Nitroimidazole group meso-substituted heptamethine cyanine dye as well as preparation method and application thereof

Abstract

The invention relates to a nitroimidazole group meso-substituted heptamethine cyanine dye as well as a preparation method and application thereof. According to the dye, a nitroimidazole group is introduced into the middle position of a heptamethine cyanine parent nucleus, so that the dye has excellent absorption characteristics in a near-infrared region, the excitation wavelength is 730-780 nm, the emission wavelength is 800-860 nm, and the dye has good tissue penetrating power. The nitroimidazole group can effectively quench fluorescence in an excited state through a light-induced electron transfer effect, energy is promoted to be released in a non-radiative transition mode, the photothermal conversion efficiency is remarkably improved, and meanwhile high light stability and low fluorescence quantum yield are kept. The dye can be further prepared into a nano preparation and shows excellent biocompatibility and tumor targeting ability. The compound has wide application prospects in the fields of non-diagnostic or therapeutic protein labeling, bioimaging, photothermal therapy and the like, is especially suitable for preparing efficient photosensitive reagent drugs for tumor photothermal therapy, and provides a new strategy for design of near-infrared photothermal diagnosis and treatment materials.

Description

Technical Field

[0001] This invention relates to the fields of fine chemicals and biomedicine, and in particular to a class of heptamethine dyes with nitroimidazole groups substituted at the mesone, their preparation methods and applications. Background Technology

[0002] Cancer has become one of the leading causes of death and morbidity worldwide. Treatment methods typically include surgery, radiotherapy, chemotherapy, and phototherapy. Photothermal therapy (PTT) is an emerging tumor treatment that uses photothermal conversion materials to convert light energy into heat energy, thereby achieving precise ablation of tumor cells. This technology has several advantages, including low invasiveness, precise targeting of tumor tissue, a relatively gentle treatment process, and minimal damage to surrounding normal tissues. Photosensitizing agents are one of the core components of photothermal therapy.

[0003] Biological tissues absorb and scatter light less in the near-infrared window, thus near-infrared photosensitizers exhibit good penetration and light utilization in biological tissues, while significantly reducing photothermal activity in the tissues themselves. Among these, heptamethine cyanide dyes possess a high molar extinction coefficient in the near-infrared window, demonstrate good biocompatibility, and are easily modified molecularly, making them excellent near-infrared photosensitizers. However, heptamethine cyanide dyes still face some challenges in clinical applications, such as inherently low photothermal conversion efficiency and poor photostability. Existing methods generally improve their performance by adjusting molecular structure, adding functional groups, or using carriers. However, a dilemma faced by those skilled in the art is that any molecular modification aimed at enhancing one property often exacerbates other problems; this seesaw effect makes synergistic performance improvement extremely difficult.

[0004] Therefore, there is an urgent need for a novel molecular design strategy that can fundamentally reconstruct the nonradiative transitions and vibrational relaxations of heptamethrin dyes, break the inherent constraints between their performance parameters, and thus simultaneously achieve a balance between high photothermal conversion efficiency, excellent photostability, and good bioavailability. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned technical problems by providing a nitroimidazolium-substituted heptamethrin dye photosensitizer, its preparation method, and its applications. This aims to enable the dye to more efficiently convert light energy into heat energy after absorption, reducing energy loss and improving the efficacy of photothermal therapy for tumors. In the ground state, the nitroimidazolium group of the dye exhibits a near-perpendicular conformation to the heptamethrin dye. In the excited state, the nitroimidazolium group can quench the fluorescence signal of the heptamethrin dye through photoinduced electron transfer. The torsion of the nitroimidazolium group relative to the heptamethrin dye consumes the energy absorbed by the dye; that is, the excited-state energy is mainly released through non-radiative transitions, thus the dye exhibits high photothermal conversion performance. This invention provides a new approach for the design of photosensitizers for photothermal therapy.

[0006] The specific technical solution of this application is as follows: First, a type of nitro-substituted cyanine dye fluorescent probe for photothermal therapy of tumors is disclosed, having the following general formula 1 structure:

[0007] In general formula 1: L is selected from one of the substituents described in formulas L1 to L3:

[0008] R1 is selected from one of the substituents described in formulas A1 to A5 below:

[0009] R2 is selected from one of the substituents described in formulas A1 to A5 below:

[0010] P1 and P2 are each independently selected from one of the following groups: sulfonate group, carboxyl group, amino group, hydrogen atom, and halogen atom; X is selected from one of methylene, secondary amino, N-methyl, and N-ethyl.

[0011] In formula A1, n is selected from any integer from 0 to 15; for example: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15; More preferably, the dye is the compound Cy-IMZ(NO2) or IR783-IMZ(NO2) from the examples.

