Comparative Study On Imaging Of ⁹⁹ Tc^m -survivin MRNA Antisense PNA In Tumor And Inflammation Animal Models

Objective: To prepare the antisense peptic nucleic acid (PNA) molecular probe targeting survivin mRNA labeled with 99Tcm and analyze the biodistribution and imaging of 99Tcm-survivin mRNA antisense PNA in nude mice bearing lung carcinomar A549 xenogafts and Kunming mice bearing staphylococcus aureus inflammation. To investigate the value of 99Tcm -survivin mRNA antisense PNA as an imaging agent in specific diagnosis of carcinoma.Methods: The antisense PNA fragment aimed directly at survivin mRNA 240～251 base sequence was synthesized and purified. The sequence of survivin mRNA antisense PNA is 5′-CCCAGCCTTCCA-3′, a four amino acid sequence Gly-(D)Ala-Gly-Gly- and an Aba (4-aminobutyric acid) were linked to the 5′end of PNA by chemical synthesize to be used as a chelator and provided an N4 configuration for strong and efficient chelation of 99Tcm,then it was labeled directly with 99Tcm by the ligands exchange method. The labeling efficiency and stability of survivin mRNA antisense PNA fragment was measured with high performance liquid chromatography (HPLC) and paper chromatography. To cultivate A549 human pulmonary adenocarcinoma cells, then inject it subcutaneously in the right upper limbs of BALB/c nude mice. The mice were housed under specific pathogen free (SPF) environmental conditions, and the tumors were allowed to grow to a diameter of 1.0～1.5cm for experiment. The Kunming mice were inoculated with suspensions consists of staphylococcus aurous in the muscle of right upper limbs to induce inflammation and used for tissue distribution and imaging studies. Twenty BALB/c nude mice with A549 xenografts were randomly divided into 4 groups of 5 mice each. Three groups (1h group, 2h group, 3h group) were selected randomly and used for tissue distribution study. Each mouse was injected intravenously via the tail vein with 555KBq (15μCi) of 99Tcm-survivin mRNA antisense PNA, then the mice were sacrificed by cervical dislocation 1, 2 and 4h post injection, respectively. The mice were dissected and tissues of interest (blood, heart, liver, lung, kidney, spleen, intestine, stomach, bone, skeletal muscle) were weighted and their radioactivity was measured along with two standard sources. The percentage activity of injected dose per gram of tissue (%ID/g) and target to non-target (T/NT) ratios were calculated. The rest group was injected intravenously via the tail vein with 37MBq (1mCi) of 99Tcm-survivin mRNA antisense PNA, then imaging in vivo was performed 1, 2 and 4h post injection, periodically. To process the inflammation group with the same method. All data were analyzed by SPSS 13.0 statistical software, P < 0.05 was considered significant.Result: The labeling efficiencies of 99Tcm-survivin mRNA antisense PNA reached (94.89±2.73) % (n=6) and the labeled compound showed complete stability at room temperature and the labeling efficiencies >95% after 4 hours. The result of HPLC confirmed that the 99Tcm was joined to the PNA with high radiochemical purity. Results of biodistribution in vivo showed that the highest radioactivity levels were in liver and kidney in nude mice bearing A549 xenografts, and the similar results were obtained in Kunming mice. The radioactivity ratios of tumor to contralateral muscles or inflammatory lesions to contralateral muscles were 3.69±1.13 and 2.03±0.47 respectively after injected 99Tcm-survivin mRNA antisense PNA 4 hours later, and there was significant difference between tumor and inflammation groups (t=3.01,p=0.02). The xenografts were clearly imaged at 0.5h after injection of 99Tcm-survivin mRNA antisense PNA and the clearest image was showed at 4h after injection, whereas the inflammatory lesions were not clearly imaged at any time after injection of 99Tcm-survivin mRNA antisense PNA.Conclusion: 99Tcm-survivin mRNA antisense PNA displays excellent biological characteristics. This in vivo study shows that 99Tcm-survivin mRNA antisense PNA can be uptaked specifically by tissues of tumor and may be used to distinguish neoplasm from inflammation, and provides evidences that 99Tcm-survivin mRNA antisense PNA has potential value in early specific diagnosis of lung cancer.