The experimental group was irradiated with a 660 nm laser for 10 minutes, while the control group was kept in the dark throughout the process. Finally, confocal microscopy was used to acquire cell fluorescence images, and the intracellular ROS production levels of each group were compared.

[0095] 1.2 Detection of photodynamic killing effect of cells by Calcein-AM / PI double staining

[0096] The efficacy of MB-BIO photodynamic therapy was evaluated at the cellular level using Calcein-AM / PI live / dead cell double staining. A549 cells were stained at a concentration of 6 × 10⁻⁶ cells. 4 Cells were seeded per well in confocal microscopy dishes and allowed to adhere overnight. Cells were incubated in fresh medium containing gradient concentrations of MB-BIO (0–10 μM) at 37°C for 1 h. The experimental group was irradiated with a 660 nm laser for 10 min, while the control group was protected from light. After irradiation, cells were incubated for another 2 h and stained with Calcein-AM / PI mixed staining solution. Cell fluorescence images were acquired using a laser confocal microscope to visually evaluate the MB-BIO-mediated photodynamic tumor-suppressing effect.

[0097] 1.3 In vivo fluorescence imaging of zebrafish

[0098] This experiment used 6-day-old wild-type zebrafish of strain AB, provided by the Biomedical Research and Development Platform for Novel Zebrafish Models of Human Diseases at Guangxi Medical University. The zebrafish were cultured at a constant temperature of 28 ℃ in a GZX-250E light incubator. Phenylacetyl thiourea (PTU) was added to the culture medium to inhibit melanin synthesis and eliminate interference from pigments on fluorescence imaging.

[0099] Juvenile zebrafish were randomly divided into 5 groups of 3 fish each, and the treatments were as follows: 1) Probe-only group: incubated with 20 μM MB-BIO for 1 h; 2) Low-concentration exogenous HClO group: pretreated with 5 μM HClO for 1 h, then incubated with 20 μM MB-BIO for 1 h; 3) High-concentration exogenous HClO group: pretreated with 10 μM HClO for 1 h, then incubated with 20 μM MB-BIO for 1 h; 4) Inflammatory endogenous HClO group: treated with 1 μg / mL LPS and 1 μg / mL PMA for 3 h to induce inflammation, then incubated with 20 μM MB-BIO for 1 h; 5) Inflammation + HClO scavenger group: pretreated with LPS / PMA for 3 h, then incubated with 200 μM ABH for 1 h, and finally incubated with 20 μM MB-BIO for 1 h. All incubation operations were performed at 28 ℃ in the dark to prevent probe photodegradation. After treatment, zebrafish juveniles were rinsed with pre-cooled PBS to remove residual probes and impurities, anesthetized with 0.03% tricaine (MS-222) solution for 2-3 min, and fixed in a confocal imaging dish. Fluorescence images were acquired using an FV3000 multiphoton laser confocal microscope, with the MB-BIO excitation wavelength set to 640 nm and the fluorescence emission acquisition range to 650-750 nm.

[0100] Evaluation of in vivo fluorescence imaging and photodynamic therapy for tumor suppression in tumor-bearing mice: All animal experiments used SPF-grade BALB / c mice, provided by the Experimental Animal Center of Guangxi Medical University. In vivo fluorescence imaging of small animals was performed using the Chinese AniView600 multi-mode small animal imaging system, with an excitation wavelength of 600 nm and a fluorescence detection wavelength of 680 nm.

[0101] When the tumor volume of A549 tumor-bearing mice grows to approximately 100 mm 3 At that time, 100 μL of 200 μM MB-BIO solution was injected intratumorally. Some mice were then irradiated with a 660 nm laser (power density 0.8 W / cm²) after injection. 2 The probe is activated to collect in vivo fluorescence images at preset time points, and the changes in fluorescence signal intensity at the tumor site with light exposure time are dynamically tracked.