[0012] The dye has an excitation wavelength of 730-780 nm and an emission wavelength of 800-860 nm.

[0013] This invention discloses a method for preparing the nitroimidazolium-substituted cyanine dyes described above, and the synthetic route is as follows:

[0014] In a dry organic solvent under alkaline conditions, the dye shown in general formula S-1 is mixed evenly with the nitroimidazolium compound shown in L, heated at 70-120℃ for 8-24 h, and after multiple recrystallizations, dried to obtain the dye shown in general formula 1.

[0015] More preferably, the organic solvent used in the above synthetic route is selected from one of N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, and 1,4-dioxane.

[0016] More preferably, the molar ratio of the dye shown in the above synthetic route S-1 to the nitroimidazolium compound shown in L is 1:(1-10).

[0017] More preferably, the alkaline agent providing alkaline conditions in the above synthetic route is selected from at least one of sodium hydride, sodium hydroxide, potassium hydroxide, sodium methoxide, N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, and cesium carbonate.

[0018] More preferably, the reaction temperature of the above synthetic route is 70-90℃ and the reaction time is 8-16 h.

[0019] More preferably, the solvent for recrystallization in the above synthetic route is selected from at least one of water, methanol, ethanol, acetonitrile, isopropanol, ethyl acetate, and tert-butyl methyl ether.

[0020] This invention discloses a method for preparing the dye represented by the general formula S-1 described above, and the synthetic route is as follows:

[0021] In an anhydrous organic solvent, the compound of general formula S-2 is mixed evenly with the compound of general formula S-3 and the compound of general formula S-4. Under catalytic conditions, the mixture is heated at 25-120℃ for 8-24 h. After multiple recrystallizations, the mixture is dried to obtain the dye of general formula S-1.

[0022] More preferably, the organic solvent in the above synthetic route is selected from at least one of acetic anhydride, methanol, acetonitrile, ethanol, and toluene.

[0023] More preferably, the molar ratio of the compound of general formula S-2 to the compound of general formula S-3 and the compound of general formula S-4 is 1-1.5:1:(1-1.5).

[0024] More preferably, the catalyst providing alkaline conditions in the above synthetic route is selected from sodium acetate, potassium acetate, and calcium acetate.

[0025] More preferably, the reaction temperature of the above synthetic route is 50-100℃ and the reaction time is 8-16 h.

[0026] More preferably, the solvent for recrystallization in the above synthetic route is selected from at least one of water, methanol, ethanol, acetonitrile, isopropanol, ethyl acetate, and tert-butyl methyl ether.

[0027] The above-mentioned nitroimidazolium-substituted cyanine dyes are used in protein labeling, bioimaging, and preparation of photothermal therapy photosensitizing agents for non-diagnostic or therapeutic purposes; especially in the preparation of drugs for photothermal therapy of tumors.

[0028] Furthermore, the above applications include, but are not limited to: cell photoacoustic imaging and thermal imaging; bacterial photoacoustic imaging and thermal imaging; tumor photoacoustic imaging and thermal imaging; preparation of photosensitizer drugs for photothermal therapy; and application in basic research on photothermal therapy.

[0029] The nitro-substituted cyanine dye for enhancing photothermal therapy of tumors described in this invention has an absorption wavelength of approximately 730-780 nm and an emission wavelength of approximately 800-860 nm. It possesses strong tissue penetration, low fluorescence quantum yield, excellent photostability, and high photothermal conversion efficiency. By quenching fluorescence, it can enhance the effective thermal ablation of tumor tissue, precisely heating the tumor while minimizing damage to surrounding normal tissues. This localized heating characteristic makes photothermal therapy safer and more effective during treatment, showing promising clinical translational applications in biomedical fields such as tumor diagnosis and treatment.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Excellent near-infrared absorption characteristics: The nitro-substituted cyanine dye fluorescent probe for tumor photothermal therapy disclosed in this invention has a maximum absorption wavelength of 730-780 nm and an emission wavelength of 800-860 nm, which are in the near-infrared region. Compared with ultraviolet light or short-wavelength light, it has a deeper tissue penetration depth and usually causes a lower degree of tissue damage. In addition, it has less scattering in biological tissues, which can effectively reduce the attenuation of light in tissues and ensure that more light energy can reach the tumor site.

[0031] 2. Low Fluorescence Quantum Yield: The nitro-substituted cyanine dye fluorescent probe for tumor photothermal therapy disclosed in this invention has a significantly lower fluorescence quantum yield than the control example without nitro substitution. This means that after light absorption, more energy is used for photothermal conversion rather than fluorescence emission. Under the same illumination conditions, it produces a higher temperature rise and a faster heating rate, reaching the temperature threshold that causes irreversible damage to tumor tissue in a shorter time, thus significantly improving the efficacy of photothermal therapy. Simultaneously, the lower fluorescence quantum yield also means that the fluorescent probe is less prone to photobleaching during photothermal therapy, further enhancing its photostability and prolonging its effective duration of action in vivo.

