Novel nuclear magnetic resonance contrast agent Gd-DOTA-PP for realizing neutrophil extracellular trap net imaging by targeting citrullinated histone as well as preparation method and application of novel nuclear magnetic resonance contrast agent Gd-DOTA-PP

Abstract

The invention discloses a novel nuclear magnetic resonance contrast agent Gd-DOTA-PP for targeting citrullinated histone to achieve neutrophil extracellular trap net imaging and a preparation method and application thereof.The method comprises the steps that citrullinated histone targeting polypeptide PP and ligand molecules DOTA-NHS are combined, and a compound DOTA-PP is obtained; and then gadolinium (Gd) ions are introduced into the DOTA-PP through a coordination reaction, and the novel contrast agent Gd-DOTA-PP is prepared. The contrast agent can be selectively combined with an important marker, namely citrullinated histone, of neutrophil extracellular trapping nets (NETs), and T1 weighted imaging signals of a target area are remarkably enhanced. The preparation method is simple and convenient to operate, the preparation process is highly controllable, and the obtained contrast agent has excellent biocompatibility and targeting characteristic. The accurate targeting ability of the probe enables the influence signal of the probe in NETs enrichment areas such as tumor early metastasis, tumor infiltration lymph nodes and the like to be obviously enhanced, a powerful tool is provided for tumor diagnosis, especially accurate diagnosis of the early metastasis, and the probe has important clinical application value.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of materials science, oncology, and medical imaging, and in particular to a gadolinium-based contrast agent for nuclear magnetic resonance imaging, Gd-DOTA-PP, its preparation method, and its application. Background Technology

[0002] Metastasis is a leading cause of cancer-related death, and early identification of metastatic lesions is crucial for improving patient prognosis. While currently widely used imaging methods, such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), play important roles in tumor metastasis assessment, their ability to detect small lesions is limited, severely impacting the accuracy of early diagnosis and patient outcomes. Contrast-enhanced MRI offers a potential solution for detecting small lesions. However, traditional contrast agents (such as Gd-DOTA) lack the ability to specifically identify pathological features, which limits the effectiveness of MRI in imaging the tumor microenvironment.

[0003] In recent years, neutrophil extracellular trapping networks (NETs) have gradually attracted research attention as an important component of the tumor metastatic microenvironment. NETs are extracellular network structures composed of DNA, histones, etc., which are squeezed out by neutrophils under inflammatory stimulation. Initially, NETs were thought to be a mechanism for defending against infection, but more and more studies have found that NETs play a pro-cancer role in tumor metastasis. Specifically, NETs can enhance the invasiveness of tumor cells by promoting epithelial-mesenchymal transition (EMT); at the same time, NETs can protect circulating tumor cells (CTCs) from immune clearance by trapping them and promote their deposition in target organs, thereby promoting the formation of metastatic lesions. Therefore, NETs have been considered a key biomarker for the early diagnosis of tumor micrometastases.

[0004] Histone citrullination is a key step in NET formation and is recognized as a specific biomarker for NETs. Targeting citrullinated histones can provide new insights for specific imaging of NETs. However, there is currently no effective in vivo imaging method to achieve precise visualization of NETs, ​​which has become a bottleneck in the field of tumor metastasis imaging. Based on this, this invention targets citrullinated histones and identifies and optimizes several stable and highly specific peptide molecules through high-throughput library screening. Subsequently, the most specific peptide sequence PP (QSLVDSDGKTY) was prepared using the standard BOC solid-phase peptide synthesis method, and DOTA-PP was obtained by covalently binding it to DOTA-NHS. Finally, it was synthesized by reacting with gadolinium ions (Gd³⁺). + The chelation of ) successfully prepared the NETs-specific MRI contrast agent Gd-DOTA-PP.

[0005] Compared to traditional MRI contrast agents, Gd-DOTA-PP exhibits significant targeting ability and enhanced T1 imaging signal in tumor metastases and tumor infiltration areas, enabling precise localization of NETs. This targeted imaging capability significantly improves the detection rate of micrometastases, providing a novel and effective tool for clinical use and solving the problem of insufficient detection capability of existing technologies for early metastases.