[0102] Evaluation of in vivo photodynamic therapy for tumor suppression and biosafety: Tumor volume approximately 100 mm 3 BALB / c mice bearing tumors were randomly divided into three groups: a PBS control group, an MB-BIO monotherapy group, and an MB-BIO combined with 660nm laser photodynamic therapy group. Mice in the MB-BIO monotherapy and combined therapy groups received intratumoral injection of the drug at a dose of 7 mg / kg; the control group received an equal volume of sterile PBS. One hour after injection, mice in the combined therapy group underwent 660nm laser irradiation at a power density of 0.8 W / cm². 2The irradiation time was 5 minutes. The complete treatment cycle lasted 10 days, and the mouse body weight and tumor volume were recorded every two days. The overall health status and weight fluctuations of the mice were continuously observed throughout the treatment to evaluate the in vivo biosafety of MB-BIO.

[0103] 2. Results and Discussion

[0104] 2.1 In vitro optical response of MB-BIO to HClO and near-infrared light

[0105] First, the optical properties of MB-BIO were investigated in PBS buffer (pH 7.4, containing 0.1% dimethyl sulfoxide) to evaluate its detection capability for HClO. Figure 2 As shown in a and 2b, MB-BIO alone exhibits almost no UV-Vis absorption and fluorescence emission; upon the addition of HClO, a significant absorption peak appears, and the fluorescence intensity increases dramatically. With increasing HClO concentration, the fluorescence intensity of MB-BIO at 685 nm gradually increases, enhancing by approximately 69 times. Figure 2 b).

[0106] Within the range of 0–10 μM, the fluorescence intensity showed a good linear relationship with the HClO concentration, and the regression equation was y = 73.146x + 33.548 (R²). 2 = 0.9919) Figure 2 c), with a limit of detection (LOD) of 15.6 nM. The results indicate that MB-BIO can achieve qualitative and quantitative detection of HClO in vitro.

[0107] The response kinetics to HClO were then investigated. (The text continues with further details on the response kinetics of MB-BIO to HClO.) Figure 2 As shown in Figure d, after the addition of HClO, the fluorescence intensity reached its maximum value within 5 seconds, demonstrating a rapid response; and the fluorescence intensity remained stable for 60 minutes, exhibiting excellent stability. In the control group without HClO, MB-BIO remained in a fluorescence-quenched state without spontaneous activation, confirming its good stability under physiological conditions.

[0108] Response characteristics to near-infrared light. Further investigation will be conducted on the response of MB-BIO to near-infrared light (660 nm). Figure 2 As shown in e and 2f, under near-infrared illumination, a significant absorption peak appears at 660 nm, and significant fluorescence emission occurs at 685 nm. With increasing irradiation time (0–10 min), the absorption and emission intensities gradually increase, reaching a plateau after 10 min and remaining stable thereafter. Figure 2 g).

[0109] This fluorescence activation originates from a photoinduced breakage mechanism: upon photon absorption, the urea bonds in MB-BIO undergo irreversible breakage, generating the fluorophore MB and its corresponding amine derivatives. This photoactivation property endows MB-BIO with photocontrolled activation potential, offering new possibilities for the synergistic application of fluorescence imaging and photodynamic therapy.

[0110] Photostability. Photostability experiments showed that after MB-BIO reacted with HClO, there was no significant fluorescence attenuation under continuous 660nm laser irradiation, demonstrating excellent resistance to photobleaching. This is beneficial for achieving low background and high signal-to-noise ratio imaging in complex biological systems. Figure 2 g).

[0111] pH stability. The pH stability of the probe is crucial for bioimaging. For example... Figure 2 As shown in h, MB-BIO maintains a stable low fluorescence background ("off") over a wide pH range of 4 to 12; and even with the addition of HClO within this pH range, the probe can still effectively detect HClO and produce significant fluorescence. It exhibits good stability under various physiologically relevant pH conditions, and its detection performance is minimally affected by interference.

