Molecular Imaging Of^99mTc-labeled EGFR MRNA Antisense Peptide Nucleic Acid （PNA） In Nude Mice Bearing Ovary Carcnoma Xenografts

Objective: To provide a new method of molecular imaging to diagnosis,therapy tumor specifically in the early stage and evaluate the therapeutic effect,^99mTc labeled EGFR mRNA antisense or mismatch PNA were prepared, theradiolabeling rate and radiochemistry purity were detected, the stability invivo and vitro were evaluated and expriments of distributions and molecularimaging in mude mice bearing EGFR-expressing SKOV3human ovarycarcinoma xenografts were administrated.Methods: EGFR-targeted antisense PNA （5’-AATGAGGACATAACCA-G-3’）,specially targeting the gene sequence of EGFR mRNA from503-519（Gene Bank No:201283.1） and mismatch PNA （5’-AGTAGAGATGTGAC-GAT-3’） which targeted none of the human genes were synthesized. On the5’terminus of EGFR mRNA antisense or mismatch PNA special segement, fouramino acid （Gly-（D）-Ala-Gly-Gly）, forming a similar N4structure, will beused as a linker for coupling of^99mTc and EGFR mRNA antisense ormismatch PNA segment, and one4-aminobutyric acid （Aba） was introducedas a barrier to eliminate the steric hindrance, then^99mTc labeled EGFR mRNAantisense and mismatch probes were prepared. The two probe labeled by^99mTcwere analyzed by high pressure liquid chromatography （HPLC）. To evaluatethe lableling stability in vitro, the^99mTc labeled antisense or mismatch PNAwas incubated at temperature, in normal saline and in fresh human serum（equivalence with the probes）37°C for1h,2h,4h,6h,12h, and24h,respectively. The in vivo stability was evaluated by HPLC analysis of theurine samples collected from SKOV3tumor mice injected the probes at30minand2-3h for radiochemical purity determination. The human ovary carcinomaSKOV3cells were incubated by standing adherent. Cells were cultured in 6-well plates with3×10⁶cells in each well. We changed fresh culturemedium after the cells arrived89%-90%of the well and30μL^99mTc-EGFRmRNA antisense or mismatch PNA were added into each well. The C_outandC_inwere harvested and counted after the cells incubated1,2,4,6,12, and24h.Cellular uptake ratio was calculated by the formula C_in/(C_in+C_out). We changedthe culture medium quickly after the cells incubated6h (involved30μL^99mTc-EGFR mRNA antisense or mismatch PNA were added into eachwell)（3×10⁶cells each well）, then the C_inand C_outwere harvested after thecells incubated1,2,4,6,12,24h. Cellular retention kinetics ratio wascalculated by the formula C_in/(C_in+C_out).The cells were harvested and the micewere injected subcutaneously with6×10⁶SKOV3cells of each line （200μL）in the right anterior superior limbs. The expreiments were performed when thetumors had been reached a diameter of1.0-1.5cm. The experiments of cellularuptake and retention kinetics in SKOV3cells is to study the pharmacokineticsof antisense PNA. In the biodistribution studies,48BALB/c nude mice withSKOV3tumor xenografts were randomly divided into2groups （one group forantisense PNA and the other for mismatch PNA）.^99mTc labeled EGFR mRNAantisense or mismatch PNA probe was injected through the tail into micebearing SKOV3xenografts （n=6for each group and they were killed atdifferent times after injection1h,2h,4h and6h）. Tumors and normal tissueswere excised and weighed and the radioactivity uptake in the tumor andnormal tissues was expressed as the percentage of injected dose per gram oforgan （%ID/g）. Ten BALB/c nude mice with SKOV3tumor xenografts wererandomly divided into2groups （one for antisense probe and the other formismatch probe）. At1h,2h,4h,6h,8h and10h, the mice were imaged tobeserved the radionuclide accumulation in tumors of two groups with time.The expression of EGFR mRNA in human ovary carcioma ovary carcinomaSKOV3cells by reverse transcription-polymerase chain reaction （RT-PCR）.All numeric data are expressed as average±SD by the SAS9.1. The resultswere analysis by T test. P value of less than0.5was considered significant.Results: The result of HPLC showed the average labeling efficiency of the^99mTc labeled EGFR mRNA antisense or mismatch PNA was98.80%±1.14%and98.63±1.36%, respectively, and the radiochemistry purity>95%.The radiochemical purity reached85%when the probes mixtured with normalsaline and fresh human serum at37°C even at24h and the urine of HPLCshowed the radiochemical purity reached89%when injected2-3h. The uptakeand retention kinetics showed that^99mTc labeled EGFR mRNA antisense PNAwas higher than the mismatched one at any time point （P<0.05）. Thebiodistribution confirm fast blood clearance and the high uptake were foundprimarily in the liver and the kidneys, followed by spleen, except for thetumor. High uptake of^99mTc labeled EGFR mRNA antisense PNA wasobserved in tumor, with1.55±0.12%ID/g observed at1h increasing to3.93±0.74%ID/g at6h after injection. For imaging, a clear localization of theimage was seen shortly after injection^99mTc labeled EGFR mRNA antisensePNA, but no tumor image was observed at any time using^99mTc labeled EGFRmRNA mismatch PNA.Conclusions:^99mTc labeled EGFR mRNA mRNA antisense PNA hashigh radiochemistry purity and good stability. It can specifically uptake inEGFR expressing positive tumors and can be used as a promising tracer formolecular imaging to detect the EGFR expressing in tumors.