Novel probes targeting elastin and biomedical applications thereof

Abstract

This invention discloses a novel probe for targeting and binding elastin and its biomedical applications. The novel probe for targeting and binding elastin is constructed by linking glycopeptides or lipoglycopeptides to functional groups via linking groups. The method for preparing the novel probe for targeting and binding elastin using this invention is simple, highly specific, exhibits excellent binding ability to elastin, has a high signal-to-noise ratio, good biocompatibility, and a convenient and simple experimental procedure. The discovery of this probe provides a method for observing changes in elastin at the molecular level and has great biomedical application prospects in the diagnosis and imaging of important diseases closely related to elastin, such as cancer, organ fibrosis, and cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of medical imaging or diagnostics and relates to a novel probe that targets and binds to elastin, which is a conjugate of glycopeptides and lipoglycopeptides. Background Technology

[0002] Elastin is a major component of elastic fibers and one of the main components of the extracellular matrix, primarily found in connective tissue, vascular tissue, lungs, and skin. It provides the repetitive stretching and elastic recoil properties required by vertebrate tissues. The elastin peptide chain contains more than 713 amino acid residues. While the amino acid sequence of elastin does not contain a continuous, repetitive structure running the entire peptide chain, it does contain alternating hydrophobic and hydrophilic segments. Under normal circumstances, elastin is not as widely distributed as collagen, but it is still an essential matrix component of many tissues. However, in many diseases such as organ fibrosis and tumors, the balance of elastin synthesis is disrupted, leading to excessive proliferation and fibrosis within organs and tumors. This affects the normal physiological function of organs and the delivery and penetration of chemotherapy drugs into tumors. Therefore, elastin is a key biomarker for many important diseases, and establishing efficient and specific detection methods for elastin is crucial for exploring the pathological mechanisms of these diseases and for accurate diagnosis.

[0003] Currently, there are few reported elastin probes, such as the ESMA probe with the following structure (Sun Q, et al., Elastin imaging enables noninvasive staging and treatment monitoring of kidney fibrosis, Science Translational Medicine, 2019, 11, 486, eaat4865. DOI:10.1126 / scitranslmed.aat4865.PMID:30944168;PMCID:PMC7115882):

[0004]

[0005] Currently, the main methods for detecting elastin are tissue staining methods, including H&E staining, Weigert's van Geison method, Verhoeff-Van Gieson method, and Gomori trichrome staining. Other methods include elastin antibody immunoimaging, autofluorescence imaging, and imaging using fluorescent dyes that bind to elastin (such as sulforhodamine B and Alexa Fluor 633hydrazide). These methods have drawbacks such as complex operation, weak probe binding ability, low specificity, low resolution, low signal-to-noise ratio, and the ability to detect elastin only in vitro. Therefore, there is an urgent need to develop a detection method that can simultaneously achieve strong binding ability, high specificity, high signal-to-noise ratio, and high biocompatibility at both the in vitro tissue level and the in vivo animal level. The development of such a method will be of great significance for the diagnosis and imaging of important diseases closely related to elastin, such as cancer, organ fibrosis, and cardiovascular diseases. Summary of the Invention

[0006] To address existing needs and problems, this invention aims to provide novel probes targeting elastin based on glycopeptides and lipoglycopeptides, and their biomedical applications. The materials used are simple and readily available, the preparation method is straightforward, the probes exhibit high specificity, excellent binding affinity to elastin, a high signal-to-noise ratio, good biocompatibility, and a convenient and simple experimental procedure. The discovery of this probe provides a method for observing changes in elastin at the molecular level, and it holds immense promise for applications in the diagnosis and imaging of important diseases closely related to elastin, such as cancer, organ fibrosis, and cardiovascular diseases.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides the application of a fluorescent probe based on glycopeptide antibiotics or lipoglycopeptide antibiotics in the preparation of a drug, wherein the drug is used for imaging or diagnosis of tissue sections, and the fluorescent probe based on glycopeptide antibiotics or lipoglycopeptide antibiotics comprises a glycopeptide antibiotic or lipoglycopeptide antibiotic portion and a fluorescence imaging portion.

[0009] In a preferred aspect of the invention, the fluorescent probe based on glycopeptide antibiotics or lipoglycopeptide antibiotics comprises a compound or a pharmaceutical salt thereof with the structure shown in formula (I):

[0010] T-(B1-L-B2-R)n(I)

[0011] Where T is the targeting group that targets elastin, and R is the functional group;

[0012] B1 is a linker or direct bond between L and the target group T;

[0013] L represents a connecting arm or is not present;

[0014] B2 is a linker or direct bond between functional groups R and L;

[0015] B2 is a direct bond, meaning that L is part of the functional group R or does not exist;

[0016] B1 is a direct bond, meaning that L is part of the target group T or does not exist;

[0017] When L does not exist, R is connected to T through B1 or B2;

[0018] T represents a glycopeptide antibiotic or a lipoglycopeptide antibiotic molecule;

[0019] R represents the fluorescence imaging component;

[0020] n is an integer from 1 to 10.