[0032] 3. High photostability: The nitro-substituted cyanine dye fluorescent probe for tumor photothermal therapy disclosed in this invention can still essentially reach its initial temperature after six heating and cooling cycles. Furthermore, compared to the unsubstituted control, it exhibits significantly superior photostability after three heating and cooling cycles. This stable light absorption ensures that the fluorescent probe can efficiently ablate tumors during photothermal therapy, while avoiding fluctuations in treatment efficacy due to unstable light absorption characteristics.

[0033] 4. Excellent photothermal conversion performance: The aqueous solution of the nitro-substituted cyanine dye fluorescent probe for photothermal therapy of tumors disclosed in this invention exhibits a significantly higher temperature rise rate during laser irradiation than the control fluorescent probe. After quantification, the photothermal conversion efficiency (PCE) is also significantly higher than that of the control molecule. It can more efficiently convert absorbed light energy into heat energy, thereby achieving higher heating efficiency and realizing effective thermal ablation of tumor tissue in a short time, thereby reducing thermal damage to surrounding normal tissues and improving the precision and safety of treatment.

[0034] 5. High biocompatibility: The nitro-substituted cyanine dye fluorescent probe for photothermal therapy of tumors disclosed in this invention exhibits extremely low cytotoxicity. During photothermal therapy, the fluorescent probe can efficiently convert light energy into heat energy to ablate tumor tissue, while avoiding significant damage to surrounding normal cells and tissues. This significantly improves the safety of treatment and reduces adverse reactions caused by the treatment. Attached Figure Description

[0035] Figure 1A B is the NMR of Cy-IMZ (NO2) 1 H and 13 C spectrum Figure 2 High-resolution mass spectrometry of Cy-IMZ (NO2) Figure 3 IR783-IMZ (NO2) NMR 1 H spectrum Figure 4 AB shows the particle size distribution and transmission electron microscopy image of Cy-IMZ (NO2) nanoparticles. Figure 5A B is the NMR of Cy-IMZ (H) 1 H and 13 C spectrum Figure 6 High-resolution mass spectrometry of Cy-IMZ (H) Figure 7 AB represents the absorption and fluorescence spectra of Cy-IMZ(NO2) in different solvents. Figure 8 AB represents the UV absorption and fluorescence spectra of Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2). Figure 9 Comparison of the basic optical properties of ICG, Cy-Cl, Cy-IMZ(H) and Cy-IMZ(NO2) Figure 10. UV absorption and fluorescence spectra of ABCy-IMZ (NO2) nanoparticles. Figure 11abcd represents ICG, Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2). 1 Comparison of O2 generation capabilities Figure 12 Heating curves of Cy-Cl, Cy-IMZ(H), Cy-IMZ(NO2), and pure water under laser irradiation Figure 13 The relationship between the heating performance of Cy-IMZ (NO2) and dosage and light intensity for AB Figure 14 Comparison of photothermal stability of ICG, Cy-Cl, Cy-IMZ (H), and Cy-IMZ (NO2) Figure 15 Photothermal stability evaluation of Cy-IMZ (NO2)NPs Figure 16 abcd represents the photothermal conversion efficiency test of ICG, Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2). Figure 17 Evaluation of cellular uptake of Cy-IMZ (NO2) nanoparticles Figure 18 Evaluation of phototoxicity and dark toxicity of Cy-IMZ (NO2) nanoparticles on cells (AB) Figure 19 ABCD represents the evaluation of the photothermal killing performance of Cy-IMZ (NO2) nanoparticles against cancer cells. Detailed Implementation The following specific examples illustrate the nitroimidazolium-substituted cyanine dyes, their preparation methods, and applications described in this invention: Example 1 Synthesis of Cy-IMZ (NO2) The synthetic route for Cy-IMZ(NO2) is shown below:

