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

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

A snapshot of the vast array of diamagnetic CEST MRI contrast agents.

Since the inception of CEST MRI in the 1990s, a number of compounds have been identified as suitable for generating contrast, including paramagnetic lanthanide complexes, hyperpolarized atom cages and, most interesting, diamagnetic compounds. In the past two decades, there has been a major emphasis in this field on the identification and application of diamagnetic compounds that have suitable biosafety profiles for usage in medical applications. Even in the past five years there has been a tremendous growth in their numbers, with more and more emphasis being placed on finding those that can be ultimately used for patient studies on clinical 3 T scanners. At this point, a number of endogenous compounds present in tissue have been identified, and also natural and synthetic organic compounds that can be administered to highlight pathology via CEST imaging. Here we will provide a very extensive snapshot of the types of diamagnetic compound that can generate CEST MRI contrast, together with guidance on their utility on typical preclinical and clinical scanners and a review of the applications that might benefit the most from this new technology.

Exploring the potential of the novel imidazole-4,5-dicarboxyamide chemical exchange saturation transfer scaffold for pH and perfusion imaging.

Here, we describe and assess the potential of 14 newly synthesized imidazole-4,5-dicarboxyamides (I45DCs) for pH and perfusion imaging. A number of these aromatic compounds possess large labile proton chemical shifts (up to 7.7 ppm from water) because of their intramolecular hydrogen bonds and a second labile proton to allow for chemical exchange saturation transfer (CEST) signal ratio-based pH measurements. We have found that the contrast produced is strong for a wide range of substitutions and that the inflection points in the CEST signal ratio versus pH plots used to generate concentration-independent pH maps can be adjusted based on these subsitutions to tune the pH range that can be measured. These I45DC CEST agents have advantages over the triiodobenzenes currently employed for tumor and kidney pH mapping, both preclinically and in initial human studies. Finally, as CEST MRI combined with exogenous contrast has the potential to detect functional changes in the kidneys, we evaluated our highest performing anionic compound (I45DC-diGlu) on a unilateral urinary obstruction mouse model and observed lower contrast uptake in the obstructed kidney compared with the unobstructed kidney and that the unobstructed kidney displayed a pH of ~ 6.5 while the obstructed kidney had elevated pH and an increased range in pH values. Based on this, we conclude that the I45DCs have excellent imaging properties and hold promise for a variety of medical imaging applications, particularly renal imaging.

Intramolecular Hydrogen Bonding Based CEST MRI Contrast Agents As an Emerging Design Strategy: A Mini-Review.

Intramolecular hydrogen bonding-based chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) contrast agents represent an innovative design strategy aiming to overcome limitations in diamagnetic CEST (diaCEST) MRI contrast agent specificity and also those associated with traditional metal-based MRI contrast agents. Ward and Balaban's proposal of small diamagnetic compounds marked a paradigm shift in contrast-based radiologic research, inspiring extensive investigations since 2000. These contrast agents leverage labile hydrogen bonds, serving as chemical exchange sites to induce saturation of water. The selective manipulation of radiofrequency (RF) allows for optimized signal contrast in soft tissue, with a significant signal amplification even at low probe concentrations, mitigating concerns about dose-dependent toxicities. This mini-review delves into the evolution of CEST MRI, its classification, and the strategic design principles of synthetic small molecules containing intramolecular hydrogen bonds. With a focus on applications and potential clinical relevance, the authors highlight the promising role of intramolecular hydrogen bonding-based CEST MRI in diverse medical contexts, especially renal imaging and pH mapping, paving the way for enhanced molecular imaging capabilities. Ongoing research endeavors aim to further optimize and expand the utility of these contrast agents, underscoring their transformative potential in clinical diagnostics and imaging.

The Proton Resonance Enhancement for CEST imaging and Shift Exchange (PRECISE) family of RF pulse shapes for Chemical Exchange Saturation Transfer MRI.

