Near-infrared immunofluorescence conjugate and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a near-infrared immunofluorescence conjugate and application thereof. The invention finds that a near-infrared fluorescent dye can be coupled with an antibody to form a conjugate, and the conjugate can be loaded into a modified chitosan carrier, so that the near-infrared immunofluorescence probe which has an imaging function and can be orally taken is prepared. The conjugate of the near-infrared fluorescent dye and the antibody is covered by emission wavelength in a near-infrared region within the range of 700-1700nm, and has lower light scattering, higher in-vivo imaging resolution and deeper penetration depth; and the conjugate of the near-infrared fluorescent dye and the antibody has the advantages of low cost, biosafety, suitability for patent medicine and the like. The conjugate of the modified chitosan loaded near-infrared fluorescent dye based on the formula I and the antibody can be administered through oral delivery, intravenous injection and other modes, and has good compliance.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a near-infrared immunofluorescence conjugate and an application thereof. Background Art

[0002] Due to the complexity of the gastrointestinal environment, many drugs face problems such as low bioavailability and poor stability when administered orally. The development of an effective oral delivery system is of great significance for improving drug efficacy. As the most commonly used in vivo delivery route for small molecule preparations, the oral delivery system has the advantages of convenience, high patient compliance and low cost compared to injection (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 been an important research direction in the medical field in recent years. However, peptide drugs face multiple challenges in the process of oral delivery, including proteins with large molecular weight (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, mucus barrier, epithelial permeation and liver 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 can usually 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 system, bioadhesion system, 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 the body (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]. Chinese Journal of Pharmaceutical Industry, 2024.). These nanocarriers not only improve the solubility and stability of drugs, but also enhance their ability to penetrate the 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 an important role in oral drug delivery. For example, polymer nanocarriers can enhance the solubility and permeability of hydrophobic chemotherapy drugs, overcome the gastrointestinal absorption barrier, and reduce the first-pass effect of the metabolic system, thereby improving the bioavailability of drugs (Le Zhicheng, Liu Zhijia, Chen Yongming. Research progress on oral delivery of chemotherapy drugs mediated by polymer nanocarriers [J]. Polymer Materials Science and Engineering, 2019.). In addition, nanodrug delivery systems help to build more effective oral nanodrug delivery systems by simulating cell models of small intestinal absorption and research on endocytosis mechanisms (Dong Andi, Ji Fengqi, Zhang Chunpeng, et al. Development of in vitro absorption models of oral nanodrug delivery systems and analysis of endocytosis mechanisms [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 the types of tumors involved are also relatively numerous. Traditional endoscopic examinations include gastroscopy and colonoscopy, which can directly observe the internal conditions of the digestive tract and perform biopsy and other operations (Zhang Yu, Chen Xiaochang, Zhong Min. Discussion on digestive endoscopy and gastrointestinal diseases with video [J]. Chinese and Foreign Medical Treatment, 2010.). However, traditional endoscopic examinations have certain limitations, such as possible patient discomfort and cross-infection risks (Wang Weiwei. Research on in vitro magnetically driven diagnostic and therapeutic capsule system [D]. Shanghai Jiaotong University, 2013.). Although traditional high-resolution white light endoscopy (WLE) is the most popular detection method in clinical practice, its missed detection rate is relatively high. In high-risk patients with gastrointestinal cancer, especially inflammatory patients, the missed diagnosis rate of adverse lesions is three times that of healthy people. In order to overcome these shortcomings, wireless capsule endoscopy (WCE) technology came into being.

[0007] WCE is an endoscope that does not require insertion. Patients only need to swallow a capsule equipped with a micro camera. The capsule takes images while moving in the digestive tract and transmits them to an external receiver via wireless signals (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, and requiring no anesthesia and no special preparation. It is particularly suitable for small intestinal areas that are difficult to examine with traditional endoscopes (Zhang Sijie. Research on MEMS-based digestive tract wireless endoscope [D]. Chongqing University, 2005.). The development of micro gastrointestinal endoscopes also provides new possibilities for the diagnosis of gastrointestinal diseases. Due to their smaller size, micro endoscopes can more easily reach various parts of the digestive tract, including hard-to-reach areas of the small intestine (Xue Chunxiang. Development and prospects of micro gastrointestinal endoscopes [J]. Continuing Medical Education, 2022.). These micro-endoscopes can not only be used for diagnosis, but also for some treatment operations, such as polypectomy (Department of Gastroenterology, Shaoyang Second People's Hospital. Clinical analysis of 80 cases of gastrointestinal diseases treated with digestive endoscopy [J]. Chinese and Foreign Medical, 2014.). Although wireless capsule endoscopes and micro-endoscopes offer many advantages, they also have some limitations. For example, wireless capsule endoscopes cannot perform biopsy or treatment operations, and the operation of micro-endoscopes may require higher technical requirements and equipment support (Wang Weiwei. Research on in vitro magnetically driven diagnostic and therapeutic capsule system [D]. Shanghai Jiaotong University, 2013.). In addition, the image quality of wireless capsule endoscopes may be affected by the capsule movement speed and the digestive tract environment (Fu Yan'an. Research on image processing technology of wireless capsule endoscopes [D]. Shandong University, 2013.). Therefore, it is of great clinical significance to develop a detection method that can specifically identify micro-tumor lesions in the complex gastrointestinal mucosal system and has good patient compliance.

