A polypeptide probe for detecting collagen in tumor lesions and its preparation method and application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a polypeptide probe for detecting tumor lesion collagen and a preparation method and application thereof. The sequence of the polypeptide is: (Gly-Pro-Hyp) a ‑(Gly‑Amp‑Pro) b ‑(Gly‑Pro‑Hyp) c , wherein a is an integer between 3 and 10, b is an integer between 1 and a, and c is an integer between a‑1 and a+1; the polypeptide probe can maintain a stable triple helix structure at physiological pH, and become a single-stranded structure at the weakly acidic pH of the tumor, thereby achieving specific recognition of tumor lesion collagen. The polypeptide probe has a simple preparation method, convenient detection, good specificity, and strong binding ability. It has broad application prospects in the fields of early diagnosis, efficacy evaluation, and treatment of tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a polypeptide probe for detecting collagen in tumor lesions, and a preparation method and application thereof. Background Art

[0002] Collagen is an important biomarker in the process of tumor development and is involved in regulating the proliferation, differentiation, invasion and metastasis of tumor cells. Overexpression and cross-linking of fibroblast collagen lead to tissue hardening, while promoting the proliferation, invasion and spread of surrounding tumor cells. During tumor invasion, tumor cells secrete a large amount of matrix metalloproteinases, which lead to pathological remodeling of the tumor cell extracellular matrix to form tumor lesion collagen. The production of a large amount of tumor lesion collagen in the tumor microenvironment is a hallmark event of tumor metastasis and invasion. Therefore, the specific detection of tumor lesion collagen is of great significance for the accurate diagnosis, pathological analysis, targeted treatment and prognosis evaluation of tumors.

[0003] At present, some short peptides and antibodies have been developed for the recognition and imaging of collagen. Using the domains of other proteins binding to collagen, collagen targeting peptides such as TKKTLRT and LRELHLNNN have been identified. The peptide KLWVLPK that specifically binds to type IV collagen has been successfully screened using phage display technology. The above studies mainly focus on the targeted recognition of natural collagen, and due to the special structure of the three peptide chains of natural collagen tightly stacked, the above peptides often have the defects of low affinity and non-specific binding.

[0004] The triple helix structure of diseased collagen will partially unwind, and the exposed unfolded part provides a target for identifying diseased collagen. (Gly-Pro-Hyp) n and (Gly-Hyp-Hyp) n The peptide sequence can specifically bind to the unfolded collagen chain, which points out a new direction for the design of probes targeting diseased collagen. n Derived probes have promoted the in vivo imaging study of diseased collagen. At the same time, a completely single-chain diseased collagen targeting peptide probe (Gly-Hyp-Pro) constructed based on sequence reconstruction strategy n , achieving in vitro imaging of tumor tissue. However, the above single-stranded probes will non-selectively bind to diseased collagen in any disease, lack specificity for tumor diseased collagen, and cannot meet the high specificity and accuracy requirements of tumor diagnosis.

[0005] In view of the problems existing in the prior art, the inventors have found through experiments that (Gly-Pro-Hyp) a -(Gly-Amp-Pro)b -(Gly-Pro-Hyp) c The polypeptide with the sequence can maintain a stable triple helix structure at physiological pH, but becomes a single-stranded structure at the weakly acidic pH of the tumor. This property is used to achieve specific recognition of tumor lesion collagen without binding to lesion collagen in other environments. The probe can be effectively used for specific imaging of tumor lesion collagen in vivo and in vitro. It has broad application prospects in the fields of early diagnosis, efficacy evaluation and treatment of tumors. Summary of the invention

[0006] The primary purpose of the present invention is to provide a polypeptide for detecting collagen in tumor lesions, the sequence of which is: (Gly-Pro-Hyp) a -(Gly-Amp-Pro) b -(Gly-Pro-Hyp) c , where a is an integer between 3 and 10, b is an integer between 1 and a, and c is an integer between a-1 and a+1.

[0007] The second object of the present invention is to provide a polypeptide probe for detecting collagen in tumor lesions, wherein the polypeptide probe comprises the polypeptide and a signal molecule F, and the signal molecule F is connected to the N-terminus or side chain of the polypeptide sequence.

[0008] Preferably, the signal molecule F is connected to the polypeptide sequence N via a linker amino acid Ahx.

