Preparation method of protein carboxymethyl transferase responsive polypeptide assembled supramolecular fluorescent probe

Abstract

The invention discloses a preparation method of a protein carboxymethyl transferase responsive polypeptide assembled supramolecular fluorescent probe, and the imaging capability of the probe on bladder cancer is researched. The principle that PCMT enzyme can catalyze conversion of isoaspartic acid into aspartic acid is utilized, a main body polypeptide isoDV responded by the PCMT enzyme is designed and synthesized, a fluorescent molecule 7-nitro-2, 1, 3-benzoxadiazole (NBD) with hydrophobic environment responsiveness is modified to a main body polypeptide molecule isoDV, and the main body polypeptide molecule isoDV and the fluorescent molecule NBD are blended to obtain the fluorescent probe NisoDV. NisoDV catalyzes and repairs isoaspartic acid in the fluorescent probe to be converted into aspartic acid through PCMT enzyme overexpressed in bladder cancer cells, so that polypeptide is promoted to be assembled to form nanofibers, and NBD units in the nanofibers are subjected to fluorescence enhancement due to hydrophobicity dependence, so that PCMT detection is realized. In-vivo and in-vitro studies prove the effective PCMT detection capability of the supramolecular probe.

Description

Technical Field

[0001] The invention relates to a polypeptide-assembled supramolecular fluorescent probe responsive to protein carboxylmethyl transfer, and belongs to the field of biomedicine. Background Art

[0002] Protein carboxylmethyltransferase (PCMT) plays a key role in protein repair and can restore aspartate isomers in proteins. Its expression is closely related to cancer prognosis, so PCMT can be used as a biomarker for cancer diagnosis. However, there are currently limited methods for directly detecting PCMT, mainly due to the limited ability of methylation to regulate the optical properties of the dye.

[0003] The assembly of peptides to form nanostructures provides the possibility for the development of biomaterials with specific functions. Stimuli-responsive peptide assembly in living cells has become an emerging strategy for the precise creation of biomedical agents at pathological sites to achieve the diagnosis and treatment of diseases. The present invention aims to address the challenge of direct detection of PCMT and develop a peptide assembly supramolecular fluorescent probe based on aspartic acid isomerization regulation for imaging PCMT in cancer cells. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that the existing PCMT fluorescent probe is difficult to detect directly in cells and living bodies, and to provide a PCMT enzyme-responsive polypeptide-assembled supramolecular fluorescent probe. A preparation method of the probe is also provided.

[0005] The technical solution of the present invention is:

[0006] The purpose of the first aspect of the present invention is to provide a PCMT-responsive polypeptide-assembled supramolecular fluorescent probe, wherein the polypeptide supramolecular fluorescent probe is NisoDV, which is prepared by blending polypeptide isoDV and NBD-isoDV, and the structural formulas of the components are as follows:

[0007]

[0008] The volume ratio of polypeptide isoDV and NBD-isoDV is 98:2.

[0009] The second aspect of the present invention is to provide the preparation steps of the above-mentioned PCMT enzyme-responsive polypeptide supramolecular fluorescent probe as follows:

[0010] Step 1: Synthesis of peptide isoDV and NBD-isoDV;

[0011] (1) For the peptide isoDV, the peptide isoDV was synthesized according to the structural formula by conventional Fmoc solid phase synthesis method. Specifically, Rink resin was weighed and placed in a reactor, dichloromethane was added to swell the resin, 4 times the equivalent of Fmoc-protected amino acid, 4 times the equivalent of HBTU and 6 times the equivalent of DIEA relative to the Rink resin were dissolved in DMF and reacted with the resin for 1.5-2 hours, and 25% piperidine was used to remove the Fmoc protecting group to obtain the peptide isoDV;

[0012] (2) For the peptide NBD-isoDV, after the amino acids are connected according to the structural formula by conventional Fmoc solid phase synthesis, the protecting group of the last amino acid is removed, and 4 times the equivalent of NBD-β-Ala, 4 times the equivalent of HBTU and 6 times the equivalent of DIEA relative to the Rink resin are added. After the reaction is completed, the peptide is cleaved from the resin using a cleavage agent to obtain the NBD-labeled peptide NBD-isoDV;

[0013] Step 2: preparing NisoDV by blending peptide isoDV and NBD-isoDV;

[0014] First, freeze-dried powders of polypeptides isoDV and NBD-isoDV were used to obtain polypeptide mother solutions of isoDV and NBD-isoDV with a pH of 7.4 and the same concentration; and NisoDV mother solutions were prepared by mixing the mother solutions of polypeptides isoDV and NBD-isoDV in a volume ratio of 98:2.

