TGM2 specific binding near-infrared probe and application thereof

Abstract

The invention discloses a near-infrared probe specifically bound with TGM2 and application of the near-infrared probe, the probe comprises a polypeptide compound, the polypeptide compound comprises a polypeptide sequence, the polypeptide sequence is CGNSNPKSC, a PEG5-CH2CH2COOH group is connected to an amino group at an N terminal of cysteine of the polypeptide sequence, the PEG5-CH2CH2COOH group is connected to a Phe-OH group, and the Phe-OH group is connected to an amino group at an N terminal of cysteine of the polypeptide sequence. The Phe-OH group is connected with 4-p-methylphenyl butyric acid, and the lysine of the polypeptide sequence is connected with a near-infrared dye ICG. According to the invention, the optimized polypeptide is reacted and combined with the near-infrared dye ICG to form the fluorescent probe capable of specifically targeting gastric cancer, and the fluorescent probe has good affinity and targeting property and is high in combination efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a preparation method and application of a near-infrared probe specifically binding to TGM2. Background Art

[0002] The inventors previously successfully screened a cyclic peptide GX1 (CGNSNPKSC) that specifically targets gastric cancer microvessels of TGM2, but there is a problem of low binding efficiency when using it to develop a near-infrared probe specifically binding to TGM2. Summary of the Invention

[0003] Aiming at the defects or deficiencies of the prior art, the present invention provides a near-infrared probe specifically binding to TGM2.

[0004] To this end, the near-infrared probe specifically binding to TGM2 provided by the present invention includes a polypeptide compound, the polypeptide compound includes a polypeptide sequence, the polypeptide sequence is CGNSNPKSC, a PEG5-CH2CH2COOH group is connected to the amino group at the N-terminus of cysteine of the polypeptide sequence, a Phe-OH group is connected to the PEG5-CH2CH2COOH group, a 4-p-tolylbutyric acid is connected to the Phe-OH group, and a near-infrared dye ICG is connected to the lysine of the polypeptide sequence.

[0005] An optional solution is that the structure of the near-infrared probe specifically binding to TGM2 is as shown in Formula I:

[0006]

[0007] The present invention also provides a preparation method of the above-mentioned near-infrared probe specifically binding to TGM2, and the method steps include:

[0008] S1, preparing a polypeptide compound;

[0009] S2, dissolving the polypeptide compound, EDC and NHS in an organic solvent, reacting at room temperature and collecting the product, reacting the product with NH2-Reactive ICG at room temperature, and then obtaining the near-infrared probe specifically binding to TGM2 through dialysis and freeze-drying.

[0010] In addition, the present invention also protects the application of the above-mentioned near-infrared probe specifically binding to TGM2 in the preparation of reagents, reagent kits or chips for gastric cancer diagnosis and prognosis evaluation.

[0011] Based on the reaction and combination of the optimized polypeptide and the near-infrared dye ICG, the present invention forms a fluorescent probe that can specifically target gastric cancer, has good affinity and targeting ability, and high binding efficiency.

[0012] The binding efficiency is defined as follows: After the probe is injected into mice via the tail vein, the binding efficiency of the relevant probe is evaluated according to the tumor-to-background contrast at 2 h and 8 h. The higher the contrast, the higher the binding efficiency of the probe. Binding Figure 5 and 6 As shown, the tumor-to-background contrast of the probe of the present invention can reach more than 5 at 2 h and more than 15 at 8 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a structural diagram of the GX1-P5-ICG probe of the present invention.

[0014] Figure 2 It is a mass spectrum diagram of the GX1-P5-ICG probe (P5-ICG probe) of the present invention and each control probe in the examples.

