Screening For Hepatocellular Carcinoma Cell Specific Binding Peptides Using Phage Display Peptide Library

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world because of the lack of targeting drugs which only kill carcinoma cells but not normal cells in chemotherapy. Therefore, tumor-associated moleculars based research have become one of the most promising and actively explored fields for the development of therapeutic agents against HCC and a variety of other tumor types.Chapter one Screening for hepatocellular carcinoma cell specific binding peptidesObjective :To screening the peptides binding specifically to the hepatoma cells using phage display peptides library and study the affinity of these peptides to hepatoma carcinoma cells.Methods :(1)Whole-cell subtractive screening with phage display 7^TM peptide library was performed on HepG2 cells and normal L-02 cells, and the latter were used as controls.(2)After 4 rounds of panning in vitro, 50 phage clones picked randomly were sequenced to identify the consensus sequence.(3)The affinity of 7 phages with hepatoma cells was examined by enzyme-linked immunosorbent assay (ELISA) to exclude false positive clones and those binding with equal affinity to target cells and controls. According to the sequence analysis and ELISA results, one phage clone was selected to program further identifications.Results:(1)After 4 rounds of panning in vitro, phages binding to the HepG2 cells were enriched from 0.6×10⁴pfu at the first round to 5.856×10⁶pfu at the end of the fourth round of panning (with an increase of the 976-fold); the output/input ratio was also increased from 0.3×10^-7 at the first round to 292.8×10^-7 at the end of the fourth round of panning. Binding of these phages to wild type L-02 cells were decreased.(2) After panning from hepatoma cells in vitro, 50 phage clones were randomly picked and sequenced. 7 sequences were obtained from these 50 clones. S1, S4, S13 were repeated found in the sequences. Among them, S1 was the one with most repeats. Low homology between the peptide sequences displayed by the phages and no known proteins was identified by BLAST analysis.(3) ELISA results suggested that S1, S4, S13, S8, S9, S14, S20 phages could bind differentially to hepatoma cells and control cells. S1 phage was chosen for further analysis.Chapter Two Identification of binding specificity of HCBP1 to Hepatoma cellsObjective : To identify the affinity of S1 phage and peptide HCBP1 to hepatoma carcinoma cells in order to make nano-drug carrier with high target to hepatoma in the future.Methods:(1) In vitro binding assay and immunocytochemical stain were performed to determine the specificity of the phages to hepatoma cells, and immunohistochemical staining was used to examine the binding specificity of the phages to hepatoma cells.(2)Peptides displayed on the phages were synthesized and competitive binding assay was performed to observe the competitive inhibition effect of the peptides with their phage counterparts.(3)Immunoflurescence microscopy was used to study the binding of synthesized peptides to hepatoma cells.Results:(1)In vitro binding assay, immunocytochemical staining and immunohistochemical staining suggested that S1 phage preferably binds to hepatoma cells rather than controls, and phage was also found to be able to bind to hepatoma tissue sections.(2)Competition binding assay revealed a significant competition between the synthesized peptide HCBP1 displayed on the S1 phage and the phage itself while binding to targets.(3)Using immunofluorescence microscopy, fluorescence labeled HCBP1 peptide was seen on the membrane and in the perinuclear cytoplasm of hepatoma cells.Conclusions:(1)7 phage clones binding preferably with hepatoma cells were identified. HCBP1 was binding site of the S1 phage with targets.(2)Synthesized peptide has specificity to hepatoma cells. It may become the targeting molecular to hepatoma and can be used to make nano-drug carriers with high specificity to hepatome in the future.