A molecular probe for detecting senile chronic atrophic gastritis and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and discloses a molecular probe for detecting old chronic atrophic gastritis and application, wherein the molecular probe is a magnetic-optical dual-mode composite nanoparticle coupled with Mcemp1; a preparation method of the molecular probe comprises the following steps: Fe3O4@OA solution is prepared by taking acetylacetone iron, oleylamine and dibenzyl ether as raw materials; after being modified by SPE-PEG2000-NH2 polymer, the magnetic core Fe3O4-PEG-NH2 NPs are obtained through purification; after the magnetic core is coupled with Mcemp1 and purification, the Fe3O4-PEG-Mcemp1 NPs are obtained; then, near-infrared fluorescent dye ICG is labeled, and the magnetic-optical dual-mode composite nanoparticle coupled with Mcemp1 is obtained after dialysis. The application realizes non-invasive continuous monitoring by intravenous administration of the magnetic particle probe, combines the dual-mode technology, quantifies the density of the gland in real time, dynamically tracks the gastric mucosa, solves the problems of insufficient penetration depth of the fluorescent probe and low quantitative precision, and fills the technical gap of dynamic evaluation and cancer warning.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, more particularly, it relates to a molecular probe for detecting chronic atrophic gastritis in the elderly and application. BACKGROUND

[0002] The lesion monitoring and therapeutic effect evaluation of chronic atrophic gastritis (CAG) in the elderly face multiple technical challenges. Current clinical diagnosis and follow-up mainly rely on endoscopic biopsy and serological markers (such as pepsinogens PG I, II, and gastrin-17). However, endoscopic examination requires invasive operation and the review cycle is as long as 1-3 years, which is difficult to capture the dynamic evolution of gastric mucosa atrophy or intestinalization in real time (such as 10-day-level fluctuations in gland regeneration and destruction), and although serological indicators can reflect the functional state of the gastric mucosa, their sensitivity in differentiating between mild and severe atrophy is only 62%-78%, and they lag behind the pathological process, and cannot monitor the activity of inflammation or the activation of cancer-related molecular pathways. Elderly patients have significantly reduced tolerance to repeated endoscopic procedures due to physical function decline, comorbid cardiovascular and cerebrovascular diseases, and multiple drug use (such as non-steroidal anti-inflammatory drugs), combined with the existing problems of insufficient tissue penetration depth and low quantitative accuracy of the target probe based on fluorescence imaging, resulting in the lack of a continuous dynamic evaluation system for the atrophy degree and repair capacity of CAG mucosa.

[0003] Magnetic particle probe technology provides a new direction to solve the above problems. Compared with traditional fluorescent probes, magnetic particle probes have deep tissue penetration, signal stability, and multi-modal quantitative potential, which can break through the physical limitations of fluorescence imaging. The current clinical demand for visualization of dynamic pathological evolution (such as focal distribution of intestinal metaplasia and rapid progression of low-grade intraepithelial neoplasia) and therapeutic effect of CAG is urgent, but there is no research on the application of magnetic particle probes in this field. The existing static evaluation system based on OLGA / OLGIM staging system cannot quantitatively evaluate the real-time changes of key markers (such as gland density and gland spacing) in the "inflammation-cancer" transformation of gastric mucosa, and the conventional endoscopic review cycle is seriously mismatched with the speed of molecular events (such as 35% of low-grade intraepithelial neoplasia progressing within 6 months).

[0004] Based on the inventors' previous research results that Mcemp1 may be a promising biomarker for specific and targeted CAG imaging, and the discovery that Mcemp1 is related to gastric mucosal lesions, the present application proposes a magnetic particle probe that can be administered intravenously, supports 10-day-level continuous monitoring, and can quantitatively evaluate the therapeutic effect, which has important clinical value for realizing precise intervention for elderly CAG patients and early diagnosis of gastric cancer. SUMMARY

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

[0006] To achieve the above object, the present application provides the following technical solutions:

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

[0008] The preparation method of the magnetic-optical dual-mode composite nanoparticle coupled with Mcemp1 comprises the following steps:

