Application of CNN2-B probe in specific recognition of atherosclerotic plaque

Abstract

The application provides application of a CNN2-B probe in specific identification of an atherosclerotic plaque, and relates to the technical field of fluorescent probes. ‑ The structural formula of the CNN2-B probe is as follows: the CNN2-B probe is an ONOO ‑ / lipid double response type probe, which has very weak fluorescence in the case of existing alone, can only make fluorescence sharply increase in the case of existing of lipid and ONOO ‑ , and has lipid droplet positioning function. The probe can be used in specific identification of an atherosclerotic plaque, so that the position of the atherosclerotic plaque can be more accurately judged.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and particularly to the application of the CNN2-B probe in the specific identification of atherosclerotic plaques. Background Technology

[0002] Atherosclerosis (AS) is a major cause of fatal cardiovascular disease. Due to the limitations of traditional diagnostic imaging techniques, researchers are currently focusing on studying new fluorescence imaging modalities for atherosclerotic plaques, thus requiring the development of plaque-specific recognition probes.

[0003] Existing small-molecule organic probes for plaque identification typically rely on lipid labeling, such as Oil Red O. However, lipid probes lack selectivity for other adipocytes and tissue cells; for example, they cannot distinguish between foam cells and adipocytes. Consequently, adipose tissue near the arterial wall can severely interfere with plaque detection, leading to false positives. Therefore, developing a highly selective fluorescent probe for plaque identification is of great significance for the specific identification of atherosclerotic plaques. Summary of the Invention

[0004] The purpose of this invention is to provide the application of a CNN2-B probe in the specific identification of atherosclerotic plaques. This CNN2-B probe is ONOO. - This lipid-responsive probe, with lipid droplet localization capabilities, can be used to more accurately determine the location of atherosclerotic plaques in their specific identification.

[0005] The technical problem solved by this invention is achieved by the following technical solution.

[0006] This invention proposes the application of the CNN2-B probe in the specific identification of atherosclerotic plaques. The structural formula of the CNN2-B probe is as follows:

[0007] .

[0008] The beneficial effects of applying the CNN2-B probe in the specific identification of atherosclerotic plaques according to embodiments of the present invention are as follows:

[0009] The CNN2-B probe of this invention is ONOO. - / Lipid-responsive probes, in ONOO - When present alone, the fluorescence is very weak; it only appears in lipids and ONOO. - The fluorescence is dramatically enhanced only when both conditions are present, and the probe also possesses lipid droplet localization capabilities. Using this probe for the specific identification of atherosclerotic plaques allows for more precise determination of their location. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1A This is a comparison of the fluorescence intensity of the CNN2-B probe in different response systems according to Example 1 of the present invention;

[0012] Figure 1B This is a trend graph showing the effect of reaction time on the fluorescence intensity of the CNN2-B probe in Example 1 of the present invention;

[0013] Figure 2A A trend graph showing the effect of changes in LDs mimics concentration on the fluorescence intensity of CNN2-B in Example 1 of this invention;

[0014] Figure 2B For ONOO - Trend graph showing the effect of concentration change on the fluorescence intensity of CNN2-B in Example 1 of the present invention;

[0015] Figure 3 This is a comparison diagram of the selectivity of CNN2-B for organic bioactive molecules and inorganic ions in Example 1 of the present invention;

[0016] Figure 4A This is a fluorescence colocalization map of CNN2-B and its response product CNN2 in Embodiment 1 of the present invention;

[0017] Figure 4B The images show the fluorescence distribution of A549 cells after staining with CNN2-B and Mito-Tracker according to Example 1 of this invention.

[0018] Figure 5 The images show the fluorescence distribution of mouse macrophages stained with probes CNN2-B and HPF, respectively, under different stimuli, as described in Example 1 of this invention.

[0019] Figure 6 This is a graph showing the change in fluorescence intensity of CNN2-B over time in Example 1 of the present invention;

[0020] Figure 7 The image shows the fluorescence distribution of the patches after staining with different fluorescent probes. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The application of the CNN2-B probe in the specific identification of atherosclerotic plaques according to embodiments of the present invention will be described in detail below.

[0023] This invention proposes the application of the CNN2-B probe in the specific identification of atherosclerotic plaques. The structural formula of the CNN2-B probe is as follows:

[0024] .

