Fluorescein Redirects a Ruthenium−Octaarginine Conjugate to the Nucleus

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

Exploring the cellular accumulation of metal complexes

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

Targeting a ruthenium complex to the nucleus with short peptides

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 d-octaarginine 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

Methods to Explore Cellular Uptake of Ruthenium Complexes

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)_2dppz^(2+) (20 Å diameter), this complex is readily transported inside the cell compared to smaller and more hydrophilic complexes, such as Ru(bpy)_2dppz^(2+). Accumulation in the cellular interior is confirmed by confocal microscopy

Mechanism of cellular uptake of a ruthenium polypyridyl complex

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)2dppz2+ (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)2dppz2+ 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)2dppz2+ into the cell

Inorganic−Organic Hybrid Luminescent Binary Probe for DNA Detection Based on Spin-Forbidden Resonance Energy Transfer

We describe the design of new fluorescent binary probe sensors for DNA detection based on spin-forbidden resonance energy transfer (SF-RET). Binary probes consist of a donor and acceptor fluorophores that are attached to two different oligonucleotides and serve as a resonance energy transfer (RET) donor−acceptor pair when hybridized to adjacent sites of a target sequence. In the absence of target, excitation of the donor results in fluorescence only from the donor, but when the probes hybridize to the target, the fluorophores are brought into close proximity favoring RET, yielding fluorescence mainly from the acceptor fluorophore. These new binary probes use the metal complex Ru(bpy‘)(DIP)_2^(2+) as the energy donor and an organic fluorophore (Cy5) as the energy acceptor. Energy transfer from the MLCT state of the Ru complex to singlet Cy5 is spin forbidden and produces a delayed fluorescence of Cy5. This paper demonstrates that fluorescence delay of Cy5 can be used to time resolve the emission of the probe from the intense fluorescence background of a model system for cellular background; this provides the reported system to overcome intense autofluorescence, an important and general advantage over “classical” spin-allowed steady-state probes

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG)

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2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153772/1/acr24131.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153772/2/acr24131_am.pd