CN108623661B

CN108623661B - Bispecific polypeptide molecular probe targeting pancreatic cancer tumor cells and application thereof

Info

Publication number: CN108623661B
Application number: CN201810454453.XA
Authority: CN
Inventors: 王倩; 赵心明
Original assignee: Cancer Hospital and Institute of CAMS and PUMC
Current assignee: Cancer Hospital and Institute of CAMS and PUMC
Priority date: 2018-05-14
Filing date: 2018-05-14
Publication date: 2021-04-27
Anticipated expiration: 2038-05-14
Also published as: CN108623661A

Abstract

The invention discloses a bispecific polypeptide molecular probe for targeting pancreatic cancer tumor cells, wherein the structural formula of the molecular probe is shown as the formula (I):

Description

Bispecific polypeptide molecular probe targeting pancreatic cancer tumor cells and application thereof

Technical Field

The invention discloses a polypeptide, and more particularly discloses a bispecific polypeptide.

Background

Pancreatic Cancer (PC) is the most fatal cancer, with a 5-year survival rate of approximately 7%, with one important cause being that the majority (over 85%) of patients are diagnosed with advanced disease with a poor prognosis. Pancreatic Ductal Adenocarcinoma (PDAC) accounts for the vast majority of the types of PC pathologies, progressing from pre-pancreatic lesions to pancreatic tumors. Successful diagnosis and resection of early PDACs, including pancreatic intraepithelial neoplasia (PanIN), can result in 4-year survival rates as high as 78%. Thus, early diagnosis of PDAC may lead to better prognosis and improved patient care.

The basic imaging principle of molecular imaging is to introduce prepared molecular probe into living tissue cell to make the labeled molecular probe interact with target molecule, to detect the information from the molecular probe with proper imaging system, and to generate molecular image or functional metabolic image of living tissue after computer processing. The most important of in vivo molecular imaging is the selection of imaging target and suitable imaging technology, namely high specificity molecular probe and high sensitivity detection technology. Molecular imaging in vivo must satisfy the following conditions: high specificity, high sensitivity, biological barrier permeability, biological signal amplifiability.

In recent years, the advent of molecular imaging has identified a new breakthrough for the early diagnosis of pancreatic cancer, providing an opportunity for early diagnosis of PDACs, even pre-cancerous lesions from individuals without overt symptoms. Furthermore, molecular imaging, combined with current imaging systems and binding affinities, can be directed to disease-specific molecular biomarkers that appear several years before symptoms appear. Therefore, the molecular imaging agent with the PDAC early diagnosis capability, treatment monitoring and precise surgery real-time guidance is very promising clinically. The current technologies for molecular imaging mainly include nuclear medicine imaging (PET, SPECT), Magnetic Resonance Imaging (MRI), optical imaging, and ultrasound imaging.

The selection of a target with high tumor-related property in the application of molecular imaging technology is the key point for ensuring the specificity of diagnosis. The traditional high-expression pancreatic cancer cell membrane surface receptors and cytokines related to pancreatic cancer reported in the past research include Epidermal Growth Factor Receptor (EGFR), urokinase-type plasminogen activator receptor (uPAR), carcinoembryonic antigen cell adhesion molecule (CEACAM), Vascular Endothelial Growth Factor Receptor (VEGFR), pancreatic cancer targets discovered in the latest pancreatic cancer mechanism research, novel biomarker clathrin plectin-1 and the like.

One of the characteristics of PDACs is that they present a dense fibrous matrix, low blood vessels and poorly perfused tumor vessels, lack the enhanced permeability and retention effect (EPR effect) of solid tumors, and increase the interstitial fluid pressure within the tumor, rendering the transport of most nanoparticles ineffective. Recent analysis of the past 10 years literature has shown that the nanoparticle dose delivered into dense PDACs is no more than 0.4%, well below 0.7% (median) for all types of cancer. Based on the excellent biocompatibility of the small-molecule polypeptide and considering the long-term safety and clinical transformation prospect in vivo, the small-molecule polypeptide image tracer agent is probably more suitable for PDAC diagnosis. Among the various imaging modalities, Magnetic Resonance Imaging (MRI) is widely used in stand-alone imaging clinics that can provide high soft tissue contrast. Near infrared fluorescence imaging (NIRF) can show the pharmacokinetics and biodistribution of the whole body in real time, has high sensitivity and high specificity, and the NIRF molecular probe has high specificity in the application of surgical navigation. However, NIRF molecular probes have low spatial resolution and poor tissue penetration.

Plectin1 is a traditional scaffold cytoskeletal protein that is present in almost all mammalian tissues and cell types, particularly in tissues that are resistant to mechanical stress. Under normal physiological conditions, plectin1 is normally expressed only in the cytoplasm and not on the cell membrane. However, if the tissue is cancerous, plectin1 changes in cellular localization and may be present on both the cytoplasm and cell membrane. Plectin1 was found in both murine and human PDAC cells. Plectin1 has attracted considerable interest to developers due to its specific aberrant localization on the surface of pancreatic cancer epithelial cells. In 2008, a section of targeting peptide PTP of which the functional short peptide contains 7 amino acids (KTLLPTP) is screened out by using a gene mouse PDAC model and a phage display library technology, and pancreatic cancer can be specifically targeted. Through proteomics analysis, the targeting peptide PTP realizes targeting effect through specific binding with plectin-1 which is wrongly positioned on the cell membrane surface during pancreatic cancer occurrence.

However, pancreatic cancer is genetically very complex, with tumor mass being caused by 63 genetic changes and abnormalities in the 12 core signaling pathways in patients, with heterogeneity in tumor biomarker expression among different cancer patients. Thus, effective targeting may require the use of a combinatorial approach to attack multiple targets simultaneously.

