Publications

The reliance of modern technology growth on lanthanides presents dual challenges: securing sustainable sources from natural or recycled materials and reducing environmental harm from waste discharge. However, the similar ionic radii, oxidation states, and binding affinities of Ln³⁺ ions hinder their nondestructive detection in mixtures. Furthermore, the overlap of spectroscopic signals and the inapplicability for opaque solutions limit the harness of luminescent sensors for differentiating one Ln³⁺ from another. Here, we introduce ¹⁹F-paramagnetic guest exchange saturation transfer magnetic resonance fingerprinting (¹⁹F-paraGEST MRF), a rapid signal acquisition, encoding, and analysis approach for detecting specific Ln³⁺ in mixtures. Based on a small-sized experimental ¹⁹F-paraGEST data set, we generated a de novo dictionary of ∼2500 combinations of Ln³⁺ mixtures, resulting in ∼7,000,000 simulated ¹⁹F-paraGEST MRF patterns of different Ln³⁺ concentrations. This dictionary was later used for computational pattern recognition of experimental NMR signal evolutions (\u201cfingerprints\u201d), utilizing a rapid computational approach executable on a standard laptop within seconds. Hence, fast and reliable multiplexed lanthanide detection in complex mixtures was enabled. Demonstrated through the analysis of lanthanides content of permanent magnets from a hard disk drive, this MR-based method paves the way for broader applications of lanthanide detection in murky, nontransparent mixtures and further exploration of supramolecular sensors in diverse scenarios.

Polymeric fluorinated nanoparticles (PFNPs) are useful materials in many applications, especially in the field of ¹⁹F magnetic resonance imaging (MRI). Despite the development of numerous PFNPs with diverse chemical compositions and structures, those with high fluorine content and capable of highly sensitive ¹⁹F MRI remain scarce. Here we report an elegantly designed aqueous photo-polymerization-induced self-assembly (photo-PISA) system for the synthesis of PFNPs with high fluorine content for effective ¹⁹F MRI applications. This innovative photo-PISA system is enabled by two analogous fluorinated monomers, allowing efficient production of PFNPs with different morphologies and high fluorine content (25 wt %) in aqueous solution. These PFNPs exhibit favorable ¹⁹F MRI properties and morphology-dependent biological behavior, and have potential as advanced polymeric nanomaterials for imaging and drug delivery applications.

Purpose: Despite growing evidence of the link between elevated levels of trimethylamine-N-oxide (TMAO) and multiple diseases, there is no method with which to spatially monitor its hepatic formation from the interstitially produced trimethylamine (TMA). This study aimed to develop a deuterium metabolic spectroscopy (DMS) and imaging (DMI) approach to detect the TMA-to-TMAO metabolism in vivo. Methods: The metabolism of ²H₉-TMA (TMA-d₉) to ²H₉-TMAO (TMAO-d₉) in cells overexpressing the hepatic enzyme flavin-dependent monooxygenase 3 (FMO3) was monitored in vitro with ²H-NMR. Using an ultrahigh-field (15.2T) MRI scanner, the hepatic metabolism of the orally administered TMA-d₉ to TMAO-d₉ was studied in mice with DMS and DMI. Results: The spectrally resolved ²H-NMR peaks of intracellularly produced TMAO-d₉ (3.1 ppm) from that of supplemental TMA-d₉ (2.7 ppm) could be detected only in cells that overexpressed FMO3. In vivo, DMS and DMI experiments performed after oral administration of TMA-d₉ revealed the conversion to high TMAO-d₉ levels in the liver of females, which express high levels of FMO3. In contrast, there was no indication of TMAO-d₉ production in the liver of males, in agreement with reports of the role of testosterone in downregulating the expression of FMO3. Conclusion: This work shows the ability to use ²H-MR-based methodologies to spatially monitor the TMA-to-TMAO metabolic pathway in vivo, and thus should be explored further to investigate the role of TMAO in diverse pathologies.

Elucidating structural information of supramolecular hostguest systems is pivotal for understanding molecular recognition and designing functional materials. This study explores the binding modes of fluorinated benzylamine guests in cyclodextrin-based paramagnetic cavitands, employing Gd(III)-capped cyclodextrins (Gd-a-CD and Gd-b-CD, comprising six and seven glucopyranoside units, respectively) and high-field ¹⁹F electron-nuclear double resonance (ENDOR). The ¹⁹F ENDOR spectra revealed distinct behaviors based on the fluorine position and cyclodextrin cavity size. For para-fluorinated benzylamine guests, Gd-b-CD displayed a bimodal distribution of GdF distances, corresponding to two distinct binding modes, whereas Gd-a-CD exhibited a single binding mode. In contrast, meta-fluorinated benzylamines demonstrated a single binding mode for both Gd-a-CD and Gd-b-CD, underscoring the influence of cavity size and fluorine substitution in the guest on binding specificity. ENDOR measurements performed at the EPR central transition of Gd(III) are generally expected to yield GdF distances without orientation-specific details. Surprisingly, in Gd-CDs systems, an unexpected orientation selectivity was observed, enabling the extraction of both GdF distances and orientation of the guest molecule relative to the cavitands Gd(III) zero-field splitting (ZFS) tensor. This two-faceted capability of ¹⁹F-ENDOR allows for determining hostguest complexation geometry and provides insights into ZFS orientation within the cavitand structure.

The \u201cVoices\u201d under this Perspective underline the importance of interdisciplinary collaboration and partnerships across several disciplines, such as medical science and technology, medicine, bioengineering, and computational approaches, in bridging the gap between research, manufacturing, and clinical applications. Effective communication is key to bridging team gaps, enhancing trust, and resolving conflicts, thereby fostering teamwork and individual growth toward shared goals. Drawing from the success of the COVID-19 vaccine development, we advocate the application of similar collaborative models in other complex health areas such as nanomedicine and biomedical engineering. The role of digital technology and big data in healthcare innovation is highlighted along with the necessity for specialized education in collaborative practices. This approach is decisive in advancing healthcare solutions, leading to improved treatment and patient outcomes.

