Revealing bactericidal events on graphene oxide nano films deposited on metal implant surfaces.

At the time when pathogens are developing robust resistance to antibiotics, the demand for implant surfaces with microbe-killing capabilities has significantly risen. To achieve this goal, profound understanding of the underlying mechanisms is crucial. Our study demonstrates that graphene oxide (GO) nano films deposited on stainless steel (SS316L) exhibit superior antibacterial features. The physicochemical properties of GO itself play a pivotal role in influencing biological events and their diversity may account for the contradictory results reported elsewhere. However, essential properties of GO coatings, such as oxygen content and the resulting electrical conductivity, have been overlooked so far. We hypothesize that the surface potential and electrical resistance of the oxygen content in the GO-nano films may induce bacteria-killing events on conductive metallic substrates. In our study, the GO applied contains 52 wt% of oxygen, and thus exhibits insulating properties. When deposited as a nano film on an electrically conducting steel substrate, GO flakes generate a Schottky barrier at the interface. This barrier, consequently, impedes the transfer of electrons to the underlying conductive substrate. As a result, this creates reactive oxygen species (ROS), leading to bacterial death. We confirmed the presence of GO coatings and their hydrolytic stability by using X-ray photoelectron spectroscopy (XPS), µRaman spectroscopy, scanning electron microscopy (SEM), and Kelvin probe force microscopy (KPFM) measurements. The biological evaluation was performed on the MG63 osteoblast-like cell line and two selected bacteria species: S. aureus and P. aeruginosa, demonstrating both the cytocompatibility and antibacterial behavior of GO-coated SS316L substrates. We propose a two-step bactericidal mechanism: electron transfer from the bacteria membrane to the substrate, followed by ROS generation. This mechanism finds support in changes observed in contact angle, surface potential, and work function, identified as decisive factors. By addressing overlooked factors and effectively bridging the gap between understanding and practicality, we present a transformative approach for implant surfaces, combating microbial resistance, and offering new application possibilitie.

Design of an anticancer organoruthenium complex as the guest and polydiacetylene-coated fluorogenic nanocarrier as the host: engineering nanocarrier using ene-yne conjugation for sustained guest release, enhanced anticancer activity and reduced in vivo toxicity.

Despite the enormous efforts made over the past two decades to develop metallodrugs and nanocarriers for metallodrug delivery, there are still few precise strategies that aim to optimize the design of both metallodrugs and metallodrug carriers jointly in a concerted effort. In this work, three half-sandwich ruthenium(II) complexes with pyridylimidazo[1,5-a]pyridine ligand functionalized with polycyclic aromatic moiety (Ru(nap), Ru(ant), Ru(pyr)) are evaluated as possible anticancer candidates and polydiacetylene (PDA)-coated amino-functionalized mesoporous silica nanoparticles (AMSNs) are designed as a functional nanocarrier for drug delivery. Ru(pyr) exhibits higher cytotoxicity in HT-29 colorectal cancer cells compared to other complexes and cis-platin, but it does not exhibit better cellular uptake. Ru(pyr) is found to be preferentially accumulated in plasma, mitochondria, and ER-Golgi membrane. The complex induces cell cycle arrest in the G0/G1 phase, while higher concentrations cause programmed cell death via apoptosis. Ru(pyr) influences cancer cell adhesion property and acts as an antioxidant in HT-29 cells. In order to modulate the anticancer potency of Ru(pyr), AMSNs are used to encapsulate the complex, and then diacetylene self-assembly is allowed to deposit on the surface of the nanoparticles. Subsequently, the nanoparticles undergo topopolymerization, which results in π-conjugated PDA-Ru(pyr)@AMSNs. Owing to the ene-yne polymeric skeleton in the backbone, the non-fluorescent AMSNs turn into red-emissive particles, which are exploited for cell imaging applications. The release profile analysis reveals that such a π-conjugated polymer prevents the premature release of the complex from porous silica nanoparticles with the accelerated release of the complex in an acidic medium compared to physiological conditions. The PDA gatekeepers have also been proven to enhance the cellular internalization of Ru(pyr) with slow continuous release from the nanoformulation. Zebrafish embryo toxicity analysis suggests that the PDA-coated nanocarriers could be suitable candidates for in vivo investigations.

