The cellular uptake of a series of dipyridophenazine (dppz) complexes of Ru(II) was examined by flow cytometry. The complexes, owing to their facile synthesis, stability, and luminescence, provide a route to compare and contrast systematically factors governing cellular entry. Substituting the ancillary ligands in the dppz complexes of Ru(II) permits variation in the overall complex charge, size, and hydrophobicity. In HeLa cells, cellular uptake appears to be facilitated by the lipophilic 4,7-diphenyl-1,10-phenanthroline (DIP) ligand. Despite the large size of Ru(DIP) 2 dppz 2+ (20 Å diameter), this complex is readily transported inside the cell compared to smaller and more hydrophilic complexes such as Ru(bpy) 2 dppz 2+ . Accumulation in the cellular interior is confirmed by confocal microscopy.The cellular uptake characteristics of a small molecule are critical to its application as a therapeutic or diagnostic agent. However, our understanding of the chemical rules governing uptake is rudimentary. 1,2 Although transition metal complexes have increasingly been applied for biological applications, 3-5 their uptake properties are even less well developed. Here, we exploit flow cytometry to provide statistics on the uptake of ruthenium complexes into HeLa cells. These ruthenium complexes, owing to their facile synthesis, stability, and luminescence, provide a route to compare and contrast factors governing cellular uptake.A series of dipyridophenazine (dppz) complexes of Ru(II) was synthesized for systematic comparison. [6][7][8] Substituting the ancillary ligands on the dppz complex permits variation in the overall complex charge, size, and hydrophobicity ( Figure 1). Furthermore since these dppz Email: jkbarton@caltech.edu. Supporting Information Available. Flow cytometry for cell nuclei. This material is available free of charge via the Internet at http://pubs.acs.org. HeLa cells were prepared for flow cytometry analysis after incubation with the Ru complexes at various concentrations and times. 12 Flow cytometry was performed on a BD FACS Aria using ~20,000 cells per sample. The ruthenium complexes were excited at 488 nm, with the emission observed at 600-620 nm. Live cells were distinguished by their low To-Pro-3 emission. Figure 2 illustrates results of the flow cytometry. Cells not treated with complex exhibit some background luminescence. Incubation with 10 μM Ru(bpy) 2 dppz 2+ or Ru(phen) 2 dppz 2+ for 2 h causes only a small change in the luminescence profile. When cells are incubated with 10 μM Ru(DIP) 2 dppz 2+ , however, the luminescence intensity of the cell population increases dramatically. NIH Public AccessUptake for the different Ru complexes may be compared based upon the mean luminescence intensity of the cell population (Table 2). Below 1 μM, Ru(DIP) 2 dppz 2+ is taken up appreciably above background. At higher concentrations, Ru(bpy) 2 dppz 2+ , Ru(CO 2 Et-bpy) 2 dppz 2+ , and Ru(phen) 2 dppz 2+ are taken up to some extent, but even at 20 μM Ru, little luminescence is evident for Ru(...