[0008] Immunofluorescence probes for early screening of gastrointestinal tumors are a technology that uses specific fluorescent markers to identify and locate gastrointestinal tumor cells. The design of probes used for intraoperative tumor lesion edge marking is mostly based on the combination of monoclonal antibodies and small molecules, resulting in their half-life, immunogenicity, and tumor uptake rate being significantly affected by the properties of the monoclonal antibodies themselves. Fully humanized monoclonal antibodies are generally in the form of IgG, with a relatively large molecular weight (150kDa), weak tissue or tumor penetration, and difficulty in targeting target epitopes with steric hindrance. However, the target content in tissue lesions during the early stage of tumor infiltration is relatively low, and the properties of the cell microenvironment are also very different from those of solid tumors. For the above reasons, so far, only one probe for early screening of gastrointestinal tumors has entered clinical phase I, which is a c-MET targeting peptide combined with NIR dyes and is 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 such tumor-targeted tracer probes relies on intravenous injection, which requires entry into the blood circulation. This poses a great safety hazard and poor patient compliance, which does not meet the clinical application requirements for early physical examination and diagnosis. Therefore, the design difficulty of fluorescent immunoprobes for early gastrointestinal tumor screening lies mainly in their ability to be delivered to early micro-lesions in a high-efficiency and high-dose manner in the gastrointestinal environment, so as to achieve high TBR values ​​in mucosal tissues to identify target lesions.

[0009] A variety of fluorescent probes have been developed for early diagnosis of gastric cancer. For example, water-soluble quantum dot fluorescent probes have been used to detect gastric cancer cell-associated antigen CA242, showing higher photostability and sensitivity than traditional methods (Fu Zhiying, Li Zhaohui, He Xiaoxiao, et al. Water-soluble quantum dot fluorescent probes for detection of gastric cancer cell-associated antigen CA242 [J]. Analytical Chemistry, 2006.). In addition, 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. Preparation of fluorescent nanoprobes and induced pluripotent stem cells and their application in targeted imaging and treatment of gastric cancer [D]. Shanghai Jiaotong University, 2012.). Studies on nuclear magnetic resonance-fluorescence multifunctional molecular probes have shown that such probes can not only provide imaging information of tumors at the molecular level, but also directly participate in the treatment process through chemotherapy or photodynamic therapy (Zhou Jia. Construction of nuclear magnetic resonance-fluorescence multifunctional molecular probes and their application in gastric cancer diagnosis and treatment [D]. Shanghai Jiaotong University, 2018.). In addition, methods based on fluorescence hyperspectral imaging and machine learning have also been proposed for the early diagnosis of gastric cancer. This method improves 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] The present invention finds that near-infrared fluorescent dyes can be coupled with antibodies to form a conjugate, which can be loaded into a modified chitosan carrier, thereby preparing an orally ingestible near-infrared immunofluorescent probe with imaging function. Based on this, the present invention is completed.

[0011] In a first aspect, the present invention provides a near-infrared immunofluorescent conjugate ABC, wherein A is a near-infrared fluorescent dye, B is an anti-CEA antibody, and its amino acid sequence is 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 structure of the chitosan carrier is shown in Formula I:

[0012]

[0013] Wherein R1 is independently selected from hydrogen atom, alkyl group, aromatic hydrocarbon group, acyl form of polysaccharide compound (such as acyl form of hyaluronic acid), phenyl group, trifluoromethyl group, trifluorobenzyl modified phenyl group or fluorine modified phenyl group, etc.; wherein n represents the number of chitosan polymers, and the overall molecular weight is measured, and the molecular weight of chitosan can be modified to 3KDa, 10KDa, 100KDa or 1000KDa, etc.

[0014] Furthermore, the particle size of the chitosan carrier is 10-1000 nm.

[0015] Furthermore, the aromatic hydrocarbon group is preferably hyaluronic acid.

[0016] Furthermore, the trifluoromethyl group is preferably trifluoroacetic acid.

[0017] Furthermore, the trifluorobenzyl group is preferably trifluorobenzoic acid.

[0018] Furthermore, the chitosan carrier is obtained by amide coupling reaction, and the reaction formula is shown in Formula II or Formula III:

[0019]

[0020]

[0021] Furthermore, the base used for the 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-lutidine, imidazole or N-methylimidazole (NMI).