[0009] Preferably, the signal molecule F is one or more of fluorescein dyes, coumarin dyes, rhodamine dyes, cyanine dyes, BODIPY dyes, tetraphenylethylene dyes (TPE), hexaphenylsilane dyes (HPS), distyryl anthracene dyes (DSA), and rare earth ion complexes.

[0010] Preferably, the signal molecule F is carboxyfluorescein.

[0011] The third object of the present invention is to provide a method for preparing the polypeptide probe, which comprises the following steps:

[0012] (1) 80-120 mg of the resin was added to a reactor with a sieve plate, and the resin was swollen with 2-8 mL of dichloromethane; the N-terminal Fmoc protecting group was removed with a piperidine / N,N-dimethylformamide solution;

[0013] (2) The amino acid with Fmoc protection at the N-terminus, HOBt and HBTU are dissolved in DMF, activated at low temperature for 10-30 min, and DIEA is added dropwise to the solution. After the solution is mixed evenly, it is added to the reactor and reacted for 1-6 hours;

[0014] (3) After the reaction is completed, the reaction solution is extracted from the reactor, the resin is washed with DMF and DCM 2-4 times, the resin is treated with 15-25% piperidine / DMF solution 3 times, and the resin is washed with DMF and DCM 3 times, until the protecting group is completely removed;

[0015] (4) repeating steps (2) and (3) until a polypeptide of the target sequence is synthesized;

[0016] (5) The resin was washed with DCM and methanol alternately for 2-4 times, then the resin was drained, the cutting solution was added, and the reaction was carried out for 1-6 hours;

[0017] (6) The reaction solution was precipitated with icy ether, the precipitate was collected by centrifugation and washed three times with icy ether, and dried to obtain a crude polypeptide product;

[0018] (7) The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column, lyophilized and stored at -20°C;

[0019] (8) When preparing the functionalized peptide, after step (4), the resin is dispersed in DMF, the signal molecule, HOBt and HBTU are weighed, dissolved in DMF, activated at low temperature for 10-30 min, DIEA is added to the solution and mixed thoroughly, the mixture is added to the resin dispersion, and the reaction is carried out in the dark for 12-48 hours. The resin is treated in the same manner as in step (5), and the peptide is purified in the same manner as in steps (6) and (7).

[0020] Preferably, the cutting fluid in step (5) is TFA:TIS:water=95:2.5:2.5.

[0021] The fourth object of the present invention is to provide the use of the polypeptide probe in preparing a detection reagent, and / or a kit, and / or an imaging reagent for detecting a tumor.

[0022] The fifth object of the present invention is to provide a detection reagent containing the polypeptide probe.

[0023] The sixth object of the present invention is to provide a detection kit containing the polypeptide probe.

[0024] The seventh object of the present invention is to provide an in vitro imaging agent containing the polypeptide probe.

[0025] The eighth object of the present invention is to provide a living imaging agent containing the polypeptide probe.

[0026] The beneficial effects of the present invention are as follows: the present invention provides a polypeptide probe for detecting collagen in tumor lesions, wherein the polypeptide probe can maintain a stable triple helix structure in physiological pH, and change into a single-stranded structure in the weakly acidic pH of the tumor, thereby realizing specific recognition of collagen in tumor lesions. The polypeptide probe has a simple preparation method, convenient detection, good specificity, and strong binding ability, and has broad application prospects in the fields of early diagnosis, efficacy evaluation, and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0028] Figure 1 Characterization of the thermal stability of the probe of the present invention Note: a: thermal transition temperatures of the probe at pH 7.4 (blue) and pH 6.5 (red); b: thermal transition temperature difference of the probe at pH 7.4 and pH 6.5.

[0029] Figure 2 The fluorescence spectrum of the present invention characterizes the unfolding rate of the probe

[0030] Figure 3 To characterize the responsiveness of the probe of the present invention

[0031] Figure 4 Specific cell imaging for the probe of the present invention Note: P3-8 co-imaging of 4T1-3D cells cultured with FITC-labeled type I collagen

[0032] Figure 5 Imaging of 3D tumor models using the probe of the present invention Note: Probe FAM-P3-8 images HeLa (ac), HuH7 (df), and AGS (gi) 3D cells.

[0033] Figure 6 The in vivo imaging diagram of the probe of the present invention is DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following.

