Near-infrared immunofluorescent conjugate and use thereof

Abstract

Provided are a near-infrared immunofluorescent conjugate and use thereof. It has been found that a near-infrared fluorescent dye can be conjugated with an antibody to form a conjugate, which can be loaded into a modified chitosan carrier, thereby preparing a near-infrared immunofluorescent probe with an imaging function that can be administered orally. The conjugate of the near-infrared fluorescent dye and the antibody has emission wavelength coverage in a range of 700-1700 nm in a near-infrared region, and has relatively low light scattering, relatively high in-vivo imaging resolution, and relatively deep penetration depth. In addition, the conjugate of the near-infrared fluorescent dye and the antibody has the advantages of low cost, biological safety, suitability for drug formation, and the like. The conjugate of the modified chitosan-loaded near-infrared fluorescent dye and the antibody based on formula I can be administered by means of oral delivery, intravenous injection, and the like, and has good compliance.

Description

A near-infrared immunofluorescence conjugate and its application Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a near-infrared immunofluorescence conjugate and its applications. Background Technology

[0002] Due to the complexity of the gastrointestinal environment, many drugs face problems such as low bioavailability and poor stability when administered orally. Developing effective oral delivery systems is of great significance for improving drug efficacy. Oral delivery systems are the most commonly used in vivo delivery route for small molecule formulations. Compared with injection, they have advantages such as convenience, high patient compliance, and low cost (Zhu, W, et al., Oral Delivery of Therapeutic Antibodies with a Transmucosal Polymeric Carrier[J].ACS Nano 2023,17(5):4373-4386.).

[0003] Oral delivery of peptide drugs has become an important research direction in the pharmaceutical field in recent years. However, peptide drugs face multiple challenges in oral delivery, including the presence of proteins with large molecular weights (such as antibodies) (Li, G, et al., Fluorinated Chitosan To Enhance Transmucosal Delivery of Sonosensitizer-Conjugated Catalase for Sonodynamic Bladder Cancer Treatment Post-intravesical Instillation[J]. ACS Nano 2020,14(2):1586-1599.), the biochemical environment of the gastrointestinal tract, the mucus barrier, epithelial penetration, and hepatic elimination (Huang Kan, Sun Minjie. Research progress in oral delivery technology of peptide drugs[J]. Chinese Journal of New Drugs, 2022.). These factors lead to low oral bioavailability of peptide drugs, which usually can only be administered by injection (Li Qi, Chen Hongda, Zhou Tianhua, et al. Research progress in oral delivery of peptide drugs[J]. Acta Pharmaceutica Sinica, 2022.). To overcome these obstacles, researchers have developed a variety of strategies and technologies. Such as chemical modification, carrier systems, bioadhesion systems, structural modification, and nanotechnology.

[0004] Nanocarrier technology is widely used to improve the stability and bioavailability of peptide drugs. For example, materials such as nanoparticles, lipid nanocarriers, and mesoporous silica can effectively protect peptide drugs from gastrointestinal degradation and promote their absorption in vivo (Hu Qingyuan, Lu Xiaohong, Gao Dongxu, et al. Research progress on the use of mesoporous silica for oral delivery of peptide and protein drugs with video [J]. China Pharmaceutical Industry Journal, 2024.). These nanocarriers not only improve the solubility and stability of drugs, but also enhance their ability to penetrate mucosa through surface modification (Hao Xiaoli, Ji Rimutu, He Jing. Research progress on oral delivery of bioactive peptides using nanocarriers [J]. Food Science, 2020.).

[0005] Nanotechnology plays a crucial role in oral drug delivery. For example, polymer nanocarriers can enhance the solubility and permeability of hydrophobic chemotherapeutic drugs, overcome the gastrointestinal absorption barrier, and reduce the first-pass effect of the metabolic system, thereby improving drug bioavailability (Le Zhicheng, Liu Zhijia, Chen Yongming. Research progress on oral delivery of chemotherapeutic drugs mediated by polymer nanocarriers [J]. Polymer Materials Science and Engineering, 2019.). Furthermore, studies on cell models simulating small intestinal absorption and endocytosis mechanisms of nanodrug delivery systems can help construct more effective oral nanodrug delivery systems (Dong Andi, Ji Fengqi, Zhang Chunpeng, et al. Development of in vitro absorption models and analysis of endocytosis mechanisms for oral nanodrug delivery systems [J]. Journal of Shenyang Pharmaceutical University, 2022.).

[0006] Endoscopy is the gold standard for examining gastrointestinal diseases. The gastrointestinal organs have a very large internal mucosal surface area and a variety of complex physiological microenvironments, and are involved in a wide range of tumor types. Traditional endoscopic examinations include gastroscopy and colonoscopy, which allow direct observation of the digestive tract and the performance of procedures such as biopsies (Zhang Yu, Chen Xiaochang, Zhong Min. Discussion on Digestive Endoscopy and Gastrointestinal Diseases with Video [J]. Sino-Foreign Medical Treatment, 2010.). However, traditional endoscopy has certain limitations, such as potential patient discomfort and the risk of cross-infection (Wang Weiwei. Research on Extracorporeal Magnetic Driven Diagnostic Capsule System [D]. Shanghai Jiaotong University, 2013.). While traditional high-resolution white light endoscopy (WLE) is the most widely used clinical diagnostic method, its false negative rate is relatively high. In high-risk patients with gastrointestinal cancer, especially those with inflammatory conditions, the false negative rate is three times that of healthy individuals. To overcome these shortcomings, wireless capsule endoscopy (WCE) technology has emerged.

