A tumor-specific logic-gated polypeptide, probe, preparation method and application

Abstract

The present invention belongs to the technical field of tumor detection, and specifically relates to a tumor-specific logic-gated polypeptide, including an enzyme gating sequence E, a pH gating sequence A, and a pathological collagen gating sequence P. Sequence E contains at least 2 repeated Gly-Xaa-Yaa; sequence A contains at least 1 Gly-Amp-Pro; sequence P contains at least 6 repeated Gly-Zaa-Vaa; Xaa, Yaa, Zaa, and Vaa are any different amino acids; sequence E and sequence A can be located at the C-terminus, N-terminus, or the middle of sequence P. The polypeptide probe has the ability to detect multiple factors of the tumor microenvironment and can be used for specific detection of tumors. The probe preparation method is simple, and can be used as a tumor imaging reagent, and has broad application prospects in specific detection of tumors, prognosis evaluation, and drug screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor detection, and in particular relates to a logic-gated polypeptide, a probe, a preparation method and an application thereof with tumor specificity. Background Art

[0002] The tumor microenvironment (TME) is a complex biochemical system that plays a decisive role in the occurrence, progression and metastasis of tumors. There are obvious differences between TME and physiological microenvironment, including pathological extracellular matrix (ECM), new blood vessels and lymphatic vessel networks, abnormal cell phenotypes, and weakly acidic and highly enzymatic environments. Changes in the composition, structure and biochemical properties of TME are key factors affecting tumor diversity and invasiveness. The tumor-specific microenvironment has become a natural target for tumor detection and treatment. Therefore, targeted analysis of TME is of great significance for pathological research, early diagnosis and treatment of tumors.

[0003] In recent years, a large number of tumor microenvironment-specific probes have been developed for specific imaging of tumors. For example, Chinese patent CN112370534A discloses a tumor microenvironment-responsive degradation-type integrated diagnosis and treatment nanoprobe, which responds to glutathione highly expressed in the tumor microenvironment to achieve specific imaging of tumor model mice. However, due to the complexity of the tumor microenvironment, it is difficult for a single factor to accurately identify the tumor microenvironment to achieve accurate diagnosis and treatment of tumors. At present, researchers have developed multi-target probes that simultaneously detect multiple abnormal features in the tumor microenvironment. For example, Chinese patent CN114010803B discloses a multifunctional targeted molecular probe for detecting Treg cells in a living tumor microenvironment. A nanoprobe that can respond to the tumor microenvironment is formed by co-coating CD-25 fluorescent antibodies, chemotherapeutic drugs, and immune checkpoint inhibitors IDO. Under the action of EPR, the nanoprobe accumulates at the tumor site, and in the presence of glutathione, the CD-25 fluorescent antibodies, chemotherapeutic drugs, and immune checkpoint inhibitors IDO are released. This type of probe can effectively improve the specificity and accuracy of tumor detection. The method of simultaneously detecting multiple factors of the tumor microenvironment provides a new idea for the specific detection of tumors and is of great significance for the accurate diagnosis of tumors.

[0004] During the research, the inventors discovered a tumor-specific logic-gated polypeptide and a polypeptide probe prepared by the polypeptide probe, which includes a pH gating sequence A, an enzyme gating sequence E, a diseased collagen gating sequence P and a signal molecule X. The polypeptide probe can specifically identify the tumor after simultaneously identifying the pH gating, enzyme gating and diseased collagen gating related to the tumor microenvironment. When the pH gating, enzyme cleavage response gating and diseased collagen gating input (1,1,1), the output signal is 1 and it is judged as a tumor. The polypeptide probe can be used for specific imaging of 3D tumor models, providing a new method for specific detection of tumors. Summary of the invention

[0005] In view of the above technical problems, the object of the present invention is to provide a logic-gated polypeptide with tumor specificity, wherein the logic-gated polypeptide comprises a pH gating sequence A, an enzyme gating sequence E and a pathological collagen gating sequence P;

[0006] Wherein, the sequence E comprises at least 2 repeats of Gly-Xaa-Yaa;

[0007] The sequence A contains at least one Gly-Amp-Pro;

[0008] The sequence P comprises at least 6 repeats of Gly-Zaa-Vaa;

[0009] The Xaa, Yaa, Zaa and Vaa are any different / same amino acids;

[0010] The sequence E and sequence A can be located at the C-terminus, N-terminus or in the middle of the polypeptide sequence P.

