Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding In Vivo(2022) ACS Nano. Abstract[All authors]
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.
(2022) NMR in Biomedicine. Abstract
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.
(2022) Nature Biotechnology. Abstract[All authors]
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.MRI detection of transgene expression in animals is expanded to two colors.
Cation-ligand Complexation Mediates the Temporal Evolution of Colloidal Fluoride Nanocrystals through Transient Aggregation(2021) Nano Letters. Abstract
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(2021) Journal of the American Chemical Society. Abstract
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.
(2021) Nature Communications. 12, 1, 3072. Abstract
AbstractMulticolor luminescent portrayal of complexed arrays is indispensable for many aspects of science and technology. Nevertheless, challenges such as inaccessible readouts from opaque objects, a limited visible-light spectrum and restricted spectral resolution call for alternative approaches for multicolor representation. Here, we present a strategy for spatial COlor Display by Exploiting Host-guest Dynamics (CODE-HD), comprising a paramagnetic cavitand library and various guests. First, a set of lanthanide-cradled α-cyclodextrins (Ln-CDs) is designed to induce pseudo-contact shifts in the 19F-NMR spectrum of Ln-CD-bound guest. Then, capitalizing on reversible host-guest binding dynamics and using magnetization-transfer 19F-MRI, pseudo-colored maps of complexed arrays are acquired and applied in molecular-steganography scenarios, showing CODE-HD’s ability to generate versatile outputs for information encoding. By exploiting the widely shifted resonances induced by Ln-CDs, the guest versatility and supramolecular systems' reversibility, CODE-HD provides a switchable, polychromatic palette, as an advanced strategy for light-free, multicolor-mapping.
Single fluorinated agent for multiplexed 19F-MRI with micromolar detectability based on dynamic exchange(2021) Angewandte Chemie (International ed.). Abstract
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.
Glyconanofluorides as Immunotracers with a Tunable Core Composition for Sensitive Hotspot Magnetic Resonance Imaging of Inflammatory Activity(2021) ACS Nano. 15, 4, p. 7563-7574 Abstract[All authors]
Nature-inspired nanosized formulations based on an imageable, small-sized inorganic core scaffold, on which biomolecules are assembled to form nanobiomimetics, hold great promise for both early diagnostics and developed therapeutics. Nevertheless, the fabrication of nanobiomimetics that allow noninvasive background-free mapping of pathological events with improved sensitivity, enhanced specificity, and multiplexed capabilities remains a major challenge. Here, we introduce paramagnetic glyconanofluorides as small-sized (<10 nm) glycomimetics for immunotargeting and sensitive noninvasive in vivo19F magnetic resonance imaging (MRI) mapping of inflammation. A very short T1 relaxation time (70 ms) of the fluorides was achieved by doping the nanofluorides’ solid crystal core with paramagnetic Sm3+, resulting in a significant 8-fold enhancement in their 19F MRI sensitivity, allowing faster acquisition and improved detectability levels. The fabricated nanosized glycomimetics exhibit significantly enhanced uptake within activated immune cells, providing background-free in vivo mapping of inflammatory activity, demonstrated in both locally induced inflammation and clinically related neuropathology animal models. Fabricating two types of nanofluorides, each with a distinct chemical shift, allowed us to exploit the color-like features of 19F MRI to map, in real time, immune specificity and preferred targetability of the paramagnetic glyconanofluorides, demonstrating the approach’s potential extension to noninvasive multitarget imaging scenarios that are not yet applicable for nanobiomimetics based on other nanocrystal cores.<br />
In situ NMR reveals real-time nanocrystal growth evolution via monomer-attachment or particle-coalescence(2021) Nature Communications. 12, 1, 229. Abstract[All authors]
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.