[0112] In vitro reactive oxygen species (ROS) generation capacity. The ROS generation performance of MB-BIO was evaluated using DPBF as a detection indicator. Figure 2 i and Figure 13 As shown in Figure a, under the test condition of continuous near-infrared laser irradiation for 10 minutes, the absorbance of DPBF at 420 nm in the MB-BIO+HClO group continuously decreased, and the decay rate was comparable to that of the methylene blue positive control group. After MB-BIO alone was irradiated with near-infrared light, the absorbance of DPBF also continuously decreased. This is mainly because near-infrared light can directly activate MB-BIO and release methylene blue, which has the ability to generate reactive oxygen species (ROS). Therefore, even if there is a lack of sufficient HClO in the tumor microenvironment, MB-BIO under near-infrared light irradiation can still maintain its ROS generation capacity. Figure 13 b). The above results demonstrate that MB-BIO possesses excellent reactive oxygen species generation efficiency and has promising application prospects in the field of photodynamic therapy.

[0113] In vitro photoacoustic imaging capability. Leveraging the near-infrared emission wavelength and excellent photoacoustic imaging potential of MB, the photoacoustic signal generation capability of MB-BIO after activation with HClO was further evaluated. Different concentrations (0–8 mM) of MB-BIO were dispersed in PBS, and photoacoustic spectra were recorded after adding HClO (5 mM). Figure 2 As shown in Figure j, MB-BIO is activated and generates a photoacoustic signal in the presence of HClO. The signal intensity increases linearly with increasing MB-BIO concentration, confirming its excellent photoacoustic imaging performance and providing experimental evidence for in vivo tumor photoacoustic imaging.

[0114] Selectivity of MB-BIO for HClO and near-infrared light. To verify the specific response of MB-BIO to HClO and near-infrared light, its fluorescence behavior in the presence of various potential interfering substances was systematically investigated. Under the same test conditions, a total of 51 substances that may induce fluorescence response were detected, including various ions, small molecules, amino acids, and enzymes, numbered 1 to 51 as follows: control group, Fe... 2+ Fe 3 + K + Ca 2+ Mg 2+ Na + Al 3+ Hg + Zn 2+ Cu 2+ Pb 2+ Ag + Cs 2+ Cl - CO3 2- HCO3 - I - COOH - NO2 - HSO4 - SO4 2- H - OH - HS - NO3 - SCN - H2O2, ONOO - SO4 2- SCN - OH - NO2 - Homocysteine ​​(Hcy), glutamine (Gln), glutathione (GSH), L-phenylalanine (L-Phe), histidine (His), threonine (Thr), serine (Ser), cysteine ​​(Cys), valine (Val), isoleucine (Iso), proline (Pro), aspartic acid (ASP), lysine (Lys), glycine (Gly), L-tyrosine (L-Tyr), β-galactosidase (β-gal), lysozyme (Lyso), glucose oxidase (GOD), trypsin (Tryp), β-glucosidase (β-Glu), laser, and HClO. Only HClO and near-infrared light significantly activated the fluorescence signal of MB-BIO; the other interfering substances did not induce significant fluorescence changes. Figure 2 The results conclusively demonstrate that MB-BIO exhibits ultra-high specificity and excellent selectivity for HClO and near-infrared light stimulation, effectively avoiding false positive signal interference in complex biological systems.

[0115] 2.2 Recognition, activation, and cancer cell targeting mechanisms

[0116] To elucidate the mechanism by which MB-BIO is activated by HClO and near-infrared light, the reaction products were first analyzed by high-resolution mass spectrometry. MB-BIO showed a molecular ion peak at m / z 699.3143. Figure 10 After treatment with HClO or near-infrared light, new strong peaks appeared at m / z 248.1227 and 248.1210, respectively, which perfectly matched the molecular weight of the fluorophore MB. Figure 11 , Figure 12 This confirms the theoretical hypothesis of the MB-BIO response mechanism. The speculated mechanism is as follows: HClO attacks the electron-deficient carbon atom of the urea bond, causing bond breakage and releasing the MB-Cl intermediate; MB-Cl is rapidly oxidized to MB, generating a strong fluorescent signal ( Figure 1 ).

