CN110075322A - A kind of near-infrared fluorescence imaging probe and the preparation method and application thereof targeting GnRH receptor - Google Patents

Abstract

The invention provides a GnRH receptor-targeted near-infrared fluorescence imaging probe GnRHa-ICG, a preparation method and application thereof. The near-infrared fluorescent imaging probe GnRHa-ICG targeting the GnRH receptor consists of a GnRH polypeptide (sequence D-2-Nal-D-4-Cl-Phe-D-3-Pal-Ser-Tyr-D-Cit- Leu‑Arg‑Pro‑D‑Ala‑NH ₂ ) coupled with activated near-infrared fluorescent dye ICG‑NHS. The present invention is based on the principle of specific binding between GnRH polypeptides and GnRH receptors, and utilizes the high expression of GnRH receptors in reproductive system tumors such as ovarian cancer, and the advantages of deeper penetration depth of near-infrared fluorescent dye ICG and weaker background tissue autofluorescence. Targeted recognition of tumor cells and tumor foci that highly express GnRH receptors has a good application prospect in fluorescence imaging and fluorescence-guided surgery.

Description

A near-infrared fluorescent imaging probe targeting GnRH receptors and its preparation method and application

technical field

The invention relates to the field of biotechnology, in particular to a preparation method and application of a tumor-specific fluorescent probe for treating reproductive system malignant tumors.

Background technique

Malignant tumors are an important threat to human health. It is estimated that there will be more than 18 million new cancer cases and 9.6 million cancer deaths worldwide in 2018. Among reproductive system-related malignant tumors, prostate cancer accounted for 13.5% of new tumor cases in men, and breast cancer, uterine body tumors and ovarian cancer accounted for 24.2%, 4.4% and 3.4% of new cases in women, respectively.

Surgery is an important treatment for solid tumors, and the thoroughness of surgery is closely related to prognosis. In order to achieve complete resection, accurate identification of the lesion during operation is the key. Existing imaging methods such as ultrasound, CT, and MRI are non-specific for tumor imaging, and it is difficult to provide real-time imaging during surgery. Fluorescence-guided surgery uses tumor-targeted fluorescent probes to guide lesion resection through intraoperative real-time fluorescence imaging, which helps to improve surgical thoroughness and prognosis. The key to realizing intraoperative imaging lies in the development of tumor-specific fluorescent probes, which are mostly composed of targeting groups and imaging probes.

Malignant tumors of the reproductive system are closely related to hormones related to the hypothalamus-pituitary-gonad axis, such as gonadotropin-releasing hormone (GnRH). Under physiological conditions, the distribution of GnRH receptors is relatively limited to the pituitary and reproductive system, including ovary, endometrium, placenta, breast, prostate, etc. Studies have confirmed that GnRH receptors are highly expressed in malignant tumors related to the reproductive system, among which the expression rate of prostate cancer is 86%, that of ovarian cancer and endometrial cancer is 80%, and that of breast cancer is 50%. A series of advances have been made in tumor targeted therapy targeting GnRH receptors, so GnRH receptors may also become targets for tumor-specific imaging.

GnRH polypeptide fragments that specifically bind to GnRH receptors can be used as targeting groups for imaging probes. At present, a variety of agonists or antagonists of GnRH receptors have entered clinical application, which are structurally similar to natural GnRH polypeptides. Using small molecular peptides as targeting groups is a commonly used targeting strategy, which has been widely used in the field of tumor targeting therapy, and the research on tumor targeting imaging is also gradually increasing. Compared with monoclonal antibodies, small molecule peptides have the advantages of weak immunogenicity, rapid distribution in vivo, strong penetrability, and easy synthesis and modification. Some tumor-targeted imaging probes based on peptides have entered the clinical trial stage. For example, the probe BLZ-100 is composed of peptide Chlorotoxin coupled with Indocyanine green (Indocyanine green, ICG), and the probe GE-137 is targeted by cMET. The peptide is coupled with Cy5.