[0015] Furthermore, the concentration of the prepared NisoDV stock solution was 500 μM.

[0016] The third aspect of the present invention aims to provide the use of the supramolecular fluorescent probe NisoDV described above in protein carboxylmethyltransferase response.

[0017] After the PCMT enzyme-responsive polypeptide supramolecular fluorescent probe NisoDV prepared by the present invention enters bladder cancer cells, under the catalytic action of the PCMT enzyme overexpressed in bladder cancer cells, the isoaspartic acid in the polypeptide molecule is recognized and repaired, thereby forming nanofibers in situ in the cells, and the fluorescent unit NBD in the fiber can achieve strong fluorescence enhancement to achieve direct detection of PCMT. In vivo and in vitro studies have jointly confirmed the ability of the polypeptide supramolecular fluorescent probe to effectively detect PCMT in bladder cancer.

[0018] The characterization of the polypeptide-assembled supramolecular fluorescent probe NisoDV obtained by the present invention includes characterization by circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy, as detailed in the specific implementation manner.

[0019] Advantages and beneficial effects of the present invention:

[0020] (1) The polypeptide-assembled supramolecular fluorescent probe designed in the present invention has the advantages of good biocompatibility and low immunogenicity.

[0021] (2) All the reaction conditions of the present invention are very mild, the preparation method is simple, and the operation is easy.

[0022] (3) The polypeptide-assembled supramolecular fluorescent probe obtained by the present invention can realize the direct detection of the bladder cancer tumor marker PCMT enzyme.

[0023] (4) The present invention provides a method for regulating polypeptide assembly under physiological conditions, thereby providing a chemical tool for disease diagnosis.

[0024] The advantages and effects of the present invention will be described below through examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .The chemical structural formulas of the component molecules isoDV and NBD-isoDV of the polypeptide self-assembled supramolecular fluorescent probe NisoDV of the present invention and the chemical structural formulas of the natural sequence control molecules DV and NBD-DV.

[0026] Figure 2 .(AD) AFM (A) and TEM (B) images of peptide isoDV. AFM (C) and TEM (D) images of peptide DV. (E) Circular dichroism spectra of peptide isoDV and its natural counterpart DV. (F, G) UPLC traces of peptide isoDV at different time points after PCMT enzyme treatment (F), and the mass spectra corresponding to the reaction intermediates observed in the UPLC trace (G). (H) TEM image of peptide isoDV after incubation with PCMT enzyme and S-adenosylmethionine and aging for 24 hours.

[0027] Figure 3 .Fluorescence spectra of NBD-DV (A) and NBD-isoDV (B) co-assembled with DV or isoDV, respectively. (C) Maximum fluorescence intensity of NDV and NisoDV composed of fixed peptide DV or isoDV at 1 mM concentration and different ratios of NBD derivatives. Fluorescence spectra of different total peptide concentrations with fixed NBD-DV (D) and NBD-isoDV (E) at a constant molar ratio of 98:2 with DV or isoDV. (F) Maximum fluorescence intensity of NDV and NisoDV containing 2% NBD derivatives at different total concentrations. (G) CD spectra of NDV and NisoDV. (H) Maximum fluorescence intensity of NisoDV in the presence of PCMT and SAM and other interfering agents.

