Molecular probe for detecting senile chronic atrophic gastritis and application

Abstract

The invention belongs to the technical field of biomedical materials, and discloses a molecular probe for detecting senile chronic atrophic gastritis and application of the molecular probe. The molecular probe is coupled Mcemp1 magnetic-optical bimodal composite nanoparticles; the preparation method comprises the following steps: preparing a Fe3O4 (at) OA solution by taking ferric acetylacetonate, oleylamine and dibenzyl ether as raw materials, modifying the Fe3O4 (at) OA solution through an SPE-PEG2000-NH2 polymer, and purifying the Fe3O4 (at) OA solution to obtain a magnetic core Fe3O4-PEG-NH2 NPs; the preparation method comprises the following steps: coupling a magnetic core with Mcomp1, and then purifying, so as to obtain Fe3O4-PEG-Mcomp1 NPs (N < 3 > O34-PEG-Mcomp1); and then carrying out near-infrared fluorescent dye ICG labeling, and dialyzing to obtain the magnetic-optical dual-mode composite nanoparticles coupled with the Memp1. Non-invasive continuous monitoring is achieved through the intravenous administration magnetic particle probe, the bimodal technology is combined, gland density is quantified in real time, the gastric mucosa condition is dynamically tracked, the problems that a fluorescent probe is insufficient in penetration depth and low in quantification precision are solved, and the technical blank of dynamic evaluation and canceration early warning is filled up.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and more particularly to a molecular probe for detecting chronic atrophic gastritis in the elderly and its application. Background Art

[0002] Chronic atrophic gastritis (CAG) in the elderly faces multiple technical challenges in monitoring lesions and evaluating treatment efficacy. Currently, clinical diagnosis and follow-up rely primarily on endoscopic biopsy and serological markers (such as pepsinogen PG I and II, and gastrin-17). However, endoscopic examinations are invasive procedures with a long follow-up period of 1-3 years, making it difficult to capture the dynamic evolution of gastric mucosal atrophy or intestinal metaplasia (such as the 10-day fluctuations in glandular regeneration and destruction) in real time. While serological markers can reflect the functional status of the gastric mucosa, their sensitivity for distinguishing mild from severe atrophy is only 62%-78%, and they lag behind the pathological process, making it impossible to monitor inflammatory activity or activation of cancer-related molecular pathways. Elderly patients have significantly reduced tolerance for repeated endoscopic procedures due to factors such as declining physical function, coexisting cardiovascular and cerebrovascular diseases, and polypharmacy (such as nonsteroidal anti-inflammatory drugs). Furthermore, existing fluorescence imaging-based targeted probes suffer from insufficient tissue penetration depth and low quantitative accuracy, resulting in the lack of a continuous dynamic assessment system for the degree of mucosal atrophy and repair capacity in CAG.

[0003] Magnetic particle probe technology provides a new direction for solving the above problems. Compared with traditional fluorescent probes, magnetic particle probes can break through the physical limitations of fluorescence imaging by virtue of their deep tissue penetration, signal stability and multimodal quantitative potential. Currently, there is an urgent need for visual evaluation of the dynamic pathological evolution of CAG (such as the distribution of intestinal metaplasia foci and the rapid progression of low-grade intraepithelial neoplasia) and therapeutic efficacy in clinical practice, but no studies have reported the application of magnetic particle probes in this field. The existing static evaluation system based on the OLGA / OLGIM staging system is difficult to quantify the real-time changes in key markers of gastric mucosal "inflammation-cancer" transformation (such as glandular density and glandular spacing), and the conventional endoscopic review cycle is seriously mismatched with the rate of molecular events (such as 35% of low-grade intraepithelial neoplasia progresses within 6 months).

[0004] Based on the inventors' previous research results showing that Mcemp1 may be a promising biomarker for specific and targeted MI / R imaging, and the discovery that Mcemp1 is associated with gastric mucosal lesions, the present invention proposes a magnetic particle probe that can be administered intravenously, supports 10-day continuous monitoring, and can quantitatively evaluate therapeutic efficacy. It has important clinical value for achieving precise intervention in elderly CAG patients and early diagnosis of gastric cancer. Summary of the Invention

[0005] In order to solve the problems of insufficient tissue penetration depth and low quantitative accuracy of existing fluorescent probes, the present invention provides a molecular probe for detecting chronic atrophic gastritis in the elderly and its application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A molecular probe for detecting chronic atrophic gastritis in the elderly, wherein the molecular probe is a magnetic-optical dual-modal composite nanoparticle coupled with Mcemp1;

[0008] The preparation method of the coupled Mcemp1 magneto-optical dual-modal composite nanoparticles comprises the following steps:

[0009] Step 1: Synthesis and modification of magnetic core

[0010] Fe3O4@OA solution was prepared from ferric acetylacetonate, oleylamine and dibenzyl ether, and then modified with SPE-PEG2000-NH2 polymer and purified to obtain magnetic core Fe3O4-PEG-NH2NPs.