Investigation of the Immunogenic Properties of Ovalbumin Modified by Urban Airborne Particulate Matter.

Exposure to air particulate matter (PM) is linked to the blood oxidative stress and systemic inflammation. The aim of this study was to elucidate whether oxidative PM modification of ovalbumin (OVA), the major antioxidant serum protein, may alter its antigenicity and/or immunogenicity. Ovalbumin was exposed via dialysis to the standard urban PM (SRM 1648a) or to PM with removed organic content (encoded as LAP). Both structural changes and biological properties of PM-modified OVA were measured. T lymphocytes and dendritic cells (the major antigen-presenting cells) isolated from C57BL/6 and OT-II (323-339 epitope) OVA-specific T cell receptor (TCR)-transgenic mice were used to test the effect of PM on OVA immunogenicity. The immunogenicity of both SRM 1648a and LAP-modified OVA was significantly higher than that of control OVA, as measured by the epitope-specific T cell proliferation and interferon γ production by the stimulated cells. This effect was associated with mild oxidative changes in the carrier molecule outside the structure of the OVA epitope and with increased resistance to proteolysis of PM-modified OVA. Interestingly, dendritic cells showed enhanced capacity for the uptake of proteins when the cells were cultured with PM-modified OVA. Our results suggest that the enhanced immunogenicity of PM-modified OVA is not associated with altered antigenicity or antigen presentation. However, it may result from slower degradation and longer persistence of modified antigens in dendritic cells. Whether this phenomenon is associated with enhanced risk prevalence of autoimmune diseases observed in the areas with high urban PM pollution needs to be explained.

Impact of Polypyridyl Ru Complexes on Angiogenesis-Contribution to Their Antimetastatic Activity.

The use of polypyridyl Ru complexes to inhibit metastasis is a novel approach, and recent studies have shown promising results. We have reported recently that Ru (II) complexes gathering two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and the one being 2,2'-bipyridine (bpy) or its derivative with a 4-[3-(2-nitro-1H-imidazol-1-yl)propyl (bpy-NitroIm) or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moieties can alter the metastatic cascade, among others, by modulating cell adhesion properties. In this work, we show further studies of this group of complexes by evaluating their effect on HMEC-1 endothelial cells. While all the tested complexes significantly inhibited the endothelial cell migration, Ru-bpy additionally interrupted the pseudovessels formation. Functional changes in endothelial cells might arise from the impact of the studied compounds on cell elasticity and expression of proteins (vinculin and paxillin) involved in focal adhesions. Furthermore, molecular studies showed that complexes modulate the expression of cell adhesion molecules, which has been suggested to be one of the factors that mediate the activation of angiogenesis. Based on the performed studies, we can conclude that the investigated polypyridyl Ru (II) complexes can deregulate the functionality of endothelial cells which may lead to the inhibition of angiogenesis.

Nanoencapsulation of Ru(p-cymene) Complex Bearing Ginger-based Natural Product into Liposomal Nanoformulation to Improve Its Cellular Uptake and Antiproliferative Activity.