[0028] Figure 4(A) WB detection of PCMT levels in normal cells SV-HUC-1, HUVEC, L02 or cancer cell lines HeLa and 5637 cells. (B, C) Cell viability of 5637 cells (B) or SV-HUC-1 cells (C) after incubation with different concentrations of DV or isoDV for 24 hours. (D) Fluorescence confocal images of 5637 cells incubated with NisoDV or DV for 24 hours, or confocal images of siRNA-transfected 5637 cells incubated with NisoDV for 24 hours. (E) Flow cytometry analysis of the median fluorescence intensity of 5637 cells incubated with NDV and NisoDV. Statistical analysis was performed using one-way analysis of variance (ANOVA). *p<0.05, **p<0.01, ***p<0.001. (F) Biological transmission electron microscopy images and magnified areas of 5637 cells incubated with isoDV for 24 hours. (G, H) Biological transmission electron microscopy images of SV-HUC-1 (G) cells and 5637 (H) cells incubated with isoDV or PBS for 24 h, respectively.

[0029] Figure 5 .(A) In vivo fluorescence imaging of 5637 bladder cancer-bearing mice after injection of NDV or NisoDV. (B) Quantitative mean fluorescence intensity of in vivo signals at tumor sites. Statistical analysis was performed using one-way analysis of variance (ANOVA). *p<0.05, **p<0.01, ***p<0.001. (C) Ex vivo fluorescence imaging of tumor tissues and major organs dissected from mice 24 hours after injection of NDV or NisoDV. (D) Quantitative mean fluorescence intensity of ex vivo signals at tumor sites or major organs of mice injected with NDV or NisoDV. H: heart, Li: liver, S: spleen, Lu: lung, K: kidney, T: tumor. Statistical analysis was performed using one-way analysis of variance (ANOVA). *p<0.05, **p<0.01, ***p<0.001. (EH) Confocal images of frozen sections of tumor tissues from mice injected with NisoDV (E, F) or NDV (G, H). (I) Hemolysis photos (inset) and hemolysis rates of NDV or NisoDV (n=3). (J) H&E staining of isolated major organs (heart, liver, spleen, lung and kidney) collected from mice injected with NDV or NisoDV.

[0030] Figure 6.Schematic diagram of in situ assembly of supramolecular fluorescent probes regulated by aspartate isomerization in living cells for direct detection of PCMT in vivo. Top: Chemical structures of peptides isoDV, DV, NBD-isoDV, and NBD-DV. PCMT and catalytic isoDV are converted to DV and subsequently assembled into nanofibers. Bottom: After tail vein injection, peptide NisoDV undergoes PCMT-induced in situ conversion from monomeric NisoDV to NDV to form nanofibers. Peptide NDV exhibits a strong fluorescence signal due to the hydrophobicity-dependent fluorescence enhancement of the NBD fluorescent unit, thereby directly detecting PCMT. DETAILED DESCRIPTION

[0031] Embodiment 1:

[0032] The preparation of a PCMT enzyme-responsive polypeptide self-assembled supramolecular fluorescent probe mainly involves the following steps:

[0033] 1) Weigh 720 mg of Rink resin and place it in a peptide synthesis tube. Add dichloromethane to swell the resin for 15 minutes, filter and discard the solvent, add the DMF solution of Fmoc-Glu(OtBu)-OH amino acid to the reactor and shake for 1.5 hours. After the reaction is completed, wash the resin 3 times with dichloromethane and DMF alternately; use 25% piperidine to remove the Fmoc protecting group to obtain the peptide isoDV. Repeat the above steps and connect all amino acids according to the structural formula. After the connection is completed, remove the protecting group of the last amino acid, use a cleavage agent to cleave the peptide from the resin to obtain the peptide NBD-isoDV. The structural formulas of the obtained peptides isoDV and NBD-isoDV are as follows:

[0034]

[0035] 2) The obtained lyophilized powders of polypeptides isoDV and NBD-isoDV were dissolved to obtain polypeptide mother solutions of isoDV and NBD-isoDV with a pH of 7.4 and a concentration of 4 mM; a NisoDV mother solution with a concentration of 500 μM was prepared by mixing the mother solutions of polypeptides isoDV and NBD-isoDV at a volume ratio of 98:2.

[0036] The final solution obtained is NisoDV.

[0037] The chemical structural formulas of the component molecules isoDV and NBD-isoDV of the polypeptide self-assembled supramolecular fluorescent probe NisoDV of the present invention and the chemical structural formulas of the natural sequence control molecules DV and NBD-DV are as follows: Figure 1 shown.

