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.

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.

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.

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.

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

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.

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.

Quantifying Guest Exchange in Supramolecular Systems.

The ability to accurately determine and quantitatively evaluate kinetic phenomena associated with supramolecular assemblies, in real time, is key to a better understanding of their defined architectures and diverse functionalities. Therefore, analytical tools that can precisely assess a wide range of exchange rates within such systems are of considerable importance. This study demonstrates the ability to use an NMR approach based on saturation transfer for the determination of rates of guest exchange from molecular capsules. By using cavitands that assemble into distinct dimeric assemblies, we show that this approach, which we term guest exchange saturation transfer (GEST), allows the use of a conventional NMR setup to study and quantitatively assess a wide range of exchange rates, from 35 to more than 5000 s-1 .

Imaging the DNA Alkylator Melphalan by CEST MRI: An Advanced Approach to Theranostics.

Brain tumors are among the most lethal types of tumors. Therapeutic response variability and failure in patients have been attributed to several factors, including inadequate drug delivery to tumors due to the blood-brain barrier (BBB). Consequently, drug delivery strategies are being developed for the local and targeted delivery of drugs to brain tumors. These drug delivery strategies could benefit from new approaches to monitor the delivery of drugs to tumors. Here, we evaluated the feasibility of imaging 4-[bis(2-chloroethyl)amino]-l-phenylalanine (melphalan), a clinically used DNA alkylating agent, using chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), for theranostic applications. We evaluated the physicochemical parameters that affect melphalan's CEST contrast and demonstrated the feasibility of imaging the unmodified drug by saturating its exchangeable amine protons. Melphalan generated a CEST signal despite its reactivity in an aqueous milieu. The maximum CEST signal was observed at pH 6.2. This CEST contrast trend was then used to monitor therapeutic responses to melphalan in vitro. Upon cell death, the decrease in cellular pH from ∼7.4 to ∼6.4 caused an amplification of the melphalan CEST signal. This is contrary to what has been reported for other CEST contrast agents used for imaging cell death, where a decrease in the cellular pH following cell death results in a decrease in the CEST signal. Ultimately, this method could be used to noninvasively monitor melphalan delivery to brain tumors and also to validate therapeutic responses to melphalan clinically.

Single (19)F probe for simultaneous detection of multiple metal ions using miCEST MRI.

The local presence and concentration of metal ions in biological systems has been extensively studied ex vivo using fluorescent dyes. However, the detection of multiple metal ions in vivo remains a major challenge. We present a magnetic resonance imaging (MRI)-based method for noninvasive detection of specific ions that may be coexisting, using the tetrafluorinated derivative of the BAPTA (TF-BAPTA) chelate as a (19)F chelate analogue of existing optical dyes. Taking advantage of the difference in the ion-specific (19)F nuclear magnetic resonance (NMR) chemical shift offset (Δω) values between the ion-bound and free TF-BAPTA, we exploited the dynamic exchange between ion-bound and free TF-BAPTA to obtain MRI contrast with multi-ion chemical exchange saturation transfer (miCEST). We demonstrate that TF-BAPTA as a prototype single (19)F probe can be used to separately visualize mixed Zn(2+) and Fe(2+) ions in a specific and simultaneous fashion, without interference from potential competitive ions.

Tumor-specific expression and detection of a CEST reporter gene.

PURPOSE: To develop an imaging tool that enables the detection of malignant tissue with enhanced specificity using the exquisite spatial resolution of MRI. METHODS: Two mammalian gene expression vectors were created for the expression of the lysine-rich protein (LRP) under the control of the cytomegalovirus (CMV) promoter and the progression elevated gene-3 promoter (PEG-3 promoter) for constitutive and tumor-specific expression of LRP, respectively. Using those vectors, stable cell lines of rat 9L glioma, 9L(CMV-LRP) and 9L(PEG-LRP) , were established and tested for CEST contrast in vitro and in vivo. RESULTS: 9L(PEG-LRP) cells showed increased CEST contrast compared with 9L cells in vitro. Both 9L(CMV-LRP) and 9L(PEG-LRP) cells were capable of generating tumors in the brains of mice, with a similar growth rate to tumors derived from wild-type 9L cells. An increase in CEST contrast was clearly visible in tumors derived from both 9L(CMV-LRP) and 9L(PEG-LRP) cells at 3.4 ppm. CONCLUSION: The PEG-3 promoter:LRP system can be used as a cancer-specific, molecular-genetic imaging reporter system in vivo. Because of the ubiquity of MR imaging in clinical practice, sensors of this class can be used to translate molecular-genetic imaging rapidly.

Variable delay multi-pulse train for fast chemical exchange saturation transfer and relayed-nuclear overhauser enhancement MRI.

PURPOSE: Chemical exchange saturation transfer (CEST) imaging is a new MRI technology allowing the detection of low concentration endogenous cellular proteins and metabolites indirectly through their exchangeable protons. A new technique, variable delay multi-pulse CEST (VDMP-CEST), is proposed to eliminate the need for recording full Z-spectra and performing asymmetry analysis to obtain CEST contrast. METHODS: The VDMP-CEST scheme involves acquiring images with two (or more) delays between radiofrequency saturation pulses in pulsed CEST, producing a series of CEST images sensitive to the speed of saturation transfer. Subtracting two images or fitting a time series produces CEST and relayed-nuclear Overhauser enhancement CEST maps without effects of direct water saturation and, when using low radiofrequency power, minimal magnetization transfer contrast interference. RESULTS: When applied to several model systems (bovine serum albumin, crosslinked bovine serum albumin, l-glutamic acid) and in vivo on healthy rat brain, VDMP-CEST showed sensitivity to slow to intermediate range magnetization transfer processes (rate < 100-150 Hz), such as amide proton transfer and relayed nuclear Overhauser enhancement-CEST. Images for these contrasts could be acquired in short scan times by using a single radiofrequency frequency. CONCLUSIONS: VDMP-CEST provides an approach to detect CEST effect by sensitizing saturation experiments to slower exchange processes without interference of direct water saturation and without need to acquire Z-spectra and perform asymmetry analysis.