[0117] Density functional theory (DFT) calculations further revealed the differences in photophysical properties between MB and MB-BIO. Frontier molecular orbital analysis showed that MB has a narrow band gap (2.48 eV), while the band gap of MB-BIO is significantly increased (4.09 eV). After MB-BIO is converted into MB in response to HClO or near-infrared light, the π electron cloud is highly delocalized on the MB framework, and the conjugated system is effectively expanded. The calculation results confirmed that the band gap decreases after the probe recognizes HClO, realizing a switch from "off" to "on", and completing target detection.

[0118] Natural transition orbital (NTO) analysis highlights the HONTO-LUNTO pairs, which vividly describe the electronic transition behavior from the ground state to the first excited state. Figure 3 a). The MB oscillator has a high intensity (0.819), explaining its strong light absorption response; the MB-BIO oscillator has an extremely weak intensity (0.001), corresponding to a low molar extinction coefficient ( Figure 3 a). The results are in high agreement with experimental observations: after MB-BIO is converted to MB, the absorption and fluorescence emission intensities of the system are significantly enhanced.

[0119] Subsequently, molecular docking simulations were used to study the molecular recognition mechanism between MB-BIO and the biotin transporter of human cancer cells, elucidating its cancer cell targeting ability. Figure 3As shown in Figure b, the three-dimensional model reveals that MB-BIO forms three hydrogen bonds with four amino acid residues (ARG-601, ARG-64, and GLU-131) of the biotin transporter. The surface model shows that the ligand is located within a cavity on the protein surface, and the cavity and ligand structures are complementary, facilitating binding. The binding energy of the docking interaction is -8.28 kcal / mol, indicating that MB-BIO has a strong affinity for the biotin transporter of cancer cells. The two-dimensional diagram shows that the ligand interacts with the amino acid residues through van der Waals forces, which are essential for the stability of the binding site. Figure 14 The above results provide a molecular basis for confirming MB-BIO's ability to target cancer cells.

[0120] 2.3 Cell-level fluorescence imaging

[0121] 2.3.1 Cytotoxicity of MB-BIO

[0122] The cytotoxicity of MB-BIO was evaluated in A549 and BEAS-2B cells using the CCK-8 assay. Figure 15 As shown, even at a concentration of 50 μM, the survival rate of all cell lines treated with MB-BIO remained above 70%, indicating good biosafety and supporting subsequent bioimaging applications. Notably, MB-BIO exhibited lower toxicity to normal BEAS-2B cells than to A549 cancer cells, demonstrating superior selectivity and safety in normal cells.

[0123] 2.3.2 Detection of HClO at the cellular level using MB-BIO

[0124] First, we evaluated MB-BIO's ability to visualize HClO in live cells. For example... Figure 4 As shown in a and 4b, BEAS-2B cells incubated with MB-BIO showed almost no fluorescence signal, indicating that the probe remained stably "off" in a normal cellular environment with low HClO and low background interference.

[0125] Cells pretreated with exogenous HClO and then incubated with MB-BIO showed significantly enhanced fluorescence, demonstrating that MB-BIO can effectively visualize intracellular HClO.

[0126] To further evaluate its ability to detect endogenous HClO, BEAS-2B cells were stimulated with phorbol ester (PMA) and lipopolysaccharide (LPS) to induce endogenous HClO production. The results showed that intracellular fluorescence was significantly enhanced compared with the unstimulated group, indicating that MB-BIO can effectively monitor endogenous HClO fluctuations triggered by inflammatory stimulation.

[0127] To verify that the fluorescence signal originates from the specific response of MB-BIO to endogenous HClO, the HClO scavenger 4-aminobenzoyl hydrazine (ABH) was introduced for inhibition experiments. After ABH pretreatment, the PMA / LPS-induced fluorescence signal was significantly weakened, further confirming that the fluorescence activation of MB-BIO originates from the increased intracellular HClO level.