Elucidating dissociation activation energies in host-guest assemblies featuring fast exchange dynamics(2021) Chemical Science. 12, 3, p. 865-871 Abstract
The ability to mediate the kinetic properties and dissociation activation energies (Ea) of bound guests by controlling the characteristics of “supramolecular lids” in host-guest molecular systems is essential for both their design and performance. While the synthesis of such systems is well advanced, the experimental quantification of their kinetic parameters, particularly in systems experiencing fast association and dissociation dynamics, has been very difficult or impossible with the established methods at hand. Here, we demonstrate the utility of the NMR-based guest exchange saturation transfer (GEST) approach for quantifying the dissociation exchange rates (kout) and activation energy (Ea,out) in host-guest systems featuring fast dissociation dynamics. Our assessment of the effect of different monovalent cations on the extractedEa,outin cucurbituril:guest systems with very fastkouthighlights their role as “supramolecular lids” in mediating a guest's dissociationEa. 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.
Inducing defects in 19 F-nanocrystals provides paramagnetic-free relaxation enhancement for improved in-vivo hotspot MRI(2020) Nano Letters. 20, 10, p. 7207-7212 Abstract
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.
(2019) Organic Chemistry Frontiers. 6, 9, p. 1503-1512 Abstract
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.
(2019) Chemistry-A European Journal. 25, 7, p. 1687-1690 Abstract
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) <k(ex) <5000 s(-1)), which play a critical role in receptor-anion binding dynamics. The signal amplification obtained with this approach allows for the identification of different types of intermolecular interactions between the receptor and the anion, something that could not have been detected by techniques hitherto used to study molecular assemblies. These findings, which are supported by a computational molecular dynamic study, demonstrate the uniqueness and added value of this NMR method.
(2018) Angewandte Chemie - International Edition. 57, 25, p. 7478-7482 Abstract
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 “hot-spot” display of their distribution.
(2018) Magnetic Resonance in Medicine. 79, 2, p. 1010-1019 Abstract
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.
(2017) Angewandte Chemie - International Edition. 56, 48, p. 15314-15318 Abstract
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).
(2017) Israel Journal of Chemistry. 57, 9, p. 843-853 Abstract
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.
(2017) Magnetic Resonance Imaging. 35, p. 46-53 Abstract
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 0.653-0.905) and 0.763 (95% CI 0.605-0.921), 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. (C) 2016 Elsevier Inc. All rights reserved.
(2016) Molecular Pharmaceutics. 13, 9, p. 3043-3053 Abstract[All authors]
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 similar to 7.4 to similar 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.
(2016) Chemical Science. 7, 12, p. 6905-6909 Abstract
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 (Delta omega) 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 F-19-NMR framework, one can detect mu M concentrations of the complex and study the effect of different solutes on the resulting host-guest system. The results expand the "NMR toolbox" available to explore a wider range of dynamic host-guest systems in which NMR signals cannot be detected.
(2015) Stem cells translational medicine. 4, 12, p. 1472-1481 Abstract
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 (F-19) 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 radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% 15.8% CD45+, 24.6% +/- 12.5% CD34+, and 7.5% +/- 3.3% CD31+ cells, with 2.1 +/- 0.7 x 10(5) cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% +/- 13.5% of CD34+ progenitor cells compared with 47.8% +/- 18.5% of hematopoietic CD45+ cells, with an average of 2.8 +/- 2.0 X 10(12) F-19 atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% +/- 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% +/- 22.3% of CD45-/CD31-/CD34- (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of F-19 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 silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 x 10(6) 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.
(2015) Magnetic Resonance in Medicine. 74, 2, p. 544-549 Abstract
PurposeTo develop an imaging tool that enables the detection of malignant tissue with enhanced specificity using the exquisite spatial resolution of MRI. MethodsTwo 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. Results9L(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. ConclusionThe 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. (c) 2015 Wiley Periodicals, Inc.
(2015) ACS Chemical Biology. 10, 5, p. 1160-1170 Abstract
Recent advancements in molecular and synthetic 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 MM. 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,1 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.
Advances in using MRI probes and sensors for in vivo cell tracking as applied to regenerative medicine(2015) Disease Models & Mechanisms. 8, 4, p. 323-336 Abstract
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.
(2015) Nature Communications. 6, 6719. Abstract
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.[All authors]
(2015) Biomaterials. 42, p. 144-150 Abstract
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 in vivo. 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 in vivo. The CEST properties of the three individual hydrogel components (HA, GelinS, and polyethylene glycol diaciylate) were first measured in vitro. 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. In vitro, GelinS gave the strongest CEST signal at 3.6 ppm offset from the water peak, originating from the amide protons present in gelatin. In vivo, 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. (C) 2014 Elsevier Ltd. All rights reserved.