[0015] Figure 3 It is an immunofluorescence verification diagram of cell-specific binding of the probe of the present invention to the co-culture system of highly expressed TGM2 gastric cancer cells and vascular endothelial cells; (a) Immunoblot screening diagram of the co-cultured tumor-endothelial cell line with highly expressed TGM2. This diagram shows that among the gastric cancer cell lines (MKN45 cells, AGS cells, SGC7901 cells, MKN28 cells, HGC27 cells, BGC28 cells) co-cultured with HUVEC cells, the co-cultured cell line of MKN45 and HUVEC has the highest TGM2 expression level. Therefore, this co-cultured cell line is selected for subsequent verification; (b) Immunofluorescence binding diagrams of P5-ICG, GX1-ICG probe, URP-P5-ICG probe, and URP-ICG probe with Co-HUVEC (co-cultured with MKN 45). This diagram shows that compared with other control groups, the P5-ICG probe has better affinity for gastric cancer MKN45 cells and tumor vascular endothelial cells, and very low affinity for normal vascular endothelial cells, which is highly consistent with the expression of TGM2; (c) Quantitative diagram of the average fluorescence intensity of Co-HUVEC in Figure (b). This diagram shows that P5-ICG is highly taken up by the tumor-endothelial cell line within 1 h and has a high binding efficiency; (d) Quantitative diagram of the average fluorescence intensity of -HUVEC in Figure (b). This diagram shows the high specificity of P5-ICG for tumor-endothelial cells.

[0016] Figure 4 It is a flow cytometry verification of cell-specific binding of the probe of the present invention to the co-culture system of highly expressed TGM2 gastric cancer cells and vascular endothelial cells; This diagram shows that P5-ICG has better binding efficiency for tumor vascular endothelial cells.

[0017] Figure 5Verification diagram of the specific binding ability of the probe of the present invention to nude mice bearing orthotopic gastric cancer; (a) IVIS system imaging diagrams of tumor-bearing mice at 0-24 h after injecting 20 nmol of P5-ICG, GX1-ICG, URP-P5-ICG, and URP-ICG probes respectively. This diagram shows that compared with other near-infrared probes, the probe can accumulate in the tumor within 2 h for P5-ICG, the highest contrast is achieved at 8 h, and it can remain in the tumor for 24 hours; (b) Quantitative diagram of tumor / background signal values of the four groups of probes at 0-24 h. This diagram shows that compared with other groups, the tumor-to-background contrast of P5-ICG reaches 8 at 2 h and 18 at 8 h, and the binding efficiency to the tumor is the highest; (c) IVIS system imaging of various organs of tumor-bearing mice at 8 h. This diagram shows that compared with other groups, the contrast between the tumor and the kidney is the highest for P5-ICG, with excellent high contrast; (d) Quantitative diagram of tumor / background signal values in diagram (c). This diagram shows that compared with other groups, the contrast between the tumor and the kidney is 5 for P5-ICG.

[0018] Figure 6 Comparison diagram of the probe of the present invention with another gastric cancer-targeting cyclic peptide GEBP11 labeled with ICG and ICG; (a) Immunofluorescence intensity comparison diagram of binding to co-cultured cells of highly expressed TGM2 gastric cancer cells and vascular endothelial cells, verification diagram of the specific binding ability of orthotopic gastric cancer-bearing nude mice. This diagram shows that compared with other groups, the tumor-to-background contrast of P5-ICG is the highest at 8 h; (b) Comparison diagram of the specific binding ability to orthotopic gastric cancer-bearing nude mice within 8 h. This diagram shows that compared with other groups, the binding efficiency of P5-ICG to the tumor is the highest.

[0019] Figure 7 Verification diagram of the specific binding ability of the probe of the present invention to clinical gastric cancer tissue specimens by immunofluorescence technique; (a) Immunofluorescence diagrams of P5-ICG, GX1-ICG, URP-P5-ICG, and URP-ICG binding to tumor and adjacent tissues. This diagram shows that in human gastric cancer specimens, within 1 h, the binding site of P5-ICG is consistent with TGM2, with excellent tumor binding efficiency, and it also confirms the value of P5-ICG as a targeting probe for clinical auxiliary diagnosis of tumors; (b) Quantitative diagram of the average fluorescence intensity in (a). This diagram shows that within 1 h, P5-ICG highly accumulates at the site of TGM2 expression in the tumor, with high binding efficiency.