[0038] Embodiment 2:

[0039] 1. The structural characterization of the obtained peptide self-assembled supramolecular fluorescent probe NisoDV, including atomic force microscopy, transmission electron microscopy, circular dichroism spectroscopy and various characterization methods are as follows:

[0040] 1) Atomic force microscopy: AFM images were taken by a CSPM 5500 instrument in tapping mode; AFM samples of all peptides were prepared by diluting the peptide stock solution; 10 μL of peptide solution was dropped onto the surface of a freshly cut mica sheet and left for 5 minutes, then the remaining solution was removed with filter paper and dried before use for sample testing;

[0041] 2) Transmission electron microscopy: TEM images were recorded by a HITACHI HT7700 Exalens microscope at an accelerating voltage of 200 kV; TEM samples of all peptides were prepared by diluting the mother solution to a concentration of 500 μM; 10 μL of the peptide solution was dropped onto a carbon-coated copper grid and left for 5 minutes. After the remaining solution was removed with filter paper, it was stained with 2% uranyl acetate for 5 minutes. After the remaining stain was removed with filter paper, the copper grid was placed in a desiccator to dry for later testing.

[0042] 3) Circular dichroism (CD) spectra: CD spectra were recorded by Biologic MOS-500 spectrometer at 25°C using 1 mm quartz cuvettes; CD samples of all peptides were prepared by diluting 500 μM peptide stock solutions, and CD spectral scans were recorded at a wavelength interval of 1.0 nm and a wavelength range of 190 to 260 nm;

[0043] 2. The enzyme responsiveness of the designed peptide isoDV PCMT is studied as follows:

[0044] The peptide isoDV (100 μM) was mixed with PCMT (5 μM) and SAM (1 mM) in Tris-HCl buffer (100 mM, pH 7.4) and incubated at 37° C. The enzyme response process was monitored by UPLC-MS.

[0045] 3. TEM characterization of the designed peptide isoDV PCMT enzyme response assembly:

[0046] TEM images were taken after the peptide isoDV (500 μM) was co-incubated with PCMT (15 μM) and SAM (3 mM) and aged for 24 h.

[0047] Experimental results: Figure 2As shown, AFM and TEM showed that no ordered nanostructure was observed for the peptide isoDV, which existed in the form of a monomer, while the corresponding natural sequence DV formed bundled nanofibers. Circular dichroism spectroscopy studies showed that the peptides DV and isoDV formed secondary structures of β-folding and random coiling, respectively. The morphological and conformational results indicate the key role of isoaspartic acid in regulating the assembly tendency of amphiphilic peptides, as the incorporation of isoaspartic acid causes the twisting of the hydrophobic surface of the peptide, which affects the assembly of the peptide. We then studied the assembly reactivity induced by the peptide isoDV and PCMT enzyme. After adding PCMT enzyme and methyl donor SAM, the UPLC trace showed that isoaspartic acid was methylated in the peptide isoDV, and a succinimide intermediate was formed, and isoDV was gradually converted into DV( Figure 2 , F and G). TEM images of peptide isoDV treated with PCMT and SAM show the formation of nanofibers ( Figure 2 H), directly demonstrating PCMT-induced transformation and assembly of isoDV.

[0048] Embodiment 3:

[0049] 1. Characterization of fluorescence enhancement and fluorescence interference of the prepared supramolecular fluorescent probe. The fluorescence enhancement is determined by fluorescence spectrometer. The specific methods are as follows:

[0050] 1) Dilute the peptide master solutions DV and isoDV to different concentrations with PBS buffer and mix with NBD-DV or NBD-isoDV to prepare fluorescent samples. For samples with a fixed total peptide concentration, the total concentration of fixed peptide DV and isoDV is 1 mM, and the content of NBD-modified peptides ranges from 0.25% to 8%. Record the fluorescence spectrum in the range of 500nm to 700nm on an Agilent Cary Eclipse fluorescence spectrophotometer, with an excitation wavelength of 464nm.

