Basic Study Of Specific Binding Peptide Screened By Phage Peptide Library Technique In Tumor Imaging

Objective:The aim of this study was to screen a twelve animo-acid peptide with highspecificity and binding affinity for florescence imaging and radionuclide imaging. Thephage peptide library technique was introduced in the primary screening, and thesurface magnetic resonance technique was added to verify and re-screen the bindingpeptides. Florescence and radionuclide were used to label the winner peptide. Tumortargeted ability and biodisturibution of each labeled fashion were evaluated in thetumor-bearing animal model.Method:Through bioinformatics methods, we obtained a conservative sequence on thesurface of MT1-MMP cytilic protein. The peptide was synthesized through solidphase method. Using this peptide as the target, the phage peptide library techniquewas adopted to screen the binding peptides.The phage clones with preferable bindingaffinity to MT1-MMP conservative peptide were obtained via four rounds ofscreening. Seven clones were selected and were sequenced based on thesingle-strained DNA. ELISA was used to test the binding affinity of the selectedphages to the conservative peptide.Three phages with favorable KD value wereobtained. We synthesized the three peptides through solid phase method. The surfaceresonance technique was introduced to assess the interaction between the screenedpeptide and MT1-MMP cytilic protein in solution. A direct method was adopted, thatis, to attach the protein to the surface of the chip, when the sample solution flowsthrough the surface of the chip, the molecular being analyzed would bind to thesurface of the chip. The KD value and other kinetic parameters between the threepeptides and MT1-MMP protein were obtained using software Biaevaluation. Wetherefore chose AF7P peptide with high binding affinity and specificity to MT1-MMPprotein for imaging study. Cy5.5-AF7P was obtained by labeling AF7P with florescence. We chose MDA-MB-435tumor cell with high MT1-MMP expressionand A549tumor cell with low MT1-MMP expression to prepare tumor bearing modelseparately. Nine mouse were prepared for each model,and were divided into threegroups for further use. We performed tumor imaging study2h,4h, and24h afterinjecting100μL Cy5.5-AF7P through the tail vein, the targeting ability ofCy5.5-AF7P towards MDA-MB-435and A549tumor tissue were compared. A groupof tumor bearing mouse was sacrificed after the4h imaging point, the liver, lung,kidney, spleen, intestine, musle, heart, bone, tumor and blood were obtained andspreaded on a black paper. By using a unique software Maestro for qualitativelyanalyzing the OD value of the interested zone, we compared the biodistribution ofCy5.5-AF7P in each organ between MDA-MB-435and A549tumor bearing model.To obtain an radionuclide molecular probe,We radiolabelled AF7P with^99mTc byusing HYNIC as bi-functional linker and by using tricine and TPPTS as collaborativeligands. Twelve MDA-MB-435mouse were injected with100μL^99mTc-HYNIC-AF7P(11.1MBq/kg), through eye blood sampling2,5,10,15,20,30,45and60min afterthe injection, we obtained the radioactive counts on a γ counter. The%ID/g value wascalculated and a blood discharge curve was drawn.The tumor-bearing mouse modelwas sacrificed at the05h,1h,2h and4h after injection of100μL^99mTc-HYNIC-AF7P(11.1MBq/kg). The liver, lung, kidney, spleen, intestine, muscle, heart, bone, tumorand blood was obtained and weighed. Each organ was placed on a γ counter to obtainradioactive counts. After calibration, the%ID/g and T/NT value was calculated. Thebiodistribution of^99mTc-HYNIC-AF7P in MDA-MB-435tumor bearing mouse wasevaluated. We performed tumor imaging study2h after injecting100μL^99mTc-HYNIC-AF7P (11.1MBq/kg) into MDA-MB-435tumor bearing model throughtail vein, the targeting ability of^99mTc-HYNIC-AF7P to the MDA-MB-435tumortissue was evaluated.Result:(1) The highly conservative sequence on the surface of MT1-MMP protein cytalyiczone was synthesized: R160EVPYAYIREGHEKQ174. Using this peptide as thetarget, we screened the phage display twelve peptide library and obtained threepeptides with preferably binding affinity. HWKHLHNTKTFL (AF7P),HMLSRHDHSYSL(AF4P)and HGKYLGAHLHKY(AF3P).The KD value between the three phage clones and the conservative peptide was0.075,0.188and2.92nM, respectively.(2) The interaction of the three peptide with MT1-MMP cytilic protein was tested bysurface resonance technique, The KD value between the three peptides and theMT1-MMP was109μmol (AF7P),132μmol (AF4P) and444μmol(AF3P),respectively.(3) By labeling AF7P with florence, molecular probe Cy5.5-AF7P was obtained,2hafter injection in MDA-MB-435tumor bearing mouse with high MT1-MMPexpression, the tumor was clearly imaged,4h after injection the tumor can stillbe clearly seen. The whole-body radioactive background decreased. The tumortargeting ability of Cy5.5-AF7P to MAD435tumor was significantly better thanA549tumor. By qualitative analysis of biodistribution of Cy5.5-AF7P in eachorgan of MDA-MB-435tumor bearing model.We found that Cy5.5-AF7Pdemonstrate strong optical signal in the kidney, which indicates Cy5.5-AF7Pmetabolized through kidney.(4) We radiolabelled AF7P with^99mTc by using HYNIC as bi-functional linker andby using tricine and TPPTS as collaborative ligands.The radionuclide molecularprobe^99mTc-HYNIC-AF7P (tricine)(TPPTS) was obtained. The labeling rate of^99mTc-HYNIC-AF7P was above95%tested by radio-HPLC. Under37℃,theradio-purification of^99mTc-HYNIC-AF7P was constantly above93%for6h in0.9salty water, which demonstrated a favorable stabilization quality. Thepharmacokinetics in MAD435tumor-bearing model indicated that10min,20minand60min after injection, the radioactive count percentage in the blood was19.32%,7.21%and1.27%, respectively,which indicated the probe dischargeswiftly in the body.(5) Biodistribution of AF7P in MDA-MB-435tumor-bearing mouse indicated: After0.5h,1h,2h and4h injection of^99mTc-HYNIC-AF7P, the uptake value of theprobe in the MAD435tumor cell was3.37,2.59,2.18and1.05%ID/g,respectively. The kidney and liver was constantly with high uptake value.4h afterinjection, the uptake value of kidney and liver were (4.33±0.20)%ID/g and(2.55±0.10)%ID/g, respectively. The SPECT imaging demonstrated that2hafter injection, the tumor was with favorable imaging quality. Conclusion:MT1-MMP is one of the most important members of MMPs family. It has anessential role in the tumor angiogenesis and tumor development. To our bestknowledge, there has been not any peptide-based probe being reported for specificallytargeting MT1-MMP until now. Our study demonstrates that adopting emergingscreening techniques-phage display library and surface magnetic resonancetechnique, specific peptide-based probe can be developed and can pose greatsignificance in the diagnosis and therapy of tumors with high MT1-MMP expression.