[0011] Preferably, the Zaa and Vaa are amino acids Pro and / or Hyp, respectively.

[0012] Preferably, Xaa and Yaa in the sequence E are any one of the amino acids Pro, Hyp, Leu, Val, Arg, Gln, Ala, Ile, Phe, Asp, Lys, and Ser.

[0013] Preferably, the sequence E comprises the sequence shown below:

[0014] Gly-Pro-Leu-Gly-Val-Arg;

[0015] and / or Gly-Pro-Gln-Gly-Leu-Arg;

[0016] and / or Gly-Ala-Ile-Gly-Gln-Pro;

[0017] and / or Gly-Leu-Leu-Gly-Gln-Pro;

[0018] and / or Gly-Ala-Leu-Gly-Gln-Pro;

[0019] and / or Gly-Ala-Ile-Gly-Leu-Pro;

[0020] and / or Gly-Pro-Arg-Gly-Leu-Pro;

[0021] and / or Gly-Pro-Gln-Gly-Phe-Gln;

[0022] and / or Gly-Leu-Asp-Gly-Ala-Lys;

[0023] and / or Gly-Pro-Gln-Gly-Val-Arg;

[0024] and / or Gly-Pro-Ser-Gly-Ala-Ser;

[0025] and / or Gly-Pro-Arg-Gly-Pro-Pro;

[0026] and / or Gly-Pro-Gln-Gly-Phe-Gln;

[0027] and / or Gly-Leu-Lys-Gly-Pro-Gln;

[0028] and / or Gly-Ala-Arg-Gly-Ser-Asp;

[0029] and / or Gly-Arg-Ala-Gly-Pro-Pro;

[0030] and / or Gly-Ser-Lys-Gly-Pro-Lys.

[0031] Preferably, sequence A contains 2 Gly-Amp-Pro.

[0032] Preferably, the sequence P comprises 8 repeats of Gly-Hyp-Hyp or Gly-Pro-Hyp.

[0033] The second object of the present invention is to provide a logic-gated polypeptide probe with tumor specificity, wherein the logic-gated polypeptide probe with tumor specificity comprises the logic-gated polypeptide with tumor specificity and a signal molecule X; the signal molecule X is located at the N-terminus, C-terminus or the middle of the polypeptide.

[0034] Preferably, the signal molecule X is connected to the middle side chain of the polypeptide sequence via the linker amino acid Ahx-Lys.

[0035] Preferably, the signal molecule X is one or more of fluorescein dyes, coumarin dyes, rhodamine dyes, cyanine dyes, BODIPY dyes, tetraphenylethylene dyes, hexaphenylsilane dyes, distyryl anthracene dyes, and rare earth ion complexes.

[0036] Preferably, the signal molecule X is carboxyfluorescein FAM.

[0037] Preferably, the tumor-specific logic-gated polypeptide probe sequence is Ac-(Gly-Xaa-Yaa)2-(Gly-Zaa-Vaa)4-(Gly-Amp-Pro)2-(Gly-Zaa-Vaa)4-Ahx-K(X)-NH2.

