A intestinal tumor marker based on laser confocal endoscopy and its preparation method, use method and application

Abstract

The present invention discloses an intestinal tumor marker based on laser confocal endoscopy and its preparation method, use method and application, which belongs to the technical field of imaging agents. Taking into account the impact and safety of labeling dyes on human health, the present invention uses DSPE‑PEG‑FITC nanoliposomes as dyes to label UEA‑I for the first time, and studies the expression level of UEA‑I in normal intestinal epithelium and colon cancer in this way, and successfully verifies the feasibility of fluorescently labeled DSPE‑PEG‑FITC‑UEA‑I as an intestinal imaging agent, which can be applied to the detection of colon cancer through optical imaging. The present invention combines the advantages of confocal endoscopy and the advantages of specific molecular markers, and can perform real-time imaging of the luminal surface, which is non-invasive and minimally invasive, reduces the risk of inspection, and specifically labels tumor markers, which can achieve early and accurate diagnosis and treatment of neoplastic lesions.

Description

Technical Field

[0001] The present invention belongs to the technical field of imaging agents, and in particular relates to an intestinal tumor marker based on laser confocal endoscopy and a preparation method, a use method and an application thereof. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, and its early diagnosis and treatment are crucial to improving patient survival. At present, although white light endoscopy is a commonly used technology for detecting gastrointestinal tumors, it has some limitations, especially for the detection of multifocal and flat tumors and the judgment of tumor demarcation lines. These limitations may affect the complete resection of the tumor and the patient's prognosis. Combining cancer-specific molecular imaging technology and optical imaging agents is an exciting new technology. By using fluorescently labeled cancer-specific molecular imaging agents, the distinction between tumors and normal tissues can be enhanced, thereby improving the visualization of tumors. Early diagnosis and treatment are crucial to improving the survival rate of patients with colorectal cancer, so the development of this technology is of great significance.

[0003] Over the past two decades, new endoscopic techniques such as narrow-band imaging (NBI), ultra-high magnification endoscopy (EC), and confocal laser endoscopy (CLE) have emerged as promising tools to improve cancer identification and guide endoscopic resection. These techniques provide more detailed and accurate images compared to conventional white-light endoscopy. NBI uses narrow bandwidth filters to highlight structural mucosal patterns and mucosal / submucosal vasculature, which helps improve the visibility of cancer. EC has ultra-high magnification, which allows for more detailed observation of structures and cellular atypia during routine endoscopy, helping to improve the identification of lesions. CLE is a new enhanced technology that uses fluorescein as an imaging agent to magnify the mucosa up to 1000 times at the subcellular level, providing more detailed image information. Although these new technologies provide enhanced images, their application still has some limitations. For example, NBI may have difficulty distinguishing inflammatory lesions from neoplastic lesions in some cases, resulting in a high false-positive rate. EC requires longer procedure time, and its recognition theory system is not yet perfect, which may require further optimization and standardization. With CLE, because fluorescein stains all cell types equally, both normal and cancerous, distinguishing the live-image version of cancer can be a challenge.

[0004] Confocal laser endoscopy (CLE) using fluorescently labeled molecular probes has been reported to identify colon dysplasia and cancer. And previous studies have shown that there are differences in fucosylation levels between colon cancer and normal colon. Fucosylation is a glycosylation modification that adds fucose (6-deoxy-L-galactose) to oligosaccharides / proteins. It has been reported that lack of fucosylation may lead to dysplasia and eventually develop into cancer that mainly affects the right colon and cecum. Ulex oleracea agglutinin 1 (UEA-I) is a specific lectin that specifically binds to α1-2-fucose glycans and can therefore be used to detect fucosylation levels. Most importantly, they are abundant in plants and animals and are a safe protein that may be suitable for human use. In addition, since the colorectum is a luminal organ, UEA-I is administered by local spraying and can be excreted from the body by endoscopic suction after observation, so it will not be completely absorbed. Therefore, UEA-I is a safe and feasible molecular imaging target.

