Developing The Novel Molecular Probes Targeting To Glypican3 Receptor And Evaluating Its Efficacy By The Fluorescent And PET Imaging Study In The Hepatocellular Carcinoma

BackgroundEarly detection and treatment of primary hepatocellular carcinoma (HCC) lesions, can significantly improve the prognosis of patients.Conventional diagnostic imaging technologies B ultrasound, CT and MRI, displayed cases of hepatocellular carcinoma based on morphological changes, but all above Conventional diagnostic imaging technologies have deficiencies in the differential diagnosis, staging and early diagnosis and treatment of systemic evaluation.18F-FDG PET/CT, as the new medical imaging technology, has some clinical application in the diagnosis and staging of hepatocellular carcinoma, but it cannot meet the clinical work because of its low diagnostic sensitivity.Glypcian3 (GPC3) was selected as a novel molecular target for HCC.GPC3 is heparan sulfate proteoglycan essential in regulating embryonal cell growing. While its expression is absent in normal adult tissues,GPC3 is significantly over-expressed in up to 80% of human HCC’s. L5 (sequence:RLNVGGTYFLTTRQ) is a novel GPC3 receptor targeting peptide. It was reported that the L5 phage bound to immobilized GPC3 receptor.Development of the fluoresceit and 18F Labeled L5 peptide might have a potential to be the good tracers for imaging of hepatocellular carcinoma.Objective:1. To synthesis the GPC3 targeting peptide of L5 and identify its combining capacity with hepatocellular carcinoma cells, making preparation for the further targeting probe synthesis and tumor targeting imaging.2. To develop a novel fluorescent probe by labeling the L5 with 5-carboxyfluorescein (FAM), then verify its ability to "paint" the hepatocellular carcinoma and assess its potential as a fluorescent molecular probe for hepatocellular carcinoma.3. To develop a novel PET molecular probe using 18F-labeled L5 peptide and to investigate its potential for the diagnosis and delineation of the hepatocellular carcinoma by PET/CT.Methods1. Synthesis the GPC3-targeting peptide of L51). Synthesis of the GPC3-targeting peptide of L5L5 peptide was synthesized using standard solid phase peptide chemistry from Fmoc-protected amino acids. Symmetric anhydride method was used for by coupling of the first amino acid at the carboxyl terminus to 2-Chlorotrityl chloride resin. At the end of the reaction, sufficient pyridine and acetic anhydride were added to block remained active site on the resin. Amide bond coupling of next amino acid was achieved with HOBt/HBTU/DIEA as the peptide coupling reagent and repeated until the last amino acid was coupled to peptide resin. After the reaction was complete, triflouoroacetic acid as main cleavage reagent was used to harvest the peptide from the resin. Removal of the solvent by rotary evaporation gave a crude oil that was triturated with cold ether. The crude mixture obtained was then centrifuged, the ether was removed by decantation, and the resulting white solid was purified by high performance liquid chromatogram (HPLC). The product was isolated by lyophilization and characterized by electrospray mass spectrometry.2). Labeling the L5 and the control peptides with 5-carboxyfluorescein (FAM)5-FAM contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. During the synthesis of L5,Fmoc-Lys(Dde)-OH was coupled to the peptide to end chain of the peptide. After the completion of the labeling, the resulting solid was purified by HPLC. The product was isolated by lyophilization and characterized by electrospray mass spectrometry.3) The peptide L5 labeled NOTA was synthesized by the company of Chinese Peptide.2.The combining capacity of GPC3 targeting peptide in vitro1) Indirect immunofluorescence assay verify GPC3 expression on HepG2 cells and the normal liver cells-HL7720Cells were cultured on six-chamber slides. After fixed with 4% paraformaldehyde solution(PSF), the slides were incubated with anti-GPC3 first antibody (diluted 100 times with 5% bull serum albumin) in 4℃ for all night,then washed and incubated with Cys3-conjugated second antibody.Imaging was obtained from fluorescent microscope.2).In vitro binding and block test of FAM-L5 with HepG2 cells and HL-7702HepG2 cells and HL-7702 cells were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI1640 containing 10% fetal bovine serum at 4℃ in a humidified 5% carbon dioxide-containing atmosphere.HepG2 cells were the experiment cells, while HL-7702 