Preparation method and application of novel polypeptide fluorescent probe

Abstract

The invention discloses a novel polypeptide fluorescent probe as well as a preparation method and application thereof, and belongs to the field of biomedical technology and fluorescence imaging. The novel polypeptide fluorescent probe is sequentially connected with a targeted recognition module, an enzyme response module, a self-assembly module and a functional module through amido bonds. The targeted recognition module is used for recognizing a CD44v6 specific receptor highly expressed on bladder cancer cells, the enzyme response module is a specific polypeptide sequence of MMP2 enzyme, and the functional module is a biological orthogonal group. The polypeptide fluorescent probe disclosed by the invention is targeted on the surface of a bladder cancer cell through a specific recognition sequence, and nano-fibers are formed through enzyme response shearing and in-situ self-assembly and are adhered on the surface of the cell to form an artificial receptor similar to the surface of the cell; then, fluorescent molecules are delivered to be subjected to a click chemical reaction with biological orthogonal groups exposed outside the nanofibers, so that high-imaging signal-to-noise ratio visual tumor positioning of a tumor area is realized, and a new method is provided for surgical precise excision of bladder cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel polypeptide fluorescent probe and a preparation method and application thereof. Background Art

[0002] Bladder cancer originates from urothelial cells or umbrella cells in the bladder cavity. It is one of the most common malignant tumors of the urinary system and is more common in men (risk of disease 1.1%) and the elderly (>55 years old). Its incidence rate gradually increases with age, with new cases and deaths accounting for 4% and 6% of all cancer cases, respectively. In addition, bladder cancer is also a heterogeneous disease with different clinical outcomes. Generally, newly diagnosed cases are non-muscle-invasive bladder cancer (accounting for about 75%), and the treatment method is transurethral resection. However, because the surgery will miss and ignore small tumors, about 80% of patients will have varying degrees of recurrence; in addition, bladder cancer is diagnosed in the late stage, so its effective treatment results depend to a large extent on its early detection. Therefore, it is of great clinical significance to develop a method that can accurately identify bladder cancer early.

[0003] At present, the traditional diagnostic methods for bladder cancer mainly include CT, MRI and cystoscopy, but they have limitations such as long acquisition time, radiation risk, difficulty in seeing lesions, and false positives. Fluorescence imaging technology has attracted much attention due to its advantages such as high resolution, high specificity and high sensitivity, and can non-invasively monitor in real time and guide precise resection of cancer surgery. Previously, fluorescent contrast agents approved by the US Food and Drug Administration have been widely used, such as 5-aminolevulinic acid and indocyanine green, which are approved for surgical imaging (including intracranial aneurysms, vascular bypass and bladder tumors). In addition, antibody / small molecule conjugation or nanoparticle encapsulation as tumor-targeting carriers have shown great prospects in the in vivo imaging of solid tumors, and studies have shown that targeted fluorescent contrast agents can also effectively reduce the risk of recurrence of transurethral resection.

[0004] Although previous studies have achieved relatively satisfactory results, there are still some shortcomings in traditional fluorescent probes that need to be addressed, such as: ① The recognition characteristics of traditional fluorescent contrast agents mainly rely on a limited number of natural receptors (~10 6 ), resulting in low efficiency in identifying tumor cells; ② The size of antibodies or nanoparticles is usually large, which will affect the penetration rate in tumor tissue; ③ During the operation, repeated saline washing or photobleaching of contrast agents leads to loss of fluorescence signal; ④ The fluorescent molecules themselves have the characteristics of short half-life (2-4min), photobleaching and non-specific targeting. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a novel polypeptide fluorescent probe and a preparation method and application thereof to solve the above problems.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a novel polypeptide fluorescent probe and a preparation method and application thereof.

[0008] The novel polypeptide fluorescent probe is composed of a target recognition polypeptide module, an enzyme trigger response module, a self-assembly module and a functional module;

[0009] The polypeptide fluorescent probe specifically recognizes the targeted peptide segment, adheres to the cell surface through the proximity adhesion effect after enzyme response shearing to form a fiber network that increases the receptors on the cell surface, and then infuses the azidated fluorescent molecules to produce a bioorthogonal reaction to achieve specific imaging.

[0010] Furthermore, the receptors recognized by the targeting recognition module are targeting receptors highly expressed on bladder cancer cells, and the targeting receptors include integrins, fibroblast growth factor receptor 1 and CD44v6 receptors;

[0011] The specific sequence of the CD44v6 receptor is NRWHEDC or CSDRIMRGC.