(2015) Journal of the American Chemical Society. 137, 1, p. 78-81 Abstract
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 F-19 chelate analogue of existing optical dyes. Taking advantage of the difference in the ion-specific F-19 nuclear magnetic resonance (NMR) chemical shift offset (Delta omega) 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.
(2015) Chemical Communications. 51, 23, p. 4869-4871 Abstract
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.
(2015) ACS Macro Letters. 4, 1, p. 34-38 Abstract
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 (Keq) 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.
(2014) Magnetic Resonance in Medicine. 72, 3, p. 823-828 Abstract
Purpose: Demonstrate applicability of natural D-glucose as a T-2 MRI contrast agent. Methods: D-glucose solutions were prepared at multiple concentrations and variable pH. The relaxation rate (R-2 = 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 22 degrees 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 T-1 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. (C) 2014 Wiley Periodicals, Inc.
(2014) NMR in Biomedicine. 27, 7, p. 774-783 Abstract
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 250 mu M 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 T-2 relaxation of the water protons in their vicinity. The new alginatecoated SPIO NP formulations, which have no effect on cell viability for 24 h, 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. Copyright (C) 2014 John Wiley & Sons, Ltd.[All authors]
Variable Delay Multi-Pulse Train for Fast Chemical Exchange Saturation Transfer and Relayed-Nuclear Overhauser Enhancement MRI(2014) Magnetic Resonance in Medicine. 71, 5, p. 1798-1812 Abstract
PurposeChemical 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. MethodsThe 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. ResultsWhen 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. ConclusionsVDMP-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. Magn Reson Med 71:1798-1812, 2014. (c) 2013 Wiley Periodicals, Inc.
(2014) ACS Chemical Biology. 9, 1, p. 134-138 Abstract[All authors]
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 acid-long 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.2013 Wiley Periodicals, Inc.
Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI(2013) NATURE PROTOCOLS. 8, 12, p. 2380-2391 Abstract
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.
(2013) Journal of the American Chemical Society. 135, 33, p. 12164-12167 Abstract
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 F-19 NMR spectroscopy to obtain magnetic resonance images of Ca2+. We exploited the chemical shift change (Delta omega) of F-19 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 F-19 frequency and detection of the label transfer to the Ca2+-free F-19 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.
(2013) Journal of the American Chemical Society. 135, 4, p. 1617-1624 Abstract[All authors]
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.
Early in vivo MR spectroscopy findings in organophosphate-induced brain damageupotential biomarkers for short-term survival(2012) Magnetic Resonance in Medicine. 68, 5, p. 1390-1398 Abstract
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 (46% 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. Magn Reson Med, 2012. (c) 2012 Wiley Periodicals, Inc.
(2012) Magnetic Resonance in Medicine. 68, 6, p. 1919-1923 Abstract
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. Magn Reson Med, 2012. (c) 2012 Wiley Periodicals, Inc.
Use of MR Cell Tracking to Evaluate Targeting of Glial Precursor Cells to Inflammatory Tissue by Exploiting the Very Late Antigen-4 Docking Receptor(2012) Radiology. 265, 1, p. 175-185 Abstract
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 6 11.7 vs 36.4 cells per field of view +/- 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[All authors]
Detection of rapidly exchanging compounds using on-resonance frequency-labeled exchange (FLEX) transfer(2012) Magnetic Resonance in Medicine. 68, 4, p. 1048-1055 Abstract
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. (c) 2012 Wiley Periodicals, Inc.
(2012) Magnetic Resonance in Medicine. 68, 4, p. 1074-1086 Abstract
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 B0 artifacts. Magn Reson Med, 2012. (c) 2012 Wiley Periodicals, Inc.[All authors]
(2011) Journal of Controlled Release. 156, 2, p. 239-245 Abstract
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. (C) 2011 Elsevier B. V. All rights reserved.[All authors]
Monitoring Enzyme Activity Using a Diamagnetic Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agent(2011) Journal of the American Chemical Society. 133, 41, p. 16326-16329 Abstract[All authors]
Chemical exchange saturation transfer (CEST) is a new approach for generating magnetic resonance imaging (MM) 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 MM 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 MM. Consequently, CDase activity was imaged with high-resolution CEST MM. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MM has the potential to follow the kinetics of multiple enzymes in real time in living tissue.