Integrin is a cell adhesion molecule involved in tumorigenesis and tumor progression and is also a useful target or biomarker for detecting PDAC. Integrin beta 4(ITGB4), one of the integrin families, has been shown to play an indispensable role in PDAC with changes in plectin localization. In addition, plectin and integrin beta 4 directly interact in synergy during PDAC growth, invasion and migration, playing an equally important role. The most common ligand recognition site for most integrins is the RGD short peptide sequence (Arg-Gly-Asp). RGD short peptide mediates the adhesion of integrin and ECM components, and the beta subunit is connected with cytoskeleton through an intracellular segment to form an ECM-integrin-cytoskeleton transmembrane complex. Extracellular signals are transmitted to cytoskeletal proteins through the complex to induce cytoskeletal reconstruction, so that cell deformation and movement are caused, and biological behaviors such as survival, growth, proliferation, differentiation, invasion, migration and the like of cells are influenced.

The invention aims to provide a bispecific small molecular probe aiming at pancreatic cancer tumor cells, which can overcome the defects of the prior art, and further provides the application of the bispecific small molecular probe in the molecular imaging technology. More specifically, the invention aims to accurately discover early-stage pancreatic cancer by using a molecular imaging MRI (magnetic resonance imaging) method and a near-infrared fluorescence bimodal imaging method; and the aim of overcoming the high heterogeneity of pancreatic cancer by the superposition of double-target targeting effects, improving the characteristic of limited targeting capability of a single target and improving the sensitivity of detecting early-stage micro pancreatic cancer is fulfilled so as to achieve the aim of detecting micro pancreatic cancer foci by MRI and fluorescence bimodal detection.

Disclosure of Invention

Based on the above-mentioned objects, the inventors have conducted intensive studies on plectin-1 and integrin beta 4 as synergistic bispecific targets by considering the preferred aspects of conventional pancreatic cancer target proteins, affinity vectors. Based on the research result, the invention firstly provides a bispecific polypeptide molecular probe for targeting pancreatic cancer tumor cells, and the structural formula of the molecular probe is shown as the formula (I):

wherein X, Y is an optional amino acid, X is selected from lysine, cysteine, aspartic acid, Y is selected from phenylalanine or tyrosine, wherein the amino acid residue at position a and/or the amino acid residue at position b marks one imaging molecule for molecular imaging, and the amino acid residue at position c marks another imaging molecule for molecular imaging.

In a preferred embodiment, said X is lysine and said Y is phenylalanine.

In a preferred embodiment, the amino acid residue at the a site and the amino acid residue at the b site are both labeled DOTA @ Gd (III), the amino acid residue at the c site is labeled Cy7, and the structural formula of the molecular probe is shown in the formula (II):

preferably, Cy7 is attached to the polypeptide in a 1:1 molar ratio.

The invention also provides the application of the molecular probe in preparing a tumor diagnosis reagent.

In a preferred embodiment, the probe is prepared as an injection.

Finally, the invention also provides a method for preparing the probe, which comprises the following steps:

(1) synthesizing a main peptide chain Lys-Thr-Leu-Leu-Pro-Thr-Pro-Lys-Lys of the molecular probe;

(2) the Lys at the 8 th position on the main peptide chain is deprotected, and the accessory peptide chain Arg-Gly-Asp-Phe is synthesized by taking the Lys as the starting point;

(3) coupling the peptide chain obtained in the step (2) with Cyanine7 monosuccinate;

(4) conjugating the peptide chain obtained in step (3) with DOTA (tBu) 3-ester;

(5) chelating the peptide chain obtained in the step (4) with a gadolinium compound;

(6) and (5) harvesting the product obtained in the step (5).

In a preferred embodiment, the coupling of step (3) is carried out using a coupling agent of HATU: DIEA (N-tetramethyluronium hexafluorophosphate: N, N-diisopropylethylamine) at a ratio of 3:10 and reacting the peptide chain with Cyanine7 monosuccinate in a weight ratio of 3: 1.

In another preferred embodiment, the conjugation in step (4) is performed by conjugating DOTA-NHS ester protected by three tbus with lysine at

positions

9 and 10 in the main peptide chain deprotected by hydrazine hydrate as one activated carboxyl group.

In yet another preferred embodiment, the gadolinium compound in step (5) is Gd hexahydrate, and the peptide chain is chelated with the gadolinium compound at a weight ratio of 4: 1.

The invention combines PTP and RGD to provide a bispecific polypeptide, which improves the specificity of pancreatic cancer detection. Wherein the PTP targets a protein plectin overexpressed on the surface of pancreatic cancer ductal epithelial cells; RGD as ligand peptide of wide targeting integrin family can target not only integrin 3, which is a protein with high expression of tumor new vessels, but also integrin 4 which is interacted with plectin expression. The bispecific targeting not only improves the specificity of targeting pancreatic cancer (plectin1 and integrin 4) after PTP and RGD are combined, but also improves the sensitivity of detecting malignant tissues by simultaneously targeting pancreatic cancer ductal epithelial cells and neovessels in pancreatic cancer mesenchyme.

The invention utilizes a polypeptide solid phase chemical synthesis method to prepare bispecific targeting peptide (DOTA @ Gd (III)) marked with DOTA @ Gd and Cy7]₂and/Cy 7-PTP/RGD, labeling two peptides (PTP/RGD) as plectin/integrin targets, respectively, for MRI/NIRF dual-mode imaging and surgical navigation. The tumor targeted MRI/fluorescence bimodal imaging purpose is achieved by improving the binding capacity with pancreatic cancer tumor tissues through the bispecific targeted peptide and carrying out high sensitivity, high specificity and real-time NIRF imaging represented by MRI, and the whole tumor localization of mice can be carried out.

More specifically, the invention replaces the original nano material design by a small molecule ring type chelating gadolinium agent (DOTA @ Gd). The design of a macromolecular imaging component is strived to improve the sensitivity of pancreatic cancer MRI molecular imaging.

The invention provides an MRI/NIRF dual-model agent of a bispecific double peptide DOTA @ Gd/Cy7, and evaluates the characteristics, in-vivo in-vitro toxicity and a binding target point on a cell level of a bispecific probe for pancreatic cancer. The high binding affinity of bispecific targeting was demonstrated by confocal imaging techniques and validated for molecular imaging and optical guidance of mouse models of pancreatic cancer. The molecularly imaged dipeptide DOTA @ Gd/Cy7 provided a significant T1 weighted signal in PDAC tumors and long-term cycling. The double peptide DOTA @ Gd/Cy7 is successfully applied to the NIRF image-guided resection of tumor lesions. CFL imaging "histopathology in vivo" and "fluorescent staining in vitro" were used to verify the binding mechanism of the dipeptide to PDAC. Through experiments, the dual-specificity small molecule probe successfully improves the signal intensity of fluorescence and magnetic resonance imaging, and enhances the specificity of PDAC. In conclusion, the double peptide DOTA @ Gd/Cy7 is a promising drug which can directly attack malignant tumors specifically expressing plectin-1 and ITGB4, in particular PDAC.