[0035] The polypeptide probes P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 prepared in the following examples can specifically bind to tumor lesion collagen, and imaging studies were carried out using P2-8 and P3-8 as examples. However, the polypeptide probes described in the present invention are not limited to P2-8 and P3-8, and other probes also have good staining ability and can specifically bind to tumor lesion collagen.

[0036] Example 1. Preparation of polypeptide probe P1-6

[0037] 1. Design of peptide probes

[0038] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3, where FAM is carboxyfluorescein.

[0039] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3

[0040] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0041] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0042] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0043] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0044] (5) Steps (3) and (4) were repeated until the target peptides (Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 were synthesized. (Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 was capped by treating with 25% acetic anhydride for 30 minutes and used to prepare unmodified P1-6.

[0045] 3. Signal molecule modification peptide sequence

[0046] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 resin and reacted in the dark for 24 h.

[0047] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0048] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)3-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3-NH2. The crude polypeptide product was passed through a C18 chromatographic column and purified by reverse phase high performance liquid chromatography to obtain polypeptide probe P1-6.

[0049] Example 2. Preparation of polypeptide probe P1-7

[0050] 1. Design of peptide probes

[0051] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3, where FAM is carboxyfluorescein.

[0052] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3

[0053] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0054] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0055] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0056] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0057] (5) Steps (3) and (4) were repeated until the target peptides (Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 were synthesized. (Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 was capped by treating with 25% acetic anhydride for 30 minutes and used to prepare unmodified P1-7.

[0058] 3. Signal molecule modification peptide sequence

[0059] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3 resin and reacted in the dark for 24 h.

[0060] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0061] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)3-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column to obtain polypeptide probe P1-7.

[0062] Example 3. Preparation of polypeptide probe P1-8

[0063] 1. Design of peptide probes

[0064] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4, where FAM is carboxyfluorescein.

[0065] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4

[0066] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0067] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0068] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0069] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0070] (5) Steps (3) and (4) were repeated until the target sequence polypeptides (Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 were synthesized. (Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 was capped by treatment with 25% acetic anhydride for 30 minutes and used to prepare unmodified P1-8.

[0071] 3. Signal molecule modification peptide sequence

[0072] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4 resin and reacted in the dark for 24 h.

[0073] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0074] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)1-(Gly-Pro-Hyp)4-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column to obtain polypeptide probe P1-8.

[0075] Example 4. Preparation of polypeptide probe P2-7

[0076] 1. Design of peptide probes

[0077] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3, where FAM is carboxyfluorescein.

[0078] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3

[0079] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0080] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0081] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0082] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0083] (5) Steps (3) and (4) were repeated until the target peptides (Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 were synthesized. (Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 was capped by treating with 25% acetic anhydride for 30 minutes and used to prepare unmodified P2-7.

[0084] 3. Signal molecule modification peptide sequence

[0085] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3 resin and reacted in the dark for 24 h.

[0086] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0087] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)3-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography on a C18 column to obtain polypeptide probe P2-7.

[0088] Example 5. Preparation of polypeptide probe P2-8

[0089] 1. Design of peptide probes

[0090] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4, where FAM is carboxyfluorescein.

[0091] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4

[0092] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0093] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0094] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0095] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0096] (5) Steps (3) and (4) were repeated until the target sequence polypeptides (Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 were synthesized. (Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 was capped by treatment with 25% acetic anhydride for 30 minutes and used to prepare unmodified P2-8.

[0097] 3. Signal molecule modification peptide sequence

[0098] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 resin and reacted in the dark for 24 h.

[0099] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0100] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4-NH2. The crude polypeptide product was passed through a C18 chromatographic column and purified by reverse phase high performance liquid chromatography to obtain polypeptide probe P2-8.

[0101] Example 6. Preparation of polypeptide probe P2-9

[0102] 1. Design of peptide probes

[0103] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4, where FAM is carboxyfluorescein.

[0104] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4

[0105] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0106] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0107] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0108] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0109] (5) Steps (3) and (4) were repeated until the target sequence polypeptides (Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 were synthesized. (Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 was capped by treating with 25% acetic anhydride for 30 minutes and used to prepare unmodified P2-9.

[0110] 3. Signal molecule modification peptide sequence

[0111] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4 resin and reacted in the dark for 24 h.

[0112] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0113] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)2-(Gly-Pro-Hyp)4-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column to obtain the polypeptide probe P2-9.

