A rare earth nano fluorescent probe targeting endoplasmic reticulum of tumors and its preparation method and application

Abstract

The present invention provides a tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe and its preparation method and application. The tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe comprises rare earth inorganic nanocrystals and water-soluble ligands loaded on the surface of rare earth inorganic nanocrystals, water-soluble ligands modified by tumor targeting molecules and water-soluble ligands modified by endoplasmic reticulum targeting molecules; the rare earth inorganic nanocrystals have a core-shell-shell structure, and the core-shell-shell structure is a rare earth luminescent core NaGdF4:Yb / Er / Ce, a rare earth ion shell NaYbF4 and a rare earth inert protective layer NaYF4 in sequence. The tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe provided by the present invention has good dispersibility, stability and tumor endoplasmic reticulum targeting ability, can realize dynamic sorting of tumor cells and normal cells, and perform high-resolution rare earth nano fluorescent medical imaging on solid tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biofluorescent probes, and in particular relates to a tumor endoplasmic reticulum-targeted rare earth nano-fluorescent probe and a preparation method and application thereof. Background Art

[0002] As we all know, cancer treatment has always been a difficult problem to be overcome, and the lack of selectivity and specific targeting of drugs is the main obstacle to systemic drug delivery. To overcome this problem, researchers are exploring strategies that can precisely act on specific tissues and cells. In addition, the transport and mechanism of action of drugs within cells are also key links in drug delivery. After entering the cell, many drugs need to interact with molecular targets in organelles, but random organelle interactions may lead to unnecessary toxicity. Therefore, achieving precise drug delivery at the organelle level to maximize the therapeutic effect and minimize toxic side effects is the key to improving treatment efficiency.

[0003] The endoplasmic reticulum is a central organelle with many important functions, such as synthesizing and folding proteins, modifying secretory and transmembrane proteins, regulating lipid synthesis and metabolism, storing calcium, and mediating signal transduction. Therefore, the strict regulation of endoplasmic reticulum function is crucial for maintaining cell homeostasis and determining cell fate. In view of this, targeted regulation of the endoplasmic reticulum with higher controllability and efficiency is expected to provide a new perspective for the treatment of endoplasmic reticulum-related diseases or cancer. However, the existing endoplasmic reticulum-targeted fluorescent probes are usually poorly water-soluble and highly cytotoxic, which may interfere with the physiological processes of proteins or cells. Some of the lipophilic fluorescent probes that have been developed face the problems of poor photostability and easy quenching. These shortcomings limit the effectiveness of endoplasmic reticulum-targeted probes in biomedical research and clinical applications.

[0004] Rare earth-doped inorganic nanofluorescent materials have become a strong candidate for the new generation of biological fluorescent probes due to their low biological toxicity, no background autofluorescence, high stability and excellent imaging resolution. Therefore, the development of tumor endoplasmic reticulum-targeted probes based on rare earth inorganic nanofluorescent materials not only has important scientific research value, but also has clinical application potential, and is expected to bring new progress in the diagnosis and treatment of diseases such as cancer. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a tumor endoplasmic reticulum targeted rare earth nanofluorescent probe and its preparation method and application. The tumor endoplasmic reticulum targeted rare earth nanofluorescent probe provided by the present invention can accurately target the tumor endoplasmic reticulum for imaging, realize real-time dynamic sorting of tumor cells and normal cells, and perform tumor resection surgery navigation.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe, which comprises rare earth inorganic nanocrystals and water-soluble ligands loaded on the surface of the rare earth inorganic nanocrystals, water-soluble ligands modified with tumor targeting molecules, and water-soluble ligands modified with endoplasmic reticulum targeting molecules;

[0008] The rare earth inorganic nanocrystal has a core-shell-shell structure, and the core-shell-shell structure is composed of a rare earth luminescent core NaGdF4:Yb / Er / Ce, a rare earth ion shell NaYbF4 and a rare earth inert protective layer NaYF4.

[0009] The tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe provided by the present invention has good dispersibility, medium stability, photostability, biocompatibility and tumor endoplasmic reticulum-targeting ability; under the irradiation of 980nm excitation light, it simultaneously generates luminescence in the visible light region (400-700nm) and the near-infrared light region (800-1700nm), can perform laser confocal imaging of tumor cells, realize cell level visualization, perform dynamic sorting of tumor cells and normal cells, and perform high-resolution rare earth nanofluorescent medical imaging of living organisms in solid tumor lesions or micro-metastatic tumor lesions.

[0010] Preferably, the particle size of the rare earth inorganic nanocrystals is 20-50 nm, for example, it can be 20 nm, 22 nm, 25 nm, 28 nm, 30 nm, 32 nm, 35 nm, 38 nm, 40 nm, 42 nm, 45 nm, 48 nm, 50 nm, etc.

[0011] Preferably, the mass ratio of the rare earth luminescent core, the rare earth ion shell and the rare earth inert protective layer is 1:(1.5-2.5):(3.5-4.5); the specific point values ​​in (1.5-2.5) can be 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, etc.; the specific point values ​​in (3.5-4.5) can be 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, etc.

