Release of Toxic Gd3+ Ions to Tumour Cells by Vitamin B12 Bioconjugates

Two probes consisting of vitamin B12 (CNCbl) conjugated to Gd chelates by esterification of the ribose 5-OH moiety, Gd-DTPA-CNCbl (1; DTPA=diethylenetriamine-N,N,N,N,N-pentaacetic acid) and Gd-TTHA-CNCbl (2; TTHA=triethylenetetramine-N,N,N,N,N,N-hexaacetic acid), have been synthesised and characterised. The crystal structure of a dimeric form of 1, obtained by crystallisation with an excess of GdCl3, has been determined. The kinetics of binding to and dissociation from transcobalamin II show that 1 and 2 maintain high-affinity binding to the vitamin B12 transport protein. Complex 2 is very stable with respect to Gd3+ release owing to the saturated co-ordination of the Gd3+ ion by four amino and five carboxylate groups. Hydrolysis of the ester functionality occurs on the time scale of several hours. The lack of saturation and the possible involvement of the ester functionality in co-ordination result in lower stability of 1 towards hydrolysis and in a considerable release of Gd3+ in vitro. Gd3+ ions released from 1 are avidly taken up by the K562 tumour cells to an extent corresponding to approximately 1010 Gd3+ per cell. The internalisation of toxic Gd3+ ions causes a marked decrease in cell viability as assessed by Trypan blue and WST-1 tests. On the contrary, the experiments with the more stable 2 did not show any significant cell internalisation of Gd3+ ions and any influence on cell viability. The results point to new avenues of in situ generation of cytotoxic pathways based on the release of toxic Gd3+ ions by vitamin B12 bioconjugates.