[0021] In some embodiments, B1 is a covalent bond, preferably an amide bond or an ester bond.

[0022] In some embodiments, B2 is a linker formed in a covalent or non-covalent manner. Preferably, the covalent linking methods include: click chemistry, cross-linking, and condensation. Preferably, the non-covalent linking methods include: electrostatic interaction, hydrophobic interaction, affinity interaction, antibody-antigen binding, and biotin-avidin binding.

[0023] In some embodiments, L has about 1 to about 100 connecting atoms and optionally includes an ethoxy ((CH2CH2O)) moiety, amine, ester, amide, ketone, urea, carbamate and carbonate functional groups;

[0024] Preferably, L is a straight-chain or branched alkyl group with 2-20 carbon atoms;

[0025] Preferably, L is a polyethylene glycol group with 2-10 ethylene oxide ((CH2CH2O)) units.

[0026] In some embodiments, the fluorescence imaging portion is a fluorescent compound or nanoaggregate excited by one radiation wavelength and detected by a second, different radiation wavelength, including visible or near-infrared fluorescent probes. Preferred optical probes include fluorescein molecules, coumarin molecules, rhodamine molecules, cyanine dyes (preferably cyanine dyes or indocyanine green dyes, ICG dyes), BODIPY molecules, squaric acid molecules, squaric acid cyanine molecules, phosphorescent molecules, semiconductor molecules, carbon quantum dots, and silicon. The material comprises one or more of the following: quantum dots, sulfur quantum dots, selenium quantum dots, tellurium quantum dots, phosphorus quantum dots, perovskite quantum dots, upconversion rare earth nanomaterials, long afterglow nanomaterials, and chelating agents, preferably such as Cy5.5, Cy3, Cy5, Alexa 680, DiD (1,1'-di(octadecyl)-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate) and DiR (1,1'-di(octadecyl)-3,3,3',3'-tetramethylindole tricarbocyanine iodide).

[0027] In some embodiments, the glycopeptide antibiotic or lipoglycopeptide antibiotic molecule is preferably:

[0028] Vancomycin antibiotic, structure: Teicoplanin antibiotic, structure: Where R' is

[0029] The structure of the antibiotic tervacin is: Dabavancin antibiotic, structure: Orivacin antibiotic, structure: Itonamycin antibiotic, structure: Where R” is H or Cl;

[0030] Balhemycin antibiotic, structure: Ristoxin antibiotic, structure: The antibiotic ramoranine has the following structure: Where R”' is In the above definition, T represents the linkage site of the group.

[0031] In some embodiments, the probe is preferably:

[0032] The structure of the vancomycin-based antibiotic probe is as follows: The probe based on the antibiotic teicoplanin has the following structure: Where R' is

[0033] The probe based on the antibiotic tervacin has the following structure: The probe based on the antibiotic dabavancin has the following structure: The probe based on the antibiotic orivancin has the following structure: The probe based on the antibiotic irhinomycin has the following structure: Where R” is H or Cl;

[0034] The probe based on the balhexamethylenetetramine antibiotic has the following structure: The probe based on ristomycin has the following structure:

[0035] A probe based on remolanine. The structure is as follows:

[0036] Where R”' is

[0037] In the above formulas, T represents the group linkage site, and R1 to R... 27 Each of the above formulas independently represents an optical dye modification or H, provided that at least one optical dye modification is present in each of the above formulas.

[0038] In some embodiments, the probe may be present in the form of a pharmaceutically acceptable salt, such as an acid addition salt including acetate, adipic acidate, alginate, citrate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, butyrate, camphorate, camphorsulfonate; digluconate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, trimethylbenzenesulfonate, methanesulfonate, naphthylsulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectin ester, persulfate, 3- Phenylacetate, picrate, pentanoate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate; or basic addition salts, wherein the cations of the basic addition salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as non-toxic quaternary ammonium cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylmethylamine, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenylethylamine, and N,N'-dibenzylethylenediamine.

[0039] Furthermore, the tissue sections include, but are not limited to, animal tissue sections and human pathological tissue sections; preferably, the tissue sections include, but are not limited to, benign tumor tissue, malignant tumor tissue, orthopedic tissue disease, fibrotic tissue, inflamed tissue, proliferative tissue, atherosclerotic tissue, cirrhotic tissue, skin photodamaged tissue, skin thermally damaged tissue, skin acid and alkali corroded tissue, or radiation-damaged tissue; preferably, the tissue sections are frozen sections or paraffin sections.

