A cell fluorescence probe anchoring integrin and a preparation method and application thereof
By using fluorescent probes that are modified cyclodextrin and peptide-bonded, the problems of insufficient endocytosis and fluorescence duration are solved, enabling long-lasting cell fluorescence imaging, which is suitable for tumor cell targeting and nuclear imaging.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN SECOND PEOPLES HOSPITAL (SHENZHEN INST OF TRANSLATIONAL MEDICINE)
- Filing Date
- 2023-10-23
- Publication Date
- 2026-06-26
AI Technical Summary
Existing fluorescent imaging reagents suffer from severe intracellular endocytosis and insufficient fluorescence duration, making them unable to effectively label cells for extended periods.
By linking the carbon-carbon double bond functional group of modified cyclodextrin to the thiol group of the terminal cysteine of a peptide via a click reaction, a fluorescent probe for anchoring cytokines is formed, which binds to cyclodextrin units to reduce endocytosis and prolong fluorescence duration.
It reduces endocytosis, significantly prolongs the fluorescence duration of the fluorescent imaging reagent, and maintains non-cytotoxicity, making it suitable for tumor cell-targeted fluorescence imaging and nuclear imaging.
Smart Images
Figure CN117431056B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical applications, and more specifically, relates to a cell fluorescent probe for anchoring cell integrins, its preparation method, and its application. Background Technology
[0002] Integrins are widely distributed on the cell membrane surface and belong to a class of transmembrane proteins that regulate important physiological activities such as cell migration and proliferation. Normally, integrins are rarely expressed in normal vascular endothelial and epithelial cells, but they are highly expressed on the surface of various solid tumor cells, including lung cancer, osteosarcoma, neuroblastoma, breast cancer, prostate cancer, bladder cancer, glioblastoma, and invasive melanoma. Furthermore, they are highly expressed on the membranes of newly formed vascular endothelial cells in all tumor tissues.
[0003] Molecular imaging is a crucial component of medical molecular imaging and a key focus and hot topic in medical research in recent years. In cell research, fluorescence imaging techniques, which use fluorescent dyes to label and trace receptors on cell membranes, are effective methods for studying cell membrane structure. A necessary condition for using live-cell fluorescent dyes is their non-cytotoxicity. Currently available commercially available live-cell fluorescent dyes include cell-permeable cytoplasmic markers (such as thiol-reactive CellTracker probes), polar tracers (such as fluorescein CH), and membrane tracers (such as the DiD and FM families). These dyes all have certain limitations and limitations. For example, the DiD family of membrane tracers is easily endocytosed by the cell membrane, leading to lateral diffusion within the membrane and ultimately contaminating the entire cell. Secondly, the timeliness of fluorescence imaging reagents is also quite important. Therefore, designing novel fluorescence imaging reagents that reduce endocytosis and have long-lasting effects is crucial.
[0004] CN108815537A discloses a tumor cell-targeting specific fluorescent probe, its preparation method, and its application. Specifically, the fluorescent probe contains a portion consisting of a polypeptide complex and a fluorescent molecule linked together. The polypeptide complex is composed of a targeting polypeptide, a membrane-penetrating peptide, a peptide containing a bonding group, and a nuclear-targeting peptide linked in any order. The polypeptide complex is covalently linked to the fluorescent molecule through the bonding group. The fluorescent molecule is an aggregation-induced emission compound. This fluorescent probe can target tumor cells that highly express αⅤβ3 and / or CD13 proteins and directionally transfer the carried polypeptide and fluorescent molecule into the cell. However, there is still room for improvement in extending the fluorescence duration.
[0005] CN109701035A discloses a novel preparation process for fluorescent amphiphilic peptide self-assembled nanomicelles for molecular diagnosis of breast cancer. Specifically, it discloses a composite amphiphilic peptide nanomicelle targeting integrin αvβ3, using a breast cancer-specific targeting amphiphilic peptide on its surface as the building matrix, and encapsulating the fluorescent substance indocyanine green. The targeting group is a C18-GRRRRRRRRGDS (C18GR7RGDS) amphiphilic peptide containing the arginine-glycine-aspartic acid (RGD) tripeptide sequence. This technique overcomes the shortcomings of commonly used fluorescent substances such as indocyanine green (used alone), including instability, easy photobleaching, and limited long-term use, achieving multifunctional applications of amphiphilic peptide nanomicelles. However, there is still room for improvement in extending the fluorescence duration.
