Screening Peptides Targeting Pancreatic Cancer Cells Using Phage Display Technology And Inhibiting Pancreatic Cancer Growth

Background: Pancreatic cancer(PC)is a highly malignant tumor of the digestive system.Early pancreatic cancer is often difficult to diagnose due to its atypical clinical symptoms and lack of specificity for early screening indicators,thus losing the opportunity for radical surgical resection.Advanced pancreatic cancer is less sensitive to clinical radiation therapy and chemotherapy,and the side effects of radiotherapy and chemotherapy have a greater impact on the patient’s body.The prognosis of patients with pancreatic cancer is poor.We want to increase the targeting of anti-tumor drugs to improve the efficacy of anti-tumor drugs on pancreatic cancer and reduce damage to normal cells in the body.On the issue of targeting,we use phage display technology for screening.For anti-tumor drugs,we selected KLA((KLAKLAK)2)apoptotic peptide as a therapeutic peptide.Compared with chemotherapeutic drugs,KLA apoptotic peptide has a small molecular weight,strong tumor penetration ability,and low immunogenicity,good biocompatibility,low toxicity,easy synthesis and modification[12-13],is an ideal anti-tumor drug.However,KLA apoptotic peptides are difficult to penetrate the cell membrane,and after entering the cell,they cannot destroy the mitochondrial membrane to induce apoptosis [16].Therefore,the purpose of this study is to use phage display technology to screen out targeted peptides that can specifically act on pancreatic cancer cells.The targeting of the peptide is used to realize imaging diagnosis of pancreatic cancer.And the peptide is combined with the apoptotic peptide KLA,in order to realize that the fusion peptide is targeted to pancreatic cancer cells and inhibits the growth of pancreatic cancer cells and tumors,and the related mechanism was studied.Methods: In this study,human pancreatic cancer cell line PANC-1 was selected as the target cell.First,we used phage display technology to screen out phages that targeted PANC-1 cells.Second,the screened phages were amplified,DNA was extracted and sequenced.The amino acid sequence was deduced by comparison with the brief genetic code table provided by the kit.The targeting peptide HMNPWSD was synthesized according to the obtained amino acid sequence.The peptide was labeled with fluoresceinisothiocyanate(FITC)and incubated with PANC-1 cells.Its affinity was evaluated by using cell immunofluorescence experiments and flow cytometry.The target peptide HMNPWSD was conjugated with the KLA peptide,and the cell immunofluorescence experiment and in vivo imaging system(IVIS)were used to assess the affinity of target peptide for the cell in vitro and Tumors in vivo.The effect of HMNPWSD-KLA fusion peptide on the proliferation function of PANC-1 cells was test by MTT assay.Flow cytometry analysis,Western Blot,and mitochondrial membrane potential assessment were performed to study the mechanism of PANC-1 cell growth inhibition.Finally,we established a nude mouse model bearing PANC-1 tumors,and then observed the effects of the fusion peptide HMNPWSD-KLA on the growth of PANC-1 tumors in vivo through animal experiments.The side effects of the fusion peptide HMNPWSD-KLA were evaluated by organ tissue sections and related hematological parameters of nude mice.Results: We screened out the peptide HMNPWSD targeting PANC-1 cells by phage display technology,and the results of cell immunofluorescence and flow cytometry analysis confirmed its specificity for PANC-1 cells.At the same time,the specific affinity of the fusion peptide HMNPWSD-KLA to PANC-1 cells and tumors was confirmed by immunofluorescence and IVIS experiments.The inhibitory effect of the fusion peptide on the growth and proliferation of PANC-1 cells was verified by the MTT assay.The result of flow cytometry analysis,Western Blot,and mitochondrial membrane potential evaluation prove that the mechanism for inhibiting the growth of PANC-1 cells is through the destruction of cell mitochondria and inducing apoptosis.The experimental results of a nude mouse model bearing the PANC-1 tumor proved that the fusion peptide can significantly inhibit the growth of PANC-1 tumor.In addition,it has been proved that the fusion peptide has little effect on nude mice by examining organ tissue sections and hematological parameters of nude mice.Conclusion: The fusion peptide HMNPWSD-KLA has targeting specificity for pancreatic cancer cell line PANC-1 cells and tumors.Moreover,the fusion peptide induces apoptosis by destroying the mitochondria of PANC-1 cells,which significantly inhibits the growth of cells and tumors.In addition,the fusion peptide has little side effects on important organs and hematological parameters of nude mice.