The organometallic compounds are prospective candidates in the row of developing metallochemotherapeutics with the aim of overcoming the limitations of platinum drugs. In order to explore the anticancer properties of organometallic compounds with the natural medicines, two Ru(II)-p-cymene complexes containing the natural products, viz., 6-gingerol (6G) and benzylated-6-gingerdione (B-6GD) have been synthesized and characterized well. The phenolic group of the Ru(6G) complex facilitates its higher cell-free antioxidant activity than its analogue complex. Also, the same complex shows higher cytotoxicity toward A549 lung and HeLa-S3 cervical cancer cells than the Ru(B-6GD) complex but lower cytotoxicity toward A2058 metastatic melanoma cancer cells. Both complexes are shown to easily accumulate in melanoma cancer cells, and their degree of cytotoxicity in the same cells is found to be positively correlated with cell uptake. The cytotoxicity of complexes arises from their intracellular activity, mainly due to the induction of singlet oxygen production in cancer cells. The subcellular fractionation study shows that mitochondria and ER-Golgi membranes might be their predominant targets. Also, the mechanistic investigation revealed that Ru(B-6GD) induces caspase-dependent non-apoptotic cell death whereas Ru(6G) can induce caspase-independent non-apoptotic cell death. Furthermore, both complexes are found to moderately alter the adhesion properties of cancer cells, which is beneficial for antimetastatic treatment. Despite the potential pharmacological activity, Ru(6G) is encapsulated into polymer-supported liposomes to reduce its toxicity and further improve its anticancer potency. The π-conjugated yne-ene chain of polydiacetylene aids in the development of a stable nanoformulation, which achieved a slow release of the complex. Most importantly, the cancer cell uptake of the liposome-encapsulated Ru(6G) complex is 20 times enhanced and the total ROS formation in cancer cells is significantly increased compared to the non-encapsulated complex. However, the nanoformulation does not alter the antimetastatic potency of the encapsulated complex.

Inhibition of Metastasis by Polypyridyl Ru(II) Complexes through Modification of Cancer Cell Adhesion - In Vitro Functional and Molecular Studies.

The effect of polypyridyl Ru(II) complexes on the ability of cancer cells to migrate and invade, two features important in the formation of metastases, is evaluated. In vitro studies are carried out on breast cancer cell lines, MDA-MB-231 and MCF-7, as well as melanoma cell lines A2058 and A375. Three Ru(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and as a third ligand 2,2'-bipyridine (bpy), or its derivative with either 4-[3-(2-nitro-1H-imidazol-1-yl)propyl] (bpy-NitroIm), or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moiety attached are examined. The low sub-toxic doses of the studied compounds greatly affected the cancer cells by inhibiting cell detachment, migration, invasion, transmigration, and re-adhesion, as well as increasing cell elasticity. The molecular studies revealed that the Ru(II) polypyridyl complexes impact the activity of the selected integrins and upregulate the expression of focal adhesion components such as vinculin and paxillin, leading to an increased number of focal adhesion contacts.

Significance of Specific Oxidoreductases in the Design of Hypoxia-Activated Prodrugs and Fluorescent Turn off-on Probes for Hypoxia Imaging.

Hypoxia is one of the hallmarks of the tumor microenvironment and can be used in the design of targeted therapies. Cellular adaptation to hypoxic stress is regulated by hypoxia-inducible factor 1 (HIF-1). Hypoxia is responsible for the modification of cellular metabolism that can result in the development of more aggressive tumor phenotypes. Reduced oxygen concentration in hypoxic tumor cells leads to an increase in oxidoreductase activity that, in turn, leads to the activation of hypoxia-activated prodrugs (HAPs). The same conditions can convert a non-fluorescent compound into a fluorescent one (fluorescent turn off-on probes), and such probes can be designed to specifically image hypoxic cancer cells. This review focuses on the current knowledge about the expression and activity of oxidoreductases, which are relevant in the activation of HAPs and fluorescent imaging probes. The current clinical status of HAPs, their limitations, and ways to improve their efficacy are briefly discussed. The fluorescence probes triggered by reduction with specific oxidoreductase are briefly presented, with particular emphasis placed on those for which the correlation between the signal and enzyme expression determined with biochemical methods is achievable.

Relevance of the electron transfer pathway in photodynamic activity of Ru(II) polypyridyl complexes containing 4,7-diphenyl-1,10-phenanthroline ligands under normoxic and hypoxic conditions.