[0006] In summary, current technologies still lack a fluorescent imaging probe that reduces cellular endocytosis and has a long-lasting effect. Summary of the Invention
[0007] The purpose of this invention is to address the shortcomings of existing fluorescent imaging reagents, such as insufficient fluorescence duration, by providing a cellular fluorescent probe for anchoring integrins. This is achieved by linking the double-bonded functional group of modified cyclodextrin to the thiol group of the terminal cysteine residue of a polypeptide via a click reaction, thereby enhancing the fluorescence duration of the fluorescent imaging reagent by protecting the fluorescent molecule with cyclodextrin. Furthermore, the cyclodextrin unit is a relatively large hydrophilic unit, which can reduce the amount of reagent endocytosis. Therefore, a fluorescent imaging reagent that anchors integrins, reduces endocytosis, and prolongs fluorescence duration is obtained. The detailed technical solution of this invention is described below.
[0008] The first objective of this invention is to provide a cellular fluorescent probe for anchoring cellular integrins, comprising a polypeptide complex, a fluorescent molecule, and a cyclodextrin unit. The polypeptide complex comprises a peptide containing cysteine, a peptide containing a bonding group, and a targeting peptide linked in any order. The cyclodextrin unit contains a carbon-carbon double bond functional group and binds to the polypeptide complex via a click reaction with the peptide containing cysteine. The polypeptide complex is covalently linked to the fluorescent molecule via the bonding group. The fluorescent molecule is an aggregation-induced emission compound.
[0009] Preferably, the targeting peptide contains an Arg-Gly-Asp sequence; more preferably, the Arg-Gly-Asp sequence is located at the middle position of the polypeptide complex and accounts for more than 30% of the number of amino acid units.
[0010] Preferably, the cyclodextrin unit is formed by the reaction of cyclodextrin molecules and modified molecules, wherein the modified molecules are compounds containing isocyanate and carbon-carbon double bond functional groups.
[0011] Preferably, the modified molecule includes ethyl isocyanate methacrylate or ethyl isocyanate acrylate.
[0012] Preferably, the cyclodextrin molecule includes one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.
[0013] Preferably, the fluorescent molecules include fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC).
[0014] Preferably, the cyclodextrin is β-cyclodextrin, and the fluorescent molecule is fluorescein isothiocyanate.
[0015] The second objective of this invention is to protect a method for preparing a cellular fluorescent probe that anchors cellular integrins, comprising the following steps:
[0016] (1) Binding of peptides to fluorescent molecules: After dissolving the peptide in an organic solvent, it is covalently linked to the fluorescent molecule to obtain a peptide intermediate labeled with the fluorescent molecule.
[0017] (2) Cyclodextrin unit: After fully dissolving and mixing cyclodextrin molecules and modified molecules in an organic solvent, a catalyst is added and the reaction is carried out under the protection of nitrogen atmosphere, so that the cyclodextrin molecules are grafted with double bond structure to obtain cyclodextrin unit.
[0018] (3) Click reaction of polypeptide with cyclodextrin: Under the protection of inert gas, the cyclodextrin unit is dissolved in an organic solvent, and the polypeptide intermediate labeled with fluorescent molecules obtained in step (1) is added. The reaction is stirred under UV light so that the cyclodextrin unit and the peptide containing cysteine undergo a click reaction to obtain the cell fluorescent probe according to any one of claims 1-7.
[0019] Preferably, in step (1), 6-aminohexanoic acid is introduced before the fluorescent molecule reaction.
[0020] 6-Aminocaproic acid (Acp), also known as an alkyl spacer, provides six carbon atoms in a straight chain, reducing steric hindrance and improving reaction efficiency. Secondly, the addition of Acp reduces the probability of FITC reacting with -SH and the side chain -NH2 in the peptide structure.
[0021] This invention also protects the use of the aforementioned cell fluorescence probe for anchoring cell integrins in the preparation of tumor cell-targeting fluorescence imaging reagents, tumor cell tracing reagents, tumor cell nuclear imaging reagents, or drug carriers.