[0007] WCE is a non-invasive endoscope where the patient simply swallows a capsule containing a miniature camera. As the capsule moves through the digestive tract, it captures images and transmits them wirelessly to an external receiver (Liu Jianqing. Experimental Study on the Design of Human Wireless Capsule Endoscope Based on OV6920 [D]. South China University of Technology, 2010.). This technology has the advantages of being non-invasive, painless, requiring no anesthesia, and requiring no special preparation, making it particularly suitable for the small intestine, which is difficult to examine with traditional endoscopes (Zhang Sijie. Research on Micro-MEMS-Based Wireless Endoscope for the Digestive Tract [D]. Chongqing University, 2005.). The development of miniature gastrointestinal endoscopes has also provided new possibilities for the diagnosis of gastrointestinal diseases. Due to their smaller size, miniature endoscopes can more easily reach various parts of the digestive tract, including the hard-to-reach small intestine (Xue Chunxiang. Development and Prospect of Miniature Gastrointestinal Endoscopes [J]. Continuing Medical Education, 2022.). These miniature endoscopes can be used not only for diagnosis but also for some therapeutic procedures, such as polyp removal (Department of Gastroenterology, Second People's Hospital of Shaoyang City. Clinical analysis of 80 cases of gastrointestinal diseases treated by digestive endoscopy [J]. Sino-Foreign Medical Treatment, 2014.). Although wireless capsule endoscopy and miniature endoscopy offer many advantages, they also have some limitations. For example, wireless capsule endoscopy cannot perform biopsies or therapeutic procedures, while the operation of miniature endoscopes may require higher technical skills and equipment support (Wang Weiwei. Research on extracorporeal magnetically driven diagnostic and therapeutic capsule system [D]. Shanghai Jiaotong University, 2013.). In addition, the image quality of wireless capsule endoscopy may be affected by the capsule's movement speed and the digestive tract environment (Fu Yan'an. Research on image processing technology of wireless capsule endoscopy [D]. Shandong University, 2013.). Therefore, developing a detection method that can specifically identify small tumor lesions in the complex gastrointestinal mucosal system and has good patient compliance is of great clinical significance.

[0008] Immunofluorescent probes for early screening of gastrointestinal tumors are a technique that uses specific fluorescent markers to identify and locate gastrointestinal tumor cells. Probes used for intraoperative tumor lesion margin labeling are often designed based on the binding of monoclonal antibodies and small molecules, resulting in significant influences on their half-life, immunogenicity, and tumor uptake by the monoclonal antibody itself. Fully human monoclonal antibodies are generally in IgG form, with a relatively large molecular weight (150 kDa), resulting in weak tissue or tumor penetration and difficulty in targeting sterically hindered epitopes. Furthermore, the target content in early-stage tumor lesions is low, and the cellular microenvironment differs significantly from that of solid tumors. For these reasons, to date, only one probe for early screening of gastrointestinal tumors has entered Phase I clinical trials: a c-MET targeting peptide bound to an NIR dye, used for fluorescent endoscopic detection of human adenomas. However, due to the inherent expression of c-MET in normal mucosa, the tumor-to-background ratio (TBR) is relatively low (maximum TBR = 1.51). On the other hand, the in vivo delivery of these tumor-targeting tracer probes all rely on intravenous injection, requiring them to enter the bloodstream, which poses significant safety risks and leads to poor patient compliance, failing to meet the clinical application requirements for early physical examination and diagnosis. Therefore, the main challenge in designing fluorescent immunoassay probes for early gastrointestinal tumor screening lies in their ability to deliver high-efficiency, high-dose delivery to early, small lesions within the gastrointestinal environment, thereby achieving high TBR values ​​in the mucosal tissue to identify target lesions.

[0009] Various fluorescent probes have been developed for the early diagnosis of gastric cancer. For example, water-soluble quantum dot fluorescent probes have been used to detect the gastric cancer cell-associated antigen CA242, showing higher photostability and sensitivity than traditional methods (Fu Zhiying, Li Zhaohui, He Xiaoxiao, et al. Detection of gastric cancer cell-associated antigen CA242 using water-soluble quantum dot fluorescent probes [J]. Analytical Chemistry, 2006.). Furthermore, fluorescent nanoprobes have also been used for targeted imaging and treatment of gastric cancer. These probes can specifically identify tumors through specific molecular markers such as BRCA1 and HER2 (Ruan Jing. Research on the preparation of fluorescent nanoprobes and induced pluripotent stem cells and their application in targeted imaging and treatment of gastric cancer [D]. Shanghai Jiao Tong University, 2012.). Studies on NMR-fluorescence multifunctional molecular probes have shown that these probes can not only provide tumor imaging information at the molecular level, but also directly participate in the treatment process through chemotherapy or photodynamic therapy (Zhou Jia. Construction of NMR-fluorescence multifunctional molecular probes and their application in the diagnosis and treatment of gastric cancer [D]. Shanghai Jiao Tong University, 2018.). In addition, methods based on fluorescence hyperspectral imaging and machine learning have been proposed for the early diagnosis of gastric cancer. These methods improve the accuracy and efficiency of diagnosis by analyzing fluorescence spectra and spatial information (Li Yuanpeng. Research on early diagnosis methods of gastric cancer based on fluorescence hyperspectral imaging and machine learning [D]. Jinan University, 2019.). Summary of the Invention

[0010] This invention discovers that near-infrared fluorescent dyes can couple with antibodies to form conjugates, which can then be loaded into modified chitosan carriers to prepare orally administered near-infrared immunofluorescent probes with imaging capabilities. Based on this, this invention was completed.

