We report on the first dual nanosensors for imaging of pH values and oxygen partial pressure in cells. The sensors have a unique nanostructure in that a soft core structure is rigidized with a silane reagent, while poly(ethylene glycol) chains form an outer shell. Lipophilic oxygen-sensitive probes and reference dyes are encapsulated inside the hydrophobic core, while a pH-sensitive probe is covalently attached to the poly(ethylene glycol) end-group on the shell. The core/shell structure renders the nanosensors well dispersed and highly stable in various kinds of aqueous media. Their average size is 12 nm, and they respond to both pH and oxygen in the physiological range. They do not pass cell membranes, but can be internalized into the cellular cytosol by electroporation, upon which they enable sensing and imaging of pH values and oxygen with high spatial resolution. The nanosensor strategy shown here is expected to be applicable to the development of various other kinds of multiple nanosensors for in vivo studies.

Hydrogel nanoparticles (nanogels) are appealing probes for use in chemical and biochemical sensing because of their stability, biocompatibility, and softness.[1] Nanogels have been reported [2,3] for detection of several analytes, but mostly for the macrorealm. Recently, fluorescent nanogels have been reported [4] that are capable of transducing volume changes into a change in fluorescence intensity, and nanoscale sensing of temperature in the cytoplasm of living cells was demonstrated.[5] Fluorescence is by far the most powerful method for detecting the cellular dynamics of low-molecular-weight species, including protons (pH), oxygen, and ions such as calcium(II) and chloride.[6] Most sensing methods, including those based on microscopy, are based on the measurement of fluorescence intensity. Unfortunately, single-intensity-based sensing is compromised by the local distribution of probes, which often bind to proteins, and by drifts of light sources and detectors. More robust signals can be obtained by twowavelength ratiometric methods, amongst others.[7] Herein we present the first ratiometric fluorescent nanogel capable of sensing pH values in the physiological range, that is, from six to eight. It can be prepared rather simply from an inert but biocompatible polyurethane polymer that was made pHsensitive by loading it with the pH indicator bromothymol blue (BTB). Furthermore, it was rendered fluorescent by addition of two standard fluorophores that undergo efficient fluorescence resonance energy transfer (FRET) inside the nanogel. The fluorophores coumarin 6 (C6) and Nile Red (NR) were chosen to give a dual (green and red) fluorescent signal that can be easily ratioed.The nanogel (NG) was obtained by a modified reprecipitation method [8] (see the Experimental Section). In essence, an ethanol solution of a hydrogel containing both hydrophilic and hydrophobic domains was dialyzed against water. As a result, the polymer chains rearrange to form a three-dimensionally stable nanostructure [1] based on mainly hydrophobic interaction. The optical probes used in this work are then entrapped into this network. The polyurethane chosen is wellsuited for making such NGs because it contains both hydrophilic and hydrophobic domains, is optically transparent down to 300 nm, commercially available, and widely used in medicine and in contact lenses. Importantly, the volume of the NG particles is hardly affected by pH, which is mandatory with respect to the efficiency of FRET and in terms of in vivo sensing as it will not disturb cellular activities.[9] Figure 1 shows a model of the chemical composition of such a NGbased pH sensor bead.The sensing capability of the NG architecture described herein relies on two specific features. The first is the spectral overlap of the absorption of the pH indicator BTB with the dual emission of the fluorophores C6 and Nile Red (Figure 2 a). The second feature is the efficient FRET (predominantly red fluorescence at pH 7) that occurs between C6 and NR in an aqueous suspension of NG, but not i...

The past 20 years have seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists, pharmacological and toxicological compounds. Most studies on barrier function use G protein coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance based techniques such as Electric Cell-Substrate Impedance Sensing (ECIS) reside in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine and Sphingosine-1-Phosphate.

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Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca 2+ entry (SOCE).