[0009] Step one, synthesis and modification of magnetic core

[0010] Fe3O4@OA solution was prepared using acetylacetone iron, oleylamine and dibenzyl ether as raw materials, and then the magnetic core Fe3O4-PEG-NH2 NPs was obtained after modification by SPE-PEG2000-NH2 polymer and purification;

[0011] Step two, preparation of Mcemp1-magnetic core nanoparticle

[0012] After the magnetic core obtained in step one was coupled with Mcemp1 and purified, Mcemp1-magnetic core nanoparticle was obtained, and Fe3O4-PEG-Mcemp1 NPs solution was obtained by placing it in PBS buffer with pH = 7.4 and a concentration of 0.1 mol / L;

[0013] Step two, preparation of Mcemp1 / ICG dual-mode probe

[0014] The Mcemp1-magnetic core nanoparticle in the Fe3O4-PEG-Mcemp1 NPs solution obtained in step two was labeled with near-infrared fluorescent dye ICG, and after dialysis, the magnetic-optical dual-mode composite nanoparticle coupled with Mcemp1, i.e. Mcemp1 / ICG dual-mode probe, was obtained.

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

[0016] (1) Acetylacetone iron, oleylamine and dibenzyl ether were stirred under nitrogen flow, heated to a first preset temperature at a heating rate of 3.0-3.5 ℃ / min and kept for more than 1 h, and then heated to a second preset temperature and kept for more than 12 h, to obtain superparamagnetic Fe3O4 nanoparticles with a particle size of 20-50 nm;

[0017] (2) using a magnet to separate the superparamagnetic Fe3O4 nanoparticles obtained in step (1) and performing ultrasonic extraction 1-3 times, 1-1.5 h / time, filtering, combining the filtrates, concentrating by rotary evaporation, and then freezing and drying after being refrigerated for 12 h or more, and then dispersing into a chloroform solution to obtain a Fe3O4@OA solution; the volume-to-mass ratio of the ethanol to the superparamagnetic Fe3O4 nanoparticles is 8-12 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-to-volume ratio of the acetylacetone iron, oleylamine, and dibenzyl ether is 0.7 g:1.7 ml:20 ml.

[0020] Preferably, after the modification of the DSPE-PEG2000-NH2 polymer, the following steps are performed: an equal volume of a citric acid solution is added to the Fe3O4@OA solution, which is concentrated by rotary evaporation to a Fe content of 0.8-1.2 mg / mL, a dilute ammonia solution with a volume fraction of 1-10% is used to adjust the pH to 10-11, and then the DSPE-PEG2000-NH2 polymer is added to make the mass concentration of the DSPE-PEG2000-NH2 in the solution consistent with the Fe content, and the solution is stirred in a water bath at 70-90°C for 24 h or more.

[0021] Preferably, the purification in steps one and two is high-speed centrifugal separation using a 25-40 kD ultrafiltration tube at a speed of 5000-6000 rpm for 15-25 min, and the dialysis in step three is dialysis in deionized water for 48 h or more using a dialysis bag with a molecular weight cut-off of 3-4 kDa.

[0022] Preferably, the coupling specifically includes the following steps: the magnetic core Fe3O4-PEG-NH2 NPs obtained in step one are added to a buffer solution to adjust the pH to 7.0-8 to obtain a Fe3O4@PEG-NH2 solution, a sulfo-SMCC bifunctional crosslinking agent solution is added and reacted for 30 min or more, and then a Mcemp1 solution is added, and the solution is shaken in the dark at 4-8°C for 12 h or more.

[0023] The mass concentration of the Fe3O4@PEG-NH2 solution and the Mcemp1 solution is 0.8-1.2 mg / mL, the molar concentration of the sulfide-SMCC bifunctional crosslinking agent in the sulfide-SMCC bifunctional crosslinking agent solution is 8-12 mM, the volume ratio of the Fe3O4@PEG-NH2 solution to the sulfide-SMCC bifunctional crosslinking agent solution is 1:1-4, and the mass ratio of the Fe3O4-PEG-NH2 NPs to the Mcemp1 is 1:1-3.