Natural D-glucose as a biodegradable MRI relaxation agent.

PURPOSE: Demonstrate applicability of natural D-glucose as a T2 MRI contrast agent. METHODS: D-glucose solutions were prepared at multiple concentrations and variable pH. The relaxation rate (R2 = 1/T2 ) was measured at 3, 7, and 11.7 T. Additional experiments were performed on blood at 11.7 T. Also, a mouse was infused with D-glucose (3.0 mmol/kg) and dynamic T2 weighted images of the abdomen acquired. RESULTS: The transverse relaxation rate depended strongly on glucose concentration and solution pH. A maximum change in R2 was observed around physiological pH (pH 6.8-7.8). The transverse relaxivities at 22°C (pH 7.3) were 0.021, 0.060, and 0.077 s(-1) mM(-1) at 3.0, 7.0, and 11.7 T, respectively. These values showed good agreement with expected values from the Swift-Connick equation. There was no significant dependence on glucose concentration or pH for T1 and the diffusion coefficient for these solutions. The transverse relaxivity in blood at 11.7 T was 0.09 s(-1) mM(-1) . The dynamic in vivo experiment showed a 10% drop in signal intensity after glucose infusion followed by recovery of the signal intensity after about 50-100 s. CONCLUSION: Glucose can be used as a T2 contrast agent for MRI at concentrations that are already approved for human use.

Alginate-coated magnetic nanoparticles for noninvasive MRI of extracellular calcium.

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.

Optogenetic-guided cortical plasticity after nerve injury.

Peripheral nerve injury causes sensory dysfunctions that are thought to be attributable to changes in neuronal activity occurring in somatosensory cortices both contralateral and ipsilateral to the injury. Recent studies suggest that distorted functional response observed in deprived primary somatosensory cortex (S1) may be the result of an increase in inhibitory interneuron activity and is mediated by the transcallosal pathway. The goal of this study was to develop a strategy to manipulate and control the transcallosal activity to facilitate appropriate plasticity by guiding the cortical reorganization in a rat model of sensory deprivation. Since transcallosal fibers originate mainly from excitatory pyramidal neurons somata situated in laminae III and V, the excitatory neurons in rat S1 were engineered to express halorhodopsin, a light-sensitive chloride pump that triggers neuronal hyperpolarization. Results from electrophysiology, optical imaging, and functional MRI measurements are concordant with that within the deprived S1, activity in response to intact forepaw electrical stimulation was significantly increased by concurrent illumination of halorhodopsin over the healthy S1. Optogenetic manipulations effectively decreased the adverse inhibition of deprived cortex and revealed the major contribution of the transcallosal projections, showing interhemispheric neuroplasticity and thus, setting a foundation to develop improved rehabilitation strategies to restore cortical functions.

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.

Identifying priority sites for whale shark ship collision management globally.

The expansion of the world's merchant fleet poses a great threat to the ocean's biodiversity. Collisions between ships and marine megafauna can have population-level consequences for vulnerable species. The Endangered whale shark (Rhincodon typus) shares a circumglobal distribution with this expanding fleet and tracking of movement pathways has shown that large vessel collisions pose a major threat to the species. However, it is not yet known whether they are also at risk within aggregation sites, where up to 400 individuals can gather to feed on seasonal bursts of planktonic productivity. These "constellation" sites are of significant ecological, socio-economic and cultural value. Here, through expert elicitation, we gathered information from most known constellation sites for this species across the world (>50 constellations and >13,000 individual whale sharks). We defined the spatial boundaries of these sites and their overlap with shipping traffic. Sites were then ranked based on relative levels of potential collision danger posed to whale sharks in the area. Our results showed that researchers and resource managers may underestimate the threat posed by large ship collisions due to a lack of direct evidence, such as injuries or witness accounts, which are available for other, sub-lethal threat categories. We found that constellations in the Arabian Sea and adjacent waters, the Gulf of Mexico, the Gulf of California, and Southeast and East Asia, had the greatest level of collision threat. We also identified 39 sites where peaks in shipping activity coincided with peak seasonal occurrences of whale sharks, sometimes across several months. Simulated collision mitigation options estimated potentially minimal impact to industry, as most whale shark core habitat areas were small. Given the threat posed by vessel collisions, a coordinated, multi-national approach to mitigation is needed within priority whale shark habitats to ensure collision protection for the species.

Metal ion sensing using ion chemical exchange saturation transfer 19F magnetic resonance imaging.

Although metal ions are involved in a myriad of biological processes, noninvasive detection of free metal ions in deep tissue remains a formidable challenge. We present an approach for specific sensing of the presence of Ca(2+) in which the amplification strategy of chemical exchange saturation transfer (CEST) is combined with the broad range of chemical shifts found in (19)F NMR spectroscopy to obtain magnetic resonance images of Ca(2+). We exploited the chemical shift change (Δω) of (19)F upon binding of Ca(2+) to the 5,5'-difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) by radiofrequency labeling at the Ca(2+)-bound (19)F frequency and detection of the label transfer to the Ca(2+)-free (19)F frequency. Through the substrate binding kinetics we were able to amplify the signal of Ca(2+) onto free 5F-BAPTA and thus indirectly detect low Ca(2+) concentrations with high sensitivity.