[0114] Example 7. Preparation of polypeptide probe P3-7

[0115] 1. Design of peptide probes

[0116] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3, where FAM is carboxyfluorescein.

[0117] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3

[0118] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0119] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0120] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0121] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0122] (5) Steps (3) and (4) were repeated until the target peptides (Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 were synthesized. (Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 was capped by treatment with 25% acetic anhydride for 30 minutes and used to prepare unmodified P3-7.

[0123] 3. Signal molecule modification peptide sequence

[0124] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After low-temperature activation for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3 resin and reacted in the dark for 24 h.

[0125] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0126] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)3-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column to obtain polypeptide probe P3-7.

[0127] Example 8. Preparation of polypeptide probe P3-8

[0128] 1. Design of peptide probes

[0129] The peptide probe sequence designed this time is: X-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4, where X is carboxyfluorescein or ROX or Cy7.5 or dansyl chloride (Dy).

[0130] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4

[0131] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0132] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0133] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0134] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0135] (5) Steps (3) and (4) were repeated until the target peptides (Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 were synthesized. (Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 was capped by treatment with 25% acetic anhydride for 30 minutes and used to prepare unmodified P3-8.

[0136] 3. Signal molecule modification peptide sequence

[0137] (1) Weigh carboxyfluorescein (10 eq) or ROX (10 eq) or Cy7.5 (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolve them in DMF, activate at low temperature for 20 min, or weigh Dy (10 eq) and dissolve it in DMF, add DIEA (16 eq) dropwise to the solution, add the mixture to the synthetic peptide Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 resin, and react in the dark for 24 h;

[0138] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0139] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain the crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2 or ROX-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2 or Cy7.5-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2 or Dy-Ahx-(Gly-Pro-Hyp)4-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2. The crude polypeptide product was passed through a C18 chromatographic column and purified by reverse phase high performance liquid chromatography to obtain polypeptide probe P3-8.

[0140] Example 9. Preparation of polypeptide probe P3-9

[0141] 1. Design of peptide probes

[0142] The peptide probe sequence designed this time is: FAM-Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4, where FAM is carboxyfluorescein.

[0143] 2. Solid phase synthesis of peptide sequences (Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4

[0144] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0145] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0146] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0147] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0148] (5) Steps (3) and (4) were repeated until the target peptides (Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 and Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 were synthesized. (Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 was capped by treatment with 25% acetic anhydride for 30 minutes and used to prepare unmodified P3-9.

[0149] 3. Signal molecule modification peptide sequence

[0150] (1) Carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq) were weighed and dissolved in DMF. After activation at low temperature for 20 min, DIEA (16 eq) was added dropwise to the solution. The mixture was added to the synthetic peptide Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4 resin and reacted in the dark for 24 h.

[0151] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0152] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)5-(Gly-Amp-Pro)3-(Gly-Pro-Hyp)4-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography on a C18 column to obtain the polypeptide probe P3-9.

[0153] Comparative Example 1: Preparation of polypeptide probe P0-8

[0154] 1. Design of peptide probes

[0155] The peptide probe sequence designed this time is: X-Ahx-(Gly-Pro-Hyp)8, where X is carboxyfluorescein or dansyl chloride.

[0156] 2. Solid phase synthesis of peptide sequence Ahx-(Gly-Pro-Hyp)8

[0157] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0158] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0159] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBt (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0160] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution 3 times, for 5 min, 5 min and 15 min respectively. The resin is washed with 10 mL DMF 3 times and 10 mL DCM 2 times, and the color reaction is used to detect that the protecting group is completely removed;

[0161] (5) Repeat steps (3) and (4) until the polypeptide of the target sequence Ahx-(Gly-Pro-Hyp)8 is synthesized.

[0162] 3. Signal molecule modification peptide sequence

[0163] (1) Weigh carboxyfluorescein (10 eq), HOBt (10 eq) and HBTU (10 eq), dissolve them in DMF, and activate them at low temperature for 20 min. Alternatively, weigh Dy (10 eq) and dissolve it in DMF. Add DIEA (16 eq) dropwise to the solution. Add the mixture to the synthesized peptide Ahx-(Gly-Pro-Hyp)8 resin and react for 24 h in the dark.