[0051] 2) For samples with a constant content of NBD-modified peptide, the proportion of NBD-modified peptide was fixed at 2% of the total concentration, and samples with different total peptide concentrations ranging from 62.5 μM to 2 mM were prepared. The fluorescence spectrum was recorded in the range of 500 nm to 700 nm on an Agilent Cary Eclipse fluorescence spectrophotometer, with an excitation wavelength of 464 nm.

[0052] 3) Fluorescence spectra of NisoDV in the presence of various interfering agents: NisoDV (500 μM) alone, NisoDV (500 μM) with PCMT, SAM, PCMT+SAM, KCl (10 mM), NaCl (10 mM), MgCl 2 (2.5mM), H 2 O 2Fluorescence spectra of 1H2O (10 μM), GSH (2 mM), vitamin C (1 mM) or glucose (10 mM).

[0053] 2. Characterization of the assembly ability of the prepared supramolecular fluorescent probe NisoDV and its control molecule NDV, and determination of the assembly status by CD, the specific method is as follows:

[0054] CD spectra: CD spectra were recorded by Biologic MOS-500 spectrometer at 25°C using 1 mm quartz cuvettes; the CD sample concentration of peptide NisoDV and its control molecule NDV was 500 μM, and CD spectral scans were recorded at a wavelength interval of 1.0 nm and a wavelength range of 190 to 260 nm;

[0055] Experimental results: NBD units are widely used as fluorescent probes in peptide assemblies because of their hydrophobic microenvironment that enhances fluorescence quantum yield. Therefore, the co-assembled peptide NDV exhibits a strong fluorescence intensity, while NisoDV only shows a weak fluorescence ( Figure 3 , A and B). The results showed that 2% NBD-DV resulted in the largest difference in fluorescence intensity between NDV and NisoDV ( Figure 3 C). To avoid the cytotoxicity of the supramolecular fluorescent probe caused by high-dose peptides, we also studied the minimum total peptide concentration and the maximum fluorescence contrast between NDV and NisoDV with fixed 2% NBD. The total peptide concentration of 500 μM basically achieved the maximum fluorescence contrast ( Figure 3 , DF). This experiment determined the supramolecular fluorescent probe NisoDV and its control molecule NDV with 2% NBD content and a total peptide concentration of 500 μM. Conformational studies confirmed that NDV maintained a β-folded secondary structure, while NisoDV maintained a random coiled secondary structure ( Figure 3 G). The selectivity of the supramolecular probe NisoDV for detecting PCMT was also evaluated. The fluorescence spectroscopic experiments with conventional biological interfering agents ( Figure 3 H), demonstrating the excellent selectivity of the peptide self-assembled supramolecular fluorescent probe for detecting PCMT.

[0056] Embodiment 4:

[0057] 1. Western blot (WB) detection: HUVEC cells, L02 cells, Hela cells, or 5637 cells were cultured at 2×10 5The cells were seeded at a density of 100 μg / ml in a six-well plate and cultured overnight. The culture medium was discarded, washed with PBS, treated with 2% SDS and collected. After measuring the protein concentration by Bradford protein quantitative detection kit, the protein was incubated with loading buffer at 95°C for 10 minutes. Subsequently, proteins from different cells were loaded onto SDS-PAGE gels and electrophoretically transferred to PVDF membranes. The membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with the corresponding specific primary antibody (Anti-PCMT) at 4°C overnight. After incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour, the protein bands were imaged using ECL chemiluminescence detection under the Tanon-5200Multi instrument.

[0058] Experimental results: Figure 4 As shown in A, 5637 bladder cancer cells overexpress PCMT enzyme.

