| [Background]Pancreatic cancer is one of the most common pancreatic malignancies, which possesses highly invasion, so early diagnosis is the basic way of improving the prognosis of patients with pancreatic cancer. At present, the diagnosis technologies for pancreatic cancer, including tumor markers detection, conventional imaging examination and endoscopic techniques, play a limited role in early diagnosis. The early diagnosis of pancreatic cancer must be at the cellular and molecular levels. Therefore, molecular imaging technology is an effective way to solve the problem of early diagnosis of pancreatic cancer. The study proposed a new PET imaging method for pancreatic cancer, which was based on that the plectin-1expression in pancreatic cancer cell membranes and intracellular, but also characterized specific expression in the cell membrane. We took the peptide PTP as a targeting ligand, which could be specifically combined with the plectin-1, labeled with a radionuclide18F as the signal, to prepare the molecular probe18F-PTP.18F-PTP combined with cationic polymer carrier polyethyleneimine (PEI), which were connected through the arm oligopeptides (-GGEEE-), to synthetic of complex probe18F-PTP/PEI, for PET imaging of the pancreas cancer. This molecular imaging method based on that the18F-PTP/PEI and pancreatic cancer cell plectin-1to bind specifically by targeting peptide PTP, making a lot of18F-PTP retention in the cell with the cell transduction of PEI and combined to plectin-1, achieving targeted overlay with the cell membrane and intracellular molecules to avoid solution and elution from the probe, improving the target/non-target ratio by the dual role of molecular targeting and cell transduction, which were favor of PET imaging in early detection of pancreatic cancer. The imaging method that jointly designed molecular targeting and cell transduction is an innovative model of molecular imaging. This study adopted cytology in vitro, pathology experiments, fluorescence imaging experiments in vivo and small animal PET/CT imaging experiment to test the feasibility of this imaging method. If this new PET imaging method for pancreatic cancer could be achieved, not only could the problem of early diagnosis of pancreatic cancer be solved, and be help to targeted therapy for pancreatic cancer, but also could other solid tumors targeted diagnosis and therapy be referred to this method.[Objective]1. To prepare targeting polypeptide PTP for pancreatic cancer with solid phase synthesis, purification and identification, then to lay the foundation for preparation of molecular probes and future clinical applications; to explore the feasibility of PTP as a probe ligand by synthesizing fluorescent probe FAM-PTP, which was purified and identified, and by verifying the targeting of PTP for pancreatic cancer with cytological in vitro, pathological and animal tomography fluorescence imaging experiments; to synthesize composites probe FAM-PTP/PEI, then to determine the optimal synthesis conditions for the preparation of radiolabeled probes18F-PTP/PEI complex; to verify whether cellular transduction function with carriers PEI is benefit to improve the sensitivity of tumor imaging by cytological in vitro, pathological and animal tomography fluorescence imaging experiments.2. To synthesize radionuclide labeled intermediate N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) by hand, then to edit module synthesis process according to the optimal manual reaction conditions to achieve automatic synthesis of production18F-SFB; to synthesize radionuclide labeled molecular probes18F-PTP and complex probe18F-PTP/PEI, and examine the molecular probe needle18F-PTP and complex probe F-PTP/PEI feasibility for pancreatic cancer diagnosis by measuring in vitro stability, in vivo pharmacokinetics of the drug, and micro PET/CT imaging study in nude mice with pancreatic cancer, then to evaluate whether the carrier PEI contributes to improve the sensitivity of PET imaging.[Methods]1. Synthesis and identification of fluorescent molecular probes FAM-PTP and FAM-PTP/PEI(1)Synthesis, purification and identification for targeting polypeptide PTP of pancreatic cancerTo facilitate to synthesize the complex probe FAM-PTP/PEI and18F-PTP/PEI, the arm oligopeptide (-GGEEE-) was directly connected to the carboxyl terminus of PTP. The whole sequence was:the amino terminus (N-side) KTLLPTPGGEEE-carboxy-terminal (C-side). To Rink resin as the carrier, with Fmoc amino protection strategy, peptide PTP was synthesized in solid-phase. Firstly, the C-terminus of the first amino acid and glutamic acid (Glu, E) of the carboxyl groups covalently linked to the resin, then the starting point for the synthesis of amino-glutamate, glutamic acid to the second amino-acylation reaction to form a peptide bond, the same method from the carboxyl end (C terminal) to the amino terminal (N terminal) amino acids were sequentially added, the reaction was completed polypeptide connected to the main cutting reagent trifluoroacetic acid peptide from the object cleaved from the resin. Synthetic products by high performance liquid chromatography (HPLC) separation, purification, analysis, were identified by mass spectral analyzes.