Deuterium (²H) MRI is an emerging tool for noninvasive imaging. We explore the integration of ²H MRI with deuterated multifunctional nanopolymers for deuterated particle imaging (DPI). To this end, amine-terminated G5-polyamidoamine (PAMAM) dendrimers were labeled with deuterated acetyl surface groups, leading to highly ²H-loaded bioparticles, making them ideal for imaging studies. The accumulation of ∼5 nm PAMAM dendrimers in the kidneys could then be seen by ²H MRI with high submillimeter resolution. The natural abundance HDO signal provided an internal concentration reference to these measurements, leading to quantitative dynamic maps showing distinct nanopolymer uptakes within the renal compartments. Further, these nanopolymers allowed us to obtain in vivo maps of activity in the lymph nodes in an inflammatory rodent leg model, demonstrating these deuterated nanopolymers potential as a novel class of contrast agents for the quantitative mapping of physiological processes.

The rapid fluctuations of metal ion levels in biological systems are faster than the time needed to map fluorinated sensors designed for the ¹⁹F-MRI of cations. An attractive modular solution might come from the activity-based sensing approach. Here, we propose a highly reactive but still ultimately specific synthetic fluorinated sensor for ¹⁹F-MRI mapping of labile Zn²⁺. The sensor comprises a dipicolylamine scaffold for Zn²⁺ recognition conjugated to a fluorophenyl acetate entity. Upon binding to Zn²⁺, the synthetic sensor is readily hydrolyzed, and the frequency of its ¹⁹F-functional group in ¹⁹F-NMR is shifted by 12 ppm, allowing the display of the Zn²⁺ distribution as an artificial MRI-colored map highlighting its specificity compared to other metal ions. The irreversible Zn²⁺-induced hydrolysis results in a \u201cturn-on\u201d ¹⁹F-MRI, potentially detecting the cation even upon a transient elevation of its levels. We envision that additional metal-ion sensors can be developed based on the principles demonstrated in this work, expanding the molecular toolbox currently used for ¹⁹F-MRI.

The ability to obtain quantitative spatial information on subcellular processes of deep tissues in vivo has been a long-standing challenge for molecular magnetic resonance imaging (MRI) approaches. This challenge remains even more so for quantifying readouts of genetically engineered MRI reporters. Here, we set to overcome this challenge with a molecular system designed to obtain quantitative ²H-MRI maps of a gene reporter. To this end, we synthesized deuterated thymidine, d₃-thy, with three magnetically equivalent deuterons at its methyl group (-CD₃), showing a singlet peak with a characteristic ²H-NMR frequency (δ = 1.7 ppm). The upfield 3.0 ppm offset from the chemical shift of the HDO signal (δ = 4.7 ppm) allows for spectrally resolving the two ²H NMR signals and quantifying the concentration of d₃-thy based on the known concentration of a tissues HDO. Following systemic administration of d₃-thy, its accumulation as d₃-thy monophosphate in cells expressing the human thymidine kinase 1 (hTK1) transgene was mapped with ²H-MRI. The data obtained in vivo show the ability to use the d₃-thy/hTK1 pair as a reporter probe/reporter gene system to quantitatively map transgene expression with MRI. Relying on a structurally unmodified reporter probe (d₃-thy) to image the expression of unmutated human protein (hTK1) shows the potential of molecular imaging with ²H-MRI to monitor gene reporters and other relevant biological targets.

MgF₂-based formulations have unique physicochemical properties, making them attractive for diverse applications. Here, we show the ability to control the morphology of Sm³⁺-doped, paramagnetic MgF₂ nanocrystals (Sm:MgF₂ NCs) for use as nanosized imaging agents for ¹⁹F-magnetic resonance imaging (¹⁹F-MRI). By reducing the temperature of the reaction mixture from 160 to 110 °C and shortening the reaction time from 16 to 3 h, the morphology of the fabricated oleate-coated Sm:MgF₂ NCs was transitioned from rod-shaped to spherical-shaped nanofluorides, both with a characteristic high-resolution liquid-state ¹⁹F-NMR resonance at −198 ppm. Further coating the surface of the spheric Sm:MgF₂ NCs with a phospholipid layer resulted in a water-soluble formulation that was used to show its detectability with ¹⁹F-MRI.

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.

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-β-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-β-CDs with fluorinated guests, revealing multiple co-existing populations of host-guest complexes exclusively in solutions containing Ln-β-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-β-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.

Nanostructured metal fluorides (nanofluorides) are an emerging type of inorganic nanocrystals (NCs) with unique physiochemical properties for advanced applications. One recent demonstration used water-dispersed ultrasmall CaF₂ nanofluorides as imaging agents that combined the advantages of inorganic NCs with the benefit of background-free ¹⁹F-magnetic resonance imaging (¹⁹F-MRI). Nevertheless, obtaining small nanofluorides with a face-centered cubic crystal structure, where all fluorides are magnetically equivalent to result in a single ¹⁹F NMR signal, is challenging for other types of nanofluorides, preventing their use in ¹⁹F-MRI. Here, we show the development of ultrasmall, water-dispersed, barium fluoride (BaF₂) NCs for bioimaging applications. By doping BaF₂ with two types of lanthanides, diamagnetic-La³⁺ and paramagnetic-Sm³⁺, we were able to control the morphology and ¹⁹F-MR properties of the final La,Sm:BaF₂ (termed LaSamBa) formulation. The fine-tuning of the La³⁺ content enabled us to obtain monodispersed 4.5 nm NCs, and control over the Sm³⁺ content provided LaSamBa with very short T₁ relaxation properties (ca. 100 ms) needed for enhanced ¹⁹F-MRI sensitivity. This type of nanofluorides was examined in two different imaging modalities (i.e., ¹⁹F-MRI and CT), benefiting from their single ¹⁹F-NMR signal and the high atomic number of barium atoms, respectively. As their ¹⁹F chemical shift significantly differs from that of other nanofluorides (e.g., CaF₂ and SrF₂), LaSamBa expanded the nanofluoride library for future multitarget ¹⁹F-MRI studies.

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 didn't last too long as paraCEST agents showed limited translational potential. In 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 past two decades tremendous progress was 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 will give 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 will discuss the strong influence of synthetic biology on the development of the next generation of protein based CEST contrast agents.

