Preparation method and application of polarity and viscosity dual-response type NIR-II fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes. In particular to a polarity and viscosity double-response type NIR-II fluorescent probe as well as a preparation method and application thereof. The invention provides a method for synthesizing a near-infrared two-region emission fluorescent probe by taking 5-tert-butyl-2-chloro-3-hydroxycyclohexane-1-ene-1-formaldehyde and 6-bromo-2-methyl-1-ethyl quinoline-1-onium as raw materials, and a preparation method of the near-infrared two-region emission fluorescent probe. The probe can detect polarity and viscosity with high sensitivity and high selectivity, has a photo-thermal treatment effect, can emit fluorescence in a near-infrared second region, and has a good photo-thermal killing effect on breast cancer cells. The probe provides a precise tool for polarity and viscosity detection and tumor diagnosis and treatment in the field of biomedicine, and is expected to be widely applied to disease diagnosis and drug research and development.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a polarity and viscosity dual-response NIR-II fluorescent probe, its preparation method, and its application. Background Technology

[0002] Breast cancer is the most common cancer among women worldwide, ranking first in both incidence and mortality among female cancers, seriously threatening women's health and lives (Liu XH, Wang ZC, Shen L., Shen SL, and Zhang XF Viscosity-Responsive Cell-Membrane-Anchored Fluorescent Probe for Visualization of Tumor Cell Membranes and Tumors. Analytical Chemistry, 2025, 97, 26759-26769). The clinical management of precancerous and malignant breast cancer lesions places a significant burden on healthcare systems. Although significant progress has been made in the diagnosis and treatment of breast cancer, reliable biomarkers are still needed to further improve women's screening, triage, and management. In recent years, using changes in the cancer cell microenvironment to achieve accurate cancer diagnosis has become a hot research area. Fluorescent probes can be activated or quenched under specific biological stimuli associated with cancer cells. These probes respond to changes in the subcellular microenvironment, such as viscosity, polarity, pH, and oxygen content. These subcellular microenvironmental factors are considered key biomarkers for cancer development (Qian M., Ye Y., Ren TB, Xiong B., Yuan L., and Zhang XB Cancer-Targeting and Viscosity-Activatable Near-Infrared Fluorescent Probe for Precise Cancer Cell Imaging. Analytical Chemistry, 2024, 96, 13447-13454). Among these, viscosity, as an important parameter in the cellular microenvironment, influences signal transduction, metabolite diffusion, and interactions between biomolecules. Increased tumor-associated viscosity is associated with lactate production and elevated levels of proteins, enzymes, and lipids.Polarity is another key microenvironmental parameter that plays a crucial role in maintaining cellular homeostasis, and its abnormalities are often closely associated with cellular dysfunction and cancer (Narotamo H., Franco CA, Silveira M. 3D CellPol: Joint Detection and Pairing of Cell Structures to Compute Cell Polarity. Biomedical Signal Processing and Control, 2025, 104, 107537). Abnormalities in viscosity and polarity are closely related to cellular dysfunction and cancer. Therefore, monitoring cancer biomarkers could provide potential strategies for the precise diagnosis and treatment of cancer.

[0003] Near-infrared II (NIR-II) fluorescent imaging probes, with their longer emission wavelengths (900-1700 nm), can effectively reduce photon scattering and autofluorescence, thereby improving the penetration ability and imaging resolution of deep tissues (Xu LZ, Zhang Q., Wang X., Lin WY Biomedical Applications of NIR-II Organic SmallMolecule Fluorescent Probes in Different Organs. Coordination ChemistryReviews, 2024, 519, 216122). Existing NIR-II probes are either in a "normal" mode or respond to only a single stimulus, leading to low signal-to-noise ratios and potential false positives (Dou K., Lu J., Xiu Y. L, Wang R., Won M., Kim J., Yu FB, and Kim JS Metabolic Acidity / H2O2 Dual‐Cascade‐Activatable Molecular Imaging Platform toward Metastatic Breast Tumor Malignancy. Angewandte Chemie International Edition, 2024, 64, e202419191). Fluorescent probes capable of detecting multiple biomarkers in a single system offer significant advantages, such as reduced detection complexity and consistent in vivo localization and metabolism. Multi-biomarker responsive fluorescent probes have attracted widespread attention due to their ability to improve detection specificity and enable multiplex analysis. This approach can reduce false positives and improve imaging resolution (Deng M., Wang PP, Zhai ZB, Liu Y., Cheng D., He L. W., and Li S. J. A Triple-Responsive and Dual-NIR Emissive Fluorescence Probe for Precise Cancer Imaging and Therapy by Activating Pyroptosis Pathway. Analytical Chemistry, 2025, 97, 2998-3008). Furthermore, the expression of polarity and viscosity in breast cancer cells differs from that in normal cells, making it a reliable marker of the microenvironment.Based on the aberrant expression of polarity and viscosity, NIR-II fluorescent probes can be precisely activated in hypoxic tumor environments, enabling targeted imaging of breast cancer and thus providing higher diagnostic sensitivity and accuracy. Therefore, the rational design and synthesis of polarity- and viscosity-responsive NIR-II fluorescent probes is crucial for achieving early diagnosis, deep localization, and personalized treatment planning for breast cancer.