[0024] Preferably, the near-infrared fluorescent dye ICG labeling is specifically dissolving ICG-NHS ester in a pH 8.5 carbonate buffer, adding a Fe3O4-PEG-Mcemp1 NP solution under light shielding conditions, stirring and reacting at 15-35 DEG C for 12 h or more, and the molar ratio of the ICG-NHS ester to the Mcemp1-magnetic core nanoparticle is 1:4-6.

[0025] The application also provides a use of the above-mentioned conjugated Mcemp1 magnetic-optical dual-mode composite nanoparticle in dual-mode imaging for detecting elderly chronic atrophic gastritis.

[0026] The probe realizes real-time, dynamic and quantitative evaluation of the lesion degree and treatment effect of chronic atrophic gastritis through the dual-mode synergistic mechanism of magnetic particle imaging (MPI) and near-infrared fluorescence imaging (IVIS). The MPI technology is based on the magnetic properties of superparamagnetic Fe3O4 nanoparticles. Under an external alternating magnetic field, the harmonic signal strength generated by the nuclear magnetic moment flip is linearly related to the local probe concentration, and the atrophic degree of the gastric mucosa gland can be directly quantified. Pathological studies have shown that the decrease in gland density (such as severe atrophy corresponding to a decrease of >2 / 3 of the glands) leads to a decrease in probe retention density, and the MPI signal strength is significantly negatively correlated with the gland density, which can be used to classify the atrophy grade.

[0027] Meanwhile, the ICG near-infrared fluorescent dye (emission wavelength 750-810 nm, penetration depth 5-10 mm) carried by the probe can be used to analyze the inflammation 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 in the fluorescence hotspot range (ROI) and intensity can directly reflect the mucosal repair process. This technology combines magnetic quantification and optical dynamic tracking, breaks through the limitations of traditional endoscopy for static evaluation, and provides a multi-dimensional tool for long-term efficacy management and precise treatment navigation for elderly patients.

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

[0029] 1. The application breaks through the defects of invasiveness and hysteresis of traditional monitoring. The prior art relies on invasive endoscopic biopsy (review cycle 1-3 years) and serum markers (PG I / PG II), which cannot capture the dynamic evolution of lesions (such as 10-day fluctuations in gland regeneration and destruction). The present application realizes non-invasive continuous monitoring for more than 10 days by intravenous administration of magnetic particle probes, and combines MPI (magnetic particle imaging) and IVIS (near-infrared fluorescence) dual-mode technology to quantitatively measure gland density and dynamically track gastric mucosa.

[0030] 2. The application solves the problems of insufficient penetration depth and low quantitative accuracy of the fluorescent probe. The tissue penetration depth of the existing fluorescent probe (such as NIR-I organic fluorescent group imaging technology) is 1-3 mm, and the mucosa repair capacity cannot be evaluated. The present application adopts a superparamagnetic Fe3O4 core + ICG near-infrared fluorescence dual-mode 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 micro lesions (≤2 mm) and quantify the degree of gland atrophy.

[0031] 3. Fill the technical gap of dynamic evaluation and cancer warning. The existing technology cannot track the key events of "inflammation-cancer" transformation in real time. The present application uses magnetic-optical signal correlation molecular markers to warn about the gastric mucosa in advance, reducing the window period of traditional pathological diagnosis. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0035] Figure 4 Hydrated particle size detection of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe;

[0036] Figure 5 Correspondence between Mcemp1-Fe3O4-ICG probe concentration and fluorescence signal value;

[0037] Figure 6 Correspondence between Mcemp1-Fe3O4-ICG probe concentration and MPI signal value;

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

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

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

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

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

[0043] Figure 12 Results of phagocytosis of IgG-Fe3O4-ICG probe and Mcemp1-Fe3O4-ICG probe by mast cells. DETAILED DESCRIPTION

[0044] In order to enable the personnel in the technical field to better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by the personnel in the art without creative labor should belong to the protection scope of the present application.

[0045] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below in combination with the embodiments.