[0022] The beneficial effects of this invention are:
[0023] (1) The prepared fluorescent imaging reagent has no obvious cytotoxicity. By binding to integrins on the cell membrane via the RGD sequence, peptides and fluorescent molecules can be anchored to integrin sites on the cell membrane. A cyclodextrin unit is covalently linked to the peptide tail, which can fix the cyclodextrin unit around the fluorescent molecule, increasing the binding probability. The cyclodextrin protects the fluorescent molecule and prolongs the fluorescence duration of the fluorescent imaging reagent. Furthermore, the cyclodextrin unit is a large hydrophilic unit, which can reduce endocytosis of the reagent. Therefore, a fluorescent imaging reagent that anchors cell integrins, reduces endocytosis, and prolongs fluorescence duration is obtained.
[0024] (2) Adding 6-aminocaproic acid before the fluorescent molecule reaction provides a 6-carbon straight-chain space, reducing steric hindrance and improving reaction efficiency. Secondly, the addition of Acp reduces the probability of FITC reacting with -SH and side chain -NH2 in the peptide structure. Attached Figure Description
[0025] Figure 1 A schematic diagram of the structure of the cell fluorescent probe in Example 1 of this invention.
[0026] Figure 2 The fluorescence intensity test diagram of the cell fluorescent probe prepared in Example 1 of this invention, wherein, Figure 2 In the image, 'a' represents the fluorescence spectrum of a fluorescent molecule with a concentration of 10 mg / mL, stored at room temperature for one day, under 405 nm light illumination. Figure 2 In the image, b represents the fluorescence spectrum of a fluorescent molecule with a concentration of 10 mg / mL, stored at room temperature for 10 days under 405 nm light irradiation. Figure 2 In the diagram, 'c' represents the green fluorescence produced by the fluorescent molecule after being excited by blue light in a well plate at a concentration of 1 mg / mL and left at room temperature for 30 days. Figure 2 In the figure, d represents the green fluorescence produced by ordinary FITC at a concentration of 1 mg / mL after being placed at room temperature for 30 days when excited by blue light in a well plate.
[0027] Figure 3 The fluorescence spectrum test diagram of the cell fluorescent probe of the present invention is shown in the figure. The black solid line and the black dashed line are the fluorescence spectrum curves of the cell fluorescent probe prepared in Example 1 after being placed at room temperature for 0 days and 30 days, respectively. The red solid line and the red dashed line are the fluorescence spectrum curves of ordinary FITC (without CD-RDG modification) after being placed at room temperature for 0 days and 30 days, respectively.
[0028] Figure 4 A schematic diagram of the fluorescent imaging reagent of the present invention binding to integrins on the cell membrane.
[0029] Figure 5 The cell fluorescence imaging diagram of the present invention, wherein, Figure 5In the image, a is a 100μm image of the fluorescent molecule prepared in Example 1, b is a 200μm image of the fluorescent molecule prepared in Example 1, c is a 100μm image of the fluorescent molecule prepared in Example 1, and d is a 200μm image of the fluorescent molecule prepared in Example 1.
[0030] Figure 6 The cell fluorescence imaging images of fluorescent molecules in Example 1 of this invention are shown in Figure a, where Figure a is a cell fluorescence imaging image after 2 hours of staining and culture, and Figure b is a cell fluorescence imaging image after 8 days of staining and culture. Detailed Implementation
[0031] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings:
[0032] Example
[0033] Example 1
[0034] The molecular structure diagram of the cell fluorescent probe in this embodiment is shown below. Figure 1 As shown.
[0035] The reagent consists of a cyclodextrin unit and a fluorescent molecule at each end, with a polypeptide chain as the backbone in the middle. The specific sequence of the polypeptide is Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly-Cys, with the RGD sequence located at the middle position of the short-chain polypeptide. The polypeptide backbone and cyclodextrin unit exhibit good biocompatibility and water solubility. The fluorescent molecule is FITC. The isothiocyanate group reacts with the amino group of the polypeptide to bind to the head of the polypeptide chain, yielding a 5-FITC-polypeptide. The cyclodextrin unit is grafted with a carbon-carbon double bond functional group by reacting with isocyanoethyl methacrylate under anhydrous conditions. The cyclodextrin unit after the double bond functional group reacts with the thiol group of the terminal cysteine residue of the polypeptide, binding to the short-chain polypeptide via a click reaction. This binding increases the probability of binding to the fluorescent FITC molecule on the polypeptide. The cyclodextrin protects FITC, prolonging the fluorescence duration of the fluorescent imaging reagent, and increases the reagent's hydrophilicity, reducing cellular endocytosis of the reagent. Therefore, a fluorescent imaging reagent that anchors integrins, reduces intracellular phagocytosis, and prolongs fluorescence duration was obtained.
