Macrophage reprogramming monitoring probe as well as preparation and application thereof

Abstract

The invention provides a macrophage reprogramming monitoring probe as well as preparation and application thereof, and the macrophage reprogramming monitoring probe has a structure as shown in a formula I which is described in the specification. The monitoring probe provided by the invention can accurately monitor the concentration of the TAM phenotype conversion marker NO, and visualizes the immune regulation effect of the antitumor traditional Chinese medicine in real time.

Description

Technical Field

[0001] This invention belongs to the field of monitoring technology, specifically relating to a macrophage reprogramming monitoring probe and its preparation and application. Background Technology

[0002] Tumor macrophages (TAMs) are among the most abundant immune cell types in the tumor immune microenvironment, comprising two phenotypes: tumor suppressor M1 macrophages and tumor-promoting M2 macrophages. During tumor development, the number of M2 macrophages increases significantly, becoming the dominant group within TAMs. These M2 cells not only fail to kill tumor cells but also contribute to tumor angiogenesis, facilitate tumor evasion of the immune system, and exacerbate tumor invasion and metastasis. In contrast, M1 macrophages exhibit strong anti-tumor properties, directly eliminating tumor cells through the release of anti-tumor active substances or phagocytosis. Furthermore, M1 macrophages can activate tumor-specific T cells, further stimulating the body's anti-tumor immune response. Therefore, targeted immunotherapy that reprograms TAMs from M2 macrophages to M1 macrophages has become a new paradigm in tumor immunotherapy.

[0003] Macrophage reprogramming monitoring is crucial for real-time assessment of the body's immune function and for revealing the effects of reprogramming drugs on improving immune function. Currently, the main biological methods for monitoring macrophage reprogramming include the following: (1) Flow cytometry: By detecting macrophage surface markers (such as CD11b, CD14, CD68, etc.), macrophages are measured to assess their phagocytic capacity and oxidative capacity, and the ratio of M1 to M2 macrophages is analyzed; (2) Immunohistochemistry and immunofluorescence: Macrophages and their activation status are detected using specific antibodies in tissue sections, and the spatial relationship between macrophages and other cell types or markers is observed; (3) Western blotting and qPCR: The expression of key proteins (such as CD86 and iNOS) is studied by analyzing the changes in the whole-genome expression profile of macrophages under different conditions. Although these methods have been applied in macrophage reprogramming research, they are highly dependent on tissue biopsy and gene detection, are complex to operate, and have limited sensitivity, making it difficult to achieve real-time in situ monitoring of macrophage immune responses. Therefore, there is an urgent need to develop a precise, non-invasive, and real-time tool for monitoring the tumor immune activation status of macrophages.

[0004] Fluorescence imaging technology, with its superior sensitivity, high resolution, deep tissue penetration, and favorable biometabolic properties, has demonstrated broad application value in the medical field, particularly in predicting immunotherapy response rates. By innovatively combining fluorescence imaging with specific molecular probes, we can non-invasively monitor the activity of immune cells, the dynamic changes in the immune microenvironment, and the subtle transformations of this microenvironment after immunotherapy. When macrophage phenotypes change, M1 macrophages, in addition to phagocytizing and killing tumor cells, also highly express antitumor active substances such as TNF and inducible nitric oxide synthase, and secrete high levels of NO. NO also possesses high bactericidal and antitumor activity. Therefore, developing monitoring probes that can accurately monitor the concentration of NO, a marker of TAM phenotype transformation, is of great significance for real-time visualization of the immunomodulatory effects of TAM reprogramming drugs, such as antitumor traditional Chinese medicine, and for elucidating their mechanisms of action. Summary of the Invention

[0005] In view of this, the present invention provides a macrophage reprogramming monitoring probe, its preparation and application, which can accurately monitor the concentration of NO, a TAM phenotypic conversion marker, and visualize the immunomodulatory effect of anti-tumor traditional Chinese medicine in real time.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a macrophage reprogramming monitoring probe having the structure shown in Formula I:

[0007] Formula I.