Fluorine-containing materials have enriched the field of molecular and cellular MRI with unambiguous and quantitative detection capabilities. The background-free \u201chot-spot\u201d 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 (

Luminescent materials with their rich color palettes have revolutionized the field of bioimaging through the ability to distinguish between spectrally resolved colors and, thus, to map the complexity of biological systems. Yet, advanced solutions to overcome the restricted tissue penetration of light are still needed to allow in vivo mapping of tissue multiplexity in both health and disease. Among the diverse capabilities and many advantages of MRI, the ability to encode specific frequencies of imaging agents and, by that, to allow pseudo-color display of MRI maps, is unique. Here, I summarize our recently developed molecular probes that are capable of generating artificial MR-based colors. To this end, the use of nanofabrication, supramolecular chemistry, and protein engineering approaches to generate novel molecular formulations (inorganic nanocrystals, supramolecular assemblies, and enzyme/substrate pairs) as MRI sensors with unique multicolor display characteristics is reviewed.

NA

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 CaF₂and 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.

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.

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.

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.

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.

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-HDs 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.

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 s1) 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.

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 ¹⁹F-MRI raises the need to further enhance this platform's capabilities. Here, we employ the principles of CEST-MRI in ¹⁹F-MRI to obtain a 900-fold signal amplification of a biocompatible fluorinated agent, which can be presented in a \u201cmulticolor\u201d fashion. Capitalizing on the dynamic interactions in hostguest supramolecular assemblies in an approach termed GEST, we demonstrate that an inhalable fluorinated anesthetic can be used as a single ¹⁹F-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 ¹⁹F-MRI to the mapping of targets that have so-far remained non-detectable.

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 (

The ability to mediate the kinetic properties and dissociation activation energies (E_a) of bound guests by controlling the characteristics of \u201csupramolecular lids\u201d 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 extractedE_a,outin cucurbit[7]uril:guest systems with very fastk_outhighlights their role as \u201csupramolecular lids\u201d in mediating a guest's dissociationE_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.

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.

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 ten-fold shorter T1 of the NCs fluorides. Such paramagnetic-free relaxation enhancement of CaF2 NCs, gained without affecting neither their size nor their colloidal characteristics, improved 4-fold the obtained 19F-MRI signal-to-noise ratio, allowing their use, in-vivo, with enhanced hot-spot MRI sensitivity.

Dynamic processes in host-guest systems, such as the exchange between bound and free guests, can endow such systems with unique properties and function. However, both the dynamic exchange and the relatively low concentration of a studied complex reduce the sensitivity and limit the efficiency of the currently available analytical tools to explore such processes. In this highlight, we present an NMR approach based on saturation transfer, termed GEST - Guest Exchange Saturation Transfer. This technique can be used to detect micromolar concentrations of complexes with the sensitivity of millimolar concentrations, thus offering the ability to amplify otherwise undetected signals in NMR spectra. In addition, by performing the GEST experiment and fitting the data using computational simulations, the exchange rate of free and bound guest and the fractional occupancy of the host can be extracted. We further present several examples of how GEST can be employed to extract important information from host-guest systems. This method can expand the NMR toolbox available to study dynamic host-guest systems in solution without any special expertise or dedicated hardware and can assist in developing more advanced host-guest systems that can be used for many applications, including in molecular and cellular MRI.

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)

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 ¹⁹F-ionic NCs can average out homonuclear dipolar interactions, enabling one to obtain high-resolution ¹⁹F NMR signals in solution that reflect the MR properties of F ⁻ in the crystal core. Decorating ¹⁹F-NC surfaces with a biocompatible poly(ethylene glycol) coating maintains colloidal stability in water while preserving the NC high-resolution ¹⁹F 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 ¹⁹F MRI by facilitating a \u201chot-spot\u201d display of their distribution.

PurposeGenetically 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.MethodsWe 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.ResultsWe 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.ConclusionThis 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. (c) 2017 International Society for Magnetic Resonance in Medicine.

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 5000s(-1).

Although much is known about the diverse roles of metal ions in biology, most of the acquired knowledge was obtained with fluorescent dyes or electrophysiological approaches. However, the ability to non-invasively monitor variation in metal ions and to assess their physiological distribution in health and disease is very limited. Recent advances in the field of molecular magnetic resonance imaging (MRI) have offered new capabilities through the design and development of MRI-responsive sensors for a wide range of applications, including the ability to sense and spatially map metal ions. Here, we briefly summarize the recent progress in the development and performance of MRI sensors designed to monitor metal ions in biology while emphasizing their in vivo uses, their limitations, and remaining challenges. Among the proposed MRI-sensors, Zn²⁺ and Ca²⁺ responsive agents are those that have already been used in live intact subjects, and therefore, these will be emphasized here.

Background The mouse embryo is ideal for studying human cardiac development. However, laboratory discoveries do not easily translate into clinical findings partially because of histological diagnostic techniques that induce artifacts and lack standardization. Aim To present a step-wise approach using 17.6 T MRI, for evaluation of mice embryonic heart and accurate identification of congenital heart defects. Subjects 17.5-embryonic days embryos from low-risk (non-diabetic) and high-risk (diabetic) model dams. Study design Embryos were imaged using 17.6 Tesla MRI. Three-dimensional volumes were analyzed using ImageJ software. Outcome measures Embryonic hearts were evaluated utilizing anatomic landmarks to locate the four-chamber view, the left- and right-outflow tracts, and the arrangement of the great arteries. Inter- and intra-observer agreement were calculated using kappa scores by comparing two researchers evaluations independently analyzing all hearts, blinded to the model, on three different, timed occasions. Each evaluated 16 imaging volumes of 16 embryos: 4 embryos from normal dams, and 12 embryos from diabetic dams. Results Inter-observer agreement and reproducibility were 0.779 (95% CI 0.6530.905) and 0.763 (95% CI 0.6050.921), respectively. Embryonic hearts were structurally normal in 4/4 and 7/12 embryos from normal and diabetic dams, respectively. Five embryos from diabetic dams had defects: ventricular septal defects (n = 2), transposition of great arteries (n = 2) and Tetralogy of Fallot (n = 1). Both researchers identified all cardiac lesions. Conclusion A step-wise approach for analysis of MRI-derived 3D imaging provides reproducible detailed cardiac evaluation of normal and abnormal mice embryonic hearts. This approach can accurately reveal cardiac structure and, thus, increases the yield of animal model in congenital heart defect research.

One of the most attractive features of chemical exchange saturation

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.