Near-infrared fluorescent dyes have the advantages of deeper penetration depth and weaker background tissue autofluorescence, making them more suitable for in vivo imaging. ICG is a near-infrared fluorescent dye approved by the U.S. FDA for clinical use. It is used for angiography, tumor sentinel lymph node imaging, and liver function testing. It has been reported in the literature that ICG can also accumulate in tumor tissue due to increased vascular permeability of tumor tissue and obstruction of lymphatic flow. However, ICG cannot specifically bind tumor cells, and the false positive rate when it is directly used for imaging ovarian cancer lesions is as high as 62%.

Therefore, it is necessary to increase its specific imaging ability based on the imaging advantages of near-infrared fluorescent dye ICG. The invention couples the GnRH polypeptide that can specifically bind to the GnRH receptor and the near-infrared fluorescent dye ICG to prepare a near-infrared fluorescent imaging probe targeting the GnRH receptor. The probe can target and recognize tumor cells and tumor foci that highly express GnRH receptors in vivo and in vitro, and has good application prospects in fluorescence imaging and fluorescence-guided surgery.

SUMMARY OF THE INVENTION

The present invention provides a near-infrared fluorescent imaging probe targeting GnRH receptors and its preparation method and application. The near-infrared fluorescent imaging probe GnRHa-ICG targeting GnRH receptors is composed of GnRH polypeptide (GnRHa) and activated The near-infrared fluorescent dye ICG-NHS is coupled, and the structural formula of GnRHa-ICG is as follows:

The structural formula of the near-infrared fluorescent dye ICG-NHS is as follows:

The GnRH polypeptide only uses the amino acid sequence of the clinical drug GnRH receptor antagonist Cetrorelix, without the modification group of the drug, and the sequence formula is D-2-Nal-D-4-Cl-Phe-D- 3-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH ₂ , the structural formula is as follows:

A method for preparing a near-infrared fluorescent imaging probe targeting GnRH receptors, comprising the following steps:

(1) Use solid phase carrier to synthesize polypeptide GnRHa;

(2) Add 3 mg of the polypeptide GnRHa obtained in step (1), 20 μL N, N-diisopropylethylamine and 0.5 mL ultra-dry DMF to a 5 mL reaction bottle, and react for 10 min under nitrogen protection; mg ICG-NHS was added to the DMF reaction liquid, and the stirring reaction was continued at room temperature for 12 h;

(3) The reaction solution obtained in step (2) was settled with 5 mL of ether to obtain a green solid;

(4) After dissolving the solid obtained in step (3) with a small amount of methanol, it was separated, purified and freeze-dried by high performance liquid chromatography to obtain 2 mg of a green solid product with a yield of 80%.

The near-infrared fluorescence imaging probe targeting GnRH receptors of the present invention is applied to: using human tumor cell lines and nude mouse peritoneal implanted tumor models to detect the binding ability of probes and tumor cells, and the targeting probe GnRHa-ICG can be used in vivo and in vitro Targeted recognition of tumor cells and tumor foci that highly express GnRH receptors.

The human tumor cell line was used to test the binding ability of the probe to tumor cells, and the results showed that the binding force of the targeting probe GnRHa-ICG was stronger than that of ICG; the in vivo imaging of the probe was detected by the nude mouse peritoneal implanted tumor model of human ovarian cancer cell line The results show that the targeting probe GnRHa-ICG can specifically bind to the peritoneal implants, and the effect is better than ICG.

The advantages of the present invention are:

1. The present invention utilizes the high expression of GnRH receptors in reproductive system tumors such as ovarian cancer, and targets and recognizes tumor cells based on the principle of specific binding between GnRH polypeptides and GnRH receptors.

2. The present invention utilizes the advantages of deeper penetration depth of near-infrared fluorescent dye ICG and weaker background tissue autofluorescence, and has good application prospects in fluorescence imaging and fluorescence-guided surgery.