[0038] The third object of the present invention is to provide a method for preparing the tumor-specific logic-gated polypeptide probe, the method comprising:

[0039] (1) solid phase synthesis of a resin containing the polypeptide Ac-(Gly-Xaa-Yaa)2-(Gly-Zaa-Vaa)4-(Gly-Amp-Pro)2-(Gly-Zaa-Vaa)4-Ahx-K-NH2;

[0040] (2) dissolving the signal molecule, HOBt and HBTU in DMF, activating at low temperature for 10-30 min, and then adding DIEA dropwise to the solution to obtain a mixed solution;

[0041] (3) adding the mixed solution prepared in step (2) to the resin described in step (1) and reacting in the dark for 12-48 hours;

[0042] (4) The resin after the reaction in step (3) was washed alternately with DCM and methanol, treated with a cutting solution (TFA:TIS:water=95:2.5:2.5) for 2-4 hours, and then added with icy ether. The resulting precipitate was the crude product of the polypeptide probe.

[0043] (5) The crude product was purified by reverse phase HPLC through a C18 column, lyophilized and stored at -20°C.

[0044] The fourth object of the present invention is to provide the use of the logic-gated polypeptide probe in the preparation of a detection reagent, and / or a kit, and / or an imaging reagent for detecting a tumor.

[0045] The fifth object of the present invention is to provide a detection reagent containing the logic-gated polypeptide probe.

[0046] The sixth object of the present invention is to provide a detection kit containing the logic-gated polypeptide probe.

[0047] The seventh object of the present invention is to provide a cell imaging reagent containing the logic-gated polypeptide probe.

[0048] The eighth object of the present invention is to provide a living imaging agent containing the logic-gated polypeptide probe.

[0049] The beneficial effects of the present invention are as follows: (1) The present invention provides a logic-gated polypeptide probe with tumor specificity, wherein the logic-gated polypeptide probe comprises a pH gating sequence A, an enzyme gating sequence E, a pathological collagen gating sequence P and a signal analysis X; the polypeptide probe has good tumor microenvironment specificity and can target and identify tumors; (2) the logic-gated polypeptide probe with tumor specificity has a simple preparation method and can be used as a living imaging agent, and has broad application prospects in the specific detection of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Thermal stability study of peptide probe Log111. Figure legend: a: The first derivative of the thermal curve of Log111 when gated at (0,0,0); b: The first derivative of the thermal curve of Log111 when pH gated at 1; c: The thermal curve of Log111 when both pH gated and MMPs gated at 1; d: The first derivative of the thermal curve of Log111 when both pH gated and MMPs gated at 1

[0051] Figure 2 Logic gating diagram of peptide probe solution

[0052] Figure 3 3D cell imaging of the logic-gated peptide probe Log111. Figure captions: 3D cells stained with Log111 and Hoechst33342 under (ac) [0,0,0], (df) [1,0,0], (gi) [1,1,0] and (jl) [1,1,1].

[0053] Figure 4 3D tumor model imaging of peptide probes;

[0054] Note: Log111 and Hoechst33342 stain the microenvironment and cell nucleus of (ac) HuH7 and (df) AGS-3D tumor models, respectively. DETAILED DESCRIPTION

[0055] 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 contents described below.

[0056] The polypeptide probe Log111 prepared in the following examples:

[0057] Ac-GPLGVR(GOO)4-(GAmpP)2-(GOO)4-Ahx-K(FAM)-NH2 can bind to tumors by logic gating, and 3D cell imaging was performed using this polypeptide probe as an example. However, the polypeptide probe described in the present invention is not limited to 3D cell imaging, and can specifically bind to the tumor site of tumor model mice.

[0058] Example 1 Preparation of polypeptide probe Log111

[0059] 1. Design of peptide probes

[0060] The polypeptide probe sequence designed in this example is:

[0061] Ac-GPLGVR(GOO)4-(GAmpP)2-(GOO)4-Ahx-K(FAM)-NH2;

[0062] FAM is carboxyfluorescein.

[0063] 2. Solid phase synthesis of peptide sequence Ac-GPLGVR(GOO)4-(GAmpP)2-(GOO)4-Ahx-K-NH2

[0064] (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;

[0065] (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;

[0066] (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.