The characteristics of host-guest systems, such as molecular recognition, complexation, encapsulation, guest composition, and dynamic exchange, are manifested by changes in the chemical shifts (Δω) in the NMR spectrum. However, in cases where NMR signals cannot be detected, due to low concentrations, poor solubility, or relatively fast exchange, an alternative is needed. Here, we show that by using the magnetization transfer (MT) method, the undetectable NMR signals of host-guest assemblies can be amplified by two orders of magnitude. It is shown that the binding kinetics characteristics of a fluorinated guest and cucurbit[n]uril (CB[n]) hosts in aqueous solutions determine the NMR signal amplification of host-guest assemblies. In addition, by using the MT technique within the ¹⁹F-NMR framework, one can detect μM concentrations of the complex and study the effect of different solutes on the resulting host-guest system. The results expand the \u201cNMR toolbox\u201d available to explore a wider range of dynamic host-guest systems in which NMR signals cannot be detected.

Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated (¹⁹F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiationinduced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0%6 15.8% CD45+, 24.6%612.5% CD34+, and 7.5%63.3% CD31+ cells, with 2.160.7310⁵ cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% 6 13.5% of CD34+ progenitor cells compared with 47.8% 6 18.5% of hematopoietic CD45+ cells, with an average of 2.8 6 2.0 3 10^{12 19}F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7%65.0%) of CD31+ cellswere also labeled, althoughmost coexpressed CD34.Only16%622.3%of CD452/CD312/CD342(triple-negative) cellswere labeledwith CS-ATMDM Green. After induction of cell death by either apoptosis or necrosis, >95% of¹⁹F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a siliconebreastphantomcould be visualizedwith a clinical 3-Teslamagnetic resonance imaging scanner at a sensitivity of approximately 2310⁶ cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients.

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. Magn Reson Med 74:544-549, 2015.

Recent advancements in molecular and synthetic biology, combined with synthetic chemistry and biotechnology, have opened up new opportunities to engineer novel platforms that can monitor complex biological processes with various noninvasive imaging modalities. After decades of using gadolinium- or iron-based metallic sensors for MRI, the recently developed chemical exchange saturation transfer (CEST) contrast mechanism has created an opportunity for rational design, in silico, of nonmetallic biosensors for MRI. These biomolecules are either naturally occurring compounds (amino acids, sugars, nucleosides, native proteins) or can be artificially engineered (synthetic probes or recombinant proteins). They can be administered either as exogenous agents or can be genetically (over)expressed. Moreover, they can be precisely engineered to achieve the desired biochemical properties for fine tuning optimized imaging schemes. The availability of these agents marks the dawn of a new scientific era for molecular and cellular MRI. (Chemical Equation Presented).

The field of molecular and cellular imaging allows molecules and cells to be visualized in vivo non-invasively. It has uses not only as a research tool but in clinical settings as well, for example in monitoring cell-based regenerative therapies, in which cells are transplanted to replace degenerating or damaged tissues, or to restore a physiological function. The success of such cell-based therapies depends on several critical issues, including the route and accuracy of cell transplantation, the fate of cells after transplantation, and the interaction of engrafted cells with the host microenvironment. To assess these issues, it is necessary to monitor transplanted cells non-invasively in real-time. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task, given its ability to image deep inside tissue with high temporal resolution and sensitivity. Extraordinary efforts have recently been made to improve cellular MRI as applied to regenerative medicine, by developing more advanced contrast agents for use as probes and sensors. These advances enable the non-invasive monitoring of cell fate and, more recently, that of the different cellular functions of living cells, such as their enzymatic activity and gene expression, as well as their time point of cell death. We present here a review of recent advancements in the development of these probes and sensors, and of their functioning, applications and limitations.

Alterations in mucin expression and glycosylation are associated with cancer development. Underglycosylated mucin-1 (uMUC1) is overexpressed in most malignant adenocarcinomas of epithelial origin (for example, colon, breast and ovarian cancer). Its counterpart MUC1 is a large polymer rich in glycans containing multiple exchangeable OH protons, which is readily detectable by chemical exchange saturation transfer (CEST) MRI. We show here that deglycosylation of MUC1 results in >75% reduction in CEST signal. Three uMUC1⁺ human malignant cancer cell lines overexpressing uMUC1 (BT20, HT29 and LS174T) show a significantly lower CEST signal compared with the benign human epithelial cell line MCF10A and the uMUC1^- tumour cell line U87. Furthermore, we demonstrate that in vivo CEST MRI is able to make a distinction between LS174T and U87 tumour cells implanted in the mouse brain. These results suggest that the mucCEST MRI signal can be used as a label-free surrogate marker to non-invasively assess mucin glycosylation and tumour malignancy.

Superpositively charged mutants of green fluorescent protein (GFP) demonstrated a dramatically improved chemical exchange saturation transfer (CEST) MRI contrast compared to their wild type counterparts. The mutants +36 GFP and +48 GFP were successfully expressed in mammalian cells and retained part of their fluorescence, making them a new potential bimodal reporter gene.

Composite hyaluronic acid (HA) hydrogels containing gelatin are used in regenerative medicine as tissue-mimicking scaffolds for improving stem cell survival. Once implanted, it is assumed that these biomaterials disintegrate over time, but at present there is no non-invasive imaging technique available with which such degradation can be directly monitored invivo. We show here the potential of chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) as a label-free non-invasive imaging technique to monitor dynamic changes in scaffold composition invivo. The CEST properties of the three individual hydrogel components (HA, GelinS, and polyethylene glycol diacrylate) were first measured invitro. The complete hydrogel was then injected into the brain of immunodeficient rag2^-/- mice and CEST MR images were obtained at day 1 and 7 post-transplantation. Invitro, GelinS gave the strongest CEST signal at 3.6ppm offset from the water peak, originating from the amide protons present in gelatin. Invivo, a significant decrease in CEST signal was observed at 1 week post-implantation. These results were consistent with the biodegradation of the GelinS component, as validated by fluorescent microscopy of implanted hydrogels containing Alexa Fluor 488-labeled GelinS. Our label-free imaging approach should be useful for further development of hydrogel formulations with improved composition and stability.