[0022] Furthermore, the condensing agent used for the amide coupling in the 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).

[0023] Furthermore, the amide reaction time is 1-24 hours.

[0024] Furthermore, the reaction temperature of the amide reaction is 0-60°C.

[0025] Furthermore, the purification method of the amide reaction is selected from column chromatography, dialysis or ultrafiltration centrifugation.

[0026] Furthermore, the absorption spectrum and emission spectrum of the near-infrared fluorescent dye range from 600 to 1000 nm.

[0027] Furthermore, the near-infrared fluorescent dye is an asymmetric near-infrared fluorescent dye.

[0028] Furthermore, the asymmetric near-infrared fluorescent dye is selected from one or more of cyanine dyes, rhodamine dyes and / or BODIPY dyes.

[0029] In a second aspect, the present invention provides a method for preparing the near-infrared immunofluorescent conjugate as described in the first aspect, the method comprising the following steps:

[0030] M1. Preparing an immunofluorescence preparation, mixing an anti-CEA antibody, a reducing agent, and a near-infrared fluorescent dye dissolved in an organic solvent to obtain a conjugate of the near-infrared fluorescent dye and the antibody;

[0031] M2. dissolving the modified chitosan and the near-infrared immunofluorescence preparation as shown in Formula I at different molar ratios;

[0032] M3. Shake the solution obtained in step M1 and concentrate to the required volume.

[0033] Furthermore, the reducing agent in step M1 includes but is not limited to one or more of tris(2-chloroethyl) phosphate, diethyltriaminepentaacetic acid and / or 5,5'-dithiobis(2-nitrobenzoic acid); preferably tris(2-chloroethyl) phosphate and DTPA.

[0034] Furthermore, the reaction temperature in step M1 is 4-60°C, preferably 30°C.

[0035] 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.

[0036] Furthermore, in step M2, the molar ratio of the modified chitosan to the near-infrared immunofluorescence preparation is selected from 0.5:1-20:1.

[0037] Furthermore, the oscillation reaction in step M3 includes but is not limited to reacting by one or more of a cell crusher, a high pressure homogenizer and / or an ultrasonicator.

[0038] Furthermore, the oscillation reaction temperature in step M3 is selected from 4-40°C.

[0039] Furthermore, the oscillation reaction time in step M3 is selected from 5-60 min.

[0040] In a third aspect, the present invention provides an imaging agent, comprising the near-infrared immunofluorescent conjugate ABC described in the first aspect, wherein A is a near-infrared fluorescent dye, B is an anti-CEA antibody, and its amino acid sequence is 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 structure of the chitosan carrier is shown in Formula I:

[0041]

[0042] Wherein R1 is independently selected from hydrogen atom, alkyl group, aromatic hydrocarbon group, acyl form of polysaccharide compound (such as acyl form of hyaluronic acid), phenyl group, trifluoromethyl group, trifluorobenzyl modified phenyl group or fluorine modified phenyl group, etc.; wherein n represents the number of chitosan polymers, and the overall molecular weight is measured, and the molecular weight of chitosan can be modified to 3KDa, 10KDa, 100KDa or 1000KDa, etc.

[0043] In a fourth aspect, the present invention provides a use of the near-infrared immunofluorescent conjugate ABC as described in the first aspect in the preparation of an imaging agent; wherein A is a near-infrared fluorescent dye, B is an anti-CEA antibody, and its amino acid sequence is 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 structure of the chitosan carrier is shown in Formula I:

[0044]

[0045] Wherein R1 is independently selected from hydrogen atom, alkyl group, aromatic hydrocarbon group, acyl form of polysaccharide compound (such as acyl form of hyaluronic acid), phenyl group, trifluoromethyl group, trifluorobenzyl modified phenyl group or fluorine modified phenyl group, etc.; wherein n represents the number of chitosan polymers, and the overall molecular weight is measured, and the molecular weight of chitosan can be modified to 3KDa, 10KDa, 100KDa or 1000KDa, etc.

[0046] Furthermore, the imaging is in vivo imaging and / or ex vivo imaging.

[0047] Furthermore, the delivery methods of the imaging agent include oral delivery and parenteral delivery.

[0048] Furthermore, the parenteral delivery includes, but is not limited to, one or more of intravenous, subcutaneous, intramuscular or intraperitoneal injection, rectal delivery via suppository, transdermal delivery, and / or intraocular delivery.

[0049] Beneficial Effects

[0050] Compared with the prior art, the present invention has the following advantages:

[0051] (1) The conjugates of near-infrared fluorescent dyes and antibodies have emission wavelengths covering the range of 700-1700 nm in the near-infrared region, with low light scattering, high in vivo imaging resolution, and deep penetration depth;

[0052] (2) Conjugates of near-infrared fluorescent dyes and antibodies have the advantages of low cost, biosafety, and suitability for drug development.