[0012] Furthermore, the enzyme-triggered response module is a response shearing enzyme highly expressed in the tumor region, and the response shearing enzyme includes caspase-3 / 7, glucose hydrolase, alkaline phosphatase, cathepsin B and MMP2 enzyme;

[0013] Among them, the response cleavage peptide sequence of the MMP2 enzyme includes PLGYLG, PLGVRG, PLGLAG, PVGLIG, PLGLAG, GPLGIAGQC and GPLGVRGS.

[0014] Furthermore, the self-assembling peptide module can spontaneously assemble ordered aggregates under the condition of hydrophilic-hydrophobic imbalance, and the sequence of the self-assembling module is any one of YLGFFC, KLVFF, KLKLKL, and FF.

[0015] Furthermore, the novel polypeptide fluorescent probe also contains a hydrophilic substance, and the hydrophilic substance is polyethylene glycol-4.

[0016] Furthermore, the azide is modified to form an azidated fluorescent molecule, and the fluorescent molecule to be modified is any one of Cyanine5, Cyanine7, Indocyanine Green and Fluorescein Isothiocyanate.

[0017] Furthermore, a method for preparing a novel polypeptide fluorescent probe is also provided, comprising the following steps:

[0018] S1. Connecting the targeting peptide, the enzyme-responsive cleavage peptide, the self-assembling peptide and the functional peptide in sequence through amide bonds by solid phase synthesis to obtain a polypeptide fluorescent probe;

[0019] S2. Cy5-N3 is connected to the peptide fluorescent probe through click reaction to obtain a new peptide fluorescent probe.

[0020] The synthesis steps of the polypeptide fluorescent probe are as follows:

[0021] P1, select the first amino acid resin for swelling;

[0022] P2, remove the Fmoc group in step P1 to expose the amino group, and undergo an amide condensation reaction with the carboxyl group of the next amino acid, so that the amino acids on the peptide fluorescent probe are sequentially connected and fixed on the resin;

[0023] P3, removing the Fmoc group of PEG4 in step P2 to expose NH2;

[0024] P4, coupling reaction of the peptide fluorescent probe with NH2 in step P3;

[0025] P5, cleaving and separating the polypeptide in step P4 from the resin to obtain a purified polypeptide fluorescent probe;

[0026] P6. The polypeptide fluorescent probe in step P5 undergoes a click reaction with the fluorescent molecule Cy5-N3 to obtain a novel polypeptide fluorescent probe.

[0027] Further, in step P1, the amino acid is aspartic acid, and the resin swelling time is 9h-12h;

[0028] In the steps P2 and P3, the deprotecting agent is composed of hexahydropyridine and N,N-dimethylformamide;

[0029] The main component of the coupling agent in step P4 is N,N-dimethylformamide;

[0030] The main components of the lysate in step P5 are trifluoroacetic acid and triisopropylsilane;

[0031] In the step P6, the reaction ratio of the polypeptide fluorescent probe to Cy5-N3 is 1:1, and the reaction time is 5 min-90 min.

[0032] Furthermore, it is applied to in vivo imaging of cells and small animals, using a peptide concentration of 50 μM-100 μM and an incubation time of 0.5 h-12 h;

[0033] and / or applied in surgical resection of bladder cancer.

[0034] The beneficial effects of the present invention are:

[0035] 1. The fluorescent probe of the present invention can specifically target the surface of bladder cancer, and self-assemble on the cell surface to form nanofibers through enzyme-responsive shearing and self-assembly enhanced proximity adhesion effect to construct a fiber network of "artificial receptors" (DBCO). The azidated fluorescent molecules undergo click chemistry reaction with the artificial receptors to achieve targeted imaging of bladder cancer. The long-term retention and strong fluorescence signal in the body significantly improve the signal-to-noise ratio of the bladder cancer site, avoid the photobleaching problem of fluorescent molecules, and provide a new method for the early diagnosis of bladder cancer.

[0036] 2. The polypeptide fluorescent probe of the present invention can accurately target bladder cancer, form nanofibers on the cell surface through enzyme response shearing, and present a high signal-to-noise ratio in the tumor area after reacting with fluorescent molecules, thus achieving efficient enrichment and imaging of the tumor area, solving the problem that traditional fluorescent molecules have no targeting.

[0037] 3. The polypeptide fluorescent probe of the present invention forms nanofibers on the cell surface in advance to increase the number of receptors on the cell surface. It can not only deliver different fluorescent molecules or drugs on demand, but also the formed nanofiber network is resistant to erosion, solving the problems of limited number of cell surface receptors and short retention time of traditional fluorescent probes.