[0067] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed three times with 5 mL of DMF and DCM, respectively. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution three times for 5 min, 5 min and 15 min, respectively. The resin is washed three times with 5 mL of DMF and DCM, respectively, and the color reaction is used to detect that the protecting group is completely removed;

[0068] (5) Repeat steps (3) and (4) until the polypeptide of the target sequence Ac-GPLGVR(GOO)4-(GAmpP)2-(GOO)4-Ahx-K-NH2 is synthesized.

[0069] 3. Signal molecule modification peptide sequence

[0070] (1) using 1% TFA in DCM solution to remove the amine protecting group of the side chain of amino acid K;

[0071] (2) 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 synthesized peptide Ac-GPLGVR(GOO)4-(GAmpP)2-(GOO)4-Ahx-K-NH2 resin and reacted in the dark for 24 h.

[0072] (3) 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 3 h;

[0073] (4) The reaction solution was added to icy ether for precipitation. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and centrifuged. The precipitate was washed twice with icy ether and air-dried to obtain the crude polypeptide product Ac-GPLGVR(GOO)4-(GAmpP)2-(GOO)4-Ahx-K(FAM)-NH2;

[0074] (5) The crude polypeptide product was purified by reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column, and then freeze-dried and stored at -20°C.

[0075] Example 2 Study on the responsiveness of peptide probe Log111

[0076] The polypeptide probe prepared in Example 1 was used for responsiveness experiment.

[0077] Take pH 6.5 and pH 7.4 phosphate buffers, and prepare 1 mg / mL Log111 probe solutions respectively; characterize the thermal curve of the probe by circular dichroism and perform first-order derivative to obtain its thermal transition temperature. The thermal transition temperatures of the peptide probe Log111 at pH 6.5 and pH 7.4 are 47°C and 61°C, respectively ( Figure 1 a and b). The thermal stability results of the probes show that when the pH gate of the probe Log111 changes from 0 to 1, the thermal stability of the probe decreases.

[0078] Fluorescence spectroscopy was used to characterize the thermal transition temperature of the peptide probe Log111 in the presence of enzyme gating and pH gating. The fluorescence intensity of the fluorescence spectrum at 525nm was monitored in real time during the process of Log111 heating from 0°C to 65°C. The probe fluorescence intensity gradually increased and gradually stabilized with the increase in temperature. The change in fluorescence intensity corresponds to the transition of the polypeptide triple helix structure to a single-stranded state. The first-order derivative of the temperature was calculated using the thermal transition curve, and the extreme value of the first-order derivative curve was the thermal transition temperature of the probe. When the enzyme gating and pH gating were both 1, a new peak of 34°C ( Figure 1 c and d). The results of thermal transition temperature demonstrate that the ability of peptide probe FAM-Log111 to change enzyme gating from 0 to 1 reduces the thermal transition temperature of the probe by 13°C, and there is no interference between pH gating and enzyme gating.

[0079] Example 3 Logic gating study of polypeptide probes

[0080] This patent studies the logic gating of the probe Log111 at the solution level and the cell level, with 1 and 0 defined as the presence and absence of the input signal, respectively. First, the logic gating performance of the probe at the solution level was characterized by a 96-well plate combined experiment. We used collagen or pathological collagen to coat the 96-well plate and detect the output signal of the Log111 probe under different input signals. Among them, pH 7.4, collagen and no enzyme were recorded as [0,0,0], pH 6.5, collagen and no enzyme were recorded as [1,0,0], pH 7.4, diseased collagen and no enzyme were recorded as [0,1,0], pH 7.4, collagen and enzyme digestion were recorded as [0,0,1], pH 6.5, collagen and enzyme digestion were recorded as [1,0,1], pH 6.5, diseased collagen and no enzyme were recorded as [1,1,0], pH7.4, diseased collagen and enzyme digestion were recorded as [0,1,1], and pH 6.5, diseased collagen and enzyme digestion were recorded as [1,1,1]. The results are as follows Figure 2 As shown in the figure, only when the input is [1,1,1], a strong fluorescence signal is output, while under other inputs, the fluorescence signal is weak. The results show that only when the pH, diseased collagen and enzyme gating outputs are 1 at the same time, can the output be 1, that is, the tumor microenvironment.