The purpose of this study was to investigate the correlation between the spectroscopic and photophysical properties of Ru(II) polypyridyl complexes and their photodynamic activity in vitro. A series of Ru(II) polypyridyl complexes with 4,7-diphenyl-1,10-phenanthroline (dip) and 2,3-bis(2-pyridyl)quinoxaline (dpq) and its derivatives were synthesized and characterized regarding their photophysical, biological, and photodynamic properties. The complexes were evaluated not only in the context of 1O2 generation but also regarding other types of reactive oxygen species (ROS) to assess the possibility of Ru(II) complexes to induce phototoxicity via various ROS using fluorescence and EPR spectroscopy. The compounds were found to be moderately cytotoxic with IC50 values ranging from 1 to 35 µM and retained their cytotoxic activity under hypoxic conditions. The unraveled phototoxic activity is based mainly on the generation of H2O2 and 1O2, highlighting the importance of electron-transfer processes in the observed photodynamic activity of Ru polypyridyl complexes. A combination of photodynamic activity with cytotoxicity under decreased dioxygen concentrations may help overcome the current photodynamic therapy (PDT) limitation. The findings highlight the need for broadening the scope of tested Ru-based photosensitizers.

Moving Ru polypyridyl complexes beyond cytotoxic activity towards metastasis inhibition.

In recent years, Ru polypyridyl complexes have been intensively studied for their anticancer activity. The vast majority of research focuses on assessing their cytotoxic activity, as well as targeting cancer cells with them. Since the formation of metastases poses a greater risk than primary tumors, scientists recently began evaluating these compounds as potential metastasis inhibitors. This review highlights the latest achievements in this field with particular attention to the identification of the target proteins responsible for such activity. Cell migration, invasion, and adhesion are key components of metastasis, therefore understanding how they are affected by Ru polypyridyl complexes is of great importance. KEYWORDS: Ruthenium polypyridyl complexes Antimetastatic Migration Invasion Adhesion Metalloproteinases.

Inhibition of Matrix Metalloproteinases and Cancer Cell Detachment by Ru(II) Polypyridyl Complexes Containing 4,7-Diphenyl-1,10-phenanthroline Ligands-New Candidates for Antimetastatic Agents.

Primary tumor targeting is the dominant approach in drug development, while metastasis is the leading cause of cancer death. Therefore, in addition to the cytotoxic activity of a series of Ru(II) polypyridyl complexes of the type [Ru(dip)2L]2+ (dip: 4,7-diphenyl-1,10-phenanthroline while L = dip; bpy: 2,2'-bipyridine; bpy-SC: bipyridine derivative bearing a semicarbazone 2-formylopyridine moiety; dpq, dpq(CH3)2, dpb: quinoxaline derivatives) their ability to inhibit cell detachment was investigated. In vitro studies performed on lung cancer A549 cells showed that they accumulate in cells very well and exhibit moderate cytotoxicity with IC50 ranging from 4 to 13 µM. Three of the studied compounds that have dip, bpy-SC, or dpb ligands after treatment of the cells with a non-toxic dose (<1/2IC50) enhanced their adhesion properties demonstrated by lower detachment in the trypsin resistance assay. The same complexes inhibited both MMP-2 and MMP-9 enzyme activities with IC50 ranging from 2 to 12 µM; however, the MMP-9 inhibition was stronger. More detailed studies for [Ru(dip)2(bpy-SC)]2+, which induced the greatest increase in cell adhesion, revealed that it is predominately accumulated in the cytoskeletal fraction of A549 cells. Moreover, cells treated with this compound showed the localization of MMP-9 to a greater extent also in the cytoskeleton. Taken together, our results indicate the possibility of a reduction of metastatic cells escaping from the primary lesion to the surrounding tissue by prevention of their detachment and by influencing the activity of MMP-2 and MMP-9.

Unexplored features of Ru(ii) polypyridyl complexes - towards combined cytotoxic and antimetastatic activity.