[0011] In a first aspect, the present invention provides a near-infrared immunofluorescence conjugate ABC, wherein A is a near-infrared fluorescent dye, B is an anti-CEA antibody with an amino acid sequence as shown in SEQ ID NO:1, and C is a chitosan carrier; the chitosan carrier is a covalent conjugate based on chitosan and a fluorine-containing group; the chemical structural formula of the chitosan carrier is shown in Formula I.

[0012] R1 is independently selected from hydrogen atoms, alkyl groups, aromatic hydrocarbon groups, acyl forms of polysaccharide compounds (such as the acyl form of hyaluronic acid), phenyl, trifluoromethyl, trifluorobenzyl-modified phenyl or fluorinated phenyl, etc.; n represents the number of polymers of chitosan. The molecular weight of the modified chitosan is 3 kDa, 10 kDa, 100 kDa or 1000 kDa, etc.

[0013] Furthermore, the particle size of the chitosan carrier is 10-1000 nm.

[0014] Furthermore, the aromatic hydrocarbon group is preferably hyaluronic acid.

[0015] Furthermore, the trifluoromethyl group is preferably trifluoroacetic acid.

[0016] Furthermore, the trifluorobenzyl group is preferably trifluorobenzoic acid.

[0017] Furthermore, the chitosan carrier is obtained through an amide coupling reaction, as shown in Formula II or Formula III:

[0018] Furthermore, the bases used for amide coupling in Formula II include, but are not limited to, triethylamine (TEA), N-methylmorpholine (NMM) and N,N-diisopropylethylamine (DIPEA, Hunig's base), pyridine, DBU, 2,6-dimethylpyridine, imidazole or N-methylimidazolium (NMI).

[0019] Furthermore, the condensing agents used for amide coupling in Formula III include, but are not limited to, N,N'-carbonyldiimidazole (CDI), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), N,N'-dicyclohexylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), phosphorus pentachloride, acetic anhydride (Ac2O), cyanuric chloride (TCT), boric acid (B(OH)3), N-benzyl-9-(tetrahydro-2H-pyran-2-yl)adenine (PBA) or n-(2-[4-nitro-2-(trifluoromethyl)phenyl]amino}ethyl)benzamide (3-NPBA).

[0020] Furthermore, the reaction time of the amide is 1-24 hours.

[0021] Furthermore, the reaction temperature for the amide reaction is 0-60℃.

[0022] Furthermore, the purification method for the amide reaction is selected from column chromatography, dialysis, or ultrafiltration centrifugation.

[0023] Furthermore, the absorption and emission spectra of the near-infrared fluorescent dye are in the range of 600-1000 nm.

[0024] Furthermore, the near-infrared fluorescent dye is an asymmetric near-infrared fluorescent dye.

[0025] Furthermore, the asymmetric near-infrared fluorescent dye is selected from one or more of cyanine dyes, rhodamine dyes, and / or BODIPY dyes.

[0026] In a second aspect, the present invention provides a method for preparing the near-infrared immunofluorescence conjugate as described in the first aspect, the method comprising the following steps:

[0027] M1. To prepare an immunofluorescent agent, an anti-CEA antibody, a reducing agent, and a near-infrared fluorescent dye dissolved in an organic solvent are mixed to obtain a conjugate of the near-infrared fluorescent dye and the antibody.

[0028] M2. Dissolve the modified chitosan and near-infrared immunofluorescence reagent as shown in Formula I at different molar ratios;

[0029] M3. Shake the solution obtained in step M1 to concentrate it to the required volume.

[0030] Furthermore, the reducing agent in step M1 includes, but is not limited to, one or more of tri(2-chloroethyl) phosphate, diethyltriaminepentaacetic acid and / or 5,5'-dithiobis(2-nitrobenzoic acid); preferably tri(2-chloroethyl) phosphate and DTPA.

[0031] Furthermore, the reaction temperature in step M1 is 4–60°C, preferably 30°C.

[0032] Furthermore, the organic solvent in step M1 includes, but is not limited to, one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and / or acetonitrile.

[0033] Furthermore, in step M2, the molar ratio of modified chitosan to near-infrared immunofluorescence agent is selected from 0.5:1 to 20:1.

[0034] Furthermore, the oscillation reaction in step M3 includes, but is not limited to, being carried out by one or more of a cell disruptor, a high-pressure homogenizer, and / or an ultrasonic device.

[0035] Furthermore, the oscillation reaction temperature in step M3 is selected from 4-40℃.

[0036] Furthermore, the oscillation reaction time in step M3 is selected from 5-60 min.

[0037] Thirdly, the present invention provides an imaging agent comprising the near-infrared immunofluorescence conjugate ABC described in the first aspect, wherein A is a near-infrared fluorescent dye, B is an anti-CEA antibody with an amino acid sequence as shown in SEQ ID NO:1, and C is a chitosan carrier; the chitosan carrier is a covalently coupled compound based on chitosan and a fluorine-containing group; the chemical structural formula of the chitosan carrier is shown in Formula I.

