Application of leucine aminopeptidase in preparation of product for diagnosing atherosclerosis or evaluating vulnerability of atherosclerotic plaque

Abstract

The invention discloses application of leucine aminopeptidase in preparation of a product for diagnosing atherosclerosis or evaluating atherosclerotic plaque vulnerability, and discloses direct association between LAP and AS plaque vulnerability and application of LAP as a cardiovascular and cerebrovascular disease diagnosis biomarker for the first time. The invention relates to application of the LAP activity in preparation of a product for diagnosing AS or evaluating AS plaque vulnerability, further provides a first method for in-vivo, real-time and dynamic visual imaging of the LAP activity in a living body AS plaque, in addition, the feasibility of diagnosing AS based on the LAP activity is verified in various human source clinical samples for the first time, the perfect distinction degree is achieved, and finally, the application of the LAP activity in preparation of a product for diagnosing AS or evaluating AS plaque vulnerability is achieved. A complete evidence chain is formed through multi-modal means such as in-vivo imaging, in-vitro imaging and histopathology; the method has the advantages that a brand new diagnosis and research direction is opened up for AS diagnosis or AS plaque vulnerability evaluation, the LAP activity in the AS plaque can be specifically identified and imaged and displayed in a living body environment, and a more accurate and timely disease monitoring means is provided.

Description

Technical Field

[0001] This invention relates to the field of cardiovascular and cerebrovascular disease diagnosis, and in particular to the application of a leucine aminopeptidase in the preparation of products for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques. Background Technology

[0002] Atherosclerosis (AS) is a complex chronic arterial disease characterized by lipid deposition, inflammation, endothelial cell damage, and plaque formation, changes that can ultimately lead to narrowing of blood vessels and thrombosis. This disease is one of the leading causes of cardiovascular disease worldwide, encompassing serious health problems such as myocardial infarction and stroke. The development of atherosclerosis is closely linked to various metabolic diseases such as diabetes, kidney disease, and metabolic syndrome, which together constitute a significant part of the global health burden.

[0003] Existing atherosclerosis detection technologies mainly rely on imaging and biochemical indicators, but these methods often have problems such as the inability to monitor in real time and the inability to directly reflect enzyme activity in atherosclerotic plaques.

[0004] In the pathophysiology of atherosclerosis, leucine aminopeptidase (LAP) is a key biomarker and potential therapeutic target. LAP is a membrane-bound metalloproteinase widely distributed in various cell types, including endothelial cells, smooth muscle cells, and macrophages, which play a central role in the development of atherosclerosis. LAP not only participates in protein metabolism but also directly affects the structure and function of the vascular wall by regulating cell proliferation, migration, and apoptosis. Furthermore, LAP plays an important role in regulating local and systemic inflammatory responses and oxidative stress, processes that are key regulators of atherosclerotic plaque stability and rupture risk. However, the specific role and value of LAP in atherosclerosis, particularly in the formation and rupture of vulnerable plaques in the cardiovascular and cerebrovascular systems, remains completely unknown. There are no reports from academia or industry of using LAP as a biomarker for in vivo imaging studies of atherosclerosis. Current technology lacks methods for real-time, dynamic, and precise visualization of LAP activity within atherosclerotic plaques in vivo.

[0005] Furthermore, accurate detection of LAP and its activity remains a significant challenge in clinical practice. Existing detection methods primarily rely on in vitro biochemical analyses, such as enzyme-linked immunosorbent assays (ELISA) and Western blotting. While these methods provide useful information, they typically have the following limitations: 1. Sample processing and time delays: These methods require complex sample pretreatment and lengthy analysis times, making them unsuitable for rapid diagnosis or real-time monitoring. 2. Inability to reflect actual activity in tissues: In vitro tests cannot directly reflect the actual activity status of LAP in plaques or other tissues in vivo, limiting their application in disease monitoring and treatment efficacy evaluation. 3. Insufficient specificity: Some body fluids may contain interfering substances that affect the accurate detection of leucine aminopeptidase, reducing the specificity of the method.

[0006] Currently, there are no publicly available reports, either domestically or internationally, on the use of LAP biopsy in the diagnosis of cardiovascular diseases. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an application of leucine aminopeptidase in the preparation of products for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques. By using leucine aminopeptidase as a biomarker for the diagnosis of cardiovascular and cerebrovascular diseases, a leucine aminopeptidase-responsive probe is designed that can specifically identify and image leucine aminopeptidase activity in atherosclerotic plaques in an in vivo environment, thereby providing a more accurate and timely means of disease monitoring.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: the application of leucine aminopeptidase in the preparation of products for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques.