[0036] (1) Synthesis of compound I 2,3,3-trimethyl-3H-indole (4.00 g, 25.00 mmol), 2-iodoethane (4.70 g, 30.00 mmol), and acetonitrile were used as solvents and refluxed for 12 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed and the temperature was allowed to drop to room temperature, excess ice-cold diethyl ether was added. The volume ratio of ice-cold diethyl ether to the reaction liquid was approximately 1.5:1, and the product precipitated. After centrifugation in 50 mL centrifuge tubes, the clearly separated solvent was poured off, washed three times with diethyl ether, dissolved in a mild solvent, dichloromethane, and evaporated under reduced pressure to obtain compound I (4.20 g, 22.00 mmol, Y = 88%), which was directly used in the next reaction. (2) Synthesis of Compound II: Anhydrous N,N-dimethylformamide (17.50 mL, 23 mmol) was dissolved in anhydrous dichloromethane (17.50 mL, 27 mmol), and 17.50 mL of POCl3 (17.50 mL, 19 mmol) was slowly added dropwise to a mixed solution of DMF and DCM. The mixture was stirred at room temperature for 0.5 h, and cyclohexanone (5.00 g, 23.00 mmol) was added to the reaction solution. The mixture was reacted at 80 °C for 5 h, and then cooled to room temperature before being poured into ice water and stirred for 12 h. The resulting yellow solid, Compound II, was used directly in the next step of the reaction (8.00 g, 46 mmol, Y = 91%). (3) Synthesis of compound Cy-Cl: Intermediate I (3.00 g, 16.00 mmol) and intermediate II (1.38 g, 8 mmol) were added to 10 mL of acetic anhydride, and sodium acetate (1.31 g, 16 mmol) was added. The mixture was reacted at 80 °C for 8 h, recrystallized three times with methanol and tert-butyl methyl ether, and dried to obtain a green solid Cy-Cl (5.20 g, 10.00 mmol, Y = 63%), which was directly used in the next step of the reaction.

[0037] (4) Synthesis of compound Cy-IMZ (NO2): Cy-Cl (1.00 g, 2 mmol), 5-nitro-1H-imidazole (2.21 g, 20 mmol) and sodium hydride (234.28 mg, 10 mmol) were added to 10 mL of N,N-dimethylformamide solution and reacted at 80 °C for 12 h. The mixture was recrystallized three times with methanol and tert-butyl methyl ether and dried to obtain green solid Cy-IMZ (NO2) (0.50 g, 0.85 mmol, Y = 43%).

[0038] As shown in Figures 1 and 3, ESI-HRMS: m / z calcd. M + for C37 H 42 N5O2 + , 588.3333, found, 588.3341. 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.29 (s, 1H), 7.57 (d, J =7.4 Hz, 2H), 7.43 (dd, J = 16.7, 7.6 Hz, 4H), 7.24 (t, J = 7.3 Hz, 2H), 6.79(d, J = 14.1 Hz, 2H), 6.39 (d, J = 14.0 Hz, 2H), 4.26 (q, J = 8.7, 8.0 Hz,4H), 2.78 (t, J = 6.3 Hz, 4H), 1.96 (p, J = 5.8 Hz, 2H), 1.27 (d, J = 11.4Hz, 18H). 13 C NMR (101 MHz, DMSO-d6) δ 172.12, 148.14, 141.95, 141.58, 140.60,139.54, 129.13, 126.89, 125.87, 123.98, 123.13, 111.97, 102.26, 49.23, 27.56,27.39, 24.55, 20.57, 12.82. Example 2 Synthesis of IR783--IMZ (NO2) The synthesis route is shown below:

[0039] Compound IR783 (2 mmol), 5-nitro-1H-imidazole (2.21 g, 20 mmol), and sodium hydride (234.28 mg, 10 mmol) were added to 20 mL of N,N-dimethylformamide solution and reacted at 80 °C for 16 h. The mixture was recrystallized three times with methanol and ethyl acetate, and dried to obtain a green solid IR783-IMZ(NO2) (0.85 mmol, Y = 43%). (See Figure 5.) 1 H NMR (400 MHz, DMSO-d6).

[0040] Example 3 Synthesis of Cy-IMZ (NO2)NPs nanoparticles 1.0 mg of the photothermal reagent Cy-IMZ (NO2) was fully dissolved in 0.5 mL of dimethyl sulfoxide (DMSO), and 5 mg of DSPE-PEG2000 was also fully dissolved in 0.5 mL of DMSO. The mixture was then placed in an ultrasonic cleaner and sonicated for 15 min to ensure complete dissolution. The mixture was then sonicated in an ultrasonic homogenizer (25 W), and the DMSO solution was rapidly added to 9.0 mL of deionized water in a single batch, followed by continuous sonication for 10 min. Afterward, the prepared crude nanoparticle product was dialyzed in a dialysis bag (Mw = 3500) for 48 h, with the water changed every 6 h. The solution in the dialysis bag was then collected and filtered through a 220 nm filter membrane to prepare Cy-IMZ (NO2) nanoparticles (NPs).

[0041] Characterization of nanoparticles: Dynamic light scattering (DLS) was used for testing. The nanoparticle dialysis solution was diluted appropriately, and 2.5 μL was dropped onto a copper grid covered with a carbon film. The nanoparticle solution on the grid was allowed to dry naturally, either at room temperature or using a drying oven to accelerate the drying process. After the sample dried, the copper grid was placed under a transmission electron microscope (TEM) to observe the sample by electron beam irradiation.