[0038] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art may be taught from the practice of the present invention. The objectives and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to make the purpose, technical solution and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0040] Figure 1 The chemical structure diagram of DBCO-TPP prepared in Example 1 of the present invention;

[0041] Figure 2 The scanning electron microscopy and transmission electron microscopy images of Example 1 of the present invention co-incubated with MMP2 enzyme;

[0042] Figure 3 This is a laser confocal image of Example 1 of the present invention after co-incubation with different cells;

[0043] Figure 4 This is a scanning electron microscopic image of Example 1 of the present invention after co-incubation with different cells;

[0044] Figure 5 (A) is a laser confocal image of the novel polypeptide fluorescent probe of Example 1 of the present invention after co-incubation with azide-Cy5 and TP-Cy5 in EJ tumor spheres;

[0045] Figure 5 (B) is a quantitative graph of the fluorescence intensity of the novel polypeptide fluorescent probe of Example 1 of the present invention after co-incubation with azide-Cy5 and TP-Cy5 in EJ tumor spheres;

[0046] Figure 6 (A) is a small animal in vivo imaging image of Cy5 of the present invention in ectopic transplanted bladder cancer;

[0047] Figure 6 (B) is a small animal in vivo imaging image of ectopic transplanted bladder cancer after intratumoral injection of the novel polypeptide fluorescent probe of Example 1 and azide-Cy5 of the present invention;

[0048] Figure 7 (A) is a fluorescence imaging diagram of the novel polypeptide fluorescent probe of Example 1 of the present invention and azide-Cy5 in micro-tumors of different sizes in the air-sac bladder cancer model;

[0049] Figure 7 (B) is a graph showing the correlation between the novel polypeptide fluorescent probe of Example 1 of the present invention and azide-Cy5 in micro-tumors of different sizes in the air-sac bladder cancer model;

[0050] Figure 8 (A) is a fluorescence imaging diagram of the novel polypeptide fluorescent probe of Example 1 of the present invention and azide-Cy5, TP-Cy5 and Cy5 in human in vitro tissue specimens;

[0051] Figure 8 (B) is a quantitative graph of the fluorescence intensity of the novel polypeptide fluorescent probe of Example 1 of the present invention and azide-Cy5, TP-Cy5 and Cy5 in human in vitro tissue specimens. DETAILED DESCRIPTION

[0052] like Figure 1-8 As shown, the present invention provides a novel polypeptide fluorescent probe and a preparation method and application. The following is a brief expression of each substance:

[0053] DBCO-TPP intermediate fluorescent probe,

[0054] HATU stands for 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0055] HOBT stands for 1-hydroxybenzotriazole.

[0056] Example 1

[0057] S1. Swelling resin and deprotection: Weigh 0.1 mmol of aspartic acid Wang resin into a peptide synthesis tube, add N,N-dimethylformamide (DMF) and swell on a shaker for 9h-12h, filter and add deprotection agent (composed of hexahydropyridine and N,N-dimethylformamide) to react for 15min, rinse with DMF and dichloromethane alternately three times, use Kaiser reagent to detect whether deprotection is successful, if the resin shows color, it means that deprotection is successful and the amino group is exposed;

[0058] S2. Amino acid coupling: When the previous amino acid is successfully deprotected, weigh the next amino acid, HATU and HOBT, add the coupling agent and react for 10 minutes, then add it to the product obtained in step S1 and react on a shaking table for 1 hour, then rinse it alternately with N,N-dimethylformamide and dichloromethane three times, and use Kaiser reagent to detect whether the coupling is successful. If the resin does not show color, it means that the coupling is successful.

[0059] S3, continue to repeat the order of step S1 and step S2 to connect the amino acids of DBCO-TPP to the resin in sequence;

[0060] S4, cleavage, drying and purification: add cleavage solution (composed of trifluoroacetic acid and triisopropylsilane) to DBCO-TPP, stir in an ice bath for 2.5 h, and blow nitrogen to volatilize the cleavage solution;

[0061] S5. Add ice ether, quickly perform ultrasonic centrifugation, collect the precipitate, vacuum dry, and obtain a novel polypeptide fluorescent probe after purification. Store at -20℃ for future use. The molecular structure is shown in the figure below. Figure 1 shown.

[0062] verify

[0063] 1. Verify at the solution level whether MMP2 can cleave DBCO-TPP and self-assemble to form nanofibers.

