HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Comparison of the structural, electrochemical, and spectroscopic properties of two cryptates of trivalent uranium.

We describe a study of the influence of cryptand denticity on the structural, electronic, and electrochemical properties of UIII-containing cryptates. Two cryptands (2.2.2 and 2.2.1) are reported. The cryptand with the smaller denticity leads to negative electrochemical potentials and shorter bond lengths that are consistent with a better fit for UIII than the larger cryptand. These studies provide insight into the rational design of cryptand-based ligands for trivalent uranium.

Three derivatives of phenacyldiphenylphosphine oxide: influence of aromatic and alkyl substituents on the luminescence sensitization of four Ln(NO3)3 salts.

A series of four ß-carbonylphosphine oxide compounds have been synthesized, and their complexes with the nitrate salts of Sm3+, Eu3+, Tb3+ and Dy3+ have been characterized in solution and in the solid state. Analysis of the complexes using IR and NMR suggests that metal-ligand binding occurs mainly through the phosphine oxide group of the ligand, with some involvement of the carbonyl group. All 16 complexes luminesce in solutions of acetonitrile, albeit with varying degrees of intensity. The highest quantum yield values obtained for this series are those where the ligand contains an aryl carbonyl group paired with an electron rich phosphine oxide group (29.8 and 11% for the Tb3+ and Eu3+ complexes, respectively). In contrast, the longest emission lifetime values were found for complexes where the ligand contains a bulky substituent on the carbonyl group paired with an electron rich phosphine oxide (1.86, 1.402, 0.045 ms for the Tb3+, Eu3+ and Sm3+ complexes, respectively).

HaloTag display enables quantitative single-particle characterization and functionalization of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells, and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity, and other properties. Measuring how incorporation varies across a population of EVs is important for characterizing such materials and understanding their function, yet it remains challenging to quantitatively characterize the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterization platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labeling to antibody-labeling of EVs using single vesicle flow cytometry, enabling us to quantify the substantial degree to which antibody labeling can underestimate the absolute number of proteins present on an EV. Finally, we demonstrate use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterization tool which complements and expands existing methods.

Magnetic resonance thermometry using a GdIII-based contrast agent.

The complexes described here serve as contrast agents for magnetic resonance imaging thermometry. The complexes differentially enhance contrast between 275 and 325 K. The basis of the temperature response of the fluorinated contrast complex is the modulation of water exchange caused by trifluoromethyl groups that can be chemically controlled.

Rational Design of High-Relaxivity EuII -Based Contrast Agents for Magnetic Resonance Imaging of Low-Oxygen Environments.

Metal-based contrast agents for magnetic resonance imaging present a promising avenue to image hypoxia. EuII -based contrast agents have a unique biologically relevant redox couple, EuII/III , that distinguishes this metal for use in hypoxia imaging. To that end, we investigated a strategy to enhance the contrast-enhancing capabilities of EuII -based cryptates in magnetic resonance imaging by controlling the rotational dynamics. Two dimetallic, EuII -containing cryptates were synthesized to test the efficacy of rigid versus flexible coupling strategies. A flexible strategy to dimerization led to a modest (114 %) increase in contrast enhancement per Eu ion (60 MHz, 298 K), but a rigid linking strategy led to an excellent (186 %) increase in contrast enhancement despite this compound's having the smaller molecular mass of the two dimetallic complexes. We envision the rigid linking strategy to be useful in the future design of potent EuII -based contrast agents for magnetic resonance imaging.

Systematic Tuning of the Optical Properties of Discrete Complexes of EuII in Solution Using Counterions and Solvents.

We describe a systematic study of the influence of halides and solvents on the optical properties of EuII-containing complexes in solution starting from well-defined crystalline precursors. Anionic halides, chloride and bromide, blue-shift the spectroscopic properties of EuII, whereas neutral ligands, methanol and acetonitrile, cause a red shift. This system provides evidence that EuII has a stronger affinity for chloride, and to some extent bromide, relative to acetonitrile but not methanol. We also describe a simple procedure using an ion-exchange resin for the exchange of iodide counterions to hexafluorophosphate. These findings are a step toward designing ligands that can tune the optical properties of EuII-containing complexes for solution-based applications.

Spectroscopic and Electrochemical Trends in Divalent Lanthanides through Modulation of Coordination Environment.

Due to the importance of both visible-light luminescence and lanthanides in modern society, the influence of the ligand environment on complexes of YbII were studied and compared with analogous complexes of EuII. Four ligands with systematically varied electronic and steric characteristics were used to probe the coordination environment and electronic and redox properties of the corresponding YbII-containing complexes. Strong-field nitrogenous donors gave rise to bathochromic shifts, leading to visible-light absorption by YbII. Trends in properties across the series of YbII-containing complexes were compared to trends reported for the analogous EuII-containing complexes, revealing the translatability of coordination environment effects across the divalent lanthanide series. These studies provide valuable information regarding the behavior of small and medium-sized divalent lanthanides outside of the solid state.