[0011] Step 2: Preparation of Mcemp1-magnetic core nanoparticles

[0012] The magnetic core obtained in step 1 was coupled with Mcemp1 and then purified to obtain Mcemp1-magnetic core nanoparticles, which were placed in a PBS buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L to obtain a Fe3O4-PEG-Mcemp1 NPs solution;

[0013] Step 2: Preparation of Mcemp1 / ICG dual-modality probe

[0014] The Mcemp1-magnetic core nanoparticles in the Fe3O4-PEG-Mcemp1 NPs solution obtained in step 2 were labeled with the near-infrared fluorescent dye ICG, and after dialysis, Mcemp1-coupled magneto-optical dual-modal composite nanoparticles, namely Mcemp1 / ICG dual-modal probes, were obtained.

[0015] Preferably, the preparation steps of the Fe3O4@OA solution are as follows:

[0016] (1) stirring ferric acetylacetonate, oleylamine, and dibenzyl ether under a nitrogen flow, heating to a first preset temperature at a heating rate of 3.0 to 3.5°C / min, maintaining the temperature for more than 1 hour, and further heating to a second preset temperature, maintaining the temperature for more than 12 hours, to obtain superparamagnetic Fe3O4 nanoparticles with a particle size of 20 to 50 nm;

[0017] (2) Using magnetic separation combined with ethanol, the superparamagnetic Fe3O4 nanoparticles obtained in step (1) were ultrasonically extracted 1 to 3 times, 1 to 1.5 hours per time, filtered, the filtrate was combined, concentrated by rotary evaporation, refrigerated for more than 12 hours, and then freeze-dried, and then dispersed into a chloroform solution to obtain a Fe3O4@OA solution; the volume mass ratio of the ethanol to the superparamagnetic Fe3O4 nanoparticles was 8 to 12 mL: 1 mg.

[0018] Preferably, the first preset temperature is 200-220°C, and the second preset temperature is 280-320°C.

[0019] Preferably, the mass volume ratio of the ferric acetylacetonate, oleylamine and dibenzyl ether is 0.7 g:1.7 ml:20 ml.

[0020] Preferably, after modification of the SPE-PEG2000-NH2 polymer, the following steps are performed: adding an equal volume of citric acid solution to the Fe3O4@OA solution and concentrating the solution by rotary evaporation until the iron content is 0.8-1.2 mg / mL; adjusting the pH to 10-11 with a dilute ammonia solution with a volume fraction of 1-10%; then adding the DSPE-PEG2000-NH2 polymer so that the mass concentration of DSPE-PEG2000-NH2 in the solution is consistent with the iron content; and stirring the reaction in a water bath at 70-90°C for more than 24 hours.

[0021] Preferably, the purification in step 1 and step 2 is: high-speed centrifugation using a 25-40 kD ultrafiltration tube at a speed of 5000-6000 rpm and a centrifugation time of 15-25 min; in step 3, the dialysis is performed in deionized water for more than 48 h through a dialysis bag with a molecular weight cutoff of 3-4 kDa.

[0022] Preferably, the coupling is specifically as follows: the magnetic core Fe3O4-PEG-NH2NPs obtained in step 1 are added to a buffer solution and the pH is adjusted to 7.0-8 to obtain a Fe3O4@PEG-NH2 solution, and a sulfo-SMCC bifunctional crosslinker solution is added to react for more than 30 minutes; then, Mcemp1 solution is added and shaken in the dark at 4-8°C for more than 12 hours.

[0023] The mass concentrations of the Fe3O4@PEG-NH2 solution and the Mcemp1 solution are 0.8~1.2 mg / mL, the molar concentration of the sulfo-SMCC bifunctional crosslinker in the sulfo-SMCC bifunctional crosslinker solution is 8~12 mM, the volume ratio of the Fe3O4@PEG-NH2 solution to the sulfo-SMCC bifunctional crosslinker solution is 1:1~4, and the mass ratio of the Fe3O4-PEG-NH2NPs and Mcemp1 is 1:1~3.

