Self-assembly polypeptide probe for enhancing tumor magnetic resonance imaging

Abstract

The invention discloses a self-assembled polypeptide probe for enhancing tumor magnetic resonance imaging. Belongs to the technical field of biotechnology and new medicine, the contrast agent Asp-Asp-Asp-Asp-Lys-Cys (StBu)-Lys (DOTA (tBu))-CBT (Gd-AS) comprises a peptide sequence Asp-Asp-Asp-Asp-Lys-C (StBu)-Lys bound to a CBT part, and a DOTA (Gd) motif connected to a lysine (Lys) side chain; asp-Asp-Asp-Lys (DDDDK) is used as a high-specificity polypeptide substrate of the enterokinase, and the Asp-Asp-Asp-Lys (DDDDK) can be specifically cut; after the Gd-AS is taken by cells, the Gd-AS can be reduced in a tumor microenvironment, then the Gd-AS is cut by overexpressed enterokinase through a CBT-Cys click reaction to form cyclic dimers (namely AS-Dimers), and the cyclic dimers are further self-assembled to form gadolinium-containing nanoparticles (namely AS-NPs); by means of the self-assembly method, T1 weighted magnetic resonance imaging of enhanced enterokinase in tumor cells is achieved under a low magnetic field.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology and new pharmaceutical technology, and relates to a self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging. Background Art

[0002] Enterokinase (ENTK), also known as enteropeptidase, is mainly produced in the brush border of duodenal enterocytes and is a membrane-bound protease. Enterokinase is very stable over a wide range of temperatures (such as 4 - 45 °C) and pH values (4.5 - 9.5). Trypsinogen propeptide contains an acidic sequence (DDDDK) composed of four aspartic acids followed by one lysine, which can be recognized and hydrolyzed by enterokinase to regulate the biological processes of the intestinal visceral mucosa. In addition to its role in normal physiological processes, the expression of enterokinase in cancer cell lines not only originates from the duodenum but also exists on the mitochondria of various cancer cells (such as cervical cancer cell line HeLa, liver cancer cell line HepG2, osteosarcoma cell line Saos-2, and oral squamous cell carcinoma cells). The role of enterokinase in the activation of tumor-related trypsin-2 is related to the directional degradation of the extracellular matrix and may promote the invasion and metastasis of cancer cells; therefore, its specific expression in tumors can be used to design various multifunctional molecules for tumor imaging;

[0003] However, there is currently no method for detecting the activity of enterokinase clinically. Summary of the Invention

[0004] In view of the above problems, the object of the present invention is to propose a polypeptide probe that specifically responds to enterokinase and self-assembles into nanoparticles to improve the magnetic resonance imaging effect of tumors with high specific expression of enterokinase.

[0005] The technical solution of the present invention is: A self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging, the self-assembling polypeptide probe includes the peptide sequence Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys and a DOTA motif linked to the lysine side chain conjugated with a CBT moiety; the self-assembling polypeptide probe self-assembles into nanoparticles under the action of enterokinase.

[0006] Further, the self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging has the following sequence:

[0007] Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys(DOTA(Gd))-CBT.

[0008] Furthermore, Asp-Asp-Asp-Asp-Lys in the self-assembled polypeptide probe is specifically cleaved as a highly specific polypeptide substrate of enterokinase; Cys-Lys(DOTA(Gd))-CBT undergoes a click reaction to generate nanoparticles.

[0009] Furthermore, after the magnetic resonance polypeptide probe (abbreviated as Gd-AS) is taken up by cells, it can be reduced to AS-Red in the tumor microenvironment, and then cleaved by overexpressed enterokinase. Subsequently, it forms a cyclic dimer (i.e., AS-Dimers) through a CBT-Cys click reaction and further self-assembles into gadolinium-containing nanoparticles (i.e., AS-NPs).

[0010] Furthermore, after the polypeptide probe self-assembles into nanoparticles, the relaxation rate is enhanced, thereby realizing T1-weighted magnetic resonance imaging of enterokinase.

