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Abstract
This thesis reports the development of a series of inert dinuclear polypyridylruthenium(II) complexes from DNA binding agents to anticancer drugs. The complexes of the general form ΔΔ/ΛΛ-[{Ru(phen)2}2{µ-bbn}]4+ {“Rubbn” where phen = 1,10-phenanthroline; bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16)}, were tested for inherent cytotoxicity, mechanism of cytotoxicity and cellular uptake in the L1210 murine leukaemia cell line, and DNA-binding affinity. Poly(amidoamine) dendrimers and cucurbit[10]uril have also been evaluated as potential delivery vehicles for the Rubbn series of complexes.
The Rubbn series of complexes and corresponding mononuclear complexes containing the bbn ligands were tested for anticancer activity. The ΔΔ-Rubb16 complex displayed the highest cytotoxicity of the series, with an IC50 value of 5 μM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Isothermal titration calorimetry experiments showed that all complexes bound to CT-DNA with binding affinities (K) ranging from 6.7 – 28.0 x 104 M-1, and that the binding was entropically favoured and enthalpically opposed. Confocal microscopy and flow cytometry studies indicated that the complexes accumulated in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bbn bridge of the metal complex. ΔΔ-Rubb16 entered the L1210 cells by passive diffusion (with a minor contribution from protein-mediated active transport) while inducing cell death via apoptosis. Additionally, metal complex uptake in leukaemia cells was approximately 16-times that observed in healthy B cells.
Rubbn was used as a model compound to evaluate the potential of anionic half-generation poly(amidoamine) (PAMAM) dendrimers and cucurbit[10]uril as delivery vehicles for large cationic ruthenium compounds. Given the ability of each class of delivery molecule to act as a host for metal-based drugs, the suitability of each delivery molecule was evaluated with a suitable platinum compound in addition to Rubbn.
The suitability of PAMAM dendrimers as drug delivery vehicles for Rubb7 was studied by nuclear magnetic resonance (NMR) spectroscopy and evaluated against [Pt(S,S-dach)(5,6-Me2phen)]2+ (56MESS) (where; S,S-dach = 1S,2S-diaminocyclohexane). From one- and two-dimensional 1H NMR spectra both 56MESS and Rubb7 were found to bind to the surface of generation 3.5, 4.5, 5.5 and 6.5 dendrimers through electrostatic interactions. The higher charge and larger size of Rubb7 resulted in stronger binding to all dendrimer generations (Kb ≥ 2 x 105 M-1) compared with 56MESS (Kb ≥ 1 x 104 M-1). Interestingly, there appeared to be no observable trend between dendrimer size and binding constant strength. The relatively low binding constants (104 – 105 M-1) indicate that anionic dendrimers are not useful drug delivery vehicles for small, charged cationic transition metal-based complexes.
The binding of Rubb5 to Q[10] was studied by 1H NMR and luminescence spectroscopy, and molecular modelling. The 1H NMR resonances of the methylene protons in the bridging ligand exhibited large upfield chemical shift changes upon addition of Q[10]. These shifts are indicative of encapsulation of the bridging ligand within the Q[10] cavity, with the metal centres positioned outside the portals. The large metal centres of Rubb5 provided a restriction to the movement of the complex in and out of the cavity and resulted in binding kinetics that were slow on both the 1H NMR and biological timescales. This result was consistent with molecular modelling simulations. Due to the strong binding affinity (1.9 x 109 M-1) and slow exchange rates Q[10] showed considerable promise as a delivery mechanism for large dinuclear ruthenium(II) complexes.
These studies highlight the utility of the Rubbn series of complexes to act as anticancer agents. The results of this study may be used in the future to assist in the design of further derivatives of dinuclear polypyridylruthenium(II) complexes for biological application.