cells were the control cells. For fluorescence microscopy, cells were seeded on cover slips in 24-well plates and incubated in RPMI1640 (0.5 ml/well) overnight. HepG2 cells were incubated at 4℃ for 1 h with different concentration of OμM,1 μM,4 μM,10 μM,20 μM and 40 μM solution of a FAM-L5 in PBS/1%BSA.For the blocking experiment, the HepG2 cells of two wells were first treated with 20μM non-conjugated L5 peptide either and then incubated with 1 μM and 4μM FAM-L5 at 4℃ for 1 h. After that, the cells were washed three times with phosphate-buffered saline (PBS). Slides were visualized under a blue light using a fluorescent inversion microscope (Olympus IX71).2) The relative intensity of fluorescence comparison of quantitative analysis between HepG2 cells and HL-7702 cells.①HepG2 cells and HL-7702 cells were cultured in log phase, and resuspended after centrifugation digest. Cells were passaged in 6-well plates.②The adherent cells were incubated at 4℃ for 1 hr with different concentration of 0μM,5 μM,10 μM,20 μM,40 μM,60 μM and 80 μM,100 μM, 120 μM solution of a FAM-L5 in PBS/1% BSA. All cells were washed 3 times,5 minutes once,then digested by pancreatic enzymes. Cells were collected to analysis cell fluorescence intensity relative by Flow cytometric.3. Fluorescent Imaging of FAM-L5 in the tumor modal1)Tumor modalTo produce HepG2 tumors, a total of 1×106 cells(HepG2) were injected intramuscularly into the left flank of BALB/C athymic nude mice (purchased from the Laboratory Animal Center of the Southern Medical University).Tumor isografts were monitored until tumor size was≈1 cm in largest diameter after 4-6 weeks later. To confirm the successful establishment of the tumor modal, the histopathologic examination was performed. HepG2 xenograft models were sacrified and the tumor was removed. Tumor tissue was collected and fixed with formalin. Tissue sections of 3μm thinness were prepared and placed on the clean glass slide. HE staining was performed routinely.The same condition to produce U87MG tumors, a total of 1×106 cells(U87MG) were injected intramuscularly into the left flank of BALB/C athymic nude mice (purchased from the Laboratory Animal Center of the Southern Medical University). Tumor isografts were monitored until tumor size was≈1 cm in largest diameter after 4-6 weeks later. To confirm the successful establishment of the tumor modal, the histopathologic examination was performed. U87MG xenograft models were sacrified and the tumor was removed. Tumor tissue was collected and fixed with formalin. Tissue sections of 3 μm thinness were prepared and placed on the clean glass slide. HE staining was performed routinely.2)The tumor tissue and liver tissue fluorescent imagingHepG2 tumor-bearing nude mice and U87MG tumor-bearing nude mice were intravenously injected with 150μl of 1mM FAM-L5 peptides. After 1 h circulation, mice were killed and tumor and liver were removed. The tumor tissue and normal liver tissue sections were prepared and mounted in DAPI (4’,6-diamidino-2-phenylindole)-containing mounting media. Some of the sections were prepared for immunofluorescence test. After that, they were collected and viewed under blue light, green light and purple light using an Olympus DP71 fluorescent microscope (Olympus America, Center Valley, PA, USA).3). Fluorescent biodistribution in the HepG2 hepatocellular carcinoma xenograft model.HepG2 hepatocellular carcinoma bearing nude mice were intravenously injected with 150μl of 1mM FAM-L5 peptides.After 1 hr circulation, mice were sacrificed and tumor and the normal organs were removed. After washing with the saline several times, the tumor and the normal organs were collected. The uptake of FAM-L5 in the tumor and normal organs were observed under blue light with exposure times of 60 s, using the Kodak in-Vivo Imaging System F (Kodak, American) and processed for fluorescence intensity analysis, comparing the uptake of organs from U87MG-tumor-bearing injected FAM-L5.4). Fluorescence images analysisUsing the Kodak MI analysis software, the regions of interest (ROI) were drawn around the border of the tumor or normal organs on the fluorescence images and fluorescence intensity was measured. The tumor/non-tumor ratios (T/NT ratios) were calculated by dividing the fluorescence intensity in the tumor to that of the normal organs.4. Synthesis of PET molecular probe of 18F-NOTA-L5 and in vivo PET/CT imaging1) 18F-AlF-NOTA-L5 was radio labeled by chelating NOTA-L5 with Al18F. 18F-L5 