(2)Synthesis, purification and identification of fluorescent molecular probes FAM-PTPAt the terminal of synthesis polypeptide of PTP, that was after the condensation reaction of the last bit of amino acid lysine was completed, the amino terminal lysine ε-NH2was protected, activated, and bound covalently to fluorescein FAM. After ligation completed, the targeting peptide segments was cleaved from the resin with trifluoroacetic acid-base. Synthetic products were separated, purified, analyzed by high performance liquid chromatography (HPLC), and were identified by mass spectral analyzes. (3) Synthesis, identification and determination of biological toxicity of Complex probe FAM-PTP/PEIWith EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) as a cross-linking agent, FAM-PTP connected to PEI, then obtained the solution of composite probe FAM-PTP/PEI, which was saved in4℃refrigerator. Infrared spectrophotometric method and ninhydrin color reaction were used to verify whether the connection of product of FAM-PTP and PEI was successful; the toxicity of the composites probe FAM-PTP/PEI was tested by cytotoxicity assays (MTT) and hemolytic assay.2. Evaluate of the biological function of the molecular probes FAM-PTP and FAM-PTP/PEI in vitro(1)In vitro binding experiments of fluorescent molecular probes FAM-PTP, FAM-PTP/PEI with pancreatic cancer PANC-1cellsConventional cultured pancreatic cancer PANC-1cells were recovered to the logarithmic growth phase. PANC-1cells were incubated under the conventional culture conditions with freshly prepared cell culture medium solution containing the fluorescent probe FAM-PTP and FAM-PTP/PEI cells, which the concentration of the fluorescent molecules was60uM. The distribution of fluorescein in living cells was observed placed under an inverted fluorescence microscopy, the relative fluorescence intensity was analyzed with the flow cytometry, and the impact of the concentration of the fluorescent probe and cell incubation time on cellular uptake was also examined.(2)The animal imaging of fluorescent molecular probes FAM-PTP, FAM-PTP/PEI targeting to pancreatic cancer cells15pancreatic PANC-1xenograft nude mouse models were established by conventional methods, when the tumor diameter of more than0.5cm,12of them were removed randomly as experimental animals, then were randomly divided into two groups, six every group. Nude mice were injected FAM-PTP, FAM-PTP/PEI (1mM,150μl) via the tail vein, were taken off the neck to death after40min, rapidly frozen at-80℃for30min. Nude mice were cutted into coronal slices with a microtome, and the slices of animals were flatted on a clean glass plate, with a fluorescence imaging device for imaging, to observe fluorescence uptake of tumor and organs, measure tumor fluorescent uptake, and compare the differences in uptake of the fluorescent tumors in two groups of nude mice. After separation of the organizations organs were separated after imaging, rapidly frozen at-80℃for30min, to make frozen sections. After DAPI-stained, green fluorescence distribution in organs was observed in inverted fluorescence microscope.3. Synthesis and identification of probes18F-PTP and18F-PTP/PEI(1) Synthesis, identification and determination of biological toxicity of Radio nuclide-labeled intermediates18F-SFBRadiolabeled intermediates manual18F-SFB was synthesized manually according to the literature to explore the best synthesis conditions.18F-SFB was synthesized automatically by using the GE module TRACERlab FXFN nucleophilic fluorination reaction synthesis system for automatic synthesis of18F-SFB, editing module synthesis procedure in accordance with the manual synthesis of optimal reaction conditions, adding various reagents in the module to open the program. After obtaining the product, the radiochemical purity was measured by TLC and HPLC, the radioactivity was measured by activity meter, and radiochemical yield was calculated.(2)Synthesis of Radionuclide molecular probe18F-PTP and18F-PTP/PEI; determination of stability in vitro and pharmacokinetics in vivoIntermediate18F-SFB was dissolved in50μL of acetonitrile, which was added100μg PTP (is dissolved in200μL pH8.50.1M borax-boric acid buffer solution), then reacted at room temperature for30min. After filtered, the reaction liquid was separated through a HPLC, then the peak of tR=8～19.5min was collected. The product solution was dissolved, evaporated rotated, and again was dissolved with0.9%NaCl injection solution. The desired product compound18F-PTP was evaluated by Radio-TLC and Radio-HPLC. The optimal reaction conditions were determined through observing the effects of pH, temperature and reaction time on the labeling rate.18F-PTP and PEI were coupled according preparation of a fluorescent compound probe FAM-PTP/PEI. The stability of the two probes in PBS and in calf serum was measured at37℃; in vivo pharmacokinetic parameters in Kunming mice of the two probes was determined.4. The study of micro PET/CT imaging of pancreatic cancer bearing nude mice15pancreatic