[0036] Preparation method of fluorescence imaging reagents:
[0037] The binding of peptides to fluorescent molecules: Fluorescein isothiocyanate (FITC) has relatively high activity. FITC reacts with the N-terminus of the peptide. In this case, 6-aminocaproic acid (Acp) is added before the FITC reaction. Acp, also known as an alkyl spacer, provides a 6-carbon straight-chain space, reducing steric hindrance and improving reaction efficiency. Secondly, the addition of Acp reduces the probability of FITC reacting with -SH and the side chain -NH2 in the peptide structure. The specific reaction is as follows: the peptide is dissolved in piperazine (PIP) and DMF (8:2) solvent, and an equimolar amount of Fmoc-Apc-OH and standard coupling reagent (DIC / HOBt) is added and reacted for 1 h; FITC and DIEA are added, and the reaction is carried out in TFA / TIS / H2O for 2 h, with a TFA / TIS / H2O volume ratio of 95:2.5:2.5, to obtain the peptide labeled with the fluorescent molecule.
[0038] Modification of cyclodextrin: β-cyclodextrin (1 eq) completely dried to remove moisture under vacuum and ethyl isocyanate methacrylate (1 eq) were thoroughly dissolved and mixed in DMF solvent. A trace amount of Sn(Oct)₂ (3 μM) was added as a catalyst, and the reaction was carried out at room temperature for 4 h under a nitrogen atmosphere. The product was then precipitated in 10 volumes of ice-cold acetone and dried under vacuum to obtain the β-cyclodextrin double bond modified product: β-CD-AOI.
[0039] Click reaction of peptides with cyclodextrin: After purging 20 mL of anhydrous DMF with N2 for 30 min to remove oxygen, 78 mg of carbon-carbon double-bond modified cyclodextrin was added to a round-bottom flask and stirred thoroughly to dissolve. 100 mg of FITC-grafted short-chain peptide was dissolved in 5 mL of deoxygenated dimethylformamide (DMF) and added dropwise to the flask (oxygen removal was necessary during peptide dissolution). 15.5 mg of dimethyl benzoate (DMPA) was then added, and the reaction proceeded for 1 h, followed by 1 h of reaction under UV irradiation. The resulting solution was poured into a dialysis bag with a molecular weight cutoff of 2000 and dialyzed against deionized water for 3 days. After lyophilization, the solution was collected and stored at -20°C to obtain the cell fluorescent probe.
[0040] Example 2
[0041] The method in this embodiment is basically the same as that in Example 1, the main difference being the different polypeptide complexes.
[0042] In this embodiment, the arrangement and number of RGD sequences in the polypeptide are increased, and the sequence of the short chain polypeptide becomes: Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly-Arg-Gly-Asp-Ser-Pro-Gly-Cys.
[0043] Example 3
[0044] This embodiment is basically the same as the method in Example 1, the main difference being the difference in fluorescent molecules and cyclodextrin units.
[0045] TRITC was chosen as the fluorescent molecule. The cyclodextrin unit was selected from γ-cyclodextrin, whose cavity size is well-matched to TRITC.
[0046] Example 4
[0047] The method in this embodiment is basically the same as that in Example 1, the main difference being the different modification reaction of cyclodextrin.
[0048] The reactant for the modified grafting was changed to ethyl isocyanate acrylate, and the ratio of β-cyclodextrin to ethyl isocyanate acrylate was changed to (1:2). On average, β-cyclodextrin was grafted with 2 double bond structures.
[0049] Test Implementation Examples
[0050] 1. Fluorescence intensity test.
[0051] The cell fluorescent probe prepared in Example 1 was used to prepare a cell integrin fluorescent probe staining solution with a concentration of 10 mg / mL using PBS. The fluorescent probes with and without cyclodextrin units were compared. The fluorescence time-efficacy was tested after the same concentration solution was placed at room temperature for 30 days using a fluorometer.