[0046] Embodiment 1

[0047] A molecular probe for detecting old chronic atrophic gastritis, the molecular probe being a Mcemp1 magnetic-optical dual-mode composite nanoparticle, and a preparation method thereof comprises the following steps:

[0048] Step one, synthesis and modification of magnetic core (Fe3O4-PEG-NH2 NPs)

[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 flow of nitrogen (N2) and heated to 220°C (nucleation temperature) at a heating rate of 3.3°C / min for 1 h.

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

[0051] 3) After the reaction was completed, the product was separated using a magnet and extracted 3 times with 10 times the amount of ethanol under ultrasonic conditions for 1 h each time, filtered, the filtrate was combined, concentrated by rotary evaporation, placed at -20°C for 12 h and then freeze-dried for 48 h to remove unreacted OAm and dibenzyl ether, and 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, concentrated by rotary evaporation to a Fe content of 1 mg / mL, and the pH was adjusted to 11 with a dilute ammonia solution (a sodium tripolyphosphate solution or an ethylenediaminetetraacetic acid disodium salt solution can also be used) with a volume fraction of 1%. DSPE-PEG2000-NH2 polymer was added to the citric acid-coated iron oxide to give a final concentration of 1 mg / ml of DSPE-PEG2000-NH2 polymer, and the reaction was stirred at 80°C for 24 h to form hydrophilic amino-modified magnetic cores (Fe3O4-PEG-NH2 NPs) by covalent modification through amide bonds.

[0053] 5) The reaction product was separated using a 30 kD ultrafiltration tube in a high-speed centrifuge at a speed of 6000 rpm for 20 min to purify the reaction product and remove residual citric acid, ammonia solution and DSPE-PEG2000-NH2 polymer to obtain Fe3O4@PEG-NH2 (Fe3O4-PEG-NH2 NPs), which was verified by dynamic light scattering (DLS) to have a particle size of 31.715 ± 3.309 nm and a Zeta potential of -0.22 mV.

[0054] Step two, preparation of Fe3O4-PEG-Mcemp1 NPs

[0055] 1) Thiolated Mcemp1 peptide (thiol introduced by Traut's reagent) was dissolved in 100 μΐ^DMSO per 1 mg Mcemp1 and added to 900 μΐ^PBS buffer with pH = 7.4 and a concentration of 0.1 mol / L to obtain a Mcemp1 solution with a mass concentration of 1 mg / ml.

[0056] 2) The Fe3O4@PEG-NH2 prepared in step one was added to PBS buffer to obtain a Fe3O4@PEG-NH2 solution, and the pH was adjusted to 7.4 using a dilute ammonia solution with a volume fraction of 1% to obtain a final concentration of Fe3O4@PEG-NH2 solution of 1 mg / mL. A 10 mM sulfide-SMCC bifunctional crosslinking agent with a purity of >95% was added in a volume ratio of 1:1~4 of Fe3O4@PEG-NH2 solution to sulfide-SMCC bifunctional crosslinking agent (solution) to react the maleimide group of sulfide-SMCC with the amino group on the surface of the magnetic core for 30 min to form an activated intermediate.

[0057] 3) The Mcemp1 solution was then added (volume ratio of Fe3O4@PEG-NH2 solution to Mcemp1 solution of 1:1~3), and the mixture was shaken in the dark at 4 °C for 12 h to covalently link the targeting peptide to the surface of the magnetic core via thiol-maleimide click chemistry. After the reaction was complete, a 30 kDa ultrafiltration tube was used to purify the mixture by centrifugation at 5000 rpm to remove unbound peptides to obtain Fe3O4-PEG-Mcemp1 NPs (Mcemp1-magnetic core nanoparticles); the Fe3O4-PEG-Mcemp1 NPs were placed in PBS buffer with pH = 7.4 and a concentration of 0.1 mol / L to obtain a Fe3O4-PEG-Mcemp1 NPs solution for later use.