was radio labeled by coupling 18F-NFP with-L5 peptide.2). Micro PET/CT imagingMicro-PET/CT scan was performed on a SIEMENS Inveon scanner (Siemens, Germany). The micro-PET/CT studies of HEPG2 hepatocellular carcinoma xenograft model were performed by tail-vein injection of 3.7 MBq (100 μCi) of 18F-L5. Micro PET/CT images were acquired as 10-min static images at 60min after the injection with the mice under isoflurane anesthesia. The images were reconstructed by a 3-dimensional ordered subsets expectation maximum (OSEM) algorithm and CT correction was necessary for attenuation correction.Using the Syngo analysis software, the regions of interest (ROI) were drawn around the border of the tumor or normal organs on the images of micro PET/CT and the radioactivity was measured. The tumor/non-tumor ratios (T/NT ratios) were calculated by dividing the radioactivity in the tumor to that of the normal organs.Statistical AnalysisThe descriptive data were expressed as mean ± standard deviation. Statistical Package for the Social Sciences, version 19.0 (SPSS Inc.), was used for statistical analysis. The independent samples t test was used for the statistical comparison of the relative intensity of fluorescence that HepG2 cells and HL-7702 cells combined with FAM-L5, respectively. The paired sample t test was used for the statistical comparison of T/non-NT ratios of the relative intensity of fluorescence between experimental tumor HepG2 and control tumor U87MG after intravenous injection. A P value of less than 0.05 was considered statistically significant.Results1. Synthesis of the GPC3-targeting peptide of L51).Synthesis of the GPC3-targeting peptide of L5The obtained L5 peptide by solid phase synthesis showed as the white powder. The molecular mass (m/z:Da) determined by Mass chromatographic analysis (ESI) was 1626.1, which was in accordance with calculated 1625.86. It indicated GPC3-targeting peptide of L5 was correctly synthesized. The final product was purified by preparative HPLC. The purity of L5 peptide was 99.15% as determined by analytical HPLC.2). Synthesis of FAM-L5The final products showed as pallide-flavens solid powder. The molecular mass (m/z:Da) determined by Mass chromatographic analysis (ESI) was 1985.0, which was in accordance with calculated 1984.18. The final products were purified by preparative HPLC. The purity of tLyP-1 peptide was 98.44% as determined by analytical HPLC.3) Synthesis of NOTA-L5The NOTA-L5 peptide by solid phase synthesis showed as the white powder. The molecular mass (m/z:Da) determined by Mass chromatographic analysis (ESI) was 1985.0, which was in accordance with calculated 2131.0. It indicated NOTA-L5 was correctly synthesized. The final product was purified by preparative HPLC. The purity of NOTA-L5 peptide was 95.4% as determined by analytical HPLC.2.The combining capacity of GPC3 targeting peptide in vitro1) Indirect immunofluorescence assay verify GPC3 expression on HepG2 cells and the normal liver cells-HL7720.The result showed that GPC3 was highly expressed on HepG2 cells and it was poorly expressed on HL-7702 cells by confocal microscopy.2).In vitro imaging of combining capacity of FAM-L5 peptides with HepG2 cellsHepG2 cells were incubated with different concentration of FAM-L5 peptide at 4 ℃ for 1 hr. The present study showed that FAM-L5 can be strongly uptaked by the HepG2, even at a very low concentration of 1 μM. The uptake of FAM-L5 in HepG2 cells seemed to increase slightly with the increase of concentrations. The cells which treated with PBS as a control did not show any green fluorescence. This result suggested that FAM-L5 can be intensely uptaked by the HepG2 cells.For the blocking experiment, the cells of two wells were first treated with 20μM non-conjugated L5 peptide either and then with 1 μM and 4Mm L5. The study showed that the uptake of FAM-L5 in HepG2 cells was dramatically inhibited by incubation with excessive quantity of non-conjugated L5 peptide. The FAM-15 uptake in HepG2 cells blocked by 20 times excessive quantity of non-conjugated L5 peptide was lower than that of 5 times block. The result suggested that FAM-L5 and non-conjugated L5 peptide have the relationship of competitive inhibition, which is on the accordance with the principle of the receptor-ligand competitive binding.3. Fluorescent Imaging of FAM-L5 in the tumor modal1).Tumor modalThe tumor models were generated by intramuscularly injection of HepG2 cells into the left flank of BALB/C athymic nude