[0052] Figure 2 The fluorescence intensity test diagram of the cell fluorescent probe prepared in Example 1 of this invention, wherein, Figure 2 In the image, 'a' represents the fluorescence spectrum of a fluorescent molecule with a concentration of 10 mg / mL, stored at room temperature for one day, under 405 nm light illumination. Figure 2 In the image, b represents the fluorescence spectrum of a fluorescent molecule with a concentration of 10 mg / mL, stored at room temperature for 10 days under 405 nm light irradiation. Figure 2 In the diagram, 'c' represents the green fluorescence produced by the fluorescent molecule after being excited by blue light in a well plate at a concentration of 1 mg / mL and left at room temperature for 30 days. Figure 2 In the figure, d represents the green fluorescence produced by ordinary FITC at a concentration of 1 mg / mL after being placed at room temperature for 30 days when excited by blue light in a well plate.
[0053] Figure 3 The fluorescence spectrum test diagram of the cell fluorescent probe prepared in Example 1 of this invention is shown in the figure. The black solid line and the black dashed line are the fluorescence spectrum curves of the cell fluorescent probe after 0 days and 30 days of incubation at room temperature, respectively. The red solid line and the red dashed line are the fluorescence spectrum curves of ordinary FITC (without CD-RDG modification) after 0 days and 30 days of incubation at room temperature, respectively.
[0054] Depend on Figure 2 and Figure 3It can be seen that the fluorescence intensity of the fluorescent molecules containing cyclodextrin units is significantly stronger than that of the control group, and cyclodextrin protects FITC and prolongs the fluorescence duration of the fluorescence imaging reagent.
[0055] 2. Cell staining fluorescence test.
[0056] A schematic diagram of the binding of fluorescent imaging reagents to integrins on the cell membrane is shown below. Figure 4 As shown.
[0057] Cell staining steps:
[0058] (1) Add the cytotine fluorescent probe (0.1 mg / 1 mL diluted 100 times with PBS) to the surface of the seeded cells (adipose-derived mesenchymal stem cells, ADSCs). Add 0.5 mL to each well of a 24-well plate. After staining and culturing for 2 hours, observe the cells under a fluorescence microscope at 500–550 nm using a 488 nm laser (e.g., ...). Figure 5 ).
[0059] (2) Use this fluorescent probe solution (PBS diluted 200 times, 0.05 mg / 1 mL) to immerse cells (human umbilical vein endothelial cells, HUVECs) in a cell plate. Add 0.5 mL to each well of a 24-well plate, stain and incubate for 2 hours, then take images under a fluorescence microscope (e.g., ...). Figure 6 a). After observation, continue culturing, and take photos directly after 8 days of culturing (e.g., Figure 6 b).
[0060] Figure 5 The cell fluorescence imaging diagram of the present invention, wherein, Figure 5 In the image, a is a 100μm image of the fluorescent molecule prepared in Example 1, b is a 200μm image of the fluorescent molecule prepared in Example 1, c is a 100μm image of the fluorescent molecule prepared in Example 1, and d is a 200μm image of the fluorescent molecule prepared in Example 1.
[0061] Figure 6 The cell fluorescence imaging images of fluorescent molecules in Example 1 of this invention are shown in Figure a, where Figure a is a cell fluorescence imaging image after 2 hours of staining and culture, and Figure b is a cell fluorescence imaging image after 8 days of staining and culture.
[0062] Depend on Figure 5 It is known that ordinary FITC cannot bind specifically to cells (e.g., Figure 5 Fluorescent probes that combine peptides and CD molecules can anchor to integrin sites on the cell membrane (e.g., CD). Figure 5 ab), while the fluorescent probe does not excessively increase the fluorescence background of the material.
[0063] Depend on Figure 6It can be seen that in cell staining experiments, fluorescent probes containing cyclodextrin units can also prolong the fluorescence duration in cell labeling, and the cells can still be clearly observed under a fluorescence microscope after at least 8 days of labeling.
[0064] The test results for Examples 1, 2, 3 and 4 are shown in Table 1.
[0065] Results and Discussion: The preparation of corresponding fluorescent probes using short-chain peptides with added RGD sequences showed that, compared to Example 1, the fluorescent probe staining solution exhibited a longer cell labeling time, reaching 242 hours (approximately 10 days) (as shown in Table 1). The staining time for this fluorescent probe during cell culture labeling was reduced to only 30 minutes.