[0006] In summary, the development of this contrast agent not only expands the application of MRI from anatomical imaging to precise targeted imaging, but also shows broad application prospects in the early diagnosis of tumor metastases and the study of the tumor microenvironment, providing important support for the development of precision medicine. Summary of the Invention

[0007] The purpose of this invention is to provide a magnetic resonance imaging (MRI) contrast agent, Gd-DOTA-PP, its preparation method, and its applications. This contrast agent integrates the MRI advantages of Gd-based contrast agents with the targeting ability of PP peptides to citrullinated histones. By modifying Gd-DOTA with PP peptides as ligands, this invention achieves specific imaging of NETs (Net-Induced Tissue Organisms). This innovative method not only enhances the MRI imaging effect on NET-accumulated regions, such as tumor tissues, but also opens up new avenues for MRI diagnosis of clinical tumors.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the present invention, a magnetic resonance imaging contrast agent Gd-DOTA-PP is provided, the structural formula of which is shown below:

[0009] In a second aspect of the present invention, a method for preparing the above-mentioned magnetic resonance imaging contrast agent Gd-DOTA-PP is provided, wherein the reaction formula for preparation is as follows, specifically including: (1) Compound PP polypeptide, DOTA-NHS and DIEA were added to anhydrous DMF, reacted at room temperature, and then dialyzed and lyophilized to obtain compound DOTA-PP; (2) The compound DOTA-PP was dissolved in pure water, and GdCl3·6H2O was added to carry out a chelation reaction. After pH adjustment, dialysis and freeze drying, the nuclear magnetic resonance contrast agent Gd-DOTA-PP was obtained.

[0010]

[0011] In step (1), the amounts of PP peptide, DOTA-NHS, and DIEA are preferably 60 mg, 25 mg, and 10 μL, respectively, and the amount of anhydrous DMF required is preferably 5 mL.

[0012] In step (1), the preferred reaction conditions are stirring at room temperature and a preferred reaction time of 24 hours.

[0013] In step (1), the molecular weight cutoff of the dialysis bag required in the dialysis step is preferably 500, the dialysis solution is preferably deionized water, and the dialysis time is preferably 48 hours.

[0014] In step (2), the preferred ratio of the compound DOTA-PP to GdCl3·6H2O is 4:1, the preferred amount of pure water is 5 mL, and the preferred pH-adjusting substance is Na2CO3. 3。

[0015] In step (2), the reaction temperature is preferably room temperature and the reaction time is preferably 24 hours.

[0016] In step (2), the molecular weight cutoff of the dialysis bag required in the dialysis step is preferably 500, the dialysis solution is preferably deionized water, and the dialysis time is preferably 48 hours.

[0017] In a third aspect of the invention, the contrast agent proposed in this invention has been verified to have a good diagnostic effect on subcutaneous tumors.

[0018] A subcutaneous tumor model was constructed in male C57BL / 6 mice using the B16 cell line. After successful modeling, the mice were anesthetized and fixed, and scanned using a 3.0T MRI scanner to obtain images of the subcutaneous tumor before contrast agent injection. Gd-DOTA-PP was then injected into the subcutaneous tumor model mice via the tail vein, and scans were performed 30 minutes later to record signal changes at the subcutaneous tumor site after contrast agent injection. This allowed for the diagnosis of subcutaneous tumors in mice and the evaluation of imaging results. The contrast agent used is a magnetic resonance contrast agent, specifically targeting the MR detection of subcutaneous tumors and suitable for tumor-specific imaging. In a fourth aspect of the invention, the contrast agent proposed in this invention has been verified to have good diagnostic effects on tumor-infiltrating lymph nodes (2 mm). A popliteal metastatic tumor model was constructed in male C57BL / 6 mice using the Hepa1-6 mouse hepatocellular carcinoma cell line. After successful modeling, the mice were anesthetized and fixed, and scanned using a 3.0T MRI scanner to obtain lymph node imaging images before contrast agent injection. Then, the contrast agent was injected into the lymph node metastatic tumor model mice via the tail vein. Scanning was performed 30 minutes later to record signal changes in the subcutaneous tumor site after contrast agent injection, enabling diagnosis of the popliteal lymph nodes in mice and evaluation of the imaging results.

[0019] Compared with the prior art, the present invention has the following technical effects or advantages: (1) This invention develops a novel targeted contrast agent that can perform molecular imaging of citrullinated histone, a marker of NETs, ​​to achieve specific imaging of NETs-rich regions and significantly improve the diagnostic effect of tumor regions. (2) The present invention selects NETs in metastatic tumors as targets, which can improve the diagnostic effect of micrometastatic tumors and improve detection performance; (3) The method of the present invention is simple, has low production cost, and is easy to scale up; (4) The synthesis and modification methods of the contrast agent Gd-DOTA-PP in this invention are mature and the conditions are easy to control; (5) The required reaction conditions and reagents are all readily available and commonly used. Attached Figure Description

[0020] Figure 1 This is the synthesis roadmap for DOTA-PP and Gd-DOTA-PP.

