Assessing the Role of Membrane Interactions and the <i>In Vitro</i> Biological Activity of Novel Bismuth Flavonol Complexes

<p dir="ltr">Flavonols (Flavs) are a diverse group of naturally occurring polyphenolic compounds with well-documented anti-oxidant, anti-microbial and anti-cancer properties. Their ability to interact with biological membranes and modulate cellular processes has led to increased interest in their therapeutic potential. Metal-flavonol complexes have emerged as promising candidates for enhancing the biological activity of flavonoids. Recently, studies of Bi-flavonoid complexes (BiFlavs) have been performed to determine if their properties can be harnessed as a novel strategy to overcome drug resistance and improve bioavailability in cancer therapy. This Thesis systematically investigates the bioactivity of BiFlavs by assessing their membrane interactions, uptake in cancer cells, cytotoxicity, and binding to serum proteins using combined biophysical, biochemical, and cellular techniques.</p><p dir="ltr">The ability of Flavs and BiFlavs to interact with membrane mimics was examined using electrical impedance spectroscopy (EIS), quartz crystal microbalance with dissipation (QCM-D) monitoring and neutron reflectometry (NR). Flavs transiently increased membrane conductance in EIS studies, indicative of pore formation, with interactions largely reversible. In contrast BiFlavs, particularly BiPh(Flav)₂ and BiPh(BrFlav)₂, exhibited stronger and more persistent membrane interactions which was evident by significant mass deposition on lipid bilayers in QCM-D experiments. NR data indicated that the free Flavs interacted with the head group region of the lipid membrane mimic. The BiFlavs, BiPh(Flav)₂ and BiPh(BrFlav)₂, were found to interact with the hydrophobic core and the polar head groups of the membrane mimic, while Bi(BrFlav)₃ primarily disrupted the organisation of the head group region. These findings suggested that Bi coordination influences membrane penetration and stability, potentially affecting cellular uptake and bioactivity.</p><p dir="ltr">The cellular accumulation of BiFlavs was assessed in a range of cancer and noncancer cell lines using graphite furnace atomic absorption spectroscopy (GFAAS). The accumulation of Bi was assessed in colorectal (HCT-8, HCT-116 and HT-29) and pancreatic (MIA PaCa-2 and BxPC-3) cancer cells as well as leukemia (K562) cells, osteosarcoma (U-2 OS) cells and human peripheral blood mononuclear cells (PBMCs). Intracellular Bi concentrations of the different BiFlavs were consistent with the extent of membrane disruption observed in model membrane studies, suggesting that permeability may influence uptake efficiency. However, differences observed between cancer cell lines indicated that alternative transport mechanisms, such as receptor-mediated transport, may also contribute.</p><p dir="ltr">The <i>in vitro</i> anti-cancer activity of BiFlavs was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Free flavonoids exhibited limited cytotoxicity, with Flav and BrFlav displaying particularly weak anti-cancer effects. The BiFlavs were found to have significantly greater cytotoxicity compared to the free ligands. Among the BiFlav complexes, BiPh(Flav)₂ exhibited the greatest cytotoxicity across multiple cancer cell lines. Despite promising uptake studies, BiFlavs exhibited cytotoxicity in non-cancerous PBMCs, raising concerns about selectivity. Flow cytometry assays of apoptotic and necrotic markers confirmed that apoptosis was the predominant mode of cell death in all cell lines studied.</p><p dir="ltr">To understand the potential<i> in vivo</i> transport and bioavailability of BiFlavs, the binding interactions of BiFlavs and the free ligands with major serum transport proteins, human serum albumin (HSA), human lactoferrin (hLf) and human transferrin (hTf), were investigated using UV-visible spectroscopy. BiFlavs exhibited significantly stronger protein binding than free flavonoids. BiPh(Flav)₂ and BiPh(BrFlav)₂ demonstrated preferential binding to hTf. This aligned with cellular uptake trends, where transferring receptor (TfR)-rich leukemia (K562) and osteosarcoma (U-2 OS) cells displayed elevated BiFlav accumulation, suggesting a potential mechanism for targeted delivery.</p><p dir="ltr">The studies of BiFlav complexes in this Thesis demonstrated a promising relationship between membrane interactions and<i> in vitro </i>activity. Consequentially future investigations are warranted to optimise selectivity, minimise off-target toxicity and improve bioavailability. Additionally, <i>in vivo</i> studies are required to validate the biological relevance of these findings by assessing the biodistribution and <i>in vivo</i> safety profile of the BiFlavs, particularly in cancer and inflammatory disease animal models.</p>