[0004] Photodynamic therapy (PDT) has demonstrated immense potential in cancer treatment due to its remarkable therapeutic effects, high specificity, and minimally invasive nature. This therapy generates reactive oxygen species (ROS) through photosensitizers (PS), light irradiation, and molecular oxygen, thereby inducing local chemical damage (Nguyen VN, Nguyen MV, Pham TH, Vu AT, and Nguyen TX: Recent Advances in Near-Infrared Organic Photosensitizers for Photodynamic Cancer Therapy. Biomaterials Science, 2025, 13, 1179-1188). Currently, PDT is widely used in cancer treatment, particularly showing promising clinical applications in targeted tumor therapy and local treatment. Given the low light absorption characteristics of biological samples, near-infrared (NIR) lasers are often chosen as the induction light source for PDT. In particular, NIR-II lasers (900-1700 nm) offer better phototherapy (PDT) effects compared to near-infrared I (750-900 nm) lasers due to less scattering, allowing for deeper tissue penetration. Furthermore, compared to traditional fluorescent probes, highly photodynamic activated fluorescent probes not only possess imaging capabilities but can also generate photodynamic effects through light triggering, thus achieving the dual benefits of photodynamic therapy.

[0005] Given the limited research on polarity and viscosity dual-response NIR-II fluorescent probes in photodynamic therapy, it is urgent to develop an NIR-II fluorescent probe that can both achieve early diagnosis of breast cancer and possess photodynamic therapy capabilities. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a polarity and viscosity dual-response NIR-II fluorescent probe, its preparation method, and its photodynamic applications. The fluorescent probe achieves dual response to polarity and viscosity, exhibits strong fluorescence emission in NIR-II, and demonstrates excellent photodynamic targeting (PDT) at tumor sites, effectively killing cancer cells, thus solving the problems existing in the prior art.

[0007] The technical solution of this invention is to provide a polarity and viscosity dual-response NIR-II fluorescent probe, the structural formula of which is: .

[0008] This invention provides a method for preparing a polarity and viscosity dual-response NIR-II fluorescent probe, the synthesis steps of which include:

[0009] 6-Bromo-1-ethyl-2-methylquinoline-1-onium salt and 5-tert-butyl-2-chloro-3-hydroxycyclohexyl-1-ene-1-carboxaldehyde were added to a round-mouth flask and dissolved in n-butanol. Piperidine was then added, and the mixture was heated and stirred. After the reaction was complete, the solvent was removed by rotary evaporation, and the sample was purified by silica gel column chromatography to obtain the fluorescent probe.

[0010] Preferably, the molar ratio of 6-bromo-1-ethyl-2-methylquinoline-1-onium salt and 5-tert-butyl-2-chloro-3-hydroxycyclohex-1-ene-1-carboxaldehyde is 2-3:1.

[0011] Preferably, the solvent n-butanol content is 10-20 mL.

[0012] Preferably, the piperidine content is 80-200 μL.

[0013] Preferably, the reaction temperature is 90-110 ℃.

[0014] Preferably, the stirring reaction time is 9-12 hours.

[0015] Preferably, the eluent used in the silica gel column chromatography purification is dichloromethane:methanol = 20-10:1.

[0016] The polarity and viscosity dual-response NIR-II fluorescent probe of this invention can respond to abnormal changes in polarity and viscosity in the tumor microenvironment. NIR-II fluorescence is activated in a hypoxic tumor environment, achieving highly sensitive and selective detection of polarity and viscosity in breast cancer cells. Simultaneously, under 808 nm laser irradiation, the probe generates a large amount of reactive oxygen species, effectively killing 4T1 breast cancer cells, thus possessing the dual functions of precise detection and photodynamic therapy.