[0064] S1. First, weigh DBCO-TPP and prepare a 5 mM stock solution with DMSO;

[0065] S2. The mother solution was taken out and added into ultrapure water to prepare a 50 μM working solution. MMP2 (2 μg / mL) was added for co-incubation. The specific morphology was observed by scanning electron microscopy and transmission electron microscopy.

[0066] The results are as follows Figure 2 As shown, the results of scanning electron microscopy and transmission electron microscopy both showed nanofibers, indicating that DBCO-TPP can be sheared and self-assembled by MMP2 to form nanofibers.

[0067] 2. Verify the targeting of DBCO-TPP at the cellular level.

[0068] S1. First, two bladder cancer cells (EJ and RT112) with high expression of CD44v6 and two normal cells (HUVEC and MSC) were selected;

[0069] S2. Then, prepare rhodamine-labeled DBCO-TPP as follows: prepare rhodamine into 10 mg / mL (1 mL) with buffer solution (MES:NaCl=1:5), add EDC (40 mg) and NHS (60 mg) and mix for 15 min; dissolve DBCO-TPP in DMSO to prepare a 10 mg / mL working solution, react DBCO-TPP and rhodamine at a ratio of 1:1 for 2 h, then dialyze for 24 h and freeze-dry; finally, incubate the above rhodamine-labeled DBCO-TPP (50 μM) with the above four cells for 2 h, and observe the co-localization of rhodamine-labeled DBCO-TPP in the cells by laser confocal microscopy.

[0070] The results are as follows Figure 3 As shown, red fluorescence of rhodamine can be seen on the surface of EJ and RT112 cells, but no fluorescence is seen in HUVEC and MSC cells, which proves that the DBCO-TPP of the present invention has high specificity.

[0071] 3. Verify the cleavage ability of MMP2 at the cellular level.

[0072] S1. First, select EJ and RT112 cells with high expression of CD44v6, MDA-MB-231 cells with low expression of CD44v6, and HUVEC cells without CD44v6 expression;

[0073] S2. DBCO-TPP (50 μM) was co-incubated with the above four types of cells for 12 h, and then washed three times with PBS, and then observed by scanning electron microscopy to see whether nanofibers were formed on the cell surface.

[0074] The results are as follows Figure 4 As shown, nanofibers are formed on the surface of EJ and RT112 cells, but not in MDA-MB-231 and HUVEC, indicating that DBCO-TPP can be precisely targeted to the surface of EJ and RT112 cells, and self-assemble to form nanofibers attached to the cell surface after MMP2 cleavage; while in MDA-MB-231 and HUVEC, there is no or less CD44v6, so it cannot be specifically targeted to the cell surface, and nanofibers formed by MMP2 cleavage will be washed away and not adhere to the cell surface regardless of whether there is MMP2 cleavage. The above results prove that DBCO-TPP can be cleaved by MMP2 enzyme to self-assemble into nanofibers.

[0075] 4. Verify the increased number of “artificial receptors” in the pre-targeting nanoplatform.

[0076] S1. Prepare the CD44v6 targeting peptide labeled with Cy5 (referred to as TP-Cy5), the specific steps are as follows: 1 mg of Cy5 NHS (MW: 753.88) is dissolved in 400 μL of DMSO and added to 1 mL of ultrapure water; 10 mg of DBCO-TPP is dissolved in 400 μL of DMSO and added to 1 mL of ultrapure water; the above Cy5 solution is added to the DBCO-TPP solution and mixed, and then 15 μL of triethylamine is added, and the mixture is stirred in an ice bath in the dark for 8 h, and TP-Cy5 is obtained by vacuum drying and placed at -20°C for use;

[0077] S2. Cultivate EJ tumor spheres and plate 1×10 4 EJ cells, tumor spheres can be successfully cultured after 5-7 days of culture;

[0078] S3. Finally, EJ tumor spheres were pretreated with DBCO-TPP (50 μM) for 2 h, the culture medium was discarded, and the PBS was washed once;

[0079] S4. Then, different concentrations of azide-Cy5 (0.25 μM-10 μM) were incubated for 30 min, and the control group was incubated with different concentrations of TP-Cy5 (0.25 μM-10 μM) for 30 min. The fluorescence intensity of the tumor spheres was observed by laser confocal microscopy.