[0021] Figure 2 These are mass spectrometry results for DOTA-PP and Gd-DOTA-PP. Figure A shows the mass spectrum of DOTA-PP, and Figure B shows the mass spectrum of Gd-DOTA-PP.

[0022] Figure 3 This image shows Gd-DOTA-PP used for MRI imaging of subcutaneous tumors in mice. Figure A shows an MRI scan of the subcutaneous tumor before contrast agent injection, and Figure B shows an MRI scan of the subcutaneous tumor after Gd-DOTA-PP injection.

[0023] Figure 4 This image shows Gd-DOTA-PP used for MRI imaging of tumor lymph node metastases in mice. Figure A shows the MRI scan of the lymph nodes before contrast agent injection, and Figure B shows the MRI scan of the lymph nodes after Gd-DOTA-PP injection. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0027] As an optional embodiment of the present invention, the present invention provides a method for preparing the nuclear magnetic resonance imaging contrast agent Gd-DOTA-PP, comprising the following steps:

[0028] (1) PP polypeptide, DOTA-NHS, anhydrous DMF and DIEA were added to a dry single-necked flask. After stirring at room temperature for a period of time, the mixture was dialyzed and freeze-dried to obtain compound DOTA-PP.

[0029] (2) Add compound DOTA-PP and GdCl3·6H2O to pure water, adjust the pH and react at room temperature for a period of time, then dialyze and freeze dry to obtain Gd-DOTA-PP.

[0030] As one of the preferred solutions in the specific implementation plan, in step (1), the stirring reaction time at room temperature is 24 hours.

[0031] As one of the preferred solutions in the specific implementation plan, in step (1), the dialysis bag used for dialysis has a molecular weight cutoff of 500, the dialysis solution is deionized water, and the dialysis time is 48 hours.

[0032] As one of the preferred embodiments of the specific implementation plan, in step (1), the ratio of the amount of PP peptide, DOTA-NHS, anhydrous DMF and DIEA is 60 mg: 25 mg: 5 mL: 10 μL.

[0033] As one of the preferred options for the specific implementation plan, in step (2), the reaction time at room temperature is 24 hours.

[0034] As one of the preferred solutions in the specific implementation plan, in step (2), the dialysis bag used for dialysis has a molecular weight cutoff of 500, the dialysis solution is deionized water, and the dialysis time is 48 hours.

[0035] As one of the preferred embodiments of the specific implementation plan, in step (2), the ratio of the compound DOTA-PP and GdCl3·6H2O is 4:1, the amount of pure water required is 5 mL, and the substance used to adjust the pH is Na2CO3.

[0036] The following will provide a detailed description of the nuclear magnetic resonance imaging contrast agent Gd-DOTA-PP, its preparation method, and its application, in conjunction with embodiments, comparative examples, and experimental data.

[0037] Example 1: A novel nuclear magnetic resonance contrast agent, Gd-DOTA-PP, for imaging neutrophil extracellular traps by targeting citrullinated histones, and its preparation method: 1. First, a water-soluble DOTA-PP with citrullinated histone targeting ability was prepared, and its structure was characterized by mass spectrometry. (See [link to documentation]). Figure 2 As shown. The preparation method of the DOTA-PP is as follows: 60 mg of PP peptide, 25 mg of DOTA-NHS, 5 mL of anhydrous DMF, and 10 μL of DIEA were added to a dry flask. After stirring at room temperature for 24 hours, the mixture was dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 500 and then lyophilized to obtain DOTA-PP.

[0038] 2. Compound DOTA-PP and GdCl3·6H2O were added to 5 mL of pure water at a ratio of 4:1. The pH was then adjusted to 6-7 with Na2CO3. The mixture was reacted at room temperature for 24 hours. After that, it was dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 500 and then lyophilized to obtain the contrast agent Gd-DOTA-PP.

[0039] Example 1: Characterization of Gd-DOTA-PP The mass spectrometry samples of DOTA-PP and Gd-DOTA-PP prepared in Example 1 were tested, and the actual molecular weight of the samples was compared with the theoretical molecular weight. For example... Figure 2 As shown in Figure A, mass spectrometry analysis revealed that the molecular weight of DOTA-PP was 1598.1334, consistent with the molecular weight of the synthesized structure. Figure 2 As shown in Figure B, the mass spectrometry analysis of Gd-DOTA-PP showed a molecular weight of 1751.1528, which is consistent with the molecular weight of the synthesized structure.

