In vitro and in vivo evaluation of ^99mTc-DO3A-EA-Folate for receptor-mediated targeting of folate positive tumors

Abstract

Background: The cell membrane folate receptor is a potential molecular target for tumor selective drug delivery via receptor-mediated endocytosis, including delivery of radiolabeled folate-chelate conjugates for diagnostic imaging.

Method: A new radiopharmaceutical, ^99mTc-1,4,7-tris (carboxymethyl)-10-(4-aminoethyl)-1,4,7,10-tetraazacyclododecane (DO3A-EA)-Folate has been synthesized introducing DO3A-EA to the γ-carboxyl group of folic acid and was characterized by different spectroscopic techniques. Cytotoxicity was determined by macrocolony and MTT assay on three different cell lines. Cell uptake studies and receptor binding assay were performed using ^99mTc-DO3A-EA-Folate. Tumor imaging was performed in KB cell line implanted tumor bearing nude mice, and uptake of the radiotracer was estimated.

Results: The synthesized conjugate binds with ^99mTc at high efficiency at ambient temperature. The resulting conjugate is stable under physiological conditions for 24 h after radiocomplexation. Using an in vitro receptor binding assay, the conjugate showed K_d in μM range on human tumor cell lines (KB, U-87MG and OAW). The pharmacokinetic data revealed rapid wash out of the more than 75% activity within 5 min from the circulation with hepato-biliary clearance. Data from γ scintigraphic and biodistribution studies performed in KB tumor bearing nude mice revealed major accumulation of radiotracer at tumor site. High tumor uptake was shown in the tumor bearing mice; tumor to blood ratios reached 2.27 ± 0.32 and 6.05 ± 1.02 at 1 and 4 h after post injection, respectively.

Conclusion: These results suggest that ^99mTc-DO3A-EA-Folate may be clinically useful as a noninvasive radiodiagnostic imaging agent for the detection of FR-positive human cancers.

Keywords::

• Folate receptor
• ^99mTc-Folate
• tumor imaging
• bifunctional chelating agent

Acknowledgments

We are grateful to Dr. R. P. Tripathi, Institute of Nuclear Medicine and Allied Sciences, University of Delhi, for providing support and commendable research facilities.

Declaration of interest

The work was supported by Defence Research and Development Organization, Ministry of Defence, under R&D Project INM-311.