[0005] By utilizing the specific binding of dyes and UEA-I, it can help distinguish the differences in fucosylation levels in colon tissues, thereby helping to identify the differences between tumor lesions and normal tissues. The types of labeling dyes currently combined with UEA-I include DyLight 649, DyLight 594, fluorescein (FITC), rhodamine, etc. The structure of SPE-PEG-FITC is a main chain composed of phosphatidylethanolamine (DSPE) and polyethylene glycol (PEG), and the fluorescent dye FITC (fluorescent isothiocyanate) is obtained by copolymerization as an end group. DSPE-PEG-FITC has a hydrophobic phospholipid part and a hydrophilic PEG part, which allows it to form self-assembled nanoparticles in aqueous solution, which are called nanoliposomes. FITC molecules are covalently bonded to DSPE-PEG, so that DSPE-PEG-FITC has a fluorescent labeling function that can be used in cell imaging, protein tracking, drug delivery and other fields. At the same time, DSPE-PEG-FITC has good biocompatibility, has no toxic side effects on organisms, and can exist stably under physiological conditions. DSPE-PEG-FITC can also be combined with other functional ligands to achieve targeted delivery to specific cells or tissues. However, it is not known in the art whether DSPE-PEG-FITC can be used in combination with UEA-I and applied to intestinal tumor labeling based on laser confocal endoscopy. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an intestinal tumor marker based on laser confocal endoscopy and its preparation method, use method and application. Taking into account the impact and safety of labeling dyes on human health, the present invention uses DSPE-PEG-FITC nanoliposomes as a dye to label UEA-I for the first time, and studies the expression level of UEA-I in normal intestinal epithelium and colon cancer in this way, and successfully verifies the feasibility of fluorescently labeled DSPE-PEG-FITC-UEA I as an intestinal imaging agent, which can be applied to the detection of colon cancer through optical imaging. The present invention combines the advantages of confocal endoscopy and the advantages of specific molecular markers, and can perform real-time imaging of the luminal surface, which is non-invasive and minimally invasive, reduces the risk of inspection, and specifically labels tumor markers, which can achieve early and accurate diagnosis and treatment of neoplastic lesions.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide an intestinal tumor marker based on confocal laser endoscopy (CLE), wherein the intestinal tumor marker is Ulex oleraceus agglutinin 1 (UEA-I) nanoliposome encapsulation, wherein the proportion of UEA-I is: 1 / (10-30) mg / ml.

[0009] Furthermore, the nanoliposome is DSPE-PEG-FITC.

[0010] The second object of the present invention is to provide a method for preparing the intestinal tumor marker, the preparation method comprising the following steps:

[0011] (1) Preparation of DSPE-PEG-FITC: 100-110 mg, 0.2-0.3 mmol of fluorescein isothiocyanate (FITC) and 490-510 mg, 0.2-0.3 mol of distearylphosphatidylethanolamine-polyethylene glycol amino (DSPE-PEG-NH2) were mixed in 8-12 ml of water, and the reaction solution was stirred at 55-65° C. for 1-3 days; then dialyzed and freeze-dried to obtain a brown product, which is the DSPE-PEG-FITC;

[0012] (2) Preparation of DSPE-PEG-FITC-UEA I: The DSPE-PEG-FITC solution was mixed with the UEA-I solution, and then heated, stirred, cooled and filtered to obtain the DSPE-PEG-FITC-UEAI, wherein the proportion of UEA-I was 1 / (10-30) mg / ml.

[0013] Furthermore, in step (1), the reaction solution was stirred at 60° C. for 2 days; the dialysis was performed 3 times using a MW 1000 dialysis bag; and the freeze-drying time was 2 days.

[0014] Furthermore, in step (2), the concentration of the DSPE-PEG-FITC solution is 10-30 mg / ml, the concentration of the UEA-I solution is 0.08-0.12 mg / ml, and the mixed volume ratio of the two is (0.8-1.2): (0.8-1.2); heated to 58-62° C.; and stirred for 25-35 minutes.

[0015] Furthermore, in step (2), the concentration of the DSPE-PEG-FITC solution is 20 mg / ml, the concentration of the UEA-I solution is 0.10 mg / ml, and the mixing volume ratio of the two is 1:1; the mixture is heated to 60° C. and stirred for 30 minutes.

[0016] The third object of the present invention is to provide a method for using the intestinal tumor marker, the method comprising the following steps:

[0017] (1) Use white light endoscopy to initially observe and mark the location of suspicious lesions;

[0018] (2) Clearing intestinal fluid;

[0019] (3) using CLE for further observation and applying the intestinal tumor markers to the lesions for staining;

[0020] (4) When the dye enters the cell nucleus and the glandular structure can be clearly outlined in the normal area, it can be judged that the staining is good. Then, focused observation is performed, video data is retained, and CLE is used to predict pathological results.