[0020] Figure 8 Diagram of precise resection of gastric cancer tissue in mice guided by the probe of the present invention under an optical imaging system. (a) Diagram (a) shows the process of guiding the resection of gastric cancer tissue in mice by the IVIS system. (b) The left and right diagrams respectively show the HE verification of the tumor tissues resected for the first and second times. (c) The left and right diagrams respectively show the TGM2 immunofluorescence verification and GX1-P5 binding site verification of the tumor tissues resected for the first and second times.

[0021] Figure 9 It is a schematic structural diagram of the control substance in the examples. Detailed implementation manners

[0022] Unless otherwise specified, scientific and technical terms in this article are understood according to the knowledge of those of ordinary skill in the relevant fields. The polypeptide compounds described in this article are the compounds disclosed in CN2024102202533. The GX1-P5 polypeptide compounds used in the following examples were prepared by the method disclosed in CN2024102202533.

[0023] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and examples. The raw materials, cells and other experimental materials used in the following examples are all commercially available products.

[0024] Example:

[0025] Take 10 mg of the GX1-P5 polypeptide compound and dissolve it in 1 ml of dichloromethane. Add EDC (0.4 mg) and NHS (0.6 mg), and react at room temperature for 5 hours; after the reaction is completed, use the chloroform precipitation method for purification. Dissolve the purified compound and NH2-Reactive ICG (10 mg, purchased from Xi'an Ruixi Biotechnology Co., Ltd.) in a phosphate standard buffer solution with a pH of 9.18 to form a suspension, and react at room temperature for 24 hours. After the reaction is completed, desalt by dialysis and freeze-dry at -50 °C to obtain the dark green solid of the probe GX1-P5-ICG of the present invention.

[0026] Further research was carried out on the probe GX1-P5-ICG, GX1-ICG probe, URP-P5-ICG probe, URP-ICG probe, and GEBP11-ICG probe of the present invention in Example 1 as follows:

[0027] Binding Figure 9 As shown, where: the structure of the GX1-ICG probe is that ICG is linked at the lysine of the polypeptide sequence CGNSNPKSC. The structure of the URP-P5-ICG probe is that ICG is linked at the lysine of the polypeptide sequence C11H13O2-Phe-OH-PEG5-CH2CH2CO-CNKSPSGNC. The structure of the URP-ICG probe is that ICG is linked at the lysine of the polypeptide sequence CNKSPSGNC. The structure of the GEBP11-ICG probe is that ICG is linked at the cysteine of the polypeptide sequence CTKNSYLMC, and the method of labeling ICG for the GEBP11-ICG is the same as the method in Example 1. The above Example 1 and each control probe were commissioned to be synthesized by Suzhou Jingguan Medical Co., Ltd., and the mass spectra of each probe are as Figure 2 shown.

[0028] Verification of the binding efficiency of GX1-P5-ICG to tumor-endothelial cell lines:

[0029] (1) Screening of co-cultured tumor-endothelial cell lines with high expression of TGM2:

[0030] Gastric cancer cell lines (MKN45 cells, AGS cells, SGC7901 cells, MKN28 cells, HGC27 cells, BGC28 cells) and HUVECs were routinely cultured. The above gastric cancer cell lines were cultured in the logarithmic growth phase, the original medium was discarded, and after washing, the special medium for endothelial cells without LSGS was added, and the cells were continuously cultured routinely for 24 h. The media in the culture flasks of MKN45 cells, AGS cells, SGC7901 cells, MKN28 cells, HGC27 cells, and BGC28 cells were collected, centrifuged, the supernatant was filtered, and mixed with the freshly prepared special medium for endothelial cells at a ratio of 1:4 and added to HUVEC cells for co-culture for 72 h to obtain 6 co-cultured tumor-endothelial cells Co-HUVECs.

