ABSTRACT
Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.
Subject(s)
Extracellular Vesicles , Flow Cytometry , Monocytes , Humans , Extracellular Vesicles/metabolism , Flow Cytometry/methods , Monocytes/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Nucleic Acids/metabolism , Staining and Labeling/methods , THP-1 Cells , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Lipopolysaccharides/pharmacology , Cell Line, Tumor , A549 CellsABSTRACT
The metal-ligand charge transfer (3MLCT) and phosphorescence-quenching metal-centered (3MC) states of the helicate and mesocate diastereoisomers of a double-stranded dinuclear polypyridylruthenium(II) complex have been investigated using ultrafast transient absorption spectroscopy. At 294 K, transient signals of the helicate decayed significantly slower than those of the mesocate, whereas at 77 K, no clear contrast in kinetics was observed. Contributions to excited-state decay from high-lying 3MLCT states were identified at both temperatures. Spectroscopic data (294 K) suggest that the 3MC state of the helicate lies above the 3MLCT and that the reverse is true for the mesocate; this was further validated by density functional theory calculations. The stabilization of the 3MC state relative to the 3MLCT state in the mesocate was explained by a reduction in ligand field strength due to distortion near the ligand bridge, which causes further deviation from octahedral geometry compared to the helicate. This work illustrates how minor structural differences can significantly influence excited state dynamics.
ABSTRACT
A new photoluminescent polypyridylruthenium(II) stain for extracellular vesicles (EVs) released from lipopolysaccharide-stimulated THP-1 monocytes enabled important new insights into how the bacteria-induced immune system affects the blood-brain barrier (BBB). These included previously unknown aspects of EV interactions with BBB microvascular endothelial cells and the extracellular matrix relevant to human brain diseases.
Subject(s)
Endothelial Cells , Extracellular Vesicles , Humans , Endothelium , Brain , Blood-Brain BarrierABSTRACT
Linear and non-linear tetranuclear ruthenium(ii) complexes containing the bridging ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-heptane have been synthesised and their biological properties examined. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the ruthenium(ii) complexes were determined against six strains of bacteria: Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA); and the Gram-negative Escherichia coli (E. coli) strains MG1655, APEC, UPEC and Pseudomonas aeruginosa (P. aeruginosa). The results showed that both tetranuclear complexes had significant antimicrobial activity, with the non-linear (branched) species (Rubb7-TNL) having slightly higher activity than the corresponding linear analogue (Rubb7-TL). The corresponding toxicity against three eukaryotic cell lines - BHK (baby hamster kidney), Caco-2 (heterogeneous human epithelial colorectal adenocarcinoma) and Hep-G2 (liver carcinoma) - have also been determined. Interestingly, both Rubb7-TNL and Rubb7-TL were as toxic to the eukaryotic cells as they were to the bacteria, a rarity for kinetically-inert cationic polypyridylruthenium(ii) complexes, and exhibited lower IC50 values than cisplatin over 24-, 48- or 72-hour incubation times. Fluorescence spectroscopy was used to study the binding of the ruthenium complexes with human serum albumin (HSA). Rubb7-TNL and Rubb7-TL exhibited strong HSA binding, with equilibrium binding constants in the order of 107 M-1. Confocal microscopy was used to examine the cellular localisation of Rubb7-TNL in BHK cells. The results indicated that the ruthenium complex localised in the nucleolus. Significant accumulation was also observed in the cytoplasm, but not in the mitochondria. Taken together, the results of this study suggest that Rubb7-TNL is an unlikely candidate as an antimicrobial agent, but may have potential as an anticancer drug.