[0040] A second aspect of the present invention provides a tissue section imaging kit, comprising a fluorescent probe based on a glycopeptide antibiotic or a lipoglycopeptide antibiotic, and a reagent selected from at least one of a tissue cleanser, a tissue clearing agent, a tissue immobilization agent, a blocking agent, or an antifluorescence quenching mounting medium.

[0041] Furthermore, in the tissue section imaging kit of the present invention, the fluorescent probe based on glycopeptide antibiotics or lipoglycopeptide antibiotics is a compound or its pharmaceutical salt as defined in the first aspect of the present invention.

[0042] The specific method for in vitro tissue section imaging of the present invention includes:

[0043] The tissue sections used in the in vitro tissue section imaging are frozen sections or paraffin sections, and the concentration of the target probe in the solution containing the target probe is 0.1 μmol / L-2 mol / L. For the tissue sections after removing the embedding reagent, 500 μL of blocking solution is added, and the liquid is removed after blocking for 30 min. First, 200-500 μL of probe solution is used to stain the tissue sections at room temperature for 15-30 min, and then the staining is washed off. Then, 200-500 μL of nuclear staining agent is used to stain the cell nuclei for 10 min. After washing off the nuclear staining agent, blocking solution is added, a coverslip is placed on top, and the tissues are observed and photographed using a fluorescence microscope. The nuclear staining agent is DAPI or Hoechst 33342.

[0044] Preferably, the tissue sections include, but are not limited to, animal tissue sections and human pathological tissue sections. These tissue sections include, but are not limited to, benign tumor tissue, malignant tumor tissue, orthopedic tissue, fibrotic tissue, inflamed tissue, proliferative tissue, atherosclerotic tissue, cirrhotic tissue, skin damaged by light, skin damaged by heat, skin damaged by acid or alkali corrosion, or radiation-damaged tissue.

[0045] Technical terms

[0046] elastin

[0047] Elastin is the main component of elastic fibers. Elastic fibers are primarily found in ligaments and blood vessel walls. Elastic fibers coexist with collagen fibers, giving tissues elasticity and tensile strength. Like collagen, elastin is rich in glycine and proline, but unlike collagen, elastin has a low degree of hydroxylation and lacks hydroxylysine. Elastin molecules cross-link with each other through covalent bonds formed by lysine residues; the cross-linked network they form can generate elasticity through conformational changes.

[0048] In numerous diseases, such as benign tumors, malignant tumors, orthopedic diseases, organ fibrosis (e.g., pulmonary fibrosis), inflammation, tissue hyperplasia, atherosclerosis, cirrhosis, skin damage from light, heat, acid or alkali corrosion, or radiation, the balance of elastin synthesis is disrupted, leading to excessive proliferation and fibrosis within organs and tumors. This affects the normal physiological function of organs and the delivery and penetration of chemotherapy drugs into the tumor. Therefore, elastin is a key biomarker for many important diseases.

[0049] Fluorescent probes based on glycopeptide or lipoglycopeptide antibiotic molecules / probes targeting elastin

[0050] Fluorescent probes based on glycopeptide or lipoglycopeptide antibiotic molecules are known in the prior art. For example, CN108982430B reported the use of corresponding probes for microbial labeling, which are optically dye-labeled glycopeptide or lipoglycopeptide antibiotic molecules.

[0051] The present invention preferably uses one or more fluorescent probes based on glycopeptide antibiotics or lipoglycopeptide antibiotic molecules, wherein the fluorescent probes based on glycopeptide antibiotics or lipoglycopeptide antibiotic molecules include fluorescent probes based on vancomycin, fluorescent probes based on teicoplanin, fluorescent probes based on tervavancin, fluorescent probes based on dapavancin, fluorescent probes based on orivancin, fluorescent probes based on iridocin, fluorescent probes based on balhemycin, and fluorescent probes based on ristocetin.

[0052] This invention discovers the characteristic of fluorescent probes based on glycopeptide or lipoglycopeptide antibiotic molecules targeting elastin. In this invention, fluorescent probes based on glycopeptide or lipoglycopeptide antibiotic molecules have the same meaning as probes targeting elastin.

[0053] Glycopeptide antibiotics or lipoglycopeptide antibiotics

[0054] Glycopeptide antibiotics share a highly modified heptapeptide backbone and target D-alanyl-D-alanine, a component of the bacterial cell wall. Based on their amino acid composition, they can be divided into four families: vancomycin, ristocetin, avoparcin, and synmonicin.

[0055] Lipoglycopeptide antibiotics are molecules formed by attaching hydrophobic groups to the sugar ring or amino acid side chain of glycopeptide antibiotics.

[0056] Preferred glycopeptide or lipoglycopeptide antibiotics include teicoplanin, vancomycin, tervacin, dapavancin, orivin, iridocin, balhemycin, and ristoctocin.