The protamines are a low-molecular-weight, arginine-rich family of nuclear proteins that protect chromosomal DNA in germ cells by packing it densely using electrostatic interactions. Human protamine-1 (hPRM1) has been developed as a magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) reporter gene, based on a sequence that is approximately 50% arginine, which has a side chain with rapidly exchanging protons. In this study, we have synthesized hPRM1 and determined how its CEST MRI contrast varies as a function of pH, phosphorylation state, and upon noncovalent interaction with nucleic acids and heparin (as antagonist). CEST contrast was found to be highly sensitive to phosphorylation on serine residues, intra- and intermolecular disulfide bridge formation, and the binding of negatively charged nucleotides and heparin. In addition, the nucleotide binding constants (K_eq) for the protamines were determined through plotting the molar concentration of heparin versus CEST contrast and compared between hPRM1 and salmon protamine. Taken together, these findings are important for explaining the CEST contrast of existing arginine-rich probes as well as serving as a guideline for designing new genetic or synthetic probes.

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 ¹⁹F chelate analogue of existing optical dyes. Taking advantage of the difference in the ion-specific ¹⁹F nuclear magnetic resonance (NMR) chemical shift offset (δ ω) 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 ¹⁹F probe can be used to separately visualize mixed Zn²⁺ and Fe²⁺ ions in a specific and simultaneous fashion, without interference from potential competitive ions.

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/T ₂) 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 22C (pH 7.3) were 0.021, 0.060, and 0.077 s^-1mM^-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 ^-1mM^-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 T ₂ contrast agent for MRI at concentrations that are already approved for human use. Magn Reson Med 72:823-828, 2014.

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 (Ca²⁺), are essential ingredients for life. Despite the tremendous importance of Ca²⁺ for the well-being of living systems, the noninvasive determination of the changes in Ca²⁺ levels in general, and extracellular Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ concentrations in the range of 250μm to 2.5mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca²⁺, 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 24h, allow the detection of Ca²⁺ levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca²⁺ 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 Ca²⁺ levels in myriad in vitro and in vivo biomedical applications.

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.

Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acidlong hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells.

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 Ca²⁺ in which the amplification strategy of chemical exchange saturation transfer (CEST) is combined with the broad range of chemical shifts found in ¹⁹F NMR spectroscopy to obtain magnetic resonance images of Ca²⁺. We exploited the chemical shift change (Δω) of ¹⁹F upon binding of Ca²⁺ 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 Ca²⁺-bound ¹⁹F frequency and detection of the label transfer to the Ca ²⁺-free ¹⁹F frequency. Through the substrate binding kinetics we were able to amplify the signal of Ca²⁺ onto free 5F-BAPTA and thus indirectly detect low Ca²⁺ concentrations with high sensitivity.

Synthetic chemistry has revolutionized the understanding of many biological systems. Small compounds that act as agonists and antagonists of proteins, and occasionally as imaging probes, have contributed tremendously to the elucidation of many biological pathways. Nevertheless, the function of thousands of proteins is still elusive, and designing new imaging probes remains a challenge. Through screening and characterization, we identified a thymidine analogue as a probe for imaging the expression of herpes simplex virus type-1 thymidine kinase (HSV1-TK). To detect the probe, we used chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI), in which a dynamic exchange process between an exchangeable proton and the surrounding water protons is used to amplify the desired contrast. Initially, five pyrimidine-based molecules were recognized as putative imaging agents, since their exchangeable imino protons resonate at 5-6 ppm from the water proton frequency and their detection is therefore less affected by endogenous CEST contrast or confounded by direct water saturation. Increasing the pK_a value of the imino proton by reduction of its 5,6-double bond results in a significant reduction of the exchange rate (k_ex) between this proton and the water protons. This reduced k_ex of the dihydropyrimidine nucleosides fulfills the "slow to intermediate regime" condition for generating high CEST-MRI contrast. Consequently, we identified 5-methyl-5,6-dihydrothymidine as the optimal probe and demonstrated its feasibility for in vivo imaging of HSV1-TK. In light of these findings, this new approach can be generalized for designing specific probes for the in vivo imaging of a variety of proteins and enzymes.

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.

Purpose: Protein kinases including protein kinase A (PKA) underlie myriad important signaling pathways. The ability to monitor kinase activity in vivo and in real-time with high spatial resolution in genetically specified cellular populations is a yet unmet need, crucial for understanding complex biological systems as well as for preclinical development and screening of novel therapeutics. Methods: Using the hypothesis that the natural recognition sequences of protein kinases may be detected using chemical exchange saturation transfer magnetic resonance imaging, we designed a genetically encoded biosensor composed of eight tandem repeats of the peptide LRRASLG, a natural target of PKA. Results: This sensor displays a measurable change in chemical exchange saturation transfer signal following phosphorylation by PKA. The natural PKA substrate LRRASLG exhibits a chemical exchange saturation transfer-magnetic resonance imaging contrast at +1.8 and +3.6 ppm, with a >50% change after phosphorylation with minutes-scale temporal resolution. Expression of a synthetic gene encoding eight monomers of LRRASLG yielded two peaks at these chemical exchange saturation transfer frequencies. Conclusion: Taken together, these results suggest that this gene may be used to assay PKA levels in a biologically relevant system. Importantly, the design strategy used for this specific sensor may be adapted for a host of clinically interesting protein kinases.

Organophosphates are highly toxic substances, which cause severe brain damage. The hallmark of the brain injury is major convulsions. The goal of this study was to assess the spatial and temporal MR changes in the brain of paraoxon intoxicated rats. T2-weighted MRI and ¹H-MR-spectroscopy were conducted before intoxication, 3 h, 24 h, and 8 days postintoxication. T2 prolongation mainly in the thalami and cortex was evident as early as 3 h after intoxication (4-6% increase in T2 values, P < 0.05). On spectroscopy, N-acetyl aspartate (NAA)/creatine and NAA/choline levels significantly decreased 3 h postintoxication (>20% decrease, P < 0.005), and 3 h lactate peak was evident in all intoxicated animals. On the 8th day, although very little T2 changes were evident, NAA/creatine and choline/creatine were significantly decreased (>15%, P < 0.05). Animals who succumbed had extensive cortical edema, significant higher lactate levels and a significant decrease in NAA/creatine and NAA/choline levels compared to animals which survived the experiment. Organophosphates-induced brain damage is obvious on MR data already 3 h postintoxication. In vivo spectroscopic changes are more sensitive for assessing long-term injury than T2-weighted MR imaging. Early spectroscopic findings might be used as biomarkers for the severity of the intoxication and might predict early survival.