[0053] (3) The conjugate of the modified chitosan loaded with near-infrared fluorescent dye and antibody based on formula I can be administered by oral delivery, intravenous injection, etc., and has good compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the nuclear magnetic hydrogen spectrum characterization diagram of the trifluoromethyl-modified chitosan CF3DC prepared in Example 1.

[0055] Figure 2 This is the NMR fluorine spectrum characterization graph of the trifluoromethyl-modified chitosan CF3DC prepared in Example 1 and the NMR fluorine spectrum characterization graph with the addition of internal reference trifluoroacetic acid for labeling efficiency analysis.

[0056] Figure 3 This is the general synthetic formula of the hyaluronic acid-modified chitosan DC-HA prepared in Example 3.

[0057] Figure 4 This is the nuclear magnetic hydrogen spectrum characterization diagram of the hyaluronic acid modified chitosan DC-HA prepared in Example 2.

[0058] Figure 5 This is the general synthesis formula of the trifluorobenzoyl-modified chitosan BFDC prepared in Example 2.

[0059] Figure 6 This is the nuclear magnetic hydrogen spectrum characterization diagram of trifluorobenzoyl-modified chitosan BFDC prepared in Example 2.

[0060] Figure 7 This is the NMR fluorine spectrum characterization diagram of the trifluorobenzoyl-modified chitosan BFDC prepared in Example 2 and the NMR fluorine spectrum characterization diagram after adding reference trifluoroacetic acid for quantification.

[0061] Figure 8 The ultraviolet absorption and fluorescence spectra of the near-infrared immunofluorescence preparation ICGM-B9 prepared in Example 1, wherein ICGM is a heptamethine cyanine dye containing a unilateral maleimide group, and B9 is a single-domain antibody that can target CEACAM5.

[0062] Fig. 9 The particle size distribution diagram and potential diagram prepared in Example 1 as the molar ratio of ICGM-B9 and CF3DC changes, and the scanning transmission electron microscopy image of ICGM-B9 and the conjugate of near-infrared fluorescent immunoreagent and chitosan ICGM-B9-DC.

[0063] Fig.10 This is the near-infrared two-window imaging of intestinal metastasis tumor model mice after intravenous injection of ICGM-B9.

[0064] Fig.11 Diagram of the anatomical process for removing tumors and tumors near the gastrointestinal tract.

[0065] Fig.12 This is a HE staining analysis of the removed tumor and the tumor near the gastrointestinal tract.

[0066] Fig.13 These are the bioluminescence, near-infrared one-window imaging, and near-infrared two-window imaging images of intestinal metastasis tumor model mice after oral administration of ICGM-B9-DC.

[0067] Fig.14 The images include the anatomical process of removing the tumor and the tumor near the gastrointestinal tract, and the fluorescent imaging of the removed tumor, that is, the fluorescent imaging of the remaining organs after the tumor is removed.

[0068] Fig.15 This is a picture of HE staining analysis of the tumor. DETAILED DESCRIPTION

[0069] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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.

[0070] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0071] Explanation of terms

[0072] Covalent coupling: a coupling method in which several adjacent atoms form stable chemical bonds by sharing electrons.

[0073] CEA: A common broad-spectrum tumor marker in clinical practice, widely used in the differential diagnosis, disease monitoring and prognosis evaluation of multiple tumors such as colon cancer, rectal cancer, gastric cancer, and lung cancer.

[0074] Antibody conjugate: In the present invention, a small molecule with biological activity is connected to an antibody through a chemical link, and the antibody acts as a carrier to transport the small molecule into the target cell.

[0075] 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). It is customary to divide the near infrared region into two regions: near infrared region 1 (750-900nm) and near infrared region 2 (1000-1700nm). The near infrared region is the first non-visible light region discovered by humans.

[0076] Mouse tumor-bearing: refers to the process of transplanting tumor cells into mice to form tumor tissue in the mice. This experimental method is widely used in tumor research to evaluate the proliferation ability of tumor cells, drug efficacy, and immune system response. Tumor-bearing operation: Inject the treated tumor cell suspension into the subcutaneous or other parts of the mouse. Common inoculation sites include the flank, armpit, or back. Before inoculation, the injection site needs to be disinfected with an alcohol cotton ball, and the tumor cell suspension should be injected slowly to prevent leakage or cell overflow caused by too fast injection. Among them, the intraperitoneal injection method is mostly used to construct a metastatic tumor model, and the number of lesions is random, which is determined based on the specific number of lesions after the mouse is dissected.