[0057] Linker or spacer

[0058] In this invention, the terms "linker" and "spacer" have the same meaning.

[0059] Representative linkers may have about 1 to about 100 linking atoms and may include ethylene oxide moieties, amines, esters, amides, ketones, ureas, carbamates, and carbonate functional groups. Other linkers used in the methods of the present invention may have about 1 to about 50 linking atoms, or about 1 to about 10 linking atoms, or about 5 to about 10 linking atoms.

[0060] Preferably, L is a straight-chain or branched alkyl group with 2-20 carbon atoms;

[0061] Preferably, L is a polyethylene glycol group with 2-10 ethylene oxide ((CH2CH2O)) units.

[0062] In some embodiments of the invention, the linker or spacer is part of a functional group, for example, using a probe formed using Cy5-NHS, where the alkyl group in the Cy5 molecule acts as the linker.

[0063]

[0064] Connector

[0065] In this invention, a linker refers to a covalent or non-covalent connection structure formed between a linker and a target group, between a linker and a functional group, or between a target group and a functional group when a linker is absent.

[0066] Preferably, the linker B1 formed between the linker and the target group is a covalent linker, preferably an amide bond or an ester bond.

[0067] Preferably, the B2 formed between the Linker and the functional group is a covalent or non-covalent connection. Preferably, the covalent connection includes click chemistry, cross-linking, and condensation. Preferably, the non-covalent connection includes electrostatic interaction, hydrophobic interaction, affinity interaction, antibody-antigen binding, and biotin-avidin binding.

[0068] In some embodiments of the present invention, the linker described in the present invention is a direct linker bond, which is a covalent bond formed between two non-hydrogen atoms of an organic compound (e.g., between C and C, between CN and CO). In these embodiments, the group used as a spacer arm is connected to the main structure of the functional molecule or the target group through a direct linker bond.

[0069] Pharmaceutically acceptable salts

[0070] The probes may be present in the form of pharmaceutically acceptable salts. When used herein, the term "pharmaceutically acceptable salt" means a salt or zwitterionic form of the probe that is soluble in water or oil or dispersible, suitable for contact with a patient's tissues without excessive toxicity, irritation, allergic response, or other problems or complications, within reasonable medical judgment, commensurate with a reasonable benefit / risk ratio, and effective for their intended use. The salts may be prepared during the final separation and purification of the compounds and / or diagnostic agents, or separated by reacting a suitable nitrogen atom with a suitable acid. Typical acid addition salts include acetates, adipicates, alginates, citrates, aspartates, benzoates, benzenesulfonates, bisulfates, butates, camphorates, camphorsulfonates; digluconates, glycerol phosphates, hemisulfates, heptanoates, hexanoates, formates, fumarates, hydrochlorides, hydrobromide, hydroiodates, 2-hydroxyethanesulfonate, lactates, maleates, succinate, methanesulfonate, naphthylsulfonate, nicotinate, 2-naphthalenesulfonate, oxalates, palmitates, pectinates, persulfates, 3-phenylpropionates, picrates, pentanoates, propionates, succinates, tartrates, trichloroacetate, trifluoroacetate, phosphates, glutamates, bicarbonates, p-toluenesulfonates, and undecanoates. Examples of acids that can be used to form pharmaceutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid.

[0071] Basic addition salts can be prepared during the final separation and purification of the probe by reacting the carboxyl group with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a metal cation, or with ammonia or an organic primary, secondary, or tertiary amine. The pharmaceutically acceptable cations of the salt include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as non-toxic quaternary ammonium cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylmethylamine, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, and N,N'-dibenzylethylenediamine. Other typical organic amines that can be used to form basic addition salts include ethylenediamine, ethanolamine, diethanolamine, meglumine, piperidine, and piperazine.

[0072] Optical dyes

[0073] The optical dye is a fluorescent compound or nano-aggregate that is excited by one radiation wavelength and detected by a second or different radiation wavelength. Preferred optical probes include fluorescein molecules, coumarin molecules, rhodamine molecules, cyanine dyes (preferably cyanocyanate dyes or indocyanine green dyes (ICG dyes), BODIPY molecules, squaric acid molecules, phosphorescent molecules, semiconductor molecules, carbon quantum dots, silicon quantum dots, sulfur quantum dots, phosphorus quantum dots, perovskite quantum dots, upconversion rare earth nanomaterials, long afterglow nanomaterials, and chelating agents, preferably such as Cy5.5, Cy3, Cy5, Alexa 680, DiD (1,1'-di(octadecyl)-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate) and DiR (1,1'-di(octadecyl)-3,3,3',3'-tetramethylindole tricarbocyanine iodide).