[0024] Preferably, the near-infrared fluorescent dye ICG labeling is specifically performed by dissolving ICG-NHS ester in a pH 8.5 carbonate buffer, adding Fe3O4-PEG-Mcemp1 NPs solution under light-proof conditions, stirring and reacting at 15-35°C for more than 12 hours, and the molar ratio of the ICG-NHS ester and Mcemp1-magnetic core nanoparticles is 1:4-6.

[0025] The present invention also provides the use of the above-mentioned Mcemp1-coupled magneto-optical dual-modal composite nanoparticles in dual-modal imaging for detecting chronic atrophic gastritis in the elderly.

[0026] This probe uses a dual-modality synergistic mechanism of magnetic particle imaging (MPI) and near-infrared fluorescence imaging (IVIS) to achieve real-time, dynamic, and quantitative assessment of the extent of chronic atrophic gastritis lesions and the therapeutic efficacy. MPI technology is based on the magnetic properties of superparamagnetic Fe3O4 nanoparticles. Under an external alternating magnetic field, the harmonic signal intensity generated by the reversal of its nuclear magnetic moment is linearly related to the local probe concentration, and can directly quantify the degree of atrophy of gastric mucosal glands. Pathological studies have shown that a decrease in glandular density (such as severe atrophy corresponding to a reduction of >2 / 3 of the glands) leads to a decrease in probe retention density. The MPI signal intensity is significantly negatively correlated with glandular density, and the atrophy grade can be divided.

[0027] At the same time, the ICG near-infrared fluorescent dye (emission wavelength 750-810nm, penetration depth 5-10mm) carried by the probe analyzes inflammatory activity in real time through IVIS imaging. The fluorescence intensity is highly matched with the histological score (such as OLGA / OLGIM staging). During the treatment process, the reduction of the fluorescent hotspot range (ROI) and the decrease in intensity can intuitively reflect the progress of mucosal repair. This technology combines magnetic quantification with optical dynamic tracking, breaking through the limitations of static evaluation of traditional endoscopy, and providing a multi-dimensional tool for long-term efficacy management and precise treatment navigation for elderly patients.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention overcomes the defects of invasiveness and hysteresis of traditional monitoring. Existing technologies rely on invasive endoscopic biopsy (review cycle 1-3 years) and serum markers (PGⅠ / PGⅡ), which cannot capture the dynamic evolution of lesions (such as the 10-day fluctuation of gland regeneration and destruction). The present invention achieves non-invasive continuous monitoring for ≥10 days through intravenous administration of magnetic particle probes, combined with MPI (magnetic particle imaging) and IVIS (near-infrared fluorescence) dual-modality technology, to quantify glandular density in real time and dynamically track the condition of the gastric mucosa.

[0030] 2. The present invention solves the problems of insufficient penetration depth and low quantitative accuracy of fluorescent probes. Existing fluorescent probes (such as NIR-I organic fluorophore imaging technology) have a tissue penetration depth of 1 to 3 mm and cannot assess the ability of mucosal repair. The present invention adopts a superparamagnetic Fe3O4 core + ICG near-infrared fluorescence dual-modality design, with a penetration depth of ≥5 mm (IVIS) and a magnetic signal stability of at least 1 μg Fe / mL (MPI), which can simultaneously locate microscopic lesions (≤2 mm) and quantify the degree of glandular atrophy.

[0031] 3. Filling the technological gap in dynamic assessment and cancer early warning. Existing technologies are unable to track key events in the "inflammation-to-cancer" transformation in real time. This invention uses magnetic-optical signals linked to molecular markers to provide early warning of gastric mucosal conditions, shortening the diagnostic window compared to traditional pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Scanning electron microscopy images of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe;

[0033] Figure 2 Fluorescence spectra of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe;

[0034] Figure 3 The UV spectra of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe;

[0035] Figure 4 This is the hydration particle size detection diagram of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe;

[0036] Figure 5 is the corresponding relationship between the concentration of Mcemp1-Fe3O4-ICG probe and the fluorescence signal value;

[0037] Figure 6 is the corresponding relationship between the Mcemp1-Fe3O4-ICG probe concentration and the MPI signal value;