[0042] DLS results showed that Cy-IMZ(NO2) encapsulated in DSPE-PEG2000 formed uniform nanoparticles with an average hydrodynamic size of approximately 127 nm and a PDI of 0.237. Furthermore, TEM analysis revealed that the self-assembled Cy-IMZ(NO2) nanoparticles had a diameter of approximately 80 nm. Figure 6 All of the above results demonstrate the successful preparation of Cy-IMZ(NO2)NPs.

[0043] The synthetic route for the control example Cy-IMZ (H) is shown below:

[0044] Compound Cy-Cl (1.00 g, 2.00 mmol) and imidazole (1.33 g, 20 mmol) were added to 5 mL of N,N-dimethylformamide solution and refluxed at 80 °C for 5 h. After the reaction was allowed to cool to room temperature, it was recrystallized three times with methanol and tert-butyl methyl ether and dried to obtain the green solid compound Cy-IMZ(H) (0.63 g, 1.00 mmol, Y = 59%).

[0045] like Figure 3 , Cy-IMZ(H)ESI-HRMS: m / z calcd. M + for C 37 H 43 N4+ , 543.3482,found,543.3486. 1 H NMR (400 MHz, DMSO-d6)δ7.89 (s, 1H), 7.54 (d, J = 7.4 Hz, 2H), 7.48(s, 1H), 7.40(d, J= 7.7 Hz, 4H), 7.32 (s, 1H), 7.23 (t,J= 7.2 Hz, 2H), 6.85(d, J= 14.1 Hz, 2H), 6.31 (d,J = 14.1 Hz, 2H), 4.22 (q,J= 7.2 Hz, 4H), 2.74(t, J = 6.4 Hz, 4H), 1.94 (p,J = 6.5, 6.1 Hz, 2H), 1.26 (t,J= 10.8 Hz, 18H). 13 CNMR(101MHz,DMSO-d6)δ171.91,149.04,142.02,141.84,141.55,139.23,129.69,129.07, 127.39, 125.56, 123.04, 122.68, 111.74, 101.61, 49.12, 27.82,27.59, 24.46, 20.79, 12.74. Example 4: Spectral Performance Testing of Fluorescent Dyes The dyes Cy-Cl, Cy-IMZ (H) and Cy-IMZ (NO2) were dissolved in chromatographic grade DMSO to prepare a stock solution with a concentration of 50 mg / mL, and the Cy-IMZ (NO2) NPs dialysate was stored in a refrigerator at 4°C for later use.

[0046] (1) Absorption and emission spectra of dye Cy-IMZ (NO2) in different solvents During testing, the mother liquor was added to different solvents, mixed thoroughly, and then poured into quartz dishes. Tests were performed using a UV-spectrum spectrophotometer and a fluorescence spectrometer. All tests were conducted at room temperature. Figure 7 As shown, a shoulder peak was observed at a short wavelength of the dye's absorption peak, which may be related to the aggregation of the dye in solution. Simultaneously, the dye's emission decreased with increasing solvent polarity. This may be because when the dye is in a low-polarity environment, its ionization is relatively small, resulting in weaker interaction with the solvent and thus strong fluorescence; while in a polar solvent, the enhanced dipole moment leads to rapid dissipation of its excited-state energy, thereby weakening the fluorescence.

[0047] (2) UV absorption and fluorescence spectra of Cy-Cl, Cy-IMZ(H) and Cy-IMZ(NO2) Based on the results of the ultraviolet absorption spectrum, an appropriate amount of dye stock solution was added to control the absorbance of the dye in different solvents to around 0.3. The prepared solution was added to a long-necked quartz dish, and the corresponding excitation wavelength was set. The sample was tested in an absolute fluorescence quantum yield analyzer. Each sample was tested three times, and the final test result was the average of the three test results.

[0048] The results are as follows Figure 8 As shown, the UV absorption and fluorescence emission spectra of Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2) in aqueous solution were compared. It can be seen that after the introduction of imidazole and nitroimidazole, the maximum absorption wavelengths of Cy-IMZ(H) and Cy-IMZ(NO2) in aqueous solution exhibit a redshift compared to Cy-Cl. Near-infrared light has a deeper tissue penetration depth than ultraviolet light or shorter wavelength light and generally leads to a lower degree of tissue damage. Under the same testing conditions, the fluorescence of Cy-IMZ(NO2) was significantly quenched compared to Cy-Cl and Cy-IMZ(H).

