CN110075322B - Near-infrared fluorescence imaging probe targeting GnRH receptor and preparation method and application thereof - Google Patents

Abstract

The invention provides a GnRH receptor targeted near-infrared fluorescence imaging probe GnRHa-ICG and a preparation method and application thereof. The near-infrared fluorescence imaging probe GnRHA-ICG targeting GnRH receptor consists of GnRH polypeptide (sequence D-2-Nal-D-4-Cl-Phe-D-3-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH)₂) And the activated near-infrared fluorescent dye ICG-NHS. Based on the principle of specific combination of GnRH polypeptide and GnRH receptor, the invention utilizes the advantages of high expression of GnRH receptor in reproductive system tumors such as ovarian cancer and the like, deeper penetration depth of near-infrared fluorescent dye ICG and weaker autofluorescence of background tissues, can identify tumor cells and tumor foci of the high-expression GnRH receptor in a targeted manner, and has good application prospect in fluorescence imaging and fluorescence guided surgery.

Description

Near-infrared fluorescence imaging probe targeting GnRH receptor and preparation method and application thereof

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 tumor.

Background

Malignant tumors are an important threat to human health. Over 1800 million new cancer cases and 960 ten thousand cancer death cases are expected to occur globally in 2018. Among reproductive system-related malignancies, prostate cancer accounts for 13.5% of new cases of tumor in men, and breast, uterine and ovarian cancers account for 24.2%, 4.4% and 3.4% of new cases, respectively, in women.

Surgery is an important treatment mode for solid tumors, and the thoroughness of surgery is closely related to prognosis. Accurate identification of lesions is critical in the art to achieve complete resection. The existing imaging means such as ultrasound, CT, MRI and the like are tumor non-specific imaging, and real-time imaging is difficult to provide in operation. Fluorescence-guided surgery uses a tumor-targeted fluorescent probe to guide lesion excision through intraoperative real-time fluorescence imaging, and is helpful for improving the thoroughness of surgery and further improving prognosis. The key point for realizing the intraoperative imaging is to develop a tumor-specific fluorescent probe, and the probe mostly consists of a targeting group and an imaging probe.

Malignant tumors of the reproductive system are closely related to hormones related to the hypothalamic-pituitary-gonadal axis, such as Gonadotropin-releasing hormone (GnRH). Under physiological conditions, the distribution of GnRH receptors is relatively restricted to the pituitary and reproductive systems, including ovary, endometrium, placenta, breast, prostate, and the like. Studies have demonstrated high expression of GnRH receptors in reproductive-related malignancies, with 86% expression of prostate cancer, 80% of ovarian and endometrial cancers, and 50% of breast cancer. Tumor targeted therapies targeting GnRH receptors have made serial advances, and therefore GnRH receptors may also be targets for tumor-specific imaging.

Fragments of GnRH polypeptides that specifically bind to GnRH receptors can be used as targeting groups for imaging probes. There are a number of agonists or antagonists of the GnRH receptor currently in clinical use, structurally similar to the native GnRH polypeptide. The small molecular polypeptide is a common targeting strategy, has more applications in the field of tumor targeted therapy, and the research on tumor targeted imaging is gradually increased. Compared with monoclonal antibody, the small molecular polypeptide has the advantages of weak immunogenicity, fast in-vivo distribution, strong penetrability, easy synthesis and modification and the like. Tumor-targeted imaging probes based in part on polypeptides have entered clinical trials, for example, probe BLZ-100 is formed by coupling the polypeptide Chlorotoxin with Indocyanine green (ICG), and probe GE-137 is formed by coupling the cMET-targeted polypeptide with Cy 5.

The near-infrared fluorescent dye has the advantages of deeper penetration depth and weaker autofluorescence of background tissues, and is more suitable for in-vivo imaging. ICG is a near infrared fluorescent dye approved by FDA for clinical use in the united states for angiography, tumor sentinel lymph node imaging, liver function testing, and the like. The literature reports that ICG can also accumulate in tumor tissue due to increased vascular permeability in tumor tissue and impaired lymphatic return. However, ICG does not specifically bind to tumor cells and has a false positive rate as high as 62% when used directly for ovarian cancer lesion imaging.

Therefore, it is necessary to increase the specific imaging ability of the near-infrared fluorescent dye ICG based on its imaging advantages. The invention couples GnRH polypeptide capable of specifically binding GnRH receptors with near-infrared fluorescent dye ICG to prepare a near-infrared fluorescent imaging probe targeting the GnRH receptors. The probe can identify tumor cells and tumor foci of a high-expression GnRH receptor in a targeted manner in vivo and in vitro, and has good application prospects in fluorescence imaging and fluorescence guided surgery.