[0128] In summary, MB-BIO can specifically and dynamically visualize exogenous and endogenous HClO at the cellular level.

[0129] 2.3.3 Differentiating between cancer cells and normal cells

[0130] To explore the potential of MB-BIO in distinguishing between cancer cells and normal cells, the probe was co-incubated with normal cell lines (BEAS-2B, THLE-3) and cancer cell lines (HepG2, A549), and the fluorescence response was detected. Figure 4 As shown in c and 4d, normal cells exhibited only weak fluorescence signals, confirming that MB-BIO remained quenched in the normal cellular environment, resulting in low background fluorescence. Both cancer cell lines showed significantly enhanced fluorescence, with A549 cells showing the most prominent signal. These results indicate that MB-BIO can effectively distinguish between cancer cells and normal cells, demonstrating potential value in tumor diagnosis.

[0131] 2.3.4 Lysosomal specific localization ability

[0132] To verify whether MB-BIO possesses lysosome-specific localization ability, HepG2 cells were co-incubated with MB-BIO and a commercially available lysosomal fluorescent probe (Lyso-Tracker Green), and subcellular distribution was observed using confocal microscopy. Figure 5 As shown in figure a, the red fluorescence signal of MB-BIO highly overlaps with the green fluorescence signal of Lyso-Tracker Green-labeled lysosomes, with a Pearson correlation coefficient of 0.70, indicating that MB-BIO has a strong co-localization relationship with lysosomes and can selectively target lysosomes with high affinity.

[0133] The specific localization mechanism of lysosomes may be related to the dimethylamino group in its molecular structure: under physiological pH (about 7.4), the dimethylamino group mainly exists in a non-ionized state, which can efficiently penetrate the cell membrane and organelle membranes; after entering the acidic lysosomal microenvironment (pH 4.5~6.0), the group is protonated and converted to a positively charged form; since the lysosomal membrane has extremely low permeability to charged molecules, the protonated MB-BIO is effectively "captured" in the lysosome and enriched.

[0134] 2.3.5 Intracellular reactive oxygen species generation capacity

[0135] To evaluate the potential of MB-BIO for photodynamic therapy, 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), a commercially available ROS indicator, was systematically investigated in its ability to induce ROS generation in A549 and HepG2 cells. Figure 5 As shown in b and 5c, cells incubated with MB-BIO and then irradiated with a 660nm laser showed significantly enhanced H2DCFDA fluorescence compared to the unirradiated group or the light-only group. This demonstrates that MB-BIO efficiently generates ROS under 660nm laser excitation and exhibits excellent photosensitivity, laying the foundation for subsequent photodynamic therapy applications.

[0136] 2.3.6 Effects of Extracorporeal Photodynamic Therapy

[0137] Double staining with calcein-AM and propidium iodide (PI) was used to distinguish between live and dead cells, and the photodynamic therapy effect of MB-BIO at the cellular level was evaluated. In the laser irradiation group, significant cancer cell apoptosis was observed at a MB-BIO concentration of 5 μM, and the killing effect reached saturation at 10 μM, with almost all cancer cells being eliminated. In contrast, cells incubated with MB-BIO alone (without laser irradiation) showed almost no apoptosis or cytotoxicity, demonstrating excellent biocompatibility. Figure 5 d).

[0138] In summary, in vitro photodynamic experiments have demonstrated that MB-BIO possesses potent photodynamic therapeutic effects and excellent biosafety, providing a solid experimental basis and promising prospects for subsequent in vivo photodynamic therapy research.

[0139] 2.4 MB-BIO-based fluorescence imaging of HClO in zebrafish

[0140] Further evaluation of MB-BIO's ability to detect HClO in vivo using a zebrafish model will explore its potential application in in vivo tumor detection. Figure 6 As shown, zebrafish incubated with MB-BIO showed almost no fluorescence signal, indicating that MB-BIO remained stably "off" under physiological conditions, with low background interference, making it suitable for in vivo imaging.