[0038] R1 is independently selected from hydrogen atoms, alkyl groups, aromatic hydrocarbon groups, acyl forms of polysaccharide compounds (such as the acyl form of hyaluronic acid), phenyl, trifluoromethyl, trifluorobenzyl-modified phenyl or fluorinated phenyl, etc.; n represents the number of polymers of chitosan. The molecular weight of the modified chitosan is 3 kDa, 10 kDa, 100 kDa or 1000 kDa, etc.

[0039] Fourthly, the present invention provides the application of the near-infrared immunofluorescence conjugate ABC as described in the first aspect in the preparation of imaging agents; wherein, A is a near-infrared fluorescent dye, B is an anti-CEA antibody with an amino acid sequence as shown in SEQ ID NO:1; C is a chitosan carrier; the chitosan carrier is a covalent conjugate based on chitosan and a fluorine-containing group; the chemical structural formula of the chitosan carrier is shown in Formula I:

[0040] R1 is independently selected from hydrogen atoms, alkyl groups, aromatic hydrocarbon groups, acyl forms of polysaccharide compounds (such as the acyl form of hyaluronic acid), phenyl, trifluoromethyl, trifluorobenzyl-modified phenyl or fluorinated phenyl, etc.; n represents the number of polymers of chitosan. The molecular weight of the modified chitosan is 3 kDa, 10 kDa, 100 kDa or 1000 kDa, etc.

[0041] Furthermore, the imaging is in vivo imaging and / or ex vivo imaging.

[0042] Furthermore, the imaging agent can be delivered orally or via parenteral delivery.

[0043] Furthermore, the parenteral delivery includes, but is not limited to, one or more of the following: intravenous, subcutaneous, intramuscular or intraperitoneal injection, rectal delivery via suppository, percutaneous delivery and / or intraocular delivery. Beneficial effects

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] (1) The conjugate of near-infrared fluorescent dye and antibody has emission wavelength coverage in the near-infrared region of 700-1700nm, with low light scattering, high in vivo imaging resolution and deep penetration depth.

[0046] (2) The conjugates of near-infrared fluorescent dyes and antibodies have advantages such as low cost, biosafety, and suitability for drug development.

[0047] (3) The near-infrared fluorescent dye and antibody conjugate loaded with chitosan based on Formula I can be administered orally or intravenously, with good compliance. Attached Figure Description

[0048] Figure 1 shows the 1H NMR spectrum of the trifluoromethyl-modified chitosan CF3DC prepared in Example 1.

[0049] Figure 2 shows the NMR fluorine spectrum characterization of the trifluoromethyl-modified chitosan CF3DC prepared in Example 1, and the NMR fluorine spectrum characterization of the chitosan CF3DC with added internal reference trifluoroacetic acid for labeling efficiency analysis.

[0050] Figure 3 shows the general formula for the synthesis of hyaluronic acid-modified chitosan DC-HA prepared in Example 3.

[0051] Figure 4 shows the 1H NMR spectrum of the hyaluronic acid-modified chitosan DC-HA prepared in Example 2.

[0052] Figure 5 shows the general formula for the synthesis of trifluorobenzoyl-modified chitosan BFDC prepared in Example 2.

[0053] Figure 6 shows the 1H NMR spectrum of the trifluorobenzoyl-modified chitosan BFDC prepared in Example 2.

[0054] Figure 7 shows the NMR fluorine spectrum characterization of the trifluorobenzoyl-modified chitosan BFDC prepared in Example 2, and the NMR fluorine spectrum characterization after adding reference trifluoroacetic acid.

[0055] Figure 8 shows the ultraviolet absorption and fluorescence spectra of the near-infrared immunofluorescence preparation ICGM-B9 prepared in Example 1, where ICGM is a heptamethrin dye containing a single maleimide group, and B9 is a single-domain antibody that can target CEACAM5.

[0056] Figure 9 shows the particle size distribution, potential diagram, and scanning transmission electron microscopy (STEM) image of ICGM-B9 and the conjugate ICGM-B9-DC of near-infrared immunofluorescence reagents and chitosan prepared in Example 1, as the molar ratio of different near-infrared immunofluorescence reagents ICGM-B9 and CF3DC changes.

[0057] Figure 10 shows a near-infrared two-window image of a mouse model of intestinal metastatic tumors after intravenous injection of ICGM-B9.

[0058] Figure 11 shows the anatomical process of removing tumors and tumors near the gastrointestinal tract.

[0059] Figure 12 shows the HE staining analysis of the removed tumor and tumors near the gastrointestinal tract.

[0060] Figure 13 shows the bioluminescence, near-infrared one-window imaging, and near-infrared two-window imaging of mice with intestinal metastatic tumors after oral administration of ICGM-B9-DC.

[0061] Figure 14 shows the anatomical process of removing the tumor and the tumor near the gastrointestinal tract, as well as the fluorescence imaging of the removed tumor and the fluorescence imaging of the remaining organs after tumor removal.

[0062] Figure 15 shows the HE staining analysis of the extracted tumor. Detailed Implementation

[0063] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0064] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0065] Terminology Explanation

[0066] Covalent coupling: A coupling method in which several adjacent atoms share electrons and form stable chemical bonds with each other.

[0067] CEA: A common broad-spectrum tumor marker in clinical practice, widely used in the differential diagnosis, disease monitoring, and prognostic assessment of various tumors such as colon cancer, rectal cancer, gastric cancer, and lung cancer.