Purpose: To determine if glial precursor cells can be targeted to inflamed brain through overexpression of very late antigen-4 (VLA-4) and whether this docking process can be monitored with magnetic resonance (MR) cell tracking after intraarterial injection. Materials and Methods: All experimental procedures were performed between August 2010 and February 2012 and were approved by the institutional animal care and use committee. Human glial precursor cells (hGPs) were transfected with VLA-4 and labeled with superparamagnetic iron oxide that contained rhodamine. A microfluidic adhesion assay was used for assessing VLA-4 receptor-mediated cell docking in vitro. A rat model of global lipopolysaccharide (LPS)-mediated brain inflammation was used to induce global vascular cell adhesion molecule-1 (VCAM-1) expression. hGPs were infused into the carotid artery in four animal cohorts (consisting of three rats each): rats that received VLA-4-naive hGPs but did not receive LPS, rats that received VLA-4-expressing hGPs but not LPS, rats that received VLA-4-naive hGPs and LPS, and rats that received VLA-4-expressing hGPs and LPS. MR imaging was performed at 9.4 T before and 1, 10, 20, and 30 minutes after injection. Brain tissue was processed for histologic examination. Quantification of low-signal-intensity pixels was performed with pixel-by-pixel analysis for MR images obtained before and after cell injection. Results: With use of the microfluidic adhesion assay, cell binding to activated brain endothelium significantly increased compared with VLA-4-naive control cells (71.5 cells per field of view ± 11.7 vs 36.4 cells per field of view 6 3.3, respectively; P < .05). Real-time quantitative in vivo MR cell tracking revealed that VLA-4-expressing cells docked exclusively within the vascular bed of the ipsilateral carotid artery and that VLA-4-expressing cells exhibited significantly enhanced homing as compared with VLA-4-naive cells (1448 significant pixels ± 366.5 vs 113.3 significant pixels ± 19.88, respectively; P < .05). Furthermore, MR cell tracking was crucial for correct cell delivery and proper ligation of specific arteries. Conclusion: Targeted intraarterial delivery and homing of VLA-4-expressing hGPs to inflamed endothelium is feasible and can be monitored in real time by using MR imaging in a quantitative, dynamic manner.

Frequency-labeled exchange transfer is a promising MRI technique for labeling and detecting exchanging protons of low-concentration solutes through the water signal. Early frequency-labeled exchange studies have used off-resonance excitation-based labeling schemes that are well suited to study rapidly exchanging protons or molecules far from the water resonance (e.g., water in paramagnetic contrast agents) or slowly exchanging protons close to the water resonance (e.g., some amide protons). However, off-resonance labeling is not efficient for rapidly exchanging protons close to water. Here, we show that a new frequency-labeled exchange labeling scheme with excitation pulses applied on the water resonance gives much higher exchange contrast for rapidly exchanging protons resonating close to the water resonance frequency. This labeling scheme is particularly suited for studying rapidly exchanging hydroxyl, amine, and imino protons in diamagnetic chemical exchange saturation transfer agents. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.

Chemical exchange saturation transfer MRI is a promising new technique for cellular and molecular imaging. This contrast allows the detection of tumors and ischemia without the use of gadolinium as well as the design of microenvironment-sensitive probes that can be discriminated based on their exchange contrast properties and saturation frequency. Current acquisition schemes to detect and analyze this contrast suffer from sensitivity to spatial B₀ inhomogeneity and low contrast-to-noise-ratio, which is an obstacle to widespread adoption of the technology. A new method to detect chemical exchange saturation transfer contrast is proposed here, termed "length and offset varied saturation" which acquires a set of images with the saturation parameters varied so as to modulate the exchange contrast. Either fast fourier transform or the general linear model can be employed to decompose the modulation patterns into separate sources of water signal loss. After transformation, a length and offset varied saturation phase map is generated, which is insensitive to B₀ inhomogeneity. When collected on live mice bearing 9L gliosarcomas, and compared to the conventional asymmetry in the magnetization transfer ratio map using offset increment correction, the results show that length and offset varied saturation phase mapping obtains about three to four times contrast-to-noise-ratio and exhibits less B ₀ artifacts.

The potential of poly(propylene fumarate) (PPF) scaffolds as drug carriers was investigated and the kinetics of the drug release quantified using magnetic resonance imaging (MRI) and optical imaging. Three different MR contrast agents were used for coating PPF scaffolds. Initially, iron oxide (IONP) or manganese oxide nanoparticles (MONP) carrying the anti-cancer drug doxorubicin were absorbed or mixed with the scaffold and their release into solution at physiological conditions was measured with MRI and optical imaging. A slow (hours to days) and functional release of the drug molecules into the surrounding solution was observed. In order to examine the release properties of proteins and polypeptides, protamine sulfate, a chemical exchange saturation transfer (CEST) MR contrast agent, was attached to the scaffold. Protamine sulfate showed a steady release rate for the first 24 h. Due to its biocompatibility, versatile drug-loading capability and constant release rate, the porous PPF scaffold has potential in various biomedical applications, including MR-guided implantation of drug-dispensing materials, development of drug carrying vehicles, and drug delivery for tumor treatment.

Chemical exchange saturation transfer (CEST) is a new approach for generating magnetic resonance imaging (MRI) contrast that allows monitoring of protein properties in vivo. In this method, a radiofrequency pulse is used to saturate the magnetization of specific protons on a target molecule, which is then transferred to water protons via chemical exchange and detected using MRI. One advantage of CEST imaging is that the magnetizations of different protons can be specifically saturated at different resonance frequencies. This enables the detection of multiple targets simultaneously in living tissue. We present here a CEST MRI approach for detecting the activity of cytosine deaminase (CDase), an enzyme that catalyzes the deamination of cytosine to uracil. Our findings suggest that metabolism of two substrates of the enzyme, cytosine and 5-fluorocytosine (5FC), can be detected using saturation pulses targeted specifically to protons at +2 ppm and +2.4 ppm (with respect to water), respectively. Indeed, after deamination by recombinant CDase, the CEST contrast disappears. In addition, expression of the enzyme in three different cell lines exhibiting different expression levels of CDase shows good agreement with the CDase activity measured with CEST MRI. Consequently, CDase activity was imaged with high-resolution CEST MRI. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MRI has the potential to follow the kinetics of multiple enzymes in real time in living tissue.