[0141] The fluorescence intensity of zebrafish treated with exogenous HClO was significantly increased and positively correlated with HClO concentration; the fluorescence of zebrafish induced by PMA / LPS stimulation to generate endogenous HClO was also significantly enhanced, demonstrating that MB-BIO can effectively respond to exogenous and endogenous HClO, specifically visualizing HClO fluctuations in living organisms and PMA / LPS-induced inflammatory responses.

[0142] To further verify the specificity of the fluorescence response, zebrafish were pretreated with the endogenous HClO generation inhibitor ABH. Subsequently, the fluorescence signal decreased significantly, confirming that the fluorescence enhancement specificity was due to the increase in endogenous HClO levels, highlighting the ability of MB-BIO to monitor the dynamic changes in HClO generation.

[0143] In summary, MB-BIO is an ideal tool for visualizing HClO in living organisms, has imaging feasibility in biological systems, and can detect tumors by specifically responding to the high expression of HClO in cancer cells, showing broad application potential.

[0144] Fluorescence imaging and photodynamic therapy effects of 2.5 MB-BIO in tumor-bearing mice

[0145] The in vivo activation characteristics and imaging performance of MB-BIO were investigated in a tumor-bearing mouse model using fluorescence imaging technology. After intratumoral injection of MB-BIO into mice, the tumor areas of mice not irradiated with laser showed only weak fluorescence signals. This result indicates that although the tumor microenvironment can activate MB-BIO to a small extent, the overall activation efficiency of the probe is low due to the limitation of endogenous HClO concentration. In stark contrast, in mice irradiated with a 660 nm laser, the fluorescence signal at the tumor site significantly increased with prolonged irradiation time, demonstrating that exogenous light irradiation can effectively activate MB-BIO and promote the release of the highly fluorescent photosensitizer methylene blue (MBBIO). Figure 7 b). Unlike traditional detection probes that rely solely on endogenous HClO activation, MB-BIO possesses a dual regulatory mechanism combining HClO response and exogenous laser activation, enabling simultaneous tumor-specific fluorescence imaging and photodynamic therapy, thus demonstrating significant advantages in precision tumor diagnosis and treatment.

[0146] To further evaluate the in vivo antitumor efficacy and biosafety of MB-BIO, BALB / c mice subcutaneously inoculated with A549 tumors were randomly divided into three groups: a PBS control group, an MB-BIO-only group, and an MB-BIO combined with laser therapy group (MB-BIO+L). Tumors were allowed to grow to approximately 100 mm². 3 At that time, mice in each group were injected intratumorally with either PBS or MB-BIO (7 mg / kg); 1 hour after injection, the MB-BIO+L group was treated with a 660 nm near-infrared laser at a power density of 0.8 W / cm². 2 The irradiation time was 5 minutes. During the 10-day treatment period, mouse body weight and tumor volume were recorded every two days; at the end of the experiment, tumors in each group were dissected, weighed, and photographed. Biosafety results showed that mice in all groups exhibited normal behavior and activity and no significant fluctuations in body weight throughout the experimental period, indicating that mice tolerated MB-BIO-mediated photodynamic therapy well. Figure 7 c). For example Figure 7As shown in d and 7e, compared with the PBS control group and the MB-BIO alone group, tumor growth was significantly inhibited in the MB-BIO combined with laser group; large areas of necrotic foci and granulation tissue were visible on the tumor surface, and the underlying tumor tissue was completely removed after peeling off the surface eschar. The above results fully demonstrate that 660nm laser excitation can induce a strong photodynamic antitumor effect.

[0147] Based on the above data, MB-BIO not only possesses excellent photodynamic therapy effects, but also exhibits extremely low systemic toxicity and good in vivo biocompatibility, fully demonstrating that this probe has broad application prospects in the field of integrated tumor diagnosis and photodynamic therapy.

