Further Screening And Identification Of Human Liver Cancer Specific Binding Peptides

Background and ObjectiveHepatocellular carcinoma(HCC)is a common solid malignant tumor and the important killer to people in worldwide.So far,especially in China,most of HCC patients lost their chance to detect HCC early,and their medical treatments are just depended on the surgical operation with a high recurrence rate and chemical therapy with the severe fatal side effects.It is difficult to diagnose HCC early that results in the lost of surgical operation when HCC was detected.The selection and identification of cancer targeting molecular fragment is hopeful approach to have people to diagnose HCC early and treat HCC patient by a targeting delivery way,and phage displayed peptide library technology is a useful method to meet this need.Comparing monoclonal antibody,targeting peptide is easier to penetrate tumor tissue and more specific to bind targeted cells because peptide is a type of small and short molecule.Therefore,cancer targeting peptide is of the important potential to be used a probe or targeting element of vector or nanomedicine for the early diagnosis and targeted therapy of cancers.Early diagnosis of HCC is the key to improve survival rates of HCC patients.Based on the previous experimental data in our lab,the current study selected and characterized the positive phage clones further to confirm their specificity and sensitivity to HCC,and the peptide probe was then synthesized and characterized.We believe that this study will be an important basic study to establish a novel approach for the early diagnosis and targeting therapy of HCC.Methods1.The biopanning of a PhD 12 peptide library was used to screen the positive phage clones with the good affinity to HepG2 cells.This job was previously finished in our lab,and two phage clones(PC6?PC28)were selected.2.Cell immunofluorescence and immunocytochemical methods were used to compare and confirm the specificity/sensitivity of the two phage clones.3.The probe synthesizes.The specific probe was synthesized following the peptide sequence in the best phage clone and labeled at N terminal of the peptide with FITC.4.The specificity/sensitivity of probe was characterized by using immunofluorescence,immunocytochemistry,flow cytometry and competitive inhibition methods on the cultured cells of different cancers from digestive tract.5.The MTT assay was performed to test the cytotoxicity of the peptide to cells.6.The peptide binding was characterized under a laser confocal microscope.7.The assay of clinical HCC tissue chips were used to characterize the probe's affinity to the HCC tissues from patients with different pathological stage.8.The analysis of the peptide bioinformatics to explore the specific affinity depended motif spatial structural frame.9.The prediction analysis to explore the target molecule of Hepa probe on HCC cell surface by related dabase.Results1.The result of immunofluorescence and immunocytochemical assay indicates that the phage clone,PC28 is best to bind HCC cells.2.The peptide probe Hepa probe was synthesized based on the sequencing result of PC28.3.The specificity and sensitivity of the FITC labeled peptide probe were showed as great by the results of immunofluorescence,competitive inhibition assay,and flow cytometry.4.The result of MTT assay shown that the probe binding has no significant cytotoxicity to cells.5.The probe was showed to bind to the cell membrane under laser confocal microscope.6.The results of HCC tissue chips indicated that the probe can bind to the most common HCC tissues types,especially to the tissues differentiated at grade ?.7.The analysis of the peptide bio-information explored an important motif spatial structural frame for this specific affinity.8.The results of prediction and analysis support CK-2 may be the target molecule of Hepa probe on HCC cell surface.Summary and conclusionsWe selected and identificated a peptide buinding to HCC specifically and sensitively by using a biopanning process of phage displayed peptide library.The best positive phage clone and the FITC labeled synthetic probe were characterized using a variety of methods.Our results demonstrated that Hepa probe binds to HCC cells specifically and sensitively.Its target molecule on the HCC cell surface may be CK-2.Therefore,the peptide fragment presents the great potential applications in the early diagnosis and targeted therapy of HCC.