[0077] As used herein, the term "delivery" or "administration" with respect to a medicament means a method of providing a medicament to a subject using any of the delivery formulations or formulation conjugates or delivery systems known to those skilled in the art. In this system. Delivery modes include, but are not limited to, oral delivery, parenteral delivery such as intravenous, subcutaneous, intramuscular or intraperitoneal injection, rectal delivery via suppositories, transdermal delivery, intraocular delivery, or administration by any route or method of delivering a therapeutically effective drug. The amount of the drug or conjugate that reaches the cells or tissues to which it is targeted. Alternatively, routine experimentation will determine other acceptable routes of administration.

[0078] Experimental Materials

[0079] Standard recombinant DNA techniques and molecular cloning techniques used in the following examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience), and materials and methods suitable for growing microorganisms are well known in the art.

[0080] The main chemical and biological reagents were purchased from the official website or can be prepared according to methods known in the art (a) ALA Antaris, H. Chen, K. Cheng, Y. Sun, G. Hong, C. Qu, S. Diao, Z. Deng, X. Hu, B. Zhang, X. Zhang, OK Yaghi, ZR Alamprambil, X. Hong, Z. Cheng H. Dai, Nat. Mater. 2016, 15, 235-242.; (b) KK Maiti, A. Samanta, M. Vendrell, KS Soh, M. Olivo Y. T. Chang, Chem. Commun. 2011, 47, 3514-3516.

[0081] Example 1 Trifluoromethyl modified chitosan (CF3DC)

[0082] A. Test Methods

[0083] (1) Trifluoroacetic acid (154 μL, 2 mmol) was dissolved in 5 mL of ultra-dry DMSO;

[0084] (2) adding (1) to EDCI (0.575 mg, 3 mmol) and N-hydroxysuccinimide (0.345 mg, 3 mmol) for reaction;

[0085] (3) Add chitosan (100 KDa, 100 mg) to (2) and react;

[0086] (4) adding (3) into a 7000Da dialysis bag and dialyzing the solution;

[0087] (5) Then, the liquid in the dialysis bag was taken out and freeze-dried to obtain a white powder.

[0088] B. Test Results

[0089] Figure 1 is the hydrogen spectrum of CF3DC. From the figure, we can get: 1 H NMR (400MHz, D2O) δ3.77–3.44(m,3H),2.86(s,2H),2.53(s,1H),1.87(s,1H).

[0090] Figure 2 The measured value of CF3DC 10 mg before and after adding 11.47 μL trifluoroacetic acid (equivalent to the number of theoretical trifluoromethyl groups in CF3DC 10 mg) as internal standard 19 F NMR spectrum. The chemical shift of CF3DC before adding trifluoroacetic acid is 75.84. After adding trifluoroacetic acid, a new peak appears at 75.85, which can be determined as the chemical shift of the fluorine atom in trifluoroacetic acid. According to the calculation of its integrated area, the probability of successful trifluoromethyl labeling of chitosan in 10mg CF3DC is about 34%.

[0091] Example 2 Hyaluronic acid modified chitosan (DC-HA)

[0092] A. Test Methods

[0093] (1) Hyaluronic acid (0.2 g, 100 mmol) was dissolved in 5 mL of ultra-dry DMSO;

[0094] (2) Add (1) to DCC (0.102 mg, 100 mmol) and DMAP (0.060 mg, 100 mmol) for reaction;

[0095] (3) Add chitosan (100 KDa, 500 mg) to (2) and react;

[0096] (4) adding (3) into a 7000Da dialysis bag and dialyzing the solution;

[0097] (5) Then, the liquid in the dialysis bag was taken out and freeze-dried to obtain a white powder.

[0098] B. Test Results

[0099] like Figure 3 As shown, hyaluronic acid was introduced into chitosan by the same method to obtain hyaluronic acid-modified chitosan.

[0100] like Figure 4 As shown, the hydrogen spectrum of the reaction product was measured. Since D2O was used as a deuterated reagent, the hydrogen spectrum peak of the hydroxyl group could not be observed, but a doublet peak appeared at the position of chemical shift of 4.62, which was consistent with the chemical shift of quaternary amine hydrogen. Secondly, a single peak hydrogen spectrum appeared in the high field region, which was consistent with the chemical shift of the imino hydrogen of hyaluronic acid. Therefore, the coupling of chitosan and hyaluronic acid was successful.

[0101] Example 3 Trifluorobenzyl modified chitosan (BFDC)

[0102] A. Test Methods

[0103] (1) Trifluorobenzoic acid (308 μL) was dissolved in 5 mL of ultra-dry DMSO;

[0104] (2) adding (1) to EDCI (0.575 mg, 3 mmol) and N-hydroxysuccinimide (0.345 mg, 3 mmol) for reaction;

[0105] (3) Add chitosan (100 KDa, 100 mg) to (2) and react;

[0106] (4) adding (3) into a 7000Da dialysis bag and dialyzing the solution;

[0107] (5) Then, the liquid in the dialysis bag was taken out and freeze-dried to obtain a white powder.