[0017] The beneficial effects of the present invention compared with the prior art;

[0018] (1) After the probe reacts with polarity / viscosity, the fluorescence intensity at 900 nm-1200 nm is significantly enhanced, and it shows a good linear relationship with polarity and viscosity, resulting in high detection sensitivity. In the presence of various interfering substances, it only produces a significant fluorescence response to polarity and viscosity, demonstrating outstanding selectivity.

[0019] (2) Because the molecular structure contains two bromine atoms, the probe has a high efficiency in generating reactive oxygen species (ROS) and can achieve significant photodynamic therapy on breast cancer cells under 808 nm laser irradiation, providing a new tool for NIR-II photodynamic therapy of in situ breast cancer. Attached Figure Description

[0020] Figure 1 This is a synthetic route diagram for the NIR-II fluorescent probe.

[0021] Figure 2 For NIR-II fluorescent probe molecules 1 H NMR spectrum.

[0022] Figure 3 This is a high-resolution mass spectrum of the NIR-II fluorescent probe.

[0023] Figure 4 The absorption spectrum of the NIR-II fluorescent probe (10 μM) in pure water and a mixed solution of 1,4-dioxane:water = 50:50 is shown.

[0024] Figure 5 The fluorescence spectra of the NIR-II fluorescent probe (10 μM) in different concentrations of 1,4-dioxane are shown.

[0025] Figure 6 The linear relationship between the fluorescence intensity of the NIR-II fluorescent probe and different concentrations of 1,4-dioxane is shown.

[0026] Figure 7 The absorption spectrum of the NIR-II fluorescent probe (5 μM) in a mixed solution of pure water and glycerol:water = 90:10 is shown.

[0027] Figure 8 The fluorescence spectra of the NIR-II fluorescent probe (10 μM) at different viscosity values ​​(6.5 - 226 cp) are shown.

[0028] Figure 9 This represents the linear relationship between the fluorescence intensity of the NIR-II fluorescent probe and the logarithm of its viscosity value.

[0029] Figure 10 The fluorescence intensity at 930 nm is shown for the NIR-II fluorescent probe (10 μM) in response to different analytes (100 μM). (1: Blank; 2: Mg) 2+ ; 3:Ca 2+ ; 4:NH 4+ ; 5:Fe 3+ ; 6:Cu 2+ 7:K + ; 8:Na+ 9:HPO 4- ; 10:CH3COO - ; 11:Br - ; 12:Cl - ; 13:ClO - ; 14:NO 2- ; 15:H2O2; 16:NO 3- ; 17:CO3 2- ; 18:S2O4 2- ;19:HS - ; 20:SO4 2- ; 21:HSO 3- ; 22: Val; 23: Thr; 24: Met; 25: pHe; 26: Leu; 27: Ile; 28: Tvp; 29: Cys;

[0030] Figure 11 This is a diagram of a cytotoxicity experiment using the NIR-II fluorescent probe.

[0031] Figure 12 The fluorescence spectra of the reactive oxygen species probe DCFH in aqueous solution are shown for different durations of irradiation with an 808 nm laser by the NIR-II fluorescent probe.

[0032] Figure 13 Images of confocal fluorescence generated by the reactive oxygen species (ROS) probe DCFH-DA in 4T1 cells under different conditions (probe DCFH-DA was used for ROS staining, scale bar: 40 µm).

[0033] Figure 14 Image showing staining of live and dead cells with NIR-II fluorescent probe (scale bar: 100 µm). Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to the prior art, and reagents or instruments used without specifying the manufacturer are considered to be conventional products that can be purchased commercially.

[0035] Example 1:

[0036] Synthesis of NIR-II fluorescent probes

[0037] 0.750 g (3 mmol) of 6-bromo-1-ethyl-2-methylquinoline-1-onium salt and 0.228 g (1 mmol) of 5-tert-butyl-2-chloro-3-hydroxycyclohex-1-en-1-carboxaldehyde were added to a round-mouth flask and dissolved in n-butanol (10 mL). Piperidine (100 μL) was then added, and the mixture was heated to 110 °C and stirred for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with dichloromethane:methanol = 10:1 as the eluent, yielding a dark red solid, which was the fluorescent probe (0.416 g), with a yield of approximately 41.6%.