Drawings

FIG. 1A is a high performance liquid chromatography analysis of bispecific molecular probes;

FIG. 1B is a mass spectrometry diagram of a bispecific molecular probe;

FIG. 2A is a graph of UV absorption spectroscopy of a bispecific molecular probe with free Cy 7;

FIG. 2B is a graph of dual specificity molecular probe fluorescence intensity and UV absorption time;

FIG. 2C is a graph of the fluorescence intensity of bispecific molecular probes plotted against the concentration of free Cy 7;

FIG. 2D is a graph of the change in relaxation rate of T1 after binding of bispecific probe with different concentrations of Gd (III);

FIG. 3 detection of expression levels of human pancreatic cancer cell plectin-1 and ITGB4 by bispecific molecular probe

FIG. 4 is a diagram showing the affinity detection result of FITC-labeled dipeptide for PC cells;

FIG. 5 cellular fluorescence profiles comparing the affinity of di-and mono-peptides for PC cells;

FIG. 6 is a diagram of the targeting distribution mechanism of bispecific molecular probes in PC tumors

FIG. 7 shows the optical metabolic distribution of dual specificity molecular probe in vivo and MRI imaging, Gd quantitative mapping;

FIG. 8 is a graph of surgical navigation and histological evaluation under NIR guidance of a dual-specificity molecular probe

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 preparation example

1. Backbone peptide Synthesis (L1)

The main peptide ligand (L1) was synthesized by standard solid phase peptide synthesis and the fmoc (9-fluorenylmethyloxycarbonyl) -protected amino acid was synthesized using 2-chlorotrifluoroethylene resin (15mL/g, shaker shake for 30min, dissolved in DCM in an isolation column). Fmoc-Lys (Dde) -OH (0.53g,1.0mmol) was dissolved in DMF (13mL), the suspension was stirred over the resin for 60 min, the resin was washed with DCM and further stirred with methanol for 30min to eliminate residual chloride in the resin. The fmoc protected end was eluted with 25% of piperidine three times in DMF. This process is repeated from C-to N-terminus as Fmoc-Lys (Dde) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Pro-OH, Fmoc-Thr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH. The experiments on the resin succeeded in fixing each amino acid. After each reaction cycle, the resin was thoroughly cleaned for subsequent processing.

The amino acid sequence of the synthesized main chain peptide is as follows:

Boc-Lys(Boc)-Thr(tBu)-Leu-Leu-Pro-Thr(tBu)-Pro-Lys(Alloc)-

Lys(Dde)-Lys(Dde)-Resin。

2. side chain peptide Synthesis (L2)

After the main peptide ligand (L1), the main peptide chain, was synthesized, the allyl protection (PPh3) on lys (alloc) at position 8 on L1 was eliminated with palladium (Pd) and triphenylphosphine (PPh3), thereby exposing the binding site. Thereafter, Fmoc standard solid phase polypeptide synthesis was repeated in the following sequence:

Fmoc-arg(Pbf)-OH,Fmoc-gly-OH,Fmoc-asp(OtBu)-OH,Fmoc-Phe-OH

the amino acid sequences of the synthesized main chain and the auxiliary chain are as follows:

Boc-Lys(Boc)-Thr(tBu)-Leu-Leu-Pro-Thr(tBu)-Pro-Lys

【Arg(Pbf)-Gly-Asp(OtBu)-Phe】-Lys(Dde)-Lys(Dde)-Resin

3. coupling of polypeptide ligand to Cyanine7 Monosuccinate (Cy7-NHS) (L3)

Peptide ligands were chemically synthesized using standard solid phase peptides, as described above. Cyanine7 monocytidine acid ester (5mg) was added to the resin under the action of a coupling agent (HATU: DIEA,3:10) and bound to the side chain of the peptide ligand on the resin (15 mg).

4. Synthesis of [ DOTA (tBu) 3-ester ] and L3 conjugate (L4)

[DOTA(tBu)₃Esters of (A) with (B) and (C)]Is prepared by a multi-step synthesis, which begins with a macrocyclic polyamine hydrochloride (cyclen hydrochloride). Three tBu protecting groups were attached to DOTA-NHS ester to keep one activated carboxyl group reacting with hydrazine hydrate-deprotected lysine on L4 ligand (DDE-deprotected Lys).

After synthesis of the L4 ligand, all protecting groups on the peptide complex were removed by reaction with thallium (III) trifluoroacetate (1.03 g, 2.5mmol) at 0 ℃ in DMF (10 ml) for 2h and washed thoroughly with DMF. The peptide complex is cleaved from the resin. The filtered solution was mixed with cold diethyl ether (100 ml). The crude product was isolated by centrifugation and washed to yield L4(16.45mg, 34.27%), characterized by MS [ m/z [ m + H ] +) 2934.56(obsd.), 2933.18 (calcd.).

Chelate preparation of L4 with gadolinium (L5)

Ligand L5(20.4mg,6.4umol) was dissolved in demineralised water (5 ml). 0.25% NH₄OH is added into the water solution to adjust the pH value to 6.4-6.9. Subsequently, 5.0mg of Gd hexahydrate (13.45. mu. mol) was added to 1.0ml of water (the weight ratio of 20.4mg of ligand L5 to 5.0mg of Gd hexahydrate was approximately 4: 1). The mixture was stirred at room temperature overnight. The mixed solution was purified with a dialysis bag (molecular cut, 1000da), then concentrated in the dark and lyophilized. By MS (m/z [ m + H ]]+):3676.97(obsd.)，3676.58(calcd.)]A crude product (13.94mg, 28.46%) was obtained. Finally, blue freeze-dried powder (22.18mg, yield 95.3%) is used to obtain the high-purity double-target probe.