[0009] Furthermore, the product is a formulation or a kit.

[0010] Furthermore, the formulation is a leucine aminopeptidase-responsive probe.

[0011] Furthermore, the leucine aminopeptidase response probe is obtained by mixing hemicyanine, LAP-specific peptides, and dichloromethane, then adding a condensing agent and a base to carry out a condensation reaction, followed by washing, drying, and purification.

[0012] Furthermore, the specific preparation process of the leucine aminopeptidase-responsive probe is as follows: Step 1: Perform the condensation linkage reaction, the specific process is as follows: Step S1.1: Under inert gas protection, dissolve 1 mmol of LAP-specific peptide and 1.0-1.5 mmol of hemicyanine in 40-50 mL of anhydrous dichloromethane and stir until completely dissolved to obtain the reaction system solution. Step S1.2: Under the temperature conditions of 0℃-25℃, add 1.2-1.8 mmol of condensing agent and 2.0-4.0 mmol of organic base to the reaction system solution to carry out the condensation reaction. After the carboxyl group of the LAP specific peptide and the amino group of hemicyanine react completely to form an amide bond, the condensation reaction solution is obtained. Step 2: Purification is performed, and the specific process is as follows: Step S2.1: Wash the condensation reaction solution with a 0.1 mol / L dilute hydrochloric acid aqueous solution to remove excess alkali and separate the organic phase; Step S2.2: After drying with anhydrous sodium sulfate, the solvent is removed by concentration under reduced pressure to obtain the crude product of the LAP response probe; Step S2.3: Purify the crude product of the LAP response probe by column chromatography or preparative high performance liquid chromatography to obtain the leucine aminopeptidase response probe.

[0013] Furthermore, when purifying the crude product of the LAP response probe using column chromatography: a chromatography column is packed with 200-300 mesh silica gel as the stationary phase, and a mixed solvent of dichloromethane and methanol is used as the eluent, wherein the volume ratio of dichloromethane to methanol is 20:1. The eluent is then concentrated under reduced pressure to remove the solvent, thereby obtaining the leucine aminopeptidase response probe.

[0014] Furthermore, when purifying the crude product of the LAP response probe using preparative high-performance liquid chromatography: a reversed-phase C18 preparative column is used, and the mobile phase is a mixture of water and acetonitrile, wherein the volume ratio of water to acetonitrile is 1:1. Elution is performed to obtain an eluent, and then the eluent is concentrated under reduced pressure to remove the solvent, thereby obtaining the leucine aminopeptidase response probe.

[0015] Furthermore, an in vitro detection method for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques for non-therapeutic purposes uses the leucine aminopeptidase-responsive probe to react with clinical samples from subjects, and assesses LAP activity in the clinical samples by detecting the optical signal after the reaction.

[0016] Furthermore, the clinical sample can be any one of serum, cerebrospinal fluid, brain tissue, and cerebral blood vessels.

[0017] Compared with existing technologies, the advantages of this invention are as follows: This invention reveals for the first time the direct association between LAP and atherosclerotic plaque vulnerability, and the application of LAP as a diagnostic biomarker for cardiovascular and cerebrovascular diseases in the preparation of products for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques, opening up a completely new diagnostic and research direction for diagnosing AS or assessing the vulnerability of AS plaques; Furthermore, this invention also provides the first in vivo, real-time, and dynamic visualization imaging method for LAP activity in living AS plaques, which can specifically identify and image LAP activity in atherosclerotic plaques in a living environment, thereby providing a more accurate and timely means of disease monitoring; In addition, this invention systematically validates the feasibility of diagnosing AS based on LAP activity in multiple human clinical samples for the first time, and has perfect discrimination, with an AUC (Area Under Curve) of 1.0, showing broad prospects for clinical translation; Finally, this invention forms a complete chain of evidence through multimodal methods such as in vivo imaging, ex vivo imaging, and histopathology, with high reliability. Attached Figure Description