[0008] Secondly, the present invention provides a method for preparing the macrophage reprogramming monitoring probe, comprising the following steps: S1. Obtain compound 1; S2. Mix the compound 1 with o-phenylenediamine, an activating reagent, and a first solvent to obtain a monitoring probe; Compound 1 has the structure shown in Formula 1:

[0009] Formula 1.

[0010] It should be noted that in step S2, after mixing compound 1 with o-phenylenediamine, the activating reagent, and the first solvent, the solvent is removed by evaporation under reduced pressure. The crude product is then purified by silica gel column chromatography to obtain the final product, an orange solid monitoring probe. In some embodiments, the eluent for the silica gel column chromatography purification is dichloromethane / methanol = (20-40):1 (v / v), exemplarily 20:1, 30:1, and 40:1.

[0011] The synthesis route of the monitoring probe is as follows:

[0012] Preferably, step S1 includes: S11. Mix 4-(diethylamino)-2-hydroxybenzaldehyde, 8-hydroxyjuloridin, a second solvent and anion exchange reagent to obtain compound 2; S12. Compound 2, octathionecyclooctane, alkaline solution and third solvent are mixed to obtain compound 1; Compound 2 has the structure shown in Formula 2:

[0013] Formula 2.

[0014] It should be noted that in step S12, after mixing the compound 2, octathionecyclooctane, alkaline solution and third solvent, the reaction is carried out under nitrogen protection at a temperature of 85-100°C.

[0015] It should be noted that in step S12, after mixing compound 2, octathionecyclooctane, alkaline solution, and a third solvent, the solvent is removed under reduced pressure, and the mixture is purified to obtain compound 1. Purification can be performed using column chromatography. Preferably, the eluent for column chromatography is petroleum ether / ethyl acetate = 20-40):1 (v / v), with examples of 20:1, 30:1, and 40:1.

[0016] The synthetic route for compound 1 is as follows:

[0017] It should be noted that 4-(diethylamino)-2-hydroxybenzaldehyde has the following structure:

[0018] In step S11, 4-(diethylamino)-2-hydroxybenzaldehyde, 8-hydroxyjuloridin, and the second solvent are mixed and reacted at a temperature of 80-160°C. After the reaction is complete, an anion exchange reagent is added to form a precipitate, which is then separated to obtain a solid product. For example, the solid product can be obtained by vacuum filtration.

[0019] Preferably, the activating agent comprises trifluoromethanesulfonic anhydride; and / or, The first solvent includes water (DCM); and / or, The second solvent includes concentrated phosphoric acid; and / or, The anion exchange reagent includes ammonium hexafluorophosphate; and / or, The alkaline solution includes potassium tert-butoxide; and / or, The third solvent is tetrahydrofuran.

[0020] Preferably, the molar ratio of compound 1 to o-phenylenediamine is 1:(5-10), for example, the molar ratio of compound 1 to o-phenylenediamine can be 1:5, 1:7.5, or 1:10; and / or, The molar ratio of compound 1 to the activating agent is (0.5-2):1, exemplarily 0.5:1, 1:1, or 2:1; and / or, The molar ratio of compound 2 to the alkaline solution is 1:(2-3). Exemplarily, the molar ratio of the alkaline solution to compound 2 can be 2:1, 2.5:1, or 3:1, etc.; and / or, The molar ratio of the anion exchange reagent to 4-(diethylamino)-2-hydroxybenzaldehyde is 1:(0.5-2), and the molar ratio of the anion exchange reagent to 4-(diethylamino)-2-hydroxybenzaldehyde can be 1:0.5, 1:1, or 1:2, etc.

[0021] In some embodiments, the synthetic route of compound 2 is as follows: The synthesis route for the monitoring probe can be as follows:

[0022] Thirdly, the present invention provides a kit comprising the monitoring probe described herein, or a monitoring probe prepared according to the preparation method described herein.

[0023] Fourthly, the present invention provides a biosensor, including the monitoring probe described above, or the monitoring probe prepared according to the preparation method described above.