[0031] For the 6 co-cultured gastric cancer tumor-endothelial cells Co-HUVECs collected, 100 μL of lysis buffer was added per 1×106 cells, and the cells were lysed on ice for 30 min. The expression level of TGM2 in the 6 co-cultured tumor-endothelial cells Co-HUVECs was detected by Western blot. The results are as Figure 3 shown in

[0032] Figure 3 a. The results in a show that the cell line co-cultured with MKN45 and HUVEC has the highest expression level of TGM2. Therefore, this co-cultured cell line was selected for verification in the subsequent experiments.

[0033] (2) Detection of the binding efficiency of the immunofluorescence probe to Co-HUVECs (MKN45)

[0034] MKN45, HUVEC, and Co-HUVECs (MKN45) in the logarithmic growth phase were taken and washed 3 times with sterile PBS; to prevent specific binding, 10% BSA was used to incubate with the cells. After washing with PBS, GX1-P5-ICG, GX1-ICG probe, URP-P5-ICG probe, and URP-ICG probe with a concentration of 5 nmol / ml were added, and the cells were incubated at 37 °C for 1 h. After washing 3 times with PBS, the cells were fixed with pre-cooled acetone at 4 °C for 15 min, and the binding effect was observed under a confocal microscope. The fluorescence binding effect can be seen in Figure 3 b.

[0035] Figure 3b shows the binding imaging effects of the fluorescent probes P5-ICG, GX1-ICG, URP-P5-ICG, and URP-ICG with MKN4, HUVEC, and Co-HUVEC5 cells captured by a near-infrared confocal microscope. The results of this figure show that P5-ICG has better affinity for Co-HUVECs (MKN45), is highly consistent with the expression of TGM2, is highly taken up by tumor-endothelial cell lines within 1 h, and has a high binding efficiency.

[0036] (3) Flow cytometry was used to detect the binding efficiency of the probe to Co-HUVECs (MKN45).

[0037] MKN45, HUVEC, and Co-HUVECs (MKN45) in the logarithmic growth phase were digested with type I collagenase to prepare single-cell suspensions; the supernatant was discarded by centrifugation; 2 ml of pre-cooled PBS solution containing 0.5-1% BSA was added to the centrifuge tubes respectively, and the cells were incubated on ice for 1 h for blocking, the supernatant was discarded by centrifugation, and fluorescent probes P5-ICG, GX1-ICG, URP-P5-ICG, and URP-ICG with a concentration of 5 nmol / ml were added. The cell suspensions were pipetted and mixed evenly, and incubated on ice for 1 h; the supernatant was removed by centrifugation, and after resuspension with PBS, the binding ability of the fluorescent probe was detected by flow cytometry, and the results are shown in Figure 4.

[0038] Figure 4 Confirmed by flow cytometry results, compared with GX1-ICG, P5-ICG has better affinity for gastric cancer MKN45 cells and tumor vascular endothelial cells, and has very low affinity for normal vascular endothelial cells, with better targeting.

[0039] Verification of the binding efficiency of GX1-P5-ICG to gastric cancer:

[0040] The concentration of gastric cancer MKN45 cells was adjusted to 1.0×10 6 cells / mL, 200 μL per nude mouse, and injected subcutaneously into the gastric mucosa; after 4 weeks, the formation of tumors was observed, the tumor size was measured, and according to the tumor size, they were randomly divided into 4 groups, with 6 mice in each group; 4 groups of nude mice were injected via the tail vein with 20 nmol of near-infrared fluorescent probes GX1-P5-ICG (also referred to as P5-ICG in this article), GX1-ICG, URP-P5-ICG, and URP-ICG, and near-infrared imaging was performed at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h respectively; the nude mice were sacrificed at 8 h and 24 h, and the heart, liver, spleen, lung, kidney, stomach, intestine, muscle, and tumor tissue organs were taken to detect the fluorescence signal distribution in each organ tissue, and the results are shown in Figure 5 Figure.