[0059] 2. Characterization of in vitro cytotoxicity of the prepared peptide supramolecular fluorescent probe NisoDV;

[0060] The cytotoxicity of the peptides to 5637 human bladder cancer cells and SV-HUC-1 human urothelial cells was determined by using a standard MTT assay as follows:

[0061] 1) The 5637 human bladder cancer cells and SV-HUC-1 human urothelial cells were respectively seeded at a density of 5000 cells per well in a 96-well plate containing 1640 culture medium containing 10% FBS and 1% penicillin-streptomycin;

[0062] 2) After incubation for 24 hours, the cells were incubated with different concentrations of polypeptide for 24 hours; wherein the different concentrations were: 0, 62.5, 125, 250, 500, 1000 and 2000 μM;

[0063] 3) Discard the culture medium, wash each well three times with PBS, add culture medium containing MTT and incubate for further 4 hours;

[0064] 4) Finally, the supernatant was removed, 100 μL of DMSO was injected into each well to dissolve the purple crystals, and the viability of the cells was measured by recording the absorption intensity at 495 nm on a microplate reader.

[0065] Experimental results: Figure 4 As shown in A, B and C, peptides DV and isoDV have good biocompatibility against 5637 bladder cancer cells and SV-HUC-1 at a concentration of 500 μM.

[0066] 3. Characterization of intracellular fluorescence imaging of the prepared peptide supramolecular fluorescent probe NisoDV. The intracellular fluorescence enhancement was observed by confocal microscopy and quantified by flow cytometry. The method is as follows;

[0067] 1) 5637 human bladder cancer cells or SV-HUC-1 urothelial cells were cultured at 1×10 per well 5 The cells were seeded at a density of 100 cells / mL in a confocal dish and cultured at 37°C for 24 h. Then, 500 μM of NisoDV supramolecular fluorescent probe and its control molecule NDV were added to the cells. After 12 h, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, stained with DAPI for 20 min, washed three times with PBS, and imaged using a confocal laser scanning microscope.

[0068] 2) For the negative control experiment of 5637 human bladder cancer cells, small interfering RNA was used to inhibit the expression of PCMT enzyme in bladder cancer cells and added to the cell culture medium. The transfection step was performed according to the standard protocol provided by the supplier, and the fluorescence signal of the polypeptide in the transfected cells was measured using the same protocol as above.

[0069] 3) 5637 human bladder cancer cells or SV-HUC-1 urothelial cells were cultured at 1×10 5 Cells were seeded in six-well plates at a density of 10 cells / mL and cultured at 37°C for 24 hours. The cells were washed with PBS, and fresh serum-free medium containing NDV or NisoDV (500 μM) was added and incubated for 2, 4, 8, 12, or 24 hours. The cells were digested with trypsin and washed thoroughly with PBS. The incubated cells were resuspended in 500 μL PBS and analyzed by flow cytometry.

[0070] Experimental results: Confocal laser scanning microscopy images of 5637 cells treated with NisoDV showed strong fluorescence signals, while incubation with NDV produced weak fluorescence signals ( Figure 4 D). Quantification of median fluorescence intensity by flow cytometry analysis showed that tumor cells treated with NisoDV for 24 h displayed a 4.5-fold higher signal intensity than NDV ( Figure 4 E). Normal SV-HUC-1 cells or siRNA-transfected 5637 cells incubated with NisoDV to inhibit PCMT expression did not show strong fluorescence signals ( Figure 4 D).

[0071] 4. Biological transmission electron microscopy: 5637 cells and SV-HUC-1 cells were cultured at 1×10 5The cells were seeded in 6-well plates at a density of 10 cells / mL overnight. The cells were incubated with isoDV (500 μM) or PBS for 24 hours. The cells were trypsinized and washed thoroughly with PBS, and the cells were collected and then fixed in 2.5% glutaraldehyde overnight. After removing the fixative, they were washed three times with PBS and fixed with 1% citric acid solution for 2 hours. Subsequently, they were washed three times with PBS, dehydrated with gradient ethanol-PBS solution (from 30%, 50%, 70%, 80%, 90% to 95%) for 15 minutes each time, and treated with 100% ethanol or acetone for 20 minutes. The cell samples were treated with a mixture of embedding medium and acetone (volume ratio 1:1) for 1 hour, another mixture of embedding medium and acetone (volume ratio 3:1) for 3 hours, and pure embedding medium overnight. After embedding, they were heated at 70°C overnight to obtain embedded macrophage samples. The samples were sliced ​​with a LEICA EMUC7 ultramicrotome to obtain slices with a width of approximately 70-90 nm. Finally, the cells were stained with lead citrate solution and uranyl acetate solution (50% ethanol) for 5-10 min, respectively. Biological transmission electron microscopy images were recorded by FEI Tecnai Spirit at 80 kV accelerating voltage.