[0081] We used the peptide probe Log111 to stain 4T1 tumor cells and fibroblast HFF-13D cells for cell logic gating studies. 4T1 cells and HFF-1 cells were cultured in DMEM high-glucose medium containing 1% double antibody and 10% (v / v) fetal bovine serum. The cells were placed in a cell culture incubator at 37°C and cultured in an atmosphere of 5% CO2. Cells in exponential growth were digested using 0.25% trypsin-EDTA (1x) cell dissociation medium. 5 mg / mL type I collagen dissolved in 0.1 M acetic acid was adjusted to a neutral pH on ice. 1×10 4 Cells at a concentration of 1 cell / mL were added to a neutral collagen solution. After complete mixing, the cell-collagen mixture was immediately added to a sterile confocal culture dish and incubated at 37°C for 1 hour to ensure that the cell-collagen mixture was completely gelled. Serum-free culture medium was added to the gel and cultured for 24 hours. MMPs inhibitor (GM6001) was added during cell culture to establish a condition where the MMPs signal output was 0. Fix with 4% paraformaldehyde fixative for 4 hours, and then wash with PBS 3 times (10 minutes / time). The peptide probe was incubated with the fixed 3D cells in a dark room at 37°C for 4 hours. The cells were washed with PBS 3 times (20 minutes / time). Hoechst 33342 solution was added, and after incubation at 37°C in the dark for 20 minutes, the cells were washed twice with PBS solution. Imaged on a laser confocal microscope (inverted), using a wavelength of 405nm to excite blue fluorescence to observe the cell nucleus, and a wavelength of 488nm to excite green fluorescence to observe the pathological collagen degraded by tumor cells. The cell imaging results showed that when MMPs ( Figure 3 ac)、pH( Figure 3 df) and pathological collagen ( Figure 3 gi) When any gate is 0, there is no obvious fluorescence signal, that is, the output is 0. Only when the pH, pathological collagen and enzyme gates are 1 at the same time, will strong green fluorescence be displayed ( Figure 3 jl). The above experimental results consistently show that the output is 1, that is, a tumor, only when the three gating inputs of pH, diseased collagen and enzyme are all 1.

[0082] Example 4 3D tumor model imaging study using peptide probes

[0083] The peptide probe Log111 was further used for cell imaging of HuH7 and AGS 3D tumor models. AGS cells were cultured in 1640 medium containing 1% double antibody and 10% (v / v) fetal bovine serum; HuH7 cells were cultured in DMEM high glucose medium containing 1% double antibody and 10% (v / v) fetal bovine serum. The cells were placed in a cell culture incubator at 37°C and cultured in an atmosphere of 5% CO2. Cells in exponential growth were digested using 0.25% trypsin-EDTA (1x) cell dissociation medium. 5 mg / mL type I collagen dissolved in 0.1 M acetic acid was adjusted to neutral pH on ice. A mixture containing 1×10 4 Cells at a concentration of 1 cell / mL were added to a neutral collagen solution. After complete mixing, the cell-collagen mixture was immediately added to a sterile confocal culture dish and incubated at 37°C for 1 hour to ensure that the cell-collagen mixture was completely gelled. Serum-free medium was added to the gel and cultured for 24 hours. Fix with 4% paraformaldehyde fixative for 4 hours, then wash 3 times with PBS (10 minutes / time). The peptide probe was incubated with the fixed 3D cells in a dark room at 37°C for 4 hours. Wash the cells 3 times with PBS (20 minutes / time). Hoechst 33342 solution was added, incubated at 37°C in the dark for 20 minutes, and then the cells were washed twice with PBS solution. Imaged on a laser confocal microscope (inverted), using a wavelength of 405nm to excite blue fluorescence to observe the cell nucleus, and a wavelength of 488nm to excite green fluorescence to observe the pathological collagen degraded by tumor cells.

