Proteolytic Vesicles Derived from Salmonella enterica Serovar Typhimurium-Infected Macrophages: Enhancing MMP-9-Mediated Invasion and EV Accumulation.

Proteolysis of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a crucial role in the immune response to bacterial infections. Here we report the secretion of MMPs associated with proteolytic extracellular vesicles (EVs) released by macrophages in response to Salmonella enterica serovar Typhimurium infection. Specifically, we used global proteomics, in vitro, and in vivo approaches to investigate the composition and function of these proteolytic EVs. Using a model of S. Typhimurium infection in murine macrophages, we isolated and characterized a population of small EVs. Bulk proteomics analysis revealed significant changes in protein cargo of naïve and S. Typhimurium-infected macrophage-derived EVs, including the upregulation of MMP-9. The increased levels of MMP-9 observed in immune cells exposed to S. Typhimurium were found to be regulated by the toll-like receptor 4 (TLR-4)-mediated response to bacterial lipopolysaccharide. Macrophage-derived EV-associated MMP-9 enhanced the macrophage invasion through Matrigel as selective inhibition of MMP-9 reduced macrophage invasion. Systemic administration of fluorescently labeled EVs into immunocompromised mice demonstrated that EV-associated MMP activity facilitated increased accumulation of EVs in spleen and liver tissues. This study suggests that macrophages secrete proteolytic EVs to enhance invasion and ECM remodeling during bacterial infections, shedding light on an essential aspect of the immune response.

NMR exchange dynamics studies of metal-capped cyclodextrins reveal multiple populations of host-guest complexes in solution.

Metal-capped molecular hosts are unique in supramolecular chemistry, benefitting from the inner cavity's hydrophobic nature and the metal center's electrochemical properties. It is shown here that the paramagnetic properties of the metals in lanthanide-capped cyclodextrins (Ln-α-CDs and Ln-ß-CDs) are a convenient NMR indicator for different populations of host-guest complexes in a given solution. The paramagnetic guest exchange saturation transfer (paraGEST) method was used to study the exchange dynamics in systems composed of Ln-α-CDs or Ln-ß-CDs with fluorinated guests, revealing multiple co-existing populations of host-guest complexes exclusively in solutions containing Ln-ß-CDs. The enhanced spectral resolution of paraGEST, achieved by a strong pseudo contact shift induction, revealed that different molecular guests can adopt multiple orientations within Ln-ß-CDs' cavities and, in contrast, only a single orientation inside Ln-α-CDs. Thus, paraGEST, which can significantly improve NMR detectability and spectral resolution of host-guest systems that experience fast exchange dynamics, is a convenient tool for studying supramolecular systems of metal-capped molecular hosts.

Frontiers in 19F-MR imaging: nanofluorides and 19F-CEST as novel extensions to the 19F-MRI toolbox.

Fluorine-containing materials have enriched the field of molecular and cellular MRI with unambiguous and quantitative detection capabilities. The background-free "hot-spot" display and the large range of chemical shifts of the broad palette of 19F-formulations are now used for a variety of applications. The common features of these formulations are: (i) they are based on organic molecular backbones (i.e., organofluorines); and (ii) their 19F-MRI detectability relies on a well-defined and clearly observed 19F-MR signal. During the last few years, our lab aimed to expand the 19F-MR toolbox with new capabilities that were, thus far, not used in molecular and cellular 19F-MRI. This Feature Article summarizes our developments and implementations in the field of 19F-MRI emphasizing (i) the introduction of ultrasmall inorganic fluoride-based nanocrystals (nanofluorides) as nano-sized (<10 nm) agents for 19F-MRI, and (ii) the use of Chemical Exchange Saturation Transfer (CEST) in the 19F-MRI framework to indirectly amplify 19F-MR signals of otherwise-undetected fluorinated entities.

Protein and peptide engineering for chemical exchange saturation transfer imaging in the age of synthetic biology.

At the beginning of the millennium, the first chemical exchange saturation transfer (CEST) contrast agents were bio-organic molecules. However, later, metal-based CEST agents (paraCEST agents) took center stage. This did not last too long as paraCEST agents showed limited translational potential. By contrast, the CEST field gradually became dominated by metal-free CEST agents. One branch of research stemming from the original work by van Zijl and colleagues is the development of CEST agents based on polypeptides. Indeed, in the last 2 decades, tremendous progress has been achieved in this field. This includes the design of novel peptides as biosensors, genetically encoded recombinant as well as synthetic reporters. This was a result of extensive characterization and elucidation of the theoretical requirements for rational designing and engineering of such agents. Here, we provide an extensive overview of the evolution of more precise protein-based CEST agents, review the rationalization of enzyme-substrate pairs as CEST contrast enhancers, discuss the theoretical considerations to improve peptide selectivity, specificity and enhance CEST contrast. Moreover, we discuss the strong influence of synthetic biology on the development of the next generation of protein-based CEST contrast agents.