[0066] Discussion of Results in Example 3: Using the γ-CD-TRITC fluorescent probe molecule, compared to Example 1, the fluorescent probe staining solution was excited under 561 nm green light, and the receiving wavelength was 570–610 nm, which was observed by fluorescence microscopy. Because TRITC is a derivative of the rhodamine family, its properties are relatively stable, and the labeled cells have a longer duration of action, up to 476 h (approximately 20 days) (as shown in Table 1).
[0067] Discussion of results in Example 4: Compared with Example 1, the fluorescent probe staining solution for cell labeling has a shorter time limit (120h) (as shown in Table 1).
[0068] Table 1. Results of Cell Staining Fluorescence Test
[0069] Experimental results Cell staining time Fluorescence aging time Excitation wavelength Example 1 2h 197 488nm Example 2 0.5h 242 488nm Example 3 0.5h 476 561nm Example 4 2h 120 488nm
[0070] In summary, the cellular fluorescent probe for anchoring cell integrins provided by this invention recognizes and labels cells by binding to integrins on the cell membrane via the RGD sequence, whereas ordinary FITC cannot specifically bind to cells. This cellular fluorescent probe, by modifying cyclodextrin units to reduce endocytosis and by protecting fluorescent molecules with cyclodextrin units, significantly prolongs the fluorescence duration of fluorescent imaging reagents. Simultaneously, under the condition of the material being present, this fluorescent probe does not excessively increase the fluorescence background of the material.
[0071] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.
Claims
1. A cellular fluorescent probe for anchoring cellular integrins, characterized in that, The device comprises a polypeptide complex, a fluorescent molecule, and a cyclodextrin unit. The polypeptide complex consists of a cysteine-containing peptide, a peptide containing a bonding group, and a targeting peptide linked in any order. The cyclodextrin unit contains a carbon-carbon double bond functional group and binds to the polypeptide complex via a click reaction with the cysteine-containing peptide. The polypeptide complex is covalently linked to the fluorescent molecule through the bonding group. The targeting peptide contains an Arg-Gly-Asp sequence. The Arg-Gly-Asp sequence is located at the middle position of the polypeptide complex and accounts for more than 30% of the amino acid units. The fluorescent molecule is fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. The specific sequence of the polypeptide complex is Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly-Cys or Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly-Arg-Gly-Asp-Ser-Pro-Gly-Cys.
2. The cell fluorescent probe according to claim 1, characterized in that, The cyclodextrin unit is formed by the reaction of cyclodextrin molecules and modified molecules, wherein the modified molecules are compounds containing isocyanate and carbon-carbon double bond functional groups.
3. The cell fluorescent probe according to claim 2, characterized in that, The modified molecules include ethyl isocyanate methacrylate or ethyl isocyanate acrylate.
4. The cell fluorescent probe according to claim 2 or 3, characterized in that, The cyclodextrin molecules include one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.
5. The cell fluorescent probe according to claim 4, characterized in that, The cyclodextrin is β-cyclodextrin, and the fluorescent molecule is fluorescein isothiocyanate.
6. A method for preparing a cellular fluorescent probe for anchoring cellular integrins according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Binding of peptides to fluorescent molecules: After dissolving the peptide in an organic solvent, it is covalently linked to the fluorescent molecule to obtain a peptide intermediate labeled with the fluorescent molecule; (2) Cyclodextrin unit: After fully dissolving and mixing cyclodextrin molecules and modified molecules in an organic solvent, a catalyst is added and the reaction is carried out under the protection of nitrogen atmosphere, so that the cyclodextrin molecules are grafted with double bond structure to obtain cyclodextrin unit; the modified molecule is a compound containing isocyanate and carbon-carbon double bond functional groups. (3) Click reaction of peptide with cyclodextrin: Under the protection of inert gas, the cyclodextrin unit is dissolved in an organic solvent, and the peptide intermediate labeled with fluorescent molecules obtained in step (1) is added. The reaction is stirred under UV light so that the cyclodextrin unit and the peptide containing cysteine can undergo a click reaction to obtain a cell fluorescent probe.
7. The method for preparing the cell fluorescent probe according to claim 6, characterized in that, In step (1), 6-aminohexanoic acid is introduced before the fluorescent molecule reaction.
8. The use of the cell fluorescent probe for anchoring cell integrins according to any one of claims 1-5 in the preparation of tumor cell-targeting fluorescent imaging reagents, tumor cell tracking reagents, tumor cell nuclear imaging reagents or drug carriers.