Two-Pronged Dormant Photosensitizer-Antibiotic Bacterial Inactivation: Mechanism, Dosage, and Cellular Evolution Visualized at the Single-Cell Level.

Innovative therapeutic approaches are required to battle the rise of antibiotic-resistant bacterial strains. Tapping on reactive oxygen species (ROS) generation in bacteria induced by bactericidal antibiotics, here we report a two-pronged strategy for bacterial inactivation relying on the synergistic combination of a bactericidal antibiotic and newly designed dormant photosensitizers (DoPSs) that activate in the presence of ROS. Intramolecular quenching renders DoPS inert in the presence of light. ROS trapping by DoPS aborts the quenching mechanism unmasking, in equal proportions, singlet oxygen (1O2) sensitization and fluorescence emission. Juxtaposed antioxidant-prooxidant activity built within our DoPS enables (i) initial activation of a few molecules by ROS and (ii) subsequent rapid activation of all DoPS in a bacterium via a domino effect mediated by photogenerated 1O2. Bulk colony forming unit studies employing the minimum inhibitory concentration of the antibiotic illustrate rapid and selective inactivation of Escherichia coli and Pseudomonas aeruginosa only in the presence of light, antibiotic, and DoPS. Single-cell, real-time imaging studies on E. coli reveal an autocatalytic progression of DoPS activation from focal points, providing a unique amplification system for sensing. Single-cell analysis further illustrates the impact of DoPS cellular loading on the rate of DoPS activation and cell death times and on the 1O2 dosing necessary for cell death to occur. Our two-pronged therapy discriminates based on cell metabolites and has the potential to result in lower toxicity, pave the way to reduced drug resistance, and provide insightful mechanistic information about bacterial membrane response to 1O2.

Singlet Oxygen Flux, Associated Lipid Photooxidation, and Membrane Expansion Dynamics Visualized on Giant Unilamellar Vesicles.

The physical properties of lipid membranes depend on their lipid composition. Photosensitized singlet oxygen (1O2) provides a handle to spatiotemporally control the generation of lipid hydroperoxides via the ene reaction, enabling fundamental studies on membrane dynamics in response to chemical composition changes. Critical to relating the physical properties of the lipid membrane to hydroperoxide formation is the availability of a sensitive reporter to quantify the arrival of 1O2. Here, we show that a fluorogenic α-tocopherol analogue, H4BPMHC, undergoes a >360-fold emission intensity enhancement in liposomes following a reaction with 1O2. Rapid quenching of 1O2 by the probe (kq = 4.9 × 108 M-1 s-1) ensures zero-order kinetics of probe consumption. The remarkable intensity enhancement of H4BPMHC upon 1O2 trapping, its linear temporal behavior, and its protective role in outcompeting membrane damage provide a sensitive and reliable method to quantify the 1O2 flux on lipid membranes. Armed with this probe, fluorescence microscopy studies were devised to enable (i) monitoring the flux of photosensitized 1O2 into giant unilamellar vesicles (GUVs), (ii) establishing the onset of the ene reaction with the double bonds of monounsaturated lipids, and (iii) visualizing the ensuing collective membrane expansion dynamics associated with molecular changes in the lipid structure upon hydroperoxide formation. A correlation was observed between the time for antioxidant H4BPMHC consumption by 1O2 and the onset of membrane fluctuations and surface expansion. Together, our imaging studies with H4BPMHC in GUVs provide a methodology to explore the intimate relationship between photosensitizer activity, chemical insult, membrane morphology, and its collective dynamics.

Impact of transitioning patients to oral diuretics 24 hours before discharge from heart failure hospitalization on 30 day outcomes.

BACKGROUND: Patients hospitalized for heart failure (HF) are at high risk for post-discharge events. Although transition from intravenous to oral diuretics for ≥24 h is commonly practiced to reduce post-discharge risk, evidence supporting this strategy is limited. We investigated the impact of this practice on 30 day post-discharge outcomes following HF hospitalization at our institution. METHODS: Retrospective chart review of patients hospitalized with a primary HF diagnosis, discharged on oral diuretic, and followed at our institution. Admission, in-hospital, and pre-discharge characteristics of patients discharged with ≥24-h observation were compared to those of patients observed for <24-h on oral diuretics. Differences between groups in composite 30 day all-cause mortality and rehospitalization, each component, and HF rehospitalization were assessed. RESULTS: Of 285 patients meeting entry criteria, 178 received oral diuretics ≥24 h prior to discharge and 107 were discharged <24 h after transitioning to oral diuretics. Baseline characteristics were similar between groups. Patients with ≥24 h observation on oral diuretics had longer in-hospital stays and greater weight and net volume loss than those observed <24 h. Patients receiving oral diuretics for <24 h were more likely to have had neurohormonal drugs and diuretic dose changed within 24-h of discharge. Oral diuretic treatment for ≥24 h failed to reduce any study endpoint. CONCLUSIONS: Transitioning patients to oral diuretics for ≥24 h prior to discharge following HF hospitalization failed to improve 30-day outcomes. These results question this strategy for all patients hospitalized for worsening HF.