[0108] B. Test Results

[0109] The 4-trifluorobenzoyl group was introduced into the amino group to modify chitosan. The reaction process is as follows: Figure 5 shown.

[0110] like Figure 6 and Figure 7As shown in the figure, 4-trifluorobenzoyl group was introduced into chitosan by the same method, and the hydrogen spectrum and fluorine spectrum of the product were measured. According to the hydrogen spectrum analysis, there was no trifluorobenzoic acid residue in the obtained trifluorobenzyl-chitosan, and its fluorine spectrum proved that the trifluoromethyl group was indeed present in the obtained product. This synthetic route is generally suitable for the modification of chitosan.

[0111] In Example 1-3, different groups are introduced into chitosan, such as fluorinated groups (such as trifluoromethyl, trifluorobenzoyl), acidic polysaccharide groups (hyaluronic acid acylation), etc. Wherein, the introduction of hyaluronic acid and the introduction of trifluorobenzoyl cannot effectively improve the water solubility of chitosan, and the chitosan modified by trifluoromethyl has good dispersibility in water; and trifluoromethyl has a strong electron-withdrawing effect, and it is extremely easy to form hydrogen bonds with negatively charged protein structures, so as to be effectively embedded in antibody-dye conjugate ICGM-B9, and assist in forming stable chitosan micelles. In addition, it is proved in animal experiments that chitosan modification based on fluorinated groups can effectively promote the uptake rate of epithelial cells of the mesentery to a certain extent, open the intercellular space, thereby further promoting the uptake of intestinal tumors for ICGM-B9-DC. Therefore, in subsequent experiments, trifluoromethyl chitosan is used for the load and delivery of near-infrared immunofluorescence conjugate ICGM-B9.

[0112] Example 4 Near-infrared immunofluorescent dye and antibody conjugate (ICGM-B9)

[0113] The heptamethine cyanine dye (ICGM) containing a single-sided maleimide group is coupled to the single-domain antibody B9 that can target CEACAM5, comprising the following steps:

[0114] (1) mixing the antibody nbB9 targeting CEA with TCEP (3.0 equiv) and DTPA (5.0 equiv) and shaking the mixture for reaction; (2) adding ICGM (10.0 equiv) to (1) and reacting the mixture;

[0115] (3) adding the system after the reaction in (2) into a dialysis bag, wherein the dialysis fluid is PBS and 0.5 M sodium chloride solution, and performing a dialysis reaction;

[0116] (4) combining the system after the reaction in (3) with Ni-NTA overnight and then purifying;

[0117] (5) After elution with Buffer C, SDS-PAGE and reversed phase chromatography-mass spectrometry were used to confirm the successful coupling.

[0118] like Figure 8As shown, the antibody coupled with the dye was characterized by SDS-Page, and its corresponding band was found to be at a molecular weight of 15KDa. The UV-visible spectrum and fluorescence spectrum of the antibody-dye conjugate ICGM-B9 were further characterized, and its absorption spectrum peak was measured at 780nm, the emission wavelength was about 808nm, and its tail peak could extend to a wavelength range of 1300nm.

[0119] like Fig. 9 As shown in Figure 2, as the molar ratio of ICGM-B9 in different near-infrared immunofluorescence preparations changes, its particle size and potential also change accordingly. Fig. 9 As shown in A, the particle size is tested by a dynamic particle size analyzer (DLS); Fig. 9 As shown in B, the zeta potential is used to analyze the change of its surface charge; Fig. 9 As shown in C, the actual particle size was confirmed by TEM, and the negative staining salt used was uranyl acetate. The particle size was adjusted to a uniform form (hydrated particle size 200nm) by adjusting the molar ratio of the antibody dye conjugate ICGM-B9 to chitosan (4 / 3, 1 / 3, 1 / 6, 1 / 9, 1 / 12, 1 / 15). At the same time, the zeta potential of its surface was measured, and it was found that chitosan was positively charged (7.07) and ICGM-B9 was negatively charged (-8.94). With the adjustment of the molar ratio, when the particle size was uniform, it tended to be neutral. At this time, the uniform sample particle size was tested by transmission electron microscopy, and it was found that the particle size was a uniform circular structure of about 11nm ( Fig. 9 G). This particle size has a significant advantage in penetrating capillaries, because the intercellular space of the capillary wall is about 50nm, which is one of the main reasons why it is more easily taken up by the microvessels formed by intestinal tumor cells in the later stage.

[0120] Example 5 Preparation of chitosan-loaded near-infrared immunofluorescence conjugate (ICGM-B9-DC)

[0121] The CF3DC prepared in Example 1 and the near-infrared immunofluorescent dye prepared in Example 4 are coupled with the antibody coupling preparation ICGM-B9, and the method comprises the following steps:

[0122] (1) CF3DC and ICGM-B9 were added into deionized water at different molar ratios (3 / 4; 3 / 1; 6 / 1; 9 / 1; 12 / 1; 15 / 1);

[0123] (2) Oscillate the reaction in a cell crusher, remove the cells and centrifuge, collect the precipitate and disperse it into a tube.