[0012] Preferably, the molar percentage of Er in the rare earth luminescent core NaGdF4:Yb / Er / Ce to all rare earth elements is (1-5) mol%, for example, it can be 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, etc.

[0013] Preferably, the molar ratio of the water-soluble ligand, the water-soluble ligand modified with the tumor targeting molecule and the water-soluble ligand modified with the endoplasmic reticulum targeting molecule is (0.1-0.8):(0.02-0.1):(0.02-0.1); the specific point values ​​in (0.1-0.8) can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, etc.; the specific point values ​​in (0.02-0.1) can be 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc.

[0014] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0015] Preferably, the water-soluble ligand is distearoylphosphatidylethanolamine-polyethylene glycol.

[0016] Preferably, the number average molecular weight of the polyethylene glycol is 1000-5000, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, etc.

[0017] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0018] Preferably, the tumor targeting molecule is a cell-targeting penetrating peptide.

[0019] Preferably, the cell-targeting penetrating peptide is selected from any one or a combination of at least two of iRGD, cRGD, TAT, R9 or LMWP.

[0020] Preferably, the endoplasmic reticulum targeting molecule is p-toluenesulfonamide.

[0021] Preferably, the particle size of the tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe is 50-150 nm, for example, it can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, etc.

[0022] Preferably, the Zeta potential of the tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe is 10-50 mV, for example, it may be 10 mV, 15 mV, 20 mV, 25 mV, 30 mV, 35 mV, 40 mV, 45 mV, 50 mV, etc.

[0023] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0024] In a second aspect, the present invention provides a method for preparing the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe as described in the first aspect, the preparation method comprising:

[0025] (1) preparing rare earth luminescent core;

[0026] (2) using a layer-by-layer epitaxial growth method to sequentially grow a rare earth ion shell layer and a rare earth inert protective layer on the surface of the rare earth luminescent core to obtain a rare earth inorganic nanocrystal;

[0027] (3) Loading water-soluble ligands, water-soluble ligands modified with tumor targeting molecules, and water-soluble ligands modified with endoplasmic reticulum targeting molecules on the surface of the rare earth inorganic nanocrystals to obtain the tumor endoplasmic reticulum targeting rare earth nano fluorescent probe.

[0028] The method for preparing the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe provided by the present invention is simple to operate and has good repeatability. The prepared fluorescent probe has good biosafety and biocompatibility, exhibits excellent luminescence performance and photostability, and ensures its reliability and durability in practical applications.

[0029] Preferably, the specific steps of preparing the rare earth luminescent core include:

[0030] The gadolinium salt, ytterbium salt, erbium salt, cerium salt, sodium trifluoroacetate and solvent are mixed, the temperature is raised to the reaction temperature, the reaction is carried out, the mixture is cooled, and the mixture is centrifuged to obtain the product.

[0031] Preferably, the anions of the gadolinium salt, ytterbium salt, erbium salt or cerium salt are each independently selected from any one of trifluoroacetate ion, acetate ion, nitrate ion, chloride ion or sulfate ion.

[0032] Preferably, the gadolinium salt, ytterbium salt, erbium salt and cerium salt are gadolinium trifluoroacetate, ytterbium trifluoroacetate, erbium trifluoroacetate and cerium acetate, respectively.

[0033] Preferably, the molar ratio of the gadolinium salt, ytterbium salt, erbium salt, cerium salt and sodium trifluoroacetate is (0.25-1.25):(0.05-0.75):(0.005-0.05):(0.005-0.05):(0.5-1.5); the specific point values ​​in (0.25-1.25) can be 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, etc.; (0.05-0.75) The specific point values ​​can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, etc.; the specific point values ​​in (0.005-0.05) can be 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, etc.; the specific point values ​​in (0.5-1.5) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0034] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0035] Preferably, the solvent comprises any one or a combination of at least two of 1-octadecene, oleylamine or oleic acid.

[0036] Preferably, the solvent comprises 1-octadecene, oleylamine and oleic acid.

[0037] Preferably, the volume ratio of 1-octadecene, oleylamine and oleic acid is (1-10):(1-10):(1-10); the specific point values ​​in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0038] Preferably, the reaction is carried out under nitrogen, and the reaction temperature is 290-320°C, for example, 290°C, 295°C, 300°C, 305°C, 310°C, 315°C, 320°C, etc.; the reaction time is 0.5-1.5h, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, etc.

[0039] Preferably, the cooling temperature is 20-30°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.

[0040] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0041] Preferably, washing is performed after the centrifugation.

[0042] Preferably, the specific steps of growing the rare earth ion shell layer include:

[0043] The ytterbium salt, sodium trifluoroacetate and solvent are mixed, the temperature is raised to the reaction temperature, the reaction is carried out, the mixture is cooled and centrifuged to obtain the product.

[0044] Preferably, the anion of the ytterbium salt is selected from any one of trifluoroacetate ion, acetate ion, nitrate ion, chloride ion or sulfate ion.

[0045] Preferably, the ytterbium salt is ytterbium trifluoroacetate.