[0148] 3. Conclusion

[0149] In summary, this application successfully designed and synthesized a novel dual-mode activatable fluorescent probe, MB-BIO, which possesses both tumor cell targeting and lysosomal localization capabilities. It can achieve tumor-specific imaging and photodynamic therapy under HClO or near-infrared light stimulation. MB-BIO achieves active tumor targeting through biotin-mediated recognition and precisely anchors lysosomes via a dimethylcarbamoyl group. Experimental results show that under activation by high HClO expression in the tumor microenvironment or exogenous 660nm NIR irradiation, MB-BIO exhibits rapid response (HClO response in only 5 seconds, NIR response in 10 minutes), high sensitivity (HClO detection limit of 15.6 nM), and excellent selectivity, while also possessing outstanding near-infrared fluorescence and photoacoustic imaging performance. The methylene blue released after probe activation is a highly efficient photosensitizer, which can generate a large amount of reactive oxygen species under light irradiation, strongly inducing tumor cell apoptosis. In vitro experiments confirmed that MB-BIO can clearly distinguish between tumor cells and normal cells, precisely locate lysosomes, and exhibit a photocontrolled, potent tumor-suppressive effect. Furthermore, in vivo fluorescence imaging experiments in zebrafish and tumor-bearing mice validated the probe's dual-mode activation characteristics for HClO and NIR, enabling high-contrast tumor imaging and demonstrating excellent photodynamic therapy efficacy in vivo. In summary, MB-BIO is the first multifunctional therapeutic platform integrating tumor cell targeting, lysosomal localization, and HClO / NIR dual-mode activation, efficiently combining specific tumor imaging with photodynamic therapy. This research provides a novel approach to overcoming core challenges in precision oncology and possesses extremely high clinical translational potential.

Claims

1. A dual-mode response probe, denoted as compound MB-BIO, has the following structure: 。 2. The method for preparing the dual-mode response probe according to claim 1, comprising the following steps: 1) Take compound MB-Cl and 1,3-diamino-2-propanol in a first organic solvent, and carry out a substitution reaction with or without the addition of a basic substance to obtain compound MB-NH2-OH; 2) Take compound MB-NH2-OH and (+)-biotin-N-hydroxysuccinimide ester in a second organic solvent, and carry out a substitution reaction with or without the addition of a basic substance to obtain compound MB-BIO-OH; 3) Place the compound MB-BIO-OH in a third organic solvent, add an alkaline substance, and then add dimethylformyl chloride to react and obtain the crude product of the target compound; 、 、 。 3. The preparation method according to claim 2, characterized in that, In steps 1) to 3), the alkaline substance is an organic base and / or an inorganic base.

4. The preparation method according to claim 3, characterized in that, The organic base is selected from one or more of triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, potassium tert-butoxide and sodium tert-butoxide; The inorganic base is selected from one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, cesium hydroxide, and cesium carbonate.

5. The preparation method according to claim 2, characterized in that, In steps 1) to 3), the reaction temperature is room temperature, or between room temperature and the boiling point of the organic solvent.

6. The preparation method according to claim 2, characterized in that, The first organic solvent is selected from one or more of dichloromethane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, dichloroethane, and tetrahydrofuran; The second organic solvent is one or a combination of two or more selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and N-methylpyrrolidone; The third organic solvent is selected from one or more combinations of dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile and tetrahydrofuran.

7. The preparation method according to claim 2, characterized in that, The compound MB-NH2-OH obtained in step 1) or the compound MB-BIO-OH obtained in step 2) is purified before being used in subsequent steps.

8. The preparation method according to any one of claims 2 to 7, characterized in that, It also includes a step of purifying the crude target product obtained.

9. The use of the dual-mode response probe of claim 1 in at least one of the following (a1) to (a4): (a1) Preparation of drugs for photodynamic therapy; (a2) Cellular or biological imaging not for diagnostic or therapeutic purposes; (a3) Preparation of in vivo tumor imaging drugs; (a4) Preparation of reactive oxygen species.

10. A product comprising the dual-mode response probe of claim 1.

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