Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients.

Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O2-sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited "inverted" oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered "normal" gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.

Tunable Self-Referenced Molecular Thermometers via Manipulation of Dual Emission in Platinum(II) Pyridinedipyrrolide Complexes.

Optical temperature sensors based on self-referenced readout schemes such as the emission ratio and the decay time are crucial for a wide range of applications, with the former often preferred due to simplicity of instrumentation. This work describes a new group of dually emitting dyes, platinum(II) pincer complexes, that can be used directly for ratiometric temperature sensing without an additional reference material. They consist of Pt(II) metal center surrounded by a pyridinedipyrrolide ligand (PDP) and a terminal ligand (benzonitrile, pyridine, 1-butylimidazol or carbon monoxide). Upon excitation with blue light, these complexes exhibit green to orange emission, with quantum yields in anoxic toluene at 25 °C ranging from 13% to 86% and decay times spanning from 8.5 to 97 µs. The emission is attributed to simultaneous thermally activated delayed fluorescence (TADF) and phosphorescence processes on the basis of photophysical investigations and DFT calculations. Rather uniquely, simple manipulations in substituents of the PDP ligand and alteration of the terminal ligand allow fine-tuning of the ratio between TADF and phosphorescence from almost 100% TADF emission (Pt(MesPDPC6F5(BN)) to over 80% of phosphorescence (Pt(PhPDPPh(BuIm)). Apart from ratiometric capabilities, the complexes also are useful as decay time-based temperature indicators with temperature coefficients exceeding 1.5% K-1 in most cases. Immobilization of the dyes into oxygen-impermeable polyacrylonitrile produces temperature sensing materials that can be read out with an ordinary RGB camera or a smartphone. In addition, Pt(PhPDPPh)Py can be incorporated into biocompatible RL100 nanoparticles suitable for cellular nanothermometry, as we demonstrate with temperature measurements in multicellular colon cancer spheroids.

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

In situ spatiotemporal solute imaging of metal corrosion on the example of magnesium.

Visualization and quantification of corrosion processes is essential in materials research. Here we present a new approach for 2D spatiotemporal imaging of metal corrosion dynamics in situ. The approach combines time-integrated Mg2+ flux imaging by diffusive gradients in thin films laser ablation inductively coupled plasma mass spectrometry (DGT LA-ICP-MS) and near real-time pH imaging by planar optodes. The parallel assessment of Mg2+ flux and pH distributions on a fine-structured, bare Mg alloy (b-WE43) showed intense Mg dissolution with Mg2+ flux maxima up to 11.9 ng cm-2 s-1 and pH increase >9 during initial corrosion (≤15 min) in aqueous NaNO3 solution (c = 0.01 mol L-1). The techniques visualized the lower initial corrosion rate in buffered synthetic body fluid (Hank's balanced salt solution; pH 7.6) compared to unbuffered NaNO3 (pH 6.0), but precise localization of Mg corrosion remains challenging under these conditions. To further demonstrate the capability of DGT LA-ICP-MS for spatiotemporal metal flux imaging at the microscale, a coated Mg alloy (c-WE43) with lower reactivity was deployed for ≤120 min. The high spatial resolution (∼10 µm × 80 µm) and low limits of detection (≤0.04 ng cm-2 s-1, t = 60 min) enabled accurate in situ localization and quantification (Urel = 20%, k = 2) of distinct Mg2+ flux increase, showing micro-confined release of Mg2+ from surface coating defects on c-WE43 samples. The presented approach can be extended to other metal species and applied to other materials to better understand corrosion processes and improve material design in technological engineering.

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

Vapochromism and Magnetochemical Switching of a Nickel(II) Paddlewheel Complex by Reversible NH3 Uptake and Release.

Reaction of [NiCl2 (PnH)4 ] (1) (PnH=6-tert-butyl-pyridazine-3-thione) with NiCl2 affords the binuclear paddlewheel (PW) complex [Ni2 (Pn)4 ] (2). Diamagnetic complex 2 is the first example of a PW complex capable of reversibly binding and releasing NH3 . The NH3 ligand in [Ni2 (Pn)4 (NH3 )] (2⋅NH3 ) enforces major spectroscopic and magnetic susceptibility changes, thus displaying vapochromic properties (λmax (2)=532 nm, λmax (2⋅NH3 )=518 nm) and magnetochemical switching (2: S=0; 2⋅NH3 : S=1). Upon repeated adsorption/desorption cycles of NH3 the PW core remains intact. Compound 2 can be embedded into thin polyurethane films (2P ) under retention of its sensing abilities. Therefore, 2 qualifies as reversible optical probe for ammonia. The magnetochemical switching of 2 and 2⋅NH3 was studied in detail by SQUID measurements showing that in 2⋅NH3 , solely the Ni atom coordinated the NH3 molecule is responsible for the paramagnetic behavior.

Amphiphilic Rhenium-Oxo Corroles as a New Class of Sensitizers for Photodynamic Therapy.