Hydrogel nanoparticles (nanogels) are appealing probes for use in chemical and biochemical sensing because of their stability, biocompatibility, and softness.[1] Nanogels have been reported [2,3] for detection of several analytes, but mostly for the macrorealm. Recently, fluorescent nanogels have been reported [4] that are capable of transducing volume changes into a change in fluorescence intensity, and nanoscale sensing of temperature in the cytoplasm of living cells was demonstrated.[5] Fluorescence is by far the most powerful method for detecting the cellular dynamics of low-molecular-weight species, including protons (pH), oxygen, and ions such as calcium(II) and chloride.[6] Most sensing methods, including those based on microscopy, are based on the measurement of fluorescence intensity. Unfortunately, single-intensity-based sensing is compromised by the local distribution of probes, which often bind to proteins, and by drifts of light sources and detectors. More robust signals can be obtained by twowavelength ratiometric methods, amongst others.[7] Herein we present the first ratiometric fluorescent nanogel capable of sensing pH values in the physiological range, that is, from six to eight. It can be prepared rather simply from an inert but biocompatible polyurethane polymer that was made pHsensitive by loading it with the pH indicator bromothymol blue (BTB). Furthermore, it was rendered fluorescent by addition of two standard fluorophores that undergo efficient fluorescence resonance energy transfer (FRET) inside the nanogel. The fluorophores coumarin 6 (C6) and Nile Red (NR) were chosen to give a dual (green and red) fluorescent signal that can be easily ratioed.The nanogel (NG) was obtained by a modified reprecipitation method [8] (see the Experimental Section). In essence, an ethanol solution of a hydrogel containing both hydrophilic and hydrophobic domains was dialyzed against water. As a result, the polymer chains rearrange to form a three-dimensionally stable nanostructure [1] based on mainly hydrophobic interaction. The optical probes used in this work are then entrapped into this network. The polyurethane chosen is wellsuited for making such NGs because it contains both hydrophilic and hydrophobic domains, is optically transparent down to 300 nm, commercially available, and widely used in medicine and in contact lenses. Importantly, the volume of the NG particles is hardly affected by pH, which is mandatory with respect to the efficiency of FRET and in terms of in vivo sensing as it will not disturb cellular activities.[9] Figure 1 shows a model of the chemical composition of such a NGbased pH sensor bead.The sensing capability of the NG architecture described herein relies on two specific features. The first is the spectral overlap of the absorption of the pH indicator BTB with the dual emission of the fluorophores C6 and Nile Red (Figure 2 a). The second feature is the efficient FRET (predominantly red fluorescence at pH 7) that occurs between C6 and NR in an aqueous suspension of NG, but not i...

Intraocular pressure (IOP) is mostly regulated by aqueous humor outflow through the human trabecular meshwork (HTM) and represents the only modifiable risk factor of glaucoma. The lack of IOP-modulating therapeutics that targets HTM underscores the need of engineering HTM for understanding the outflow physiology and glaucoma pathology in vitro. Using a 3D HTM model that allows for regulation of outflow in response to a pharmacologic steroid, a fibrotic state has been induced resembling that of glaucomatous HTM. This disease model exhibits HTM marker expression, ECM overproduction, impaired HTM cell phagocytic activity and outflow resistance, which represent characteristics found in steroid-induced glaucoma. In particular, steroid-induced ECM alterations in the glaucomatous model can be modified by a ROCK inhibitor. Altogether, this work presents a novel in vitro disease model that allows for physiological and pathological studies pertaining to regulating outflow, leading to improved understanding of steroid-induced glaucoma and accelerated discovery of new therapeutic targets.

Endothelial barrier function is tightly regulated by plasma membrane receptors and is crucial for tissue fluid homeostasis; its dysfunction causes disease, including sepsis and inflammation. The ubiquitous activation of Ca 2؉ signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca 2؉ signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and proposes the requirement of G protein-coupled receptor (GPCR)-generated Ca 2؉ signaling in activating the endothelial contractile apparatus and generating interendothelial gaps. Notwithstanding endothelia being non-excitable in nature, the hypothesis of Ca 2؉ -induced endothelial contraction has been invoked to explain actions of GPCR agonists that either disrupt or stabilize endothelial barrier function. Here, we challenge this correlative hypothesis by showing a lack of causal link between GPCR-generated Ca 2؉ signaling and changes in human microvascular endothelial barrier function. We used three endogenous GPCR agonists: thrombin and histamine, which disrupt endothelial barrier function, and sphingosine-1-phosphate, which stabilizes barrier function. The qualitatively different effects of these three agonists on endothelial barrier function occur independently of Ca 2؉ entry through the ubiquitous store-operated Ca 2؉ entry channel Orai1, global Ca 2؉ entry across the plasma membrane, and Ca 2؉ release from internal stores. However, disruption of endothelial barrier function by thrombin and histamine requires the Ca 2؉ sensor stromal interacting molecule-1 (STIM1), whereas sphingosine-1-phosphate-mediated enhancement of endothelial barrier function occurs independently of STIM1. We conclude that although STIM1 is required for GPCR-mediated disruption of barrier function, a causal link between GPCR-induced cytoplasmic Ca 2؉ increases and acute changes in barrier function is missing. Thus, the cytosolic Ca 2؉ -induced endothelial contraction is a cum hoc fallacy that should be abandoned.The endothelial layer of blood vessels is a highly regulated barrier between the bloodstream and the interstitial tissue, controlling transvascular passage of fluids, solutes, and cells. A significant contribution to endothelial permeability resides in the paracellular diffusion pathway facilitated via intercellular gaps (1-3). Paracellular permeability is essentially mediated by cellcell junctional proteins, which are in turn regulated by intracellular signaling pathways that impact on cytoskeletal architecture (2, 4). The balance between competing tethering and disassembling mechanisms determines the degree of endothelial barrier function and thus the extent of vascular leakage. Disruption of endothelial barrier function causes increased vascular permeability and is associated with reorganization of the actin cytoskeleton and disassembly of adherens junctions which are contributed by vascular endothelial cadherin (VEcadherin)⅐catenin 2 complexes (2, 4). These ev...