Targeting Study And Screening Of Three Novel Peptides For High Expression Of Epidermal Growth Factor Receptor In Oral Epidermoid Carcinoma

Objective:To develop new potential targeted peptide drugs for molecular imaging and treatment of overexpressed EGFR human tumors,three PEG-modified peptides were designed,synthesized and identified in vitro and in vivo.Methods:(1)The main binding region of the peptide sequences was designed to recognize the extra-membrane binding domain of EGFR,at one end of the peptide,three molecules of PEG with-NH2 group were connected for improving their hydrophilicity.The amino-sequence of the three PEG-modified peptides has a triple tyrosine structure as a substrate of EGFR receptor tyrosine protein kinase to improve affinity and binding force with EGFR receptor,reduce its receptor activation effect,and also reserve three labeling molecules for radioactive iodine labeling.In addition,the fluorophore FITC was attached to the side chain amino group of lysine residue.The three PEG-modified peptides were named as SPW,Pep4 and Pep6 respectively.The purity of three PEG-modified peptides was identified by HPLC-MS.(2)The EGFR expression of KB as well as L929 cells was detected with Western blot and immunofluorescence.The effect of three PEG-modified peptides on the cell proliferation ability was detected by CCK-8 assay.The binding affinity of the three PEG-modified peptides to KB cells and L292 cells was observed with confocal microscopy.For locating the binding position of peptides to KB cells,the binding site of the three PEG-modified peptides to KB cells membrane was compared with immunofluorescence of EGFR monoclonal antibody.And the distribution of peptides in cells was observed by confocal microscopy.(3)Human Oral cancer-bearing mice model was established by subcutaneous injection of KB cells.Three PEG-modified peptides were injected into the tail vein of mice and imaged in IVIS Spectrum respectively,fluorescent images of mice were collected at 90 min,120 min and 150 min,and the fluorescence signal of tumor site was observed intensely for screening the suitable peptide with obvious binding distribution in tumor site.After screening,the Pep4 was selected for further experiments.The fluorescent images of tumor-bearing mice were obtained at 0 min,30 min,60 min,70 min,90 min,100 min,110 min,120 min,150 min,180 min and 210 min after injection of Pep4 through caudal vein.Perform spectral separation data processing and observe the distribution of Pep4 fluorescence signal in mice over time?The ROI of tumor site and normal tissue was measured at the above time point,and the T/NT ratio was calculated by quantitative analysis.Results:(1)The three new peptides were synthesized succesfully,and the structure sequences were(NH2)PEG3-X-X-X-X-X-Tyr-Tyr-Tyr-Lys(FITC)CO-NH2[SPW],(COOH)Ser-X-X-X-X-X-Tyr-Tyr-Tyr-Lys(FITC)-PEG3(NH2)[Pep4]and X-X-X-X-X-X-Tyr-Tyr-Tyr-Lys(FITC)-PEG3(NH2)[Pep6]respectively.The purity of the three PEG-modified peptides was over 97%.(2)The high expression of EGFR in KB cells and low expression of EGFR in L929 cells were observed with western blot band and fluorescence image.The survival rate of KB and L929 cells treated with different concentration has no significant change(p?0.05).All of three PEG-modified peptides have no cytotoxicity and cell proliferation effect.The binding affinity of the three PEG-modified peptides to KB,4T1 and U251 cells was significantly higher than that of L929 cells.The peptides were bind to EGFR on the cell membrane and enter to the cytoplasm and nucleus by endocytosis.(3)The human oral cancer-bearing mice model was successfully established with100%tumor formation rate.There was no obvious fluorescence signal at the site of tumor nodules in tumor-bearing mice after tail vein injection of SPW and Pep6.The EGFR targeting ability of SPW and Pep6 was poor in vivo.However,there was obvious fluorescence signal at the site of tumor nodules in tumor-bearing mice after tail vein injection of Pep4.The strongest fluorescent signal can be observed at the tumor site during 90-150 minutes after injection because the Pep4 was quickly cleared from blood and tissues,which was excreted through urine.At the 90 and 150 min after injection,the T/NT ratio reached 15.68 and 30.89 respectively.From 180 to 210 minutes after injection,there was no signal in blood and normal tissues due to the completely cleared of Pep4.Conclusion:We obtained a PEG-modified peptide Pep4 suitable for targeted imaging and treatment of human tumor over-expressed EGFR.It has good solubility,tissue permeability,affinity,specificity,rapid clearance properties.It may be a potential targeted drugs worth further study.