Diffusion 19F-NMR of Nanofluorides: In Situ Quantification of Colloidal Diameters and Protein Corona Formation in Solution.

The NMR-detectability of elements of organic ligands that stabilize colloidal inorganic nanocrystals (NCs) allow the study of their diffusion characteristics in solutions. Nevertheless, these measurements are sensitive to dynamic ligand exchange and often lead to overestimation of diffusion coefficients of dispersed colloids. Here, we present an approach for the quantitative assessment of the diffusion properties of colloidal NCs based on the NMR signals of the elements of their inorganic cores. Benefiting from the robust 19F-NMR signals of the fluorides in the core of colloidal CaF2 and SrF2, we show the immunity of 19F-diffusion NMR to dynamic ligand exchange and, thus, the ability to quantify, with high accuracy, the colloidal diameters of different types of nanofluorides in situ. With the demonstrated ability to characterize the formation of protein corona at the surface of nanofluorides, we envision that this study can be extended to additional formulations and applications.

Self-assembly of an MRI responsive agent under physiological conditions provides an extended time window for in vivo imaging.

An MRI-responsive agent that spontaneously self-assembles to a large supramolecular structure under physiological conditions was designed. The obtained assembly provides an extended time window for in vivo studies, as demonstrated for a fluorine-19 probe constructed to sense Zn2+ with 19F-iCEST MRI, in the future.

Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding In Vivo.

The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of a pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible and, thus, allows mapping of the binding of RBD to ACE2 receptors noninvasively in live subjects. Moreover, we show that EVsRBD can be modified to display mutants of the RBD of SARS-CoV-2, allowing rapid screening of currently raised or predicted variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner. Relying on MRI for visualization, the presented approach could be considered in the future to map ligand-receptor binding events in deep tissues, which are not accessible to luminescence-based imaging.

Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression.

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes ('reporter genes') that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides ('reporter probes'). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.

Cation-Ligand Complexation Mediates the Temporal Evolution of Colloidal Fluoride Nanocrystals through Transient Aggregation.

Colloidal inorganic nanofluorides have aroused great interest for various applications with their development greatly accelerated thanks to advanced synthetic approaches. Nevertheless, understanding their colloidal evolution and the factors that affect their dispersion could improve the ability to rationally design them. Here, using a multimodal in situ approach that combines DLS, NMR, and cryogenic-TEM, we elucidate the formation dynamics of nanofluorides in water through a transient aggregative phase. Specifically, we demonstrate that ligand-cation interactions mediate a transient aggregation of as-formed CaF2 nanocrystals (NCs) which governs the kinetics of the colloids' evolution. These observations shed light on key stages through which CaF2 NCs are dispersed in water, highlighting fundamental aspects of nanofluorides formation mechanisms. Our findings emphasize the roles of ligands in NCs' synthesis beyond their function as surfactants, including their ability to mediate colloidal evolution by complexing cationic precursors, and should be considered in the design of other types of NCs.

Fast Ion-Chelate Dissociation Rate for In Vivo MRI of Labile Zinc with Frequency-Specific Encodability.

Fast ion-chelate dissociation rates and weak ion-chelate affinities are desired kinetic and thermodynamic features for imaging probes to allow reversible binding and to prevent deviation from basal ionic levels. Nevertheless, such properties often result in poor readouts upon ion binding, frequently result in low ion specificity, and do not allow the detection of a wide range of concentrations. Herein, we show the design, synthesis, characterization, and implementation of a Zn2+-probe developed for MRI that possesses reversible Zn2+-binding properties with a rapid dissociation rate (koff = 845 ± 35 s-1) for the detection of a wide range of biologically relevant concentrations. Benefiting from the implementation of chemical exchange saturation transfer (CEST), which is here applied in the 19F-MRI framework in an approach termed ion CEST (iCEST), we demonstrate the ability to map labile Zn2+ with spectrally resolved specificity and with no interference from competitive cations. Relying on fast koff rates for enhanced signal amplification, the use of iCEST allowed the designed fluorinated chelate to experience weak Zn2+-binding affinity (Kd at the mM range), but without compromising high cationic specificity, which is demonstrated here for mapping the distribution of labile Zn2+ in the hippocampal tissue of a live mouse. This strategy for accelerating ion-chelate koff rates for the enhancement of MRI signal amplifications without affecting ion specificity could open new avenues for the design of additional probes for other metal ions beyond zinc.

Versatile non-luminescent color palette based on guest exchange dynamics in paramagnetic cavitands.