[0018] Figure 1 This is a synthetic route diagram for the LAP-responsive probe; Figure 2 The reaction principle of the LAP-responsive probe and its molecular docking simulation diagram with LAP; Figure 3 The image shows the in vitro photoacoustic spectrum of the LAP-responsive probe. Figure 4 The image shows the in vitro fluorescence spectrum of the LAP-responsive probe. Figure 5 Fluorescence signal of atherosclerotic plaques at different locations after injection of LAP-responsive probes into atherosclerotic mice; Figure 6 for Figure 5 Histological and immunohistochemical staining images of AS plaques in different locations in the middle; Figure 7 Photoacoustic images and photoacoustic signal intensity comparison of the aortic region at different time points after injecting LAP-responsive probes into healthy mice and atherosclerotic mice; Figure 8 Quantitative fluorescence intensity graphs of cerebrospinal fluid from healthy individuals and patients with atherosclerosis after co-incubation with LAP-responsive probes; Figure 9 Quantitative fluorescence intensity graphs of serum from healthy individuals and patients with atherosclerosis after co-incubation with LAP-responsive probes; Figure 10Quantitative fluorescence intensity maps of brain tissue and LAP-responsive probes co-incubated in healthy individuals and patients with atherosclerosis, and quantitative fluorescence intensity maps of cerebral blood vessels and LAP-responsive probes co-incubated in healthy individuals and patients with atherosclerosis. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0020] This invention, through extensive and in-depth research, unexpectedly discovered for the first time a direct association between LAP and atherosclerotic plaque vulnerability. Therefore, LAP can serve as a diagnostic biomarker for cardiovascular and cerebrovascular diseases and can be used in the preparation of products for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques.

[0021] Atherosclerosis is a crucial pathological process leading to cardiovascular disease. This invention aims to overcome the limitations of existing technologies. For example, current atherosclerosis detection techniques mainly rely on imaging and biochemical indicators, but these methods often suffer from problems such as the inability to monitor in real time and the inability to directly reflect enzyme activity in atherosclerotic plaques. In particular, for the detection of LAP activity in atherosclerotic plaques, traditional methods mostly rely on in vitro measurements and cannot provide direct information about the in vivo LAP activity status.

[0022] To overcome these limitations, this invention synthesizes a leucine aminopeptidase-responsive probe, namely the LAP-responsive probe, abbreviated as L-RP. This LAP-responsive probe binds to a specific LAP substrate peptide and hemicyanine (HD), enabling it to bind to and be cleaved by active LAP with high specificity, releasing a signal detectable by optical imaging technology. This design allows the LAP-responsive probe to directly monitor LAP activity in vivo under physiological conditions, significantly improving the diagnostic and therapeutic monitoring capabilities for atherosclerosis and related diseases.

[0023] The following section will detail the functional verification process of the LAP response probe.

[0024] Example 1: Synthesis and in vitro optical property characterization of LAP-responsive probes.

[0025] This embodiment details the synthesis, purification, and basic in vitro optical response characteristics of the LAP-responsive probe.

[0026] I. Synthesis and purification (e.g.) Figure 1 (as shown) Step 1: Perform the condensation linkage reaction, the specific process is as follows: Step S1.1: Under inert gas protection, 1 mmol of LAP-specific peptide and 1.0-1.5 mmol of hemicyanine were dissolved in 40-50 mL of anhydrous dichloromethane and stirred until completely dissolved to obtain the reaction system solution; wherein, hemicyanine and LAP-specific peptide were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. Step S1.2: Under the temperature conditions of 0℃-25℃, add 1.2-1.8 mmol of condensing agent HATU and 2.0-4.0 mmol of organic base DIPEA to the reaction system solution to carry out the condensation reaction. During the condensation reaction, monitor the progress of the condensation reaction by thin layer chromatography or high performance liquid chromatography until the carboxyl group of the LAP specific peptide and the amino group of hemicyanine react completely to form an amide bond, and then obtain the condensation reaction solution. Step 2: Purification is performed, and the specific process is as follows: Step S2.1: Wash the condensation reaction solution with a 0.1 mol / L dilute hydrochloric acid aqueous solution to remove excess alkali and separate the organic phase; Step S2.2: After drying with anhydrous sodium sulfate, the solvent is removed by concentration under reduced pressure to obtain the crude product of the LAP response probe; Step S2.3: Purify the crude product of the LAP response probe by column chromatography or preparative high performance liquid chromatography to obtain the leucine aminopeptidase response probe.