[0038] Figure 7 Fluorescence imaging of mice in different groups after injection of IgG-Fe3O4-ICG probe;

[0039] Figure 8 Fluorescence imaging of mice in different groups after injection of Mcemp1-Fe3O4-ICG probe;

[0040] Figure 9MPI images of mice in different groups after Mcemp1-Fe3O4-ICG probe injection (A is the in vivo imaging of mice, B is the in vitro tissue imaging);

[0041] Figure 10 MPI images of mice in different groups after gG-Fe3O4-ICG probe injection (A is the in vivo imaging of mice, B is the in vitro tissue imaging);

[0042] Figure 11 HE staining of gastric antrum tissue and transplanted tumor tissue after Mcemp1-Fe3O4-ICG probe injection in each group of mice;

[0043] Figure 12 The results of mast cell phagocytosis of IgG-Fe3O4-ICG probe and Mcemp1-Fe3O4-ICG probe are shown in the figure. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0045] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0046] Example 1

[0047] A molecular probe for detecting chronic atrophic gastritis in the elderly is provided. The molecular probe is a magnetic-optical dual-modal composite nanoparticle coupled with Mcemp1. The preparation method thereof comprises the following steps:

[0048] Step 1, synthesis and modification of magnetic core (Fe3O4-PEG-NH2NPs)

[0049] 1) 0.7 g of iron acetylacetonate (Fe(acac)3), 1.7 ml of oleylamine (OAm), and 20 ml of dibenzyl ether were added to a 50 mL three-necked flask to obtain a mixture. The mixture was stirred under a nitrogen (N2) stream and heated to 220°C (nucleation temperature) at a heating rate of 3.3°C / min and held for 1 hour.

[0050] 2) The system is then heated to 300°C and maintained for 12 hours to generate superparamagnetic Fe3O4 nanoparticles with a particle size of 20-50 nm through high-temperature thermal decomposition.

[0051] 3) After the reaction, magnetic separation was used, and ultrasonic extraction was performed three times with 10-fold ethanol, each time for 1 hour. The filtrates were filtered, combined, and concentrated by rotary evaporation. The mixture was placed at -20°C for 12 hours and freeze-dried for 48 hours to remove unreacted OAm and dibenzyl ether. The mixture was then dispersed in 10 mL of chloroform to obtain a Fe3O4@OA solution.

[0052] 4) An equal volume of 10% citric acid standard solution was added to the Fe3O4@OA solution, and the solution was concentrated by rotary evaporation to an iron content of 1 mg / mL. The pH was adjusted to 11 with a 1% dilute ammonia solution (sodium tripolyphosphate solution or disodium ethylenediaminetetraacetic acid solution could also be used). DSPE-PEG2000-NH2 polymer was added to the citric acid-coated iron oxide to a final concentration of 1 mg / mL. The reaction was stirred at 80°C for 24 hours to form hydrophilic amino-modified magnetic cores (Fe3O4-PEG-NH2NPs) covalently modified via amide bonds.

[0053] 5) The reaction products were separated by high-speed centrifugation at 6000 rpm using a 30 kD ultrafiltration tube for 20 min to remove residual citric acid, ammonia solution and DSPE-PEG2000-NH2 polymer to obtain Fe3O4@PEG-NH2 (Fe3O4-PEG-NH2NPs). Dynamic light scattering (DLS) analysis confirmed that the particle size was 31.715±3.309 nm and the zeta potential was -0.22 mV.

[0054] Step 2: Preparation of Fe3O4-PEG-Mcemp1 NPs

[0055] 1) Dissolve thiolated Mcemp1 peptide (thiol groups were introduced by Traut's reagent) in 100 μL DMSO per 1 mg of Mcemp1, and add 900 μL of 0.1 mol / L PBS buffer (pH = 7.4) to obtain a Mcemp1 solution with a mass concentration of 1 mg / ml.

[0056] 2) The Fe3O4@PEG-NH2 prepared in step 1 was added to PBS buffer to obtain a Fe3O4@PEG-NH2 solution. The pH was adjusted to 7.4 with 1% dilute ammonia solution to a final concentration of 1 mg / mL. A 10 mM solution of sulfo-SMCC bifunctional crosslinker (>95% purity) was added to a volume ratio of 1:1 to 4 between the Fe3O4@PEG-NH2 solution and the solution. The maleimide groups of the sulfo-SMCC reacted with the amino groups on the surface of the magnetic core for 30 minutes to form an activated intermediate.