6. In vitro characterization

The dipeptide DOTA @ Gd/Cy7(abbr, [ DOTA @ Gd (III) ]2/Cy7-ptp/RGD) was synthesized by solid phase reaction. Both analytical high performance liquid chromatography (FIG. 1A) and mass spectrometry (FIG. 1B) confirmed their successful synthesis. The synthesized product without chelating Gd (III) is synthesized for the first time, the molecular weight is confirmed to be 2933.18, and the purity is 99.33%. Then, Gd (III) was chelated to DOTA in a molar ratio of 1: 3. The final product after conjugation with gd (iii) was 3676.94 molecular weight, and the purity of the synthesized material was 95.38%. In addition, DOTA @ gd (iii) was also synthesized by a similar method.

The absorption spectra of the bispecific molecular probe and free Cy7 were analyzed using an ultraviolet-visible spectrophotometer (UV-2450, Shimadzu, Japan). The bispecific probe labeled with Cy7NHS ester produced a characteristic peak at 748nm, similar to the absorption waveform of free Cy7 by uv absorption spectroscopy (fig. 2A). The change in the ultraviolet absorption of the bispecific probe after binding to Cy7 was negligible, which means that Cy7 was efficiently and stably attached to the side chain of lysin at a molar ratio of 1:1 in solid phase synthesis.

The serum stability of bispecific molecular probes in a mixed solution containing equal amounts of Fetal Bovine Serum (FBS) and Phosphate Buffered Saline (PBS) was evaluated by detecting 48h fluorescence excitation and emission spectra and changes in UV absorbance of the bispecific molecular probes by fluorescence spectroscopy (F-7000; Hitachi). As shown in FIG. 2B, the fluorescence intensity and UV absorption of the 50% FBS/PBS mixture decreased only slightly within 48 hours, indicating that the dual specific probes had better serum stability.

As shown in fig. 2C, the correlation between the change in fluorescence intensity of the bispecific probe and the change in Cy7 concentration was evaluated. The results showed that the fluorescence intensity was positively correlated with the concentration of free Cy 7. The fluorescence intensity of the bispecific probe increased with increasing concentration of Cy7, but decreased when the concentration of Cy7 reached 20.4ug/ml, which means that the fluorescence aggregation quenching effect consisted of the dipeptide DOTA @ Gd/Cy7, similar to free Cy 7.

The Gd (III) content was measured by means of inductively coupled plasma optical emission spectrometer (ICP-OES, Jiangsu Tianguan instruments, Ltd.). The relaxation times (T1) of the detectors were measured with a 7T MR (Bruker BioSpec70/20, Germany) scanner (national center for Nano science and technology, China).

FIG. 2D shows the change in relaxation rate of T1 (7.0-T MRI) after binding of bispecific probes with different concentrations of Gd (III). The experimental results show that at room temperature, the T1 relaxation rate of the dipeptide DOTA @ Gd/Cy7 per molecule is 6.88mM^-1s^-1While the T1 relaxation rate of DOTA @ Gd/Cy7 was 3.57mM^-1s^-1. This means that the bispecific probe has a more significant T1 relaxation rate than DOTA @ Gd. This result may be associated with a complex, complex spatial structure of the polypeptide, affecting the nuclear angular precession frequency of the surrounding water molecules. Therefore, the in vitro characterization result of the dipeptide DOTA @ Gd/Cy7 shows that the fluorescence property and the ultraviolet property of the probe are not influenced by the conjugated specific targeting element, and compared with the DOTA @ Gd, the T1 relaxation capacity of the probe is greatly improved.

7. Biosafety evaluation of bispecific molecular probes

For biomedical applications, probe toxicity testing is an important prerequisite. Therefore, the dual specificity polypeptide molecular probe DOTA @ Gd/Cy7 and the free monomer thereof need to be evaluated for cytotoxicity on normal and PC cells. Panc1(PC cells, ITGB4 and Plectin-1 double transition), T3M4(PC cells, mildly expressed) and HDPE6-C7 (normal human pancreatic ductal cells, rarely expressed) were incubated with bispecific polypeptide molecular probes DOTA @ Gd/Cy7DOTA @ Gd and Cy7 (

concentrations

0,30, 60, 90, 120, 150ug/ml) for 48h for MTT colorimetric (cell proliferation and cytotoxicity assay equipment). From Panc1 and T3M4 cells, it was observed that significant cytotoxicity was shown in vitro and in vivo experiments at probe concentrations as high as 90 ug/ml. In HPDE6-C7 cells, there was no significant cytotoxicity even at concentrations as high as 150 ug/ml. All cell lines maintained 86% viability at the tested concentrations. In addition, in vivo toxicity experiments were performed in healthy mice injected with the bispecific polypeptide DOTA @ Gd/Cy7 (concentration 60 ug/ml). Histopathology, shown in figure 6, confirmed toxicity in vivo, indicating that there was no significant acute inflammation (24 hours post-i.v.) and chronic inflammatory response (30 days post-i.v.) in major organs, including liver, spleen, kidney, pancreas and muscle. The results of in vitro and in vivo probe toxicity experiments show that the bispecific polypeptide DOTA @ Gd/Cy7 is stable and safe at the tested dose.

Example 2 detection of plectin-1 and ITGB4 in human pancreatic cancer cells by bispecific molecular probes

Western Blotting assay

The expression levels of plectin-1 and ITGB4 were examined by Western Blotting. Six pancreatic cancer cell harvest index stages and cell lysis 1 XPBS, 1% Nonidet detergent p40, 2. mu.g/ml aprotinin, 50. mu.g/ml phenylmethylisulfonyl fluoride (PMSF). The cell lysate centrifuge was used at 12000 Xg for 30min at 4 ℃. Total protein was extracted from the supernatant and then analyzed using pierce BCA quantification. Proteins were isolated from each cell line by applying SDS-PAGE (10% lysis gel) and electroblotting to a polyvinylidene fluoride (PVDF) membrane using a semi-dry blotting system (biorad). The membrane was incubated for 30 minutes with 1 XPBST buffer to allow specific binding of antibody antigen and binding of anti-mouse or anti-rabbitt secondary antibody to horseradish peroxidase. Chemiluminescent signals were detected using LAS 4000(GE Healthcare Life Sciences).