[0124] The near-infrared immunofluorescence conjugate ICGM-B9-DC ( Fig. 9 A, B is the process of optimizing the synthesis path. Fig. 9G is the transmission electron microscopy image of ICGM-B9-DC that was finally used in subsequent experiments).

[0125] Example 6 Fluorescence imaging of near-infrared immunofluorescent dye and antibody-coupled preparation ICGM-B9 in mice

[0126] The near-infrared immunofluorescent dye obtained in Example 4 was used to couple the antibody to the preparation ICGM-B9 for the experiment.

[0127] A. Near infrared two-window imaging

[0128] (1) Eight-week-old tumor-bearing Balb / c mice were selected and intraperitoneally injected with 2×10 6 LS174T cell line;

[0129] (2) D-luciferin (20 mg / mL × 200 μL) was injected intraperitoneally and imaged on a near-infrared two-window imager;

[0130] (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.

[0131] like Fig.10 As shown in A, it is the bright field image of the model mouse in steps (1) (2); Fig.10 B shows the bioluminescent imaging of the model mouse. Four circular bright spots were observed, indicating the locations of four tumors. After intraperitoneal injection, the number of tumors is not fixed, and the specific tumors are determined by the results of later dissection.

[0132] like Fig.10 C is the bright field image of the model mouse in step (3); Fig.10 D shows the near-infrared two-window imaging image of the mouse based on intravenous injection. The optimal time is 10min-120min, and more than 8 irregular bright spots are observed, which means that there are at least 8 tumors in the near-infrared two-window imaging image.

[0133] B. Anatomical observation

[0134] like Fig.11 As shown, the mice were dissected and observed under white light, and fluorescent signals were seen in the abdomen near the stomach under the liver, as well as in the intestines and near the colon. After laparotomy, small white tumors were found in the following locations.

[0135] C. Immunohistochemical Analysis

[0136] like Fig.12 As shown, the colon tumors, liver, spleen and stomach tumors were removed and sent for immunohistochemical analysis to confirm the presence of tumors or early lesions of tumor infiltration in the corresponding areas.

[0137] Conventional intravenous injection has proven that the probe ICGM-B9 has tumor targeting, which is also the premise for the feasibility of oral delivery of the probe. However, ICGM-B9 can only be imaged by injection, and the patient compliance of the injection method in clinical applications is poorer than that of oral administration.

[0138] Example 7 Fluorescence imaging of near-infrared immunofluorescence conjugate ICGM-B9-DC in mice

[0139] A. Near infrared two-window imaging

[0140] (1) Eight-week-old tumor-bearing Balb / c mice were selected and intraperitoneally injected with 2×10 6 LS174T cell line;

[0141] (2) D-luciferin (20 mg / mL × 200 μL) was injected intraperitoneally and imaged on a near-infrared two-window imager;

[0142] (3) The same mice as in (1) were orally gavaged with 0.5 mg / mL × 200 μL of ICGM-B9-DC, and near-infrared two-window imaging of the mice was performed within 0-24 hours.

[0143] like Fig.13 As shown in the figure, these are images of the same mouse under different imaging conditions. The lower left corner image is the superposition of the bright field image and the fluorescent field image. Fig.13 As shown in A, it is a bioluminescent image of the model mouse in steps (1) and (2) 10 minutes after D(-) luciferin injection, which is used to mark the tumor location visible based on this method; Fig.13 As shown in B, it is a near-infrared one-window imaging image of the mouse based on oral reagent (imaging area is 845-945nm), as shown in FIG. Fig.13 As shown in C, it is a near-infrared two-window imaging image of the mouse based on oral reagents. The laser is an 808-nm laser, the collected imaging band is 1300nm-1700nm, and the optimal time is 10min-120min. Fig.13 As shown in D, this is the near-infrared two-window imaging image of the mouse based on oral reagent. The laser is a 980-nm laser, the collected imaging band is 1100nm-1700nm, the optimal time is 10min-120min, and more than 6 irregular bright spots are observed, which means that the imaging image based on the near-infrared two-window has at least 6 concentrated tumors or tumor infiltration lesions.

[0144] B. Anatomical observation

[0145] like Fig.14 As shown, there are fluorescent signals under the liver and near the spleen, as well as in the intestines and near the colon. After laparotomy, white tumors were found in the following locations: Fig.14 A), and after opening the abdomen, fluorescent signals can be seen in the white tumors ( Fig.14 B and Fig.14 C).