Description of drawings

Fig. 1 Synthesis and HPLC characterization of GnRHa-ICG probe;

Fig. 2 UV absorption spectrum and fluorescence spectrum of GnRHa-ICG and ICG;

Fig. 3 Combination of GnRHa-ICG and ICG with tumor cells;

Fig. 4 Imaging of GnRHa-ICG and ICG in nude mouse peritoneal implanted tumor model.

Detailed ways

The present invention provides a novel near-infrared fluorescent imaging probe GnRHa-ICG targeting GnRH receptors, which is formed by coupling GnRH polypeptide (GnRHa) and near-infrared fluorescent dye ICG. The preparation method is as follows:

(1) The peptide GnRHa was synthesized on a solid-phase carrier with the sequence D-2-Nal-D-4-Cl-Phe-D-3-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala- _NH2 ;

(2) Add 3 mg of the polypeptide GnRHa obtained in step (1), 20 μL N, N-diisopropylethylamine and 0.5 mL ultra-dry DMF to a 5 mL reaction bottle, and react for 10 min under nitrogen protection; mg ICG-NHS was added to the DMF reaction liquid, and the stirring reaction was continued at room temperature for 12 h;

(3) The reaction solution obtained in step (2) was settled with 5 mL of ether to obtain a green solid;

(4) After dissolving the solid obtained in step (3) with a small amount of methanol, it was separated, purified and freeze-dried by high performance liquid chromatography to obtain 2 mg of a green solid product with a yield of 80%.

The synthetic route and HPLC characterization of the GnRHa-ICG probe are shown in Figure 1.

Prepare GnRHa-ICG and ICG solutions with a concentration of 2.5 μM (the solvent is DMSO), use a spectrophotometer to detect the ultraviolet absorption spectrum of the probe, and use a fluorescence spectrometer to detect the fluorescence spectrum of the probe. The results are shown in Figure 2.

Example

The specific implementation of the application of the above-mentioned near-infrared fluorescent probe in tumor targeting imaging is as follows:

Inoculate the human ovarian cancer cell line A2780 (high expression of GnRH receptor) and human lung cancer cell line H1299 (low expression of GnRH receptor) in the logarithmic growth phase on the chamber slide, and start the experiment when the cell confluency reaches 50% . Aspirate the culture medium and wash once with PBS buffer. Add 60 μM GnRHa-ICG and ICG respectively, and incubate with tumor cells for 30 min at 37° C. Aspirate the supernatant and wash 3 times with PBS. Add 4% paraformaldehyde and fix at room temperature for 10 min. Aspirate the fixative and wash once with PBS. Add cell membrane dye WGA488 and incubate at room temperature for 10 min. Aspirate membrane dye and wash 3 times with PBS. The slides were sealed with fluorescent mounting medium containing DAPI, and the fluorescence intensity was observed under a confocal microscope.

The human ovarian cancer cell line A2780 in the logarithmic growth phase was inoculated in a 12-well plate, and the experiment was started when the cell confluence reached 60%. Aspirate the culture medium and wash once with PBS. Add 5 μM GnRHa-ICG and ICG respectively, and incubate with tumor cells for 30 min at 37°C. Aspirate the supernatant and wash 3 times with PBS. Trypsin was digested to make cell suspension, and the fluorescence intensity was detected by flow cytometry.

The above test results are shown in Figure 3. GnRHa-ICG and ICG probes show red fluorescence, cell membranes show green fluorescence, and cell nuclei show blue fluorescence. The fluorescence intensity of the targeting probe GnRHa-ICG was higher than that of ICG, and the fluorescence intensity of the A2780 cell line with high GnRH receptor expression was higher than that of the H1299 cell line with low receptor expression (Figure 3A). The results of flow cytometry were consistent with those observed by fluorescence microscopy, and the average fluorescence intensity of GnRHa-ICG was higher than that of ICG (Figure 3B). The results showed that the targeting probe GnRHa-ICG had stronger binding ability to tumor cells expressing GnRH receptor than ICG.