The well-documented cytotoxic activity of coordinatively saturated and substitutionally inert polypyridyl Ru(ii) complexes substantiates their high potency as antiproliferative agents against primary tumors. However, the primary cause of cancer morbidity and mortality responsible for about 90% of cancer deaths is the occurrence of metastasis. Therefore, scientists have to concentrate their efforts on designing compounds affecting not only the primary tumor, but also efficiently inhibiting metastasis. Herein, we report two families of Ru(ii) polypyridyl complexes bearing 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety as one of the ligands and 4,4'-di-tert-butyl-2,2'-dipyridyl or 4,7-diphenyl-1,10-phenanthroline as auxiliary ligands. These complexes strengthen cells' adherent properties and inhibit the activity of metalloproteinases (MMPs) in vitro, which is relevant in anti-metastatic treatment. The in vitro studies were performed on human lung adenocarcinoma (A549) and human pancreatic cancer (PANC-1) cells, which have a well-documented invasive potential. The induced alteration of the tumor cells' adhesion properties correlated with the high cytotoxic effect exerted by the complexes and their excellent cellular uptake. It was also proved that both complexes directly inhibit M-MP2 and M-MP9 enzyme activities, which are essential for the development of tumor metastasis. The results of this study indicate that the biological properties of polypyridyl Ru(ii) complexes extend beyond the standard cytotoxic activity and represent an important step towards designing new anti-metastatic agents.

Variations in Reactive Oxygen Species Generation by Urban Airborne Particulate Matter in Lung Epithelial Cells-Impact of Inorganic Fraction.

Air pollution is associated with numerous negative effects on human health. The toxicity of organic components of air pollution is well-recognized, while the impact of their inorganic counterparts in the overall toxicity is still a matter of various discussions. The influence of airborne particulate matter (PM) and their inorganic components on biological function of human alveolar-like epithelial cells (A549) was investigated in vitro. A novel treatment protocol based on covering culture plates with PM allowed increasing the studied pollutant concentrations and prolonging their incubation time without cell exposure on physical suffocation and mechanical disturbance. PM decreased the viability of A549 cells and disrupted their mitochondrial membrane potential and calcium homeostasis. For the first time, the difference in the reactive oxygen species (ROS) profiles generated by organic and inorganic counterparts of PM was shown. Singlet oxygen generation was observed only after treatment of cells with inorganic fraction of PM, while hydrogen peroxide, hydroxyl radical, and superoxide anion radical were induced after exposure of A549 cells to both PM and their inorganic fraction.

Versatile Impact of Serum Proteins on Ruthenium(II) Polypyridyl Complexes Properties - Opportunities and Obstacles.

Ruthenium(II) polypyridyl complexes have been extensively studied for the past few decades as promising anticancer agents. Despite the expected intravenous route of administration, the interaction between Ru(II) polypyridyl compounds and serum proteins is not well characterized and vast majority of the available literature data concerns determination of the binding constant. Ru-protein adducts can modify the biological effects of the Ru complexes influencing their cytotoxic and antimicrobial activity as well as introduce significant changes in their photophysical properties. More extensive research on the interaction between serum proteins and Ru(II) polypyridyl complexes is important for further development of Ru(II) polypyridyl compounds towards their application in anticancer therapy and diagnostics and can open new opportunities for already developed complexes.

New ruthenium compounds bearing semicarbazone 2-formylopyridine moiety: Playing with auxiliary ligands for tuning the mechanism of biological activity.

Two ruthenium(II) complexes Ru1 and Ru2 bearing as a one ligand 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety (bpySC: 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone) and as the others 2,2'-bipyridine (bpy) and 4,7-diphenyl-1,10-phenanthroline (dip), respectively, as auxiliary ligands have been prepared. Their biological activity has been studied on murine colon carcinoma (CT26) and human lung adenocarcinoma (A549) cell lines. The anti-proliferative activity was dependent on the presence of bpy or dip in the complex, with one order of magnitude higher cytotoxicity for Ru2 (dip ligands). Ru1 (bpy ligands) exhibited a distinct increase in cytotoxicity going from 24 to 72h of incubation with cells as was not observed for Ru2. Even though both studied compounds were powerful apoptosis inducing agents, the mechanism of their action was entirely different. Ru1-incubated A549 cells showed a notable increase in cells number in the S-phase of the cell cycle, with concomitant decrease in the G2/M phase, while Ru2 promoted a cell accumulation in the G0/G1 phase. In contrast, Ru1 induced marginal oxidative stress in A549 cell lines even upon increasing the incubation time. Even though Ru1 preferably accumulated in lysosomes it triggered the apoptotic cellular death via an intrinsic mitochondrial pathway. Ru1-incubated A549 cells showed swelling and enlarging of the mitochondria. It was not observed in case of Ru2 for which mitochondria and endoplasmic reticulum were found as primarily localization site. Despite this the apoptosis induced by Ru2 was caspase-independent. All these findings point to a pronounced role of auxiliary ligands in tuning the mode of biological activity.