[0021] Furthermore, when CLE is used to predict the pathological results in step (4), the excitation wavelength is 495 nm and the emission wavelength is 519 nm.

[0022] Furthermore, the judgment criteria of the CLE pathological results are as follows:

[0023] 1) The diagnostic features of normal mucosa include hexagonal honeycomb appearance, regular circular cavities, uniformly arranged epithelial cells, normal glandular epithelial cell polarity, normal goblet cell secretion, orderly capillary network and regular crypt architecture, and UEA-I development is uniform and regular;

[0024] 2) The diagnostic features of adenoma are unclear structural boundaries, mild irregularity of crypt morphology due to compression, progression to villous transformation, destruction of crypts or even complete change of morphology, mild deformation and pseudostratification of cell nuclei, absence of goblet cells, and regular distribution of UEA-I imaging;

[0025] 3) The diagnostic features of adenocarcinoma are distorted crypts, reduced or even absent goblet cells, irregular stroma, and abnormal ductal structure. UEA-I development is irregular, with most areas being absent, but irregular ductal structure can still be observed.

[0026] The fourth object of the present invention is to provide an application of the intestinal tumor marker, the intestinal tumor marker preparation method and the intestinal tumor marker use method in the preparation of intestinal tumor diagnosis or treatment products.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention can accurately identify the tumor site by observing the expression of UEAI reflecting the fucosylation level under laser confocal endoscopy;

[0029] 2. The present invention uses laser confocal endoscopy to observe the lesion in real time, which can save time for pathological examination and optimize the diagnosis and treatment steps;

[0030] 3. The present invention detects intestinal tumors through DSPE-PEG-FITC-UEAI labeling, and can diagnose the lesion site without biopsy and histopathological examination, which is non-invasive;

[0031] 4. The DSPE-PEG-FITC-UEAI dye in the present invention has good biocompatibility, has no toxic side effects on organisms, can exist stably under physiological conditions, can be rapidly excreted from the body, and does not cause harm to human health while improving diagnostic efficiency.

[0032] 5. The present invention combines the advantages of confocal endoscopy and specific molecular markers, and can perform real-time imaging of the inner surface of the cavity, which is non-invasive and minimally invasive, reduces the risk of examination, specifically marks tumor markers, and improves the early detection rate of intestinal tumors. This method is used for screening of intestinal tumors, and can achieve accurate early diagnosis and treatment of neoplastic lesions. It is real-time, non-invasive, accurate, and suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are the expression results of UEA-1 encapsulated by DSPE-PEG in different tissues observed in mice under CLE in Example 2 of the present invention, wherein the tissues are normal intestinal mucosa, intestinal adenoma and intestinal adenocarcinoma. DETAILED DESCRIPTION

[0034] Example 1

[0035] The purpose of this embodiment is to explore the method of identifying the site of intestinal tumors by laser confocal endoscopy using DSPE-PEG-FITC-UEAI specific markers, in the hope of accurately marking tumor lesions using cancer-specific molecular imaging agents and improving the early detection rate of intestinal tumors. This method is used for screening of intestinal tumors and has the advantages of real-time, non-invasive, and accuracy, and is suitable for promotion and application.

[0036] This embodiment includes the following steps:

[0037] 1. Preparation of DSPE-PEG-FITC-UEAI

[0038] (1) Preparation of DSPE-PEG-FITC: FITC (107 mg, 0.275 mmol) and DSPE-PEG-NH2 (500 mg, 0.275 mol) were mixed in water (10 ml). The reaction solution was stirred at 60°C for 2 days. After three dialysis against deionized water (MW 1000), the solution was freeze-dried for two days, and a brown product (DSPE-PEG-FITC) was obtained.

[0039] (2) Preparation of DSPE-PEG-FITC-UEA I: First, dilute with distilled water to prepare a 20 mg / ml DSPE-PEG-FITC solution and a 0.1 mg / ml UEA-1 solution. Then, mix the DSPE-PEG-FITC solution and the UEA-1 solution in a volume ratio of 1:1. Heat the mixture to 60°C and slowly stir for 30 minutes. Then, cool the solution and filter through a membrane (0.22 μm) to make the ratio of UEA-I therein be 1 / 20 mg / ml, thereby obtaining DSPE-PEG-FITC-UEA I.