[0074] tissue section

[0075] Tissue sections are thin slices made from biological tissue and applied to glass slides for microscopic observation. They are an important tool and method in the development of histology and have often developed alongside the advancement of microscopy and improvements in slide preparation techniques. Tissue sections are widely used in fields such as biology and medicine (pathology, infectious diseases, etc.).

[0076] Preferably, the tissue section described in this invention is a tissue section containing elastin.

[0077] Preferably, the tissue section described in this invention is a pathological section, and more preferably, it is a section showing abnormal elastin production due to disease.

[0078] This invention provides a novel probe targeting elastin and its biomedical applications. The selected raw materials have well-defined molecular structures, and the preparation method is simple, highly biocompatible, and without significant toxic side effects, employing covalent or non-covalent linkage techniques. This probe exhibits high specificity and binding ability to elastin, a high signal-to-noise ratio, and high spatiotemporal resolution. It can be effectively applied to the diagnosis and imaging of some elastin-related diseases, providing a new method for the diagnosis of elastin-related diseases. Attached Figure Description

[0079] Figure 1 Structure and synthetic route of targeting elastin probes ( Figure 1 A represents the synthetic route for Vanco-Cy5. Figure 1 B is the general synthetic formula for Vanco-Cy3, Vanco-TAMRA, Vanco-Rhodamine, Teico-Cy5, Orita-Cy5, Ramo-Cy5, and Tela-Cy5.

[0080] Figure 2 Mass spectrometry characterization of the probe targeting elastin ( Figure 2 (AE represents the mass spectrometry characterization of Vanco-Cy5, Teico-Cy5, Orita-Cy5, Ramo-Cy5, and Tela-Cy5, respectively).

[0081] Figure 3 Images showing the co-localization of Vanco-Cy5, a probe targeting elastin, with elastin in mouse lungs. Figure 3 A shows the staining results of mouse lung sections using the Vanco-Cy5 probe. Figure 3 B represents the imaging result of elastin autofluorescence. Figure 3 C represents the staining results of mouse lung sections using the elastin-targeting dye Sulforhodamine B. Figure 3 D is a superimposed image of Vanco-Cy5 probe staining, elastin autofluorescence imaging, and elastin SRB staining. Figure 3 E represents the trend of fluorescence intensity changes along the arrow direction after Vanco-Cy5 probe staining, elastin autofluorescence imaging, and elastin dye SRB staining.

[0082] Figure 4 The staining effect of the probe Vanco-Cy5, which targets elastin, on different tissue sections. Figure 4 A represents mouse lung tissue. Figure 4 B represents mouse kidney tissue. Figure 4 C represents mouse bladder tissue. Figure 4 D represents mouse ear cartilage tissue. Figure 4E represents mouse vascular tissue. Figure 4 F represents human vascular tissue.

[0083] Figure 5 The staining results of elastin in mouse lung sections using probes based on different glycopeptides or lipoglycopeptides. Figure 5 (AD images show staining of mouse lung sections with Teico-Cy5, Orita-Cy5, Ramo-Cy5, and Tela-Cy5, respectively).

[0084] Figure 6 Staining results of the probe Vanco-Cy5, which targets elastin, on lung tissue sections from normal and fibrotic mice. Figure 6 A shows the staining results of Hematoxylin-eosin staining, Masson's trichrome staining, and Vanco-Cy5 probe staining on lung sections of normal mice; 6B shows the staining results of Hematoxylin-eosin staining, Masson's trichrome staining, and Vanco-Cy5 probe staining on lung sections of fibrotic mice.

[0085] Figure 7 The staining and diagnostic results of the Vanco-Cy5 probe targeting elastin on pleural invasion in lung sections from patients with non-small cell lung cancer (top image shows Hematoxylin-eosin staining results, middle image shows the staining results of Vanco-Cy5 probe on non-small cell lung cancer sections and TTF-1 antibody on tumor cells, bottom image shows a magnified comparison of Hematoxylin-eosin and Vanco-Cy5 probe staining results in region BD). Detailed Implementation

[0086] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0087] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0088] Example 1: 6.25 mg of vancomycin powder and 3.5 mg of Cy5-NHS powder were dissolved in 200 μL of dimethylformamide, and the mixture was shaken to ensure homogeneity. The two solutions were then mixed under stirring, and 4 μL of triethylamine was added. The mixture was stirred overnight at room temperature to obtain the reaction mixture. The successfully synthesized probe Vanco-Cy5 was separated using high-performance liquid chromatography (HPLC). Eluent A was deionized water containing 0.1% formic acid, and eluent B was acetonitrile containing 0.1% formic acid. The molecular weight of Vanco-Cy5 was characterized using matrix-assisted laser desorption / ionization imaging mass spectrometry (MAMS).

[0089] The above synthetic route is attached. Figure 1 As shown, the mass spectra of the prepared elastin-targeting probe vancomycin-Cy5 (Vanco-Cy5) and the probe prepared using the same method are as follows. Figure 2 As shown.