[0057] 3) Subsequently, Mcemp1 solution (volume ratio of Fe3O4@PEG-NH2 solution to Mcemp1 solution is 1:1-3) was added, and the mixture was oscillated in dark at 4°C for 12 h to covalently connect the targeting peptide to the surface of the magnetic core through thiol-maleimide click chemistry. After the reaction, 30 kDa ultrafiltration tube was used to centrifuge at 5000 rpm to purify and remove the unbound peptide, thereby obtaining Fe3O4-PEG-Mcemp1 NPs (Mcemp1-magnetic core nanoparticle); the Fe3O4-PEG-Mcemp1 NPs were placed in a PBS buffer with a pH of 7.4 and a concentration of 0.1 mol / L, thereby obtaining a Fe3O4-PEG-Mcemp1 NPs solution for standby.

[0058] Step three, preparation of Mcemp1 / ICG bimodal probe (Fe3O4-PEG-Mcemp1-ICG NPs)

[0059] 1) ICG-NHS ester (molar ratio of ICG-NHS ester to Fe3O4-PEG-Mcemp1 NPs is 1:5) was dissolved in a pH 8.5 carbonate buffer, and Fe3O4-PEG-Mcemp1 NPs solution was added under dark conditions. The mixture was stirred at room temperature for 12 h to realize the covalent binding of NHS ester to the residual amino groups on the surface of the magnetic core and achieve fluorescent labeling.

[0060] 2) After the reaction, the mixture was dialyzed in a dialysis bag with a molecular weight cut-off of 3.5 kDa for 48 h in deionized water to remove free dye, thereby obtaining Mcemp1 magnetic-optical bimodal composite nanoparticles, i.e., Mcemp1 / ICG bimodal probe (Fe3O4-PEG-Mcemp1-ICG NPs, also referred to as Mcemp1-Fe3O4-ICG probe).

[0061] The Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe (the preparation steps are the same as those of the Mcemp1-Fe3O4-ICG, except that the Mcemp1 is replaced by an IgG antibody solution) were subjected to electron microscope scanning, fluorescence spectrum, ultraviolet spectrum, infrared spectrum, and DLS / zeta hydration particle size and potential detection, and the detection results are shown in Figure 1-6 .

[0062] Test example:

[0063] I. Experimental method

[0064] 1. Animal preparation

[0065] Sixty SPF-grade, 6- to 8-week-old, Balb / c male mice and 20 SPF-grade, 6- to 8-week-old, Balb / c-nu athymic male nude mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0066] 2. Cell Preparation

[0067] NCI-N87 (N87) human gastric cancer cells (product number: CL-0169) were purchased from Wuhan Pronocell Life Science Co., Ltd. They were cultured in RPMI-1640 + 10% FBS + 1% P / S complete medium. At the 4th to 5th passage, 1×10 7 The density of cells / 1mL was used, and 0.1mL was injected subcutaneously in the right axilla of mice to form tumors.

[0068] 3. Modeling and grouping

[0069] Two gastric disease models, CSG and CAG, were established using 6- to 8-week-old male Balb / c mice, and the gastric tumor inoculation group was constructed using Balb / c-nu athymic male nude mice.

[0070] In the CSG group, gastric mucosal lesions were induced using MNNG (N-methyl-N'-nitro-N-nitrosoguanidine). At the start of the experiment, MNNG was dissolved in drinking water to a concentration of 80-200 μg / mL, and the mice were allowed to drink freely. The CSG group maintained the model for three months, during which time the MNNG solution was replaced weekly to ensure concentration stability.

[0071] For the CAG group, the same method was used for MNNG induction, but the modeling time was extended to 6 months to better simulate the chronic development process of the disease from chronic gastritis to atrophic gastritis.

[0072] The mice in the blank group were given only normal drinking water and managed under the same environmental conditions and used as the control group.

[0073] For the gastric transplant tumor inoculation group (transplant tumor group), this experiment used a human gastric cancer cell line for xenograft modeling. First, N87 cells were cultured to the logarithmic growth phase, the cells were collected and prepared at a concentration of 1×10 7 cells / 1mL of cell suspension. The cell suspension was then inoculated subcutaneously into the right axilla of the mice. Following inoculation, the mice's health was regularly observed, tumor growth was measured, and tumor volume and progression were assessed.