Photophysical characterization of a highly luminescent divalent-europium-containing azacryptate.

We report a new luminescent EuII-containing complex. The complex is excited with visible light, leading to emission centered at 447 nm with a lifetime of 1.25 µs. Computational studies suggest that the steric bulk of the ligand is a major factor influencing the wavelength of emission.

First use of a divalent lanthanide for visible-light-promoted photoredox catalysis.

We report the first catalytic use of a divalent lanthanide in visible-light-promoted bond-forming reactions. Our new precatalyst uses europium in the +2 oxidation state and is active in the presence of blue light from light-emitting diodes. The use of low-energy visible light reduces the occurrence of potential side reactions that might be induced by higher-energy UV light. The system described here uses zinc metal as a sacrificial reductant and is tolerant to wet, protic solvents. The catalyst can be made in situ from relatively inexpensive and air-stable EuCl3·6H2O, and the ligand can be synthesized in large quantities in two steps. With 0.5% loading of precatalyst, an average of 120 turnovers was observed in six hours for reductive coupling of benzyl chloride. We expect that the results will initiate the study of visible-light-promoted photoredox catalysis using divalent europium in a variety of reactions.

Fluorinated EuII-based multimodal contrast agent for temperature- and redox-responsive magnetic resonance imaging.

Magnetic resonance imaging (MRI) using redox-active, EuII-containing complexes is one of the most promising techniques for noninvasively imaging hypoxia in vivo. In this technique, positive (T1-weighted) contrast enhancement persists in areas of relatively low oxidizing ability, such as hypoxic tissue. Herein, we describe a fluorinated, EuII-containing complex in which the redox-active metal is caged by intramolecular interactions. The position of the fluorine atoms enables temperature-responsive contrast enhancement in the reduced form of the contrast agent and detection of the oxidized contrast agent via MRI in vivo. Positive contrast is observed in 1H-MRI with Eu in the +2 oxidation state, and chemical exchange saturation transfer and 19F-MRI signal are observed with Eu in the +3 oxidation state. Contrast enhancement is controlled by the redox state of Eu, and modulated by the fluorous interactions that cage a bound water molecule reduce relaxivity in a temperature-dependent fashion. Together, these advancements constitute the first report of in vivo, redox-responsive imaging using 19F-MRI.

Unique Eu(II) Coordination Environments with a Janus Cryptand.

Two new Eu(II)-containing cryptates were prepared with a new nitrogenous cryptand functionalized with three benzo groups. The introduction of three aromatic rings into the ligand backbone imparts lopsided geometrical features on the resulting Eu(II) coordination environments. In both complexes, the interactions between Eu and the amines on the aromatic side of the molecule are weaker than those on the nonaromatic side, resulting in one discrete unit with two distinct faces. One of the new complexes is, to the best of our knowledge, the first direct observation of a bis-aquo Eu(II)-containing cryptate with two nonadjacent inner-sphere water molecules. In addition to solid-phase structure, the electronic UV-visible and emission spectra of the new complexes were studied in acetonitrile. Experimental results show that the decreased Lewis basicity of the aromatic face hypsochromically shifts absorbances and emissions from a structurally related compound without the benzo groups.

StratBAM: A Discrete-Event Simulation Model to Support Strategic Hospital Bed Capacity Decisions.

The ability to accurately measure and assess current and potential health care system capacities is an issue of local and national significance. Recent joint statements by the Institute of Medicine and the Agency for Healthcare Research and Quality have emphasized the need to apply industrial and systems engineering principles to improving health care quality and patient safety outcomes. To address this need, a decision support tool was developed for planning and budgeting of current and future bed capacity, and evaluating potential process improvement efforts. The Strategic Bed Analysis Model (StratBAM) is a discrete-event simulation model created after a thorough analysis of patient flow and data from Geisinger Health System's (GHS) electronic health records. Key inputs include: timing, quantity and category of patient arrivals and discharges; unit-level length of care; patient paths; and projected patient volume and length of stay. Key outputs include: admission wait time by arrival source and receiving unit, and occupancy rates. Electronic health records were used to estimate parameters for probability distributions and to build empirical distributions for unit-level length of care and for patient paths. Validation of the simulation model against GHS operational data confirmed its ability to model real-world data consistently and accurately. StratBAM was successfully used to evaluate the system impact of forecasted patient volumes and length of stay in terms of patient wait times, occupancy rates, and cost. The model is generalizable and can be appropriately scaled for larger and smaller health care settings.