[0090] Example 2: This invention demonstrates the imaging test of co-localization of Vanco-Cy5 sample from Example 1 with elastin in mouse lung sections. The test steps are as follows: 8 μm thick frozen sections were prepared from lung tissue samples of C57BL / 6 mice (Animal experiment ethics review approval number 2022R0011). The embedding reagent was washed off with PBS, and the sections were blocked with 5% BSA for 30 min. The Vanco-Cy5 probe from Example 1 was added at a concentration of 1 μmol / L, and after incubation for 15 min, the sections were washed twice with PBS. Subsequently, the sections were stained with the elastin-targeting dye Sulforhodamine B at a concentration of 10 mg / mL, incubated for 15 min, and then washed twice with PBS. After mounting with coverslips, the staining was observed under a confocal microscope.

[0091] The images of the Vanco-Cy5 probe targeting elastin and the co-localization of elastin in mouse lung sections are attached. Figure 3 As shown, elastin autofluorescence was excited at a high power at 488 nm, with a receiving and emission wavelength range of 500-560 nm; among which... Figure 3 A shows the staining results of mouse lung tissue sections using the Vanco-Cy5 probe. Figure 3 B represents the imaging result of elastin autofluorescence. Figure 3 C represents the staining results of mouse lung tissue sections with the elastin-targeting dye Sulforhodamine B (SRB). Figure 3 D is a superimposed image of Vanco-Cy5 probe staining, elastin autofluorescence imaging, and elastin SRB staining. Figure 3E represents the fluorescence intensity trends along the arrow directions after Vanco-Cy5 probe staining, elastin autofluorescence imaging, and elastin SRB staining. The staining results indicate that the prepared Vanco-Cy5 probe has the ability to selectively bind elastin, exhibiting high specificity and a high signal-to-noise ratio.

[0092] Example 3: This invention uses the Vanco-Cy5 sample from Example 1 for in vitro tissue section staining imaging testing. The testing steps are as follows: human lung vascular tissue was collected from Zhejiang Cancer Hospital (medical ethics review approval number IRB-2021-447 (Department)); mouse tissue was obtained from C57BL / 6 mice (animal experiment ethics review approval number 2022R0011); frozen sections of the above different tissue samples were prepared with a thickness of 8 μm; the embedding reagent was washed away with PBS; the sections were blocked with 5% BSA for 30 min; the Vanco-Cy5 probe from Example 1 was added at a concentration of 1 μmol / L; after incubation for 15 min, the sections were washed twice with PBS; after mounting with coverslips, the staining was observed under a confocal microscope.

[0093] The staining of the prepared elastin-targeting probe Vanco-Cy5 on different tissue sections is shown in the attached figure. Figure 4 As shown, A represents mouse lung tissue, B represents mouse kidney tissue, C represents mouse bladder tissue, D represents mouse ear cartilage tissue, E represents mouse vascular tissue, and F represents human vascular tissue—all confocal micrographs. The staining results indicate that the Vanco-Cy5 probe can stain elastin in tissues from different species, with a simple operation method, high specificity, and a high signal-to-noise ratio.

[0094] Example 4: This invention applies the staining imaging test of mouse lung tissue sections from the sample in Example 1. The test steps are as follows: The mouse lung tissue was obtained from C57BL / 6 mice, and the animal experiment ethics review approval number is 2022R0011. Frozen sections of the lung tissue sample were prepared with a thickness of 8 μm. The embedding reagent was washed away with PBS, and the sections were blocked with 5% BSA for 30 min. Different probes from Example 1 were added for staining at a concentration of 1 μmol / L. After incubation for 15 min, the sections were washed twice with PBS, sealed with coverslips, and observed under a confocal microscope.

[0095] The staining of mouse lung tissue sections with the prepared elastin-targeting probes Teico-Cy5, Orita-Cy5, Ramo-Cy5, and Tela-Cy5 is shown in the attached figure. Figure 5 As shown, Figure 5AD images show the staining of elastin in mouse lung sections by Teico-Cy5, Orita-Cy5, Ramo-Cy5, and Tela-Cy5, respectively. The staining results indicate that the four probes—Teico-Cy5, Orita-Cy5, Ramo-Cy5, and Tela-Cy5—can also stain lung sections, and the morphology and distribution are similar to those of Vanco-Cy5 staining (see attached image). Figure 4 A) indicates that the probes prepared by this method based on glycopeptide antibiotics and lipoglycopeptide antibiotics can both bind to elastin efficiently and stain elastin on tissue sections.