Nitroimidazole derivatives of polypyridyl ruthenium complexes: Towards understanding their anticancer activity and mode of action.

The mechanism of cell death induced by the ruthenium polypyridyl complexes comprising two 4,7-diphenyl-1,10-phenanthroline ligands as well as one unmodified 2,2'-bipyridyl or modified with 2-nitroimidazole moiety attached by shorter (C3H6) or longer (C6H12) linker was investigated. Cytotoxicity and proliferation assays revealed that the studied Ru polypyridyl complexes are more toxic against human pancreas carcinoma PANC-1 cell line than normal human keratinocytes HaCaT with IC50 of 3-5µM. The Ru complexes despite accumulation in mitochondria do not lead to mitochondrial disfunction, though decreasing of mitochondrial Ca2+ causes mitochondria membrane hyperpolarization. The Ru polypyridyl conjugates induce some phenotypical characteristic of apoptosis, such as condensation of chromatin or phosphatidylserine translocation, however no caspase or calpain activation in the studied cell lines was observed, indicating that detected cell death does not occur via mitochondria- or ER-activated pathways. Caspase-independent cell death is caused by enormous ROS formation, mainly hydrogen peroxide and peroxyl radicals as well as by intracellular Ca2+ homeostasis disruption. Accumulation of the Ru compounds inhibits the completion of DNA synthesis, arresting cells in S-phase of cell cycle.

The biological effect of the nitroimidazole derivative of a polypyridyl ruthenium complex on cancer and endothelial cells.

The ruthenium polypyridyl complexes [Ru(dip)2(bpy/bpy-2-nitroIm)](2+) (dip = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2'-bipyridine, bpy-2-nitroIm = 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]) were found to be ca. ten times more cytotoxic against breast cancer (4T1) and human lung adenocarcinoma epithelial cells (A549) than a well-known anticancer drug, cisplatin. Even though the Ru complexes were quite cytotoxic towards FVB mouse lung microvascular endothelial cells (MLuMEC FVB) their efflux from these non transformed cells was much more efficient than from cancer ones. Both Ru complexes accumulated in cells. The cellular uptake of both Ru complexes occurs through passive diffusion while the nitroimidazole derivative is also endocytosed. They arrest cell growth in the S-phase and induce apoptosis. Such cell response can result from activation of oxidative stress by Ru complexes. The modulation of the mRNA expression profile for genes which might be involved in metastasis and angiogenesis processes by Ru complexes was analyzed for both cancer (4T1) and endothelial (MLuMEC FVB) cells. Ru complexes appeared to have a distinct impact on cell adhesion and migration as well as they affect endothelial cell vasculature. They are not only cytotoxic but are also potentially invasive and anti-metastatic agents. This work illustrates the putative future development of polypyridyl ruthenium.

Multifaceted interplay between lipophilicity, protein interaction and luminescence parameters of non-intercalative ruthenium(II) polypyridyl complexes controlling cellular imaging and cytotoxic properties.