[0024] Fifthly, the present invention provides the application of the monitoring probe described above, or the monitoring probe prepared according to the preparation method described above, in screening immunologically active species of traditional Chinese medicine.

[0025] Preferably, the kit, or the biosensor described herein, is used in the detection of NO concentration.

[0026] Preferably, it includes the following steps: SA, mix the monitoring probe with NO or a NO-containing sample to obtain a mixture; SB, Detect the fluorescence intensity of the mixture; SC, by referring to the NO concentration-dependent standard curve, determine the concentration of NO or the NO-containing test sample, wherein the NO concentration-dependent standard curve is obtained by reacting NO solutions of different concentrations with the monitoring probe, and the NO concentration range is 1-600 μM.

[0027] Preferably, in step SA, the concentration of the monitoring probe in the test sample is 0.005-2 mM. Exemplarily, the concentration of the monitoring probe in the test sample is 0.005 mM, 1 mM, or 2 mM. In some embodiments, in step A1, the monitoring probe is mixed with NO or a test sample containing NO, and then incubated to obtain a mixture at a temperature of 30-50°C. Further, the incubation temperature is 35-40°C. Preferably, in step A1, the incubation time is 0.5-30 min. Further, the incubation time is 0.5-10 min.

[0028] Preferably, in step SA, the method for detecting NO or a NO-containing sample using the detection probe specifically includes the following steps: SA01, Prepare the fluorescent probe solution; SA02, prepare test samples of different concentrations of NO or containing NO; SA03. Plot a concentration-dependent standard curve for NO or the test sample containing NO.

[0029] Preferably, in step SA01, the monitoring probe solution is obtained by dissolving the probe in a mixed solvent of acetonitrile and water.

[0030] Preferably, in step SA02, the solution of NO or the test sample containing NO is obtained by mixing the NO or the test sample containing NO with a buffer solution. Further, the pH of the buffer solution is 7-11. The buffer solution can be an aqueous solution of HEPES, MES buffer, Tris-HCl buffer, NaOH-H3BO3 buffer, NaCO3-NaHCO3 buffer, or phosphate buffer (PBS buffer).

[0031] Preferably, in step SA02, the solution of NO or the test sample containing NO is obtained by mixing a saturated NO solution with a PBS solution; wherein the concentration of the saturated NO solution is less than or equal to 600 μM. Further, the concentration of the NO solution is 1-600 μM.

[0032] Preferably, step SA03 includes: taking the detection probe solution, using one group of solutions as a blank sample, adding a solution of NO or a test sample containing NO of known concentration to the remaining groups of solutions respectively, mixing, incubating, measuring the fluorescence intensity of the mixture, and plotting a concentration-dependent standard curve of NO or the test sample containing NO with the fluorescence intensity y of each group of mixtures as the ordinate and the concentration x of the solution of NO or the test sample containing NO as the abscissa.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The monitoring probe synthesized in this invention has fluorescence emission in the near-infrared region and long-wavelength fluorophores, and has high fluorescence quantum efficiency and better tissue penetration ability.

[0034] (2) The monitoring probe provided by the present invention has good affinity for NO or test samples containing NO, and has high specificity and sensitivity for NO sensing.

[0035] (3) The monitoring probe provided by the present invention can accurately monitor the concentration of NO, a TAM phenotypic conversion marker, and visualize the immunomodulatory effect of antitumor traditional Chinese medicine in real time. Attached Figure Description

[0036] Figure 1 This is a synthesis route diagram of the fluorescent probe provided in Example 1 of the present invention; Figure 2 The above is a hydrogen NMR spectrum characterization of the fluorescent probe provided in Example 1 of this invention; Figure 3 The image shows the carbon NMR spectrum of the fluorescent probe provided in Example 1 of this invention. Figure 4 This is a mass spectrometry characterization of the fluorescent probe provided in Example 1 of the present invention; Figure 5 The UV spectra of the fluorescent probe before and after the reaction with NO are provided in the performance test and results of this invention; Figure 6 The fluorescence change curves and linear correlation diagrams of the fluorescent probes after binding with NO or NO-containing test samples provided in the performance test and results of this invention are shown. Figure 7 This is a cytotoxicity evaluation diagram of the fluorescent probe provided in the performance test and results of this invention; Figure 8 This is an evaluation diagram showing the effectiveness of the fluorescent probe in distinguishing between endogenous and exogenous NO, as provided in the performance test and results of this invention. Figure 9 The graphs show the evaluation of the effects of different drugs on TAM phenotypic conversion at the cellular level using the fluorescent probes provided in the performance tests and results of this invention, as well as the expression of key proteins.