[0049] (3) Test of absolute fluorescence quantum yield of dye like Figure 9 Cy-IMZ(NO2) exhibits an absolute fluorescence quantum yield of 0.1%, with fluorescence almost completely quenched. In contrast, Cy-Cl (Φf = 1.7%) and Cy-IMZ(H) (Φf = 2.6%) show strong fluorescence emission at 800 nm. The decay of the singlet excited-state energy of Cy-IMZ(NO2) along non-fluorescent pathways suggests that it can convert absorbed energy into heat non-radiatively, rather than releasing energy through fluorescence emission.

[0050] (4) UV absorption and fluorescence spectroscopy of Cy-IMZ (NO2) nanoparticles As shown in Figure 10, Cy-IMZ(NO2)NPs exhibit an absorption peak of approximately 728 nm in aqueous solution, indicating that the synthesis of DSPE-PEG... 2000 The encapsulating dye underwent H-aggregation. Comparison revealed that the ratio of the maximum absorbance of the H-aggregates to the monomers (A1 / A2≈0.78) was almost negligible in the Cy-IMZ(NO2) aqueous solution, with barely any absorption peak shifting to shorter wavelengths (A1 / A2≈0.39). This indicates that Cy-IMZ(NO2) aggregated to a relatively small extent in the aqueous solution. Simultaneously, the Cy-IMZ(NO2) NPs showed almost no fluorescence in the aqueous solution (undetectable).

[0051] Example 5: Dyes 1 Comparison of O2 generation capabilities DPBF was used as a singlet oxygen scavenger. 3 mL of MeOH was added to a sealable quartz cuvette, followed by an appropriate amount of DPBF stock solution. After thorough mixing, the absorbance at 415 nm was adjusted to approximately 1.0. Then, dye stock solution was added, and the absorbance at the maximum absorption wavelength of the dye molecules was adjusted to be between 0.3 and 0.4. This experiment used 808 nm light irradiation (10 mW / cm²). 2 The test time interval was set to 30 s for all tests, allowing us to obtain the change in spectral intensity with illumination time. The singlet oxygen quantum yield of the tested dye can then be calculated as follows:

[0052] in, This represents the singlet oxygen quantum yield of the dye being tested. The singlet oxygen quantum yield of indocyanine green is represented by K, which represents the slope of the decrease in absorbance of DPBF with increasing illumination time at a wavelength of 415 nm. The subscript ICG indicates the reference substance indocyanine green, and Ps indicates the dye to be tested.

[0053]

[0054] Where F is the correction factor for absorbance, and A 808 The absorbance at 808 nm.

[0055] As shown in Figure 11, the absorbance of the Cy-Cl and ICG groups at 414 nm decreased significantly with increasing illumination time, indicating that both Cy-Cl and ICG can produce [the desired light output] under 808 nm illumination. 1 O2 bleaching of DPBF. Under the same conditions, the rate of decrease in absorbance of Cy-IMZ(H), Cy-IMZ(NO2), and DPBF solutions was significantly slower, indicating that Cy-IMZ(H) and Cy-IMZ(NO2) reacted more readily under the aforementioned light conditions. 1 Its ability to generate O2 is very weak.

[0056] Example 6: In vitro photothermal properties of dyes (1) Temperature rise of different dyes Prepare aqueous solutions of Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2) at a concentration of 25 μg / mL. The test light source conditions were 808 nm and 0.3 W / cm². 2The laser power was used for a total irradiation of 10 minutes, and the temperature was recorded every 30 seconds using an infrared camera. The results (e.g.) Figure 12 The results show that Cy-IMZ(NO2) exhibits the largest temperature increase (ΔT ≈ 22℃), significantly greater than Cy-IMZ(H) (ΔT ≈ 8℃) and Cy-Cl (ΔT ≈ 5.5℃). Under the same conditions, the temperature of pure water only increases by 0.5℃, which is negligible.

[0057] (2) Effects of concentration and optical power density on temperature Aqueous solutions of Cy-IMZ (NO2) at concentrations of 5, 25, and 50 μg / mL were prepared. The test light source conditions were 808 nm and 0.3 W / cm². 2 The laser power was controlled, and the irradiation lasted for a total of 10 minutes, with the temperature recorded every 30 seconds using an infrared camera. Simultaneously, an aqueous solution of 50 μg / mL Cy-IMZ (NO2) was prepared, and the test light source conditions were 808 nm, 0.1, 0.25, and 0.5 W / cm². 2 The laser power was used for a total irradiation of 10 minutes, and the temperature was recorded every 30 seconds using an infrared camera. For example... Figure 13 The results shown all indicate that the photothermal conversion capability of Cy-IMZ(NO2) is highly dependent on concentration and optical power density.