[0025] Furthermore, in a preferred embodiment of the present invention, the CNN2-B probe is lipid and ONOO. - Dual-response probe. The existing probe KC-ONOO is a probe that can react with peroxynitrite (ONOO). - This invention relates to a probe that reacts with drug-induced liver injury and has been applied to monitor such injury. The inventors of this invention discovered that the probe KC-ONOO is an ONOO... - / Lipid-responsive probes rather than simple ONOO - Response probe, which is in ONOO - When present alone, the fluorescence is very weak; it only appears in lipids and ONOO. - The fluorescence is dramatically enhanced only when both conditions are met, and it also has lipid droplet localization capabilities, thus it can be used as a specific recognition probe for atherosclerotic plaques.

[0026] Furthermore, in a preferred embodiment of the present invention, the application includes in vitro testing of the CNN2-B probe, cellular testing of the CNN2-B probe, and animal testing of the CNN2-B probe.

[0027] Further, in a preferred embodiment of the present invention, the probe solution for the in vitro testing of the CNN2-B probe is selected from the PBS buffer of the CNN2-B probe, wherein the PBS buffer of the CNN2-B probe further contains dimethyl sulfoxide, the concentration of the CNN2-B probe in the PBS buffer of the CNN2-B probe is 9.5~10.5 μmol / L, the pH of the PBS buffer is 7.4, and the mass fraction of the dimethyl sulfoxide is 0.08~0.12%. Preferably, the concentration of the CNN2-B probe in the PBS buffer of the CNN2-B probe is 10 μmol / L, the pH of the PBS buffer is 7.4, and the mass fraction of the dimethyl sulfoxide is 0.1%.

[0028] Furthermore, in a preferred embodiment of the present invention, the CNN2-B probe cell-level test includes the following steps:

[0029] The CNN2-B probe solution was incubated with the cells on the culture medium for 50-70 minutes. After removing the culture medium containing the probe, the cells were observed using a fluorescence microscope.

[0030] Furthermore, in a preferred embodiment of the present invention, the concentration of the CNN2-B probe solution is 9.5~10.5 μmol / L, and the excitation channel of the fluorescence microscope is selected as λ. ex = 488 nm. Preferably, the concentration of the CNN2-B probe solution is 10 μmol / L.

[0031] Furthermore, in a preferred embodiment of the present invention, the CNN2-B probe animal-level testing includes the following steps:

[0032] Prepare a PBS solution for the CNN2-B probe as the probe solution. Immerse filter paper in the probe solution for 8-12 seconds, remove it, and apply it to the surface of the tissue to be observed. After standing for 4-6 minutes, remove the filter paper. Continuous observation can then be performed in the imaging system. The excitation channel selected is λ. ex = 460 nm.

[0033] Further, in a preferred embodiment of the present invention, the PBS buffer for the CNN2-B probe further comprises dimethyl sulfoxide (DMSO). The concentration of the CNN2-B probe in the PBS buffer is 9.5–10.5 μmol / L, the pH of the PBS buffer is 7.4, and the mass fraction of the DMSO is 0.08–0.12%. Preferably, the concentration of the CNN2-B probe in the PBS buffer is 10 μmol / L, the pH of the PBS buffer is 7.4, and the mass fraction of the DMSO is 0.1%.

[0034] The CNN2-B fluorescent probe of the present invention, which is dual-responsive to peroxynitrite and lipid, can more accurately determine the location of atherosclerotic plaques by cross-recognizing the two markers of lipid and peroxynitrite.

[0035] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0036] Example 1

[0037] This embodiment provides a CNN2-B probe, the synthesis method of which is described in "a selective fluorescent turn-on probe for imaging peroxynitrite in living cells and drug-damaged liver tissues"; Peng Wang et al., Talanta, (204) 2019, pp. 431-437. The structural formula of the CNN2-B probe is as follows:

[0038] .

