| Objective: To describe and evaluate a novel molecular probe of99Tcm-ZEGFR:1907in vitro and vivo, analyze its targeting binding to epidermalgrowth factor receptor and the correlation between99Tcm-ZEGFR:1907and EGFRexpression level. Build two kinds of mice exnografts whose EGFR expressionlevels are different. In vivo, biodistribution and visualization in micexenografts were performed and compared to determine the feasibility indiagnosing primary and specific EGFR-expressing positive carcinoma.Methods: Affibody molecule ZEGFR:1907was assembled using Fmoc/tBusolid phase peptide synthesis. On the N-terminus of ZEGFR:1907sequence, fouramino acids (Gly-(D) Ala-Gly-Gly), forming a similar N4structure, was usedas a linker for coupling of99Tcmand ZEGFR:1907, and one Aba was introduced asa spacer to eliminate the steric hindrance, thus the affibody molecule would belabeled with99Tcmvia ligand exchange method. The labeling rate of themolecular probe99Tcm-ZEGFR:1907was analyzed by reverse-phase highperformance liquid chromatography (RP-HPLC). To assess the labelingstability in vitro,99Tcm-ZEGFR:1907was incubated with normal saline or freshhuman serum at37℃respectively, and theradiochemical purity were detectedby RP-HPLC at1,2,4,6,12and24h respectively. The human ovarian cancercells SKOV3(EGFR overexpression) and human breast cancer cellsMDA-MB-435S (low EGFR expression) were incubated largely by standingadherent at37℃. In logarithmic growth phase, the two kind of cells wereharvested and cultivated in Roswell Park Memorial Institute1640(RPMI1640) medium respectively, then the cell suspension were injectedsubstaneously into the right anterior superior limbs of the mice with a matterof1x107cells in0.2ml every mouse. The mice were fed in specific pathogenfree environment. After4-5weeks, the size of tumors grew to a diameter of 1-1.5cm, and the exnografts without necrosis could be used in experiment. Theurine of the BABL/c nude mice bearing SKOV3tumor xenografts within2-3hafter injects the molecular probe through tail vein was collected to evaluate themetabolic stability in vivo of99Tcm-ZEGFR:1907. In vitro, cellular uptake andretention of99Tcm-ZEGFR:1907were performed in SKOV3cells andMDA-MB-435S cells to research the targeting binding between99Tcm-ZEGFR:1907and EGFR and find the correlation between99Tcm-ZEGFR:1907and EGFR expression level. To study the binding specificity between99Tcm-ZEGFR:1907and EGFR about100-fold excess unlabeled ZEGFR:1907wereinjected into SKOV3cells20min early before injecting molecular probe. Inbiodistribution studies, sixteen mice from every kind of xenografts wererandomly divided into four groups respectively, and about37KBq in150ul99Tcm-ZEGFR:1907were injected into each mouse through its tail vein. At1,2，4,and6h after injection, four mice were killed at every time point aftercollecting their eye venous blood, and their organs or tissue samples of interest(blood, heart, liver, spleen, kidney, lung, stomach, small intestine, brain, bone,muscle and tumor) were taken out to measure radioactivity and weigh andcalculate their percentage of injected activity per gram of tissue (%ID/g) andthe ratios of tumor to contralateral muscles (T/M) respectively. In blockingstudy,100-fold excess ZEGFR:1907was separately injected into additional fourmice with SKOV3exnografts2h before99Tcm-ZEGFR:1907were injected, and themice were used in biodistribution studies4hours after injecting the molecularprobe. Then, analyze and compare the difference between two kinds ofexnogrfts and the effect of block. In molecule imaging studies, five mice wererandomly chosen from every kind of exnogrfts to inject99Tcm-ZEGFR:1907about37MBq (1mCi) in0.1ml every mouse, and performed to image. In addition,five mice bearing EGFR expressing positive SKOV3xenografts were used inblocking study,100-fold excess unlabeled ZEGFR:1907was injected into lateraltail vein of every mouse at2hours before inject99Tcm-ZEGFR:1907. At1,2,4,6,8and10h after injection, the mice were anesthetized and imaged. To analyzeand compare the radioactive accumulation in tumor region, the ratio of radioactive counts between the tumor part and the contralateral muscle part(T/NT) at each time point was calculated through drawing regions of interest(ROI). All experimental data are shown as average±SD and analyzed withTwo-sample t (or t′) test and Wilcoxon rank sum test by SAS9.1. P value ofless than0.05was considered significant.Results: The molecular probe99Tcm-ZEGFR:1907was identified byRP-HPLC, the analysis showed a single radiation peak with retention time atabout23min and a high labeling rate (96.485±1.42%), and displayed a stablelabeling rate (>95%)within6h. The molecular probe were incubated in twokinds of medium at37℃and detected by RP-HPLC, the result displayed thatthe radiochemical purity were all above90%within6h and the radiation peakswere stable without drift. Urine was collected1h after injection the probe, itsradiochemical purity was above90%within4h, the analysis of urine byRP-HPLC revealed a main radiation peak at about23min and a small peak ofthe probe’s degradation products at19min and without the peak of free99TcmO-4.The cellular uptake of the molecular probe99Tcm-ZEGFR:1907inSKOV3cells was significantly higher than that in MDA-MB-435S cells atevery time point(t=3.28-24.05,t′=16.21,Z=2.80,all P<0.05) except at1h(t=0.32,P=0.7573＞0.05). A maximum (9.97％) of the cellular uptake inSKOV3cells was found after12h incubation with99Tcm-ZEGFR:1907, then, amoderate decrease of the capacity occurred but not a significant, and adifference (from (5.52±0.43)%to (2.69±0.26)%) of the cellular uptake inSKOV3cells was detected before and after the management of block at4h andthe difference was statistically significant (P<0.0001).The cellular retentionkinetics of99Tcm-ZEGFR:1907in SKOV3cells was obviously higher than that inMDA-MB-435S cells at each time point, and gradually reached stability. Theresults of biodistribution showed that radioactive accumulations in tumorregion were obviously higher than in muscle region, and the ratio of T/Mincreased from2.60±0.06at1h to7.05±0.14at6h. More radionuclide wasuptaken by the kidney and liver than other tissues. In addition, a rapidclearance of99Tcm-ZEGFR:1907from most of the normal organs except the liver and kidneys was found in biodistribution study. The ratio of T/M in EGFRoverexpressing SKOV3xenografts was significantly （Z=2.00-2.01， allP<0.05） higher than that in low EGFR expressin MDA-MB-435S xenograftsat each time point. The ratio of T/M in SKOV3xenografts decreased from4.95±0.09to1.34±0.04after the management of blocking at4h, and thedifference was statistically significant (Z=2.0016, P=0.0453<0.05). As earlyas1h, radioactive concentration of99Tcm-ZEGFR:1907in SKOV3xenografts werevisualized and grew gradually with time. The ratio of T/NT achievedmaximum (6.61±0.09) at10h. However, there was no obvious uptake inMDA-MB-435S xenografts and the SKOV3xenografts in blocked groupthroughout the imaging process and their ratio of T/NT were statisticallysignificantly lower than the ratio of T/NT in SKOV3xenografts at each timepoint.Conclusions: The molecular probe of99Tcm-ZEGFR:1907had a high labelingefficiency and a good stability in vivo and in vitro. It was specifically taken upby EGFR-positive tumors, and the higher EGFR levels the more uptaken bytumors. The molecular probe99Tcm-ZEGFR:1907may be developed as apotentially new method of molecular imaging for early and specific diagnosisof EGFR-expressing tumors. |
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