Multicolor luminescent portrayal of complexed arrays is indispensable for many aspects of science and technology. Nevertheless, challenges such as inaccessible readouts from opaque objects, a limited visible-light spectrum and restricted spectral resolution call for alternative approaches for multicolor representation. Here, we present a strategy for spatial COlor Display by Exploiting Host-guest Dynamics (CODE-HD), comprising a paramagnetic cavitand library and various guests. First, a set of lanthanide-cradled α-cyclodextrins (Ln-CDs) is designed to induce pseudo-contact shifts in the 19F-NMR spectrum of Ln-CD-bound guest. Then, capitalizing on reversible host-guest binding dynamics and using magnetization-transfer 19F-MRI, pseudo-colored maps of complexed arrays are acquired and applied in molecular-steganography scenarios, showing CODE-HD's ability to generate versatile outputs for information encoding. By exploiting the widely shifted resonances induced by Ln-CDs, the guest versatility and supramolecular systems' reversibility, CODE-HD provides a switchable, polychromatic palette, as an advanced strategy for light-free, multicolor-mapping.

Glyconanofluorides as Immunotracers with a Tunable Core Composition for Sensitive Hotspot Magnetic Resonance Imaging of Inflammatory Activity.

Nature-inspired nanosized formulations based on an imageable, small-sized inorganic core scaffold, on which biomolecules are assembled to form nanobiomimetics, hold great promise for both early diagnostics and developed therapeutics. Nevertheless, the fabrication of nanobiomimetics that allow noninvasive background-free mapping of pathological events with improved sensitivity, enhanced specificity, and multiplexed capabilities remains a major challenge. Here, we introduce paramagnetic glyconanofluorides as small-sized (<10 nm) glycomimetics for immunotargeting and sensitive noninvasive in vivo19F magnetic resonance imaging (MRI) mapping of inflammation. A very short T1 relaxation time (70 ms) of the fluorides was achieved by doping the nanofluorides' solid crystal core with paramagnetic Sm3+, resulting in a significant 8-fold enhancement in their 19F MRI sensitivity, allowing faster acquisition and improved detectability levels. The fabricated nanosized glycomimetics exhibit significantly enhanced uptake within activated immune cells, providing background-free in vivo mapping of inflammatory activity, demonstrated in both locally induced inflammation and clinically related neuropathology animal models. Fabricating two types of nanofluorides, each with a distinct chemical shift, allowed us to exploit the color-like features of 19F MRI to map, in real time, immune specificity and preferred targetability of the paramagnetic glyconanofluorides, demonstrating the approach's potential extension to noninvasive multitarget imaging scenarios that are not yet applicable for nanobiomimetics based on other nanocrystal cores.

Single Fluorinated Agent for Multiplexed 19 F-MRI with Micromolar Detectability Based on Dynamic Exchange.

The weak thermal polarization of nuclear spins limits the sensitivity of MRI, even for MR-sensitive nuclei as fluorine-19. Therefore, despite being the source of inspiration for the development of background-free MRI for various applications, including for multiplexed imaging, the inability to map very low concentrations of targets using 19 F-MRI raises the need to further enhance this platform's capabilities. Here, we employ the principles of CEST-MRI in 19 F-MRI to obtain a 900-fold signal amplification of a biocompatible fluorinated agent, which can be presented in a "multicolor" fashion. Capitalizing on the dynamic interactions in host-guest supramolecular assemblies in an approach termed GEST, we demonstrate that an inhalable fluorinated anesthetic can be used as a single 19 F-probe for the concurrent detection of micromolar levels of two targets, with potential in vivo translatability. Further extending GEST with new designs could expand the applicability of 19 F-MRI to the mapping of targets that have so-far remained non-detectable.

In situ NMR reveals real-time nanocrystal growth evolution via monomer-attachment or particle-coalescence.

Understanding inorganic nanocrystal (NC) growth dynamic pathways under their native fabrication environment remains a central goal of science, as it is crucial for rationalizing novel nanoformulations with desired architectures and functionalities. We here present an in-situ method for quantifying, in real time, NCs' size evolution at sub-nm resolution, their concentration, and reactants consumption rate for studying NC growth mechanisms. Analyzing sequential high-resolution liquid-state 19F-NMR spectra obtained in-situ and validating by ex-situ cryoTEM, we explore the growth evolution of fluoride-based NCs (CaF2 and SrF2) in water, without disturbing the synthesis conditions. We find that the same nanomaterial (CaF2) can grow by either a particle-coalescence or classical-growth mechanism, as regulated by the capping ligand, resulting in different crystallographic properties and functional features of the fabricated NC. The ability to reveal, in real time, mechanistic pathways at which NCs grow open unique opportunities for tunning the properties of functional materials.

Inducing Defects in 19F-Nanocrystals Provides Paramagnetic-free Relaxation Enhancement for Improved In Vivo Hotspot MRI.