An evolutionary conserved detoxification system for membrane lipid-derived peroxyl radicals in Gram-negative bacteria.

How double-membraned Gram-negative bacteria overcome lipid peroxidation is virtually unknown. Bactericidal antibiotics and superoxide ion stress stimulate the transcription of the Burkholderia cenocepacia bcnA gene that encodes a secreted lipocalin. bcnA gene orthologs are conserved in bacteria and generally linked to a conserved upstream gene encoding a cytochrome b561 membrane protein (herein named lcoA, lipocalin-associated cytochrome oxidase gene). Mutants in bcnA, lcoA, and in a gene encoding a conserved cytoplasmic aldehyde reductase (peroxidative stress-associated aldehyde reductase gene, psrA) display enhanced membrane lipid peroxidation. Compared to wild type, the levels of the peroxidation biomarker malondialdehyde (MDA) increase in the mutants upon exposure to sublethal concentrations of the bactericidal antibiotics polymyxin B and norfloxacin. Microscopy with lipid peroxidation-sensitive fluorescent probes shows that lipid peroxyl radicals accumulate at the bacterial cell poles and septum and peroxidation is associated with a redistribution of anionic phospholipids and reduced antimicrobial resistance in the mutants. We conclude that BcnA, LcoA, and PsrA are components of an evolutionary conserved, hitherto unrecognized peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals.

Fluorescence-Amplified Detection of Redox Turnovers in Supported Lipid Bilayers Illuminates Redox Processes of α-Tocopherol.

Electron-transfer processes in lipid membranes are key to biological functions, yet challenging to study because of the intrinsic heterogeneity of the systems. Here, we report spectro-electrochemical measurements on indium tin oxide-supported lipid bilayers toward the selective induction and sensing of redox processes in membranes. Working at neutral pH with a fluorogenic α-tocopherol analogue, the dynamics of the two-electron oxidation of the chromanol to a chromanone and the rapid thermal decay of the latter to a chromoquinone are recorded as a rapid surge and drop in intensity, respectively. Continuous voltage cycling reveals rapid chromoquinone two-electron, two-proton reduction to dihydrochromoquinone at negative bias, followed by slow regeneration of the former at positive bias. The kinetic parameters of these different transitions are readily obtained as a function of applied potentials. The sensitivity and selectivity afforded by the reported method enables monitoring signals equivalent to femtoampere currents with a high signal-to-background ratio. The study provides a new method to monitor membrane redox processes with high sensitivity and minimal concentrations and unravels key dynamic aspects of α-tocopherol redox chemistry.

Modification of amyloid-beta peptide aggregation via photoactivation of strained Ru(ii) polypyridyl complexes.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by progressive and irreversible damage to the brain. One of the hallmarks of the disease is the presence of both soluble and insoluble aggregates of the amyloid beta (Aß) peptide in the brain, and these aggregates are considered central to disease progression. Thus, the development of small molecules capable of modulating Aß peptide aggregation may provide critical insight into the pathophysiology of AD. In this work we investigate how photoactivation of three distorted Ru(ii) polypyridyl complexes (Ru1-3) alters the aggregation profile of the Aß peptide. Photoactivation of Ru1-3 results in the loss of a 6,6'-dimethyl-2,2'-bipyridyl (6,6'-dmb) ligand, affording cis-exchangeable coordination sites for binding to the Aß peptide. Both Ru1 and Ru2 contain an extended planar imidazo[4,5-f][1,10]phenanthroline ligand, as compared to a 2,2'-bipyridine ligand for Ru3, and we show that the presence of the phenanthroline ligand promotes covalent binding to Aß peptide His residues, and in addition, leads to a pronounced effect on peptide aggregation immediately after photoactivation. Interestingly, all three complexes resulted in a similar aggregate size distribution at 24 h, forming insoluble amorphous aggregates as compared to significant fibril formation for peptide alone. Photoactivation of Ru1-3 in the presence of pre-formed Aß1-42 fibrils results in a change to amorphous aggregate morphology, with Ru1 and Ru2 forming large amorphous aggregates immediately after activation. Our results show that photoactivation of Ru1-3 in the presence of either monomeric or fibrillar Aß1-42 results in the formation of large amorphous aggregates as a common endpoint, with Ru complexes incorporating the extended phenanthroline ligand accelerating this process and thereby limiting the formation of oligomeric species in the initial stages of the aggregation process that are reported to show considerable toxicity.

