Two-photon fluorescent probe and application thereof in monitoring of tumor microenvironment

Abstract

The application discloses a two-photon fluorescent probe and uses the same for tumor microenvironment monitoring. The application opens the two-photon fluorescent characteristics of the material by responding to the overexpression of SO2 and H2O2 at the tumor site in stages, monitors and indicates the changes of cell oxidative stress and homeostasis balance, and is further used for early diagnosis of diseases caused by internal environment homeostasis imbalance. The application regulates the cancer cell uptake capacity of the molecule by introducing suitable lipophilic and hydrophilic groups, increases the recognition site of biological thiols by introducing conjugated olefin bonds, and further realizes the step-by-step recognition of endogenous signal molecules by using the easiness of substitution reaction, enhances the fidelity of the signal, and is expected to realize the in-situ activated two-photon fluorescence, early and accurate diagnosis of a series of diseases such as cardiovascular diseases, Alzheimer's disease and the like induced by internal environment homeostasis imbalance.

Description

Technical Field

[0001] This invention belongs to the field of life science technology, specifically relating to a two-photon fluorescent probe and its application in tumor microenvironment monitoring. By responding stepwise to the overexpression of SO2 and H2O2 in tumor sites, the two-photon fluorescence properties of the material are activated to monitor and indicate changes in cellular oxidative stress and homeostasis. Background Technology

[0002] Oxidative stress typically refers to the failure of intracellular defenses against reactive oxygen species (ROS) or the excessive production of ROS, leading to cellular dysfunction and tissue damage. Hydrogen peroxide (H2O2), a typical reactive oxygen species, is an essential oxygen metabolite. Physiologically, it is considered a ubiquitous intracellular messenger, playing a crucial role in the innate immune system, host defense, oxidative biosynthesis, metabolism, oxidative stress, and signal transduction. Abnormal levels of endogenous H2O2 in cells are associated with various diseases, such as cancer, diabetes, neurodegenerative diseases, inflammation, and cardiovascular diseases. Besides excessive ROS production, imbalances in antioxidant metabolism can also lead to cellular oxidative stress. Reduced sulfur dioxide (SO2) is a reactive sulfur compound, typically presenting as sulfite (SO32-). 2- ) and hydrogen sulfate (HSO3) - Endogenous SO2 exists in neutral fluids or blood plasma and plays a crucial role in maintaining cellular homeostasis and regulating redox states. Abnormalities in endogenous SO2 concentration are often closely associated with Alzheimer's disease and cardiovascular disease. Endogenous H2O2 and SO2 are closely linked, maintaining intracellular dynamic balance, and their levels reflect oxidative stress and disease.

[0003] Fluorescence imaging technology, with its advantages of high sensitivity, low detection limit, simple operation, fast response speed, and low invasiveness, is widely used for imaging and detecting bioactive molecules in living cells. It can provide high-resolution imaging of biological samples and real-time tracking of the location, activity, interactions, and metabolism of biomolecules, providing a powerful tool for studying changes in life activities. Small molecule fluorescent probes have shown great promise in fields such as bioimaging, drug screening, and medical diagnostics. Their combination with fluorescence confocal imaging technology makes it easier to apply fluorescent probes to the non-destructive detection of bioactive molecules in vivo. Detection of multiple signal molecules in organisms often involves multiple fluorescent probes. However, multiple probe combinations often have different biological and chemical properties in vivo, such as emission wavelength and localization, and can interfere with each other, complicating the detection process and sometimes rendering quantitative analysis methods ineffective. This limitation is particularly pronounced when considering imaging applications in cell and animal models. Therefore, designing a single fluorescent probe to simultaneously identify endogenous biological signal molecules is of great significance, especially cascade recognition, which is crucial for monitoring the body's internal environmental homeostasis and for the early diagnosis of diseases caused by homeostasis imbalances.

