Oxygen Assessment in Tumors In Vivo Using Phosphorescence Lifetime Imaging Microscopy.

The oxygen level in a tumor is a crucial factor for its development and response to therapies. Phosphorescence lifetime imaging (PLIM) with the use of phosphorescent oxygen probes is a highly sensitive, noninvasive optical technique for the assessment of molecular oxygen in living cells and tissues. Here, we present a protocol for microscopic mapping of oxygen distribution in a mouse tumor model in vivo. We demonstrate that PLIM microscopy, in combination with an Ir(III)-based probe, enables visualization of cellular-level heterogeneity of tumor oxygenation.

Biocompatible Phosphorescent O2 Sensors Based on Ir(III) Complexes for In Vivo Hypoxia Imaging.

In this work, we obtained three new phosphorescent iridium complexes (Ir1-Ir3) of general stoichiometry [Ir(N^C)2(N^N)]Cl decorated with oligo(ethylene glycol) fragments to make them water-soluble and biocompatible, as well as to protect them from aggregation with biomolecules such as albumin. The major photophysical characteristics of these phosphorescent complexes are determined by the nature of two cyclometallating ligands (N^C) based on 2-pyridine-benzothiophene, since quantum chemical calculations revealed that the electronic transitions responsible for the excitation and emission are localized mainly at these fragments. However, the use of various diimine ligands (N^N) proved to affect the quantum yield of phosphorescence and allowed for changing the complexes' sensitivity to oxygen, due to the variations in the steric accessibility of the chromophore center for O2 molecules. It was also found that the N^N ligands made it possible to tune the biocompatibility of the resulting compounds. The wavelengths of the Ir1-Ir3 emission maxima fell in the range of 630-650 nm, the quantum yields reached 17% (Ir1) in a deaerated solution, and sensitivity to molecular oxygen, estimated as the ratio of emission lifetime in deaerated and aerated water solutions, displayed the highest value, 8.2, for Ir1. The obtained complexes featured low toxicity, good water solubility and the absence of a significant effect of biological environment components on the parameters of their emission. Of the studied compounds, Ir1 and Ir2 were chosen for in vitro and in vivo biological experiments to estimate oxygen concentration in cell lines and tumors. These sensors have demonstrated their effectiveness for mapping the distribution of oxygen and for monitoring hypoxia in the biological objects studied.

Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor.

Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response.

Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses. Each of the degrees has a characteristic set of quantitatively expressed features. We focus on the identification and description of early, initial changes of the dermis as the least specific. The results obtained by us and the proposed classification of the degrees of the disease can be used to objectify the dynamics of tissue changes during treatment.

Red Light-Emitting Water-Soluble Luminescent Iridium-Containing Polynorbornenes: Synthesis, Characterization and Oxygen Sensing Properties in Biological Tissues In Vivo.

New water-soluble polynorbornenes P1-P4 containing oligoether, amino acid groups and luminophoric complexes of iridium(III) were synthesized by ring-opening metathesis polymerization. The polymeric products in organic solvents and in water demonstrate intense photoluminescence in the red spectral region. The polymers P1 and P3 with 1-phenylisoquinoline cyclometalating ligands in iridium fragments reveal 4-6 fold higher emission quantum yields in solutions than those of P2 and P4 that contain iridium complexes with 1-(thien-2-yl)isoquinoline cyclometalating ligands. The emission parameters of P1-P4 in degassed solutions essentially differ from those in the aerated solutions showing oxygen-dependent quenching of phosphorescence. Biological testing of P1 and P3 demonstrates that the polymers do not penetrate into live cultured cancer cells and normal skin fibroblasts and do not possess cytotoxicity within the concentrations and time ranges reasonable for biological studies. In vivo, the polymers display longer phosphorescence lifetimes in mouse tumors than in muscle, as measured using phosphorescence lifetime imaging (PLIM), which correlates with tumor hypoxia. Therefore, preliminary evaluation of the synthesized polymers shows their suitability for noninvasive in vivo assessments of oxygen levels in biological tissues.

Biocompatible Ir(III) Complexes as Oxygen Sensors for Phosphorescence Lifetime Imaging.

Synthesis of biocompatible near infrared phosphorescent complexes and their application in bioimaging as triplet oxygen sensors in live systems are still challenging areas of organometallic chemistry. We have designed and synthetized four novel iridium [Ir(N^C)2(N^N)]+ complexes (N^C-benzothienyl-phenanthridine based cyclometalated ligand; N^N-pyridin-phenanthroimidazol diimine chelate), decorated with oligo(ethylene glycol) groups to impart these emitters' solubility in aqueous media, biocompatibility, and to shield them from interaction with bio-environment. These substances were fully characterized using NMR spectroscopy and ESI mass-spectrometry. The complexes exhibited excitation close to the biological "window of transparency", NIR emission at 730 nm, and quantum yields up to 12% in water. The compounds with higher degree of the chromophore shielding possess low toxicity, bleaching stability, absence of sensitivity to variations of pH, serum, and complex concentrations. The properties of these probes as oxygen sensors for biological systems have been studied by using phosphorescence lifetime imaging experiments in different cell cultures. The results showed essential lifetime response onto variations in oxygen concentration (2.0-2.3 µs under normoxia and 2.8-3.0 µs under hypoxia conditions) in complete agreement with the calibration curves obtained "in cuvette". The data obtained indicate that these emitters can be used as semi-quantitative oxygen sensors in biological systems.

Novel group of tyrosyl-DNA-phosphodiesterase 1 inhibitors based on disaccharide nucleosides as drug prototypes for anti-cancer therapy.

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors based on disaccharide nucleosides was identified. TDP1 plays an essential role in the resistance of cancer cells to currently used antitumour drugs based on Top1 inhibitors such as topotecan and irinotecan. The most effective inhibitors investigated in this study have IC50 values (half-maximal inhibitory concentration) in 0.4-18.5 µM range and demonstrate relatively low own cytotoxicity along with significant synergistic effect in combination with anti-cancer drug topotecan. Moreover, kinetic parameters of the enzymatic reaction and fluorescence anisotropy were measured using different types of DNA-biosensors to give a sufficient insight into the mechanism of inhibitor's action.

Mutants of Ca2+ -regulated Photoprotein Obelin for Site-specific Conjugation.

Color variants of Ca2+ -regulated photoprotein obelin were shown to be an important tool for dual-analyte binding assay. To provide site-directed conjugation with biospecific molecules, several obelin color mutants carrying unique cysteine residues were obtained and characterized for their novel properties. A pair of obelins Y138F,A5C and W92F,H22E,D12C was found to be most suitable (in terms of high bioluminescent activity and stability) as reporters in simultaneous assay of two targets in a sample. Availability of SH-groups, accessible for chemical modification, essentially simplifies the synthesis of biospecific conjugates, increases their yield and conserves obelins' bioluminescence activity. Conjugates with immunoglobulin and oligonucleotide were produced and successfully applied in single nucleotide polymorphism genotyping.

Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs.

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19.