The relative expression of plectin1 and ITGB4 in human pancreatic cancer cells, Aspc1, Bxpc3, Panc1, SW1990, Capan2 and T3M4 lines, was evaluated. Actin (muscle protein) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as expression controls for the relative abundance of Western blotting. In these pancreatic cancer cell lines, expression of Plectin-1 was very high. Whereas ITGB4 is overexpressed in most pancreatic cancer cell lines. As shown in FIG. 3, in Panc1 cells, plectin-1 and ITGB4 were doubly overexpressed, thus selecting Panc1 cells as positive cells in vitro and in vivo experiments. Theoretically, this also means that the bispecific polypeptide DOTA @ Gd/Cy7(PTP/RGD) is capable of high binding to Panc1 tumor cell growth models in vitro or in vivo.

2. Bispecific molecular probe detection

Human pancreatic cancer cells (Panc1) were purchased from ATCC and normal human pancreatic ductal epithelial cell line (HPDE6-C7) was purchased from Cobioer, Inc. and labeled with d-fluorescein by stable transfection. In DMEM medium supplemented with 10U/ml penicillin, streptomycin 10. mu.g/ml, and 10% FBSPanc1 and HPDE6-C7 cells were cultured. Bxpc3, Capan2, SW1990, T3M4 cells were cultured in RPMI 1640 medium supplemented with 10U/ml penicillin, streptomycin 10. mu.g/ml, and 15% bovine serum albumin. All cells were cultured in complete medium and 5% carbon dioxide incubator at 37 ℃ and 95% humidity (Heracell, germany). Cell status and counts were assessed using Countess C10227 (Invitrogen). All the above cell lines satisfy the requirement of cell activity. Cell proliferation and cytotoxicity assessment MTS assay (WI Promega, usa) was used. Panc1, T3M4HPDE6-C7 cells were seeded in 96-well plates at a density of 10 per well⁴Cells were cultured for 24h per 100. mu.L of medium. Thereafter, medium replacement was performed using an equal volume of fresh medium containing 6 different concentrations of probe (0-200. mu.g/ml), cells were washed three times with PBS after 24h of culture, and then 100ml of medium and MTS (20. mu.L) were mixed and added to each well. Cell viability was assessed by measuring absorbance at 490nm using a Synergy HT microplate reader (BioTek, USA).

Panc1 and HPDE6-C7 cells were plated in four 6-well plates at a density of 10 cells per well⁴The cells were incubated in the medium for 24 hours. Thereafter, medium replacement was performed using fresh medium containing six concentrations (0-2.0. mu.g/ml) of FITC-labeled bispecific peptide, and incubation was performed for 4 h. After three washes with PBS, the cells were digested with EDTA and centrifuged for 100g × 3 min. Samples were evaluated using BD Accuri C6 and flow cytometer data was analyzed using FlowJo 7.6 software.

The in vitro affinity of Panc1 cells and HPDE6-C7 cells for the bispecific peptides (PTP and RGD) was determined using confocal microscopy and flow cytometry. First, 10⁵The individual cells were seeded into confocal dishes (D35-10-1-N, 35 mm dishes) and incubated in a carbon dioxide incubator at 37 ℃ for 24 hours, followed by fixation with paraformaldehyde. After three PBS washes, cells were incubated in the dark with Alex flow 488-labeled PTP and Alex flow 647-labeled RGD (peptide/dye molar ratio, 1/1.5) at room temperature for 4 hours. Thereafter, cell nuclei were labeled with DAPI for 5 minutes. For blocking purposes, cells were preincubated and blocked with 1ml (20. mu.g/ml) of anti-plectin-1 mAb and anti-integrin-4 mAb for 4 hours. Using image J softThe target co-located target is analyzed and a color image is drawn. Second, the above method was used for Panc1 and HPDE6-C7 cells. Cells were fixed and incubated with 50nm FITC-labeled bispecific peptide for 4 hours, and for blocking assays, anti-plectin-1, anti-ITGB 4, or both antibodies were incubated with Panc1 cells for 2h, and HPDE6-C7 cells as negative controls were incubated directly with FITC-labeled bispecific peptide. Oil Len PE fluorescence confocal microscope (PE ultrasview) was used for cell imaging and Image J2X software determined the fluorescence intensity.

After the safety of the bispecific probes in vitro and in vivo was defined, the affinity of the probes to the cellular target was evaluated by fluorescence microscopy and flow cytometry. The affinity of the single targeting peptide to the over-expressed target of Panc1 cells was first assessed. AF647(AlexaFluor647) -labeled RGD and AF488(AlexaFluor488) -labeled PTP were co-cultured with Panc1 cells and the mean fluorescence intensity was quantified using Image J. Compared with blocking control pre-incubated with plectin-1/ITGB4 monoclonal antibody for 4 hours, both AF 647-labeled RGD and AF 488-labeled PTP showed stronger extracellular fluorescence intensity, with significant difference in fluorescence intensity. In the AF 647-labeled RGD and AF 488-labeled PTP groups, significant fluorescence differences were observed by single blocking, which was more evident in the double-blocking group. Thus, the single targeting peptide has good binding affinity to Panc1 cells. Further combination of the two single peptides revealed more single peptide binding to Panc1 cells and stronger fluorescence intensity. The results show that the single peptide (PTP/RGD) co-localizes well in Panc1 cells simultaneously.

Secondly, to test the high affinity of FITC-labeled dual-targeting peptides for PC cells, fluorescence microscopy and flow cytometry qualitative and quantitative evaluation of the binding of the dual-targeting peptides to the target were performed in Panc1 cells (plectin-1/ITGB4 overexpressed) and HPDE6-C7 cells (rarely expressed). The experimental grouping was that the double targeting peptide labeled FITC was incubated with Panc1 cells (positive cells), HPDE6-C7 cells (negative cells) were incubated with bispecific probe as negative control, and free FITC was used as non-specific control. As shown in fig. 4A and 4B, incubation of Panc1 cells with FITC-labeled dual-targeting peptide showed stronger fluorescence intensity, indicating that FITC-labeled dual-targeting peptide has higher target affinity for Panc1 cells than HPDE6-C7 cells.