[0037] Note: Figure 6 In the middle, the fluorescence change curve is as follows Figure 6 (a) The linear correlation plot is as follows Figure 6 (b). Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0039] Example 1 This embodiment provides a monitoring probe, referring to... Figure 1 The preparation method includes the following steps: First, 386 mg (1 mmol) of 4-(diethylamino)-2-hydroxybenzaldehyde and 378 mg (2 mmol) of 8-hydroxyjuloridin were weighed and dissolved in 10 mL of concentrated phosphoric acid. The mixture was heated at 160 °C with stirring and refluxed for 5 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction system was poured into 30 mL of water. NaOH solution was added to adjust the pH of the solution to 8. Then, ammonium hexafluorophosphate (652 mg) was added to precipitate the solid. The solid was filtered, washed three times with water, and dried under vacuum to obtain compound 2, with a yield of 86% (where: yield = (actual yield / theoretical yield) * 100%). Next, compound 2 (347.5 mg, 1 mmol), octathionecyclooctane (478.72 mg, 1.87 mmol), and t-BuOK (330.4 mg, 2.95 mmol) were weighed and dissolved in 50 mL of THF under nitrogen protection at 85 °C with stirring and reflux for 10 h. After the reaction was completed, the mixture was cooled to room temperature. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was eluent by silica gel column chromatography with petroleum ether / ethyl acetate = 20 / 1 to obtain compound 1, with a yield of 26%. Finally, 80 mg of compound 1 was dissolved in 10 mL of anhydrous DCM, and then trifluoromethanesulfonic anhydride (Tf₂O, 90 μL) was slowly added. The reaction mixture was stirred at room temperature for 30 minutes, followed by the addition of 290 mg of o-phenylenediamine, and the mixture was stirred for another 10 hours. After the reaction was complete, the solvent was removed by evaporation under reduced pressure. The crude product was purified by silica gel column chromatography using methanol / dichloromethane = 1 / 20 as the eluent to give the final product as an orange solid in 43% yield. The fluorescence 1H NMR spectrum is shown below. Figure 2 The characterization by carbon NMR is shown in [reference needed]. Figure 3 Mass spectrometry characterization diagram is shown below. Figure 4 .

[0040] Performance Tests and Results (1) Ultraviolet-visible spectrum of the monitoring probe for detecting NO Weigh 1.0 mol of the monitoring probe prepared in Example 1 and dissolve it in 1 mL of DMSO to obtain a 1 mM monitoring probe stock solution (fluorescent probe stock solution), which is stored at -20°C. The NO required for the experiment was prepared from sodium nitrite and concentrated sulfuric acid, purified with NaOH aqueous solution, and stored in deionized water under an N2 atmosphere. Its concentration (1.92 mM) was determined using a Griess kit. Add 50 μL of the above saturated NO aqueous solution to 1 mL of a mixed solution (CH3CN:PBS = 3:7), then add 2 μL of the above monitoring probe stock solution and mix well to obtain the detection solution. Record the UV absorption spectrum of the monitoring probe (fluorescent probe) in the 400-680 nm range using a UV spectrophotometer. The results are shown in [Figure number missing]. Figure 5 .

[0041] like Figure 5 As shown, without the addition of NO saturated solution, the absorption peak is at 470 nm. After the addition of NO saturated solution, the absorption peak significantly red-shifts to approximately 580 nm, accompanied by a significant increase in absorption intensity. This indicates that the probe exhibits excellent response performance and selectivity to NO.

