Further Identification Of Cervical Cancer Targeting Polypeptides

Background:Cervical cancer is the second most common malignant tumor after breast cancer in female worldwide,and the incidence gradually tend to be younger.Cervical cancer has a more clear pathogenesis,closely related to lifestyle,such as early marriage and pregnancy,prolific,premature or frequent sexual life,smoking,oral contraceptives and pathogens,etc,but human papillomavirus(HPV)infection is the most dangerous etiological agent.Early cervical cancer can be treated by surgery,advanced and recurrent cervical cancer mainly through radiotherapy and chemotherapy,but the clinical outcomes is not ideal.It is urgent to find a low toxicity and effective treatment for advanced and recurrent cervical cancer,and molecular targeted therapy provides a new idea for this problem.The crux of targeted therapy is the identification of targeting molecules with high binding specificity and sensitivity to tumor cells and tissues,and then development of targeted molecular probes and targeted molecular-directed antineoplastic drug carriers.Phage display technology is a popular approach to select the new tumor targeting molecules.It is to insert a gene of an exogenous polypeptide or protein into the phage capsid protein gene and present it on the phage surface in the form of a fusion protein,and then using affinity enrichment to screen the phage expressing a specific peptide or protein.The specific peptides have the advantages of strong targeting,small size,large tissue penetration,easy synthesis,low immunogenicity,and small side effects.They are expected to be coupled with radionuclides and fluorescent markers as a molecular probe for early cancer diagnostic imaging,and most importantly,coupled with antitumor drugs,nanoparticles andchemotherapeutic drug carriers such as liposomes for cancer targeted therapy,which not only greatly improve the efficacy of chemotherapy drugs but also significantly reduce its side effects.Therefor,it has a very wide,attractive social and economic prospects.Our previous experiment screened a 12-peptide phage clone targeted cervical cancer SiHa cells using biopanning,which laid the foundation for further research.Purpose:In this study,we continue to confirm thebinding specificity of these six clones at multi-level and multi-angle,and obtain the best positive clone.Peptide probe is synthesized according to the peptide sequence displayed by the best clone,and identifying its binding specificity and sensitivity to cervical cancer cells and tissus,which will lay the foundation for the further development of cervical cancer targeted Probe.Methods:1.Previous experiment screened six positive phage clones from a 12-mer peptide display library using SiHa and HEK293 as target cells and absorber cells,respectively.2.The binding specificity of positive phage clones to SiHa cells was evaluated by cyto-immunofluorescence,flow cytometryand immunocytochemistry,and the best positive clone was identified.3.Analysis of best positive clone binding specificity by cervical cancer clinical tissuespecimens.4.Synthesizing unlabeled probe peptide,control peptide and FITC labeled probe peptide,control peptide according to the peptide sequence displayed by best positive clone.5.The binding specificity and sensitivity of the peptide probe to SiHa cells was detected by competitive inhibition,laser scanning confocal microscopy and flow cytometry.6.Analysis of peptide probe binding specificity and sensitivity by cervical cancer clinical tissue specimens and tissue ship.7.Peptide probe binding location assay.8.MTT assay for peptide probe cytotoxicity.9.Analysis of physicochemical properties and secondary structure of the peptide by bioinformatics.10.The prediction of peptide probe targeted molecules,and molecular docking using AutoDock4.2.6 software.Results:1.Positive phage clone S7 was identified as the best by flow cytometry,cyto-immunofluorescence and immunocytochemistry.2.Immunohistofluorescence showed that S7 clone homes to cervical cancer tissues with high specificity.3.Unlabeled probe peptide,control peptide and FITC-labeled probe peptide,control peptide were synthesized,and named as CSP3 Probe,Ctrl Probe,FITC-CSP3 Probe,FITC-Ctrl Probe,respectively.4.CSP3 Probe binds to SiHa cells with high specificity and sensitivity through the experiments competitive inhibition,laser scanning confocal microscopy and flow cytometry with various synthetic peptides.5.CSP3 Probe binds to cervical cancer tissues with high specificity and sensitivity through clinical tissue specimens and tissue ship.6.The binding site of CSP3 Probe was located on the cell membrane.7.MTT assay showed that CSP3 has no significant cytotoxicity.8.Through bioinformatics analysis,CSP3 is a weakly hydrophobic molecule,and its secondary structure is random coil.9.ADF-H,a molecule that may interact with CSP3,was searched using ScanProsite,and it may be a CSP3-targeted molecule by molecule docking.Conclusions:Multi-level and multi-angle identification result show that CSP3 Probe targets cervical cancer cells andtissues with significant specificity and sensitivity,and it is expected to be used as a molecular probe for early diagnostic imaging and targeted therapy of cervical cancer by coupling fluorescent markers,nanoparticles,antitumor drugs and chemotherapeutic drug carriers such as liposomes.