The results of flow cytometry were consistent with confocal imaging results. Fig. 4C and 4D show that as the concentration of the dipeptide label increases, the visible fluorescence changes significantly and the fluorescence intensity of the probe increases significantly. In FIG. 4E, the mean fluorescence intensity of Panc1 cells was significantly higher than that of HPDE6-C7 cells. The affinity of the dual targeting peptide for panc1 cells and HPDE6-C7 cells was determined by fluorescence flow cytometry. However, no significant change in fluorescence intensity was observed in HPDE6-C7 cells or free FITC groups. In FIGS. 4F and G, the flow cytometry quantification is shown, the FITC concentration is increased, and the fluorescent signal intensity of the double peptide labeled FITC can reach 9.125X 10⁴(approximately saturated 2.0 ug/ml FITC). However, the intensity of the fluorescence signal of Panc1 cells incubated with free FITC did not exceed 2X 10⁴. Whereas HPDE6-C7 cells, due to the lack of binding sites for PTP and RGD peptides, have FITC-labeled dipeptides with relatively low fluorescence intensity, not more than 1.2X 10⁴. Thus, confocal imaging and flow cytometry demonstrated that bispecific probes could bind efficiently to Panc1 cells in vitro, depending on the overexpression of plectin-1 and ITGB4 in Panc1 cells. That is, the bispecific probe, effectively targeting plectin-1 and ITGB4, resulted in more effective binding affinity to Panc1 cells and cells.

Finally, the invention carries out comparative test on the targeting of the double-targeting peptide and the single peptide, and verifies the synergistic effect of the double-targeting peptide. As shown in fig. 5A, immunofluorescence staining of Panc1 cells was significantly enhanced with FITC-labeled dual targeting peptides compared to multiple blocking controls (Panc1 cell targets were effectively blocked with saturating doses of anti-plectin-1, anti-ITGB 4, or both antibodies prior to cell staining). Therefore, after PTP and RGD are combined, the targeting effect of PDAC is obviously enhanced and amplified, and the combined design of the PTP and the RGD produces a remarkable synergistic effect.

In summary, confocal imaging and flow cytometry data show a significant increase in fluorescence intensity when the dipeptide (PTP/RGD) is labeled with FITC compared to multiple blocking controls. These results indicate that the bispecific PTP/RGD polypeptides have the ability to target cells with high targeting and specificity. In vitro experiments, the bispecific PTP/RGD polypeptides showed high affinity and specificity.

Example 3 use of bispecific molecular probes for animal tumor models NIRF and MRI imaging

1. Establishment of animal model

The animal experiment program is approved by the animal ethics committee of tumor hospital of Chinese medical academy of sciences [ NCC2016A002]. Strict compliance with animal care and use protocols was ensured throughout the study. The tumor model was established from 6-week female BALB/C nude mice purchased from Beijing Wittingle laboratory animal technology Co., Ltd. (China). Subcutaneous and in situ tumor models were established with Panc1 cells overexpressing plectin-1 and ITGB4 proteins to assess the specificity of the bispecific probes. Panc1 cells were implanted into BALB/C nude mice by mass implantation and cell injection methods. Total 10⁶Individual cells were injected into the skin fold of the left axilla to form a subcutaneous tumor model. In addition, when the diameter of the subcutaneous tumor was 10mm, the tumor mass was peeled off and cut into 1mm³And (5) blocking. After three washes with sterile saline, freshly transected tumor masses and tissue sealant were evenly embedded on the surface of normal pancreatic tissue. The tumor mass embedding method can reduce detectable leakage of the pancreas and shorten the tumor cycle compared to cell implantation. Tumor growth was monitored and after about 3 weeks a clear tumor and an in vivo in situ tumor of 6-8 mm in diameter were obtained.

2. In vivo fluorescence imaging and biodistribution

Fluorescence imaging and biological profiles were monitored using an animal imaging Spectrum System (USA). To reveal the potential and dynamic biological distribution of bispecific probes in vivo targeting tumors and major metabolic organs, 24 panc 1-bearing tumor mice were divided into 4 groups (6 per group). The targeting probe and free dye were injected via tail vein. For the blocking group, mouse anti-human plectin-1 and ITGB4 monoclonal antibodies were injected via the rat tail vein two hours ago to block their specific binding sites.

Fluorescent Molecular Imaging (FMI) was performed within 48 hours after injection. Subcutaneous images were obtained in the left lateral decubitus position of the mice to assess the metabolism of the tumor. Region of interest (ROI) analysis was used to determine the selective accumulation of probes within tumors, dissecting tumors and major organs including brain, lung, liver, spleen, pancreas, kidney, intestine, etc. to assess fluorescence signal intensity. Tumors were excised and fixed with OCT gel for pathological analysis. The frozen portion (20 μm thick) was exposed to white light and near infrared spectroscopy and come CM1950 machine (800 nm). The frozen sections were then observed with an automated inverted fluorescence microscope to determine the NIR band.

3. Fiber confocal fluorescence microscopy imaging

After anesthesia, mice bearing tumor-in-situ pancreatic cancer were injected with 100 μ L of nonspecific FITC (concentration, 1 mg/ml) via tail vein for morphological analysis of the pancreatic tumor and normal pancreatic tissue angiography. Thereafter, other tumor-bearing mice were sequentially injected with AF 488-labeled RGD and AF 647-labeled PTP. A 2 cm incision is made at the midline of the abdomen. Pancreatic tumors and peritoneal regions were well exposed 3 hours after injection. Using a fiber dual-frequency confocal fluorescence microscope system (FCFM) model S1500

In vivo tumor imaging was performed. The mean fluorescence signal intensity was measured at 4 random Regions (ROI) of 1mm diameter. Fluorescence intensities greater than 1000 were determined to be effective. After image acquisition, the tumor mass is rapidly excised and fixed to a tissue cryomatrix

In (1). After freezing at-80 ℃, the fixed tumors were cut into 5- μm sections. The frozen tissues were washed three times with PBS to remove OCT gel and stained with DAPI (100. mu.g/ml) for 5 min. Tissue sections containing the two-band fluorescently labeled peptide and stained with DAPI were visualized in 3D HISTECH using fluorescence bands 405, 488, and 647 nm. Images were scanned, merged and displayed using Pannorviewer software. Finally, the bispecific polypeptide labeled with AF647 was injected into the tail vein. After two hours, non-specific FITC was injected, and 20 minutes later, FITC fluorescence was detected with an optical fiber having a wavelength of 488nm, red fluorescence, and a wavelength of 647nm, and an image was obtained.