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

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

[0060] 2) After the reaction, remove the free dye by dialysis in deionized water for 48 h through a dialysis bag with a molecular weight cutoff of 3.5 kDa, and finally obtain the Mcemp1 magnetic-optical dual-mode composite nanoparticles, i.e., the Mcemp1 / ICG dual-mode probe (Fe3O4-PEG-Mcemp1-ICG NPs, also referred to as the Mcemp1-Fe3O4-ICG probe).

[0061] The Mcemp1-Fe3O4-ICG probe and the IgG-Fe3O4-ICG probe (the preparation steps are the same as those of the Mcemp1-Fe3O4-ICG, except that the Mcemp1 is replaced with 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 Figures 1-6 .

[0062] Test Example:

[0063] I. Experimental Methods

[0064] 1. Animal Preparation

[0065] 60 SPF level, 6-8 weeks old, Balb / c male mice, and 20 SPF level, 6-8 weeks old, Balb / c-nu athymic male nude mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0066] 2. Cell Preparation

[0067] NCI-N87 (N87) human gastric cancer cells (product code: CL-0169) were purchased from Wuhan Ponsure Biotechnology Co., Ltd. They were cultured in RPMI-1640 + 10% FBS + 1% P / S complete medium. When the culture reached 4-5 passages, they were injected subcutaneously into the right armpit of mice at a density of 1×10 7 cells / 1mL, and 0.1 mL was injected subcutaneously into each nude mouse to form a tumor.

[0068] 3. Modeling and Grouping

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

[0070] For the CSG group, MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) was used to induce gastric mucosal lesions. At the beginning of the experiment, MNNG was dissolved in drinking water to prepare a mouse drinking solution with a concentration of 80-200 μg / mL, and the mice were allowed to drink freely. The modeling time for the CSG group was 3 months, and during this period, the MNNG solution was replaced once a week to ensure the stability of the solution concentration.

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

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

[0073] For the gastric grafted tumor inoculation group (grafted tumor group), the present test example used a human gastric cancer cell line for xenotransplantation modeling. First, N87 cells were cultured to the logarithmic growth phase, and the cells were collected and prepared into a cell suspension with a concentration of 1 x 10 7 cells / 1 mL. Then, the cell suspension was inoculated into the subcutaneous tissue of the right armpit of the mouse by subcutaneous injection. After inoculation, the health status of the mouse was observed regularly, the growth of the tumor was measured regularly, and the volume and progression of the tumor were evaluated.

[0074] 3. Imaging experiment

[0075] (1) MPI imaging

[0076] Each model group mouse (blank group (CON), CSG group, CAG group, grafted tumor group) was injected with 100 μL of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe at a concentration of 1 mg / mL by tail vein injection, and was scanned and imaged 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 gastric 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 group 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] The mice were subjected to in vivo fluorescence imaging using the IVIS Spectrum imaging system (PerkinElmer, USA). The mice in each model group (blank group, CSG group, CAG group, and gastric tumor graft group) were injected with 100 μL of Mcemp1-Fe3O4-ICG probe and IgG-Fe3O4-ICG probe at a concentration of 1 mg / mL via the tail vein, and fluorescence imaging was performed at different time points (0 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, and 240 h) after injection. Before imaging, the mice were anesthetized with isoflurane to ensure that the mice remained in a stable state during imaging, and were placed on the imaging platform in a supine position to ensure that the imaging area (abdomen) was fully exposed. During imaging, the excitation wavelength was set to 740 nm, the emission wavelength was 790 nm, the exposure time was 3-5 seconds, the Binning was set to Medium, and the f / stop was 2. After the imaging data was collected by the IVIS Spectrum imaging system, image analysis was performed using Living Image software (PerkinElmer). This software can generate a heat map of the region of interest (ROI) and quantitatively analyze the fluorescence signal intensity of different regions of the mouse abdomen at different time points. Changes in fluorescence signal intensity reflect the distribution and clearance of nanoparticles. The data were further analyzed by GraphPad Prism software to compare the signal intensity differences between groups and ensure the statistical significance of the results. After 240 hours (10 days), the last in vivo imaging was performed. After imaging was completed, the mice were anesthetized and euthanized, and the stomach, pancreas, liver, heart, spleen, lungs, small intestine, and tumor graft (if applicable) were removed for ex vivo imaging. The same imaging conditions (740 nm excitation, 790 nm emission) were used to image each organ individually, and the corresponding fluorescence signal intensity was recorded. Through this combination of in vivo and ex vivo imaging, the targeting ability, drug release, and distribution characteristics of Mcemp1 and IgG magnetic-optical composite nanoparticles in various tissues can be comprehensively evaluated.

