Polypyridyl-Based Copper Phenanthrene Complexes: Combining Stability with Enhanced DNA Recognition.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp ) rising from 105 to 107 m-1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine-cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

Multiparametric nanoparticle-induced toxicity readouts with single cell resolution in HepG2 multicellular tumour spheroids.

The use of nanomaterials as therapeutic delivery vehicles requires their careful pre-clinical evaluation. Of particular importance in this regard is measurement of cellular toxicity, ideally assessing multiple parameters in parallel from various relevant subcellular organelles. In recent years it has become evident that in vitro monolayer-grown cells do not always accurately predict any toxicity response seen in vivo, and so there is a need for more sophisticated in vitro cell models, employing a greater depth of characterisation. In this work we present an automated high-content screening microscopy approach for quantifying nanoparticle-induced toxicity in a three-dimensional multicellular tumour spheroid (MCTS) cell model. As a proof-of-principle, we perform a comparative toxicity profile study of carboxylate- versus amine-modified polystyrene nanoparticles in HepG2 spheroids. Following treatment with these nanoparticle types, we demonstrate that several hundred spheroids, of various sizes, can be morphologically profiled in a single well using automated high-content image analysis. This provides a first level of information about spheroid health in response to nanoparticle treatment. Using a range of fluorescent reporters assessing membrane permeability, lysosome function and mitochondrial activity, we also show that nanoparticle-induced toxicity information can be obtained from individual cells with subcellular resolution. Strikingly, our work demonstrates that individual cells do not all behave in a consistent manner within a spheroid structure after exposure to nanoparticles. This highlights the need for toxicity studies to not only assess an appropriate number of spheroids, but also the importance of extracting information at the subcellular level.