4. In vivo NIRF imaging

The tumor targeting characteristics of the dipeptide DOTA @ Gd/Cy7, as well as bispecific targeting and biological distribution in vivo were explored by non-invasive NIRF and MRI imaging techniques. The subcutaneous tumors of mice were injected intravenously with the dipeptide DOTA @ Gd/Cy7(150ul/mouse,400ug/ml probe). The biological distribution of the double peptide DOTA @ Gd/Cy7 in the mice is observed by a fluorescence imaging technology.

NIRF imaging of mice injected with the dipeptide DOTA @ Gd/Cy7, grouped into bispecific probe sets, plectin-1mAb blocking, ITGB4mAb blocking, and free Cy 7. Significant changes in tumor area and TBR (tumor background ratio) were seen for the bispecific probe set 30min after injection. The fluorescence intensity of the tumor gradually increased after 4h, and the TBR reached 9.46 and reached a maximum value, indicating that the bi-specific probe continuously accumulated in the tumor region. The fluorescence signal in the tumor remained high after 24 hours, but the fluorescence signal of the free Cy7 group was not obtained 8 hours after tail vein injection, indicating that the bispecific probe had better retention time and higher retention capacity in the PC tumor compared to the free dye. Plectin-1 and ITGB4 blocked alone maintained stronger fluorescence intensity and longer cycle than free Cy7, but the fluorescence was low and shorter time than the dual-specificity target probe. This suggests that bispecific probe affinity is more prominent than single target and free Cy 7.

For efficient molecular imaging, good TBR and long cycle times are necessary. Good TBR depends not only on high tumors but also on low background, which is why targeting probes select small molecule probes for PDACs. Small molecule probes have incomparable advantages with respect to metabolic properties. It not only utilizes simple permeability, but also utilizes EPR effect, and is based on small molecular weight and particle size, and is convenient for excretion. Both the permeability and the EPR effect contribute to high probe concentration and good fluorescence intensity at the tumor site. On the other hand, due to the non-immunogenic nature of small molecule size and targeting peptides, small molecule probes can be easily detached from RES (reticuloendothelial system) monitoring, reducing background noise absorption by the liver and spleen, and increasing renal excretion function-related uptake.

5. In vivo MRI imaging and Gd quantification

MRI imaging was performed using a 7.0T magnetic resonance scanner. Bispecific Gd/Cy7-PTP/RGD was injected at 0.03mM Gd/kg (100ul) in the tail vein of mice, followed by 1mL of heparin-phosphate saline to reduce probe residues in the tail vein. One group of 9 mice was used for each drug. As a control, the same procedure was performed as for the DOTA @ Gd group. T1 weighted images were obtained using a mouse body volume coil (body volume coil). Mice with pancreatic tumors (n ═ 3 per group) were euthanized at either peak or end of metabolism. Dissecting out tumor and main tumor organs such as liver, spleen, kidney, muscle, etc. Tissue samples were homogenized and digested with concentrated nitric acid (1.0mL, 70%, EMD, Gibbstown, NJ, USA). The sample was liquefied to 4d, the solution was centrifuged at 15,000rpm for 8 minutes per minute, and the supernatant was diluted with deionized water (1: 5). The Gd concentration in the supernatant was determined by the ICP-OES method. The Gd content was determined.

According to our design, the double peptide DOTA @ Gd/Cy7 is expected to improve in vivo tumor fluorescence and magnetic resonance imaging. The increased concentration of gd (iii) in the tumor region is critical for the success of MRI molecular imaging. The small molecule probe adopts an MR module, makes full use of simple permeability and EPR effect, and more effectively improves Gd (III) concentration at a tumor site instead of relying on the EPR effect. It is very important for high-density malignancies, especially in pancreatic cancer, with extremely low perfusion, mainly low vascular and high interstitial pressure. However, the targeting module and the NIFR module expand the probe molecular weight, reducing the gd (iii) concentration per probe. Therefore, in order to overcome the disadvantage, the loading rate of Gd is increased, the T1 relaxation effect of the bi-specific molecular probe is improved, and the bi-DOTAs is designed to load bi-Gd (III) s. Next, in vivo magnetic resonance imaging, the dual peptide DOTA @ Gd/Cy7 was injected at different time points to demonstrate targeting probe performance, DOTA @ Gd as a control group.

As shown in fig. 6A, two groups of injection via tail vein, bispecific probe and DOTA @ Gd were performed, and T1 weighted images of mice were collected at 2,4,8,12, and 24 hours after injection, respectively, to observe the metabolic characteristics of the probe in the tumor at different time points.