[0074] 3. Imaging Experiment

[0075] (1) MPI imaging

[0076] Each model group of mice (blank group (CON), CSG group, CAG group, transplanted tumor group) was injected with 100 μL of Mcemp1-Fe3O4-ICG probe or IgG-Fe3O4-ICG probe at a concentration of 1 mg / mL by tail vein injection, and scanning and imaging were performed after isoflurane anesthesia. 2D MPI scanning was performed at different time points (0 h, 12 h, 24 h, 48 h, 120 h, 240 h) after injection, and the scanning field of view was 6 x 10 cm. The scanning mode was isotropic, and the scanning time was 3 min. The MPI signal was the average MPI signal in the stomach region of interest (ROI). Momentum MPI scanner with a magnetic field gradient strength of 6 T / m was used for analysis. The Momentum MPI scanner was used to scan the model mice injected with nanoparticles, and the excitation field was the peak value along the z axis: 45 kHz, 20 mT. VivoQuant v.4.0 (Invicro, Boston, MA, USA) software was used to analyze the MPI images.

[0077] (2) IVIS imaging

[0078] In vivo fluorescence imaging of mice was performed using an IVIS Spectrum imaging system (PerkinElmer, USA). Mice in each model group (control group, CSG group, CAG group, and gastric tumor group) were injected via the tail vein with 100 μL of 1 mg / mL Mcemp1-Fe₃O₄-ICG probe or IgG-Fe₃O₄-ICG probe. Fluorescence imaging was performed at various time points after injection (0 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, and 240 h). Mice were anesthetized with isoflurane before imaging to ensure stability during imaging. They were placed on the imaging platform in the supine position, ensuring full exposure of the imaging area (abdomen). For imaging, the excitation wavelength was set to 740 nm, the emission wavelength was 790 nm, the exposure time was 3–5 seconds, the binning setting was medium, and the f / stop was 2. Imaging data were acquired using the IVIS Spectrum imaging system and analyzed using Living Image software (PerkinElmer). The software can draw a heat map of the region of interest (ROI) and quantitatively analyze the fluorescence signal intensity in different areas of the mouse abdomen at different time points. The change in fluorescence signal intensity reflects the distribution and clearance of the nanoparticles. The data were further statistically analyzed using GraphPadPrism software to compare the differences in signal intensity between groups and ensure the statistical significance of the results. After 240 hours (10 days), the last in vivo imaging was performed. After the imaging was completed, the mice were anesthetized and euthanized, and tissues such as the stomach, pancreas, liver, heart, spleen, lung, small intestine and transplanted tumors (if applicable) were removed for ex vivo imaging. Each organ was imaged separately using the same imaging conditions (740nm excitation, 790nm emission), and its corresponding fluorescence signal intensity was recorded. Through this combination of in vivo and ex vivo imaging, the targeting of Mcemp1 and IgG magneto-optical composite nanoparticles, drug release and their distribution characteristics among various tissues can be comprehensively evaluated.

[0079] 4. HE staining of gastric tissue

[0080] Forty-eight hours after injection of the Mcemp1-Fe3O4-ICG probe, mice in each group were fasted for 12 hours without food or water. Eyeballs were removed before recovery from anesthesia, and mice were killed by exsanguination. The abdominal cavity was opened, and the intestines and pancreas were removed. The stomach was exposed and cut open at the upper and lower openings, along the lesser curvature of the stomach. Gastric contents were cleared with 5 mL of pre-chilled saline. After washing, the samples were fixed in 4% paraformaldehyde for subsequent pathological staining. Fixed gastric tissue was routinely dehydrated and paraffin-embedded. 5-μm-thick tissue sections were cut serially from the gastric antrum. After being fished and dried, the sections were sequentially immersed in xylene I, xylene II, anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, followed by water washing. After staining with hematoxylin solution, the sections were washed, differentiated, washed with water, reversed with blue, and rinsed. The sections were then dehydrated in 70%, 80%, 95%, and anhydrous ethanol, stained with eosin solution, and dehydrated in anhydrous ethanol. The sections were transparentized with xylene and mounted with neutral gum. The morphology of the mucosal epithelium, gastric glands, chief cells, parietal cells, and muscularis mucosa was observed and images were collected under an upright microscope.