[0027] When purifying the crude LAP response probe using column chromatography: a 200-300 mesh silica gel column was packed as the stationary phase, and a mixed solvent of dichloromethane and methanol (volume ratio of dichloromethane to methanol 20:1) was used as the eluent. The eluent was then concentrated under reduced pressure to remove the solvent, yielding the leucine aminopeptidase response probe. The elution process was monitored by thin-layer chromatography, and the fraction rich in the target product was collected as the eluent.

[0028] When purifying the crude LAP response probe using preparative high-performance liquid chromatography (HPLC): a reversed-phase C18 column (250 mm × 21.2 mm, 5 μm particle size) was used. The mobile phase was a mixture of water and acetonitrile (1:1 volume ratio). Elution was performed to obtain the eluent. The eluent was then concentrated under reduced pressure to remove the solvent, yielding the leucine aminopeptidase response probe. The characteristic absorption of the target product at 650 nm was monitored using a UV detector, and the fraction corresponding to the target peak was automatically or manually collected as the eluent.

[0029] II. Molecular docking simulation: Molecular docking software (such as AutoDock Vina) is used to simulate the binding mode between the recognition peptide in the LAP response probe and the active site of the LAP protein, such as... Figure 2 As shown in the figure. The calculation results show that there is a strong interaction between the two, with a binding energy as low as -8.5 kcal / mol, indicating that the LAP response probe has a high affinity for the target enzyme, and theoretically predicts its good specificity.

[0030] III. In vitro optical response characteristics: After incubating the LAP-responsive probe (5 μM) and LAP (60 U / L) together at 37°C for 1-2 hours, a spectral scan was performed. The specific scan results are shown in the figure. Figure 3 and Figure 4 As shown.

[0031] Photoacoustic spectrum: such as Figure 3 As shown, the photoacoustic signal of the unactivated LAP response probe is weak, but after LAP activation, the photoacoustic signal intensity in the 685-720 nm band is significantly enhanced.

[0032] Fluorescence spectroscopy: such as Figure 4 As shown, the fluorescence signal of the unactivated LAP response probe is extremely weak, but after LAP activation, it exhibits a strong near-infrared fluorescence emission peak at a wavelength of 745 nm.

[0033] The above results fully demonstrate that the LAP-responsive probe can be specifically activated by LAP, thereby generating a significantly enhanced dual-modal (photoacoustic / fluorescence) signal, and possesses the core function of an LAP activity imaging probe.

[0034] Example 2: Validation of the in vitro targeting performance of LAP-responsive probes and its correlation analysis with biomarkers of AS plaque vulnerability.

[0035] This embodiment aims to verify the targeting ability of LAP-responsive probes to atherosclerotic plaques at the in vitro level and to explore the correlation between their imaging signals and classic plaque vulnerability pathological features, thereby establishing the value of LAP as a vulnerability biomarker.

[0036] Animal model establishment and treatment: To study the application of LAP response probes in atherosclerosis (AS) models, an atherosclerosis mouse model was first established using mice with gene knockout of apolipoprotein E (ApoE- / -) and given a high-fat diet to induce the development of atherosclerosis.

[0037] Probe injection and sample collection: The LAP response probe (2 mg / kg dissolved in PBS) was injected into AS model mice via the tail vein. 90 minutes after injection, the mice were anesthetized and euthanized, and the aorta was completely removed and cleaned in ice-cold PBS.

[0038] In vitro fluorescence imaging: The acquired aortic sample was laid flat in a culture dish, and fluorescence imaging was performed using a small animal in vivo optical imaging system (IVIS Spectrum or similar device) at an emission wavelength of 745 nm (Ex = 640 nm) to obtain the overall fluorescence signal distribution map of the aorta.

[0039] Image Analysis and Segmentation: Quantitative analysis of the fluorescence signal intensity of the aorta was performed using the software integrated into the small animal in vivo optical imaging system. Based on the relative intensity of the fluorescence signal, each aorta was divided into three representative segments: the weakest signal region (location 1), the intermediate signal region (location 2), and the strongest signal region (location 3). These segments were then extracted for subsequent analysis, such as... Figure 5 As shown.

