Photophysical Properties and DNA Binding of Two Intercalating Osmium Polypyridyl Complexes Showing Light-Switch Effects.

The synthesis and photophysical characterization of two osmium(II) polypyridyl complexes, [Os(TAP)2dppz]2+ (1) and [Os(TAP)2dppp2]2+ (2) containing dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dppp2 (pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) intercalating ligands and TAP (1,4,5,8-tetraazaphenanthrene) ancillary ligands, are reported. The complexes exhibit complex electrochemistry with five distinct reductive redox couples, the first of which is assigned to a TAP-based process. The complexes emit in the near-IR (1 at 761 nm and 2 at 740 nm) with lifetimes of >35 ns with a low quantum yield of luminescence in aqueous solution (∼0.25%). The Δ and Λ enantiomers of 1 and 2 are found to bind to natural DNA and with AT and GC oligodeoxynucleotides with high affinities. In the presence of natural DNA, the visible absorption spectra are found to display significant hypochromic shifts, which is strongly evident for the ligand-centered π-π* dppp2 transition at 355 nm, which undergoes 46% hypochromism. The emission of both complexes increases upon DNA binding, which is observed to be sensitive to the Δ or Λ enantiomer and the DNA composition. A striking result is the sensitivity of Λ-2 to the presence of AT DNA, where a 6-fold enhancement of luminescence is observed and reflects the nature of the binding for the enantiomer and the protection from solution. Thermal denaturation studies show that both complexes are found to stabilize natural DNA. Finally, cellular studies show that the complexes are internalized by cultured mammalian cells and localize in the nucleus.

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications.

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.