[0004] Compared to traditional single-photon fluorescence imaging, two-photon fluorescence imaging offers deeper tissue penetration and lower photodamage. The two-photon absorption cross section (δ) is a crucial parameter for evaluating two-photon performance. Generally, δ(ω) can be increased by adjusting three structural units: the electron donor group (D), the electron acceptor group (A), and the conjugated π-connector linking the electron donor and acceptor. For example, increasing the degree of intramolecular charge transfer (ICT) and increasing the electron intensity or conjugation degree of the donor / acceptor (D-π-A) can significantly improve the δ value of common two-photon fluorophores. However, for conjugated systems with rotor structures, distorted intramolecular charge transfer (TICT) is prone to occur, which is detrimental to the development of two-photon activity. Furthermore, from a molecular design perspective, rigid or highly conjugated structures typically face problems such as high cost, complex synthesis, low economic efficiency, and poor solubility, which also hinders their further biological applications. Therefore, there is an urgent need to develop simple, convenient, and biosafety-efficient organic small molecule dyes that can activate the two-photon activity of signaling molecules overexpressed at tumor sites in situ through fluorescence response, enabling real-time and precise early diagnosis of diseases caused by homeostasis imbalance, thus achieving "early detection and early treatment". Summary of the Invention

[0005] To address the problems of the prior art, this invention provides a two-photon fluorescent probe for tumor microenvironment monitoring. This invention relates to a cascade-responsive near-infrared fluorescent probe for the tumor microenvironment and its preparation. Specifically, it activates the two-photon fluorescence properties of a material by responding sequentially to the overexpression of SO2 and H2O2 at tumor sites, monitoring and indicating changes in cellular oxidative stress and homeostasis, and further applying it to the early diagnosis of diseases caused by internal environment homeostasis imbalance. To achieve this objective, this invention employs the following technical solution:

[0006] Firstly, this invention provides a two-photon fluorescent probe CVS, with the molecular formula C... 25 H 26 N2O5S2, named as: (E)-3-(2-(2-(7-(diethylamino)-2-oxo-2H-benzopyran-3-yl)vinyl)benzo[d]thiazolyl-3-onthiol-3-yl)propane-1-sulfonic acid inner salt, with the following structural formula:

[0007]

[0008] Secondly, the present invention also provides a method for preparing the two-photon fluorescent probe, which includes the following steps:

[0009] 2-Methylbenzothiazolyl propane sulfonate and 7-(diethylamino)-2-oxo-2H-coumarin-3-carboxaldehyde were dissolved in a solvent, an initiator was added, and the mixture was refluxed at 60℃~90℃ for 20~30h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure, separated by column chromatography, and dried to obtain a purple-red solid, CVS.

[0010] The preparation process involves the following reactions:

[0011]

[0012] The preparation of the intermediate 2-methylbenzothiazolium propane sulfonate in the above preparation method is described in the reference. [1,2] get.

[0013] In the above preparation method, the preparation of 7-(diethylamino)-2-oxo-2H-coumarin-3-carboxaldehyde (CQ) is described in the reference. [3]The molar ratio is 1:1 to 3, preferably 1:1.08 to 1.8, more preferably 1.09 to 1.2. The reflux reaction temperature is preferably 75℃ to 85℃. The solvent is preferably acetonitrile, ethanol, methanol, or acetic acid (sodium acetate), preferably acetonitrile, with a concentration of 98% (v / v) or higher, and anhydrous acetonitrile is preferred; the initiator can be some organic basic substances such as organic amines including but not limited to piperidine, pyridine, piperazine, triethylamine, and trimethylamine, and its amount is generally small, such as 1 to 2 drops, so no special requirements are made; the TLC thin-layer chromatography solvent can be dichloromethane:methanol, with a ratio of 50 to 5:1 (v / v). In the above preparation method, 2-methylbenzothiazolium propane sulfonate salt can also be activated first, and then reacted with CQ. The activation method can be to add or not add an initiator to the solution of 2-methylbenzothiazolium propane sulfonate salt and reflux the reaction.

[0014] The fluorescent molecule synthesized in this invention uses coumarin as the basic backbone and electron-donating groups, and connects to thiazole salts modified with sulfonate endonucleates to form coumarin-based salt derivatives. By introducing suitable lipophilic and hydrophilic groups to regulate the molecule's uptake ability by cancer cells, by introducing conjugated olefin bonds to increase the recognition sites of biothiols, and by further utilizing the ease of substitution reactions, the tiered recognition of endogenous signal molecules can be achieved, enhancing the signal fidelity. This is expected to achieve in-situ activated two-photon fluorescence, enabling early and accurate diagnosis of a series of diseases induced by imbalances in the internal environment, such as cardiovascular disease and Alzheimer's disease.