[0040] Histopathological analysis: The arterial segments obtained above were embedded in paraffin and sectioned, and then stained as follows: Hematoxylin and eosin (H&E) staining: used to observe the overall morphology and structure of plaques, the degree of luminal narrowing, and the size of the necrotic core, such as... Figure 5 As shown.

[0041] Masson Trichrome staining: Used to show the content and distribution of collagen fibers within plaques; collagen is stained blue, such as... Figure 5 As shown.

[0042] Immunohistochemical (IHC) staining: Anti-CD68 antibody was used to label macrophage infiltration within the plaques; anti-MMP2 antibody was used to detect the expression level of matrix metalloproteinase 2 (MMP2), such as... Figure 5 As shown.

[0043] Experimental results: LAP response probe signals are correlated with plaque load and distribution: such as Figure 5 As shown, in vitro aortic imaging revealed a non-uniform distribution of fluorescence signals from the LAP-responsive probe, consistent with the expected distribution of AS plaques. Quantitative analysis indicated significant differences in fluorescence intensity across different segments: location 3 > location 2 > location 1.

[0044] The intensity of LAP-responsive probe signals is positively correlated with plaque vulnerability, and histopathological results correspond perfectly with fluorescence signal intensity. Location 1: H&E staining showed normal vessel wall structure with no typical plaque formation; Masson staining showed intact and dense collagen structure; CD68 and MMP2 were only expressed in trace amounts or not at all. This indicates that this segment is normal vascular tissue with low background signal from LAP response probes.

[0045] Location 2: H&E staining revealed obvious plaque formation, but the necrotic core was relatively small; Masson staining showed a partial reduction in collagen content; CD68 and MMP2 expression were positive. This indicates that this segment is a stable plaque, and the LAP response probe showed a moderate intensity signal.

[0046] Location 3: H&E staining shows large, typical vulnerable plaque features, including a large necrotic core (e.g., Figure 5 (Indicated by the black arrow) and a thin fibrous cap; Masson staining showed significant loss of collagen content and sparse, broken collagen arrangement; CD68 staining showed extensive macrophage infiltration; MMP2 expression was strong. This indicates that this segment is a highly vulnerable plaque, with the strongest LAP response probe signal, such as... Figure 5 As shown.

[0047] Experimental conclusions: This embodiment, through rigorous comparison with in vitro imaging and multimodal histopathology, confirmed the following points: (1) LAP-responsive probes can effectively target atherosclerotic plaques, and their signal intensity is related to plaque burden. (2) The signal intensity of LAP-responsive probes is significantly positively correlated with the classic pathological features of plaque vulnerability (huge necrotic core, collagen degradation, massive macrophage infiltration, and high MMP2 expression). This result is the first pathological demonstration that LAP activity is a key molecular feature of atherosclerotic plaque vulnerability, thus establishing the scientific basis for using LAP-responsive probes for plaque risk stratification.

[0048] Example 3: LAP response probes were used to detect LAP activity in a mouse model of atherosclerosis using photoacoustic imaging.

[0049] Animal Model Establishment and Treatment: To investigate the application of LAP-responsive probes in atherosclerosis (AS) models, two mouse models were first established. One group served as the atherosclerosis model, using mice with apolipoprotein E knockout (ApoE- / -) and fed a high-fat diet to induce the development of atherosclerosis. The other group served as the control group, using C57BL / 6 mice fed a standard diet as the healthy group.

[0050] Implementation of photoacoustic imaging: Before conducting the photoacoustic imaging experiment, all mice were first administered LAP-responsive probes via intravenous injection. Following injection, photoacoustic images of the aortic region were collected using a specific photoacoustic imaging system. In healthy mice, the photoacoustic signal in the aortic region was very weak after injection of the LAP-responsive probes. This indicates that under normal physiological conditions, the LAP-responsive probes are not cleaved by LAP and remain in their original state.

[0051] Photoacoustic signal analysis: In the atherosclerotic mouse group, the photoacoustic signal rapidly increased after injection of the LAP-responsive probe, exceeding that of the healthy mouse group. This indicates that the LAP-responsive probe can reflect changes in LAP levels in plaques, further validating the detection of LAP activity in plaques by the LAP-responsive probe (e.g., Figure 7 (As shown).

[0052] The method described in this embodiment successfully enables the imaging of LAP in plaques at the in vivo level using LAP-responsive probes, providing new imaging tools and strategies for the early diagnosis and treatment of atherosclerosis in the future.