A set of rhenium(V)-oxo meso-triarylcorroles bearing ester and carboxylic acid functionalities were synthesized with a view to determining their potential for photodynamic therapy. Toward this end, we measured their near-IR phosphorescence and their ability to sensitize singlet oxygen formation. The two esters studied, ReVO 5,10,15-tris(meta-carbomethoxyphenyl)corrole and ReVO 5,10,15-tris(para-carbomethoxyphenyl)corrole, were found to exhibit phosphorescence quantum yields of around 1% and fairly long phosphorescence lifetimes of about 60 µs in toluene. The corresponding carboxylic acids, which were examined in ethanolic/aqueous media, in contrast, showed much lower phosphorescence quantum yields on the order of 0.01% and somewhat shorter phosphorescent lifetimes. The quantum yields for singlet oxygen formation, on the other hand, turned out to be equally high (0.72 ± 0.02) for the esters and corresponding carboxylic acids. For the two carboxylic acids, we also carried out photocytotoxicity measurements on rat bladder cancer cells (AY27) and human colon carcinoma cells (WiDr). Cell viability measurements (MTT assays) indicated 50% cell death (LD50) for AY27 cells upon 5 min of blue light exposure with the meta carboxylic acid and upon 7 min of exposure with the para carboxylic acid; complete cell death resulted after 20 min for both compounds. The WiDr cells proved less sensitive, and LD50 values were reached after 8 and 12 min illumination with the meta and para carboxylic acids, respectively.

Multiparametric Optical Bioimaging Reveals the Fate of Epoxy Crosslinked Biomeshes in the Mouse Subcutaneous Implantation Model.

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants.

Airway surface liquid pH is not acidic in children with cystic fibrosis.

Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans. In order to address this deficiency, ASL pH was measured in vivo in children using a novel luminescent technology integrated with fibre-optic probes. Here we show that ASL pH in children with CF is similar to that of children without CF. Findings were supported by highly controlled direct pH measurements in primary human airway epithelial cell culture models, which also suggest that the potential ASL pH gradient produced by defective apical ion transport is balanced out by paracellular shunting of acid/base. Thus, reduced baseline ASL pH is unlikely to be an important pathobiological factor in early CF lung disease.

Complexes of Ir(III)-octaethylporphyrin with peptides as probes for sensing cellular O2.

Ir(III)-porphyrins are a relatively new group of phosphorescent dyes that have potential for oxygen sensing and labeling of biomolecules. The requirement of two axial ligands for the Ir(III) ion permits simple linkage of biomolecules by a one-step ligand-exchange reaction, for example, using precursor carbonyl chloride complexes and peptides containing histidine residue(s). Using this approach, we produced three complexes of Ir(III)-octaethylporphyrin with cell-penetrating (Ir1 and Ir2) and tumor-targeting (Ir3) peptides and studied their photophysical properties. All of the complexes were stable and possessed bright, long-decay (unquenched lifetimes exceeding 45 µs) phosphorescence at around 650 nm, with moderate sensitivity to oxygen. The Ir1 and Ir2 complexes showed positive staining of a number of mammalian cell types, thus demonstrating localization similar to endoplasmic reticulum and ATP- and temperature-independent intracellular accumulation (direct translocation mechanism). Their low photo- and cytotoxicity allows intracellular oxygen to be probed.

Preparation and Characterization of Chromium(III)-Activated Yttrium Aluminum Borate: A New Thermographic Phosphor for Optical Sensing and Imaging at Ambient Temperatures.

A new thermographic phosphor based on chromium(III)-doped yttrium aluminum borate (YAB) is obtained as single crystals by high temperature flux growth and as a microcrystalline powder via solution combustion synthesis. The phosphor is excitable both in the blue (lambda(max) 422 nm) and in the red part of the spectrum (lambda(max) 600 nm) and shows bright NIR emission. The brightness of the phosphor is comparable to that of a well-known lamp phosphor Mn(IV)-doped magnesium fluorogermanate. At ambient temperatures, the Cr(III)-doped YAB shows high temperature dependence of the luminescence decay time, which approaches 1% per deg. The material shows no decrease in luminescence intensity at higher temperatures. The new phosphor is particularly promising for applications in temperature-compensated optical chemosensors (including those based on NIR-emitting indicators) and in pressure-sensitive paints.

Ultrabright oxygen optodes based on cyclometalated iridium(III) coumarin complexes.

Cyclometalated iridium(III) coumarin complexes represent new types of probes for optical oxygen sensing. In comparison to the most commonly used ruthenium(II) polypyridyl dyes and porphyrin complexes with platinum group metals, they possess much more efficient visible absorption and higher quantum yields, which results in much higher brightnesses. Spectral properties and oxygen sensitivity can be fine-tuned by varying the nature of the coumarin ligand and using respective monomeric or dimeric complexes. When incorporated in a model polystyrene film the probes show optimal dynamics of luminescence decay time for oxygen monitoring in the range from 0% to 100% air saturation. Cross-sensitivity to temperature is significantly lower than for the commonly used ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline oxygen probe. The probes, however, exhibit significantly lower photostability, which restricts their application. If long-term measurements are not required, the probes can be successfully used for reliable monitoring of oxygen concentration. High brightness of the complexes makes them particularly attractive for application in thin films (for monitoring of fast processes) and various types of nano- and microparticles, including magnetic beads. If temperature compensation is not applied, the novel optodes result in the lower errors in determination of oxygen content.