[0068] Antibody-conjugate: In this invention, it refers to a small molecule with biological activity that is linked to an antibody through a chemical link, and the antibody acts as a carrier to target and transport the small molecule to the target cell.

[0069] Near-infrared: In this invention, it refers to infrared light (NIR), which is an electromagnetic wave between visible light (VIS) and mid-infrared light (MIR). Conventionally, the near-infrared region is further divided into two areas: near-infrared region I (750–900 nm) and near-infrared region II (1000–1700 nm). The near-infrared region is the earliest discovered non-visible light region.

[0070] Tumor bearing in mice: This refers to the process of transplanting tumor cells into mice, allowing them to form tumor tissue. This experimental method is widely used in tumor research to assess the proliferative capacity of tumor cells, drug efficacy, and immune system response. Tumor bearing procedure: A prepared suspension of tumor cells is injected subcutaneously or at other sites into the mouse. Common injection sites include the flank, axilla, or back. Before injection, the injection site should be disinfected with an alcohol swab, and the tumor cell suspension should be injected slowly to prevent leakage or premature extravasation. Intraperitoneal injection is often used to construct metastatic tumor models, and the number of lesions is randomized, depending on the specific number of lesions found after dissection of the mouse.

[0071] As used herein, the terms “delivery” or “administration” in relation to a pharmaceutical agent mean the administration of a pharmaceutical agent to a subject using any of the methods known to those skilled in the art for delivering a formulation or formulation conjugate or delivery system. Delivery methods include, but are not limited to, oral delivery, parenteral delivery such as intravenous, subcutaneous, intramuscular, or intraperitoneal injection, rectal delivery via suppository, percutaneous delivery, intraocular delivery, or any route or method of administration by which a therapeutically effective drug is delivered. The amount of the drug or conjugate reaches the cells or tissues to which it is targeted. Alternatively, routine experiments will determine other acceptable routes of administration.

[0072] Experimental materials

[0073] The standard recombinant DNA and molecular cloning techniques used in the following examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience), and the materials and methods suitable for microbial growth are well known in the art.

[0074] The main chemical and biological reagents were purchased from the official website or could be prepared according to methods known in the art. (a) AL Antaris, H. Chen, K. Cheng, Y. Sun, G. Hong, C. Qu, S. Diao, Z. Deng, X. Hu, B. Zhang, X. Zhang, OK Yaghi, ZRA Lamparambil, X. Hong, Z. Cheng, H. Dai, Nat. Mater. 2016, 15, 235-242.; (b) KK Maiti, A. Samanta, M. Vendrell, KSSoh, M. Oliva, Y. T. Chang, Chem. Commun. 2011, 47, 3514-3516.

[0075] Example 1: Trifluoromethyl-modified chitosan (CF3DC)

[0076] A. Test methods

[0077] (1) Dissolve trifluoroacetic acid (154 μL, 2 mmol) in 5 mL of ultra-dry DMSO;

[0078] (2) Add (1) to EDCI (0.575 mg, 3 mmol) and N-hydroxysuccinimide (0.345 mg, 3 mmol) and react;

[0079] (3) Add chitosan (100 kDa, 100 mg) to (2) and react;

[0080] (4) Add (3) to a 7000 Da dialysis bag and change the dialysis solution;

[0081] (5) The liquid in the dialysis bag was then removed and freeze-dried to obtain a white powder.

[0082] B. Test Results

[0083] Figure 1 shows the proton spectrum of CF3DC. From the figure, we can see that... 1 H NMR(400MHz,D2O)δ3.77–3.44(m,3H),2.86(s,2H),2.53(s,1H),1.87(s,1H).

[0084] Figure 2 shows the results of adding 11.47 μL of trifluoroacetic acid (equivalent to the theoretical number of trifluoromethyl groups in 10 mg of CF3DC) as an internal standard to CF3DC 10 mg before and after addition. 19 F-NMR spectrum. The chemical shift of CF3DC before the addition of trifluoroacetic acid was 75.84. After the addition of trifluoroacetic acid, a new peak appeared at 75.85, which can be identified as the chemical shift of the fluorine atom in trifluoroacetic acid. Based on the integral area calculation, the probability of successful chitosan trifluoromethyl labeling in 10 mg CF3DC is approximately 34%.

[0085] Example 2: Hyaluronic acid-modified chitosan (DC-HA)

[0086] A. Test methods

[0087] (1) Hyaluronic acid (0.2g, 100mmol) was dissolved in 5mL of ultra-dry DMSO;

[0088] (2) Add (1) to DCC (0.102 mg, 100 mmol) and DMAP (0.060 mg, 100 mmol) and react;

[0089] (3) Add chitosan (100 kDa, 500 mg) to (2) and react;

[0090] (4) Add (3) to a 7000 Da dialysis bag and change the dialysis solution;

[0091] (5) The liquid in the dialysis bag was then removed and freeze-dried to obtain a white powder.

[0092] B. Test Results

[0093] As shown in Figure 3, hyaluronic acid was introduced into chitosan using the same method to obtain hyaluronic acid-modified chitosan.

[0094] As shown in Figure 4, the proton NMR spectrum of the reaction product was measured. Since D₂O was used as the deuterated reagent, the proton NMR peak of the hydroxyl group could not be observed. However, a doublet appeared at a chemical shift of 4.62, which coincides with the chemical shift of the quaternary ammonium hydrogen. Furthermore, a singlet proton NMR appeared in the high-field region, which coincides with the chemical shift of the imino hydrogen in hyaluronic acid. Therefore, the chitosan-hyaluronic acid coupling was successful.