[0038] The prepared compounds were characterized using proton nuclear magnetic resonance spectroscopy: 1 1H NMR (400 MHz, Acetone) δ 8.25 (d, J = 13.6 Hz, 2H), 8.02 (dd, J = 5.9, 3.7 Hz, 4H), 7.97 (d, J = 9.5 Hz, 2H), 7.87 – 7.79 (m, 4H), 6.43 (d, J = 13.6 Hz, 2H), 4.63 (s, 4H), 3.03 (d, J = 12.7 Hz, 2H), 2.29 (t, J = 13.8 Hz, 2H), 1.55 (t, J = 7.1 Hz, 6H), 1.40 (d, J = 6.8 Hz, 1H), 1.08 (s, 9H). The 1H NMR spectrum is shown below. Figure 2 As shown, this confirms that the compound is the NIR-II fluorescent probe described above.

[0039] Furthermore, the prepared compound was further validated using high-resolution mass spectrometry: HR-MS (ESI, m / s): The theoretically calculated molecular mass-to-charge ratio is [M]. + C 36 H 38 Br₂ClN₂⁺: 693.1065, the actual molecular weight obtained is 693.1065; high-resolution mass spectrum as shown below. Figure 3 As shown, this further illustrates that the structure of the prepared compound conforms to expectations.

[0040] Example 2:

[0041] NIR-II fluorescent probe and polarity response

[0042] First, the spectroscopic properties of the NIR-II fluorescent probe for polarity were investigated in a H2O / 1,4-dioxane mixed solution. For example... Figure 5As shown, the maximum absorption wavelength of the NIR-II fluorescent probe in H2O is 630 nm. Upon addition of 1,4-dioxane (50%), the maximum absorption wavelength of the probe redshifts to 880 nm. The NIR-II fluorescence spectra of the NIR-II fluorescent probe in solvents of different polarities were then investigated. Figure 6 As shown, the fluorescence emission spectra of the NIR-II fluorescent probe in solutions of different polarities were detected. A series of solutions with different polarities were obtained by adjusting the ratio of water to 1,4-dioxane. The data show that the NIR-II fluorescent probe exhibits significant fluorescence emission intensity in media with low dielectric constants. Figure 7 As shown, when the proportion of 1,4-dioxane in the 1,4-dioxane / H2O mixture increases from 30% to 85%, the fluorescence emission intensity at 930 nm gradually increases, and the fluorescence intensity shows a good linear relationship with the concentration of 1,4-dioxane (R²=0.9809), indicating that the NIR-II fluorescent probe can be used to detect polarity.

[0043] Example 3:

[0044] Response of NIR-II fluorescent probe to viscosity

[0045] First, the spectral response of the NIR-II fluorescent probe to viscosity was detected in an H2O / Glycerol mixed solution. For example... Figure 7 As shown, the maximum absorption wavelength of the NIR-II fluorescent probe in H2O is 630 nm. Upon addition of glycerol (90%), the maximum absorption wavelength of the probe exhibits a significant red shift, with new absorption peaks appearing at 760 nm and 875 nm. The NIR-II fluorescence spectra of the NIR-II fluorescent probe in solvents of different viscosities were then investigated. Figure 8 and 9 As shown, the fluorescence emission spectra of the NIR-II fluorescent probe in solutions of different viscosities were detected. Solutions with viscosities ranging from 6 cp to 219 cp were obtained by adjusting the ratio of water to glycerol. The data show that in solutions of different viscosities (6 cp–219 cp), the fluorescence intensity of the NIR-II fluorescent probe is stronger at higher viscosities, and the position of the fluorescence emission peak remains stable. This is because the high-viscosity environment restricts the movement of probe molecules, reducing non-radiative energy loss and thus enhancing the fluorescence signal. At an emission wavelength of 930 nm, the fluorescence intensity shows a good linear relationship with viscosity (R² = 0.9423), indicating that the NIR-II fluorescent probe can be used for viscosity detection.

[0046] Example 4:

[0047] Selectivity of NIR-II fluorescent probes

[0048] To verify the selectivity of the probe for polarity and viscosity, the fluorescence spectrum of the NIR-II fluorescent probe was first measured in an H2O system. Figure 10 As shown, various analytes were added to the NIR-II fluorescent probe, including metal ions (Mg²⁺, Ca²⁺, NH₄⁺, Na⁺, Fe³⁺, Cu²⁺, K⁺), anions (Cl⁻, Br⁻, CH₃COO⁻, HPO₄⁻), reactive nitrogen species (NO₂⁻, NO₃⁻), reactive sulfur species (HSO₃⁻, SO₄²⁻, HS⁻, S₂O₄²⁻), reactive oxygen species (ClO⁻, CO₃²⁻), biothiols (Cys, Hcy, GSH), and amino acids (Val, Thr, Met, pHe, Leu, Ile, Tvp). At 930 nm, the fluorescence signals of Glycerol (85%) and 1,4-dioxane (85%) were significantly enhanced, while the responses of other components were negligible. The results show that the NIR-II fluorescent probe exhibits high selectivity for polarity and viscosity, enabling accurate detection in complex biological environments.