[0080] The fluorescence of TP-Cy5 represents the number of CD44v6 receptors on the cell surface, while the DBCO-TPP group shows an increased number of "artificial receptors". Figure 5 The results showed that the DBCO-TPP group showed strong fluorescence at 0.25μM-10μM, while the TP-Cy group only showed weak fluorescence at 100μM, and the 3D graph also had an obvious trend; in addition, the fluorescence intensity quantification graph also showed a significant statistical difference (p < 0.0001), which indicated that the novel polypeptide fluorescent probe of the present invention indeed increased the expression of cell surface receptors.

[0081] 5. Verify the long-term retention of DBCO-TPP in vivo.

[0082] S1, 5×10 6 EJ cells were mixed with matrix gel and injected into the right armpit of BALB / C nude mice, and tumors were observed after 20 days.

[0083] S2. 50 μL of DBCO-TPP (50 μM) was pre-injected intratumorally for 2 hours, and then azide-Cy5 (10 μM, 50 μL) was injected intratumorally for 30 minutes. The control group was injected with pure Cy5 (10 μM, 50 μL) intratumorally for 30 minutes. Finally, the two groups were placed under the IVIS imaging system to capture the fluorescence attenuation at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 14h.

[0084] The results are as follows Figure 6 As shown, after intratumoral injection of the same dose of Cy5, a stronger fluorescence signal was observed in the tumor of the DBCO-TPP group, and the fluorescence signal intensity remained almost unchanged within 8 hours, which was significantly higher than that of the pure Cy5 group, proving the long-term retention of DBCO-TPP in vivo.

[0085] 6. Verify the ability of DBCO-TPP to identify micro-tumors in the airbag model.

[0086] S1. Select 6-week-old BALB / C female nude mice as the model, inject 2-3 mL of sterile air into the back of the nude mice, and supplement 1 mL of sterile air into the back every other day;

[0087] S2. One week later, EJ cell suspension (5×10 6 cells), nude mice were fed normally for different periods of time and then given DBCO-TPP, azide-Cy5 and pure Cy5, and images were taken under IVIS to identify microtumors.

[0088] The results are as follows Figure 7 As shown, tumors of different volumes (30.0±1.5, 35±1.2, 45±3.0, 55±1.0, 75±4.2, and 110±2.3 mm) can be observed from the bright field. 3 ), the fluorescence field also showed obvious fluorescence in the tumor tissue, which could be clearly distinguished from adjacent tissues, and the correlation diagram between tumor volume and fluorescence intensity also showed that the fluorescence intensity increased with the increase of volume (Pearson's r = 0.9926). This result proves that the novel polypeptide fluorescent probe of the present invention can identify micro-tumors and expand its application in the field of bladder cancer imaging.

[0089] 7. Verify the imaging effect of DBCO-TPP on in vitro tissues in clinical samples.

[0090] S1. Bladder cancer tissue and normal urothelial tissue removed during surgery were rinsed with saline and immediately placed in RNALater tissue solution at 4°C.

[0091] S2. The cancer tissue and normal tissue were pre-immersed in DBCO-TPP (50 μM) for 2 h, and then continued to be immersed in azide-Cy5 (10 μM) for 30 min; the TP-Cy5 (20 μM) and pure Cy5 (10 μM) of the control group were incubated for the same time (30 min), and the imaging effect was captured under the IVIS system.

[0092] The results are as follows Figure 8 As shown, for tumor tissue, DBCO-TPP can be observed to be able to target the tumor site and show strong fluorescence, TP-Cy5 can also target with slightly weaker fluorescence intensity, while the fluorescence intensity of the non-targeted tumor site and the adjacent site of the cancer in the pure Cy5 group is not much different; for normal tissue, the three groups are not much different. The average fluorescence quantitative graph also shows that the DBCO-TPP group is higher, and there is a significant statistical difference with the other two control groups (p < 0.0001), indicating that the novel polypeptide fluorescent probe of the present invention has a highly sensitive recognition ability and can visually guide bladder cancer imaging.

[0093] In summary, the novel polypeptide fluorescent probe disclosed in the present invention can accurately target and identify tumor cells, and can be sheared and self-assembled under the response of MMP2 enzyme to form a nanofiber network that adheres to the surface of bladder cancer cells, which not only increases the number of receptors expressed on the cell surface and prolongs the retention time in the body, but also can identify micro-tumors and improve surgical resection rates. In addition, the novel polypeptide fluorescent probe of the present invention was verified in human ex vivo tissues, and good imaging effects were also achieved, further verifying its application prospects in future clinical transformation.

