Screening Of An Affinity Peptide Specific To MT-loop Of Membrane Type-1 Matrix Metalloproteinase And Its Application In Tumor Imaging

Molecular imaging techniques can be applied to the non-invasive real-time imaging of targets during physiological and pathological processes at a molecular level.These techniques help us understand the expression site,level,distribution,and duration of specific genes and proteins in vivo.Targets of molecular imaging are often biomarkers such as proteins,receptors,and nucleic acids that are abnormally expressed in the body with diseases.Membrane type I matrix metalloproteinase?MT1-MMP?plays an important role in many physiological and pathological processes,such as tissue growth,differentiation,repair,and angiogenesis,especially tumor occurrence,development,invasion,and metastasis.It can degrade many components of the extracellular matrix and regulate the synthesis and secretion of cytokines,hormones,and cell adhesion molecular receptors.The MT1-MMP expression is significantly increased in some histologically destructive diseases,such as rheumatoid arthritis and atherosclerosis,and on the surface of various tumor cells.With the important role of MT1-MMP,this biomarker should be detected and imaged to enhance our understanding of its physiological and biochemical functions.Its detection and imaging are also a good way for the early diagnosis of diseases and the targeted treatment of tumors.The targeting specificity of molecular probes is important in molecular imaging.We compared the structure of matrix metalloproteinases?MMPs?and found that an MT-loop is a unique area of membrane-type matrix metalloproteinases?MT-MMPs?.MT-loop is located on the protein surface,far from the catalytic active site,but it is not involved in catalytic action.The mutation and deletion of MT-loop have no effect on the activity and conformation of MT1-MMP.After comparing the MT-loop amino acid sequences of MT-MMPs,we found that their MT-loop sequences are totally different.With these characteristics,the MT-loop region is an ideal target for exploring MT1-MMP-specific probes.Peptide probes have been widely considered for molecular imaging because of their good pharmacokinetic characteristics,strong tissue penetration,fast blood clearance,and high safety.In our previous study,AF7p,a peptide that targets the MT-loop region,was identified from a 12-mer peptide library by using a traditional phage display?Ph.D.?technology.AF7p performed high binding affinity and specificity to MT1-MMP and can be successfully used to image MT1-MMP expression in vivo.However,the target of AF7p during screening is a synthetic linear structural sequence,which lacks the natural conformation of the MT-loop region.Thus,its binding specificity should be further improved.In addition,MT-loop is a sequence with eight amino acids.The short targeting peptide could reduce the non-specific binding to the region except MT-loop and improve the specificity of the peptide.Besides,the small probe is also better for in vivo metabolism.In this study,we were aimed to screen the 7-mer peptide targeting the natural conformation of the MT-loop region of MT1-MMP,and to explore the application of the peptide for in vivo imaging.At the beginning of the research,we constructed the recombinant protein WT-MT1-MMP,which is the catalytic domain of MT1-MMP,and the mutant recombinant protein D₂-MT1-MMP,which is MT-loop deleted WT-MT1-MMP.The expression,purification,renaturation and activity assays of the two recombinant proteins were performed.Homologous modeling was conducted to verify the different between two proteins in their natural conformation is only MT-loop region.Then,an optimized"subtraction biopanning"strategy was applied by using these two recombinant proteins and the pool in favor of the MT-loop was enriched.ELISA was performed and HS7 peptide with the highest affinity was selected.After the peptide was synthesized and characterized in vitro,ELISA and biolayer interference?BLI?were performed showing that the binding ability of HS7 to WT-MT1-MMP was better than that of AF7p.The binding specificity of HS7 was also verified.The interaction between HS7 and MT1-MMP was explored by computer simulation,verifying that HS7 is a superior probe.This study is the first to apply"subtraction biopanning"to screen the affinity peptides of a MMP family member.In experiments at a cellular level,the cell lines HT1080 with a high expression of MT1-MMP,A549 with a moderate expression of MT1-MMP,and MCF7 with a low expression of MT1-MMP were selected.The cell adhesion experiment indicated the affinity of HS7 to MT1-MMP at the cellular level.Then,the fluorescent dye FITC was labeled with HS7 and used as an imaging probe.After verifying the safety of FITC-HS7 probe by MTT assay,the labeling ability of the probe to the cell lines with different expressing levels of MT1-MMP was determined.With the increased expression of MT1-MMP in the cell lines,the fluorescence on the images was also enhanced.The colocalization experiment of the cells showed that the place where the FITC-peptide was labeled was where MT1-MMP was expressed,suggesting that this binding profile was mainly due to the MT1-MMP expression level of the cells.For in vivo imaging,the near-infrared fluorescent dye Cy5.5,with a high tissue penetrability was conjugated with HS7 and formed Cy5.5-HS7 probe.The tumor bearing mice models of the three cell lines were also prepared.Cy5.5-HS7 probe was injected through the tail vein,and the distribution of the probe was observed at different time points.The results implied that Cy5.5-HS7 could effectively image MT1-MMP-positive tumor in vivo,and the half-life of Cy5.5-HS7 was longer than that of AF7p.The imaging and immunohistochemical analysis of ex vitro organs also confirmed the targeting ability of Cy5.5-HS7.H&E staining of primary organs from xenograft mice model confirmed the safety of the probe.In conclusion,a 7-mer peptide library was used to screen a specific peptide targeting the natural conformation of the MT-loop region of MT1-MMP by constructing a mutant protein and by using the optimized screening strategy.In vitro binding experiments showed that the affinity of HS7 to WT-MT1-MMP was better than that of AF7p,indicating the possibility and advantage of"subtraction biopanning"for screening of peptides that target the region with a native conformation.HS7,the affinity peptide of MT-loop,could be conjugated with a fluorescent dye to form a peptide probe,used for the non-invasive labeling of a MT1-MMP overexpression cell line,and utilized for the imaging of MT1-MMP-positive tumor models in vivo,indicating that HS7 was a superior probe for in vivo imaging.These results suggested that the development of HS7 provided an effective tool for exploring the distribution and activity of MT1-MMP in vivo and played an important role in the prevention,diagnosis and treatment of diseases,particularly tumors.