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.

Purpose: To assess, by MR spectroscopy (MRS) and diffusion weighted imaging (DWI), the ability of electrical stimulation of the sphenopalatine ganglion (SPG) to augment stroke recovery in transient middle cerebral artery occluded (t-MCAO) rats, when treatment is started 18 ± 2 h post-occlusion. Materials and Methods: ¹H-MRS imaging (¹H-MRSI) and DWI were used to evaluate ischemic brain tissue after SPG stimulation in rats subjected to 2 h of t-MCAO. Rats were examined by ¹H-MRSI, DWI, and behavioral tests at 16 ± 2 h, 8 days, and 28 days post-MCAO. Results: N-Acetyl-aspartate (NAA) levels of the stimulated and control rats were the same 16 ± 2 h post-MCAO (0.52 ± 0.03, 0.54 ± 0.03). At 28 days post-occlusion, NAA levels were significantly higher in the treated group (0.60 ± 0.04) compared with those of the untreated animals (0.50 ± 0.04; P < 0.05). This effect was more pronounced for regions with low NAA values (0.16 ± 0.03) that changed to 0.32 ± 0.03 (P = 0.04) for the treated group and to 0.10 ± 0.03 (P = 0.20) for the controls. DWI data showed better ischemic tissue condition for the treated rats, but the measured parameters showed only a trend of improvement. The MR results were corroborated by behavioral examinations. Conclusion: Our findings suggest that SPG stimulation may ameliorate MR tissue characteristics following t-MCAO even if treatment is started 18 h post-occlusion.

High b-value q-space diffusion imaging (QSI) and conventional DTI methodologies were used to study the MRI diffusion characteristics of excised brains of 21-day-old myelin-deficient (md) rats and their age-matched controls. Three different indices were calculated from the QSI data, i.e., Displacement, Probability and Kurtosis, for the purpose of evaluating the effect of the myelin sheaths on the MR diffusion characteristics in white matter (WM) ROIs of the md versus control brains. The examined WM ROIs were the corpus callosum, the external capsule, and the internal capsule. In all examined WM ROIs, significant differences were observed between the md and control brains for all QSI indices. These differences reveal that myelin sheaths surrounding the axons in WM ROIs mostly affect the component exhibiting restricted diffusion, which is manifested by low mean displacement values and high probability and kurtosis values. Such differences were found to be more pronounced in long diffusion times, i.e., Δ = 200 ms. Conventional DTI performed with relatively low b-values (b < 1500 s/mm²) was also used to study md versus control brains. Interestingly, the fractional anisotropy (FA) index, which was calculated from DTI data, did not reveal any significant difference between the groups in the examined WM ROIs. However, some distinctions were revealed by the three eigenvalues (λ₁, λ₂, and λ₃) obtained from the tensor analysis. These findings were supported by Voxel-based analysis using SPM. Finally, MRI-guided histology showed very good agreement between myelin-stained regions and regions with highly restricted diffusion detected by QSI.

Theoretical and experimental studies of restricted diffusion have been conducted for decades using single pulsed field gradient (s-PFG) diffusion experiments. In homogenous samples, the diffusion-diffraction phenomenon arising from a single population of diffusing species has been observed experimentally and predicted theoretically. In this study, we introduce a composite bi-compartmental model which superposes restricted diffusion in microcapillaries with free diffusion in an unconfined compartment, leading to fast and slow diffusing components in the NMR signal decay. Although simplified (no exchange), the superposed diffusion modes in this model may exhibit features seen in more complex porous materials and biological tissues. We find that at low q-values the freely diffusing component masks the restricted diffusion component, and that prolongation of the diffusion time shifts the transition from free to restricted profiles to lower q-values. The effect of increasing the volume fraction of freely diffusing water was also studied; we find that the transition in the signal decay from the free mode to the restricted mode occurs at higher q-values when the volume fraction of the freely diffusing water is increased. These findings were then applied to a phantom consisting of crossing fibers, which demonstrated the same qualitative trends in the signal decay. The angular d-PGSE experiment, which has been recently shown to be able to measure small compartmental dimensions even at low q-values, revealed that microscopic anisotropy is lost at low q-values where the fast diffusing component is prominent. Our findings may be of importance in studying realistic systems which exhibit compartmentation.

q-Space diffusion MRI (QSI) provides a means of obtaining microstructural information about porous materials and neuronal tissues from diffusion data. However, the accuracy of this structural information depends on experimental parameters used to collect the MR data. q-Space diffusion MR performed on clinical scanners is generally collected with relatively long diffusion gradient pulses, in which the gradient pulse duration, δ, is comparable to the diffusion time, Δ. In this study, we used phantoms, consisting of ensembles of microtubes, and mathematical models to assess the effect of the ratio of the diffusion time and the duration of the diffusion pulse gradient, i.e., Δ/δ, on the MR signal attenuation vs. q, and on the measured structural information extracted therefrom. We found that for Δ/δ ∼ 1, the diffraction pattern obtained from q-space MR data are shallower than when the short gradient pulse (SGP) approximation is satisfied. For long δ the estimated compartment size is, as expected, smaller than the real size. Interestingly, for Δ/δ ∼ 1 the diffraction peaks are shifted to even higher q-values, even when δ is kept constant, giving the impression that the restricted compartments are even smaller than they are. When phantoms composed of microtubes of different diameters are used, it is more difficult to estimate the diameter distribution in this regime. Excellent agreement is found between the experimental results and simulations that explicitly account for the use of long duration gradient pulses. Using such experimental data and this mathematical framework, one can estimate the true compartment dimensions when long and finite gradient pulses are used even when Δ/δ ∼ 1.