Subject(s)
2,2'-Dipyridyl/pharmacology , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Coordination Complexes/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Heptanes/pharmacology , Pyridines/chemistry , Ruthenium/pharmacology , 2,2'-Dipyridyl/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Caco-2 Cells , Cell Proliferation/drug effects , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Drug Screening Assays, Antitumor , Hep G2 Cells , Heptanes/chemistry , Humans , Ligands , Ruthenium/chemistryABSTRACT
Herein we report the separation of the three stereoisomers of the DNA light-switch compound [{Ru(bpy)2}2(tpphz)]4+ (tpphz = tetrapyrido[3,2-a:2',3'-c:3â³,2â³-h:2â´,3â´-j]phenazine) by column chromatography and the characterization of each stereoisomer by X-ray crystallography. The interaction of these compounds with a DNA octanucleotide d(GCATATCG).d(CGATATGC) has been studied using NMR techniques. Selective deuteration of the bipyridyl rings was needed to provide sufficient spectral resolution to characterize structures. NMR-derived structures for these complexes show a threading intercalation binding mode with slow and chirality-dependent rates. This represents the first solution structure of an intercalated bis-ruthenium ligand. Intriguingly, we find that the binding site selectivity is dependent on the nature of the stereoisomer employed, with Λ RuII centers showing a better intercalation fit.
Subject(s)
DNA, B-Form/chemistry , Intercalating Agents/chemistry , Organometallic Compounds/chemistry , Pyridines/chemistry , Ruthenium/chemistry , Base Sequence , DNA, B-Form/genetics , Kinetics , Models, Molecular , Nucleic Acid Conformation , StereoisomerismABSTRACT
The toxicity (IC50) of a series of mononuclear ruthenium complexes containing bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (bbn) as a tetradentate ligand against three eukaryotic cell lines-BHK (baby hamster kidney), Caco-2 (heterogeneous human epithelial colorectal adenocarcinoma) and Hep-G2 (liver carcinoma)-have been determined. The results demonstrate that cis-α-[Ru(Me4phen)(bb7)]2+ (designated as α-Me4phen-bb7, where Me4phen = 3,4,7,8-tetramethyl-1,10-phenanthroline) showed little toxicity toward the three cell lines, and was considerably less toxic than cis-α-[Ru(phen)(bb12)]2+ (α-phen-bb12) and the dinuclear complex [{Ru(phen)2}2{µ-bb12}]4+. Fluorescence spectroscopy was used to study the binding of the ruthenium complexes with human serum albumin (HSA). The binding of α-Me4phen-bb7 to the macrocyclic host molecule cucurbit[10]uril (Q[10]) was examined by NMR spectroscopy. Large upfield 1H NMR chemical shift changes observed for the methylene protons in the bb7 ligand upon addition of Q[10], coupled with the observation of several intermolecular ROEs in ROESY spectra, indicated that α-Me4phen-bb7 bound Q[10] with the bb7 methylene carbons within the cavity and the metal center positioned outside one of the portals. Simple molecular modeling confirmed the feasibility of the binding model. An α-Me4phen-bb7-Q[10] binding constant of 9.9 ± 0.2 × 106 M-1 was determined by luminescence spectroscopy. Q[10]-encapsulation decreased the toxicity of α-Me4phen-bb7 against the three eukaryotic cell lines and increased the binding affinity of the ruthenium complex for HSA. Confocal microscopy experiments indicated that the level of accumulation of α-Me4phen-7 in BHK cells is not significantly affected by Q[10]-encapsulation. Taken together, the combined results suggest that α-Me4phen-7 could be a good candidate as a new antimicrobial agent, and Q[10]-encapsulation could be a method to improve the pharmacokinetics of the ruthenium complex.