[0046] Preferably, the molar ratio of the ytterbium salt to sodium trifluoroacetate is (0.5-1.5):(0.5-1.5); the specific value of (0.5-1.5) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0047] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0048] Preferably, the solvent comprises 1-octadecene and / or oleic acid.

[0049] Preferably, the solvent comprises 1-octadecene and oleic acid.

[0050] Preferably, the volume ratio of 1-octadecene to oleic acid is (1-10):(1-10); the specific point values ​​in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0051] Preferably, the reaction is carried out under nitrogen, and the reaction temperature is 290-320°C, for example, 290°C, 295°C, 300°C, 305°C, 310°C, 315°C, 320°C, etc.; the reaction time is 0.5-1.5h, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, etc.

[0052] Preferably, the cooling temperature is 20-30°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.

[0053] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0054] Preferably, washing is performed after the centrifugation.

[0055] Preferably, the specific steps of growing the rare earth inert protective layer include:

[0056] The yttrium salt, sodium trifluoroacetate and solvent are mixed, the temperature is raised to the reaction temperature, the reaction is carried out, the mixture is cooled and centrifuged to obtain the product.

[0057] Preferably, the anion of the yttrium salt is selected from any one of trifluoroacetate ion, acetate ion, nitrate ion, chloride ion or sulfate ion.

[0058] Preferably, the yttrium salt is yttrium trifluoroacetate.

[0059] Preferably, the molar ratio of the yttrium salt to sodium trifluoroacetate is (0.5-1.5):(0.5-1.5); the specific value of (0.5-1.5) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0060] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0061] Preferably, the solvent comprises 1-octadecene and / or oleic acid.

[0062] Preferably, the solvent comprises 1-octadecene and oleic acid.

[0063] Preferably, the volume ratio of 1-octadecene to oleic acid is (1-10):(1-10); the specific point values ​​in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0064] Preferably, the reaction is carried out under nitrogen, and the reaction temperature is 290-320°C, for example, 290°C, 295°C, 300°C, 305°C, 310°C, 315°C, 320°C, etc.; the reaction time is 0.5-1.5h, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, etc.

[0065] Preferably, the cooling temperature is 20-30°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.

[0066] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0067] Preferably, washing is performed after the centrifugation.

[0068] Preferably, the specific steps of loading the water-soluble ligand, the water-soluble ligand modified with the tumor targeting molecule and the water-soluble ligand modified with the endoplasmic reticulum targeting molecule include:

[0069] The rare earth inorganic nanocrystals are dispersed in an organic solvent, water-soluble ligands, water-soluble ligands modified by tumor targeting molecules and water-soluble ligands modified by endoplasmic reticulum targeting molecules are added, and stirred for reaction to obtain the tumor endoplasmic reticulum targeting rare earth nano fluorescent probe.

[0070] Preferably, a post-treatment step is performed after the reaction.

[0071] Preferably, the post-processing includes washing and sterilization.

[0072] Preferably, the aseptic treatment comprises re-dispersing the product into physiological saline or sterile ultrapure water and performing aseptic filtration and / or ultraviolet irradiation sterilization.

[0073] Preferably, the organic solvent is selected from any one of chloroform, dichloromethane or cyclohexane.

[0074] In a third aspect, the present invention provides a use of the tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe as described in the first aspect in preparing a tumor cell visualization laser confocal imaging agent or in preparing a solid tumor fluorescent imaging agent.

[0075] The tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe provided by the present invention is more inclined to target gastric cancer cells. The solid tumor includes micro-metastatic tumor lesions, and the micro-metastatic tumor lesions refer to metastatic tumor lesions with a size less than 1 mm.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] (1) The tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe provided by the present invention has good dispersibility, medium stability, photostability, biocompatibility and tumor endoplasmic reticulum targeting ability, and can accurately target the location of the endoplasmic reticulum of tumor cells;

[0078] (2) The tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe provided by the present invention can simultaneously generate light in the visible light region (400-700nm) and the near-infrared light region (800-1700nm) under irradiation with 980nm excitation light, and can implement visualized laser confocal cell imaging of tumor cells, realize real-time dynamic sorting of tumor cells and normal cells, and perform high-resolution rare earth nanofluorescent medical imaging of solid tumor lesions or micro-metastatic tumor lesions in living organisms as well as tumor resection surgery navigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1X-ray diffraction pattern of the rare earth inorganic nanocrystal NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 prepared in Example 1;

[0080] Figure 2 This is a transmission electron microscope image of the rare earth inorganic nanocrystal NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 prepared in Example 1;

[0081] Figure 3 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Transmission electron microscope image in water;

[0082] Figure 4 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Particle size distribution diagram;

[0083] Figure 5 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Fluorescence spectrum under 980nm excitation light;

[0084] Figure 6 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER , Fourier transform infrared spectra of water-soluble rare earth inorganic nanocrystals LnNPs without tumor endoplasmic reticulum targeting capability, tumor targeting molecule iRGD and endoplasmic reticulum targeting molecule PTSC prepared in Comparative Example 1;