[0015] Thirdly, the two-photon fluorescent probe described in this invention can be applied to SO2 detection, as well as to the cascade detection of SO2 and H2O2. Furthermore, the two-photon fluorescent probe described in this invention can be applied to the monitoring of the tumor or cancer cell microenvironment. The tumor microenvironment-activated two-photon fluorescent probe described in this invention can be used for imaging endogenous biomolecules within cells; more specifically, the cell imaging is high-fidelity cascade imaging of SO2 and H2O2 overexpression within tumor cells or cancer cells; the cancer cells include, but are not limited to, cervical cancer cells, liver cancer cells, etc.

[0016] Furthermore, the two-photon fluorescent probe described in this invention can be applied to the preparation of systems for monitoring imbalances in the body's internal environment or microenvironment. Even further, the two-photon fluorescent probe described in this invention can be used in the preparation of systems for monitoring diseases induced by imbalances in the body's internal environment or microenvironment.

[0017] Cell imaging experiments showed that with increasing incubation time, the CVS probe responded more readily to endogenous SO2 and H2O2. Under single and two-photon excitation, cancer cells were gradually illuminated and attached well to mitochondria. By observing the relationship between fluorescence trends and incubation time, the levels of SO2 and H2O2 in cells were qualitatively analyzed to preliminarily assess the abnormality of the organism. Subsequently, the fluorescence data were quantified to compare and determine whether the levels of the two indicators (SO2 and H2O2) increased individually or simultaneously, thus indicating the possibility of pathological changes. The developed probe is economical, has good photostability, low dark cytotoxicity, and a wide range of applications, suggesting that this fluorescent probe can serve as an early diagnostic tool for cardiovascular diseases and Alzheimer's disease.

[0018] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0019] 1. The probe of this invention uses a rigid planar coumarin structure as the electron-donating group (D), C=C as the π-bridge, and a thiazole salt group as the electron-withdrawing group (A), introducing a heavy atom (such as SO). - The 3-ion (or equivalent) internal salt reduces cellular dark toxicity and modulates the hydrophilicity / hydrophobicity of the molecule, synthesizing the target product CVS through nucleophilic substitution and condensation reactions. The probe CVS possesses excellent properties of cascade recognition of endogenous SO2 and H2O2, activating two-photon fluorescence. Furthermore, due to its cationic nature, it can effectively target the mitochondria of cancer cells, achieving high-fidelity fluorescence response imaging. This research finding has significant reference value for developing two-photon fluorescent markers specifically indicating body homeostasis and disease development.

[0020] 2. The probe material of this invention is free of heavy atoms and has cationic fluorescence properties. It is a water-soluble optical material with low dark toxicity and good biocompatibility. It has a strong affinity for cancer cells such as cervical cancer cells and liver cancer cells, while showing no significant response changes to normal cells such as human renal epithelial cells and human umbilical vein endothelial cells. It has significant application value.

[0021] 3. The preparation method of the present invention uses readily available raw materials, has low cost, simple synthesis steps, and is easy to operate. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the response mechanism of the two-photon fluorescent probe of the present invention.

[0023] Figure 2 This is a diagram showing the specific cascade response of CVS to SO2 and H2O2 in vitro.

[0024] Figure 3 This is a high-resolution mass spectrum of CVS in vitro in response to SO2 and H2O2.

[0025] Figure 4 It includes subcellular colocalization, imaging of different cell lines, and dark toxicity maps.

[0026] Figure 5 This is an in vitro two-photon fluorescence image.

[0027] Figure 6 This is a single / two-photon diagram of the intracellular cascade response to SO2 and H2O2. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0029] Example 1

[0030] A method for preparing a tumor microenvironment cascade-activated two-photon fluorescent probe includes the following steps:

[0031] A. Preparation of intermediate 2-methylbenzothiazolium propane sulfonate (Reference) [1,2] get.

[0032] B. Preparation of raw material 7-(diethylamino)-2-oxo-2H-coumarin-3-carboxaldehyde (CQ) (Reference) [3] Preparation of C and CVS