ABSTRACT
A series of mononuclear ruthenium(ii) complexes containing the tetradentate ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-heptane have been synthesised and their biological properties examined. In the synthesis of the [Ru(phen')(bb7)]2+ complexes (where phen' = 1,10-phenanthroline and its 5-nitro-, 4,7-dimethyl- and 3,4,7,8-tetramethyl- derivatives), both the symmetric cis-α and non-symmetric cis-ß isomers were formed. However, upon standing for a number of days (or more quickly under harsh conditions) the cis-ß isomer converted to the more thermodynamically stable cis-α isomer. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the ruthenium(ii) complexes were determined against six strains of bacteria: Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA); and the Gram-negative Escherichia coli (E. coli) strains MG1655, APEC, UPEC and Pseudomonas aeruginosa (P. aeruginosa). The results showed that the [Ru(5-NO2phen)(bb7)]2+ complex had little or no activity against any of the bacterial strains. By contrast, for the other cis-α-[Ru(phen')(bb7)]2+ complexes, the antimicrobial activity increased with the degree of methylation. In particular, the cis-α-[Ru(Me4phen)(bb7)]2+ complex showed excellent and uniform MIC activity against all bacteria. By contrast, the MBC values for the cis-α-[Ru(Me4phen)(bb7)]2+ complex varied considerably across the bacteria and even within S. aureus and E. coli strains. In order to gain an understanding of the relative antimicrobial activities, the DNA-binding affinity, cellular accumulation and water-octanol partition coefficients (log P) of the ruthenium complexes were determined. Interestingly, all the [Ru(phen')(bb7)]2+ complexes exhibited stronger DNA binding affinity (Ka ≈ 1 × 107 M-1) than the well-known DNA-intercalating complex [Ru(phen)2(dppz)]2+ (where dppz = dipyrido[3,2-a:2',3'-c]phenazine).
ABSTRACT
Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb12-mono, which was among the most potent in its ability to kill L3 (IC50 = 3.1 ± 0.4 µM) and adult (IC50 = 5.2 ± 0.3 µM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb12-mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significant decreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance.
Subject(s)
Acetylcholinesterase/drug effects , Antinematodal Agents/administration & dosage , Antinematodal Agents/pharmacology , Trichuris/drug effects , Administration, Oral , Animals , Antinematodal Agents/chemistry , Cholinesterase Inhibitors/administration & dosage , Cholinesterase Inhibitors/pharmacology , Disease Models, Animal , Larva/drug effects , Male , Mice , Parasite Egg Count , Ruthenium/administration & dosage , Ruthenium/chemistry , Ruthenium/pharmacology , Trichuriasis/drug therapy , Trichuriasis/parasitology , Trichuris/enzymologyABSTRACT
The cis-α isomer of [Ru(bb7 )(dppz)]2+ (dppz=dipyrido[3,2-a:2',3'-c]phenazine; bb7 =bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-alkane) has been synthesised. The minimum inhibitory concentrations and the minimum bactericidal concentrations of [Ru(bb7 )(dppz)]2+ and its parent complex [Ru(phen)2 (dppz)]2+ (phen=1,10-phenanthroline) were determined against a range of bacteria. The results showed that both ruthenium complexes exhibited good activity against Gram-positive bacteria, but [Ru(bb7 )(dppz)]2+ showed at least eightfold better activity against the Gram-negative bacteria than [Ru(phen)2 (dppz)]2+ . Luminescence assays demonstrated that [Ru(bb7 )(dppz)]2+ accumulated in a Gram-negative bacterium to the same degree as in a Gram-positive species, and assays with liposomes showed that [Ru(bb7 )(dppz)]2+ interacted more strongly with membranes than the parent [Ru(phen)2 (dppz)]2+ complex. The DNA binding affinity for [Ru(bb7 )(dppz)]2+ was determined to be 6.7 × 106 m-1 . Although more toxic to eukaryotic cells than [Ru(phen)2 (dppz)]2+ , [Ru(bb7 )(dppz)]2+ exhibited greater activity against bacteria than eukaryotic cells.
ABSTRACT
BACKGROUND: Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)-the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri-68%, Rubb7-tnl-56%) and were significantly morphologically altered (Rubb12-tri-62% abnormal, Rubb7-tnl-35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. CONCLUSIONS/SIGNIFICANCE: Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes.