[0085] Figure 7 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Zeta potential diagram of water-soluble rare earth inorganic nanocrystals LnNPs without tumor endoplasmic reticulum targeting capability prepared in Comparative Example 1;

[0086] Figure 8 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER and near-infrared photostability profile of the near-infrared fluorescent probe ICG;

[0087] Fig. 9 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Media stability profiles in fetal bovine serum and saline;

[0088] Fig.10 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Endoplasmic reticulum-targeted fluorescence imaging of normal human gastric cells and human gastric cancer cells;

[0089] Fig.11 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER The endoplasmic reticulum-targeted fluorescence imaging of human gastric cancer cells by water-soluble rare earth inorganic nanocrystals LnNPs prepared in Comparative Example 1 that do not have the ability to target the tumor endoplasmic reticulum;

[0090] Fig.12 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Cell survival rate results after incubation with normal human gastric cells for 12h, 24h, and 48h;

[0091] Fig.13 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Tumor fluorescence imaging images (a), (b) and tumor tissue pathology detection image (c) in the human gastric cancer cell nude mouse abdominal metastasis model under 980nm excitation light;

[0092] Fig.14 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER Fluorescence imaging of micro-tumors in a nude mouse abdominal metastasis model of human gastric cancer cells under 980nm excitation light (a) and tumor tissue pathology detection (b);

[0093] Fig.15 Tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 ER HE staining of the heart, liver, spleen, lung, and kidney of nude mice injected with human gastric cancer cells and healthy mice without any injection. DETAILED DESCRIPTION

[0094] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0095] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0096] The sources of the instruments used in the following specific embodiments are as follows:

[0097] name factory model X-ray powder diffractometer Rigaku MiniFlex2 Transmission electron microscopy FEI TECNAI G2F20 Fourier Transform Infrared Spectrometer Bruker VERTEX 70 Spectrometer Edinburgh FLS980 Laser particle size analyzer Malvern Zetasizer Ultra Confocal imaging microscopy Nikon A1RHD25 Full-spectrum small animal in vivo fluorescence imaging system RayLight NirVivo Mix Small Animal Fluorescence Microscopy System RayLight NirVivo Pro

[0098] Example 1

[0099] This embodiment provides a tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe LnNPs ER , and its preparation method is as follows:

[0100] (1) Preparation of rare earth luminescent core (NaGdF4:Yb / Er / Ce): weigh 0.136 g sodium trifluoroacetate, 0.3673 g gadolinium trifluoroacetate, 0.1024 g ytterbium trifluoroacetate, 0.0101 g erbium trifluoroacetate and 0.0127 g cerium acetate, add 6.5 mL 1-octadecene, 3.5 mL oleylamine and 3.2 mL oleic acid, mix well, heat to 310°C in nitrogen, react for 30 min, stop heating, cool to 25°C, collect precipitate by centrifugation and wash, and obtain;

[0101] (2) Preparation of core-shell structured nanocrystals (abbreviated as NaGdF4:Yb / Ce / Er@NaYbF4): Add the rare earth luminescent core NaGdF4:Yb / Er / Ce to 6.5 mL of 1-octadecene and 6.5 mL of oleic acid, add 0.136 g of sodium trifluoroacetate and 0.512 g of ytterbium trifluoroacetate, mix well, heat to 310°C in nitrogen, react for 30 min, stop heating, cool to 25°C, collect the precipitate by centrifugation and wash, and obtain the nanocrystals;

[0102] (3) Preparation of core-shell-shell structured rare earth inorganic nanocrystals (abbreviated as NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4): Add core-shell structured nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4 to 6.5 mL of 1-octadecene and 6.5 mL of oleic acid, add 0.136 g of sodium trifluoroacetate and 0.428 g of yttrium trifluoroacetate, mix well, heat to 310°C in nitrogen, react for 30 min, stop heating, cool to 25°C, collect the precipitate by centrifugation and wash, and obtain the product;

[0103] (4) Preparation of tumor endoplasmic reticulum-targeted rare earth nanofluorescent probes (abbreviated as LnNPs) ER ): 10 mg of rare earth inorganic nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 was dispersed in 20 mL of chloroform, 30 mg of DSPE-PEG, 10 mg of DSPE-PEG-iRGD, and 10 mg of DSPE-PEG-PTSC were added, and the mixture was stirred for reaction. After the organic solvent was completely evaporated, deionized water was added for redispersion, and the precipitate was collected by centrifugation and washed. Finally, the product was redispersed in physiological saline for sterile filtration and storage to obtain tumor endoplasmic reticulum-targeted rare earth nano fluorescent probes LnNPs. ER The concentration is 10 mg / mL.