[0033] Weigh 0.20 g (0.74 mmol) of 2-methylbenzothiazolyl propane sulfonate into a 25 mL single-necked round-bottom flask, add 10 mL of acetonitrile to dissolve it, add 2 drops of piperidine, and heat to 80 °C for 30 min. Then weigh 0.17 g (0.81 mmol) of CQ, dissolve it in 10 mL of acetonitrile, add it to the above reaction system, and continue to reflux at 80 °C for 24 h. Monitor the reaction by thin-layer chromatography (TLC). After the reaction is complete, cool to room temperature and concentrate under reduced pressure. Separate by column chromatography and purify the crude product (eluent: dichloromethane: methanol = 25:1 (v:v)) to give 0.24 g of purple solid, with a yield of 64%. 1H NMR (400MHz, DMSO-d6), δ (ppm) 8.87 (s, 1H), 8.36 (d, J = 8.0Hz, 2H), 8.13 (d, J =8.0Hz,1H),8.02(d,J=8.0Hz,1H),7.83(t,J=8.0Hz,1H),7.73(t,J=7.6Hz,1 H),7.55(d,J=8.0Hz,1H),6.90(d,J=8.0,1H),6.69(s,1H),4.99(t,J=4.0,2H ),3.55-3.52(m,4H),2.65(t,J=8.0Hz,2H),2.21(s,2H),1.23-1.16,(m,6H).

[0034] Example 2

[0035] Specific cascade response diagram of the target product CVS to SO2 and H2O2 in vitro

[0036] The probe possesses a C=C double bond structure and has been reported to provide addition sites for some reactive sulfur compounds. First, we screened a series of compounds, including reactive oxygen species and reactive sulfur compounds (H₂O₂, SO₃). 2- (SO2 donor), ClO - O2 ·- S2O3 2- ), metal cations (Cu) 2+ Fe 3+ Mg 2+ Na + Biomolecules (DNA, RNA), proteins (BSA, HSA), amino acids (Lys, Ser, Arg, Leu, Asp), etc. Figure 2 As shown in Figure a, under UV-Vis excitation, the target compound CVS showed no significant fluorescence response to the aforementioned metal ions, biomolecules, and amino acids, but exhibited approximately a 3-fold fluorescence enhancement to SO2. Interestingly, when the solution of CVS reacted with SO2 was titrated with H2O2, the fluorescence intensity at the same position increased by approximately 11-fold. Figure 2 b) The calculated detection limits (LODs) for SO2 and H2O2 were 3.17 nM and 40.4 nM, respectively. Figure 2 (d and 2e), while compared to titrating only H2O2, the fluorescence intensity still did not change significantly ( Figure 2c), therefore, we hypothesize that CVS exhibits a cascaded fluorescence response to SO2 and H2O2, with sulfite ions first adding to the double bond, followed by the competitive substitution of sulfite ions by OH groups in H2O2. As the UV titration spectrum shows, with increasing SO2 concentration, the absorption at 540 nm gradually decreases, while the absorption around 405 nm gradually increases, accompanied by the appearance of an equivalent absorption point. Further titration of H2O2 showed no significant change in the absorption peak, indicating that the addition of H2O2 did not significantly alter the molecular framework or the ICT process. Finally, the above reaction process was also confirmed by high-resolution mass spectrometry. Figure 3 ).

[0037] Example 3

[0038] Subcellular colocalization of CVS, imaging of different cell lines, and dark cytotoxicity assay.

[0039] Considering that cancer cells typically carry more negative charges on their surface than normal cells, the positively charged thiazole salt probe prepared in this invention theoretically has a higher affinity for cancer cells. Based on this, we screened several common cell lines, including two cancer cell lines (HeLa cervical cancer cells and HepG2 liver cancer cells) and two normal cell lines (293T human renal epithelial cells and HUVEC human umbilical vein endothelial cells). HeLa and HepG2 cells were purchased from BeNa Culture Collection; the other cells were from the American Type Culture Collection (ATCC). The staining of chloroform (MO) in different cell lines was investigated. Interestingly, as expected, only cancer cells showed a strong uptake capacity for CVS and were able to respond in situ to endogenous SO2 and H2O2, exhibiting a fluorescence "off-on" process under 405nm excitation. In contrast, normal cells showed weak or no uptake of CVS, and the cells were essentially non-fluorescent. Figure 4 a). Next, the spatial distribution of CVS in cells was assessed using a commercial mitochondrial red dye. Figure 4 Figures b and 4c show that CVS can be well localized to the mitochondrial sites of cancer cells, with an overlap of 0.96. Subsequently, to quantitatively assess the cytotoxicity of the target product, this invention used standard 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) to measure the cytotoxicity of cancer cell lines. Experimental results showed that CVS has good biocompatibility and safety; even at a concentration of 20 μM, its cell viability remained above 85% under dark conditions, indicating its potential for further application in bioimaging. Figure 4 d).