Transition metal complexes provide a promising avenue for the design of therapeutic and diagnostic agents, but the limited understanding of their cellular uptake is a roadblock to their effective application. Here, we examine the mechanism of cellular entry of a luminescent ruthenium(II) polypyridyl complex, Ru(DIP) 2 dppz 2+ (where DIP = 4,7-diphenyl-1,10-phenanthroline and dppz = dipyridophenazine), into HeLa cells, with the extent of uptake measured by flow cytometry. No diminution of cellular uptake is observed under metabolic inhibition with deoxyglucose and oligomycin, indicating an energy-independent mode of entry. The presence of organic cation transporter inhibitors also does not significantly alter uptake. However, the cellular internalization of Ru(DIP) 2 dppz 2+ is sensitive to the membrane potential. Uptake decreases when cells are depolarized with high potassium buffer and increases when cells are hyperpolarized with valinomycin. These results support passive diffusion of Ru(DIP) 2 dppz 2+ into the cell.Transition metal complexes have tremendous potential as diagnostic and therapeutic agents. They can be exploited for their modularity, reactivity, imaging capabilities, redox chemistry, and their precisely defined three-dimensional structure. An increasing number of biological applications have been explored (1-3). Complexes that are currently in clinical use include the platinum anticancer drugs, radiodiagnostic agents containing 99m Tc, and gadolinium(III) magnetic resonance imaging contrast agents.In order to design new metal-based drugs more rationally, an understanding of the physiological processing of metal complexes is required. Though cellular uptake is critical to the success of a drug or probe, few mechanistic details are known regarding metal complex uptake. Different entry mechanisms may be preferred depending on the application, as the mode of entry affects cell-type specificity, the rate of internalization, and the fate of the compound once inside the cell. For example, entry by diffusion affords broad cell-type specificity, a great advantage in the use of fluorescent probes for live cell imaging. Conversely, medicinal chemists may seek to deliver drugs to target organs, taking advantage of tissue-specific transporters (4) or receptors (5). For each mode of entry, there are also drawbacks. With protein-mediated transport, the degree of modification of the molecule is limited because transport relies on recognition. With endocytosis, molecules are often trapped in endosomes and face degradation by lysosomal enzymes.Ruthenium(II) polypyridyl complexes are useful for studying cellular uptake due to their facile synthesis, stability in aqueous solution, and luminescence. Using confocal microscopy and flow cytometry, we have examined the uptake of a series of dipyridophenazine (dppz)

The cellular uptake and localization of a Ru-octaarginine conjugate with and without an appended fluorescein are compared. The inherent luminescence of the Ru(II) dipyridophenazine complex allows observation of its uptake without the addition of a fluorophore. Ru-octaarginine-fluorescein stains the cytosol, nuclei and nucleoli of HeLa cells under conditions where the Ru-octaarginine conjugate without fluorescein shows only punctate cytoplasmic labeling. At higher concentrations, however, Ru-octaarginine without the fluorescein tag does exhibit cytoplasmic, nuclear, and nucleolar staining. Attaching fluorescein to Ru-octaarginine lowers the threshold concentration required for diffuse cytoplasmic labeling and nuclear entry. Hence, the localization of the fluorophore-bound peptide cannot serve as a proxy for that of the free peptide.

Transition metal complexes offer great potential as diagnostic and therapeutic agents, and a growing number of biological applications have been explored. To be effective, these complexes must reach their intended target inside the cell. Here we review the cellular accumulation of metal complexes, including their uptake, localization, and efflux. Metal complexes are taken up inside cells through various mechanisms, including passive diffusion and entry through organic and metal transporters. Emphasis is placed on the methods used to examine cellular accumulation, to identify the mechanism (s) of uptake, and to monitor possible efflux. Conjugation strategies that have been employed to improve the cellular uptake characteristics of metal complexes are also described.

In an effort to develop octahedral metal complexes as chemotherapeutic and diagnostic agents targeted to DNA, it is critical to optimize the properties of their cellular uptake. Appending Doctaarginine has been found to improve both the uptake and nuclear localization efficiency of these complexes, but the increased positive charge interferes with selective DNA binding and hence activity. Herein, we evaluate the nuclear entry of a series of luminescent ruthenium peptide conjugates of shorter sequence and lower charge. As is the case for the D-octaarginine conjugate (Ru-D-R8), the tetrapeptide RrRK (where r = D-arginine) facilitates nuclear localization of the ruthenium complex above a threshold concentration, though the threshold is higher for this conjugate (Ru-RrRK) than for Ru-D-R8. Furthermore, appended fluorescein, which lowers the threshold concentration for Ru-D-R8, does not improve nuclear entry of Ru-RrRK, indicating that fluorescein conjugation is not a general strategy for modulating the distribution of cell-penetrating peptides. Similarly, the concentration required for nuclear entry of Ru-RrRK is much higher than has been reported for a thiazole orange RrRK conjugate, demonstrating the influence of payload on the efficiency of uptake and localization of cell-penetrating peptides.