PANC-1xenograft nude mouse models were established by conventional methods, when the tumor diameter of more than0.5cm,12of them were removed randomly as experimental animals, then were randomly divided into two groups, six every group. After injected200μCi of18F-PTP and18F--PTP/PEI via the tail vein, waiting for40mins, nude mice exposed to anesthesia with isoflurane after, then conducted static micro PET/CT imaging. Using Inveon Research workplace (IRW2.2) workstation for data processing, PET data was analyzed after CT attenuation correction and reconstruction for coronal plane, cross-sectional, sagittal images through filtered back projection OSEM3D. PET images were fused with the CT images, in which the tumor regions of interest were manually outlined. SUVave was calculated by the workstation automatically.[Results]1. Synthesis and identification of fluorescent molecular probes FAM-PTP and FAM-PTP/PEI(1)Synthesis, purification and identification for targeting polypeptide PTP of pancreatic cancer:PTP synthesized by solid phase was a white powder. The purity of PTP measured by HPLC analysis was98.3%. The main peak molecular weight by mass spectrometry analysis was1270.6, in according with the theoretical molecular weight1270.5. This high purity suggested that the synthesis was correct to prepare molecular probes, and the fluorescent molecule probe PTP could be used to mark fluorescein and radioactive nuclides.(2)Synthesis, purification and identification of fluorescent molecular probes FAM-PTPFAM-PTP synthesized by solid phase was a yellow powder. The purity of FAM-PTP measured by HPLC analysis was98.1%. The main peak molecular weight by mass spectrometry analysis was1628.7, in according with the theoretical molecular weight1629.7. This high purity suggested that the synthesis was correct to prepare molecular probes, and the fluorescent molecule probe FAM-PTP could be used to verify in vivo and in vitro experiment.(3) Synthesis, identification and determination of biological toxicity of Complex probe FAM-PTP/PEITwo experiments of Infrared spectrophotometric method and ninhydrin color reaction confirmed free amino groups of PEI significantly reduced due to a combination of FAM-PTP, and then it proved FAM-PTP had been successfully grafted onto PEI chain; Cytotoxicity assays (MTT) showed that the complex biological probes FAM-PTP/PEI had no significant toxicity, and then FAM-PTP/PEI could be used for in vivo experiments.2. Evaluate of the biological function of the molecular probes FAM-PTP and FAM-PTP/PEI in vitro()In vitro binding experiments of fluorescent molecular probes FAM-PTP, FAM-PTP/PEI with pancreatic cancer PANC-1cellsIn the FAM-PTP group, fluorescence microscopy showed that speckled green fluorescent appeared in PANC-1cell membrane, and a small amount of green fluorescence in cytoplasm; In FAM-PTP/PEI group, fluorescence microscopy showed that green fluorescence appeared in both of PANC-1cell membrane and cytoplasm. It suggested that the probe not only could connect with the cell membrane, but also could turn into the cell with the help of transduction of PEI cells. Flow cytometry analysis:With the increase of the probe concentration and the extension of incubation time, the fluorescence intensity of FAM-PTP group cells gradually increased up firstly, to certain intensity, then appeared a saturation phenomenon. However, the fluorescence intensity of FAM-PTP group cells continued to increase, and did not appeared a saturation within the experimental setup probe concentration. It suggested that the transduction of vector could help the pancreatic cancer cells to increase uptake of the molecular probe PEI.(2)The animal imaging of fluorescent molecular probes FAM-PTP, FAM-PTP/PEI targeting to pancreatic cancer cells The coronal slices fluorescence imaging of pancreatic cancer bearing nude mice showed that the green fluorescence distribution were seen in both groups of FAM-PTP and FAM-PTP/PEI, and the fluorescence intensity of FAM-PTP/PEI group was higher than that of FAM-PTP group; The medium green fluorescence was seen in muscles, skin and intestinal of two group, but only a small number of green fluorescence in other organizations. The tumor fluorescence uptake values of two group were compared quantitatively:the tumor fluorescent uptake value of FAM-PTP was (3273.50±901.68), and that of FAM-PTP/PEI was (6634.83±662.08). The fluorescent tumor uptake values of two group were significantly difference (F=-7.360; P=0.000).The results of fluorescence microscopy imaging of Frozen sections were similar to that of coronal slices.3. Synthesis and identification of probes18F-PTP and18F-PTP/PEI(1) Synthesis, identification and determination of biological toxicity of radionuclide-labeled intermediates18F-SFB18F-SFB was synthesized automatically by Tracerlab FXFN module, and the whole process took about50min. The product18F-SFB through TLC scanning, the value of product was Rf=0.79, in according with Rf=0.80of the cold synthetic reference material. Through HPLC analysis, the product was consistent with the cold synthesized determined product SFB, and other radioactive impurity peak did not appear. By Bio-Chrom Lite program analysis, radiochemical purity was greater than99%.