[0079] 4. H-E staining of gastric tissue

[0080] After 48 h of injection of the Mcemp1-Fe3O4-ICG probe, the mice in each group were fasted for 12 h without water restriction. Before the mice recovered from anesthesia, the eyeballs were removed to take blood, and the mice were bled to death. The mouse abdominal cavity was opened, the intestines and pancreas were removed, and the stomach was exposed. The stomach was cut from the upper and lower stomach ports, respectively, and the stomach tissue was cut along the lesser curvature. The stomach contents were washed with 5 mL of pre-cooled normal saline. After washing, the sample was fixed in 4% paraformaldehyde fixing solution for later pathological staining. After the fixed stomach tissue was routinely dehydrated, it was paraffin-embedded. 5-μm-thick tissue sections were continuously cut at the gastric antrum, and the sections were placed in xylene I, xylene II, anhydrous ethanol, 90% alcohol, 80% alcohol, 70% alcohol, and then washed with water. After staining with hematoxylin staining solution, washing, differentiation, washing, counterstaining, and washing, the sections were dehydrated with 70%, 80%, 95%, and anhydrous ethanol, respectively, and then stained with eosin staining solution. The sections were dehydrated with anhydrous ethanol, transparentized with xylene, and sealed with neutral resin. The morphology of the mucosal epithelium, gastric glands, chief cells, parietal cells, and mucosal muscle layer was observed under a microscope, and images were collected.

[0081] 5. In vitro cell phagocytosis

[0082] The HMC-1 human mast cells in the logarithmic growth phase were digested, counted, and then inoculated into a 6-well plate at a density of 1×10 5 cells / well, and cultured in complete medium containing 10% fetal bovine serum overnight. At the same time, the cell adhesion agent was diluted with PBS at a ratio of 1:200, and then spread on the surface of a glass culture dish at a dose of 1 mL / well, and incubated at 37 °C for 30 min with occasional shaking. The liquid in the dish was completely discarded, and the dish was washed once with sterile PBS buffer and then directly seeded with HMC-1 human mast cells for adhesion. The Mcemp1-Fe3O4-ICG probe and the IgG-Fe3O4-ICG probe previously dissolved in serum-free medium were added to the respective dishes, and the cells were completely covered. The culture plates were placed in a 37 °C, 5% CO2 cell incubator and incubated in the dark for 1, 2, 4, 8, 12, and 24 h. After incubation, the medium containing the probe was immediately aspirated, and the cells were washed with pre-cooled sterile PBS for 3-5 times to completely remove the free probe that was not internalized and attached to the cell surface. After each washing, the liquid was gently aspirated. Then, Hoechst 33342 live cell staining solution was added at a ratio of 1:100, and the cells were stained for 10 min. 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 cells.

[0083] II. 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 Figures 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 CAG mouse model of Mcemp1-Fe3O4 uptake compared with IgG-Fe3O4-ICG uptake, there is a significant difference.

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

[0087]

[0088] Note: * represents the signal intensity of Mcemp1-Fe3O4-ICG probe compared with the signal intensity of IgG-Fe3O4-ICG probe, P<0.01.

[0089] 2, H-E staining of gastric tissue

[0090] 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 gland arranged in order, no inflammatory cell infiltration. Atrophic gastritis (CAG) group of mice, the gastric antral gland arrangement is uneven, the nucleus and cytoplasm is abnormal, the mucosal epithelium disappears, the number of chief cells and parietal cells is significantly reduced, and the number of intrinsic glands is reduced by more than 2 / 3. Chronic gastritis (CSG) group of mice, the gastric antral gland is relatively uniform, but the mucosal epithelium is thin. Gastric cancer transplanted tumor (GC) group of mice showed obvious gastric cancer cell infiltration.