[0058] (3) Photothermal stability test Aqueous solutions of 25 μg / mL ICG, Cy-Cl, Cy-IMZ(H), Cy-IMZ(NO2), and Cy-IMZ(NO2)NPs were prepared. The test light source conditions were 808 nm and 0.3 W / cm². 2 The laser power was applied for irradiation for 10 minutes, followed by cooling for 10 minutes, and this cycle was repeated 3 to 6 times. The temperature was recorded every 30 seconds using an infrared camera.

[0059] The results are as follows Figure 14 As shown, the dye molecule Cy-IMZ(NO2) exhibited significantly higher photothermal stability (808 nm, 0.3 W / cm²) than ICG, Cy-Cl, and Cy-IMZ(H) after three heating and cooling cycles. 2 Excellent performance. Simultaneously, the stability of Cy-IMZ(NO2) NPs was tested, including temperature changes during six heating and cooling cycles under continuous laser irradiation (808 nm, 0.3 W / cm²). 2The results showed that Cy-IMZ(NO2) NPs not only maintained a similar temperature rise as the monomeric Cy-IMZ(NO2), but also, even after six heating and cooling cycles, the Cy-IMZ(NO2) NPs could still essentially reach the initial temperature. Figure 15 This indicates that Cy-IMZ(NO2) NPs possess excellent photothermal stability.

[0060] (4) Photothermal conversion efficiency test and calculation The photothermal conversion capabilities of Cy-IMZ(H) and Cy-IMZ(NO2) were further quantified and compared with those of Cy-Cl and ICG. The photothermal conversion efficiency (PCE) of ICG, Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2) was tested and calculated. Aqueous solutions of 25 μg / m³ of ICG, Cy-Cl, Cy-IMZ(H), and Cy-IMZ(NO2) were prepared. The test light source conditions were 808 nm and 0.3 W / cm². 2 The laser power was applied for irradiation for 10 minutes, then cooled to room temperature, with the temperature recorded every 30 seconds using an infrared camera. The PCE of the dye was calculated using the following formula:

[0061] The heat transfer coefficient h is calculated using the following formula, where s is the surface area of ​​the container, and Q is the heat transfer coefficient. Dis I represents the heat dissipated by the solvent and container, and A represents the laser power. 808 The absorbance at 808 nm.

[0062]

[0063] Where m is the mass of the photothermal reagent solution, and C H2O The specific heat capacity of the solution is 4.2 J / (g·℃), and the relevant time constant τ is... s Calculated as follows

[0064] Wherein, the driving force temperature θ is a dimensionless parameter.

[0065] Among them, T Max For the maximum steady-state temperature, T Surr The ambient temperature.

[0066] Data such as Figure 16The results show that the PCE order of the four dyes is Cy-IMZ(NO2) > Cy-IMZ(H) > ICG > Cy-Cl, with Cy-IMZ(NO2) having a PCE as high as 73%.

[0067] Example 7: Evaluation of cellular uptake of Cy-IMZ (NO2) nanoparticles 4T1 cells were used for cell uptake experiments to determine the maximum enrichment time of nanoparticles in cells and to identify the optimal treatment time for subsequent photothermal therapy. Since Cy-IMZ (NO2) NPs are non-fluorescent, they were encapsulated with the commercially available fluorescent dye Cy5 to create NPs for imaging. Nanoparticles loaded with the fluorescent dye were co-incubated with 4T1 cells for different incubation times, and then observed using confocal laser scanning microscopy (CLSM). The results showed that after incubation, the nanoparticles in the cytoplasm emitted strong red fluorescence, indicating that the nanoparticles had been effectively uptaken by the cells and reached saturation at 3 hours. Figure 17 ).

[0068] Example 8: Evaluation of phototoxicity and dark toxicity of Cy-IMZ (NO2) nanoparticles on cells The first step was cell seeding and pretreatment. 4T1 and MCF-7 cells were seeded into 96-well plates, with each well containing 1×10⁶ cells. 4 Cells were incubated in culture medium for 24 hours to allow them to attach and recover. Next, in the cell treatment stage, culture medium containing different concentrations of Cy-IMZ(NO2) NPs (0, 1, 2, 3, 4, 5, 6 μg / mL) was added to each well, and the cells were incubated for 3 hours to allow time for cell uptake of the nanoparticles. Afterwards, the Cy-IMZ(NO2) NPs-treated cells were exposed to laser irradiation at 808 nm, 0.3 W / cm². 2 The cells were irradiated with high intensity laser light for 10 minutes. Finally, cell viability was assessed. After laser irradiation, the cells were incubated for another 12 hours, and then MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol) solution was added to each well to react with the live cells and produce purple formazan crystals. After incubation at 37°C for 4 hours, the solution was carefully removed, and 100 μL of DMSO was added to each well. The absorbance at 490 nm was measured using a microplate reader.