Characterizing diffusion of gases and liquids within pores is important in understanding numerous transport processes and affects a wide range of practical applications. Previous measurements of the pulsed gradient stimulated echo (PGSTE) signal attenuation, E(q), of water within nerves and impermeable cylindrical microcapillary tubes showed it to be exquisitely sensitive to the orientation of the applied wave vector, q, with respect to the tube axis in the high-q regime. Here, we provide a simple three-dimensional model to explain this angular dependence by decomposing the average propagator, which describes the net displacement of water molecules, into components parallel and perpendicular to the tube wall, in which axial diffusion is free and radial diffusion is restricted. The model faithfully predicts the experimental data, not only the observed diffraction peaks in E(q) when the diffusion gradients are approximately normal to the tube wall, but their sudden disappearance when the gradient orientation possesses a small axial component. The model also successfully predicts the dependence of E(q) on gradient pulse duration and on gradient strength as well as tube inner diameter. To account for the deviation from the narrow pulse approximation in the PGSTE sequence, we use Callaghan's matrix operator framework, which this study validates experimentally for the first time. We also show how to combine average propagators derived for classical one-dimensional and two-dimensional models of restricted diffusion (e.g. between plates, within cylinders) to construct composite three-dimensional models of diffusion in complex media containing pores (e.g. rectangular prisms and/or capped cylinders) having a distribution of orientations, sizes, and aspect ratios. This three-dimensional modeling framework should aid in describing diffusion in numerous biological systems and in a myriad of materials sciences applications.

In recent years, diffusion tensor imaging (DTI) and its variants have been used to describe fiber orientations and q-space diffusion MR was proposed as a means to obtain structural information on a micron scale. Therefore, there is an increasing need for complex phantoms with predictable microcharacteristics to challenge different indices extracted from the different diffusion MR techniques used. The present study examines the effect of diffusion pulse sequence on the signal decay and diffraction patterns observed in q-space diffusion MR performed on micron-scale phantoms of different geometries and homogeneities. We evaluated the effect of the pulse gradient stimulated-echo, the longitudinal eddy current delay (LED) and the bipolar LED (BPLED) pulse sequences. Interestingly, in the less homogeneous samples, the expected diffraction patterns were observed only when diffusion was measured with the BPLED sequence. We demonstrated the correction ability of bipolar diffusion gradients and showed that more accurate physical parameters are obtained when such a diffusion gradient scheme is used. These results suggest that bipolar gradient pulses may result in more accurate data if incorporated into conventional diffusion-weighted imaging and DTI.

Axonal degeneration is an important determinant of progressive neurological disability in multiple sclerosis (MS). Thus, therapeutic approaches promoting neuroprotection could aid the treatment of progressive MS. Here, we used what we believe is a novel water-soluble fullerene derivative (ABS-75) attached to an NMDA receptor antagonist, which combines antioxidant and anti-excitotoxic properties, to block axonal damage and reduce disease progression in a chronic progressive EAE model. Fullerene ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice immunized with myelin-oligodendrocyte glycoprotein (MOG). Reduced disease progression in ABS-75-treated mice was associated with reduced axonal loss and demyelination in the spinal cord. Fullerene ABS-75 halted oxidative injury, CD11b⁺ infiltration, and CCL2 expression in the spinal cord of mice without interfering with antigen-specific T cell responses. In vitro, fullerene ABS-75 protected neurons from oxidative and glutamate-induced injury and restored glutamine synthetase and glutamate transporter expression in astrocytes under inflammatory insult. Glutamine synthetase expression was also increased in the white matter of fullerene ABS-75-treated animals. Our data demonstrate the neuroprotective effect of treatment with a fullerene compound combined with a NMDA receptor antagonist, which may be useful in the treatment of progressive MS and other neurodegenerative diseases.

High b-value q-space diffusion MRS was used to study the diffusion characteristics of formalin-fixed swine optic and sciatic nerves over a large range of diffusion times (3.7-99.3 ms). The very short diffusion time range was studied with a 1 ms resolution. The displacement distribution profiles obtained were fitted to a bi-Gaussian function, and structural parameters were extracted from the q-space diffusion MRS data. This structural information was correlated with axon sizes obtained by histological examination. It was found that high b-value q-space diffusion MRS can easily distinguish between the two nerve types. The root mean square displacements (rmsds) of both the slow and fast diffusing components of the optic nerves were found to be smaller than those of the sciatic nerves. When the rmsd was plotted against the square root of the diffusion time (t_d^1/2), it was found that all four components showed an increase in rmsd; this increase was significantly smaller than expected from the Einstein equation. However, the most restricted component is the slow diffusing component of the optic nerve. This is also the only diffusing component that shows a large change in the slope (i.e. a 'breaking point') of the plot of rmsd as a function of t_d^1/2. This rmsd is very similar to the mean axon size of these optic nerves determined histologically. Such a change in slope was less apparent for the slow diffusing component of sciatic nerves, which showed a wider distribution of axon size in histological images. The fast diffusing components of both nerve types showed only a small gradual change in the slope of rmsd plotted against t_d^1/2. These findings are discussed in the context of component assignment, origin of restriction, and relationships between the structural information extracted from q-space diffusion MRS and histological examination.

In recent years, much effort has been made to increase our ability to infer nerve fiber direction through the use of diffusion MR. The present study examines the effect of the rotational angle (α), i.e. the angle between the diffusion sensitizing gradients and the main axis of the fibers in the nerves, on different NMR indices. The indices examined were the apparent diffusion coefficient (ADC), extracted from low b-values (b_max ≈ 1200 s/mm²), and the root mean square (rms) displacement of the fast and the slow-diffusing components extracted from high b-value q-space diffusion MR data. In addition, the effect of both the diffusion time and myelination was evaluated. We found that the most sensitive index to the rotational angle is the rms displacement of the slow-diffusing component extracted from the high b-value q-space diffusion MR experiment. For this component the rms displacement was nearly constant for α values ranging from -10° to +80° (where α = 0° is the z direction), but it changed dramatically when diffusion was measured nearly perpendicular to the nerve fiber direction, i.e., for α = 90 ± 10°. The ADC and the rms displacement of the fast-diffusing component exhibited only gradual changes, with a maximal change at α = 45 ± 15°. The sensitivity of the rms displacement of the slow-diffusing component to the rotational angle was found to be higher at longer diffusion times and in mature fully myelinated nerves. The relevance of these observations for determining the fiber direction is briefly discussed.

(Chemical Equation Presented) A series of new adamantyl- oligoethyleneglycol-fullerene hybrids was prepared via Bingel-Hirsch functionalization of the C₆₀ fullerene with various adamantyl-oligoethyleneglycol malonates. As NMDA-targeted antioxidants, these compounds may have the potential to be developed as therapeutic agents for the treatment of neurological disorders.