Subject(s)
Acetylcholinesterase/drug effects , Cholinesterase Inhibitors/pharmacology , Organometallic Compounds/pharmacology , Ruthenium/pharmacology , Schistosoma haematobium/drug effects , Schistosoma mansoni/drug effects , Animals , Biological Transport/drug effects , Disease Models, Animal , Female , Glucose/metabolism , Larva , Male , Mice , Praziquantel/therapeutic use , Schistosoma haematobium/enzymology , Schistosoma mansoni/enzymology , Schistosomiasis haematobia/drug therapy , Schistosomiasis haematobia/parasitology , Schistosomiasis mansoni/drug therapy , Schistosomiasis mansoni/parasitologyABSTRACT
The relatively non-toxic family of cucurbit[n]uril, Q[n], have shown considerable potential in vitro as drug delivery agents, with only a few examples of pharmacokinetic (PK) studies for drugâQ[n]. Drug-free Q[n] PK studies are the next step in determining the pharmacological applicability in their drug delivery potential. The results for the first PK and bio-distribution of drug-free 14C-Q[7] are described for administration via intravenous (i.v.) and intraperitoneal (i.p.) dosing. A study of oral administration of drug-free 14C-Q[8] has also been undertaken to determine the time course for the gastrointestinal tract (GIT), absorption and subsequent bio-distribution. Q[10], a potential drug carrier for larger drugs, was evaluated for its effect on the PK profile of a dinuclear ruthenium complex (Rubb12), a potential antimicrobial agent. The Rubb12âQ[10] complex and free Rubb12 were administered by i.v. to determine differences in Rubb12 plasma concentrations and organ accumulation. Interestingly, the PK profiles and bio-distribution observed for Q[7] showed similarities to those of Rubb12âQ[10]. Drug-free Q[7] has a relatively fast plasma clearance and a generally low organ accumulation except for the kidneys. Drug-free Q[8] showed a low absorption from the GIT into the blood stream but the small percentage absorbed reflected the organ accumulation of Q[7]. These results provide a better understanding of the probable PK profile and bio-distribution for a drugâQ[n] through the influence of the drug delivery vehicle and the positive clearance of drug-free Q[n] via the kidneys supports its potential value in future drug delivery applications.
Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacokinetics , Imidazoles/chemistry , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacokinetics , Ruthenium/chemistry , Animals , Bridged-Ring Compounds/chemistry , Capsules , Mice , Tissue DistributionABSTRACT
Condensation studies of chromosomal DNA in E. coli with a tetranuclear ruthenium complex are carried out and images obtained with wide-field fluorescence microscopy. Remarkably different condensate morphologies resulted, depending upon the treatment protocol. The occurrence of condensed nucleoid spirals in live bacteria provides evidence for the transertion hypothesis.
Subject(s)
Chromosomes, Bacterial , DNA, Bacterial , Escherichia coli/genetics , Chromosomes, Bacterial/drug effects , DNA, Bacterial/drug effects , Escherichia coli/drug effects , Escherichia coli/metabolism , Microscopy, Fluorescence , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Ribosomes/metabolism , Ruthenium/chemistryABSTRACT
Dinuclear polypyridylruthenium(II) complexes bridged by a flexible methylene linker have received considerable interest as potential antibacterial agents. Their potency and uptake into bacterial cells is directly modulated by the length of the bridging linker, which has implicated membrane interactions as an essential feature of their mechanism of action. In this work, a combination of molecular dynamics (MD) simulations and solid-state NMR was used to present an atomistic model of a polypyridylruthenium(II) complex bound and incorporated into a bacterial membrane model. The results of 31P, 2H, 1H, and 13C NMR studies revealed that the antibacterial [{Ru(phen)2}2(µ-bb12)]4+ complex (Rubb12), where phen = 1,10-phenanthroline and bb12 = bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane), incorporated into a negatively charged model bacterial membrane, but only associated with the surface of a charge-neutral model of a eukaryotic membrane. Furthermore, an inactive [{Ir(phen)2}2(µ-bb12)]6+ (Irbb12) analogue, which is not taken up by bacterial cells, maintained only a surface-bound association with both bacterial and eukaryotic model membranes according to 31P and 2H NMR. The effects of Rubb12 on 31P chemical shift anisotropy and 2H acyl chain order parameters for negatively charged membranes correlated with a membrane-spanning state of the complex according to MD simulation-in which the metal centers embed in the lipid head group region and the central void, created by the biconic shape of the complex, resulting in increasing disorder of lipid acyl chains and membrane-thinning. A transbilayer mechanism and membrane-spanning may be essential for the cellular uptake and antibacterial activity of this class of compounds.