[0049] Example 5:

[0050] Cytotoxicity assays using NIR-II fluorescent probes

[0051] The toxicity of different concentrations of NIR-II fluorescent probes to 4T1 cells was detected using the CCK-8 assay. After adding different concentrations (0-50 μM) of the fluorescent probe to 4T1 cells and culturing for 24 hours, the cell viability was greater than 90%. Figure 11 This indicates that the NIR-II fluorescent probe has low cytotoxicity.

[0052] Example 6:

[0053] Photodynamic properties determination of NIR-II fluorescent probe

[0054] The ability of a NIR-II fluorescent probe to generate reactive oxygen species under 808 nm laser irradiation was investigated using the reactive oxygen species probe DCFH-DA. Figure 12 It can be seen that using an 808 nm laser (0.6 W cm⁻¹) -2 Irradiation of the NIR-II fluorescent probe for 0-7 min showed that the fluorescence of the reactive oxygen species (ROS) probe DCFH-DA at 540 nm increased with increasing irradiation time, indicating that the NIR-II fluorescent probe can generate a large amount of ROS. The ability of the NIR-II fluorescent probe to generate ROS in 4T1 cells was further investigated. Figure 13 As shown, when treated with the NIR-II fluorescent probe alone, the cells showed no fluorescence, meaning no reactive oxygen species were produced; however, when treated with an 808 nm laser (0.6 W cm⁻¹), the cells showed no fluorescence.-2 After 5 minutes of irradiation, the fluorescence intensity in the cells was significantly enhanced, proving that the NIR-II fluorescent probe can generate a large amount of reactive oxygen species under 808 nm laser irradiation, and has the potential for application in NIR-II photodynamic therapy.

[0055] Example 7:

[0056] Live and dead cell staining with NIR-II fluorescent probe

[0057] Finally, the ability of the NIR-II fluorescent probe to kill breast cancer 4T1 cells was further investigated. 4T1 cells, after different treatments, were co-stained with calcein AM (green) and propidium iodide (PI, red) for confocal fluorescence imaging. The experimental results are as follows: Figure 14 As shown, only when 4T1 cells were incubated with the NIR-II fluorescent probe (10 μM) and treated with an 808 nm laser (0.6 W cm⁻¹) did the effect occur. -2 A large number of cells died only after 5 minutes of irradiation. The experimental results further validated the highly efficient photodynamic therapy effect of the NIR-II fluorescent probe on breast cancer cells.

Claims

1. A polarity and viscosity dual-response NIR-II fluorescent probe, characterized in that, Its chemical structure is as follows:

2. The method for preparing the polarity and viscosity dual-response NIR-II fluorescent probe according to claim 1, characterized in that, Its synthetic route is as follows:

3. The method for preparing the polarity and viscosity dual-response NIR-II fluorescent probe according to claim 2, characterized in that, The reaction steps include the following: 6-Bromo-1-ethyl-2-methylquinoline-1-onium salt and 5-tert-butyl-2-chloro-3-hydroxycyclohexyl-1-ene-1-carboxaldehyde were added to a round-mouth flask and dissolved in n-butanol. Piperidine was then added, and the mixture was heated to 110 °C and stirred for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with dichloromethane:methanol = 10:1 as the eluent, yielding a deep red solid, which is the fluorescent probe.

4. The method for preparing the polarity and viscosity dual-response NIR-II fluorescent probe according to claim 2, characterized in that, The molar ratio of 6-bromo-1-ethyl-2-methylquinoline-1-onium salt to 5-tert-butyl-2-chloro-3-hydroxycyclohex-1-ene-1-carboxaldehyde is 3:

1.

5. The application of the polarity and viscosity dual-response NIR-II fluorescent probe according to claim 1, characterized in that, The fluorescent probe has two bromine atoms, exhibiting excellent photodynamic therapy effects and effectively killing cancer cells.

Images