Disclosure of Invention

The invention provides a near-infrared fluorescence imaging probe of a targeted GnRH receptor and a preparation method and application thereof, wherein the GnRHa-ICG of the targeted GnRH receptor is formed by coupling GnRH polypeptide (GnRHa) and activated near-infrared fluorescent dye ICG-NHS, and the structural formula of the GnRHa-ICG is as follows:

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

the GnRH polypeptide only selects an amino acid sequence of a clinical medicine GnRH receptor antagonist Cetrorelix without a modification group of the medicine, and the sequence formula is D-2-Nal-D-4-Cl-Phe-D-3-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH₂The structural formula is as follows:

a preparation method of a near-infrared fluorescence imaging probe targeting a GnRH receptor comprises the following steps:

(1) synthesizing the polypeptide GnRHa by adopting a solid phase carrier;

(2) adding 3mg of the polypeptide GnRHA obtained in the step (1), 20 mu L N, N-diisopropylethylamine and 0.5 mL of ultra-dry DMF into a 5 mL reaction bottle, and reacting for 10min under the protection of nitrogen; adding 1 mg ICG-NHS into the DMF reaction solution, and continuously stirring at room temperature for reaction for 12 h;

(3) settling the reaction solution obtained in the step (2) by using 5 mL of diethyl ether to obtain a green solid;

(4) and (4) dissolving the solid obtained in the step (3) with a small amount of methanol, and separating, purifying and freeze-drying by using a high performance liquid chromatography to obtain a green solid product 2 mg with the yield of 80%.

The near-infrared fluorescence imaging probe of the target GnRH receptor is applied to: the combination ability of the probe and the tumor cells is detected by utilizing a human tumor cell line and a nude mouse abdominal cavity tumor implantation model, and the target probe GnRHa-ICG can identify the tumor cells and tumor foci of the high-expression GnRH receptor in a targeted manner in vitro and in vivo.

The binding capacity of the probe and the tumor cells is detected through a human tumor cell line, and the result shows that the binding capacity of the target probe GnRha-ICG is stronger than that of the ICG; the in-vivo imaging ability of the probe is detected by a nude mouse abdominal cavity tumor implantation model of the human ovarian cancer cell line, and the result shows that the target probe GnRha-ICG can be specifically combined with an abdominal cavity tumor implantation area, and the effect is superior to that of ICG.

The invention has the advantages that:

1. the invention utilizes the high expression of GnRH receptor in reproductive system tumors such as ovarian cancer and the like, and targets and identifies tumor cells based on the principle of specific combination of GnRH polypeptide and GnRH receptor.

2. The invention has good application prospect in fluorescence imaging and fluorescence guided surgery by utilizing the advantages of deeper penetration depth of near-infrared fluorescent dye ICG and weaker autofluorescence of background tissues.

Drawings

FIG. 1 Synthesis and HPLC characterization of the GnRHA-ICG probe;

FIG. 2 UV absorption and fluorescence spectra of GnRHA-ICG and ICG;

FIG. 3 GnRHa-ICG and ICG binding to tumor cells;

FIG. 4 visualization of GnRHA-ICG and ICG in the intraperitoneal hybridoma model in nude mice.

Detailed Description

The invention provides a novel near-infrared fluorescence imaging probe GnRHa-ICG of a targeted GnRH receptor, which is formed by coupling GnRH polypeptide (GnRHa) and near-infrared fluorescence dye ICG. The preparation method comprises the following steps:

(1) synthesizing polypeptide GnRHA with solid phase carrier and sequence D-2-Nal-D-4-Cl-Phe-D-3-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH₂；

(2) Adding 3mg of the polypeptide GnRHA obtained in the step (1), 20 mu L N, N-diisopropylethylamine and 0.5 mL of ultra-dry DMF into a 5 mL reaction bottle, and reacting for 10min under the protection of nitrogen; adding 1 mg ICG-NHS into the DMF reaction solution, and continuously stirring at room temperature for reaction for 12 h;

(3) settling the reaction solution obtained in the step (2) by using 5 mL of diethyl ether to obtain a green solid;

(4) and (4) dissolving the solid obtained in the step (3) with a small amount of methanol, and separating, purifying and freeze-drying by using a high performance liquid chromatography to obtain a green solid product 2 mg with the yield of 80%.

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

GnRHA-ICG and ICG solutions (DMSO is a solvent) with the concentration of 2.5 mu M are prepared, the ultraviolet absorption spectrum of the probe is detected by using a spectrophotometer, and the fluorescence spectrum of the probe is detected by using a fluorescence spectrometer. The results are shown in FIG. 2.

Examples

The specific implementation mode of the near-infrared fluorescent probe in the tumor targeted imaging application is as follows:

the human ovarian carcinoma cell line a2780 (high-expression GnRH receptor) and the human lung carcinoma cell line H1299 (low-expression GnRH receptor) in the logarithmic growth phase were seeded on chamber slides and the experiment was started until the cell confluence reached 50%. The culture medium was aspirated and washed 1 time with PBS buffer. 60 μ M GnRHA-ICG and ICG were added separately and incubated with tumor cells at 37 ℃ for 30 min. The supernatant was aspirated and washed 3 times with PBS. 4% paraformaldehyde was added and fixed at room temperature for 10 min. The fixative was aspirated and washed 1 time with PBS. Cell membrane dye WGA488 was added and incubated for 10min at room temperature. The membrane dye was blotted and washed 3 times with PBS. The fluorescence intensity was observed by confocal microscopy using a fluorescent mounting medium containing DAPI.