[0072] Experimental results: BioTEM images showed that 5637 cells treated with isoDV showed a large number of long bundles of nanofibrils ( Figure 4 F), while treated SV-HUC-1 cells showed no detectable nanofibrils ( Figure 4 G), which is similar to 5637 cells treated with PBS ( Figure 4 H). These results suggest that PCMT plays a key role in the assembly of peptides into nanofibers in living cells and in the generation of fluorescent signals following enzyme-induced aspartate isomerization.

[0073] Embodiment 5:

[0074] 1. In vivo fluorescence imaging study of a PCMT enzyme-responsive peptide self-assembled supramolecular fluorescent probe;

[0075] 1) In vivo fluorescence imaging: Female BALB / c nude mice aged 6-8 weeks were purchased from Weitong Lihua Laboratory Animal Co., Ltd. All in vivo experiments were performed in accordance with the protocol approved by the Animal Care and Use Committee of Nankai University. 5637 cells (1×10 7 cells, 150 μL of suspension mixed with Matrigel, 1:1) was subcutaneously inoculated in the right abdomen of mice. When the tumor diameter was about 6 mm, 200 μL of NDV (2 mM) or NisoDV (2 mM) was injected through the tail vein. 2, 4, 8, 12, and 24 hours after injection, the mice were anesthetized with isoflurane. Fluorescence images were collected using a live imaging system with an excitation wavelength of 488 nm.

[0076] 2) To detect the biodistribution of the fluorescent probe, mice were sacrificed 24 h after injection, and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected for ex vivo imaging and analysis.

[0077] Experimental results: In mice injected subcutaneously with NisoDV, obvious fluorescent signals were detected around tumor tissue for a long time ( Figure 5 , A and B). The enhanced accumulation of NisoDV at tumor sites compared with NDV was attributed to enhanced cellular uptake and PCMT-induced assembly. Ex vivo fluorescence imaging of dissected organs and tumor tissues confirmed the improved tumor accumulation of NisoDV ( Figure 5 , C and D).

[0078] 2. Fluorescence Imaging of Tumor Cryosections

[0079] Tumor-bearing mice were injected with 200 μL of NDV (2 mM) or NisoDV (2 mM) solution via the tail vein.

[0080] Twelve hours after injection, tumor tissues of mice were dissected and cryosectioned. Fluorescence imaging of cryosections was performed using confocal microscopy, where nuclei were stained with DAPI and tumor imaging was indicated by fluorescence signals associated with NBD.

[0081] Experimental conclusion: Confocal images of frozen sections of tumor tissue from mice injected with NisoDV showed strong fluorescence signals over a large area ( Figure 5 , E and F). However, tumor tissues from mice treated with NDV showed rather weak fluorescence signals around the tumors ( Figure 5 , G and H). These results indicate that the tumor accumulation and penetration of the probe NisoDV were improved. PCMT-induced NisoDV assembly was confirmed by high-resolution confocal imaging, and nanofibers were detected within tumor tissues ( Figure 5 F). Overall, the in vivo and in vitro fluorescence imaging studies demonstrated the ability of the supramolecular probe NisoDV to efficiently detect PCMT based on enzyme-induced peptide assembly.

[0082] 3. Hematoxylin and eosin (H&E) staining and hemolysis assay of isolated organs

[0083] 1) The main organs (heart, liver, spleen, lung, and kidney) were collected and fixed with 4% paraformaldehyde, and then stained with hematoxylin and eosin (H&E).

[0084] 2) Fresh mouse red blood cells (RBCs) were separated by centrifugation. The red blood cells were resuspended in PBS to obtain a 2% red blood cell suspension. Different concentrations of NDV or NisoDV were then added to the resulting red blood cell suspension. Red blood cells in PBS and deionized water were used as negative and positive controls. After incubation at 37°C for 12 hours, all samples were centrifuged at 2000rpm for 5 minutes. The absorbance of the supernatant at 540nm was detected by a microplate reader. The hemolysis rate was calculated according to the following formula:

[0085] Hemolysis (%) = (A sample -A 0 ) / (A 1 -A 0 )×100%

[0086] Among them A 1 and A 0 are the absorbance of the positive control sample and the negative control sample, respectively.