[0011] Furthermore, a method for preparing the magnetic resonance polypeptide probe described above, the preparation steps are as follows:

[0012] Step (1): Synthesize the polypeptide sequence Boc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Cys(StBu) containing the enterokinase-specific cleavage polypeptide substrate by solid-phase peptide synthesis method;

[0013] Step (2): Liquid-phase synthesize the amino acid Lys linked with a CBT group and a gadolinium-based contrast agent, and combine it with the polypeptide sequence synthesized by solid-phase peptide synthesis to form Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys(DOTA(Gd))-CBT (Gd-AS);

[0014] Step (3): Purify Gd-AS by high-performance liquid chromatography (HPLC).

[0015] Furthermore, when the magnetic resonance polypeptide probe is applied in vivo or in vitro, polypeptide cleavage is triggered by the action of enterokinase, and nanoparticle generation is realized to enhance magnetic resonance imaging.

[0016] When triggered by enterokinase, Gd-AS is cleaved and reduced to a new product Cys-Lys(DOTA(Gd))-CBT, which assembles into nanoparticles under the action of CBT-Cys click, thereby effectively increasing the relaxation rate of the gadolinium-based contrast agent.

[0017] The beneficial effects of the present invention are as follows: The "intelligent" response assembly strategy disclosed in the present invention triggers the formation of nanoparticles through enterokinase, combining the advantages of simple synthesis and rapid cell uptake of small molecule probes with the characteristics of signal amplification and long retention time of nanoprobes, showing enhanced tumor imaging effects in in vivo studies; in addition, such nanoparticles have good biocompatibility and biodegradability and are suitable for clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a specific mechanism diagram of the polypeptide magnetic resonance probe for enhanced tumor imaging in the embodiments of the present invention;

[0019] Figure 2 is the electron microscopy image of AS-NPs after the enzymatic cleavage reaction in Example 2 of the present invention;

[0020] Figure 3 is the high performance liquid chromatography diagram of the reaction solution before and after the enzymatic cleavage reaction in Example 2 of the present invention;

[0021] Figure 4 is Figure 3 the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry diagram at 11.5 min in

[0022] Figure 5 is the relaxation rate schematic diagram before and after the assembly of the assembled polypeptide probe Gd-AS in Example 3 of the present invention;

[0023] Figure 6 is the cell viability statistical chart of HeLa cells after co-incubation with Gd-AS in Example 4 of the present invention;

[0024] Figure 7 is the cell magnetic resonance imaging diagram of HeLa cells after co-incubation with Gd-AS in Example 4 of the present invention;

[0025] Figure 8 is the magnetic resonance imaging of tumors and the statistical chart of the gray scale ratio of tumors to references after intraperitoneal injection of drugs to HeLa tumor-bearing nude mice in Example 5 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following further elaborates on the specific technical solutions of the present invention with reference to specific examples.

[0027] As shown in the figure, the present invention discloses a self-assembled polypeptide probe for enhancing tumor magnetic resonance imaging, including the peptide sequence Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys; and a DOTA(Gd) motif connected to the side chain of lysine (Lys) combined with the CBT part; the contrast agent molecule can self-assemble to form nanoparticles under the action of enterokinase.

[0028] Furthermore, the magnetic resonance polypeptide probe (abbreviated as Gd-AS) has the following sequence:

[0029] Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys(DOTA(Gd))-CBT.

[0030] Furthermore, Asp-Asp-Asp-Asp-Lys (DDDDK) in the magnetic resonance contrast agent is a highly specific polypeptide substrate of enterokinase and can be specifically cleaved; Cys-Lys(DOTA(Gd))-CBT can undergo a click reaction to generate nanoparticles.

[0031] Furthermore, after Gd-AS is taken up by cells, it can be reduced to AS-Red in the tumor microenvironment, then cleaved by overexpressed enterokinase, and then cleaved through the CBT-Cys click reaction to form a cyclic dimer (i.e., AS-Dimers), and further self-assembled into gadolinium-containing nanoparticles (i.e., AS-NPs).

[0032] Furthermore, after the polypeptide probe self-assembles into nanoparticles, the relaxation rate is enhanced, thereby realizing T1-weighted magnetic resonance imaging of enterokinase.

[0033] Furthermore, the magnetic resonance polypeptide probe has high biosafety, fast cell uptake rate, and can improve the imaging signal and retention time after in-situ assembly into nanoparticles.