[0164] (2) The resin was washed three times with DCM and methanol respectively, the resin was drained, and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 2.5 h;

[0165] (3) The reaction solution was added to icy ether to precipitate the polypeptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and collected by centrifugation. The precipitate was washed twice with icy ether and air-dried to obtain a crude polypeptide product FAM-Ahx-(Gly-Pro-Hyp)8-NH2 or Dy-Ahx-(Gly-Pro-Hyp)8-NH2. The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column to obtain the polypeptide probe P0-8.

[0166] Example 10. Determination of polypeptide probe performance

[0167] 1. Stability test

[0168] (1) Circular dichroism: Unmodified peptides were prepared in 50 mM phosphate buffer at pH 6.5 and pH 7.4 at a concentration of 1 mg / mL, heated at 85°C for 30 minutes, and then equilibrated at 4°C for more than 24 hours. The circular dichroism spectra were characterized by an Aviv 400 spectrophotometer (Applied Photophysics Ltd, England). The cuvette slit width was 10 mm, the wavelength scanning range was from 190 to 280 nm, the increment was 0.5 nm, and each measurement was repeated three times. The thermal curve of the peptide was obtained by monitoring the signal at 225 nm as a function of temperature. The temperature was adjusted using a Peltier temperature controller, the temperature range was set to 4°C to 85°C, the average heating rate was 1.0°C / min, and the equilibration time was 2 min. The first-order derivative of the thermal curve was calculated and smoothed to obtain the thermal transition temperature, where the thermal transition temperature (Tm) was the extreme value of the first-order derivative. The Tm of P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 at pH 6.5 and 7.4 were obtained, respectively, and the Tm difference (ΔTm) under the two environments was obtained.

[0169] The experimental results are as follows Figure 1 As shown, the thermal shift temperatures of polypeptides P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 at pH 6.5 were 17, 31, 49, 19, 31, 42, 17, 27 and 36°C, respectively, while the thermal shift temperatures of polypeptides P1-6, P1-7, P1-8, P2-7, P2-8, P2-9, P3-7, P3-8 and P3-9 at pH 7.4 were 22, 36, 54, 30, 41, 52, 33, 43 and 51°C, respectively. Figure 1 a). By comparing the thermal transition temperatures and their differences of the above polypeptide sequences at two pH values ​​( Figure 1b), it is proved that as the number of repeating sequences (Gly-Pro-Hyp) increases, the thermal transition temperature of the polypeptide increases; and the more (Gly-Amp-Pro) sequences there are, the greater the difference in thermal transition temperature between pH 6.5 and pH 7.4. Among them, the thermal transition temperatures of P2-8 and P3-8 at pH 6.5 are 31 and 27°C, respectively, both less than 37°C; while at pH 7.4, the thermal transition temperatures are 41 and 43°C, respectively, both greater than 37°C.

[0170] (2) Fluorescence spectroscopy: FAM-modified peptide probes P0-8, P2-8 and P3-8 were prepared into 300 μM probe solutions in pH 6.5 and pH 7.4 buffers, respectively. The probe solutions were kept constant at 85°C in a water bath for 20 min, and the heated probe solutions were placed in a 0°C ice-water mixture for 12 h. The fluorescence spectra were measured again at 37°C by a fluorescence spectrophotometer. The excitation wavelength was 497 nm, and the emission spectrum scanning range was 500-700 nm.

[0171] The experimental results are as follows Figure 2 As shown, the fluorescence intensity of the probes at pH 6.5 and pH 7.4 at a wavelength of 525 nm was monitored by fluorescence spectroscopy at 37°C, and the folding rate of the probe was calculated to further study the unfolding ability of the probe ( Figure 2 ). Compared with P2-8, the unfolding ratio of the triple helix of P3-8 at pH 6.5 is much greater than that of P3-8 at pH 7.4, and greater than that of P2-8 at pH 6.5. The above results indicate that the thermal stability of P2-8 and P3-8 is regulated by pH, and the sequence length affects the degree of influence of pH on their thermal stability.

[0172] 2. pH response performance

[0173] The fluorescence spectrum of the Dy-modified peptide probe was recorded on a RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan), with an excitation wavelength of 330 nm, an emission spectrum scanning range of 450 to 650 nm, and a scanning interval of 1 nm. The peptide probes that need to be pH-fluorescent titrated were prepared as solutions of the same concentration at pH 7.8, pH 7.6, pH 7.4, pH 7.25, pH 7.15, pH 7.05, pH 6.95, pH 6.85, pH 6.72, pH 6.63, pH 6.55, pH 6.41, pH 6.24, and pH 6.01, incubated at 37°C before each measurement, balanced for 5 minutes, and the titration curve of the probe fluorescence intensity changing with pH was completed by measuring the fluorescence spectrum. Each group of experiments was repeated three times.