[0087] Experimental conclusion: Both H&E staining test and hemolysis test showed that the supramolecular probe has reliable biosafety ( Figure 5 , I and J).

[0088] Based on the above embodiments, the application of the supramolecular fluorescent probe NisoDV provided by the present invention in the protein carboxymethyltransferase response is shown in the attached Figure 6 Schematic diagram of supramolecular fluorescent probe for direct detection of PCMT in vivo. After the PCMT enzyme-responsive polypeptide supramolecular fluorescent probe NisoDV prepared by the present invention enters bladder cancer cells, under the catalysis of the PCMT enzyme overexpressed in bladder cancer cells, the isoaspartic acid in the polypeptide molecule is recognized and repaired, thereby forming nanofibers in situ in the cells, and the fluorescent unit NBD in the fiber can achieve strong fluorescence enhancement to achieve direct detection of PCMT.

[0089] It should be further explained that the above implementation modes are only used to understand the technical solution of the present invention, and are not used to limit the protection scope of the present invention. Any obvious adjustments and modifications made to the technical solution of the present invention that belong to the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A polypeptide-assembled supramolecular fluorescent probe responsive to protein carboxylmethyltransferase, wherein the polypeptide-assembled supramolecular fluorescent probe is NisoDV, which is prepared by blending polypeptide isoDV and NBD-isoDV, and the structural formulas of the components are as follows: The volume ratio of polypeptide isoDV and NBD-isoDV is 98:

2.

2. The method for preparing the protein carboxymethyltransferase-responsive polypeptide-assembled supramolecular fluorescent probe according to claim 1, characterized in that: Here are the steps: Lyophilized powders of polypeptides isoDV and NBD-isoDV are used to obtain polypeptide mother solutions of isoDV and NBD-isoDV with a pH of 7.4 and the same concentration; a mother solution of polypeptide-assembled supramolecular fluorescent probe NisoDV is prepared by mixing the mother solutions of polypeptides isoDV and NBD-isoDV at a volume ratio of 98:2; wherein the polypeptides isoDV and NBD-isoDV have the structures shown in claim 1.

3. The method for preparing a polypeptide-assembled supramolecular fluorescent probe responsive to protein carboxylmethyltransferase according to claim 2, characterized in that: The polypeptide isoDV is obtained by the following method: the polypeptide isoDV is synthesized by a conventional Fmoc solid phase synthesis method, and the specific operation is: weighing a Rink resin and placing it in a reactor, adding dichloromethane to swell the resin, dissolving 4 times the equivalent of Fmoc-protected amino acids, 4 times the equivalent of HBTU and 6 times the equivalent of DIEA relative to the Rink resin in DMF and reacting with the resin for 1.5-2 hours, and removing the Fmoc protecting group with 25% piperidine; the polypeptide isoDV is obtained.

4. The method for preparing a polypeptide-assembled supramolecular fluorescent probe responsive to protein carboxylmethyltransferase according to claim 2, characterized in that: The peptide NBD-isoDV is obtained by the following method: after the amino acids are connected by conventional Fmoc solid phase synthesis, the protecting group of the last amino acid is removed, and 4 times the equivalent of NBD-β-Ala, 4 times the equivalent of HBTU and 6 times the equivalent of DIEA relative to the Rink resin are added. After the reaction is completed, the peptide is cleaved from the resin using a cleavage agent to obtain the NBD-labeled peptide NBD-isoDV.

5. The method for preparing a polypeptide-assembled supramolecular fluorescent probe responsive to protein carboxylmethyltransferase according to claim 2, characterized in that: The prepared polypeptide-assembled supramolecular fluorescent probe NisoDV had a mother solution concentration of 500 μM.

6. Use of the supramolecular fluorescent probe NisoDV according to claim 1 in protein carboxymethyltransferase response.

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