Strained, Photoejecting Ru(II) Complexes that are Cytotoxic Under Hypoxic Conditions.

A series of strained Ru(II) complexes were studied for potential anticancer activity in hypoxic tissues. The complexes were constructed with methylated ligands that were photolabile and an imidizo[4,5-f][1,10]phenanthroline ligand that contained an appended aromatic group to potentially allow for contributions of ligand-centered excited states. A systematic variation of the size and energy of the aromatic group was performed using systems containing 1-4 fused rings, and the photochemical and photobiological behaviors of all complexes were assessed. The structure and nature of the aromatic group had a subtle impact on photochemistry, altering environmental sensitivity, and had a significant impact on cellular cytotoxicity and photobiology. Up to 5-fold differences in cytotoxicity were observed in the absence of light activation; this rose to 50-fold differences upon exposure to 453 nm light. Most significantly, one complex retained activity under conditions with 1% O2 , which is used to induce hypoxic changes. This system exhibited a photocytotoxicity index (PI) of 15, which is in marked contrast to most other Ru(II) complexes, including those designed for O2 -independent mechanisms of action.

Photophysical Properties and Photobiological Activities of Ruthenium(II) Complexes Bearing π-Expansive Cyclometalating Ligands with Thienyl Groups.

A new family of cyclometalated ruthenium(II) complexes [Ru(N^N)2(C^N)]+ derived from the π-extended benzo[h]imidazo[4,5-f]quinolone ligand appended with thienyl groups (n = 1-4, compounds 1-4) was prepared and its members were characterized for their chemical, photophysical, and photobiological properties. The lipophilicities of 1-4, determined as octanol-water partition coefficients (log Po/w), were positive and increased with the number of thienyl units. The absorption and emission bands of the C^N compounds were red-shifted by up to 200 nm relative to the analogous Ru(II) diimine systems. All of the complexes exhibited dual emission with the intraligand fluorescence (1IL, C^N-based) shifting to lower energies with increasing n and the metal-to-ligand charge transfer phosphorescence (3MLCT, N^N-based) remaining unchanged. Compounds 1-3 exhibited excited state absorption (ESA) profiles consistent with lowest-lying 3MLCT states when probed by nanosecond transient absorption (TA) spectroscopy with 532 nm excitation and had contributions from 1IL(C^N) states with 355 nm excitation. These assignments were supported by the lifetimes observed (<10 ns for the 1IL states and around 20 ns for the 3MLCT states) as well as a noticeable ESA for 3 with 355 nm excitation that did not occur with 532 nm excitation. Compound 4 was the only member of the family with two 3MLCT emissive lifetimes (15, 110 ns), and the TA spectra collected with both 355 and 532 nm excitation was assigned to the 3IL state, which was corroborated by its 4-6 µs lifetime. The ESA for 4 had a rise time of approximately 10 ns and an initial decay of 110 ns, which suggests a possible 3MLCT-3IL excited state equilibrium that results in delayed emission from the 3MLCT state. Compound 4 was nontoxic toward human skin melanoma cells (SKMEL28) in the dark (EC50 = >300 µM); 1-3 were cytotoxic and yielded EC50 values between 1 and 20 µM. The photocytotoxicites with visible light ranged from 87 nM with a phototherapeutic index (PI) of 13 for 1 to approximately 1 µM (PI = >267) for 4. With red light, EC50 values varied from 270 nM (PI = 21) for 3 to 12 µM for 4 (PI = >25). The larger PIs for 4, especially with visible light, were attributed to the much lower dark cytotoxicity for this compound. Because the dark cytotoxicity contributes substantially to the observed photocytotoxicity for 1-3, it was not possible to assess whether the 3IL state of 4 led to a much more potent phototoxic mechanism in the absence of dark toxicity. There was no stark contrast in cellular uptake and accumulation by laser scanning confocal and differential interference contrast microscopy to explain the large differences in dark toxicities between 1-3 and 4. Nevertheless, the study highlights a new family of Ru(II) C^N complexes where π-conjugation beyond a certain point results in low dark cytotoxicity with high photocytotoxicity, opposing the notion that cyclometalated Ru(II) systems are too toxic to be phototherapeutic agents.

