Preparation And Characterization Of ⁹⁹Tc^m Labeled ZHER2:V2-Pemetrexed In Vitro

Objective:Human epidermal growth factor receptor 2?HER2?expression in malignant tumors provides important information for the diagnosis and treatment of diseases.Radiolabeled HER2 targeting molecules?antibodies,antibody fragments,short peptides,and scaffold proteins?that can used to diagnose and treatment HER2-positivedisease.In this study,the aim is to investigate the method of ⁹⁹Tc^m labeled epidermal growth factor receptor2 small molecule ZHER2:V2 with pemetrexed,an antitumor drug,and to analyze its binding properties to HER2 receptor in vitro.Method:A small molecule targeting binding protein ZHER2:V2 was synthesized by solid-phase synthesis of Fmoc/tBu polypeptide.The C-terminus was linked to four amino acids,Gly-Gly-Gly-Cys-,forming a strong chelating group of N₃S structure.A variant ZHER2:V2 was formed,and the N-terminus was coupled with the antitumor drug pemetrexed,⁹⁹Tc^m was labeled by a ligand exchange method,and a ⁹⁹Tc^m-ZHER2:V2-pemetrexed molecular imaging probe was prepared.The labeling rate,radiochemical purity,and in vitro stability of the molecular probe were analyzed and determined by high liquid chromatography and paper chromatography.Real-time PCR was used to detect HER2 expression in human lung adenocarcinoma H2009 and H23 cells.The probe was combined with HER2high expression human lung adenocarcinoma H2009,A549 cells and HER2low expression human lung adenocarcinoma H23 cells and 1,2,4,6,12,12and 24h were selected for in vitro cell uptake,retention,internalization and block experiment.Cell uptake experiment,three types of cells grown at logarithmic phase were inoculated in three replicates,and 30?l/well of molecular probe was added.The supernatant was collected at each time point,then the cells were digested and the precipitate was collected to determine the uptake rate.The retention experiment culture method and design were the same as above,and the culture was continued after changing the liquid for 4hours,and then the retention rate was measured.After the supernatant was collected in the internalization assay,p H 2.5 was added to the acetic acid to rinse the wells,and the rinse was collected.The cells were then digested and the precipitate was collected to determine the internalization rate.Blocking experiment.At the time point of 4h,HER2 high-expressing H2009 and A549cells were blocked with an excess of unlabeled ZHER2:V2-pemetrexed,and compared with the unblocked group to observe the uptake rate.The specificity of this molecular probe for targeted binding to HER2 was studied.All measurement data were represented by x±s,and were processed by two-sample t test.The data was analyzed by SPSS 21 statistical software.P<0.05 was statistically significant.Result:The ⁹⁹Tc^m-ZHER2:V2-pemetrexed had an immediate marking rate of?97.11±0.24?%?n=6?and the radiochemical purity was>97%.The stability was good in physiological saline and serum.At most of the time,radiochemical purity was>92%.Real-time PCR showed 2^-??Ct=1.06437.The expression level of HER2 gene receptor on cell surface of H2009 was greater than that of H23 cells.The uptake rate of ⁹⁹Tc^m-ZHER2:V2-pemetrexed molecular probe in H2009 and A549 HER2 high-expressing cells at different time points gradually increased with time,and the uptake rate of H2009 and A549 cells was much higher than that of H23 cells?t=7.846 to 30.793,P=0.010 to 0.000;t=7.794 to 5.353,P=0.001 to 0.032?.The molecular probes had a longer residence time in HER2-overexpressing H2009 and A549cells.The half-clearing time was 12h of both cells,and the retention rate gradually decreased from 1h?84.71±2.21?%to 12h?43.87±3.39?%;from 1h?85.62±2.61?%to 12h?43.74±3.87?%,respectively;The H2009 and A549cells were slowly internalized,and the internalization rates were?45.5±0.94?%and?45.54±4.64?%respectively after 24 h,and the cell membrane binding capacity was strong and the affinity was high.H2009 cells and A549 cell uptake⁹⁹Tc^m-ZHER2:V2-pemetrexed canbe blockedby excess unlabeled ZHER2:V2-pemetrexed,the uptake rates of H2009 cells and A549 cells were significantly decreasedfrom?2.03±0.39?%to?0.01±0.00?%,?t=9.000,P=0.012?and from?3.76±0.15?%to?0.89±0.02?%?t=33.452,P=0.000?,respectively.Conclusion:The ⁹⁹Tc^m-ZHER2:V2-pemetrexed molecular probe has a simple labeling method,high labeling rate and good stability in vitro.And it has a high uptake rate of in vitro HER2 high-expression lung adenocarcinoma cells,long residence time,and specific binding to HER2,which enableittobea novel HER2-targeted molecular imaging probe for HER2 positive tumor imaging and treatment.