[0104] Example 2

[0105] This embodiment provides a tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe LnNPs ER , and its preparation method is as follows:

[0106] (1) Preparation of rare earth luminescent core (NaGdF4:Yb / Er / Ce): weigh 0.109 g of sodium trifluoroacetate, 0.2978 g of gadolinium trifluoroacetate, 0.0512 g of ytterbium trifluoroacetate, 0.0151 g of erbium trifluoroacetate and 0.010 g of cerium acetate, add 6.5 mL of 1-octadecene, 3 mL of oleylamine and 3.5 mL of oleic acid, mix well, heat to 300°C in nitrogen, react for 40 min, stop heating, cool to 24°C, collect the precipitate by centrifugation and wash, and obtain;

[0107] (2) Preparation of core-shell structured nanocrystals (abbreviated as NaGdF4:Yb / Ce / Er@NaYbF4): rare earth luminescent core NaGdF4:Yb / Er / Ce was added to 7 mL of 1-octadecene and 6 mL of oleic acid, and 0.109 g of sodium trifluoroacetate and 0.4609 g of ytterbium trifluoroacetate were added. The mixture was mixed evenly, and the mixture was heated to 300 °C in nitrogen. After reacting for 40 min, the mixture was stopped from heating and cooled to 24 °C. The precipitate was collected by centrifugation and washed.

[0108] (3) Preparation of core-shell-shell structured rare earth inorganic nanocrystals (abbreviated as NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4): Add core-shell structured nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4 to 7 mL of 1-octadecene and 6 mL of oleic acid, add 0.109 g of sodium trifluoroacetate and 0.3852 g of yttrium trifluoroacetate, mix well, heat to 300°C in nitrogen, react for 40 min, stop heating, cool to 24°C, collect the precipitate by centrifugation and wash, and obtain;

[0109] (4) Preparation of tumor endoplasmic reticulum-targeted rare earth nanofluorescent probes (abbreviated as LnNPs) ER ): 10 mg of rare earth inorganic nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 was dispersed in 25 mL of dichloromethane, 35 mg of DSPE-PEG, 15 mg of DSPE-PEG-iRGD, and 15 mg of DSPE-PEG-PTSC were added, and the mixture was stirred for reaction. After the organic solvent was completely evaporated, deionized water was added for redispersion, and the precipitate was collected by centrifugation and washed. Finally, the product was redispersed in physiological saline for sterile filtration and storage to obtain tumor endoplasmic reticulum-targeted rare earth nano fluorescent probes LnNPs. ER The concentration is 10 mg / mL.

[0110] Example 3

[0111] This embodiment provides a tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe LnNPs ER , and its preparation method is as follows:

[0112] (1) Preparation of rare earth luminescent core (NaGdF4:Yb / Er / Ce): weigh 0.1496 g of sodium trifluoroacetate, 0.3473 g of gadolinium trifluoroacetate, 0.0768 g of ytterbium trifluoroacetate, 0.0202 g of erbium trifluoroacetate and 0.009 g of cerium acetate, add 6 mL of 1-octadecene, 4 mL of oleylamine and 4 mL of oleic acid, mix well, heat to 320°C in nitrogen, react for 50 min, stop heating, cool to 26°C, collect precipitate by centrifugation and wash, and obtain;

[0113] (2) Preparation of core-shell structured nanocrystals (abbreviated as NaGdF4:Yb / Ce / Er@NaYbF4): Add the rare earth luminescent core NaGdF4:Yb / Er / Ce to 6 mL of 1-octadecene and 7 mL of oleic acid, add 0.1496 g of sodium trifluoroacetate and 0.622 g of ytterbium trifluoroacetate, mix well, heat to 320°C in nitrogen, react for 50 min, stop heating, cool to 26°C, collect the precipitate by centrifugation and wash, and obtain the nanocrystals;

[0114] (3) Preparation of core-shell-shell structured rare earth inorganic nanocrystals (abbreviated as NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4): Add core-shell structured nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4 to 6 mL of 1-octadecene and 7 mL of oleic acid, add 0.1496 g of sodium trifluoroacetate and 0.513 g of yttrium trifluoroacetate, mix well, heat to 320°C in nitrogen, react for 50 min, stop heating, cool to 26°C, collect the precipitate by centrifugation and wash, and obtain the product;

[0115] (4) Preparation of tumor endoplasmic reticulum-targeted rare earth nanofluorescent probes (abbreviated as LnNPs) ER ): 10 mg of rare earth inorganic nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 was dispersed in 15 mL of cyclohexane, 30 mg of DSPE-PEG, 15 mg of DSPE-PEG-iRGD, and 15 mg of DSPE-PEG-PTSC were added, and the mixture was stirred for reaction. After the organic solvent was completely evaporated, deionized water was added for redispersion, and the precipitate was collected by centrifugation and washed. Finally, the product was redispersed in physiological saline for sterile filtration and storage to obtain tumor endoplasmic reticulum-targeted rare earth nanofluorescent probes LnNPs. ER The concentration is 10 mg / mL.

[0116] Comparative Example 1

[0117] This comparative example provides a water-soluble rare earth inorganic nanocrystal (abbreviated as LnNPs) that does not have the ability to target the tumor endoplasmic reticulum. The only difference between it and Example 1 is that only 50 mg DSPE-PEG is added in step (4), and DSPE-PEG-iRGD and DSPE-PEG-PTSC are not added. The other steps remain unchanged.