Subject(s)
2,2'-Dipyridyl/pharmacology , Anti-Bacterial Agents/pharmacology , Molecular Dynamics Simulation , Polymers/pharmacology , Ruthenium/pharmacology , Staphylococcus aureus/drug effects , 2,2'-Dipyridyl/chemistry , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Cell Death/drug effects , Cell Line , Magnetic Resonance Spectroscopy , Mice , Microbial Sensitivity Tests , Polymers/chemical synthesis , Polymers/chemistry , Ruthenium/chemistry , Staphylococcus aureus/cytologyABSTRACT
The biological processing - mechanism of cellular uptake, effects on the cytoplasmic and mitochondrial membranes, intracellular sites of localisation and induction of reactive oxygen species - of two dinuclear polypyridylruthenium(ii) complexes has been examined in three eukaryotic cells lines. Flow cytometry was used to determine the uptake of [{Ru(phen)2}2{µ-bb12}](4+) (Rubb12) and [Ru(phen)2(µ-bb7)Ru(tpy)Cl](3+) {Rubb7-Cl, where phen = 1,10-phenanthroline, tpy = 2,2':6',2''-terpyridine and bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane} in baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (HepG2) cell lines. The results demonstrated that the major uptake mechanism for Rubb12 and Rubb7-Cl was active transport, although with a significant contribution from carrier-assisted diffusion for Rubb12 and passive diffusion for Rubb7-Cl. Flow cytometry coupled with Annexin V/TO-PRO-3 double-staining was used to compare cell death by membrane damage or apoptosis. Rubb12 induced significant direct membrane damage, particularly with HepG2 cells, while Rubb7-Cl caused considerably less membrane damage but induced greater levels of apoptosis. Confocal microscopy, coupled with JC-1 assays, demonstrated that Rubb12 depolarises the mitochondrial membrane, whereas Rubb7-Cl had a much smaller affect. Cellular localisation experiments indicated that Rubb12 did not accumulate in the mitochondria, whereas significant mitochondrial accumulation was observed for Rubb7-Cl. The effect of Rubb12 and Rubb7-Cl on intracellular superoxide dismutase activity showed that the ruthenium complexes could induce cell death via a reactive oxygen species-mediated pathway. The results of this study demonstrate that Rubb12 predominantly kills eukaryotic cells by damaging the cytoplasmic membrane. As this dinuclear ruthenium complex has been previously shown to exhibit greater toxicity towards bacteria than eukaryotic cells, the results of the present study suggest that metal-based cationic oligomers can achieve selective toxicity against bacteria, despite exhibiting a non-specific membrane damage mechanism of action.
Subject(s)
Eukaryotic Cells/metabolism , Ruthenium/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Apoptosis , Biological Transport , Cation Transport Proteins/antagonists & inhibitors , Cation Transport Proteins/metabolism , Cell Line , Cell Membrane/metabolism , Cell Membrane Permeability , Eukaryotic Cells/drug effects , Flow Cytometry , Humans , Membrane Potential, Mitochondrial , Molecular Structure , Reactive Oxygen Species/metabolism , Ruthenium/chemistry , TemperatureABSTRACT
OBJECTIVES: The objectives of this study were to: (i) determine the in vitro activities of a series of di-, tri- and tetra-nuclear ruthenium complexes (Rubbn, Rubbn-tri and Rubbn-tetra) against a range of Gram-positive and -negative bacteria and compare the antimicrobial activities with the corresponding toxicities against eukaryotic cells; and (ii) compare MIC values with achievable in vivo serum concentrations for the least toxic ruthenium complex. METHODS: The in vitro activities were determined by MIC assays and time-kill curve experiments, while the toxicities of the ruthenium complexes were determined using the Alamar blue cytotoxicity assay. A preliminary pharmacokinetic study was undertaken to determine the Rubb12 serum concentration in mice as a function of time after administration. RESULTS: Rubb12, Rubb12-tri and Rubb12-tetra are highly active, with MIC values of 1-2 mg/L (0.5-1.5 µM) for a range of Gram-positive strains, but showed variable activities against a panel of Gram-negative bacteria. Time-kill experiments indicated that Rubb12, Rubb12-tri and Rubb12-tetra are bactericidal and kill bacteria within 3-8 h. The di-, tri- and tetra-nuclear complexes were â¼50 times more toxic to Gram-positive bacteria and 25 times more toxic to Gram-negative