[0174] The experimental results are as follows Figure 3As shown, probe P3-8 responds rapidly to pH around 6.8, and at pH 6.5, the probe almost completely changes from a triple helical structure to a single strand structure. However, the fluorescence intensity of probe P0-8 hardly changes in the pH range of 6 to 8. The titration results of pH-fluorescence intensity prove that probe P3-8 responds sensitively and rapidly to pH in the range from neutral to weakly acidic, while the negative control probe P0-8 does not respond.

[0175] Example 11: Imaging Experiment

[0176] 1. Staining of 3D tumor models with peptide probes

[0177] The type I collagen dissolved by acetic acid was fully mixed with fluorescein isothiocyanate (FITC) solution at a molar ratio of 18:1 and reacted at 4°C in the dark for 24 hours. The completely reacted FITC-type I collagen solution was dialyzed in a 20mM pH 7.4 PBS solution through a dialysis bag with a molecular weight cutoff of 1000 until the dialysate could not detect fluorescence, indicating that the FITC small molecules that did not participate in the reaction had been completely removed. FITC-type I collagen was used for 3D cell culture of tumor cells, and 3D tumor cells were stained with ROX-modified P3-8 probe. 3D cells were imaged by laser confocal microscopy (inverted) (Olympus FV3000, Japan), and the cell nucleus was observed by 405nm wavelength excitation of blue fluorescence, 488nm wavelength excitation of green fluorescence, and 561nm wavelength excitation of red fluorescence to observe tumor lesion collagen.

[0178] The experimental results are as follows Figure 4 As shown, FITC-I collagen showed strong green fluorescence, while the tumor extracellular matrix showed obvious red fluorescence, which partially overlapped with the green fluorescence of FITC-I collagen. Hoechst 33342 staining showed the distribution of cell nuclei. The imaging results proved that probe P3-8 specifically recognized the pathological collagen in the tumor.

[0179] At the same time, the probe FAM-P3-8 was further used for 3D tumor model imaging of HeLa, HuH7 and AGS cells, and Hoechst 33342 was used to stain the cell nucleus. Figure 5 As shown, the extracellular matrix in the three models showed strong green fluorescence ( Figure 5 a, d and g). Blue fluorescence clearly locates the cell distribution ( Figure 5 b, e and h), the green fluorescence is distributed around the blue fluorescence ( Figure 5 c, f and i). The above results indicate that probe P3-8 has the ability to target tumor lesion collagen produced by different tumor types, further demonstrating the versatility of the P3-8 polypeptide probe.

[0180] 2. In vivo imaging of tumor model mice

[0181] Before the in vivo imaging of mice, the mice were anesthetized by intraperitoneal injection of pentobarbital (10% w / v), and then the near-infrared imaging photos of the mice were taken as the images before imaging. 40nmol of Cy7.5-modified peptide probe P3-8 in PBS solution was injected subcutaneously into 4T1 tumor-bearing mice, and the imaging images at different time points were collected using the VISQUE compact small animal in vivo real-time imaging system. Each mouse was placed in the same position and instrument scanning parameters.

[0182] The experimental results are as follows Figure 6 As shown in the figure, an obvious fluorescence signal was observed 20 minutes after the probe was injected. As time went on, the fluorescence signal at the tumor site gradually increased. A strong fluorescence signal was observed at the tumor site 30 hours after injection, and the location of the fluorescence signal coincided with the actual location of the tumor, and there was no fluorescence signal at other sites; while normal mice had no obvious fluorescence signal. The results of in vivo imaging of mice showed that the probe P3-8 could specifically target the tumor site of tumor mice and achieve specific imaging of the tumor.