[0095] Example 3: Trifluorobenzyl-modified chitosan (BFDC)

[0096] A. Test methods

[0097] (1) Trifluorobenzoic acid (308 μL) was dissolved in 5 mL of ultra-dry DMSO;

[0098] (2) Add (1) to EDCI (0.575 mg, 3 mmol) and N-hydroxysuccinimide (0.345 mg, 3 mmol) and react;

[0099] (3) Add chitosan (100 kDa, 100 mg) to (2) and react;

[0100] (4) Add (3) to a 7000 Da dialysis bag and change the dialysis solution;

[0101] (5) The liquid in the dialysis bag was then removed and freeze-dried to obtain a white powder.

[0102] B. Test Results

[0103] A 4-trifluorobenzoyl group was introduced at the amino group to modify chitosan. The reaction process is shown in Figure 5.

[0104] As shown in Figures 6 and 7, a 4-trifluorobenzoyl group was introduced into chitosan using the same method, and the 1H and 1F spectra of the product were measured. The 1H spectrum analysis showed that no trifluorobenzoic acid residue was found in the obtained trifluorobenzyl-chitosan, and the 1F spectrum confirmed the presence of trifluoromethyl groups in the product. This synthetic route is generally applicable to the modification of chitosan.

[0105] In Examples 1-3, different functional groups were introduced into chitosan, such as fluorinated groups (e.g., trifluoromethyl, trifluorobenzoyl) and acidic polysaccharide groups (hyaluronic acid acylation). The introduction of hyaluronic acid and trifluorobenzoyl failed to effectively improve the water solubility of chitosan, while the trifluoromethyl-modified chitosan exhibited good dispersibility in water. Furthermore, trifluoromethyl has a strong electron-withdrawing effect, readily forming hydrogen bonds with negatively charged protein structures, thus effectively embedding into the antibody-dye conjugate ICGM-B9 and aiding in the formation of stable chitosan micelles. In addition, animal experiments demonstrated that fluorinated chitosan modification can effectively promote the uptake rate of mesenteric epithelial cells to a certain extent, opening intercellular spaces and further promoting the uptake of ICGM-B9-DC by intestinal tumors. Therefore, in subsequent experiments, trifluoromethyl chitosan was used for loading and delivery of the near-infrared immunofluorescence conjugate ICGM-B9.

[0106] Example 4: Near-infrared immunofluorescent dye and antibody conjugate (ICGM-B9)

[0107] The conjugation of heptamethrin dye (ICGM) containing a single-sided maleimide group to a single-domain antibody B9 that targets CEACAM5 includes the following steps:

[0108] (1) Mix the antibody nbB9 targeting CEA with TCEP (3.0 equiv) and DTPA (5.0 equiv) and shake to react;

[0109] (2) Add ICGM (10.0 equiv) to (1) and carry out the reaction;

[0110] (3) Add the system after reaction (2) into a dialysis bag. The dialysis solution is PBS and 0.5M sodium chloride solution. Perform the dialysis reaction.

[0111] (4) The system after reaction (3) was combined with Ni-NTA and purified overnight;

[0112] (5) After elution with Buffer C, the coupling success was confirmed by SDS-PAGE and reverse chromatography-mass spectrometry.

[0113] As shown in Figure 8, the antibody-dye conjugate was characterized by SDS-Page analysis, and the corresponding band was found to be at a molecular weight of 15 kDa. Further characterization of the UV-Vis and fluorescence spectra of the antibody-dye conjugate ICGM-B9 revealed that its absorption peak was at 780 nm, its emission wavelength was approximately 808 nm, and its tail peak could extend to a wavelength range of 1300 nm.

[0114] As shown in Figure 9, the particle size and potential change with the molar ratio of different near-infrared immunofluorescence reagents ICGM-B9. As shown in Figure 9A, the particle size was measured using a dynamic particle size analyzer (DLS); as shown in Figure 9B, the surface charge change was analyzed using zeta potential; and as shown in Figure 9C, the true particle size was confirmed using TEM, with uranium acetate as the negative staining salt. The particle size was adjusted to a uniform morphology (hydrated particle size 200 nm) by varying the molar ratio of the antibody-dye conjugate ICGM-B9 to chitosan (4 / 3, 1 / 3, 1 / 6, 1 / 9, 1 / 12, 1 / 15). Simultaneously, the zeta potential of the surface was measured, revealing that chitosan is positively charged (7.07) and ICGM-B9 is negatively charged (-8.94). With further adjustment of the molar ratio, the particle size tended towards electron neutrality when uniform. Transmission electron microscopy (TEM) analysis of the uniform sample revealed a uniform spherical structure with a particle size of approximately 11 nm (Figure 9G). This particle size offers a significant advantage in penetrating capillaries, as the intercellular space of the capillary wall is approximately 50 nm, which is one of the main reasons why it is more easily taken up by microvessels formed by intestinal tumor cells later on.

[0115] Example 5: Preparation of chitosan-supported near-infrared immunofluorescence conjugate (ICGM-B9-DC)

[0116] The CF3DC prepared in Example 1 was conjugated with the near-infrared immunofluorescent dye and antibody conjugation preparation ICGM-B9 prepared in Example 4. The method includes the following steps:

[0117] (1) CF3DC and ICGM-B9 were added to deionized water at different molar ratios (3 / 4; 3 / 1; 6 / 1; 9 / 1; 12 / 1; 15 / 1);

[0118] (2) The reaction was shaken in a cell disruptor, then removed and centrifuged. The precipitate was collected and dispersed into a tube.