strains, classified as susceptible, than to liver and kidney cells. Preliminary pharmacokinetic experiments established that serum concentrations higher than MIC values can be obtained for Rubb12 with an administered dose of 32 mg/kg. CONCLUSIONS: The ruthenium complexes, particularly Rubb12, have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complex can be readily further modified in order to increase the selectivity for bacteria over eukaryotic cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Eukaryotic Cells/drug effects , Organometallic Compounds/pharmacology , Ruthenium/pharmacology , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/toxicity , Cell Survival/drug effects , Colorimetry/methods , Female , Male , Mice, Inbred BALB C , Microbial Sensitivity Tests , Microbial Viability/drug effects , Organometallic Compounds/pharmacokinetics , Organometallic Compounds/toxicity , Oxazines/analysis , Ruthenium/pharmacokinetics , Ruthenium/toxicity , Serum/chemistry , Xanthenes/analysisABSTRACT
Ruthenium(II) complexes containing the tetradentate ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ("bbn "; n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bbn )](2+) (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-α-[Ru(phen)(bb12 )](2+) species showed good activity against Gram-negative species. In particular, the cis-α-[Ru(phen)(bb12 )](2+) complex was two to four times more active than the cis-ß-[Ru(phen)(bb12 )](2+) complex against the Gram-negative strains. The cis-α- and cis-ß-[Ru(phen)(bb12 )](2+) complexes readily accumulated in the bacteria but, significantly, showed the highest level of uptake in Pseudomonas aeruginosa. Furthermore, the accumulation of the cis-α- and cis-ß-[Ru(phen)(bb12 )](2+) complexes in P.â aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-α-[Ru(phen)(bb12 )](2+) complex into live P.â aeruginosa was confirmed by using fluorescence microscopy. The water/octanol partition coefficients (log P) were determined to gain understanding of the relative cellular uptake. The cis-α- and cis-ß-[Ru(phen)(bbn )](2+) complexes exhibited relatively strong binding to DNA (Kb ≈10(6) M(-1) ), but no significant difference between the geometric isomers was observed.
Subject(s)
DNA/chemistry , Gram-Negative Bacteria/chemistry , Gram-Positive Bacteria/chemistry , Organometallic Compounds/chemical synthesis , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/metabolism , Cell Membrane Permeability , DNA/metabolism , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Ligands , Microbial Sensitivity Tests , Organometallic Compounds/chemistry , Ruthenium/chemistryABSTRACT
One of the major advances in medical science has been the development of antimicrobials; however, a consequence of their widespread use has been the emergence of drug-resistant populations of microorganisms. There is clearly a need for the development of new antimicrobials--but more importantly, there is the need for the development of new classes of antimicrobials, rather than drugs based upon analogues of known scaffolds. Due to the success of the platinum anticancer agents, there has been considerable interest in the development of therapeutic agents based upon other transition metals--and in particular ruthenium(II/III) complexes, due to their well known interaction with DNA. There have been many studies of the anticancer properties and cellular localisation of a range of ruthenium complexes in eukaryotic cells over the last decade. However, only very recently has there been significant interest in their antimicrobial properties. This review highlights the types of ruthenium complexes that have exhibited significant antimicrobial activity and discusses the relationship between chemical structure and biological processing--including site(s) of intracellular accumulation--of the ruthenium complexes in both bacterial and eukaryotic cells.
Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Ruthenium/chemistry , Ruthenium/pharmacology , Animals , Bacteria/cytology , Bacteria/drug effects , HumansABSTRACT
Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand ("bb12") showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ΔΔ and ΛΛ enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ΔΔ enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ΔΔ and ΛΛ enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes.