[0118] Test Example 1 Composition and particle size of rare earth inorganic nanocrystals, LnNPs ER Particle size and fluorescence performance analysis

[0119] X-ray diffraction and transmission electron microscopy analysis were performed on the rare earth inorganic nanocrystals NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 prepared in Example 1; the tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe LnNPs prepared in Example 1 ER Analysis by transmission electron microscopy and fluorescence detection was performed.

[0120] Test results: Figure 1 This is the X-ray diffraction pattern of NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 prepared in Example 1. Figure 2 The transmission electron microscope image of NaGdF4:Yb / Ce / Er@NaYbF4@NaYF4 prepared in Example 1 shows that the particle size of the rare earth inorganic nanocrystals is 30 nm; Figure 3 LnNPs prepared in Example 1 ER The transmission electron microscope image in water shows that there is no agglomeration and it has good dispersibility. Figure 4 LnNPs prepared in Example 1 ER The particle size distribution diagram shows that the particle size is 100nm and the PDI value is 0.234, indicating that the size is uniform. Figure 5 LnNPs prepared in Example 1 ER The fluorescence spectrum under 980nm excitation light can produce good upconversion and near-infrared luminescence under 980nm excitation light.

[0121] Test Example 2 Loading of Tumor Endoplasmic Reticulum Targeting Molecules

[0122] The tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 were tested respectively. ER , Comparative Example 1 prepared water-soluble rare earth inorganic nanocrystals LnNPs without tumor endoplasmic reticulum targeting ability, tumor targeting molecule iRGD and endoplasmic reticulum targeting molecule PTSC Fourier transform infrared spectra, comparative analysis of LnNPs ER Whether there are characteristic peaks of iRGD and PTSC in LnNPs ER And the changes of Zeta potential of LnNPs.

[0123] Test results: Figure 6 It can be seen that LnNPs ER There are -OH vibration peaks of iRGD and benzene ring vibration peaks and S=O vibration peaks of PTSC; Figure 7 It can be seen that LnNPs ER Compared with the Zeta potential of LnNPs, the potential changed from negative to positive, indicating that the tumor endoplasmic reticulum targeting molecules were successfully loaded on the rare earth inorganic nanocrystals.

[0124] Test Example 3 Near-infrared light stability

[0125] The tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 after freeze-drying were continuously irradiated with a 980 nm laser ER The powder or the FDA-approved commercial near-infrared fluorescent dye ICG was continuously irradiated with a xenon lamp, and its luminescence intensity at 1532 nm was detected by a fluorescence spectrometer every 5 minutes within 2 hours to analyze the photochemical stability.

[0126] Test results: Figure 8 It can be seen that LnNPs ER It has good near-infrared light stability.

[0127] Test Example 4: Medium Stability

[0128] 200 μg of the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 were added ER Mix with 3 mL of normal saline (abbreviated as NS) or fetal bovine serum (abbreviated as FBS), incubate at 37°C, take out 40 μL of the solution at intervals within 3 days, and analyze the medium stability of the material by laser particle size analyzer.

[0129] Test results: Fig. 9 It can be seen that LnNPs ER It has good medium stability in both normal saline and fetal bovine serum.

[0130] Test Example 5 Targeting to Normal Human Gastric Cells and Human Gastric Cancer Cells

[0131] Human normal gastric cells GES-1 and human gastric cancer cells MGC-803 were purchased from Fenghui Biotechnology Co., Ltd. (China) and cultured in 1640 medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37°C and 5% CO2. GES-1 cells and MGC-803 cells were cultured at 2×10 4 / well in a confocal culture dish, and after they are completely attached to the wall, 10 μL of 10 mg / mL tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 is added. ERAfter incubation at 37 °C for 6 h, the old culture medium was discarded, the cells were washed twice with PBS buffer, commercial endoplasmic reticulum dye was added and incubated at 37 °C for 15 min, washed three times with PBS buffer, fixed with 4% paraformaldehyde for 5 min, washed twice with PBS buffer, stained with DAPI for 5 min, and washed three times with PBS buffer before observing the cell targeting under a confocal microscope.

[0132] Test results: Fig.10 It can be seen that compared with normal human gastric cells, LnNPs ER It is more likely to target human gastric cancer cells.

[0133] Test Example 6 Tumor endoplasmic reticulum targeting of human gastric cancer cells

[0134] MGC-803 cells were cultured at 2×10 4 / well were spread in a confocal culture dish, and after they were completely attached to the wall, 10 μL of 10 mg / mL tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 were added. ER Or the water-soluble rare earth inorganic nanocrystals LnNPs without tumor endoplasmic reticulum targeting ability prepared in Comparative Example 1 were incubated at 37° C. for 6 hours, the old culture medium was discarded, the cells were washed twice with PBS buffer, commercial endoplasmic reticulum dye was added and incubated at 37° C. for 15 min, the cells were washed three times with PBS buffer, and then fixed with 4% paraformaldehyde for 5 min, washed twice with PBS buffer, stained with DAPI for 5 min, and washed three times with PBS buffer before observing the tumor endoplasmic reticulum targeting under a confocal microscope.