[0040] 2. Using laser confocal endoscopy to explore DSPE-PEG-FITC-UEAI specific labeling to identify intestinal tumor sites:

[0041] 1. First, use a white light endoscope to observe from the ileocecal region to the rectum and anus. Observation points: Pay attention to the location and size of suspicious lesions and mark the location of suspicious lesions;

[0042] 2. Clean the local intestine. If necessary, the intestinal mucosa can be thoroughly rinsed with physiological saline solution or simethicone and pronase solution to remove intestinal fluid;

[0043] 3. Use laser confocal endoscopy to observe and use a spray tube to drop a small amount of DSPE-PEG-FITC-UEAI dye on the lesion (observe from the normal area to the suspected lesion site step by step);

[0044] 4. Wait for about 2 to 3 minutes after dyeing;

[0045] 5. After 3 minutes of staining, the dye gradually enters the cell nucleus, and the glandular structure can be clearly outlined in the normal area;

[0046] 6. If the staining is good, you can make a judgment on the observed area and start collecting video data. It should be pointed out that you should pay attention to multiple areas when collecting data, and collect video data from different areas;

[0047] 7. For suspicious lesions, they can be observed in detail. If the staining is not satisfactory, they can be rinsed and stained again. After the staining results are satisfactory, video data can be retained; the endoscope system with an excitation wavelength of 495nm and an emission wavelength of 519nm is used for exploration, and the image display module receives the images;

[0048] 8. Use CLE to predict possible pathological results based on diagnostic criteria. The CLE result judgment criteria are as follows:

[0049] 1) The diagnostic features of normal mucosa include a hexagonal honeycomb appearance, regular circular cavities, evenly arranged epithelial cells, normal glandular epithelial cell polarity, normal goblet cell secretion, an orderly capillary network, and regular crypt architecture. UEA-I development is uniform and regular.

[0050] 2) The diagnostic features of adenoma are unclear structural boundaries, mild irregularity of crypt morphology due to compression, progression to villous transformation, destruction of crypts, and even complete change of morphology. The nuclei are slightly deformed and pseudostratified, and goblet cells are absent. UEA-I still shows regular distribution.

[0051] 3) The diagnostic features of adenocarcinoma are distorted crypts, reduced or even absent goblet cells, irregular stroma, and abnormal ductal structure. UEA-I shows irregular development, with most areas missing, but irregular ductal structures can still be observed.

[0052] Example 2

[0053] In mice, white light endoscopy was used to observe the intestines for lesions, and then CLE was used to observe the surrounding mucosa of the suspected lesions, and then the suspicious lesions were observed and judged (see Example 1 for specific methods). The expression results of UEA-1 encapsulated by DSPE-PEG in normal intestinal mucosa, intestinal adenomas and intestinal adenocarcinomas observed under CLE in mice are shown in Figure 1. Figure 1 shown.

[0054] The results showed that in normal colon tissue, we can clearly see the uniform distribution of DSPE-PEG-wrapped UEA-1, which is completely consistent with the glandular structure. Under CLE, each independent gland can be clearly seen, there is no fusion between glands, and the crypts are clearly visible; in adenoma tissue, the distribution of fluorescently labeled UEA-1 is uneven and flaky, but the distribution of fluorescently labeled UEA-1 is still consistent with the glandular structure, and there is fusion in some areas. Under CLE, the glands are still independent, and some areas are fused, and the crypt structure is not clear; in adenocarcinoma tissue, the distribution of fluorescently labeled UEA-1 is uneven and irregular, and fluorescently labeled UEA-1 is missing in some areas. The glandular structure cannot be identified under CLE, and the glands are completely fused and disappeared. Record the characteristics of CLE lesions, make a preliminary judgment on the results, and take biopsies from key suspicious areas for pathological examination.

[0055] Example 3

[0056] Previously reported: Confocal images were prospectively used to predict histopathology. CLE was used to evaluate all sites before biopsy, and the histological findings were compared with CLE judgment. The sensitivity and specificity of CLE for the detection of malignant lesions were 95.6% and 97.7%, respectively. The positive and negative predictive values ​​were 91.6% and 95.6%, respectively. The overall correction rate of CLE for all sites was 95.6%.