[0042] (2) Fluorescence spectra of fluorescent probes under different NO concentrations Dissolve 2 μL of the fluorescent probe stock solution in 1 mL of a mixed solution (CH3CN:PBS = 3:7). Then add NO solutions of different concentrations (0, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 55 μM, 60 μM) to the mixed solution. Incubate at 37 °C for 0.5 h. Detect and record the fluorescence intensity of the mixed solution using a fluorescence spectrometer. The results are shown below. Figure 6 (a) Then perform curve fitting, and the results are shown in [the table]. Figure 6 (b).

[0043] like Figure 6 As shown in (b), the linear fit between the probe and the fluorescence intensity is approximately 1, and the detection limit for NO calculated from the fitted curve is 0.25 μM. This demonstrates the high sensitivity of the probe to NO detection. When the added NO reaches 55 μM, the fluorescence intensity reaches saturation and no longer increases.

[0044] (3) Biosafety and tumor cell toxicity detection of fluorescent probes: HepG2 cells were cultured for at least three passages in a 37°C oven using cell culture medium (10% fetal bovine serum, 1% penicillin, 1% streptomycin). After cell dispersal and centrifugation, the supernatant was discarded, and the cells were diluted to 5000-8000 cells / 100μL. The cells were then added to 96-well plates and cultured for 24 hours. Different concentrations (0, 1μM, 5μM, 10μM, 15μM, 20μM) of the fluorescent probe prepared in Example 1 were added to each well, and the cells were cultured for another 24 hours. The culture medium was removed, and 10μL of CCK8 solution and 90μL of fresh culture medium were added to each well. The cells were cultured for another 1 hour, and the absorbance at 570nm was recorded using a microplate reader. The cell viability of each sample was calculated using the formula (As / A0×100%, where As is the absorbance of the test well, and A0 is the absorbance of the test well with a concentration of 0 fluorescent probe). The results are shown in [Figure number missing]. Figure 7 .

[0045] like Figure 7 As shown, the cell viability of the fluorescent probe is above 75%. This indicates that the fluorescent probe provided by this invention has very low toxicity and good biosafety.

[0046] (4) Evaluation diagram of the effectiveness of fluorescent probes in distinguishing between endogenous and exogenous NO; healthy RAW264.7 cells were divided into two groups of 2 × 10⁻⁶ cells. 5 The wells were divided into four groups and inoculated into 35 mm sterile laser confocal microscopy dishes (BS-15-GJM, Biosharp). After incubation for 24 h, the culture medium was discarded and the samples were washed three times with PBS buffer. The first group was co-incubated with the fluorescent probe (1 μM) prepared in Example 1 for 0.5 h and then imaged using laser scanning confocal microscopy (Blank). The second group was incubated with LPS (200 ng / mL) for 24 h, then 1 μM probe was added and incubated for another 0.5 h before laser scanning confocal microscopy (M1). The third group was incubated with sodium nitroprusside (0.05 mM) for 2 h and then imaged using laser scanning confocal microscopy (MO + sodium nitroprusside). The fourth group was incubated with sodium nitroprusside (0.05 mM) for 2 h, then aminoguanidine (AG, 10 μg / mL) was added before laser scanning confocal microscopy (MO + sodium nitroprusside + AG). The results are shown in [Figure number missing]. Figure 8 .

[0047] like Figure 8 As shown, the fluorescence of M1 increased significantly after the addition of nitroprusside, while the fluorescence decreased after the addition of the NO scavenger (AG). This indicates that the fluorescent probe can be used to image nitroprusside-induced exogenous NO and LPS-induced endogenous NO, demonstrating its ability to detect NO at the biological level.