[0096] Example 5: This invention uses the Vanco-Cy5 sample from Example 1 to test the in vitro tissue section diagnosis of a mouse model of pulmonary fibrosis. The test steps are as follows: A mouse model of pulmonary fibrosis was established, with the animal experiment ethics review approval number 2022R0011. Bleomycin solution was administered intratracheally to the lungs of C57BL / 6 mice, and sterile saline was perfused into the lungs of the mice as a control group. After 14 days of continued feeding, the mouse lungs were removed and 8μm thick frozen sections were prepared. Commercially available Hematoxylin-eosin and Masson's trichrome staining kit were used for staining to determine the occurrence of pulmonary fibrosis. For Vanco-Cy5 staining, the embedding reagent was washed off with PBS, the sections were blocked with 5% BSA for 30 min, the Vanco-Cy5 probe from Example 1 at a concentration of 1μmol / L was added, and after incubation for 15 min, the sections were washed twice with PBS, mounted with coverslips, and observed under a confocal microscope.

[0097] The staining of lung tissue sections from normal and fibrotic mice by the prepared elastin-targeting probe Vanco-Cy5 is shown in the attached figure. Figure 6 As shown, Figure 6 A and B are staining images of lung sections from normal mice and fibrotic mice, respectively, stained with Hematoxylin-eosin, Masson's trichrome, and Vanco-Cy5 probe staining. The staining results indicate that fibrotic lung sections have a higher proportion of elastin compared to normal lung sections (see attached image). Figure 6 Furthermore, the structure of the elastin fibers is more disordered, indicating that the targeted elastin probe prepared by this method can more clearly diagnose pulmonary fibrosis.

[0098] Example 5: This invention uses the Vanco-Cy5 sample from Example 1 to test for diagnosing pleural invasion of lung tumors in patients with non-small cell lung cancer. The test steps are as follows: Lung sections from patients with non-small cell lung cancer were collected from Zhejiang Cancer Hospital, with medical ethics review approval number IRB-2021-447 (Department). The sections were first stained with a commercial Hematoxylin-eosin staining kit to observe the morphology of the tumor tissue. For Vanco-Cy5 staining, the sections were dewaxed and rehydrated, and blocked with 5% BSA for 60 min; 150 μL of thyroid transcription factor 1 (TTF-1) antibody diluted 1:100 was incubated overnight at 4°C, followed by washing twice with PBS; staining was performed with the corresponding secondary antibody for 60 min, followed by washing twice with PBS again; the Vanco-Cy5 probe from Example 1 was added at a concentration of 1 μmol / L, incubated for 15 min, washed twice with PBS, and then the slides were mounted with coverslips and observed under a confocal microscope.

[0099] The staining of lung sections from non-small cell lung cancer patients with the prepared elastin-targeting probe Vanco-Cy5 is shown in the attached figure. Figure 7 As shown in the image (top image shows Hematoxylin-eosin staining, middle image shows Hoechst, Vanco-Cy5 (elastin), and TTF-1 antibody staining (tumor cells), bottom image shows magnified comparison of regions B, C, and D), the staining results indicate that the Vanco-Cy5 probe accurately displays the elastin layer of the visceral pleura. The TTF-1 assay shows that tumor cells gradually invade the pleura (regions B and C), and then break through the pleura to the outside of the pleura (region D), proving that the tumor cells have breached the visceral pleura of the lung. Similarly, it can be observed that the elastin layer of the pleura thickens and the elastin fibers proliferate and thicken to resist tumor cell invasion (region D). This indicates that the novel probe targeting elastin prepared by this method can assist in the diagnosis of visceral pleural invasion of lung tumors in patients with non-small cell lung cancer, and the diagnostic results are relatively intuitive and have high accuracy.

[0100] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that the invention and its improvements, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of fluorescent probes based on glycopeptide or lipoglycopeptide antibiotics in the preparation of drugs, wherein the drugs are used for imaging or diagnosis of tissue sections, and the fluorescent probes based on glycopeptide or lipoglycopeptide antibiotics are compounds having the structure shown in formula (I) below or pharmaceutical salts thereof: T-(B1-L-B2-R)n (I) Where T is the targeting group that targets elastin, and R is the functional group; B1 is a linker or direct bond between L and the target group T; L represents a connecting arm or is not present; B2 is a linker or direct bond between functional groups R and L; B2 is a direct bond, meaning that L is part of the functional group R or does not exist; B1 is a direct bond, meaning that L is part of the target group T or does not exist; When L does not exist, R is connected to T through B1 or B2; T represents a glycopeptide antibiotic or a lipoglycopeptide antibiotic molecule; R represents the fluorescence imaging component; n is an integer from 1 to 10.

2. The application as described in claim 1, Where B1 is a covalent bond; B2 is a linker formed in a covalent or non-covalent manner. The covalent linking method is selected from click chemistry, cross-linking, or condensation reaction. The non-covalent linking method is selected from electrostatic interaction, hydrophobic interaction, affinity interaction, antibody-antigen binding, or biotin-avidin binding.