[0057] At the same time, the existing technology ClinicalTrials.gov ID: NCT02156557 also reported that even though it used a peptide sequence KCCFPAQ with high sensitivity and specificity and fluorescently labeled it for wide-area imaging of proximal colon lesions, it only had a sensitivity of 89% and a specificity of 92%.

[0058] In contrast, the DSPE-PEG-FITC-UEA I marker of the present invention was used to explore the intestine, CLE was applied to evaluate all sites before biopsy, and the histological results were compared with the CLE judgment. The sensitivity and specificity of CLE for the detection of malignant lesions were 98.0% and 98.9%, respectively. The positive and negative predictive values ​​were 97.1% and 98.0%, respectively. The overall correction rate of CLE for all locations was 98.0%. The specific results are shown in Table 1 below:

[0059] Table 1 Specific test results using the method of the present invention

[0060]

[0061] Therefore, DSPE-PEG-FITC-UEA I has important prospects and significance as a marker for intestinal tumors.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A intestinal tumor marker based on confocal laser endoscopy (CLE), characterized in that: The intestinal tumor marker is a nanoliposome containing UEA-I, wherein the proportion of UEA-I is 1 / (10-30) mg / ml, and the nanoliposome is DSPE-PEG-FITC. The preparation method of the intestinal tumor marker comprises the following steps: (1) Preparation of DSPE-PEG-FITC: 100-110 mg, 0.2-0.3 mmol of fluorescein isothiocyanate (FITC) and 490-510 mg, 0.2-0.3 mol of distearylphosphatidylethanolamine-polyethylene glycol amino (DSPE-PEG-NH2) were mixed in 8-12 ml of water, and the reaction solution was stirred at 55-65° C. for 1-3 days; then dialyzed and freeze-dried to obtain a brown product, which is the DSPE-PEG-FITC; (2) Preparation of DSPE-PEG-FITC-UEA I: The DSPE-PEG-FITC solution was mixed with the UEA-I solution, and then heated, stirred, cooled and filtered to make the ratio of UEA-I therein be: 1 / (10-30) mg / ml, thereby obtaining the DSPE-PEG-FITC-UEA I.

2. The method for preparing an intestinal tumor marker according to claim 1, characterized in that: The following steps are involved: (1) Preparation of DSPE-PEG-FITC: 100-110 mg, 0.2-0.3 mmol of fluorescein isothiocyanate (FITC) and 490-510 mg, 0.2-0.3 mol of distearylphosphatidylethanolamine-polyethylene glycol amino (DSPE-PEG-NH2) were mixed in 8-12 ml of water, and the reaction solution was stirred at 55-65° C. for 1-3 days; then dialyzed and freeze-dried to obtain a brown product, which is the DSPE-PEG-FITC; (2) Preparation of DSPE-PEG-FITC-UEA I: The DSPE-PEG-FITC solution was mixed with the UEA-I solution, and then heated, stirred, cooled and filtered to make the ratio of UEA-I therein be: 1 / (10-30) mg / ml, thereby obtaining the DSPE-PEG-FITC-UEA I.

3. The preparation method according to claim 2, characterized in that: In step (1), the reaction solution was stirred at 60° C. for 2 days; the dialysis was performed 3 times using a MW 1000 dialysis bag; and the freeze-drying time was 2 days.

4. The preparation method according to claim 2, characterized in that: In the step (2), the concentration of the DSPE-PEG-FITC solution is 10-30 mg / ml, the concentration of the UEA-I solution is 0.08-0.12 mg / ml, and the volume ratio of the two is (0.8-1.2): (0.8-1.2); the mixture is heated to 58-62° C. and stirred for 25-35 minutes.

5. The preparation method according to claim 4, characterized in that: In the step (2), the concentration of the DSPE-PEG-FITC solution is 20 mg / ml, the concentration of the UEA-I solution is 0.10 mg / ml, and the volume ratio of the two is 1:1; the mixture is heated to 60° C. and stirred for 30 minutes.

6. Use of the intestinal tumor marker according to claim 1 or the method for preparing the intestinal tumor marker according to any one of claims 2 to 5 in preparing intestinal tumor diagnosis or treatment products.

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