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.
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 Puckett, Cindy A; Ernst, Russell J; Barton, Jacqueline K
Dalton transactions : an international journal of inorganic chemistry,
01/2010, Letnik:
39, Številka:
5
Journal Article
Recenzirano
Odprti dostop
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.
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)2dppz2+ (20 Å diameter), this complex is readily transported inside the cell compared to smaller and more hydrophilic complexes, such as Ru(bpy)2dppz2+. Accumulation in the cellular interior is confirmed by confocal microscopy.
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.
Transition metal complexes have enormous potential as diagnostic and therapeutic agents, but their internalization and distribution in living cells are only poorly understood. Here, we perform one of ...the few systematic explorations of the uptake efficiency and mechanism of a class of metal complexes: luminescent dipyridophenazine (dppz) complexes of ruthenium(II). Substitution of the ancillary ligands permits variation in the overall complex charge, size, and hydrophobicity. We find that internalization of these complexes occurs mostly through passive diffusion, driven by the membrane potential, and that hydrophobicity, rather than size, is the most important determinant of compound accumulation. Across different cell types with all compounds, mostly uneven cytoplasmic staining is observed with near exclusion from the nucleus. Conjugation to cell-penetrating peptides, such as D-octaarginine, increases uptake efficiency, but leads to trapping in endosomes below a threshold concentration. Above this threshold concentration, substantial staining of the nucleus as well as the cytosol is observed. An appended fluorescein tag lowers the threshold concentration, indicating the importance of payload to the internalization and distribution of cell-penetrating peptides. Shorter peptides, including the nuclear targeting signal RrRK (where r = D-arginine), are also studied, though none have as high a degree of uptake nor as low a threshold concentration as the octaarginine conjugate. These studies provide a basis for the future design and optimization of metal complexes for biological application.
Transgender and gender diverse (TGD) people experience high rates of stigma and marginalization that are theorized to exacerbate substance use and psychological distress. However, little research has ...examined the role of various minority stressors in relation to substance use in TGD populations.
In this sample of 181 TGD individuals in the U.S. who reported substance use or binge drinking over the past month (M age = 25.6; SD = 5.6), we evaluated whether enacted stigma predicted alcohol use, substance use, and psychological distress.
Participants endorsed a high rate of exposure to enacted stigma over the past 6 months (e.g., 52% had been verbally insulted). Furthermore, 27.8% of the sample was classified as having moderate or higher severity drug use, and 35.4% were classified as having hazardous drinking levels. We found that enacted stigma was significantly related to moderate-high drug use and psychological distress. There were no significant associations between stigma variables and hazardous levels of drinking. Enacted stigma had an indirect effect on psychological distress via increased expectations of stigma.
This study adds to the growing literature exploring minority stressors in relation to substance use and mental health. Subsequent research is needed to examine TGD-specific factors that may more fully explain how TGD people cope with enacted stigma or that may influence substance use, particularly alcohol use.
•Transgender and gender diverse (TGD) people commonly experience minority stressors.•27.8% of the sample was classified as having moderate or higher severity drug use.•35.4% were classified as having hazardous drinking levels.•Enacted stigma was associated with moderate-high drug use and distress.•Neither enacted or expected stigma was associated with alcohol use severity.
Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, ...radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope-Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope-Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to "snap" functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.
Thesis (Ph. D.) -- California Institute of Technology, 2010.
Title from home page (viewed 03/22/2010). Advisor and committee chair names found in the thesis' metadata record in the digital ...repository. Includes bibliographical references.
The coronavirus (COVID-19) pandemic and associated shelter-in-place ordinances rapidly limited access to in-person social interactions, raising concerns of diminishing social support and community ...cohesion while psychological stressors increased. For LGBTQIA+ people, connectedness to the LGBTQIA+ community buffers against the harmful effects of stressors and decreases risks for poor psychological and behavioural health outcomes. The current study uses qualitative cross-sectional (between-person) and trajectory (within-person) analysis methods to characterise how LGBTQIA+ people's perceptions of community connectedness shifted during the first year of the pandemic. A convenience sample of LGBTQIA+ people in the U.S. completed an initial online survey in September 2020 (n = 298 and a follow-up survey in September 2021; n = 129). The survey included questions about changes in connectedness to the LGBTQIA+ community since the pandemic's beginning. Eight cross-cutting themes (related to identity shifts/exploration, disconnection, online connections, and increased awareness of social justice issues) were identified and then organised within each level of the LGBTQIA+ Social-Ecological Model (i.e. the individual-, couple-, interpersonal-, organisational-, community-, and chronosystem- level). Given the importance of social support for LGBTQIA+ wellbeing, more longitudinal research is needed to determine whether these changes persist after the resolution of the acute phase of the pandemic.