[0084] Log111 and Hoechst 33342 staining of HuH7 ( Figure 4 ac) and AGS( Figure 4 df) Imaging results of tumor models Figure 4 As shown, the 3D extracellular matrix of the HuH7 tumor model showed strong green fluorescence, and the blue fluorescence showed the distribution of cells. The imaging showed that HuH7 had a weakly acidic pH, high enzyme activity, and a tumor microenvironment rich in diseased collagen. The 3D extracellular matrix of the AGS tumor model showed strong green fluorescence similar to that of the HuH7 tumor model, and the blue fluorescence showed the distribution of tumor cells. The imaging results of the AGS tumor model showed that AGS tumors also had a weakly acidic pH, high enzyme activity, and a tumor microenvironment rich in diseased collagen. The above results indicate that the logic-gated tumor-targeting peptide probe Log111 is widely applicable to the specific detection of different types of tumors.

[0085] In summary, the present invention provides a logic-gated polypeptide probe with tumor specificity, wherein the logic-gated polypeptide probe comprises a pH gating sequence A, an enzyme gating sequence E, a pathological collagen gating sequence P and a signal analysis X; the polypeptide probe has good tumor microenvironment specificity and can target and identify tumors; the logic-gated polypeptide probe with tumor specificity has a simple preparation method, can be used as a living imaging reagent, and has broad application prospects in the specific detection of tumors.

Claims

1. A tumor-specific logic-gated polypeptide probe, characterized in that: The logic gated polypeptide sequence is Ac-GPLGVR(Gly-Hyp-Hyp)4-(GAmpP)2-(Gly-Hyp-Hyp)4-Ahx-K(X)-NH2, X is a signal molecule selected from one of fluorescein dyes, coumarin dyes, rhodamine dyes, cyanine dyes, BODIPY dyes, tetraphenylethylene dyes, hexaphenylsilane dyes, distyryl anthracene dyes, and rare earth ion complexes.

2. The method for preparing a tumor-specific logic-gated polypeptide probe according to claim 1, characterized in that: The method comprises: (1) Solid phase synthesis of a resin containing the peptide Ac-GPLGVR(Gly-Hyp-Hyp)4-(GAmpP)2-(Gly-Hyp-Hyp)4-Ahx-K-NH2; (2) dissolving the signal molecule X, HOBt and HBTU in DMF, activating at low temperature for 10-30 min, and then adding DIEA dropwise to the solution to obtain a mixed solution; (3) adding the mixed solution prepared in step (2) to the resin described in step (1) and reacting in the dark for 12-48 hours; (4) The resin after the reaction in step (3) was washed with DCM and methanol alternately, and a cutting solution (TFA:TIS:water=95:2.5:2.5) was added for 2-4 hours, and then the reaction solution was added to ice ether, and the resulting precipitate was a crude product of the polypeptide probe; (5) The crude product was purified by reverse phase HPLC through a C18 column, lyophilized and stored at -20°C; The signal molecule X is one of fluorescein dyes, coumarin dyes, rhodamine dyes, cyanine dyes, BODIPY dyes, tetraphenylethylene dyes, hexaphenylsilane dyes, distyryl anthracene dyes, and rare earth ion complexes.

3. Use of the logic gated polypeptide probe according to claim 1 in the preparation of a detection reagent, and / or a kit, and / or an imaging reagent for detecting a tumor, characterized in that: The tumor is breast cancer, liver cancer or gastric cancer.

4. A detection reagent, a cell imaging reagent, or a living body imaging reagent comprising the logic-gated polypeptide probe according to claim 1.

5. A detection kit containing the logic-gated polypeptide probe according to claim 1.

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