[0053] Example 4: Validation of the diagnostic performance of LAP-responsive probes for atherosclerosis in clinical human samples.

[0054] This embodiment aims to validate the application value of LAP-responsive probes at the clinical sample level. By detecting LAP activity in human serum and cerebrospinal fluid (CSF) samples, its potential for diagnosing atherosclerosis (AS) is assessed. Considering the crucial role of LAP in vascular homeostasis and endothelial function regulation, and the close relationship between abnormal LAP activity and the progression of atherosclerotic lesions, monitoring serum LAP levels is of great significance for understanding the pathophysiological process of atherosclerosis, serving as a biomarker for disease progression, and for diagnosis and treatment.

[0055] Experimental Methods: Clinical samples were collected from clinically diagnosed patients with atherosclerosis and healthy volunteers. All procedures were conducted in accordance with ethical guidelines and informed consent was obtained. Serum and cerebrospinal fluid samples were diluted 1:1 with PBS. 100 μL of the diluted sample was mixed with an equal volume of 10 μM LAP response probe working solution and incubated at 37°C in the dark for 1 hour. After the reaction, the mixture was transferred to 96-well black plates, and fluorescence imaging and intensity quantification were performed using an in vivo optical imaging system (IVIS) or a fluorescence microplate reader at an excitation wavelength of 640 nm and an emission wavelength of 745 nm. Receiver operating characteristic (ROC) curve analysis was used to evaluate diagnostic efficacy.

[0056] Experimental results: Cerebrospinal fluid (CSF) test results: such as Figure 8As shown, after incubation with the LAP-responsive probe, cerebrospinal fluid samples from the atherosclerosis patient group (sample number 4, data shown in the figure are the mean and standard deviation of the 4 sample values) exhibited strong near-infrared fluorescence signals, while the signals from the healthy control group (sample number 4, data shown in the figure are the mean and standard deviation of the 4 sample values) were significantly weaker. Quantitative analysis showed that the fluorescence intensity of the atherosclerosis patient group was significantly higher than that of the healthy control group (p<0.001). Further ROC curve analysis showed that the area under the curve (AUC) for distinguishing between atherosclerosis patients and healthy individuals based on cerebrospinal fluid fluorescence signals was 1.000 (95% CI: 1.000-1.000), indicating that it has perfect diagnostic and differential diagnostic capabilities.

[0057] Serum test results: such as Figure 9 As shown, a trend consistent with that observed in cerebrospinal fluid was observed in serum samples. The fluorescence signal produced by serum from patients with atherosclerosis (sample size 3; data shown in the figure represent the mean and standard deviation of the 3 samples) after incubation with the LAP-responsive probe was significantly higher than that from the healthy control group (sample size 3; data shown in the figure represent the mean and standard deviation of the 3 samples), with a highly significant difference (p < 0.001). ROC analysis also yielded an AUC value of 1.000 (95% CI: 1.000–1.000), further demonstrating the perfect accuracy of this method for diagnosing atherosclerosis based on serum samples.

[0058] Experimental conclusion: This embodiment systematically validates the application value of LAP-responsive probes in clinical human samples for the first time. Results show that LAP activity in the serum and cerebrospinal fluid of patients with atherosclerosis is significantly higher than in healthy individuals. The LAP-responsive probes can efficiently and specifically detect this activity difference, achieving perfect differentiation of atherosclerotic patients in both different types of clinical samples (AUC = 1.0). This indicates that LAP activity in the circulatory system can serve as a novel molecular diagnostic biomarker, laying a solid foundation for the development of non-invasive or minimally invasive in vitro diagnostic kits for atherosclerosis and demonstrating broad prospects for clinical translation.

[0059] Example 5: Imaging verification of LAP-responsive probes in human ex vivo tissue samples.

[0060] This embodiment aims to further verify the ability of LAP-responsive probes to detect LAP activity in atherosclerotic lesions at the human tissue level, providing more direct histological evidence for its clinical application.

[0061] Brain tissue and cerebral blood vessel samples were collected from patients with atherosclerosis and healthy volunteers. All samples were obtained in accordance with ethical guidelines. Tissue samples were cut into appropriate sizes and incubated with 10 μM LAP-responsive probes at 37°C for a period of time. After incubation, the samples were thoroughly washed with PBS to remove unbound LAP-responsive probes. Fluorescence imaging was then performed using a small animal in vivo optical imaging system at an excitation wavelength of 640 nm and an emission wavelength of 745 nm, and the fluorescence intensity was quantitatively analyzed.