mice. Fourteen days after inoculation, the tumors were visualized. At 4-6 weeks after inoculation, the tumors were found to grow up to about 1.0cm in diameter. There were 6 groups of tumor modals generated in different time with 5 mice in each group. The achievement ratio of tumor modal generation was 83.3%(25/30).The tumor of the above animal modal was confirmed to be the hepatocellular carcinoma by the histopathologic examination.2).The tumor tissue fluorescent imagingAfter 1 hr circulation of FAM-L5 peptides in the HepG2 tumor-bearing nude mice, the mice were sacrificed and the tumor tissue fluorescent imaging was performed. The confocal microscope images showed FAM-L5 was mostly accumulated in the tumor tissue at 1 hr after intravenous injection of FAM-L5. FAM-L5 uptake in the normal liver tissue was found to be very low and the distribution of fluorescence was not in the blood vessels. The low uptake of FAM-L5 in the brain was suggested to be benefit for the detection of hepatocellular carcinoma.3). The fluorescent biodistribution in the HepG2 xenograft modelAfter 1 h circulation of FAM-L5 in the HepG2 tumor-bearing nude mice. The mice were sacrified and the fluorescent biodistribution of FAM-L5 was studied. The study demonstrated that FAM-L5 was intensely uptaked by the tumor, whereas the uptake of FAM-L5 in the liver was minimal. The uptake in the tumor was significantly higher than that in the normal liver with Tumor/brain ratios was 14.28±37.90. Meanwhile, except those of gallbladder and intestine, T/NT ratios of other organs were all larger than 2.0. The present study showed that the fluorescent distributions in the gallbladder and intestine were very high which indicated that FAM-L5 excreted from the body through the hepato-biliary tract. The uptake of FAM-L5 in other organs, such as brain heart, lungs, spleen, muscle and kidney was also very low.FAM-L5 in the control tumor U87MG (T/NT=1.44±0.53)was lower than that in HepG2 tumor (t=3.69,P=0.008).4. Synthesis of PET molecular probe of 18F-L5 and in vivo micro PET/CT imaging1) Synthesis of PET molecular probe of 18F-L5With the guidance of Pro. Tang Ganghua,18F-FP-L5 was produced.18F-NOTA-L5 was radio labeled by chelating NOTA-L5 with 18F-AlF. The total reaction time was about 60 min. The overall radiochemical yield without decay correction was 14.6%-69.8%.2) The in-vivo PET/CT and micro PET/CT scan.2) in vivo micro PET/CT imagingThe in-vivo micro PET/CT imaging of subcutaneous HEPG2 xenograft model was performed at 60min after injection of 18F-L5.The study showed 18F-NPF-L5 accumulated a lot in the tumor at 60min after injection. The radioactivity distribution in the gallbladder,intestine, kidney and the bladder were very high. No obviously radioactivity was found in the brain, lung muscle and bones. The Tumor/liver ratios at the time point of 60 min was 1.46.The study showed 18F-NOTA-L5 accumulated a lot in the tumor at 60min after injection. The radioactivity distribution in the gallbladder,intestine, kidney and the bladder were very high. No obviously radioactivity was found in the brain, lung muscle and bones.Conclusions:1. In the present study, we successfully synthesized a GPC3 targeting peptide of L5 with a high purity of 99.15%.2. We successfully synthesized a fluorescent molecular probe by labeling the L5 with 5-carboxyfluorescein (FAM) with a high purity of 98.44%.3. In vitro imaging study confirmed that FAM-L5 can be intensely uptaked by the HepG2 hepatocellular carcinoma cells. The uptake of FAM-L5 in the HepG2 cells was in accordance with the principle of the receptor-ligand competitive binding.4. The tumor tissue fluorescent imaging study indicated the main coaggregation sites of FAM-L5 is in the hepatocellular carcinoma tumor, while the uptake in the liver tissue and glioma were low. Meanwhile, the immunofluorescence test showed GPC3 was highly expressed in hepatocellular carcinoma.5. The fluorescent biodistribution study indicated that FAM-L5 was excreted through hepato-biliary tract.6. We successfully synthesized PET molecular probe of 18F-L5 by labeling the 18F.7. The in-vivo PET/CT and micro PET/CT scans indicated that 18F-L5 can target to the tumor. Uptake of 18F-NFP-L5 and 18F-NOTA-L5 in the tumor was very intense with the high T/B ratios. The biodistribution of 18F-NFP-L5 and 18F-NOTA-L5 was similar to that of FAM-L5.8.FAM-L5、18F-NFP-L5 and 18F-NOTA-L5 showed a potential to become a good PET tracer for imaging of hepatocellular carcinoma.