3. The application as described in claim 2, wherein B1 is an amide bond or an ester bond.

4. The application as described in claim 1, L has 1-100 connecting atoms and optionally includes an ethyleneoxy ((CH2CH2O)) moiety, amine, ester, amide, ketone, urea, carbamate or carbonate functional group.

5. The application as described in claim 4, Where L is a straight-chain or branched alkyl group with 2-20 carbon atoms; or a polyethylene glycol group with 2-10 ethyleneoxy ((CH2CH2O)) units.

6. The application of claim 1, wherein the fluorescence imaging portion is a fluorescent compound or nanoaggregate excited by a first radiation wavelength and detected by a second radiation wavelength, the first radiation wavelength and the second radiation wavelength being different, and the fluorescent compound or nanoaggregate being selected from visible light or near-infrared fluorescent probes.

7. The application as described in claim 6, wherein the fluorescence imaging component is selected from one or more of the following: fluorescein molecules, coumarin molecules, rhodamine molecules, anthocyanin dyes, indocyanine green dyes, BODIPY molecules, squaric acid molecules, squaric acid cyanine molecules, phosphorescent molecules, semiconductor molecules, carbon quantum dots, silicon quantum dots, sulfur quantum dots, selenium quantum dots, tellurium quantum dots, phosphorus quantum dots, perovskite quantum dots, upconversion rare earth nanomaterials, long afterglow nanomaterials, or chelating agents.

8. The application as claimed in claim 1, wherein the glycopeptide antibiotic or lipoglycopeptide antibiotic molecule is selected from: Vancomycin antibiotic, structure: ; Teicoplanin antibiotic, structure: , Where R' is , , , or ; The structure of the antibiotic tervacin is: ; Dabavancin antibiotic, structure: ; Orivacin antibiotic, structure: ; Itonamycin antibiotic, structure: , Where R'' is H or Cl; Balhemycin antibiotic, structure: ; Ristoxin antibiotic, structure: ; Or ramoranine antibiotic, with the following structure: , Where R''' is , or In the above substituents, T represents the linking site of the group.

9. The application as described in claim 1, The probe is selected from: The structure of the vancomycin-based antibiotic probe is as follows: ; The probe based on the antibiotic teicoplanin has the following structure: , Where R' is , , , or ; The probe based on the antibiotic tervacin has the following structure: ; The probe based on the antibiotic dabavancin has the following structure: ; The probe based on the antibiotic orivancin has the following structure: ; The probe based on the antibiotic irhinomycin has the following structure: , Where R'' is H or Cl; The probe based on the balhexamethylenetetramine antibiotic has the following structure: ; The probe based on ristomycin has the following structure: ; Alternatively, a probe based on remolanine may be constructed as follows: , Where R''' is , or ; in, In the above substituents, T represents the group linkage site, and R1 to R... 27 Each of the above formulas independently represents an optical dye modification or H, provided that at least one optical dye modification is present in each of the above formulas.

10. The application according to any one of claims 1-2, wherein the probe is present in the form of a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is an acid addition salt selected from acetate, adipic acidate, alginate, citrate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, trimethylbenzenesulfonate, methanesulfonate, naphthylsulfonate, nicotinate, etc. 2-Naphthalenesulfonate, oxalate, palmitate, pectin ester, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, or undecanoate; or the pharmaceutically acceptable salt is a basic addition salt, wherein the cation of the basic addition salt is selected from lithium, sodium, potassium, calcium, magnesium, and aluminum, or a non-toxic quaternary ammonium cation.

11. The application as described in any one of claims 1-2, wherein the tissue section is selected from animal tissue sections or human pathological tissue sections, and the imaging and diagnosis are performed by staining elastin with the probe.

12. The application as described in any one of claims 1-2, wherein the tissue section is selected from benign tumor tissue, malignant tumor tissue, orthopedic disease tissue, fibrotic tissue, inflammatory tissue, proliferative tissue, atherosclerotic tissue, cirrhotic tissue, skin photodamaged tissue, skin thermally damaged tissue, skin acid and alkali corroded tissue, or radiation-damaged tissue.

13. The application as described in any one of claims 1-2, wherein the tissue section is a frozen section or a paraffin section.

14. A tissue section imaging kit comprising a fluorescent probe based on a glycopeptide antibiotic or a lipoglycopeptide antibiotic, and a reagent selected from at least one of a tissue cleanser, a tissue clearing agent, a tissue immobilization agent, a blocking agent, or an antifluorescence quenching mounting medium, wherein the fluorescent probe based on the glycopeptide antibiotic or the lipoglycopeptide antibiotic is the probe described in any one of claims 1-9 or a pharmaceutically acceptable salt thereof.

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