[0135] Test results: Fig.11 It can be seen that LnNPs ER It has a colocalization effect with commercial endoplasmic reticulum dyes, and the Pearson colocalization coefficient is 0.86, which proves that it can be well targeted to the endoplasmic reticulum of tumor cells and used for endoplasmic reticulum imaging of tumor cells.

[0136] Test Example 7 In vitro biological safety

[0137] GES-1 cells were cultured at 4 × 10 4 / well in a 96-well plate, and after they are completely attached to the wall, the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe LnNPs prepared in Example 1 are added. ERThe concentrations were 2 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 600 μg / mL, and three replicate wells were set for each drug addition group. After incubation at 37°C for 12 h, 24 h, and 48 h, the old culture medium was discarded, and fresh culture medium was replaced. 10 μL CCK-8 working solution was added to each well, incubated in a 37°C incubator for 1 h, and then the absorbance at 450 nm was detected by an enzyme marker to calculate the cell activity of each group. At the same time, a blank group and a control group were set on a 96-well plate, where the control group refers to the group without the addition of LnNPs. ER , the blank group refers to only CCK-8 working solution; cell activity (%) = [(OD value of drug-added group-OD value of blank group) / (OD value of control group-OD value of blank group)] × 100%.

[0138] Test results: Fig.12 It can be seen that in the range of 0-600 μg / mL LnNPs ER Within this range, the survival rate of normal human gastric cells remained above 80% even 48 hours after administration, and no obvious toxicity was observed.

[0139] Test Example 8: Fluorescence localization of living tumors

[0140] The nude mouse experiment was approved by the Experimental Animal Ethics Committee of Fujian Medical University. MGC-803 tumor cells (approximately 3×10 6 The human gastric cancer cell nude mouse abdominal cavity metastasis model was established in the present invention, and the experiment was carried out about 5 weeks after the tumor model was established.

[0141] Example 1 Preparation of tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe LnNPs ER The injection dose was 100 mg / kg. LnNPs were observed by full-spectrum small animal in vivo imaging system at the time of injection and 24 hours after injection. ER Distribution in the abdominal cavity of nude mice ( Fig.13 In (a), the tumor is located according to the fluorescent signal, and surgical dissection is performed to remove the tumor and major organs ( Fig.13 (b)), and HE pathological section test was performed on the spleen, an organ with partial fluorescence signal ( Fig.13 (c)) confirmed that the fluorescence signal on it came from tumor tissue, indicating that LnNPs ER It can accurately target tumor tissue while avoiding normal organ tissue.

[0142] Furthermore, the LnNPs were observed using a small animal fluorescence microscopy system. ERAble to accurately locate smaller abdominal metastases in the abdominal cavity of nude mice ( Fig.14 (a)), HE pathological section test after surgical navigation resection ( Fig.14 In (b), the resected tissue was confirmed to be tumor tissue, with a tumor diameter as low as 126 μm.

[0143] Test Example 9 In vivo biological safety

[0144] After the imaging of Test Example 8 was completed, the dissected organs, including heart, liver, spleen, lung, and kidney, were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced. They were then stained with HE for histopathological examination and compared with histopathological sections of untreated normal healthy nude mice.

[0145] Test results: Fig.15 It can be seen that after the imaging, the pathological sections of the nude mice were compared with those of normal healthy nude mice, and no abnormal lesions were found, indicating that LnNPs ER It has good biosafety.

[0146] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate a tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe and its preparation method and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0147] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0148] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe, characterized in that: The tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe comprises rare earth inorganic nanocrystals and water-soluble ligands loaded on the surface of the rare earth inorganic nanocrystals, water-soluble ligands modified with tumor targeting molecules and water-soluble ligands modified with endoplasmic reticulum targeting molecules; The rare earth inorganic nanocrystal has a core-shell-shell structure, and the core-shell-shell structure is composed of a rare earth luminescent core NaGdF4:Yb / Er / Ce, a rare earth ion shell NaYbF4 and a rare earth inert protective layer NaYF4.

2. The tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe according to claim 1, characterized in that: The particle size of the rare earth inorganic nanocrystals is 20-50nm; Preferably, the mass ratio of the rare earth luminescent core, the rare earth ion shell and the rare earth inert protective layer is 1:(1.5-2.5):(3.5-4.5); Preferably, the molar percentage of Er in the rare earth luminescent core NaGdF4:Yb / Er / Ce to all rare earth elements is (1-5) mol%.

3. The tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe according to claim 1 or 2, characterized in that: The molar ratio of the water-soluble ligand, the water-soluble ligand modified with the tumor targeting molecule and the water-soluble ligand modified with the endoplasmic reticulum targeting molecule is (0.1-0.8):(0.02-0.1):(0.02-0.1); Preferably, the water-soluble ligand is distearoylphosphatidylethanolamine-polyethylene glycol; Preferably, the number average molecular weight of the polyethylene glycol is 1000-5000; Preferably, the tumor targeting molecule is a cell-targeting penetrating peptide; Preferably, the cell-targeting penetrating peptide is selected from any one or a combination of at least two of iRGD, cRGD, TAT, R9 or LMWP; Preferably, the endoplasmic reticulum targeting molecule is p-toluenesulfonamide.