Subject(s)
Coordination Complexes/chemistry , DNA/chemistry , RNA/chemistry , Ruthenium/chemistry , Animals , Apoptosis/drug effects , Cell Line , Circular Dichroism , Coordination Complexes/chemical synthesis , Coordination Complexes/pharmacology , Cricetinae , Gram-Positive Bacteria/drug effects , HEK293 Cells , Hep G2 Cells , Humans , Microscopy, Fluorescence , StereoisomerismABSTRACT
A series of inert tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ligand ("bb(n)" for n = 10, 12 and 16) have been synthesised and their potential as antimicrobial agents examined. Due to the modular nature of the synthesis of the oligonuclear complexes, it was possible to make both linear and non-linear tetranuclear ruthenium species. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria--Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In order to gain an understanding of the relative antimicrobial activities, the cellular uptake and water-octanol partition coefficients (log P) were determined for a selection of the ruthenium complexes. Although the trinuclear complexes were the most lipophilic based upon log P values and showed the greatest cellular uptake, the linear tetranuclear complexes were generally more active, with MIC values <1 µM against the Gram positive bacteria. Similarly, although the non-linear tetranuclear complexes were slightly more lipophilic and were taken up to a greater extent by the bacteria, they were consistently less active than their linear counterparts. Of particular note, the cellular accumulation of the oligonuclear ruthenium complexes was greater in the Gram negative strains compared to that in the Gram positive S. aureus and MRSA. The results demonstrate that the lower antimicrobial activity of polypyridylruthenium(II) complexes towards Gram negative bacteria, particularly P. aeruginosa, is not strongly correlated to the cellular accumulation but rather to a lower intrinsic ability to kill the Gram negative cells.
Subject(s)
Anti-Bacterial Agents/chemistry , Coordination Complexes/chemistry , Ruthenium/chemistry , 1-Octanol/chemistry , Anti-Bacterial Agents/pharmacology , Coordination Complexes/pharmacology , Electrochemistry , Escherichia coli/drug effects , Escherichia coli/growth & development , Escherichia coli/metabolism , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/metabolism , Ruthenium/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Staphylococcus aureus/metabolism , Water/chemistryABSTRACT
The homogenous water oxidation catalysis by [Ru(terpy)(bipy)Cl](+) (1) and [Ru(terpy)(Me2bipy)Cl](+) (2) (terpy = 2,2':6',2''-terpyridine, bipy = 2,2'-bipyridine, Me2bipy = 4,4'-dimethyl-2,2'-bipyridine) under the influence of two redox mediators [Ru(bipy)3](2+) (3) and [Ru(phen)2(Me2bipy)](2+) (4) (phen = 1,10-phenanthroline) was investigated using Ce(4+) as sacrificial oxidant. Oxygen evolution experiments revealed that mixtures of both 2-4 and 2-3 produced more molecular oxygen than catalyst 2 alone. In contrast, the combination of mediator 4 and catalyst 1 resulted in a lower catalytic performance of 1. Measurements of the temporal change in the intensity of a UV transition at 261 nm caused by the addition of four equivalents of Ce(4+) to 2 revealed three distinctive regions-suggested to correspond to the stepwise processes: (i) [Ru(IV)=O](2+) â [Ru(V)=O](3+); (ii) [Ru(V)=O](3+) â [Ru(III)-(OOH)](2+); and (iii) [Ru(III)-(OOH)](2+) â [Ru(II)-OH2](2+). UV-Visible spectrophotometric experiments on the 1-4 and 2-4 mixtures, also carried out with four equivalents of Ce(4+), demonstrated a faster [Ru(phen)2(Me2bipy)](3+) â [Ru(phen)2(Me2bipy)](2+) reduction rate in 2-4 than that observed for the 1-4 combination. Cyclic voltammetry data measured for the catalysts and the mixtures revealed a coincidence in the potentials of the Ru(II)/Ru(III) redox process of mediators 3 and 4 and the predicted [Ru(IV)=O](2+)/[Ru(V)=O](3+) potential of catalyst 2. In contrast, the [Ru(IV)=O](2+)/[Ru(V)=O](3+) process for catalyst 1 was found to occur at a higher potential than the Ru(II)/Ru(III) redox process for 4. Both the spectroscopic and electrochemical experiments provide evidence that the interplay between the mediator and the catalyst is an important determinant of the catalytic activity.