[0041] Figure 5This is a graph showing the affinity and targeting ability of the P5-ICG near-infrared probe for mouse in situ cancer. The results of this graph show that in the imaging of the mouse gastric cancer model, compared with other control groups, and compared with other near-infrared probes, the probe can accumulate in the tumor within 2 hours, with the highest contrast at 8 hours, and can remain in the tumor for 24 hours. It has good affinity and targeting ability for mouse gastric cancer in situ, and the optimal imaging time in the mouse body is 8 hours after tail vein injection. For GX1-ICG, the tumor / background ratio is 3 at 2 hours and the contrast is 11 at 8 hours; for URP-P5-ICG and URP-ICG, the tumor / background ratio is 1 within 2 hours and they fail to bind to gastric cancer.

[0042] Strengthen the verification of the binding rate and targeting affinity of GX1-P5 to gastric cancer:

[0043] Take Co-HUVECs (MKN45) in the logarithmic growth phase and wash them 3 times with sterile PBS; to prevent specific binding, incubate the cells with 10% BSA, and after washing with PBS, add P5-ICG, GX1-ICG, URP-P5-ICG, URP-ICG, GEBP11-ICG (another gastric cancer targeting cyclic peptide), and ICG at a concentration of 5 nmol / ml, incubate at 37 °C for 1 hour, wash 3 times with PBS, then fix the cells with pre-cooled acetone at 4 °C for 15 min, and observe the binding effect under a confocal microscope. The fluorescence binding effect can be seen Figure 6 (a).

[0044] Figure 6 (a) shows the binding imaging effects of the near-infrared fluorescent probes P5-ICG, GX1-ICG, URP-P5-ICG, URP-ICG, GEBP11-ICG, and ICG to Co-HUVECs captured by a near-infrared confocal microscope. The results of this graph show that in the in vitro cell immunofluorescence experiment, it is confirmed that compared with other control groups, the probe of the present invention has a higher binding efficiency to gastric cancer MKN45 cells and tumor vascular endothelial cells within 1 hour, and has better affinity and targeting ability.

[0045] Adjust the concentration of gastric cancer MKN45 cells to 1.0×106 cells / mL, inject 200 μL per nude mouse subcutaneously into the gastric mucosa; observe the formation of tumors after 4 weeks, measure the tumor size, and randomly divide them into 4 groups according to the tumor size, with 6 mice in each group. Inject 20 nmol of the near-infrared fluorescent probes P5-ICG, GX1-ICG, URP-P5-ICG, URP-ICG, GEBP11-ICG (another gastric cancer targeting cyclic peptide), and 20 nmol of ICG into the tail veins of the 4 groups of nude mice respectively, and perform near-infrared imaging at 8 hours. The results are as shown in Figure 6 (b).

[0046] Figure 6(b) The binding imaging effects of fluorescence probes P5-ICG, GX1-ICG, URP-P5-ICG, URP-ICG, GEBP11-ICG, ICG and the orthotopic carcinoma mouse model were captured by the IVIS system. The results of this figure show that: in the orthotopic gastric cancer mouse model, compared with other control groups, the tumor-to-background contrast is the highest at 8 h, the binding efficiency to the tumor is the highest, less probes are metabolized by the liver and kidney, and it has high clinical application value.

[0047] Verify the value of the probe for clinical gastric cancer diagnosis

[0048] (1) Clinical pathological data of human ex vivo tissues:

[0049] Fresh gastric cancer tissues and adjacent tissues from 10 patients who underwent gastric cancer resection surgery at Xijing Digestive Disease Hospital from October to December 2024 were collected. The patients were diagnosed with gastric cancer by gastroscopy plus biopsy pathology before surgery and did not receive any treatment before surgery. The gastric cancer tissues of 10 cases were confirmed as gastric cancer tissues by the pathology department, and the adjacent tissues were gastric mucosal tissues more than 5 cm away from the cancer. This part of the experimental operation was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University.