(2)Synthesis of Radionuclide molecular probe18F-PTP and18F-PTP/PEI; determination of stability in vitro and pharmacokinetics in vivoThe coupling reaction of Polypeptide and18F-SFB depended on the set of pH, temperature, reaction time, and the optimum parameters were:temperature was42℃,18F-SFB was dissolved in50μL of acetonitrile,100μg polypeptide was dissolved in150μL pH8.5borate buffer, the reaction time was30min. By Radio-TLC and Radio-HPLC characterization, the peptide PTP and18F-SFB coupling were successful The radiochemical purity of18F-PTP product was higher than98%. The composite probe18F-PTP/PEI was synthesized according to preparation methods of the fluorescent composites probe FAM-PTP/PEI. According to the activity of the final product, by adding an appropriate volume of medical0.9%NaCl solution,2μCi/μL solution of18F-PTP and18F-PTP/PEI was dubbed, with pH value of7.0-7.4, which the appearance was colorless, transparent, clarifying. By determination of Radio-TLC, the stabilities of the two probes in fetal calf serum in PBS were good, and after360min, their radiochemical purity remained above80%. The pharmacokinetics of18F-PTP and18F-PTP/PEI in vivo mice were according with a simple two-compartment model, and pharmacokinetic parameters were calculated through evaluated pharmacokinetics software:the half-lives of distribution phase were1.98min and2.75min, which suggested that these two probes could rapidly distribute throughout the body, and be conducive to its rapid uptake in tumor tissue; The half-lives of clear phase were15.48min and23.62min, which suggested that the clearance rate of the both probes described in the blood was fast so as to reduce the blood background and help improve the imaging results.4. The study of micro PET/CT imaging of pancreatic cancer bearing nude mice18F-PTP and18F-PTP/PEI micro PET/CT imaging results of pancreatic cancer bearing nude mice were similar to the results of animal tomography fluorescence imaging. Tumor tissues of the both groups showed apparently radioactive uptake, and the latter was significantly higher than the former; the uptake values of intestinal and kidney were higher, which suggested that indicating that18F-PTP and18F-PTP/PEI mainly metabolized in the liver and gallbladder and urinary systems; the uptake values of heart and lung was low, indicating that18F-PTP and18F-PTP/PEI cleared faster in blood. No brain distribution of radioactivity, indicating that18F-PTP and18F-PTP/PEI may not pass the blood brain barrier. SUVave of the two groups of tumors was compared quantitatively:the tumor SUVave of18F-PTP group was (4.37±0.93), and that of18F-PTP/PEI was (6.88±1.10), which was significant difference (t=4.274; P=0.002).[Conclusion]1. Our research prepares targeting polypeptides PTP with high purity by the solid phase synthesis method. After separated and purified by high performance liquid chromatography (HPLC), the purity is98.3%. Polypeptide PTP is labeled with fluorescein FAM, then is used to synthesize fluorescent probe FAM-PTP. After separated and purified by high performance liquid chromatography (HPLC), the purity is98.3%. And the complex molecular probes FAM-PTP/PEI is built, which is confirmed to successfully constructed by infrared spectrophotometric method and ninhydrin color reaction, and has no toxicity by cytotoxicity assays (MTT), so as to could be used for in vivo experiments.2. Through confirmed in vitro cytology experiments, the fluorescent probe FAM-PTP can bind with pancreatic cancer PANC-1cell, but only a small amount turn into the tumor cells; but with the help of the transduction of PEI, FAM-PTP/PEI can turn into tumor cells; through confirmed by the fluorescence biodistribution experiment of pancreatic cancer bearing nude mice, the molecular probes FAM-PTP can specifically bind to pancreatic cancer tissues in vivo, which can target to pancreatic cancer; the transduction of PEI can further enhance the FAM-PTP uptake of pancreatic cancer cells in vivo.3. According to the optimum reaction conditions confirmed by manual explore,18F-SFB is automatically synthesized by Tracerlab FXFN module, which the radiochemical purity is greater than99%. The entire process takes about50min. This method obtains Curie equivalent intermediates, and reduces the radiation dose to operators. Our study prepares the radiolabeled molecular probe18F-PTP and the complex probe18F-PTP/PEI, and determine the optimal reaction conditions. The tests of in vitro stability and in vivo pharmacokinetic experiment show that these two probes can be used to the in vivo imaging experiments.4. The micro PET/CT imaging study of pancreatic cancer bearing nude mice shows that the molecular probe18F-PTP can specifically target to pancreatic cancer tissues, and can be used for PET imaging in the diagnosis of pancreatic cancer; And the complex probe18F-PTP/PEI with the help of cellular transduction of PEI can further improve the PET imaging sensitivity. |
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