Paramagnetic relaxation enhancement (PRE) is the current strategy of choice for enhancing magnetic resonance imaging (MRI) contrast and for accelerating MRI acquisition schemes. Yet, debates regarding lanthanides' biocompatibility and PRE-effect on MRI signal quantification have raised the need for alternative strategies for relaxation enhancement. Herein, we show an approach for shortening the spin-lattice relaxation time (T1) of fluoride-based nanocrystals (NCs) that are used for in vivo 19F-MRI, by inducing crystal defects in their solid-crystal core. By utilizing a phosphate-based rather than a carboxylate-based capping ligand for the synthesis of CaF2 NCs, we were able to induce grain boundary defects in the NC lattice. The obtained defects led to a 10-fold shorter T1 of the NCs' fluorides. Such paramagnetic-free relaxation enhancement of CaF2 NCs, gained without affecting either their size or their colloidal characteristics, improved 4-fold the obtained 19F-MRI signal-to-noise ratio, allowing their use, in vivo, with enhanced hotspot MRI sensitivity.

Elucidating dissociation activation energies in host-guest assemblies featuring fast exchange dynamics.

The ability to mediate the kinetic properties and dissociation activation energies (E a) of bound guests by controlling the characteristics of "supramolecular lids" in host-guest molecular systems is essential for both their design and performance. While the synthesis of such systems is well advanced, the experimental quantification of their kinetic parameters, particularly in systems experiencing fast association and dissociation dynamics, has been very difficult or impossible with the established methods at hand. Here, we demonstrate the utility of the NMR-based guest exchange saturation transfer (GEST) approach for quantifying the dissociation exchange rates (k out) and activation energy (E a,out) in host-guest systems featuring fast dissociation dynamics. Our assessment of the effect of different monovalent cations on the extracted E a,out in cucurbit[7]uril:guest systems with very fast k out highlights their role as "supramolecular lids" in mediating a guest's dissociation E a. We envision that GEST could be further extended to study kinetic parameters in other supramolecular systems characterized by fast kinetic properties and to design novel switchable host-guest assemblies.

Dynamic Interactions in Synthetic Receptors: A Guest Exchange Saturation Transfer Study.

The accumulated knowledge regarding molecular architectures is based on established, reliable, and accessible analytical tools that provide robust structural and functional information on assemblies. However, both the dynamicity and low population of noncovalently interacting moieties within studied molecular systems limit the efficiency and accuracy of traditional methods. Herein, the use of a saturation transfer-based NMR approach to study the dynamic binding characteristics of an anion to a series of synthetic receptors derived from bambusuril macrocycles is demonstrated. The exchange rates of BF4 - are mediated by the side chains on the receptor (100 s-1

Calcium Fluoride Nanocrystals: Tracers for In Vivo 19 F Magnetic Resonance Imaging.

Inorganic nanocrystals (NCs) have been extensively developed for a variety of uses. The ability to obtain high-resolution NMR signals from the core nuclei of NCs in solution could offer new opportunities in materials sciences and MR imaging. Herein, we demonstrate that small, water-soluble 19 F-ionic NCs can average out homonuclear dipolar interactions, enabling one to obtain high-resolution 19 F NMR signals in solution that reflect the MR properties of F- in the crystal core. Decorating 19 F-NC surfaces with a biocompatible poly(ethylene glycol) coating maintains colloidal stability in water while preserving the NC high-resolution 19 F NMR properties, even after further functionalization. The high content and magnetic equivalence of the fluorides within the NCs enable their use as imaging tracers for in vivo 19 F MRI by facilitating a "hot-spot" display of their distribution.

Quantification and tracking of genetically engineered dendritic cells for studying immunotherapy.

PURPOSE: Genetically encoded reporters can assist in visualizing biological processes in live organisms and have been proposed for longitudinal and noninvasive tracking of therapeutic cells in deep tissue. Cells can be labeled in situ or ex vivo and followed in live subjects over time. Nevertheless, a major challenge for reporter systems is to identify the cell population that actually expresses an active reporter. METHODS: We have used a nucleoside analog, pyrrolo-2'-deoxycytidine, as an imaging probe for the putative reporter gene, Drosophila melanogaster 2'-deoxynucleoside kinase. Bioengineered cells were imaged in vivo in animal models of brain tumor and immunotherapy using chemical exchange saturation transfer MRI. The number of transduced cells was quantified by flow cytometry based on the optical properties of the probe. RESULTS: We performed a comparative analysis of six different cell lines and demonstrate utility in a mouse model of immunotherapy. The proposed technology can be used to quantify the number of labeled cells in a given region, and moreover is sensitive enough to detect less than 10,000 cells. CONCLUSION: This unique technology that enables efficient selection of labeled cells followed by in vivo monitoring with both optical and MRI. Magn Reson Med 79:1010-1019, 2018. © 2017 International Society for Magnetic Resonance in Medicine.