The human ovarian cancer cell line A2780 in the logarithmic growth phase is inoculated into a 12-well plate, and the experiment is started when the cell confluency reaches 60%. The culture medium was aspirated and washed 1 time with PBS. 5 μ M GnRHA-ICG and ICG were added separately and incubated with tumor cells at 37 ℃ for 30 min. The supernatant was aspirated and washed 3 times with PBS. Pancreatin digestion is carried out to prepare cell suspension, and the fluorescence intensity is detected by a flow cytometer.

The above detection results are shown in FIG. 3. The GnRHa-ICG and ICG probes fluoresce red, the cell membrane green, and the nucleus blue. The fluorescence intensity of the target probe GnRHa-ICG is higher than that of ICG, and the fluorescence intensity of the A2780 cell line with high GnRH receptor expression is higher than that of the H1299 cell line with low receptor expression (FIG. 3A). The flow cytometry results were consistent with the fluorescence microscopy observations, with the average fluorescence intensity of GnRHa-ICG being higher than that of ICG (FIG. 3B). The result shows that the target probe GnRha-ICG has stronger binding force to tumor cells expressing GnRH receptors than ICG.

Digesting human ovarian cancer cell line A2780 to prepare single cell suspension, and adding each single cell suspension at a ratio of 1x10⁷100ul was inoculated into the abdominal cavity of female nude mice. The tumor cells are sown and grown in the abdominal cavity, and after 2 weeks, the number of the planting foci can exceed 10, and the size is about 0.1cm-1 cm.

The experiment group tumor-bearing mice were injected with 0.03mg (1.5 mg/kg) of the targeting probe GnRHA-ICG in the peritoneal cavity, and the control group tumor-bearing mice were injected with 0.03mg (1.5 mg/kg) of ICG in the peritoneal cavity. The mice were sacrificed 2h after the injection of the imaging probe, and the abdominal cavity was opened for near-infrared fluorescence in vivo imaging detection (excitation wavelength 780nm, emission wavelength 845 nm). Major organs and tumor tissues were then removed for in vitro fluorescence imaging detection. Tumor, liver (positive control) and skeletal muscle (negative control) cryosections of experimental mice were taken, encapsulated with fluorescent encapsulating tablets containing DAPI, and fluorescence intensity was observed with a confocal microscope.

The imaging results are shown in figure 4. The probe GnRHa-ICG of the experimental group has obvious signals and no signals in the intestinal tract in the abdominal cavity implantation range (figure 4A), and the observation of a frozen section confocal microscope is consistent with the result of in vivo imaging (figure 4B); the ICG probe in the control group showed strong signal in the digestive tract, and could not distinguish tumor foci (FIG. 4C). By comparing the imaging results of the target probes GnRha-ICG and ICG, the targeting probes are prompted to have higher selectivity on in-vivo tumor lesions, can obviously weaken the nonspecific imaging of the ICG in digestive systems such as intestinal tracts and the like, and have better in-vivo imaging effect on tumor tissues.

Claims (3)

1. The near-infrared fluorescence imaging probe for the targeted GnRH receptor is characterized in that the GnRHa-ICG is formed by coupling GnRH polypeptide GnRHa and activated near-infrared fluorescence dye ICG-NHS, and the structural formula of the GnRHa-ICG is as follows:

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

the GnRH polypeptide only selects an amino acid sequence of a clinical medicine GnRH receptor antagonist Cetrorelix without a modifying group of the medicine, and the GnRH polypeptide has the following structural formula:

。

2. a method of making a GnRH receptor targeted near infrared fluorescence imaging probe of claim 1, comprising the steps of:

(1) synthesizing the polypeptide GnRHa by adopting a solid phase carrier;

(2) adding 3mg of the polypeptide GnRHA obtained in the step (1), 20 mu L N, N-diisopropylethylamine and 0.5 mL of ultra-dry DMF into a 5 mL reaction bottle, and reacting for 10min under the protection of nitrogen; adding 1 mg ICG-NHS into the DMF reaction solution, and continuously stirring at room temperature for reaction for 12 h;

(3) settling the reaction solution obtained in the step (2) by using 5 mL of diethyl ether to obtain a green solid;

(4) and (4) dissolving the solid obtained in the step (3) with a small amount of methanol, and separating, purifying and freeze-drying by using a high performance liquid chromatography to obtain a green solid product 2 mg with the yield of 80%.

3. Use of the near-infrared fluorescence imaging probe targeting a GnRH receptor according to claim 1 in the preparation of a near-infrared fluorescence imaging drug.

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