[0048] (5) Fluorescent probes were used to assess and validate changes in NO concentration during TAM-induced phenotypic transition: healthy RAW264.7 cells were divided into two groups of 2 × 10⁻⁶ cells. 5 Five groups were inoculated into 35mm sterile laser confocal microscopy dishes (BS-15-GJM, Biosharp) at different well densities and incubated for 24 hours. After incubation, the culture medium was discarded, and the samples were washed three times with PBS buffer. Interleukin-4 (IL-4, 40 ng / mL) was added to each group, and incubation continued for another 24 hours. The culture medium was then discarded, and the samples were washed three times with PBS buffer. One group was incubated with a fluorescent probe for 0.5 hours before laser scanning confocal microscopy imaging (denoted as M2). The other four groups were given diosgenin (2 μM), β-elemene (10 μg / mL), baicalin (12.5 μg / mL), and Lycium barbarum polysaccharide (12.5 μg / mL), and then incubated with a fluorescent probe for 0.5 hours before laser scanning confocal microscopy imaging (denoted as M2+diosgenin, M2+β-elemene, M2+baicalin, M2+Lycium barbarum polysaccharide). The results are shown in [Figure number missing]. Figure 9 (a), and the fluorescence intensity was analyzed, the results are shown in [figure missing]. Figure 9 (b).

[0049] like Figure 9 As shown in (b), the fluorescence intensity increased after drug administration. Subsequently, we used Western blot to detect the expression levels of related proteins such as IL-4, CD86, and iNOS. Figure 9 As shown in (c), the expression of M1 protein increased after administration of all four drugs. This indicates that the fluorescent probe provided by this invention has the potential to screen drugs.

[0050] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0051] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0052] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A macrophage reprogramming monitoring probe, characterized in that, It has the structure shown in Equation I: Formula I.

2. The method for preparing the macrophage reprogramming monitoring probe according to claim 1, characterized in that, Includes the following steps: S1. Obtain compound 1; S2. Mix the compound 1 with o-phenylenediamine, an activating reagent, and a first solvent to obtain a monitoring probe; Compound 1 has the structure shown in Formula 1: Formula 1.

3. The preparation method according to claim 2, characterized in that, Step S1 includes: S11. Mix 4-(diethylamino)-2-hydroxybenzaldehyde, 8-hydroxyjuloridin, a second solvent and anion exchange reagent to obtain compound 2; S12. Compound 2, octathionecyclooctane, alkaline solution and third solvent are mixed to obtain compound 1; Compound 2 has the structure shown in Formula 2: Formula 2.

4. The preparation method according to claim 3, characterized in that, The activating agent includes trifluoromethanesulfonic anhydride; and / or, The first solvent includes water (DCM); and / or, The second solvent includes concentrated phosphoric acid; and / or, The anion exchange reagent includes ammonium hexafluorophosphate; and / or, The alkaline solution includes potassium tert-butoxide; and / or, The third solvent is tetrahydrofuran.

5. The preparation method according to claim 3, characterized in that, The molar ratio of compound 1 to o-phenylenediamine is 1:(5-10); and / or, The molar ratio of compound 1 to the activating agent is (0.5-2):1; and / or, The molar ratio of compound 2 to the alkaline solution is 1:(2-3); and / or, The molar ratio of the anion exchange reagent to 4-(diethylamino)-2-hydroxybenzaldehyde is 1:(0.5-2).

6. A reagent kit, characterized in that, Includes the monitoring probe according to claim 1, or the monitoring probe prepared by the preparation method according to any one of claims 1-5.

7. A biosensor, characterized in that, Includes the monitoring probe according to claim 1, or the monitoring probe prepared by the preparation method according to any one of claims 1-5.

8. The application of the monitoring probe according to claim 1, or the monitoring probe prepared by the preparation method according to any one of claims 1-5, in screening immunologically active species of traditional Chinese medicine.

9. The application of the kit according to claim 6 or the biosensor according to claim 7 in the detection of NO concentration.

10. The application according to claim 9, characterized in that, Includes the following steps: SA, mix the monitoring probe with NO or a NO-containing sample to obtain a mixture; SB, Detect the fluorescence intensity of the mixture; SC, by referring to the NO concentration-dependent standard curve, determine the concentration of NO or the NO-containing test sample, wherein the NO concentration-dependent standard curve is obtained by reacting NO solutions of different concentrations with the monitoring probe, and the NO concentration range is 1-600 μM.

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