[0039] Experimental Example 1

[0040] This experimental example demonstrates the ONOO of the CNN2-B probe in Example 1. - The analysis of lipid dual-response performance included the following steps:

[0041] ONOO - Preparation: Add 10 mL of 0.7 M H2O2 to 10 mL of 0.6 M NaNO2, stir at 0°C for 5 minutes, quickly add 10 mL of 0.6 M HCl, then quickly add 20 mL of 1.5 M NaOH, continue stirring for 5 minutes and mix thoroughly. Add 500 mg of manganese dioxide and stir for 10 minutes to remove excess H2O2. Filter the mixture to remove manganese dioxide. Finally, ONOO is obtained. - After repackaging, store at -20℃ for later use. Use an extinction coefficient ε = 1670 cm⁻¹. -1 M -1 ONOO was determined by measuring the absorption of the solution at 302 nm before use. - The concentration.

[0042] Prepare a 1 mM stock solution of the CNN2-B probe from Example 1 for later use. To achieve a final concentration of 10 μM for CNN2-B in a 3 mL system, add 10 μM of ONOO. - The emission spectrum and fluorescence intensity of the probe were detected under excitation light at 430 nm and / or with 200 μg / mL lipid droplet mimicry. The reaction time was 0–250 minutes.

[0043] like Figure 1A The image shows a comparison of the fluorescence intensity of the CNN2-B probe from Example 1 in different response systems. Figure 1A It can be seen that the probe at 10μM ONOO - They showed almost no fluorescence when present alone with 200 μg / mL lipid droplet mimics (LDs mimics), but exhibited fluorescence in ONOO.- Fluorescence can be enhanced by 364 times when both LDs and mimics are present.

[0044] like Figure 1B This is a trend graph showing the effect of reaction time on the fluorescence intensity of the CNN2-B probe in Example 1. From... Figure 1B It can be seen that the dual response of the CNN2-B probe is time-dependent, with fluorescence gradually being released over a period of 250 minutes.

[0045] Experimental Example 2

[0046] This experimental example demonstrates the ONOO of the CNN2-B probe in Example 1. - The analysis of lipid concentration-dependent responses includes the following steps:

[0047] Prepare a 1 mM stock solution of the CNN2-B probe from Example 1 for later use. In a 3 mL system, adjust the final concentration of CNN2-B to 10 μM by adding 0–10 μM of ONOO. - The probe was reacted with 0~200 μg / mL LDs mimics for 250 minutes, and the emission spectrum and fluorescence intensity of the probe were detected under 430 nm excitation light.

[0048] like Figure 2A The graph shows the trend of the effect of LDs mimics concentration changes on the fluorescence intensity of CNN2-B in Example 1. From... Figure 2A It can be seen that, with a fixed input of 10 μM ONOO - Under these conditions, the fluorescence of the probe increases with increasing LDs mimics concentration (0~200 μg / mL), showing a linear correlation in the range of 0~150 μg / mL.

[0049] like Figure 2B The image shown is ONOO. - A trend graph showing the effect of concentration changes on the fluorescence intensity of CNN2-B in Example 1. From... Figure 2B It can be seen that, under the condition of a fixed input of 200 μg / mL LDs mimics, the fluorescence of the probe increases with ONOO. - The effect is enhanced with increasing concentration (0~1μM) and shows a linear correlation in the 4~10μM range.

[0050] Experimental Example 3

[0051] This experimental example analyzes the response selectivity of CNN2-B in Example 1 in vitro, specifically including the following steps:

[0052] Prepare a 1 mM stock solution of the CNN2-B probe from Example 1 for later use. In a 3 mL system, bring the final concentration of CNN2-B to 10 μM, and then... - Under the presence of these substances, 200 μg / mL of LDs mimics, BSA, HSA, DNA, RNA, Cys, GSH, ADP, ATP, and Glucose were added respectively. Additionally, 100 μM ClO₂ was added under the presence of 200 μg / mL LDs mimics. , •OH, •O2 - , 1 O2, BuOO - H2O2, ClO IO4 NO, NO2 Ca 2+ Cu 2+ Fe 3 + , K + , and Zn 2+ The reaction was carried out for 250 minutes, and the emission spectrum and fluorescence intensity of the probe were detected under excitation light at 430 nm.