[0034] Furthermore, a method for preparing the magnetic resonance polypeptide probe; the preparation includes the following steps:

[0035] (1): Synthesize the polypeptide sequence Boc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Cys(StBu) containing the enterokinase-specific cleavage polypeptide substrate by solid-phase peptide synthesis (SPPS);

[0036] (2): Liquid-phase synthesize the amino acid Lys linked with a CBT group and a gadolinium-based contrast agent, and combine it with the polypeptide sequence synthesized by solid-phase peptide synthesis to form Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys(DOTA(Gd))-CBT (Gd-AS);

[0037] (3): Purify Gd-AS by high-performance liquid chromatography (HPLC).

[0038] Furthermore, when the magnetic resonance polypeptide probe is applied in vivo or in vitro, polypeptide cleavage is triggered by the action of enterokinase and nanoparticle generation is achieved to enhance magnetic resonance imaging.

[0039] Example 1: Synthesis of self-assembled polypeptide probe Gd-AS:

[0040] 1. Materials and equipment:

[0041] (1) Required reagents: Fmoc-protected amino acids, resin, HBTU, HOBt, DMF, DCM, TFA, TES, TIS, ether, etc.;

[0042] (2) Equipment: Solid-phase peptide synthesizer, high-performance liquid chromatography (HPLC), mass spectrometer (MS), refrigerated centrifuge, ultrapure water system, etc.;

[0043] 2. Steps:

[0044] (1) Liquid-phase synthesis:

[0045] The amino acid Fmoc-Lys is first combined with CBT solution, and after removing the side-chain protection, DOTA is combined through the HBTU / HOBt coupling reaction;

[0046] (2) Deprotection and purification:

[0047] Use the Piperidine / DMF mixed solution to remove the Fmoc protecting group in Fmoc-Lys(DOTA(tBu))-CBT;

[0048] (3) Solid-phase peptide synthesis: Synthesize a polypeptide with the sequence Boc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Cys(StBu) using a solid-phase peptide synthesizer; the Fmoc protecting group is removed after each step of the reaction, and the amino acids are gradually connected to the resin through the HBTU / HOBt coupling reaction;

[0049] (4) Liquid-phase synthesis: Lys(DOTA(tBu))-CBT is connected to the peptide chain Boc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Cys(StBu) through the HBTU / HOBt coupling reaction, and all protecting groups are removed using the TFA / DCM mixed solution;

[0050] (5) Chelate contrast agent: Asp-Asp-Asp-Asp-Lys-Cys(StBu)-K(DOTA)-CBT chelates with gadolinium ions in an ultrapure aqueous solution with a pH of 6 - 7 to obtain the final product Gd-AS;

[0051] (6) Purify Gd-AS with high purity through HPLC;

[0052] (7) Structure confirmation: Confirm the molecular structure and purity of Gd-AS by mass spectrometry and nuclear magnetic resonance (NMR);

[0053] Among them, the synthesis route of Lys(DOTA(tBu))-CBT is as follows:

[0054]

[0055] The synthesis route of Gd-AS is as follows:

[0056]

[0057] Example 2: In vitro enzymatic cleavage assembly of self-assembled polypeptide probe Gd-AS

[0058] 1. Materials and equipment:

[0059] (1) Required reagents: Enterokinase, TCEP, PBS buffer (pH 7.4), etc.;

[0060] (2) Equipment: Ultrasonic, transmission electron microscope (TEM), high performance liquid chromatography, etc.;

[0061] 2. Steps:

[0062] (1) Preparation of enzymatic cleavage sample: Dissolve Gd-AS in PBS buffer at a concentration of 800 μM; add 8 mM TCEP, after ultrasonic treatment for 1 hour, add 10 U / mL enterokinase, and react in a constant temperature shaker at 37 °C for 12 h to form nanoparticles (AS-NPs) of uniform size;

[0063] (2) Structure characterization: Observe the nanostructure after enzymatic cleavage reaction by TEM, and determine the formation of its dimer (AS-Dimer) by high performance liquid chromatography and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0064] Example 3: Change in relaxation rate of self-assembled polypeptide probe Gd-AS before and after assembly

[0065] 1. Materials and equipment:

[0066] (1) Required reagents: Enterokinase, TCEP, PBS buffer (pH 7.4), etc.;

[0067] (2) Equipment: 0.5T MR scanner (NMI20-015 V-I, NIUMAG), etc.;

[0068] 2. Steps:

[0069] (1) Preparation of digested samples: Dissolve Gd-AS in PBS buffer at a concentration of 800 μM. Add 8 mM TCEP, and after sonication for 1 hour, add enterokinase at 10 U / mL and react in a constant temperature shaker at 37 °C for 12 h to form nanoparticles (AS-NPs) of uniform size;

[0070] (2) Relaxivity experiment: Dilute the reaction solution of 800 μM to different concentrations (25, 50, 100, 200, and 400 μM), and quantitatively analyze the relaxation time by a 0.5T MR scanner (NMI20-015 V-I, NIUMAG).