[0146] And further imaging of organs under different imaging conditions ( Fig.14 D), found that under the condition of 808nm laser excitation, the signals other than tumors were mostly concentrated in the intestines and stomach, and no signals were left in other organs ( Fig.14 E); Under 980nm laser excitation conditions, no signal remains in organs other than tumors ( Fig.14 F) This experiment demonstrated that the material can be precisely targeted to the tumor area via oral delivery without interference from other metabolic signals.

[0147] C. Immunohistochemical Analysis

[0148] like Fig.15 As shown, the intestinal tumors, liver, spleen and stomach tumors were removed and sent for immunohistochemical analysis, confirming that the areas with fluorescence shown in the near-infrared two-window imaging did indeed have tumors or early lesions of tumor infiltration.

[0149] The above data prove that ICGM-B9-DC, an antibody-fluorescent dye conjugate loaded with chitosan, has successfully achieved accurate identification and delivery of tumor lesions in the gastrointestinal tract and even tumor lesions metastasized to distant organs (such as the liver, spleen, etc.). This is mainly due to the high tumor delivery efficiency of the antibody-fluorescent dye conjugate ICGM-B9 in intravenous injection and the acid and alkali resistance of its oral delivery medium chitosan in the gastrointestinal environment. In clinical applications, patient compliance is high.

Claims

1. A near-infrared immunofluorescence conjugate ABC, wherein: A is a near-infrared fluorescent dye, B is an anti-CEA antibody, and its amino acid sequence is shown in SEQ ID NO: 1; C is a chitosan carrier; the chitosan carrier is a covalent coupling product based on chitosan and a fluorine-containing group; the chemical structure of the chitosan carrier is shown in Formula I: Wherein R1 is independently selected from hydrogen atom, alkyl group, aromatic hydrocarbon group, acyl form of polysaccharide compound (such as acyl form of hyaluronic acid), phenyl group, trifluoromethyl group, trifluorobenzyl modified phenyl group or fluorine modified phenyl group, etc.; wherein n represents the number of chitosan polymers, and the overall molecular weight is measured, and the molecular weight of chitosan can be modified to 3KDa, 10KDa, 100KDa or 1000KDa, etc.

2. The near-infrared fluorescent dye as claimed 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 according to claim 1 is obtained by amide coupling reaction, and the reaction formula is shown in Formula II or Formula III:

4. The amide coupling reaction as claimed in claim 2, wherein the base used for the 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-lutidine, imidazole or N-methylimidazole (NMI).

5. The amide coupling reaction as claimed in claim 2, wherein the condensing agent used for the 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 according to claim 1, comprising the steps of: M1. Preparing an immunofluorescence preparation, mixing an anti-CEA antibody, a reducing agent, and a near-infrared fluorescent dye dissolved in an organic solvent to obtain a conjugate of the near-infrared fluorescent dye and the antibody; M2. dissolving the modified chitosan and the near-infrared immunofluorescence preparation as shown in Formula I at different molar ratios; M3. Shake the solution obtained in step M1 and concentrate to the required volume.

7. An imaging agent, comprising the near-infrared immunofluorescent conjugate ABC according to claim 1, wherein: A is a near-infrared fluorescent dye, B is an anti-CEA antibody, and its amino acid sequence is shown in SEQ ID NO: 1; C is a chitosan carrier; the chitosan carrier is a covalent coupling product based on chitosan and a fluorine-containing group; the chemical structure of the chitosan carrier is shown in Formula I: Wherein R1 is independently selected from hydrogen atom, alkyl group, aromatic hydrocarbon group, acyl form of polysaccharide compound (such as acyl form of hyaluronic acid), phenyl group, trifluoromethyl group, trifluorobenzyl modified phenyl group or fluorine modified phenyl group, etc.; wherein n represents the number of chitosan polymers, and the overall molecular weight is measured, and the molecular weight of chitosan can be modified to 3KDa, 10KDa, 100KDa or 1000KDa, etc.

8. Use of the near-infrared immunofluorescent conjugate ABC as claimed in claim 1 in the preparation of an imaging agent; wherein, A is a near-infrared fluorescent dye, B is an anti-CEA antibody, and its amino acid sequence is shown in SEQ ID NO: 1; C is a chitosan carrier; the chitosan carrier is a covalent coupling product based on chitosan and a fluorine-containing group; the chemical structure of the chitosan carrier is shown in Formula I: Wherein R1 is independently selected from hydrogen atom, alkyl group, aromatic hydrocarbon group, acyl form of polysaccharide compound (such as acyl form of hyaluronic acid), phenyl group, trifluoromethyl group, trifluorobenzyl modified phenyl group or fluorine modified phenyl group, etc.; wherein n represents the number of chitosan polymers, and the overall molecular weight is measured, and the molecular weight of chitosan can be modified to 3KDa, 10KDa, 100KDa or 1000KDa, 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 described in any one of claims 7 or 8, the delivery method of the imaging agent includes 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, transdermal delivery and / or intraocular delivery.

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