As shown in FIG. 6B, it was clearly demonstrated that in mouse tumors, the T1-weighted signal was gradually increased in mouse tumorsThe changes were made and treated with the dipeptide DOTA @ Gd/Cy7 as a control, and treatment with the dipeptide DOTA @ Gd/Cy7 brought more valuable details into the probe distribution than fluorescence imaging, which only provided two-dimensional information for probe metabolism. The T1 weighted signal gradually goes from well perfused periphery to non-perfused pancreatic tumor central region. Significantly, the brighter T1-weighted signal and long retention time, resulting from target probe accumulation, increased gradually to 12h, but the T1-weighted signal for DOTA @ Gd decreased 4 hours after injection. In order to perform the necessary metabolism of the target probe, quantification of gd (iii) retention at the tumor site was analyzed in fig. 6C. Tumor tissues were collected from mice injected at different time intervals and lysed with lysis solution. The homogeneous lysate was diluted and measured by the ICP-OES method, and the Gd (III) concentration in the tumor tissue was quantified. Higher Gd (III) concentrations, including liver, spleen and kidney, were found in tumors 4h after injection, consistent with the in vivo fluorescence results described above. However, gd (iii) uptake was shown to be superior to that of tumors in the kidney after 24 hours of injection, suggesting that renal excretion is the major metabolic pathway for small molecules. At different times, the absolute T1 and the signal-to-noise ratio (snr) are determined in fig. 7. Absolute T1 represents R1 (T1)^-1Ms) relaxation. The relaxation of R1 of the dual specific target probe is obviously larger than DOTA @ Gd, and the SNR of the dual specific target probe is also obviously enhanced and prolonged. This is because the bispecific molecular probe has longer blood circulation time and better tumor penetration ability, so that the Gd (III) concentration in the tumor is higher, the retention time is longer, and the signal intensity of the nuclear magnetic T1 modality is higher than that of the small molecule DOTA @ Gd. This conclusion is consistent with the fluorescence modality.

Example 4 Dual targeting peptide Probe NIR guided surgical navigation and histological evaluation

Subcutaneous and orthotopic tumor-transplanted nude mice with the average tumor diameter of more than 5mM are taken, and are injected with a double-target probe (1mM, 200ul) through a tail vein, metabolized for 2 hours, and then surgically excised under the guidance of a near-infrared fluorescence microscope. And (3) laser irradiation excitation, and collecting probe fluorescent signals by using a 800nm fluorescent wave band channel. The fluorescence signal of the tumor tissue is strong, and the tumor tissue can be completely excised. Excised tumor tissue was fixed with 4% paraformaldehyde and HE stained.

A mouse model of panc1 cells for in situ pancreatic tumors was first established. Before surgery, bioluminescence imaging/tomography (BLI/BLT) was performed to locate tumors and assess tumor invasiveness. After resection of the pancreatic carcinoma in situ, bispecific Gd (III)/Cy7-PTP RGD was injected for 4 h. Mice were anesthetized with 2% sodium pentobarbital by intraperitoneal infusion at a dose of 30 μ g/g and mounted on the operating platform. NIRF-fluorescence imaging guidance systems surgically resect tumors. After the first resection, the presence of residual tumors was examined. The excised specimens were fixed in 4% paraformaldehyde and paraffin for pathological analysis.

Fluorescence and magnetic resonance imaging were performed with the dipeptide DOTA @ Gd/Cy7, and NIRF guidance was performed in mice, and islet transplantation with tumor growth in situ was performed in mice. BLI (bioluminescence imaging) of the orthotopic xenograft model showed significant fluorescence intensity and volume after 27 days of panc1-luc cell implantation at the tail end of normal pancreas. While BLT (bioluminescence tomography) further pinpoints the orthotopic xenograft in three dimensions in the left abdominal region (as shown in fig. 8A). The tumor was successfully and completely resected under NIRF imaging guidance, and the resected tumor tissue was histologically analyzed as shown in figures 8B and C. Staining of dissected tumors by HE (hepatoxin-eosin) further confirmed that the excision margins of FIG. 8Da were negative. Paraffin sections of tumor fractions were used to observe IHC Immumohistochemical) stained tumors heterogeneity of PNCA (proliferating cell nuclear antigen). Nuclear staining was shown as a medium, dark brown color, suggesting that the excised nodules were panc1 cells with high heterogeneity, as shown in figure 8Dc, under the characteristics of pancreatic tumors. The frozen sections from which the other part of the tumor was excised were cut into 20 pieces by an inverted fluorescence microscope. NIRF imaging showed that in figure 8De, most of the double targeting peptide positive regions expressed human plectin-1 and ITGB4, and a strong NIR signal was found throughout the tumor region, indicating that the double peptide DOTA @ Gd/Cy7 has the ability to effectively track PDAC xenograft tumors.

Optical imaging has certain limitations in deep tissue and image reconstruction and quantification that can be easily and significantly adapted to intraoperative guidance. The double peptide DOTA @ Gd/Cy7 is used for carrying out good intraoperative guidance, and the value of clinical transformation is verified. A highly specific NIRF probe can improve the intra-operative localization and assessment of intra-operative suspicious lesions and provide a rapid assessment of resection margins to avoid tumor residue and recurrence. Similar approaches to ICG-guided surgery have successfully clinically discovered and resected SLNs from breast cancer.

Claims

1. A bispecific polypeptide molecular probe targeting pancreatic cancer tumor cells is characterized in that the structural formula of the molecular probe is shown as the formula (I):

（I），

the imaging molecule used for molecular imaging is marked by the amino acid residue at the site a and/or the amino acid residue at the site b, the imaging molecule used for molecular imaging is marked by the amino acid residue at the site c, X is lysine, and Y is phenylalanine.

2. The probe of claim 1, wherein the amino acid residues at the a site and the b site are both labeled DOTA @ Gd (III), and the amino acid residue at the c site is labeled Cy7, and the structural formula of the molecular probe is shown in formula (II):

（II）。

3. the probe of claim 2, wherein Cy7 is attached to the polypeptide in a 1:1 molar ratio.

4. Use of the molecular probe of claim 3 for preparing a tumor diagnostic reagent.

5. The use according to claim 4, wherein the probe is prepared as an injection.

6. A method of making the probe of claim 3, the method comprising the steps of:

(4) conjugating the peptide chain obtained in step (3) with DOTA (tBu) 3-ester;

(6) and (5) harvesting the product obtained in the step (5).

7. The method as claimed in claim 6, wherein the coupling agent used in the coupling in step (3) is HATU: DIEA at a ratio of 3:10, and the reaction ratio of the peptide chain to Cyanine7 monosuccinate is 3:1 by weight.

8. The method of claim 6, wherein the conjugation in step (4) is performed by conjugating DOTA-NHS ester protected by three tbus with lysine at positions 9 and 10 in the main peptide chain deprotected by hydrazine hydrate as an activated carboxyl group.

9. The method according to claim 6, wherein the gadolinium compound in step (5) is Gd hexahydrate, and the weight ratio of the peptide chain to the gadolinium compound is 4: 1.