Screening Of Membrane Type-2 Matrix Metalloproteinase Specific Affinity Peptide And Application In Fluorescence Imaging Of Lung Cancer

Background:Lung cancer is one of the most common malignancies in China currently.According to data reported by the National Cancer Center in 2017,lung cancer is the leading cause of morbidity and mortality.More than 67.6%of newly diagnosed lung cancer cases are located in stage ? and ?.In patients with stage IA and stage I B subjected to comprehensive treatment,the 5 year survival rate was about 60%-80%.While 5 year survival rate for stage? A,? B,and IV was only 14%,5%,and 1%,respectively.Radical surgery of early detected tumor is most likely to cure lung cancer and increase five year survival rate.Based on existing medical imaging techniques,molecular imaging can visualize living biological processes at the cellular and molecular levels,and special molecular events that play an important role in the occurrence and progression of disease.Thus it can be used in the early diagnosis of diseases,intraoperative imaging,drug development,and monitoring of therapeutic responses.In molecular imaging studies,tumor targeting peptides have become a hot topic,because of their high specific binding ability to target sites and their good pharmacokinetics and biosafety.The molecular markers of different biological behaviors of tumors are potentially ideal targets for molecular imaging diagnosis of tumors.Membrane type-2 matrix metalloproteinase(MT2-MMP)is overexpressed in lung cancer.MT2-MMP is associated with the degradation of extracellular matrix,and promotes the epithelial-mesenchymal transition of tumor cells.In addition,as an anti-apoptotic factor and angiogenesis-inducing factor,MT2-MMP may be a potential target for molecular imaging diagnosis of lung cancer.At present,no peptide ligands that specifically target MT2-MMP have been discovered.Therefore,in this study,we used MT2-MMP as a target to screen out the specifical target of MT2-MMP binding peptide(MT2-AF5p)by phage display peptide library screening technology.Then we verified affinity of MT2-AF5p in vitro and specificity by using ELISA,atomic force microscope and isothermal titration calorimetry.Finally,MT2-AF5p was fluorescently labeled for molecular imaging both in vivo and in vitro.Method1.Using MT2-MMP surface specific peptide as target,four rounds of phage display peptide libraries were strictly screened for reduction,and through the continuous optimization of screening strategies,positive phage clones that specifically bind to target molecules were screened out.2.Positive phage clones were screened by ELISA to remove non-specific binding and false positive phage.3.Positive phage clones were subjected to DNA sequencing to compare the corresponding peptide sequences and to evaluate the presence or absence of a common motif.4.Catalytic domain of MT2-MMP was expressed,purificated and refolded.ELISA was performed to identify the targeting of MT2-AF5p to structural proteins subsequently.5.Positive phage displayed peptides were synthesized by solid phase synthesis.The force spectrum curves of preponderant peptides and target molecules were evaluated by atomic force microscopy.Affinity was evaluated by recording the magnitude of statistical dissociation force of hundreds of force spectrum curve.Meanwhile,blocking experiment was performed to examine the binding specificity.6.Isothermal titration calorimetry further determined the binding affinity and specificity of MT2-AF5p with target peptide or target protein,and selected better targeted superior peptides.7.The ONCOMINE database was used to search for microarray data of mRNA expression levels in clinical specimens of lung cancer MT2-MMP,and post-evaluation of differential expression was performed.8.Immunohistochemistry,Western Blot and immunocytochemistry were used to detect the expression of MT2-MMP in lung cancer tissues and different lung cancer cell lines.9.FITC and quantum dots were labeled with MT2-AF5p(FITC-MT2-AF5p,QD@MT2-AF5p),and cell imaging was used to evaluate the targeting of peptide probes to high expression for MT2-MMP in lung cancer cells.10.Cell adhesion assays evaluated the binding of different concentrations of peptides to MT2-MMP high expression lung cancer cell lines.11.MTT was utilized to examine the proliferation of cells of FITC-MT2-AF5p and QD@MT2-AF5p and cytotoxicity was detected.12.Lung cancer bearing mice were constructed to evaluate the tumor targeting capacity of QD@MT2-AF5p in vivo.Result:1.Phage display technology obtained a series of positive clones that specifically bind to target molecule.The final round of yield can reach 8.46×10-1.A total of 16 candidate polypeptides were obtained by ELISA screening.The common motif would be PPX.2.Six high affinity peptides with MT2-MMP catalytic domain were screened by ELISA,and the Kd values were in the nanomolar range.3.MT2-AF4p and MT2-AF5p were the dominant peptides in the candidate peptides by atomic force microscopy.The highest frequency of interactions with target peptides occurred at 240 pN and 275 pN,respectively.The highest frequency of interaction with target proteins was 225 pN and 250 pN.Blocking experiments confirmed the intermolecular interactions were specific binding excepting MT2-AF6p.4.Isothermal titration calorimetry confirmed that MT2-AF4p and MT2-AF5p have good binding specificity and sensitivity to target molecules.The Ka value of MT2-AF4p was 4.19×104±8.8×103 and the binding site was 0.59.The Ka value of MT2-AF5p was 5.36×104±5.52×103,and the binding site was 0.64.Depending on in vitro binding test,MT2-AF5p is the best affinity peptide.5.Validation experiments confirmed that MT2-MMP was highly expressed in lung cancer tissues compared with adjacent normal tissues,as well as in lung cancer cell lines.6.Fluorescence labeled cell imaging and cell adhesion experiments showed that MT2-AF5p can specifically bind to the highly expressed MT2-MMP lung cancer cell lines.MTT and hemolysis experiments confirmed that the fluorescently labeled MT2-AF5p was not cytotoxic.In vivo imaging of small animals confirmed that fluorescently labeled MT2-AF5p can be used for in vivo imaging of MT2-MMP high expression lung cancer.Conclusion:1.In this study,phage displayed peptide technology was used,and the screening strategy was continuously optimized.MT2-MMP specific affinity peptide MT2-AF5p(HHRLHSAPPPQA)was screened by a variety of different identification techniques.2.Fluorescently labeled MT2-AF5p can specifically bind to MT2-MMP high expression lung cancer cell lines and tumor bearing mice with lung cancer.3.Molecular imaging peptide probe screening strategy targeting specific markers in lung cancer biology opens up a new way for lung cancer early diagnosis,intraoperative optical imaging,therapeutic evaluation,and has important application prospects.