[0071] Example 4: In vitro magnetic resonance imaging experiment

[0072] 1. Materials and equipment:

[0073] (1) Required reagents: HeLa cells, CCK-8 kit, PBS buffer (pH 7.4), trypsin, etc.;

[0074] (2) Equipment: Cell culture incubator, microplate reader, fluorescence microscope, etc.;

[0075] 2. Procedures:

[0076] (1) Cell culture: Culture HeLa cells in DMEM medium containing 10% FBS;

[0077] (2) Cell viability assay: Add Gd-AS to HeLa cells and measure cell viability at different time points using the CCK-8 kit;

[0078] (3) Cell imaging: Observe and record images using a 0.5T MR scanner (NMI20-015 V-I, NIUMAG).

[0079] Example 5: In vivo anti-cancer experiment

[0080] 1. Materials and equipment:

[0081] (1) Required reagents: HeLa cells, BALB / c nude mice, PBS buffer (pH 7.4), etc.;

[0082] (2) Equipment: Animal experiment equipment, 1T MR scanner (Bruker ICON TM ) etc.;

[0083] 2. Procedures:

[0084] (1) Establishment of animal model: Subcutaneously inoculate HeLa cells into the back of nude mice to establish a tumor model;

[0085] (2) Administration experiment: After the tumor volume reaches a certain size, intraperitoneal injection of Gd-AS is administered;

[0086] (3) Effect evaluation: Observe the tumor imaging of tumor-bearing mice and record the results of the small animal magnetic resonance imaging instrument respectively, and draw the gray-scale ratio between the tumor area and the quantitative reference.

Claims

1. A self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging, characterized in that: The self-assembling polypeptide probe comprises a peptide sequence Asp-Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys and a DOTA motif connected to a lysine side chain combined with a CBT part; the self-assembling polypeptide probe self-assembles to form nanoparticles under the action of enterokinase.

2. A self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging according to claim 1, characterized in that: The self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging has the following sequence: Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys(DOTA(Gd))-CBT.

3. A self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging according to claim 2, characterized in that: In the self-assembling polypeptide probe, Asp-Asp-Asp-Asp-Lys is specifically cleaved as a highly specific polypeptide substrate of enterokinase; and Cys-Lys(DOTA(Gd))-CBT undergoes a click reaction to generate nanoparticles.

4. A self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging according to claim 3, characterized in that: After being taken up by cells, the magnetic resonance polypeptide probe can be reduced to AS-Red in the tumor microenvironment, and then cleaved by overexpressed enterokinase, and then cut through the CBT-Cys click reaction to form cyclic dimers, namely AS-Dimers; and further self-assemble into gadolinium-containing nanoparticles, namely AS-NPs.

5. A self-assembling polypeptide probe for enhancing tumor magnetic resonance imaging according to claim 4, characterized in that: After the polypeptide probe is self-assembled into nanoparticles, the relaxation rate is enhanced, thereby realizing T1-weighted magnetic resonance imaging of enterokinase.

6. A method for preparing the magnetic resonance polypeptide probe according to claim 1, characterized in that: The preparation steps are as follows: Step (1): synthesizing a polypeptide sequence Boc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Cys(StBu) containing an enterokinase-specific cleavage polypeptide substrate by solid phase peptide synthesis; Step (2): liquid phase synthesis of the amino acid Lys that connects the CBT group and the gadolinium-based contrast agent, and combining it with the peptide sequence synthesized by solid phase peptide synthesis to form Asp-Asp-Asp-Asp-Lys-Cys(StBu)-Lys(DOTA(Gd))-CBT; Step (3): Purify Gd-AS by high performance liquid chromatography.

7. The method according to claim 6, characterized in that When the magnetic resonance polypeptide probe is used in vivo or in vitro, the polypeptide is sheared by the action of enterokinase and nanoparticles are generated to enhance magnetic resonance imaging.

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