Purpose: To optimize a 100 msec pulse for producing CEST MRI contrast and evaluate in mice. Methods: A gradient ascent algorithm was employed to generate a family of 100 point, 100 msec pulses for use in CEST pulse trains ('PRECISE'). Gradient ascent optimizations were performed for exchange rates (k ca ) = 500 s -1 , 1,500 s -1 , 2,500 s -1 , 3,500 s -1 and 4,500 s -1 and offsets (Δω) = 9.6, 7.8, 4.2 and 2.0 ppm. 7 PRECISE pulse shapes were tested on an 11.7 T scanner using a phantom containing three representative CEST agents with peak saturation B 1 = 4 µT. The pulse producing the most contrast in phantoms was then evaluated for CEST MRI pH mapping of the kidneys in healthy mice after iopamidol administration. Results: The most promising pulse in terms of contrast performance across all three phantoms was the 9.6 ppm, 2500 s -1 optimized pulse with ∼2.7 x improvement over Gaussian and ∼1.3x's over Fermi pulses. This pulse also displayed a large improvement in contrast over the Gaussian pulse after administration of iopamidol in live mice. Conclusion: A new 100 msec pulse was developed based on gradient ascent optimizations which produced better contrast compared to standard Gaussian and Fermi pulses in phantoms. This shape also showed a substantial improvement for CEST MRI pH mapping in live mice over the Gaussian shape and appears promising for a wide range of CEST applications.

Modulation of protein-graphene oxide interactions with varying degrees of oxidation.

The degree of oxidation of graphene oxide (GO) has been shown to be important for its toxicity and drug-loading efficiency. However, the effect of its variations on GO-protein interaction remains unclear. Here, we evaluate the effect of the different oxidation degrees of GO on its interaction with human ubiquitin (8.6 kDa) using solution state nuclear magnetic resonance (NMR) spectroscopy in combination with other biophysical techniques. Our findings show that the interaction between the protein and the different GO samples is weak and electrostatic in nature. It involves fast dynamic exchange of the protein molecules from the surface of the GO. As the oxidation degree of the GO increases, the extent of the interaction with the protein changes. The interaction of the protein with GO can thus be modulated by tuning the degree of oxidation. This study opens up new avenues to design appropriate graphenic materials for use in various biomedical fields such as drug delivery, biomedical devices and imaging.

Influence of Oxidation Degree of Graphene Oxide on Its Nuclear Relaxivity and Contrast in MRI.

Graphene oxide (GO) serves as a versatile platform for various applications, with the oxygen content of GO playing an important role in governing its properties. In the present study, different GO types covering a wide range of oxidation degree were prepared using our newly developed two-step method involving ball milling of graphite followed by its oxidation to GO. In addition to the variations in their physicochemical properties, the different GO types exhibited differences in proton relaxivity due to their paramagnetic nature. Nuclear magnetic resonance spectroscopy studies showed that the degree of oxidation of GO perturbs its nuclear relaxation properties and, together with intercalated Mn2+ ions, provides large contrast variation in magnetic resonance imaging (MRI). The study for the first time reveals that the surface chemistry of GO affects its relaxivity and opens up new avenues for developing tunable GO-based contrast agents in magnetic resonance imaging for diagnostics and therapies.

Correlation between defect density in mechanically milled graphite and total oxygen content of graphene oxide produced from oxidizing the milled graphite.

It has been reported that defect density in ball-milled graphite lattice increases with the milling time. Guided by this, we hypothesized that the oxygen content of graphene oxide can be substantially enhanced by oxidizing ball-milled graphite and also, the oxygen content would monotonically increase with the milling time as the defect sites would be preferred sites for oxidation. Interestingly, we observed that this correlation was not directly proportional for all milling hours. Even though, the defect density of graphite monotonically increased with milling time, the oxygen content of graphene oxide initially increased and then decreased. This was due to milling time dependent change in the size of the graphite plates and consequent relative abundance of the different oxygen containing functional groups on graphene oxide (GO) produced from the milled graphite.