The human ovarian cancer cell line A2780 was digested to make a single cell suspension, and inoculated into the peritoneal cavity of female nude mice at 1x10 ⁷ /100ul per mouse. Tumor cells spread and grow in the peritoneal cavity. After 2 weeks, the number of implanted lesions can exceed 10, and the size is about 0.1cm-1cm.

The tumor-bearing mice in the experimental group were intraperitoneally injected with 0.03 mg (1.5 mg/kg) of the targeting probe GnRHa-ICG, and the tumor-bearing mice in the control group were intraperitoneally injected with 0.03 mg (1.5 mg/kg) of ICG. The mice were sacrificed 2 hours after injection of the imaging probe, and the abdominal cavity was opened for near-infrared fluorescence in vivo imaging detection (excitation wavelength 780nm, emission wavelength 845nm). Major organs and tumor tissues were then removed for in vitro fluorescence imaging testing. Frozen sections of the tumor, liver (positive control) and skeletal muscle (negative control) of the mice in the experimental group were taken, sealed with DAPI-containing fluorescent mounting medium, and the fluorescence intensity was observed with a confocal microscope.

The imaging results are shown in Figure 4. The probe GnRHa-ICG in the experimental group had obvious signals in the peritoneal implants and liver, but no signal in the intestinal tract (Figure 4A), and the confocal microscope observation of frozen sections was consistent with the results of in vivo imaging (Figure 4B); the signal of the digestive tract of the probe ICG in the control group Strong, tumor foci could not be distinguished (Fig. 4C). By comparing the imaging results of the targeting probe GnRHa-ICG and ICG, it is suggested that the targeting probe has higher selectivity for living tumor lesions, which can significantly weaken the non-specific imaging of ICG in the digestive system such as the intestinal tract, and has a higher effect on tumor tissue. It has better in vivo imaging effect.

Claims (3)

1. A near-infrared fluorescent imaging probe targeting GnRH receptors, characterized in that, the near-infrared fluorescent imaging probe GnRHa-ICG targeting GnRH receptors is composed of GnRH polypeptide GnRHa and activated near-infrared fluorescent dyes ICG-NHS coupling, the structural formula of GnRHa-ICG is as follows: The structural formula of the near-infrared fluorescent dye ICG-NHS is as follows: The GnRH polypeptide only uses the amino acid sequence of the clinical drug GnRH receptor antagonist Cetrorelix instead of the modification group of the drug. The structural formula of the GnRH polypeptide is as follows: . 2. by the preparation method of the near-infrared fluorescence imaging probe of a kind of targeting GnRH receptor described in claim 1, comprises the following steps: (1) Use solid phase carrier to synthesize polypeptide GnRHa; (2) Add 3 mg of the polypeptide GnRHa obtained in step (1), 20 μL N, N-diisopropylethylamine and 0.5 mL ultra-dry DMF to a 5 mL reaction bottle, and react for 10 min under nitrogen protection; mg ICG-NHS was added to the DMF reaction liquid, and the stirring reaction was continued at room temperature for 12 h; (3) The reaction solution obtained in step (2) was settled with 5 mL of ether to obtain a green solid; (4) After dissolving the solid obtained in step (3) with a small amount of methanol, it was separated, purified and freeze-dried by high performance liquid chromatography to obtain 2 mg of a green solid product with a yield of 80%. 3. according to the application of a kind of near-infrared fluorescence imaging probe of targeting GnRH receptor as claimed in claim 1, utilize human tumor cell line and nude mouse peritoneal implantation tumor model to detect the binding ability of probe and tumor cell, target probe GnRHa-ICG can target and recognize tumor cells and tumor foci that highly express GnRH receptors in vivo and in vitro.