Space invaders: Organometallic fragments such as the ferrocenyl group (shown in red in the picture) help to enhance cellular entry of NLS peptides. Eventually, these nontoxic conjugates find their way to the cellular nucleus as shown by fluorescence microscopy studies in this work.
Intracellular delivery to biomolecular targets is still a major challenge in molecular and cell biology. We recently found that attaching an organometallic group, namely the cobaltocenium cation, to the SV 40 large T antigen nuclear localisation signal (NLS) greatly enhances cellular uptake of the conjugate (Noor et al., Angew. Chem. Int. Ed.2005, 45, 2429). In addition, nuclear localisation of the conjugate was observed. In this work, we present a thorough investigation of this novel cellular delivery system with respect to the nature of the metal complex and the peptide sequence. A number of ferrocene ((FeII), neutral metal complex) and cobaltocenium ((CoIII), cationic metal complex) bioconjugates with both the NLS wild-type sequence PKKKRKV and a scrambled sequence (NLSscr, KKVKPKR) were prepared by solid-phase peptide synthesis (SPPS). Cellular and nuclear uptake of these bioconjugates was studied by fluorescence microscopy on living Hep G2 cells. In addition, cytotoxicity screening on the conjugates was carried out, as the toxic effects of several simple metallocenes have been noted previously. Rapid cellular uptake as well as nuclear localisation was observed for the metal–NLS conjugates, but not for any dipeptide controls, the metal–NLSscr conjugates or any metal-free conjugates. It thus appears that the presence of a metallocene, but not its charge, and the correct NLS sequence is essential for cellular uptake. Fluorescence microscopy co-localisation studies did not reveal a significant endosomal entrapment of the conjugates. The metallocene not only provides a hydrophobic handle for membrane translocation but also facilitates the localisation and distribution of the conjugate in the cytoplasm. The NLS peptide on the other hand is responsible for the nuclear localisation of the bioconjugate. Finally, none of the conjugates were found to be toxic up to the highest concentrations that was tested (1 mM) against the Hep G2 cells that were used in this study. In conclusion, this work supports metallocene–NLS bioconjugates, in particular with the very robust cobaltocenium group, as a simple but potent, nontoxic system for cellular uptake and nuclear delivery. Concurrently, our finding is relevant to the still-unresolved question of cytotoxicity of metallocenes because it excludes binding and/or damage to the DNA as a mechanism of metallocene cytotoxicity. This finding is confirmed by a combined yeast cytotoxicity/genotoxicity assay, which also shows very little toxic effects for all organometal–NLS conjugates that were tested.