[0091] 3, in vitro cell phagocytosis

[0092] The results of mast cell phagocytosis of two kinds of probes are shown in Figure 12 It can be seen from Figure 12 that the mast cell phagocytosis of IgG-Fe3O4-ICG probe is faster, mainly concentrated in the 4-8 hour period, but the persistence is poor. However, the phagocytosis of Mcemp1-Fe3O4-ICG probe can be identified 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.

[0093] The embodiments are only used for explaining the present application, and are not used for limiting the present application, and the person skilled in the art can make the modification of the embodiments without the creative contribution according to the need after reading the description, and as long as the modification is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A molecular probe for detecting chronic atrophic gastritis in the elderly, characterized in that, The molecular probe is a coupled Mcemp1 magneto-optical dual-mode composite nanoparticle. The preparation method of the coupled Mcemp1 magneto-optical dual-mode composite nanoparticles includes the following steps: Step 1: Synthesis and Modification of Magnetic Nuclei Fe3O4@OA solution was prepared using acetylacetone iron, oleylamine and dibenzyl ether as raw materials, and then purified by SPE-PEG2000-NH2 polymer to obtain magnetic core Fe3O4-PEG-NH2 NPs; Step 2: Preparation of Mcemp1-magnetic core nanoparticles The magnetic nuclei obtained in step one were added to a buffer solution and the pH was adjusted to 7.0-8 to obtain a 0.8-1.2 mg / mL Fe3O4@PEG-NH2 solution. An 8-12 mM sulfonyl-SMCC bifunctional cross-linking agent solution was added at a volume ratio of 1:1-4 (Fe3O4@PEG-NH2 solution to sulfonyl-SMCC bifunctional cross-linking agent solution) and reacted for at least 30 minutes. Then, a 0.8-1.2 mg / mL Mcemp1 solution was added at a mass ratio of 1:1-3 (Fe3O4-PEG-NH2NPs to Mcemp1). The mixture was shaken at 4-8°C in the dark for at least 12 hours. After coupling and purification, Mcemp1-magnetic nucleus nanoparticles were obtained and placed in a 0.1 mol / L PBS buffer solution at pH 7.4 to obtain a Fe3O4-PEG-Mcemp1 NPs solution. Step 3: Fabrication of the Mcemp1 / ICG dual-modal probe The Mcemp1-magnetic nanoparticles in the Fe3O4-PEG-Mcemp1 NPs solution obtained in step two were labeled with the near-infrared fluorescent dye ICG. Specifically, ICG-NHS ester was dissolved in pH 8.5 carbonate buffer, and Fe3O4-PEG-Mcemp1 NPs solution was added under light-protected conditions. The mixture was stirred at 15~35℃ for more than 12 h. The molar ratio of ICG-NHS ester to Mcemp1-magnetic nanoparticles was 1:4~6. After dialysis, coupled Mcemp1 magneto-optical dual-mode composite nanoparticles were obtained, namely Mcemp1 / ICG dual-mode probes.

2. The 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 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 1~10% (v / v) dilute ammonia solution; adding DSPE-PEG2000-NH2 polymer to make the mass concentration of DSPE-PEG2000-NH2 in the solution consistent with the iron content; and reacting in a water bath at 70~90℃ for more than 24 h with stirring.

3. The molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that, In steps one and two, the purification is performed by high-speed centrifugation using a 25-40 kD ultrafiltration tube at a speed of 5000-6000 rpm for 15-25 min.

4. The molecular probe for detecting chronic atrophic gastritis in the elderly according to claim 1, characterized in that, In step three, the dialysis is performed by dialysis in deionized water for more than 48 hours using a dialysis bag with a molecular weight cutoff of 3-4 kDa.

5. The application of the coupled Mcemp1 magneto-optical dual-modal composite nanoparticles as described in any one of claims 1-4 in the preparation of a reagent for detecting chronic atrophic gastritis in the elderly in dual-modal imaging.

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