[0040] Example 4

[0041] Two-photon fluorescence image of the target probe in vitro

[0042] Using a fully automated coordinated femtosecond TIS sapphire pulsed laser (Coherent ultra II) as the light source (680–1080 nm, 80 MHz, 140 fs), two-photon fluorescence measurements were performed in vitro on the CVS, CVS+SO2, and CVS+SO2+H2O2 systems. The two-photon fluorescence spectra from the three sets of experiments show that CVS did not exhibit significant two-photon fluorescence under different excitation wavelengths. For CVS+SO2 and CVS+SO2+H2O2, the two-photon fluorescence intensity reached its peak at an excitation wavelength of 800 nm, and the trend of the two-photon fluorescence intensity was consistent with that of the single-photon fluorescence intensity. Comparison of the single / two-photon emission positions of CVS+SO2 and CVS+SO2+H2O2 revealed no significant redshift or blueshift. Subsequently, with the excitation wavelength fixed at 800 nm, two-photon verification was conducted by varying the laser power (300 mW–700 mW). The results showed that the logarithmic ratio of the input / output intensity for both groups was approximately 2, indicating significant two-photon optical activity. Figure 5 ).

[0043] Example 5

[0044] Single / two-photon diagram of the target probe's cascade response to SO2 and H2O2 within the cell.

[0045] Based on the excellent two-photon optical activity of CVS in in vitro cascade responses to SO2 and H2O2, we are prompted to further investigate single / two-photon activation imaging of compounds in response to endogenous SO2 and H2O2 signaling molecules at the cellular level. Figure 6 As shown, HepG2 cells were co-incubated with CVS (10 μM) for 0 min, 10 min, 20 min, and 30 min, respectively, and then imaged using single-photon and two-photon methods. At 0 min, the cells showed no fluorescence regardless of whether excited by a 405 nm or pulsed 800 nm light source. As the incubation time was gradually extended to 10 min, the target probe CVS gradually recognized endogenous SO2, activating fluorescence and two-photon activity, and the cells brightened from dark. Continuous observation of the imaging channel at 20 min and 30 min showed significant enhancement in both single-photon and two-photon fluorescence. Combined with the in vitro experimental results, we infer that this may be attributed to the interaction between CVS and endogenous H2O2 after CVS recognizes SO2. Therefore, CVS possesses high sensitivity and optical stability, as well as deep tissue penetration, playing an important role in reflecting the body's redox homeostasis and analyzing physiological and pathological conditions.

[0046] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

[0047] References

[0048] [1]Abeyrathna, N.; Liao, YJAm.Chem.Soc.2015,137,11282–11284.

[0049] [2]Li, Y.; Huang, Y.; Sun, X.; Zhong, K.; Tang, L. Talanta.2023,258,124412.

[0050] [3] Sun, YQ.; Liu, J.; Zhang, J.; Yang, T.; Guo, W. Chemical Communications. 2013, 49, 2637.

Claims

1. A two-photon fluorescent probe, with the molecular formula C 25 H 26 N2O5S2, structural formula as follows: ， The preparation method of the two-photon fluorescent probe includes the following steps: 2-Methylbenzothiazolium propane sulfonate and 7-(diethylamino)-2-oxo-2H-coumarin-3-carboxaldehyde were dissolved in a solvent, an initiator was added, and the mixture was refluxed at 60℃~90℃ for 20~30 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure, separated by column chromatography, and dried to obtain the final product. The molar ratio of 2-methylbenzothiazolium propane sulfonate to 7-(diethylamino)-2-oxo-2H-coumarin-3-carboxaldehyde was 1:1.08~1.

8. The solvent was acetonitrile, and the initiator was at least one selected from piperidine, pyridine, piperazine, triethylamine, and trimethylamine.

2. The application of the two-photon fluorescent probe according to claim 1 in the preparation of an SO2 detection system.

3. The application of the two-photon fluorescent probe according to claim 1 in the preparation of a cascade detection system for SO2 and H2O2.

4. The application of the two-photon fluorescent probe according to claim 1 in the preparation of a monitoring system for cancer cell microenvironment.

5. The application of the two-photon fluorescent probe of claim 1 in the preparation of a system for monitoring the homeostasis imbalance of the internal environment or microenvironment.

6. The application of the two-photon fluorescent probe of claim 1 in the preparation of a system for monitoring diseases induced by imbalances in the internal environment or microenvironment.

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