[0069] Cell viability = (OD experimental group) OD blank control / (OD negative control) (OD blank control) × 100%, where the negative and blank control groups were the no-drug group and the blank culture medium group, respectively.

[0070] As shown in Figure 18, Cy-IMZ(NO2) NPs exhibited good biocompatibility in both cell types under non-illuminated conditions. Since the photothermal effect of the dye is concentration- and power-dependent, phototoxicity experiments were conducted at different concentrations and power levels. The results showed that the dye has good phototoxicity, and its biocompatibility can be maintained at 0.3 W / cm². 2 Effectively kills cells.

[0071] Example 9: Evaluation of the photothermal killing performance of Cy-IMZ (NO2) nanoparticles on cancer cells This invention uses Calcein AM and PI (propidium iodide) staining agents to distinguish between live and dead cells. 4T1 cells were seeded in 35 mm confocal culture dishes and incubated for 24 h, then divided into four groups: (1) Control group: Cells were given fresh culture medium and no light treatment was applied; (2) Light group: Cells were replaced with fresh culture medium, using 808 nm, 0.3 W / cm 2 Laser irradiation for 10 minutes; (3) Cy-IMZ (NO2) NPs: Cells were added to DMEM medium containing 6 μg / mL Cy-IMZ (NO2) NPs, without light treatment; (4) Cy-IMZ (NO2) NPs + Light: Cells were added to DMEM medium containing 6 μg / mL Cy-IMZ (NO2) NPs, incubated for 3 h, and then exposed to 808 nm, 0.3 W / cm 2 Laser irradiation for 10 minutes.

[0072] After incubating the cells for another 2 hours, they were stained with Calcein AM and PI (excitation wavelength 488 nm, Calcein AM channel 505 nm-545 nm, PI channel 600 nm-700 nm) and imaged using a confocal microscope. Figure 19 As shown, strong green fluorescence was observed in the first three groups, indicating cell survival. However, after laser irradiation, strong red fluorescence signal was observed in 4T1 cells incubated with Cy-IMZ (NO2) NPs, with almost no green fluorescence signal, indicating that the 4T1 cells were almost completely killed.

[0073] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention. Various modifications and equivalent substitutions can be made to the invention by those skilled in the art without departing from its spirit and scope. For example, the order of steps can be adjusted, the concentration of components, reaction conditions, etc., can be varied within reasonable ranges, and the materials used can be replaced by functionally equivalent substances. Therefore, the scope of protection of this invention should be determined by the claims, and not limited to the above embodiments.

Claims

1. A class of heptamethine cyanine dyes with a meso-substituted nitroimidazole group, characterized in that: It has the following structural formula as shown in General Formula 1: In general formula 1: L is selected from one of the substituents described in formulas L1 to L3: R1 is selected from one of the substituents described in formulas A1 to A5 below: R2 is selected from one of the substituents described in formulas A1 to A5 below: P1 and P2 are each independently selected from one of the following groups: sulfonate group, carboxyl group, amino group, hydrogen atom, and halogen atom; X is selected from one of methylene, secondary amino, N-methyl, and N-ethyl; In formula A1, n is selected from any integer from 0 to 15.

2. The dye according to claim 1, characterized in that: The dye has an excitation wavelength of 730-780 nm and an emission wavelength of 800-860 nm.

3. The method for preparing the dye according to claim 1, characterized in that: The synthesis route is as follows: The dye of general formula S-1 is mixed evenly with the nitroimidazolium compound of L in a dry organic solvent under alkaline conditions, heated at 70-120℃ for 8-24 h, crystallized, and dried to obtain the product.

4. The method according to claim 3, characterized in that: The organic solvent is selected from one of N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, and 1,4-dioxane.

5. The method according to claim 3, characterized in that: The molar ratio of the compound with general formula S-1 to the nitroimidazolium group shown in compound L is 1:(1-10).

6. The method according to claim 3, characterized in that: The alkaline agent for the alkaline conditions is selected from at least one of sodium hydride, sodium hydroxide, potassium hydroxide, sodium methoxide, N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, and cesium carbonate.

7. The method according to claim 3, characterized in that: The reaction temperature of the synthetic route is 70-90℃, and the reaction time is 8-16 h.

8. The method according to claim 3, characterized in that: The solvent used for recrystallization in the synthetic route is selected from at least one of water, methanol, ethanol, acetonitrile, isopropanol, ethyl acetate, and tert-butyl methyl ether.

9. The use of the dye as described in claim 1 in protein labeling, bioimaging, and preparation of photothermal therapy photosensitizing agents for non-diagnostic or therapeutic purposes.

10. The application according to claim 9, characterized in that: The dye is used in the preparation of drugs for photothermal therapy of tumors.

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