[0094] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A novel polypeptide fluorescent probe, characterized in that: The novel polypeptide fluorescent probe is composed of a target recognition polypeptide module, an enzyme trigger response module, a self-assembly module and a functional module; The polypeptide fluorescent probe specifically recognizes the targeted peptide segment, adheres to the cell surface through the proximity adhesion effect after enzyme response shearing to form a fiber network that increases the receptors on the cell surface, and then infuses the azidated fluorescent molecules to produce a bioorthogonal reaction to achieve specific imaging.

2. A novel polypeptide fluorescent probe according to claim 1, characterized in that: The receptors recognized by the targeting recognition module are targeting receptors highly expressed on bladder cancer cells, and the targeting receptors include integrins, fibroblast growth factor receptor 1 and CD44v6 receptors; The specific sequence of the CD44v6 receptor is NRWHEDC or CSDRIMRGC.

3. A novel polypeptide fluorescent probe according to claim 2, characterized in that: The enzyme trigger response module is a response shear enzyme highly expressed in the tumor area, and the response shear enzyme includes caspase-3 / 7, glucose hydrolase, alkaline phosphatase, cathepsin B and MMP2 enzyme; Among them, the response cleavage peptide sequence of the MMP2 enzyme includes PLGYLG, PLGVRG, PLGLAG, PVGLIG, PLGLAG, GPLGIAGQC and GPLGVRGS.

4. A novel polypeptide fluorescent probe according to claim 3, characterized in that: The self-assembling peptide module can spontaneously assemble ordered aggregates under the condition of hydrophilic-hydrophobic imbalance, and the sequence of the self-assembling module is any one of YLGFFC, KLVFF, KLKLKL, and FF.

5. A novel polypeptide fluorescent probe according to claim 4, characterized in that: The novel polypeptide fluorescent probe also contains a hydrophilic substance, and the hydrophilic substance is polyethylene glycol-4.

6. A novel polypeptide fluorescent probe according to claim 5, characterized in that: The functional module is a group that can undergo a bioorthogonal reaction, specifically any one or a combination of two of diphenylcyclooctyne, azide, tetrazine and trans-cyclooctene.

7. A novel polypeptide fluorescent probe according to claim 6, characterized in that: After the azide is modified, an azidated fluorescent molecule is formed, and the fluorescent molecule to be modified is any one of Cyanine5, Cyanine7, Indocyanine Green and Fluorescein Isothiocyanate.

8. A novel polypeptide fluorescent probe according to any one of claims 1 to 7, further comprising a method for preparing the novel polypeptide fluorescent probe, characterized in that: The following steps are included: S1. Connecting the targeting peptide, the enzyme-responsive cleavage peptide, the self-assembling peptide and the functional peptide in sequence through amide bonds by solid phase synthesis to obtain a polypeptide fluorescent probe; S2. Cy5-N3 is connected to the peptide fluorescent probe through click reaction to obtain a new peptide fluorescent probe. The synthesis steps of the polypeptide fluorescent probe are as follows: P1, select the first amino acid resin for swelling; P2, remove the Fmoc group in step P1 to expose the amino group, and undergo an amide condensation reaction with the carboxyl group of the next amino acid, so that the amino acids on the peptide fluorescent probe are sequentially connected and fixed on the resin; P3, removing the Fmoc group of PEG4 in step P2 to expose NH2; P4, coupling reaction of the peptide fluorescent probe with NH2 in step P3; P5, cleaving and separating the polypeptide in step P4 from the resin to obtain a purified polypeptide fluorescent probe; P6. The polypeptide fluorescent probe in step P5 undergoes a click reaction with the fluorescent molecule Cy5-N3 to obtain a novel polypeptide fluorescent probe.

9. The method for constructing a novel polypeptide fluorescent probe according to claim 8, characterized in that: In the step P1, the amino acid is aspartic acid, and the resin swelling time is 9h-12h; In the steps P2 and P3, the deprotecting agent is composed of hexahydropyridine and N,N-dimethylformamide; The main component of the coupling agent in step P4 is N,N-dimethylformamide; The main components of the lysate in step P5 are trifluoroacetic acid and triisopropylsilane; In the step P6, the reaction ratio of the polypeptide fluorescent probe to Cy5-N3 is 1:1, and the reaction time is 5 min-90 min.

10. The polypeptide fluorescent probe according to any one of claims 1 to 7 is used for in vivo imaging of cells and small animals, the polypeptide concentration used is 50 μM-100 μM, and the incubation time is 0.5h-12h; and / or applied in surgical resection of bladder cancer.

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