Influence of different chelators (HYNIC, MAG(3) and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisenseDNA

We have shown recently that cell accumulation in culture of antisense DNA i s strongly influenced by the presence of a Tc-99m-MAG(3) group for radiolab eling. We have now compared the in vitro and mouse in vivo behavior of Tc-9 9m when radiolabeled to one antisense phosphorothioate DNA by three differe nt methods. The 18-mer antisense DNA against the RI alpha subunit of PKA wa s conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG(3) and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native D NA. Size exclusion HPLC showed rapid and quantitative protein binding for a ll three chelators upon incubation of labeled DNAs in 37 degreesC serum and cell culture medium. However, in each case, radiolabeled and intact oligon ucleotide was still detectable after 24 h. Cellular uptake was tested in an RI alpha mRNA-positive cancer cell line. The order of cellular accumulatio n of Tc-99m was DTPA>HYNIC(tricine) > MAG(3), with the differences increasi ng with time between 4 and 24 h. The rate of Tc-99m egress from cells was f ound to be MAG(3) > HYNIC > DTPA. which may explain the order of cellular a ccumulation. The biodistribution in normal mice was heavily influenced by t he labeling method and followed a pattern similar to that seen previously b y us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one antisense DNA in one cell line, the result s suggest that the success of antisense imaging may depend, in part, on the method of radiolabeling.