(2011) Proceedings of the National Academy of Sciences of the United States of America. 108, 21, p. 8838-8843 Abstract[All authors]
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.
Late Stimulation of the Sphenopalatine-Ganglion in Ischemic Rats: Improvement in N-Acetyl-Aspartate Levels and Diffusion Weighted Imaging Characteristics as Seen by MR(2010) Journal of Magnetic Resonance Imaging. 31, 6, p. 1355-1363 Abstract
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: (1)H-MRS imaging ((1)H-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 (1)H-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.
(2009) NeuroImage. 48, 1, p. 109-116 Abstract
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., Delta = 200 ms. Conventional DTI performed with relatively low b-values (b
Observation of restricted diffusion in the presence of a free diffusion compartment: Single- and double-PFG experiments(2009) Journal of Magnetic Resonance. 200, 2, p. 214-225 Abstract
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. (C) 2009 Elsevier Inc. All rights reserved.
The effect of the diffusion time and pulse gradient duration ratio on the diffraction pattern and the structural information estimated from q-space diffusion MR: Experiments and simulations(2008) Journal of Magnetic Resonance. 194, 2, p. 230-236 Abstract
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, delta, is Comparable to the diffusion time, Delta. In this study, we used phantoms, consisting of ensembles Of MiCFOtubes, and mathematical models to assess the effect of the ratio of the diffusion time and the duration of the diffusion pulse gradient, i.e., Delta/delta, on the MR signal attenuation vs. q, and on the measured structural information extracted therefrom. We found that for Delta/delta similar to 1, the diffraction pattern obtained from q-space MR data are shallower than when the short gradient pulse (SGP) approximation is satisfied. For long delta the estimated compartment size is, as expected, smaller than the real size. Interestingly, for Delta/delta similar to 1 the diffraction peaks are shifted to even higher q-values, even when delta 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 Delta/delta similar to 1. (C) 2008 Elsevier Inc. All rights reserved.
(2008) NMR in Biomedicine. 21, 8, p. 888-898 Abstract
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 disappearance when the gradient orientation possesses a small axial component. The model also successfully predicts the dependence of E(q) oil gradient pulse duration and oil gradient strength as well as tube inner diameter. To account for the deviation front 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 complex media containing pores 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. Copyright (C) 2008 John Wiley & Sons. Ltd.
Crossing fibers, diffractions and nonhomogeneous magnetic field: correction of artifacts by bipolar gradient pulses(2008) Magnetic Resonance Imaging. 26, 6, p. 801-808 Abstract
In recent years, diffusion tenser 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. (C) 2008 Elsevier Inc. All rights reserved.
(2008) Journal of Clinical Investigation. 118, 4, p. 1532-1543 Abstract[All authors]
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 of nerves: structural information and comparison with histological evidence(2008) NMR in Biomedicine. 21, 2, p. 165-174 Abstract
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 I 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. Copyright (C) 2007 John Wiley & Sons, Ltd.
The effect of the rotational angle on MR diffusion indices in nerves: Is the rms displacement of the slow-diffusing component a good measure of fiber orientation?(2008) Journal of Magnetic Resonance. 190, 1, p. 33-42 Abstract
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 (alpha), 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)approximate to 1200 s/mm(2)), 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 alpha values ranging from -10 degrees to +80 degrees (where alpha=0 degrees is the z direction), but it changed dramatically when diffusion was measured nearly perpendicular to the nerve fiber direction, i.e., for alpha=90 +/- 10 degrees. The ADC and the rms displacement of the fast-diffusing component exhibited only gradual changes, with a maximal change at alpha=45 +/- 15 degrees. 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.
(2005) Journal of Organic Chemistry. 70, 7, p. 2660-2666 Abstract
A series of new adamantyl-oligoethyleneglycol-fullerene hybrids was prepared via Bingel-Hirsch functionalization of the C-60 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.