Cyclometalated Ruthenium(II) Complexes Derived from α-Oligothiophenes as Highly Selective Cytotoxic or Photocytotoxic Agents.

The photophysical and photobiological properties of a new class of cyclometalated ruthenium(II) compounds incorporating π-extended benzo[ h]imidazo[4,5- f]quinoline (IBQ) cyclometalating ligands (C^N) bearing thienyl rings ( n = 1-4, compounds 1-4) were investigated. Their octanol-water partition coefficients (log Po/w) were positive and increased with n. Their absorption and emission energies were red-shifted substantially compared to the analogous Ru(II) diimine (N^N) complexes. They displayed C^N-based intraligand (IL) fluorescence and triplet excited-state absorption that shifted to longer wavelengths with increasing n and N^N-based metal-to-ligand charge transfer (MLCT) phosphorescence that was independent of n. Their photoluminescence lifetimes (τem) ranged from 4-10 ns for 1IL states and 12-18 ns for 3MLCT states. Transient absorption lifetimes (τTA) were 5-8 µs with 355 nm excitation, ascribed to 3IL states that became inaccessible for 1-3 with 532 nm excitation (1-3, τTA = 16-17 ns); the 3IL of 4 only was accessible by lower energy excitation, τTA = 3.8 µs. Complex 4 was nontoxic (EC50 > 300 µM) to SK-MEL-28 melanoma cells and CCD1064-Sk normal skin fibroblasts in the dark, while 3 was selectively cytotoxic to melanoma (EC50= 5.1 µM) only. Compounds 1 and 2 were selective for melanoma cells in the dark, with submicromolar potencies (EC50 = 350-500 nM) and selectivity factors (SFs) around 50. The photocytotoxicities of compounds 1-4 toward melanoma cells were similar, but only compounds 3 and 4 displayed significant phototherapeutic indices (PIs; 3, 43; 4, >1100). The larger cytotoxicities for compounds 1 and 2 were attributed to increased cellular uptake and nuclear accumulation, and possibly related to the DNA-aggregating properties of all four compounds as demonstrated by cell-free gel mobility-shift assays. Together, these results demonstrate a new class of thiophene-containing Ru(II) cyclometalated compounds that contain both highly selective chemotherapeutic agents and extremely potent photocytotoxic agents.

Organometallic Ru(II) Photosensitizers Derived from π-Expansive Cyclometalating Ligands: Surprising Theranostic PDT Effects.

The purpose of the present study was to investigate the influence of π-expansive cyclometalating ligands on the photophysical and photobiological properties of organometallic Ru(II) compounds. Four compounds with increasing π conjugation on the cyclometalating ligand were prepared, and their structures were confirmed by HPLC, 1D and 2D (1)H NMR, and mass spectrometry. The properties of these compounds differed substantially from their Ru(II) polypyridyl counterparts. Namely, they were characterized by red-shifted absorption, very weak to no room temperature phosphorescence, extremely short phosphorescence state lifetimes (<10 ns), low singlet oxygen quantum yields (0.5-8%), and efficient ligand-centered fluorescence. Three of the metal complexes were very cytotoxic to cancer cells in the dark (EC50 values = 1-2 µM), in agreement with what has traditionally been observed for Ru(II) compounds derived from small C^N ligands. Surprisingly, the complex derived from the most π-expansive cyclometalating ligand exhibited no cytotoxicity in the dark (EC50 > 300 µM) but was phototoxic to cells in the nanomolar regime. Exceptionally large phototherapeutic margins, exceeding 3 orders of magnitude in some cases, were accompanied by bright ligand-centered intracellular fluorescence in cancer cells. Thus, Ru(II) organometallic systems derived from π-expansive cyclometalating ligands, such 4,9,16-triazadibenzo[a,c]napthacene (pbpn), represent the first class of potent light-responsive Ru(II) cyclometalating agents with theranostic potential.