Here, we examine the photophysical properties of five ruthenium(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and functionalized bipyridine (R1bpy-R2, where R1= H or CH3, R2= H, CH3, COO⁻,4-[3-(2-nitro-1H-imidazol-1-yl)propyl] or 1,3-dicyclohexyl-1-carbonyl-urea) towards development of luminescence probes for cellular imaging. These complexes have been shown to interact with albumin and the formed adducts exhibited up to eightfold increase in the luminescence quantum yield as well as the average lifetime of emission. It was demonstrated that they cannot bind to DNA through the intercalation mode and its luminescence in the presence of DNA is quenching. Cell viability experiments indicated that all complexes possess significant dose-dependent cytotoxicity (with IC50 5-19 µM) on 4T1 breast cancer cell line and their anti-proliferative activity correlates very well with their lipophilicity. Cellular uptake was studied by measuring the ruthenium content in cells using ICP-MS technique. As expected, the better uptake is directly related to higher lipophilicity of doubly charged ruthenium complexes while uptake of monocationic one is much lower in spite of the highest lipophilicity. Additionally staining properties were assessed using flow cytometry and fluorescence microscopy. These experiments showed that complex with 1,3-dicyclohexyl-1-carbonyl-urea substituent exhibits the best staining properties in spite of the lowest luminescence quantum yield in buffered solution (pH 7.4). Our results point out that both the imaging and cytotoxic properties of the studied ruthenium complexes are strongly influence by the level of internalization and protein interaction.

2-Nitroimidazole-ruthenium polypyridyl complex as a new conjugate for cancer treatment and visualization.

A novel long-lifetime highly luminescent ruthenium polypyridyl complex containing 2-nitroimidazole moiety [Ru(dip)2(bpy-2-nitroIm)]Cl2 (dip=4,7-diphenyl-1,10-phenanthroline, bpy-2-nitroIm=4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine) has been designed cancer treatment and imaging. The luminescence properties of the synthesized compound strongly depend on the oxygen concentration. Under oxygen-free conditions quantum yield of luminescence and the average lifetime of emission were found to be 0.034 and 1.9 µs, respectively, which is ca. three times higher in comparison to values obtained in air-equilibrated solution. The binding properties of the investigated ruthenium complex to human serum albumin have been studied and the apparent binding constant for the formation of the protein-ruthenium adduct was determined to be 1.1×10(5)M(-1). The quantum yield and the average lifetime of emission are greatly enhanced upon binding of ruthenium compound to the protein. The DNA binding studies revealed two distinguished binding modes which lead to a decrease in luminescence intensity of ruthenium complex up to 60% for [DNA]/[Ru]<2, and enhancement of emission for [DNA]/[Ru]>80. Preliminary biological studies confirmed fast and efficient accumulation of the ruthenium complex inside cells. Furthermore, the ruthenium complex was found to be relatively cytotoxic with LD50 of 12 and 13 µM for A549 and CT26 cell lines, respectively, under normoxic conditions. The retention and cellular uptake of ruthenium complex is enhanced under hypoxic conditions and its LD50 decreases to 8 µM for A549 cell line.

Interaction of apo-transferrin with anticancer ruthenium complexes NAMI-A and its reduced form.

NAMI-A i.e. (ImH)[trans-RuCl(4)(DMSO)(Im)] (where Im is imidazole) is a ruthenium(III) complex with promising antimetastatic activity, which has been classified for II phase clinical trial. In this study, its binding properties toward apo-transferrin (apo-Tf) with regard to its hydrolytic and redox behavior are systematically investigated by the use of fluorescence spectroscopy. The reaction of NAMI-A and its reduced form with apo-Tf is proceeded by formation of aqua derivatives and the presence of at least one labile aqua ligand is sufficient to form adducts. It is found that presence of bicarbonate is not necessary for interaction of studied ruthenium complexes with apo-Tf. The calculated association constants for both NAMI-A and its reduced form are very similar with the values of 1.28 × 10(4)M(-1) and 1.36 × 10(4)M(-1) at 37 °C, respectively however, the reduced derivatives reach the equilibrium ca. 8-10 times slower. The percentage of ruthenium content in protein fractions separated from protein-unbounded ruthenium by using FPLC (fast protein liquid chromatography) method is rather high and depends on redox state of the complex, for most samples is found higher for reduced species.