[0183] In summary, the present invention provides a polypeptide probe for detecting tumor lesion collagen. The polypeptide probe can maintain a stable triple helix structure at physiological pH, and become a single-stranded structure at the weakly acidic pH of the tumor, thereby achieving specific recognition of tumor lesion collagen. Experimental results show that the polypeptide probe can specifically identify lesion collagen in tumors, and has the ability to target tumor lesion collagen produced by different tumor types. The results of in vivo imaging of mice show that the polypeptide probe can specifically target the tumor site of tumor mice and achieve specific imaging of the tumor. The polypeptide probe has a simple preparation method, convenient detection, good specificity, and strong binding ability. It has broad application prospects in the fields of early diagnosis, efficacy evaluation, and treatment of tumors.

[0184] The above embodiments are only used to understand the method and core concept of the present invention. It should be noted that for those skilled in the art, other implementations based on the patent concept of the present invention will also fall within the scope of protection of the claims of the present invention without departing from the principles of the present invention.

Claims

1. A polypeptide for detecting collagen in tumor lesions, characterized in that: The sequence of the polypeptide is: (Gly-Pro-Hyp) a -(Gly-Amp-Pro) b -(Gly-Pro-Hyp) c , wherein a is 4, b is 2, and c is 4; or a is 4, b is 3, and c is 4; or a is 5, b is 3, and c is 4; the Pro is proline, the Hyp is hydroxyproline, and the Amp is 4-aminoproline.

2. A polypeptide probe for detecting collagen in tumor lesions, characterized in that: The polypeptide probe comprises the polypeptide according to claim 1 and a signal molecule F, wherein the signal molecule F is connected to the N-terminus or the side chain of the polypeptide sequence.

3. The polypeptide probe according to claim 2, characterized in that The signal molecule F is connected to the N-terminus of the polypeptide sequence via the linker amino acid Ahx.

4. The polypeptide probe according to claim 2, characterized in that The signal molecule F is one of fluorescein dyes, coumarin dyes, rhodamine dyes, cyanine dyes, BODIPY dyes, tetraphenylethylene dyes, HPS, distyryl anthracene dyes, and rare earth ion complexes.

5. The method for preparing a polypeptide probe according to any one of claims 2 to 4, characterized in that: The preparation method comprises the following steps: (1) 80-120 mg of the resin was added to a reactor with a sieve plate, and the resin was swollen with 2-8 mL of dichloromethane; the N-terminal Fmoc protecting group was removed with a piperidine / N,N-dimethylformamide solution; (2) The amino acid with Fmoc protection at the N-terminus, HOBt and HBTU are dissolved in DMF, activated at low temperature for 10-30 min, and DIEA is added dropwise to the solution. After the solution is mixed evenly, it is added to the reactor and reacted for 1-6 hours; (3) After the reaction is completed, the reaction solution is extracted from the reactor, the resin is washed with DMF and DCM 2-4 times, the resin is treated with 15%-25% piperidine / DMF solution 3 times, and the resin is washed with DMF and DCM 3 times, until the protecting group is completely removed; (4) repeating steps (2) and (3) until a polypeptide of the target sequence is synthesized; (5) The resin was washed with DCM and methanol alternately for 2-4 times, then the resin was drained, the cutting solution was added, and the reaction was carried out for 1-6 hours; (6) The reaction solution was precipitated with icy ether, the precipitate was collected by centrifugation and washed three times with icy ether, and dried to obtain a crude polypeptide product; (7) The crude polypeptide product was purified by reverse phase high performance liquid chromatography through a C18 column, lyophilized and stored at -20°C; (8) When preparing the functionalized peptide, after step (4), the resin is dispersed in DMF, the signal molecule, HOBt and HBTU are weighed, dissolved in DMF, activated at low temperature for 10-30 minutes, DIEA is added to the solution and mixed thoroughly, the mixture is added to the resin dispersion, and the reaction is carried out in the dark for 12-48 hours; the resin is treated in the same manner as step (9), and the peptide is purified in the same manner as steps (6) and (7).

6. The preparation method according to claim 5, characterized in that: The cutting solution described in step (5) is TFA:TIS:water=95:2.5:2.

5.

7. Use of the polypeptide probe according to any one of claims 2 to 4 in the preparation of a detection reagent, and / or a kit, and / or an imaging reagent for detecting a tumor.

8. A detection reagent comprising the polypeptide probe according to any one of claims 2 to 4.

9. A detection kit comprising the polypeptide probe according to any one of claims 2 to 4.

10. An in vitro / in vivo imaging reagent comprising the polypeptide probe according to any one of claims 2 to 4.

Images