Solid Phase Synthesis and Biological studies on metal conjugates of bioactive peptides for targeted delivery

Abstract

Summary Peptide-based drug delivery systems and therapeutics have gained an enormous attention during the last ten years. Progress in this field will lead to site specific delivery, improved receptor affinities, efficient cellular uptake and/or nuclear targeting. Thus, in this research project, metal conjugates of some bioactive peptides were synthesized and biologically tested in order to evaluate the effects of these metal moieties on the biological activities such as the cellular uptake, nuclear targeting and binding affinity, of these selected peptides. In this study, the two redox active metallocenes, ferrocene and cobaltocenium and in addition the cytotoxic platinum were chosen as metal labels. The investigated peptides were the Simian Virus 40 nuclear localisation signal (NLS), the HIV transactivator of transcription (TAT) peptide and the small regulatory peptide, the neurotensin (NT). Metal conjugates of these bioactive peptides were successfully synthesized by solid phase peptide synthesis (SPPS) which was used not only for the covalent bonding of the metallocenes to the peptides but also for the complexation of platinum to the peptides and fluorescence labelling with FITC. Comprehensive characterisation of the synthesized bioconjugates was carried out by various techniques such as NMR, RP-HPLC, MS and electrochemistry. The cellular uptake and nuclear localisation of the metallocene-NLS and -TAT bioconjugates was monitored by fluorescence microscopy in living liver cancer cells (Hep G2). The metallocene-NLS conjugates were efficiently internalized by the cells and were localised in the nuclei of the Hep G2 cells. The metallocene moiety is responsible for the enhanced cellular uptake of these bioconjugates and the NLS transports the organometallic species into the nuclei. This is the first example of the directed nuclear delivery of ferrocene and the cobaltocenium cation, by conjugation to the NLS peptide. The use of the scrambled NLS sequence (NLSscr) abolishes the nuclear targeting property of the conjugates. All metallocene-NLS bioconjugates were found to be non-toxic in concentrations up to 1mM in the WST-1 proliferation assay. In case of the metallocene-TAT bioconjugates, the ferrocene moiety plays a role in the escape of the conjugate from the endosomes, which is an advantage because the utility of TAT peptide as a vector for cellular delivery is limited by its inability to escape from the endosomes. Moreover, these peptides are toxic in higher concentration due to cell membrane perturbation. This was also demonstrated in the present study using the WST-1 proliferation assay. In the last part of the project, metal conjugates of wild type NT(8-13) and the modified Pseudoneurotensin (pNT) were synthesized and tested for their binding affinity to the NTR1 receptors in the HT 29 adenocarcinoma cell line. Replacement of Arg8-Arg9 with Lysines in pNT led to a significant decrease in the binding affinity. The metal-NT bioconjugates showed good receptor affinity especially the cobaltocenium-NT conjugate (IC50=2.3nM). In this case, the lipophilicity of the metallocene bioconjugate may facilitate the crossing of the blood brain barrier which is a limiting factor in any centrally intended therapy. The Pt-NT bioconjugate also showed good affinity (IC50=6.8nM) for the receptors. Thus, such bioconjugates may be specifically and selectively delivered to the tumour tissues that overexpress the neurotensin receptors. In conclusion, the optimised synthesis procedures for the studied metals and the peptides were established. The biological studies demonstrate a great potential of these metals for the improvement of the biological functions of the tested peptides especially for use as vectors for cellular uptake and targeted nuclear delivery. This represents a novel application of bioorganometallic chemistry in biological systems.