Synthesis of [99mTc]chitosan-folate and its in vitro evaluation as a folate-receptor targeted radiopharmaceutical

1881 Objectives: The folate receptor is overexpressed in a variety of human tumors. In this study, 99mTc-chitosan-folate, was synthesized and evaluated for its efficacy as a targeted agent for the imaging of tumor that overexpress the folate receptor. Methods: The carboxylate group of folic acid was activated by NHS and DCC. Briefly, 50 mg of folic acid dissolved in 1 ml of dimethylsulfoxide (DMSO) was reacted with NHS, DCC, and triethylamine (TEA) under nitrogen atmosphere at room temperature for 12 h (folic acid/NHS/DCC molar ratio=1:2:2). The activated folic acid was reacted with chitosan (water-soluble, Mv=5000) dissolved in 1 ml of DMSO. The reaction was performed under nitrogen atmosphere at room temperature for 5 days. The resultant solution was diluted with deionized water and dialyzed against deionized water six times (SpectraPor 2, MW cutoff=3500) and freeze-dried. Conjugation was determined by 400 MHz 1H NMR spectorymetry. Chitosan-folate was also conjugated FITC. KB and A549 cells were maintained in RPMI 1640 media with or without folate. Chitosan-folate was labeled with 99mTc. 99mTc labeled conjugate was incubated with KB and A549 cells. 99mTc uptake was identified using COBRA gamma counter. KB and A549 cells were incubated with FITC-Chitosan-folate and FITC-Chitosan for 20 min and 1 hr respectively. For a competition study unconjugated folic acid was added to the incubation medium at 2~6 μmol/L. Cellular uptake was performed by fluorescence-activated cell sorting using a FACScan. Results: Conjugation was performed successively. This was confirmed by NMR. Technetium labeling efficiency of chitosan-folate was above 90% up to 6 hours. 99m Tc- chitosan-folate was specifically uptaked by KB cells. Moreover, only FITC-Chitosan-folate was internalization by KB cells. Competitive binding studies revealed that only the chitosan-folate is recognized by folate receptor on KB cells. Conclusions: These results suggest that 99m Tc-chitosan-folate might be useful as a diagnostic agent for noninvasive imaging of folate-receptor positive cells.