Abstract
The preparation of two polyarginine conjugates of the complex Os(II) [bis-(4′-(4-carboxyphenyl)-2,2′:6′,2″-terpyridine)] [Os-(Rn)2]x+ (n = 4 and 8; x = 10 and 18) is reported, to explore whether the R8 peptide sequence that promotes cell uptake requires a contiguous amino acid sequence for membrane permeation or if this can be accomplished in a linearly bridged structure with the additive effect of shorter peptide sequences. The conjugates exhibit NIR emission centered at 754 nm and essentially oxygen-insensitive emission with a lifetime of 89 ns in phosphate-buffered saline. The uptake, distribution, and cytotoxicity of the parent complex and peptide derivatives were compared in 2D cell monolayers and a three-dimensional (3D) multicellular tumor spheroid (MCTS) model. Whereas, the bis-octaarginine sequences were impermeable to cells and spheroids, and the bis-tetraarginine conjugate showed excellent cellular uptake and accumulation in two 2D monolayer cell lines and remarkable in-depth penetration of 3D MCTSs of pancreatic cancer cells. Overall, the data indicates that cell permeability can be promoted via non-contiguous sequences of arginine residues bridged across the metal centre.
Highlights
The Os(II) parent complex [Os(tpybenzCOOH)2]2+ was conjugated to two polyarginine chains of varying lengths (R4 and R8), and we explore whether the optimal R8 requires a contiguous peptide structure for uptake or whether it can be accomplished in a bridged structure with a shorter peptide sequence
We explore the impact of polyarginine chains of different lengths to each carboxyl termini of the Os(II) complex, to understand in such an arrangement, if the octaarginine sequence we have observed to be so efficient in driving related metal complexes across the cell membrane and into the cytoplasm require that the arginine residues are contiguous and if longer arginine chains further improve the uptake
Using an achiral Os(II) bisterpyridinyl-coordinated complex with linear bilateral conjugation sites, we demonstrate that assembly of polyarginine at opposing ends of the structure exhibits an additive effect in terms of cargo cellular permeation
Summary
Most widely classified as phosphors, have emerged in the past decade as feasible alternatives to organic fluorophores for intracellular imaging and sensing.[1,2] The attractive photophysical properties of such complexes have been widely reported, and for complexes of ruthenium, these include good photostability, long emission lifetimes, and Stokes-shifted emission in the red spectral region.[3−6] While the emission maxima of complexes of Ir(III) and Ru(II) can be tuned toward the NIR, it can be synthetically challenging and such tuning may compromise photostability, exacerbated in the physiological conditions of temperature and buffered media, as well as emission quantum yield.[7−9] osmium (II) polypyridyl complexes exhibit similar advantages to Ru(II) for imaging but with the additional benefits of outstanding photostability and deep-red to NIR emission in the 700−850 nm spectral region, making them attractive candidates, in particular for tissue imaging ( still prone to the impact of the energy gap law). Reported ruthenium octaarginine conjugates have generally been found to be nuclear excluding with non-specific distribution throughout the cytoplasm.[38,39] Uptake of an osmium polypyridyl phenanthroline imidazole complex conjugated to R8, [Os(bpy)2(picarg8)]10+ showed comparable uptake to its ruthenium analogue, unlike the Ru analogue, nuclear penetration of the osmium complex was observed in CHO cells under photoirradiation.[11] The increased lipophilic character of the osmium complex compared to its ruthenium analogue was suggested as a reason for its nuclear permeation.[11] Given the punctate distribution of the complex at later time points, to establish distribution of [Os-(R4)2]10+, co-localization studies were carried out using Lysotracker Green for the lysosomes and MitoTracker Deep Red for the mitochondria. Redistribution of nona-argininemodified fluorescent dyes was noted after fixation where nucleolus migration was observed.[76] as shown in the PLIM image of punctuate staining of [Os-(R4)2]10+ in fixed HPAC spheroids (Figure S27), the lifetime distribution is very uniform. The lifetime of [Os-(R4)2]10+ and its uniformity are likely to reflect the sample fixation, which causes extensive cross-linking of protein structures into a gel state within the cell.[77,78]
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