Preparation Of ^99mTc-Z_HER2:2891 And Imaging In Malignant Tumors

Objective: This study was to develop an improved method labeling a decorated Affibody molecule Z_HER2:2891 with Technetium-99m(^99mTc)using-G?Gly?GGC?Cys?as a chelator and to detect the radiolabeling rate and evaluate the in vivo and vitro stability.The research of cell,which includes cellular uptake,retention kinetics,internalization,and blocking studies,biodistribution and molecular imaging were performed in SKOV3?high HER2 expression?model.The molecular imaging was also performed in MCF-7?low HER2 expression?model.The aim of this study was to evaluate the feasibility of ^99mTc-Z_HER2:2891 used for visualization of HER2 expression in vivo.Methods: The HER2-binding Z_HER2:2891 Affibody molecule with a C-terminal chelating sequence-G?Gly?GGC?Cys?was synthesized by Fmoc/tBu solid phase synthesis.-GGGC,forming a similar N3 S structure,served as a chelating moiety for strong chelation of ^99mTc.The Z_HER2:2891 was labeled with ^99mTc using the ligand exchange method.The labeling efficiency was analyzed by reverse-phase high performance liquid chromatography?RP-HPLC?and paper chromatography.To evaluate the labeling stability in vitro,^99mTc-Z_HER2:2891 was incubated with normal saline and fresh human serum at 37?,respectively,and the radiochemistry purity was detected by RP-HPLC at 1,2,4,6 and 8h.The human ovarian cancer SKOV3 cells?high HER2 expression?were incubated by standing adherent.In logarithmic growth phase,the SKOV3 cells were harvested and cultivated in Roswell Park Memorial Institute 1640?RPMI 1640?medium,then the cell suspension were injected subcutaneously into the right anterior superior limbs of the mice with nearly 1x10⁷ cells in 0.2 ml every mouse.The mice were fed in specific pathogen free environment.The tumors were allowed to be used in experiment when grew to reached a diameter of 1.5-2.0 cm for about 4-6weeks.MCF-7 cells were injected into the left anterior superior limbs of the mice with the same method.To evaluate the stability in vivo of^99mTc-Z_HER2:2891,the urine of the BALB/c nude mice bearing SKOV3 tumor xenografts was collected within 1.5-2h after be injected the molecular probe through tail vein.Cellular uptake,retention kinetics,internalization,and blocking studies of the probe were investigated in the SKOV3 cell lines.For biodistribution studies,16 BALB/c nude mice bearing high HER2-expressing SKOV3 xenografts were randomly divided into 4 groups with 4 mice in each group.Each mouse was injected with labeled conjugates.At 1,2,4 and 6 h after injection,one group of the mice were euthanized.Blood,heart,liver,spleen,lung,kidney,stomach,small intestine,brain,bone,muscle and tumor were collected,weighed and measured for radioactivty.The tissue or organs uptake values were calculated as the percent injected dose per gram tissue?%ID/g?.Ratios of radioactivity in the tumor to that in the opposite side muscle?T/M?were also calculated to evaluate tumor uptake.Then,the biodistribution state in SKOV3 exnografts were analyzed.In molecular imaging studies,five BALB/c nude mice with SKOV3 or MCF-7 xenografts were randomly chosen and injected with ^99mTc-Z_HER2:2891 via the tail vein.In addition,5 mice bearing the SKOV3 tumor xenografts were pre-injected with excess of non-labelled Z_HER2:2891,to saturate the HER2 receptors of tumours.At 1,2,4,6 and 8h after injection,the mice were anesthetized and imaged.The ratio of radioactive counts in the tumor to that in the contralateral equivalent region?target to nontarget ratio [T/NT]?were calculated by drawing regions of interest?ROI?at each time point.All numeric data were expressed as mean ± SD and were performed with A Student t?or t??test or Wilcoxon rank sum test using SPSS?version 13.0?and GraphPad Prism?version 5.0?.P values of less than 0.05 were considered significant.Results: In the RP-HPLC analysis,the retention time of ^99mTc-Z_HER2:2891was observed at about 11.66 min and the labeling rate at room temperature provided yields of?99.29±0.43?% after 20 min?n=6?.The labeling rate was high and needed no purity.The radiochemical purity of ^99mTc-Z_HER2:2891 wasover 96%?n=6?,within 8 h at room temperature,indicating that no significant degradation and off-labeling occurred in fresh human serum or normal saline at 37 ?.RP-HPLC analysis of the urine samples from nude mice administered ^99mTc-Z_HER2:2891 at 1.5-2 h showed 2 peaks and no peak corresponding to free ^99mTc was seen.The peak detected before the retention time may be the degradation fragments of the peptide.The cellular uptake of the molecular probe 99 m Tc-Z_HER2:2891 in SKOV3 cells were?5.27±0.32?%,?5.38±0.74?%,?5.74±1.03?%,?4.50±1.45?%,?5.68±0.95?%,?12.41±1.28?%,?10.79±2.46?%,at 1,2,4,6,8,12 and 24 h,respectively,indicating that the uptake ratios increased as time gone by,except for 6 and 24 h.A maximum of the cellular uptake in SKOV3 cells was found after 12 h incubation with^99mTc-Z_HER2:2891,then,a moderate decrease of the capacity occurred but not significantly.The cellular retention kinetics demonstrated that ^99mTc-Z_HER2:2891residual in the SKOV3 cell was as high as 51.30%.The internalization studies showed that ^99mTc-Z_HER2:2891 combined with the HER2 receptors could move from the cell membrane into the cell.In the cellular blocked studies an excess amount?500 times and 100 times?of unlabeled Z_HER2:2891 was added at the same time with the probe.The blocked studies showed that the uptake rate was decreased from?5.74±1.03?% to?0.45±0.05?% ??0.38±0.04?%,at 500 times and 1000 times,respectively.The results of biodistribution showed that radioactive accumulations in SKOV3 xenografts were very high,and the tumor uptake was?4.14±1.61?,?6.47±2.09?,?5.04±2.58?,?10.07±0.33?% ID/g at 1,2,4 and 6 h,respectively.The ratio of T/M increased from 4.87±0.47 at1 h to 29.94±18.35 at 6h.In addition,a rapid clearance of ^99mTc-Z_HER2:2891 from most of the normal organs except the kidneys.In vivo imaging,SKOV3tumor-bearing mice were visualized as early as 1 h and were shown most clearly at 6 h after the administration of ^99mTc-Z_HER2:2891.There was no significant difference between 6 h and 8 h.There was no obvious tumor uptake in low HER2-expressing MCF-7 xenografts at 1h and afterwards light radioactivity uptake were visualized,but had no radioactivity uptake peak.The tumors in SKOV3 tumor-bearing mice accumulated obviously moreradioactivity than did the MCF-7 xenografts or the blocked SKOV3 xenografts,with significantly difference T/NT ratios between the two types of xenografts at all time points?p<0.05,n=5?.Conclusions: This study showed that ^99mTc-Z_HER2:2891 is a promising imaging agent for HER2 expression in tumors.The label method was simple,and the labeled efficiency was high.Moreover,it had a good stability in vivo and in vitro so this molecular probe could be used in animal imaging directly.This molecular probe showed high specificity for HER2 overexpressing cell,and could be used in high HER2-expression tumor imaging.^99mTc-Z_HER2:2891maybe developed as a potentially new method of tracer for precision diagnosis for high HER2-expressing tumors.