[0050] (2) Fluorescence imaging of human ex vivo tissues:

[0051] After the tissue samples were frozen, embedded in OCT and sectioned at a thickness of 4 μm, they were first fixed with formalin to maintain the tissue morphology, and then treated with Triton X-100 for cell permeabilization to facilitate the penetration of subsequent antibodies and fluorescence probes; subsequently, non-specific binding was reduced by BSA blocking, and a mixture of rabbit anti-human TGM2 antibody and different fluorescence-labeled probes (including P5-ICG, FITC-GX1, URP-P5-ICG and URP-ICG, with a concentration of 5 nmol / ml) was incubated overnight at 4°C. The next day, incubation was carried out with an AF488-labeled secondary antibody, and unbound antibodies and probes were washed with PBST; subsequently, the sections were mounted with a mounting medium containing DAPI to protect the sections and enhance the stability of the fluorescence signal. Fluorescence images of each sample were collected using a confocal microscope, and the average fluorescence intensity in 3 20×20 μm 2 regions in each image was measured using Image J software.

[0052] The results are as Figure 7 shown. The results of this figure show that: in human gastric cancer specimens, P5-ICG is consistent with the binding site of TGM2, has excellent tumor targeting, and also confirms the value of P5-ICG as a targeting probe for clinical auxiliary diagnosis of tumors.

[0053] Optical imaging combined with GX1-P5-ICG-guided resection of orthotopic gastric tumors in mice

[0054] The gastric submucosa of 4-week-old nude mice was injected with gastric cancer MKN45 cells at a cell concentration of 1.0×10 6 cells / mL, at a dose of 200 μL per mouse. After 4 weeks of feeding, 3 nude mice successfully developed tumors. Subsequently, 20 nmol of the GX1-P5-ICG near-infrared probe was injected into the 3 tumor-bearing nude mice via the tail vein. After 8 hours, imaging was performed using the IVIS system, and the results showed obvious fluorescence in the tumor area, while almost no fluorescence was observed in normal organs.

[0055] Based on this significant fluorescence contrast, the first resection of the tumor was successfully completed with the aid of fluorescence-guided technology. Imaging was performed again after the operation, and residual fluorescence imaging was found at the tumor margin. Therefore, under the guidance of the fluorescence signal, a second resection was performed. After the operation, the fluorescence in the original tumor area completely disappeared, indicating that the tumor had been completely resected (see Figure 8 ).

[0056] The results are as Figure 8 shown. Based on the visualization tumor imaging indication of GX1-P5-ICG, the margin of in situ gastric cancer in mice can be accurately delineated and precise surgical resection of tumor tissue can be achieved.

[0057] The above description is only the preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A near-infrared probe specifically binding to TGM2, comprising a polypeptide compound, wherein the polypeptide compound comprises a polypeptide sequence, wherein the polypeptide sequence is CGNSNPKSC, wherein a PEG5-CH2CH2COOH group is connected to the N-terminal amino group of cysteine ​​in the polypeptide sequence, wherein the PEG5-CH2CH2COOH group is connected to a Phe-OH group, wherein the Phe-OH group is connected to 4-p-tolylbutyric acid, wherein: The lysine of the polypeptide sequence is connected with a near-infrared dye ICG.

2. The TGM2-specific near-infrared probe according to claim 1, characterized in that: The structure of the near-infrared probe specifically binding to TGM2 is shown in Formula I:

3. The method for preparing the TGM2-specific near-infrared probe according to claim 1, characterized in that: The method steps include: S1, preparation of polypeptide compounds; S2, the peptide compound, EDC and NHS are dissolved in an organic solvent, the product is collected after reaction at room temperature, the product is reacted with NH2-Reactive ICG at room temperature, and then dialyzed and freeze-dried to obtain a TGM2-specific near-infrared probe.

4. Use of the TGM2-specific near-infrared probe according to claim 1 or 2 for preparing gastric cancer diagnosis and prognosis evaluation reagents, kits or chips.

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