4. The tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe according to any one of claims 1 to 3, characterized in that: The particle size of the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe is 50-150 nm; Preferably, the Zeta potential of the tumor endoplasmic reticulum-targeted rare earth nanofluorescent probe is 10-50 mV.

5. The method for preparing a tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe according to any one of claims 1 to 4, characterized in that: The preparation method comprises: (1) preparing rare earth luminescent core; (2) using a layer-by-layer epitaxial growth method to sequentially grow a rare earth ion shell layer and a rare earth inert protective layer on the surface of the rare earth luminescent core to obtain a rare earth inorganic nanocrystal; (3) Loading water-soluble ligands, water-soluble ligands modified with tumor targeting molecules, and water-soluble ligands modified with endoplasmic reticulum targeting molecules on the surface of the rare earth inorganic nanocrystals to obtain the tumor endoplasmic reticulum targeting rare earth nano fluorescent probe.

6. The method for preparing the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe according to claim 5, characterized in that: The specific steps of preparing the rare earth luminescent core include: The gadolinium salt, ytterbium salt, erbium salt, cerium salt, sodium trifluoroacetate and solvent are mixed, heated to the reaction temperature, reacted, cooled, and centrifuged to obtain; Preferably, the anions of the gadolinium salt, ytterbium salt, erbium salt or cerium salt are each independently selected from any one of trifluoroacetate ion, acetate ion, nitrate ion, chloride ion or sulfate ion; Preferably, the molar ratio of the gadolinium salt, ytterbium salt, erbium salt, cerium salt and sodium trifluoroacetate is (0.25-1.25):(0.05-0.75):(0.005-0.05):(0.005-0.05):(0.5-1.5); Preferably, the solvent comprises any one or a combination of at least two of 1-octadecene, oleylamine or oleic acid; Preferably, the reaction is carried out under nitrogen, the reaction temperature is 290-320° C., and the reaction time is 0.5-1.5 h; Preferably, the cooling temperature is 20-30°C; Preferably, washing is performed after the centrifugation.

7. The method for preparing the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe according to claim 5 or 6, characterized in that: The specific steps of growing the rare earth ion shell layer include: The ytterbium salt, sodium trifluoroacetate and solvent are mixed, heated to the reaction temperature, reacted, cooled, and centrifuged to obtain; Preferably, the anion of the ytterbium salt is selected from any one of trifluoroacetate ion, acetate ion, nitrate ion, chloride ion or sulfate ion; Preferably, the molar ratio of the ytterbium salt to sodium trifluoroacetate is (0.5-1.5):(0.5-1.5); Preferably, the solvent comprises 1-octadecene and / or oleic acid; Preferably, the reaction is carried out under nitrogen, the reaction temperature is 290-320° C., and the reaction time is 0.5-1.5 h; Preferably, the cooling temperature is 20-30°C; Preferably, washing is performed after the centrifugation.

8. The method for preparing a tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe according to any one of claims 5 to 7, characterized in that: The specific steps of growing the rare earth inert protective layer include: The yttrium salt, sodium trifluoroacetate and solvent are mixed, heated to the reaction temperature, reacted, cooled, and centrifuged to obtain; Preferably, the anion of the yttrium salt is selected from any one of trifluoroacetate ion, acetate ion, nitrate ion, chloride ion or sulfate ion; Preferably, the molar ratio of the yttrium salt to sodium trifluoroacetate is (0.5-1.5):(0.5-1.5); Preferably, the solvent comprises 1-octadecene and / or oleic acid; Preferably, the reaction is carried out under nitrogen, the reaction temperature is 290-320° C., and the reaction time is 0.5-1.5 h; Preferably, the cooling temperature is 20-30°C; Preferably, washing is performed after the centrifugation.

9. The method for preparing a tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe according to any one of claims 5 to 8, characterized in that: The specific steps of loading the water-soluble ligand, the water-soluble ligand modified with the tumor targeting molecule and the water-soluble ligand modified with the endoplasmic reticulum targeting molecule include: Dispersing the rare earth inorganic nanocrystals in an organic solvent, adding water-soluble ligands, water-soluble ligands modified by tumor targeting molecules, and water-soluble ligands modified by endoplasmic reticulum targeting molecules, and stirring to react, thereby obtaining the tumor endoplasmic reticulum targeting rare earth nano fluorescent probe; Preferably, a post-treatment step is also performed after the reaction; Preferably, the post-treatment includes washing and sterilization; Preferably, the aseptic treatment comprises re-dispersing the product into physiological saline or sterile ultrapure water for aseptic filtration and / or ultraviolet irradiation sterilization; Preferably, the organic solvent is selected from any one of chloroform, dichloromethane or cyclohexane.

10. Use of the tumor endoplasmic reticulum-targeted rare earth nano fluorescent probe according to any one of claims 1 to 4 in preparing a tumor cell visualization laser confocal imaging agent or in preparing a solid tumor fluorescent imaging agent.

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