[0119] Dialysis yielded the near-infrared immunofluorescence conjugate ICGM-B9-DC (Figure 9A, B shows its optimized synthesis route, and Figure 9G shows the transmission electron microscope image of ICGM-B9-DC finally used in subsequent experiments).

[0120] Example 6: Fluorescence imaging of near-infrared immunofluorescent dye and antibody-conjugated formulation ICGM-B9 in mice.

[0121] The near-infrared immunofluorescent dye obtained in Example 4 and the antibody-conjugated formulation ICGM-B9 were used for the experiment.

[0122] A. Near-infrared two-window imaging

[0123] (1) Eight-week-old tumor-bearing Balb / c mice were selected and injected intraperitoneally with 2×10⁻⁶ g of urea 21 days prior to the event. 6 LS174T cell line;

[0124] (2) Intraperitoneal injection of D-fluorescein (20mg / mL×200μL) and imaging on a near-infrared two-window imager;

[0125] (3) The same mice as in (1) were intravenously injected with 0.5 mg / mL × 200 μL of ICGM-B9, and near-infrared two-window imaging of the mice was performed within 0-24 h.

[0126] Figure 10A shows the bright-field image of the model mouse in steps (1) and (2); Figure 10B shows the bioluminescence image of the model mouse, where four circular bright spots are observed, indicating the approximate locations of four tumors. The number of tumors after intraperitoneal injection is not fixed and is determined by subsequent dissection.

[0127] Figure 10C shows the bright field image of the model mouse in step (3); Figure 10D shows the near-infrared two-window imaging image of the mouse based on intravenous injection. The optimal time is 10min-120min. More than 8 irregular bright spots were observed, which means that there are at least 8 tumors in the near-infrared two-window imaging image.

[0128] B. Anatomical observation

[0129] As shown in Figure 11, the mouse was dissected and observed under white light. Fluorescent signals were observed in the abdomen near the stomach, intestines and near the colon below the liver. After laparotomy, small white tumors were indeed found in the following locations.

[0130] C. Immunohistochemical analysis

[0131] As shown in Figure 12, tumors in the colon, liver, spleen, and stomach were removed and sent for immunohistochemical analysis to confirm the presence of tumors or early lesions with tumor infiltration in the corresponding areas.

[0132] The tumor-targeting properties of the ICGM-B9 probe were demonstrated through routine intravenous injection, which is a prerequisite for the feasibility of oral delivery. However, ICGM-B9 can only be used for imaging via injection, and patient compliance with injection is worse than with oral administration in clinical applications.

[0133] Example 7: In vivo fluorescence imaging of near-infrared immunofluorescence conjugate ICGM-B9-DC in mice.

[0134] A. Near-infrared two-window imaging

[0135] (1) Eight-week-old tumor-bearing Balb / c mice were selected and injected intraperitoneally with 2×10⁻⁶ g of urea 21 days prior to the event. 6 LS174T cell line;

[0136] (2) Intraperitoneal injection of D-fluorescein (20mg / mL×200μL) and imaging on a near-infrared two-window imager;

[0137] (3) The same mice as in (1) were orally administered 0.5 mg / mL × 200 μL of ICGM-B9-DC, and then near-infrared two-window imaging of the mice was performed within 0-24 hours.

[0138] Figure 13 shows images of the same mouse under different imaging conditions. The lower left corner image is a superposition of the bright field image and the fluorescence field image. Figure 13A shows the bioluminescence image of the model mouse 10 minutes after D(-) fluorescein injection in steps (1) and (2), used to mark the tumor location visible based on this method. Figure 13B shows the near-infrared one-window imaging image of the mouse based on the oral reagent (imaging area is 845-945nm). Figure 13C shows the near-infrared two-window imaging image of the mouse based on the oral reagent. The laser used was an 808-nm laser, and the collected imaging band was 1300nm-1700nm. The optimal time was 10min-120min. As shown in Figure 13D, this is a near-infrared two-window imaging image of the mouse based on an oral reagent. The laser used was a 980-nm laser, and the collected imaging band was 1100nm-1700nm. The optimal time was 10min-120min. More than 6 irregular bright spots were observed, which means that there are at least 6 concentrated tumors or tumor-infiltrating lesions in the near-infrared two-window imaging image.

[0139] B. Anatomical observation

[0140] As shown in Figure 14, there are fluorescent signals in the abdomen below the liver and near the spleen, as well as in the intestines and near the colon. After laparotomy, white tumors were indeed found in the following locations (Figure 14A), and fluorescent signals could be seen at all the white tumor sites after opening the abdomen (Figure 14B and Figure 14C).

[0141] Furthermore, imaging of organs under different imaging conditions (Figure 14D) revealed that under 808nm laser excitation, signals other than those from the tumor were concentrated in the intestines and stomach, with no signal residue in other organs (Figure 14E); under 980nm laser excitation, no signal residue was found in organs other than the tumor (Figure 14F). This experiment demonstrates that the material can be precisely targeted to the tumor region via oral delivery without interference from other metabolic signals.

[0142] C. Immunohistochemical analysis

[0143] As shown in Figure 15, tumors in the intestines, liver, spleen, and stomach were removed and sent for immunohistochemical analysis. This confirmed that the areas showing fluorescence in the near-infrared two-window imaging did indeed have tumors or early lesions with tumor infiltration.