[0081] 5. In vitro cell phagocytosis

[0082] HMC-1 human mast cells in the logarithmic growth phase were digested and counted, and 1×10 5 Cells were seeded at a density of 10 cells / well in a 6-well plate and cultured overnight in complete culture medium supplemented with 10% fetal bovine serum. Meanwhile, cell adhesion reagent was diluted 1:200 with PBS and plated onto the surface of a glass-bottomed culture dish at a dose of 1 mL / well. Incubated at 37°C for 30 min with occasional agitation. The liquid in the dish was completely discarded, the bottom of the dish was washed once with sterile PBS buffer, and HMC-1 human mast cells were directly plated and allowed to adhere. Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe, pre-dissolved in serum-free culture medium, were added to each dish, ensuring complete coverage of the cell layer. The culture plates were incubated in a 37°C, 5% CO2 incubator in the dark for 1, 2, 4, 8, 12, and 24 h. After the incubation period, the probe-containing medium was immediately aspirated and the cells were washed 3-5 times with pre-chilled sterile PBS to completely remove any free probe that had not been internalized and adhered to the cell surface. Gently aspirate the liquid after each wash. Then, Hoechst 33342 live cell staining solution was added at a ratio of 1:100. After staining for 10 minutes, the intracellular fluorescent spots (phagocytic vacuoles) were observed and photographed using a fluorescence microscope, and Z-Stack scanning was used to confirm that the probe was located inside the cell.

[0083] 2. Experimental Results

[0084] 1. In vivo imaging results

[0085] MPI in vivo imaging MPI detection and IVIS in vivo fluorescence imaging results are shown in Figure 7-10 and Table 1, the results show that: Mcemp1-Fe3O4-ICG probe in mice stay longer than IgG-Fe3O4-ICG probe, after injection of Mcemp1-Fe3O4-ICG probe, the mouse stomach relative MPI signal is 3.39 times of the injection of IgG-Fe3O4-ICG probe group, and there is a statistically significant difference. This means that the MI / R mouse model has a significant difference in the uptake of CRT@Fe3O4 compared with the uptake of Fe3O4.

[0086] Table 1 MPI signal of probe after 48 hours

[0087]

[0088]

[0089] Note: * represents the signal intensity of Mcemp1-Fe3O4-ICG probe compared with the signal intensity of IgG-Fe3O4-ICG probe, P<0.01.

[0090] 2, H-E staining of gastric tissue

[0091] The H-E staining of the gastric antral tissue of each group of mice was observed (see Figure 11 ). The blank control (CON) group of mice, the gastric antral tissue glands arranged in order, no inflammatory cell infiltration. Atrophic gastritis (CAG) group of mice, the gastric antral glands arranged unevenly, the nucleus and cytoplasm ratio was abnormal, the mucosal epithelium disappeared, the number of chief cells and parietal cells decreased significantly, and the number of intrinsic glands decreased more than 2 / 3. While the chronic gastritis (CSG) group of mice, the gastric antral glands were relatively evenly arranged, but the mucosal epithelium was thin. Gastric cancer transplanted tumor (GC) group of mice showed obvious gastric cancer cell infiltration.

[0092] 3, in vitro cell phagocytosis

[0093] The results of mast cell phagocytosis of two kinds of probes are shown in Figure 12 . As can be seen from Figure 12 , the mast cell phagocytosis of IgG-Fe3O4-ICG probe is faster, mainly concentrated in the period of 4-8 hours, but the persistence is poor. However, the phagocytosis of Mcemp1-Fe3O4-ICG probe can be recognized from 8 hours to 48 hours, which suggests that the probe has a longer duration in the cell, and it is of great significance for long-term observation of the probe at multiple time points.

[0094] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A molecular probe for detecting chronic atrophic gastritis in the elderly, characterized in that: The molecular probe is a magnetic-optical dual-modal composite nanoparticle coupled with Mcemp1; The preparation method of the coupled Mcemp1 magneto-optical dual-modal composite nanoparticles comprises the following steps: Step 1: Synthesis and modification of magnetic core Fe3O4@OA solution was prepared from ferric acetylacetonate, oleylamine and dibenzyl ether, and then modified with SPE-PEG2000-NH2 polymer and purified to obtain magnetic core Fe3O4-PEG-NH2NPs. Step 2: Preparation of Mcemp1-magnetic core nanoparticles The magnetic core obtained in step 1 was coupled with Mcemp1 and then purified to obtain Mcemp1-magnetic core nanoparticles, which were placed in a PBS buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L to obtain a Fe3O4-PEG-Mcemp1 NPs solution; Step 2: Preparation of Mcemp1 / ICG dual-modality probe The Mcemp1-magnetic core nanoparticles in the Fe3O4-PEG-Mcemp1 NPs solution obtained in step 2 were labeled with the near-infrared fluorescent dye ICG, and after dialysis, Mcemp1-coupled magneto-optical dual-modal composite nanoparticles, namely Mcemp1 / ICG dual-modal probes, were obtained.

2. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that: The preparation steps of the Fe3O4@OA solution are as follows: (1) stirring ferric acetylacetonate, oleylamine, and dibenzyl ether under a nitrogen flow, heating to a first preset temperature at a heating rate of 3.0 to 3.5°C / min, maintaining the temperature for more than 1 hour, and further heating to a second preset temperature, maintaining the temperature for more than 12 hours, to obtain superparamagnetic Fe3O4 nanoparticles with a particle size of 20 to 50 nm; (2) Using magnetic separation combined with ethanol, the superparamagnetic Fe3O4 nanoparticles obtained in step (1) were ultrasonically extracted 1 to 3 times, 1 to 1.5 hours per time, filtered, the filtrate was combined, concentrated by rotary evaporation, refrigerated for more than 12 hours, and then freeze-dried, and then dispersed into a chloroform solution to obtain a Fe3O4@OA solution; the volume mass ratio of the ethanol to the superparamagnetic Fe3O4 nanoparticles was 8 to 12 mL: 1 mg.

3. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 2, characterized in that: The first preset temperature is 200-220°C, and the second preset temperature is 280-320°C.

4. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 2, characterized in that: The mass volume ratio of the ferric acetylacetonate, oleylamine and dibenzyl ether is 0.7 g:1.7 mL:20 mL.

5. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that: The SPE-PEG2000-NH2 polymer modification includes: adding an equal volume of 10% citric acid solution to the Fe3O4@OA solution and concentrating it by rotary evaporation until the iron content is 0.8-1.2 mg / mL, adjusting the pH to 10-11 with a dilute ammonia solution with a volume fraction of 1-10%, and then adding DSPE-PEG2000-NH2 polymer to make the mass concentration of DSPE-PEG2000-NH2 in the solution consistent with the iron content, and reacting at 70-90°C in a water bath with stirring for more than 24 hours.

6. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that: In step 1 and step 2, the purification is: high-speed centrifugation separation using a 25-40 kD ultrafiltration tube, a rotation speed of 5000-6000 rpm, and a centrifugation time of 15-25 min. In step 3, the dialysis is performed in deionized water for more than 48 hours through a dialysis bag with a molecular weight cutoff of 3-4 kDa.

7. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that: The coupling is specifically as follows: the magnetic core obtained in step 1 is added to a buffer solution and the pH is adjusted to 7.0-8 to obtain a Fe3O4@PEG-NH2 solution, and a sulfo-SMCC bifunctional crosslinker solution is added to react for more than 30 minutes; then, the Mcemp1 solution is added and the mixture is shaken at 4-8°C in the dark for more than 12 hours.

8. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 7, characterized in that: The mass concentrations of the Fe3O4@PEG-NH2 solution and the Mcemp1 solution are both 0.8-1.2 mg / mL, the molar concentration of the sulfo-SMCC bifunctional crosslinker solution is 8-12 mM, the volume ratio of the Fe3O4@PEG-NH2 solution to the sulfo-SMCC bifunctional crosslinker solution is 1:1-4, and the mass ratio of the Fe3O4-PEG-NH2NPs to Mcemp1 is 1:1-3.

9. A molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that: The near-infrared fluorescent dye ICG labeling is specifically performed by dissolving ICG-NHS ester in a pH 8.5 carbonate buffer, adding Fe3O4-PEG-Mcemp1 NPs solution under light-proof conditions, and stirring the reaction at 15-35°C for more than 12 hours. The molar ratio of the ICG-NHS ester and Mcemp1-magnetic core nanoparticles is 1:4-6.

10. Use of the Mcemp1-coupled magneto-optical dual-modal composite nanoparticles according to any one of claims 1 to 9 for detecting chronic atrophic gastritis in the elderly in dual-modal imaging.

Images