[0053] like Figure 3 The image shows a comparison of the selectivity of CNN2-B for organic bioactive molecules and inorganic ions in Example 1. Among them, Figure 3 A is a comparison diagram of the selectivity of CNN2-B for organic bioactive molecules in Example 1. Figure 3 B is a comparison diagram of the selectivity of CNN2-B for inorganic ions in Example 1. From Figure 3 It can be seen that the CNN2-B probe is basically only associated with ONOO. - It reacts with LDsmimics but is not sensitive to other bioactive substances.

[0054] Test Example 4

[0055] This experimental example analyzes the organelle localization of CNN2-B in Example 1. A549 cells were used to assess the organelle localization of the probe. Specific steps included:

[0056] A549 cells were cultured. The concentration of CNN2-B administered in Example 1 was 10 μM. First, the cells were co-incubated with mitochondrial probes or lipid droplet probes for 1 hour, then fixed, stained with DAPI, and subjected to confocal imaging. Next, 100 μM SIN-1 was pre-administered for 30 minutes, followed by the addition of 10 μM CNN2-B and other organelle probes for 1 hour. The cells were then fixed, stained with DAPI, and subjected to confocal imaging.

[0057] like Figure 4A The image shows the fluorescence colocalization map of CNN2-B and its response product CNN2 from Example 1. Figure 4A It can be seen that CNN2-B is mainly located in mitochondria, while the response product of CNN2-B, CNN2, is mainly located in lipid droplets.

[0058] like Figure 4B The image shows the fluorescence distribution of A549 cells after staining with CNN2-B and Mito-Tracker as described in Example 1. Figure 4B It can be seen that CNN2-B responds to ONOO in cells. - It then transfers to lipid droplets and significantly enhances fluorescence.

[0059] Experimental Example 5

[0060] This experimental example studies the CNN2-B specific recognition of foam cells in Example 1, specifically including the following steps:

[0061] Mouse macrophages (Raw 264.7) were cultured and stimulated with 4 μg / mL oxidized low-density lipoprotein for 24 hours to induce foam cell formation. Alternatively, 4 μg / mL LPS and 100 Nm PMA were added to induce inflammatory states in the cells. The cells were then incubated with 10 μM CNN2-B or HPF for 1 hour, removed and fixed, stained with DAPI, and then subjected to confocal imaging.

[0062] like Figure 5 The image shows the fluorescence distribution of mouse macrophages stained with probes CNN2-B and HPF (from Example 1) under different stimuli. Figure 5 A shows fluorescence images of mouse macrophages stained with probes CNN2-B and DAPI from Example 1 under different stimuli. Figure 5 Figure B shows fluorescence images of mouse macrophages stained with probes HPF and DAPI under different stimuli. Figure 5 It can be seen that CNN2-B can specifically image foam cells without being interfered with by other inflammatory signals.

[0063] Experimental Example 6

[0064] This experimental case studies the specific recognition of atherosclerotic plaques using CNN2-B in Example 1. A carotid atherosclerosis model was established using carotid artery ligation combined with a high-fat diet. The specific steps include:

[0065] Prepare a 10 μM PBS solution of CNN2-B from Example 1. Take a small piece of filter paper, stick it with the CNN2-B solution, and incubate it on the outside of the blood vessel for 10 minutes. Remove the filter paper and perform imaging observation.

[0066] like Figure 6 The figure shown is a graph illustrating the change in fluorescence intensity over time for CNN2-B in Example 1. From... Figure 6 It can be seen that after CNN2-B is administered in situ, the location of plaques on the blood vessel wall gradually becomes apparent over time.

[0067] like Figure 7 The image shows the fluorescence distribution of the patches after staining with different fluorescent probes. Figure 7 It can be seen that CNN2-B is mainly localized to lipid droplets inside the plaque.

[0068] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. The application of the CNN2-B probe, characterized in that, The structural formula of the CNN2-B probe is as follows: ； The CNN2-B probe is composed of lipids and ONOO. - A dual-response probe; the application is a CNN2-B probe cell-level assay; the CNN2-B probe cell-level assay includes the following steps: co-incubating the CNN2-B probe solution and cells on a culture medium for 50-70 min, removing the culture medium, and observing using a fluorescence microscope; in the CNN2-B probe cell-level assay, the concentration of the CNN2-B probe solution is 9.5-10.5 μmol / L, and the excitation channel of the fluorescence microscope is selected as λ. ex = 488nm.

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