[0144] The above data demonstrate that the chitosan-loaded antibody-fluorescent dye conjugate ICGM-B9-DC successfully achieves precise identification and delivery of tumor lesions in the gastrointestinal tract and even those metastasized to distant organs (such as the liver and spleen). This is mainly due to the high tumor delivery efficiency of the antibody-fluorescent dye conjugate ICGM-B9 during intravenous injection and the acid- and alkali-resistant properties of its oral delivery medium, chitosan, in the gastrointestinal environment. Patient compliance is high in clinical applications.

Claims

1. A near-infrared immunofluorescence conjugate ABC, wherein, A is a near-infrared fluorescent dye; B is an anti-CEA antibody, the amino acid sequence of which is shown in SEQ ID NO:1; C is a chitosan carrier; the chitosan carrier is a covalently coupled compound based on chitosan and a fluorine-containing group; the chemical structural formula of the chitosan carrier is shown in Formula I. R1 is independently selected from hydrogen atoms, alkyl groups, aromatic hydrocarbon groups, acyl forms of polysaccharide compounds (such as the acyl form of hyaluronic acid), phenyl, trifluoromethyl, trifluorobenzyl-modified phenyl or fluorinated phenyl, etc.; n represents the number of polymers of chitosan. The molecular weight of the modified chitosan is 3 kDa, 10 kDa, 100 kDa or 1000 kDa, etc.

2. The near-infrared fluorescent dye as described in claim 1 is an asymmetric near-infrared fluorescent dye, selected from one or more of cyanine dyes, rhodamine dyes, and / or BODIPY dyes.

3. The chitosan carrier as described in claim 1 is obtained via an amide coupling reaction, as shown in Formula II or Formula III:

4. The amide coupling reaction as described in claim 2, wherein the base used for amide coupling in Formula II includes, but is not limited to, triethylamine (TEA), N-methylmorpholine (NMM) and N,N-diisopropylethylamine (DIPEA, Hunig's base), pyridine, DBU, 2,6-dimethylpyridine, imidazole or N-methylimidazolium (NMI).

5. The amide coupling reaction as described in claim 2, wherein the condensing agent used for amide coupling in Formula III includes, but is not limited to, N,N'-carbonyldiimidazole (CDI), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), N,N'-dicyclohexylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), phosphorus pentachloride, acetic anhydride (Ac2O), cyanuric chloride (TCT), boric acid (B(OH)3), N-benzyl-9-(tetrahydro-2H-pyran-2-yl)adenine (PBA) or n-(2-[4-nitro-2-(trifluoromethyl)phenyl]amino}ethyl)benzamide (3-NPBA).

6. The method for preparing the near-infrared immunofluorescence conjugate as described in claim 1, wherein the method comprises the following steps: M1. To prepare an immunofluorescent agent, an anti-CEA antibody, a reducing agent, and a near-infrared fluorescent dye dissolved in an organic solvent are mixed to obtain a conjugate of the near-infrared fluorescent dye and the antibody. M2. Dissolve the modified chitosan and near-infrared immunofluorescence reagent as shown in Formula I at different molar ratios; M3. Shake the solution obtained in step M1 to concentrate it to the required volume.

7. An imaging agent comprising the near-infrared immunofluorescence conjugate ABC of claim 1, wherein, A is a near-infrared fluorescent dye; B is an anti-CEA antibody, the amino acid sequence of which is shown in SEQ ID NO:1; C is a chitosan carrier; the chitosan carrier is a covalently coupled compound based on chitosan and a fluorine-containing group; the chemical structural formula of the chitosan carrier is shown in Formula I. R1 is independently selected from hydrogen atoms, alkyl groups, aromatic hydrocarbon groups, acyl forms of polysaccharide compounds (such as the acyl form of hyaluronic acid), phenyl, trifluoromethyl, trifluorobenzyl-modified phenyl or fluorinated phenyl, etc.; n represents the number of polymers of chitosan. The molecular weight of the modified chitosan is 3 kDa, 10 kDa, 100 kDa or 1000 kDa, etc.

8. The application of the near-infrared immunofluorescence conjugate ABC as described in claim 1 in the preparation of imaging agents; wherein, A is a near-infrared fluorescent dye; B is an anti-CEA antibody, the amino acid sequence of which is shown in SEQ ID NO:1; C is a chitosan carrier; the chitosan carrier is a covalently coupled compound based on chitosan and a fluorine-containing group; the chemical structural formula of the chitosan carrier is shown in Formula I. R1 is independently selected from hydrogen atoms, alkyl groups, aromatic hydrocarbon groups, acyl forms of polysaccharide compounds (such as the acyl form of hyaluronic acid), phenyl, trifluoromethyl, trifluorobenzyl-modified phenyl or fluorinated phenyl, etc.; n represents the number of polymers of chitosan. The molecular weight of the modified chitosan is 3 kDa, 10 kDa, 100 kDa or 1000 kDa, etc.

9. As described in any one of claims 7 or 8, the imaging is in vivo imaging and / or ex vivo imaging.

10. As claimed in any one of claims 7 or 8, the imaging agent is delivered via oral delivery and parenteral delivery; parenteral delivery includes, but is not limited to, one or more of intravenous, subcutaneous, intramuscular or intraperitoneal injection, rectal delivery via suppository, percutaneous delivery and / or intraocular delivery.

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