[0062] Experimental results are as follows Figure 10 As shown, after incubation with the LAP-responsive probe, the brain tissue and cerebral blood vessels of patients with atherosclerosis exhibited strong near-infrared fluorescence signals, while the corresponding tissues of the healthy control group showed significantly weak signals. Quantitative analysis of the fluorescence intensity showed that the signal intensity of the patient group tissues was higher than that of the healthy control group. These experimental results demonstrate that LAP activity is indeed highly expressed in human tissues with atherosclerotic lesions. The LAP-responsive probe can specifically recognize and respond to this activity, effectively distinguishing between diseased and normal tissues even in complex human tissue environments. This not only corroborates the findings in mouse models but also strongly supports the conclusion that LAP is a key biomarker for atherosclerosis from a human pathological perspective, further highlighting the application potential of the LAP-responsive probe of this invention in clinical pathological diagnosis.

Claims

1. Application of leucine aminopeptidase in the preparation of products for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques.

2. The application according to claim 1, characterized in that: The product is a formulation or a reagent kit.

3. The application according to claim 2, characterized in that: The formulation is a leucine aminopeptidase-responsive probe.

4. The application according to claim 3, characterized in that: The leucine aminopeptidase response probe is obtained by mixing hemicyanine, LAP-specific peptides, and dichloromethane, then adding a condensing agent and alkali to carry out a condensation reaction, followed by washing, drying, and purification.

5. The application according to claim 4, characterized in that: The specific preparation process of the leucine aminopeptidase-responsive probe is as follows: Step 1: Perform the condensation linkage reaction, the specific process is as follows: Step S1.1: Under inert gas protection, dissolve 1 mmol of LAP-specific peptide and 1.0-1.5 mmol of hemicyanine in 40-50 mL of anhydrous dichloromethane and stir until completely dissolved to obtain the reaction system solution. Step S1.2: Under the temperature conditions of 0℃-25℃, add 1.2-1.8 mmol of condensing agent and 2.0-4.0 mmol of organic base to the reaction system solution to carry out the condensation reaction. The carboxyl group of the LAP specific peptide and the amino group of hemicyanine react completely to form an amide bond to obtain the condensation reaction solution. Step 2: Purification is performed, and the specific process is as follows: Step S2.1: Wash the condensation reaction solution with a 0.1 mol / L dilute hydrochloric acid aqueous solution to remove excess alkali and separate the organic phase; Step S2.2: After drying with anhydrous sodium sulfate, the solvent is removed by concentration under reduced pressure to obtain the crude product of the LAP response probe; Step S2.3: Purify the crude product of the LAP response probe by column chromatography or preparative high performance liquid chromatography to obtain the leucine aminopeptidase response probe.

6. The application according to claim 5, characterized in that: When purifying the crude product of the LAP response probe using column chromatography: a chromatography column is packed with 200-300 mesh silica gel as the stationary phase, and a mixed solvent of dichloromethane and methanol is used as the eluent, wherein the volume ratio of dichloromethane to methanol is 20:

1. The eluent is then concentrated under reduced pressure to remove the solvent, and the leucine aminopeptidase response probe is obtained.

7. The application according to claim 5, characterized in that: When purifying the crude product of the LAP response probe using preparative high-performance liquid chromatography: a reversed-phase C18 preparative column is used, and the mobile phase is a mixture of water and acetonitrile, wherein the volume ratio of water to acetonitrile is 1:

1. Elution is performed to obtain the eluent, and then the eluent is concentrated under reduced pressure to remove the solvent, thereby obtaining the leucine aminopeptidase response probe.

8. An in vitro detection method for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques for non-therapeutic purposes, characterized in that: The leucine aminopeptidase response probe according to any one of claims 4 to 7 is used to react with clinical samples from subjects, and the LAP activity in the clinical samples is assessed by detecting the optical signal after the reaction.

9. The in vitro detection method for diagnosing atherosclerosis or assessing the vulnerability of atherosclerotic plaques for non-therapeutic purposes according to claim 8, characterized in that: The clinical sample can be any one of serum, cerebrospinal fluid, brain tissue, or cerebral blood vessels.

Images