[0040] Experimental Example 2: MRI Contrast Imaging Effect of Subcutaneous Tumors in Mice A mouse subcutaneous tumor model was constructed using the B16-F10 melanoma cell line. The tumors were allowed to grow to a suitable volume (100 mm²). 3 Mice were then scanned using a 3.0T MRI scanner, followed by an intravenous injection of 100 µL of Gd-DOTA-PP contrast agent (0.02 mmol / kg Gd). Figure 3 As shown, using a subcutaneous tumor mouse model, an MRI scan was performed 30 minutes after Gd-DOTA-PP was injected into the tail vein. The difference compared to before the injection was observed ( Figure 3 -A), the T1 signal in the subcutaneous tumor area was significantly enhanced ( Figure 3 -B).

[0041] Experimental Example 3: MRI contrast agent imaging effect of lymph node metastases in mice Hepa1-6 hepatocellular carcinoma cells were injected into the left paw pad of mice to establish a mouse model of popliteal lymph node metastasis. The injection volume was 1 million cells per mouse. Two weeks after injection, the cells were scanned using a 3.0T MRI scanner. Subsequently, 100 µL of Gd-DOTA-PP contrast agent (0.02 mmol / kg Gd) was injected via the tail vein. MRI scans were repeated 30 minutes later to observe changes in the T1 signal of the popliteal lymph nodes. Figure 4 As shown in Figure -B, using a mouse model of popliteal lymph node metastases, an MRI scan was performed 30 minutes after Gd-DOTA-PP was injected via the tail vein. The difference compared to before the injection was observed ( Figure 4 -A), the T1 signal of the popliteal lymph node metastases was significantly enhanced ( Figure 4 -B).

[0042] The above results indicate that the compound described in this invention application has a significant targeted contrast effect; the compound, as a contrast agent, has advantages such as good water solubility, controllable conditions, low cost, non-toxicity, and easy metabolism, and can avoid gadolinium deposition and renal systemic fibrosis, making it an ideal T1-enhanced contrast agent for tumor tissue.

[0043] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0044] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0045] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A gadolinium-based nuclear magnetic resonance imaging contrast agent based on citrullinated histone-targeting peptide PP, characterized in that, The structural formula of the contrast agent is shown below: 。 2. A method for preparing the nuclear magnetic resonance imaging contrast agent Gd-DOTA-PP as described in claim 1, characterized in that, The method includes: Compound PP peptide, DOTA-NHS, and DIEA were added to anhydrous DMF. After reaction, the compound DOTA-PP was obtained by dialysis and lyophilization. The compound DOTA-PP was dissolved in pure water, and GdCl3·6H2O was added to carry out a chelation reaction. After adjusting the pH, the reaction proceeded. The resulting product was then dialyzed and lyophilized to obtain the nuclear magnetic resonance imaging contrast agent Gd-DOTA-PP. The reaction formula is as follows: 。 3. The method for preparing the nuclear magnetic resonance imaging contrast agent Gd-DOTA-PP according to claim 2, characterized in that, The preparation method of the compound DOTA-PP includes: PP peptide, DOTA-NHS, and DIEA were mixed, and the mixture was reacted at room temperature for 24 hours. The resulting solution was then dialyzed and lyophilized to obtain compound DOTA-PP. The reaction formula for preparing the compound DOTA-PP is as follows: 。 4. The preparation method according to claim 3, characterized in that, In the compound DOTA-PP, the ratio of the compound PP polypeptide, DOTA-NHS, anhydrous DMF and DIEA is 60 mg: 25 mg: 5 mL: 10 μL.

5. The preparation method according to claim 3, characterized in that, In the compound DOTA-PP, the dialysis bag used in the dialysis process has a molecular weight cutoff of 500, the dialysis solution is deionized water, and the dialysis time is 48 hours.

6. The method for preparing a magnetic resonance imaging contrast agent Gd-DOTA-PP according to claim 2, characterized in that, The preparation method of the compound Gd-DOTA-PP includes: Compound DOTA-PP and GdCl3·6H2O were added to pure water, and the pH was adjusted to 6-7. After reacting at room temperature for 24 hours, Gd-DOTA-PP was obtained by dialysis and lyophilization.

7. The preparation method according to claim 6, characterized in that, The ratio of the compound DOTA-PP to GdCl3·6H2O is 4:1, the amount of pure water used is 5 mL, and the pH is adjusted by Na2CO3.

8. The preparation method according to claim 6, characterized in that, In the compound Gd-DOTA-PP, the dialysis bag used in the dialysis process has a molecular weight cutoff of 500, the dialysis solution is deionized water, and the dialysis time is 48 hours.

9. A magnetic resonance imaging contrast agent Gd-DOTA-PP prepared by the preparation method according to any one of claims 1-8.

10. The use of the magnetic resonance imaging contrast agent Gd-DOTA-PP according to claim 9 in the preparation of imaging tools for tumor diagnosis.

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